AU766787B2 - Meth1 and meth2 polynucleotides and polypeptides - Google Patents

Meth1 and meth2 polynucleotides and polypeptides Download PDF

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AU766787B2
AU766787B2 AU24641/99A AU2464199A AU766787B2 AU 766787 B2 AU766787 B2 AU 766787B2 AU 24641/99 A AU24641/99 A AU 24641/99A AU 2464199 A AU2464199 A AU 2464199A AU 766787 B2 AU766787 B2 AU 766787B2
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amino acids
polypeptide
meth2
garni
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Gregg A Hastings
Luisa Iruela-Arispe
Steven M. Ruben
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Human Genome Sciences Inc
Beth Israel Deaconess Medical Center Inc
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Beth Israel Deaconess Medical Center Inc
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Description

WO 99/37660 PCT/US99/01313 METH1 and METH2 Polynucleotides and Polypeptides Background of the Invention Federally-Sponsored Research and Development Part of the work performed during development of this invention utilized U.S. Government funds. The U. S. Government has certain rights in this invention.
Field of the Invention The present invention relates to novel anti-angiogenic proteins, related to thrombospondin. More specifically, isolated nucleic acid molecules are provided encoding human METH1 and METH2 (ME, for metalloprotease, and TH, for thrombospondin). METH1 and METH2 polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the same. Also provided are diagnostic methods for the prognosis of cancer and therapeutic methods for treating individuals in need of an increased amount of METH1 or METH2.
Related Art Angiogenesis, the formation of new blood vessels from pre-existing vasculature, is a tightly regulated process in normal adults. Under physiological circumstances, growth of new capillaries is tightly controlled by an interplay of growth regulatory proteins which act either to stimulate or to inhibit blood vessel growth. Normally, the balance between these forces is tipped in favor of inhibition and consequently blood vessel growth is restrained. Under certain pathological circumstances, however, local inhibitory controls are unable to restrain the increased activity of angiogenic inducers. Angiogenesis is a key step in the metastasis of cancer (Folkman, Nature Med. 1:27-31 (1995)) and in abnormal wound healing, inflammation, rheumatoid arthritis, psoriasis, and diabetic retinopathy, it is integral to the pathology (Folkman et al., Science 235:442-447 (1987)), engendering the hope that these pathological entities could be regulated WO 99/37660 PCT/US99/01313 -2by pharmacological and/or genetic suppression of blood vessel growth (Iruela- Arispe et al., Thromb. Haem. 78:672-677 1997)).
Thrombospondin-1 (TSP-I) is a 450 kDa, anti-angiogenic adhesive glycoprotein released from activated platelets and secreted by growing cells (reviewed in Adams, Int. J. Biochem. Cell. Biol. 29:861-865 (1997)). TSP-1 is a homotrimer, with each subunit comprised of a 1152 amino acid residue polypeptide, post-translationally modified by N-linked glycosylation and betahydroxylation of asparagine residues.
TSP-1 protein and mRNA levels are regulated by a variety of factors.
TSP-1 protein levels are downregulated by IL-1 alpha and TNF alpha. TSP-1 mRNA and protein levels are upregulated by polypeptide growth factors including PDGF, TGF-beta, and bFGF (Bornstein, Faseb J. 6: 3290-3299 (1992)) and are also regulated by the level of expression of the p5 3 tumor suppressor gene product (Dameron et al., Science 265:1582-1584 (1994)). At least four other members of the thrombospondin family have been identified: TSP-2, TSP-3, TSP-4, and (also called COMP). There is a need in the art to identify other molecules involved in the regulation of angiogenesis.
Summary of the Invention The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the METH1 polypeptide having the amino acid sequence shown in SEQ ID NO:2 or the amino acid sequence encoded by the cDNA clone deposited in a bacterial host as ATCC Deposit Number 209581 on January 15, 1998.
The present invention also provides isolated nucleic acid molecules comprising a polynucleotide encoding the METH2 polypeptide having the amino acid sequence shown in SEQ ID NO:4 or the amino acid sequence encoded by the cDNA clone deposited in a bacterial host as ATCC Deposit Number 209582 on January 15, 1998.
WO 99/37660 PCT/US99/01313 -3- The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of METHI or METH2 polypeptides or peptides by recombinant techniques.
The invention further provides an isolated METH1 or METH2 polypeptide having an amino acid sequence encoded by a polynucleotide described herein.
The invention further provides a diagnostic method useful during diagnosis or prognosis of cancer.
An additional aspect of the invention is related to a method for treating an individual in need of an increased level ofMETH1 or METH2 activity in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of an isolated METHI or METH2 polypeptide of the invention or an agonist thereof.
Brief Description of the Figures Figure 1 shows the nucleotide (SEQ ID NO:1) and deduced amino acid (SEQ ID NO:2) sequences of METH1. The protein has a predicted leader sequence of about 28 amino acid residues (underlined).
Figure 2 shows the nucleotide (SEQ ID NO:3) and deduced amino acid (SEQ ID NO:4) sequences of METH2. The protein has a predicted leader sequence of about 23 amino acid residues (underlined).
Figure 3 shows a comparison of the amino acid sequence ofMETH 1 (SEQ ID NO:2) and METH2 (SEQ ID NO:4) with that of their closest homologue, a bovine metalloprotease (pNPI) (SEQ ID NO:5). Identical amino acids are boxed.
Functional domains predicted by sequence and structural homology are labeled, including the signal peptide (single line), the potential cleavage site for mammalian subtilisin (double underlined), the zinc-binding-site (dotted line) in the metalloprotease domain, and the putative disintegrin loops (arrows).
WO 99/37660 PCT/US99/01313 -4- Figure 4 shows the primary structure of METH METH2 and pNPI which includes a prodomain, a catalytic metalloprotease domain, a cysteine rich disintegrin domain, a TSP-like domain, a spacer region and a different number of TSP-like domains, three for METH1, two for METH2, and four for pNPI.
Figure 5 shows a comparison of the TSP-like domain ofMETH1 (SEQ ID NO:2) and METH2 (SEQ ID NO:4) with those ofTSPl (SEQ ID NOs:6, 7, and 8) and TSP2 (SEQ ID NOs:9, 10, and 11), cysteines are numbered I to 6, tryptophans are marked by asterisks.
Figure 6 shows that peptides and recombinant protein derived from the TSP-like domain of METH1 and METH2 block VEGF-induced angiogenesis.
Angiogenesis was induced on CAMs from 12-14-day-old embryos using a nylon mesh containing VEGF casted on matrigel and in the presence or absence of the peptides or recombinant protein. Capillary density was evaluated as described in Example 4. Positive and negative control included VEGF alone and vehicle alone, respectively. Quantification of the angiogenic response induced by VEGF in the presence of recombinant proteins. TSPI, purified platelet TSP1, GST, purified GST, GST-TSP1, GST-METH1, and GST-METH2 are described in Example 4. Quantification of the angiogenic response induced by VEGF in the presence or absence of the peptides; P-TSP1, P-METH1, and P-METH2 (peptide derived from the Type I repeats of TSP, METH1 and METH2, respectively); SCI and SC2 are scramble peptides used as controls. Doseresponse of the VEGF-induced angiogenesis in the presence of GST-METH1.
Dose-response of the VEGF-induced angiogenesis in the presence of GST- METH2. The angiogenic index was expressed considering the vascular response from the VEGF-matrigel as 100% and subtracting the background levels (matrigel alone). Assays were repeated, at least, twice. Each treatment was done in triplicate. Values represent the mean, bars indicate standard deviations. *p<0.001.
Figure 7 shows the effect of METHI and METH2 recombinant proteins on bFGF-stimulated cell proliferation. Cells were cultured on 24-well plates in media containing bFGF and the recombinant protein to be tested (3 pg/ml, unless WO 99/37660 PCTIS99/01313 indicated in the graph). Controls included vehicle or GST recombinant protein alone. HDEC, human dermal endothelial cells; HMEC, human mammary epithelial cells; HDF, human dermal fibroblasts; SMC, smooth muscle cells; Dose-response of GST-METHI and GST-METH2 on HDEC proliferation. Experiments were repeated, at least, twice. Each treatment was done in triplicate. Values represent the mean, bars indicate standard deviations.
*p<0.01.
Figure 8 shows a schematic representation of the pHE4-5 expression vector (SEQ ID NO:12) and the subcloned METH1 or METH2 cDNA coding sequence. The locations of the kanamycin resistance marker gene, the METH or METH2 coding sequence, the oriC sequence, and the laclq coding sequence are indicated.
Figure 9 shows the nucleotide sequence of the regulatory elements of the pHE promoter (SEQ ID NO:13). The two lac operator sequences, the Shine-Delgarno sequence and the terminal HindII and NdeI restriction sites (italicized) are indicated.
Figure 10 shows an analysis of the METH1 amino acid sequence. Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown, and all were generated using the default settings. In the "Antigenic Index or Jameson-Wolf" graph, the positive peaks indicate locations of the highly antigenic regions of the METHI or METH2 protein, regions from which epitopebearing peptides of the invention can be obtained. The domains defined by these graphs are contemplated by the present invention. Tabular representation of the data summarized graphically in Figure 10 can be found in Table 1.
Figure 11 shows an analysis of the METH2 amino acid sequence. Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown, and all were generated using the default settings. In the "Antigenic Index or Jameson-Wolf" graph, the positive peaks indicate locations of the highly antigenic WO 99/37660 PCT/US99/01313 -6regions of the METHI or METH2 protein, regions from which epitopebearing peptides of the invention can be obtained. The domains defined by these graphs are contemplated by the present invention. Tabular representation of the data summarized graphically in Figure 11 can be found in Table 2.
S 10 S 15 Table 1 Res Pos. Garni.. Chou-... Garni.. Chou-... Garni... Chou-... Garni.. Kyte-... Eisen.. Eisen.. Karpi James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro,... Alpha Beta Flexi Antig Surra...
Met I A A 0.41 0.60 Gly 2 .A C 0.91 *.0.50 0.81 Asn 3 A A 0.71 *.0.75 1.24 Ala 4 A A 0.89 *.1.09 1.26 Glu 5 A A 0.93 *F 1.58 1.97 Arg 6 .A B 1.23 F 1.92 1.21 Ala 7 B T .1.69 F 2.66 1.61 Pro 8 .T T .1.39 F 3.40 1.82 Gly 9 .T T .1.28 F 3.06 1.25 Ser 10 T T .0.93 F 2.42 1.07 Arg I I .T T .0.61 1.93 0.68 Ser 12 .T T .0.34 *F 1.74 1.07 Phe 13 B .T .0.34 *F 0.25 0.59 Gly 14 B T .0.38 0.25 0.47 Pro 15 B B -0.13 *F -0.45 0.50 Val 16 B B -1.06 -0.45 0.48 Pro 17 B B 1.57 F -0.45 0.40 Thr 18 .A B 1.68 F -0.45 0.21 Leu 19 .A B 1.92 0.60 0.24 Leu 20 A A 2.30 -0.60 0.15 Leu 21 A A -2.03 0.60 0.I11 Res Pos. Garni.. Chou-... Garni.. Chou-... Garni... Chou-... Garni.. Kyte-... Eisen.. Eisen.. Karpl James Emini Alpha Alpha Beta Beta Turn Turn Coil ilydro... Alpha Beta Flexi Antig Surfa...
Leu 22 A A 2.63 0.60 0.13 Ala 23 A A 3.13 0.60 0.13 Ala 24 A A 2.91 0.60 0.13 Ala 25 A A 2.96 0.60 0.16 Leu 26 A A B3. -2.44 0.60 0.12 Leu 27 A A B3. -1.63 0.60 0.16 Ala 28 A A B 1.63 0.30 0.26 Val 29 A A B -1.86 0.30 0.32 Ser 30 A A 1.61 0.32 Asp 31 A A 0.69 *F -0.15 0.31 Ala 32 A A 0.09 *F 0.75 0.83 Leu 33 .A C 0.20 1.55 0.96 Gly 34 .A C 1.06 F 1.85 0.77 Arg 35 T C 1.36 F 2.70 1.32 Pro 36 T C 1.36 3.00 2.76 Scr 37 T C 1.94 2.70 4.66 Glu 38 A T .2.76 2.20 4.12 Glu 39 A A 2.29 F 1.50 4.61 Asp 40 A A 1.32 F 1.20 2.84 Glu 41 A A .0.68 F 0.90 1.22 Glu 42 A A 0.77 F 0.75 0.52 Leu 43 A A 0.77 .060.4 .Ln 10 15 Res Pos. Garni.. Chou,.. Garni.. Chou,.. Garni... Chou-... Garni.. Kyte-... Eisen.. Eisen.. Karpi James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi Antig Surfa...
Val 44 A A 0.04 0.60 0.48 Val 45 A A 0.04 0.23 Pro 46 A A 0.07 0.48 Glu 47 A 0.52 1.10 1.27 Leu 48 A 0.08 1.41 1.73 Glu 49 A 0.59 *F 1.72 1.73 Arg 50 A 1.41 *F 1.88 0.99 Ala 51 A T .1.28 2.24 1.64 Pro 52 T T .0.97 3.10 0.93 Gly 53 T T .1.47 F 2.49 0.69 His 54 T C 1.58 F 1.38 0.98 Gly 55 C 0.66 1.62 1.25 Thr 56 C 1.36 0.71 1.04 Thr 57 .A B 0.76 0.60 1.49 Arg 58 .A B .1.07 0.60 1.25 Leu 59 .A B 0.51 1.17 Arg 60 .A B 0.16 0.30 0.821 Leu 61 .A B 0.47 .0.30 0.36 His 62 .A B 0.78 .0.30 0.74 Ala 63 A A .0.67 0.65 Phe 64 A 0.67 .15 1.37 Asp 65 A A .0.56 -0.15 0.83
N
Ln. 10 Res Pos. Garni.. Chou-... Garni.. Chou-... Garni.. Chou-... Garni.. Kyte-... Eisen.. Eisen.. Karpi James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi Antig Surfa...
Gin 66 A A 0.56 0.60 1.37 Gin 67 A A 0.59 0.60 1.30 Leu 68 A A 0.37 I *F 0.90 1.35 Asp 69 A A 1.18 .0.30 0.64 Leu 70 .A B 0.97 0.73 Glu 71 A B 0.97 1.37 Leu 72 .A B 0.67 1.77 1.37 Arg 73 T C 1.18 I *F 2.86 2.22 Pro 74 T T .0.48 $F 3.40 1.72 Asp 75 .T T .0.48 2.76 1.80 Ser 76 T C -0.11 *F 2.07 0.76 Ser 77 B 0.49 I *F 0.73 0.50 Phe 78 .B 0.03 .0.24 0.46 Leu 79 B 0.46 .0.40 0.34 Ala 80 B .T 0.77 .0.20 0.22 Pro 81 B T 1.28 0.20 0.37 Gly 82 T T 0.98 0.20 0.37 Phe 83 B .T 0.28 .0.20 0.63 Thr 84 B B 0.32 .0.60 0.65 Leu 85 B B -0.08 .0.60 0.49 GIn 86 B B 0.24 0.56 Asn 87 B T .0.63 .87 0.76 p 10 Res Pos. Garni.. Chou-... Garni.. Chou-... Garni... Chou-... Garni.. Kyte-... Eisen.. Eisen.. Karpl... James... Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi... Antig... Surfa...
Val 88 B T 1.03 F 1.93 1.84 Gly 89 T C 1.00 F 2.74 1.42 Arg 90 T T 1.51 F 3.10 0.87 Lys 91 T C 1.51 F 2.74 1.58 Ser 92 T C 1.20 F 2.43 2.76 Gly 93 T C 1.84 F 2.38 2.04 Ser 94 T C 1.38 F 2.33 1.57 Glu 95 C 1.06 F 1.63 0.97 Thr 96 C 1,01 F 2.04 1.51 Pro 97 C 1.00 F 2.60 1.96 Leu 98 C 1.34 F 2.04 1.63 Pro 99 A 0.83 F 1.58 1.89 Glu 100 A A 0.24 F 1.12 1.01 Thr 101 A A 0.52 F 0.86 1.23 Asp 102 A A 0.07 F 0.60 1.08 Leu 103 A A 0.18 0.30 0.34 Ala 104 A A 0.14 -0.60 0.20 His 105 A B -0.16 -0.60 0.19 Cys 106 A B -0.19 -0.60 0.31 Phe 107 A B -0.50 -0.60 0.30 Tyr 108 B T -0.54 -0.20 0.32 Ser 109 T T 0.04 F 0.35 0.44 Res Pos. Garni.. Chou-... Garni.. Chou-... Garni... Chou,.. Garni.. Kyte-... Eisen.. Eisen.. Karpi James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi... Antig Surfa...
Gily 110 T T 0.27 *F 0.35 0.82 Thr IlII. T T .0.40 *F 0.59 0.52 Val 112 B B 0.89 0.93 0.65 Asn 113 B T 0.83 1.72 1.01 Gly 114 B C 0.83 *F 1.61 0.94 Asp 115 T C 0.59 2.40 1.69 Pro 116 T C 0.31 2.16 1.06 Ser 117 T C 0.58 1.92 1.08 Ser 118 A .T 0.23 F 1.33 0.66 Ala 119 A A 0.19 .0.06 0.35 Ala 120 A A -1.00 .0.30 0.35 Ala 121 A A 1.46 .0.60 0.22 Leu 122 A A -1.16 .0.60 0.11 Ser 123 A A 1.20 .0.30 0.20 Leu 124 A A -1.47 .0.30 0.19 Cys 125 .A B -0.77 .0.30 0.17 Glu 126 .A B -0.52 *.0.30 0.25 Gly 127 A -0.30 0.65 0.30 Val 128 A 0.70 F 0.65 0.57 Arg 129 B 0.13 F 0.65 0.29 Gly 130 B B 0.28 .0.60 0.45 Ala 131 B B -1.09 .0.60 0.50 Res Pos. Garni.. Chou-... Garni.. Chou,.. Garni... Chou-... Garni.. Kyte-... Eisen.. Eisen.. .Karpi James Emini Alpha Alpha Beta Beta Turn Turn Coil Hlydro... Alpha Beta Flexi... Antig Surfa...
Phe 132 B B 1.09 0.60 0.21 Tyr 133 B B 0.23 0.60 0.21 Leu 134 .A B B 0.93 0.60 0.36 Leu 135 .A B B 0.83 *-0.60 0.42 Gly 136 A A .B 0.94 0.60 0.42 Glu 137 A A 1.13 0.60 0.44 Ala 138 A A .B 0.89 0.60 0.38 Tyr 139 B 13 0.29 0.60 0.66 Phe 140 B B 0.29 -0.60 0.59 lie 141 B B 0.16 0.60 0.48 Gin 142 B B 0.74 0.60 0.48 Pro 143 B B 0.74 0.60 0.55 Leu 144 .A C -0.80 0.80 Pro 145 .A C -0.10 0.62 Ala 146 A A 0.90 .0.30 0.69 Ala 147 A A 0.09 1.64 Ser 148 A A 0.29 *F 0.75 0.88 Glu 149 A A. 0.21 *F 0.45 0.88 Arg 150 A A -0.17 0.60 1.25 Leu 151 A A -0.17 0.30 0.94 Ala 152 A A .0.21 .30 0.55 FThr 153 A A .0.17 .0.04 0.43 Res Pos. Garni.. Chou-... Garni.. Chou-... Garni... Chou-... Garni.. Kyte-... Eisen.. Eisen.. Karpi James... Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi Antig... Surfa...
Ala 154 A A 0.17 0.08 0.52 Ala 155 T C 0.10 2.07 0.89 Pro 156 T 0.70 F 2.86 1.24 Gly 157 T T .1.08 F 3.40 1.90 Glu 158 T C 0.80 F 2.86 2.90 Lys 159 1 .18 F 2.32 1.90 Pro 160 C 0.96 *F 1.98 2.97 Pro 161 C 1.37 1.64 3.41 Ala 162 A A 0.831 F 0.60 3.22 Pro 363 A A 0.78 0.68 Leu 364 A A 0.08 0.60 Gin 165 A A 0.68 0.60 0.49 Phe 166 .A B -0.36 0.26 His 167 .A B 0.34 0.62 Leu 168 .A B 0.56 0.70 Leu 369 .A B3. 1.48 .0.87 1.31 Arg 170 T T 1 .48 3.06 1.88 Arg 171 .T T .1.83 *F 3.40 3.96 Asn 172 .r T 1 .87 3.06 4.75 Arg 173 .T T 1 .82 2.72 4.05 Gin 174 .T .9 1 24.53 Gly 175 T 1 .83 I .F 2.19 0.94 ~s 15 Res Pos. Garni.. Chou-... Garni.. Chou-... Garni... Chou-... Garni.. Kyte-... Eisen.. Eisen.. Karpl... James... Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi... Antig... Surfa...
Asp 176 T T 1.41 F 2.30 0.48 Val 177 B T 0.74 F 1.85 0.40 Gly 178 T T 0.29 F 2.50 0.22 Gly 179 B T -0.57 F 1.85 0.13 Thr 180 B B -1.08 F 0.30 0.13 Cys 181 B B -1.08 -0.10 0.10 Gly 182 B B -0.22 -0.05 0.16 Val 183 B B 0.12 0.30 0.19 Val 184 B B 0.26 0.90 0.60 Asp 185 B T 0.68 F 1.75 0.94 Asp 186 B T 1.13 F 2.20 2.49 Glu 187 B T 1.17 F 2.50 5.18 Pro 188 T C 1.68 F 3.00 4.48 Arg 189 T C 2.58 F 2.70 2.66 Pro 190 T C 1.99 F 2.40 3.07 Thr 191 T C 1.99 F 2.10 2.00 Gly 192 T C 1.68 F 1.80 1.77 Lys 193 A A 1.89 F 0.90 1.65 Ala 194 A A 1.78 F 0.90 1.98 Glu 195 A A 1.99 F 0.90 3.35 Thr 196 A A 2.30 F 0.90 2.90 Glu 197 A A 2.64 F 0.90 4.79 10 t<
J;
Res Pos. Garni.. Chou-... Garni.. Chou-... Garni... Chou-... Garni.. Kyte-... Eisen.. Eisen.. Karpl... James... Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi... Antig... Surfa...
Asp 198 A A 2.26 F 0.90 4.79 Glu 199 A A 2.53 F 0.90 3.29 Asp 200 A T 2.53 F 1.30 2.74 Glu 201 A T 2.50 F 1.30 2.84 Gly 202 A T 2.50 F 1.30 1.62 Thr 203 A T 2.50 F 1.30 1.68 Glu 204 A A 2.50 F 0.90 1.62 Gly 205 A A 2.16 F 1.20 2.84 Glu 206 A A 1.94 F 1.50 1.95 Asp 207 A T 2.29 F 2.20 1.74 Glu 208 A C 2.31 F 2.30 3.04 Gly 209 T C 2.01 F 3.00 1.85 Pro 210 T T 2.14 F 2.60 1.48 Gin 211 T T 2.14 F 2.30 1.32 Trp 212 T C 2.14 F 1.44 2.32 Ser 213 C 1.93 F 1.78 2.50 Pro 214 T T 1.69 F 2.12 2.23 Gin 215 T C 1.09 F 1.56 2.15 Asp 216 T. C 1.09 F 2.40 1.32 Pro 217 T C 1.03 F 2.16 1.48 Ala 218 T 0.48 F 1.77 0.85 Leu 219 B 0.34 F 0.53 0.38 Ea 10 Res Pos. Garni.. Chou-... Garni.. Chou-... Garni... Chou-... Garni.. Kyte-... Eisen.. Eisen.. Karpl... James... Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi... Antig... Surfa...
Gin 220 B 0.34 F -0.01 0.24 Gly 221 B T 0.13 F -0.05 0.41 Val 222 B T 0.03 F -0.05 0.77 Gly 223 B T 0.28 F 0.25 0.64 Gin 224 B T 0.78 F 0.25 0.64 Pro 225 B 0.43 F 0.20 1.25 Thr 226 T 0.48 F 0.60 1.25 Gly 227 T C 0.44 F 0.45 0.97 Thr 228 B T 0.90 F 0.25 0.44 Gly 229 B T 0.94 F 0.85 0.60 Ser 230 B T 1.20 F 1.30 1.20 lie 231 A B 1.62 F 0.90 1.67 Arg 232 A B 1.27 F 0.90 3.30 Lys 233 A B 0.72 F 0.90 2.13 Lys 234 A B B 0.77 F 0.90 2.26 Arg 235 A B B 0.77 F 0.90 1.55 Phe 236 B B 1.62 0.75 1.04 Val 237 B B 1.62 0.30 0.71 Ser 238 B T 1.33 0.70 0.71 Ser 239 T C 0.43 0.15 1.28 His 240 T C 0.32 0.45 1.28 Arg 241 T C 0.71 1.05 1.65 Res Pos. Garni.. Chou-... Garni.. Chou-... Garni... Chou-... Garni.. Kyle-... Eisen.. Eisen.. Karpi .James... Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi Antig Surfa...
Tyr 242 A B 0.97 *.0.45 1.78 Val 243 A B 0.46 *.0.45 1.29 Glu 244 B B 0.10 0.54 Thr 245 B B 0.66 0.26 Met 246 A .B B 0.77 0.35 Leu 247 A B 0.52 0.30 0.34 Val 248 A B .0.03 0.30 0.41 Ala 249 A B 0.57 0.30 0.55 Asp 250 A T 0.84 F 0.25 0.66 Gln 251 A .T 0.24 F 0.25 0.90 Ser 252 A .T 0.13 F 1.30 1.54 Met 253 A .T .0.69 0.70 0.80 Ala 254 A 0.93 0.63 Glu 255 A 0.63 -0.10 0.46 Phe 256 A 0.29 -0.10 0.63 His 257 A .T 0.22 *.0.10 0.61 Gly 258 A .T .0.42 *F 0.25 0.29 Ser 259 A .T .0.98 F 0.25 0.68 Gly 260 A .T .0.73 F 0.85 0.68 Leu 261 A A 0.62 F 0.00 1.07 Lys 262 A A 0.16 0.60 0.66 His 263 .A B 0.12 -0.60 0.55 Res Pos. Garni.. Chou-... Garni.. Chou,.. Garni... Chou-.. Garni.. Kyte-... Eisen.. Eisen.. Karpi James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi Antig Surfa...
Tyr 264 .A B 0.63 0.60 0.96 Leu 265 .A B 0.99 0.40 Leu 266 .A B 0.48 0.25 Thr 267 .A B 1.38 0.22 Leu 268 .A B 1.93 0.19 Phe 269 A A 2.28 .0.60 0.24 Ser 270 A A 1.36 0.60 0.17 Val 271 A A 1.36 0.39 Ala 272 A A 1.29 0.60 0.38 Ala 273 A A 0.43 0.44 Arg 274 A A .0.23 0.15 1.18 Leu 275 A A .0.32 1.59 Tyr 276 .T 0.88 .1.39 2.44 Lys 277 B .0.58 F 1.48 1.67 His 278 B T .1.28 1.12 1.42 Pro 279 B T .1.17 .F 2.36 1.77 Ser 280 .T T .1.68 3.40 1.43 lie 281 B .T .1.07 2.36 1.41 Arg 282 B B .0.72 *F 1.47 0.67 Asn 283 B B -0.06 1.13 0.67 Ser 284 B B -0.70 F 0.19 0.79 Val 285 B B 1.26 0.30 0.30 £2 10 '9 Res Pos. Garni.. Chou-... Garni.. Chou-... Garni... Chou-... Garni.. Kyte-... Eisen.. Eisen.. Karpl... James... Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi... Antig... Surfa...
Ser 286 B B -1.22 -0.60 0.14 Leu 287 B B -1.29 -0.60 0.08 Val 288 B B -2.18 -0.60 0.21 Val 289 B B -2.69 -0.60 0.11 Val 290 B B -2.69 -0.60 0.11 Lys 291 B B -3.28 -0.60 0.11 lie 292 B B -2.50 -0.60 0.10 Leu 293 B B -1.64 -0.60 0.19 Val 294 B B -0.79 -0.30 0.16 lie 295 B B 0.07 0.00 0.39 His 296 A B 0.07 0.90 0.81 Asp 297 A B 0.61 F 1.80 2.19 Glu 298 A 1.21 F 2.30 3.09 Gin 299 T 2.07 F 3.00 3.51 Lys 300 C 2.10 F 2.50 3.64 Gly 301 T C 1.82 F 2.40 1.56 Pro 302 T C 1.52 F 2.10 1.30 Glu 303 B T 1.52 F 1.45 0.87 Val 304 A T 0.93 F 1.00 1.42 Thr 305 A T 0.30 F 0.85 0.93 Ser 306 A T -0.17 F 0.85 0.54 Asn 307 A T -0.27 F -0.05 0.60 S 10 Res Pos. Garni.. Chou-... Garni.. Chou-... Garni... Chou,.. Garni.. Kyte-... Eisen.. Eisen.. Karpl James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi Antig Surfa...
Ala 308 A T 1.08 0.60 Ala 309 A 0.11 0.40 0.37 Leu 310 A 0.20 0.10 0.45 Thr 311 B 0.20 0.10 0.72 Leu 312 B 0.87 0.40 0.61 Arg 313 B 0.28 0.40 0.40 Asn 314 .T 0.02 0.44 Phe 315 .T T .0.83 *.0.20 0.57 Cys 316 .T T .1.19 .0.20 0.50 Asn 317 .T T .2.00 0.62 Trp 318 .T T .1.86 0.35 1.25 Gin 319 T 1.86 0.45 3.16 Lys 320 T 2.34 *F 0.60 3.16 GIn 321 T..2.80 F 0.94 4.65 His 322 .C 2.50 1.68 4.15 Asn 323 C 2.79 *F 2.02 2.78 Pro 324 T C 2.90 F 2.56 2.68 Pro 325 T T .2.86 3.40 3.86 Ser 326 T C 2.27 F 2.86 4.01 Asp 327 T IC 2.30 F 2.52 2.62 Arg 328 A A .2.27 F 1.58 2.94 Asp 329 T A A 2.23 1.24 2.98 Res Pos. Garni.. Chou-... Garni.. Chou-... Garni... Chou-... Garni.. Kyte-... Eisen.. Eisen.. Karpi James... Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi Antig... Surfa...
Ala 330 A A 2.44 2.80 Glu 331 A A 2.43 *.0.75 2.38 His 332 A T .1.84 *1.15 2.06 Tyr 333 A T .0.84 *.0.85 2.06 Asp 334 A T .0.03 0.70 0.83 Thr 335 A T 0.08 -0.20 0.51 Ala 336 A A 0.39 0.28 lic 337 A A 0.24 -0.60 0.24 Leu 338 .A B 0.00 0.60 0.33 Phe 339 .A B 0.00 -0.60 0.56 Thr 340 .A B 0.50 F 0.00 1.34 Arg 341 .A B 0.58 *F 0.25 1.34 Gln 342 .A T 0.03. 1.35 0.83 Asp 343 .A T 0.48 1.60 0.57 Leu 344 .A .T 1.18 *F 2.15 0.39 Cys 345 .T T .1.18 2.50 0.39 Gly 346 T T 0.40 2.25 0.34 Ser 347 T T .0.40 F 1.10 0.22 Gln 348 B .T .0.09 F 1.35 0.68 Thr 349 B .0.09 F 0.90 0.99 Cys 350 B 0.41 F 0.05 0.61 Asp 351 B .T .0.16 *F 0.25 0.35] Res Pos. Garni.. Chou,.. Garni.. Chou-... Garni... Chou,.. Garni.. Kyte-... Eisen.. Eisen.. Karpi James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro,... Alpha Beta Flexi Antig Surfa...
Thr 352 .B T 0.13 F 0.25 0.24 Leu 353 B T 0.13 0.10 0.45 Gly 354 B T 0.68 0.70 0.45 Met 355 B 0.36 .0.10 0.23 Ala 356 B 0.67 .0.10 0.28 Asp 357 B T 1.21 0.10 0.41 Val 358 B T 1.07 0.10 0.30 Gly 359 B .T 0.72 0.10 0.16 Thr 360 B .T -0.33 0.70 0. 16_ Val 361 B 0.04 0.34 Cys 362 B 0.07 *.1.18 0.46 Asp 363 B .T .0.62 *F 1.87 0.62 Pro 364 .r T .0.30 3.06 1.12 Ser 365 T T .0.31 *F 3.40 1.12 Arg 366 .T T .0.31 F 2.91 0.90 Ser 367 B T 0.09 *F 1.87 0.43 Cys 368 B B .0.09 *.0.38 0.22 Scr 369 B B 0.30 0.64 0.20 Val 370 B B 0.60 *.0.30 0.25 Ile 371 B B 0.14 *.0.60 0.77 Glu 372 B B 0.37 0.60 0.57 Asp 1 373 1 A .T .0.30 F 1.15 0.63 S 10 z 15 Res Pos. Garni.. Chou-... Garni.. Chou,.. Garni... Chou,.. Garni.. Kyte-... Eisen.. Eisen.. Karpl James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi Antig Surfa...
Asp 374 A T .0.01 1.56 Gly 375 A T .0.28 1.00 0.91 Leu 376 A T .0.47 0.55 Gin 377 A A 0.16 .0.30 0.29 Ala 378 A A 0.16 0.42 Ala 379 A A 0.74 0.60 0.73 Phe 380 A A 0.43 0.43 Thr 381 A A .0.38 .0.60 0.57 Thr 382 A A -0.43 0.98 Ala 383 A A 0.19 0.94 His 384 A A 0.37 -0.30 0.64 Glu 385 A A 0.21 -0.30 0.61 Leu 386 A A -0.18 -0.30 0.45 Gly 387 A .B 0.13 -0.60 0.28 His 388 A .B 0.12 0.26 Val 389 A .B -0.06 -0.60 0.32 Phe 390 A .B -0.09 -0.60 0.49 Asn 391 B B 0.72 0.49 Met 392 B T .1.07 0.25 1.11 Pro 393 A T .0.51 *.0.85 2.14 His 394 T T .1.41 *F 1.70 L.34 Asp 395 A T .2.11 *F 1.30 2.72 S 10
I
15 Res Pos. Garni.. Chou-... Garni.. Chou-... Garni... Chou-... Garni.. Kyte-... Eisen.. Eisen.. Karpl... James... Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi... Antig... Surfa...
Asp 396 A A 1.44 F 0.90 3.04 Ala 397 A A 1.46 F 0.90 1.20 Lys 398 A A 1.37 F 0.75 0.73 Gin 399 A A 0.59 0.60 0.58 Cys 400 A B 0.59 -0.30 0.48 Ala 401 A B 0.24 0.30 0.38 Ser 402 B T -0.02 0.10 0.22 Leu 403 B T -0.07 0.04 0.30 Asn 404 T T -0.07 0.68 0.48 Gly 405 T T 0.60 F 1.37 0.62 Val 406 C 0.89 F 1.96 1.26 Asn 407 T C 1.16 F 2.40 1.05 Gin 408 A T 1.37 F 1.96 1.44 Asp 409 A T 0.77 F 1.72 1.92 Ser 410 A T 0.52 1.33 1.18 His 411 A A 1.08 -0.06 0.69 Met 412 A A 0.48 0.30 0.55 Met 413 A A -0.33 -0.60 0.41 Ala 414 A A -0.63 -0.60 0.25 Ser 415 A A -0.33 -0.60 0.34 Met 416 A A -1.11 -0.60 0.55 Leu 417 A T -0.51 -0.20 0.45 Res Pos. Garni.. Chou-... Garni.. Chou-... Garni... Chou,.. Garni.. Kyte-... Eisen.. Eisen.. Karpi James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi Antig Surfa...
Ser 418 A T .0.06 *.0.38 0.56 Asn 419 A T .0.34 .0.66 0.76 Leu 420 .T C 0.64 1.29 1.24 Asp 421 T T .1.03 .2.37 1.60 His 422 T T .1.56 F 2.80 1.54 Ser 423 T C 1.56 F 1.72 1.97 Gln 424 T C 1.34 F 1.44 1.58 Pro 425 1, 1.49 I .F 0.86 1.79 Trp 426 T 1.19 F 0.43 0.72 Ser 427 T C 0.63 I .F 0.15 0.55 Pro 428 T T .0.69 F 0.35 0.36 Cys 429 T T 1 0.09 0.20 0.54 Ser 430 B T 0.59 0.20 0.40 Ala 431 B B 0.61 0.60 0.18 Tyr 432 B B -0.61 0.60 0.49 Met 433 B B -1.10 -0.60 0.49 Ile 434 B B 1.24 0.42 Thr 435 B B 0.94 0.22 Ser 436 B B 0.36 0.37 Phe 437 B B 0.46 -0.60 0.85 Leu 438 B T .0.11 *F 0.56 0.58 Asp 439 T T T .0.66 *F 1.27 0.59 S 10 Res Pos. Garni.. Chou-... Garni.. Chou-... Garni... Chou-... Garni.. Kyte-... Eisen.. Eisen.. Karpi James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi Antig Surfa...
Asn 440 T C 0.97 F 1.38 0.68 Gly 441 T T .0.60 .F 2.94 1.42 His 442 T T .0.49 F 3.10 0.46 Gly 443 A T .0.70 F 1.49 0.23 Glu 444 A T .0.70 1.03 0.23 Cys 445 B .T .0.74 0.29 Leu 446 .A B 0.88 1.25 0.58 Met 447 .A B 0.91 *.1.28 0.52 Asp 448 .A T 1.26 *F 2.02 1.67 Lys 449 .A C 1.04 *F 2.16 3.26 Pro 450 .T T .0.82 5*F 3.40 5.10 Gin 451 T T .1.63 F 3.06 2.14 Asn 452 B .T .1.42 S*F 2.02 1.85 Pro 453 B .T .1.21 0.63 0.99 lie 454 B .0.82 F 0.09 0.88 Gin 455 B 1.03 -0.25 0.54 Leu 456 B .T .0.22 *F 0.25 0.59 Pro 457 B .T .0.01 *F 0.25 0.69 Gly 458 B .T 0.12 0.25 0.62 Asp 459 B .17 0.46 .F 0.25 0.74 Leu 460 T C 0.16 1.05 0.69 Pro 461 B .T .0.72 .F 0.85 0. 9 3 S 10 Res Pos. Garni.. Chou-... Garni.. Chou-... Garni... Chou-... Garni.. Kyte-... [isen.. Eisen.. Karpi James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi Antig Surfa...
Gly 462 B T .0.93 F 0.25 0.88 Thr 463 .B T .0.69 .F 0.74 1.78 Ser 464 B 0.69 .F 1.48 1.16 Tyr 465 .T 1.61 *F 2.22 1.88 Asp 466 T T .1.82 2.61 2.56 Ala 467 .T T .1.50 *F 3.40 3.31 Asn 468 .T T .1.81 .F 2.76 1.13 Arg 469 B .T .1.41 2.32 1.17 Gin 470 B B .1.34 .0.53 1.01 Cys 471 B B 0.64 *.0.04 0.90 Gin 472 B B 0.89 -0.60 0.40 Phe 473 B B 0.89 0.26 0.23 Thr 474 B B 0.78 .0.08 0.74 Phe 475. B T 0.48 1.72 0.71 Gly 476 T T 1.19 2.76 1.10 Glu 477 .T T .1.16 3.40 1.52 Asp 478 .T T 19 *F 3.06 2.39 Ser 479 .T T .1.29 *F 2.72 1.30 Lys 480 .T 1.99 *F 2.43 1.16 His 481 T 1.74 *F 2.34 1.16 Cys 482 T C 1.16 *F 2.10 0.87 rPro 483 A .T .0.86 FO 2.5 04 S 10 Res Pos. Garni.. Chou-... Garni.. Chou-... Garni... Chou,... Gari.. Kyte-... Eisen.. Eisen.. Karpi James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi Antig Surfa...
Asp 484 T T .0.84 2.50 0.43 Ala 485 A T .0.13 2.00 1.17 Ala 486 A 0.13 F 1.40 0.41 Ser 487 .B T T .0.22 F 1.75 0.33 Thr 488 .B .T 0.38 0.50 0.46 Cys 489 .B .T 0.67 -0.05 0.38 Ser 490 B T 0.74 F -0.05 0.30 Thr 491 B B 0.47 0.60 0.11 Leu 492 B B 0.51 0.60 0.30 Trp 493 B B -0.51 0.60 0.22 Cys 494 B B 0.14 0.60 0.22 Thr 495 B B T -0.19 F -0.05 0.36 Gly 496 B T 0.22 F -0.05 0.34 Thr 497 T T 0.27 F 0.65 0.62 Ser 498 T T 0.79 F 0.65 0.32 Gly 499 .T T 0.98 F 0.35 0.27 Gly 500 T T 1.33 F 0.35 0.14 Val 501 B B 0.99 -0.60 0.05 Leu 502 B B 0.99 0.60 0.10 Val 503 B B 0.64 -0.60 0.14 Cys 504 B -0.20 0.38 Gin 505 .B T -06 0.0.2 10 Res Pos. Garni.. Chou-... Garni.. Chou-... Garni... Chou,.. Garni.. Kyte-... Eisen.. Eisen.. Karpi James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi Antig Surfa...
Thr 506 B T 0.04 F 0.25 0.72 Lys 507 B T .0.48 F 0.40 2.09 His 508 C 0.74 0.05 1.27 Phe 509 .B 1.41 0.40 0.89 Pro 510 T 1 .07 0.30 0.74 Trp 511 T T .1.07 0.20 0.54 Ala 512 .T T .0.72 0.51 0.90 Asp 513 T T .0.09 F 1.27 0.78 Gly 514 T T .0.44 F 1.58 0.40 Thr 515 T T .0.66 I .F 2.49 0.39 Ser 516 T T .0.60 *F 3.10 0.40 Cys 517 T T 1 .23 ~F 2.49 0.40 Gly 518 .T T .0.94 2.48 0.56 Glu 519 T 0.62 1.67 0.44 Gly 520 T 0.04 I *F 1.36 0.44 Lys 521 T 0.34 *F 0.45 0.31 Trp 522 T 0.67 .*0.90 0.29 Cys 523 B .T 1 .06 -0.20 0.29 Ile 524 B .T .0.39 0.70 0.29 Asn 525 T T 0.12 0.15 Gly 526 T T 0.37 0.65 0.20 Lys 52 T 0.17 *F _r 0.45 0.47 9 10 t\J S 15 Res Pos. Garni.. Chou-... Garni.. Chou-... Garni... Chou-... Garni.. Kyte-... Eisen.. Eisen.. Karpl... James... Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi... Antig... Surfa...
Cys 528 T T 0.52 1.40 0.58 Val 529 B T 1.41 1.04 0.85 Asn 530 B T 1.52 F 1.83 0.71 Lys 531 B 1.91 F 2.32 2.58 Thr 532 B T .1.83 F 2.66 6.96 Asp 533 T T 1.80 F 3.40 5.89 Arg 534 T T 2.66 F 3.06 2.55 Lys 535 B T 2.34 F 2.32 2.95 His 536 B 2.09 F 1.78 2.55 Phe 537 B 1.70 F 1.44 2.01 Asp 538 B 1.67 F 0.65 0.87 Thr 539 B 1.21 F -0.25 0.87 Pro 540 C 0.87 F -0.05 1.00 Phe 541 T 0.61 F 0.45 0.80 His 542 T T 0.97 0.20 0.58 Gly 543 T T 0.37 0.20 0.37 Ser 544 T T 0.39 0.20 0.43 Trp 545 T T 0.26 0.20 0.33 Gly 546 C 0.74 -0.20 0.33 Met 547 T 0.49 0.00 0.38 Trp 548 T 0.49 0.00 0.38 Gly 549 T C 0.79 0.00 0.38 Res Pos. Garni.. Chou-... Garni.. Chou-... Garni... Chou-... Garni.. Kyte-... Eisen.. Eisen.. lKarpl James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi Antig Surfa...
Pro 550 T T .0.41 F 0.35 0.64 Trp 551 T T .0.46 0.66 0.33 Gly 552 T T .1.17 1.27 0.44 Asp 553 T 1.14 1.98 0.56 Cys 554 T T .0.82 .F 2.49 0.77 Ser 555 T T .0.69 .F 3.10 0.42 Arg 556 T T .0.63 2.79 0.25 Thr 557 T T .0.63 2.18 0.46 Cys 558 T T 0.22 .F 1.87 0.34 Gly 559 T T .0.44 .F 1.56 0.13 Gly 560 T T .0.50 *F 0.65 0.15 Gly 561 T T .0.08 0.35 0.45 Val 562 B B -0.21 .0.60 0.65 Gin 563 B B 0.57 .0.60 0.65 Tyr 564 B B 0.91 1.29 Thr 565 B B .0.59 3.01 Met 566 B B 0.93 .0.98 0.93 Arg 567 B B 1.79 .1.62 0.99 Glu 568 T 1.58 *5F 2.86 1.11 Cys 569 T T .0.97 *F 3.40 1.73 Asp 570 T T .1.07 *F 2.91 0.66 S 10 le= 15 Res Pos. Garni.. Chou-... Garni.. Chou-.. Garni... Chou-... Garni.. Kyte-... Eisen.. Eisen.. Karpl James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi Antig Surfa...
Asn 571 T C 1.71 *F 2.37 0.59 Pro 572 T C 1.60 *F 2.52 2.18 Val 573 C 1.26 *F 2.32 2.10 Pro 574 T T .1.58 2.42 1.29 Lys 575 T .1.62 *F 2.61 0.83 Asn 576 T T .1.38 .3.40 2.23 Gly 577 T T .0.92 3.06 2.26 Gly 578 .T T .1.78 *F 2.27 0.61 Lys 579 B .T .1.64 F 1.53 0.65 Tyr 580 B .T .1.64 F 1.19 0.65 Cys 581 8 T .1.76 F 1.30 1.32 Glu 582 B 1.24 1.10 1.29 Gly 583 B B 1.70 0.75 0.61 Lys 584 B B .1.41 0.90 2.24 Arg 585 B B3. 1.77 1.15 2.02 Tyr 586 B B 2.13 1.25 4.01 Arg 587 B B 1.47 *.1.50 2.68 Tyr 588 B .T .1.81 0.73 Arg 589 T T .0.96 .2.50 1.59 Ser 590 T T .2.10 0.67 Cys 591 .T T .1.70 0.74 Asn 592 .A T 0.92 1.50 0.63 t 10 kNj z- 15 Res Pos. Garni.. Chou-... Garni.. Chou-... Garni... Chou,.. Garni.. IKyte-... Eisen.. Eisen.. Karpi James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi Antig Surfa...
Leu 593 .A B 0.96 0.89 0.25 Glu 594 .A B 0.84 F 1.13 0.73 Asp 595 .A T 1.17 F 2.17 0.76 Cys 596 B .T .1.81 F 2.66 1.48 Pro 597 T T .1.47 F 3.40 1.37 Asp 598 T T .2.32 F 2.91 0.81 Asn 599 T T .2.01 3.02 3.03 Asn 600 T T .1.31 *F 2.98 2.83 Gly 601 T T .2.09 *F 2.94 1.47 Lys 602 T C 2.30 F 2.70 1.79 Thr 603 T C 2.30 *F 3.00 1.92 Phe 604 A A 2.30 2.10 3.37 Arg 605 A A 1.63 F 1.80 2.91 Glu 606 A A 1.98 *F 1.50 1.08 Glu 607 A A 1.34 .F 1.20 2.17 Gin 608 A A 1.62 *F 0.90 1.12 Cys 609 A A .2.32 0.88 Glu 610 A A .2.21 0.81 Ala 611 A A .1.51 0.81 His 612 A A .1.21 *.0.45 1.32 Asn 613 A j A j 1.26 .0.45 1.02 G~ 64 A A .3 *.0.45 2.02d
INJ
Res Pos. Garni.. Chou-... Garni.. Chou-... Garni... Chou-... Garni.. Kyte-... Eisen.. Eisen.. Karpi James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi Antig Surfa...
Phe 615 A A 1.03 F 0.60 1.50 Ser 616 A A 0.92 F 0.90 1.25 Lys 617 A A 0.61 F 0.45 0.62 Ala 618 .A T 0.31 F 0.25 0.71 Ser 619 .A T 0.03 F 0.85 0.71 Phe 620 T 0.46 F 1.26 0.35 Gly 621 T T .0.17 F 1.07 0.54 Ser 622 .r C -0.73 *F 1.08 0.41 Gly 623 T C -0.14 F 0.99 0.35 Pro 624 T C -0.13 F 2.10 0.61 Ala 625 .A C -0.32 F 0.89 0.48 Val 626 .A B 0.19 *.0.03 0.34 Glu 627 .A B .0.16 0.34 Trp 628 .A B 0.26 0.67 lie 629 B -0.12 1.42 Pro 630 B .T .0.12 *.0.10 0.83 Lys 631 T T .0.12 *.0.20 0.78 Tyr 632 T T 0.18 *.0.20 0.82 Ala 633 T 0.10 *.0.84 0.71 Gly 634 T 0.83 0.98 0.55 Val 635 B 1.04 0.92 0.70 Ser 636 T B T .1.11 2.66 1.17 Res Pos. Garni.. Chou-... Garni.. Chou,.. Garni... Chou-... Garni.. Kyte-... Eisen.. Eisen.. Karpi James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi Antig Surfa...
Pro 637 T T .0.69 I *F 3.40 2.31 Lys 638 T T .1.32 .I*F 3.06 1.67 Asp 639 T T .0.86 *F 2.72 2.49 Arg 640 A A 0.82 I *F 1.58 1.33 Cys 641 A A 0.46 I *F 1.09 0.46 Lys 642 .A B 0.67 .0.30 0.15 Leu 643 .A B 0.03 0.13 lie 644 A B 0.08 .0.60 0.25 Cys 645 .A B 0.38 0.25 Gin 646 .A B 0.60 .0.30 0.30 Ala 647 .A B -0.99 .0.30 0.30 Lys 648 .A B -0.42 F -0.15 0.55 Gly 649 T T 0.23 *F 0.65 0.50 lie 650 T T 0.27 0.20 0.43 Gly 651 B .T 1.12 .0.20 0.18 Tyr 652 B .T 1.34 .0.20 0.14 Phe 653 B B 1.39 .0.60 0.16 Phe 654 B B 1.26 0.60 0.29 Val 655 B B -0.32 .0.60 0.28 Leu 656 B B 0.83 .0.60 0.65 Gin 657 B T 1.44 .0.20 0.56 Pro 658 B T 0.74 -0.05 0.56 0
LJ
-I
C
C',
'0
C
L.J
Res Pos. Carni.. Chou-... Garni.. Chou-... Garni... Chou-... Garni.. Kyte-... Eisen.. Eisen.. Karpi James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi Antig Surfa...
Lys 659 T T 0.39 1.40 1.13 Val 660 B T 0.16 F 0.85 0.65 Val 661 B T .0.76 0.85 0.60 Asp 662 B .T .0.09 F 1.06 0.47 Gly 663 B .T .0.00 0.67 0.34 Thr 664 B .T 0.26 SF 1.48 0.61 Pro 665 B 0.60 F 1.49 0.56 Cys 666 .T 1.16 F 2.10 0.95 Ser 667 .T C 0.84 F 1.89 0.88 Pro 668 T T .0.89 F 1.88 0.82 Asp 669 T T .0.34 F 1.82 2.06 Ser 670 T T 0.11 F 1.61 1.14 Thr 671 B T 0.30 0.85 0.39 Set 672 B B 0.00 -0.15 0.18 Val 673 B B 0.13 -0.60 0.23 Cys 674 B B -0.13 .0.60 0.16 Val 675 B B 0.50 .0.60 0.20 Gin 676 B B 1.04 -0.45 0.15 Gly 6'77 B B -0.70 -0.45 0.20 Gin 678 B B 0.43 -0.15 0.54 Cys 679 B B 0.11 0.30 0.32 Val 680 B B 0.08 .0.30 0.32 Res Pos. Garni.. Chou-... Garni.. Chou-... Garni... Chou-... Carni.. Kyte-... Eisen.. Eisen.. Karpi James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi Antig Surfa...
Lys 681 .B T .0.08 *.0.10 0.10 Ala 682 B T .0.53 *.0.70 0.30 Gly 683 B T 0.36 *.1.00 0.80 Cys 684 B T 0.58 *.1.00 0.28 Asp 685 A .B 0.28 0.20 Arg 686 A .B 0.07 0.60 0.33 lie 687 A .B 0.57 *.0.60 0.82 lie 688 A B 0.96 *F 0.75 0.99 Asp 689 A T .1.67 F 1.30 1.01 Ser 690 A .T .0.97 F 1.30 2.88 Lys 691 A .T .0.86 1.61 3.55 Lys 692 .T T .1.79 F 2.32 3.55 Lys 693 .T 2.01 F 2.43 5.30 Phe 694 .T 1.67 F 2.74 1.42 Asp 695 .T T .1.11 *F 3.10 0.70 Lys 696 B .T .0.40 *F 2.39 0.26 Cys 697 B .T .0.01 1.63 0.16 Gly 698 B .T 0.38 1.32 0.10 Val 699 B 0.32 *.0.21 0.05 Cys 700 T -0.02 0.00 0.14 Gly 701 T T 0.37 F 0.65 0.14 Gl-y 702 T T 0.01 F 0.65 0.26 2 0 Res Pos. Garni.. Chou-... Garni.. Chou-... GarniL.. Chou-... Garni.. Kyte-... Eisen.. Eisen.. Karpl James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi Antig Surfa...
Asn 703 T T 0.33 F 0.65 0.69 Gly 704 T T .0.57 F 0.65 0.37 Ser 705 T T 1.28 F 1.25 0.76 Thr 706 B T .0.73 F 1.41 0.94 Cys 707 B T .0.78 1.37 0.67 Lys 708 B T .0.43 *F 1.63 0.67 Lys 709 B 0.48 F 1.69 0.46 lie 710 B .T 0.08 *F 2.60 1.14 Scr 711 .B .T 0.08 *F 1.89 0.42 Gly 712 B .T .0.29 F 1.03 0.31 Ser 713 B .T 0.34 F 0.77 0.58 Val 714 B B 0.34 0.11 0.44 Thr 715 B B 0.33 F 0.73 0.89 Ser 716 B B 0.29 F 1.16 1.03 Ala 717 B .0.39 F 1.64 1.37 Lys 718 T C 0.66 F 2.32 1.49 Pro 719 T T .1.51 2.80 1.51 Gly 720 T T .0.93 *F 2.52 2.50 Tyr 721 B .T .0.34 *.1.54 0.88 His 722 B B .0.62 -0.04 0.40 Asp 723 B B 0.31 0.58 Ile 724 B B 0.31 *-0.60 0.26] Res Pos. Garni.. Chou-... Garni.. Chou-... Garni... Chou-... Garni.. Kyte-... Eisen.. Eisen.. Karpl... James... Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi... Antig... Surfa...
lie 725 B B -0.28 -0.60 0.29 Thr 726 B B -0.38 -0.60 0.25 lie 727 B T -0.93 -0.20 0.36 Pro 728 B T -1.24 F -0.05 0.52 Thr 729 T C -0.36 F 0.15 0.52 Gly 730 T C -0.36 F 0.30 1.19 Ala 731 B C -0.04 F -0.25 0.54 Thr 732 B C -0.01 F 0.65 0.65 Asn 733 B B 0.24 F -0.15 0.48 lie 734 B B 0.56 F 0.45 0.96 Glu 735 B B 1.01 F 0.60 1.15 Val 736 B B 1.60 F 0.90 1.40 Lys 737 B B 1.91 F 1.24 3.21 Gin 738 B 2.02 F 1.78 3.21 Arg 739 B 2.57 F 2.12 8.48 Asn 740 B T 2.27 F 2.66 4.20 Gin 741 T T 3.23 F 3.40 3.25 Arg 742 T T 3.19 F 3.06 3.25 Gly 743 T T 3.19 F 3.00 3.25 Ser 744 T 2.73 F 2.74 3.02 Arg 745 C 2.43 F 2.48 1.52 Asn 746 T T 1.73 F 2.82 2.06 Res Pos. Garni.. Chou-... Garni.. Chou-.. Garni... Chou-... Garni.. Kyte-... Eisen.. Eisen.. Karpi... James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi... Antig Surfa...
Asn 747 T T .0.81 *F 2.80 1.33 Gly 748 .T C 0.57 *F 1.57 0.56 Ser 749 B T 0.02 *F 0.79 0.35 Phe 750 .A B 0.09 0.15 Leu 751 .A B -0.68 .0.32 0.31 Ala 752 .A B 1.27 0.23 lie 753 .A B 0.92 .0.60 0.27 Lys 754 A A 0.97 0.30 0.55 Ala 755 A A 0.58 0.30 0.54 Ala 756 A A -0.01 F 0.60 1.12 Asp 757 A T 0.31 F 0.85 0.87 Gly 758 B .T 0.23 0.25 0.61 Thr 759 B .T 0.28 F -0.05 0.50 Tyr 760 B .T 0.03 .0.20 0.48 lie 761 B 0.56 .0.40 0.48 Leu 762 B 0.31 .0.40 0.55 Asn 763 B T .0.34 -0.50 0.55 Gly 764 T T 0.16 F 0.50 1.14 Asp 765 .T T 0.21 *F 0.50 1.14 Tyr 766 T C 0.37 0.45 0.95 Thr 767 B B 0.37 .0.15 1.38 Leu 1 768 BB 0.37 .0.60 0.68 Res Pos. Garni.. Chou-... Garni.. Chou-... Garni... Chou,.. Garni.. Kyte-... Eisen.. Eisen.. Karpi James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi Antig Surra...
Ser 769 B B 0.71 .F -0.45 0.75 Thr 770 B B 0.71 -0.15 0.90 Leu 771 A B 0.07 0.60 1.83 Glu 772 A B 0.22 0.45 0.96 Gln .773 A B 0.34 F 0.45 0.66 Asp 774 A B 0.69 0.00 1.25 Ile 775 A B 0.66 1.44 Met 776 A B 3 0.61 0.30 0.82 Tyr 777 B B 0.24 0.37 Lys 778 B B 1.06 -0.60 0.39 Gly 779 B B 0.94 0.32 Val 780 13. B 0.30 0.40 Val 781 B B 0.00 0.32 Leu 782 B B 0.10 0.43 Arg 783 B B 0.44 0.57 Tyr 784 B .T 0.40 .0.25 1.03 Ser 785 T T 0.13 0.80 1.68 Gly 786 T C 0.13 *F 1.05 0.86 Ser 787 T C 0.13 0.45 0.56 Ser 788 .A C 0.02 .F 0.05 0.34 Ala 789 A A 0.38 0.45 0.60 Ala 790 A A 0.21 .0.60 0.88 15 Res Pos. Garni.. Chou-... Garni.. Chou,.. Garni... Chou,.. Garm.. Kyte-... Eisen.. Eisen.. Karpi James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi... Antig Surfa...
Leu 791 A A 0.24 .0.30 0.46 Glu 792 A A 0.24 *.0.60 0.89 Arg 793 .A B B 0.16 F 0.90 1.18 Ile 794 A A B 0.13 F 0.60 1.24 Arg 795 A A B 0.51 F 0.75 0.96 Ser 796 .A T 0.51 1.13 0.76 Phe 797 C 0.56 0.81 0.89 Ser 798 T C 0.44 .F 1.89 0.91 Pro 799 T C 1.12 F 2.32 1.17 Leu 800 .T T .0.20 F 2.80 2.10 Lys 801 T C 0.19 2.32 1.29 Glu 802 C 0.00 1.84 1.20 Pro 803 A B 0.30 1.16 1 .02 Leu 804 A B -0.34 0.73 0.89 Thr 805 .B B 0.34 0.38 Ile 806 B B -0.70 -0.60 0.20 Gin 807 B B -1.56 -0.60 0.35 Val 808 B B 1.69 0.18 Leu 809 B B -0.88 -0.60 0.26 Thr 810 B B -1.16 -0.60 0.24 Vai 8i11 B B -1.08 -0.60 0.33 Gly 1 812 B B 0.97 0.3 2 9 9 3 1 9
I~'
'S 15 Res Pos. Garni.. Chou-... Garni.. Chou-... Garni... Chou-... Garni.. Kyte-... Eisen.. Eisen.. Karpl... James... Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi... Antig... Surfa...
Asn 813 A -0.32 0.12 0.44 Ala 814 A 0.53 0.34 0.92 Leu 815 A -0.04 F 1.76 1.86 Arg 816 B 0.86 F 1.53 0.81 Pro 817 B 0.96 F 2.20 1.61 Lys 818 B B 0.64 F 1.48 3.05 lie 819 B B 0.99 F 1.56 2.25 Lys 820 B B 1.10 F 0.44 2.28 Tyr 821 B B 0.13 -0.38 0.99 Thr 822 B B 0.39 -0.45 1.04 Tyr 823 A B 0.39 -0.45 1.04 Phe 824 A B 1.32 -0.45 1.33 Val 825 A B 1.32 0.45 1.85 Lys 826 A B 1.57 F 0.90 2.36 Lys 827 A A 1.58 F 0.90 4.71 Lys 828 A A 1.12 F 0.90 8.51 Lys 829 A A 1.82 F 0.90 3.68 Glu 830 A A 2.09 F 0.90 2.96 Ser 831 A A 1.16 F 0.90 1.50 Phe 832 A A 0.90 0.30 0.52 Asn 833 A B 0.54 -0.30 0.47 Ala 834 B -0.20 -0.40 0.50 Res Pos. Garni.. Chou-... Garni.. Chou-... Garni... Chou-... Carni.. Kyte-... Eisen.. Eisen.. Karp[i... James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi Antig Surfa...
lie 835 C -0.50 0.50 Pro 836 T C -0.79 0.00 0.42 Thr 837 T T 0.38 *4.0.20 0.42 Phe 838 A T 1.23 0.63 Ser 839 T C -1.53 *.0.00 0.30 Ala 840 .A B B -0.64 0.15 Trp 841 A B B 0.43 0.60 0.29 Val 842 A A .B 0.41 0.30 0.38 Ile 843 A A .B 0.06 0.40 Glu 844 A A .B 0.24 0.37 Glu 845 A A 0.17 *.0.30 0.87 Trp 846 A A 0.16 .0.61 0.66 Gly 847 A A 1.06 1.37 0.51 Glu 848 .A T 1.64 2.08 0.59 Cys 849 .A T 0.98 .F 2.09 0.76 Ser 850 T T .0.98 F 3.10 0.41 Lys 851 T T .0.46 F 2.79 0.41 Ser 852 T T .0.46 F 2.18 0.63 Cys 853 T T .0.17 0.47 Glu 854 A A 0.83 *.0.61 0.24 Leu 855 A A 1.24 0.30 0.32 Gly 856 .A 1.31 0.85 1.16 S 10 Res Pos. Garni.. Chou-... Garni.. Chou-... Garni... Chou-... Garni.. Kyte-... Eisen.. Eisen.. Karpl... James... Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi... Antig... Surfa...
Trp 857 A A 0.80 0.75 1.31 Gin 858 A A 0.61 -0.15 1.31 Arg 859 A A 0.61 -0.30 0.98 Arg 860 A B 0.76 0.45 1.61 Leu 861 A B 1.21 0.60 0.50 Val 862 A B 1.50 0.60 0.50 Glu 863 A B. 0.61 0.94 0.43 Cys 864 A B 0.50 0.98 0.36 Arg 865 A T 0.04 F 2.17 0.78 Asp 866 T T 0.86 F 2.91 0.45 lie 867 T T 1.50 F 3.40 1.45 Asn 868 T T 0.91 F 3.06 1.14 Gly 869 T C 1.28 F 2.07 0.69 Gin 870 T C 1.17 F 1.28 1.32 Pro 871 T C 0.50 F 1.54 1.42 Ala 872 T C 0.80 F 1.05 0.77 Ser 873 A T 0.84 F 0.85 0.45 Glu 874 A A 1.19 F 0.75 0.58 Cys 875 A A 0.33 0.60 1.00 Ala 876 A A 0.59 0.60 0.55 Lys 877 A A 0.97 F 0.75 0.64 Glu 878 A A 0.68 F 0.90 1.84 S 10 Res Pos. Garni.. Chou,.. Carol.. Chou-... Garni... Chou-... Garni.. Kyte-... Eisen.. Eisen.. Karpl James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi Antig Surfa...
0 Val 879 A A 0.38 0.90 1.84 Lys 880 A A 0.73 0.90 1.23 Pro 881 A T .1.43 1.30 1.03 Ala 882 .T T .1.18 2.01 2.71 Ser 883 T T .0.51 2.32 2.10 Thr 884 T T .0.78 2.18 0.73 Arg 885 B .T .0.73 2.09 0.73 Pro 886 .T T .0.91 3.10 0.91 Cys 887 .T T .1.29 0.85 Ala 888 T T .0.92 2.43 0.67 Asp 889 T 1 1.02 0.23 His 890 T C 0.91 1.51 0.67 Pro 891 T T .0.83 1.65 1.16 Cys 892 T -T 1.50 1.00 0.73 Pro 893 .T T .1.28 0.60 0.93 Gin 894 .A T 0.93 0.10 0.49 Trp 895 .A B 0.97 0.40 0.91 Gin 896 .A B .0.89 0.05 1.02 Leu 897 .A B 1.26 0.60 0.62 Gly 898 T 17 0.00 0.79 Glu 899 .T 0.50 F 0.45 0.61 Trp 900 1 1 1 T 0.49 F 0.45 0.40 Res Pos. Garni.. Chou-... Garni.. Chou,.. Garni... Chou-... Garni.. Kyte-... Eisen.. Eisen.. Karpi James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi Antig Surfa...
Ser 901 T T .0.53 F 0.65 0.54 Ser 902 T T 1.03 F 1.25 0.62 Cys 903 T T 0.71 .F 0.65 0.85 Ser 904 .T T 0.37 *F 1.25 0.34 Lys 905 T 0.70 1.05 0.25 Thr 906 T 0.66 1.69 0.94 Cys 907 T 0.71 *F 2.03 0.69 Gly 908 T T .1.42 F 2.27 0.54 Lys 909 T 2.61 0.75 Gly 910 T T .1.83 F 3.40 2.81 Tyr 911 T T .1.84 F 3.06 5.57 Lys 912 .A B 1.70 1.92 3.73 Lys 913 .A B 2.09 1.58 3.11 Arg 914 .A B .1.38 1.24 3.97 Ser 915 .A B 0.91 *F 0.90 1.06 Leu 916 .A B 0.86 0.75 0.44 Lys 917 .A B 0.78 0.30 0.30 Cys 918 A B 0.73 0.30 Leu 919 .A B 0.28 0.62 Ser 920 B 0.23 0.50 0.31 Hlis 921 B .T .0.19 .F 0.85 0.56 Asp 922] T T 0.67 0.65 0.51 S 10 a 15 Res Pos. Garni.. Chou-... Garni.. Chou-... Garni... Chou-... Garni.. Kyte-... Eisen.. Eisen.. Karpl... James... Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi... Antig... Surfa...
Gly 923 T T -0.30 F 0.65 0.31 Gly 924 T T 0.48 F 0.65 0.31 Val 925 B 0.78 -0.10 0.25 Leu 926 B 0.51 -0.10 0.44 Ser 927 B -0.16 -0.10 0.59 His 928 B T 0.19 0.10 0.43 Glu 929 B T 0.32 F 0.85 0.87 Ser 930 A T 0.37 F 1.30 1.00 Cys 931 A T 1.22 F 0.85 0.61 Asp 932 A T 1.57 F 1.15 0.70 Pro 933 A T 1.39 F 1.30 1.05 Leu 934 A T 1.43 F 1.30 3.02 Lys 935 A T 1.70 F 1.30 3.62 Lys 936 A A 1.67 F 0.90 3.18 Pro 937 A A 0.78 F 0.90 3.34 Lys 938 A A 0.99 F 0.90 1.17 His 939 A A 1.10 0.60 0.98 Phe 940 A B 0.39 -0.30 0.55 lie 941 A B 0.03 -0.30 0.15 Asp 942 A A 0.36 -0.60 0.16 Phe 943 A A -0.99 -0.60 0.18 Cys 944 A A -0.96 -0.60 0.26 Res Pos. Garni.. Chou,.. Garni.. Chou-... Garni... Chou-... Garni.. Kyte-... Eisen.. Eisen.. Karpi James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi Antig Surfa...
Thr 945 A A 0.92 0.27 Met 946 A A 0.33 .0.60 0.16 Ala 947 A A 0.72 .0.30 0.41 Glu 948 A A 0.41 0.30 0.36 Cys 949 A A 0.13 0.30 0.47 Ser 950 A A 0.21 0.30 06 Table 2 Lo Ln 04 c
N
M
Res Pos. Garni.. Chou-... Garni.. Chou-... Garni.. Chou-... Garni.. Kyte-... Eisen... Eisen... Karpi... James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi... Antig Surfa...
Met I B 0.37 .0.40 0.50 Phe 2 B 0.57 .0.40 0.61 Pro 3 B 0.77 .0.40 0.48 Ala 4 C -0.59 0.49 Pro 5 C -0.09 0.87 Ala 6 C 0.22 .0.85 1.11 Ala 7 T C 0.11 1.15 Pro 8 A T .0.11 0.61 Arg 9 T T .0.00 *.0.20 0.94 Trp 10 B .T 0.60 0.81 Leu I I .A B -0.82 0.43 Pro 12 .A B -1.04 0.18 Phe 13 .A B 1.64 0.14 Leu 14 A A -2.57 0.60 0.14 Leu 15 A A -3.09 0.60 0.08 Leu 16 A A -3.09 -0.60 0.07 Leu 17 A A 3.69 -0.60 0.07 Leu 18 A A 3.80 -0.60 0.07 Leu 19 A A -3.20 0.60 0.07 Leu 20 A A -3.20 0.60 0.14 Res Pos. Garni.. Chou,.. Garni.. Chou-... Garni.. Chou-... Garni.. Kyte-... Eisen... Eisen... Karpi James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro,... Alpha Beta Flexi Antig Surfa...
Leu 21 A A 2.98 0.14 Leu 22 .A B 2.06 0.60 0.17 Pro 23 .A B 1.59 0.39 Leu 24 A A 1.37 0.47 Ala 25 A A 0.77 0.58 Arg 26 .A B 0.54 *.0.82 0.58 Gly 27 .A B 0.38 F 0.63 0.71 Ala 28 B 0.38 f 2.14 1.37 Pro 29 C 0.60 F 2.60 1.08 Ala 30 B 0.60 F 1.84 1.11 Arg 31 B 0.14 F 1.58 1.11 Pro 32 B 0.14 *F 1.17 0.71 Ala 33 B T .0.73 1.11 0.69 Ala 34 A T .0.36 0.85 0.61 Gly 35 T C 0.64 0.45 0.40 Gly 36 .T C 0.53 0.45 0.53 Gln 37 A 0.07 F 0.65 0.91 Ala 38 B -0.33 F 0.65 0.76 Ser 39 B B B -0.60 F -0.15 0.57 Glu 40 B B B 0.47 F -0.15 0.24 Leu 41 B B B -0.43 -0.30 0.37 Val 42 B B B 0.32 -0.30 0.40 S 10 tNtj 15 Res Pos. Garni.. Chou-... Garni.. Chou,.. Garni.. Chou,.. Garni.. Kyte-... Eisen... Eisen... Karpi James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi Antig Surfa...
Val 43 B B3 B 0.54 0.46 Pro 44 .B B) B 0.46 -0.24 0.46 Thr 45 B B B 0.80 0.27 0.95 Arg 46 .B B B 0.29 *F 0.63 1.26 Leu 47 T C -0.02 2.04 1.10 Pro 48 T C 0.49 F 2.10 0.77 Gly 49 T C 0.70 F 1.89 0.39 Ser 50 .T C 0.20 F 1.68 0.81 Ala 51 A A -0.50 F 0.87 0.43 Gly 52 A A -0.50 F 0.66 0.44 Glu 53 A A -0.32 -0.30 0.27 Leu 54 A A 0.79 -0.30 0.37 Ala 55 A A 0.79 0.31 Leu 56 A A -0.79 -0.60 0.24 His 57 A A 1.14 0.29 Leu 58 A A 1.49 0.60 0.25 Ser 59 A A 0.63 0.60 0.30 Ala 60 A A -0.39 0.30 0.44 Phe 61 A A 0.28 0.30 0.53 Gly 62 T T 1.10 *.0.50 0.34 Lys 63 A T 1.10 *F -0.05 0.251 Gy 64 B T I -06 .I 0.0 02 Res Pos. Garni.. Chou-... Garni.. Chou,.. Garni.. Chou-.. Garni.. Kyte-... Eisen... Eisen... Karp[i... James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi Antig Surfa...
Phe 65 B T 0.91 0.47 Val 66 B B 0.80 .0.30 0.19 Leu 67 .B B 0.67 .0.30 0.20 Arg 68 .B B 0.71 0.35 Leu 69 B B 0.37 .1.20 0.80 Ala 70 T C 0.03 1.61 Pro 71 T C 0.19 F 3.00 1.10 Asp 72 .T T .0.19 .F 2.60 1.16 Asp 73 A T 0.51 F 1.75 0.95 Ser 74 A A 0.09 0.90 0.62 Phe 75 A -A 0.68 .0.60 0.57 Leu 76 A A .0.19 0.30 0.59 Ala 77 A A 0.23 -0.60 0.38 Pro 78 A A 0.66 -0.30 0.89 Glu 79 A A 0.36 F -0.15 0.75 Phe 80 A A .0.46 0.90 1.29 Lys 81 A A .0.46 0.90 1.63 lie 82 A A 0.70 *F 0.75 0.78 Glu 83 A A 0.57 F 0.45 0.89 Arg 84 A A 0.27 F 0.75 0.44 Leu 85 .A T 0.62 0.85 0.84 Gly 86 A T -I T 0.69 F 1.15 0.48 Res Pos. Garni.. Chou-... Carni.. Chou-... Garni.. Chou-... Garni.. Kyte-... Eisen... Eisen... Karpi James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi Antig Surfa...
Gly 87 T C 0.99 F 1.35 0.48 Ser 88 T C 0.68 *F 1.05 0.59 Gly 89 T C 0.22 F 1.05 0.86 Arg 90 B .T .0.69 1.19 0.86 Ala 91 T C 1.03 1.73 0.63 Thr 92 B .T .1.49 2.32 1.11 Gly 93 .B .T .1.44 2.66 1.11 Gly 94 T T .0.98 F 3.40 1.09 Glu 95 B 0.98 *F 2.31 0.62 Arg 96 B 1.22 *F 2.12 1.23 Gly 97 T 0.87 F 2.18 1.23 Leu 98 B .T .0.51 *F 1.49 0.38 Arg 99 B .T .0.16 *.0.70 0.17 Gly 100 B .T 0.14 0.15 Cys 101 B .T 0.60 0.24 Phe 102 B -0.57 0.12 Phe 103 B T 0.61 .0.20 0.18 Ser 104 .B T 0.72 *F -0.05 0.24 Gly 105 T C -0.72 0.15 0.45 Thr 106 T C -0.06 F 0.45 0.52 Val 107 1 j B I C 0.43 .F 1.25 0.67 Asn 1108 1 1 B I C 1.13 F 1.70 1.05 P9 Res Pos. Garni.. Chou-... Garni.. Chou,.. Garni.. Chou-... Garni.. Kyte-... Eisen... Eisen... Karpi James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi Antig Surfa...
Gly 109 B C 1.13 2.30 1.26 Glu 110 T C 0.67 .F 3.00 2.27 Pro III A T .0.39 2.50 1.16 Glu 112 A .T .0.66 2.20 1.19 Ser 113 A T 0.20 F 1.75 0.69 Leu 114 A A .B 0.16 0.00 0.33 Ala 115 A A .B 0.97 .0.30 0.26 Ala 116 A A .B 1.42 .0.60 0.16 Val 117 A A .B -1.31 .0.60 0.10 Ser 118 .A B B -1.36 0.20 Leu 119 B B 1.36 S.-0.30 0.20 Cys 120 B T 1.07 0.10 0.22 Arg 121 B T 0.82 0.10 0.22 Gly 122 T T 0.27 *F 0.65 0.26 Leu 123 T T 0.67 .F 1.25 0.65 Ser 124 .T C -0.67 .F 0.45 0.29 Gly 125 B T 0.81 .F -0.05 0.24 Ser 126 B T 0.92 -0.05 0.24 Phe 127 B T 0.92 0.10 0.30 Leu 128 .A B C -0.11 -0.30 0.30 Leu 129 .A 0.19 .F 0.65 0.39 Asp 130 A A 0.17 F 0.45 0.77 Res Pos. Garni.. Chou-... Garni.. Chou-... Garni.. Chou-... Garni.. Kyte-... Eisen... Eisen... Karpi James Etnini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi Antig Surfa...
Gly 131 A A 0.18 F 0.45 0.81 Glu 132 A A 0.37 0.90 1.42 Glu 133 A A 0.44 0.75 0.60 Phe 134 A A 1.04 1.04 Thr 135 .A B 1.04 0.93 lie 136 B 1.04 0.05 0.93 Gin 137 B 0.46 .F -0.10 1.06 Pro 138 C 0.11 0.25 0.75 Gin 139 .T 0.47 .F 0.60 1.05 Gly 140 T C 0.48 .F 0.45 0.60 Ala 141 .T T .0.56 1.25 0.52 Gly 142 T C -0.03 F 0.45 0.25 Gl Iy 143 T C 0.18 I .F 0.65 0.25 Ser 144 C -0.03 F 0.65 0.43 Leu 145 B .0.28 *F 0.65 0.68 Ala 146 B 0.98 *F 0.85 0.93 GIn 147 B T .0.51 2.00 1.36 Pro 148 B .T .0.86 .1.05 1.36 His 149 B T .1.27 *.1.45 2.34 Arg 150 B T .1.79 *.1.55 2.64 Leu 151 B 2.03 *.0.85 1.80 Gin 152 B 1.82 *.0.65 1.31 Res Pos. Garni.. Chou-... Garni.. Chou,.. Garni.. Chou-... Garni.. Kyte-... Eisen... Eisen... Karpi James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi... Antig Surfa...
Arg 153 T 1.44 *.1.05 1.03 Trp 154 T 1.13 *F 0.84 1.26 Gly 155 T C 0.43 *F 0.93 0.72 Pro 156 T C 1.36 *F 1.17 0.37 Ala 157 T T .1.14 1.61 0.69 Gly 158 T C 0.22 2.40 1.08 Ala 159 C 0.30 F 1.81 0.58 Arg 160 .13B. 0.76 F 1.37 0.89 Pro 161 B 0.62 F 1.58 1.75 Leu 162 C 1.00 F 1.84 1.72 Pro 163 .C 1.34 1.90 1.35 Arg 164 C 1.64 F 2.20 1.52 Gly 165 T .IC 1.53 2.40 1.93 Pro 166 T C 0.89 3.00 2.17 Glu 167 T C 1.70 2.55 0.82 Tmp 168 A T .1.60 1.44 Glu 169 A .1.14 *.1.85 1.34 Val 170 A 1.49 .F 1.85 0.77 Glu 171 A 1.36 *F 2.00 1.26 Thr 172 A 1.36 *F 2.15 0.72 Gly 173 .T C 1.76 .F 3.00 1.68 Glu 174 A T .1.76 .F 2.50 1.90 Res Pos. Garni.. Chou-... Garni.. Chou-... Garni.. Chou-... Garni.. Kyte-... Eisen... Eisen... Karpl James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi Antig Surfa...
Gly 175 A T 2.61 .F 2.20 2.28 Gin 176 A T .2.72 .F 1.90 4.00 Arg 177 A A 2.69 *F 1.54 4.52 Gin 178 A A 3.03 F 1.58 4.52 Giu 179 .A .T 3.00 F 2.32 4.36 Arg 180 .A T 3.34 2.66 3.03 Gly 181 T T .3.34 3.40 3.03 Asp 182 T C 3.23 F 2.86 3.03 His 183 T C 2.93 2.52 2.58 Gin 184 T C 2.93 *F 2.18 3.50 Glu 185 .A C 2.82 1.44 3.63 Asp 186 A A 3.17 F 0.90 4.61 Ser 187 A A .2.87 .F 0.90 4.61 Glu 188 A A 2.90 F 0.90 3.57 Glu 189 A A 2.90 .F 0.90 3.70 Giu 190 A A 2.90 F 0.90 4.79 Ser 191 A A 2.90 .F 0.90 4.79 Gin 192 A A 2.61 F 0.90 4.79 Glu 193 A A 2.61 F 0.90 2.79 Gfu 194 A A 2.27 F 0.90 3.61 Glu 195 A A 1.68 F 0.90 2.06 Ala 1196 A A 1.68 F 1.16 1.20 z 5 Res Pos. Garni.. Chou-... Garni.. Chou-... Garni.. Chou-... Garni.. Kyte-... Eisen... Eisen... Karp[i... James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi Antig Surfa...
Glu 197 A A 1.68 F 1.27 0.93 Gly 198 A A 1.47 F 1.53 0.93 Ala 199 .A T .1.26 .F 2.34 1.42 Ser 200 C 1.04 F 2.60 1.27 Glu 201 C 1.42 *F 2.04 1.99 Pro 202 .C 0.61 *F 1.78 3.04 Pro 203 C 0.61 F 1.52 1.87 Pro 204 T C0.61 F 1.46 1.07 Pro 205 .T C 0.60 F 0.45 0.70 Leu 206 .T C 0.30 F 0.45 0.65 Gly 207 B T .0.62 *F 0.51 0.57 Ala 208 B 0.52 1.17 0.72 Thr 209 B 0.78 F 1.58 1.25 Ser 210 B T .1.10 *F 2.34 2.53 Arg 211 B T .1.21 *F 2.60 4.91 Thr 212 B T .0.70 *F 2.34 2.95 Lys 213 B .T .0.99 2.08 1.63 Arg 214 B B 1.30 *F 1.42 1.12 Phe 215 B B 1.01~ 1.01 1.34 Val 216 B BI. 1.01 .0.60 0.68 Ser 217 A B .0.62 .0.60 0.68 Glu 218 A A 0.28 .0.30 0.68 Res Pos. Garni.. Chou-... Garni.. Chou-... Garni.. Chou,.. Garni.. Kyte-... Eisen... Eisen... Karpi James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi Antig Surfa...
Ala 219 A A .B 0.39 .0.30 0.68 Arg 220 A A .B 0.00 *0.60 0.87 Phe 221 A A .B 0.04 *0.60 0.73 Val 222 A A .B 0.47 0.30 0.59 Glu 223 A A .B 1.32 0.30 0.25 Thr 224 A A .B 1.32 0.60 0.21 Leu 225 A A .B 1.43 0.60 0.29 Leu 226 A A B 3 -1.32 0.30 0.28 Val 227 A A .B 0.77 0.60 0.20 Ala 228 A A .B 1.37 0.30 0.32 Asp 229 A A .B 1.64 .0.30 0.38 Ala 230 A A 1.42 0.30 0.52 Ser 231 A A 1.31 .0.30 0.52 Met 232 A A 0.70 0.30 0.27 Ala 233 A A 0.46 -0.60 0.42 Ala 234 A A -1.04 -0.60 0.31 Phe 235 A A -0.46 -0.60 0.32 Tyr 236 A A -0.97 -0.60 0.52 Gly 237 A A -0.37 .0.60 0.43 Ala 238 A A 0:22 .0.60 0.86 Asp 239 A A 0.78 0.30 0.88 Leu 240 A A 0.59 *.0.45 1.21 Con Res Pos. Garni.. Chou-... Garni.. Chou,.. Garni.. Chou,.. Garni.. Kyte-... Eisen... Eisen... Karp[i... James Eniini Alpha Alpha Beta Beta Turn Turn Coil Hlydro... Alpha Beta Flexi Antig Surfa...
Gin 241 A A .B 0.02 0.30 0.84 Asn 242 A A .B 0.06 0.41 His 243 .A B B 0.17 0.72 Ilie 244 .A B B 0.77 0.35 Leu 245 .A B B 0.26 0.21 Thr 246 .A B B -1.11 -0.60 0.21 Leu 247 .A B B 1.70 0.60 0.22 Met 248 A A 13 B -2.26 0.27 Ser 249 A A B 1.26 0.60 0.19 Val 250 A A .B 1.33 0.30 0.45 Ala 251 A A .B -1.27 0.30 0.32 Ala 252 A A B 0.41 0.60 0.37 Arg 253 A A B 0.16 0.15 1.01 Ilie 254 A A B. 0.24 1.36 Tyr 255 A .0.80 .0.99 2.08 Lys 256 B 0.50 1.42 His 257 B .T .1.13 1.12 1.42 Pro 258 .T C 1.02 2.56 1.81 Scr 259 .T T .1.61 3.40 1.46 Ile 260 .T T .0.97 *F 2.76 1.44 Lys 261 B 0.92 *F 1.67 0.65 Asn 262 .T .0.14 F 1.73 0.78 0 0 Res Pos. Garni.. Chou-... Garni.. Chou-... Garni.. Chou,.. Garni.. Kyte-... Eisen... Eisen... Karpl James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi Antig Surfa...
Ser 263 B B 0.24 *F 0.19 0.92 Ile 264 B B 0.80 .0.30 0.45 Asn 265 B B 0.77 .0.60 0.21 Leu 266 B B 0.77 0.60 0.12 Met 267 A B 1.62 0.33 Val 268 B B 2.13 0.15 Val 269 B B 2.13 0.60 0.15 Lys 270 A B 2.99 0.60 0.11 Val 271 B B -2.18 -0.60 0.11 Leu 272 B B -1.58 -0.30 0.25 Ile 273 A B 0.72 0.30 0.21 Val 274 A B 0.18 0.49 Glu 275 A B 0.16 0.75 1.19 Asp 276 A A 0.36 F 0.90 1.79 GIU 277 A A 0.96 *F 0.90 2.39 Lys 278 A T 1.84 *F 1.30 2.13 Trp 279 .A .C 1.84 1.10 2.21 Gly 280 .T C 1.54 *F 1.35 0.95 Pro 281 .T C 1.54 F 1.36 0.64 Glu 282 B .T .1.54 F 1.62 1.01 Val 283 B .T 1 .16 F 2.23 1.64 284 F T C 1.10 2.74 1.05 Res Pos. Garni.. Chou-... Garni.. Chou-... Garni.. Chou-.. Garni.. Kyte-... Eisen... Eisen... Karpi James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flex!i... Antig Surfa...
Asp 285 T T .0.63 F 3.10 0.60 Asn 286 T T .0.53 2.49 0.67 Gly 287 T T 0.28 F 2.18 0.72 Gly 288 T 0.69 1.07 0.35 Leu 289 .B 0.99 F 0.36 0.43 Thr 290 .B 0.29 .0.10 0.70 Leu 291 B 0.38 .0.40 0.61 Arg 292 B 0.03 .0.40 0.40 Asn 293 B 0.02 .0.10 0.44 Phe 294 T T .0.83 .0.20 0,57 Cys 295 .T T .1.26 .0.20 0.50 Asn 296 .T T .2.18 0.61 Trp 297 T T .1.37 .0.65 1.38 Gin 298 .T 1.37 *.0.45 2.23 Arg 299 .T 2.07 *.1.05 2.23 Arg 300 T 2.52 *F 1.20 3.67 Phe 301 .T 2.22 F 1.84 3.28 Asn 302 T 2.51 F 2.18 2.24 Gin 303 T C 2.62 *F 2.52 1.91 Pro 304 .T C 2.48 2.86 4.33 Ser 305 T T .2.16 F 3.40 3.66 Asp 306 T T .2.86 3.06 3.27 Res Pos. Garni..
Alpha Arg 307 His 308 Pro 309 Glu 310 His 311
A
Tyr 312
A
Asp 313
A
Thr 314
A
Ala 315
A
Ile 316 Leu 317 Leu 318 Thr 319 FArg 320 GI n 321 Asn 322 Phe 323 Cys 324 Gly 325 Gin 326 Glu 327 Gly 328 Chou-... Garni.. Chou-... Garni.. Chou-... Garni..
K
Alpha Beta Beta Turn Turn Coil
B:
C
C
T
T
T
T
T
B
B
B
B
B
B
B
B
B
B
B
B
T
T
T
T
T
T
T
T
T
T
T
T
yte-...
ydro...
2.82 2.58 2.79 2.78 2.19 1.19 0.41 -0.19 -0.50 -0.36 -0.51 -0.11 -0.0~ 0.7 1.5 1.5 0.6 -0.0 -0.0 .3 Eisen... Eisen... Karpl James Emini Alpha Beta Flexi Antig Surfa...
*F 2.32 3.66 *F 1.98 3.72 *F 1:.64 3.49 *F 1.5 2.97 *F 1.00 3.15 *F 1.00 2.06 F 0.85 0.83 -0.20 0.51 *-0.60 0.27 F 0.00 1.23 8 F -0.08 1.29 3 *F 0.69 0.84 8 *F 1.31 0.57 9 .1.98 0.51 6 *2.20 0.51 3 .F 1.53 0.31 3 .F 1.11 0.30 F 0.89 0.30 16 .F 1.27 0.50 0 .4 0% 0
-J
0% 0%
C
S Re o. an..Co-..Grn. ho-. Gri.Chou-... Garni.. Kyte-... Eisen... Eisen... Karpi James Em in i Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Bea Fx.. Ang.. Sr.. 0 lie 351 B B 01 .0 08 -0.37 0.60 0.59 AB .3 0.60 0.66 D e 35 A A0.2-0.60 0.385 Res Pos. G Pro 373A His 374 Asp 375 Asp 376 Ser 377 Lys 378 Pro 379 Cys 380 Thr 381 Arg 382 Leu 383 Phe 384 Gly 385 Pro 386 Met 387 Gly 388 Lys 389 is 3 9 0 His 391 Val 392 Met 393 arni..
ltpha
A
A
A
A
Chou-... G Alpha B
A
A
A
A
A
arni..
eta
B
B
B
B
B
B
B
Chou-...
G
Beta
T
B
B
B
B
arni..
urn
T
T
T
T1
T
T
Chou,.. G Turn C
T
T
T
T
T
T
T
T
T
T
T
oil
C
C
Kyte-... Eisen... Eisen... K Hydro... Alpha Beta F 0.39 1.29 1.89 1.812 2.13 1.36 0.66 0.31 0.40 E -0.242 0.52 0.28 0.281 0.07 -0.298 t-1.9 nrpl Ji lexi
A
F
F
F
F
F
ames E ntig S 2.41 3.40 30 0.0 -0.60 mini urfa...
1.78 1.78 3.61 3.61 1.42 1.23 1.44 0.89 0.38 0.25 0.71 0.49 0.25 0.59 0.93 1.29 0.62 0.62 0.63 0.49 030 0 '0
-J
0% 0% 0 0 '0 -4 0% 0% 0 00 e~ Res Leu Leu Asp Gly Gly His Gly Asp Gys Leu Leu Asp Ala Pro Gly Ala Ala Let Pro Le Pr Tli Pos. Garni.. Chou-...
G
Alpha Alpha Bi 417 A
A
418
A
419 420 421 422 423 424 425 426 427 428 429 430
A
431 432 433 434 435 u 436 0 437 r 438 arni..
eta
B
B
B
B
B
B
B
B
B
B
B
B
Garni.. Kyte-... Eisen... Eisen... Karpl james Emini Chou-... Garni.. Chou-..- Hydro... Alpha Beta Flexi Antig Surfa...
Beta Turn Turn Coil -0.18 IF 0.76 0.64 T 0.03 F 1.47 0.44 T T 0.50 F 2.18 0.35 T T 0.81 F 1.89 0.42 T T 0.14 F 3.10 0.84 T T 0.14 F 2.79 0.27 T T 0.14 T 0.14 0.72 0.1 T -0.10 1.01 0.03 -0.28 0.50 -0.52 -0.10 0.40 -1.11 F 0.25 0.49 T -1-26 F 0.25 0.60 F 0.65 0.30 T T -0.66 -0.20 0.46 T -0.66 -0.87 -0.40 0.24 -0.59 -0.40 0.38 -0.72 -0.40 0.54 -1.19 -0.20 0.53 T -0.81 F 0.00 0.53 T T -0.57 F 0.45 0.53 0 '0 -4 0% 0% 0 0 9 9 9 9 0% Res Phe Arg His Cys rPro As n Thr Ser Ala Gin Asp Val Cys Gin Leu Trp Cys His Thr Asj Pos. Garni.. Chou-... G 4E 1 Alp .ha Alpha
B
462 462 477
A
478
A
479 480 47 48 482 arni..
eta
B
B
B
B
B
B
B
B
B
Chou-...
G
Bets T arni..
urn
T
T
T
T
T
T
T
T
T
T
T
T
Chou-... Garni.. Kyte-... E Turn Coil Hydro... A T .0.74 1.44 1.48 T C 1.39 T 0.80 T 1.50 T 1.39 0.57 0.57 0.19 -0.72 0.13 0.46 T .0.46 T 0.46 T 1.06 T 10.53 isen... Eisen... K lpha Beta F arpi lexi
F
F
F
F
F
F
F
F
James Emini Antig Surfa...
1.35 0.73 1.40 0.73 1.65 0.71 1.45 1.18 2.50 0.80 IA.6 5 0.60 1.55 1.93 1.50 2.08 1.10 0.96 0.45 0.36 0.45 0.27 -0.30 0.46 -0.30 0.22 -0.60 0.14 -0.60 0.10 -0.60 0.25 -0.60 0.36 -0.26 0.35 0.88 0.42 1.52 0.40 3.06 1.30 3.40 1.48 0
-J
ON
cw Res Pos. G
A
Ala 483 Glu 484 Pro 485 Leu 486 Cys ;487 His 488 Thr 489 Lys 490 Asn 491 Gly 492 Ser 493 Leu 494 Pro 495 Trp 496 Ala 497 Asp 498 Gly 499 jThr 500 Pro 501 Cys 502 Gly 503 P~ro 504 arni.. C Ipho A
A
A
A
A
Ipha Garni.. Chou-..-
G
Beta Beta
T
B
B
B
urn
T
T
T
T
T
T
T
T
T
F
T
rurn
T
T
T
T
T
T
T
T
T
T
T
T
Garni.. Kyte-...
E
Coil Hydro...
A
C 0.53 0.53 0.53 0.58 0.92 1.17 0.87 0.27 0.87 1.24 c 0.69 c 1.00 0.61 0.30 0.43 0.07 0.53 C 0.53 0.48 1.03 C 0.22 -0.10 isen...
Ipha Eisen... Karpi Ja Beta Flexi
A
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F-
mes Emini ntig Surfa...
2.41 0.85 1.67 0.27 0.73 0.37 0.24 0.53 0.78 0.62 0.61 0.64 1.49 0.77 2.52 1.92 2.80 1.16 2.52 1.25 1.09 0.66 0.36 0.41 0.18 0.69 0.50 0.51 0.05 0.90 1.15 0.90 1.40 0.46 2.05 0.45 2.50 0.42 1.65 0.42 1.20 0.39 0.65 0.21 0 '.0 .4 0~.
0% 0 Res Pos. Garni.. Chou-... Garni.. Chou,.. Garni.. Chou-... Garni.. Kyte-... Eisen... Eisen... Karpl James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi Antig Surfa...
Gly 505 T 0.09 0.25 0.21 His 506 B 0.12 0.40 0.28 Leu 507 .B 0.44 0.50 0.32 Cys 508 B T .0.49 0.32 Ser 509 T T .0.03 F 1.67 0.31 Glu 510 .T T 0.43 F 1.28 0.20 Gly 511 T T 0.61 1.49 0.31 Ser 512 T 1 0.20 *F 2.10 0.36 Cys 513 A C 0.87 .F 1.79 0.36 Leu 514 A C 1.17 F 1.58 0.63 Pro 515 A A .0.31 F 1 .17 0.81 Glu 516 A A 0.66 *F 1.11 1.13 Glu 517 A A 1.07 F 0.90 2.37 Glu 518 A A 1.52 F 0.90 3.00 Val 519 A A 2.38 F 0.90 2.68 Glu 520 A A 2.38 *F 0.90 3.09 Arg 521 A .T 1.52 3027 Pro 522 A T 0.67 1.30 2.76 Lys 523 A T 0.67 *F 1.30 1.18 Pro 524 B T 0.8 *F 1.30 1.01 Val 525 B .0.83 0.65 0.65 EVal 526 B 0.43T 0.65 03 Res Pos. Garni.. Chou-... Garni.. Chou-... Garni.. Chou,.. Garni.. Kyte-... Eisen... Eisen... Karpi James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi Antig Surfa...
Asp 527 .B T .0.06 -0.05 0.22 Gly 528 B T 0.20 *F -0.05 0.30 Gly 529 T T 0.28 F 0.65 0.62 Trp 530 T C 0.23 0.00 0.39 Ala 531 C 0.88 .0.20 0.39 Pro 532 .T 0.59 0.00 0.61 Trp 533 .T 0.59 0.00 0.61 Gly 534 T C 1 0.93 0.00 0.59 Pro 535 TT .0.56 F 0.35 0.66 Trp 536 T T .0.84 *F 0.66 0.34 Gly 537 T C 1.17 *F 1.07 0.46 Glu 538 .T 1.14 *F 1.98 0.58 Cys 539 .T T .0.82 *F 2.49 0.80 Ser 540 T T .0.69 *F 3.10 0.43 Arg 541 T T .0.63 2.79 0.25 Tlir 542 .T T .0.63 *F 2.18 0.46 Cys 543 .T T 0.22 *F 1.87 0.34 Gly 544 .T T .0.44 1.56 0.13 Gly 545 .T T .0.04 F 0.65 0.15 Gly 546 .T T 0.37 *F 0.35 0.25 Val 547 B B 0.09 0.60 0.33 GIn 548 B B .0.69 0.60 0.46 Res Pos. Garni.. Chou-... Garni.. Chou-... Garni.. Chouw... Garni.. Kyte-... Eisen... Eisen... Karpi James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi Antig Surfa...
Phe 549 B B 1.03 .0.30 0.91 Ser 550 .B B 0.71 .0.79 2.13 His 551 B 1.10 *.1.18 0.66 Arg 552 T .1.96 .2.37 1.52 Glu 553 T 1.74 F 2.86 1.89 Cys 554 T T .2.44 3.40 2.15 Lys 555 T T .2.53 3.06 1.90 Asp 556 T C 2.57 2.52 1.70 Pro 557 T C 2.46 *F 2.52 5.49 Glu 558 C 2.11 F 2.32 4.41 Pro 559 T T .2.43 2.72 2.62 Gin 560 T T .2.50 .F 2.76 1.67 Asn 561 .T T .2.26 F 3.40 1.89 Gly 562 T T .1.80 F 2.76 1.92 Gly 563 T T .0.99 F 2.27 0.59 Arg 564 B T .0.86 0.93 0.30 Tyr 565 B T .0.97 0.44 0.30 Cys 566 B T .1.08 1.00 0.60 Leu 567 B 0.83 1.40 0.60 Gly 568 B .1.22 1.55 0.39 Arg 569 B 0.87 2.30 1.45 Arg 570 .1 3.00 27 Res Pos. Garni.. Chou-... Garni.. Chou-... Garni.. Chou-.. Garni.. Kyte-... Eisen... Eisen... Karpl James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi Antig Surfa...
Ala 571 T 1.48 F 2.70 4.82 Lys 572 T 1.62 2.40 3.30 Tyr 573 T T .1.93 1.85 0.90 Gin 574 T T .1.51 F 1.10 1.22 Ser 575 T T .1.40 0.50 0.88 Cys 576 T T .1.99 .0.50 0.97 His 577 .A B 1.28 0.60 0.97 Thr 578 .A T 1 .31 F 0.85 0.39 Glu 579 .A .T 1 .10 F 1.00 1.12 Glu 580 .A .T 1.40 F 1.64 1.27 Cys 581 .A B 1.72 1.58 1.47 Pro 582 T C 1.80 .F 2.37 0.84 Pro 583 T T .1.81 I .F 2.91 0.97 Asp 584 T T .1.11 3.40 2.43 Gly 585 T T .1.22 *F 3.06 1.36 Lys 586 .A T 1.89 *F 2.32 1.72 Ser 587 A A .2.10 F 1.58 1.79 Phe 588 A A .2.31 F 1.24 3.13 Arg 589 A A 1.64 F 0.90 2.71 Glu 590 A A 1.99 F 0.60 1.08 Gin 591 A A .1.99 F 0.90 2.17 Gin 592 A A .2.04 *F 0.90 2.21 0 c~J -a 0~.
0
C.,
ri~ 0 S 10 15 Res Pos. Garni.. Chou-... Garni.. Chou,.. Garni.. Chou,.. Garni.. Kyte-... Eisen... Eisen... Karpi... James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi... Antig Surfa...
Cys 593 A A 2.74 1.15 2.00 G Iu 594 .A T 2.04 F 1.50 1.86 Lys 595 .A T 1.80 F 1.75 1.08 Tyr 596 .T 1.80 2.05 3.17 Asn 597 .T T .1.56 2.50 2.94 Ala 598 .T T .1.91 1.35 2.30 Tyr 599 B .T .1.9!1 0.70 2.12 Asn 600 B .T .1.27 2.20 Tyr 601 B 1.51 0.25 2.16 Thr 602 B 1.17 0.70 2.30 Asp 603 B T .1.76 1.75 1.42 Met 604 B T .1.19 2.00 1.45 Asp 605 .T T .0.38 *F 2.50 0.83 Gly 606 B .T .0.62 F 1.85 0.41 Asn 607 B B 0.64 F 0.60 0.72 Leu 608 A B -0.21 0.45 Leu 609 B B 0.18 0.35 0.34 Gin 610 B B 0.22 0.33 Trp 611 B B .0.32 -0.60 0.79 Val 612 B B 0.27 1.50 Pro 613 B T .0.20 0.88 0 "0 ~0 .4 a" 0'~ 0 Res Pos. Garni.. Chou-... Garni.. Chou-. Garni.. Chou-... Garni.. Kyte-... Eisen... Eisen... Karpi James Emini Alpha Alpha Bets Beta Turn Turn Coil Hydro... Alpha Beta Flexi Antig Surfa...
Lys 614 B T .0.16 .0.20 0.82 Tyr 615 B T 0.14 0.10 0.82 Ala 616 T T 0.07 0.71 Gly 617 T 0.90 *.0.64 0.55 Val 618 .B 1.11 0.69 Ser 619 B .T .1.18 2.32 1.14 Pro 620 B .T .0.76 2.66 2.26 Arg 621 .T T .1.39 3.40 1.63 Asp 622 .T T .0.92 3.06 2.43 Arg 623 .A T 1.08 2.32 1.30 Cys 624 .A B 0.71 F 1.43 0.57 Lys 625 .A B 1.03 .0.64 0.18 Leu 626 .A B 0.33 0.18 Phe 627 .A B 0.44 0.04 0.35 Cys 628 .A B -0.01 0.98 0.34_ Arg 629 .A B 0.77 0.41 Ala 630 A .T .0,42 .2.36 0.92 Arg 631 T T .1.23 3.40 2.31 Gly 632 .T T .1.23 3.06 2.04 Arg 633 .T T .1.94 2.72 1.75 Ser 634 A A 0.98 F 1.58 1.79 Res Pos. Garni.. Chou-... Garni.. Chou,.. Garni.. Chou-... Garni.. Kyte-... Eisen... Eisen... Karpi James Emini Alpha Alpha Beta Beta Turn Turn Coil H~ydro... Alpha Beta Flexi Antig Surfa...
Glu 635 A A 0.87 F 1.24 1.34 Phe 636 A A .0.76 S*F 0.45 0.59 Lys 637 A A 0.51 .0.30 0.77 Val 638 A A 0.44 0.45 Phe 639 A A 0.11 0.45 1.03 Glu 640 A A 1.00 0.38 Ala 641 A .B 0.30 -0.30 0.36 Lys 642 A B -0.69 .0.30 0.70 Val 643 A B 0.14 0.60 0.40 lie 644 A B 0.26 *F 0.45 0.57 Asp 645 B B 0.92 F 0.45 0.23 Gly 646 B B -0.68 *F -0.45 0.17 Thr 647 B B 0.93 *F -0.15 0.24 Leu 648 B C-0.08 F 0.05 0.22 Cys 649 B T 0.50 5*.0.10 0.39 Gly 650 .T C -0.31 F 0.45 0.39 Pro 651 T T 0.56 F 0.65 0.39 Glu 652 A .T 1.13 F 0.25 0.73 Thr 653 A .T 0.99 F 0.25 0.52 Leu 654 A .B 1.18 0.18 Ala 655 B B -0.72 0.60 0.08 Ile 1656 7 B I I -0.861 1 -0.60 F 0.10 S 10 Res Pos. Garni.. Chou,.. Garni.. Chou-... Garni.. Chou-... Garni.. Kyte-... Eisen... Eisen... Karp! James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi Antig Surfa...
Cys 657 B B 0.86 0.13 Val 658 A B 1.21 0.21 Arg 659 B B 1.26 0.30 0.16 Gly 660 B T T 0.62 0.25 0.23 Gin 661 B B 0.32 0.45 0.61 Cys 662 B B 0.00 0.30 0.32 Val 663 B B 0.19 0.32 Lys 664 B 0.08 0.10 Ala 665 B T .0.39 0.70 0.30 Gly 666 B T 0.47 0.70 0.56 Cys 667 B T 0.66 .0.70 0.21 Asp 668 B B 0.20 0.15 His 669 B B 0.14 .0.30 0.26 Va! 670 B B 0.23 0.64 Va! 671 B B 0.69 *.0.64 0.59 Asp 672 B B 1.40 1.13 0.86 Ser 673 B T .0.59 *F 2.32 2.31 Pro 674 A T .0.62 *F 2.66 2.56 Arg 675 .T T .1.52 .F 3.40 2.56 Lys 676 .T T 1.l'71 3.06 3.82 Leu 677 T 1.37 .F 2.52 1.33 Asp 678 .T T .8!1 F 2.23 0. 67] Z- 15 Res Pos. Garni.. Chou-... Garni.. Chou-... Garni.. Chou-... Garni.. Kyte-... Eisen... Eisen... Karpl... James... Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi... Antig... Surfa...
Lys 679 B T 0.36 F 1.49 0.25 Cys 680 B T -0.10 0.70 0.16 Gly 681 B T -0.49 0.70 0.10 Val 682 B 0.37 -0.10 0.05 Cys 683 B T 0.02. 0.10 0.18 Gly 684 T T -0.02 F 1.59 0.18 Gly 685 T T 0.34 F 1.93 0.38 Lys 686 T T 0.02 F 2.27 0.96 Gly 687 T 0.99 F 2.41 0.52 Asn 688 T T 1.70 F 3.40 1.03 Ser 689 B T 1.19 F 2.66 1.03 Cys 690 B T 1.23 F 2.34 0.77 Arg 691 B T 0.84 F 2.17 0.64 Lys 692 B 0.89 F 1.80 0.47 Val 693 B T 0.08 F 1.98 1.18 Ser 694 B T 0.07 F 1.70 0.50 Gly 695 B T 0.52 F 0.93 0.36 Ser 696 B T 0.10 F 0.46 0.75 Leu 697 B 0.06 F 0.39 0.81 Thr 698 B 0.67 F 0.37 1.31 Pro 699 B T 0.62 F 0.10 1.53 Thr 700 T T 0.72 F 0.50 1.84 Res Pos. Garni.. Chou-... Garni.. Chou-... Garni.. Chou-.. Garni.. Kyte-... Eisen... Eisen... Karpi James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi... Antig Surfa...
Asn 701 B T .1.02 F 0.10 2.00 Tyr 702 T T .1.83 *.0.35 2.08 Gly 703 T T .1.26 *.0.65 2.41 Tyr 704 T T .0.61 1.05 Asn 705 .B .T .0.61 0.50 Asp 706 B T -0.28 *.0.10 0.72 lie 707 B B 0.24 0.32 Val 708 B 13. -0.49 0.30 0.31 Thr 709 B B -0.59 0.19 Ilie 710 B B -1.18 -0.60 0.27 Pro 711 B T 1.49 -0.20 0.36 Ala 712 B T -0.60 0.36 Gly 713 T C -0.63 0.00 0.83 Ala 714 .T C -0.32 0.15 0.38 Thr 715 B B -0.29 0.45 0.62 Asn 716 B B -0.03 I *F -0.15 0.47 Ile 717 B B 0.56 *F 0.45 0.92 Asp 718 B B3. 1.01 0.60 1.11 Val 719 B B 1.30 I *F 0.90 1.35 Lys 720 B B 1.58 0.90 2.58 Gin 721 B 1.37 1.10 2.10 Arg 72 1 1.10 4.38 Res Pos. Garni.. Chou-... Garni.. Chou-... Garni.. Chou-... Garni.. Kyte-... Eisen... Eisen... Karpi James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi Antig Surfa...
Ser 723 C 1.06 1.30 2.17 724 T C 1.91 *F 1.05 0.93 His Pro 725 T C 1.87 1.33 0.82 Gly q726 T T .1.87 F 1.21 0.99 Val 727 B .T .1.41 F 1.84 1.21 Gin 728 B 1.71 1.77 0.77 Asn 729 B T T .1.50 *F 2.80 1.26 Asp 730 .T T .0.90 1.92 2.66 Gly 731 T T .0.66 F 1.64 1.27 Asn 732 B T .0.70 *F 0.81 0.80 Tyr 733 .A B .0.74 0.32 0.39 Leu 734 .A B 0.43 0.79 Ala 735 .A B 0.16 0.71 Leu 736 .A B 0.19 -0.40 0.46 Lys 737 .A B -0.16 F 0.85 0.93 Thr 738 B .T .0.09 F 1.45 0.91 Ala 739 A .T .0.66 F 2.10 1.91 Asp 740 B T .0.43 F 2.00 1.50 Gly 741 B .T .0.43 *F 1.05 0.86 Gin 742 B 0.39 *F 0.35 0.70 Tyr 1743 B 0.36 0.30 0.67 Res Pos. Garni.. Chou,.. Garni.. Chou,.. Garni.. Chou-.. Garni.. Kyte-... Eisen... Eisen... Karpi James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi Antig Surfa...
Leu 744 B 0.94 0.67 Leu 745 B 0.13 0.40 0.63 Asn 746 B T 0.11 *F -0.05 0.33 Gly 747 T T 1.00 *F 0.35 0.40 Asn 748 T C -1.06 0.34 Leu 749 T C -0.83 0.29 Ala 750 A A B 0.91 0.29 Ile 751 .A B 0.91 0.13 Ser 752 .A B 0.57 0.27 Ala 753 A A -0.57 0.30 0.46 Ile 754 A A 0.64 0.45 1.09 Glu 755 A A 0.87 0.45 0.57 Gin 756 A B 0.83 *F 0.45 0.47 Asp 757 A .B -0.49 *F -0.15 0.49 Ile 758 A B -0.24 0.60 0.57 Leu 759 A B 0.33 0.30 0.33 Val 760 A B -0.56 0.30 0.28 Lys 761 A B 1.37 -0.45 0.28 Gly 762 B B -1.32 *F -0.45 0.28 Thr 763 B B -0.68 0.45 0.76 Ile 764 B B -0.17 F -0.15 0.59 Leu 765 B B 0.34 -0.60 0.80 Res Pos. Garni.. Chou-... Garni.. Chou,.. Garni.. Chou-... Garni.. Kyte-... Eisen... Eisen... Karpi... James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi... Antig Surta...
Lys 766 B B 0.00 -0.45 0.55 Tyr 767 B T 0.54 *F 0.40 1.05 Ser 768 T C -0.82 F 0.45 0.89 Gly 769 T C -0.24 *F 0.45 0.45 Ser 770 T C -0.24 0.15 0.42 fie 771 .A B 0.29 0.60 0.26 Ala 772 .A B 0.07 0.45 1Thr 773 .A B 0.44 0.66 Leu 774 .A B -0.10 -0.30 0.77 Glu 775 A A -0.10 *.0.45 1.32 Arg 776 .A B 0.09 F 0.60 1.23 Leu 777 .A T .0.79 I .F 1.00 1.29 Gln 778 .A .T 0.89 F 1.30 1.46 Ser 779 .A .T .0.89 F 1.00 1.15 Phe 780 B .0.68 *F 0.41 1.15 Arg 781 C 0.57 *F 0.82 1.03 Pro 782 C 1.17 1.63 1.33 Leu 783 T C 0.36 *F 2.04 2.37 Pro 784 T C 0.34 2.10 1.00 Glu 785 .T C 0.19 F 1.29 0.93 FPro 786 .B T .0.08 0.88 0.84 Leu 787 .B B -0.52 .F 0.27 0.94 Res Pos. Garni.. Chou-... Garni.. Chou-.. Garni.. Chou,.. Garni.. Kyte-... Eisen... Eisen... Karpi James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi Antig Surfa...
Thr 788 B B 0.52 0.45 Val 789 B B 0.62 0.60 0.24 Gln 790 B B 1.48 0.60 0.42 Leu 791 B B 1.48 0.60 0.21 Leu 792 .B B 1.01 0.45 Thr 793 .B B 0.70 0.26 Val 794 B .T 0.70 .F 0.25 0.54 Pro 795.. B T 1.40 F 0.25 0.48 Gly 796 B .T 0.80 -0.05 0.29 Glu 797 B .T 0.20 0.25 0.60 Val 798 B 0.16 *F 0.05 0.60 Phe 799 B 0.16 *F 1.00 1.22 Pro 800 B .T .0.41 *F 1.25 0.52 Pro 801 .T T .0.51 2.00 1.41 Lys 802 T T .0.20 F 1.60 2.55 Val 803 B T .0.36 2.00 2.38 Lys 804 B B 0.36 0.80 1.33 Tyr 805 B B 0.29 0.00 0.58 Thr 806 B B 0.29 0.58 Phe 807 B B -0.33 -0.40 0.45 Phe 808 B B 0.52 0.46 Va 89 .B .T 0.38 4 020 0.53- Res Pos. Garni.. Chou-... Garni.. Chou-... Garni.. Chou-... Garni.. Kyte-... Eisen... Eisen... Karpi James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi Antig Surfa...
Pro 810 B T 0.13 I *F -0.05 0.45 Asn 811 T T 0.52 *F 1.25 0.88 Asp 812 T T 0.12 1.40 1.02 Val 813 A -0.02 *F 0.65 0.89 Asp 814 A 0.83 .0.50 0.54 Phe 815 A 0.74 0.57 Ser 816 A 0.44 1.02 Met 817 A 0.49 0.82 GIn 818 A T .1.34 2.20 1.89 Ser 819 T C 1.46 3.00 2.44 Ser 820 T C 1.57 2.70 4.84 Lys 821 A .T .1.56 2.20 2.82 Glu 822 A 1.84 I *F 1.70 3.04 Arg 823 A .B 1.84 F 1.20 3.27 Ala 824 A .B 1.26 F 0.90 2.63 1Thr 825 B B 0.67 F 0.60 1.06 Thr 826 B B 062 F -0.15 0.38 Asn 827 B B 0.41 0.65 Ile 828 B 1 B -0.51 0.70 Ile 829 B B -0.73 0.40 GIn 830 .A B -0.46 0.21 LPro 831 A B F -0.73 0.40 Res Pos. Garni.. Chou-... Garni.. Chou-... Garni.. Chou-... Garni.. Kyte-... Eisen... Eisen... Karpl James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi Antig Surfa...
Leu 832 .A B 0.73 0.57 Leu 833 A B 0.13 0.60 0.57 Hius 834 .A B 0.10 0.39 Ala 835 .A B B 0.91 0.35 Gin 836 .A B B 1.04 0.60 0.35 Trp 837 .A B B 0.23 0.60 0.26 Val 838 .A B B 0.29 0.42 Leu 839 B T .0.02 0.20 0.26 Gly 840 T T .0.61 *.0.45 0.33 Asp 841 T T 0.06 I .F 1.15 0.76 Trp 842 T T 0.07 F 2.00 0.50 Ser 843 T C 0.49 I F 2.05 0.67 Glu 844 .T T .0.99 F 2.50 0.54 Cys 845 T T .0.67 F 1.65 0.74 Ser 846 .T T .0.32 F 2.00 0.30 Ser 847 .T 0.02 F 1.55 0.17 Thr 848 T 1 -0.02 F 0.70 0.32 Cys 849 T -0.31 F 0.45 0.24 Gly 850 T T .0.36 *.0.20 0.18 Ala 851 T T .0.77 0.20 0.22 Gly 852 .T T .1.18 0.50 0.81 Trp 853 .T T .8 1.25 1.60] Res Pos. Garni.. Chou-... Garni.. Chou-... Garni.. Chou-.. Garni.. Kyte-... Eisen... Eisen... Karpi James Emini Alpha Alpha Beta Beta Turn Turn Coil Hlydro... Alpha Beta Flexi Antig Surfa...
Gin 854 B B 0.99 .F 0.60 2.29 Arg 855 B B 1.33 *F 0.60 1.72 Arg 856 B B 1.26 0.90 2.83 Thr 857 B B 1.71 F 1.05 0.87 Val 858 B B 2.00 1.20 0.87 Glu 859 B B 1.79 1.50 0.75 Cys 860 T 1.38 0.80 Arg 861 T 0.92 F 3.00 1.44 Asp 862 T C 1.23 2.55 0.82 Pro 863 T T .1.50 2.60 2.66 Ser 864 .T T .1.20 2.30 1.37 Gly 865 T T .1.28 F 1.70 1.10 GIn 866 A 0.86 0.05 0.72 Ala 867 B 0.19 *F 0.05 0.78 Ser 868 B .0.40 -0.10 0.42 Ala 869- A .0.74 -0.10 0.39 Trhr 870 A T .0.50 *.0.70 0.77 Cys 871 A T 0.31 0.58 Asn 872 A T .0.32 *.0.10 0.48 Lys 873 A T .0.41 F 0.85 0.66 Ala 874 A 1.00. .tO F 0.80 1.90 Leu 875 A 1.31 1.10 2.05] Res Pos. Garni.. Chou-... Garni.. Chou-... Garni.. Chou-... Garni.. Kyte-... Eisen... Eisen... Karpi James Emini Alpha Alpha Beta Beta Turn Turn Coil Hydro... Alpha Beta Flexi Antig Surfa...
Lys 876 A T .1.39 F 1.30 1.71 Pro 877 A T .1.43 F 1.30 1.71 Glu 878 A T .1.18 F 1.30 4.14 Asp 879 A T .1.10 F 1.30 3.20 Ala 880 A 1.91 F 1.10 1.11 Lys 881 A T .1.57 F 1.30 1.11 Pro 882 A T .1.78 .F 1.15 0.89 Cys 883 A .T .0.97 1.30 1.53 Glu 884 A .T .0.30 F 1.15 0.63 Ser 885 A A 0.68 -0.15 0.22 Gin 886 .A B 0.18 .F -0.15 0.63 Leu 887 .A B 0.36 -0.30 0.30 Cys 888 .A B -0.08 -0.60 0.29 Pro 889 .A B -0.47 -0.60 0.21 Leu 890 B 0.56 -0.40 0.33 WO 99/37660 PCT/US99/01313 -32- Detailed Description By screening cDNA libraries with cDNA encoding the anti-angiogenic domain of TSP-1, the present inventors have identified two novel proteins, METHI and METH2 (also called VEGA-1 and VEGA-2, respectively, for vascular endothelial growth antagonist) which contain the anti-angiogenic domain ofTSP-1, a metalloproteinase domain, and a disintegrin-like domain. The present inventors have demonstrated that both METH1 and METH2 have anti-angiogenic activity.
Thus, the present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding a METH1 polypeptide having the amino acid sequence shown in SEQ ID NO:2, which was determined by sequencing a cloned cDNA. The METH1 protein of the present invention shares sequence homology with thrombospondin-1 and pNPI. The nucleotide sequence shown in SEQ ID NO: 1 was obtained by sequencing a cDNA clone, which was deposited on January 15, 1998 at the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110-2209, and given accession number 209581.
The cDNA clone contained in ATCC Deposit No. 209581 contains a METHI sequence, encoding amino acids 1 to 950 of SEQ ID NO:2.
The present invention also provides isolated nucleic acid molecules comprising a polynucleotide encoding a METH2 polypeptide having the amino acid sequence shown in SEQ ID NO:4, which was partially determined by sequencing a cloned cDNA. The METH2 protein of the present invention shares sequence homology with thrombospondin-1 and pNPI. The nucleotide sequence shown in SEQ ID NO:3 was partially obtained by sequencing a cDNA clone, which was deposited on January 15, 1998 at the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110-2209, and given accession number 209582. The cDNA clone contained in ATCC Deposit No. 209582 contains a partial METH2 sequence, encoding amino acids 112-890 of SEQ ID NO:4.
WO 99/37660 PCTfUS99/01313 -33- Nucleic Acid Molecules Some of the nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer (such as the Model 373 from Applied Biosystems, Inc.), and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA sequence determined as above. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art. As is also known in the art, a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion.
Using the information provided herein, such as the nucleotide sequence in SEQ ID NO: 1 or SEQ ID NO:3, a nucleic acid molecule of the present invention encoding a METHI or METH2 polypeptide may be obtained using standard cloning and screening procedures, such as those for cloning cDNAs using mRNA as starting material. Illustrative of the invention, the nucleic acid molecule described in SEQ ID NO: 1 was discovered in a cDNA library derived from human heart and the nucleic acid molecule described in SEQ ID NO:3 was discovered in a cDNA library derived from human lung. The determined nucleotide sequence of the METH1 cDNA of SEQ ID NO: 1 contains an open reading frame encoding WO 99/37660 PCT/US99/01313 -34a protein of about 950 amino acid residues, including a predicted leader sequence of about 28 amino acid residues. The present inventors have determined that the nucleotide sequence of the METH2 cDNA of SEQ ID NO:3 contains an open reading frame encoding a protein of about 890 amino acid residues, including a predicted leader sequence of about 23 amino acid residues.
The present invention also provides the mature form(s) of the METH I and METH2 proteins of the present invention. According to the signal hypothesis, proteins secreted by mammalian cells have a signal or secretory leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Most mammalian cells and even insect cells cleave secreted proteins with the same specificity. However, in some cases, cleavage of a secreted protein is not entirely uniform, which results in two or more mature species on the protein. Further, it has long been known that the cleavage specificity of a secreted protein is ultimately determined by the primary structure of the complete protein, that is, it is inherent in the amino acid sequence of the polypeptide. Therefore, the present invention provides a nucleotide sequence encoding the mature METHI polypeptide having the amino acid sequence encoded by the cDNA clone contained in the host identified as ATCC Deposit No. 209581 and as shown in SEQ ID NO:2. The present invention also provides a nucleotide sequence encoding the mature METH2 polypeptide having the amino acid sequence as shown in SEQ ID NO:4. By the mature METHI protein having the amino acid sequence encoded by the cDNA clone contained in the host identified as ATCC Deposit No. 209581 is meant the mature form(s) of the METHI protein produced by expression in a mammalian cell COS cells, as described below) of the complete open reading frame encoded by the human DNA sequence of the clone contained in the vector in the deposited host. As indicated below, the mature METHI having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 209581 may or may not differ from the predicted "mature" METH 1 protein shown in SEQ ID NO:2 (amino acids from about 29 to about 950) depending on the accuracy of WO 99/37660 PCT/S99/01313 the predicted cleavage site based on computer analysis; and the mature METH2 may or may not differ from the predicted "mature" METH2 protein shown in SEQ ID NO: 4 (amino acids from about 24 to about 890) depending on the accuracy of the predicted cleavage site based on computer analysis.
Methods for predicting whether a protein has a secretory leader as well as the cleavage point for that leader sequence are available. For instance, the methods of McGeoch (Virus Res. 3:271-286 (1985)) and von Heinje (Nucleic Acids Res. 14:4683-4690 (1986)) can be used. The accuracy of predicting the cleavage points of known mammalian secretory proteins for each of these methods is in the range of 75-80%. von Heinje, supra. However, the two methods do not always produce the same predicted cleavage point(s) for a given protein.
In the present case, the predicted amino acid sequence of the complete METHI and METH2 polypeptides of the present invention were analyzed by a computer program ("PSORT") Nakai and M. Kanehisa, Genomics 14:897-911 (1992)), which is an expert system for predicting the cellular location of a protein based on the amino acid sequence. As part of this computational prediction of localization, the methods of McGeoch and von Heinje are incorporated. The analysis by the PSORT program predicted the cleavage site between amino acids 28 and 29 in SEQ ID NO:2 and amino acids 23 and 24 in SEQ ID NO:4.
Thereafter, the complete amino acid sequences were further analyzed by visual inspection, applying a simple form of the rule of von Heinje. von Heinje, supra. Thus, the leader sequence for the METH1 protein is predicted to consist of amino acid residues from about 1 to about 28 in SEQ ID NO:2, while the mature METH1 protein is predicted to consist of residues from about 29 to about 950; and the leader sequence for the METH2 protein is predicted to consist of amino acid residues from about 1 to about 23 in SEQ ID NO:4, while the mature METH2 protein is predicted to consist of residues from about 24 to about 890.
An alternative predicted mature METH protein consists of residues 30 to 950 in SEQ ID NO:2.
WO 99/37660 PCT/US99/01313 -36- As one of ordinary skill would appreciate, due to the possibilities of sequencing errors, as well as the variability of cleavage sites for leaders in different known proteins, the predicted METH1 polypeptide encoded by the deposited cDNA comprises about 950 amino acids, but may be anywhere in the range of 910-990 amino acids; and the predicted leader sequence of this protein is about 28 amino acids, but may be anywhere in the range of about 18 to about 38 amino acids. Also, the predicted METH2 polypeptide comprises about 890 amino acids, but may be anywhere in the range of 850 to about 930 amino acids; and the predicted leader sequence of this protein is about 23 amino acids, but may be anywhere in the range of about 13 to about 33 amino acids.
As indicated, nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced synthetically. The DNA may be double-stranded or single-stranded. Single-stranded DNA or RNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand.
By "isolated" nucleic acid molecule(s) is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment. For example, recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution.
Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.
Isolated nucleic acid molecules of the present invention include DNA molecules comprising an open reading frame (ORF) shown in SEQ ID NO: 1; DNA molecules comprising the coding sequence for the mature METH 1 protein; and DNA molecules which comprise a sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode WO 99/37660 PCT[S99/01313 -37the METH1 protein. Also included are DNA molecules comprising an open reading frame (ORF) shown in SEQ ID NO:3; DNA molecules comprising the coding sequence for the mature METH2 protein; and DNA molecules which comprise a sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode the METH2 protein. Of course, the genetic code is well known in the art. Thus, it would be routine for one skilled in the art to generate such degenerate variants.
In another aspect, the invention provides isolated nucleic acid molecules encoding the METH1 or METH2 polypeptides having an amino acid sequence as encoded by the cDNA clones contained in the plasmids deposited as ATCC Deposit No. 209581 on January 15, 1998 or ATCC Deposit No. 209582 on January 15, 1998, respectively. In a further embodiment, nucleic acid molecules are provided encoding the mature METH1 or METH2 polypeptide or the fulllength METH1 or METH2 polypeptide lacking the N-terminal methionine. The invention also provides an isolated nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:3 or the nucleotide sequence of the METH 1 or METH2 cDNA contained in the above-described deposited clones, or a nucleic acid molecule having a sequence complementary to one of the above sequences. Such isolated molecules, particularly DNA molecules, are useful as probes for gene mapping, by in situ hybridization with chromosomes, and for detecting expression of the METH1 or METH2 gene in human tissue, for instance, by Northern blot analysis.
The present invention is further directed to fragments of the isolated nucleic acid molecules described herein. By a fragment of an isolated nucleic acid molecule having the nucleotide sequence of the deposited cDNA or the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO:3 is intended fragments at least about 15 nt, and more preferably at least about 20 nt, still morepreferably at least about 30 nt, and even more preferably, at least about 40 nt in length which are useful as diagnostic probes and primers as discussed herein. Of course, larger fragments 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, WO 99/37660 PCT/US99/01313 -38- 750, 800, 850, 900, 950, 1000, 1050, 1100, 1200, 1300, 1400,1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, or 3000 nt in length are also useful according to the present invention as are fragments corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA or as shown in SEQ ID NO: 1 or SEQ ID NO:3. By a fragment at least 20 nt in length, for example, is intended fragments which include 20 or more contiguous bases from the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in SEQ ID NO:1 or SEQ ID NO:3.
Preferred nucleic acid fragments of the present invention include nucleic acid molecules encoding epitope-bearing portions of the METHI or METH2 protein. Methods for determining epitope-bearing portions of the METH1 and METH2 proteins are described in detail below.
Other preferred nucleic acid fragments of the present invention include nucleic acid molecules encoding: the metalloprotease domain ofMETH1, amino acids 235 to 459 in SEQ ID NO:2; the disintegrin domain of METH1, amino acids 460 to 544 in SEQ ID NO:2; the first TSP-like domain ofMETHI, amino acids 545 to 598 in SEQ ID NO:2; the second TSP-like domain ofMETH1, amino acids 841 to 894 in SEQ ID NO:2; the third TSP-like domain of METH1, amino acids 895 to 934 in SEQ ID NO:2; amino acids 536 to 613 in SEQ ID NO:2; amino acids 549 to 563 in SEQ ID NO:2; the metalloprotease domain of METH2, amino acids 214 to 439 in SEQ ID NO:4; the disintegrin domain of METH2, amino acids 440 to 529 in SEQ ID NO:4; the first TSP-like domain of METH2, amino acids 530 to 583 in SEQ ID NO:4; the second TSP-like domain of METH2, amino acids 837 to 890 in SEQ ID NO:4; amino acids 280 to 606 in SEQ ID NO:4; and amino acids 529 to 548 in SEQ ID NO:4.
In addition, the present inventors have identified the following cDNA clones related to portions of the sequence shown in SEQ ID NO: 1: HOUCQ 17RA (SEQ ID NO: 14), HPLBMIIR (SEQ ID NO:15), HGBI07R (SEQ ID NO:16), HNTMA49R (SEQ ID NO: 17), HNALE27R (SEQ ID NO: 18), and (SEQ ID NO:19).
WO 99/37660 PCTIUS99/01313 -39- The following public ESTs, which relate to portions of SEQ ID NO: 1, have also been identified: D67076 (SEQ DD NO:20), ABO01735 (SEQ ID NO:2 X14787 (SEQ ID NO:22), U64857 (SEQ ID NO:23), X04665 (SEQ ID NO:24), M64866 (SEQ ID NO:25), L07803 (SEQ ID NO:26), U08006 (SEQ ID NO:27), M16974 (SEQ ID NO:28), L13855 (SEQ ID NO:29), AL021 529 (SEQ ID NO:30), D86074 (SEQ ID NO:3 L05390 (SEQ ID NO:32), Z69361 (SEQ DD NO:33), X99599 (SEQ ID NO:34), AF018073 (SEQ ID NO:.35), L23760 (SEQ I7D NO:36), Z46970 (SEQ lID NO:-37), AC004449 (SEQ ID NO:38), Z69589 (SEQ ID NO:39), Z22279 (SEQ ID NO:40), and X17524 (SEQ ID NO:41).
The present inventors have also identified the following cDNA clones related to portions of SEQ ID NO:3: HCE4D69FPO2 (SEQ ID NO:42), (SEQ ID NO:43), HKIXH64R (SEQ ID NO:44), HIBDB45R (SEQ ID NO:19), HCE3Z95R (SEQ ID NO:45), HTLEQ90R (SEQ ID NO:46), HM4WEF4SR (SEQ ID NO:47), HTOFC34RA (SEQ ID NO:48), HH-FD120R (SEQ ID NO:49),I-HMSHY47R (SEQ ID NO: 50), HCESF9OR (SEQ IDNO :51), IICA046R (SEQ ID NO:52), HTTAQ67R (SEQ ID NO:53), HFKCF19F (SEQ lID NO:54), HMCAS31R (SEQ ID NO:55), HMvWGP26R (SEQ ID NO:56), HLHTP36R (SEQ ID NO:57), HE8ANIIR (SEQ ID NO:58), HEONN73R(SEQ ID NO:59), HBNBG53R (SEQ ID NO:60), and HMSCH94R (SEQ ID NO: 61).
The following public ESTs, which relate to portions of the sequence shown in SEQ ID NO:3, have also been identified: D67076 (SEQ ID AB001735 (SEQ ID NO:21), AB005287 (SEQ ID NO:62), X87619 (SEQ ID NO:63), X14787 (SEQ ID NO:22), X04665 (SEQ ID NO:24), M87276 (SEQ ID NO:64), M62458 (SEQ ID NO:65), AB002364 (SEQ ID NO:66), AB005297 (SEQ ID NO:67), X691 61 (SEQ ID NO:68), X1 6619 (SEQ ID NO:69), 136448 (SEQ ID NO:70), L12260 (SEQ ID NO:71), 136352 (SEQ ID NO:72), X15898 (SEQ ID NO:73), 107789 (SEQ ID NO:74), 108144 (SEQ ID NO:75) U31814 (SEQ ID NO: 76), and AF00 1444 (SEQ ID NO: 77).
WO 99/37660 PCTIUS99/01313 In specific embodiments, the polynucleotides of the invention are less than 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, or 7.5 kb in length. In a further embodiment, polynucleotides of the invention comprise at least 15 contiguous nucleotides of METHI or METH2 coding sequence, but do not comprise all or a portion of any METH1 or METH2 intron. In another embodiment, the nucleic acid comprising METH1 or METH2 coding sequence does not contain coding sequences ofa genomic flanking gene 5' or 3' to the METH1 or METH2 gene in the genome).
In another aspect, the invention provides an isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent hybridization conditions to a portion of the polynucleotide in a nucleic acid molecule of the invention described above, for instance, the cDNA clones contained in ATCC Deposit No. 209581 or ATCC Deposit No. 209582. By "stringent hybridization conditions" is intended overnight incubation at 42 0 C in a solution comprising: 50% formamide, 5x SSC (750 mM NaCI, 75mM trisodium citrate), 50 mM sodium phosphate (pH 5x Denhardt's solution, 10% dextran sulfate, and Rg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1x SSC at about 65 0
C.
By a polynucleotide which hybridizes to a "portion" of a polynucleotide is intended a polynucleotide (either DNA or RNA) hybridizing to at least about nucleotides and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably about 30, 40, 50, 60 or 70 nt of the reference polynucleotide. These are useful as diagnostic probes and primers as discussed above and in more detail below.
By a portion of a polynucleotide of"at least 20 nt in length," for example, is intended 20 or more contiguous nucleotides from the nucleotide sequence of the reference polynucleotide the deposited cDNAs or the nucleotide sequence as shown in SEQ ID NO: 1 or SEQ ID NO:3). Of course, a polynucleotide which hybridizes only to a poly A sequence (such as the 3' terminal poly(A) tract of the METHI or METH2 cDNA shown in SEQ ID NO:1 and SEQ ID NO:3, WO 99/37660 PCT/US99/01313 -41respectively) or to a complementary stretch of T (or U) resides, would not be included in a polynucleotide of the invention used to hybridize to a portion of a nucleic acid of the invention, since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly stretch or the complement thereof practically any double-stranded cDNA clone).
Also contemplated are nucleic acid molecules that hybridize to the METH 1 or METH2 polynucleotides at moderately high stringency hybridization conditions. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages offormamide result in lowered stringency); salt conditions, or temperature. For example, moderately high stringency conditions include an overnight incubation at 37 degree C in a solution comprising 6X SSPE SSPE 3M NaCl; 0.2M NaH 2
PO
4 0.02M EDTA, pH 0.5% SDS, formamide, 100 pg/ml salmon sperm blocking DNA; followed by washes at 50 C with IXSSPE, 0.1% SDS. In addition, to achieve even lower stringency, washes performed following stringent hybridization can be done at higher salt concentrations 5X SSC).
Note that variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.
Of course, a polynucleotide which hybridizes only to polyA+ sequences (such as any 3' terminal polyA+ tract of a cDNA shown in the sequence listing), or to a complementary stretch ofT (or U) residues, would not be included in the definition of"polynucleotide," since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly stretch or the complement thereof practically any double-stranded cDNA clone).
WO 99/37660 PCT/US99/01313 -42- The METH1 or METH2 polynucleotide can be composed of any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, METH1 or METH2 polynucleotides can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, the METHI or METH2 polynucleotides can be composed of triplestranded regions comprising RNA or DNA or both RNA and DNA. METHI or METH2 polynucleotides may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically, or metabolically modified forms.
"SEQ ID NO: 1" refers to a METH1 polynucleotide sequence while "SEQ ID NO:2" refers to a METH1 polypeptide sequence. "SEQ ID NO:3" refers to a METH2 polynucleotide sequence while "SEQ ID NO:4" refers to a METH2 polypeptide sequence.
As indicated, nucleic acid molecules of the present invention which encode a METH1 or METH2 polypeptide may include, but are not limited to, those encoding the amino acid sequence of the mature polypeptide, by itself; the coding sequence for the mature polypeptide and additional sequences, such as those encoding the leader or secretory sequence, such as a pre-, or pro- or preproprotein sequence; the coding sequence of the mature polypeptide, with or without the aforementioned additional coding sequences, together with additional, non-coding sequences, including for example, but not limited to introns and non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing, including splicing and polyadenylation signals, for example ribosome binding and stability of mRNA; WO 99/37660 PCTIUS99/01313 -43an additional coding sequence which codes for additional amino acids, such as those which provide additional functionalities. Thus, the sequence encoding the polypeptide may be fused to a marker sequence, such as a sequence encoding a peptide which facilitates purification of the fused polypeptide. In certain preferred embodiments of this aspect of the invention, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc.), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. The "HA" tag is another peptide useful for purification which corresponds to an epitope derived from the influenza hemagglutinin protein, which has been described by Wilson et al., Cell 37:767-778 (1984). As discussed below, other such fusion proteins include the METHI or METH2 fused to Fc at the N- or C-terminus.
The present invention further relates to variants of the nucleic acid molecules of the present invention, which encode portions, analogs or derivatives of the METH 1 or METH2 protein. Variants may occur naturally, such as a natural allelic variant. By an "allelic variant" is intended one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. Lewin, ed., Genes II, John Wiley Sons, New York (1985). Non-naturally occurring variants may be produced using art-known mutagenesis techniques.
Such variants include those produced by nucleotide substitutions, deletions or additions, which may involve one or more nucleotides. The variants may be altered in coding regions, non-coding regions, or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the METHI or METH2 protein or portions thereof Also especially preferred in this regard are conservative substitutions.
Further embodiments of the invention include isolated nucleic acid molecules comprising a polynucleotide having a nucleotide sequence at least WO 99/37660 PCT/US99/01313 -44identical, and more preferably at least 96%, 97%, 98% or 99% identical to: a nucleotide sequence encoding the polypeptide having the amino acid sequence in SEQ ID NO:2; a nucleotide sequence encoding the polypeptide having the amino acid sequence in SEQ ID NO:2, but lacking the N-terminal methionine; a nucleotide sequence encoding the polypeptide having the amino acid sequence at positions from about 29 to about 950 in SEQ ID NO:2; a nucleotide sequence encoding the polypeptide having the amino acid sequence at position from about to about 950 in SEQ ID NO:2; a nucleotide sequence encoding the polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 209581; a nucleotide sequence encoding the mature METH1 polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 209581; a nucleotide sequence encoding amino acids 235 to 459 in SEQ ID NO:2 (the metalloprotease domain of METH1); a nucleotide sequence encoding amino acids 460 to 544 in SEQ ID NO:2 (the disintegrin domain of METH1); a nucleotide sequence encoding amino acids 545 to 598 in SEQ ID NO:2 (the first TSP-like domain of METH1); a nucleotide sequence encoding amino acids 841 to 894 in SEQ ID NO:2 (the second TSP-like domain of METH1); a nucleotide sequence encoding amino acids 895 to 934 in SEQ ID NO:2 (the third TSP-like domain of METHI); a nucleotide sequence encoding amino acids 536 to 613 in SEQ ID NO:2; a nucleotide sequence encoding amino acids 549 to 563 in SEQ ID NO:2; a nucleotide sequence encoding the polypeptide having the amino acid sequence in SEQ ID NO:4; a nucleotide sequence encoding the polypeptide having the amino acid sequence in SEQ ID NO:4, but lacking the N-terminal methionine; a nucleotide sequence encoding the polypeptide having the amino acid sequence at positions from about 24 to about 890 in SEQ ID NO:4; a nucleotide sequence encoding the polypeptide having the amino acid sequence at positions from about 112 to about 890 in SEQ ID NO:4; a nucleotide sequence encoding the polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 209582; a nucleotide sequence encoding the mature METH2 polypeptide having the amino WO 99/37660 PCTIUS99/01313 acid sequence encoded by the cDNA clone contained in ATCC Deposit No.
209582; a nucleotide sequence encoding amino acids 214 to 439 in SEQ ID NO:4 (the metalloprotease domain ofMETH2); a nucleotide sequence encoding amino acids 440 to 529 in SEQ ID NO:4 (the disintegrin domain of METH2); a nucleotide sequence encoding amino acids 530 to 583 in SEQ ID NO:4 (the first TSP-like domain ofMETH2); a nucleotide sequence encoding amino acids 837 to 890 in SEQ ID NO:4 (the second TSP-like domain ofMETH2); a nucleotide sequence encoding amino acids 280 to 606 in SEQ ID NO:4; a nucleotide sequence encoding amino acids 529 to 548 in SEQ ID NO:4; or a nucleotide sequence complementary to any of the above nucleotide sequences.
By a polynucleotide having a nucleotide sequence at least, for example, "identical" to a reference nucleotide sequence encoding a METH1 or METH2 polypeptide is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the METH1 or METH2 polypeptide. In other words, to obtain a polynucleotide having a nucleotide sequence at least identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
As a practical matter, whether any particular nucleic acid molecule is at least 95%, 96%, 97%, 98% or 99% identical to, for instance, the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO:3 or to the nucleotide sequence of the deposited cDNA clones can be determined conventionally using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis WO 99/37660 PCTIUS99/01313 -46- Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711). Bestfit uses the local homology algorithm of Smith and Waterman, Advances in AppliedMathematics 2: 482-489 (1981), to find the best segment of homology between two sequences. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.
A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al., Comp. Appl. Biosci.
6:237-245 (1990). In a sequence alignment, the query and subject sequences are both DNA sequences. An RNA sequence can be compared by converting U's to T's. The result of said global sequence alignment is in percent identity. Preferred parameters used in a FASTDB alignment of DNA sequences to calculate percent identity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty= 1, Joining Penalty=3 0, Randomization Group Length=0, Cutoff Score=l, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the subject nucleotide sequence, whichever is shorter.
If the subject sequence is shorter than the query sequence because of 5' or 3' deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for 5' and 3' truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the 5' or 3' ends, relative to the query sequence, the percent identity is corrected by calculating the number of bases of the query sequence that are 5' and 3' of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. Whether a nucleotide is WO 99/37660 PCTIS99/01313 -47matched/aligned is determined by the results of the FASTDB sequence alignment.
This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This corrected score is what is used for the purposes of the present invention. Only bases outside the 5' and 3' bases of the subject sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the query sequence are calculated for the purposes of manually adjusting the percent identity score.
For example, a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity. The deletions occur at the 5' end of the subject sequence and, therefore, the FASTDB alignment does not show a match/alignment of the first 10 bases at the 5' end. The 10 unpaired bases represent 10% of the sequence (number of bases at the 5' and 3' ends not matched/total number of bases in the query sequence), so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining bases were perfectly matched the final percent identity would be 90%. In another example, a 90 base subject sequence is compared with a 100 base query sequence. This time the deletions are internal, so that there are no bases on the or 3' ends of the subject sequence which are not matched/aligned with the query.
In this case, the percent identity calculated by FASTDB is not manually corrected.
One again, only bases 5' and 3' of the subject sequence which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are to be made for the purposes of the present invention.
The present application is directed to nucleic acid molecules at least 96%, 97%, 98% or 99% identical to the nucleic acid sequence shown in SEQ ID NO: I or SEQ ID NO:3 or to the nucleic acid sequence of the deposited cDNAs, irrespective of whether they encode a polypeptide having METHI or METH2 activity. This is because even where a particular nucleic acid molecule does not encode a polypeptide having METH I or METH2 activity, one of skill in the art would still know how to use the nucleic acid molecule, for instance, as a WO 99/37660 PCT/US99/01313 -48hybridization probe or a polymerase chain reaction (PCR) primer. Uses of the nucleic acid molecules of the present invention that do not encode a polypeptide having METH1 or METH2 activity include, inter alia, isolating the METH1 or METH2 gene or allelic variants thereof in a cDNA library; in situ hybridization "FISH") to metaphase chromosomal spreads to provide precise chromosomal location of the METH1 or METH2 gene, as described in Verma et al., Human Chromosomes: A Manual ofBasic Techniques, Pergamon Press, New York (1988); and Northern Blot analysis for detecting METH1 or METH2 mRNA expression in specific tissues.
Preferred, however, are nucleic acid molecules having sequences at least 96%, 97%, 98% or 99% identical to the nucleic acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3 or to a nucleic acid sequence of the deposited cDNAs which do, in fact, encode a polypeptide having METHI or METH2 protein activity. By "a polypeptide having METH1 activity" is intended polypeptides exhibiting METH1 activity in a particular biological assay. For example, METHI protein activity can be measured using the chorioallantoic membrane assay (Iruela-Arispe et al., Thrombosis and Haemostasis 78(1):672- 677 (1997)) or the cornea pocket assay (Tolsma et al., J. Cell. Biol. 122:497-511 (1993)), both described in Example 4, below. By "a polypeptide having METH2 activity" is intended polypeptides exhibiting METH2 activity in a particular biological assay. For example, METH2 protein activity can also be measured using the chorioallantoic membrane assay (Iruela-Arispe et al., Thrombosis and Haemostasis 78(1):672-677 (1997)) or the cornea pocket assay (Tolsma et al., J.
Cell. Biol. 122:497-511 (1993)), both described in Example 4, below.
Briefly, in the chorioallantoic assay, the potentially anti-angiogenic compound of interest is added to type I collagen pellets (Vitrogen), along with an angiogenic growth factor, such as bFGF. The samples are mixed and placed onto nylon meshes, and allowed to polymerize. After polymerization is complete, the meshes are placed onto the chorioallantoic membrane of 12 day old chick embryos and placed at 37 0 C for 24 hours. The embryos then injected with a fluorescent WO 99/37660 PCT/US99/01313 -49agent, such as FITC-dextran, and the meshes are fixed and mounted for observation under a fluorescent microscope.
In the cornea pocket assay, hydron pellets containing the compound of interest and an angiogenic growth factor, such as bFGF, are implanted 1 to 2mm from the limbus of the cornea of rats or mice. Response is examined after a period of time, for example 5 days. The extent ofangiogenesis is evaluated by measuring the capillaries migrating from the limb of the cornea.
Of course, due to the degeneracy of the genetic code, one of ordinary skill in the art will immediately recognize that a large number of the nucleic acid molecules having a sequence at least 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence of the deposited cDNAs or a nucleic acid sequence shown in SEQ ID NO: 1 or SEQ ID NO:3 will encode a polypeptide "having METH1 or METH2 protein activity." In fact, since degenerate variants of these nucleotide sequences all encode the same polypeptide, this will be clear to the skilled artisan even without performing the above described comparison assay. It will be further recognized in the art that, for such nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a polypeptide having METHI or METH2 protein activity. This is because the skilled artisan is fully aware of amino acid substitutions that are either less likely or not likely to significantly effect protein function replacing one aliphatic amino acid with a second aliphatic amino acid).
For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie, J. U. et al., "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions," Science 247:1306-1310 (1990), wherein the authors indicate that proteins are surprisingly tolerant of amino acid substitutions.
WO 99/37660 PCTIUS99/01313 Vectors and Host Cells The present invention also relates to vectors which include the isolated DNA molecules of the present invention, host cells which are genetically engineered with the recombinant vectors, and the production of METH1 or METH2 polypeptides or fragments thereof by recombinant techniques.
The polynucleotides may be joined to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
The DNA insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the early and late promoters and promoters ofretroviral LTRs, to name a few.
Other suitable promoters will be known to the skilled artisan. The expression constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs will preferably include a translation initiating at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.
As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS and Bowes melanoma cells; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.
WO 99/37660 PCT/US99/01313 -51- Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNHI8A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.
Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTI and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to the skilled artisan.
In addition to the use of expression vectors in the practice of the present invention, the present invention further includes novel expression vectors comprising operator and promoter elements operatively linked to nucleotide sequences encoding a protein of interest. One example of such a vector is which is described in detail below.
As summarized in Figures 8 and 9, components of the pHE4-5 vector (SEQ ID NO: 12) include: 1) a neomycinphosphotransferase gene as a selection marker, 2) an E. coli origin of replication, 3) a T5 phage promoter sequence, 4) two lac operator sequences, 5) a Shine-Delgarno sequence, 6) the lactose operon repressor gene (laclq). The origin of replication (oriC) is derived from pUC19 (LTI, Gaithersburg, MD). The promoter sequence and operator sequences were made synthetically. Synthetic production of nucleic acid sequences is well known in the art. CLONTECH 95/96 Catalog, pages 215-216, CLONTECH, 1020 East Meadow Circle, Palo Alto, CA 94303. A nucleotide sequence encoding METH1 (SEQ ID NO:2) or METH2 (SEQ ID NO:4), is operatively linked to the promoter and operator by inserting the nucleotide sequence between the Ndel and Asp718 sites of the pHE4-5 vector.
As noted above, the pHE4-5 vector contains a laclq gene. Laclq is an allele of the lacI gene which confers tight regulation of the lac pperator. Amann, E. et al, Gene 69:301-315 (1988); Stark, Gene 51:255-267 (1987). The laclq gene encodes a repressor protein which binds to lac operator sequences and blocks transcription of down-stream sequences. However, the laclq gene WO 99/37660 PCT/US99/01313 -52product dissociates from the lac operator in the presence of either lactose or certain lactose analogs, isopropyl B-D-thiogalactopyranoside (IPTG).
METHI or METH2 thus is not produced in appreciable quantities in uninduced host cells containing the pHE4-5 vector. Induction of these host cells by the addition of an agent such as IPTG, however, results in the expression of the METHI or METH2 coding sequence.
The promoter/operator sequences of the pHE4-5 vector (SEQ ID NO: 13) comprise a T5 phage promoter and two lac operator sequences. One operator is located 5' to the transcriptional start site and the other is located 3' to the same site. These operators, when present in combination with the laclq gene product, confer tight repression of down-stream sequences in the absence of a lac operon inducer, IPTG. Expression of operatively linked sequences located downstream from the lac operators may be induced by the addition of a lac operon inducer, such as IPTG. Binding of a lac inducer to the laclq proteins results in their release from the lac operator sequences and the initiation of transcription of operatively linked sequences. Lac operon regulation of gene expression is reviewed in Devlin, TEXTBOOK OF BIOCHEMISTRY WITH CLINICAL CORRELATIONS, 4th Edition (1997), pages 802-807.
The pHE4 series of vectors contain all of the components of the vector except for the METH 1 or METH2 coding sequence. Features of the pHE4 vectors include optimized synthetic T5 phage promoter, lac operator, and Shine-Delgarno sequences. Further, these sequences are also optimally spaced so that expression of an inserted gene may be tightly regulated and high level of expression occurs upon induction.
Among known bacterial promoters suitable for use in the production of proteins of the present invention include the E. coli lacI and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters and the trp promoter. Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late promoters, the promoters ofretroviral LTRs, such as those of the Rous Sarcoma WO 99/37660 PCT/US99/01313 -53- Virus (RSV), and metallothionein promoters, such as the mouse metallothionein-1 promoter.
The pHE4-5 vector also contains a Shine-Delgarno sequence 5' to the AUG initiation codon. Shine-Delgarno sequences are short sequences generally located about 10 nucleotides up-stream from the AUG initiation codon.
These sequences essentially direct prokaryotic ribosomes to the AUG initiation codon.
Thus, the present invention is also directed to expression vector useful for the production of the proteins of the present invention. This aspect of the invention is exemplified by the pHE4-5 vector (SEQ ID NO: 12).
Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986).
The polypeptide may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art. A preferred fusion protein comprises a heterologous region from immunoglobulin that is useful to solubilize proteins. For example, EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobin molecules together with another human protein or part thereof In many cases, the Fc part in a fusion protein is WO 99/37660 PCT/US99/01313 -54thoroughly advantageous for use in therapy and diagnosis and thus results, for example, in improved pharmacokinetic properties (EP-A 0232 262). On the other hand, for some uses it would be desirable to be able to delete the Fc part after the fusion protein has been expressed, detected and purified in the advantageous manner described. This is the case when the Fc portion proves to be a hindrance to use in therapy and diagnosis, for example when the fusion protein is to be used as an antigen for immunizations. In drug discovery, for example, human proteins, such as the hIL5-receptor, have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. See, D.
Bennett et al., J. Mol. Recognition 8:52-58 (1995) and K. Johanson et al., of Biol. Chem. 270(16):9459-9471 (1995).
The METHI or METH2 protein can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.
WO 99/37660 PCT/US99/01313 METH1 and METH2 Polypeptides and Fragments The invention further provides an isolated METH1 polypeptide having the amino acid sequence encoded by the deposited cDNA, or the amino acid sequence in SEQ ID NO:2, or a peptide or polypeptide comprising a portion of the above polypeptides. The invention also provides an isolated METH2 polypeptide having the amino acid sequence encoded by the deposited cDNA, or the amino acid sequence in SEQ ID NO:4, or a peptide or polypeptide comprising a portion of the above polypeptides.
METH1 or METH2 polypeptides can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids. The METHI or METH2 polypeptides may be modified by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature.
Modifications can occur anywhere in the METH1 or METH2 polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given METH1 or METH2 polypeptide. Also, a given METH1 or METH2 polypeptide may contain many types of modifications. METHI or METH2 polypeptides may be branched for example, as a result ofubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic METHI or METH2 polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADPribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, WO 99/37660 PCTIUS99/01313 -56demethylation, formation of covalent cross-links, formation ofcysteine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (See, for instance, PROTEINS STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993); POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth Enzymol 182:626-646 (1990); Rattan et al., Ann NY Acad Sci 663:48-62 (1992).) It will be recognized in the art that some amino acid sequences of the METH1 and METH2 polypeptides can be varied without significant effect of the structure or function of the protein. If such differences in sequence are contemplated, it should be remembered that there will be critical areas on the protein which determine activity.
The present inventors have shown that METHI and METH2 inhibit angiogenesis in vitro and in vivo. METH1 and METH2 each contain a metalloprotease domain, a disintegrin domain, and TSP-like domains. The metalloprotease domain may be catalytically active. The disintegrin domain may play a role in inhibiting angiogenesis by interacting with integrins, since integrins are essential for the mediation of both proliferative and migratory signals. The present inventors have shown that peptides derived from the TSP-like domains of METH1 and METH2 inhibit angiogenesis in vitro and in vivo.
Thus, the invention further includes variations of the METH polypeptide which show substantial METH1 polypeptide activity or which include regions of METH1 protein such as the protein portions discussed below; and variations of the METH2 polypeptide which show substantial METH2 polypeptide activity or which include regions of METH2 protein such as the protein portions discussed below. Such mutants include deletions, insertions, inversions, repeats, and type WO 99/37660 PCTIUS99/01313 -57substitutions. As indicated above, guidance concerning which amino acid changes are likely to be phenotypically silent can be found in Bowie, et al., "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions," Science 247:1306-1310 (1990).
Thus, the fragment, derivative or analog of the polypeptide of SEQ ID NO:2 or SEQ ID NO:4, or that encoded by the deposited cDNA, may be one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as an IgG Fc fusion region peptide or leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.
Of particular interest are substitutions of charged amino acids with another charged amino acid and with neutral or negatively charged amino acids. The latter results in proteins with reduced positive charge to improve the characteristics of the METH1 or METH2 proteins. The prevention of aggregation is highly desirable. Aggregation of proteins not only results in a loss of activity but can also be problematic when preparing pharmaceutical formulations, because they can be immunogenic. (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes 36:838-845 (1987); Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377 (1993)).
As indicated, changes are preferably of a minor nature, such as conservative amino acid substitutions that do not significantly affect the folding or activity of the protein (see Table 3).
WO 99/37660 PCTIUS99/01313 -58- TABLE 3. Conservative Amino Acid Substitutions.
Aromatic Phenylalanine Tryptophan Tyrosine Hydrophobic Leucine Isoleucine Valine Polar Glutamine Asparagine Basic Arginine Lysine Histidine Acidic Aspartic Acid Glutamic Acid Small Alanine Serine Threonine Methionine Glycine Of course, the number of amino acid substitutions a skilled artisan would make depends on many factors, including those described above. Generally speaking, the number of amino acid substitutions for any given METHI or METH2 polypeptide will not be more than 50, 40, 30, 20, 10, 5, or 3.
Amino acids in the METH1 and METH2 proteins of the present invention that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity such as in vitro or in vivo inhibition of angiogenesis. Sites that are critical for inhibition of angiogenesis can also be determined by structural analysis such as crystallization, nuclear magnetic WO 99/37660 PCT/US99/01313 -59resonance or photoaffinity labeling (Smith etal., J. Mol. Biol. 224:899-904 (1992) and de Vos et al., Science 255:306-312 (1992)).
The polypeptides of the present invention are preferably provided in an isolated form. By "isolated polypeptide" is intended a polypeptide removed from its native environment. Thus, a polypeptide produced and/or contained within a recombinant host cell is considered isolated for purposes of the present invention.
Also intended as an "isolated polypeptide" are polypeptides that have been purified, partially or substantially, from a recombinant host cell or from a native source. For example, a recombinantly produced version of the METH1 or METH2 polypeptide can be substantially purified by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988).
The polypeptides of the present invention include the METH1 polypeptide encoded by the deposited cDNA including the leader; the mature METH1 polypeptide encoded by the deposited the cDNA minus the leader the mature protein); a polypeptide comprising amino acids about 1 to about 950 in SEQ ID NO:2; a polypeptide comprising amino acids about 2 to about 950 in SEQ ID NO:2; a polypeptide comprising amino acids about 29 to about 950 in SEQ ID NO:2; a polypeptide comprising amino acids about 30 to about 950 in SEQ ID NO:2; a polypeptide comprising the metalloprotease domain of METHI, amino acids 235 to 459 in SEQ ID NO:2; a polypeptide comprising the disintegrin domain of METH1, amino acids 460 to 544 in SEQ ID NO:2; a polypeptide comprising the first TSP-like domain ofMETHI, amino acids 545 to 598 in SEQ ID NO:2; a polypeptide comprising the second TSP-like domain of METH1, amino acids 841 to 894 in SEQ ID NO:2; a polypeptide comprising the third TSPlike domain ofMETHI, amino acids 895 to 934 in SEQ ID NO:2; a polypeptide comprising amino acids 536 to 613 in SEQ ID NO:2; a polypeptide comprising amino acids 549 to 563 in SEQ ID NO:2; the METH2 polypeptide encoded by the deposited cDNA including the leader; the mature METH2 polypeptide encoded by the deposited the cDNA minus the leader the mature protein); a polypeptide comprising amino acids about 1 to about 890 in SEQ ID NO:4; a WO 99/37660 PCT/US99/01313 polypeptide comprising amino acids about 2 to about 890 in SEQ ID NO:4; a polypeptide comprising amino acids about 24 to about 890 in SEQ ID NO:4; a polypeptide comprising amino acids about 112 to about 890 in SEQ ID NO:4; a polypeptide comprising the metalloprotease domain ofMETH2, amino acids 214 to 439 in SEQ ID NO:4; a polypeptide comprising the disintegrin domain of METH2, amino acids 440 to 529 in SEQ ID NO:4; a polypeptide comprising the first TSP-like domain of METH2, amino acids 530 to 583 in SEQ ID NO:4; a polypeptide comprising the second TSP-like domain ofMETH2, amino acids 837 to 890 in SEQ ID NO:4; a polypeptide comprising amino acids 280 to 606 in SEQ ID NO:4; a polypeptide comprising amino acids 529 to 548 in SEQ ID NO:4; as well as polypeptides which are at least 95% identical, and more preferably at least 96%, 97%, 98% or 99% identical to the polypeptides described above and also include portions of such polypeptides with at least 30 amino acids and more preferably at least 50 amino acids.
By a polypeptide having an amino acid sequence at least, for example, "identical" to a reference amino acid sequence of a METH1 or METH2 polypeptide is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of the METHI or METH2 polypeptide. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
WO 99/37660 PCT/US99/01313 -61- As a practical matter, whether any particular polypeptide is at least 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:4 or to the amino acid sequence encoded by deposited cDNA clones can be determined conventionally using known computer programs such the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711). When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, identical to a reference sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference amino acid sequence and that gaps in homology of up to 5% of the total number of amino acid residues in the reference sequence are allowed.
A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al., Comp. App. Biosci.
6:237-245 (1990). In a sequence alignment, the query and subject sequences are either both nucleotide sequences or both amino acid sequences. The result of said global sequence alignment is in percent identity. Preferred parameters used in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty= 1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score= 1, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the subject amino acid sequence, whichever is shorter.
If the subject sequence is shorter than the query sequence due to N- or Cterminal deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for N- and C-terminal truncations of the subject sequence when calculating global percent identity. For subject sequences truncated at the N- and C-termini, relative to the query sequence, the percent identity is corrected by calculating the number WO 99/37660 PCT[US99/01313 -62of residues of the query sequence that are N- and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total residues of the query sequence. Whether a residue is matched/aligned is determined by the results of the FASTDB sequence alignment.
This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of the present invention. Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N- and C-terminal residues of the subject sequence.
For example, a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity. The deletion occurs at the N-terminus of the subject sequence and therefore, the FASTDB alignment does not show a match/alignment of the first 10 residues at the N-terminus. The unpaired residues represent 10% of the sequence (number of residues at the Nand C-termini not matched/total number of residues in the query sequence) so is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched, the final percent identity would be 90%. In another example, a 90 residue subject sequence is compared with a 100 residue query sequence. This time, the deletions are internal, so there are no residues at the N- or C-termini of the subject sequence which are not matched/aligned with the query. In this case, the percent identity calculated by FASTDB is not manually corrected. Once again, only residue positions outside the N- and C-terminal ends of the subject sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are made for the purposes of the present invention.
WO 99/37660 PCT[US99/01313 -63- The polypeptides of the present invention are useful as a molecular weight marker on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art.
In another aspect, the invention provides a peptide or polypeptide comprising an epitope-bearing portion of a polypeptide of the invention. The epitope of this polypeptide portion is an immunogenic or antigenic epitope of a polypeptide described herein. An "immunogenic epitope" is defined as a part of a protein that elicits an antibody response when the whole protein is the immunogen. On the other hand, a region of a protein molecule to which an antibody can bind is defined as an "antigenic epitope." The number of immunogenic epitopes of a protein generally is less than the number of antigenic epitopes. See, for instance, Geysen et al., Proc. Nail. Acad. Sci. USA 81:3998- 4002 (1983).
As to the selection ofpeptides or polypeptides bearing an antigenic epitope that contain a region of a protein molecule to which an antibody can bind), it is well known in that art that relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked protein. See, for instance, Sutcliffe, J. G. et al., "Antibodies that react with predetermined sites on proteins", Science 219:660-666 (1983). Peptides capable of eliciting protein-reactive sera are frequently represented in the primary sequence of a protein, can be characterized by a set of simple chemical rules, and are confined neither to immunodominant regions of intact proteins immunogenic epitopes) nor to the amino or carboxyl terminals.
Antigenic epitope-bearing peptides and polypeptides of the invention are therefore useful to raise antibodies, including monoclonal antibodies, that bind specifically to a polypeptide of the invention. See, for instance, Wilson et al., Cell 37:767-778 (1984) at 777.
Antigenic epitope-bearing peptides and polypeptides of the invention preferably contain a sequence of at least seven, more preferably at least nine and WO 99/37660 PCTIUS99/01313 -64most preferably between about at least about 15 to about 30 amino acids contained within the amino acid sequence of a polypeptide of the invention.
The epitope-bearing peptides and polypeptides of the invention may be produced by any conventional means. Houghten, R. "General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids", Proc. Natl.
Acad. Sci. USA 82:5131-5135 (1985). This "Simultaneous Multiple Peptide Synthesis (SMPS)" process is further described in U.S. Patent No. 4,631,211 to Houghten et al. (1986).
As one of skill in the art will appreciate, METH1 or METH2 polypeptides of the present invention and the epitope-bearing fragments thereof described above can be combined with parts of the constant domain of immunoglobulins (IgG), resulting in chimeric polypeptides. These fusion proteins facilitate purification and show an increased half-life in vivo. This has been shown, for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins (EPA 394,827; Traunecker et al., Nature 331:84- 86 (1988)). Fusion proteins that have a disulfide-linked dimeric structure due to the IgG part can also be more efficient in binding and neutralizing other molecules than the monomeric METH1 or METH2 protein or protein fragment alone (Fountoulakis et al., J. Biochem. 270:3958-3964 (1995)).
METH1 and METH2 Polynucleotide and Polypeptide Fragments In the present invention, a "polynucleotide fragment" refers to a short polynucleotide having a nucleic acid sequence contained in the deposited clones or shown in SEQ ID NO: 1 or SEQ ID NO:3. The short nucleotide fragments are preferably at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length. A fragment "at least 20 nt in length," for example, is intended to include WO 99/37660 PCT/US99/01313 or more contiguous bases from the cDNA sequence contained in the deposited clones or the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:3.
These nucleotide fragments are useful as diagnostic probes and primers as discussed herein. Of course, larger fragments 50, 150, 500, 600, 2000 nucleotides) are preferred.
Moreover, representative examples ofMETH I or METH2 polynucleotide fragments include, for example, fragments having a sequence from about nucleotide number 1-50, 51-100, 101-150, 151-200,201-250,251-300,301-350, 351-400, 401-450, 451-500, 501-550, 551-600, 651-700, 701-750, 751-800, 800- 850, 851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151- 1200, 1201-1250, 1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500, 1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800, 1801- 1850, 1851-1900, 1901-1950, 1951-2000, or 2001 to the end of SEQ ID NO:1 or SEQ ID NO:3 or the cDNA contained in the deposited clones. In this context "about" includes the particularly recited ranges, larger or smaller by several 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Preferably, these fragments encode a polypeptide which has biological activity. More preferably, these polynucleotides can be used as probes or primers as discussed herein.
In the present invention, a "polypeptide fragment" refers to a short amino acid sequence contained in SEQ ID NO:2 or SEQ ID NO:4 or encoded by the cDNA contained in the deposited clones. Protein fragments may be "freestanding," or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region. Representative examples of polypeptide fragments of the invention, include, for example, fragments from about amino acid number 1-20,21-40,41-60, 61-80, 81-100, 102- 120,121-140,141-160,161-180, 181-200,201-220,221-240,241-260,261-280, or 281 to the end of the coding region or SEQ ID NO:2 pr SEQ ID NO:4.
Moreover, polypeptide fragments can be about 20, 30, 40, 50, 60,70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids in length. In this context "about" includes WO 99/37660 PCTIUS99/01313 -66the particularly recited ranges, larger or smaller by several 4, 3, 2, or 1) amino acids, at either extreme or at both extremes.
Preferred polypeptide fragments include the secreted METHI or METH2 protein as well as the mature form. Further preferred polypeptide fragments include the secreted METHI or METH2 protein or the mature form having a continuous series of deleted residues from the amino or the carboxy terminus, or both. For example, any number of amino acids, ranging from 1-60, can be deleted from the amino terminus of either the secreted METH 1 or METH2 polypeptide or the mature form. Similarly, any number of amino acids, ranging from 1-30, can be deleted from the carboxy terminus of the secreted METH1 or METH2 protein or mature form. Furthermore, any combination of the above amino and carboxy terminus deletions are preferred. Similarly, polynucleotide fragments encoding these METHI or METH2 polypeptide fragments are also preferred.
Particularly, N-terminal deletions of the METH1 polypeptide can be described by the general formula m-950, where m is an integer from 2 to 949, where m corresponds to the position of the amino acid residue identified in SEQ ID NO:2. Preferably, N-terminal deletions of the METH1 polypeptide of the invention shown as SEQ ID NO:2 include polypeptides comprising the amino acid sequence of residues: G-2 to S-950; N-3 to S-950; A-4 to S-950; E-5 to S-950; R-6 to S-950; A-7 to S-950; P-8 to S-950; G-9 to S-950; S-10 to S-950; R-11 to S-950; S-12 to S-950; F-13 to S-950; G-14 to S-950; P-15 to S-950; V-16 to S- 950; P-17 to S-950; T-18 to S-950; L-19 to S-950; L-20 to S-950; L-21 to S-950; L-22 to S-950; A-23 to S-950; A-24 to S-950; A-25 to S-950; L-26 to S-950; L- 27 to S-950; A-28 to S-950; V-29 to S-950; S-30 to S-950; D-31 to S-950; A-32 to S-950; L-33 to S-950; G-34 to S-950; R-35 to S-950; P-36 to S-950; S-37 to S-950; E-38 to S-950; E-39 to S-950; D-40 to S-950; E-41 to S-950; E-42 to S- 950; L-43 to S-950; V-44 to S-950; V-45 to S-950; P-46 to S-950; E-47 to S- 950; L-48 to S-950; E-49 to S-950; R-50 to S-950; A-51 to S-950; P-52 to S- 950; G-53 to S-950; H-54 to S-950; G-55 to S-950; T-56 to S-950; T-57 to S- 950; R-58 to S-950; L-59 to S-950; R-60 to S-950; L-61 to S-950; H-62 to S- WO 99/37660 PCTIUS99/01313 -67- 950; A-63 to S-950; F-64 to S-950; D-65 to S-950; Q-66 to S-950; Q-67 to S- 950; L-68 to S-950; D-69 to S-950; L-70 to S-950; E-71 to S-950; L-72 to S- 950; R-73 to S-950; P-74 to S-950; D-75 to S-950, S-76 to S-950; S-77 to S-950; F-78 to S-950; L-79 to S-950; A-SO to S-950; P-81 to S-950; G-82 to S-950; F- 83 to S-950; T-84 to S-950, L-85 to S-950; Q-86 to S-950; N-87 to S-950; V-88 to S-950; G-89 to S-950; R-90 to S-950; K-91 to S-950; S-92 to S-950; G-93 to S-950; S-94 to S-950,; E-95 to S-950; T-96 to S-950; P-97 to S-950; L-98 to S- 950; P-99 to S-950; E-I 00 to S-950; T-101 to S-950; D-102 to S-950; L-103 to S-950; A-104 to S-950; H-I 05 to S-950; C-I106 to S-950; F-I 07 to S-950; Y-I 08 to S-950; S-109 to S-950; G-1 10 to S-950; T-1 I11 to S-950; V-I 12 to S-950; N- 113 to S-950; G-1 14 to S-950; D-1 15 to S-950; P-I 16 to S-950; S-I 17 to S-950; S-1 1 to S-950; A-I 19 to S-950; A-120 to S-950; A-121 to S-950-; L-122 to 950; S-123 to S-950; L-124 to S-950; C-125 to S-950; E-126 to S-950; G-127 to S-950; V-128 to S-950; R-129 to S-950; G-130 to S-950; A-13 Ito S-950; F- 132 to S-950; Y-133 to S-950; L-134 to S-950; L-135 to S-950; G-136 to S-950; E-137 to S-950; A-138 to S-950; Y-139 to 5-950; F-140 to S-950; 1-141 to S- 950; Q-142 to S-950; P-143 to S-950; L-144 to S-950; P-145 to S-950; A-146 to S-950; A- 147 to S-950; S- 148 to S-950, E- 149 to S-950; R-I150 to S-950; L- 151 to S-950; A- 152 to S-95 0; T- 153 to S-950; A-I 154 to S-950; A-I 155 to S-950; P-156 to S-950; G-157 to S-950; E-158 to S-950; K-159 to S-950; P-160 to S- 950; P-161 to S-950; A-162 to S-950; P-163 to S-950; L-164 to S-950; Q-165 to S-950; F-166 to S-950; H-167 to S-950; L-168 to S-950; L-169 to S-950; R- 170 to S-950; R-171 to S-950; N-172 to S-950; R-173 to S-950; Q-174 to S-950; G-175 to S-950; D-176 to S-950; V-177 to S-950; G-178 to S-950, G-179 to S- 950; T-180 to S-950; C-181 to S-950; G-182 to S-950; V-183 to S-950; V-184 to S-950; D-185 to S-950; D-186 to S-950; E-187 to S-950; P-188 to S-950; R- 189to S-950; P-190Oto S-950; T-191 to S-950; G-192 to S-950; K-193 to S-950; A- 194 to S-950; E- 195 to S-950; T- 196 to S-950;- E- 197 to S-950; D- 198 to S- 950; E-199 to S-950; D-200 to S-950, E-201 to S-950; G-202 to S-950; T-203 to S-950; E-204 to S-950; G-205 to S-950; E-206 to S-950; D-207 to S-950; E- WO 99/37660 PCTIUS99/01313 -68- 208 to S-950; G-209 to S-950; P-21 0 to S-950, Q-21 I to S-950; W-212 to S-950, S-213 to S-950; P-214 to S-950; Q-215 to S-950; D-216 to S-950; P-217 to S- 950; A-218 to S-950; L-219 to S-950; Q-220 to S-950; G-221 to S-950;, V-222 to S-950; G-223 to S-950; Q-224 to S-950; P-225 to S-950; T-226 to S-950; G- 227 to S-950; T-228 to S-950; G-229 to S-950; S-230 to S-950; 1-231 to S-950;- R-232 to S-950; K-233 to S-950; K-234 to S-950; R-235 to S-950; F-236 to S- 950; V-237 to S-950; S-238 to S-950; S-239 to S-950; H-240 to S-950; R-241 to S-950; Y-242 to S-950, V-243 to S-950; E-244 to S-950, T-245 to S-950; M- 246 to S-950; L-247 to S-950; V-248 to S-950; A-249 to S-950; D-250 to S-950; Q-251 to S-950; S-252 to S-950; M-253 to S-950; A-254 to S-950; E-255 to S- 950; F-256 to S-950; H-257 to S-950; G-258 to S-950; S-259 to S-950; G-260 to S-950; L-261 to S-950; K-262 to S-950; H-263 to S-950; Y-264 to S-950; L- 265 to S-950; L-266 to S-950; T-267 to S-950; L-268 to S-950; F-269 to S-950; S-270 to S-950; V-271 to S-950; A-272 to S-950; A-273 to S-950; R-274 to S- 950; L-275 to S-950; Y-276 to S-950; K-277 to S-950; H-278 to S-950; P-279 to S-950; S-280 to S-950; 1-28 1 to S-950; R-282 to S-950; N-283 to S-950; S- 284 to S-950; V-285 to S-950; S-286 to S-950; L-287 to S-950, V-288 to S-950; V-289 to S-950; V-290 to S-950; K-291 to S-950; 1-292 to S-950; L-293 to S- 950; V-294 to S-950; 1-295 to S-950; H-296 to S-950; D-297 to S-950; E-298 to S-950; Q-299 to S-950; K-300 to S-950; G-301 to S-950; P-302 to S-950; E- 303 to S-950; V-304 to S-950; T-305 to S-950; S-306 to S-950; N-307 to S-950; A-308 to S-950; A-309 to S-950; L-3 10 to S-950; T-311I to S-950; L-312 to S- 950; R-313 to S-950; N-314 to S-950; F-315 to S-950; C-316 to S-950; N-317 to S-950; W-3 18 to S-950; Q-319 to S-950; K-320 to S-950; Q-321 to S-950; H- 322 to S-950; N-323 to 8-950; P-324 to 8-950; P-325 to 8-950; S-326 to 8-950; D-327 to S-950; R-328 to S-950; D-329 to S-950; A-330 to S-950; E-331I to 8- 950; H-332 to 8-950; Y-333 to 8-950; D-334 to 8-950; T-335 to S-950; A-336 to 8-950; 1-337 to 8-950; L-338 to 8-950; F-339 to 8-950; T-340 to S-950; R- 341 to S-950; Q-342 to 8-950; D-343 to S-950; L-344 to 8-950; C-345 to S-950, G-346 to S-950; S-347 to 8-950; Q-348 to 8-950; T-349 to S-950; C-350 to 8- WO 99/37660 PCT/US99/01313 -69- 950; D-351 to S-950; T-352 to S-950; L-353 to S-950; G-354 to S-950; M-355 to S-950; A-356 to S-950; D-357 to S-950; V-358 to S-950; G-359 to S-950; T- 360 to S-950; V-361 to S-950; C-362 to S-950;, D-363 to S-950; P-364 to S-950; S-365 to S-950; R-366 to S-950; S-367 to S-950; C-368 to S-950; S-369 to S- 950; V-370 to S-950; 1-371 to S-950) E-372 to S-950, D-373 to S-950; D-374 to S-950; G-375 to S-950; L-376 to S-950; Q-377 to S-950; A-378 to S-950; A- 379 to S-950;, F-380 to S-950; T-381 to S-950; T-382 to S-950; A-383 to S-950; H-384 to S-950; E-385 to S-950; L-386 to S-950; G-387 to S-950; H-388 to S- 950; V-389 to S-950; F-390 to S-950; N-391 to S-950; M-392 to S-950; P-393 to S-950; H-394 to S-950; D-395 to S-950; D-396 to S-950; A-397 to S-950; K- 398 to S-950;, Q-399 to S-950; C-400 to S-950; A-401 to S-950; S-402 to S-950; L-403 to S-950; N-404 to S-950; G-405 to S-950; V-406 to S-950; N-407 to S- 950; Q-408 to S-950; D-409 to S-950; S-410 to S-950; H-411 to S-950; M-412 to S-950; M-413 to S-950; A-414 to S-950; S-415 to S-950; M-416 to S-950; L- 417 to S-950; S-418 to S-950;,N-419 to S-950; L-420 to S-950; D-421 to S-950;, H-422 to S-950; S-423 to S-950; Q-424 to S-950; P-425 to S-950; W-426 to S- 950; S-427 to S-950; P-428 to S-950-, C-429 to S-950; S-430 to S-950; A-43 I to S-950; Y-432 to S-950; M-433 to S-950; 1-434 to S-950; T-435 to S-950; S-436 to S-950-, F-437 to S-950; L-438 to S-950; D-439 to S-950;, N-440 to S-950; G- 441 to S-950; H-442 to S-950; G-443 to S-950; E-444 to S-950; C-445 to S-950; L-446 to S-950; M-447 to S-950; D-448 to S-950; K-449 to S-950; P-450 to S- 950; Q-451 to S-950; N-452 to S-950; P-453 to S-950; 1-454 to S-950; Q-455 to S-950; L-456 to S-950; P-457 to S-950; G-458 to S-950; D-459 to S-950; L-460 to S-950; P-461 to S-950; G-462 to S-950; T-463 to S-950; S-464 to S-950; Y- 465 to S-950; D-466 to S-950; A-467 to S-950; N-468 to S-950; R-469 to S-950; Q-470 to S-950; C-471 to S-950; Q-472 to S-950; F-473 to S-950; T-474 to S- 950; F-475 to S-950; G-476 to S-950; E-477 to S-950; D-478 to S-950; S-479 to S-950; K-480 to S-950; H-481 to S-950; C-482 to S-950; P-483 to S-950; D- 484 to S-950; A-485 to S-950; A-486 to S-950; S-487 to S-950; T-488 to S-950., In C-489 to S-950;, S-490 to S-950; T-491 to S-950; L-492 to S-950; W-493 to S- WO 99/37660 PCT/US99/01313 950; C-494 to S-950; T-495 to S-950; G-496 to S-950; T-497 to S-950; S-498 to S-950; G-499 to S-950; G-500 to S-950; V-501 to S-950; L-502 to S-950; V- 503 to S-950; C-504 to S-950; Q-505 to S-950; T-506 to S-950; K-507 to S-950; H-508 to S-950; F-509 to S-950; P-5 10 to S-950; W-5 11 to S-950; A-S512 to S- 950; D-513 to S-950; G-514 to S-950;, T-515 to S-950; S-516 to S-950; C-517 to S-950, G-5 18 to S-950; E-5 19 to S-950; G-520 to S-950; K-521 to S-950; W- 522 to S-950; C-523 to S-950; 1-524 to S-950; IN-525 to S-950; G-526 to S-950, K-5.27 to S-950; C-528 to S-950, V-529 to S-950, N-530 to S-950; K-53 1 to S- 950; T-532 to S-950; D-533 to S-950; R-534 to S-950; K-535 to S-950; H-536 to S-950; F-537 to S-950; D-538 to S-950; T-539 to S-950; P-540 to S-950; F- 541 to S-950, H-542 to S-950; G-543 to S-950; S-544 to S-950; W-545 to S-950; G-546 to S-950; M-547 to S-950; W-548 to S-950; G-549 to S-950; P-550 to S- 950; W-551 to S-950; G-552 to S-950; D-553 to S-950; C-554 to S-950; S-555 to S-950; R-556 to S-950; T-557 to S-950; C-558 to S-950; G-559 to S-950; G- 560 to S-950; G-561 to S-950; V-562 to S-950; Q-563 to S-950; Y-564 to S-950; T-565 to S-950; M-566 to S-950, R-567 to S-950; E-568 to S-950; C-569 to S- 950; D-570 to S-950; N-571 to S-950; P-572 to S-950; V-573 to S-950; P-574 to S-950; K-575 to S-950; N-576 to S-950; G-577 to S-950; G-578 to S-950; K- 579 to S-950; Y-580 to S-950;'"C-58 I to S-950; E-582 to S-950; G-583 to S-950; K-584 to S-950; R-585 to S-950; V-586 to S-950; R-587 to S-950; Y-588 to S- 950; R-589 to S-950; S-590 to S-950; C-591 to S-950; N-592 to S-950; L-593 to S-950; E-594 to S-950; D-595 to S-950; C-596 to S-950; P-597 to S-950; D- 598 to S-950; N-599 to S-950; N-600 to S-950; G-601 to S-950; K-602 to S-950; T-603 to S-950; F-604 to S-950; R-605 to S-950; E-606 to S-950; E-607 to S- 950; Q-608 to S-950; C-609 to S-950; E-610 to S-950; A-611I to S-950; H-612 to S-950; N-613 to S-950; E-614 to S-950; F-615 to S-950; S-616 to S-950; K- 617 to S-950; A-6 18 to S-950; S-6 19 to S-950; F-620 to S-950; G-621I to S-950; S-622 to S-950; G-623 to S-950; P-624 to S-950; A-625 to S-950; V-626 to S- 950; E-627 to S-950; W-628 to S-950; 1-629 to S-950; P-630 to S-950; K-631I to S-950, Y-632 to S-950; A-633 to S-950; G-634 to S-950; V-635 to S-950, S- WO 99/37660 PCT/US99/01313 -71- 636 to S-950, P-637 to S-950; K-638 to S-950; D-639 to S-950; R-640 to S-950, C-641 to S-950; K-642 to S-950; L-643 to S-950; 1-644 to S-950, C-645 to S- 950; Q-646 to S-950;, A-647 to S-950; K-648 to S-950; G-649 to S-950; 1-650 to S-950; G-651 to S-950; Y-652 to S-950; F-653 to S-950; F-654 to S-950; V- 655 to S-950; L-656 to S-950; Q-657 to S-950; P-658 to S-950; K-659 to S-950;1 V-660 to S-950; V-661 to S-950; D-662 to S-9 50; G-663 to S-950; T-664 to S- 950; P-665 to S-950- C-666 to S-950; S-667 to S-950; P-668 to S-950; D-669 to S-950; S-670 to S-950; T-671 to S-950; S-672 to S-950; V-673 to S-950; C-674 to S-950; V-675 to S-950; Q-676 to S-950; G-677 to S-950;, Q-678 to S-950; C- 679 to S-950; V-680 to S-950; K-681 ito S-950; A-682 to S-950, G-683 to S-950; C-684 to S-950; D-685 to S-950; R-686 to S-950; 1-687 to S-950; 1-688 to S- 950; D-689 to S-950; S-690 to S-950; K-691 to S-950; K-692 to S-950; K-693 to S-950; F-694 to S-950; D-695 to S-950;, K-696 to S-950; C-697 to S-950; G- 698 to S-950; V-699 to S-950; C-700 to S-950; G-701 to S-950; G-702 to S-950; N-703 to S-950; G-704 to S-950; S-705 to S-950;, T-706 to S-950; C-707 to S- 950; K-708 to S-950; K-709 to S-950; 1-7 10 to S-950; S-711 to S-950, G-712 to S-950; S-713 to S-950; V-714 to S-950; T-715 to S-950; S-716 to S-950; A-717 to S-950; K-71 8 to S-950,; P-719 to S-950; G-720 to S-950; Y-721 to S-950; H- 722 to S-950; D-723 to S-950; 1-724 to S-950; 1-725 to S-950; T-726 to S-950; 1-727 to S-950; P-728 to S-950; T-729 to S-950; G-730 to S-950; A-731 to S- 950; T-732 to S-950; N-733 to S-950; 1-734 to S-950; E-735 to S-950; V-736 to S-950; K-737 to S-950; Q-738 to S-950; R-739 to S-950; N-740 to S-950; Q-741 to S-950; R-742 to S-950; G-743 to S-950; S-744 to S-950; R-745 to S-9 50; N- 746 to S-950; N-747 to S-950; G-748 to S-950; S-749 to S-950; F-750 to S-950; L-751 to S-950; A-752 to S-950; 1-753 to S-950; K-754 to S-950; A-755 to S- 950; A-756 to S-950; D-757 to S-950; G-758 to S-950; T-759 to S-950;) Y-760 to S-950; 1-761 to S-950; L-762 to S-950; N-763 to S-950; G-764 to S-950; D- 765 to S-950, Y-766 to S-950, T-767 to S-950, L-768 to S-950, S-769 to S-950; T-770 to S-950; L-771 to S-950; E-772 to S-950; Q-773 to S-950; D-774 to S- 950; 1-775 to S-950; M-776 to S-950; Y-777 to S-950; K-778 to S-950; G-779 WO 99/37660 PCTIUS99/013 1 3 -72to S-950; V-780 to S-950; V-781 to S-950; L-782 to S-950;I R-783 to S-950;
Y-
784 to S-950; S-785 to S-950; G-786 to S-950; S-787 to S-950; S-788 to S-950; A-789 to S-950; A-790 to S-950, L-791 to S-950; E-792 to S-950, R-793 to S- 950; 1-794 to S-950; R-795 to S-950; S-796 to S-950; F-797 to S-950; S-798 to S-950; P-799 to S-950; L-800 to S-950; K-801 to S-950; E-802 to S-950; P-803 to S-950; L-804 to S-950;, T-805 to S-950, 1-806 to S-950; Q-807 to S-950;
V-
808 to S-950; L-809 to S-950; T-8 10 to S-950; V-811 to S-950, G-812 to S-950; N-813 to S-950; A-814 to S-950; L-815 to S-950; R-816 to S-950; P-817 to S- 950; K-S818 to S-950; 1-819 to S-950; K-820 to S-950; Y-821 to S-950; T-822 to S-950; Y-823 to S-950; F-824 to S-950; V-825 to S-950; K-826 to S-950; K-827 to S-950; K-828 to S-950; K-829 to S-950; E-830 to S-950; S-831I to S-950;
F-
832 to S-950; N-833 to S-950; A-834 to*S-950; 1-83 5 to S-950; P-836 to S-950; T-837 to S-950; F-838 to S-950; S-839 to S-950; A-840 to S-950; W-841 to S- 950; V-842 to S-950; 1-843 to S-950; E-844 to S-950; E-845 to S-950; W-846 to S-950; G-847 to S-950; E-848 to S-950; C-849 to S-950; S-850 to S-950;
K-
851 to-S- 9 5 S-852 to S-950; C-853 to S-950; E-854 to S-950; L-855 to S-950., G-856 to S-950; W-857 to S-950, Q-858 to S-950; R-859 to S-950; R-860 to S- 950;, L-861 to S-950; V-862 to S-950; E-863 to S-950; C-864 to S-950; R-865 to S-950; D-866 to S-950; 1-867 to S-950;, N-868 to S-950, G-869 to S-950; Q- 870 to S-950; P-871 to S-950; A-872 to S-950; S-873 to S-950; E-874 to S-950;, C-875 to S-950; A-876 to S-950; K-877 to S-950; E-878 to S-950, V-879 to S- 950; K-880 to S-950; P-881 to S-950; A-882 to S-950; S-883 to S-950; T-884 to S-950; R-885 to S-950; P-886 to S-950; C-887 to S-950; A-888 to S-950; D- 889 to S-950; H-890 to S-950; P-891 to S-950; C-892 to S-950; P-893 to S-950; Q-894 to S-950; W-895 to S-950; Q-896 to S-950; L-897 to S-950; G-898 to S- 950; E-899 to S-950; W-900 to S-950; S-901 to S-950; S-902 to S-950; C-903 to S-950; S-904 to S-950; K-905 to S-950; T-906 to S-95,0; C-907 to S-950;
G-
908 to S-950; K-909 to S-950; G-91 10 to S-950; Y-9 Ii to S-950; K-91 2 to S-950; K-913 to S-950; R-914 to S-950; S-915 to S-950; L-916 to S-950; K-917 to S- 950;, C-918 to S-950; L-919 to S-950; S-920 to S-950; H-921 to S-950; D-922 WO 99/37660 PCT/US99/0131 3 -73to S-950; G-923 to S-950; G-924 to S-950; V-925 to S-950; L-926 to S-950; S- 927 to S-950; H-928 to S-950; E-929 to S-950; S-930 to S-950; C-93 1 to S-950; D-932 to S-950; P-933 to S-950; L-934 to S-950; K-935 to S-950; K-936 to S- 950; P-937 to S-950; K-938 to 5-950; H-939 to S-950, F-940 to S-950; 1-941 to S-950; D-942 to S-950; F-943 to S-950; C-944 to S-950; T-945 to S-950; of SEQ ID NO:2.
Moreover, C-terminal deletions of the METHI polypeptide can also be described by the general formula I where n is an integer from 2 to 9 50, where n corresponds to the position of amino acid residue identified in SEQ ID NO:2.
Preferably, C-terminal deletions of the MIETHI polypeptide of the invention shown as SEQ ID NO: 2 include polypeptides comprising the amino acid sequence of residues: M-1I to C-949; M-1I to E-948; M-1I to A-947; M-1I to M-946; M-1I to T-945; M- I to C-944; M-1I to F-943; M- I to D-942; M- I to 1-941;- M-1I to F-940; M-1I to H-93 9; M- I to K-93 8; M-1I to P-93 7; M-1I to K-93 6; M-1I to K-93 5; M- 1 to L-934; M-1I to P-93 3; M-1I to D-93 2; M-1I to C-931I; M- I to S-93 0; M-1I to E- 929; M-1 to H-928; M-1 to S-927; M-1 to L-926; M-1 to V-925; M-1 to G-924; M-1 to G-923; M-1 to D-922; M-1 to H-921;- M-1 to S-920; M-1 to L-919; M-1 to C-918; M-1 to K-917; M-1 to L-916; M-1 to S-915; M-1 to R-914; M-1 to K- 913; M-1I to K-912; M-1I to Y-911I;-M-1I to G-91 0;M-lI to K-909; M-1I to G-908; M-1I to C-907; M-lI to T-906; M-1I to K-905;- M- I to S-904; M-1I to C-903; M-1I to S-902; M-1I to S-90 1;M-1I to W-900; M-1I to E-899;-)M-1I to G-898; M-1I to L- 897; M- I to Q-896; M-1I to W- 8 9 5 M- I to Q-894; M-1I to P-893; M-1I to C-892; M-1 to P-891; M-1 to H-890; M-1 to D-889; M-1 to A-888; M-1 to C-887; M-1 to P-886; M-1 to R-885; M-1 to T-884; M-1 to S-883; M-1 to A-882; M-1 to P- 88 1; M-1I to K-880; M- I to V-879; M-1I to E-878; M-1I to K-877; M-1I to A-876; M-1I to C-875;- M-1I to E-874; M- I to S-873;) M-1I to A-872;, M- I to P-87 1; M-1I to Q-870; M-lI to G-869; M-lI to N-868; M- I to 1-867; M- I to D-866; M-1I to R- 865; M-1I to C-864; M-1I to E-863; M-1I to V-862; M-1I to L-861I; M-1I to R-860; M-1I to R-8 59; M-1I to Q-8 58; M-1I to W-85 7; M- I to G-856;- M-1I to L-8 55; M-lI to E-8 54; M-1I to C-8 53; M-1I to S-8 52; M- I to K-8 51; M-1I to S-8 50; M-lI to C- WO 99/37660 PCTJUS99/0131 3 -74- 849; M-1I to E-848; M- I to G-847; M- I to W- 8 4 6 M- I to E-845; M-1I to E-844; M-1I to 1-843;- M-1I to V-842; M- I to W-84 1; M-1I to A-840; M-I to S-83 9; M-1I to F-838; M-1 to T-837; M-1 to P-836; M-1 to 1-835;- M-1 to A-834; M-1 to N- 833; M-1 to F-832; M-1 to S-83 1; M-1 to E-830; M-1 to K-829; M-1 to K-828; M-1 to K-827; M-1 to K-826; M-1I to V-825; M-1 to F-824,; M-1 to Y-823; M-1I to T-822; M-1 to Y-821; M-1 to K-820; M-1 to 1-819; M-1 to K-818; M-1 to P- 817; M-1 to R-816; M-1 to L-8 15; M-1 to A-814;, M-1 to N-813; M-1 to G-812; M-1 to V-81 1; M-1 to T-810; M-1 to L-809; M-1 to V-808; M-1 to Q-807-, M-1 to 1-806; M-1 to T-805; M-1 to L-804; M-1 to P-803; M-1 to E-802; M-1 to K- 801; M-1 to L-800; M-1 to P-799; M-1 to S-798; M-1 to F-797; M-1 to S-796; M-1 to R-795-, M-1 to 1-794; M-1 to R-793; M-1 to E-792; M-1 to L-791; M-1 to A-790; M-1I to A-789; M-1I to S-788; M- I to S-787; M-1I to G-786; M-lI to S- 785; M-1 to Y-784; M-1 to R-783; M-1 to L-782; M-1 to V-781; M-1 to V-780-1 M- I to G-779; M-1I to K-778; M-1I to Y-777; M-1I to M-776; M- I to 1-775; M- 1 to D-774; M-1I to Q-773; M-1I to E-772; M-1I to L-771 M- I to T-770; M- I to S- 769; M- I to L-768; M-1I to T-767; M- I to Y-766; M-1I to D-765; M- I to G-764; M-1 to N-763; M-1 to L-762; M-1 to 1-761; M-1 to Y-760; M-1 to T-759; M-1 to G-758; M-1 to D-757; M-1 to A-756; M-1 to A-755; M-1 to K-754; M-1 to 1-753; M-1 to A-752; M-1 to L-75 1; M-1 to F-750; M-1 to S-749; M-1 to G-748; M-1 to N-747; M-1 to N-746; M-1 to R-745; M-1 to S-744; M-1 to G-743; M-1 to R-742; M- I to Q-741 M-1I to N-740; M- I to R-73 9; M-1I to Q-738; M-1 to K-737; M-1 to V-736; M-1 to E-735; M-1 to 1-734; M-1 to N-733; M-1 to T- 732; M-1 to A-731; M-1 to G-730; M-1 to T-729; M-1 to P-728; M-1 to 1-727; M-1 to T-726; M-1 to 1-725; M-1 to 1-724; M-1 to D-723; M-1 to H-722; M-1 to Y-72 1; M- I to G-720; M-1I to P-719; M- I to K-718; M- I to A-717; M- I to S- 716; M- I to T-715; M-1I to V-714; M-1I to S-713; M-1I to G-712; M-1I to S-71 1; M-1I to 1-7 10; M- I to K-709; M- I to K-708; M-1I to C-707; M-1I to T-706; M- I to S-705; M- I to G-704; M- I to N-703; M- I to G-702; M- I to G-70 1; M- I to C- 700; M-1I to V-699; M-1I to G-698; M-I to C-697; M-1I to K-696; M-1I to D-695;- M-1I to F-694; M- I to K-693 M-1I to K-692; M-1I to K-69 1; M- I to S-690; M-1I WO 99/37660 PCT/US99/01313 to D-689; M-1I to 1-688; M-1I to 1-687; M-1I to R-686; M- I to D-685;) M-1I to C- 684; M-1I to G-683; M- I to A-682; M-1I to K-681;- M-1I to V-680; M- I to C-679, M-1 to Q-678, M-1 to G-677,; M-1 to Q-676; M-1 to V-675; M-1 to C-674; M-1 to V-673; M- I to S-672; M-1I to T-67 1; M- I to S-670; M-1I to D-669; M- I to P- 668; M-1 to S-667, M-1 to C-666; M-1 to P-665; M-1 to T-664; M-1 to G-663-; M- I to D-662; M- I to V-66 1; M-1I to V-660; M-1I to K-659; M-1I to P-658; M-1I to Q-657; M-1 to L-656; M-1 to V-655; M-1 to F-654, M-1 to F-653; M-1 to Y- 652; M-1I to G-65 1; M- I to 1-650; M-1I to G-649; M- I to K-648; M-]I to A-647; M-1 to Q-646; M-1 to C-645; M-1 to 1-644; M-1 to L-643; M-1 to K-642; M-1 to C-64 1) M-1I to R-640; M-1I to D-63 9; M- I to K-63 8) M-lI to P-637, M-lI to S- 636; M-1 to V-635; M-1 to G-634; M-1 to A-633; M-1 to Y-632; M-1 to K-63 1, M-1I to P-630; M-1I to 1-629; M-1I to W-628; M- I to E-627; M-1I to V-626; M-1I to A-625; M-1I to P-624; M-1I to G-623; M-lI to S-622; M-1I to G-62 1; M-1I to F- 620; M-1I to S-619; M-1I to A-618; M- I to K-617; M-1I to S-616; M-1I to F-615; M-1I to E-614; M-1I to N-613; M-1I to H-612; M-1I to A-611I;-M-1I to E-6 10; M-1I to C-609; M-1I to Q-608; M-1I to E-607; M-1I to E-606; M-1I to R-605; M-1I to F- 604; M-1I to T-603; M-1I to K-602; M-1I to G-60 1; M-1I to N-600; M-1I to N-5 99; M-1 to D-598; M-1 to P-597; M-1 to C-596; M-1 to D-595; M-1 to E-594; M-1 to L-593; M-1 to N-592; M-1 to C-591; M-1 to S-590; M-1 to R-589; M-1 to Y- 588; M-1 to R-587; M-1 to V-586; M-1 to R-585; M-l to K-584; M-1 to G-583; M-1 to E-582; M-1 to C-581; M-1 to Y-580; M-1 to K-579; M-1 to G-578; M-1 to G-577; M-1 to N-576; M-1 to K-575; M-1 to P-574; M-1 to V-573; M-1 to P- 572; M-1 to N-571; M-1 to D-570; M-1 to C-569; M-1 to E-568; M-1 to R-567; M-1I to M-566; M-1I to T-565; M-1I to Y-564; M-1I to Q-563; M-1I to V-562; M- 1 to G-561; M-1 to G-560; M-1 to G-559; M-1 to C-558; M-1 to T-557; M-1 to R- 556; M-1 to S-555; M-1 to C-554; M-1 to D-553; M-1 to G-552; M-1 to W-551; M-1I to P-5 50; M-1I to G-549; M-1I to W-548; M-1I to M-547; M- I to G-546; M-1I to W-545; M-1 to S-544-, M-1 to G-543; M-1 to H-542; M-1 to F-541; M-1 to P-540, M-1 to T-539; M-I to D-538; M-1 to F-537; M-1 to H-536; M-1 to K- 535; M-1 to R-534; M-1 to D-533; M-1 to T-532; M-1 to K-531; M-1 to N-530; WO 99/37660 PCTIUS99/01313 -76- M-1 to V-529; M-1 to C-528; M-1 to K-527; M-1 to G-526;1 M-1 to N-525, M-1 to 1-524; M-1 to C-523; M-1 to W-522; M-1 to K-521; M-1 to G-520; M-1 to E- 519; M-1I to G-518;- M-]I to C-517; M- I to S-516; M-1I to T-515; M-1I to G-514; M-1I to D-513; M-1I to A-512; M-1I to W-51 1, M- I to P-5 10; M-1I to F-5 09; M- 1 to H-508; M-1I to K-507; M-1I to T-5 06; M-1I to Q-5 05;-M- I to C-5 04; M-1I to V 503; M-1I to L-502; M- I to V-50 1; M-1I to G-500; M-1I to G-499; M- I to S-498;- M-1I to T-497; M-1I to G-496; M- I to T-495; M- I to C-494; M-1I to W-493; M-1I to L-492; M-1 to T-491; M-1 to S-490; M-1 to C-489; M-1 to T-488; M-1 to S- 487; M-1 to A-486; M-1 to A-485; M-1 to D-484; M-1 to P-483;, M-1 to C-482; M-1I to H-48 1; M-1I to K-480; M-1I to S-479; M- I to D-478; M-1I to E-477; M-1I to G-476; M-1I to F-475; M-1I to T-474; M- I to F-473; M-1I to Q-472; M-1I to C- 471; M- 1 to Q-470; M- I to R-469; M- 1 to N-468; M- I to A-467; M- I to D-466; M- I to Y-465; M-1I to S-464; M-1I to T-463; M- I to G-462; M-1I to P-46 1; M-1I to L-460; M-1 to D-459; M-1 to G-458; M-1 to P-457; M-1 to L-456; M-1 to Q- 455; M-1 to 1-454; M-1 to P-453; M-1 to N-452; M-1 to Q-451; M-1 to P-450; M-1 to K-449; M-1 to D-448; M-1 to M-447; M-1 to L-446; M-1 to C-445; M-1 to E-444; M-1I to G-443;) M-1I to H-442; M-1I to G-441 M-1I to N-440; M- I to D-43 9; M- I to L-43 8; M-1I to F-43 7; M-1I to S-436; M-1I to T-43 5; M-1I to 1-434; M- I to M-43 I to Y-432; M-1I to A-43 1;M-1I to S-430; M-1I to C-429; M- 1 to P-428; M-1I to S-427; M-1I to W-426; M-1I to P-425; M-1I to Q-424; M-1I to S- 423; M-1I to H-422; M-1I to D-42 1; M- I to L-420; M-1I to N-419; M-1I to S-418; M-1I to L-417; M-1I to M-416 M-1I to S-415; M-1I to A-414; M-1I to M-413; M- I to M-412; M- I to H-41 1;M-1I to S-41 0;M- I to D-409; M- I to Q-408; M-1I to N-407; M-1 to V-406; M-1 to G-405; M-1 to N-404; M-1 to L-403; M-1 to S- 402; M-1I to A-401 M- I to C-400; M-1I to Q-3 99; M- I to K-3 98; M- I to A-3 97; M-1I to D-3 96; M-1I to D-3 95; M-1I to H-3 94; M-1I to P-3 93; M-1I to M-3 92; M-1I to N-3 91; M-1I to F-3 90; M- I to V-3 89; M-1I to H-3 8 8 M- I to G-3 87; M-1I to L- 3 86; M-1I to E-3 85; M-1I to H-3 84; M- I to A-3 83; M-1I to T-3 82; M- I to T-3 81; M-1 to F-380; M-1 to A-379; M-1 to A-378; M-1 to Q-377; M-1 to L-376; M-1 to G-375; M-1 to D-374; M-1 to D-373; M-1 to E-372; M-1 to 1-371; M-1 to V- WO 99/37660 PCTIUS99/01313 -77- 370; M-1 to S-369; M-1 to C-368; M-1 to S-367; M-1 to R-366; M-1 to S-365; M-1 to P-364; M-I to D-363; M-1 to C-362, M-1 to V-361; M-1 to T-360; M-1 to G-359; M-1 to V-358; M-1 to D-357, M-1 to A-356; M-1 to M-355; M-1 to G-354; M-1 to L-353; M-1 to T-352; M-1 to D-351; M-1 to C-350; M-1 to T- 349; M-1 to Q-348; M-1 to S-347; M-1 to G-346; M-1 to C-345; M-1 to L-344; M-1 to D-343; M-1 to Q-342; M-1 to R-341; M-1 to T-340; M-1 to F-339; M-1 to L-338; M-1 to 1-337; M-1 to A-336; M-1 to T-335; M-1 to D-334; M-1 to Y- 333, M-1 to H-332; M-1 to E-33 1; M-1 to A-330; M-1 to D-329; M-1 to R-328; M-1 to D-327; M-1 to S-326; M-1 to P-325; M-1 to P-324; M-1 to N-323; M-1 to H-3 22; M-1I to Q-3 21;- M-1I to K-3 20; M-1I to Q-3 19; M-1I to W-3 18; M-lI to N-3 17, M-1I to C-3 16; M-1I to F-3 15; M- I to N-3 14; M-1I to R-3 13; M-1I to L- 312; M- I to T-31 1; M-1I to L-3 10; M-1I to A-309; M-1I to A-3 08; M-1I to N-3 07; M-1 to S-306; M-1 to T-305; M-1 to V-304; M-1 to E-303; M-1 to P-302; M-1 to G-301; M-1 to K-300; M-1 to Q-299; M-1 to E-298; M-1 to D-297; M-1 to H-296; M-1I to 1-295; M-1I to V-294; M-1I to L-293;- M-1I to 1-292; M- I to K-29 1; M-1 to V-290; M-1 to V-289; M-1 to V-288; M-1 to L-287; M-1 to S-286; M-1 to V-285; M-1 to S-284; M-1 to N-283; M-1 to R-282; M-1 to 1-281; M-1 to S- 280; M-1I to P-279; M- I to H-278; M-1I to K-277; M-1I to Y-276; M-1I to L-275; M- I to R-274; M-1I to A-273; M-1I to A-272; M-1I to V-27 1; M-1I to S-270; M- 1 to F-269; M-1I to L-268; M-1I to T-267; M-1I to L-266; M-1I to L-265; M-1I to Y- 264; M-1I to H-263;- M-1I to K-262; M-1I to L-26 1; M-1I to G-260; M-1I to S-25 9; M-1 to G-258; M-1 to H-257; M-1 to F-256; M-1 to E-255; M-1 to A-254; M-1 to M-253; M-1 to S-252; M-1 to Q-251; M-1 to D-250; M-1 to A-249; M-1 to V-248; M-1 to L-247; M-1 to M-246; M-1 to T-245; M-1 to E-244; M-1 to V- 243; M-1I to Y-242; M-1I to R-24 1; M-1I to H-240; M-1I to S-23 9; M- I to S-23 8; M-1 to V-237; M-1 to F-236; M-1 to R-235; M-1 to K-234; M-1 to K-233; M-1 to R-232; M-1 to 1-23 1; M-1 to S-230; M-1 to G-229; M-1 to T-228; M-1 to G- 227; M-1I to T-226; M- I to P-225; M-1I to Q-224; M-1I to G-223; M-1I to V-222; M-1 to G-221; M-1 to Q-220; M-1 to L-219; M-1 to A-218; M-1 to P-217; M-1 to D-216; M-1I to Q-215 M-1I to P-214; M-1I to S-213; M-1I to W-212; M-1I to 99/37660 PCT/US99/01313 -78- 1; M-1I to P-21I0;- M- I to G-209; M-1I to E-208; M- I to D-207; M-lI to E- 106; M- 1 to G-205; M- I to E-204; M- 1 to T-203; M- 1 to G-202; M- 1 to E-20 1; 0-1 to D-200; M-1 to E-199; M-1 to D-198; M-1 to E-197; M-1 to T-196; M-1 o E-195; M-1 to A-194,M-1 to K-193; M-1 to G-192, M-1 to T-191; M-1 to P- M-1 to R-189; M-1 to P-188; M-1 to E-187; M-1 to D-186; M-1 to D-185; vi-I to V-184; M-1 to V-183; M-1 to G-182; M-1 to C-181; M-1 to T-180; M-1 o G-179; M-1 to G-178; M-1 to V-177; M-l to D-176; M-1 to G-175; M-1 to -174; M-1 to R-173; M-1 to N-172; M-1 to R-171; M-1 to R-170; M-1 to L- 69; M-1 to L-168; M-1 to H-167; M-1 to F-166; M-1 to Q-165; M-1 to L-164; Ai-i to P-163; M-1 to A-162; M-1 to P-161, M-1 to P-160; M-1 to K-159; M-1 o E-158, M-1 to G-157; M-1 to P-156; M-1 to A-155; M-1 to A-154,-M-1 to T- 53; M-1 to A-152; M-1 to L-151; M-1 to R-150; M-1 to E-149; M-1 to S-148, A-I to A-147; M-1 to A-146; M-1 to P-145; M-1 to L-144; M-1 to P-143; M-1 o Q-142; M-1 to 1-141; M-i to F-140; M-1 to Y-139; M-1 to A-138; M-1 to E- 37; M-1 to G-136; M-1 to L-135; M-1 to L-134; M-1 to Y-133; M-1 to F-132; Al-i to A-13 I; M-1 to G-130; M-1 to R-129; M-1 to V-128; M-1 to G-127; M-1 o E-126; M-1 to C-125; M-1 to L-124; M-1 to S-123; M-1 to L-122; M-1 to A- 21; M-1 to A-120; M-1 to A-]119; M-1 to S-1 18; M-1 to S-1 17; M-1 to P-1 16; A-i to D-1 15; M-1 to G-1 14; M-1 to N-1 13; M-1 to V-i 12; M-1 to T-1 11; M-1 o G-1 10; M-1 to S-109; M-1 to Y-108; M-1 to F-107; M-1 to C-106; M-1 to H- M-1 to A-104; M-1 to L-103; M-1 to D-102; M-1 to T-101; M-1 to E-100; d-I to P-99; M-1 to L-98; M-1 ito P-97; M-1 to T-96; M-1 to E-95; M-1 to S-94; A-i to G-93; M-1 to S-92; M-1 to K-91; M-1 to R-90; M-1 to G-89; M-1 to V- M-1I to N-87; M- I to Q-86; M-1I to L-85; M-1I to T-84; M-1I to F-83; M-1I to 3-82; M-1I to P-Si1; M-1I to A-SO; M- I to L-79; M- I to F-78; M-1I to S-77; M-1I o S-76; M-1I to D-75; M- I to P-74; M-1I to R-73; M- I to L-72; M-1I to E-7 1; Mto L-70; M-1I to D-69; M-1I to L-68; M- I to Q-67; M-1I to Q-66; M-lI to vi-I to F-64; M- I to A-63; M- I to H-62; M- I to L-61,- M- I to R-60; M-1I to L- M-1 to R-58; M-1 to T-57; M-1 to T-56; M-1 to G-55; M-1 to H-54; M-1 to 3-53; M-1 to P-52; M-1 to A-5SI; M-1 to R-50; M-1 to E-49; M-1 to L-48; M-1 WO 99/37660 PCTIUS99/01313 -79to E-47; M-lI to P-46; M-lI to V-45; M-1I to V-44; M-1I to L-43,- M-1I to E-42,- M- I to E-41; M-1 to D-40; M-1 to E-39; M-l to E-38,; M-1 to S-37; M-1 to P-36; M-1 to R-35; M-1 to G-34; M-1 to L-33; M-1 to A-32; M-1 to D-3I; M-1 to S- M-1 to V-29; M-1 to A-28; M-1 to L-27; M-1 to L-26; M-I to A-25; M-1 to A-24; M-1 to A-23; M-1 to L-22; M-1 to L-21 M-1 to L-20; M-1 to L-19; M-1 to T-18; M-1 to P-17; M-1 to V-16; M-1 to P-15; M-1 to G-14;,M-1 to F-13;,M- I to S-12, M-1 to R-1 1; M-1 to S-10;- M-1 to G-9; M-1 to P-8; M-1 to A-7; of SEQ ID NO:2. For example, any of the above listed N- or C-terminal deletions can be combined to produce a N- and C-terminal deleted MIETH I polypeptide.
Moreover, N-terminal deletions of the METH-2 polypeptide can be described by the general formula m-890, where m is an integer from 2 to 889, where m corresponds to the position of the amino acid residue identified in SEQ ID NO:4. Preferably, N-terminal deletions of the MLETH2 polypeptide of the invention shown as SEQ ID NO:4 include polypeptides comprising the amino acid sequence of residues: F-2 to L-890; P-3 to L-890; A-4 to L-890; P-5 to L-890; A-6 to L-890; A-7 to L-890; P-8 to L-890; R-9 to L-890, W- 10 to L-890; L- I I to L-890; P- 12 to L-890; F- 13 to L-890; L- 14 to L-890; L- 15 to L-890, L- 16 to L-890; L- 17 to L-890; L- 18 to L-890; L- 19 to L-890; L-20 to L-890; L-21 to L- 890; L-22 to L-890; P-23 to L-890; L-24 to L-890; A-25 to L-890; R-26 to L- 890; G-27 to L-890; A-28 to L-890; P-29 to L-890; A-30 to L-890; R-31I to L- 890; P-32 to L-890; A-33 to L-890; A-34 to L-890; G-35 to L-890; G-36 to L- 890; Q-37 to L-890; A-38 to L-890; S-39 to L-890; E-40 to L-890; L-41 to L- 890; V-42 to L-890; V-43 to L-890; P-44 to L-890; T-45 to L-890; R-46 to L- 890; L-47 to L-890; P-48 to L-890; G-49 to L-890; S-SO to L-890; A-51 to L- 890; G-52 to L-890; E-53 to L-890; L-54 to L-890; A-55 to L-890; L-56 to L- 890; H-57 to L-890; L-58 to L-890; S-59 to L-890; A-60 to L-890; F-61 to L- 890; G-62 to L-890; K-63 to L-890; G-64 to L-890; F-65 to L-890; V-66 to L- 890; L-67 to L-890; R-68 to L-890; L-69 to L-890; A-70 to L-890; P-71 to L- 890; D-72 to L-890; D-73 to L-890; S-74 to L-890; F-75 to L-890; L-76 to L- 890; A-77 to L-890; P-78 to L-890; E-79 to L-890; F-80 to L-890; K-81 to L- WO 99/37660 PCT/US99/01313 890; 1-82 to L-890; E-83 to L-890; R-84 to L-890; L-85 to L-890; G-86 to L- 890; G-87 to L-890; S-88 to L-890; G-89 to L-890; R-90 to L-890; A-91 to L- 890; T-92 to L-890; G-93 to L-8 90; G-94 to L-890; E-95 to L-890; R-96 to L- 890; G-97 to L-890; L-98 to L-890; R-99 to L-890; G- 100 to L-890; C-I oi to L- 890; F- 102 to L-890; F- 103 to L-890; S- 104 to L-890; G- 105 to L-890; T- 106 to L-890; V-i107 to L-890; N- 108 to L-890; G- 109 to L-890; E- 110 to L-890; P- IlIl to L-890; B-I 112 to L-890; S- 113 to L-890; L- 114 to L-890; A-I 115 to L-890; A- 116 to L-890; V- 117 to L-890; S-i118 to L-890; L- 119 to L-890,; C- 120 to L- 890; R-121 to L-890; G-122 to L-890; L-123 to L-890; S-124 to L-890; G-125 to L-890; S- 126 to L-890; F- 127 to L-890; L- 128 to L-890; L- 129 to L-890; D- 13 0 to L-890; G- 131 to L-890; E- 13 2 to L-890; E- 13 3 to L-890; F- 134 to L-890; T-135 to L-890; 1-136 to L-890; Q-137 to L-890; P-138 to L-890; Q-139 to L- 890; G-i140 to L-890; A-i141 to L-890; G- 142 to L-890; G- 143 to L-890; S- 144 to L-890; L- 145 to L-890; A- 146 to L-890; Q- 147 to L-890; P- 148 to L-890; H- 149 toL-890; R-i150to L-890; L- 151 to L-890; Q-152 to L-890; R- 153 to L-890; W- 154 to L-890; G-15 5 to L-890; P- 15 6 to L-890; A- 15 7 to L-890; G- 15 8 to L- 890; A- 15 9 to L-890; R- 160 to L-890; P- 161 to L-890; L- 162 to L-890; P- 163 to L-8 90; R- 164 to L-890; G-I 165 to L-8 90; P-i 166 to L-890; E- 167 to L-890; W- 168 to L-890; E- 169 to L-890; V- 170 to L-890; E-i171 to L-890; T- 172 to L-890; G-i173 to L-890; E-i174 to L-890; G- 175 to L-890; Q- 176 to L-890; R- 177 to L- 890; Q-178 to L-890; E-179 to L-890; R-180 to L-890; G-181 to L-890; D-i82 to L-890; H-i183 to L-890; Q-184 to L-890; E-185 to L-890; D-186 to L-890; S- 187 to L-890; B-i 88 to L-890; B-I189 to L-890; E-1 90 to L-890; S-191 to L-890; Q- 192 to L-890,- E- 193 to L-890; E- 194 to L-890; E- 195 to L-890,- A- 196 to L- 890; E- 197 to L-890; G- 198 to L-890; A- 199 to L-890; S-200 to L-890; E-201 to L-890; P-202 to L-890; P-203 to L-890; P-204 to L-890; P-205 to L-890; L- 206 to L-890; G-207 to L-890,- A-208 to L-890; T-209 to L-890; S-21i0 to L-890; R-211I to L-890; T-212 to L-890; K-213 to L-890; R-214 to L-890; F-215 to L- 890; V-216 to L-890; S-217 to L-890; E-218 to L-890; A-219 to L-890; R-220 to L-890; F-221 to L-890; V-222 to L-890; E-223 to L-890-1 T-224 to L-890; L- WO 99/37660 PCTIUS99/01313 -81- 225 to L-890; L-226 to L-890; V-227 to L-890; A-228 to L-890, D-229 to L-890; A-230 to L-890; S-231I to L-890; M-232 to L-8 90; A-233 to L-890; A-234 to L- 890; F-235 to L-890; Y-236 to L-890; G-237 to L-890; A-238 to L-890; D-239 to L-890; L-240 to L-890; Q-241 to L-890; N-242 to L-890; H-243 to L-890; I- 244 to L-890; L-245 to L-890; T-246 to L-890; L-247 to L-890; M-248 to L-890; S-249 to L-890; V-250 to L-890; A-251I to L-890; A-252 to L-890; R-253 to L- 890 1-254 to L-890; Y-255 to L-890; K-256 to L-890, H-257 to L-890; P-258 to L-890; S-259 to L-890; 1-260 to L-890; K-261 to L-890; N-262 to L-890; S- 263 to L-890; 1-264 to L-890; N-265 to L-890; L-266 to L-890; M-267 to L-890; V-268 to L-890, V-269 to L-890, K-270 to L-890; V-271 to L-890; L-272 to L- 890; 1-273 to L-890; V-274 to L-890; E-275 to L-890; D-276 to L-890; E-277 to L-890, K-278 to L-890; W-279 to L-890; G-280 to L-890, P-281 to L-890, E- 282 to L-890; V-283 to L-890; S-284 to L-890; D-285 to L-890; N-286 to L-890; G-287 to L-890; G-288 to L-890; L-289 to L-890; T-290 to L-890; L-291 to L- 890; R-292 to L-890; N-293 to L-890; F-294 to L-890; C-295 to L-890; N-296 to L-890; W-297 to L-890; Q-298 to L-890; R-299 to L-890; R-300 to L-8 90; F- 301 to L-890; N-302 to L-890; Q-303 to L-890; P-304 to L-890; S-305 to L-890;- D-306 to L-890; R-307 to L-890; H-308 to L-890; P-309 to L-890; E-3 10 to L- 890; H-3 11 to L-890; Y-3 12 to L-890; D-3 13 to L-890; T-3 14 to L-890; A-3 to L-890; 1-316 to L-890; L-3 17 to L-890; L-3 18 to L-890; T-3 19 to L-890; R- 320 to L-890; Q-321 to L-890; N-322 to L-890; F-323 to L-890; C-324 to L-890; G-325 to L-890; Q-326 to L-890; E-327 to L-890; G-328 to L-890; L-329 to L- 890; C-330 to L-890; D-33 1 to L-890; T-332 to L-890; L-333 to L-890; G-334 to L-890; V-3 35 to L-890; A-3 36 to L-890; D-3 37 to L-890; 1-3 38 to L-890; G- 339 to L-890; T-340 to L-890; 1-34 1 to L-890; C-342 to L-890; D-343 to L-890; P-344 to L-890; N-345 to L-890; K-346 to L-890; S-347 to L-890; C-348 to L- 890; S-349 to L-890; V-350 to L-890; 1-351 to L-890; E-352 to L-890; D-353 to L-890; E-354 to L-890; G-355 to L-890; L-356 to L-890; Q-357 to L-890; A- 358 to L-890; A-359 to L-890; H-360 to L-890; T-361I to L-890; L-362 to L-890; A-363 to L-890; H-364 to L-890; E-365 to L-890; L-366 to L-890; G-367 to L- WO 99/37660 PCTIUS99/01313 -82- 890; H-368 to L-890; V-369 to L-890; L-370 to L-890; S-371 to L-890; M-372 to L-890; P-373 to L-890,; H-374 to L-890; D-3'75 to L-890; D-376 to L-890; S- 3 77 to L-890, K-3 78 to L-890; P-3 79 to L-890; C-3 80 to L-890, T-3 81 to L-890, R-382 to L-890;, L-383 to L-890; F-384 to L-890; G-385 to L-890; P-386 to L- 890; M-387 to L-890; G-388 to L-890; K-389 to L-890; H-390to L-890; H-391 to L-890; V-3 92 to L-890; M-3 93 to L-890; A-3 94 to L-890; P-3 95 to L-890; L- 396 to L-890; F-397 to L-890; V-398 to L-890; H-399 to L-890; L-400 to L-890; N-401 to L-890; Q-402 to L-890; T-403 to L-890; L-404 to L-890; P-405 to L- 890; W-406 to L-890; S-407 to L-890; P-408 to L-890; C-409 to L-890; S-41 0 to L-890; A-411 to L-890; M-412 to L-890; Y-413 to L-890; L-414 to L-890; T- 415 to L-890; E-416 to L-890; L-417 to L-890; L-418 to L-890,;D-419 to L-890; G-420 to L-890; G-421 to L-890; H-422 to L-890; G-423 to L-890; D-424 to L- 890; C-425 to L-890; L-426 to L-890; L-427 to L-890; D-428 to L-890; A-429 to L-890; P-430 to L-890; G-43 1 to L-890; A-432 to L-890; A-433 to L-890; L- 434 to L-890; P-435 to L-890; L-436 to L-890; P-437 to L-890; T-438 to L-890; G-439 to L-890; L-440 to L-890; P-441 to L-890; G-442 to L-890; R-443 to L- 890; M-444 to L-890; A-445 to L-890; L-446 to L-890; Y-447 to L-890; Q-448 to L-890; L-449 to L-890; D-450 to L-890; Q-451 to L-890; Q-452 to L-890; C- 453 to L-:890; R-454 to L-890; Q-455 to L-890; 1-456 to L-890; F-457 to L-890; G-458 to L-890; P-459 to L-890; D-460 to L-890; F-461 to L-890; R-462 to L- 890; H-463 to L-890; C-464 to L-890; P-465 to L-890; N-466 to L-890; T-467 to L-890; S-468 to L-890; A-469 to L-890; Q-470 to L-890; D-471 to L-890; V- 472 to L-890; C-473 to L-890; A-474 to L-890; Q-475 to L-890; L-476 to L-890; W-477 to L-890; C-478 to L-890; H-479 to L-890; T-480 to L-890; D-48 1 to L- 890; G-482 to L-890; A-483 to L-890; E-484 to L-890; P-485 to L-890; L-486 to L-890; C-487 to L-890; H-488 to L-890; T-489 to L-890; K-490 to L-890; N- 491 to L-890; G-492 to L-890; S-493 to L-890; L-494 to L-890; P-495 to L-890;- W-496 to L-890; A-497 to L-890; D-498 to L-8 90; G-499 to L-890; T-500 to L- 890; P-501 to L-890; C-502 to L-890; G-503 to L-890; P-504 to L-890; G-505 to L-890; H-506 to L-890; L-507 to L-890; C-508 to L-890; S-509 to L-890; E- WO 99/37660 PCT/US99/01313 -83- 510 to L-890; G-5 11 to L-890; S-5 12 to L-890; C-5 13 to L-890;- L-5 14 to L-8 P-515 to L-890; E-516 to L-890; E-517 to L-890; E-518 to L-890; V-519 to L- 890; E-520 to L-890; R-521 to L-890; P-522 to L-890; K-523 to L-890; P-524 to L-890; V-525 to L-890; V-S526 to L-890; D-527 to L-890; G-528 to L-890; G- 529 to L-890; W-530 to L-890; A-53 1 to L-890; P-532 to L-890; W-533 to L- 890; G-534 to L-890; P-535 to L-890; W-536 to L-890; G-537 to L-890; E-538 to L-890; C-539 to L-890; S-540 to L-890; R-541 to L-890; T-542 to L-890; C- 543 to L-890; G-544 to L-890; G-545 to L-890; G-546 to L-890; V-547 to L- 890; Q-548 to L-890; F-549 to L-890; S-SS0 to L-890; H-551 to L-890; R-552 to L-890; E-553 to L-890; C-554 to L-890; K-555 to L-890; D-556 to L-890; P- SS7 to L-890; E-558 to L-890; P-559 to L-890; Q-560 to L-890; N-561 ito L-890', G-562 to L-890; G-563 to L-890; R-564 to L-890; Y-565 to L-890; C-566 to L- 890; L-567 to L-890; G-568 to L-890; R-569 to L-890; R-570 to L-890; A-571 to L-890; K-572 to L-890; Y-573 to L-890; Q-574 to L-890; S-S75 to L-890; C- 576 to L-890; H-S577 to L-890; T-578 to L-890; E-579 to L-890; E-580 to L-890; C-581 to L-890; P-582 to L-890; P-583 to L-890; D-584 to L-890; G-585 to L- 890; K-S586 to L-890; S-587 to L-890; F-588 to L-890; R-589 to L-890; E-590 to L-890; Q-591 to L-890; Q-592 to L-890; C-593 to L-890; E-594 to L-890; K- 595 to L-890; Y-596 to L-890; N-597 to L-890; A-598 to L-890; Y-599 to L- 890; N-600 to L-890; Y-601 to L-890; T-602 to L-890; D-603 to L-890; M-604 to L-890; D-605 to L-890; G-606 to L-890; N-607 to L-890; L-608 to L-890; L- 609 to L-890; Q-610 to L-890; W-611I to L-890; V-612 to L-890; P-613 to L- 890; K-614 to L-890; Y-615 to L-890; A-616 to L-890; G-617 to L-890; V-618 to L-890; S-6 19 to L-890; P-620 to L-890; R-621 to L-890; D-622 to L-890; R- 623 to L-890; C-624 to L-890; K-625 to L-890; L-626 to L-890; F-627 to L-890; C-628 to L-890; R-629 to L-890; A-630 to L-890; R-63 1 to L-890; G-632 to L- 890; R-633 to L-890; S-634 to L-890; E-635 to L-890; F-636 to L-890; K-637 to L-890; V-638 to L-890; F-639 to L-890; E-640 to L-890; A-641 to L-890; K- 642 to L-890; V-643 to L-890; 1-644 to L-890; D-645 to L-890; G-646 to L-890; T-647 to L-890; L-648 to L-890; C-649 to L-890; G-650 to L-890; P-651 to L- WO 99/3766 WO 9937660PCT/US99/01313 -84- 890; E-652 to L-890; T-653 to L-890; L-654 to L-890; A-655 to L-890; 1-656 to L-890; C-657 to L-890; V-658 to L-890; R-659 to L-8 90; G-660 to L-890; Q-66 1 to L-890; C-662 to L-890; V-663 to L-890; K-664 to L-890; A-665 to L-890; G- 666 to L-890; C-667 to L-890; D-668 to L-890; H-669 to L-890; V-670 to L- 890; V-671 to L-890; D-672 to L-890; S-673 to L-890; P-674 to L-890; R-675 to L-890; K-676 to L-890; L-677 to L-890; D-678 to L-890; K-679 to L-890; C- 680 to L-890; G-681 to L-890; V-682 to L-890; C-683 to L-890; G-684 to L- 890; G-685 to L-890; K-686 to L-890; G-687 to L-890; N-688 to L-890; S-689 to L-890; C-690 to L-890; R-691 to L-890, K-692 to L-890; V-693 to L-890; S.- 694 to L-890; G-695 to L-890; S-696 to L-890; L-697 to L-890; T-698 to L-890; P-699 to L-890; T-700 to L-890; N-701 to L-890; Y-702 to L-890; G-703 to L- 890; Y-704 to L-890; N-705 to L-890; D-706 to L-890; 1-707 to L-890; V-708 to L-890; T-709 to L-890; 1-7 10 to L-890; P-71 I to L-890; A-712 to L-890; G- 713 to L-890; A-71 4 to L-890; T-71 5 to L-890; N-71 6 to L-890; 1-717 to L-890; D-71 8 to L-890; V-719 to L-890; K-720 to L-890; Q-721 to L-890; R-722 to L- 890; S-723 to L-890; H-724 to L-890; P-725 to L-890; G-726 to L-890; V-727 to L-890; Q-728 to L-890; N-729 to L-890; D-730 to L-890; G-73 I to L-890; N- 732 to L-890; Y-733 to L-890; L-734 to L-890; A-73 5 to L-890; L-736 to L-890; K-737 to L-890; T-738 to L-890; A-739 to L-890; D-740 to L-890; G-741 to L- 890; Q-742 to L-890; Y-743 to L-890; L-744 to L-890; L-745 to L-890; N-746 to L-890; G-747 to L-890; N-748 to L-890; L-749 to L-890; A-750 to L-890; I- 751 to L-890; S-752 to L-890; A-753 to L-890; 1-754 to L-890; E-755 to L-890; Q-756 to L-890; D-757 to L-890; 1-758 to L-890; L-759 to L-890; V-760 to L- 890; K-761 to L-890; G-762 to L-890; T-763 to L-890; 1-764 to L-890; L-765 to L-890; K-766 to L-890; Y-767 to L-890; S-768 to L-890; G-769 to L-890; S- 770 to L-890; 1-77 1 to L-890; A-772 to L-890; T-773 to L-890; L-774 to L-890; E-775 to L-890; R-776 to L-890; L-777 to L-890; Q-778 to L-890; S-779 to L- 890; F-780 to L-890; R-78 1 to L-890; P-782 to L-890; L-783 to L-890; P-784 to L-890; E-785 to L-890; P-786 to L-890; L-787 to L-890; T-788 to L-890; V-789 to L-890; Q-790 to L-890, L-791 to L-890; L-792 to L-890; T-793 to L-890; V- WO 99/37660 PTU9111 PCT/US99/01313 794 to L-890; P-795 to L-890,; G-796 to L-890; E-797 to L-890; V-798 to L-890; F-799 to L-890; P-800 to L-890; P-801 to L-890; K-802 to L-890; V-803 to L- 890; K-804 to L-890; Y-805 to L-890; T-806 to L-890; F-807 to L-8 90; F-808 to L-890; V-809 to L-890; P-81 0 to L-890, N-81I ito L-890; D-812 to L-890; V- 813 to L-890; D-8 14 to L-890; F-S815 to L-890, S-S816 to L-890; M-81 7 to L-890; Q-818 to L-890; S-819 to L-890; S-820 to L-890; K-821 to L-890; E-822 to L- 890; R-823 to L-890; A-824 to L-890; T-825 to L-890; T-826 to L-890; N-827 to L-890; 1-828 to L-890; 1-829 to L-890; Q-830 to L-890; P-83 I to L-890; L- 832 to L-890; L-833 to L-890; H-834 to L-890; A-835 to L-890; Q-836 to L-890; W-837 to L-890; V-838 to L-890; L-839 to L-890; G-840 to L-890; D-841 to L- 890; W-842 to L-890; S-843 to L-890; E-844 to L-890; C-845 to L-890; S-846 to L-890; S-847 to L-890; T-848 to L-890; C-849 to L-890; G-850 to L-890; A- 851 to L-890; G-852 to L-890; W-853 to L-890; Q-854 to L-890; R-855 to L- 890; R-856 to L-890; T-857 to L-890; V-858 to L-890; E-859 to L-890; C-860 to L-890; R-861 to L-890; D-862 to L-890; P-863 to L-890; S-864 to L-890; G- 865 to L-890; Q-866 to L-890; A-867 to L-890; S-868S to L-8 90; A-869 to L-890; T-870 to L-890; C-871 to L-890; N-872 to L-890; K-873 to L-890; A-874 to L- 890; L-875 to L-890; K-876 to L-890; P-877 to L-890; E-878 to L-890; D-879 to L-890; A-880 to L-890; K-881 to L-890; P-882 to L-890; C-883 to L-890; E- 884 to L-890; S-885 to L-890; of SEQ ID NOA4 Moreover, C-terminal deletions of the IETH2 polypeptide can also be described by the general formula 1-n, where n is an integer from 2 to 890 where n corresponds to the position of amino acid residue identified in SEQ ID NOA4 Preferably, C-terminal deletions of the METH2 polypeptide of the invention shown as SEQ ID NOA: include polypeptides comprising the amino acid sequence of residues: M-1 to P-889; M-1 to C-888; M-1 to L-887; M-1 to Q-886; M-1 to S-885; M-1 to E-884; M-1 to C-883; M-1 to P-882; M-1 to K-881; M-1 to A- 880; M-1I to D-879; M-1I to E-878; M-1I to P-877; M- I to K-876; M-lI to L-875; M-1I to A-874; M-lI to K-873;- M-1I to N-872; M-lI to C-87 1; M-1I to T-870; M-I1 to A-869; M-1 to S-868; M-1 to A-867; M-1 to Q-866; M-1 to G-865; M-1 to S- WO 99/37660 PCTIUS99/01313 -86- 864; M-1I to P-863; M-1I to D-862; M-1I to R-86 1; M-1I to C-860; M-1I to E-859; M-1 to V-858; M-1 to T-857; M-1 to R-856; M-1 to R-855, M-1 to Q-854; M-1 to W-853; M-1 to G-852; M-1 to A-851; M-1 to G-850,; M-1 to C-849; M-1 to T-848;M-1 to S-847;M-1 to S-846;M-1 toC-845;M-l toE-844;M-1 to S-843; M-1 toW-842; M-1 to D-841; M-1 to G-840; M-1 to L-839; M-1 to V-838; M-1 to W-837; M-1 to Q-836; M-1 to A-835; M-1 to H-834; M-1 to L-833; M-1 to L-832; M-1 to P-83 M-1 to Q-830; M-1 to 1-829; M-1 to 1-828; M-1 to N-827; M-1I to T-826-; M-1I to T-825; M-1I to A-824; M-1I to R-823; M- I to E-822; M- I to K-82 1;M- I to S-820; M-1I to S-819; M-1I to Q-818; M-1I to M-817; M-1I to S- 816; M-1I to F-815; M- I to D-814; M- I to V-813;-M-1I to D-812; M-1I to N-81 1; M-1I to P-8 10; M-1I to V-809; M-1I to F-808; M-1I to F-807; M-1I to T-806; M-1I to Y-805; M-1I to K-804; M-1I to V-803; M-1I to K-802; M-1I to P-80 M-1I to P- 800; M-1I to F-799; M-1I to V-798; M-1I to E-797; M-1I to G-796; M-1I to P-795; M-1I to V-794; M-1I to T-793; M- I to L-792; M-1I to L-79 1; M-1I to Q-790; M- 1 to V-789; M-1 to T-788; M-1 to L-787; M-1 to P-786; M-1 to E-785; M-1 to P- 784; M-1I to L-783; M-1I to P-782; M- I to R-78 1; M-1I to F-780; M-1I to S-779; M-1I to Q-778; M-1I to L-777; M-1I to R-776, M-1I to E-775; M-1I to L-774; M-1I to T-773; M-lI to A-772; M-1I to 1-77 1; M-1I to S-770; M-1I to G-769; M-1I to S- 768; M-1I to Y-767; M-1I to K-766; M-1I to L-765; M-1I to 1-764; M-lI to T-763; M-1 to G-762; M-1 to K-761; M-1 to V-760; M-1 to L-759; M-1 to 1-758; M-1 to D-757; M-1 to Q-756; M-1 to E-755; M-1 to 1-754; M-1 to A-753; M-1 to S- 752; M-1 to 1-75 1, M-1 to A-750; M-1 to L-749; M-1 to N-748; M-1 to G-747; M-1 to N-746; M-1 to L-745; M-1 to L-744; M-1 to Y-743; M-1 to Q-742; M-1 to G-741; M-1 to D-740; M-1 to A-739; M-1 to T-738; M-1 to K-737; M-1 to L-736; M-1 to A-735; M-1 to L-734.; M-1 to Y-733; M-1 to N-732; M-1 to G- 731; M-1 to D-730; M-1 to N-729; M-1 to Q-728; M-1I to V-727; M-1 to G-726; M- I to P-725; M-1I to H-724; M-1I to S-723; M- I to R-722; M-1I to Q-72 1; M-1I to K-720; M-1I to V-719; M-1I to D-718; M-1I to 1-717; M-1I to N-716; M-lI to T- 715; M-1I to A-714; M-1I to G-713,- M-1I to A-712; M-1I to P-71 1; M- I to 1-7 M-1I to T-709; M-1I to V-708; M-1I to 1-707; M- I to D-706; M-1I to N-705; M-1I WO 99/37660 PCT/US99/01313 -87to Y-704; M- I to G-703;) M-1I to Y-702; M-1I to N-701;) M-1I to T-700; M-1I to P-699; M-1I to T-698; M-1I to L-697; M-1I to S-696; M-1I to G-695; M-1I to S-694; M-1I to V-693; M-1I to K-692; M-1I to R-691;- M-1I to C-690; M-1I to S-689; M-1I to N-688; M-1 to G-687; M-1 to K-686; M-1 to G-685; M-1 to G-684;- M-1 to C-683; M-1I to V-682; M-1I to G-681;- M-1I to C-680; M-1I to K-679; M-1I to D- 678; M-1I to L-677; M-1I to K-676; M-1I to R-675; M-1I to P-674; M-1I to S-673; M-1I to D-672; M-1I to V-67 1; M-1I to V-670; M-1I to H-669; M-1I to D-668; M-1I to C-667; M-1 to G-666; M-1 to A-665; M-1 to K-664; M-1 to V-663; M-1 to C-662; M-1 to Q-661; M-1 to G-660; M-1 to R-659; M-1 to V-658; M-1 to C- 657; M-1 to 1-656; M-1 to A-655; M-1 to L-654; M-1 to T-653; M-1 to E-652; M- I to P-65 1; M-1I to G-650; M-1I to C-649; M-1I to L-648; M-1I to T-647; M-1I to G-646; M-1I to D-645; M-1I to 1-644; M-1I to V-643; M-1I to K-642, M-1I to A- 641; M-1 to E-640; M-1 to F-639; M-1 to V-638; M-1 to K-637; M-1 to F-636; M-1 to E-635; M-1 to S-634; M-1 to R-633; M-1 to G-632; M-1 to R-63 1; M-1 to A-63 0;M-1I to R-629; M-1I to C-628; M-1I to F-627; M-1I to L-626; M-1I to K- 625; M-1I to C-624; M-1I to R-623;- M-1I to D-622; M-1I to R-621;- M-1I to P-620, M-1I to S-619; M-1I to V-618; M-1I to G-617; M-1I to A-616; M-1I to Y-615; M-1I to K-614; M-1I to P-613; M-1I to V-612; M-1I to W-61 1; M-1I to Q-6 10; M-1I to L-609; M-1I to L-608; M-1I to N-607; M-1I to G-606; M-1I to D-605; M-1I to M- 604; M- 1 to D-603;- M-1I to T-602; M-1I to Y-60 1; M- I to N-600; M- I to Y-599; M- I to A-598; M-1I to N-597; M-1I to Y-596; M-1I to K-595; M-1I to E-594; M- I to C-593; M-1 to Q-592; M-1 to Q-591; M-1 to E-590; M-1 to R-589; M-1 to F- 588; M-1 to S-587; M-1 to K-586; M-1 to G-585; M-1 to D-584; M-1 to P-583; M-1 to P-582; M-1 to C-581; M-1 to E-580; M-1 to E-579; M-1 to T-578; M-I to H-577; M-1I to C-576; M- 1 to S-575; M-lI to Q-574; M-1I to Y-573; M- I to K- 572; M-1 to A-571; M-1 to R-570; M-1 to R-569; M-1 to G-568; M-1 to L-567; M-1 to C-566; M-1 to Y-565; M-1 to R-564; M-1 to G-563; M-1 to G-562; M-1 to N-561; M-1 to Q-560; M-1 to P-559; M-1 to E-558; M-1 to P-557; M-1 to D- 556; M-1 to K-555; M-1 to C-554; M-1 to E-553; M-1 to R-552; M-1 to H-551; M-1I to S-5 50; M-1I to F-549; M-1I to Q-548; M- I to V-547; M-1I to G-546; M- I WO 99/37660 WO 9937660PCT/US99/01313 -88to G-545; M-1 to G-544; M- Ito C-543; M- Ito T-542; M- Ito R-541; M- Ito S- 540; M-1I to C-539; M-1I to E-53 8; M-1I to G-53 7, M-1I to W-536; M-1I to P-53 M-1I to G-534; M- I to W-53 3, M- I to P-532; M-1I to A-53 I1, M-1I to W-530; M-1I to G-529; M-1 to G-528; M-1 to D-527; M-1 to V-526; M-1 to V-525-; M-1 to P-524; M-1I to K-523; M- I to P-522; M- I to R-52 1; M-1I to E-520; M- I to V- 519; M-1I to E-518; M-1I to E-517; M-1I to E-516; M-1I to P-515; M-1I to L-514; M- I to C-513; M-1I to S-512; M- I to G-5 11; M-1I to E-5 10; M-1I to S-509,; M-1I to C-508; M- I to L-507; M-1I to H-506; M-1I to G-505; M-1I to P-504; M-1I to G- 503; M-1I to C-502; M-1I to P-501;- M-1I to T-500; M-1I to G-499; M-1I to D-498; M-1I to A-497; M-1I to W-496; M- 1 to P-495; M-1I to L-494; M-1I to S-493; M- I to G-492; M-1 to N-491; M-1 to K-490; M-1 to T-489; M-1 to H-488; M-1 to C-487; M-1 to L-486; M-1 to P-485; M-1 to E-484; M-1 to A-483; M-1 to G- 482; M-1I to D-48 1; M-1I to T-480; M-1I to H-479, M-1I to C-478; M-1I to W-477; M- I to L-476; M-1I to Q-475; M-1I to A-474; M-1I to C-473; M-1I to V-472; M- 1 to D-47 1; M-1I to Q-470, M- I to A-469; M-1I to S-468;- M-1I to T-467; M-1I to N- 466; M-1I to P-465;- M-1I to C-464; M-1I to H-463;- M-1I to R-462; M-1I to F-46 1; M-1 to D-460; M-1 to P-459; M-1 to. G-458; M-1 to F-457; M-1 to 1-456; M-1 to Q-45 5; M-1I to R-454; M-1I to C-453; M-1I to Q-452; M- I to Q-45 1; M-1I to D-450; M-1 to L-449; M-1 to Q-448; M-1 to Y-447; M-1 to L-446; M-1 to A- 445; M-1I to M-444; M-1I to R-443; M-1I to G-442; M-1I to P-44 1; M-1I to L-440; M-1 to G-439; M-1 to T-438; M-1 to P-437; M-1 to L-436; M-1 to P-435; M-1 to L-434; M-1 to A-433; M-1 to A-432; M-1 to G-43 1; M-1 to P-430; M-1 to A- 429; M-1I to D-428;- M- I to L-427; M-1I to L-426; M-1I to C-425;- M-1I to D-424; M-1I to G-423;- M-1I to H-422; M-1I to G-42 1; M-1I to G-420; M-1I to D-419; M-1I to L-418; M-1I to L-417; M-1I to E-416; M-1I to T-415; M-1I to L-414; M- I to Y- 413; M-1I to M-412; M-1I to A-41 1;M-1I to S-41 0;M-1I to C-409; M-1I to P-408; M-1I to S-467; M-1I to W-406; M-1I to P-405; M-1I to L-404; M-1I to T-403; M-1I to Q-402; M-1 to N-401; M-1 to L-400; M-1 to H-399; M-1 to V-398; M-I to F-397; M-1 to L-396; M-1 to P-395; M-1 to A-394; M-1 to M-393; M-1 to V- 3 92; M-1I to H-3 91; M-1I to H-3 90; M-1I to K-3 89; M-1I to G-3 88; M-1I to M-3 87; WO 99/37660 PCTIUS99/01313 -89- M-1 to P-386; M-1 to G-385; M-1 to F-384; M-1 to L-383; M-1 to R-382; M-1 to T-3 81; M-1I to C-3 80; M-1I to P-3 79; M- I to K-3 78; M-1I to S-3 77; M- I to D- 3 76; M-1I to D-3 75;- M-1I to H-3 74; M-1I to P-3 73; M- I to M-3 72; M-1I to S-3 71;- M-1I to L-3 70; M-1I to V-3 69; M-1I to H-3 68; M-1I to G-3 67; M- I to L-3 66; M- 1 to E-3 65; M- I to H-3 64; M-1I to A-3 63; M- I to L-3 62; M-1I to T-3 61; M-1I to H- 360; M-1 to A-359; M-1 to A-358; M-1 to Q-357; M-1 to L-356; M-1 to G-355; M-1 to E-354; M-1 to D-353; M-1 to E-352; M-1 to 1-35 1; M-1 to V-350; M-1 to S-349, M- I to C-348; M- I to S-347; M- I to K-346; M-1I to N-345; M- I to P- 344; M-1 to D-343; M-1 to C-342; M-1 to 1-341; M-1 to T-340; M-1 to G-339; M-1 to 1-338; M-1 to D-337; M-1 to A-336; M-1 to V-335; M-1 to G-334; M-1 to L-333; M-1 to T-332; M-1 to D-331; M-1 to C-330; M-1 to L-329; M-1 to G- 328; M-1 to E-327; M-1 to Q-326; M-1 to G-325; M-1 to C-324; M-1 to F-323; M-1 to N-322; M-1 to Q-321; M-1 to R-320; M-1 to T-3 19; M-1 to L-3 18; M-1 to L-317; M-1I to 1-316; M-1I to A-315; M- I to T-314; M-1I to D-313; M-1I to Y- 312; M-1I to H-31 1; M-1I to E-3 10; M-1I to P-309; M-1I to H-308; M-1I to R-3 07; M-1I to D-3 06; M-1I to S-3 05; M-1I to P-3 04; M- I to Q-3 03; M-1I to N-3 02; M-1I to F-301; M-1 to R-300; M-1 to R-299; M-1 to Q-298; M-1 to W-297; M-1 to N-296; M-1I to C-295; M-1I to F-294; M-1I to N-293; M-1I to R-292; M-1I to L- 29 1; M-1I to T-290; M-1I to L-289; M-1I to G-288; M-1I to G-287; M-1I to N-286; M-1I to D-285; M-1I to S-284; M-1I to V-283; M-1I to E-282; M-1I to P-28 1; M-1I to G-280; M-1I to W-279; M-1I to K-278; M-1I to E-277; M- I to D-276; M- I to E-275; M-1 to V-274; M-1 to 1-273; M-1 to L-272; M-1 to V-271; M-1 to K- 270; M-1 to V-269; M-1 to V-268; M-1 to M-267; M-1 to L-266; M-1 to N-265; M-1I to 1-264; M-1I to S-263; M-1I to N-262; M-1I to K-26 1; M-1I to 1-260; M- 1 to S-259; M-1 to P-258; M-1 to H-257; M-1 to K-256; M-1 to Y-255; M-1 to I- 254; M-1 to R-253; M-1 to A-252; M-1 to A-251; M-1 to V-250; M-1 to S-249; M-1 to M-248; M-1 to L-247; M-1 to T-246; M-1 to L-245; M-1 to 1-244; M-1 to H-243; M-1 to N-242; M-1 to Q-241; M-1 to L-240; M-1 to D-239; M-1 to A-238; M-1 to G-237; M-1 to Y-236; M-1 to F-235;, M-1 to A-234; M-1 to A- 233; M-1 to M-232; M-1 to S-231; M-1 to A-230; M-1 to D-229; M-1 to A-228; WO 99/37660 PCTIUS99/01313 M-1I to V-227; M-1I to L-226; M-1I to L-225; M-1I to T-224; M-1I to E-223; M-1I to V-222; M-1I to F-22 1; M-1I to R-220; M- I to A-219; M-1I to E-218;- M-1I to S- 217; M-1I to V-216; M-1I to F-215, M-1I to R-214; M-1I to K-213, M-1I to T-212, M-1I to R-21 M-1I to S-2 10; M- I to T-209; M-1I to A-208; M-1I to G-207; M- 1 to L-206; M-1I to P-205;- M-1I to P-204; M-1I to P-203; M-1I to P-202; M-1I to E- 1; M-1I to S-200; M-1I to A- 199; M-1I to G- 198; M- I to E- 197; M-1I to A- 196; M-1 to E-195; M-1 to E-194; M-1 to E-193; M-1 to Q-192; M-1 to S-191; M-1 to E-190; M-1 to E-189; M-1 to E-188; M-1 to S-187; M-1 to D-186; M-1 to E- 185; M-1 to Q-184; M-1 to H-183; M-1 to D-182; M-1 to G-181; M-1 to R-180; M-1 to E-179; M-1 to Q-178; M-1 to R-177; M-1 to Q-176; M-1 to G-175; M-1 to E-174; M-1 to G-173; M-1 to T-172; M-1 to E-171; M-1 to V-170; M-1 to E-* 169; M-1 to W-168; M-1 to E-167; M-1 to P-166; M-1 to G-165; M-1 to R-164; M-1 to P-163; M-1 to L-162; M-1 to P-161; M-1 to R-160; M-1 to A-159; M-1 to G-158; M-1 to A-157; M-1 to P-156; M-1 to G-155; M-1 to W-154; M-1 to R-153; M-1 to Q-152; M-1 to L-151; M-1 to R-150; M-1 to H-149; M-1 to P- 148; M-1I to Q- 147; M-1I to A- 146; M-1I to L- 145; M-1I to S- 144; M-1I to G- 143; M-A to G-142; M-1 to A-141; M-1 to G-140; M-1 to Q-139; M-1 to P-138; M-1 to Q-137; M-1 to 1-136; M-1 to T-135; M-1 to F-134; M-1 to E-133; M-1 to E- 132; M-1 to G-131; M-1 to D-130; M-1 to L-129; M-1 to L-128; M-1 to F-127;- M-1 to S-126; M-1 to G-125; M-1 to S-124; M-1 to L-123; M-1 to G-122; M-1 to R-121; M-1 to C-120; M-1 to L-1 19; M-1 to S-1 18; M-1 to V-1 17; M-1 to A- 116; M-1ltoA-115; M-1ltoL-114; M-1 toS-113; M-1 toE-1 12;M-1 toP-1 11; M-1 to E-1 10; M-1 to G-109; M-1 to N-108; M-1 to V-107; M-1 to T-106; M-1 to G- 105; M-1I to S- 104; M-1I to F- 103; M-1 to F-102; M-1 to C-101; M-1 to G- 100; M-1 to R-99; M-1 to L-98; M-1 to G-97; M-1 to R-96; M-1 to E-95; M-1 to G-94; M-1I to G-93; M-1I to T-92; M-1I to A- 9 1; M-1I to R-90; M-1I to G-89; M-1 to 8-88; M-1 to G-87; M-1 to G-86; M-1 to L-85; M-1 to R-84; M-1 to E- 83;- M-1I to 1-82; M-1I to K-81 M- I to F-80; M-1I to E-79; M-1I to P-78; M- I to A-77; M-1 to L-76; M-1 to F-75; M-1 to S-74; M-1 to D-73; M-1 to D-72; M-1 to P-7 1; M-1I to A-70; M-1I to L-69; M-1I to R-68; M- 1 to L-67; M-1I to V-66; M- WO 99/37660 PCT/US99/01313 -91- 1 to F-65; M-1 to G-64; M-1 to K-63; M-1 to G-62; M-1 to F-61; M-1 to M-1 to S-59; M-1 to L-58; M-l to H-57; M-l to L-56; M-1 to A-55; M-1 to L- 54; M-1 toE-53;M-1 toG-52;M-1 to A-51; M-1 to S-50; M-1 toG-49;M-1 to P-48; M-1 to L-47; M-l to R-46; M-1 to T-45; M-1 to P-44; M-1 to V-43; M-1 to V-42; M-1 to L-41; M-1 toE-40; M-1 to S-39; M-1 to A-38; M-1 to Q-37; M- 1 to G-36; M-1 to G-35; M-1 to A-34; M-1 to A-33; M-I to P-32; M-l to R-31; M-l to A-30; M-1 to P-29; M-1 to A-28; M-1 to G-27; M-1 to R-26; M-1 to A- M-1 to L-24; M-1 to P-23; M-1 to L-22; M-1 to L-21; M-l to L-20; M-1 to L-19; M-1 to L-18; M-l to L-17; M-1 to L-16; M-1 to L-15; M-1 to L-14; M-1 to F-13; M-1 to P-12; M-1 toL-ll; M-1 toW-10;M-1 toR-9; M-1 toP-8; M-1 to A-7; of SEQ ID NO:4. Preferably, any of the above listed N- or C-terminal deletions can be combined to produce a N- and C-terminal deleted METH2 polypeptide.
The invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini, which may be described generally as having residues m-n of SEQ ID NO:2 or SEQ ID NO:4, where n and m are integers as described above.
Also preferred are METHI or METH2 polypeptide and polynucleotide fragments characterized by structural or functional domains. Preferred embodiments of the invention include fragments that comprise alpha-helix and alpha-helix forming regions ("alpha-regions"), beta-sheet and beta-sheet-forming regions ("beta-regions"), turn and turn-forming regions ("turn-regions"), coil and coil-forming regions ("coil-regions"), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surfaceforming regions, substrate binding region, and high antigenic index regions. As set out in the Figures, such preferred regions include Garnier-Robson alpharegions, beta-regions, turn-regions, and coil-regions, Chou-Fasman alpha-regions, beta-regions, and turn-regions, Kyte-Doolittle hydrophilic regions and hydrophobic regions, Eisenberg alpha and beta amphipathic regions, Karplus- Schulz flexible regions, Emini surface-forming regions, and Jameson-Wolf high WO 99/37660 PCT/US99/01313 -92antigenic index regions. Polypeptide fragments of SEQ ID NO:2 falling within conserved domains are specifically contemplated by the present invention. (See Figures 10 11 and Tables 1& Moreover, polynucleotide fragments encoding these domains are also contemplated.
Other preferred fragments are biologically active METHI or METH2 fragments. Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the METH1 or METH2 polypeptide.
The biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity.
However, many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:1 or SEQ ID NO:3 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 936 of SEQ ID NO: b is an integer of 15 to 950, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:1, and where the b is greater than or equal to a 14. Moreover, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 876 of SEQ ID NO:3, b is an integer of 15 to 890, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:3, and where the b is greater than or equal to a 14.
Epitopes Antibodies In the present invention, "epitopes" refer to METHI or METH2 polypeptide fragments having antigenic or immunogenic activity in an animal, especially in a human. A preferred embodiment of the present invention relates WO 99/37660 PCTIUS99/01313 -93to a METH 1 or METH2 polypeptide fragment comprising an epitope, as well as the polynucleotide encoding this fragment. A region of a protein molecule to which an antibody can bind is defined as an "antigenic epitope." In contrast, an "immunogenic epitope" is defined as a part of a protein that elicits an antibody response. (See, for instance, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998- 4002 (1983).) Fragments which function as epitopes may be produced by any conventional means. (See, Houghten, R. Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985) further described in U.S. Patent No. 4,631,211.) In the present invention, antigenic epitopes preferably contain a sequence of at least seven, more preferably at least nine, and most preferably between about to about 30 amino acids. Antigenic epitopes are useful to raise antibodies, including monoclonal antibodies, that specifically bind the epitope. (See, for instance, Wilson et al., Cell 37:767-778 (1984); Sutcliffe, J. G. et al., Science 219:660-666 (1983).) Similarly, immunogenic epitopes can be used to induce antibodies according to methods well known in the art. (See, for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow, M. et al., Proc. Nail. Acad. Sci. USA 82:910- 914; and Bittle, F. J. et al., J. Gen. Virol. 66:2347-2354 (1985).) A preferred immunogenic epitope includes the secreted protein. The immunogenic epitopes may be presented together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse) or, if it is long enough (at least about 25 amino acids), without a carrier. However, immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide in Western blotting.) Using DNAstar analysis, SEQ ID NO:2 was found antigenic at amino acids: 2-14, 32-44, 47-60,66-78, 87-103, 109-118, 146-162, 168-180, 183-219, 223-243,275-284,296-306,314-334,341-354,357-376,392-399,401-410,418- 429,438-454,456-471,474-488, 510-522, 524-538, 550-561, 565-626,630-643, WO 99/37660 PCT/US99/01313 -94- 659-671,679-721,734-749,784-804, 813-820, 825-832, 845-854, 860-894, 899- 917, 919-924 and 928-939. Thus, these regions could be used as epitopes to produce antibodies against the protein encoded by METHI cDNA.
Using DNAstar analysis, SEQ ID NO:4 was found antigenic at amino acids: 26-38, 45-52, 69-76, 80-99, 105-113, 129-136, 138-217, 254-263, 273- 289, 294-313,321-331,339-356, 371-383, 417-427, 438-443,459-471,479-505, 507-526, 535-546, 550-607,615-640, 648-653, 660-667,669-681,683-704, 717- 732, 737-743, 775-787, 797-804, 811-825, 840-867 and 870-884. Thus, these regions could be used as epitopes to produce antibodies against the protein encoded by METH2 cDNA.
As used herein, the term "antibody" (Ab) or "monoclonal antibody" (Mab) is meant to include intact molecules as well as antibody fragments (such as, for example, Fab and F(ab')2 fragments) which are capable of specifically binding to protein. Fab and F(ab')2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody. (Wahl et al., J Nucl. Med. 24:316-325 (1983).) Thus, these fragments are preferred, as well as the products of a FAB or other immunoglobulin expression library. Moreover, antibodies of the present invention include chimeric, single chain, and humanized antibodies.
Fusion Proteins Any METHI or METH2 polypeptide can be used to generate fusion proteins. For example, the METHI or METH2 polypeptide, when fused to a second protein, can be used as an antigenic tag. Antibodies raised against the METHI or METH2 polypeptide can be used to indirectly detect the second protein by binding to the METH1 or METH2. Moreover, because secreted proteins target cellular locations based on trafficking signals, the METH1 or METH2 polypeptides can be used as a targeting molecule once fused to other proteins.
WO 99/37660 PCTIS99/01313 Examples of domains that can be fused to METHI or METH2 polypeptides include not only heterologous signal sequences, but also other heterologous functional regions. The fusion does not necessarily need to be direct, but may occur through linker sequences.
Moreover, fusion proteins may also be engineered to improve characteristics of the METHI or METH2 polypeptide. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the Nterminus of the METH1 or METH2 polypeptide to improve stability and persistence during purification from the host cell or subsequent handling and storage. Also, peptide moieties may be added to the METHI or METH2 polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the METH1 or METH2 polypeptide. The addition of peptide moieties to facilitate handling of polypeptides are familiar and routine techniques in the art.
Moreover, METH1 or METH2 polypeptides, including fragments, and specifically epitopes, can be combined with parts of the constant domain of immunoglobulins (IgG), resulting in chimeric polypeptides. These fusion proteins facilitate purification and show an increased half-life in vivo. One reported example describes chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. (EP A 394,827; Traunecker et al., Nature 331:84-86 (1988).) Fusion proteins having disulfide-linked dimeric structures (due to the IgG) can also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone.
(Fountoulakis et al., J. Biochem. 270:3958-3964 (1995).) Similarly, EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region ofimmunoglobulin molecules together with another human protein or part thereof. In many cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties. (EP-A 0232 262.) WO 99/37660 PCT[US99/01313 -96- Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired. For example, the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations.
In drug discovery, for example, human proteins, such as hIL-5, have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. (See, D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).) Moreover, the METH1 or METH2 polypeptides can be fused to marker sequences, such as a peptide which facilitates purification ofMETH1 or METH2.
In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (Q1AGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Nail. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Another peptide tag useful for purification, the "HA" tag, corresponds to an epitope derived from the influenza hemagglutinin protein.
(Wilson et al., Cell 37:767 (1984).) Thus, any of these above fusions can be engineered using the METH1 or METH2 polynucleotides or the polypeptides.
Biological Activities of METH1 or METH2 METH1 or METH2 polynucleotides and polypeptides can be used in assays to test for one or more biological activities. If METH1 or METH2 polynucleotides and polypeptides do exhibit activity in a particular assay, it is likely that METH1 or METH2 may be involved in the diseases associated with the biological activity. Therefore, METHI or METH2 could be used to treat the associated disease.
WO 99/37660 PCT/US99/01313 -97- Immune Activity METH1 or METH2 polypeptides or polynucleotides may be useful in treating deficiencies or disorders of the immune system, by activating or inhibiting the proliferation, differentiation, or mobilization (chemotaxis) of immune cells.
Immune cells develop through a process called hematopoiesis, producing myeloid (platelets, red blood cells, neutrophils, and macrophages) and lymphoid (B and T lymphocytes) cells from pluripotent stem cells. The etiology of these immune deficiencies or disorders may be genetic, somatic, such as cancer or some autoimmune disorders, acquired by chemotherapy or toxins), or infectious.
Moreover, METHI or METH2 polynucleotides or polypeptides can be used as a marker or detector of a particular immune system disease or disorder.
METHI or METH2 polynucleotides. or polypeptides may be useful in treating or detecting deficiencies or disorders ofhematopoietic cells. METHI or METH2 polypeptides or polynucleotides could be used to increase differentiation and proliferation ofhematopoietic cells, including the pluripotent stem cells, in an effort to treat those disorders associated with a decrease in certain (or many) types hematopoietic cells. Examples ofimmunologic deficiency syndromes include, but are not limited to: blood protein disorders agammaglobulinemia, dysgammaglobulinemia), ataxia telangiectasia, common variable immunodeficiency, Digeorge Syndrome, HIV infection, HTLV-BLV infection, leukocyte adhesion deficiency syndrome, lymphopenia, phagocyte bactericidal dysfunction, severe combined immunodeficiency (SCIDs), Wiskott-Aldrich Disorder, anemia, thrombocytopenia, or hemoglobinuria.
Moreover, METHI or METH2 polypeptides or polynucleotides can also be used to modulate hemostatic (the stopping ofbleeding) or thrombolytic activity (clot formation). For example, by increasing hemostatic or thrombolytic activity, METH1 or METH2 polynucleotides or polypeptides could be used to treat blood coagulation disorders afibrinogenemia, factor deficiencies), blood platelet disorders thrombocytopenia), or wounds resulting from trauma, surgery, or other causes. Alternatively, METH1 or METH2 polynucleotides or polypeptides WO 99/37660 PCTIUS99/01313 -98that can decrease hemostatic or thrombolytic activity could be used to inhibit or dissolve clotting, important in the treatment of heart attacks (infarction), strokes, or scarring.
METH or METH2 polynucleotides or polypeptides may also be useful in treating or detecting autoimmune disorders. Many autoimmune disorders result from inappropriate recognition of self as foreign material by immune cells. This inappropriate recognition results in an immune response leading to the destruction of the host tissue. Therefore, the administration of METH1 or METH2 polypeptides or polynucleotides that can inhibit an immune response, particularly the proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing autoimmune disorders.
Examples of autoimmune disorders that can be treated or detected by METH1 or METH2 include, but are not limited to: Addison's Disease, hemolytic anemia, antiphospholipid syndrome, rheumatoid arthritis, dermatitis, allergic encephalomyelitis, glomerulonephritis, Goodpasture's Syndrome, Graves' Disease, Multiple Sclerosis, Myasthenia Gravis, Neuritis, Ophthalmia, Bullous Pemphigoid, Pemphigus, Polyendocrinopathies, Purpura, Reiter's Disease, Stiff-Man Syndrome, Autoimmune Thyroiditis, Systemic Lupus Erythematosus, Autoimmune Pulmonary Inflammation, Guillain-Barre Syndrome, insulin dependent diabetes mellitis, and autoimmune inflammatory eye disease.
Similarly, allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems, may also be treated by METH 1 or METH2 polypeptides or polynucleotides. Moreover, METH 1 or METH2 can be used to treat anaphylaxis, hypersensitivity to an antigenic molecule, or blood group incompatibility.
METH1 or METH2 polynucleotides or polypeptides may also be used to treat and/or prevent organ rejection or graft-versus-host disease (GVHD). Organ rejection occurs by host immune cell destruction ofthe transplanted tissue through an immune response. Similarly, an immune response is also involved in GVHD, but, in this case, the foreign transplanted immune cells destroy the host tissues.
WO 99/37660 PCTIUS99/01313 -99- The administration of METHI or METH2 polypeptides or polynucleotides that inhibits an immune response, particularly the proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing organ rejection or GVHD.
Similarly, METH1 or METH2 polypeptides or polynucleotides may also be used to modulate inflammation. For example, METH1 or METH2 polypeptides or polynucleotides may inhibit the proliferation and differentiation of cells involved in an inflammatory response. These molecules can be used to treat inflammatory conditions, both chronic and acute conditions, including inflammation associated with infection septic shock, sepsis, or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine induced lung injury, inflammatory bowel disease, Crohn's disease, or resulting from over production of cytokines TNF or IL-I.) Hyperproliferative Disorders METH1 or METH2 polypeptides or polynucleotides can be used to treat or detect hyperproliferative disorders, including neoplasms. METH1 or METH2 polypeptides or polynucleotides may inhibit the proliferation of the disorder through direct or indirect interactions. Alternatively, METHI or METH2 polypeptides or polynucleotides may proliferate other cells which can inhibit the hyperproliferative disorder.
For example, by increasing an immune response, particularly increasing antigenic qualities of the hyperproliferative disorder or by proliferating, differentiating, or mobilizing T-cells, hyperproliferative disorders can be treated.
This immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response. Alternatively, decreasing an immune response may also be a method of treating hyperproliferative disorders, such as a chemotherapeutic agent.
WO 99/37660 PCT/US99/01313 -100- Examples of hyperproliferative disorders that can be treated or detected by METHI or METH2 polynucleotides or polypeptides include, but are not limited to neoplasms located in the: abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, and urogenital.
Similarly, other hyperproliferative disorders can also be treated or detected by METHI or METH2 polynucleotides or polypeptides. Examples of such hyperproliferative disorders include, but are not limited to: hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias, purpura, sarcoidosis, Sezary Syndrome, Waldenstron's Macroglobulinemia, Gaucher's Disease, histiocytosis, and any other hyperproliferative disease, besides neoplasia, located in an organ system listed above.
Infectious Disease METH1 or METH2 polypeptides or polynucleotides can be used to treat or detect infectious agents. For example, by increasing the immune response, particularly increasing the proliferation and differentiation of B and/or T cells, infectious diseases may be treated. The immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response. Alternatively, METH 1 or METH2 polypeptides or polynucleotides may also directly inhibit the infectious agent, without necessarily eliciting an immune response.
Viruses are one example of an infectious agent that can cause disease or symptoms that can be treated or detected by METH 1 or METH2 polynucleotides or polypeptides. Examples of viruses, include, but are not limited to the following DNA and RNA viral families: Arbovirus, Adenoviridae, Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae (such as, Cytomegalovirus, Herpes Simplex, Herpes Zoster), Mononegavirus WO 99/37660 PCTIUS99/01313 -101- Paramyxoviridae, Morbillivirus, Rhabdoviridae), Orthomyxoviridae Influenza), Papovaviridae, Parvoviridae, Picornaviridae, Poxviridae (such as Smallpox or Vaccinia), Reoviridae Rotavirus), Retroviridae (HTLV-I, HTLV-II, Lentivirus), and Togaviridae Rubivirus). Viruses falling within these families can cause a variety of diseases or symptoms, including, but not limited to: arthritis, bronchiollitis, encephalitis, eye infections conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis B, C, E, Chronic Active, Delta), meningitis, opportunistic infections AIDS), pneumonia, Burkitt's Lymphoma, chickenpox hemorrhagic fever, Measles, Mumps, Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella, sexually transmitted diseases, skin diseases Kaposi's, warts), and viremia. METH1 or METH2 polypeptides or polynucleotides can be used to treat or detect any of these symptoms or diseases.
Similarly, bacterial or fungal agents that can cause disease or symptoms and that can be treated or detected by METH1 or METH2 polynucleotides or polypeptides include, but not limited to, the following Gram-Negative and Grampositive bacterial families and fungi: Actinomycetales Corynebacterium, Mycobacterium, Norcardia), Aspergillosis, Bacillaceae Anthrax, Clostridium), Bacteroidaceae, Blastomycosis, Bordetella, Borrelia, Brucellosis, Candidiasis, Campylobacter, Coccidioidomycosis, Cryptococcosis, Dermatocycoses, Enterobacteriaceae (Klebsiella, Salmonella, Serratia, Yersinia), Erysipelothrix, Helicobacter, Legionellosis, Leptospirosis, Listeria, Mycoplasmatales, Neisseriaceae Acinetobacter, Gonorrhea, Menigococcal), Pasteurellacea Infections Actinobacillus, Heamophilus, Pasteurella), Pseudomonas, Rickettsiaceae, Chlamydiaceae, Syphilis, and Staphylococcal.
These bacterial or fungal families can cause the following diseases or symptoms, including, but not limited to: bacteremia, endocarditis, eye infections (conjunctivitis, tuberculosis, uveitis), gingivitis, opportunistic infections AIDS related infections), paronychia, prosthesis-related infections, Reiter's Disease, respiratory tract infections, such as Whooping Cough or Empyema, WO 99/37660 PCT/US99/01313 -102sepsis, Lyme Disease, Cat-Scratch Disease, Dysentery, Paratyphoid Fever, food poisoning, Typhoid, pneumonia, Gonorrhea, meningitis, Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis, Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo, Rheumatic Fever, Scarlet Fever, sexually transmitted diseases, skin diseases cellulitis, dermatocycoses), toxemia, urinary tract infections, wound infections. METH1 or METH2 polypeptides or polynucleotides can be used to treat or detect any of these symptoms or diseases.
Moreover, parasitic agents causing disease or symptoms that can be treated or detected by METHI or METH2 polynucleotides or polypeptides include, but not limited to, the following families: Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic, Giardiasis, Helminthiasis, Leishmaniasis, Theileriasis, Toxoplasmosis, Trypanosomiasis, and Trichomonas. These parasites can cause a variety of diseases or symptoms, including, but not limited to: Scabies, Trombiculiasis, eye infections, intestinal disease dysentery, giardiasis), liver disease, lung disease, opportunistic infections AIDS related), Malaria, pregnancy complications, and toxoplasmosis. METHI or METH2 polypeptides or polynucleotides can be used to treat or detect any of these symptoms or diseases.
Preferably, treatment using METH1 or METH2 polypeptides or polynucleotides could either be by administering an effective amount of METH1 or METH2 polypeptide to the patient, or by removing cells from the patient, supplying the cells with METHI or METH2 polynucleotide, and returning the engineered cells to the patient (ex vivo therapy). Moreover, the METHI or METH2 polypeptide or polynucleotide can be used as an antigen in a vaccine to raise an immune response against infectious disease.
Regeneration METH1 or METH2 polynucleotides or polypeptides can be used to differentiate, proliferate, and attract cells, leading to the regeneration of tissues.
(See, Science 276:59-87 (1997).) The regeneration of tissues could be used to WO 99/37660 PCT/US99/01313 -103repair, replace, or protect tissue damaged by congenital defects, trauma (wounds, burns, incisions, or ulcers), age, disease osteoporosis, osteocarthritis, periodontal disease, liver failure), surgery, including cosmetic plastic surgery, fibrosis, reperfusion injury, or systemic cytokine damage.
Tissues that could be regenerated using the present invention include organs pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac), vascular (including vascular endothelium), nervous, hematopoietic, and skeletal (bone, cartilage, tendon, and ligament) tissue.
Preferably, regeneration occurs without or decreased scarring. Regeneration also may include angiogenesis.
Moreover, METH1 or METH2 polynucleotides or polypeptides may increase regeneration of tissues difficult to heal. For example, increased tendon/ligament regeneration would quicken recovery time after damage. METH 1 or METH2 polynucleotides or polypeptides of the present invention could also be used prophylactically in an effort to avoid damage. Specific diseases that could be treated include of tendinitis, carpal tunnel syndrome, and other tendon or ligament defects. A further example of tissue regeneration of non-healing wounds includes pressure ulcers, ulcers associated with vascular insufficiency, surgical, and traumatic wounds.
Similarly, nerve and brain tissue could also be regenerated by using METH1 or METH2 polynucleotides or polypeptides to proliferate and differentiate nerve cells. Diseases that could be treated using this method include central and peripheral nervous system diseases, neuropathies, or mechanical and traumatic disorders spinal cord disorders, head trauma, cerebrovascular disease, and stoke). Specifically, diseases associated with peripheral nerve injuries, peripheral neuropathy resulting from chemotherapy or other medical therapies), localized neuropathies, and central nervous system diseases Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome), could all be treated using the METH1 or METH2 polynucleotides or polypeptides.
WO 99/37660 PCTIUS99/01313 -104- Chemotaxis METH 1 or METH2 polynucleotides or polypeptides may have chemotaxis activity. A chemotaxic molecule attracts or mobilizes cells monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells) to a particular site in the body, such as inflammation, infection, or site ofhyperproliferation. The mobilized cells can then fight off and/or heal the particular trauma or abnormality.
METH1 or METH2 polynucleotides or polypeptides may increase chemotaxic activity of particular cells. These chemotactic molecules can then be used to treat inflammation, infection, hyperproliferative disorders, or any immune system disorder by increasing the number of cells targeted to a particular location in the body. For example, chemotaxic molecules can be used to treat wounds and other trauma to tissues by attracting immune cells to the injured location. As a chemotactic molecule, METH1 or METH2 could also attract fibroblasts, which can be used to treat wounds.
It is also contemplated that METHI or METH2 polynucleotides or polypeptides may inhibit chemotactic activity. These molecules could also be used to treat disorders. Thus, METH1 or METH2 polynucleotides or polypeptides could be used as an inhibitor of chemotaxis.
Binding Activity METH 1 or METH2 polypeptides may be used to screen for molecules that bind to METH1 or METH2 or for molecules to which METH1 or METH2 binds.
The binding of METH1 or METH2 and the molecule may activate (agonist), increase, inhibit (antagonist), or decrease activity of the METHI or METH2 or the molecule bound. Examples of such molecules include antibodies, oligonucleotides, proteins receptors),or small molecules.
Preferably, the molecule is closely related to the natural ligand of METH 1 or METH2, a fragment of the ligand, or a natural substrate, a ligand, a structural or functional mimetic. (See, Coligan et al., Current Protocols in WO 99/37660 PCTIUS99/01313 -105- Immunology 1(2):Chapter 5 (1991).) Similarly, the molecule can be closely related to the natural receptor to which METH1 or METH2 binds, or at least, a fragment of the receptor capable of being bound by METHI or METH2 active site). In either case, the molecule can be rationally designed using known techniques.
Preferably, the screening for these molecules involves producing appropriate cells which express METH I or METH2, either as a secreted protein or on the cell membrane. Preferred cells include cells from mammals, yeast, Drosophila, or E. coli. Cells expressing METHI or METH2(or cell membrane containing the expressed polypeptide) are then preferably contacted with a test compound potentially containing the molecule to observe binding, stimulation, or inhibition of activity of either METH1 or METH2 or the molecule.
The assay may simply test binding of a candidate compound toMETH or METH2, wherein binding is detected by a label, or in an assay involving competition with a labeled competitor. Further, the assay may test whether the candidate compound results in a signal generated by binding to METHI or METH2.
Alternatively, the assay can be carried out using cell-free preparations, polypeptide/molecule affixed to a solid support, chemical libraries, or natural product mixtures. The assay may also simply comprise the steps of mixing a candidate compound with a solution containing METHI or METH2, measuring METH1 or METH2/molecule activity or binding, and comparing the METHI or METH2/molecule activity or binding to a standard.
Preferably, an ELISA assay can measure METHI or METH2 level or activity in a sample biological sample) using a monoclonal or polyclonal antibody. The antibody can measure METH1 or METH2 level or activity by either binding, directly or indirectly, to METH1 or METH2 or by competing with METH1 or METH2 for a substrate.
All of these above assays can be used as diagnostic or prognostic markers.
The molecules discovered using these assays can be used to treat disease or to WO 99/37660 PCT/US99/01313 -106bring about a particular result in a patient blood vessel growth) by activating or inhibiting the METH1 or METH2 molecule. Moreover, the assays can discover agents which may inhibit or enhance the production of METH1 or METH2 from suitably manipulated cells or tissues.
Therefore, the invention includes a method of identifying compounds which bind to METHI or METH2 comprising the steps of: incubating a candidate binding compound with METHI or METH2; and determining if binding has occurred. Moreover, the invention includes a method of identifying agonists/antagonists comprising the steps of: incubating a candidate compound with METH1 or METH2, assaying a biological activity, and (b) determining if a biological activity of METH or METH2 has been altered.
Other Activities METHI or METH2 polypeptides or polynucleotides may also increase or decrease the differentiation or proliferation of embryonic stem cells, besides, as discussed above, hematopoietic lineage.
METH 1 or METH2 polypeptides or polynucleotides may also be used to modulate mammalian characteristics, such as body height, weight, hair color, eye color, skin, percentage of adipose tissue, pigmentation, size, and shape cosmetic surgery). Similarly, METH or METH2 polypeptides or polynucleotides may be used to modulate mammalian metabolism affecting catabolism, anabolism, processing, utilization, and storage of energy.
METHI or METH2 polypeptides or polynucleotides may be used to change a mammal's mental state or physical state by influencing biorhythms, circadian rhythms, depression (including depressive disorders), tendency for violence, tolerance for pain, reproductive capabilities (preferably by Activin or Inhibin-like activity), hormonal or endocrine levels, appetite, libido, memory, stress, or other cognitive qualities.
METH1 or METH2 polypeptides or polynucleotides may also be used as a food additive or preservative, such as to increase or decrease storage WO 99/37660 PCT/US99/01313 -107capabilities, fat content, lipid, protein, carbohydrate, vitamins, minerals, cofactors or other nutritional components.
Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended as limiting.
Cancer Diagnosis and Prognosis It is believed that certain tissues in mammals with cancer express significantly diminished levels of the METHI or METH2 protein and mRNA encoding the METHI or METH2 protein when compared to a corresponding "standard" mammal, a mammal of the same species not having the cancer.
Further, it is believed that diminished levels of the METH 1 or METH2 protein can be detected in certain body fluids sera, plasma, urine, and spinal fluid) from mammals with cancer when compared to sera from mammals of the same species not having the cancer. Thus, the invention provides a diagnostic method useful during tumor diagnosis, which involves assaying the expression level of the gene encoding the METH I protein in mammalian cells or body fluid and comparing the gene expression level with a standard METH I gene expression level, whereby a decrease in the gene expression level under the standard is indicative of certain tumors. The invention also provides a diagnostic method useful during tumor diagnosis, which involves assaying the expression level of the gene encoding the METH2 protein in mammalian cells or body fluid and comparing the gene expression level with a standard METH2 gene expression level, whereby a decrease in the gene expression level under the standard is indicative of certain tumors.
Where a tumor diagnosis has already been made according to conventional methods, the present invention is useful as a prognostic indicator, whereby patients exhibiting diminished METH 1 or METH2 gene expression will experience a worse clinical outcome relative to patients expressing the gene at a lower level.
WO 99/37660 PCT/US99/01313 -108- By "assaying the expression level of the gene encoding the METH1 or METH2 protein" is intended qualitatively or quantitatively measuring or estimating the level of the METH1 or METH2 protein or the level of the mRNA encoding the METH 1 or METH2 protein in a first biological sample either directly by determining or estimating absolute protein level or mRNA level) or relatively by comparing to the METHI or METH2 protein level or mRNA level in a second biological sample).
Preferably, the METH1 or METH2 protein level or mRNA level in the first biological sample is measured or estimated and compared to a standard METH1 or METH2 protein level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the cancer. As will be appreciated in the art, once a standard METH1 or METH2 protein level or mRNA level is known, it can be used repeatedly as a standard for comparison.
By "biological sample" is intended any biological sample obtained from an individual, cell line, tissue culture, or other source which contains METHI or METH2 protein or mRNA. Biological samples include mammalian body fluids (such as sera, plasma, urine, synovial fluid and spinal fluid) which contain secreted mature METHI or METH2 protein, and adrenal, thyroid, stomach, brain, heart, placenta, lung, liver, muscle, kidney, pancreas, testis and ovarian tissue (for METH1); and prostate, small intestine, colon, brain and lung tissue (for METH2).
The present invention is useful for detecting cancer in mammals. In particular the invention is useful during diagnosis of the of following types of cancers in mammals: breast, ovarian, prostate, liver, lung, pancreatic, colon, and testicular. Preferred mammals include monkeys, apes, cats, dogs, cows, pigs, horses, rabbits and humans. Particularly preferred are humans.
Total cellular RNA can be isolated from a biological sample using the single-step guanidinium-thiocyanate-phenol-chloroform method described in Chomczynski and Sacchi, Anal. Biochem. 162:156-159 (1987). Levels of mRNA encoding the METHI or METH2 protein are then assayed using any appropriate method. These include Northern blot analysis (Harada et al., Cell 63:303-312 WO 99/37660 PCT/US99/03131 -109- (1990)), Sl nuclease mapping (Fujita el al., Cell 49:357- 367 (1987)), the polymerase chain reaction (PCR), reverse transcription in combination with the polymerase chain reaction (RT-PCR) (Makino et al., Technique 2:295-301 (1990)), and reverse transcription in combination with the ligase chain reaction
(RT-LCR).
Assaying METHI or METH2 protein levels in a biological sample can occur using antibody-based techniques. For example, METH 1 or METH2 protein expression in tissues can be studied with classical immunohistological methods (Jalkanen, el al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, et al., J.
Cell. Biol. 105:3087-3096 (1987)).
Other antibody-based methods useful for detecting METHI or METH2 protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
Suitable labels are known in the art and include enzyme labels, such as, glucose oxidase, and radioisotopes, such as iodine 121), carbon 4 sulfur tritium 3 indium (I 2 1n), and technetium 9 and fluorescent labels, such as fluorescein and rhodamine, and biotin.
Modes of administration It is recognized than an increase in the vascular supply plays a central role in tumor progression and metastasis; therefore, inhibitors of angiogenesis can prove effective as adjuvant therapy for cancer patients. Some of the currently recognized angiogenic suppressors are poor candidates for systemic treatment due to severe collateral effect. The present inventors have found that METH1 and METH2 are potent inhibitors of angiogenesis both in vitro and in vivo. The advantage of METH 1 and METH 1 is that these inhibitors are normally associated with suppression of physiological angiogenesis; therefore, they offer lack of toxicity and endothelial specificity over other angiogenic inhibitors. Furthermore, WO 99/37660 PCT/US99/01313 -110- METHI and METH2 present a restricted pattern of expression providing a possible advantage on organ specificity.
Accordingly, the polypeptides of the present invention may be employed to treat cancer. The METH and METH2 polypeptides of the present invention can also be used to treat individuals with other disorders that are related to angiogenesis, including abnormal wound healing, inflammation, rheumatoid arthritis, psoriasis, endometrial bleeding disorders, diabetic retinopathy, some forms of macula degeneration, hemangiomas, and arterial-venous malformations.
Thus, the invention provides a method of inhibiting angiogenesis in an individual comprising administering to such an individual a pharmaceutical composition comprising an effective amount of an isolated METH1 polypeptide of the invention, effective to increase the METHI activity level in such an individual. The invention also provides a method of inhibiting angiogenesis in an individual comprising administering to such an individual a pharmaceutical composition comprising an effective amount of an isolated METH2 polypeptide of the invention, effective to increase the METH2 activity level in such an individual.
METH1 polypeptides which may be used to inhibit angiogenesis in this manner include: METHI polypeptide encoded by the deposited cDNA including the leader; the mature METHI polypeptide encoded by the deposited the cDNA minus the leader the mature protein); a polypeptide comprising amino acids about 1 to about 950 in SEQ ID NO:2; a polypeptide comprising amino acids about 2 to about 950 in SEQ ID NO:2; a polypeptide comprising amino acids about 29 to about 950 in SEQ ID NO:2; a polypeptide comprising amino acids about 30 to about 950 in SEQ ID NO:2; a polypeptide comprising the metalloprotease domain of METH1, amino acids 235 to 459 in SEQ ID NO:2; a polypeptide comprising the disintegrin domain ofMETH 1, amino acids 460 to 544 in SEQ ID NO:2; a polypeptide comprising the first TSP-like domain of METH1, amino acids 545 to 598 in SEQ ID NO:2; a polypeptide comprising the second TSP-like domain of METH1, amino acids 841 to 894 in SEQ ID NO:2; a WO 99/37660 PCT/US99/01313 -111polypeptide comprising the third TSP-like domain of METH1, amino acids 895 to 934 in SEQ ID NO:2; a polypeptide comprising amino acids 536 to 613 in SEQ ID NO:2; and a polypeptide comprising amino acids 549 to 563 in SEQ ID NO:2.
METH2 polypeptides which may be used to inhibit angiogenesis in this manner include: the METH2 polypeptide encoded by the deposited cDNA including the leader; the mature METH2 polypeptide encoded by the deposited the cDNA minus the leader the mature protein); a polypeptide comprising amino acids about 1 to about 890 in SEQ ID NO:4; a polypeptide comprising amino acids about 2 to about 890 in SEQ ID NO:4; a polypeptide comprising amino acids about 24 to about 890 in SEQ ID NO:4; a polypeptide comprising amino acids about 112 to about 890 in SEQ ID NO:4; a polypeptide comprising themetalloprotease domain of METH2, amino acids 214 to 439 in SEQ ID NO:4; a polypeptide comprising the disintegrin domain ofMETH2, amino acids 440 to 529 in SEQ ID NO:4; a polypeptide comprising the first TSP-like domain of METH2, amino acids 530 to 583 in SEQ ID NO:4; a polypeptide comprising the second TSP-like domain of METH2, amino acids 837 to 890 in SEQ ID NO:4; a polypeptide comprising amino acids 280 to 606 in SEQ ID NO:4; and a polypeptide comprising amino acids 529 to 548 in SEQ ID NO:4.
As a general proposition, the total pharmaceutically effective amount of METHI or METH2 polypeptide administered parenterally per dose will be in the range of about 1 gg/kg/day to 10 mg/kg/day of patient body weight, although, as noted above, this will be subject to therapeutic discretion. More preferably, this dose is at least 0.01 mg/kg/day, and most preferably for humans between about 0.01 and 1 mg/kg/day for the polypeptide. If given continuously, the METH1 or METH2 polypeptide is typically administered at a dose rate of about 1 pg/kg/hour to about 50 pg/kg/hour, either by 1-4 injections per day or by continuous subcutaneous infusions, for example, using a mini-pump. An intravenous bag solution may also be employed.
Pharmaceutical compositions containing the METH1 or METH2 of the invention may be administered orally, rectally, parenterally, intracistemally, WO 99/37660 PCTIUS99/01313 -112intravaginally, intraperitoneally, topically (as by powders, ointments, drops or transdermal patch), bucally, or as an oral or nasal spray. By "pharmaceutically acceptable carrier" is meant a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The term "parenteral" as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
Chromosome Assays The nucleic acid molecules of the present invention are also valuable for chromosome identification. The sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome. The mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease.
In certain preferred embodiments in this regard, the cDNAherein disclosed is used to clone genomic DNA of a METHI or METH2 protein gene. This can be accomplished using a variety of well known techniques and libraries, which generally are available commercially. The genomic DNA then is used for in situ chromosome mapping using well known techniques for this purpose.
In addition, in some cases, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis of the 3' untranslated region of the gene is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes.
Fluorescence in situ hybridization ("FISH") of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step. This technique can be used with probes from the cDNA as WO 99/37660 PCT/US99/01313 -113short as 50 or 60 bp. For a review of this technique, see Verma el al., Human Chromosomes: A Manual Of Basic Techniques, Pergamon Press, New York (1988).
Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, for example, in V. McKusick, Mendelian Inheritance In Man, available on-line through Johns Hopkins University, Welch Medical Library. The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes).
Next, it is necessary to determine the differences in the cDNA or genomic sequence between affected and unaffected individuals. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease.
Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended as limiting.
Examples Example 1: Identification and cloning ofMETHi and METH2 To search for novel genes with TSP-like domains, a large human cDNA database consisting of approximately 900,00 expressed sequence tags (ESTs) was screened for sequences homologous to the second type I repeat of TSP 1. Several ESTs were predicted to encode proteins with TSP-like domains. Two cDNA clones originated from human heart and lung libraries were further sequenced and chosen for functional analysis.
WO 99/37660 PCT/US99/01313 -114- The amino-terminal end of METH1 was obtained using 5' rapid amplification of cDNA ends (RACE) PCR technique (Marathon cDNA amplification kit, Clontech) according to manufacturer instructions. The aminoterminal end of METH2 was obtained partially through 5'RACE PCR and later confirmed and completed by genomic screening. For the genomic screen, BAC clones (Genome Systems) were initially identified by PCR. Positive BAC clones containing 150-200bp of sequence were subsequently subcloned into pGEM vector as small fragments and sequenced.
Analysis and comparison of the deduced amino acid sequence with the GenBank, EMBL and SwissProt databases suggested that these genes belong to a new family of metalloproteases with homology to the reprolysin family in their NH2-terminal end and with several TSP-like motifs in the COOH-terminal end.
These cDNAs were named METH1 and METH2; ME, for metalloprotease and TH, for thrombospondin. The mouse homologue of METHI was identified and named ADAMTS1 (Kuno, etal., J. Biol. Chem. 272:556-562 (1997)). Direct comparison of the human and mouse sequences revealed a high level of conservation (83.4% amino acid identity). Thus far no homologues for METH2 have been identified.
Interestingly, a recently identified protein named pNPI (procollagen I Nproteinase; (Colidge, etal., Proc. Natl. Acad. Sci. USA 94:2374-2379 (1997)) showed a striking sequence and structural similarity to METHI and METH2 (Figure As the novel proteins described here, pNPI also contains metalloproteinase (reprolysin subfamily) and TSP domains at the carboxy-terminal end. Although the sequence for pNPI is of bovine origin, sequence alignment revealed identical structural features. The amino acid similarity between METH1 and METH2 is 51.7%, and between METHI or METH2 and pNPI the homology is lesser 33.9% and 36.3%, respectively.
Sequence analysis showed that the ORF of METHI and METH2 coded for proteins of 950 and 890 amino acids, respectively. In all three proteins, the
NH
2 terminal end contains a putative signal peptide followed by another putative WO 99/37660 PCT/US99/01313 -115transmembrane domain around amino acid 300, deduced from the hydrophilicity plots. It is not clear whether these proteins are bound to the membrane.
However, given preliminary data, it is more likely that this second transmembrane domain will consist of a hydrophobic pocket and that METH1, METH2 and pNPI are in fact secreted proteins. The NH,-terminal end past the signal peptide has homology to the superfamily of zinc metalloproteases and can be subdivided in a prodomain, a metalloprotease domain, and a cysteine-rich region.
The double underlined sequence in METHI and METH2 in Figure 3 localized at the boundary between the prodomain and the metalloprotease domain, are potential cleavage sites for mammalian subtilisins, such as furins (Barr, 1991).
Proteolytical processing occurs in SVMPs to yield soluble metalloproteases and disintegrins (Bjarnason, J.B. Fox, Methods Enzymol. 248:345-368 (1995)) and has also been detected in some ADAMs (reviewed by Wolsberg, T.G.
White, Developmental Biology 180:389-401 (1996)). At this point, preliminary experiments suggest that proteolytical processing occurs, at least in METHI. Additionally, both METH1 and METH2 present a Zn 2 -binding site (dotted line in Figure 3) that is presumed to be catalytically active due to the conservation of certain functionally important amino acids (Rawlings, N.D. Barrett, Methods Enzymol. 248:183-228 (1995)) suggesting that these proteins may be active proteases. Following the metalloprotease domain, there is a cysteine-rich region which contains two putative disintegrin loops (Wolsberg, T.G. White, Developmental Biology 180:389-401 (1996)) (marked by arrows in Figure Disintegrin domains are found within the superfamily of metalloproteases in snake venom metalloproteases (SVMPs) and ADAMs (mammalian proteins containing a disintegrin and a metalloprotease domain) and have a possible function inhibiting binding ofintegrins to their ligands in SVMPs.
Conversely, the ADAM-disintegrin-like domain, as part of membrane anchored proteins, may promote rather than disrupt, cell-cell interactions (Wolsberg, T.G.
White, Developmental Biology 180:389-401 (1996)). The TSP-like domains are located in the COOH-halfofMETH1 and METH2 proteins. METH1 WO 99/37660 PCT/US99/01313 -116contains two conserved TSP domains separated by a spacer region with unknown function, and a subdomain with less homology, and only 5 cysteines, following the second anti-angiogenic region. METH2 contains two TSP domains separated by the spacer region. The alignment of the TSP-like domains of METHI and METH2 with those of TSP and TSP2 are shown in Figure 5. The homology varies between 19.2% to 52% amino acid similarity among all the TSP repeats.
The cysteines, numbered 1 to 6, and the tryptophans, labeled by asterisks, are highly conserved.
Southern blot of human genomic DNA revealed the presence ofMETH and METH2 in the genome. METH1 and METH2 probes revealed bands of different size suggesting that they are transcribed from different genes.
The consensus sequence for the type I repeats includes 16 residues with 6 perfectly conserved cysteines. Typically it begins with the sequence motif WSXWS (SEQ ID NO:82) that has also been shown to bind to heparin (Guo, N., et al., J. Biol. Chem. 267:19349-19355 (1992)). The affinity of this region to heparin has been proposed to the part of the anti-angiogenic activity of TSP-1 (Guo, et al., J. Peptide Res. 49 (1997)). Among the five members of the TSP family of proteins, only TSP-1 and TSP-2 inhibit angiogenesis and contain the type I repeats (Tolsma, et al., J. Cell. Biol. 122:497-511 (1993); Kyriakides, et al., J. Cell Biol. 140:419-430 (1998)). The type I or properdin repeats were probably added to the precursor of TSP 1 and 2 by exon shuffling between 500 and 900 years ago (Adams, et al., The Thrombospondin Gene Family, 1 Ed. Molecular Biology Intelligence Unit (Springer, R.G. Landes Company, Germany (1995)). It is likely that the acquisition of this domain provided the precursor of TSP1 and TSP2 with functions, such as regulation of new vessel formation. More recently, BAI-I (brain angiogenic inhibitor- a protein isolated from a brain library for its ability to be regulated by p53, has also been shown to contain the type I repeat of TSP-1 and to provide anti-angiogenic potential to this molecule (Nishimori, et al., Oncogene 15:2145-2150 (1997)). Nevertheless, it appears that additional sequences or context are also important, since other WO 99/37660 PCT/US99/01313 -117proteins containing the type I repeats appear not to have clear or more established anti-angiogenic properties such as: properdin, F-spondin, and other members of the complement family.
Because of the presence of TSP-repeats in METH1 and METH2, along with their anti-angiogenic properties, these proteins were originally considered members of the TSP superfamily. Nevertheless, they have no additional homology to other TSPs, and in fact, the similarity to TSP1 and TSP2 is restricted to the type I repeats. Furthermore, the proteins also have strong sequence and structural homology to members of the ADAM family. These features led Kuno and colleagues to name ADAMTS to the mouse homolog of METHI (Kuno, et al., J. Biol. Chem. 272:556-562 (1997)). The recent identification of pNPI and its striking sequence homology to the proteins here described, prompt all these three proteins to be grouped in a subfamily named metallospondins. At this point, it is not clear whether pNIP has anti-angiogenic properties or whether METH1 and/or METH2 participate in the cleavage of the amino terminal pro-peptide of a 1(I) procollagen.
Example 2: Northern and Southern blot analysis Total RNA was purified from cells by guanidinium-isothiocyanate extraction, as previously described (Chomczynski, P. Sacchi, Anal.
Biochem. 162:156-159 (1987)) Poly(A)+RNA was extracted using a Boehringer Mannheim (BMB, Indianapolis, IN) kit according to the manufacturer conditions.
Other poly(A)+RNA blots were purchased from Clontech (Palo Alto, CA). Prehybridization was performed in a solution containing: 50% formamide, 6X SSPE, IX Denhardt's solution, 0.1% SDS and 100 g/ml of heat denatured salmon sperm DNA for 12-18h at 42°C. Hybridization with labeled cDNA probes proceeded in the same solution at 42 0 C for 12-18h. TSP1 and METHI probes corresponded to the entire human cDNAs. METH2 probe corresponded to a Kpnl-EcoRI fragment from the human cDNA. A 1.3Kb PstI fragment of the glyceraldehyde-3- WO 99/37660 PCT/US99/01313 -118phosphate-dehydrogenase (GPDH) was used to normalize for loading and transfer efficiency. Membranes were exposed to Kodak Biomax MS film (Kodak, New Haven, CT).
For Southern blots, human genomic DNA, purchased from Promega (Madison, WI), was heated at 65 °C for 10 min and digested with EcoRI and Psti overnight at 37°C. 5tg of digested DNA was separated in a 1% agarose gel, transferred to a nytran membrane and cross-linked by ultraviolet light. cDNA probes, as well as, prehybridization and hybridization conditions were identical to those described for Northern blots. Blots were washed with high stringency (0.2X SSC, 0.2% SDS at 50 0
C).
The expression pattern of METHI and METH2 was examined in both adult and embryonic tissues. Northern blot analysis was performed under highstringency conditions with blots that included poly(A)+RNA from human tissues.
METH1 and METH2 transcripts revealed a single band of 4.6 and 3.7Kb, respectively. Abundant METHI mRNA expression was observed in adrenal, heart, placenta, followed by skeletal muscle, thyroid and stomach. From the embryonic tissues analyzed, kidney showed the highest expression of METHI mRNA. Nevertheless, weaker expression of METHI mRNA was seen in all tissues analyzed. Distribution ofMETH2 mRNA was more restricted and weaker than that of METHI. The highest expression was seen in lung, both embryonic and adult. Interestingly, METH1 and METH2 expression do not appear to overlap. In combination, the structural similarities and their pattern of expression suggest functional redundancy yet different transcriptional regulation. The expression levels of TSP transcripts in the same blots were also analyzed, for purpose of comparison. TSPI mRNA highest expression was seen in the adult placenta and in all embryonic tissues analyzed. In contrast to METH1 and METH2 we observed constant levels of TSP1 transcript in all the other tissues examined.
The cell type distribution was also studied by Northern blot analysis of poly(A)+RNA. METH1 mRNA was detectable, at low levels, in dermal WO 99/37660 PCT/US99/01313 -119fibroblasts, vascular smooth muscle, endometrial stromal cells, and in two cancer cell lines, HeLa and G631, an adenocarcinoma and a melanoma, respectively.
METH2 mRNA was detected only on SW480, a colon carcinoma cell line, but no expression was seen in any other of the cell lines or primary strains analyzed.
The possibility that groups of angiogenic and anti-angiogenic factors regulate vascular network formation in specific organs has been a frequently discussed hypothesis likely to be true, yet unproven. The expression patterns of METH 1 and METH2, which are clearly distinct and almost non-overlapping, were puzzling, at least with concern to overall levels. TSPI and TSP2 also share identical structure, high level of amino acid similarity, yet their pattern of expression differs significantly (Iruela-Arispe, M.L.,Dev. Dyn. 197:40-56 (1993)).
The differences are likely based on dissimilar cis-acting elements in their promoters and different regulatory mechanisms, as previously suggested.
Although the promoters for METH1 and 2 have not been characterized, it is likely that they provide unique features for the regulation of each gene. Nevertheless, the possibility that one motif, the anti-angiogenic type I repeat, with demonstrated anti-angiogenic properties is present in several proteins with different tissue specificities is appealing. Alternatively, the small differences in sequence between closely related members of the same family could possess significance that goes beyond functional redundancy. In the case of TSP1 and TSP2, aside from the striking structural similarities and perhaps having functionally common anti-angiogenic properties, TSP1 and TSP2 also appear to display functions of their own and not likely shared by their similar relative. This became evident with the outcome of the two knock-outs for these genes. TSPI null animals exhibited primarily lung disorders (Lawler, et al., J Clin. Invest.
101:982-992 (1998)) and secondarily vascular abnormalities, but only under specific pathological settings or on a restricted set of organs. In contrast TSP2 knock-out mice exhibited unpredicted collagen assembly anomalies, with carry-on consequences to the skin, tendons, and bone (Kyriakides, et al., J. CellBiol.
140:419-430 (1998)). In addition, these animals also appear to have overall WO 99/37660 PCT/US99/01 3 13 -120increase in capillary density in the dermis. It is not understood how the resemblance between the newly described members of the metallospondin family translate functionally. Clearly, pNIP has been shown to display active proteolytic activity by cleaving the N-terminus of type 1 procollagen (Colidge, et al., Proc.
Natl. Acad. Sci. USA 94:2374-2379 (1997)).
A second region of functional interest corresponds to the disintegrin domain. This domain has been more fully characterized in related members of the snake venom metalloproteases that have been shown to bind to allbp3 and inhibit platelet interaction blocking coagulation (Pfaff, et al., Cell Adhes Commun.
2:491-501 (1994); Usami, etal.. Biochem. Biophys. Res. Commun. 201:331- 339 (1994)). The disintegrin motif consists of a thirteen to fifteen domain which frequently contain an RGD or a negatively charged residue at the position of the aspartic acid. The RGD, or equivalent, binds to integrins and serve as antagonist or signaling ligands (Wolsberg, T.G. White, Developmental Biology 180:389-401 (1996)). METH2, but not METHI, has an RGD sequence located amino-terminal to the disintegrin domain. In addition, both molecules present relatively high, but not perfect, degree of conservation of cysteines within the disintegrin motif. This appears to display an important role in the tertiary structure of this region and its ability to interact with integrins. In addition, some of these domains have been shown to act as functional adhesion molecules, particularly those with transmembrane regions (Wolsberg, T.G. White, J.M., Developmental Biology 180:389-401 (1996)). It is unlikely that this will be the case for METH1 and METH2, since both these proteins appear to be secreted.
Example 3: Expression and purification of recombinant proteins Recombinant constructs for expression of truncated fusion proteins were as follows: pRSET-METHI-Type I: METH1 nt 1605-1839 (from the start codon) was amplified by polymerase chain reaction using the following primers: TTT TGG ATC CGC CTT TTC ATG-3' (SEQ ID NO:78) and WO 99/37660 PCT/US99/01313 -121- GTG TGC TGC AGA TTG TTC C-3' (SEQ ID NO:79). The amplified fragment was then subcloned into the BamHI and PstI sites of the pRSET vector; (2) pGEX-METH I-TSP was generated by ligating the BamHI-EcoRI fragment from the pRSET-METH1-TSP into the Smal site of the pGEX-5X vector (Pharmacia Biotech Inc., Piscataway, NJ) by blunt-end ligation; pGEX-1.0-METH2: the fragment nt 838-1818 of METH2 cDNA (from the start codon) was ligated into BamHI-EcoRI sites ofpGEM-2TK. The METH2 fragment was amplified by PCR using the following primers: 5 '-GAAAAATGGGGATCCGAGGTG-3' (SEQ ID and 5'-GCAGGAGAATTCCGTCCATG-3' (SEQ ID NO:81) to generate BamHI and EcoR restriction sites; pGEX-METH2-TSP: a 0.5Kb XmaI- EcoRI fragment isolated from pGEX-1.0-METH2 was subcloned into the XmaI and EcoRl sites of pGEX-2TK vector. All constructs were sequenced to verify sequence fidelity and correct open reading frame.
The recombinant proteins were named 6H-METH1, the recombinant protein expressed with the plasmid pRSET-METH1-TSP, GST-METH1, the protein expressed with the plasmid pGEX-METH-TSP and GST-METH2, the protein expressed with the plasmid pGEX-METH2-TSP.
Expression plasmids were transformed into BL21:DE3 E. coli strain (Stratagene Cloning Systems, La Jolla, CA) and fusion proteins were induced following manufacturer recommendations. Briefly, induced bacteria pellets were resuspended in PBS and sonicated on ice for 1 min. The suspension was, subsequently, incubated at RT for 20min in the presence of 1% triton X-100 and centrifuged at 4"C. Histidine tagged fusion proteins were then purified on Ni- NTA beads (Qiagen, Chatsworth, CA) by incubating 20ml ofsupernatant with 1 ml of beads (50% slurry) for 2h at 4 0 C. The suspension was transferred into a column and washed with 10 columns volume of PBS containing 10 OM imidazole, followed by 50mM imidazole and finally 100mM imidazole. The protein was eluted with 500mM imidazole in PBS. Fractions containing the recombinant protein were dialyzed against phenol-red free DMEM. Samples were centrifuged for 30min at 4 0 C, part of the protein was not soluble and was lost during WO 99/37660 PCT/US99/01313 -122centrifugation. The supernatant was stored at -70 C and used for proliferation, cornea pocket and chorioallantoic membrane (CAM) assays.
For purification of GST-fusion proteins, the extract was cleared by centrifugation and applied to a GST-affinity column (Pharmacia). The column was washed with PBS-1% triton X-100 in the presence of 0.1mM reduced glutathione and, subsequently, with the same buffer in the presence of reduced glutathione. Fusion proteins were eluted with 10mM reduced glutathione in 50mM Tris-HC1, pH 7.5. Fractions containing the protein were dialyzed against DMEM, stored at -70 0 C and used for proliferation, cornea pocket and chorioallantoic membrane (CAM) assays.
Integrity and purity of recombinant proteins was analyzed in 12.5% or acrylamide gels stained with Coomassie blue.
A recombinant GST fusion protein containing the first two type I repeats of TSP was also dialyzed against DMEM before used in functional assays. Intact TSP1 was purified from platelets as previously described (Roberts, et al., J. Tissue Cult. Methods 16:217-222 (1994)).
To test the hypothesis that METH1 and METH2 TSP domains could function as regulators ofangiogenesis recombinant fusion proteins were generated in bacteria. The constructs included the first TSP domain of METH1 or METH2.
This domain is the most conserved, 52% amino acid similarity with the second type I repeat of TSP1, (this domain contains a putative binding site for CD36).
All recombinant proteins were isolated under native conditions to preserve their secondary structure as much as possible. 6H-METH 1 and GST-METH1 contained the first TSP-like domain of METH1 fused to a histidine tag or a GST, respectively. METHI recombinant protein was made with two different tags because of purification and structural advantages. The differences in size are due to the size of the tag, 6KDa the histidine and 27KDa the GST. GST-METH2 contained the first TSP domain of METH2 also fused to a GST. A fragment corresponding to the last two type I repeats of TSP1, also fused to a GST, and WO 99/37660 PCT/US99/01313 -123intact TSPI purified from platelets were used as positive controls. In addition, GST alone was included in all experiments as negative control.
Example 4: TSP domains in METH1 andMETH2 disrupt angiogenesis in vivo Cornea pocket assay Swiss Webster females and males, were purchased from Charles River (Boston, MA) and used between 8-10 weeks-old for implantation of the pellets.
Cornea pockets were performed as described by Kenyon and colleagues (Kenyon, et al., Invest. Ophthalmol. Vis. Sci. 37:1625-1632 (1996)) with few modifications. Briefly, a solution of 10pg of recombinant bFGF plus 5 mg of sucralfate were mixed with 10al of Hydron (200mg/ml in ethanol; New Brunswick, NJ) and the recombinant protein of interest (2tg). The suspension was then smeared onto a sterile nylon mesh square (pore size 500gm; Tetko Inc., Briarcliff Manor, NY) and allowed to dry for 30min. The fibers of the mesh were pulled to produce pellets of 500im 3 that were stored at -20 0 C. Uniformly sized pellets were selected under a microscope and used for the assays.
Mice were anesthetized with Avertin. An incision was made in the cornea using a Nikon SMZ-U dissecting microscope with the aid of a surgical blade. A single pellet was implanted into the pocket. Five days after pellet implantation, corneal angiogenesis was evaluated and photographed.
CAM assay Chorioallantoic membrane assays were performed on Leghorn chicken embryos (SPAFAS, MA) at 12-14 days of embryonic development. Matrigel (750gg/ml), VEGF (250ng/mesh) and the protein or peptide to be tested were mixed, placed onto nylon meshes (pore size 250pm; Tetko Inc.) and incubated sequentially at 37 0 C for 30min and at 4°C for 2h to induce polymerization.
A
positive (matrigel and VEGF) and a negative (VEGF alone) control were also prepared for each CAM. Polymerized meshes were placed onto the third outer WO 99/37660 PCT/US99/01313 -124region of the CAM and incubated for 24h. To visualize vessels, 400Pl of fluorescein isothiocyanate dextran (10mg/ml, SIGMA) was injected in the chick blood stream. After 5-10min incubation, the chick was topically fixed with 3.7% formaldehyde for 5min. The meshes were then dissected and mounted onto slides.
Fluorescence intensity was analyzed with a computer-assisted image program (NIH Image 1.59).
Peptides used on these assays were synthesized by Chiron (Raleigh,
NC).
Sequence corresponded to amino acids: P-TSP1, 430-447; P-METH1, 549-563; P-METH2, 529-548.
The evaluation of angiogenic or anti-angiogenic responses relies heavily on the sensitivity and specificity of the assays used to assess the response. To evaluate the anti-angiogenic activity of these fragments in vivo, two popular and well-accepted angiogenesis assays were used: the corneal pocket and the chorioallantoic membrane. The visibility, accessibility, and avascularity of the cornea are highly advantageous and facilitate the visualization of the neovascular response and the topical application of the test substances. A known amount of angiogenesis factor(s) is implanted, as a pellet, in a pocket made in the cornea eye.
To test an angiogenesis inhibitor, the molecule is implanted with the stimulator in the same pellet, and the response is compared to the stimulator alone.
In these experiments, bFGF was used as the vascularization stimulator.
Pellets containing the recombinant protein were implanted in mouse corneas and their ability to inhibit the bFGF-induced angiogenic response was compared to that of controls. When a bFGF pellet containing GST was implanted new capillary vessels grew from the cornea limbus, across the cornea and into the pellet within 5 days. In contrast, addition of GST-METH1 or GST-METH2 to the bFGF pellets completely abolished blood vessel growth. Table 4 contains a summary of the results obtained from 41 assays performed. Intact TSP purified from platelets and GST-TSP were used as positive controls. All assays were performed at identical concentrations, suggesting that METH1 and METH2 have similar potency to that of TSP in the inhibition of angiogenesis. In addition, when half WO 99/37660 PCTIUS99/01313 -125of the standard concentration was used, a weak, however noticeable response was seen, indicating a dose-dependent effect.
Table 4.
Activity of METH1 and METH2 recombinant proteins in the corneal pocket assay bFGF Pellets Vascularized corneas/Total corneas Vehicle TSPI GST 11/11 GST-TSPI-TI 1/4 GST-METH1-TSP 0/8 GST-METH2-TSP 0/8 In the CAM assay, the angiogenic response is analyzed by measuring the number of vessels that grow within a matrix polymer containing the angiogenic growth factor. To determine whether recombinant METH 1 and METH2 proteins inhibited neovascularization in the CAM assay induced by VEGF, a matrigel polymer containing VEGF and the recombinant protein were implanted in the CAM. Quantitative analysis of the experiments, which included three different polymers per treatment are shown in Figure 6A. Matrigels polymers containing VEGF plus 5gg of GST-METH1 or GST-METH2 caused greater than inhibition in blood vessel growth. A similar potency was found using the GST recombinant protein derived from the type I repeats of TSP1. Furthermore, the anti-angiogenic effect of the TSP domains in METH1 and METH2 was dosedependent with a complete inhibition of blood vessel growth when 15 gg/ml of protein was used (Figure 6C and GST alone, at identical concentrations, had no significant effect on VEGF-stimulated angiogenesis.
WO 99/37660 PCT/US99/01313 -126- Synthetic peptides from the second or the third type I repeats of human TSPI can mimic that anti-angiogenic effects of the intact TSPI (Tolsma, et al., Cell. Biol. 122:497-511 (1993)). In fact, a 19-residue polypeptide was shown to be sufficient to block in vivo neovascularization in the rat cornea and to inhibit the bFGF-induced migration of cultured endothelial cells (Vogel, et al., J. Cell. Biochem. 53:74-84 (1993); Tolsma, etal., J. Cell. Biol. 122:497-511 (1993)). To test whether the same was true for the METHI and METH2 TSP domains, peptides derived from the same region were synthesized and their antiangiogenic activity was evaluated in the CAM assay. The results are shown in Figure 6B. Peptides derived from both the TSP domain of METH1 and METH2 blocked VEGF-induced angiogenesis similarly to that of TSP1. In contrast, scramble peptides had no significant effects.
Example 5: Proliferation assays Human dermal endothelial cells (HDEC) were isolated and grown on VitrogenTM coated petri-dishes in EBM (Clonetics, San Diego, CA) supplemented with 15% fetal calf serum, 25 pg/ml cAMP, and 1l g/ml of hydrocortisone-21acetate and were used from passages 3 to 6. Cells were made quiescent by incubation of confluent monolayers with phenol red-free EBM containing 0.2% BSA for 48h. Human dermal fibroblasts were isolated from neonatal foreskin and by enzymatic dissociation. Both fibroblasts and smooth muscle cells were maintained in DMEM supplemented with 10% fetal calf serum. Human mammary epithelial cells (HMEC) were purchased from Clonetics and maintained in the recommended media (mammary epithelial growth media, MEGM).
Quiescent human dermal endothelial cells, between passage 3 and 6, were plated on Vitrogen T M coated 24-well plates in EBM supplemented with 0.2% BSA, 0.1% fetal calf serum and 1 ng/ml ofbFGF in the presence or absence ofthe recombinant protein and incubated at 5% CO 2 at 37 0 C for 48h. For vascular smooth muscle (VSM) and fibroblast proliferation assays, cells were incubated WO 99/37660 PCT/US99/01313 -127under the same conditions but using DMEM instead of EBM. Human mammary epithelial cells were incubated on their growth media. A pulse of 3 H]-Thymidine (1I Ci/il) was added during the last 4h prior harvesting. Cells were washed and fixed in 10% TCA. Incorporation of 3 H]-thymidine was determined by scintillation counting, as previously described (Iruela-Arispe, M.L. Sage, E.H., J. Cell. Biochem. 52:414 (1993)).
Statistical analysis were done using In-Stat software (Graph Pad Software) for Macintosh. Assuming normal distributions, data were analyzed by one-way ANOVA, followed by either T-test Dunnett test for comparisons between groups, or student-Newman-Kleus test for multiple comparisons between groups.
To gain insight into the mechanism by which METH1 and METH2 inhibit neovascularization, the direct effect of the purified recombinant fusion proteins on endothelial cell proliferation was tested. Serum-starved endothelial cells were plated into growth medium containing bFGF and FCS. Recombinant proteins (3 pg/ml) were added at the same time of plating. 40% (GST-METH1), 45% (6H- GST) or 36% (GST-METH2) inhibition was observed, in contrast to a nonsignificant effect when GST alone was added. The recombinant protein from the type I repeats ofTSP1 had similar inhibitory effects. (Figure 7A). Furthermore, suppression of proliferation mediated by METH1 or METH2 were dosedependent, as shown in Figure 7E. The inhibition was observed as early as one day after treatment and the inhibitory effect was not toxic and reversible since the removal of the recombinant protein and subsequent addition of growth factor alone led to the resumption of endothelial cell proliferation.
The cell specificity of the anti-proliferative effects for METHI and METH2 on the endothelium was evaluated by additional proliferation assays on a variety of non-endothelial cells. No significant inhibition of proliferation was seen on fibroblasts or smooth muscle cell cultures. In contrast, a non significant, but reproducible stimulation of proliferation for these two cell types could be observed. This result rules out the presence of any potential nonspecific inhibitor of cell growth in the recombinant protein preparations. On mammary epithelial WO 99/37660 PCT/US99/01313 -128cell, however, METH 1 and METH2 inhibited cell proliferation to the same degree as to endothelial cells. Interestingly, TSP also suppresses mammary epithelial cell proliferation both in vitro and in a transgenic model.
The possibility that METHI and METH2 might act as disintegrins is consistent with their anti-angiogenic properties. Clearly blockade of avp3 and P 1 integrins with antibodies has been shown to inhibit neovascularization both during development and in tumors (Brooks, e al., Cell85:683-693 (1996); Brooks, et al., Cell 92:391-400 (1998); Senger, et al., Proc. Natl. Acad. Sci.
USA 94:13612-13617 (1997)). Integrins are essential for the mediation of both proliferative and migratory signals (Schwartz, M.A. Ingber, Mol. Biol.
Cell 5:389-393 (1994)), therefore interference with those signals can be highly deleterious to the angiogenic process. The angiogenic functional assays were performed with recombinant protein containing only the type I repeats in METH1 and METH2.
The mechanism of action of METH1 and METH2 with regards to their angio-inhibitory activity is not known. To date we have evidence that these proteins are secreted and bind to endothelial cells. Further investigations are guided towards the identification ofreceptors and signal transduction mechanisms.
A likely hypothesis resulting from the lessons learned from TSP1 is that both METH1 and METH2 bind to CD36. Recently, this scavenger receptor has been implicated in the mediation of signals by which TSP-1 exert its anti-angiogenic effects (Dawson, et al., J. Cell. Biol. 138:707-717 (1997)). Both the CSVTCG (SEQ ID NO:83) (Asch, et al., Nature 262:1436-1439 (1993); Catimel, et al., Biochem. J. 284:231-236 (1992)) and the GCQXR (SEQ ID NO:84) sequences have been proposed as primary binding motifs to CD36 (Dawson, et al., J Cell. Biol. 138:707-717 (1997)). METH1 and METH2 have almost entire conservation in both these regions. A complementary and also likely occurrence is binding of METH1 and METH2 to bFGF. Binding to heparin and bFGF has been proposed as part of the anti-angiogenic activity ofTSP 1 (Guo, et al., J. Peptide Res. 49 (1997)). This property appears to be mediated WO 99/37660 PCT/US99/01313 -129through the WSXWS (SEQ ID NO:82) motif, also conserved in METH1 and METH2. Future efforts will focus on the signals implicated in the anti-angiogenic properties mediated by these novel proteins and on their potential as proteases of the extracellular milieu.
Example 6: Isolation of the METH1 or METH2 cDNA Clone From the Deposited Sample Two approaches can be used to isolate METHI or METH2 from the deposited sample. First, the deposited clone is transformed into a suitable host (such as XL-1 Blue (Stratagene)) using techniques known to those of skill in the art, such as those provided by the vector supplier or in related publications or patents. The transformants are plated on 1.5% agar plates (containing the appropriate selection agent, ampicillin) to a density of about 150 transformants (colonies) per plate. A single colony is then used to generate DNA using nucleic acid isolation techniques well known to those skilled in the art. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edit., (1989), Cold Spring Harbor Laboratory Press.) Alternatively, two primers of 17-20 nucleotides derived from both ends of the SEQ ID NO:1 or SEQ ID NO:3 within the region of SEQ ID NO:1 or SEQ ID NO:3 bounded by the 5' NT and the 3' NT of the clone) are synthesized and used to amplify the METHI or METH2 cDNA using the deposited cDNA plasmids as templates. The polymerase chain reaction is carried out under routine conditions, for instance, in 25 pl of reaction mixture with 0.5 ug of the above cDNA template. A convenient reaction mixture is 1.5-5 mM MgCl 2 0.01% (w/v) gelatin, 20 uM each of dATP, dCTP, dGTP, dTTP, 25 pmol of each primer and 0.25 Unit of Taq polymerase. Thirty five cycles of PCR (denaturation at 94 degree C for 1 min; annealing at 55 degree C for 1 min; elongation at 72 degree C for 1 min) are performed with a Perkin-Elmer Cetus automated thermal cycler.
The amplified product is analyzed by agarose gel electrophoresis and the DNA band with expected molecular weight is excised and purified. The PCR product WO 99/37660 PCT/US99/01313 -130is verified to be the selected sequence by subcloning and sequencing the DNA product.
Several methods are available for the identification of the 5' or 3' noncoding portions of the METHI or METH2 gene which may not be present in the deposited clones. These methods include but are not limited to, filter probing, clone enrichment using specific probes, and protocols similar or identical to 5' and 3' "RACE" protocols which are well known in the art. For instance, a method similar to 5' RACE is available for generating the missing 5' end of a desired fulllength transcript. (Fromont-Racine el al., Nucleic Acids Res. 21(7):1683-1684 (1993).) Briefly, a specific RNA oligonucleotide is ligated to the 5' ends of a population of RNA presumably containing full-length gene RNA transcripts. A primer set containing a primer specific to the ligated RNA oligonucleotide and a primer specific to a known sequence of the METH1 or METH2 gene of interest is used to PCR amplify the 5' portion of the METH1 or METH2 full-length gene.
This amplified product may then be sequenced and used to generate the full length gene.
This above method starts with total RNA isolated from the desired source, although poly-A+ RNA can be used. The RNA preparation can then be treated with phosphatase if necessary to eliminate 5' phosphate groups on degraded or damaged RNA which may interfere with the later RNA ligase step. The phosphatase should then be inactivated and the RNA treated with tobacco acid pyrophosphatase in order to remove the cap structure present at the 5' ends of messenger RNAs. This reaction leaves a 5' phosphate group at the 5' end of the cap cleaved RNA which can then be ligated to an RNA oligonucleotide using T4 RNA ligase.
This modified RNA preparation is used as a template for first strand cDNA synthesis using a gene specific oligonucleotide. The first strand synthesis reaction is used as a template for PCR amplification of the desired 5' end using a primer specific to the ligated RNA oligonucleotide and a primer specific to the known WO 99/37660 PCT/US99/03133 -131sequence of the gene of interest. The resultant product is then sequenced and analyzed to confirm that the 5' end sequence belongs to the METHI or METH2 gene.
Example 7: Bacterial Expression of METHI or METH2 A METHI or METH2 polynucleotide encoding a METHI or METH2 polypeptide invention is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' ends of the DNA sequence, as outlined in Example to synthesize insertion fragments. The primers used to amplify the cDNA insert should preferably contain restriction sites, such as BamHI and XbaI, at the 5' end of the primers in order to clone the amplified product into the expression vector.
For example, BamHI and XbaI correspond to the restriction enzyme sites on the bacterial expression vector pQE-9. (Qiagen, Inc., Chatsworth, CA). This plasmid vector encodes antibiotic resistance (Amp), a bacterial origin of replication (ori), an IPTG-regulatable promoter/operator a ribosome binding site (RBS), a 6-histidine tag (6-His), and restriction enzyme cloning sites. The pQE-9 vector is digested with BamHI and XbaI and the amplified fragment is ligated into the pQE-9 vector maintaining the reading frame initiated at the bacterial RBS.
The ligation mixture is then used to transform the E. coli strain M15/rep4 (Qiagen, Inc.) which contains multiple copies of the plasmid pREP4, which expresses the lacI repressor and also confers kanamycin resistance (Kan). Transformants are identified by their ability to grow on LB plates and ampicillin/kanamycin resistant colonies are selected. Plasmid DNA is isolated and confirmed by restriction analysis.
Clones containing the desired constructs are grown overnight in liquid culture in LB media supplemented with both Amp (100 ug/ml) and Kan ug/ml). The O/N culture is used to inoculate a large culture at a ratio of 1:100 to 1:250. The cells are grown to an optical density 600 (O.D.
6 0 0 of between 0.4 and 0.6. IPTG (Isopropyl-B-D-thiogalacto pyranoside) is then added to a final WO 99/37660 PCTIS99/01313 -132concentration of 1 mM. IPTG induces by inactivating the lad repressor, clearing the P/O leading to increased gene expression.
Cells are grown for an extra 3 to 4 hours. Cells are then harvested by centrifugation (20 mins at 6000Xg). The cell pellet is solubilized in the chaotropic agent 6 Molar Guanidine HCI by stirring for 3-4 hours at 4 degree C. The cell debris is removed by centrifugation, and the supernatant containing the polypeptide is loaded onto a nickel-nitrilo-tri-acetic acid ("Ni-NTA") affinity resin column (available from QIAGEN, Inc., supra). Proteins with a 6 x His tag bind to the Ni-NTA resin with high affinity and can be purified in a simple one-step procedure (for details see: The QIAexpressionist (1995) QIAGEN, Inc., supra).
Briefly, the supernatant is loaded onto the column in 6 M guanidine-HC1,' pH 8, the column is first washed with 10 volumes of 6 M guanidine-HCl, pH 8, then washed with 10 volumes of 6 M guanidine-HCI pH 6, and finally the polypeptide is eluted with 6 M guanidine-HC1, pH The purified METHI or METH2 protein is then renatured by dialyzing it against phosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus 200 mM NaC1. Alternatively, the METH1 or METH2 protein can be successfully refolded while immobilized on the Ni-NTA column. The recommended conditions are as follows: renature using a linear 6M-1M urea gradient in 500 mM NaCl, 20% glycerol, 20 mM Tris/HCl pH 7.4, containing protease inhibitors. The renaturation should be performed over a period of 1.5 hours or more. After renaturation the proteins are eluted by the addition of 250 mM immidazole.
Immidazole is removed by a final dialyzing step against PBS or 50 mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purified METH1 or METH2 protein is stored at 4°C or frozen at -80 0
C.
In addition to the above expression vector, the present invention further includes an expression vector comprising phage operator and promoter elements operatively linked to a METH 1 or METH2 polynucleotide, called pHE4a. (ATCC Accession Number 209645, deposited February 25, 1998.) This vector contains: 1) a neomycinphosphotransferase gene as a selection marker, 2) an E. coli origin WO 99/37660 PCT/UJS99/01313 -133of replication, 3) a T5 phage promoter sequence, 4) two lac operator sequences, a Shine-Delgarno sequence, and 6) the lactose operon repressor gene (laclq).
The origin of replication (oriC) is derived from pUC19 (LTI, Gaithersburg, MD).
The promoter sequence and operator sequences are made synthetically.
DNA can be inserted into the pHEa by restricting the vector with Ndel and XbaI, BamHI, Xhol, or Asp718, running the restricted product on a gel, and isolating the larger fragment (the stuffer fragment should be about 310 base pairs).
The DNA insert is generated according to the PCR protocol described in Example using PCR primers having restriction sites for NdeI primer) and Xbal, BamHI, XhoI, or Asp718 primer). The PCR insert is gel purified and restricted with compatible enzymes. The insert and vector are ligated according to standard protocols.
The engineered vector could easily be substituted in the above protocol to express protein in a bacterial system.
Example 8: Purification ofMETHI orMETH2 Polypeptidefrom an Inclusion Body The following alternative method can be used to purify METH1 or METH2 polypeptide expressed in E coli when it is present in the form of inclusion bodies. Unless otherwise specified, all of the following steps are conducted at 4-10 0
C.
Upon completion of the production phase of the E. coli fermentation, the cell culture is cooled to 4-10 degree C and the cells harvested by continuous centrifugation at 15,000 rpm (Heraeus Sepatech). On the basis of the expected yield of protein per unit weight of cell paste and the amount of purified protein required, an appropriate amount of cell paste, by weight, is suspended in a buffer solution containing 100 mM Tris, 50 mM EDTA, pH 7.4. The cells are dispersed to a homogeneous suspension using a high shear mixer.
The cells are then lysed by passing the solution through a microfluidizer (Microfuidics, Corp. or APV Gaulin, Inc.) twice at 4000-6000 psi. The WO 99/37660 PCT/US99/01313 -134homogenate is then mixed with NaCI solution to a final concentration of 0.5 M NaCI, followed by centrifugation at 7000 xg for 15 min. The resultant pellet is washed again using 0.5M NaCI, 100 mM Tris, 50 mM EDTA, pH 7.4.
The resulting washed inclusion bodies are solubilized with 1.5 M guanidine hydrochloride (GuHCl) for 2-4 hours. After 7000 xg centrifugation for 15 min., the pellet is discarded and the polypeptide containing supernatant is incubated at 4 degree C overnight to allow further GuHCI extraction.
Following high speed centrifugation (30,000 xg) to remove insoluble particles, the GuHCI solubilized protein is refolded by quickly mixing the GuHCI extract with 20 volumes of buffer containing 50 mM sodium, pH 4.5, 150 mM NaCI, 2 mM EDTA by vigorous stirring. The refolded diluted protein solution is kept at 4 degree C without mixing for 12 hours prior to further purification steps.
To clarify the refolded polypeptide solution, a previously prepared tangential filtration unit equipped with 0.16 um membrane filter with appropriate surface area Filtron), equilibrated with 40 mM sodium acetate, pH 6.0 is employed. The filtered sample is loaded onto a cation exchange resin Poros Perseptive Biosystems). The column is washed with 40 mM sodium acetate, pH 6.0 and eluted with 250 mM, 500 mM, 1000 mM, and 1500 mM NaCI in the same buffer, in a stepwise manner. The absorbance at 280 nm of the effluent is continuously monitored. Fractions are collected and further analyzed by SDS-PAGE.
Fractions containing the METH 1 or METH2 polypeptide are then pooled and mixed with 4 volumes of water. The diluted sample is then loaded onto a previously prepared set of tandem columns of strong anion (Poros Perseptive Biosystems) and weak anion (Poros CM-20, Perseptive Biosystems) exchange resins. The columns are equilibrated with 40 mM sodium acetate, pH Both columns are washed with 40 mM sodium acetate,, pH 6.0, 200 mM NaCI. The CM-20 column is then eluted using a 10 column volume linear gradient ranging from 0.2 M NaCI, 50 mM sodium acetate, pH 6.0 to 1.0 M NaC1, 50 mM sodium acetate, pH 6.5. Fractions are collected under constant A 280 monitoring WO 99/37660 PCTIUS99/01313 -135of the effluent. Fractions containing the polypeptide (determined, for instance, by 16% SDS-PAGE) are then pooled.
The resultant METH1 or METH2 polypeptide should exhibit greater than purity after the above refolding and purification steps. No major contaminant bands should be observed from Coomassie blue stained 16% SDS-PAGE gel when ug of purified protein is loaded. The purified METH1 or METH2 protein can also be tested for endotoxin/LPS contamination, and typically the LPS content is less than 0.1 ng/ml according to LAL assays.
Example 9: Cloning and Expression of METH1 or METH2 in a Baculovirus Expression System In this example, the plasmid shuttle vector pA2 is used to insert METH 1 or METH2 polynucleotide into a baculovirus to express METH1 or METH2.
This expression vector contains the strong polyhedrin promoter oftheAutographa californica nuclear polyhedrosis virus (AcMNPV) followed by convenient restriction sites such as BamHI, Xba I and Asp718. The polyadenylation site of the simian virus 40 ("SV40") is used for efficient polyadenylation. For easy selection of recombinant virus, the plasmid contains the beta-galactosidase gene from E. coli under control of a weak Drosophila promoter in the same orientation, followed by the polyadenylation signal of the polyhedrin gene. The inserted genes are flanked on both sides by viral sequences for cell-mediated homologous recombination with wild-type viral DNA to generate a viable virus that express the cloned METH1 or METH2 polynucleotide.
Many other baculovirus vectors can be used in place of the vector above, such as pAc373, pVL941, and pAcIM1, as one skilled in the art would readily appreciate, as long as the construct provides appropriately located signals for transcription, translation, secretion and the like, including a signal peptide and an in-frame AUG as required. Such vectors are described, for instance, in Luckow et al., Virology 170:31-39 (1989).
WO 99/37660 PCTIUS99/01313 -136- Specifically, the METHI or METH2 cDNA sequence contained in the deposited clone, including the AUG initiation codon and any naturally associated leader sequence, is amplified using the PCR protocol described in Example 5. If the naturally occurring signal sequence is used to produce the secreted protein, the pA2 vector does not need a second signal peptide. Alternatively, the vector can be modified (pA2 GP) to include a baculovirus leader sequence, using the standard methods described in Summers et al., "A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures," Texas Agricultural Experimental Station Bulletin No. 1555 (1987).
The amplified fragment is isolated from a 1% agarose gel using a commercially available kit ("Geneclean," BIO 101 Inc., La Jolla, The fragment then is digested with appropriate restriction enzymes and again purified on a 1% agarose gel.
The plasmid is digested with the corresponding restriction enzymes and optionally, can be dephosphorylated using calf intestinal phosphatase, using routine procedures known in the art. The DNA is then isolated from a 1% agarose gel using a commercially available kit ("Geneclean" BIO 101 Inc., La Jolla, Ca.).
The fragment and the dephosphorylated plasmid are ligated together with T4 DNA ligase. E. coli HB 101 or other suitable E. coli hosts such as XL- Blue (Stratagene Cloning Systems, La Jolla, CA) cells are transformed with the ligation mixture and spread on culture plates. Bacteria containing the plasmid are identified by digesting DNA from individual colonies and analyzing the digestion product by gel electrophoresis. The sequence of the cloned fragment is confirmed by DNA sequencing.
Five ug of a plasmid containing the polynucleotide is co-transfected with ug of a commercially available linearized baculovirus DNA ("BaculoGolda baculovirus DNA", Pharmingen, San Diego, CA), using the lipofection method described by Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987).
One ug of BaculoGold" virus DNA and 5 ug of the plasmid are mixed in a sterile WO 99/37660 PCTfUS99/01313 -137well of a microtiter plate containing 50 ul of serum-free Grace's medium (Life Technologies Inc., Gaithersburg, MD). Afterwards, 10 ul Lipofectin plus 90 ul Grace's medium are added, mixed and incubated for 15 minutes at room temperature. Then the transfection mixture is added drop-wise to Sf9 insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with 1 ml Grace's medium without serum. The plate is then incubated for 5 hours at 27 degrees C.
The transfection solution is then removed'from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum is added. Cultivation is then continued at 27 degrees C for four days.
After four days the supernatant is collected and a plaque assay is performed, as described by Summers and Smith, supra. An agarose gel with "Blue Gal" (Life Technologies Inc., Gaithersburg) is used to allow easy identification and isolation of gal-expressing clones, which produce blue-stained plaques. (A detailed description of a "plaque assay" of this type can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9-10.) After appropriate incubation, blue stained plaques are picked with the tip ofa micropipettor Eppendorf). The agar containing the recombinant viruses is then resuspended in a microcentrifuge tube containing 200 ul of Grace's medium and the suspension containing the recombinant baculovirus is used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supernatants of these culture dishes are harvested and then they are stored at 4 degree C.
To verify the expression of the polypeptide, Sf9 cells are grown in Grace's medium supplemented with 10% heat-inactivated FBS. The cells are infected with the recombinant baculovirus containing the polynucleotide at a multiplicity of infection of about 2. If radiolabeled proteins are desired, 6 hours later the medium is removed and is replaced with SF900 II medium minus methionine and cysteine (available from Life Technologies Inc., Rockville, MD). After 42 hours, 5 uCi of 35 S-methionine and 5 uCi 35 S-cysteine (available from Amersham) are added. The cells are further incubated for 16 hours and then are harvested by WO 99/37660 PCTIUS99/01313 -138centrifugation. The proteins in the supernatant as well as the intracellular proteins are analyzed by SDS-PAGE followed by autoradiography (if radiolabeled).
Microsequencing of the amino acid sequence of the amino terminus of purified protein may be used to determine the amino terminal sequence of the produced METH1 or METH2 protein.
Example 10: Expression of METH1 or METH2 in Mammalian Cells METH1 or METH2 polypeptide can be expressed in a mammalian cell. A typical mammalian expression vector contains a promoter element, which mediates the initiation of transcription of mRNA, a protein coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript.
Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription is achieved with the early and late promoters from SV40, the long terminal repeats (LTRs) from Retroviruses, RSV, HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV). However, cellular elements can also be used the human actin promoter).
Suitable expression vectors for use in practicing the present invention include, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2DHFR (ATCC 37146), pBC12MI (ATCC 67109), pCMVSport 2.0, and pCMVSport 3.0. Mammalian host cells that could be used include, human Hela, 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.
Alternatively, METH1 or METH2 polypeptide can be expressed in stable cell lines containing the METH1 or METH2 polynucleotide integrated into a chromosome. The co-transfection with a selectable marker such as DHFR, gpt, neomycin, hygromycin allows the identification and isolation of the transfected cells.
WO 99/37660 PCT/US99/01313 -139- The transfected METH 1 or METH2 gene can also be amplified to express large amounts of the encoded protein. The DHFR (dihydrofolate reductase) marker is useful in developing cell lines that carry several hundred or even several thousand copies of the gene of interest. (See, Alt, F. el al., J. Biol.
Chem. 253:1357-1370 (1978); Hamlin, J. L. and Ma, Biochem. et Biophys.
Aca 1097:107-143 (1990); Page, M. J. and Sydenham, M. Biotechnology 9:64-68 (1991).) Another useful selection marker is the enzyme glutamine synthase (GS) (Murphy et al., Biochem J 22 7:277-279 (1991); Bebbington et al., Bio/Technology 10:169-175 (1992). Using these markers, the mammalian cells are grown in selective medium and the cells with the highest resistance are selected. These cell lines contain the amplified gene(s) integrated into a' chromosome. Chinese hamster ovary (CHO) and NSO cells are often used for the production of proteins.
Derivatives of the plasmid pSV2-DHFR (ATCC Accession No. 37146), the expression vectors pC4 (ATCC Accession No. 209646) and pC6 (ATCC Accession No.209647) contain the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen et al., Molecular and Cellular Biology, 438-447 (March, 1985)) plus a fragment of the CMV-enhancer (Boshart et al., Cell 41:521-530 (1985).) Multiple cloning sites, with the restriction enzyme cleavage sites BamHI, XbaI and Asp718, facilitate the cloning of METH1 or METH2. The vectors also contain the 3' intron, the polyadenylation and termination signal of the rat preproinsulin gene, and the mouse DHFR gene under control of the SV40 early promoter.
If a naturally occurring signal sequence is used to produce a secreted protein, the vector does not need a second signal peptide. Alternatively, if a naturally occurring signal sequence is not used, the vector can be modified to include a heterologous signal sequence in an effort to secrete the protein from the cell. (See, WO 96/34891.) The amplified fragment is then digested with the appropriate restriction enzyme and purified on a 1% agarose gel using a commercially available kit WO 99/37660 PCT/US99/01313 -140- ("Geneclean," BIO 101 Inc., La Jolla, The isolated fragment and the dephosphorylated vector are then ligated with T4 DNA ligase. E. coli HB 101 or XL-1 Blue cells are then transformed and bacteria are identified that contain the fragment inserted into plasmid pC6 or pC4 using, for instance, restriction enzyme analysis.
Chinese hamster ovary cells lacking an active DHFR gene is used for transfection. Five pg of the expression plasmid pC6 or pC4 is cotransfected with ug of the plasmid pSVneo using lipofectin (Feigner et al., supra). The plasmid pSV2-neo contains a dominant selectable marker, the neo gene from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including G418. The cells are seeded in alpha minus MEM supplemented with 1 mg/ml G418. After 2 days, the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) in alpha minus MEM supplemented with 10, 25, or 50 ng/ml of metothrexate plus 1 mg/ml G418. After about 10-14 days single clones are trypsinized and then seeded in 6-well petri dishes or 10 ml flasks using different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM).
Clones growing at the highest concentrations of methotrexate are then transferred to new 6-well plates containing even higher concentrations ofmethotrexate (1 uM, 2 uM, 5 uM, 10 mM, 20 mM). The same procedure is repeated until clones are obtained which grow at a concentration of 100 200 uM. Expression ofMETH1 or METH2 is analyzed, for instance, by SDS-PAGE and Western blot or by reversed phase HPLC analysis.
Example 11: Construction of N-Terminal and/or C-Terminal Deletion Mutants The following general approach may be used to clone a N-terminal or Cterminal deletion METHI or METH2 deletion mutant. Generally, two oligonucleotide primers of about 15-25 nucleotides are derived from the desired and 3' positions of a polynucleotide of SEQ ID NO: 1 or SEQ ID NO:3. The and 3' positions of the primers are determined based on the desired METHI or WO 99/37660 PCT[US99/01313 -141- METH2 polynucleotide fragment. An initiation and stop codon are added to the and 3' primers respectively, if necessary, to express the METH1 or METH2 polypeptide fragment encoded by the polynucleotide fragment. Preferred METH 1 or METH2 polynucleotide fragments are those encoding the N-terminal and Cterminal deletion mutants disclosed above in the "Polynucleotide and Polypeptide Fragments" section of the Specification.
Additional nucleotides containing restriction sites to facilitate cloning of the METHI or METH2 polynucleotide fragment in a desired vector may also be added to the 5' and 3' primer sequences. The METH1 or METH2 polynucleotide fragment is amplified from genomic DNA or from the deposited cDNA clone using the appropriate PCR oligonucleotide primers and conditions discussed herein or known in the art. The METHI or METH2 polypeptide fragments encoded by the METHI or METH2 polynucleotide fragments of the present invention may be expressed and purified in the same general manner as the full length polypeptides, although routine modifications may be necessary due to the differences in chemical and physical properties between a particular fragment and full length polypeptide.
As a means of exemplifying but not limiting the present invention, the polynucleotide encoding the METH1 polypeptide fragment D-40 to S-950 or the METH2 polypeptide fragment L-20 to L-890 is amplified and cloned as follows: A 5' primer is generated comprising a restriction enzyme site followed by an initiation codon in frame with the polynucleotide sequence encoding the Nterminal portion of the polypeptide fragment beginning with D-40 or respectively. A complementary 3' primer is generated comprising a restriction enzyme site followed by a stop codon in frame with the polynucleotide sequence encoding C-terminal portion of the METHI or METH2 polypeptide fragment ending with S-950 or L-890, respectively.
The amplified polynucleotide fragment and the expression vector are digested with restriction enzymes which recognize the sites in the primers. The digested polynucleotides are then ligated together. The METH1 or METH2 WO 99/37660 PCTIUS99/01313 -142polynucleotide fragment is inserted into the restricted expression vector, preferably in a manner which places the METH1 or METH2 polypeptide fragment coding region downstream from the promoter. The ligation mixture is transformed into competent E. coli cells using standard procedures and as described in the Examples herein. Plasmid DNA is isolated from resistant colonies and the identity of the cloned DNA confirmed by restriction analysis, PCR and DNA sequencing.
Example 12: Protein Fusions of METH1 or METH2 METHI or METH2 polypeptides are preferably fused to other proteins.
These fusion proteins can be used for a variety of applications. For example, fusion of METHI or METH2 polypeptides to His-tag, HA-tag, protein A, IgG domains, and maltose binding protein facilitates purification. (See Example 7; see also EP A 394,827; Traunecker, et al., Nature 331:84-86 (1988).) Similarly, fusion to IgG-1, IgG-3, and albumin increases the halflife time in vivo. Nuclear localization signals fused to METH1 or METH2 polypeptides can target the protein to a specific subcellular localization, while covalent heterodimer or homodimers can increase or decrease the activity of a fusion protein. Fusion proteins can also create chimeric molecules having more than one function.
Finally, fusion proteins can increase solubility and/or stability of the fused protein compared to the non-fused protein. All of the types of fusion proteins described above can be made by modifying the following protocol, which outlines the fusion of a polypeptide to an IgG molecule, or the protocol described in Example 7.
Briefly, the human Fc portion of the IgG molecule can be PCR amplified, using primers that span the 5' and 3' ends of the sequence described below. These primers also should have convenient restriction enzyme sites that will facilitate cloning into an expression vector, preferably a mammalian expression vector.
For example, if pC4 (Accession No. 209646) is used, the human Fc portion can be ligated into the BamHI cloning site. Note that the 3' BamHI site WO 99/37660 PCT/US99/01313 -143should be destroyed. Next, the vector containing the human Fc portion is rerestricted with BamiHI, linearizing the vector, and METHI or IvETH2 polynucleotide, isolated by the PCR protocol described in Example 5, is ligated into this BamHi- site. Note that the polynucleotide is cloned without a stop codon, otherwise a fuision protein will not be produced.
If the naturally occurring signal sequence is used to produce the secreted protein, pC4 does not need a second signal peptide. Alternatively, if the naturally occurring signal sequence is not used, the vector can be modified to include a heterologous signal sequence. (See, WO 96/34891.) Human IgG Fc region:
GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACC
GTGCCCAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCC
CCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGT
CACATGCGTGGTGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAG
TTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAA
AGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGT
CCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAG
TGCAAGGTCTCCAACAAAGCCCTCCCAACCCCCATCGAGAAAACCA
TCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCT
GCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACC
TGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGG
AGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGT
GCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGG
ACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGAT
GCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTG
TCTCCGGGTAAATGAGTGCGACGGCCGCGACTCTAGAGGAT (SEQ ID WO 99/37660 PCTIUS99/01313 -144- Example 13: Production of an Antibody The antibodies of the present invention can be prepared by a variety of methods. (See, Current Protocols, Chapter For example, cells expressing METH1 or METH2 is administered to an animal to induce the production of sera containing polyclonal antibodies. In a preferred method, a preparation ofMETH 1 or METH2 protein is prepared and purified to render it substantially free of natural contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity.
In the most preferred method, the antibodies of the present invention are monoclonal antibodies (or protein binding fragments thereof). Such monoclonal antibodies can be prepared using hybridoma technology. (Kohler et al., Nature 256:495 (1975); Kohler et al., Eur. J. Immunol. 6:511 (1976); Kohler et al., Eur.
J. Immunol. 6:292 (1976); Hammerling et al., in: Monoclonal Antibodies and T- Cell Hybridomas, Elsevier, pp. 563-681 (1981).) In general, such procedures involve immunizing an animal (preferably a mouse) with METH1 or METH2 polypeptide or, more preferably, with a secreted METHI or METH2 polypeptide-expressing cell. Such cells may be cultured in any suitable tissue culture medium; however, it is preferable to culture cells in Earle's modified Eagle's medium supplemented with 10% fetal bovine serum (inactivated at about 56 degree and supplemented with about 10 g/l of nonessential amino acids, about 1,000 U/ml of penicillin, and about 100 ug/ml of streptomycin.
The splenocytes of such mice are extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line may be employed in accordance with the present invention; however, it is preferable to employ the parent myeloma cell line (SP20), available from the ATCC. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands et al. (Gastroenterology 80:225-232 (1981).) The hybridoma cells obtained through such a selection are then assayed to identify WO 99/37660 PCT/US99/01313 -145clones which secrete antibodies capable of binding the METHI or METH2 polypeptide.
Alternatively, additional antibodies capable of binding to METH1 or METH2 polypeptide can be produced in a two-step procedure using anti-idiotypic antibodies. Such a method makes use of the fact that antibodies are themselves antigens, and therefore, it is possible to obtain an antibody which binds to a second antibody. In accordance with this method, protein specific antibodies are used to immunize an animal, preferably a mouse. The splenocytes of such an animal are then used to produce hybridoma cells, and the hybridoma cells are screened to identify clones which produce an antibody whose ability to bind to the METH1 or METH2 protein-specific antibody can be blocked by METHI or METH2. Such antibodies comprise anti-idiotypic antibodies to the METH1 or METH2 proteinspecific antibody and can be used to immunize an animal to induce formation of further METH 1 or METH2 protein-specific antibodies.
It will be appreciated that Fab and F(ab')2 and other fragments of the antibodies of the present invention may be used according to the methods disclosed herein. Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments). Alternatively, secreted METH1 or METH2 protein-binding fragments can be produced through the application of recombinant DNA technology or through synthetic chemistry.
For in vivo use of antibodies in humans, it may be preferable to use "humanized" chimeric monoclonal antibodies. Such antibodies can be produced using genetic constructs derived from hybridoma cells producing the monoclonal antibodies described above. Methods for producing chimeric antibodies are known in the art. (See, for review, Morrison, Science 229:1202 (1985); Oi el al., BioTechniques 4:214 (1986); Cabilly e al., U.S. Patent No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature 314:268 (1985).) WO 99/37660 PCT[US99/01313 -146- Example 14: Production Of METH1 or METH2 Protein For High- Throughput Screening Assays The following protocol produces a supernatant containing METHI or METH2 polypeptide to be tested. This supernatant can then be used in the Screening Assays described in Examples 16-23.
First, dilute Poly-D-Lysine (644 587 Boehringer-Mannheim) stock solution (1mg/ml in PBS) 1:20 in PBS (w/o calcium or magnesium 17-516F Biowhittaker) for a working solution of 50ug/ml. Add 200 ul of this solution to each well (24 well plates) and incubate at RT for 20 minutes. Be sure to distribute the solution over each well (note: a 12-channel pipetter may be used with tips on every other channel). Aspirate off the Poly-D-Lysine solution and rinse with Iml PBS (Phosphate Buffered Saline). The PBS should remain in the well until just prior to plating the cells and plates may be poly-lysine coated in advance for up to two weeks.
Plate 293T cells (do not carry cells past P+20) at 2 x 10' cells/well in DMEM(Dulbecco's Modified Eagle Medium)(with 4.5 G/L glucose and Lglutamine (12-604F Biowhittaker))/10% heat inactivated FBS(14-503F Biowhittaker)/ x Penstrep(17-602E Biowhittaker). Let the cells grow overnight.
The next day, mix together in a sterile solution basin: 300 ul Lipofectamine (18324-012 Gibco/BRL) and 5ml Optimem I (31985070 Gibco/BRL)/96-well plate. With a small volume multi-channel pipetter, aliquot approximately 2ug of an expression vector containing a polynucleotide insert, produced by the methods described in Examples 10-12, into an appropriately labeled 96-well round bottom plate. With a multi-channel pipetter, add 50ul of the Lipofectamine/Optimem I mixture to each well. Pipette up and down gently to mix. Incubate at RT 15-45 minutes. After about 20 minutes, use a multi-channel pipetter to add 150ul Optimem I to each well. As a control, one plate of vector DNA lacking an insert should be transfected with each set of transfections.
Preferably, the transfection should be performed by tag-teaming the following tasks. By tag-teaming, hands on time is cut in half, and the cells do not WO 99/37660 PCT/S99/01313 -147spend too much time on PBS. First, person A aspirates off the media from four 24-well plates of cells, and then person B rinses each well with .5-1ml PBS.
Person A then aspirates off PBS rinse, and person B, using al2-channel pipetter with tips on every other channel, adds the 200ul of DNA/Lipofectamine/Optimem I complex to the odd wells first, then to the even wells, to each row on the 24-well plates. Incubate at 37 degree C for 6 hours.
While cells are incubating, prepare appropriate media, either 1%BSA in DMEM with lx penstrep, or HGS CHO-5 media (116.6 mg/L of CaCI 2 (anhyd); 0.00130 mg/L CuSO 4 -5H 2 0; 0.050 mg/L of Fe(NO) 3 -9H 2 0; 0.417 mg/L of FeSO 4 -7H 2 0; 311.80 mg/L ofKcl; 28.64 mg/L ofMgCl 2 48.84 mg/L ofMgSO 4 6995.50 mg/L of NaCl; 2400.0 mg/L of NaHCO 3 62.50 mg/L of NaH 2
PO
4
-H
2 0; 71.02 mg/L of Na 2
HPO
4 .4320 mg/L ofZnSO 4 -7H 2 0; .002 mg/L ofArachidonic Acid; 1.022 mg/L of Cholesterol; .070 mg/L of DL-alpha-Tocopherol-Acetate; 0.0520 mg/L of Linoleic Acid; 0.010 mg/L of Linolenic Acid; 0.010 mg/L of Myristic Acid; 0.010 mg/L of Oleic Acid; 0.010 mg/L of Palmitric Acid; 0.010 mg/L of Palmitic Acid; 100 mg/L of Pluronic F-68; 0.010 mg/L of Stearic Acid; 2.20 mg/L of Tween 80; 4551 mg/L of D-Glucose; 130.85 mg/ml of L- Alanine; 147.50 mg/ml of L-Arginine-HCL; 7.50 mg/ml ofL-Asparagine-H 2 0; 6.65 mg/ml of L-Aspartic Acid; 29.56 mg/ml of L-Cystine-2HCL-H 2 0; 31.29 mg/ml of L- Cystine-2HCL; 7.35 mg/ml of L-Glutamic Acid; 365.0 mg/ml of L-Glutamine; 18.75 mg/ml of Glycine; 52.48 mg/ml of L-Histidine-HCL-H 2 0; 106.97 mg/mi of L-Isoleucine; 111.45 mg/ml ofL-Leucine; 163.75 mg/ml ofL-Lysine HCL; 32.34 mg/ml of L-Methionine; 68.48 mg/ml of L-Phenylalainine; 40.0 mg/ml of L- Proline; 26.25 mg/ml of L-Serine; 101.05 mg/ml of L-Threonine; 19.22 mg/ml of L-Tryptophan; 91.79 mg/ml of L-Tryrosine-2Na-2H 2 0; and 99.65 mg/ml of L- Valine; 0.0035 mg/L of Biotin; 3.24 mg/L of D-Ca Pantothenate; 11.78 mg/L of Choline Chloride; 4.65 mg/L of Folic Acid; 15.60 mg/L of i-Ipositol; 3.02 mg/L of Niacinamide; 3.00 mg/L of Pyridoxal HCL; 0.031 mg/L of Pyridoxine HCL; 0.319 mg/L of Riboflavin; 3.17 mg/L of Thiamine HCL; 0.365 mg/L of Thymidine; 0.680 mg/L of Vitamin Bl 2 25 mM of HEPES Buffer; 2.39 mg/L of WO 99/37660 PCT/US99/01313 -148- Na Hypoxanthine; 0.105 mg/L ofLipoic Acid; 0.081 mg/L of Sodium Putrescine- 2HCL; 55.0 mg/L of Sodium Pyruvate; 0.0067 mg/L of Sodium Selenite; of Ethanolamine; 0.122 mg/L of Ferric Citrate; 41.70 mg/L of Methyl-B- Cyclodextrin complexed with Linoleic Acid; 33.33 mg/L of Methyl-B- Cyclodextrin complexed with Oleic Acid; 10 mg/L of Methyl-B-Cyclodextrin complexed with Retinal Acetate. Adjust osmolarity to 327 mOsm) with 2mm glutamine and Ix penstrep. (BSA (81-068-3 Bayer) 100gm dissolved in IL DMEM for a 10% BSA stock solution). Filter the media and collect 50 ul for endotoxin assay in 15ml polystyrene conical.
The transfection reaction is terminated, preferably by tag-teaming, at the end of the incubation period. Person A aspirates off the transfection media, while person B adds 1.5ml appropriate media to each well. Incubate at 37 degree C for or 72 hours depending on the media used: 1%BSA for 45 hours or for 72 hours.
On day four, using a 300ul multichannel pipetter, aliquot 600ul in one 1 ml deep well plate and the remaining supernatant into a 2ml deep well. The supernatants from each well can then be used in the assays described in Examples 16-23.
It is specifically understood that when activity is obtained in any of the assays described below using a supernatant, the activity originates from either the METH1 or METH2 polypeptide directly as a secreted protein) or by METH1 or METH2 inducing expression of other proteins, which are then secreted into the supernatant. Thus, the invention further provides a method of identifying the protein in the supernatant characterized by an activity in a particular assay.
Example 15: Construction of GAS Reporter Construct One signal transduction pathway involved in the differentiation and proliferation of cells is called the Jaks-STATs pathway. Activated proteins in the WO 99/37660 PCT/US99/01313 -149- Jaks-STATs pathway bind to gamma activation site "GAS" elements or interferonsensitive responsive element located in the promoter of many genes.
The binding of a protein to these elements alter the expression of the associated gene.
GAS and ISRE elements are recognized by a class of transcription factors called Signal Transducers and Activators of Transcription, or "STATs." There are six members of the STATs family. Statl and Stat3 are present in many cell types, as is Stat2 (as response to IFN-alpha is widespread). Stat4 is more restricted and is not in many cell types though it has been found in T helper class I, cells after treatment with IL-12. Stat5 was originally called mammary growth factor, but has been found at higher concentrations in other cells including myeloid cells. It can be activated in tissue culture cells by many cytokines.
The STATs are activated to translocate from the cytoplasm to the nucleus upon tyrosine phosphorylation by a set of kinases known as the Janus Kinase ("Jaks") family. Jaks represent a distinct family of soluble tyrosine kinases and include Tyk2, Jakl, Jak2, and Jak3. These kinases display significant sequence similarity and are generally catalytically inactive in resting cells.
The Jaks are activated by a wide range of receptors summarized in the Table below. (Adapted from review by Schidler and Darnell, Ann. Rev. Biochem.
64:621-51 (1995).) A cytokine receptor family, capable of activating Jaks, is divided into two groups: Class 1 includes receptors for IL-2, IL-3, IL-4, IL-6, EL-7, 1L-9, IL-11, IL-12, IL-15, Epo, PRL, GH, G-CSF, GM-CSF, LIF, CNTF, and thrombopoietin; and Class 2 includes IFN-a, IFN-g, and IL-10. The Class 1 receptors share a conserved cysteine motif(a set of four conserved cysteines and one tryptophan) and a WSXWS motif (a membrane proxial region encoding Trp- Ser-Xxx-Trp-Ser (SEQ ID NO:82)).
Thus, on binding of a ligand to a receptor, Jaks are activated, which in turn activate STATs, which then translocate and bind to GAS elements. This entire process is encompassed in the Jaks-STATs signal transduction pathway.
WO 99/37660 PCT/US99/01313 -150- Therefore, activation of the Jaks-STATs pathway, reflected by the binding of the GAS or the ISRE element, can be used to indicate proteins involved in the proliferation and differentiation of cells. For example, growth factors and cytokines are known to activate the Jaks-STATs pathway. (See Table below.) Thus, by using GAS elements linked to reporter molecules, activators of the Jaks- STATs pathway can be identified.
or SR ak fam...l. IFN-a/B +1 IS ~1~g 1,3 GA fam IL-6 (Peorohc z, A IF>y6IFP) CFNITPlitrphc 1,23 IR 1,3 IL-I (Pleiotrophic) 1,3 GA IFI>y6EP 11-11 (lmphocytes) 1,3, GA IL-4(lmtphyi) 61A,3RI=IF L6)IH I-7(lymtrphce) 1, GA IL- (lmrphc) 1, GA IL-13 (lmtphcyt) 9 61 GA IL-2(Peioropi 7 1 GAS gpl4 ILA3 (meloid) 6 GAS (IRF1AIFP >Ly6)(IH 11-5 (lmpyt,.;) 1+ 5 GAS G-CS (lmoyes) 1- +IGAS growh oron 11-P(ylodI I- 5 IA (-1-II-Jr->Ly6 GMF 19 eoi -J 5 GAS PGF CSF-l 1, WO 99/37660 PCT/US99/01313 -151- To construct a synthetic GAS containing promoter element, which is used in the Biological Assays described in Examples 16-17, a PCR based strategy is employed to generate a GAS-SV40 promoter sequence. The 5' primer contains four tandem copies of the GAS binding site found in the IRFI promoter and previously demonstrated to bind STATs upon induction with a range ofcytokines (Rothman et al., Immunity 1:457-468 although other GAS or ISRE elements can be used instead. The 5' primer also contains 18bp of sequence complementary to the SV40 early promoter sequence and is flanked with an Xhol site. The sequence of the 5' primer is: CCCCGAAATGATTTCCCCGAAATATCTGCCATCTCAATTAG:3' (SEQ ID NO:86) The downstream primer is complementary to the SV40 promoter and is flanked with a Hind III site: 5':GCGGCAAGCTTTTTGCAAAGCCTAGGC:3' (SEQ ID NO:87) PCR amplification is performed using the SV40 promoter template present in the B-gal:promoter plasmid obtained from Clontech. The resulting PCR fragment is digested with XhoI/Hind III and subcloned into BLSK2-.
(Stratagene.) Sequencing with forward and reverse primers confirms that the insert contains the following sequence:
GAAATGATTTCCCCGAAATATCTGCCATCTCAATTAGTCAGCAACCA
TAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGT
TCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCA
GAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAG
GAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTT:3' (SEQ ID NO:88) With this GAS promoter element linked to the SV40 promoter, a GAS:SEAP2 reporter construct is next engineered. Here, the reporter molecule is a secreted alkaline phosphatase, or "SEAP." Clearly, however, any reporter WO 99/37660 PCT/US99/01313 -152molecule can be instead of SEAP, in this or in any of the other Examples. Well known reporter molecules that can be used instead of SEAP include chloramphenicol acetyltransferase (CAT), luciferase, alkaline phosphatase, Bgalactosidase, green fluorescent protein (GFP), or any protein detectable by an antibody.
The above sequence confirmed synthetic GAS-SV40 promoter element is subcloned into the pSEAP-Promoter vector obtained from Clontech using HindIII and Xhol, effectively replacing the SV40 promoter with the amplified promoter element, to create the GAS-SEAP vector. However, this vector does not contain a neomycin resistance gene, and therefore, is not preferred for mammalian expression systems.
Thus, in order to generate mammalian stable cell lines expressing the GAS- SEAP reporter, the GAS-SEAP cassette is removed from the GAS-SEAP vector using Sall and NotI, and inserted into a backbone vector containing the neomycin resistance gene, such as pGFP-1 (Clontech), using these restriction sites in the multiple cloning site, to create the GAS-SEAP/Neo vector. Once this vector is transfected into mammalian cells, this vector can then be used as a reporter molecule for GAS binding as described in Examples 16-17.
Other constructs can be made using the above description and replacing GAS with a different promoter sequence. For example, construction of reporter molecules containing NFK-B and EGR promoter sequences are described in Examples 18 and 19. However, many other promoters can be substituted using the protocols described in these Examples. For instance, SRE, IL-2, NFAT, or Osteocalcin promoters can be substituted, alone or in combination GAS/NF-KB/EGR, GAS/NF-KB, II-2/NFAT, or NF-KB/GAS). Similarly, other cell lines can be used to test reporter construct activity, such as HELA (epithelial), HUVEC (endothelial), Reh (B-cell), Saos-2 (osteoblast), HUVAC (aortic), or Cardiomyocyte.
WO 99/37660 PCTIUS99/01313 -153- Example 16: High-Throughput Screening Assay for T-cell Activity The following protocol is used to assess T-cell activity of METH1 or METH2 by determining whether METHI or METH2 supernatant proliferates and/or differentiates T-cells. T-cell activity is assessed using the GAS/SEAP/Neo construct produced in Example 15. Thus, factors that increase SEAP activity indicate the ability to activate the Jaks-STATS signal transduction pathway. The T-cell used in this assay is Jurkat T-cells (ATCC Accession No. TIB-152), although Molt-3 cells (ATCC Accession No. CRL-1552) and Molt-4 cells (ATCC Accession No. CRL-1582) cells can also be used.
Jurkat T-cells are lymphoblastic CD4+ Thl helper cells. In order to generate stable cell lines, approximately 2 million Jurkat cells are transfected with the GAS-SEAP/neo vector using DMRIE-C (Life Technologies)(transfection procedure described below). The transfected cells are seeded to a density of approximately 20,000 cells per well and transfectants resistant to 1 mg/ml genticin selected. Resistant colonies are expanded and then tested for their response to increasing concentrations of interferon gamma. The dose response of a selected clone is demonstrated.
Specifically, the following protocol will yield sufficient cells for 75 wells containing 200 ul of cells. Thus, it is either scaled up, or performed in multiple to generate sufficient cells for multiple 96 well plates. Jurkat cells are maintained in RPMI 10% serum with 1%Pen-Strep. Combine 2.5 mis of OPTI-MEM (Life Technologies) with 10 ug ofplasmid DNA in a T25 flask. Add 2.5 ml OPTI- MEM containing 50 ul of DMRIE-C and incubate at room temperature for 15-45 mins.
During the incubation period, count cell concentration, spin down the required number of cells (107 per transfection), and resuspend in OPTI-MEM to a final concentration of 10' cells/ml. Then add 1 ml of I x 107 cells in OPTI-MEM to T25 flask and incubate at 37 degree C for 6 hrs. After the incubation, add ml of RPMI 15% serum.
WO 99/37660 PCT/US99/01313 -154- The Jurkat:GAS-SEAP stable reporter lines are maintained in RPMI serum, 1 mg/ml Genticin, and 1% Pen-Strep. These cells are treated with .supernatants containing METH 1 or METH2 polypeptides or METH 1 or METH2 induced polypeptides as produced by the protocol described in Example 14.
On the day of treatment with the supernatant, the cells should be washed and resuspended in fresh RPM1 10% serum to a density of 500,000 cells per ml.
The exact number of cells required will depend on the number of supernatants being screened. For one 96 well plate, approximately 10 million cells (for plates, 100 million cells) are required.
Transfer the cells to a triangular reservoir boat, in order to dispense the cells into a 96 well dish, using a 12 channel pipette. Using a 12 channel pipette, transfer 200 ul of cells into each well (therefore adding 100,000 cells per well).
After all the plates have been seeded, 50 ul of the supernatants are transferred directly from the 96 well plate containing the supernatants into each well using a 12 channel pipette. In addition, a dose of exogenous interferon gamma 1.0, 10 ng) is added to wells H9, H10, and H11 to serve as additional positive controls for the assay.
The 96 well dishes containing Jurkat cells treated with supernatants are placed in an incubator for 48 hrs (note: this time is variable between 48-72 hrs).
35 ul samples from each well are then transferred to an opaque 96 well plate using a 12 channel pipette. The opaque plates should be covered (using sellophene covers) and stored at -20 degree C until SEAP assays are performed according to Example 20. The plates containing the remaining treated cells are placed at 4 degree C and serve as a source of material for repeating the assay on a specific well if desired.
As a positive control, 100 Unit/ml interferon gamma can be used which is known to activate Jurkat T cells. Over 30 fold induction is typically observed in the positive control wells.
WO 99/37660 PCT/US99/01313 -155- Example 17: High-Throughput Screening Assay Identifying Myeloid Activity The following protocol is used to assess myeloid activity of METH1 or METH2 by determining whether METH1 or METH2 proliferates and/or differentiates myeloid cells. Myeloid cell activity is assessed using the GAS/SEAP/Neo construct produced in Example 15. Thus, factors that increase SEAP activity indicate the ability to activate the Jaks-STATS signal transduction pathway. The myeloid cell used in this assay is U937, a pre-monocyte cell line, although TF-1, HL60, or KGI can be used.
To transiently transfect U937 cells with the GAS/SEAP/Neo construct produced in Example 15, a DEAE-Dextran method (Kharbanda el. al., 1994, Cell Growth Differentiation 5:259-265) is used. First, harvest 2xl0e 7 U937 cells and wash with PBS. The U937 cells are usually grown in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum (FB S) supplemented with 100 units/ml penicillin and 100 mg/ml streptomycin.
Next, suspend the cells in 1 ml of 20 mM Tris-HCI (pH 7.4) buffer containing 0.5 mg/ml DEAE-Dextran, 8 ug GAS-SEAP2 plasmid DNA, 140 mM NaC1, 5 mM KCI, 375 uM NazHPO 4 -7H20, 1 mM MgCl 2 and 675 uM CaCl 2 Incubate at 37 degree C for 45 min.
Wash the cells with RPMI 1640 medium containing 10% FBS and then resuspend in 10 ml complete medium and incubate at 37 degree C for 36 hr.
The GAS-SEAP/U937 stable cells are obtained by growing the cells in 400 ug/ml G418. The G418-free medium is used for routine growth but every one to two months, the cells should be re-grown in 400 ug/ml G418 for couple of passages.
These cells are tested by harvesting 1x10 8 cells (this is enough for ten 96well plates assay) and wash with PBS. Suspend the cells in 200 ml above described growth medium, with a final density of 5x105 cells/ml. Plate 200 ul cells per well in the 96-well plate (or Ixl0 5 cells/well).
WO 99/37660 PCTfUS99/01313 -156- Add 50 ul of the supernatant prepared by the protocol described in Example 14. Incubate at 37 degee C for 48 to 72 hr. As a positive control, 100 Unit/ml interferon gamma can be used which is known to activate U937 cells.
Over 30 fold induction is typically observed in the positive control wells. SEAP assay the supernatant according to the protocol described in Example Example 18: High- Throughput ScreeningAssay Identifying NeuronalA ctivity When cells undergo differentiation and proliferation, a group of genes are activated through many different signal transduction pathways. One of these genes, EGR1 (early growth response gene is induced in various tissues and cell types upon activation. The promoter of EGR1 is responsible for such induction.
Using the EGRI promoter linked to reporter molecules, activation of cells can be assessed by METH1 or METH2.
Particularly, the following protocol is used to assess neuronal activity in PC12 cell lines. PC12 cells (rat phenochromocytoma cells) are known to proliferate and/or differentiate by activation with a number of mitogens, such as TPA (tetradecanoyl phorbol acetate), NGF (nerve growth factor), and EGF (epidermal growth factor). The EGRI gene expression is activated during this treatment. Thus, by stably transfecting PC12 cells with a construct containing an EGR promoter linked to SEAP reporter, activation of PC12 cells by METHI or METH2 can be assessed.
The EGR/SEAP reporter construct can be assembled by the following protocol. The EGR-1 promoter sequence (-633 to +1)(Sakamoto K et al., Oncogene 6:867-871 (1991)) can be PCR amplified from human genomic DNA using the following primers: 5' GCGCTCGAGGGATGACAGCGATAGAACCCCGG (SEQ ID NO:89) GCGAAGCTTCGCGACTCCCCGGATCCGCCTC-3' (SEQ ID WO 99/37660 PCT/US99/01313 -157- Using the GAS:SEAP/Neo vector produced in Example 15, EGRI amplified product can then be inserted into this vector. Linearize the GAS:SEAP/Neo vector using restriction enzymes XhoI/HindIII, removing the stuffer. Restrict the EGR1 amplified product with these same enzymes. Ligate the vector and the EGR1 promoter.
To prepare 96 well-plates for cell culture, two mis of a coating solution (1:30 dilution of collagen type I (Upstate Biotech Inc. Cat#08-115) in ethanol (filter sterilized)) is added per one 10 cm plate or 50 ml per well of the 96well plate, and allowed to air dry for 2 hr.
PC12 cells are routinely grown in RPMI-1640 medium (Bio Whittaker) containing 10% horse serum (JRH BIOSCIENCES, Cat. 12449-78P), 5% heatinactivated fetal bovine serum (FBS) supplemented with 100 units/ml penicillin and 100 ug/ml streptomycin on a precoated 10 cm tissue culture dish. One to four split is done every three to four days. Cells are removed from the plates by scraping and resuspended with pipetting up and down for more than 15 times.
Transfect the EGR/SEAP/Neo construct into PC12 using the Lipofectamine protocol described in Example 14. EGR-SEAP/PC 12 stable cells are obtained by growing the cells in 300 ug/ml G418. The G418-free medium is used for routine growth but every one to two months, the cells should be regrown in 300 ug/ml G418 for couple of passages.
To assay for neuronal activity, a 10 cm plate with cells around 70 to confluent is screened by removing the old medium. Wash the cells once with PBS (Phosphate buffered saline). Then starve the cells in low serum medium (RPMI- 1640 containing 1% horse serum and 0.5% FBS with antibiotics) overnight.
The next morning, remove the medium and wash the cells with PBS.
Scrape off the cells from the plate, suspend the cells well in 2 ml low serum medium. Count the cell number and add more low serum medium to reach final cell density as 5x10 5 cells/ml.
Add 200 ul of the cell suspension to each well of 96-well plate (equivalent to 1x10 5 cells/well). Add 50 ul supernatant produced by Example 14, 37 degree WO 99/37660 PCT/US99/01313 -158- C for 48 to 72 hr. As a positive control, a growth factor known to activate PC12 cells through EGR can be used, such as 50 ng/ul of Neuronal Growth Factor (NGF). Over fifty-fold induction of SEAP is typically seen in the positive control wells. SEAP assay the supernatant according to Example Example 19: High-Throughput Screening Assay for T-cell Activity NF-KB (Nuclear Factor KB) is a transcription factor activated by a wide variety of agents including the inflammatory cytokines IL-1 and TNF, CD30 and lymphotoxin-alpha and lymphotoxin-beta, by exposure to LPS or thrombin, and by expression of certain viral gene products. As a transcription factor, NF-KB regulates the expression of genes involved in immune cell activation, control of apoptosis (NF- KB appears to shield cells from apoptosis), B and T-cell development, anti-viral and antimicrobial responses, and multiple stress responses.
In non-stimulated conditions, NF- KB is retained in the cytoplasm with I- KB (Inhibitor KB). However, upon stimulation, I- KB is phosphorylated and degraded, causing NF- KB to shuttle to the nucleus, thereby activating transcription of target genes. Target genes activated by NF- KB include IL-2, IL-6, GM-CSF, ICAM-1 and class 1 MHC.
Due to its central role and ability to respond to a range of stimuli, reporter constructs utilizing the NF-KB promoter element are used to screen the supernatants produced in Example 14. Activators or inhibitors of NF-KB would be useful in treating diseases. For example, inhibitors of NF-KB could be used to treat those diseases related to the acute or chronic activation of NF-KB, such as rheumatoid arthritis.
To construct a vector containing the NF-KB promoter element, a PCR based strategy is employed. The upstream primer contains four tandem copies of the NF-KB binding site (GGGGACTTTCCC) (SEQ ID NO:91), 18 bp of sequence complementary to the 5' end of the SV40 early promoter sequence, and is flanked with an XhoI site: WO 99/37660 PCT/US99/01313 -159- GGACTTTCCATCCTGCCATCTCAATTAG:3' (SEQ ID NO:92) The downstream primer is complementary to the 3' end of the promoter and is flanked with a Hind III site: 5':GCGGCAAGCTTTTTGCAAAGCCTAGGC:3' (SEQ ID NO:93) PCR amplification is performed using the SV40 promoter template present in the pB-gal:promoter plasmid obtained from Clontech. The resulting PCR fragment is digested with Xhol and Hind III and subcloned into BLSK2-.
(Stratagene) Sequencing with the T7 and T3 primers confirms the insert contains the following sequence:
TTCCATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAA
CTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCG
CCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGC
CTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGA
GGCCTAGGCTTTTGCAAAAAGCTT:3' (SEQ ID NO:88) Next, replace the SV40 minimal promoter element present in the pSEAP2promoter plasmid (Clontech) with this NF-KB/SV40 fragment using XhoI and HindIII. However, this vector does not contain a neomycin resistance gene, and therefore, is not preferred for mammalian expression systems.
In order to generate stable mammalian cell lines, the cassette is removed from the above NF-KB/SEAP vector using restriction enzymes Sall and NotI, and inserted into a vector containing neomycin resistance.
Particularly, the NF-KB/SV40/SEAP cassette was inserted into pGFP-1 (Clontech), replacing the GFP gene, after restricting pGFP-1 with SalI and NotI.
Once NF-KB/SV40/SEAP/Neo vector is created, stable Jurkat T-cells are created and maintained according to the protocol described in Example 16.
Similarly, the method for assaying supernatants with these stable Jurkat T-cells is also described in Example 16. As a positive control, exogenous TNF alpha (0.1,1, WO 99/37660 PCT/US99/01313 -160ng) is added to wells H9, H10, and H 11, with a 5-10 fold activation typically observed.
Example 20: Assay for SEAP Activity As a reporter molecule for the assays described in Examples 16-19, SEAP activity is assayed using the Tropix Phospho-light Kit (Cat. BP-400) according to the following general procedure. The Tropix Phospho-light Kit supplies the Dilution, Assay, and Reaction Buffers used below.
Prime a dispenser with the 2.5x Dilution Buffer and dispense 15 ul dilution buffer into Optiplates containing 35 ul ofa supernatant. Seal the plates with a plastic sealer and incubate at 65 degree C for 30 min. Separate the Optiplates to avoid uneven heating.
Cool the samples to room temperature for 15 minutes. Empty the dispenser and prime with the Assay Buffer. Add 50 ml Assay Buffer and incubate at room temperature 5 min. Empty the dispenser and prime with the Reaction Buffer (see the table below). Add 50 ul Reaction Buffer and incubate at room temperature for 20 minutes. Since the intensity of the chemiluminescent signal is time dependent, and it takes about 10 minutes to read 5 plates on luminometer, one should treat 5 plates at each time and start the second set 10 minutes later.
Read the relative light unit in the luminometer. Set H 12 as blank, and print the results. An increase in chemiluminescence indicates reporter activity.
Reaction Buffer Formulation: of plates Rxn buffer diluent ml) CSPD (mn) 60 3 11 65 3.25 12 70 13 75 3.75 14 80 4 85 4.25 16 90 17 95 4.75 18 100 WO 99/37660 PCT/US99/01313 -161- 19 105 5.25 110 21 115 5.75 22 120 6 23 125 6.25 24 130 135 6.75 26 140 7 27 145 7.25 28 150 29 155 7.75 160 8 31 165 8.25 32 170 33 175 8.75 34 180 9 185 9.25 36 190 37 195 9.75 38 200 39 205 10.25 210 10.5 41 215 10.75 42 220 11 43 225 11.25 44 230 11.5 235 11.75 46 240 12 47 245 12.25 48 250 12.5 49 255 12.75 260 13 Example 21: High- ThroughputScreeningAssay Identifying Changes in Small Molecule Concentration and Membrane Permeability Binding of a ligand to a receptor is known to alter intracellular levels of small molecules, such as calcium, potassium, sodium, and pH, as well as alter membrane potential. These alterations can be measured in an assay to identify supernatants which bind to receptors of a particular cell. Although the following protocol describes an assay for calcium, this protocol can easily be modified to WO 99/37660 PCT/US99/01313 -162detect changes in potassium, sodium, pH, membrane potential, or any other small molecule which is detectable by a fluorescent probe.
The following assay uses Fluorometric Imaging Plate Reader ("FLIPR") to measure changes in fluorescent molecules (Molecular Probes) that bind small molecules. Clearly, any fluorescent molecule detecting a small molecule can be used instead of the calcium fluorescent molecule, fluo-3, used here.
For adherent cells, seed the cells at 10,000 -20,000 cells/well in a Co-star black 96-well plate with clear bottom. The plate is incubated in a CO 2 incubator for 20 hours. The adherent cells are washed two times in Biotek washer with 200 ul ofHBSS (Hank's Balanced Salt Solution) leaving 100 ul of buffer after the final wash.
A stock solution of 1 mg/ml fluo-3 is made in 10% pluronic acid DMSO.
To load the cells with fluo-3, 50 ul of 12 ug/ml fluo-3 is added to each well. The plate is incubated at 37 degree C in a CO 2 incubator for 60 min. The plate is washed four times in the Biotek washer with HBSS leaving 100 ul of buffer.
For non-adherent cells, the cells are spun down from culture media. Cells are re-suspended to 2-5x10 6 cells/ml with HBSS in a 50-ml conical tube. 4 ul of 1 mg/ml fluo-3 solution in 10% pluronic acid DMSO is added to each ml of cell suspension. The tube is then placed in a 37 degree C water bath for 30-60 min.
The cells are washed twice with HBSS, resuspended to 1xl0 6 cells/ml, and dispensed into a microplate, 100 ul/well. The plate is centrifuged at 1000 rpm for min. The plate is then washed once in Denley CellWash with 200 ul, followed by an aspiration step to 100 ul final volume.
For a non-cell based assay, each well contains a fluorescent molecule, such as fluo-3. The supernatant is added to the well, and a change in fluorescence is detected.
To measure the fluorescence ofintracellular calcium, the FLIPR is set for the following parameters: System gain is 300-800 mW; Exposure time is 0.4 second; Camera F/stop is F/2; Excitation is 488 nm; Emission is 530 nm; and Sample addition is 50 ul. Increased emission at 530 nm indicates WO 99/37660 PCT/US99/01313 -163an extracellular signaling event caused by the a molecule, either METH1 or METH2 or a molecule induced by METHI or METH2, which has resulted in an increase in the intracellular Ca" concentration.
Example 22: High- Throughput Screening Assay Identifying Tyrosine Kinase Activity The Protein Tyrosine Kinases (PTK) represent a diverse group of transmembrane and cytoplasmic kinases. Within the Receptor Protein Tyrosine Kinase RPTK) group are receptors for a range ofmitogenic and metabolic growth factors including the PDGF, FGF, EGF, NGF, HGF and Insulin receptor subfamilies. In addition there are a large family of RPTKs for which the corresponding ligand is unknown. Ligands for RPTKs include mainly secreted small proteins, but also membrane-bound and extracellular matrix proteins.
Activation of RPTK by ligands involves ligand-mediated receptor dimerization, resulting in transphosphorylation of the receptor subunits and activation of the cytoplasmic tyrosine kinases. The cytoplasmic tyrosine kinases include receptor associated tyrosine kinases of the src-family src, yes, Ick, lyn, fyn) and non-receptor linked and cytosolic protein tyrosine kinases, such as the Jak family, members of which mediate signal transduction triggered by the cytokine superfamily of receptors the Interleukins, Interferons, GM-CSF, and Leptin).
Because ofthe wide range of known factors capable of stimulating tyrosine kinase activity, identifying whether METH1 or METH2 or a molecule induced by METH1 or METH2 is capable of activating tyrosine kinase signal transduction pathways is of interest. Therefore, the following protocol is designed to identify such molecules capable of activating the tyrosine kinase signal transduction pathways.
Seed target cells primary keratinocytes) at a density of approximately 25,000 cells per well in a 96 well Loprodyne Silent Screen Plates purchased from Nalge Nunc (Naperville, IL). The plates are sterilized with two 30 minute rinses WO 99/37660 PCT/US99/01313 -164with 100% ethanol, rinsed with water and dried overnight. Some plates are coated for 2 hr with 100 ml of cell culture grade type I collagen (50 mg/ml), gelatin or polylysine (50 mg/ml), all of which can be purchased from Sigma Chemicals (St. Louis, MO) or 10% Matrigel purchased from Becton Dickinson (Bedford,MA), or calf serum, rinsed with PBS and stored at 4 degree C. Cell growth on these plates is assayed by seeding 5,000 cells/well in growth medium and indirect quantitation of cell number through use of alamarBlue as described by the manufacturer Alamar Biosciences, Inc. (Sacramento, CA) after 48 hr.
Falcon plate covers #3071 from Becton Dickinson (Bedford,MA) are used to cover the Loprodyne Silent Screen Plates. Falcon Microtest III cell culture plates can also be used in some proliferation experiments.
To prepare extracts, A431 cells are seeded onto the nylon membranes of Loprodyne plates (20,000/200ml/well) and cultured overnight in complete medium. Cells are quiesced by incubation in serum-free basal medium for 24 hr.
After 5-20 minutes treatment with EGF (60ng/ml) or 50 ul of the supernatant produced in Example 14, the medium was removed and 100 ml of extraction buffer ((20 mM HEPES pH 7.5, 0.15 M NaC1, 1% Triton X-100, 0.1% SDS, 2 mM Na3VO4, 2 mM Na4P207 and a cocktail of protease inhibitors 1836170) obtained from Boehringer Mannheim (Indianapolis, IN) is added to each well and the plate is shaken on a rotating shaker for 5 minutes at 4°C. The plate is then placed in a vacuum transfer manifold and the extract filtered through the 0.45 mm membrane bottoms of each well using house vacuum. Extracts are collected in a 96-well catch/assay plate in the bottom of the vacuum manifold and immediately placed on ice. To obtain extracts clarified by centrifugation, the content of each well, after detergent solubilization for 5 minutes, is removed and centrifuged for minutes at 4 degree C at 16,000 x g.
Test the filtered extracts for levels of tyrosine kinase activity. Although many methods of detecting tyrosine kinase activity are known, one method is described here.
WO 99/37660 PCT/US99/01313 -165- Generally, the tyrosine kinase activity of a supernatant is evaluated by determining its ability to phosphorylate a tyrosine residue on a specific substrate (a biotinylated peptide). Biotinylated peptides that can be used for this purpose include PSK1 (corresponding to amino acids 6-20 of the cell division kinase cdc2p34) and PSK2 (corresponding to amino acids 1-17 ofgastrin). Both peptides are substrates for a range of tyrosine kinases and are available from Boehringer Mannheim.
The tyrosine kinase reaction is set up by adding the following components in order. First, add 1ul of5uM Biotinylated Peptide, then 1 ul ATP/Mg 2 ATP/50mM MgC12), then 10ul of 5x Assay Buffer (40mM imidazole hydrochloride, pH7.3, 40 mM beta-glycerophosphate, ImM EGTA, 100mM MgCI 2 5 mM MnCl 2 0.5 mg/ml BSA), then 5ul of Sodium Vanadate(lmM), and then 5ul of water. Mix the components gently and preincubate the reaction mix at 30 degree C for 2 min. Initial the reaction by adding 10ul of the control enzyme or the filtered supernatant.
The tyrosine kinase assay reaction is then terminated by adding 10 ul of 120mm EDTA and place the reactions on ice.
Tyrosine kinase activity is determined by transferring 50 ul aliquot of reaction mixture to a microtiter plate (MTP) module and incubating at 37 degree C for 20 min. This allows the streptavadin coated 96 well plate to associate with the biotinylated peptide. Wash the MTP module with 300ul/well of PBS four times. Next add 75 ul ofanti-phospotyrosine antibody conjugated to horse radish to each well and incubate at 37 degree C for one hour. Wash the well as above.
Next add 100ul of peroxidase substrate solution (Boehringer Mannheim) and incubate at room temperature for at least 5 mins (up to 30 min). Measure the absorbance of the sample at 405 nm by using ELISA reader. The level of bound peroxidase activity is quantitated using an ELISA reader and reflects the level of tyrosine kinase activity.
WO 99/37660 PCTIUS99/01313 -166- Example 23: High-Throughput Screening Assay Identifying Phosphorylation Activity As a potential alternative and/or compliment to the assay of protein tyrosine kinase activity described in Example 22, an assay which detects activation (phosphorylation) of major intracellular signal transduction intermediates can also be used. For example, as described below one particular assay can detect tyrosine phosphorylation of the Erk-1 and Erk-2 kinases. However, phosphorylation of other molecules, such as Raf, JNK, p38 MAP, Map kinase kinase (MEK), MEK kinase, Src, Muscle specific kinase (MuSK), IRAK, Tec, and Janus, as well as any.
other phosphoserine, phosphotyrosine, or phosphothreonine molecule, can be detected by substituting these molecules for Erk-1 or Erk-2 in the following assay.
Specifically, assay plates are made by coating the wells of a 96-well ELISA plate with 0.1ml of protein G (lug/ml) for 2 hr at room temp, The plates are then rinsed with PBS and blocked with 3% BSA/PBS for 1 hr at RT. The protein G plates are then treated with 2 commercial monoclonal antibodies (100ng/well) against Erk-1 and Erk-2 (1 hr at RT) (Santa Cruz Biotechnology).
(To detect other molecules, this step can easily be modified by substituting a monoclonal antibody detecting any of the above described molecules.) After rinses with PBS, the plates are stored at 4 degree C until use.
A431 cells are seeded at 20,000/well in a 96-well Loprodyne filterplate and cultured overnight in growth medium. The cells are then starved for 48 hr in basal medium (DMEM) and then treated with EGF (6ng/well) or 50 ul of the supernatants obtained in Example 14 for 5-20 minutes. The cells are then solubilized and extracts filtered directly into the assay plate.
After incubation with the extract for 1 hr at RT, the wells are again rinsed.
As a positive control, a commercial preparation of MAP kinase (1Ong/well) is used in place of A431 extract. Plates are then treated with a commercial polyclonal (rabbit) antibody (lug/ml) which specifically recognizes the phosphorylated epitope of the Erk-l and Erk-2 kinases (1 hr at RT). This antibody is biotinylated by standard WO 99/37660 PCT/US99/01313 -167procedures. The bound polyclonal antibody is then quantitated by successive incubations with Europium-streptavidin and Europium fluorescence enhancing reagent in the Wallac DELFIA instrument (time-resolved fluorescence). An increased fluorescent signal over background indicates a phosphorylation by METH1 or METH2 or a molecule induced by METHI or METH2.
Example 24: Method of Determining Alterations in the METH1 or METH2 Gene RNA isolated from entire families or individual patients presenting with a phenotype of interest (such as a disease) is be isolated. cDNA is then generated from these RNA samples using protocols known in the art. (See, Sambrook.) The cDNA is then used as a template for PCR, employing primers surrounding regions of interest in SEQ ID NO: 1. Suggested PCR conditions consist of cycles at 95 degree C for 30 seconds; 60-120 seconds at 52-58 degree C; and 120 seconds at 70 degree C, using buffer solutions described in Sidransky, D. el al., Science 252:706 (1991).
PCR products are then sequenced using primers labeled at their 5' end with T4 polynucleotide kinase, employing SequiTherm Polymerase. (Epicentre Technologies). The intron-exon borders of selected exons of METH 1 or METH2 is also determined and genomic PCR products analyzed to confirm the results.
PCR products harboring suspected mutations in METH1 or METH2 is then cloned and sequenced to validate the results of the direct sequencing.
PCR products of METH1 or METH2 are cloned into T-tailed vectors as described in Holton, T.A. and Graham, Nucleic Acids Research 19:1156 (1991) and sequenced with T7 polymerase (United States Biochemical). Affected individuals are identified by mutations in METH1 or METH2 not present in unaffected individuals.
Genomic rearrangements are also observed as a method of determining alterations in the METH1 or METH2 gene. Isolated genomic clones are nicktranslated with digoxigenindeoxy-uridine 5'-triphosphate (Boehringer Manheim), WO 99/37660 PCT/US99/01313 -168and FISH performed as described in Johnson, Cg. et al., Methods Cell Biol. 35:73- 99 (1991). Hybridization with the labeled probe is carried out using a vast excess of human cot-1 DNA for specific hybridization to the METH1 or METH2 genomic locus.
Chromosomes are counterstained with 4,6-diamino-2-phenylidole and propidium iodide, producing a combination of C- and R-bands. Aligned images for precise mapping are obtained using a triple-band filter set (Chroma Technology, Brattleboro, VT) in combination with a cooled charge-coupled device camera (Photometrics, Tucson, AZ) and variable excitation wavelength filters. (Johnson, Cv. et al., Genet. Anal. Tech. Appl. 8:75 (1991).) Image collection, analysis and chromosomal fractional length measurements are performed using the ISee Graphical Program System. (Inovision Corporation, Durham, NC.) Chromosome alterations of the genomic region of METHI or METH2 (hybridized by the probe) are identified as insertions, deletions, and translocations. These METH1 or METH2 alterations are used as a diagnostic marker for an associated disease.
Example 25: Method of Detecting Abnormal Levels of METH1 or METH2 in a Biological Sample METH1 or METH2 polypeptides can be detected in a biological sample, and if an increased or decreased level of METH1 or METH2 is detected, this polypeptide is a marker for a particular phenotype. Methods of detection are numerous, and thus, it is understood that one skilled in the art can modify the following assay to fit their particular needs.
For example, antibody-sandwich ELISAs are used to detect METH1 or METH2 in a sample, preferably a biological sample. Wells of a microtiter plate are coated with specific antibodies to METH1 or METH2, at a final concentration of 0.2 to 10 ug/ml. The antibodies are either monoclonal or polyclonal and are produced by the method described in Example 13. The wells are blocked so that non-specific binding ofMETH1 or METH2 to the well is reduced.
WO 99/37660 PCT/US99/01313 -169- The coated wells are then incubated for 2 hours at RT with a sample containing METH1 or METH2. Preferably, serial dilutions of the sample should be used to validate results. The plates are then washed three times with deionized or distilled water to remove unbounded METHI or METH2.
Next, 50 ul of specific antibody-alkaline phosphatase conjugate, at a concentration of 25-400 ng, is added and incubated for 2 hours at room temperature. The plates are again washed three times with deionized or distilled water to remove unbounded conjugate.
Add 75 ul of 4-methylumbelliferyl phosphate (MUP) or p-nitrophenyl phosphate (NPP) substrate solution to each well and incubate 1 hour at room temperature. Measure the reaction by a microtiter plate reader. Prepare a standard curve, using serial dilutions of a control sample, and plot METHI or METH2 polypeptide concentration on the X-axis (log scale) and fluorescence or absorbance of the Y-axis (linear scale). Interpolate the concentration of the METH1 or METH2 in the sample using the standard curve.
Example 26: Formulating a Polypeptide The METH1 or METH2 composition will be formulated and dosed in a fashion consistent with good medical practice, taking into account the clinical condition of the individual patient (especially the side effects of treatment with the METH1 or METH2 polypeptide alone), the site of delivery, the method of administration, the scheduling of administration, and other factors known to practitioners. The "effective amount" for purposes herein is thus determined by such considerations.
As a general proposition, the total pharmaceutically effective amount of METH1 or METH2 administered parenterally per dose will be in the range of about lug/kg/day to 10 mg/kg/day of patient body weight, although, as noted above, this will be subject to therapeutic discretion. More preferably, this dose is at least 0.01 mg/kg/day, and most preferably for humans between about 0.01 and WO 99/37660 PCT/US99/01313 -170- 1 mg/kg/day for the hormone. If given continuously, METHI or METH2 is typically administered at a dose rate of about 1 ug/kg/hour to about ug/kg/hour, either by 1-4 injections per day or by continuous subcutaneous infusions, for example, using a mini-pump. An intravenous bag solution may also be employed. The length of treatment needed to observe changes and the interval following treatment for responses to occur appears to vary depending on the desired effect.
Pharmaceutical compositions containing METH1 or METH2 are administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, gels, drops or transdermal patch), bucally, or as an oral or nasal spray. "Pharmaceutically acceptable carrier" refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The term "parenteral" as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
METH1 or METH2 is also suitably administered by sustained-release systems. Suitable examples of sustained-release compositions include semipermeable polymer matrices in the form of shaped articles, films, or mirocapsules. Sustained-release matrices include polylactides Pat. No.
3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-Lglutamate (Sidman, U. et al., Biopolymers 22:547-556 (1983)), poly (2hydroxyethyl methacrylate) Langer et al., J. Biomed Mater. Res. 15:167-277 (1981), and R. Langer, Chem. Tech. 12:98-105 (1982)), ethylene vinyl acetate (R.
Langer et al.) or poly-D- (-)-3-hydroxybutyric acid (EP 133,988). Sustainedrelease compositions also include liposomally entrapped METH1 or METH2 polypeptides. Liposomes containing the METH1 or METH2 are prepared by methods known per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324.
WO 99/37660 PCT/US99/0131 3 -171- Ordinarily, the liposomes are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. percent cholesterol, the selected proportion being adjusted for the optimal secreted polypeptide therapy.
For parenteral administration, in one embodiment, METH1 or METH2 is formulated generally by mixing it at the desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a pharmaceutically acceptable carrier, one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. For example, the formulation preferably does not include oxidizing agents and other compounds that are known to be deleterious to polypeptides.
Generally, the formulations are prepared by contacting METH1 or METH2 uniformly and intimately with liquid carriers or finely divided solid carriers or both. Then, if necessary, the product is shaped into the desired formulation. Preferably the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution. Nonaqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes.
The carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such materials are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) polypeptides, polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, manose, or dextrins; chelating agents such as WO 99/37660 PCT/US99/01313 -172- EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and/or nonionic surfactants such as polysorbates, poloxamers, or PEG.
METHI or METH2 is typically formulated in such vehicles at a concentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, at a pH of about 3 to 8. It will be understood that the use of certain of the foregoing excipients, carriers, or stabilizers will result in the formation ofpolypeptide salts.
METH1 or METH2 used for therapeutic administration can be sterile.
Sterility is readily accomplished by filtration through sterile filtration membranes 0.2 micron membranes). Therapeutic polypeptide compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
METH1 or METH2 polypeptides ordinarily will be stored in unit or multidose containers, for example, sealed ampoules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution. As an example of a lyophilized formulation, 10-ml vials are filled with 5 ml of sterile-filtered 1% aqueous METH1 or METH2 polypeptide solution, and the resulting mixture is lyophilized.
The infusion solution is prepared by reconstituting the lyophilized METH1 or METH2 polypeptide using bacteriostatic Water-for-Injection.
The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In addition, METHI or METH2 may be employed in conjunction with other therapeutic compounds.
WO 99/37660 PCT/US99/01313 -173- Example 27: Method of Treating Decreased Levels ofMETH1 or METH2 The present invention relates to a method for treating an individual in need of a decreased level of METH1 or METH2 activity in the body comprising, administering to such an individual a composition comprising a therapeutically effective amount ofMETH 1 or METH2 antagonist. Preferred antagonists for use in the present invention are METH 1 or METH2-specific antibodies.
Moreover, it will be appreciated that conditions caused by a decrease in the standard or normal expression level ofMETH1 or METH2 in an individual can be treated by administering METH 1 or METH2, preferably in the secreted form.
Thus, the invention also provides a method of treatment of an individual in need of an increased level ofMETH 1 or METH2 polypeptide comprising administering to such an individual a pharmaceutical composition comprising an amount of METH1 or METH2 to increase the activity level of METHI or METH2 in such an individual.
For example, a patient with decreased levels of METH1 or METH2 polypeptide receives a daily dose 0.1-100 ug/kg of the polypeptide for six consecutive days. Preferably, the polypeptide is in the secreted form. The exact details of the dosing scheme, based on administration and formulation, are provided in Example 26.
Example 28: Method of Treating Increased Levels ofMETHI or METH2 The present invention also relates to a method for treating an individual in need of an increased level of METH1 or METH2 activity in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of METHI or METH2 or an agonist thereof.
Antisense technology is used to inhibit production ofMETHI or METH2.
This technology is one example of a method of decreasing levels of METH1 or METH2 polypeptide, preferably a secreted form, due to a variety of etiologies, such as cancer.
WO 99/37660 PCT/US99/01313 -174- For example, a patient diagnosed with abnormally increased levels of METH1 or METH2 is administered intravenously antisense polynucleotides at 1.0, 1.5, 2.0 and 3.0 mg/kg day for 21 days. This treatment is repeated after a 7-day rest period if the treatment was well tolerated. The formulation of the antisense polynucleotide is provided in Example 26.
Example 29: Method of Treatment Using Gene Therapy Ex Vivo One method of gene therapy transplants fibroblasts, which are capable of expressing METHI or METH2 polypeptides, onto a patient. Generally, fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in tissue-culture medium and separated into small pieces. Small chunks of the tissue are placed on a wet surface of a tissue culture flask, approximately ten pieces are placed in each flask. The flask is turned upside down, closed tight and left at room temperature over night. After 24 hours at room temperature, the flask is inverted and the chunks of tissue remain fixed to the bottom of the flask and fresh media Ham's F12 media, with 10% FBS, penicillin and streptomycin) is added. The flasks are then incubated at 37 degree C for approximately one week.
At this time, fresh media is added and subsequently changed every several days. After an additional two weeks in culture, a monolayer offibroblasts emerge.
The monolayer is trypsinized and scaled into larger flasks.
pMV-7 (Kirschmeier, P.T. et al., DNA 7:219-25 (1988)), flanked by the long terminal repeats of the Moloney murine sarcoma virus, is digested with EcoRI and HindIIl and subsequently treated with calf intestinal phosphatase. The linear vector is fractionated on agarose gel and purified, using glass beads.
The cDNA encoding METHI or METH2 can be amplified using PCR primers which correspond to the 5' and 3' end sequences respectively as set forth in Example 5. Preferably, the 5' primer contains an EcoRI site and the 3' primer includes a HindIII site. Equal quantities of the Moloney murine sarcoma virus WO 99/37660 PCT/US99/01313 -175linear backbone and the amplified EcoRI and HindIII fragment are added together, in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions appropriate for ligation of the two fragments. The ligation mixture is then used to transform bacteria HB101, which are then plated onto agar containing kanamycin for the purpose of confirming that the vector contains properly inserted METH1 or METH2.
The amphotropic pA317 or GP+aml 2 packaging cells are grown in tissue culture to confluent density in Dulbecco's Modified Eagles Medium (DMEM) with calf serum penicillin and streptomycin. The MSV vector containing the METH1 or METH2 gene is then added to the media and the packaging cells transduced with the vector. The packaging cells now produce infectious viral particles containing the METHI or METH2 gene(the packaging cells are now referred to as producer cells).
Fresh media is added to the transduced producer cells, and subsequently, the media is harvested from a 10 cm plate of confluent producer cells. The spent media, containing the infectious viral particles, is filtered through a millipore filter to remove detached producer cells and this media is then used to infect fibroblast cells. Media is removed from a sub-confluent plate of fibroblasts and quickly replaced with the media from the producer cells. This media is removed and replaced with fresh media. If the titer of virus is high, then virtually all fibroblasts will be infected and no selection is required. If the titer is very low, then it is necessary to use a retroviral vector that has a selectable marker, such as neo or his. Once the fibroblasts have been efficiently infected, the fibroblasts are analyzed to determine whether METHI or METH2 protein is produced.
The engineered fibroblasts are then transplanted onto the host, either alone or after having been grown to confluence on cytodex 3 microcarrier beads.
WO 99/37660 PCT/US99/01313 -176- Example 30: Method of Treatment Using Gene Therapy In Vivo Another aspect of the present invention is using in vivo gene therapy methods to treat disorders, diseases and conditions. The gene therapy method relates to the introduction of naked nucleic acid (DNA, RNA, and antisense
DNA
or RNA) METH 1 or METH2 sequences into an animal to increase or decrease the expression of the METH1 or METH2 polypeptide. The METH1 or METH2 polynucleotide may be operatively linked to a promoter or any other genetic elements necessary for the expression of the METH1 or METH2 polypeptide by the target tissue. Such gene therapy and delivery techniques and methods are known in the art, see, for example, WO90/11092, W098/11779; U.S. Patent NO.
5693622, 5705151,5580859; TabataH. etal. (1997) Cardiovasc. Res. 35(3):470- 479, Chao, J et al. (1997) Pharmacol. Res. 35(6):517-522, Wolff J.A. (1997) Neuromuscul. Disord. 7(5):314-318, Schwartz, B. el al. (1996) Gene Ther.
3(5):405-411, Tsurumi Y. et al. (1996) Circulation 94(12):3281-3290 (incorporated herein by reference).
The METH1 or METH2 polynucleotide constructs may be delivered by any method that delivers injectable materials to the cells of an animal, such as, injection into the interstitial space of tissues (heart, muscle, skin, lung, liver, intestine and the like). The METH1 or METH2 polynucleotide constructs can be delivered in a pharmaceutically acceptable liquid or aqueous carrier.
The term "naked" polynucleotide, DNA or RNA, refers to sequences that are free from any delivery vehicle that acts to assist, promote, or facilitate entry into the cell, including viral sequences, viral particles, liposome formulations, lipofectin or precipitating agents and the like. However, the METH 1 or METH2 polynucleotides may also be delivered in liposome formulations (such as those taught in Felgner P.L. etal. (1995)Ann. NYAcad. Sci. 772:126-139 and Abdallah B. el al. (1995) Biol. Cell 85(1):1-7) which can be prepared by methods well known to those skilled in the art.
WO 99/37660 PCT/US99/01313 -177- The METHI or METH2 polynucleotide vector constructs used in the gene therapy method are preferably constructs that will not integrate into the host genome nor will they contain sequences that allow for replication. Any strong promoter known to those skilled in the art can be used for driving the expression of DNA. Unlike other gene therapies techniques, one major advantage of introducing naked nucleic acid sequences into target cells is the transitory nature of the polynucleotide synthesis in the cells. Studies have shown that nonreplicating DNA sequences can be introduced into cells to provide production of the desired polypeptide for periods of up to six months.
The METHI or METH2 polynucleotide construct can be delivered to the interstitial space of tissues within the an animal, including of muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue. Interstitial space of the tissues comprises the intercellular fluid, mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic fibers in the walls of vessels or chambers, collagen fibers of fibrous tissues, or that same matrix within connective tissue ensheathing muscle cells or in the lacunae of bone. It is similarly the space occupied by the plasma of the circulation and the lymph fluid of the lymphatic channels. Delivery to the interstitial space of muscle tissue is preferred for the reasons discussed below. They may be conveniently delivered by injection into the tissues comprising these cells. They are preferably delivered to and expressed in persistent, non-dividing cells which are differentiated, although delivery and expression may be achieved in non-differentiated or less completely differentiated cells, such as, for example, stem cells of blood or skin fibroblasts. In vivo muscle cells are particularly competent in their ability to take up and express polynucleotides.
For the naked METH1 or METH2 polynucleotide injection, an effective dosage amount of DNA or RNA will be in the range of from about 0.05 g/kg body weight to about 50 mg/kg body weight. Preferably the dosage will be from about WO 99/37660 PCT/US99/01313 -178- 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill will appreciate, this dosage will vary according to the tissue site of injection. The appropriate and effective dosage of nucleic acid sequence can readily be determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration. The preferred route of administration is by the parenteral route of injection into the interstitial space of tissues. However, other parenteral routes may also be used, such as, inhalation of an aerosol formulation particularly for delivery to lungs or bronchial tissues, throat or mucous membranes of the nose. In addition, naked METH1 or METH2 polynucleotide constructs can be delivered to arteries during angioplasty by the catheter used in the procedure.
The dose response effects of injected METH1 or METH2 polynucleotide in muscle in vivo is determined as follows. Suitable METH 1 or METH2 template DNA for production of mRNA coding for METH1 or METH2 polypeptide is prepared in accordance with a standard recombinant DNA methodology. The template DNA, which may be either circular or linear, is either used as naked DNA or complexed with liposomes. The quadriceps muscles of mice are then injected with various amounts of the template DNA.
Five to six week old female and male Balb/C mice are anesthetized by intraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cm incision is made on the anterior thigh, and the quadriceps muscle is directly visualized. The METH1 or METH2 template DNA is injected in 0.1 ml of carrier in a 1 cc syringe through a 27 gauge needle over one minute, approximately 0.5 cm from the distal insertion site of the muscle into the knee and about 0.2 cm deep. A suture is placed over the injection site for future localization, and the skin is closed with stainless steel clips.
After an appropriate incubation time 7 days) muscle extracts are prepared by excising the entire quadriceps. Every fifth 15 um cross-section of the individual quadriceps muscles is histochemically stained for METH1 or METH2 protein expression. A time course for METH 1 or METH2 protein expression may ilAug. 2903 12:59 WRAY&ASSOCIAIES No.3645 P. 7 -179be done in a similar fashion except that quadriceps from different mice are hiarvested at different times. Persistence of METHI or MET-2 DNA in muscle following injection may be determined by Southern blot analysis after preparing total cellular DNA and HIRT sup ernatants from injected and control mice. The results of the above experimentation in mice can be used to extrapolate proper dosages and other treatment parameters in humans and other animals using METHi or METH2 naked DNA.
It will be clear that the invention may be practiced otherwise than as particularly described in the foregoing description and examples.
Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the appended claims.
The entire disclosure of all publications (including patents, patent applications, journal articles, laboratory manuals, books, or other documents) cited herein are hereby incorporated by reference.
Throughout the specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.
COMs ID No: SMBI-00382467 Received by IP Australia: Time 14:25 Date 2003-08-19 WO 99/37660 WO 9937660PCT/US99/01313 -179.1- Applicant's or agent's file reference number: 1488.107PC02 IInternational application
TBA
INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule l3bis) A. The indications made below relate to the microorganism referred to in the description on page 32. lines 16-17.] B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet 0 Name ot'depository institution American Type Culture Collection Address of depository institution including postal code and 10801 University Boulevard formerly.at: 12301 Parklawn Drive Manassas. Virginia 20110-2209 Rockville, Maryland 20852 United States of America United States of America C. ADDITIONAL INDICATIONS deave blank if not applicable) This information is continued on an additional sheet C3 DNA plasmid HOUCQ 17 D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE tif the indications are not for all designatedStaics) E. SEPARATE FURNISHING OF INDICATIONS (leare i hck ,Iaapplicahki The indications listed below will be submitted to the international B~ureau later u'spcifv the general nature of the indications. "Accession Number of Deposit"') lFor receiving Of'fice use onlN 0 Fhis sheet was received with the internattonal application Authorized officer For International Bureau use onl% 0 I'his sheet was received by the International Bureau on- Authorized officer I O7expsoljppct Fo1rm l1CT?'RO/134d(u% 1992) WO 99/37660 PCT/US99/01313 -179 2- Applicant's or agent's file reference number: 1488.107PC02 International application No: TBA INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule 13bis) A. The indications made below relate to the microorganism referred to in the description on page 32. lines 25-26.
B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet O Name of depository institution American Type Culture Collection Address of depository institution tincluding postal code and country 10801 University Boulevard formerly at: 12301 Parklawn Drive Manassas. Virginia 20110-2209 Rockville. Maryland 20852 United States of America United States of America Date of deposit Accession Number January 1998 209582 C. ADDITIONAL INDICATIONS (leave blank ifnot applicable) This information is continued on an additional sheet O DNA plasmid HCE4D69 D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indicatons are not /or all designated Statesi E. SEPARATE FURNISHING OF INDICATIONS hik no, ppltcauht; The indications listed below will be submitted to the international Burcau later (specif the general nature of the indications, e.g..
"Accession .umber of Deposit" Ior receiving Office use only O This sheet \\as received with the internatonal application Authorized officer For International Bureau use onl\ O This sheet was received by the International Bureau on: Authorized officer Form PCT.'RO!l34 Juts 1992) 107cxpsojp. 2pct EDITORIAL NOTE APPLICATION NUMBER 24641/99 The following Sequence Listing pages 1 to 240 are part of the description. The claims pages follow on pages 180 to 189.
PCTIUS99/01313 WO 99/37660
-I-
SEQUENCE LISTING <110> Iruela-Arispe, Luisa Hastings, Gregg A.
Ruben, Steven M.
<120> Methi and Meth2 Polynucleotides and Polypeptides <130> 1488.107PC02 <140> <141> <150> <151> 60/072,298 1998-01-23 <150> 60/098,539 <151> 1998-08-28 <160> 93 <170> Patentln Ver.
<210> <211> <212> <213> 1 3261
DNA
Homo sapiens <220> <221> CDS <222> .(2853) <220> <221> UI <222> (3 <223> Me <220> <221> U!l <222> <223> M~ <220> <221> U! <222> <223> M <220> <221> U] <222>( <223> M <400> 1 atg ggg IS URE ~095) ~y be
ISURE
3248) ay be
.SURE
3255) ay be
NSURE
3261) ay be any nucleic acid any nucleic acid any nucleic acid any nucleic acid aac gcg gag cgg gct ccg ggg tct cgg agc ttt ggg ccc gta 48 WO 99/37660 PCT/US99/01313 -2- Met Gly Asn Ala Glu Arg Ala Pro Gly Ser Arg Ser Phe Gly Pro Val 1 5 10 ccc acg etg ctg ctg ctc gee geg gcg cta ctg gcc gtg tcg gac gca 96 Pro Thr Leu Leu Leu Leu Ala Ala Ala Leu Leu Ala Val Ser Asp Ala 25 etc ggg cgc ccc tcc gag gag gac gag gag cta gtg gtg ceg gag ctg 144 Leu Gly Arg Pro Ser Glu Glu Asp Glu Glu Leu Val Val Pro Glu Leu 40 gag cgc gcc ccg gga eac ggg acc acg cgc ctc cgc ctg cac gec ttt 192 Glu Arg Ala Pro Gly His Gly Thr Thr Arg Leu Arg Leu His Ala Phe 55 gac cag cag ctg gat etg gag ctg egg ccc gac agc ago ttt ttg gcg 240 Asp Gin Gin Leu Asp Leu Glu Leu Arg Pro Asp Ser Ser Phe Leu Ala 70 75 ccc ggc ttc acg etc cag aac gtg ggg cgc aaa tcc ggg tc gag aeg 288 Pro Gly Phe Thr Leu Gln Asn Val Gly Arg Lys Ser Gly Ser Glu Thr 90 ccg ctt ccg gaa acc gac ctg gcg cae tge ttc tac too ggo aoo gtg 336 Pro Leu Pro Glu Thr Asp Leu Ala His Cys Phe Tyr Ser Gly Thr Val 100 105 110 aat ggo gat ccc age tog get geo gee etc ago otc tge gag ggc gtg 384 Asn Gly Asp Pro Ser Ser Ala Ala Ala Leu Ser Leu Cys Glu Gly Val 115 120 125 cgc gge gee tte tae ctg ctg ggg gag geg tat tte ate eag ccg etg 432 Arg Gly Ala Phe Tyr Leu Leu Gly Glu Ala Tyr Phe Ile Gin Pro Leu 130 135 140 ccc gee gee age gag cgc etc gee ace gee gee cca ggg gag aag ccg 480 Pro Ala Ala Ser Glu Arg Leu Ala Thr Ala Ala Pro Gly Glu Lys Pro 145 150 155 160 ccg gea eca eta eag tte cac ete ctg egg egg aat egg eag gge gao 528 Pro Ala Pro Leu Gin Phe His Leu Leu Arg Arg Asn Arg Gin Gly Asp 165 170 175 gta gge gge aeg tge ggg gte gtg gac gac gag ccc egg ceg act ggg 576 Val Gly Gly Thr Cys Gly Val Val Asp Asp Glu Pro Arg Pro Thr Gly 180 185 190 aaa geg gag acc gaa gae gag gae gaa ggg act gag ggc gag gao gaa 624 Lys Ala Glu Thr Glu Asp Glu Asp Glu Gly Thr Glu Gly Glu Asp Glu 195 200 205 ggg cot eag tgg teg ceg cag gae ceg gca etg caa gge gta gga eag 672 WO 99/37660 PCT/US99/01313 -3- Gly Pro Gin Trp Ser Pro Gin Asp Pro Ala Leu Gin Gly Val Gly Gin 210 215 220 ccc aca gga act gga age ata aga aag aag cga ttt gtg tcc agt cac 720 Pro Thr Gly Thr Gly Ser Ile Arg Lys Lys Arg Phe Val Ser Ser His 225 230 235 240 cgc tat gtg gaa acc atg ctt gtg gca gac cag tcg atg gca gaa ttc 768 Arg Tyr Val Glu Thr Met Leu Val Ala Asp Gin Ser Met Ala Glu Phe 245 250 255 cac ggc agt ggt cta aag cat tac ctt ctc acg ttg ttt tcg gtg gca 816 His Gly Ser Gly Leu Lys His Tyr Leu Leu Thr Leu Phe Ser Val Ala 260 265 270 gcc aga ttg tac aaa cac ccc agc att cgt aat tca gtt agc ctg gtg 864 Ala Arg Leu Tyr Lys His Pro Ser Ile Arg Asn Ser Val Ser Leu Val 275 280 285 gtg gtg aag atc ttg gtc atc cac gat gaa cag aag ggg ccg gaa gtg 912 Val Val Lys Ile Leu Val Ile His Asp Glu Gin Lys Gly Pro Glu Val 290 295 300 acc tcc aat gct gcc ctc act ctg cgg aac ttt tgc aac tgg cag aag 960 Thr Ser Asn Ala Ala Leu Thr Leu Arg Asn Phe Cys Asn Trp Gin Lys 305 310 315 320 cag cac aac cca ccc agt gac cgg gat gca gag cac tat gac aca gca 1008 Gin His Asn Pro Pro Ser Asp Arg Asp Ala Glu His Tyr Asp Thr Ala 325 330 335 att ctt ttc acc aga cag gac ttg tgt ggg tcc cag aca tgt gat act 1056 Ile Leu Phe Thr Arg Gin Asp Leu Cys Gly Ser Gin Thr Cys Asp Thr 340 345 350 ctt ggg atg gct gat gtt gga act gtg tgt gat ccg age aga agc tgc 1104 Leu Gly Met Ala Asp Val Gly Thr Val Cys Asp Pro Ser Arg Ser Cys 355 360 365 tcc gtc ata gaa gat gat ggt tta caa get gcc ttc acc aca gcc cat 1152 Ser Val Ile Glu Asp Asp Gly Leu Gin Ala Ala Phe Thr Thr Ala His 370 375 380 gaa tta ggc cac gtg ttt aac atg cca cat gat gat gca aag cag tgt 1200 Glu Leu Gly His Val Phe Asn Met Pro His Asp Asp Ala Lys Gin Cys 385 390 395 400 gcc agc ctt aat ggt gtg aac cag gat tcc cac atg atg gcg tca atg 1248 Ala Ser Leu Asn Gly Val Asn Gin Asp Ser His Met Met Ala Ser Met 405 410 415 ctt tcc aac ctg gac cac age cag cct tgg tct cct tgc agt gcc tac 1296 PCTIUS99/01313 WO 99/37660 Leu atg Met Ser att Ile Asn aca Thr 435 caa Leu 420 tca Ser aat Asp ttt Phe ccc His ctg Leu at a Se r gat Asp cag Gin aat Asn 440 etc Pro 425 ggt Gly cca Ser ggg Giy gat Pro gaa Giu etc Cys t gt Cys 445 ect Pro Ser 430 ttg Leu ggC Giy Ala atg Met ace Thr Tyr gac Asp tcg Ser 1344 1392 Lys Pro Gin Asn Pro Ile Gin Leu Pro Gly Asp Leu 450 455 4 0 tac Tyr 465 ca c His ace Thr gat Asp gt g Val1 gat A\sp tgc Cys t et Se r gge Gly aac Asn gee Ala ect Pro ggt Gly ace Thr 515 aaa Lys aac As n gat Asp ggg Gly 500 age Ser ace Thr egg Arg g ca Al a 485 gtg Val tgt Cys gac Asp eag Gin 470 gee Al a ctg Leu gga Gly aga Arg tge Cys age Ser gtg Val ga a Giu aag Lys cag Gin aca Thr tgt Cys ggg Gly 520 cat His ttt Phe tgt Cys caa Gin 505 aaa Lys ttt Phe aca Thr age Se r 490 ace Thr t gg T rp gat Asp ~ttt Phe 475 ace Thr aaa Lys t gt Cys a eq Thr ggg Gly ttg Leu cac His ate Ile cet Pro 540 gag Giu tgg Trp tte Phe aae Asn 525 ttt Phe ga e Asp t gt Cys ecg Pro 510 ggC Gly eat His tee Ser ace Thr 495 t gg Trp aag Lys gga Gly aaa Lys 480 9ge Giy geg Al a tgt Cys age Ser 530 tgg Trp 545 gga Gly gga Gly et t Leu t gt Cys geg gga Gly gte Val ggg Gly gag Giu gaa Giu 610 gt g at g Met ca g Gin aag Lys gac Asp 595 gea Al a gaa tgg Trp tac Tyr tac Tyr 580 t gt Cys eac His tgg 9gg Gly aeg Thr 565 tgt Cys eca Pro aae Asn att ect Pro 550 atg Met gaa Glu gac Asp gag Glu
CC(
tgg Trp agg Arg gge Gly aat Asn ttt Phe 615 aag jga 31y gaa Giu aaa Lys aa t Asn 600 tea Ser t ac gac Asp tgt Cys ega Arg 585 gga Gly aaa *Lys -get :gt :ys gac Asp 570 gt g Val1 aaa Lys get Al a 9gC teg z Ser 555 aac Asn ege Arg ace Thr tee *Ser -gte ~ga krg cca Pro t ac Tyr ttt Phe t tt Phe 620 tca aegI Thr gte Val aga Arg aga Arg 605 ggg Gly cca tge Zys cca Pro tee Ser 590 gag Giu agt Ser a ag ggt Gly aag Lys 575 tgt Cys gaa Giu ggg Gly gac gga Giy 560 aat aae Asn eaa Gin ect Pro agg 1440 1488 1536 1584 1632 1680 1728 1776 1824 1872 1920 PCTIUS99/01313 WO 99/37660 Ala V 625 tgc a Cys L cag c Gin P gtc t Vai C gao Asp tct Ser 705 tat T yr aaa Lys atc Ile too Ser ago Ser 785 aaa Lys ai ag 'ys cc ~ro :gt ~ys :cc Ser 690 act Thr cat His cag Gin aaa Lys aO' Th 77( gg( Gi' ga Gi Giu T cto a Leu I aag g Lys V 6 gtg c Val G 675 aaa a Lys 1 tgtE Cys gat Asp Cgg Arg gct Ala 755 ztta :Leu 0 ctac y Ser g ccc u Pro rp Ile Pro Lys Tyr Ala Gly Val Ser Pro Lys Asp Arg tc t le C 6 tt g al V 60 .aa in C ~ag KySI ~aa .ys atc Ile aac As n 740 got Ala gag Giu tct Ser ttg Leu go .ys '45 t a ral ;ga a.ag aaa Lys atc Ile 725 cag Gin caa Gin gat Asp cag Gin ttt Phe ata Ile 710 aca Thr agc Arc gcc Ala ggt Gly tgt Cys gat Asp 695 tca Ser att Ile gga Gly ~aaa Lys act Thr gt a Val 680 aa Lys gga Giy cca Pro t cc Ser ggc Gly cca Pro 665 aaa Lys tgt Cys tca Ser act Thr agg Arg 745 att Ile 650 L gt Cys gct Ala ggt Giy gtt Val1 gga Giy 730 aac Asr 9go Gly agc Ser ggt Gly gtt Val act Thr 715 gcc Ala aat Asn tac t Tyr P cca g Pro A tgt g Cys P~ 6 tgc Cys 700 agt Ser aco Thr ggo *Gly *ggt Gly gtt SVai 780 c ago g Ser 5 1 Giy to t he F at t Cp lat ~sp jgg ;iY gca ,la sac As n ago Ser gao Asp 765 gt 0 Val ttt Phe .tc ~he cc0 ~er gc krg gga aaa Lys ato Ile ttt Phe tac Tyi tt~ Lei ag Se.
gtt Val 655 aco Thr ato Ile aat Asn ct Pro gaa Giu 735 ott Leu act Thr I agc *1 Arc *coc Pr( ttg Leu tot Ser ata Ile gga Gly gga Gly 720 gt g Val gc Al a ttg Leu tao Tyr oto Leu 800 1968 2016 2064 2112 2160 2208 2256 2304 2352 2400 2448 gat ggc aca tat att ott aat Asp oaa Gin gog Al a aco Thr Gly gao Asp gca Ala 790 at 0 Ile Thr at t Ile 775 ttg Leu cag Gin T yr 760 at g Met gaa Giu gt t Val tao Tyr aga Arg ott Leu aaa Lys at t Ile act Thr ggt Gl~ cg Ar 79.
gt Va aat goc ott oga Asn Ala Leu Arg 815 805 810 cot aaa att aaa tao aco tao tto gta aag aag aag aag gaa tot tto Val Lys Lys Lys Glu Ser Phe Pro aat Lys got Ile ato Lys 820 coo Tyr act Tyr t ca 825 tgg gt c gaa gag 830 t gg ggO gaa 2496 2544 WO 9937660PCTIUS99/01313 -6- Asn Ala Ile Pro Thr Phe Ser Ala Trp Val Ile Glu Glu Trp Gly Glu 835 840 ggt Gly tgt tot Cys Ser 850 tca tgt gaa Ser Cys Glu ttg Le u 855 ct Pro tgg oag aga Trp Gin Arq aga Arg 860 gca Al a otg qta gaa t'gc Leu Val Glu Cys oga Arg 865 gac att aat gga Asp Ile Asn Gly cag Gin 870 oct Pro got tcc gag Ala Ser Glu tgt Cys 875 ccc Pro aag gaa gtg Lys Glu Val aag Lys 880 coa goo ago aoo Pro Ala Ser Thr aga Arg 885 t oa Ser tgt goa gao Cys Ala Asp oat His 890 aco Th r tgo 000 cag Cys Pro Gin tgg cag Trp Gin 895 tao aaa Tyr Lys 2592 2640 2688 2736 2784 2832 ctg ggg gag Leu Gly Glu aaa aga ago Lys Arg Ser 915 gag ago tgt Glu Ser Cys 930 tgg Trp 900 ttg Leu tca tgt tot Ser Cys Ser aag Lys 905 aag tgt ctg Lys Cys Leu too oat gat Ser His Asp 920 aaa oct aaa Lys Pro Lys tgt ggg aag Cys Gly Lys gga ggg gtg Gly Gly Val 925 cat ttc ata His Phe Ile 940 tta tot cat Leu Ser His gao ttt tgc Asp Phe Cys ggt Gly 910 gat oct Asp Pro tta aag Leu Lys 935 agt taa Se r aca atg goa gaa tgO Thr Met Ala Glu Cys 945 gtggtttaag tggtgttagO tttgaggcaa 2883 ggcaaagtga ttgcoagtaa gttgaatcat taacctctga tgttacatct aaaaaaaaaa catgfltgtca ggaagggctg coagtgaggt cagagtaaac gcagtgatat attacaagtt aaagggcggc tnagtctfl gtgcagggaa gtatcagtaa tgcCagttgC agoataataa tagaaaaaac cgctctagag agcaagaagg ggtgggatta aaatttgata anccccgggc aaagcaattg gatcctcga ctggagggat tgggggtaga ggatagttag attattatta toaaaaaaaa ggggoccaag ccagogtatc tagaaaagga tgaggattat ttatttcttt aaaaaaaaaa cttaogcgtg 2943 3003 3063 3123 3183 3243 3261 <210> 2 <211> 950 <212> PRT <213> Homo sapiens <400> 2 PCTIUS99/01313 WO 99/37660 Met Gly Asn Ala Gin Arg Ala Pro Gly Ser Arg Ser Phe Gly Pro Val Pro Leu Glu Asp Pro Pro Asn Arg Pro 145 Chr Gly Arg Gin Gly Leu Gly Giy 1 3C AiE Len Arg Ala Gin Phe Pro Asp 115 Ala Ala Len Pro Pro Leu Thr Gin 100 Prc PhE Se Len Le Ser G] Gly H: Asp Li Leu G Thr A Ser S Tyr I r Glu I~ u Gin 165 r Cys 0 r Glu p Ser ~r Gly -u Thr 245 Ly Len -u is 70 in
SF
Ala Gin Gly 55 Giu Asn Leu Ala Ala Asp 40 Thr Leu Val Ala Ala Ala 25 Glu Thr Ar g Giy His 105 Al a Leu Gin Arg Pro Arg 90 Cys Leu Leu Len Len Asp 75 Lys Phe Ser Al a Val1 Ar g Ser Ser Tyr Leu Val Val Leu Ser Gly Ser Cys Ser Pro His Phe Ser Gly 110 Gic Asp Glu Ala Len Gin Thr Gly Al a Leu Phe Ala Thr Val1 Val1 120 125 e u ~rg Leu 135 Len Gly Ala Gin Thr Ala Ala Pro Val Lys Gly Pro 225 Arg His Ala Gly Ala Pro 210 Thr Tyr Gly Pro Gly Giu 195 Gin Gly Val1 Ser Lei Th 18 Th Tr T12
GJ
?he G1y Asp Pro Ser 230 Met Lys His Val1 Giu Gin 215 Ile Leu Val Asp 200 Asp Arg Len Asp 185 Giu Pro Lys Arg 170 Asp Gly Al a Lys Tyr Phe Ile 140 Ala Pro Gly 155 Arg Asn Arg Gin Pro Arg Thr Gin Gly 205 Len Gin Gly 220 Arg Phe Val Gin Gin Pro 190 Gin Val1 Ser Pro Lys Giy 175 Thr Asp Gly Ser Leu Pro 160 Asp Gly Gin Gin His 240 Phe Ala 235 Len His Val Tyr Ala Leu Asp 250 Leu Gin Thr Ser Leu Met Phe Al a Ser Gin 255 Val 265 270 260 Ala Arg Leu Tyr Lys His Pro Ser Ile Arg Asn Ser Val Ser Len Val WO 99/37660 PCT/US99/01313 -8- 275 280 285 Val Val Lys Ile Leu Val Ile His Asp Glu Gin Lys Gly Pro Glu Val 290 295 300 Thr Ser Asn Ala Ala Leu Thr Leu Arg Asn Phe Cys Asn Trp Gin Lys 305 310 315 320 Gin His Asn Pro Pro Ser Asp Arg Asp Ala Glu His Tyr Asp Thr Ala 325 330 335 Ile Leu Phe Thr Arg Gin Asp Leu Cys Gly Ser Gin Thr Cys Asp Thr 340 345 350 Leu Gly Met Ala Asp Val Gly Thr Val Cys Asp Pro Ser Arg Ser Cys 355 360 365 Ser Val Ile Glu Asp Asp Gly Leu Gin Ala Ala Phe Thr Thr Ala His 370 375 380 Glu Leu Gly His Val Phe Asn Met Pro His Asp Asp Ala Lys Gin Cys 385 390 395 400 Ala Ser Leu Asn Gly Val Asn Gin Asp Ser His Met Met Ala Ser Met 405 410 415 Leu Ser Asn Leu Asp His Ser Gin Pro Trp Ser Pro Cys Ser Ala Tyr 420 425 430 Met Ile Thr Ser Phe Leu Asp Asn Gly His Gly Glu Cys Leu Met Asp 435 440 445 Lys Pro Gin Asn Pro Ile Gin Leu Pro Gly Asp Leu Pro Gly Thr Ser 450 455 460 Tyr Asp Ala Asn Arg Gin Cys Gin Phe Thr Phe Gly Glu Asp Ser Lys 465 470 475 480 His Cys Pro Asp Ala Ala Ser Thr Cys Ser Thr Leu Trp Cys Thr Gly 485 490 495 Thr Ser Gly Gly Val Leu Val Cys Gin Thr Lys His Phe Pro Trp Ala 500 505 510 Asp Gly Thr Ser Cys Gly Glu Gly Lys Trp Cys Ile Asn Gly Lys Cys 515 520 525 Val Asn Lys Thr Asp Arg Lys His Phe Asp Thr Pro Phe His Gly Ser 530 535 540 Trp Gly Met Trp Gly Pro Trp Gly Asp Cys Ser Arg Thr Cys Gly Gly 545 550 555 560 PCTIUS99/01 313 WO 99/37660 Gly Val Gin Tyr Thr Met Arg Giu Cys Asp Asn Pro Val Pro Lys Asn 565 i Gly Gly Lys Tyr Cys Giu Gly Leu Cys Ala 625 Cys Gin Val1 Asp Ser 705 Tyi Ly~ Giu 7 Giu2 610 Val Lys Pro Cys Ser 690 Thr His s Gin ~sp 395 kla Giu Le u Lys Val 675 Lys Cys Asp Arc Cys His T rp Ile Vai 660 Gin Lys Lys Ile Asn Pro As n Ile Cys 645 Val1 Gly Lys Lys Ile 721 Gir Asp Giu Pro 630 Gin Asp Gin Phe Ile 710 Thr Arg ks n The 615 Lys Ala Giy Cys Asp 695 Sex IlE Gl Lys A 5 Asn G 600 Ser L Tyr P LysC ThrI Val 680 Lys Gly Pro jSer r Tyr 760 e Met 5 u Glu n Val rg V 85 ly L ,ys J liaC ly Pro 665 Lys Cys Ser Thr Arg 745 Ile *ai P ,ys 1 ~la 3iY Ile 650 Cys Ala Gly Val1 Gly 730 Asn Leu ~rg ~hr 3er Val 635 31 y Ser Gly ValI Thi 71~ Al As As Tyr Phe Phe 620 Ser Tyr Pro Cys Cys 700 Ser Thi Gl G1 Arg S 5 Arq G 605 Gly S Pro L Phe I Asp Asp 685 Gly Ala Asn jSer y Asp 765 1 Vai 0 r Phe er C iu G er G ,ys A ~he Ser 670 Arg G1y Lys Ile Phe 750 Tyr Leu Ser ys iu iy ~sp al1 355 Uhr Ile is n Pro Gi.
731 Lei Thi Ar Pr Asn Gin Pro Arg 640 Leu Ser Ile Giy Giy 720 Vai a Ala r Leu g Tyr o Leu 800 740 Ile Ser Ser 785 Lys Lys Thr 770 Gly Glu Al a 755 Leu Ser Pro Aila Giu Se r Leu Asp Gly Gin Asp Ala Aia 790 Thr Ilie Th: Ii 77 Le Gi Tyr Arg Lys Ile Gly Arg 795 Leu Thr Val Gly Asn Ala Leu Arg 810 815 805 Pro Lys Ile Lys 820 Tyr Thr Tyr Phe Val 825 Lys Lys Lys Lys Glu Ser Phe 830 PCTIUS99/01313 WO 99/37660 Asn Ala Cys Se3 Arg Asi 865 Pro Al Leu Gi Lys Ar Glu Se 93 Thr Me 945 <210> <211> <212> <213> <220> <221> <222> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> 0 y r 0 3 Ile 835 Lys Ile Ser Glu Ser 915 Cys Al a Pro Ser Asn Thr T rp 900 Leu Asp Giu Thr Cys Gly Arg 885 Ser Lys Pro Cys Phe Glu Gin 870 Pro Se r Cys Leu Ser 950 Ser Le u 855 Pro Cys Cys Leu Lys 935 Al a 840 Giy Al a Ala Ser Ser 920 Lys Trp T rp Ser Asp Lys 905 His P ro Val1 Gin Glu His 890 Thr Asp Lys Ile Arg Cys 875 Pro Cys Gly His Glu Glu 845 Arg Leu 860 Ala Lys Cys Pro Gly Lys Gly Val 925 Phe Ile 940 Trp Val1 Giu Gin Gly 910 Leu Asp Gly Glu Val T rp 895 Tyr Ser Phe Giu Cys Lys 880 Gin Lys His Cys 3008
DNA
Homno sapiens
CDS
(2670)
UNSURE.
(2887) May be any nucleic acid
UNSURE
(2957) May be any nucleic acid
UNSURE
(2970) *May be any nucleic acid
UNSURE
(2981).
May be any nucleic acid WO 99/37660 PCTIUS99/01313 <400> 3 atg ttc coo gcc coo gcc gco coo cgq Met Phe Pro Ala Pro Ala Ala Pro Arg 1 ctg Leu goa Ala ggC Gly ttc Phe aag Lys ggg Gly t cg Ser ctg Leu ctg Leu 145 000 Pro agg Arg ctg Leu gco Al a agc Ser gt g Val atc Ile ctg Leu otg Leu gac Asp 130 got Al a ct c Leu cac Glr ctg Leu ggg Gly gcg Ala ttg Leu gag Glu cgc Arg gcg Al a 115 ggo Gi y ca g Gin ccg Pro gag 1Giu ctg Leu 9gg Gly ggc Gly cgc Arg ogo Arg ggc Gly 100 gog Ala gag Glu cog Pro oga Arg aga Arg 180 5 ctg Leu cag Gin gag Glu ctg Leu ct c Leu t gt Cys gt 0 Val1 gag Glu cac His gga Gly 165 gga Gly ctg L eu gcc Al a ctc Leu 9gg Ala 70 ggg Gly ttt Phe ago Ser tto Phe ogo Arg 150 oc Prc gac AsF oog Pro tog Ser gog Ala 55 000 Pro ggo Gly ttt Phe ot g Leu aoo Thr 135 otg Leu gag Glu oao His ctg Leu gag Glu 40 oto Leu gao Asp too Se r too Ser tgo Cys 120 at 0 Ile oag Gin t gg T rp oag Gin gcc U la 25 ct g Leu cac His gao Asp ggo Gly ggo Gly 105 ogo Arg oag Gin ogo Arg gag GiL gac Gb.
18~ -11tgg TrpI 10 ogo Arg gtg Val ctg Leu ago Ser ogg Arg 90 aoo Thr ggg Gly oog *Pro tgg Trp gtg 1Val 170 gao i Asp ct t Leu ggc Gly gt g Val1 too Ser tto Phe 75 gog Al a gt g Val otg Leu oag Gin ggt Gly 155 gag Glu ago Ser cog Pro gc Al a coo Pro goo Ala ctg Leu aoo Thr aat As n ago Ser ggo Gi y 140 000 Pro a og Thr gag Glu tto Phe Pro acg Thr tto Phe gog Al a ggg Gly ggg Gly ggo Giy 125 gog Al a goo Al a gga Gly gag Glu ctg gcc Ala Cgg Pirg Gly 000 Pro ggc Gly gag Glu 110 too Ser ggg Gly gga Gly gag Giu gag Gic 190 Otg Leu Cgg Arg ttg Leu aag Lys gag Giu gag Glu 000 Pro tto Phe ggo Gly goo Ala ggt Gly 175 ago Ser ctg Leu 000 Pro 000 Pro ggc Gly tt 0 Phe ogg Arg gag Giu ot g Leu too Ser ogo Arg 160 cag Gin oaa Gin 48 96 144 192 240 288 336 384 432 480 528 576 624 gaa gag gag goa gaa ggc got ago gag cog ooa oog coO ctg ggg goo Glu Giu Glu Ala Glu Gly Ala Ser Glu Pro Pro Pro Pro Leu Gly Ala I q, 200 205 PCT/US99/01313 WO 99/37660 -12acg agt Thr Ser 210 agg acc aag cgg Arg Thr Lys Arg gtg tct gag gcg Val Ser Glu Ala cgo Arg 220 tto gtg gag acg Phe Val Glu Thr ctg Leu 225 ctg gtg gOc gat Leu Val Ala Asp gcg Ala 230 tcc atg got gcc Ser Met Ala Ala tto Phe 235 tao ggg gcc gao Tyr Gly Ala Asp 672 720 768 cag aac oac ato Gin Asn His Ile ctg Leu 245 acg tta atg tct Thr Leu Met Ser gtg Va1 250 gca goo oga ato Ala Ala Arg Ile tao aag Tyr Lys 255 cac ccc ago His Pro Ser ato gta gaa Ile Val Glu 275 aag aat too ato Lys Asn Ser Ile aao Asn 265 otg atg gtg gta Leu Met Val Val aaa gtg otg Lys Val Leu 270 aat ggg ggg Asn Gly Gly 816 864 gat gaa aaa tgg Asp Glu Lys Trp gg0 Gly 280 cca gag gtg too Pro Glu Val Ser ott aod Leu Thr 290 otg ogt aao tto Leu Arg Asn Phe tgo Cys 295 aao tgg oag ogg Asn Trp Gin Arg ogt Arg 300 tto aac cag ccc Phe Asn Gin Pro ago Ser 305 gac cgo cac cca Asp Arg His Pro gag Glu 310 cac tao gao acg His Tyr Asp Thr goo Ala 315 ato ctg otc aoo Ile Leu Leu Thr aga Arg 320 912 960 1008 oag aao tto tgt Gin Asn Phe Cys oag gag ggg otg Gin Glu Gly Leu tgt Cys 330 gao aco ctg ggt Asp Thr Leu Gly gtg gca Val Ala 335 gao ato ggg Asp Ile Gly gat gag ggg Asp Glu Gly 355 att tgt gac cc Ile Cys Asp Pro aao Asn 345 aaa ago tgo too Lys Ser Cys Ser gtg ato gag Val Ile Glu 350 ota ggg cac Leu Gly His 1056 1104 otc cag gog goo Leu Gin Ala Ala ac ctg goo cat Thr Leu Ala His gaa Glu 365 gto otc Val Leu 370 ago atg cc cac Ser Met Pro His gao Asp 375 gao too aag cc Asp Ser Lys Pro tgo Cys 380 aca ogg otc tto Thr Arg Leu Phe ggg Gly 385 cc atg ggo aag Pro Met Gly Lys cac His 390 cac gtg atg gca His Val Met Ala oog Pro 395 otg tto gto cac Leu Phe Val His otg Leu 400 1152 1200 1248 aac cag acg Asn Gin Thr otg cc Leu Pro 405 tgg too cc tgo Trp Ser Pro Cys ago Ser 410 goo atg tat ctc Ala Met Tyr Leu aca gag Thr Glu 415 PCTIUS99/01313 WO 99/37660 ctt ctg gac ggc ggg cac gga gac tgt Leu Leu Asp Gly Gly His Gly Asp Cys 420 9CC C Ala L ctg g Leu 7 cccE Pro 465 gat Asp gct Ala tgt Cys ggc Gly gga Gi y 545 aat Asn cac His tgt Cys ct g :tg ~eu ;ac tsp ~ac ks n 1gg 31y Asp ct a Leu tgg Trp 530 gga Gly gga Giy a cc Thi gac Gi1 ca ccc c Pro L 435 cag c Gin C acc t Thr gct Ala ggc Gly cct Pro 515 gca Ala gt a Val gga Gly gag Giu j aag i Lys 595 g tgg t c ~eu ~ag ;ln :ct 3er gag Glu acg Thr 500 gag Giu cog Pro cag Gin aga Arg gaa Glu 580 tat Tyi gt coo Pro tgc Cys got Ala ccc Pro 485 cog Pro gag Glu tgg Trp ttt Phe tac Tyr 565 tgc Cyl aal Asi cc aca g Thr G agg C Arg C cag 470 otgI Leu tgo Cys gaa Glu gga Gly toa Ser 550 tgo Cys coo Pro 900 -i Ala caag [go ~ag ln 155 ;a 0 ksp Cys Gly gtg Val1 ccc Pro 535 cac His ct C Let cct Prc ta( Ty: oto Leu 440 atO Ile gto Val cac His cot Pro gag Giu 520 tgg Trp ogt Arg ggt 1Gly gao SAsp caat r Asn 600 125 :cg ?ro :tt Phe tgC Cys acg Thr 999 Gly 505 agg Arg gga Gly gag Git cgc Arc Gl' 58~ ta Ty: oto C Leu L 990 C Gly T 999C Gly AlaC aag Lys 490 cac His ccc Pro gaa Glu tgc Cys aga Arg 570 jaaa ~Lys 5 cact r Thr ~go trg ~cg ?ro -ag 31n 475 aat ks n cto Leu aag Lys tgt Cys a ac Lys 555
C
AlE agc Se~ ga As~ atgC Met 7 gat Asp 460 ott Leu 990 Gly tgC Cys ccc Pro tot Ser 540 gao Asp aag Lys tto Phe *atg p Met :tg gat 9CC oct ggt 909 ,eu Asp Ala Pro Gly Ala 1296 4 ;cc C lia L 145 :to C ?he I :ggt rrpC ago Ser toa Ser gtg Val 525 cgg Arg 000 Pro tao T yr agg Arg gao Asp 605 :tg t ~eu ~gC trg :90 'ys :tg Leu gaa Glu 510 gt a Val1 aco Thr gag Glu cag Gin gag Glu 590 999 Gly :ac yr ,ac iis cac His zoo Pro 495 9go Gly gat Asp t gt Cys ct Pro t ca Ser 575 cag Gin aat Asn cag Gin tgC Cys act Thr 480 tgg T rp ago Ser gga Gly gga Gly cag Gin 560 tgo Cys cag Gin oto Leu 1344 1392 1440 1488 1536 1584 1632 1680 1728 1776 1824 1872 tat got ggg gtg too ccc cgg gao ogo tgo Leu Gin 610 Trp Vai Pro Lys Tyr 615 Ala Gly Val Ser Pro 620 Arg Asp Arg Cys WO 99/37660 WO 9937660PCT/US99/01313 aag ttg tto tgc cga goc cgg ggg agg Lys Leu Phe Cys Arg Ala Arg Gly Arg 625 9CC aag Ala Lys tgt gtc Cys Vai tcg oct Ser Pro tcc tgc Ser Cys 690 aat gac Asn Asp 705 cag cgg Gin Arg aag acg Lys Thr 9CC ata Ala Ile ggc tcc Gly Ser 770 gag cot Glu Pro 785 cca aaa Pro Lys gt g Val1 cgt Arg Cgg Arg 675 agg Arg at t Ile agc Ser got Ala gag Giu 755 ato Ile ot g Le u gt c Val1 att Ile Gly 660 aag Lys aag Lys gt c Val1 cac His gat Asp 740 cag Gin 9CC Al a a ca Thr aaa Lys gat Asp 645 cag Gin ctg Le u gt C Val acc Thr cog Pro 725 ggg Gly gac Asp acc Thr gtg Val tac Tyr 805 630 990 acc Gly Thr tgt gtc Cys Val gac aaa Asp Lys tcc ggg Ser Gly 695 atc cca Ile Pro 710 ggt gtg Giy Val cag tac Gin Tyr ato ttg Ile Leu ctg gag Leu Giu 775 cag ctc Gin Leu 790 acc ttc Thr Phe ctg tgt Leu Cys aag 9CC Lys Ala 665 tgC 999 Cys Gly 680 toc otc Ser Leu gct ggt Ala Gly cag aac Gin Asn ctg ctc Leu Leu 745 gtg aag Val Lys 760 090 ctg Arg Leu ctg aca Leu Thr ttt gtt Phe Val ago gag Ser Giu 635 ggg oca Gly Pro 650 ggc tgt Gly Cys gtg tgt Val Cys acc CCC Thr Pro gcc act Ala Thr 715 gat ggg Asp Gly 730 aao 99C Asn Gly 999 aCC Gly Thr cag ago Gin Ser gtC cot Val Pro 795 Cct aat Pro Asn 810 ttc Phe ga a Giu gao Asp 999 Gly acc Thr 700 aat As n aac Asn aac As n at o Ile ttC Phe 780 99C Gly gaC Asp aaa Lys a ca Thr cat His 99C Gly 685 aat Asn att Ile taC Tyr Ct 9 Leu ctg Le u 765 C99 Arg gag Glu gtg Val gt g Val1 Ct 9 be u gt 9 Val 670 aaa Lys tat Tyr gaC Asp ct 9 Leu 9CC Ala 750 aag Lys Coo Pro gtc Val gao Asp tto Phe 9CC Ala 655 gtg Val 990 Gly 990 Gly gt 9 Val 909 Ala 735 atc Ile tao Tyr ttg Leu tto Phe ttt Phe 815 gag Giu 640 ato Ile gao Asp aao As n tao Tyr aag Lys 720 ctg Leu tot Ser ago Ser ooa Pro
CCC
Pro 800 ago Ser 1920 1968 2016 2064 2112 2160 2208 2256 2304 2352 2400 2448 2496 atg oag ago ago aaa gag aga goa aco aoo aao ato ato oag oog otg Met Gin Ser Ser Lys Giu Arg Ala Thr Thr Asn Ile Ile Gin Pro Leu 820 825 WO 99/37660PCIS9O13 PCTIUS99/01313 ctc cac gca Leu His Ala 835 tqc ggg gcc Cys Gly Ala cag tgg gtg ctg Gin Trp Val Leu ggg Gly 840 gac tgg Asp Trp ggc tgg cag Gly Trp Gin 850 ggc cag Gly Gin agg Arg 855 tgc Gys cga act gta Arg Thr Val aac aag gct Asn Lys Ala tct gag tgc tct agc acc Ser Glu Cys Ser Ser Thr 845 gag tgc agg gac ccc tcc Glu Cys Arg Asp Pro Ser 860 ctg aaa ccc gag gat gcc Leu Lys Pro Glu Asp Ala 875 880 tgattcaggg gggcaggggc gcc tct gcc Ala Ser Ala 865 aag Lys cagt tggc ct cc catc aatt aaaE acc Thr 870 ccc tgc gaa agc c Pro Cys Glu Ser G 885 :cttgtg ctcctggaca ~tccctt ggccatatca ~agtact gcacaaattc ~aactgt ccawtgnaat :cattat gatcaacaga ~aaaaaa aaaaaaaa ln ctg tgc C Leu Cys P cc ctg tgcggtactg aggcagcacg ctaaggggga ggaacttgct cctcacntca ~ro Leu 890 aggtgcagac gcccacccag agagaaaagg cgggttcaat tctgttgcan 2544 2592 2640 2690 2750 2810 2870 2930 2990 3008 aaggtctcca gcct cccatt tatggggcgg taaaggcata gatacaacta ctgtggtgac gccgcaaccc caaaacctat agttaaagta ntaaaaaaaa <210> 4 <211> 890 <212> PRT <213> Homo sapiens <400> 4 Met Phe Pro Ala Pro 1 5 Leu Leu Leu Leu Leu Ala Ala Pro Arg Trp 10 Arg Leu Pro Phe Leu Leu Leu Leu Pro Leu Gly Ala Pro Gly Ala Ala Gly Gly Ser Ala Gin Ala Ser Glu 40 Leu Val Val Pro Thr Phe Ala Arg Pro Arg Leu Pro Gly Lys Gly Gly Glu Leu Phe Val Ala Pro His Leu Ser Leu Arg Leu Lys Ala 70 Gly Asp Asp Ser Phe Ala Ala Pro Glu Ile Giu Arg Leu Gly Ser Gly Arg Thr Gly Gly Giu WO 99/37660 PCT/US99/01313 -16- 90 Gly Leu Arg Gly Cys Phe Phe Ser Gly Thr Val Asn Gly Glu Pro Glu 100 105 110 Ser Leu Ala Ala Val Ser Leu Cys Arg Gly Leu Ser Gly Ser Phe Leu 115 120 125 Leu Asp Gly Glu Glu Phe Thr Ile Gin Pro Gin Gly Ala Gly Gly Ser 130 135 140 Leu Ala Gin Pro His Arg Leu Gin Arg Trp Gly Pro Ala Gly Ala Arg 145 150 155 160 Pro Leu Pro Arg Gly Pro Glu Trp Glu Val Glu Thr Gly Glu Gly Gin 165 170 175 Arg Gin Glu Arg Gly Asp His Gin Glu Asp Ser Glu Glu Glu Ser Gin 180 185 190 Glu Glu Glu Ala Glu Gly Ala Ser Glu Pro Pro Pro Pro Leu Gly Ala 195 200 205 Thr Ser Arg Thr Lys Arg Phe Val Ser Glu Ala Arg Phe Val Glu Thr 210 215 220 Leu Leu Val Ala Asp Ala Ser Met Ala Ala Phe Tyr Gly Ala Asp Leu 225 230 235 240 Gin Asn His Ile Leu Thr Leu Met Ser Val Ala Ala Arg Ile Tyr Lys 245 250 255 His Pro Ser Ile Lys Asn Ser Ile Asn Leu Met Val Val Lys Val Leu 260 265 270 Ile Val Glu Asp Glu Lys Trp Gly Pro Glu Val Ser Asp Asn Gly Gly 275 280 285 Leu Thr Leu Arg Asn Phe Cys Asn Trp Gin Arg Arg Phe Asn Gin Pro 290 295 300 Ser Asp Arg His Pro Glu His Tyr'Asp Thr Ala Ile Leu Leu Thr Arg 305 310 315 320 Gin Asn Phe Cys Gly Gin Glu Gly Leu Cys Asp Thr Leu Gly Val Ala 325 330 335 Asp Ile Gly Thr Ile Cys Asp Pro Asn Lys Ser Cys Ser Val Ile Glu 340 345 350 Asp Glu Gly Leu Gin Ala Ala His Thr Leu Ala His Glu Leu Gly His 355 360 365 WO 99/37660 PCT/US99/01313 -17- Val Leu Ser Met Pro His Asp Asp Ser Lys Pro Cys Thr Arg Leu Phe 370 375 380 Gly Pro Met Gly Lys His His Val Met Ala Pro Leu Phe Val His Leu 385 390 395 400 Asn Gin Thr Leu Pro Trp Ser Pro Cys Ser Ala Met Tyr Leu Thr Glu 405 410 415 Leu Leu Asp Gly Gly His Gly Asp Cys Leu Leu Asp Ala Pro Gly Ala 420 425 430 Ala Leu Pro Leu Pro Thr Gly Leu Pro Gly Arg Met Ala Leu Tyr Gin 435 440 445 Leu Asp Gin Gin Cys Arg Gin Ile Phe Gly Pro Asp Phe Arg His Cys 450 455 460 Pro Asn Thr Ser Ala Gin Asp Val Cys Ala Gin Leu Trp Cys His Thr 465 470 475 480 Asp Gly Ala Glu Pro Leu Cys His Thr Lys Asn Gly Ser Leu Pro Trp 485 490 495 Ala Asp Gly Thr Pro Cys Gly Pro Gly His Leu Cys Ser Glu Gly Ser 500 505 510 Cys Leu Pro Glu Glu Glu Val Glu Arg Pro Lys Pro Val Val Asp Gly 515 520 525 Gly Trp Ala Pro Trp Gly Pro Trp Gly Glu Cys Ser Arg Thr Cys Gly 530 535 540 Gly Gly Val Gin Phe Ser His Arg Glu Cys Lys Asp Pro Glu Pro Gin 545 550 555 560 Asn Gly Gly Arg Tyr Cys Leu Gly Arg Arg Ala Lys Tyr Gin Ser Cys 565 570 575 His Thr Glu Glu Cys Pro Pro Asp Gly Lys Ser Phe Arg Glu Gin Gin 580 585 590 Cys Glu Lys Tyr Asn Ala Tyr Asn Tyr Thr Asp Met Asp Gly Asn Leu 595 600 605 Leu Gin Trp Val Pro Lys Tyr Ala Gly Val Ser Pro Arg Asp Arg Cys 610 615 620 Lys Leu Phe Cys Arg Ala Arg Gly Arg Ser Glu Phe Lys Val Phe Glu 625 630 635 WO 99/37660 PCT/US99/01313 -18- Ala Lys Val Ile Asp Gly Thr Leu Cys Gly Pro Glu Thr Leu Ala Ile 645 650 655 Cys Val Arg Gly Gln Cys Val Lys Ala Gly Cys Asp His Val Val Asp 660 665 670 Ser Pro Arg Lys Leu Asp Lys Cys Gly Val Cys Gly Gly Lys Gly Asn 675 680 685 Ser Cys Arg Lys Val Ser Gly Ser Leu Thr Pro Thr Asn Tyr Gly Tyr 690 695 700 Asn Asp Ile Val Thr Ile Pro Ala Gly Ala Thr Asn Ile Asp Val Lys 705 710 715 720 Gin Arg Ser His Pro Gly Val Gin Asn Asp Gly Asn Tyr Leu Ala Leu 725 730 735 Lys Thr Ala Asp Gly Gin Tyr Leu Leu Asn Gly Asn Leu Ala Ile Ser 740 745 750 Ala Ile Glu Gin Asp Ile Leu Val Lys Gly Thr Ile Leu Lys Tyr Ser 755 760 765 Gly Ser Ile Ala Thr Leu Glu Arg Leu Gin Ser Phe Arg Pro Leu Pro 770 775 780 Glu Pro Leu Thr Val Gin Leu Leu Thr Val Pro Gly Glu Val Phe Pro 785 790 795 800 Pro Lys Val Lys Tyr Thr Phe Phe Val Pro Asn Asp Val Asp Phe Ser 805 810 815 Met Gin Ser Ser Lys Glu Arg Ala Thr Thr Asn Ile Ile Gin Pro Leu 820 825 830 Leu His Ala Gin Trp Val Leu Gly Asp Trp Ser Glu Cys Ser Ser Thr 835 840 845 Cys Gly Ala Gly Trp Gin Arg Arg Thr Val Glu Cys Arg Asp Pro Ser 850 855 860 Gly Gin Ala Ser Ala Thr Cys Asn Lys Ala Leu Lys Pro Glu Asp Ala 865 870 875 880 Lys Pro Cys Glu Ser Gin Leu Cys Pro Leu 885 890 <210> <211> 1203 <212> PRT WO 99/37660 PTU9/11 PCT/IJS99/01313 -19- <213> Bovine <400> Met Asp Pro 1 Leu Leu Leu Ala Ala Ala Ile Leu Ala His Val Val Ala Ala Pro2 Pro Gly Gly2 His Leu Arg 115 Val Glu Trp 130 Gly Thr Cys 145 Ser Val Ala Glu Glu Glu Lys Glu Ala 195 Thr Ser Arg 210 Ala Asp Ser 225 Arg Val Asn Pro Ala 5 Leu Leu ksp Pro Jal Pro Ser Ala %lia Gin rg Leu 1.00 Ueu Arg Gln Gly Leu Tyr Leu Ser 165 G1u Phe 180 G1u Gin Pro Pro Leu Asp Ser Ser 245 Gly Ala Ala Gly Arq Leu Leu Cys Pro Ala Leu 10 Leu Pro Val1 Ala 70 Ile Phe Pro Glu Val 150 As n Phe Gly Pro Ser 230 Arg Pro Gly Arg 55 Thr Pro Tyr Asn Ser 135 Gi y Cys Ile Arg Leu 215 Le u Arg Leu Gly 40 Thr Ala Gly Asn Al a 120 Gly Asp Asp Glu Val 200 Gly Ser Arg Pro 25 Pro Asp Pro Leu Val 105 Arg Al a Val Giy Pro 185 His Gln Arg Met Ala Gin Ala Ala Ser 90 Thr Leu Thr Al a Leu 170 Leu Val1 Ala Al a Arg 250 Asp Gl y Gin Gly 75 Gly Val Val Arq Gly 155 Al a Giu Val Leu Leu 235 Ar g Al a His Gly Val1 Gi y Phe Al a Val1 140 Le u Gly Lys Tyr Asp 220 Gly His Arg Gly Arg Arg Ser Gly Pro 125 Glu Ala Leu Gly His 205 Thr Val1 Ala Leu Ala Leu Thr Glu Arg 110 Gly Pro Giu Ile Leu 190 Arg Gly Leu Ala Al a Glu Val Arg Giu Asp Ala Leu Ser Arg 175 Al a Pro Ile Glu Asp 255 Al a Arg Ser Arg Asp Le u Thr Le u Ser 160 Met Ala Thr Ser Glu 240 Asp Asp Tyr Asn Ile Glu Val Leu Leu Gly Val Asp Asp Ser Val Val Gln WO 99/37660 PCT/US99/01313 260 265 270 Phe His Gly Thr Glu His Val Gin Lys Tyr Leu Leu Thr Leu Met Asn 275 280 285 Ile Val Asn Glu Ile Tyr His Asp Glu Ser Leu Gly Ala His Ile Asn 290 295 300 Val Val Leu Val Arg Ile Ile Leu Leu Ser Tyr Gly Lys Ser Met Ser 305 310 315 320 Leu Ile Glu Ile Gly Asn Pro Ser Gin Ser Leu Glu Asn Val Cys Arg 325 330 335 Trp Ala Tyr Leu Gin Gin Lys Pro Asp Thr Asp His Asp Glu Tyr His 340 345 350 Asp His Ala Ile Phe Leu Thr Arg Gin Asp Phe Gly Pro Ser Gly Met 355 360 365 Gin Gly Tyr Ala Pro Val Thr Gly Met Cys His Pro Val Arg Ser Cys 370 375 380 Thr Leu Asn His Glu Asp Gly Phe Ser Ser Ala Phe Val Val Ala His 385 390 395 400 Glu Thr Gly His Val Leu Gly Met Glu His Asp Gly Gin Gly Asn Arg 405 410 415 Cys Gly Asp Glu Val Arg Leu Gly Ser Ile Met Ala Pro Leu Val Gin 420 425 430 Ala Ala Phe His Arg Phe His Trp Ser Arg Cys Ser Gln Gln Glu Leu 435 440 445 Ser Arg Tyr Leu His Ser Tyr Asp Cys Leu Arg Asp Asp Pro Phe Thr 450 455 460 His Asp Trp Pro Ala Leu Pro Gin Leu Pro Gly Leu His Tyr Ser Met 465 470 475 480 Asn Glu Gin Cys Arg Phe Asp Phe Gly Leu Gly Tyr Met Met Cys Thr 485 490 495 Ala Phe Arg Thr Phe Asp Pro Cys Lys Gin Leu Trp Cys Ser His Pro 500 505 510 Asp Asn Pro Tyr Phe Cys Lys Thr Lys Lys Gly Pro Pro Leu Asp Gly 515 520 525 Thr Met Cys Ala Pro Gly Lys His Cys Phe Lys Gly His Cys Ile Trp 530 535 540 WO 99/37660 PCT/US99/01313 -21- Leu Thr Pro Asp Ile Leu Lys Arg Asp Gly Asn Trp Gly Ala Trp Ser 545 550 555 560 Pro Phe Gly Ser Cys Ser Arg Thr Cys Gly Thr Gly Val Lys Phe Arg 565 570 575 Thr Arg Gin Cys Asp Asn Pro His Pro Ala Asn Gly Gly Arg Thr Cys 580 585 590 Ser Gly Leu Ala Tyr Asp Phe Gin Leu Cys Asn Ser Gin Asp Cys Pro 595 600 605 Asp Ala Leu Ala Asp Phe Arg Glu Glu Gin Cys Arg Gin Trp Asp Leu 610 615 620 Tyr Phe Glu His Gly Asp Ala Gin His His Trp Leu Pro His Glu His 625 630 635 640 Arg Asp Ala Lys Glu Arg Cys His Leu Tyr Cys Glu Ser Lys Glu Thr 645 650 655 Gly Glu Val Val Ser Met Lys Arg Met Val His Asp Gly Thr Arg Cys 660 665 670 Ser Tyr Lys Asp Ala Phe Ser Leu Cys Val Arg Gly Asp Cys Arg Lys 675 680 685 Val Gly Cys Asp Gly Val Ile Gly Ser Ser Lys Gin Glu Asp Lys Cys 690 695 700 Gly Val Cys Gly Gly Asp Asn Ser His Cys Lys Val Val Lys Gly Thr 705 710 715 720 Phe Ser Arg Ser Pro Lys Lys Leu Gly Tyr Ile Lys Met Phe Glu Ile 725 730 735 Pro Ala Gly Ala Arg His Leu Leu Ile Gin Glu Ala Asp Thr Thr Ser 740 745 750 His His Leu Ala Val Lys Asn Leu Glu Thr Gly Lys Phe Ile Leu Asn 755 760 765 Glu Glu Asn Asp Val Asp Pro Asn Ser Lys Thr Phe Ile Ala Met Gly 770 775 780 Val Glu Trp Glu Tyr Arg Asp Glu Asp Gly Arg Glu Thr Leu Gin Thr 785 790 795 800 Met Gly Pro Leu His Gly Thr Ile Thr Val Leu Val Ile Pro Glu Gly 810 815 WO 99/37660 PCT/US99/01313 -22- Asp Ala Arg Ile Ser Leu Thr Tyr Lys Tyr 820 825 Leu Asn Val Asp Asp Asn Asn Val Leu Glu 835 840 Glu Trp Ala Leu Lys Lys Trp Ser Pro Cys 850 855 Gly Ser Gin Phe Thr Lys Tyr Gly Cys Arg 865 870 Met Val His Arg Gly Phe Cys Asp Ser Val 885 890 Arg Arg Thr Cys Asn Pro Gin Glu Cys Ser 900 905 Gly Glu Trp Glu Pro Cys Ser Arg Ser Cys 915 920 Val Arg Ser Val Arg Cys Val Gin Pro Leu 930 935 Ser Val His Thr Lys His Cys Asn Asp Ala 945 950 Ala Cys Asn Arg Glu Leu Cys Pro Gly Arg 965 970 Ser Gin Cys Ser Val Thr Cys Gly Asn Gly 980 985 Leu Cys Arg Thr Ala Asp Asp Ser Phe Gly 995 1000 Pro Glu Thr Ala Arg Ile Cys Arg Leu Gly 1010 1015 Ser Asp Pro Ser Lys Lys Ser Tyr Val Val 1025 1030 Asp Pro Asn Ser Pro Val Gin Glu Thr Ser 1045 1050 Gly Asp Lys Ser Val Phe Cys Arg Met Glu 1060 1065 Ser Ile Pro Gly Tyr Asn Lys Leu Cys Cys 1075 1080 Met Asp Ser Arg 875 Ser Gin Gly His Arg 955 Trp Thr Val Pro Gin 1035 Ser Val Lys Ile His Glu Asp Ser 830 Asp Ser Val Gly Tyr 845 Lys Pro Cys Gly Gly 860 Arg Leu Asp His Lys 880 Lys Pro Lys Ala Ile 895 Pro Val Trp Val Thr 910 Arg Thr Gly Met Gin 925 Asn Asn Thr Thr Arg 940 Pro Glu Gly Arg Arg 960 Arg Ala Gly Ser Trp 975 Gin Glu Arg Pro Val 990 Cys Arg Glu Glu Arg 1005 Cys Pro Arg Asn Thr 1020 Trp Leu Ser Arg Pro 1040 Lys Gly Arg Cys Gin 1055 Leu Ser Arg Tyr Cys 1070 Ser Cys Asn Pro His 1085 Asp Asn Leu Thr Asp Val Asp Asp Arg Ala Glu Pro Pro Ser Gly Lys WO 99/37660 PCT/US99/01313 -23- 1090 1095 His Asn Asp Ile Glu Glu Leu Met 1105 1110 Val Met Glu Val Gin Pro Pro Pro 1125 Asn Thr Ser Ser Thr Asn Ala Thr 1140 Val Asp Val Pro Tyr Lys Ile Pro 1155 1160 Pro Asn Leu Ile Pro Arg Arg Pro 1170 1175 Gin Arg Ile Gin Glu Leu Ile Asp 1185 1190 Gly Lys Phe 1100 Pro Thr Leu Ser Val Pro Thr Leu 1115 1120 Gly Ile Pro Leu Glu Val Pro Leu 1130 1135 Glu Asp His Pro Glu Thr Asn Ala 1145 1150 Gly Leu Glu Asp Glu Val Gin Pro 1165 Ser Pro Tyr Glu Lys Thr Arg Asn 1180 Glu Met Arg Lys Lys Glu Met Leu 1195 1200 <210> 6 <211> <212> PRT <213> Homo sapiens <400> 6 Asp Asp Gly Trp Ser Pro Trp Ser Glu Trp Thr Ser Cys Ser Thr Ser 1 5 10 Cys Gly Asn Gly Ile Gin Gin Arg Gly Arg Ser Cys Asp Ser Leu Asn 25 Asn Arg Cys Glu Gly Ser Ser Val Gin Thr Arg Thr Cys His Ile Gin 40 Glu Cys <210> 7 <211> 57 <212> PRT <213> Homo sapiens <400> 7 Asp Gly Gly Trp Ser His Trp Ser Pro Trp Ser Ser Cys Ser Val Thr 1 5 10 WO 99/37660 PCT/US99/01313 -24- Cys Gly Asp Gly Val Ile Thr Arg Ile Arg Leu Cys Asn Ser Pro Ser 25 Pro Gin Met Asn Gly Lys Pro Cys Glu Gly Glu Ala Arg Glu Thr Lys 40 Ala Cys Lys Lys Asp Ala Cys Pro Ile <210> 8 <211> 57 <212> PRT <213> Homo sapiens <400> 8 Asn Gly Gly Trp Gly Pro Trp Ser Pro Trp Asp Ile Cys Ser Val Thr 1 5 10 Cys Gly Gly Gly Val Gin Lys Arg Ser Arg Leu Cys Asn Asn Pro Thr 25 Pro Gin Phe Gly Gly Lys Asp Cys Val Gly Asp Val Thr Glu Asn Gin 40 Ile Cys Asn Lys Gin Asp Cys Pro Ile <210> 9 <211> <212> PRT <213> Homo sapiens <400> 9 Glu Glu Gly Trp Ser Pro Trp Ala Glu Trp Thr Gin Cys Ser Val Thr 1 5 10 Cys Gly Ser Gly Thr Gin Gin Arg Gly Arg Ser Cys Asp Val Thr Ser 25 Asn Thr Cys Leu Gly Pro Ser Ile Gin Thr Arg Ala Cys Ser Leu Ser 40 Lys Cys <210> <211> 57 <212> PRT <213> Homo sapiens WO 99/37660 WO 9937660PCT/US99/01313 <400> Asp Gly Gly Trp Ser His 1 5 Cys Gly Val Gly Asn Ile Pro Gin Met Gly Gly Lys Ala Cys Gin Gly Ala Pro Trp Ser Pro Thr Arg Ile 25 Asn Cys Lys 40 Cys Pro Ile Trp Ser Ser Cys Ser Val Thr 10 Arg Leu Cys Asn Ser Pro Val Gly Ser Gly Arq Glu Thr Lys <210> 11 <211> 56 <212> PRT <213> Homto sapie <400> 11 Asp Gly Arg Trp 1 Cys Ala Gly Gly ~ns Ser Pro Trp Ser Pro Trp Ser Ala Cys Thr Val Thr 5 10 Ile Arg Glu Arg Thr Arg Val Cys Asn. Ser Pro Glu 25 Gly Lys Ala Cys Val Gly Asp Val Gin Glu Arg Gin 40 Arg Ser Cys Pro Pro Gin Tyr Met Cys Asn Gly Lys <210> 12 <211> 3974 <212> DNA <213> Hiomo sapiens <400> 12 ggtacctaag tgagtagggc ggtcatagct gtttcctgtg ccggaagcat aaagtgtaaa cgttgcgctc actgcccgct tcggccaacg cqcgqggaga ctgactcqct qcgctcggtc gtccgatcga tgaaattqtt gcctggggtg ttccagtcgg ggcggtttgc gttcggctqc cqqacgcctt atccgctcac cctaatgagt gaaacctgtc gtattgggcg ggcgagcggt ttttttgaat aattccacac gaqctaactc gtgccagctg ctcttccgct atcaqctcac tcgtaatcat aacatacqag 120 acattaattg 180 cattaatgaa 240 tcctcgctca 300 tcaaaggcgg 360 WO 99/37660 WO 9937660PCTIUS99/O1 313 taatacggtt agcaaaagqc cccctgacga tataaaqata tgccgcttac gctcacgctq acgaaccccc acccggtaag cgaggtatgt gaaqaacagt qtagctcttg agcagattac ctqacgctca caattcgcgc acctttcgcg aaaccagtaa cgcqtggtga atggcggaqc t t ct ga t tg gcqattaaat agcggcgtcg ctgatcatta aatgttccgg tcccatgaag atcgcgctgt cataaatatc atccacagaa caggaaccgt gcatcacaaa ccaggcgttt cggatacctg t aggt atct c cgttcagccc acacqactta aggcggtgct atttggtatc atccggcaaa gcgcagaaaa gtggaacgaa qcgaagqcga gtatggcatg cgttatacga accaggccag tgaattacat gcgttgccac ctcgcgccga aagcctgtaa actatccgct cgttatttct acggtacgcg tagcgggccc tcactcgcaa tcaggggata aaaaaggccg aatcgacgct ccccctggaa tccgcctttc agttcggtgt gaccgctgcg tcgccactgg acagagttct tgcgctctgc caaaccaccg aaaggatctc aactcacgtt agcggcatgc atagcgcccg tgtcgcagag ccacgtttct tcccaaccgc ctccagtctq tcaactgggt agcggcggtg ggatgaccag tgatgtctct actqggcgtg attaagttct tcaaattcag -26acgcaqgaaa cgttgctggc caagtcagag gctccctcgt tcccttcqggg aggtcgttcg ccttatccgq cagcaqccac tgaagtggtg tgaagccagt ctggtagcgg aaqaaqatcc aagggatttt atttacgttg gaagagagtc tatgccggtg gcgaaaacgc gtggcacaac gccctgcacg gccagcgtgq cacaatcttc gatgccattg gaccagacac gaqcatctgg gtctcggcgc ccgatagcgg gaacatgtga gtttttccat gtggcgaaac gcgctct cct aagcgtqgcg ctccaagctg taactatcgt tggtaacagg gcctaactac taccttcgga tggttttttt tttgatcttt ggtcatgaga acaccatcga aattcagggt tctcttatca gggaaaaagt aactggcggg cgccgtcgca tggtgtcgat tcgcgcaacg ctgtggaagc ccatcaacaq tcgcattgqg gtctgcgtct aacqggaagg gcaaaaggcc aggctccgcc cogacaggac gttccgaccc ctttctcata ggctgtgtgc cttgagtcca attagcagag ggctacacta aaaagagttq gtttgcaagc tctacggggt ttatcgtcga atggtgcaaa ggtgaatgtg gaccgtttcc ggaagcggcg caaacagtcg aattgtcgcg ggtagaacga cgtcagtggg tgcctgcact tattattttc t caccagcaa qgctggctgg cgactgqagt 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 WO 99/37660 WO 9937660PCTJUS99/0 1313 gccatgtccg atgctggttg ctgcgcgttg tatatcccgc gaccgcttgc tcactggtqa ttggccgatt gcgcaacgca ccactcctgc gacggatttg ccaactcgcg ctggaggatc ggcggcggtg gaaccccaga qaatcgggag tcttcagcaa cqgccacagt gcatcgccat aacagttcgg ccggcttcca caggtagccg tcggcaggag cagtcccttc gccagccacg qtcttgacaa caqccgattg gttttcaaca ccaacgatca gtqcggatat cgttaaccac tgcaactctc aaagaaaaac cattaatgca attaatgtaa agttcggggg cactgccggt aggggatcga at ccagccgg gaatcgaaat gtcccgctca cggcgatacc tatcacgggt cgatgaatcc gggtcacgac ctggcgcqag tccgagtacg gatcaagcgt caaggtgaga ccgcttcagt atagccgcgc aaaqaaccgg tctgttgtgc aaccatgcaa qatggcgctg ctcggtagtg catcaaacag tcagggccag caccct qgcq gctggcacga gttagcgcga catgqatgcg agaact ccgc gcccggggtg cgtcccggaa ctcgtgatgg gaagaactcg gtaaagcacg agccaacgct agaaaagcgg gagatcctcg cccctgatgc tgctcgctcg atgcagccgc tgacaggaga gacaacgtcg tgcctcgtcc gcgcccctgc ccagtcatag atgctgaatg gqcgcaatgc ggatacgacg gattttcgcc gcggtqaagg cccaatacgc caggtttccc attgtcgacc cggatagccg gaggtcgtcc ggcgaagaac aacqattccg caggttgggc tcaagaaggc aggaagcggt atqtcctgat ccattttcca ccqtcgggca tcttcgtcca atgcgatqtt cgcattgcat tcctgccccg agcacagctg tgcagttcat gctgacagcc ccgaatagcc agggcatcgt gcgccattac ataccgaaga tgctgqggca gcaatcagct aaaccgcctc gactggaaag aaagcggcca ctgctggttt agcctcaggc tccagcatga aagcccaacc gtcgcttggt gatagaaggc cagcccattc agcggtccgc ccatgatatt tgcgcgcctt gatcatcctg tcqcttggtg cagccatgat gcacttcgcc cgcaaggaac tcagggcacc ggaacacgqc tctccaccca tcccactgcg cgagtccggg cagctcatgt aaccagcgtg gttgcccgtc tccccgcgcg cgggcagtga tcgtgcctcc cctggatgcc agcagctgaa gatccccgcg tttcatagaa cggtcatttc gatgcqctgc gccqccaagc cacacccagc cggcaagcaq gaqcctggcg atcgacaaga gtcgaatgqg ggatactttc caatagcagc gcccgt cgtg ggacaggtcg qgcatcaqag aqcggccgqa 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 WO 99/37660 WO 9937660PCTUS99/O1 313 gaacctgcgt tcagatcttg ttgcagggct gtccataaaa ctctttgcgc cagcaccgt t cctgagtgct cggataacaa agaaattaca gcaatccatc atcccctgcg tcccaacctt ccqcccagtc ttgcgttttc t ctgcggact tgcggcagcg ttaagatgta tat g ttgttcaatc ccatcagatc accagagggc tagctatcgc ccttgtccag ggctttctac tgaagcttaa cccaattgtg -28atgcgaaacg cttggcggca gccccagctg catgtaagcc atagcccagt gtgttccgct aaaactgcaa agcggataac atcctcatcc agaaagccat gcaattccqg cactqcaaqc agctqacatt tcctttaqca aaaatagttt aatttcacac tgtctcttqa ccagtttact ttcgcttgct tacctgcttt catccggggt gcccttgcgc gacttgtgag attaaagagg 3540 3600 3660 3720 3780 3840 3900 3960 3974 <210> 13 <211> 112 <212> DNA <213> Homo sapiens <400> 13 aagcttaaaa aactgcaaaa aatagtttga cttgtgagcg gataacaatt aagatgtacc caattgtqag cqqataacaa tttcacacat taaagaggag aaattacata tg 112 <210> <211> <212> <213> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> 14 542
DNA
Mus rnusculus
UNSURE
(3) May be
UNSURE
(21) May be
UNSURE
(22) May be
UNSURE
(361) May be any nucleic acid any nucleic acid any nucleic acid any nucleic acid WO 99/37660 WO 9937660PCT/US99/01313 -29- <220> <221> UNSUR <222> (369) <223> May b <220> <221> UNSUR <222> (407) <223> May h <220> <221> UNSUR <222> (427) <223> May t <220> <221> UNSUP <222> (479) <223> May b <220> <221> UNSUF <222> (482) <223> May b <220> <221> UNSUF <222> (535) <223> May h <400> 14 gtncgaattt cttggggaga acaacccagt cctgtaacct aagcacacaa ccaagtacgc nttggctant cagattncac cntagattcc aa <210> <211> 320 any nucleic acid any nucleic acid any nucleic acid any nucleic acid any nucleic acid any nucleic acid cggcacgaga ctgttcgaga cccaaagaat tgaggactgt cgagttttca tggcgtctca tctttcgttt ctttgtctqt aaaaggaqtt nnttagacgc acgtgcggtq ggagggaagt ccagacaata aaagcttcct ccaaaggaca tgcagcccaa gtgcaaggac ttgttaatgt cttttcatgg gagqagtcca actgtgaagg atgqaaaaac ttgggagtgg ggtgcaagtt ggttgttagq aqtgtgttaa ggtgttttcn aagctgggga gtacacgatg caaacgagtg ctttagagag gcctgcggtg catgttgcca tgggtantcc aagttggttg gggggaatgg atgtgggggc agggaatgtg cgctacagat gaacaatgtg gaatggattc agccaaaggc atgttaggcc tgatccqcnt tctantttta 120 180 240 300 360 420 480 540 542 WO 99/37660 WO 9937660PCTIUS99/01 313 <212> DNA <213> Unknown <220> <223> Description of Unknown Organism:Unknown <400> cagagaacat tcgccccact cttcaatgac ccatgctgaa aaagtgggga tagcattgaa aqattccttc ttcttcttta cgaagtaggt gtatttaatt ttaggtcgaa qggcattgcc cacagtaaga acctggatgg tcaagggctc tttgagaggg ctaaagctqc gaattctttc caatgccgca gaggagccgc tqtacctcaa gacaacacct ttgtacataa tqtcttgctc taaggtggac aaagtqtagt caccattaaq aatatatgtg ccatcagcag ctttgatggc aagaaagctg cccttgttcc 120 180 240 300 320 <210> 16 <211> 316 <212> DNA <213> Elmeria tenella <400> 16 aatgccgaga cattaatgga gcaccagacc ttgtgcagac qttctaaqac ctqtgggaag gaggggtgtt atctcatqag tttgcacaat ggcagaatqc aaaqtgagga agggct cagcctgctt ccgaqtgtgc aaagqaagtg aagccagcca catccctgcc cccagtggca gctqggqaq tggtcatcat qgttacaaaa aaagaagctt gaagtgtctg tcccatgatg agctgtgatc ctttaaagaa acctaaacat ttcatagact agttaagtqq tttaagtggt gttagctttg agggcaaggc 120 180 240 300 316 <210> <211> <212> <213> <220> <221> <222> <223> <220> <221> <222> <223> 17 383
DNA
Caenorhabditis elegans
UNSURE
(160) May be any nucleic acid
UNSURE
(326) May be any nucleic acid WO 99/37660 PTU9/11 PCT[US99/01313 -31- <220> <221> UNSURE <222> (358) <223> May be any nucleic acid <220> <221> UNSURE <222> (366) <223> May be any nucleic acid <220> <221> UNSURE <222> (377) <223> May be any nucleic acid <220> <221> UNSURE <222> (379) <223> May be any nucleic acid <400> 17 gtcgacccac qcgtccggat qgtactccat aaqgacaqtg tgtaaaagct ggttgtqatc aatgtggtgt ttgcqgggga aatggatcta gtgcaaaacc tgggatatca tgatatcatc agtgaaacag cggaaccaga ggggatccag gctgctggat ggcacatata ttcttnaatg acattntgtg acaaagngnt tgt gtagcccaga gcatcataga cttgtaaaan acaattccaa ggaacaatgg qtgactacac ttccacctct ctccaaaaag aatatcagga ctgggagcca gcagctttct tttgtccacc gtctgtgtgc aagtttgata t cagt tact a ccaacatcga tgccat caaa ttagaganag 120 180 240 300 360 383 <210> <211> <212> <213> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> 18 404
DNA
Crotalus atrox
UNSURE
(21) May be any nucleic acid
UNSURE
(301) May be any nucleic acid
UNSURE
(335) WO 99/37660 PTU9/11 PCTIUS99/01313 -32- <223> May t <220> <221> UNSUF <222> (373) <223> May t <220> <221> UNSUF <222> (378) <223> May t <220> <221> UNSUF <222> (382) <223> May t <220> <221> UNSUF <222> (383) <223> May h <400> 18 cccacgcgtc gatggctgat tggtttacaa tgatggatgc ggcgtcaatg nacatggatt agccttcaga e any nucleic acid any nucleic acid any nucleic acid any nucleic acid any nucleic acid cgccca cggt gttqgaactg gctgccttca aaagcagtgt ctttccaacc gacatcattt atnccctnac nccgggactt tgtqtgatcc ccacaqccca gccagcctta tggqaccaca ctgggatgaa annttcccag gtgtgggtcc gagcagaagc tgaattaggc aatggtgtga gccagccttq tggtncatgg gggttctccc cagacatgtq tgctccqtca cacgtgttta accagggatt ggtcctcctt gggaatgttt tggg atactcttgg tagaagatga acatgccaca cccacatgat gcagtggcct tgattggaca <210> <211> <212> <213> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> 19 152
DNA
Homo sapiens
UNSURE
(105) May be
UNSURE
(122) May be
UNSURE
(135) any nucleic acid any nucleic acid WO 99/37660 PTU9/11 PCT/US99/01313 -33- <223> May be any nucleic acid <400> 19 atcgtagaaq atgaaaaatg gggcccagag gtqtccqaca atggggggct tacactqcgt aacttctgca actggcagcg qcgtttcaac cagcccagcg accgncaccc aqagcactac 120 gncacggcca tcctnctcac cagacagaac tt 152 <210> <211> 4180 <212> DNA <213> Unknown <220> <223> Description of Unknown Organism:Unknown <400> gcagctccga tccttgctca tctggacttg agccaccaac gtgacccgga tttaagcatc agttctctgc aqcagaagcc tgtccgcaca gcgcacacgg ccccgggcca atctgaagtt ggcgcagtcc tctactctgg gtgtgcgtgg tggccaccga tccacatcct gctaggtgct gatcgagtca ttccccagct tcggtgctgg gcttcgggtg aqgagcaggc ggt gcagggt ctgctccgac catgctgttg qcgccccacg cgattccacc gcagccggac cgggtccgag cacggtgaac tgccttctac gcgcctgqcc gaggcgaagg atcgcaaggc aatcgggcca ccgggggcgc aatgaaaaaa ctagctctgc cagcgaagct gcggtqccat atgggggacg ctgctcctcg gaggaagatg accacacgcc agcggtttct gcacaacatc qqtqatcccg ctacaaggag cctgccqtgc cggcggggca cagagcgcac aggcggagga ttctaggtcc ttcccgcgcg acgaactttc ctacgcgtct gcagccaaaa tccagcgggc cttccataac aqgagctggt ttcqtctgga tggcgcctgg tqgaccccac gctctgccgc aggagttctt ccgaggagga gtggcggcgc agcccgqcgq cgaagagtcc tgcagcagcc ccagtgcaga ccatcaaagt aaacgtctat gtcccttttg agcgagatct aatgctgcta cctgccctcg cgcctttggc cttcaccctg cggggacctg agccctcagc cattcagcca gtcatccgca caagtgcggc agagaqcaga aggct cctat agcagtgcgg atctttctaa gatcgtgaat ccagaccaag qggtcacgca cggggctctc tgtgcgcggg ctggagcgcg cagcagctac cagactqtgg gctcactqct ctctgtgaag gcqcctggag cggccgcagt gtcatggacg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 WO 99/37660 WO 9937660PCT/US99/01313 -34acgagaccct ctgtgcggga agaagcgatt tggccgactt ccaggtttta tggtcatata ggaatttctg atgacactgc tcggaatggc atgatggttt cgcacqatga tggcctcgat tggtcacgtc caatcaagct ttacattcgg ggtgcactgg atggcaccag acatgaagca actgctcaag tcccaaagaa tcgaggactg atgagttttc ccggcgtctc ttttcgtctt tctgtgtgca agtttgataa gccaaccagc ccccacgcct tgtgtccagc ccacggcagc caagcatccc cgaggagcag cagctggcag aattctgttc agatgttgga gcaagccgcc tgctaagcac gctctccagc cttcctagat cccttctgat agaggaatcc cacctccggt ctgtggagaa ttttgctact aacctgtgqt cggagggaag tccagacaat caaagcttcc gccaaaggac acagcccaag agggcagtgt gtgtggcgtt gact cgcgac caggacgcgg ccccgttatg ggtctaaagc agcattagga aagggaccag aaacaacaca accagacagg accgtatgtg ttcaccacag tgtgccagct ttagaccata aatggacacg cttcccggta aagcactgcc ggcttactgg gggaagtggt cctgttcatq ggtggagttc tactgtgaag aacggaaaaa tttgggaatg aggtgcaagc gttgtagatg gtgaaagctg tgtggaggaa ccgagaqcca gaaagccatc tggaaaccat attaccttct attcaattag aagttacctc acagccccag atttatgtgg accccagcag cccatgaatt tgaatggtgt gccagccctg gggaatgttt ccttgtacga ctgatgcagc tgtqccaaac gtgtcagtg gaagctgggg aatacacaat gcaaacgagt cgttcagaga agcccactgt tcacctgtga gcactccctg gctgtgatcg acggttccac gaacacccgg aggaccagga gctcgtagct aaccctgttc cctggtggtg caatgcagct tgaccqggat ctcccacacg gagctgctca gqccatgtg gagtqgcgat gtcaccttgc gatggacaag tgccaaccgc cagcacatgt aaaacacttc caagtgcgtg accatgqgga gagagaatgt ccgctacagg ggaqcagtgc agagtggaca agccaaaggc tagtccagac catcatagac atgcaagaag aaccagtggc agcataagga gaccagtcca tcggtggcag gtqaagatct ctcacccttc ccagagcact tgtgacactc gtcatagaag tttaacatgc tctcatctga agtgcctaca ccccagaatc cagtgtcagt act accct gt ccttgggcag aacaagacag ccgtggggag gacaacccag tcctgtaaca gaggcqcaca cccaagtacg attggctact tctacctctg tccaaaaaga atgtcaggaa 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 WO 99/37660 WO 9937660PCTIUS99/01313 tagtcactag acattgaagt ttagagccgc aagacctcac gaatccgcag atgctctccg acgccattcc gcggctcagq ccgaatgtgc cacactggca aqagaacctt ctttgaagaa cgttagagga gatccagtga catagcaaag atgaggcaca agtatctctt tgcactgtac ggtaagactg aaacaagatc ttcctatcaa aagagctcct gaaacaattc cacataaaaa aaacctgcct taatacatgt tacaagacct gaaacatcgg tgatggtacc ctacaaaggt ctttagtcca acccaaaatt cacattttct ttggcagaga aaaggaagtg ggtgggggat gaaatgtgtg gccaaagcat caaggtagcg gtcaaaccag gaggtaqatc qtagcatctg ttcttatatc taactagata ggtttcaaac attattcgaa cagtgaaaag gatttcttct atctatttat ctqaaggaaa ggggattgag tgtgccagag gggtatcatg aatcaaaggg tatattctqa actgtcttaa ctcaaagaac aaattcacct gagtgggtqa agagtagtgc aagccagcca tggtcaccat tcccacgatg tacattgact tggagggg taagcagtga aggacactac aaagaccata tatcgcccaa cttcttatca aaggcttggt gtcgctggtt ttcagcttgt tacaccatct aaaatgtaca caatgagcaa gttgtttaag ttagaatggg acattgtcac ggtccagaaa atggaaactt ggtacagtgg ccttaaccat actttatgaa ttgaagagtg agtgcagaga gtaccagacc gttccaaaac ggggcgtgtt tttgcacact ctgatacact ggtgtggcaa cctgccagtt cagagcacta ataattttca caaagattgq ttcaatcact gctgtggttt tcaacgtgac cagttcttaa ttggaaaaaa catgcctcct attatccatg gtatagagat aattcctqct caatggcagc cactctqtcc ttcctcggct ccaqgttctt gaagaagaca gggggagtgc cat taacgga ttgtgcagac ttgcgggaag atcaaatgaq qacacagtgc gagtqcaaga ggaggtgtgt acattctgat aggagcccca gagtctggca gaaaggcaaa ggaggcaagg tacggaaggt agaaaggctc ctatagttca aaagtgaagt gctttgcttc gctcacaaga cagggtccca ggagccacca t t tctggct a acactagagc gcgctggaaa atgqtaggcc gagtcattca tccaagacat caccctgctt cttccttgcc ggttacaaga agctgtgatc agttaagagg gtactggagg gtaggggata aaqgtagtta aagcactatt gaagccctgt gcagaaagat aggaggggac tgatgcatca atctccgtga tgttgaggta ttatgaggta ctcctgaggt ggcagtaaaa tgaqatgggg 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 WO 99/37660 WO 9937660PCT/US99/01313 -36aacatggtga tcactcatct cacatgggaq qctgctgcag <210> 21 <211> 9248 <212> DNA <213> Unknown <220> <223> Description of Unknown Organism:Unknown <400> 21 4180 gcagctccga t cct tgct ca ttctggactt gagccaccaa agtgacccgg ttttaagcat gagttctctg agcagaagcc tgtccgcaca gcgcacacgg ccccgggcca atctgaaqtt ggcgcagtcc tctactctgg gtgtgcgtgg tggccaccga tccacatcct acgagaccct ct gt gcqgga tctctcagtc gctaggtgct gatcgagtca gttccccagc ctcggtgctg agcttcgggt caggagcagg cggt gcagg ctgctccgac catgctgttg gcgccccacg cgattccacc qcagccggac cqggtccgag cacggtgaac tgccttctac gcgcctggcc gaggcgaagg gccaaccagc ccccacgcct tttacgaggc atcgcaaggc aatcggggcc t ccgggqgcg gaatqaaaaa gctagctctg ccaqcgaagc tgcggtgcca atgggggacg ctgctcctcg gagqaagatg accacacgcc agcggtttct gcacaacatc ggtgatcccg ctacaaggag cctgccgtgc cggcggggca gactcgcgac caggacgcgg gtgacttgg cagagcgcac aaggcggagq cttctaggtc attcccgcgc cacgaacttt tctacqcgtc tgcagccaaa tccagcgggc cttccataac aggagctqqt ttcgtctgga tqgcgcctgg tggaccccac gctctgccgc aggaqttctt ccgaggagga gtggcggcgc ccgagagcca gaaagccatc gtcacactcc agcccggcgg acgaagagtc ctgcagcagc gccagtgcag cccatcaaag taaacgtcta agtccctttg agcgagatct aatgctgcta cctgccctcg cgcctttggc cttcaccctg cggggacctg agccctcagc cattcagcca gtcatccgca caagtgcggc gaacacccgg aggtataaga aqatcgcctc agaqagcaga caggctccta caggagtgcg aatctttcta tgatcgtgaa tccagaccaa gggtcacgca cgggqctctc tgtgcgcggg ctggagcgcg cagcagctac cagactgtgg gct cactgct ctctgtgaag gcgcctggag cggccgcagt gtcatggacg aaccagtggc gtgaccccca taaatqcgaa 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 WO 99/37660 PTU9/11 PCTIUS99/01313 -37tgactcagac ttgcagtgaa ttgaagttct gggtcgtgac cttcccgctc CCCCCCCCCC 1260 aaaaaaagtg gaaaqagaga aataacccga gtatqagttc tgagcacgta tcaggagcct cccccatttg tqcttgggag tgctcgcact gactgatcct ttagttcttt cgacttgtcc ctcagcagcc attccccccg qgtcctagtg gct gagcctt ccccatgcct agtgaaaggg qtagaaatga aatctgcgga ttttgtctta tgcaaatatt ttgactttcc acttcctttt ccgttatgtg tgaccatact aagaatcgga ctccttacac actttaacta gcgctcaaag gggcaacggc aaaagctgac gtctttgctt ccctctcccg ccctgctaca ttaatcattc ctaggaqctg cgaaaaaggc gggcactggg cccggaactg acagactgag tccacgtgga tgactcaggg tagccatact cacatatata tttaaagcaa tgttttattt taatataccc ctttttacag gaaaccatgc ctqctaqaac ggtcctgtag tggagaatct ggt t gta gt t gagggcggag agtggtgatc cccgatggct ggttcgcccc gcaqaggttg cattcgcctg aaatttcttt gtattcatat cattttccaa agtgagagga gatcaagcag actcctcaag gctggatgat cgtccgcctg gcctcactca gacagtttga aagaaaaaag ttqttccagg cgagaaggtc gaccaggaag tcgtggctqa acttatttgc ataagggcta ctctccatcc tcgcgctgag tagaggagcc tcatttctgt ggtggctccg gcctccacgc gactccccag actctgccgt ttgccctttt cctactttac attggcaacc ggcaggaaaa agaaccccct ccccacccct ctcattcgg cttctcttgc ggctgtgqag tcactctgtt acttaaaaat tgtatttcag actattagga cataaggaag ccagtccatg ccgaatagtt agcgtttcct ctttaatgcc ttttgtaacg atggtgacct ggccttccqt ttggqcccct gcct cctacc cgctgtgqaa gttcagtctc ctagatttct gatttctctg ctggtttgag cactgctgqg gggacccctt tggctgagct atttcagccc caagttttta tcttcaaaga gcttgctttg aactcacagt ttttatttac gaaqgactgc aagcgatttg gccgacttcc aataatttga ccgcgaagcc tttagtgaat tcaqtccgtg ggatgtgcgt ctgtcccctt ctgcagaacc tcggcctcgt tgttagcctg taccagccag ccctcttttc accgctgagt aaaggaactt cagagtgggt gaatgagaga ccccgccctg tggcttcaat ctacagctgg ccacaaaaga ttttgttttg ttttagaaqa tttgactagg ccatgagcaa tgtccagccc acggcagcgg 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 WO 99/37660 PTU9/11 PCTIUS99/01313 tctaaagcat cattaggaat gggaccagaa acaacacaac cagacaqgta gtgttggatg tttgaaatgt tttcctttcg atctcaagtt cttcaagccc tgaagagcta tttatgtggc ccccagcagg ccacgaattg caataagtca caccagcaat ggacaggaga agtgagtatg atacagctga gatgctaagc atgctctcca tccttcctag ttccttgtgt cttcagagtt tcccaaagtc tattqaggat taccttctaa tcaattagcc gttacctcca agccccagtg agacaggagc ggatgagagg aaacaaaata gttatagqct ctacagttct agcaataatg agtatcactc tcccacacgt agctgctcag ggtaagtcgq atctgatgtg tttagaaggg aagtggtaca agccccaata atgtccttct actgtgccag gcttagacca ataatggaca agggctctct ttctctggca cattcattca aacgtctcga ccctgttctc tggtggtggt atgcagctct accgggatcc ttatcaacca gtgggggtgt accaattaaa agctcaatct attttaaaag aatggacagg tctcctactt gtgacactct tcatagaaga cttcagagta acgagagaga qtagggtata aacatttgaa caggttgtct tgtctgcttc cttqaatggt tagccagccc cggtaagatq ctggctaagt ggatcataag taaacaataa taaaaatcta ggtqgcagcc gaagatcttg cacccttcgg agagcactat tttcatcaac ggcggagagg aaaaaaaggc gtcttgctta cattacaggg cttcaaagtg aaaagggatt cgggatggca tgatggtttq caagttaagc aaacatctca ttttccacga cgttgacatt aataagagag ccaggccatq gtgactggcg tggtcacctt acagctcctc gagctccatg tagtaqatct cttctcgcca aagttcccta aqgttttaca gtcatatatg aatttctgca gacactgcaa tcaactcgga aaacccagaa atctcatctg tttctatttt aatcttgctt catgtgaaga tcccttgcct gatgttggaa caagccgcct ccaaatgcat gactatgttg tttcaagtat tttatacttg ccattgagcc tgtttaacat attctcatct gcagtgccta tttccagatg gctcttgctc t tac ct ccat ttgtaaaatc gcattgattt agcatcccag aggagcagaa actggcagaa ttctgttcac ggtcagcctt ggggatgaca tattgcctca aaacttccac agagtcagtc cacgcccaac ctttgtagga ctgtatgtqa tcaccacagc ggatacaacc ctacctcagc ggtcttacta ccctgatcaa tcactcaata gccgcacgat gatggcctcg catggtcacg gtgttcaacc atttcccctc tgcatcctgc agaagtcccc tcccaaaaat 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 WO 99/37660 PTU9/11 PCTIUS99/01313 -39gcatgatttc tattatttta tcttcccggt caagcactgc tggcttactg agggaagtgg gagttttccc tttagttcct tagcgtattt ttatagttct a ccat gggga gagagaatgt ccgctacagg ttcagctctc ctgggaacat aggcgcacaa ccaagtacgc ttggctactt ggagcctcag atctgtgttc gatttgttgt ttgcatgtgt ccctctctgt acatggcacc cgcacatac acatgtactt accaaacatg ggggaatgtt accttgtacg cctgatqcag gtgtgccaaa tgtgtcagtg aatgaaacat ttcctagaca tgagaattga aacactgata ccgtggggag gacaacccag tcctgtaaca agaaaccggg catcgccgtc tgagttttcc cggcgtct cg tttcgtctta ctgggcttgc cgttttgaat tgccttctgt ttaatattgc ataactaggt caggagtttt gagcacacta tgaccaaaga tattaataat tgatggacaa atgccaaccg ccagcacatg caaaacactt gcaagtgcgt atccgtttqc aactcctcta ttgtctgttt tctgtatctc actgctcaag tcccaaagaa tcgaggactg caaaggcggc gttctctttt aaagcttcct ccaaaggaca cagcccaagg tgccatgcca ttggaatact tgggaaggaa ttctctatcc ttcacataca agaaagtaqt actaaggtaa gactcgccat tgcctctttt gccccagaat ccagtgtcag tactaccctg cccttgggca gaacaagaca aactcagggt cctgtgtcct tacatttttc catttagact aacctgtggt cggagggaag tccagacaat gtgccacaac caggaaaaac ttgggaatga ggtgcaagct taggtgcttt tacaaatgtt tctaaatgca gcttaaatct ccaaagaatg aaaattcatg ccaaagtact gagtttgaga ttcttttggt ttcttttcct ccaatcaagc tttacattcg tggtgcactg gatggcacca gacatgaagc t ga ga agggc gtagtgggac tctgattccc cctgttcatg ggtggagttc tactgtgaag aacggtgagt atgtggttgg gttcagagag gcccactgta cacctgtgaa tacacttgaa tgggctgtct ggaacaactc agctagcact gccctttqaa gttagataaa tgttactggg attaaaaatt gttttgcaga tttttttttt tcccttctga gagaggaatc gcacctccgg gctgtggaga attttgctgt aaaqtgatgg tatgaqatgg taaaatgtct gaagctgggg aatacacaat gcaaacgagt catactggac aagttggaaa gagcagtgcg gagtggacac gccaaaggca tctttgcaaa ttacctattg cttgctttgg taaaagagtc aactcaagag ttatatatta tacctagcag catcgttgga aaggataaat 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 5520 5580 5640 5700 5760 5820 5880 WO 99/37660 PTU9/11 PCT[US99/01313 cctgctttga tataagatag taccttcaac gtgaaccctg cttacagata gacaaggaga tttaaccacc actctacctc actccaaaaa agatgtcagg cacgctgtgt atgtacctgt tcaggtcatc ctggaaacac agtaggctga agttccagta aaaacacatg gtcacattta catatacctg ggagccacca tttctggcta acactagagc gcgctggaga atggtaggcc gagtcattca tccaagacat agaagaaaat caaactatat tacagcatat cttccattga gctatatgaa ctacttgtag atggtgcatg tgtctgtgtg gaagtttgat aatagtcact tgctcagttg agtcccagtg tttgctacat aaacacacac cttgggagga taacctggc ctttggagac aaagaattgc ttttctttgt acattgaagt ttagagccgc aagacctcac gaatccgcag atgctctccg acgccattcc gcggctcagg tgaatgaaat ccaagttgtg tattctaggt cctaggccag ctgttgaaaa cctgggagga ttgtctccac caagggcagt aagtgtgqcg agtacaaggt gtccctagca tatgggagac agtgactttc aaaacaatta tgqttgctat cacaagtgaa cctgtaattt ctatttttaa ccacagacct gaaacatcgg tgatggtacc ctacaaaggt ctttagtcca acccaaaatt cacattttct ttggcagaga ttgcttaagc gatgaagtat actgaccatg caaatatata agccaaaatg qagcattccc aggttgtaga gtgtgaaagc tttgtqgagg gagtttcaga tctacaagac agagacaagt agttcacctt acccaggtac aaggcctagg ccctaaagtt tgatatacga agcaatgtga gggtatcatg aatcaaaqg tatattctga actgtcttaa ctcaaagaac aaattcacct gaqtgggtga agagtagtgc ttgtcatgta ttagcaagtq gaacaataat aaatcaagac aaagtgaaca agttgccatc tggcactccc tggctgtgat aaacggttcc acgctcactt cttgqgttca gtgacaagac ggggaacatg ttcatgtaat ttagcttggt aattaatcga ttttgtagga ttgattaact acattgtcac ggtccagaaa atggaaactt ggtacagtgg ccttaaccat actttatgaa ttgaagagtg agtgcagaga ttcttagcat atttataaag cagtgtgaca atttataagc tgtggcacgt acatcagatg tgtagtccag cgcatcatag acatgcaaga ctgcagtaga atccgcatgc ggtcagatgt aaaaacctga cccagtgttc ctacataatg cacatgaaac ctaaggaaaa cattgaaaga aattcctgct caatqgcagc cactctgtcc ttcctcggct ccaggttctt gaaqaagaca gggqgagtgc cattaatgga 5940 6000 6060 6120 6180 6240 6300 6360 6420 6480 6540 6600 6660 6720 6780 6840 6900 6960 7020 7080 7140 7200 7260 7320 7380 7440 WO 99/37660 WO 9937660PCT/US99/01313 -41caccctgctt cttccttgcc ggttacaaga agctgtgatc agttaagagg gggatccaqt tacataqcaa taatgaggca ttagtatctc gttgcactgt atggtaagac acaaacaaga cattcctatc gaaagagctc tagaaacaat gtacacataa ggtaaacctg aaataataca gggaacatgg tggcagctgg gaatatgact tactttataa cctataataa tcgcttttgt taataaaaga ccgaatgtgc cacactggca agagaacctt ctttgaagaa cgttagagga gagtcaaacc aggaggtaga cagtagcatc ttttcttata actgactaga tgggtttcaa tcattattcg aacagtgaaa ctgatttctt tcatctattt aaactgaagg cctggggatt tgttgtgcca tgatcactca tccaacagtc gtttccatat gtattggtgt catatttatt aaaagtgcat acacttttqa aaaggaagtg ggtgggggat gaaatgtgtg gccaaagcat caaggtagcg agtaagcagt tcaggacact tgaaaqacca tctatcgccc tacttcttat acaaggcttg aagtcgctgg agttcagctt cttacaccat ataaaatgta aaacaatgag gaggttgttt gagttagaat tctcacatgg gtatcctggt gtatatgtat gtctgttcct tttatacatt ataaaaatag atgtgtatgc aagccagcca tggtcaccat tcccacgatg tacattgact tggggagggg gaggtgtggc accctqccag tacagagcac aaataatttt cacaaagatt gtttctatca ttgctgtggt gttcaacgtg ctcagttctt cattggaaaa caacatgcct aagattatcc ggggtataga qaggctgctg gaatgtctgt atagtaaaat tctaagaagg tatttctaat agtatttata ctattttctg gtaccagacc gttccaaaac ggggcgtgtt tttgcacact ctgatacact aaggaggtgt ttacattctg taaggagccc cagagtctgg gggaaaggca ctggaggcaa tttacggaag acagaaaggc aactataatt aaaaaagtga cctgctttgc atggctcaca gatcagggtc cagggtagca tcagctcttc atgttactat actatagttt gataaaacct caatatatgt gagtgggatt ttgtgcagac ttgcgggaag atcaaatgag gacacagtgc gagtgctgga gtgtagggga ataaggtagt caaagcacta cagaagccct aagcagaaag ggaggagggg gttgatgcat tcatctccgt catgttgagg agt ttat gag ttcctcctga agaggcagta ccatgagatg ggtccactcc tactgagaga gaattgcatg ataataaatg ttaagttata taactagaaa aacttctggg 7500 7560 7620 7680 7740 7800 7860 7920 7980 8040 8100 8160 8220 8280 8340 8400 8460 8520 8580 8640 8700 8760 8820 8880 8940 caagaaatct gatgagacac aaacattgga cttcaagaca gttttaaaat ttgggtaaat 9000 WO 99/37660 PTU9/11 PCTIUS99/01313 -42qaactgtatt tcctgtttat agacgtacta ataaaaaaga agttgatgat gtctttagtg 9060 gtaagattgt tactaatgtg gttqgcaaat tgctqtaaag agccagataq taagcattta 9120 tggcattqta ggctatcttt cctgccacaa ccatgtqaca gtgagtgctt tgtaggactg 9180 aqagcagcca taaatgacat gtaaatgata aactgtggct gtgctttaat aaaactttat 9240 ttacaaaa 9248 <210> 22 <211> 5722 <212> DNA <213> Unknown <220> <223> Description of Unknown Organism:Unknown <400> 22 ggacgcacag cgccgcgct c gcctggggac tctggcggag tctgggcqcc gccaacctga qcagaaaagg ctgctggccc aaggcgggca gaagaagctc agggcccagc caaagcgtct qtcaatgaca gaagacatcc aacaacgtgg aaggacttgc gcattccccg cggtacacac taggcgtcct acaacagcgt gactggtgaa tcccccctgt gtttcctcct tggaqcqgaa ccctggacct tcctggcaac tgtacatcga tcaccagaga atttccaggg tcaggaacaa tgaatggttc aagccatctg cgcccctcca aggatccctg gttcctgatg gtttgacatc gggccccgac gcctgatgac tctggcatcc agaccactct cagcctgacc cgqccagtgg ctgtgaaaag cctggccagc ggtgctgcag aggctgctcc cagccctgcc cggcatctcc gccctcgccg ctgggcacca catgtgtgtg tttgaactca ccttccagcc aagttccaag ctgaqgcaga ggccaggt ct gtccaaggaa aagagcatca atggagaatg atcgccagac aatgtgaggt agctctacca atccgcacta tgtgatgagc ccctcgccac acagctccac gcaccaaccg ccggggccgc cagctttccg acctggtgga tgaaqaagac tcagcgtggt agcagcacgt ccctgtttgt ctgagttgga tccgcatcgc ttgtctttqg qtgtcctcct actacattgg tgtccagcat cgctcccggc catgqggctg cattccagag ccgcaagggg catcgaggat tgctgtgcgg ccggqcacg gtccaatggc ggtgtctgtg gcaggaagac cgtccccatc aaagqggggc aaccacacca cacccttgac ccacaagaca ggtcctggaa 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 WO 99/37660 WO 9937660PCTUS99/O1 313 -43ctcaggggcc gagaacaaaq cagtacagaa tcagttacca cctgatggag ccatggtccg cgctcctgcg cacattcagg tggtcatctt tctcccagcc tgcaagaaag tgttctgtca ccccagtttg caggactgtc agctaccctg atccagtgca ggagagcacc ttcaccggct tgcaagcccc aactacctgg ggcaatggca gtgtgcgtgg tcagqgcagg aatgataaaa gactatgaca tqcgcaccat agttggccaa ataacgagga tctgcaaaaa aatgctgtcc agtggacctc atagcctcaa agtqtgacaa gttctgtqac cccagatgaa acgcctgccc cctgtgqagq gaggcaagga caattgatgg atggcaqctg cagatgttga ggtgt qagaa cacagccctt gtaacccctg gccactatag tcatctgcgg ccaatgcgac aagactatga ttccagatga gagatgatgt tgtgaccacg tqagctgagg atggactgtt ggtgtcctgc tcgctgttgg ctgttctacg caaccgatgt aagatttaaa atgtggtgat tgggaaaccc catcaatgga aggggtacag ctgcgttggt atgcctgtcc gaaatgtggt tgagtgcaaa cacggacccc cggccagggt cacggatggg cgaccccatg ggaggacaca ttaccactgc caaggatgga cagggacaac gggagaccgc ctqcaqgaca cggcctcccc gatagctgca cccatcatgc cccagcgact agctgtggca gagggctcct caggatggtg ggtgtgatca tgtgaaggcg qgctgggtc aaacgtagtc gatgtaacag aatccctqct gcttgtcccc gaagtgcctg ggctacaact gtcgaacatg acccacgact taccgctgcg gacctggatg aaaaaggata attggtgatg tgtccattcc tgtgacaact gcatccgcaa tatgctatca ctgaqtgtca cctgctccaa ctgcggacga atggaattca cggtccagac gctggagcca caaggatccg aagcqcggga ct tggt ca cc gtctctgcaa aaaaccagat ttgccggcgt ctggttacag atgcctgctt gcctgccctg ccacggccaa gcaacaagaa agtgcaagcc gctggcccaa attgccccaa cctgtgatga attacaaccc gtccctacaa agtgactgaa caacggagtt ctgtcagaac tgccacagtt tggctggtct gcagcgcggc acggacctgc ctggtccccq gctctgcaac gaccaaagcc atgggacatc caaccccgca ctgcaacaaq qaagtgtact tggaaatggc caaccacaat ccccccacgc caaacaggtg cgccaagtgc tggctacgct tgagaacctg ccttcccaac tgacgatgac agctcagtat cc a ca accca 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 gatcaggcag acacagacaa caatggggaa ggaqacgcct gtgctqcaga cattqatgga 2520 WO 99/37660 PTU9/11 PCT/US99/01313 -44gacggtatcc actgatatgg cagctggact gaagatggcc tcaatgaacg atggggttqg ctgactcaga accagaacaa gaccatqaca aagatggcaa cctgatgaca gatggtcgag gacatctgtc cctctqgacc aaagaactcg a atgct gt gg gqatttgtct acccagtcct aaagttgtaa qgaaacaccc gatttcaccg gtgatgtatq gctggtggta aaatacgaat aatgctggta cttggcttcc tcaagaaaat atcttccaag agttgggaag catctctgag agaattagca aggacaactg gtgatgcctg ctgagaatgt ccaaaggqac tccagactgt acttcagtgg ttggctacca actgggacac act ccaccac ctggccaggt cctacagatg aagggaagaa gactagggtt gtagagatcc ttgcaccttc ttcttttctg gcagttttca catataaaca gtgcccattc cagtggactc aacaaaacca ggacaactgc agatcagtgt ccgcattgga tctggacaac gggagatgcc cagactcgtg caaagatgat tgacatcagt atcccaaaat caactqtgat caccttcttc gt ccagcagc caaccccacg agggcctggc gcgcaccctg gcgtctcagc aatcatggct gtttgtcttc ctaatcatca tggaactatg tgcttgcatc aaaacagact at t gctttg cactctgcct aaaagcattt ccctgacatc cagtacgtct gacaattgcc gatacctgtg tgtccctatg tgtgaccacg cccaatcccg tttgaccatg gagaccgatt gaccctaact cctggactcq atcaacaccg cgcttttatg agqgctcagg gagcacctgc tggcatgacc cacaggccaa gactcaggac tctcaagaaa aattgttgat ggcttgagaa agtgtggact catcagcatt tttccttttg ttgtcacaga tcaggcatgt ctccttcagg acaatgtgga ccttggaaca acaacaatca tgcccaatgc atgatqacaa accagaagga acagtgtgcc tccgccgatt ggqttgtacq ctgtaggtta aaagqacga ttgtgatgtg gatactcggg ggaacgccct ctcgtcacat agacgggttt coat ctatqa tggtgttctt tgaaagactg aacccccagg cctagaacgt cagcctccaa aaaaagcatc gcagqgtqct cagagaaggg aacacgggga ccagagagac caatccggat qgatattgat caaccaggct cqatgqcatt ctctgacggc agacatcgat ccagatgatt ccatcagggt tgatgagttt tgactatgct gaagcaagtc cctttctgtg gtggcacaca aggctggaaa cattagagtg taaaacctat ct ctgacctg atcataaacc atcacttctc gcgacctgcc tgaataagac tacttgcttc attgtgagqc aggactcact gcagaggcca 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 WO 99/37660 WO 9937660PCTIUS99/01313 aagcactaag ttacatgttc tgctgactgg ggagacaaag agtggccaga ctggtcacat aataggaaaa cttatatttt ttctcttttt ttttaaagtc aggattattc cttcgtgact taggcttcat caagaacagc atgttatggg atttgcaaat tcccttgtgc aaaagagaaa tgactgagta gctactgtag ttcccaataa tctatcatct tagaatattc agagttagta tttctttttt qggagggcgc ggt actaagt cgttagctga actggcttct attagggaat tgaaattggt cct accatct tatggttaca tccgtaatta tttacgatgt atgqaacagg gtaagattgt acggaaagtg acaaggaaaa attccttttt gttttaaatt atatttccag aaaatgacaa aagaattttt tacctaaaaa ggaaatagca ggtataccat agattgtgta tttctatttt ttgttttttt atacccgaga cattttcagg ttaacccatg ggacttcctc cagaatcaaa ggcttcattc cagtgagcac atggcacaaa ctaggtagtt aaaatattta aagaagcgta aaatacaqat tttgagagca tcagtctaat tctctgtttt gcaaagaaag ggagaaggaa aaggtgaaac ggatcaagcg gtcagtgttg ttgaaatgtt tgctttattt gatatgctat tatataatgt tttttttttt cgattgtatg ggattgaaag taaataggca cctgatcccc ccagtqt a aq tagatgtagc cagctgcctc attattatca ttctaattct ttttttactt aagactatcc tatttattaa agtagttgac aagctgctct atcttttcaa ccatgaggtc agoatataca ttacatacaa gaaagagttt tacatagcat aaatacaatt ttataaatta ttaaataatt ttgcacactg tttttttttg aaqaaaatat actattgctg cttaaataga acccttactc gcagtqctgg ttgtgcagat ccaaaggagg acctaactaa ctcttttgga attctggaag atgtcatctt ctctgttctg atttatcagc gccccttgtg gtggaattag ttcaatactg cttttttctt atattacctc aagtgtctaa aaaaactctg tctgaaagtt ttttctcatt tatcaggaaa aattgaagaa cttttgacct ggaggaactg gatttcatga agcaggaaag atcaccttgc ctgccattgc gtagcaggaa ggcagccgtg aacattcctt agtatgattt atctggctga tgttgagagt cctggaaatt aaatctcttg ct cagagtgg ttggttatcc ttttacccca tcatttttcc atttgttgtg caaacttaaa cagagaagta atgttttttt gccattggaa tactgcctgt ttgttggttt cccattttta 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 5520 5580 ctatttgcca ataccttttt ctaggaatgt gctttttttt gtacacattt ttatccattt 5640 WO 99/37660 PTU9/11 PCTIUS99/01313 -46tacattctaa agcaqtgtaa gttgtatatt actgtttctt atgtacaaqg aacaacaata 5700 aatcatatgg aaatttatat tt 5722 <210> 23 <211> 42521 <212> DNA <213> Unknown <220> <223> Description of Unknown Organism:Unknown <400> 23 gatcgttttc acaaatgacc aat aqt ttt t ctatcatctc tggagccaaa atttactcgg tttgcttttt ttctaacata tgatatagta t tct totgt g ttgcacaagt cagaagccat aagagacagg ggacaaaagg tgtggtggag agggaatttc tgcctttatg gcagatccaa taactttttt cagacatttt ttctctatct cgctatcaaa cagttctctt gtgtgcgcca gatttgcgct ttcttttttt tcagccatct tcctaactac ctccgtccca aagcaagctc accttcatcc catatcaagt aaaccggcaa gagttcaact ctagacgttc aacgtggtga ctttatcaag ctaatagctt tgttctctgt gtctgcgtct tctaaacatt ttttgttaca ttcatcggga aatttggaaa tttgctcctc tcagagtqtg agacgtcaca acgtgqgcct tcctatacct aaactcccca gtacgttgat ctggggacca cgagaagaga taaaagctta aagatcaacc agccatcgaa tacccatttt tcatttcctt cttttaactc agttgcgttg ctttagcagt aaaattgtgt caaaaattca ccatctctca tcactaaccc ccatgaggtt tctctctgtc agaatcttgt ccggagcqtg ggagatgttt tgggtqcccg cagtgcaggt ttttcaaaaa taggctagcc ttctcgtttt tatgtcaaga atcgtattca tcaccgtttg act cgacatt ggcagcagag tcttcatcaa aattcctgcc tcaaattqct ccatttttat gctgctcttC atcattcttc aaattgaaaa acccggcgag ccgttactqt agaacgagtg tcgtggactt ttttggtttc' tgtgacatac aaaqtgtttt ctgaaagcaa aaaagttatc ggacctttca tgacccctca agcaagtagg att t tgggca aattgttttg cttttttcga tcaaggttag tcggcagccc ggaagcgatg ctctaatttc ttccagaatg tgacaagtct ctcacgttcg ttcatttttt ctgatcttca attttttgaa ttttttctga tgaatcacaa 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 PCTIUS99/01313 WO 99/37660 PTU9/11 gaggctatct taatggagat agttcaaact ctatctattt ggtgactgca ggtactgatt taccacaagt tttctaattt actacaagtg tctgtgttga ttaaaactta gtgcggaaag gcgcaatctc taagattcag cttttatttg tatcttcgtt ggagctcacc gttctctatt gtgctctgta gaacaaggga cgaaaagagc ttgctcatcc attcgctaac agtaaagcag ctatttgttt a cgt t tgt tt gaaattctaa tttcaaacgg cttgcaagca ctggagcttc tccgtqctga ggactccata tcgagttttc ggctgataag tggggaatgt attcaaaatc tgcgatggag tctccaggat gaagccagaa aatggaaatg tacgatgacg gttgctcttc ccacttgagq tcatgcggaa cgcgttgagg tgtgaaactg acctgcggag ggacgtcgtg acttgcaatc atgcgatgct ttgaagctct ttttttaaaa ttattctcaa acccaccgaa agtccgctac gcaatgctcc caaqggaaag aggtgcgagc gttgttgatg cttccagttq ttcatttcat atgqttctac accatqatat agagcaccaa gattgatcca ctgaaccaqa tcttcagaaa aggaaqttga agggtgttca atgatctctg ttgactgtga atcaaggaca ttaccgttga ggtaagttga atccatatat qtaggaatca tcataatcat qaaactcaca ctcaaatctt atctcgtgta aaattcaacg aacaagtaag tcgtctgtaa gaaccaagtq gatgtgacgg gccgaatatt ctgcaagact tatcaaactt caacttggct agttgagaag aactctcagt qggaagccgt ctacatgtac aacccgtaat cgaggagaac agccgaatgg gcaataccgt ggatggaaac ttttataaat attaccagat tcttaaaaaa tactttctgt tagaatttta atccaaacta acttgaacgc atqaqgctct ttaactttct gccagaatct cgactccaag aaagcttgga tcagctctca attgaaggac ccagaaggag ctgaagaacq gaqgttqaaq gct caaggac gatactgcta aagtttgaca ctctactgta aatgccacaa ttcactggag gtcgtctact tgcaccgttg gcataaacaa ggtgttggtg ttaagtaaag 1200 attatcttca 1260 acaatttcct 1320 aacttttagt 1380 atgcgagtct 1440 tgatqgaaac 1500 tcaagatgtt 1560 ggaaacttct 1620 agcaacgata 1680 tcttgtaagt 1740 aattcgacaa 1800 gtttcgatga 1860 ccaccaacat 1920 gttccgatca 1980 tcggaggaac 2040 cactctccqa 2100 tcaagtacga 2160 actggactcc 2220 ttgatggaaa 2280 agccagagtt 2340 actgggaatc 2400 gccatcaagt 2460 agagaccacc 2520 ctctgtgaat 2580 cccaaaactt 2640 ataaacaatt attttctctt tqcagttttg cctgcccaga qtggcaagct ggtccgtgqt 2700 WO 99/37660 PTU9/11 PCTIUS99/01313 cgqcttgctc agaaggaagq agaaqttcga ctggagaatg aataagatca agactcgtga tggttgataa tgaacttgga agtqgaccgt gtcgtgttat gtcaagctga catggtacac gtgtcgctgt tcaaqccatc agttcggatg ctaactgctc ctaactccaa ccaatgctga acggagaagc atggggaact ccgaattcgg gcccatcgtt tcactcttgc tctatggatg agcgatcatc agagaagtgt agaagaggga cacagagaga gaacttggtt actttatgaa agtcacctqc ctccaccgaq ttaatttttt caqtgagtgg ctgtgccatc gaagccagaa atcttcatgg ttgcttgaac tgagaaacaa ctgcccaqat ggaaacagaa gggatgcgaa cgctgaagct tgttgatgtt tatcggaaat atgctgtcca cactcttgga tcgtggagcc ctgtaaagat cttcccatgt ggagacgcct aaactcttgg acttgcaatt agattttgca acaaataatt aaggact ccc attccaactg tcttccagat agcaagtgta caccaaaacg ggaaagacta tccgagtgta tacgataaga gattgtaacg aactctactt ttcggatgct gaattcgttg tcaggagatg gagtgtgcca gatactgatg gattggtaca ggatgtaacg aaccttgaag cgtaagacaa gagcttagcg tccaatacag ccgctgatgc tgggaccatg aaatatgggg tttagtgcac aaggaagagc atactaggtg catgcgccac ccaccgaatg gaggact cga actgcaccaa ccgctqaatg agccagttcc ttgatgactg ttgctaccgg gtgctgacaa agtctccact ctccagaact ccattgct~c cttccaatga ccgccgcctc agactcaatt gatgcggaga ttgccttcgg acttcggatg atcggtgacc ttgcccagct cqagggactt acctggggaa ccgctgcaac ctatccactc agtcattcca ccaaccacca cggacacgga ggttgttgat tgaggagaag tggtggtgga agaatggtgc cccaacttgc agaattcaac tgttaccgtt taaccttgaa ctgcagcgt c aatcactgct gaccatacac tggaaagggt cggatgttgt gccatcttgc accacactat ctgcccagat tgccgcagtg gatgagcaag acatttgtca aagcatacta gatactgagg gagaagtgtt gatatgacat tgtgagtacg cacaagactc gaaggacact tgtactggaa tcccaagatc gacgaagccg gttgactctg ttcggatgct gccactggac gct ga tgtt g acaaacgaga cttcttggag tgctcgaaga aacgaaggat cacgatgatg gctgcttccc tctggatgtg ggtgagactg 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 ctgctcttgq aaagaatgga accgqatgcg gagagaactg cttgaccacc aagttcggat 4260 WO 99/37660 PTU9/11 PCT/US99/01313 gctgccctga tcgcccaata gatgcccaga ct cgcggaga atggggagac agcacggatg caccaactac cctgtaatca attaacctca gcatggcatt ggaaatgaca ggcaagggaa ccaagatact ctcggaaatg ggaccgtacg caataccttc ccacaacagc tcaatggaag caatggttct ggaaatctca ggagct cagc ccagctcagc ccatgtaagt gaagcatocc aaggacgttq tggaaagacc cgqatgctgc aagctgcgcc gaacaaggaa tactgctctt ttgcccagat cacgccacca gacacaagaa tgaaaaagaa atgataccac acaactttgc gatgttacct attatgatca ccaacaactt atgctccaac caactccaga cacaacaaca acatctgcag acaacgcttt atcgtttccg catccgctgc caagtaacat tctttagaat catttcaqcg taagggggct accgccaagg ccagacggaa cagagccagt ggatgtccat ggaccacgca ggagagacca ttcctcatgq agtgqaaccq ttggagcaac tgagggacgt tagccaggat gccacqtqtt ttccaagaag caactctttc caccgctgct agttcaacaq gcaacagcaa atcccgccaa cagccaagaa cagcaaggat ccggcaggaa cgtctctcca gcatttattt aacagcaaac tttgactcct ggtcccacaa aatcagttgc tcgqatgctg gccagtacac atgatgqatg aqqctcttgg gaggaactgt tctgcggagc acatttcatg tgcaaccagt atgtgcgaga gatggaccac cactgtgtg gaagaatgct ccaccaccac attgagattc caacagcaac gacgccggac tgcgaaacct gaatgcgagc caagctcagc ccacaacagt cttactataa aacgcgatgc ggtactacga cgagggatgc caaggqagcc cccagacgga ccgttacqqa tgataactgc accagatgga tgccccacat cggatacaag tataaatatt tctgqtacgg ctatctgcqt tccggtgtga ccttctggtg ccatgttctg caccacagca aatctgctga aaccacagca catgcgagac tcacttatgg agcgttgttt caqcagctca cagctaqtc gtttctataa ttgccacctc agttgccacc ggatgcgagt qgattttacg a agact cgt g tgctgcccag cgctacgcca gccggatgcc aaaatcgccg cttgtaaqta tcaatttcag aggatgcqqt cgaaccacca ccaacttcag gagaqqatgt taaggacgtt aaatgctcag gcaacctcaa accacgtcaa ttactccgat aggat gtgga cttcgttcac accagctcaa agttgtggtt gttcgcatgt aacgttgacc ggatcctqcg 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 5520 5580 5640 5700 5760 tcacattcaa gtacaccgga tgcggaggaa acgccaacag atttgctagc aaggatcagt 5820 WO 99/37660 WO 9937660PCTJUS99/0131 3 gcgaqtcact gagttataga gtttgaaaat aaaatcttaa aaaaaggttt aaactgattt ccaacttttt qgaccgtgca cgattccatt aaggctgctt tctggaaagc tatggtggct tgcccgagta aattttcaaa ctccacccgc acacggcaat aaaaaaaaca gttgcgctat gcatgtaagt tttcctgaac gtgtgatttt aagtttgcca acgtqtagtc tattaagqaa ttctcactca tgtcataaca ctqtgtgaaq gaatgttcac gtttgcacca ttacactcca aatattgagg ttaaaatctt cagacggtgc ccaactttgt acggtggatg gtcaaaatca attcctatta gcctcggaaa agttctaagt tactttttgc tacaaattgt gctqcctaaa attctctcaa tttttttaaa ttgtagtgta tgcatgaaac gtttcaqaag cttgttcaag caagtgccta tcagtagcat caatcagata tccaaattta ccagcttctg tatttttgtt tgtttcaaaa gaagtacatt tttcaaatta atttttgaag agctggaatc cacgaaatgg ccaaggaacc taaggatgct ttactacaac tactaacaga taatagtgat ataattacct ttcccgtact tgaactgcct aaaattccct taaatgaata tcagtgtgtt t gt tct tat t acgaccagac aagatcctca cttctcgtat gtaaattgtg tggagtagct attattaaaa aagctgcttc aaatgtttqa tagtttttga ttaaaacatt gaaatattaa tttcgctttg aactcaqttt tactacaaca aacaatcgat tgtcaacttc actgccagtc ttcgctacca atatgctttg tat ttctatt ctctcctttc aataatattt gccgttccgc aagctgaaat tqtcgtgaaa acgttttaga tacaacaaca gccacgaccg gaccaaaaaa tggattgttc tatatgggaa agttgtgaag agccggaatt gtaaatqaga gttgaatagt tttgagaatt tatacgqgga atattcgtgc gtgacgaaqc aagccgacqg tcgacaacga caaaggttca atcaatgcga ttgaggagtq tttccccttt cccttctgtt tcactttccc atgaattttc cactgctttc agttaacagt gttttttttt tttqctgaag tcacaaccag gcattttcat tttaatataa tcctgggttg tttatttgag tttctcatta ggtatgcttt aactggctca tgaggccatg aqgtcttcaa tttgggttta aaaaaaaaaa caaggacacc aacctgcaac gcaacagtgc aggaccatgc gacgttcact tcaagcgaga attctttgac tcccattttc gtccgaagg caattttcta ttcacccatt ttctgaaatt acctgcatga tqtgctagaa aaqagctccc tgaagtaagc ggtttccaag atgggttttt aaatagaata tgtatataaa 5880 5940 6000 6060 6120 6180 6240 6300 6360 6420 6480 6540 6600 6660 6720 6780 6840 6900 6960 7020 7080 7140 7200 7260 7320 7380 WO 99/37660 PTU9/11 PCT[US99/01313 at tcgcctt t ttgagaaaat ggattttctt aaatatacac agtattcatg aqttgtaaga aataatctqt ttttaacagt ttcccactaa qcacqttccg tatgctgttc taccgtctta catqccgtca ataaatgtga cttcaaaatc atcatgatcc gagctctacc tgagatggag tatactaaaa accttcatgt attattttqt gaatacccaa cggcctctgt ttcaagagg tagaagtcga ttatggatta caaataagaa aactggttca ttcaggtcgg caaaaattat taaggttact agtatagaga ggggatagaa gattttttta actctttaag tatccgcccg cagatccagg atgattcaga attctactat aacatcgtgt tctggaagag agaagttgat tqaattatgc gtaagtcaaa gcaaaatctt attctggatg gtcctttcgt tttgagcacc attttctgag agtgagaqag tgccgttctq ggaactaaga caaaaattaa aaaagaagaa ggaaatttcg ttttaccata gtattgttac ttactgtaga tgttgaaggc agcatatcct acaatccttc tcaccat act atcttgcggg gactaccgt c ggtggatgtg gttgctaaga gttagactaa caaatcgaag atgcttccaq tcaaqaataq aaaatctttc taatccaaat ggaactggc gctqgctcac cactcttgag agaaataaac cagtatcctt aatcatcact ctqtatgaaa aaacqttgga acgattttta ataaaatacg tctagggata ttgaaaatct acaaggctta ctttcccagg qcccatagt c cctgattccg taataaatct ttgtttggca ctgggaatac ttgaagaacg cggatccaag aagaagctga aacaaagagq aaaattcgaa agattcgact gcaaatcact ccttggtttc tttttcgacg caacagtttc gtacaatttt catgtttcgt cactgcggta gagctgaatt aaatctagac tat tttcaaa qaatttcact ctacaagaat agacaaaaat taaagcacca aaatccactt gtcgccgtga aagaggaacg cacctatcca ctactctgcc gaatcqcttc cgtqqaaagt gatggattct atatqtcatt gtcttgttat aatccgaaaa ctgaaaagtc tcactaqtca taacttgatc gtgacgttcc ctcactggaa tgaagccgca agttgtttct gttgcatttt caatttqaaa gtaaatctat aatcattcac ggattactgt atttttgcaa catt t tccgt tgggaaaaaa ttcaaatata tgctccattt agttgactgt ttacaaccat acgagtaact gagacccgg gtgtcagaag cactttggat gttgataccg gataacattt actttataat tcaatqtgta ggttagtttc ttctccttcc tcaattctaq atgtttgttt catgatttgt gt agt a at tt tgtgcatgca 7440 7500 7560 7620 7680 7740 7800 7860 7920 7980 8040 8100 8160 8220 8280 8340 8400 8460 8520 8580 8640 8700 8760 8820 8880 8940 WO 99/37660 WO 9937660PCTIUS99/01313 -52tcttcccata aaagcaacaa atgcaacaac tgatagagcc gccacacaaa ttgcaataat 9000 tcgaagtcga gtgcttcttc ttttttqcat gcggtttttq tcacagtcct tttttcagta acttagtctt ttcaaatccc tqtatattqg ttctcctttg atataccttt gcacggctca aactgtaaat aagaatttta ataaaatatt aaaggtatcc ttttaaqttc accataccac atccctccag ttgtctagaa agatctagtg gtggaaaatt atttattgcg tataagagaa aagtatcaat tttctaattc ttgctgggqt tgttcacaac aaatctgaaa atgtctatat tttctataaa taagtacctg acatcacaga cattcacttc tttccgcact tccgctggcc ctqaatagaa tgaaaatttg gagttagaca taataqgatt aaaaaqgtat tacaaatctc attcaaaatg catagattcc tacttctctt agtcatctcg gtaaacagta tcttqaaag tacaaaaqat tattatctgt ctttctttac cgagctcgca ggcgt ccagt attttaaatt taaaaaaaat aactgcattc qqgatccgta t gcat ct tcc caaaatatga gaaataattc gatagtcaca ggctttccqg taattccaaa ttatttttct qtagagcttg tgagataqtt ttataacatc ttcacattta tccattacga ttgaagaata tgattgtgct catagcgtta ggtatcaatg gacgtaattg gaagacagtc tttttgtcta atgaggaaat gtatttgtct taagaacagg tatcggacat gcatttaatc aacacagaca atttctcaaa gcccacacat aatagatttt catttcacct gtcgaagcgg aaaaaaacag caaaaagttc ttagaaaaaa tcaagcaact gctacatcta tctccagtct cattcggctc cgatgcacgt t tt gt tgt tg gatactgaca agagaaatag attaatcaga acgtqactct tgctcagctq ggttcqatga gggcagtcac atctatagca taaaaaaaat ataactttta atttcatcac aacccctcga tcacatcqgt ggaagtttag ttccatccga aatccgaccg cttttgcaaa aaagttgtat taaaaqctac ctatttgtaa ctat caaact gtcccttgat tcggatcatc cgct caatat ataaaaatag aqtctactat ggagatgggt gatcagttga gactcgaact ctttttcgat gtggaccgtg acgaaagaqt gttttgccca gttttctcat atcgttaaaa aataatcgcc cacatttact cccccttcca ggcctcaaaa cttcgcaaat agagtccgtg aatcgtccaa cagttttaaa ttgaaaaaag actqtcgagt ttgaaaaaaa acaatgacaa cagagcaaca attttcatct gaagagtaaa caattgattt aaaatgcatt aaatactttt caatttgcat 9060 9120 9180 9240 9300 9360 9420 9480 9540 9600 9660 9720 9780 9840 9900 9960 10020 10080 10140 10200 10260 10320 10380 10440 10500 WO 99/37660 WO 9937660PCTIUS99/01313 -53gttgatagtt tggaatatta gatattaagt accaagagag cttttatttt gttagcaatt atataaaaac aaaaaacttt tcaccttcqt attgggtgaa cgagtcgtga agaaactctc gaattgacca acagagaaga ttttccactt tcgctgacta agaacaaaqc tgtgcggcat gaatggattc atacccgttg atacagttta catgtcgggc tttcaattaa aagaatttaa tatatttcat tctcatccat ccaatgttaa aacgtgactc tttggatcaa tctgtagttc gaaaaatttc ggtatttaaa tttgtgtaaq aatttcaagc gttaqccagt tacagcttcg cattattttt ttatagcaca aacaaaagaa tcggaagtga atttatatac acgtgtgctt aatcataagg ggcatagttt catccgatcq tgtatggctg aaataatttt gcaggcacgc aatacaatca aaaaaacatg t cgataagct ctcttgttga agaaagtctt ttctctgaca tagtcatcac cttcggcaag aatgagttgc tgttcaagct attcggtcta ttcacataat ggtttgtctc gaqccaattg gtctgaaaat gttggttaga ctqgttttca ttcatctqga ccaatagaat cttcggtttt tcgaaaatcc tatcttgctg ttgctccacg gcaactgtga ctaaactaaa taacgtgaaa actacacaat gaaatttaaa tccaattcg gctqtcattc tggqttttct aggtttgagt ttcqgaagtg aagatcgtca ttcatqttag aacgtaatta attaqaacat tctacttacc cacacatcag cacaaagtat acaaacactg aaaagatacg cttqaaatag gaagaaaatt tcacctgatt gtcatttctt catttaqaga aactctcacc gtgatctctt acagcgcct c gaaatcagt t aatagaattt ga at ga ccc g cttttccatc Cttttctctg tcttctcatt ccagatgaaa ccgtcatcac tgtccggtaa agatccggtc aatttggaat gagttattca caatttttaa ggtatcatca aacaccctta gaaaaqtgat act gat ctga gagaqgagaa aaqacgatga gagaggagca ctttccgatt attgttcatt tcaagaqgtg aattgatgag ccgccgcctt agtggaatgt tttgaaacca cgagtggtta gataaatgaa atctcccttt atcggatcgt ccatttctga caaatgattt aatcgtgata gaaggaattg ctgaaaaaaa ttttaaagat aacaagcttt cgcagctgat tcgtagagct actccagctg gaaatgcacg ccttcatcgg gtgggaaatc aagatataca acttcttgta tgtgtacatt gaaaataaac tacctttaat tatgtcagta ttcatccacc ttagtttgat gtcttgtagg actattttat atacaaatac gctggaatat acactgtgtc gcttttgctt 10560 10620 10680 10740 10800 10860 10920 10980 11040 11100 11160 11220 11280 11340 11400 11460 11520 11580 11640 11700 11760 11820 11880 11940 12000 12060 WO 99/37660 PTU9/11 PCT[US99/01313 -54ttttgtggat gaattacagc cccagcagac gcatacatct aattttcaat tcaacttctg aqtttagaac gtttacgcgt attaagaaac acgcaagttt ccaacacatq tctgaccatg aat cggtqaa actgaqcgac ctcgcgtgtg ggcagaagaa aaagaaaatg gaatgqtaaa tgaaaqataa gtttttgtat tttagagtgg aaataaagaa tgaattacat ttcaaaaacg ttcagctttt tctgttgtat aatgattcca attgtatttg tttcgttttt atgtttgctt aaattaggaa tacagqtgtg caatttattc t tgt tt ca a ctttgaaaac tttcacaagt tcatgttatc tcatcgttct aaaaccaaag tttgtctatc gaacatacga catttgagaa aataaaggtg caaaaaaaaa aqtgcaaatt gtcttgtcac actaggcqcc taaattccaa tttttaaaat ctctctctct tctttqcact gcgcagatqg acatgcttga ccaccacacc ataaaattga agtattctca aaaaaatctg gagtgttcat catttttgct aaaaacaaaa gttcaaatcg cgcacatact qaaaaatcaa tagtaatgaa gaqtqccaat actatgtcqq aatgcaaatg gatcagcaaa taaatacaga cagtagttgt gatccatatt aagaagctat caaaatagga tgttcataag ctattctcta tgcagcagca caataacgat tattacagtc agaagcaatt atttaatttt attgagagct agcaagtgaa tgtagagaaa cttttagagg tatatttttt ttcgatccaa gatactagaa taaatagtaa atagaaag at gagttttagg gctacaagat ttcggcagaa aatagttgaa aaacaatata acctgttata ttttgaacgc agtagctata aaatcatata cgccgagctg tcgtcaccta ataacagaag tgcgqcggct tggatagtac ctgaaaaaat tcaaacagtg qtttttgtat caccaacgta gttaggtcac aatttttttt atccacttac ttctacagta caattgagaa caacttgagc agaaaggtaa agtagcgaca tcttgtgttt atcgaatgga caaatatttt tgacgtattt agtacagcga aatatttgaa cgcataaatt aaaacgaagt tccccctcag tatttcatag caggcaatga gatgtgctca cagtcgaatg gtgtttttga tttcagaaac taaaagtttc ttgcatcaca cttccagaaa tagaggaaac cgagccctt g ttcttgtttc aaagaqtagt a aqtct cgt a actcaaaggg gaaataagtt actttttgaa gtttagagca gtagatttca ttcaatcatt agttatacat atccaaaaaa gtqaatacct tagttgtcaa ttttcgttta tccccttatc 12120 12180 12240 12300 12360 12420 12480 12540 12600 12660 12720 12780 12840 12900 12960 13020 13080 13140 13200 13260 13320 13380 13440 13500 13560 caaaaqtgga agtgaatgag gatggaaata tqataccgca tgcttcaaaa aaatttqctt 13620 WO 99/37660 PTU9/11 PCT[US99/01313 atgagaaace aacatttgaa aatttecagg aaaettgtga gaaatgtggc ggtatgtaet atgttgatgg acataaatta attecagaat aggctgaggt aaaatcactt tataaagaag tceeaatgtc aaageaqaet tgtacattg tcaaetcatc ttegttagea atctgcacac gaceegqtga eggttcgatt gaaagcgaga aaaacagcga atatttaaac taaettttea tcaaaataaa tceqaatceg teeteeaqtt etttaactgc agggaqtega agtgtgcgag gttgaeaeat eaaggtttta ggattaaaac ttgaaaatgt aggtaggetg gtaagcaeat atttaeattt aaatgatgat gtgaatetct aacgatcggc tegattgtaa aagagcaatg tccgcccaga teeaateetg eagagttgga gagtgggaca tttttgttgg tggteeeatg tgaaeaettt aaccttaegc atgcaacagt eggctcgcga tgacgceqgt ggttgtgate ttgeeggccg tttacaaatg aatagatgta aaaaatacta ttttetetgt tagaaagtge gaaaateaea taataataat gtggaetgga ttgeegagtt cggtceatgt gecatteact teagcagagc gccgggaccg tcatatgttc ctgcttccga gctgacgtct aaatactgta agagtttggt agccgatttt cactccactt tacggagtgc atcaetgaag qtctacactt aagagacttc aacacggaga ggatgggaag gtaactgaaa tgcggagtca ttgaaagtag et t tggt gte tgaataatga aattccagat tgcggaggaa gagacattat aeagattcta tatggaggtt tgccgtectg tgtcggctgg cattatggag egatgeteqa gcatccacgc agttattaat teattttccg agctagtttt ttgaatactt ttccaataae agaatgatga gcattgeegg ctggtcacag acgagcctgt ttgccaactt tggtcggtqa aeaaaatgac ggtgactaat aacgggacet tttgtaattt tgtaaaattt ggtaceatga atggaaacgc ggtccaacaa tttcaatgtg gccgtqqaaa gagacacaaa gact egaqaa gttgtgaggq qtgtcaaggt cagttattta tttaattega acaatagact gtttattaaa gtcaaataca atggtaeaag caetttggaa ccaggatagt aactacatct ggtaaatgga gegatteeca tcaggtggaa agatgtacat ttttctggaa tttacaaaat ttgttttcaa agaaaattag cccaaaaaca gttctcttca cactgacttc gtatttcgat teagaatcga atctgaggat atacttttac aaatgcaaac tgaagcaaqt tattgttaae agatttctta gcaaaattga atttggtaat ttttttcagt aacggaggtc attgtggatg acea at gag eq cgtccaatgc 13680 13740 13800 13860 13920 13980 14040 14100 14160 14220 14280 14340 14400 14460 14520 14580 14640 14700 14760 14820 14880 14940 15000 15060 15120 15180 WO 99/37660 PTU9/11 PCTIUS99/01313 -56tgacaccatc ctacaacacc gcccgacaag agaacggttt ttcataattt tgttttctcc ttcccctcag cttcttccta aaaaaacaat gtgccattac gtctaactct tacgccttcc cqtatattcc attttctaqg caacaqgcta aagcgtccga aggtttattt agccttacaa attacttata agctccacca atctgatttt ctgagtacaa ctcctgcacc tgactacgat gaaagtttgt caagaacttc cttccgaatc caattcctgt gtgcqggaag taaattttaa ttcataatat acaattggct tactttgqta ccacttgaaa ttccacttat gctgattgca tagtgggttc agcgacggta acaaaaattt gcttttggag aagtttttgg tqcactatta tcaagtatta caaaaacatg cgctatttaa ctaaatttaa gcaagcgatg aatqqtccca tgcatctgtt gattttagtt tccttgggaa tatacgcctg atggatgtgg aagcgatatg gtttggattt ctccattgcg aattactcgt tacataatca tccattcagg tttgcctatt agcttcccag acgtttcctg aattttcgct aaagtaggct agaaatcaaa gaatccgcta aggcgtqtag ttcatgaagg aaaaatttca ctggagaaca cttcaagctc gccccacaac ccggttactg ggaccactta aattgatctt ccccaccgac gatctgctcc gtaacgcgag gaacattctg tttgatttca agatctcttt tcgttccagt tgqcatgaaa tgtqaactaa cactaatgaa ctcagcgqag cggatgcggc ttttgttaaa tgcgcatact agtttggtgt tacactttaa gcactaggcc tcttgatttg gtttgccccg caatccagct acaataccqa cacatccagt tagttgaacc ctactcccgc tcaaqtaatt accatctcca atctgatgct cacgtgcgat ctgtcacact aattttcatt ttcccttctc aaataaatat tacaagacaa catcttactc gtctctgaqa ccgccgaaaa gagtaatttt tqaatttcaq catttccctg ctqtaaatct gattgataaa ggcaaagctc gttacagaat ccatctgaga tatggtaggc tgtgcaagga cgatgcttat acctgcaatg atcagtcggc ggatacaatt tctacagttt cgtgtaagcc ttgatcgtga tgcacccggg aatcttttaa ttcctatact gaatttattt aaaaaacaag tattaacttc attattttct cagaaatttg caaagagatg gcttcaaatt ccttacctqc aagctttccg tatttgaatc gcctacqggq tccatctaaa agattcttca ccaatttttt atgaactacg tctccaccac ccgtatgaaa ttgaagaagg tccagcatcg 15240 15300 15360 15420 15480 15540 15600 15660 15720 15780 15840 15900 15960 16020 16080 16140 16200 16260 16320 16380 16440 16500 16560 16620 16680 gaggaattgc tcaaaacttg aacgacaggt acaccagctt aacaccagaa qctcaacgtg 16740 PCTIUS99/01 313 WO 99/37660 -57ctcagaaagg qaggggtaag cgqaggattc tggtaacaat accaggacca gaaaactatg acagaaaatg tgtgctcttc gaataagctt cgaatcagtc ctgtaaaata agaggtgaac tqgtgtcaat ctagctttaa tcacaataaa aaattcgaaE aatcctgaat aacaattcaC.
ot ggcgggal tcgaccgat( gjaatctcta tactgatcc tcatacctat atgcaaqgtt tccccactct ggattcttcg aactgtttca gaagacgcqt gcaactgcag cctagaattg aaaatatcca gctgctcatt gaaatcqatg attgtcgatc ggtaccaagc agatttctgt *gttggaaat Latttaggtg( agaaggaga, j gaagaagac gtcagtttg g atcgaacct c cttgtttga g gaaatgttc acggctctgg agaacttaca cgttctttct ttccggtgcc cgaacccgtc acctggccgc tgcaagcggt aataagctta ccaaatttca cggactt cgt ggaaattcat Ictagctcatt ccacctacat atattaaagc g agqtgaaaaC.
a aagaqcctal a gtagttgcai g atgtacaaa g aaatggatt t tqtgtgtaa gcggtggaca aactcaattc aaacggcggt tgtagtcatt gggattcctt aaaagcagat qgggt ttat g caacttgaat gcaagccgaa cgcaaaaatt actagcgtac cttctCggga tgtctacggt tgaaatgtgz aqttcgaaas ;ttgatgaaql aaacagattl aaagaaaata t agaactcct t attgttgat t gcaagaatc :tctatcaa ttttacaga :aggaggta cgcctccac gctgtaacg gtgcatcac atttcattt acgacttga aaaaattcg attttgccg 'caactccaa ~ctgctgata ::ccattaaat attaattgtt ttttcaatcc aacacaattE tcaatttaci gt agaaaaai aagcataag gaaaaatat aaactcgtg tatcgqatt cgqattaag aatcgqaat aaagtccga tccatacgt agtttacttg 16800 aagqaggccc 16860 ctqgtggtgg 16920 cgccaccgcc 16980 tgacacttqa 17040 tgtgcaatgt 17100 tataatqtaa 17160 attacaactt 17220 qaacaacttt 17280 gtgacctgaa 17340 tcgccgagca 17400 aggatttgga 17460 aatqgagggt 17520 tatttgccaa 17580 1gaatgggaga 17640 iactgtgctcg 17700 icatattacac 17760 a gagtqaagqa 17820 a aacagaagaa 17880 t aaqcaattct 17940 a gtgtgattgt 18000 a cgcccacaaa 18060 gtctcaattg 18120 a gaactatgac 18180 ta qagatcacaa 18240 -t tgctqcctag 18300 gctgcttgca qcattatcaa tcgtgttctg qgtctttttg aggtagtcat taaaagctgc cqctcattcc ctgcttatcc atattqgtta tctgctcata gatcgtacgg acaagtttct agaaaagctg tggatcctcc aagcgttctc atgtaatgag cccaatggca caaaaatatt PCTIUS99/0131 3 WO 99/37660 atcctggtaa ttgcaataaa aqatccaaca tgggagttgc agtaccggaa atttcaatac caatqgtgta tatgctctga tcaaatcgaa atgacgagtg gcacgaagct cctcgttaac acgttttgaa ttcttcatct tqcaaagggc cgatcaagtc gagcaacaac cccataacct cqqcqat cca aatgttqtaa gccgagt gga gtgagatgqg 18360 18420 18480 18540 18600 aagatgtact gttctgtact ccaagaatga ttcaacaact acaatacgaa agtattacgg attttgtgag aggcagatct aagagtgcaa tgctgcatgt agactctttc cacactqctg tatcccgcat tgaaatcaac ttqccgatca cgtgtgct cg tagattactt ggttatagca actgaaagtt atgaaacaac gtctgaaagt gct cgagccg agatctcgct ccgaatCCgt ttgtgatcaa catccattta attgagtaag ttgtttgaaa tatcagaaaa ttccaatccg ccctgttgcc aagtacaaat 18660 gcatatgcag taaatattaa tggaccggcg qgttggcgtt gtaccatqat ttagtgacat tttaaattca ctgtttctcc atgcatttaq ttgtcaagcc ccggcatgtg qatggtctga tagggttttc aatttaaata tcacacttaa 18720 18780 18840 18900 18960 19020 19080 19140 atattaaagt tttqataaat tgttgtatac gacttgatta atatgtttag aactactgtg tgtttcccaa ataqtcaaca ttgaaaaatg gaaaaqtttg ttcaaataat tttaattaat taatattaaa attcacaata caqtgtaaca ttcaagatgt gtccggggaa atgggaatct gatatcttgc cctgcaacaa attctgaaaa tccgaaaatg actgatttct aatgctgatt cgaaagtttc cgtggacatc gcataattgl tctaaaaata ctgctcgaa ggtttttcat aaaaggaag caggatttgc cattttttgt ttgatqcaac ttccaaaaaE ttggtttttl agaaccttgi Sgtcagacga tcaaagaag tgagccatcg agaaaataaa tctgaataat ccaaatgtc ttctattttg ccaaaaaaaa aaaaaagtgt aaaacgaagq tttcaaaaaE agacaaaaaaC a tcttgttcql a ttcggtct& ggctagctctE tttttaattt ttccqagaaa tgagaaatag ttttttgaac tcaagaacaa tttttttttc aaaattttga Iaaagcatttt caaaattgtg gttcgcagcc a aaagacgaaa ~cttcacgaa t cacttacaaa agcacgagag tgaattttaa tttagagcaa attgaatttc gaaaaaagtg gcaaagtgtg ttcccgagtg aattttcatt ttgaaataga :tcgaatcaa 19200 ~gaaatagaa 19260 ataaaattca 19320 ttttgaaata 19380 actattatct 19440 aatgtggcaa 19500 aqttttqaaa 19560 aaaatccaaa 19620 tgcttttttt 19680 aagcccgcca 19740 tttgctgaac 19800 ttgtggaatc 19860 PCTIUS99/0131 3 WO 99/37660 -59ctttgaaatt ttc ctaaaattaa aca aaacgaaatg tag actcgggaga cgt gcgcctgaaa ttt aaacgtttta aa~ tgtgaaggga qt ctcgacgtgg at tcggaccgcc gc atgtgaaact gg ggatgaggat gc agtatttaga ac ataggggaqg c~ gggccactgt t ccaagaatgg a tgttcactac t tcatgcctac a attcttcatt t ttgtctaaat ctatttctaa cttcgtatca aatgattgtt ctgaacaatg ttatcttcgt tgacgctcac aagggaaaac ttttgac atcaaga gctccaa :agagaaa :tttagaq actttgca gatttgag ggtattga gagtagtc ctgcctga :atgccaaa aaaaaga~ agtgaaat tcagtctt aaaagtgg ttatttCg tacctgcc :tataacaE :ttcctgql gagccgta tccatctt tttctcgt cacttttc ttctactt attcgct gagaaaa' aaaagqtcac cgt taatcatgat tgt tccaaactgc gca ctttgaatat taa gcttctttca aaa ttccacccaa aa~ ggttctgtca aa agaggaccgc 9C ttcagtctac ca agcaatgcca ta agaatgaaag ga a ctaaaaag ac a gaacgagaac ac t aggtttgtag a :t catgcctatt t ~t tgtaactaat a :a cactgaacat t ag tattttcagc cc attccqtctt tg cctaatactt ag cqgtcatcgg :tt agacgaaaat cac attqctgtg tgt ttggtcacgc tattcaa aaa gggcCtg ttt tcaagac caa acatcgc cgt iagctacc cal Itqtctga aa cagtttga ct tgatcttg tg attacctg aa taataatc at aagacgct ct ;agtattgq gi ItggaatCg g atattggaa g aaatctgCC t tatgtcttc c gactttcaa t ataagcttt :tttcggtat tgaaaatgta gtttacacct tttcccccga gtccatccag gcaaaaaaaa tagaaaaaca aatacgccta ttgagat taaaata Laatata :caagttt tacaaata ttacccgt gtttcgcg ctggtcgt .aatttgac ;aataataa :tctacaac :gatggqa( agacatt agggccaa tttttttt :attatagg :ataaaact 3caaagttt aaaatctaI aaccacgg acggcagg aatattcc ccctcatt gagaaaaE ctcactc( tatcttt gqtctcgtga 199 cacttttcaa 19~ aaatttttaa 20( ccqtcagagc 20 atcataagaa 20 aaaaagaatg 20 ttcgacgtgt 20 cqtcgtcttg 2C actttttgtg 20 taatgaagag 2( accaqccgat 2( g aaagaacaca 2 t gacactagag 2 g aaggggagtt 2 t taaaatttcg 2 c aggcaaagtt 2 :t ctgaaagaaa g cgtatcaagc ag tcaatattta ta tttagacacg ca tattccagtt ca gccctttcat g.a cactctcttt ggt ggctqctgqg 380 040 100 160 220 280 '340 )400 A460 0520 0580 0640 0700 :0760 ~0820 ?0880 20940 21000 21060 21120 21180 21240 21300 21360 21420 PCTIUS99/01313 WO 99/37660 atctttatac atttttcacg gggttcaaaa acaattatga agaaaattqt ttgattaaat 21480 tagaatgtag aaaacgaaaa aaaagactaa gaacgtgagt aacgctctca atccatgttt gatcgtggga atgcaatatg caaaagcaaa tgtggtaqaa caaaatttat gtactcctta aaaaatcggg tcacgccaaa ataattcaac aatcaaaCc ggatgcttti gttcggagai att ct ttat a aagtaataaa aacgtgagac ggggaggcag ctctcttcct gttcattttt aaatagaaaa tatatttttc cgagtgtgcg ctacatagaa tgaaaaactt atgcaaaagc aatatttgcz atgactattC.
aaattttcc4 j agcatgaca, a catttgcta a aattcgaaa ttttcaatca tgagtacttg atctcacaac agacactcag cgctttgccc ataggtgaaa acaattacat tcagaaaata tctcaacgag atttttaatt atttactata caacgaaaaa ttttgaattc Staactaacac.
caaaaaaatC c tctgacacci g ttccatgac a aatattgag aaaattaatt t aaaqtgaaat g gggtcacqgg c ctgactgcct g gctctCCgCC C atttatqtaa tgtaactttt ctqtgttgggI cactaaagtt ttgatttcaa aaacgaccaa Ltcagccagtg gcactgtatc j gtacqaqaaa 3 tgaagtcqc a cgtgcgcctg a ccccatcctt a aaaataaccc a tctgaaccaa tqgaaaaat a gqgcaatta a :aagaagtac a ~gcctgacqct ~cgggtcgtc ~ggaacggaa :tggaccaag tttcgacagg cccaactaga tagcttttct cgacggat ct acttattttt qataaacaaa gggacgcttg aattctcgta aagatgtgcc tcagcttcca aaaacattct tcaacaattt actatccaa 21540 iacaaqataa 2 icqagaaatc 2 :cqctcacaa 2 agttcggtcg 2 aatgtaaagt 2 tttacccag 2 atcgatttat gcatccttgt cttgttttCt ggaaactaca tgttctggat acaccgaaga ttctacaaaa gttttacgta tgcctaccat agatgaagga gaaacattgc caaataaaat caaatgaccg 1600 1660 1720 :1780 ~1840 11900 11960 22020 22080 22140 22200 22260 22320 22380 22440 22500 22560 22620 22680 22740 gqaaaaaagt tagaaattat gtcgaatat acaaaaaaaa attggaagac cttaaatag t ctccgcccat attttggctt tacttcggaa tatggccgat ggccgtggca agacctccaa tcgtagtttt gagcggtcag taaqtgaaga gaggaactga gcgatgttaa caattctctc ttaaaatagg aaataatcga tagatcgcca gaaggcgggc aacagtaccg agaagcatga cttcgtccat cattgctatt taagatcagc taactaagc tgtcgttttg agtaaaataa ccaggtg~gq acgtggccac tttgtactaa gctaccaatt atgtgggata gttcgttttt gtctccttag ttacctttca 22800 22860 22920 22980 PCTIUS99/0131 3 WO 99/37660 atacattcat tca cactatctaa aaa ccattgattg tcc ttgcgtattc act ggagqcatga t9( acaqaagata tt tggaagatca gt actctcgagt ct actgacgtct tt tacgatacgt tt ttattagaaa at ccgtgaagta t ctttcgagaa g gtatataaaa a ctcgaaaatg c ttgaaagcgg t gatgaacaac g aacgcaattg
E
taccaccaac ttgatgcgag ccagaccgag aacttctCta ttcatcaatc tcaaatagtt cgatcaacac tacatcttgg ctqatqg .tgatgaa rtCtcCac :gtttcat caagaagc cttgagaa tttggtCg caagtgac tcgaaqtt .gggaaatt :cagtaaql jgaacaaa cagttgat tattagaq ttcaacga tcataata ~agaagtgS icgaggcc atcgtgta.
agctcCC agctaaag agtcaCtt ctaccatt gaatgatt tcatcaa tcccttt ttttcctatc aggtgatcat gtttttgctt cgcggctatt ttgtgctctt tttacgtctt ttattttttg cagaaaatga tccgattact ccacaagaat gaaaCaagaa ttcttggaga aattaatCaC attaaaaaqt: tqtgacgtaa ttaaaacatt tagtcgagca aatccaaaaa ggaatCatag ttttttaaac tttcqgaaatt atttgagaa aacaagcgga tggCtctcci catattgacJ qagttttcg *a ataaattga ataacacgg *a tqctgaagtt ctcaCggtt *a catggatagc qaccaagtt ;c gaaggaaatc gcggatgCt ga tttgctgcg gagttggtc.
at tattagaaaa tataCcag' ca gtcacgcttC cggatact aa gctgacaagg atctagaa .tc atatttaatt ttcggcta :ct ccggagattc tcctaaat :tc tgtttagCCa tttcattC aat tgtaacatat tatcgag( ctC caaaatCttc caagcat, ttttttqttc ttctcaatta 23040 tgqttgaagt gatgatatat 23100 acaacttctt tttaagtgtt 23160 gcctgttcag caaatttttc 23220 ctattcaaaa act-tcgqaa 23280 aaaaaattga cgacgtaagt 23340 gctgaaatcg aaatttttaa 23400 gctcagcatt tacattgttt 23460 agagcaataa aaatttctgC 23520 tccatttttc aaaaaataca 23580 icgtttcagga aaqcaaaatg 23640 a. gtqtttgagt aggaagaaaq 23700 ctactctcgaa catcaggttg 23760 t ttttcttcca gaqagaaacc 23820 a tqaaacttgc tagcgatgcg 23880 gtgatatgat ggataacata 23940 :a tttcaaaccc tggctttaac 24000 ;g atcttgaaCa ggttcqtcta 24060 ;a agcacttgac aaagatttgc 24120 cc caatattgca cttccagCCt 24180 ga cctcqaaagt tgggcaaact 24240 tt tttgtttcat ttgcatCCCC 24300 ca actttctaat tacqacaaat 24360 -ga aacaaatttc cccaaggcta 24420 ztt tttggaaatt tgtcatttta 24480 aca. ttaaagttcc aacttttatt 24540 PCT/US99/01 3 1 3 WO 99/37660 -62aaaaattcat tct atagttacat ctc gtttatactt ttg aatttactac tgc tattccgttt 9tc tctggaagga tac tagtggtacg 9t ccagaaqtcg qq caaaaaatqt cc tqagtttaaa aa gatgatcgaa at catctccgca at attgccaaca a( cccggaqtgq t gqttgtacaa t tccaacatat t ctttttcgat t caaatcagtt t taqagccacg aattaqttgt gtattattgc aatcttcqaa acqatttgca ataaagttaq atcgagcatt aatatggatc ggcaaac atg agatctc cct ctcattt act 3tgagttg ggc aggagtac tg9 ggccgCgC cg tacttcca at .aattgtgg cc .attctatt tc :cgctaaag at ttttttcgc t cttcttcaa c catgcattt 9 .ttattttat g :ggaatgatc t aaaaatgtggJ acatataagt ttaaaattgt tagccgtgag gaggtagagg taggaacgat gatggcatcc cactgaaaat ttatttg aacgatg aatgaat atatata :agtctat -ttagtgt tatgatta gaggtgcg tactcata aactgaaz :tgcggatc :aactaaa ttctttCg ggttctac ttcatcaa acggaact aaaacttl :ccttcat 3acaattg aatacatg gttccaat cacaggct acgaacqg cgtactg aagacct tgaqgtt taccqgttqa aaa gctcaacccc ttt ggctcattta cta tcatttatca ttt gtagtctttg tac agggatatag gg cggtagtttc ag gtccacggCt gg actcagttag cg tgctgtttgt ca tagaatattt C9 a qgtaccaatt a~ c tgattgtttC ci .a ttatccagag t :c ttcaactttC t :a ctatCataaa c tt tgagaagtga aa aataCatCtt ct gccagtacta :tC gccggcaCtg ttt ttagaacgag tga acttcttCgc ata gtagattatt cgaaaac gacgctc acttgct actatat ;tctgtga ]gtactgt aaaaatta ttttacat cgctatag .gagatgc.
~atcatctt caaaatg 1 ttttggtt cggcaggc tatgttgt gcattgtC :aatagtat itgatcagl aaaatagt aaaaatac tgagcgta cacacaaa tggcqaaE actgtct~ tgaatqa gggagct caagcgaqaa 246 atttactaat 246 gagatttttt 247 ataaagcgct 247 cgttttggCt 248 agtggtacaa 24~ gttttcagCt 24 aaggtagttc 25 ctatagcgtt 25 agctctaaaa 25 -ttgqagctga 25 t tggtqatggt 2~ t ctgaacaatt 2~ c gcttctgCtg 2 *a ctggattctt 2 :t tatcatgtta 2 :c gattcactct 2 ta ttaatgttgC ag ttactactag at tcgttqatCc ict tcacgattgC agc tggtgqCCgC tat acgatgcttg att atgatttcac 00 900 960 020 080 140 200 260 5320 5380 5440 5500 5560 5620 ~5680 25740 25800 25860 25920 25980 26040 26100 PCT/US99/01 3 l 3 Wo 99/37660 -63ccatgaagaa ctgcgtcagt aact cqttgtaatt ctatgagaaa acg acgaggcaga caaacatctg aaa aacaaaccgc ttctagca aca gcgagaagca taaatggtag aat tgtagtcttt gagataatca acc atagcctgaq aactgtgCac ac acatcqatca tgaactacca tc~ tgacaqtqat caacttgaga at tggcgccttc aaggaaaaac tt caaaga~~tqc tggatccaca aa gcttgcttgt cgactcaaaa tc cgtttgttta cgtaatcgga tc agaatgacgt ttacggggaa al tgctgaaatt ttgaaaggtt c aggtctcata gcgccgagta g ttctgagctc gatcagqaag a cgtataaact ttaaaggtgc a ttttaatcaa ttctctgggc t tccctactta aatcatatqt gaagaaataq caattttccq ttgagagacg .gqitctcgcaa gttttcagqc ctttaaaaga tttaattaaa tttattttca cattcctctt tcacccaqaa atgcaaaaaa cacaattqgt :cgtttc agal gtagaat ttg tttgcqt ttt tcagcta aac aaatqaa aga jcaatttt 9tt ;ttttgtt ctc aataacat cc( atcttccg 9t.
gttctcca tc gtactttt ga :gacctgaa ac ~cttctagg aE :cattattt al tccgaatat t ctaaacaat t aaaacttct t .tgaataaat t :agtttttat t :attttctttI ataaaatttg cgtgctcact taqittccgtc aaatcttqgt aaccgaattt atctctctac :tctCgc gtgacat ttccaca tqtgatc .tatcggt :tcagatt jaaaataa ggatataa gactcatc aagatgc~ ttaccaa ~aaaaatc ~ggtttca ttccgaaa aagcgaaa aatattt gaaacttl .tctcctt :caataac :tcctttg ttgctcta cctCggCC gtttttt aaacatt caatatt tttcatt tatccttttc aco ttgtcgagtt aaa aatgcataaa ctt atgctcgtat tcg aggttcgcgg gaa Cgtcatcagg tat atttggcatt cal *accaagagaa tt aaggatattc ag j gaaataatcc ga c aataataggc ca g aaaaattcca at t gacttgttgt cc .c gtgcttgg9c t ~a aaacttacat t )a ttacaagttt g ta gaagatgaaa t tt ggtcctgcga c ta qaaatgttgtt 'tq tcttacaggc ~tg ttaaaggcgc cg atttgttctt tct caatttctgc taa actcatatat aag attaagaaca ,tta aggctqattt gcttttt 261 caattcc 262 tcaataa 262 rgcgttta 263 tccggat 264 ttgtcgc 26' ttgaaact 26~ ~gqagaaa 26 ctgcttta 26 atttcttg 26 gtttcgaa 26 .ttaaaaaa 2( 3gctgcatt 24 tgttctgtt 2 aataatata 2 gaaagatct 2 gtgtqctac 2 gattaaact 2 tatttttgt ttttttagct atgcatttat cgtttgcgcg tgaaataagg tcagaatttt catctagaac tttgaagaaa 100 460 520 580 640 700 760 3820 6880 6940 '7000 7060 7120 ~7180 M740 2'7300 27360 27420 27480 27540 27600 27660 PCTIUS99/01 3 1 3 WO 99/37660 -64aatcatgaaa ta( gatctcttaa at( ggaatgatqa ta tcagcattaa ac tgacatcacg aa cttgcgaaga ta gtgagaggcg ca ccagccccat ca tcctttcatt cc tacagaagtt Cc gtgcatgttc ti tcaagaaggt a.
ggcgacacqc g attacgcgta
C
gactgcctct t cgatctttta t gatttagagt t ccattttagt cattatcaaa tgaaatgagc tgtgtgttcc gggttgaaaa agaaagaaac aagatcggtt atttatcatt ttccagaaga gtccatt gtggtatt attcgtat aaaacgag atgcagac acgtgtta .qatggttc ~agcctctt ~gtcaagta -tcttcttc :qgtgcccac agcattta tttttcca gcaatcgg atgctttc ~ctttggca :aagcatga aacagtata caaaagacz gtagttaa cttttCtC aaaagtat tgaagatt acaatagt tgtaagta agatctaE attgttgtat cccttqtttg cal ccgttcgaaa ttcqattgat tt caaaccaaaa ttgtcttctt tt aatattqaaa gttacgtggc gt gacaaagacc qqtgaaaaat ag cgggttgaga gagaaaacat tC agagaacact agagaaaacc gc cgggcatcga cgcatagaca ca tttcgcaact aatcgttatt 9c tacttggtcc gaccqcatca g~ acaaactcgc gcccatttac a~ a acgtqttgqc cgtgcgtctc a caatttcattc cctagggcat t a cgaatcctgc aggtttgttg g t gaaaaaagaJ aatgacagtt t a aaccttgata taagcattat c .a atgtgcaagg ctaaaataaa t *c atcagctgat agaatcacat ~t tacaaaaaca agaaaaatac at tcggaacttc aatagattat cg cgtttgcccg ctacaaaccg ca aacactgatg gttcttttt tg cgaatcgata gcgtcgtcat gt atgcgattcc caaaatccac ict aacaagagat gtgaacgtat ~ac aaaaactatc gaaatggctc :ccaaagt tg titagaagt ta cgcctttt tt ttgcatct ct tgcgctga at cgcgagac aa :gcctctgt CC Lcatcattt tc ~tcattaca al ataactggg a attttatga t aaaaattgt t ttgtatttg a tagtcaatt t .tcgctaagt a ~acagcatat :tacaaaata gcgtaatgac aatataatag catacgcgct gtgagtcgga tagggaggtt ctctcgacqc atatcaaccg ttacactcaa tcaacgtgaa actcgatt 27720 acacattC 27780 gtgcaqtg 27840 caccacga 27900 ggtgaaaa 27960 tqcttttg 28020 ~gctcacag 28080 jccccaatt 28140 tacacattt 28200 gatccagtt 28260 cgacaaccc 28320 aaaaaaCct 28380 agtaattct 28440 .tatcaagtc 28500 ~gtactaaag 28560 -atgcagatt 28620 agtccatagt 28680 aggtctaaaa 28740 aacgactatt 28800 tttaaaaaaa 28860 aggcataatc 28920 gtccagaaag 28980 cagtgaagtC 29040 gactcgtgat 29100 cattagcaaa 29160 ccgcqctqtC 29220 PCT/US99/OI 313 WO 99/37660 gctgatccat tcl aacqgaatca aa ccgatgtqta t9 tcagacccca cg aacgagcgtt. ac ggtttcatta aa cqagatcatt ga catcaagagC a~ gtctacattc al actaacattt t atcctttcaa t tccagccaaa g cgtggctqcc g cgattgqqaa c cgtgctggac e cttcttgaaa atttcagata cttgagctga atcactgaca aagaaagctc gaacttctgt gaggattcqc taaagtttat attgagaact gagtccgacg ggttttcagt taccgcta acgqtcat tttatcgc aagtactt attgtcaa aagtttgt ~attaataa igtgcaagg :catgaaga tcgattgaa tcgattttc ccgaacaa acataatc' :ctgaacca iaggatctt 3ccaaggaa gat tt ot agcaaaaa aacaaatc~ aaagatac( ctaaatca ttatttca atttacag ctcaacca atqagatt tcgqaggc caagatgccc aagctqtcag CaE ttccaacatg tctgagatcg C9~ cagttggctc gccattggaC ac~ tggctgtqag ctcggggctc aa cggcgagcat gatgccaaca aq gctttgcaaa tcatgtqaaa ac tctgtctaat tttattattt ca catqtggatq ttcgttcgac at atcctccaaa gattgatqtc qE cttatgcaaa attctgccaa a~ cgtgtgcttc cagtgcatac a~ agaatggaaa gaagacatcg gi acaacagcga caaagqcagt t g tcgagccgaa tggcatgctg t.
g agaagagcga agaacagcgt c g aaggtaagaa ttctgagcat t ra tgccaaggga caaactqctc t ;c atctctcctt ctcgcgaacg ag caaacaccgc aatcttctgc ct gttgggagga gttqagcaag gc tcacatcatt aagtcattgt tg ggqagagaag gttagtacca cc gtcgagtaaq tatgtctcca ~gt gctcaactgg ctgaaagaag :gc ggtaagaaat atcagatttg aga cgtcaaggag agtgaattcc iaagtcga aagctct aaaaataa acgaactt.
ctccagga ccqgaaac gtttgtCC .tgatctca itttttgta iattctatt aacatgcta gtgctgacC cgaatgat( cggcggga tcgacatg gataaaaa :acgtgacg :ttttcttg ttcgcttcz taattcac~ atgatgaa.
aatgqaqc aatctttt cggaaqaa tttttttt ttcgtcaz aggcaaagga 29, cgagcgtccg 29: ccattgtttt 29 cgatgccaag 29 tattttaqat. 29 tcagttggta 29 gtaaaaataa 29 aacataagct 29 agtatcgttt 29 tgcattttaa 2~ 1 ccgccgccgc 2 g acgacgacga 3 a tggqcaagct 3 c ttcacacatt 3 a agctgagaga 3 *a tgagaaagta ic qttgaatgac a acatgaagcg ga tgtctgcgaa gc caagaagatt.
.ga aaccgaagaa :tt ttcaatqgtt ica gaaggagaag 340 400 460 520 580 640 p700 3760 )820 9880 9940 0000 0060 0120 ~0180 30240 30300 30360 30420 30480 30540 30600 30660 30720 30780 PCTIUS99/0131 3 WO 99/37660 -66gctgctagag aag tccggagcaa atg tttaaaattt aec tcaactaatt tc( atcatttcat tt caattcattt at tcttgttcaa tt agatgqaac cc ttagcccatt tt aagagccgaa az tgatggattc a~ caatgtqttc t~ ttqataataa a aataggatgc t aactggtagc a tccaacggat c gaaatgtttt gtctgaaaat gagctctggc tgattcaaat tcagagatga ctgctataga aatcagaaaa attgttaact ggcttgg cgatttccct ct cagca ;atgaaqa ~tggcta gaagaagc ctgtcttc tcgtaaag cagtctta :gtcgaatt :caaatcat iccttttqC aaactgatC aaatggtC( atatgttt tggcaaaa aactagtc :qtcttatt ;aaatcatt jgctgaatl aagaatct' tacaagtt acgagaag gcacatgg tctgcgta gatgata ccqatccl aggtact aaaatcagcc aag gqacttggat att cttatqtttt ttc ctcaqttttt ttt tgtcttttct 9tl tcattactat tt tattacaaaa ac cggcggtCCg at cacccagctg at tqctcggaac ac 3 gcagcqattc tE g ttgaaaggat g t tcttttacag a t tccgtgcttc t gq aatgtagtcc g .c ttccatcgCc c :g aaatcat~ctt t :a ttataggaaaE gc tgggaaagat ag ctctggaatc aa gtggagaaac 'ac ttgatgtaca Lat tgtcgacaga lat cctacatcac :cg tgaaaaacga tgc cttgatcttt gctacaa acq .gatgaca ttt ;tgtattt ctt :tgctttC tCC :cctaCgc t9l gttgttaa tc acaatgtt ca gqagaata ca ttcatttg cg9 ta tt taagcaac tt gaaggattc gc tgggatgag a cgccatctt C ttcqataaa a :atcacaatg c :gagctgata t ~gatatgcaa tttggatgga agaaaattat1 gaatggaaaC aaqaaagagc aataacgtat accagatatt gcctattcaa aatttatcag gcaatgc tgc aattgta cac :cccatat tc( ~ccctttc aa :tttccct tc gtaaacat tt aaaaaaaa ga cattqttt tt acgaagCg at .acaataag q( :gtccgaaa a ~tgtaagtt t acaaagaag a gttatgCtC t tatgqggaa t aatcagaat c ggtgacgga ~ccqcacaat itccgagagg tattatataa tgaaagaqat aacaaaaaga tccagattgt cagaggctcc ccatcqctqc aattaacttt tgctgca 30840 3atgcttt 30900 gaaccaat 30960 tagtaagc 31020 accaaatc 31080 gqgaatat 31140 .atcacttC 31200 .tcgqaaag 31260 aaattgta 31320 ttcactat 31380 .aatgaaga 31440 taaatcagt 31500 agtgatgag 31560 ttttctcac 31620 .cggttagtt 31680 ttcaaactg 31740 agaaaaggac 31800 gggctccatt 31860 aattgtggga 31920 aattactatt 31980 tatctattca 32040 gtattgtaag 32100 tcgagqaaga 32160 ttcccgactg 32220 ctgtaataat 32280 caaattccag 32340 WO 9937660PCT/US99/01313 -67atcatctcca cttcgagcag ctgaaatatc acgtaggaag agcacatctc attcqacaag 32400 cttgagtaaa aagacgctcc aagatccgaa cccaccacga ccqaaattg aacctggcca gaagaaagac aaaacaccgt gaaaagaqaq atttttgaaa tttaatatca gat taa gta c aattacaatc ttcattct ct aaaaaaaaaa attagttttc tgacaatttc gtattgagca aaaacactac gatagaatat ctacaqtagc tatgttgcgc ctaaaaactz aggcgqtacc agacatcttt agtacatctc aaacgtccgg acaagagcac ctgagcccat cggaaaaaga tctaaaagat aaaaagaaga actagtcata aatgtctttt tggqtatatc tgtctgctgg gtaaggtttt actaaatacc cgcggcgaga ttgaaacaag gcatatttgc tgtagcgctt ttaaaaaatz catttaaagz aatacgcati iagaacaaagl t caggagcag cccgaaqtgg gaagacgttc tacgccgatc ggaqcagaac cgagaagcag aaaaatctga ccgaatccag aacgacgtcg actttaaaag caaatattct attaataaaa tgaaaattgt gttcgttttt tacagtaatg cccat cccca aacatttgta cgaqccaaat gtgtcgattt iaattcatttc iattactgtac :ctcagaattc :aaattaaagt a gagcatgaat atcgagaagt atcccgtaca cggacggcac aagaagcaga aagaagtcag aagtaacagt gaagaaacgg agaagaacgt qagaagtt cc tcataacttt ctatttattt ttacgatact acatgctatt tggaaaattc ccagaggcat caaaagtatg tattaacgaa atctcgtagc acgggctctt gaaattagac fttttcgctat Itgtcttccgt ttttttctql caattggaai ctgtccaaaa ccagctcaac tctagaagtc tccaggagca gctaaaagta aatgaaagga aagcatcatt cacagaagac act acttcag tttaaaagtc attcttcgta ttgccgaata ttcttgttcc tgagagtagc atgctcaata actcccttga acacaaaatt *gaaaactaca tgaattatca cccataaatc gagatatttt aataataqAc tcacttcaaz a gtgttcaatcj qcccggctcg 32460 ggcattccag 32520 gatctagaag 32580 ggacaccaaa 32640 gcagcacttt 32700 atttgaaaga 32760 ctaaatccag 32820 ggaaggagaa 32880 attaaacttt 32940 ataacactgg 33000 attaaactga 33060 aatcagttat 33120 tcattctttt 33180 cggaaaaaaa 33240 attatcaaaa 33300 aagtcgtaaa 33360 ccgagaatqc 33420 ttaattctta 33480 ttgatttatc 33540 gacacaaaca 33600 *gcgcatcaaa 33660 agtgqcttcg 33720 ittttacacga 33780 3caccotacag 33840 tcttgaagca aagttcaaa atatqatttc ggggcagtgt aaactacagg gcacagacat aaaaatttaa attgttgaag 33900 PCT/US99/01313 WO 99/37660 actaaaatat tat taaat qt caacctgtcg cagctgacq cgacgcgttg qgaaactatg ttggagtgca ttgcaaaggg taccctaaat tgatctctga cagagaccaa tttgaaaatc ggcggtgaqt gt taat tct t atatccacqt atgcaccagt agaatagaca aataaattta ggaagtgaat agggattaac aaattggtgj acacaatggz tqaaggtgt( aaaaaagtg( ttatqaattc aaacatatga atgcatacaa gacaacaact gctcagagac qagcacaaca tggaatttag gtacatgtaa cctqgagatt ttacgaaaat aaacttaaaa aaacactact tqtgtttt~Ct atttctattt cgcggcaaga catcatagaa ctatatgggt gtagtaatac attgagaaaz cttgagcac( Iaggcgcaaq( IacaaatagC( ittttacaat, -aatatttga -ataattcac q atqqtgqca attcaagggt ttaaatacaa ttgctgaggt gatacatcgg ctcacgatat gtggatcacc tgggcagagc ggcttqgctg tatgctaaat atatcctttt ttttcgacat cataccogga tatgaaagtg cccatcaatt Ltttgcactcg gttgqgaaac I gaagaatat iaaaacggttc tcgtaaatc tttgccactl j gaaggttagc a ggaatattg, g gtcaactqt a gattgaaat a caaaacaca cataataaat cataattatc qt cgtgtgac aagttgagat cgagtcatgt caaccaqctg attgctgcat aaagagttga taggatttta tggtagtgtg ttcctctcct aaaaaccaac qccgagattt ttctaacctc ggcttataga qaataggcag iaagaatttca jatattctttt icqggaggcgt tgaagttaaa gggaggtggc a aaatacgcat c tggtttttcc t ggaaattggi a attctacatl gtattttttt a aaatacaaat a agtcagaatc c gagtgactggt cgatgcagcg cccttcaccq ttgcatcaca tattatttct gttataatcc gcaccaaatt tgcagcgaaa aaaaacggcc ctctttttct taatctcttt tttggagcct tagttttttg taattcagat gtttaagcag acttgtacag aaataaagaa Iaqgggggaca atgggaaatc zccgatttttq cgagaaaaqa t tgtaccaaaa ~aataatatt 33960 ittataattg 34020 ~ttgtcacac 34080 :ggacattgc 34140 -tgaaactca 34200 cactqataat 34260 atcaatgaat 34320 attgatataa 34380 tcgtcacatc 34440 cgtgctgtaa 34500 aataaaattt 34560 ttgttccaaa 34620 acgccaagta 34680 ttcaacatga 34740 ttgaaagtat 34800 gaccaattqt 34860 ttcaataaaa 34920 acaagtatgc 34980 aagagagata 35040 agagacatgc 35100 ggtgcatgta 35160 ggaacagata 35220 aattttttqa 35280 ataaggagtg 35340 tgcccactaa 35400 aatgggcata ttcqcacaca ttccacacaa attgcataca tattccacaa tggggaatat 35460 PCT/US99/01313 WO 99/37660 -69tttgaatatt ttctgtttat cttgatccct tgcaacattt gaatatattg cacattgata tccattcacc aacagtacga ttctqttact tcgattttgt tcaaatctga atattttgtt tttgggtatt ttaaattatg agacttccca actatatatc gaagctgata gaccaacttc tttttgatag qaatgagtca tatgtattta gacgggagaa acgcaaactt gcatttgttc tgaaacatat tagattaata cattaattqa gcattatcag ctcgctactg gtgcccggtt acatccaaaa aaaagctggt gttgtgagcg actttttggt acagaattgc caaaaaaaaa ggaaagtttc caaaatatta aacaattaac acgcttcttg ctagtgtgtt taaagaaaaa aagacatttt aaaatqataa aattcacgta ccggactttt aggtaccaac caaactagga ggtgacttgt qaaatagtcC aagatgaaat aaatgttgaa gcaatcctac gaacacgcat ggaagttaac gttcagttat cgccatcttg tagttccact tgcaacaact acaatatatt tttttttaaa aataatttca accacacgac tcactttaca agaactattc gctcgttgca taatagaaat atatgcttaa gaaatgcgac aaacttggca caagacttta aaatatcaga Lgagctaattc ttgacagccc aggccttccz aattgagttt ttacttttta ataacctttc actggaatac aattgaattg gtatatccat tataggaatg gaaaattact ctgagaaatt ttggaatagc aggtgcctat ctacatttac cttcaataca ttttgtcctc gaaataatat acgtctagag atatttttca aatcacgtca caaaatgtgt tttgtcacag ttattattga atattgcaaa aaactttgaz attgaagga ittagagaatc.
tatttgtaat atagacgaat aacqttgtcg gatgacgatt ttgttaagcg ccgtataaat aatggagtta aaatatttag ttagttttca aagaaattqt caatttttaa tatttctttt ggaaaactgt cattccttgt aaatcttcga tatccaatat ttttttccaa cagaactata attttctccg tgtgtcagac *gatcaaacca *aaaaaattaa Latcatgttc igagaaqtact ;tgatgtttga taaaatattt 35520 catcaaagaa 35580 ttttaccaat 35640 ccaattggaa 35700 ataaagqata 35760 cttqcgatct 35820 aggatcccgt 35880 ttcaaaggtt 35940 ccaaaatttt 36000 tcagtqaatg 36060 aaatgttcta 36120 ttaggcctat 36180 caaatttttt 36240 agttaatata 36300 atttcttcct 36360 aaacccacta 36420 atgactaaat 36480 atcatgttga 36540 tttgtcctct 36600 acaaqgcaca 36660 gattacagaa 36720 aaatttcaaa 36780 ataaccggta 36840 cccgacaggc 36900 aggaaqaaca 36960 atgggacgta gagtactccg aatagaqcag taaqtccatt gatgagctqa aacagtaaat 37020 WO 99/37660 WO 9937660PCTIUS99/01313 aatogaaaag ggcgttttat ggttttttcq cgaaggctac cggggocga cgccacgtcc tacttaaaaa gacacaatgt aaaaaatttg aataaccatg ctgaaaaoaa toacactato aatggattgc caaaatgtat catgattact aacgacaato atatatgaca aggagtggtt aaaaaaatat cgaagattag tgaaagaatt cgaacattga ttaaggatat attcgaattt aaatagataa cggtatatca ttagtaaata agttttttct agttccagaa tccaaggtaa ctagatcacg gcgcttcag ttaatcgatt tgaaccgtac aataattttt aagcacaact aagagctqat cccttcgatt gattcaaaag ttagaaaaaa gtagatgoat gaattggcog ctttttaaat caaatgatto tggtgaatgt caagagtttg aaaaaaatcc gcagaagaag cacccagttc ttacagtaca tgaagctgcc ctacctggat tgttcaagga ct tt tt t t a aaaatattta gttaaccota ggttcgcgct aggagcgatt tttttaaaaa tagattgttg ttotaacttt tcataagtgg atggagcaag ttgacgcggt aaaacgtagc ctcaoqctaa agagtgagaa coat cat ctc gaattgacag ataaagggaa ataatttttt gggataaoc tttcaaattt cttttaccaa aaaatagtag ttgatcgttt aagtttaaaa attagtttta atggaagtaa cttcgttttt aaaaatcatc ctcggcaaat ccagtcaccc tgcqgagtac gtgtcataaa taaacacggt tttcttttta atcctatttt acacgtggat acagaattct ttaatctcca taaggcggag tgagcacata attcttcgac aaatctgatg tacaatttct aggggtaaag ccgtgaagaa gagattcaaa taaatccaaa ttcaaaaact cgttatttga aaatcggggc gacttcatca accggaatta cat cotatta cgaaatccga ctctcgtcct tttttttcgc cttttatgca ctttttctac cttcaaattt aaatcttacc tcgtttgaag ccagaagagt ggaatttttt gttaaagctg agtattgtct taagcgctcq ctcccgtttt ctaactacat gaatqatoaa taaataaata aaatatgaaa ttttgttcat atttgttaag cgagtcttaa tcgctttttg taaggctaat gatatttagt tccgagtatg aaatatcatc acacaaaatc ggagcgcgga gcttcttacg ttcagactgg gt ott tt tot gattttcgcg acacttagca aggcaaaata atacgcacaa tttgaacttt attttcattg gtagaaccqc gctgtttttg atttotgaaa tttaacttgt agaagaaaqa aaoaoaaggo aaaaatggtt ctgttotgtt agaatatagt ttttttoagt ataaaacaaa qqagoataoa cagaaaagta 37080 37140 37200 37260 37320 37380 37440 37500 37560 37620 37680 37740 37800 37860 37920 37980 38040 38100 38160 38220 38280 38340 38400 38460 38520 38580 WO 99/37660 PCT/US99/01313 -71cgtcaagaag tcggatacga aatqtataaa tttcttaaaa cttaaaactt cgagatatcc 38640 agactgtggc aaatttctta tagaaaacct tagaagggcg aatcaatgaa cgtacacgca ttctcctaca ggcgacgg atgtatgttt aaqagattcc aaaaagctgt cttattctga tttctttttt tgaattggaa ttcaqaagge tggagatccc tttttttaaE atttccaaaz ggttatttti ggtaaatcg, tgctccatc' ggcgtagaa tctcaagctt atttttctga tcaccacttg cgagattcag taggacaact tacatcttat tctacatttt gacgactact gcgagaqttg ccct~ctcct ggaaqggagg ctctacggaa tttgtagata agtgtcagtq atcaactage -tagattqatc iaataaacctc a acacatggti a cgcctacgti t tgcacacat a tqtgcgaaa cagtgcttgg a agaagtcgta a agaatactcc a aagtagqtqa a qagacaatgt g gttagaqaaq t ttgctccqtg a cgactctaat t agagccccac c tctgatcacc t agaaqaactt ataggaagccI caaatataaa ctcagaqgga acatttgacg ccaactttat attgtctgaa ggggtttcta caccqccggc tcaacaatta a cacatcqcct t tcacggataa gaaatagtt t aagtataaa t gtctcaaat t aattagacg g cagqtqtat 9 acgtgtqct c .gccacgccg c .ggccctaaa c :gccacgagg :cgtctttCC iccgqctaaaI tacttgtcaa aactaactgc atagccaatc cctgaaacct taaaaactaa tttctacaag ctaaaattca tcaaacttca qtttgtgcag qtaatgaaga cagagatttt aatagtcaq aatatgtttt 38700 gttgctaga t ttccctcga c aaaactctc t 'tgtatgcac a :cgctcatca t ~gaaaaacga c gqcaagtaaa t ~gaagtgggg :tttttgttc tggaaaaaaa ttaqaccgcc gtgtgatgca attgacagaa aacaagaaaa qtatacttat attgcgacca acgagaactc ttcttagtcc aaaataagta ggttcggagt tgatgtttca caaacactc 38760 gcggaaqtg 38820 caaaattga 38880 .tggcaccca 38940 .gtcttctcc 39000 ~gacgacgac 39060 :ttttaggca 39120 laagattccq 39180 catttccgtq 39240 ggaactctaa 39300 ctcgcacaga 39360 gcaqatatct 39420 atttgactac 39480 atctataatt 39540 atatatacga 39600 aattttccgt 39660 ttaacattat 39720 tctgattttt 39780 aaaagagttg 39840 cagatgacta 39900 tcagacttac 39960 gaaaattttg 40020 acgttttgga agtatgaatt qqgtctagac aacqqagtgg cagatgttcg cagaaaagag gtcattgaaa aacctaaqag cgttgatggt attttaaaat tttaaatttt ttggtgacta gcttgcagtt acgaacttaa catctttctc aagaaaattq tattaacaaa 40080 40140 WO 99/37660 PCT/US99/01313 -72aattattttg taqcttttct caatttcagg caattaaaac atttcaattt attcttctat 40200 tatgqaaqtt tgtttgaaaa ct cgtttgcg tctgatgagt cataataatt gattgagaqc tcgtttattt gaaactagag aacacaagt c acccgacattc gctggctagt aattgaaaat ggacggctgt aatggttgat tattatcaat gtagagaggq tggaaaagac ggatattgaz ctacaaaati ttttaaatal acagtgatti aaatttcaa, tatctctaat aaaaaacagt cgcaaaattg ttttcatata gaactctacc caccccaaga ccttqtataa agattctata cagagtaagg cggttctctt ctcaagaaat gttgtqtgag gtgtgcgtgt tcagtgaaca cggtaggtat fattatgtggt Igatgaagat itttgaataac.
a atctagacgi ttatttcaaw a ggaqatat~g a cattgtcaa tgaaactctc tcttttttga tagaaaaaat caaaaatgcc cacataaagt ggqattagaa ccaatagcaq gaatatgggc qcaagaaaag ctgaaactaa aatagaaaaa ctccgaataa gcqtatgcqt aaaaaagaga tgctctagac acatatagtc iggatgaggtE j gagattctaE a gtcccagttl t gctcctatai t gacgttacg' g gtagattcg caattttgat aactcgcgcg catttaaatt ctggcaattg tcgttctgaa aaacggatgt gaaaattaca gttaataatt taatgagcaa aagttgcgtg aggaaggaat ttttcaatat cggcaagaaa gaaagatatc cacacctttc Igatggaggaa igatgaattga iattatccgaa -tgcaaqqtaE Ek attttaaaa t gaggtctcqi g tacacagcc tctagctgc atttttttg caaagaacaa caaattatta tatcaaaaat ttgttttctc aaacacctta aatctgtata gctttttcta gtatgttaaa cggaaaccag tactaaacct gaagatatgg caaaaattta aagaagcgac caaacaaaat tggacactca cagatattta gaagatttta gaacacaaac iggattaatct agtaggtqcal a taaagtacgc acgttctcgt 40260 accaatcatc 40320 agtttaccat 40380 tgaaatagca 40440 caattattqt 40500 ccttcqagat 40560 agtaaqcggt 40620 qttttagaat 40680 catgcaagac 40740 taaaccagca 40800 gaataataca 40860 tgaattgtgt 40920 cgaataagaa 40980 tattcaaaac 41040 gcagacatgc 41100 *taaatactta 41160 *aaatgataat 41220 tatatcaaga 41280 taaaaggatc 41340 tctaatatgt 41400 a tattcgagct 41460 ccataaatgt tccactaaaa 41520 atgtgttgtc cttctccttt ggaacacaa atgtcaatgg gattgatatg catctagga gaactttttc ttatcaatag acttcactac tccgcat agc 41580 41640 ttgcgtaacc aatacaaccg attgtccaaa aaaatttqaa cactacaaaa cgtatttatt 41700 WO 99/37660 WO 9937660PCT/US99/01 313 attcggatac agacactcat ctgaggaaga aaa tgagaaa aaaagaacca ttgcttctgg gattggtttg agacgagatg gataggcaat gttgcaagga tctaaaaact agaattacat gcaagttaag cacatcgaqt ccgttqcatt tgactgaaaa tatgaggaca aaaaaqatga qgtccttttg attaataatq ggatqgcgta tqatcgaggc cggagaccag aaattaaagg gtggaagcac tagaaagctt ttttttcaca tcaatacaca cagacgattg taatgaattg tgaagaatgt qcaggcaatt gatgacgctt taqaaataga gtttgtagtc tqtccatctg tacagtagag gtggctttgg gcgacgctaa ataaattttt agttgatact ctcgtcgtcg atgtgtgaga acaaatgaat ttcgaaattt tgacaatggt gccggtqaga gagqcaaacc aaagagataa acaatcqaga tagcttgatg atggataaat cagaattgaa tgctqcccct tagtctgaag atgagaaaat aatcaagtag tcagatcaaa gctcaataca cqatgtgatg gaggccgcat aagttattcg cttttttcgg tcacagaaqt ctggtaacgg tagatgcatt cggggctctc gctcggagag gaaaaaggaa caatgacgag tttgtcgcca agtgcaaaca aagttctgaq atggggttcc agttgtgaat gaggacgcca tgattccata cttcctaata ttataactta 41760 41820 41880 41940 42000 42060 42120 42180 42240 42300 42360 42420 42480 42521 gggcacgttg gtggacaaga caagccccga t <210> 24 <211> 4434 <212> DNA <213> Unknown <220> <223> Description of Unknown Organism:Unknown <400> 24 gccgccctcg ccaccgctcc cggccgccgc gctccggtac acacaggatc accaacagct ccaccatggg gctggcctgg ggactaggcg tcctgttcct tgtggcacca accgcattcc agagtctgqc ggagacaaca gcgtqtttga ctcaccgggg ccgcccgcaa ggggtctgqg cqccgactqg tgaagqqccc agcccagctt tccgcatcga qgatgccaac ctgatccccc ctgtgcctga caagacctgg tggatgctgt qcqgacagaa aaggtttcc tccttctggc cagatgaaga agacccgggg cacgctgctq gccctggagc ggaaagacca cctgctggqc gatgcatgtg catctttgaa cqacccttcc tgacaagttc atccctgaqg ctctggccag 120 180 240 300 360 420 PCTIUS99/01313 WO 99/37660 gtcttcagcg ggaaagcagc atcaccctgt aatgctgagt agactccgca aggtttgtct accagtgt cc actaactaca gagctgtcca gacagcatcc cccctatgct tgcactgagt atgccctgct gactctgcgg ggcaatggaa tcctcggtcc ggtggctgga atcacaagga gcgaagcgC ggtccttggi agtcgtctcl acagaaaac( tqctttgcc( fcccctggt, tqqtgtccaa acgtggtgtc ttgtqcagga tggacgtccc tcgcaaaggg ttggaaccac tcctcaccct ttggccacaa gcatggtcct gcaaagtgac atcacaacgg gtcactgtca ccaatgccac acgatggctg ttcagcagcc agacacggac Lgccactggtc Itccggctct(.
gggagaccai :caccatgggi gcaacaacCo agatctgca, g qcgtgaagt t acagtggaa tggcaaggc tgtgqaagaa aqacaqgcc catccaaaqc gggcgtcaat accagaagac tgacaacaac gacaaaggac ggaactcagg tqaagaqaac agttcagtac gaactcagtt agttcctgat gtctccatgg cggccgctcc ctgccacatt cccgtggtca ;caactctccc i agcctgcaac :cacaccccac a caagcagga( g tactagcta( ggcaccctgg gctctcctgg cagctgtaca gtcttcacca gacaatttcc atcctcagga gtggtgaatg ttgcaagcca ggcctgcgca aaagagttg agaaataacg accatctgca ggagaatgct tccgagtgga tgcgatagcc caggagtgtc tcttgttctc agcccccaga aaagacgcct gtcacctgt(, ;tttggaggci tgtccaatt, cctgatggc acctcagcct caaccggcca tcqactgtga gagacctggc agggggtgct acaaaqgctg gttccagccc tctgcggcat ccattgtqac ccaatqagct aggaatggac aaaagqtgtc gtcctcgctg cctcctgttc tcaacaaccg Iacaagagatt Itgacatgtgq itgaacgggaz -gccccatca I qaggaggggt a aggactgcgl g atggatgccl a gctggaaat, g ttgatgaqt g agaacacgg gaccqtccaa 480 gtggaagaqc 540 aaagatggag 600 cagcatcgcc 660 gcagaatgtg 720 ctccaqctct 780 tgccatccgc 840 ctcctgtgat 900 cacgctgcag 960 gaggcqgcct 1020 tgttgatagc 1080 ctgccccatc 1140 ttgqcccagc 1200 tacgagctgt 1260 atqtqagggc 1320 taaacaggat 1380 1tgatggtgtg 1440 iaccctgtgaa 1500 itggaqgctgq 1560 :acagaaacgt 1620 :tggtgatgta 1680 gtccaatccc 1740 g tggtgcttgt 1800 g caaaqaagtg 1860 a ccccggctac 1920 tggcatccag tgcacaqat cctgatgcct gcttcaacca caatggagag caccggtgt aactgcctgc cctgcccccc acgcttcacc ggctcacagc ccttcggcca gggtgtcqaa 1980 WO 99/37660 WO 9937660PCTIUS99/01313 catgccacgg gactgcaaca tgcgagtgca qatggctggc gataattgcc gatgcctgtg ttccattaca aactgtccct gcctgtgctg gtctacaatg tgccccttgg tgtgacaaca tatgtgccca cacgatgatg cccgaccaga catgacaqtg gatttccgcc aactgggttg ctcgctgtag accgaaaggg tatgttgtga cagggatact ctgcggaacg gaccctcgtc ccaaagacgg ggacccatct ccaacaaaca agaacgccaa agcctggcta ccaatgagaa ccaaccttcc atgatgacga acccagctca acaaccacaa cagacattga tggaccagag aacacaatcc atcaqgatat atgccaacca acaacgatgg aggactctga tgccagacat gattccagat tacgccatca gttatgatga acgatgacta tgtggaagca cgqgcctttc ccctqtggca acataggctg gtttcattag atgataaaac ggtgtgcaag gtqcaactac cgctggcaat cctggtgtgc caactcaggg tgacaatgat gtatgactat cccagatcag tggagacggt agacactgat ggatcaqctg tgatgaagat ggctgaccat cattcctqat cqgcgatggt cgatgacatc gattcctctg gggtaaagaa gtttaatgct tgctggattt agtcacccag tgtgaaagtt cacaggaaac qaaaqatttc agtggtgatg ctatgctggt ccccgtaacc ctgggccact ggcatcatct gtggccaatg caggaagact aaaattccag gacagagatg qcaqacacag atcctcaatg atggatgggg gactctqact ggccaccaga gacaaagatg gacaaggaca cgaggtgatg tgtcctgaga gaccccaaag ctcgtccaga gtgqacttca gtctttggct tcctactggg gtaaactcca acccctggcc accgcctaca tatgaaggga ggtagactag cctqcacgqa ataqcgaccc gcggggagqa cgacttacca atgacaagga atgacaggqa atgtgggaga acaacaatqq aacgggacaa ttggagatca cagaccgcat acaatctgga gcaagggaga act gcagact cctgcaaaga atgttgacat ggacatccca ctgtcaactg gtggcacctt accagtccag acaccaaccc ccacagggcc aqgt gcgcac gatgqcgtct agaaaatcat ggttgtttgt tgggacccac catgtaccgc cacagacctg ctqcaaaaag tggaattggt caactgtcca ccgctgtgac ggaaggaqac ctgccagtac gtgtgacaat tggagatacc caactgtccc tgcctgtgac cgtgcccaat tgattttgac cagtgagacc aaatgaccct tgatcctgga cttcatcaac cagccgcttt cacgagggct tggcgagcac cctgtggcat cagccacagg ggctgactca cttctctcaa 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 WO 99/37660 WO 9937660PCTIUS99/01313 -76gaaatqgtqt tqattgaaag agaaaacccc gactcctaga tcagcctcca gaaaaagcat agcagggtgc t caqagaagg qaacacgggg gaagaaaata gactattgct acttaaatag cacccttact ggcagtgctg cttgtgcaga tcttctctga actgatcata cagqatcact acgtgcgacc atgaataaga ctacttgctt tattgtgagg gaggactcac aqcagaggcc tggaggaact ggatttcatq aagcaggaaa catcacctgc gctgccattg tgtagcagga cctgaaatac aaccaatgct tctccttggc tqcctcaaga catcttccaa cagttgggaa ccatctctga tagaattagc aaagcactaa gttacatqtt atgctgactg gggagacaaa agtgqccaga cctgqtcaca aaataggaaa gaatgtagaq ggtattgcac ttccttcttt aaatgcagtt qcatataaac ggtgcccatt gcagtggact aaacaaaacc ggggaqggcg cggtactaag gcgttagctg gactggcttc attagggaat ttgaaattgg acctaccatc atccctaatc cttctggaac tct gt gctt g ttcaaaaaca aattgctttg ccactctgcc caaaagcatt accctgacat catacccgag tcattttcag attaacccat tggacttcct cagaatcgaa tqgcttcatt tcagtgagca atcaaattgt tatgggcttg catcagtgtg gactcagcat gtttcctttt tttgtcacag ttcaggcatg cctccttcag acgattgtat gggattgaaa gtaaataggc ccctgatccc accaqtgtaa ctagatgtag ccag 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4434 <210> <211> 2837 <212> DNA <213> Unknown <220> <223> Description of Unknown Organism:Unknown <400> agagagccaq tccgatgtct gcagcctccc tggccaggcc tctcctctcc agtccccctc aggacagaca gagtactggc gtcggtcacc attcacttgc gqtcacgtga agaaacttcc tggtgacact caggctgtag ctgtgcactc aggttggttt tctcctaagt gtcacaggtg gagacaagat gctctqggca tggctctggg catagggcca agagcttctg ctggtqacca cgtcaaggac accttttcag catcagcaac attaaccgga agaccatcgg tgccaagcaq tqccgcagct aaacacacca ttcaaccacg ctggccctgC acttcatttg ttccgagggc 120 180 240 300 360 WO 99/37660 WO 9937660PCT/US99/01313 -77ctgaccccgg cagatgatct cccaactgaa cctcccagag actgggtaga agtggaagaa atcttatcga qcaggatgta atqtccatct acagccaggq ctcacatcac actcctgcga accagctgag ttgggggccc cagaaaatga cagtctgcca gcgagtgctg cagagtggac gtgatgtcac gcaaatgtga catgctccgt tgccccagat gtgatccgtg ttacctgtgc at ggagggaa qccctattga qgtqcccgcc caacaggatt qcaggaccgc gcagtttgag aggcaatcag tgtgactgtg cagtgtcacc cgtggccaaa cgtgtttgca agctgaagtc cacagatctc ggagttqagc caagaacctg tctgaagaca a acc tgggt t tcagatcacc tccatcctgt cgagtgttct cagcaacacc tacgagaatc gacttgtgga gggtggcaag cccaattqat tggagggatc ggactqtgtc tgqgtgctta taccgttttg gtcaagcttq aagtctcggg attgtgtcca cataccaact cagqtggcca ctggaagaac ggtgcatctc gattctgtqg aacaccatca gtggtccagg aacatgatga gagagagtgt agaaacatgt qtggatagtt tqctcacctg tcacactctg gtcacctgtg tgcctgggcc cgtcaqaatg gttggcaatg aactgcaagg ggccgctgga cgtgagcgct ggggatgtga tccaacccgt tacggtttga caaggagaaa gaacgctcct atggcccagg tcctggagga gtgacaccta cattctatga gagagagtca aagatatctt qtgaacatac gtgtggagaa atgagctctc ctagtgataa cagcctgtgt gt a ccacat g caacttgtgc cagacagtga gctctgggac cctccattca gaggctggag tcacccgcat gcagcggccg gcccctggtc cacgtgtttg cagaacacca gttttcctgg ctacatcccc ggagggcttc ggtgttggaa ggacactttg tgtgggcctq tagcctgtat gcagctagaa cttcaggggc aagcaagaaa agagactctc ggcacaggaq tggactgcac ccagttcctt gcaggagc cacctgcaag caacccatct tgagggctgg ccagcagaga gacaaggaca tcactggtca acgtctctqc ggaaaccaaa cccttggtca caacagccct aatgtgcaac agccaagtgc ccagtgaaca ttcctcacag gqccccggca gacctcaact gctgactccc gtgggctqcg qtagacagga ttgctgcaga agctgtcaac catctgagcc gtgtgtacgc gtcatggtga ttggagctca cgaatctttg aaatttaaaa tttgtggaag tctccgtggg ggccggtctt tgcagcctgg ccctgqtctt aactcaccag ccctgtcagc gcctgcacag gagccccagt aagagaagct aacagcttcc 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 WO 99/37660 WO 9937660PCTIUS99IO1313 -78ctgatgggtc gtgaggacct cccgctgtgt ggaaccaacc cagagaatcc tgggccactt ggctcatctg ctactaatgc aggagqattt gtgtgagcga acaagqatga tcqacacaga ttttcaatga gtgatggcgt atcaggacaa gccaccagaa ctggtcctgt ggatgagtgt caacaccaac cttcggtgtt atgtaaggac tagtgacccc cggggaqgac cacctaccac tgataaggat tgagaaggac ggttggagac caacaatgqc gcgagacaat gggtqaccac tgatctcgtt caaccaa ggctcctgcc gctgtggtca ccgggcttcc ggcctggag aagactcaca atgtacaagt tcaqacctgg tgcatcaagg qgaatcggag aattqccagc cgctgtgaca gagggggatg t gt cca tatg t gt gaca at t ggagaccagt cagtgggctt cagatatttg actgcctgcc atgctaqgac gctgccacaa gtgagtgcca atggctggcc acaactgCc atgcttqtga ttctcttcaa actgccccta cctgctctqt tctacaacac gtcctctgat gtgacaacaa tctgggcaat cttctcaact ttgtccacca agaaaaacaa gaatgcagag qattqqctac caacaacaac caaactgcca cgaggacgat tccccgtcaa tgtgcacaac qgacattgac tgaccagaga gcacaaccca tgaggacata ggtacccact aacaaagctc cqctacaagg qtgtgtgagc tgcatctacc qcaggtgatg ctggtgtgtg aattccgggc gacaatgacg ttagactatg ccagcacaga ggagacgatg gatacggatg gatcagatcg gatgatqacg 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2837 <210> 26 <211> 4108 <212> DNA <213> Unknown <220> <223> Description of Unknown Organisn:Unknown <400> 26 agaqagccag tccgatgtct gcaqcctccc tggccaggcc tctcctctcc tgccgcagct agtccccctc aggacagaca gagtactggc gtcggtcacc attcacttqc aaacacacca 120 gqtcacgtga agaaacttcc tggtgacact caqqctgtag ctgtgcactc ttcaaccacg 180 aggttggttt tctcctaagt gtcacaggtg gagacaagat gctctgggca ctggccctgc 240 tggctctggg catagggcca agagcttctg ctggtgacca cgtcaaqgac acttcatttg 300 WO 99/37660PCUS9O31 PCT/US99/01313 -79accttttcag ctgaccccgg cagatgatct cccaactgaa cctcccagag actggqtaga aqtggaagaa atcttatcga gcaggatgta atgtccatct acagccaggg ctcacatcac actcctgcqa accagctgag ttgggggccc cagaaaatga cagtctgcca gcgagtgctg caqagtggac gtgatgtcac gcaaatgtga catqctccgt tgccccagat gtgatccgtg ttacctgtgc catcagcaac ggtgCCCgcc caacaggatt gcaggaccgc gcagtttgag aggcaatcag tgtgactgtg cagtgtcacc cqtggccaaa cgtgtttgca agctgaagtc cacagatotc ggagttgagc caagaacctg tctgaagaca aacctgggtt tcagatcacc tccatcctgt cgagtgttct cagcaacacc tacgagaatc gacttgtgga gggtggcaag cccaattgat tggagggatc attaaccgga taccgttttg gtcaaqcttg aagtctcggg attgtgtcca cataccaact caggtggcca ctggaagaac qgtqcatctc gattctgtgg aacaccat ca qtggt ccagg aacatgatga qagagagtgt agaaacatgt gtggatagtt tgctcacctg tcacactctg gtcacctgtg tgcctgggcc cgtcagaatg gttggcaatg aactgcaagg ggccgctgqa cgtgagcgct aga ccat cgg tacggtttga caaggagaaa gaacgctcct atggcccagg tcctggagga gtgacaccta cattctatga gagagagtca aagatatctt gtgaacatac gtgtggagaa atgagctctc ctaqtgataa cagcctgtgt qt acc acat g caacttgtgc cagacagtga gctctgggac cctccattca gaggctggag tcacccgcat gcagcggccg qcccctggtc cacgtgtttg tgccaagcag ctacatocc ggagqgctt c ggtgttggaa ggacactttg tgtgggcctg tagcctgtat gcaqctagaa cttcaggggc aagcaagaaa aqagactctc ggcacaggag tggactgcac ccagttcctt gcaggagggc cacctgcaag caacccatct tgagggctgg ccagcagaga gacaaggaca tcactgqtca acgtctctgc ggaaaccaaa# cccttggtca caacagccct ttccgagggc ccagtqaaca ttcctcacag ggccccggca qacctcaact gctgactccc gtgggctgcg gtagacagga ttgctgcaga agctgtcaac catctgagcc gtgtgtacgc gtcatggtga ttggagctca cgaatctttg aaatttaaaa tttgtggaag tctccgtggg ggccggtctt tgcagcctgg ccctggtctt aactcaccag ccctgtcagc gcctgcacag gagccccagt 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 atggagggaa ggactgtgtc ggggatgtga cagaacacca aatgtgcaac aagagaagct 1860 WO 99/37660 WO 9937660PCTIUS99/01 313 gccctattga ctgatgggtc qtgaggacct cccgctqtqt ggaaccaacc cagagaatcc tqggccactt gqctcatctq ctactaatgc aggaqgattt gtgtgagcga acaaggatga tcqacacaga ttttcaatga qtgatggcgt atcaggacaa gccaccagaa acaacgacgg acagggacaa gaggtgacat gccctgagaa atcccaaggg tqgtgcagac tggacttcag tctttggcta cctactqqqa tgggtgctta ctggtcctgt ggatgagtgt caacaccaac cttcggtgtt atgtaaggac tagtgacccc cggggaggac cacctaccac tgataaggat tgagaaggac ggttggagac caacaatggc gcgagacaat gggtgaccac tgatctcgtt caaccaagac caagggcgat ctgtcggctt ttgtaaagat caatgccatc gaccacacaa aqcaaactca tggcactttc tcaqtcaagc agacaagccc tccaacccgt ggctcctgcc gctgtggtca ccgggcttcc ggcctggagg aagactcaca atgtacaagt tcagacctgg tgcatcaagg ggaatcggag aattqccagc cgctgtgaca gagggggatq tgtccatatg tgtgacaatt ggagaccagt aactgcccat gcctgcgact gtgttcaacc qactttgaca actgagacag attgatccca gaccctgqca tatgtcaaca agccgcttct agtcgggctt gttttcctgg cagtqggctt cagatatttg actgcctgcc atgctaqgac gctgccacaa gtgagtgcca atggctggcc acaactgccc atgcttgtga ttctcttcaa actgccccta cctgctctgt tctacaacac qtcctctgat gtgacaacaa acatctccaa ctgatgatga cagaccagga atgataatgt acttcagaaa actgggtaat tcgctgtagg ctgaccggga atgtggtgat acggctactc agccaagtgc tctgggcaat cttctcaact ttgtccacca agaaaaacaa gaatgcagag gattggctac caacaacaac caaactqcca cgaggacgat tccccgtcaa tgtqcacaac ggacattgac tqaccaqaga gcacaaccca tgaggacata ctccaaccag caatgatggt agact eggac cccagatatt cttccagatg tcqtcaccaa ttt cgacgag tgatgactac gtggaagcag tggtqtgtca aacaqcttcc ggtacccact aacaaagctc cgctacaagg gtgtqtgaqc tgcatctacc gcaggtgatg ctggtgtgtg aattccgggc gacaatgacg ttagactatg ccagcacaga ggagacgatg gatacggatg gatcagatcg gatgat gacg gctqaccatg gttccagatg ggtgacgqcc gatgatgtgt gtccctctgg ggcaaagagc tttgggtctg gctggctttg gtgacccaga ctcaaagtgg 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 WO 99/37660PCJS9/13 PCT/US99/01313 taaactccac cagaaggcca ctgcctacag atgaaggaaa gacggctggq agtgcaqaga cttagacaca tcttgacctt aaaggagaaa tgagatatga tttgtttaaa atggagtcga gactggtact ggtccggact gtggcacctg gcaagtcatg cctqt tt gt C tgcctagaga tcagtccatc aactgagtgg catcaattgc ggctcaccgt aagaatgacg agqagcttt ggcgagcacc ctatgqcatg attcacaggc gctgactcag ttctcccaag gcagggctcc ttggcacttg atctacacct actccaagag ggagcgaaqa tttacatgta agagcaca tgaggaatgc accccaaaaa ctaagacagg gaccaattta agatggtcta agctccagca tggcttttct.
ccttcatcag cttccagcct ccgagcattc aatgtaatta cctgtggcac cattggctgg ctacatgaga tgaccaaacc tttctcggac atgtgctgca gtcatttqgc caccaagtcc gctqctggaa cgctgtgttg cttgcagtat acgggaaaca aaagactaca gtcttagtgc tacgctggtg ctcaaqtatg aacacccctt atttcctgtt aagtqtcttc aacatttgqa ccttttcttg ttatgtgtat 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4108 <210> 27 <211> 820 <212> DNA <213> Unknown <220> <223> Description of Unknown Organism:Unknown <400> 27 tcgaccagag gaggqgaggc cagttcctct cccaagggtg ccacacaccc atcaccagac aggcccttcc ttcttagcca tatgctaacc ttctcctccc cctctgcagg agccaaagca gatqggagct qgagttgctg gagctcctgg gagcaggcat ccagqaaagq agaagagagt gtgacaatcc agcacctcag cctcgtgttc agggcggaaa gtacagacgc aggcttqctq agggcctctg ggaccagatg ctgtggatgt cgacccctgc actgactatt ggataaagac taagagaaga tgcaaatcag cacacttttt tctttgttct gccagcttcc ctaggttttg ctgtctacag ccaactattc tattagttac aaaactcaat agcaactgga tgttgactgt taactagaaq ctctgtccta cttacagcac ctccctgttC tgggaaattt tctgtatgca aatggagggg gacacaqgct ttctttcaac aggcctaaga cattttattc tttgqatcat 120 180 240 300 360 420 480 540 WO 99/37660 WO 9937660PCTIUS99/01313 -82caaaaaaata aagtaaaata gaaaactgaq aaaactcaat ccatgaccag ggagaactta 600 caggatgtta gagacaaaac aagcagacac ctgaaacaat caacgcccaa taaaacaaag 660 taggatgaaa attctcttag ttctttgata acaatttgtt cactcataga aacattatta 720 attggtaqgg taagcaqaca ctctgaaaca atgagaaaaa tactaaaaat tgacttgagt 780 tatttcaaat tgcctcattg acctqttata tcataactct 820 <210> 28 <211> 2397 <212> DNA <213> Unknown <220> <223> Description of Unknown Organism:Unknown <400> 28 tttttttttt gacatctttt ggtggcttct agcctggggt cagcaqttac acaaaaagta gtggtgacat agtgtggaca gtaatggaga ccattgacga ggtacaatat gccagtgtga aacgtctcta accactttga accttctttc catcctactt actccaattt qgctgagatq aactgcacag ctgccagctg ccgacaccgg ctgggatcaa ggatttccag ccaggactgc agactgcagc cctgacccag gacggtatac ctatggagat agccctggca caaagttaaa tgttttattg cctgaataga tttgctgttg gagaagqtqa agcaactggt agcctcttgc gccagctgct tgtaaggaga cttgatggct cagtatgaac gaagatgctc aatggggaat gatgagaaat gatactqgaa aaagacaagt ggcgttgatt tagctttatt ttttcttcat accagagagt cagagtggac agccaaacaa ccagttctac caggtcgctg ctgatgagga caattccagg a gaqtgtqt a ggagggagct actttcggaa tctcctcaga ctgactcatt gttacaggtc ccttcaaggt cttqtctttg aagacgggca agattgcttt gtttggggga aacttgtgta cctgaaacgc cqactgtgaa atcacagaag cgatgccagt tcgatatgac accctacaac gttttatgat tggagtgacc ccagcctgta aatatagtgc atgacttgtc gctacacccg ccgtgccagg accatctgca aggcaagcac caccttgtgt gatgtcaggg gcagccttgg tattatgggg tccacctgtg tttctgaagt aatgcaaatg atcggcatag 120 180 240 300 360 420 480 540 600 660 720 780 840 900 gcccagccgg cagcccttta ttggtgggtg taggtgtatc ccactcacaa gacacttcat 960 WO 99/37660 WO 9937660PCT/US99/0131 3 tcttgaacqa aggtqcagac tgcagtcatt atgactatgg tggtgattga gttttggagg caggagacca ctgtggaaga cacagaacag ttgttatcga ctctggaggc atgcctgccg gcaggtgcca qagccaaagc tccaggaaag cagggcggaa gctgtggatg atgcaaatca gctgtctaca atgttgactg aaagtaaaat agagacaaaa aattctctta attaaacaag tgcacatttt aatggagctt cacccattac caaagcaaaa ctccttgggc ttgtaaaaaa cattatttct gagcaccatt ttttgagatg caagcgccag atgtgggcct gtgccgcctg agatgggagc gagaagagag agtacagacg tcgacccctg gcacactttt gccaactatt ttaactagaa agaaaactga caagcagaca gttctttgat tataatgaga aagatqagga ccagatcagt atcacatctg atggaatccc attcaatatg tttggaggtg cgggtgcgag acataccgtt cagcctatcc aacctgcgcc tgcttcaaca ggtagcttgg tggagttgct tgtgacaatc caggcttgct cactgactat ttctttgttc ctattagtta gctctgt cct gaaaactcaa cctgaaacaa aacaatttgt agaaattcat aggatgacat acaattatgg gatccatggg ttggtattac aagacaaaat gcaaaactga gtggcagttc cctqggggag acgaggtgct gcgccttgga atggggtgcc gtgctgcctg ggagctcctg cagcacctca gagggcctct tggataaaga tgccagcttc caaaactcaa acttacagca tccatgacca tcaacgccca tcactcatag tttcacaaga tatgctggat catgtatqcc tggcatttat cagcagagat aaatgttggt aagggccagg tggctggagc gtcattaaag qcggcacaca ccagt atctg catcctcgaq tgagcaaaca gtctgtatgc gaatggaggg ggacacaggc cttctttcaa caggcctaag tcattttatt ctttggatca gggagaactt ataaaacaaa aaacattatt atcttcacaa gaaggaatgc aaqttcatca gaatatatcc atcacgacat gqaggtttat aaggccatgg ggtggcttgg tataatcctg agcctggggc atggaattca ggcaccagct cagacagaag agagcaggca gcctcgtgtc tggaccagat ctaagagaag actaggtttt cagcaactgg tcaaaaaaat acaggatgtt gtaggatgaa aattggtagg 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2397 gtaagcagac actctgaaac aatgagaaaa atactaaaaa ttgacttgag ttatttc <210> 29 <211> 4100 WO 99/37660 WO 9937660PCTIUS99/0131 3 -84- <212> DNA <213> Unknown <220> <223> Description of Unknown Organism:Unknown <400> 29 ggatcccccc tcttcctcgc ccacttcggc ctgttcctgt cqggtctcca ccggtgtctt tcggctggtt tqatgggtgq ttgtttgaga gccaccatct ggcccattag gctgccagac gtacgcggcg tgtagtaaac gggtaactgc ccgaactggg ccgactcccc tcgatgcgcc acgtggcacg ctcaatttcc gccgcgatgt qgccccggcg cccgcgcccc gctccgctac cgcggactgg tcccgctcct tcccggtcct ggcccagaca ctatggctqa ggctggctgg gcggagcgcg aaaacaaagt gagtgcccaa caccaattgg tgcaatttac gccgcaggaa tgtagctgca acccgcagta acccccccga cccgactccc ccatcgcgcc tatttccccc tctttccgga cgccgctcgt tcgcgggaaa ccactcgcca catcttcatc gqcgaccctg gctcctgctc gctccggctc aaataaaaaa gggagtctqg cgagatggac gaccggggat cata a agt gg ccgggcccgg ccagagtgtc cgcgcgatac atctactgta gtagagttac aatttactgc ctcccccccg ccccgactcc ccgt tc cgct, ccgtaaatca gataaaagcc ggcggtgctg cccccgtggg tcctcqgcgc gacctcaccc ttgctgctgc dcggccccac ccgqccccgc agatatattt ctctcggggc cgctccggcg tatatacacg cactcagaca cggttacatc gggagccacc tgcagtttac gtatttggcg tgcagcgcca agccggactt actccccccc cccccgactc tcgccacgct.
agagggatta gggacccccq gt gt tt t ttt ctcgatgcca gaggagctgg aggacgacga qgccqccqcc tcctgttcct acccacctcc tttcagtccg tctcgggtcg cgcagcgtcc atgtgcatcc gcactttgqc acccccacat gctaattgca tgcagccgcg gcggcgcgcg tcgggccggt tgtgcgctgt gactcccccc ccccgggacg ccagttgccc tgcggatgtc cqccqaaaaa cggcggcgct tcttcgaggc agtqggacga ctgagctccc gctcctgccc gttcctgttc gctcctgctg tctctcccgc gctgggcggc gcggctgctg ataattgatg ctggcgcccg gqaccatcac gtaacgcgcg gtaaactgca gtactgcaac gtgqccgcca ggagaccgcg cgactccccc cgtccgcgcc cgcccccggc tagtttatgt qgatacacca gggggttcct cccggtcacg tgaqqatcac 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 WO 99/37660 WO 9937660PCT/US99/01313 ccgctgctgg ctgccgccgg gaggtcatcc gccctggccg gtgcacaagg accctgcggc ctctaacgcc gaagccggcc ggcaacaaca gtgggacgcc cgagatcgcc cgaggaggtg ggacgtcaag gctggccttc ggcggtccgg ggcgcgccqc ctcccacgcc qacccgcccc ggacccgcgc cttcaggacc ccccgtcgac ggagggggga gcqccccggc aggaggtcga cccctccccg aggaggggaa acctcqagcc acatgaacgc tgtgggcgtc tcqqagagcc actccctgcc ctqccgcccg gccaccqccg cgaccccccg gccacggggg ttcgccgccg aagccgtacg ctgcccgcgc gtggtcatca agcgtccggc gcgacgtacc ggggtgctgc ccgct cggct aagctggtgt cgccacaagg tgcccgcact tggagcgtgg ggggggtggg cagcgcgctg gccctccggg ggagqggt g ggcgacggag gcccgtggga cgtcacgagc tggcaccgcc cttaaatgag gccgctgcgc gggtgcgcct ctgcagcagc ccgccgcccg ccgacqcctc agttcgacgt cgcgcctgct qcgaggacgt tcggccagga qcggqgtgcc cgacgctgcc tgctgaacac gggcgcggct tcatgctctg tgctcacctt ttggagaggc actgagtcqg atggacggaq ccggtcctgq ccgtgcccgg cgcgggagcg aagqaggaqa tcacqctgcc gtqgtcgtga tacctqqtca gcggccctca tataatgqag cgtcgtgcct agcccccgcc gcccgcgaag cgccccggcc ggccccctcg gctggccgag gttcgcctgg cccgtaccac gatccccccq cqcgcccgcc qacgctgacc cgtgcgcgcc gggcgcccac cagccatccg gaacgcgtac gcgtgcgcgc gagagcggat cggtgctgcg aggacqcgga aagacggaga cggaagacga atccctacat agccctactg acccctttgt aggagctcgg ggccccccgc gccgccgccg qgggccgggg ggcacgcccg cccgaccccg tggcgcqcgc taccgcggcc acgcgcctca gggccggqcc agcctggcca cacgqctgcc gtgcqgcgcg gtcgtccagc gctcaaaagg tcgccqctqg ctcgtccaga acaccgccgg qatggaqccc cgagt gggga cgcgccccgg ggggggcgtg qgaagacggg cgcgtactcg ctcgccgccg cgccatccag agaggtggcc cqagtaaqag ccqcttctgc ccgqggcct c cggcgtcggc gggcgcccac tgctqgagcc gctgcctgac cqgcgcccga aggcccacgq acatcttcgc tggagqcctg gqgtccccgg ggctctgcga cctgcgcgcc cccgcacgcc CgggCCqggc cggaggacga gcqctcgccg tgggccgtgg gagagcgcac gaagacgqcg ggggacgaag 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 i WO 99/37660 WO 9937660PCT/UJS99/O1 313 ggacgacgac ttatggtcgc acctggccgc gcggcgcgcg tcctgtgccg cccgacgccc cggtqttcat ttgcggcqcc gtt cctgcag caccggcggg gcccctgcgc cgaqccqgac ccaccaccgc cggcagcgtg cccqcgccgc ccggccccgg gagaccccgc cctcccgcgc tcgcgatcaa ctctgtccgc ggcggcqgcg gtccgtggac gqccctggc cggcccgcgc caqagcccac acgcgagccc ccacgagttc atgagcacct gccaacgaga ggcgagacgc gccggcggcc cccgtctacg tctcagcatc cacgcctcgg cgcggcagcg caacggagca cagcgcagcg ccccggaccc taaagggcga gcacgcgcqg ggcccgcgcc gagct cgggc gqcct ccccg ggcqcccccg gcggcqcgcg cgcgcgcgcc atcacccccg tcggacgcgc cggccgcccg gggccccgcg tgggcaccgc ccaccgtcgt cgcagcagca cgacggccgc tgcacgcctc tcaacgcctc tcaacgcctc cgtcccgggg gcgcccacgg ggqcgcagca gccggcggct tcgcctgcac ggcgcggcca tacgcqatca cqacgagcgg agccggtgcg gtccqtggcc gcgcctggcc gtcctcgcgc ccgcgqgcgc ccagcctacc gcaacaacag ggacggaccc ggcgacggcc gacgacggcc ggcccgcgcc ccggcgcgcg accagacaaa cgtggagttt gacggacacg caagacgtcc taaaagcctq gctccgqtcc acgggcgcgg gctgcaccgg acggtcgatq gcggCCtccc ggcccccacg tcgcqccagc caccaccacc cagcgggccc gagtcgtgcg gccccggcgg gccccgacgc gccgtcccct ccccccgcct agacacaacc gacacgctgt atccgccggg gcgtttgcgc cagatqttta qcggtgcgcg ccgqccgcgc gacaacgtgt actaccaccg gccgcgtctc acgatgaggc gcggcgcgac accagcagca cacgccgccq cggccgcacc tccagctgcc cccagctgcc cgacggccac catgctgtta ggttcggtct 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4100 <210> <211> 38734 <212> DNA <213> Unknown <220> <223> Description of Unknown Organism:Unknown <400> gatcctcgtg accgggtaca ccgacgcctc ctggacgccg ctgttcgcca tcgcgggcgg WO 99/37660 WO 9937660PCTIUS99/01313 ggtcgtcacc cgtcccgtcg cgtggtggac gggcccggcc gccgcaacac gacccgatga aaaccgctcg aacctcgtct atgaagggca tqccgggtga acctacccgg acggccgacc qccaccaagc ttctaccacg ctgtacggca cqgatcacca gaccactgcc ccgtgcatgg gacgaccaga ctgaggttcg gtgcaggacc ggcgtgaaca aagctgatcg ccggaagcgg gccggcgqac agacggggga gacatcgggt gtggtgaaca ggcgacgcgg ggggccgccg cggaccacga tccccaacca qtqtgcgccg gccagggcgc acaccatcga tcccggcgat tccgggagca tgccgccggt gctggacccg tgacctacgt ccccgagcaa accaccgggt aggaggacga tgcacgtgca acaccgagca aaccgctgcg cccaggacgt agttcatccc cggacgcctc cggggcgggg gacgcggcac cggacaagaq cgatqctctc ccaaggacgc gcacggtcga ggaccccqgc ccgcccccqc gtggtatccc catgqgccag ccgctgcccg atgcccgtac gggctccgag gttcggcctg qgcggccccg cccggtgcac gcaccgggac gttcggcctc ggagacggag ccccaccaac gaccgagcag catcctgcgc ccgtctgctg ccgcatcatg cgtcgacgag ggccgcgccc cggatcagcg caagcccaag agagcgagag gcacagttcc cacccagcgc ggtcgtcgag cgqagccacc gaggggcgga atcgtcqagg gacctcgtgc cacaagggcg cacggcttcc gcccgcatcc gtgtggatgt gccgaggtga tacacccgtt cactggttca gacggccgcg gcggagqqgc accacccact ttcgagacca tggtacgagt ccct ggqcgt gaacaccagg atgaacgcca t cgt caccgg gcccgtgcca gaggacgccg tgagagatgt atcgtggccc atcaacaccg agcgcqqaca accgactgaa ccacacccca cgcaggaggt tctggcgcga ccaacctcgg gctacggaqc ccggctcgct ggt ggggcga cggacaaccg acatcgagag actacatcga aqcggtacct agaccatccg acgtcatcac cctcctggct acgaacaggt ccctctacat aacccaagat agtacatctc cgcgggcggc cgggcaacgg ccgcgcgccc acggcggata gcgagttcca gcgacctgat ccgacccgca gccggccacc gacgggagac gggcaacgac catcgacggc cgacggccgc cgacggtgcc gcgggtaccc cgagcgcccg gaagctgtac cctgctcgag ctacctgctc ggccgccacc ctactccttc gttcaccttc ggtgcccatc caagcccgt c ggagaagtgg cagcgccggc gatgcgcgcc ggagcccgag caggggagcg gtagacccga cgacgcgtcg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 WO 99/37660 WO 9937660PCTIUS99/01313 -88accggcccca ttcctgttcg gtgaccctgc gccacgaact atctgggtcc gtgagcgcgt gtcggagt accgaaggcc tccggqttcg tgcatgttcq gaggccttcg cqcccgtcac accacagatc agatccacaq t cggagaggc ccgaggcgtt cggagctgac gggtcgacct accactccgg acagcacgca tccgtgacct tgctcggctc ccttcqagga tgcggtcgcg tgatcggctc cgaccttccc aggccctgqt gcqqcgcgga gccgggtcct tcgtgatgct tggtgatcca tcacctggct accagcgcaa tgttcaagca cgctggtcac tcttcctgaa acgagtacgc gcgcqtacqq ccacagatcc atcccctcca cgaggcgtcc cgcccgqctg cgacgactac ggagggactg caccctgccg ccgcaggctc cgcccgcaag cggcgaactg gcgctaccgg gcgccccatg ggcccccacc gctgctgggc gacggccttc caccgtggcc gctggcgacc ccaqcgcaag cggcacqatg gggcgtcqtq actctggaag ctcgatgcac gggcaccccg catccccatg gtcccggacg cggcctcccc ccacaqatcc cagatccgag ctgaccgccg acctccgacq ctgctccccg gagaacgtqc ctcgacgccc cggctqctcg gccggccacg gtcggcgtga ctgcagcgct gtgccgtgct ctgqcccqga qcgctgggcc aacaccgtcg gactggttcg ctgtcggtgc atggagtggt gcgtacttcg ctgtacctcc aagcgcccgg atcaactact tgggccttcg ctcgacaagt gcgaagttcg gggcgagggg ccacagagcc acqaggcacg aggtgctqcg aggqcqccgt ccctgcgcct cggccgaggg tgatgctcca ccgccgacct tacgcgcctg tgccggaggg tcggccgggg cgatcgtcgg tgctgaagct tgat cccgat ccgtggccgg gcaccgactt tctacctgct cggagtcggc gcggcaccaa tcgggtccta aqaacaaggg tcggtgacgc ccatccccct acctcgccga tcccctacgt qgccgccgta cctccacaga tatgaccgga cgaggtcctg cgacgacttc gctgtacgag gqgcgcactc ggtggcgctg tgccttcgac ccccgagaac ctacaaqgg aggcttcgcg cgccgaggag gccgtacttc gccgaccaag cgccgtgtgg cgacgaggcg gcgcttcatc caccatcggg cgacgccggc cctgaacacg caagctctac cgtgctcttc gatcatggtc gcgatacggc gtactgaccc ccgggtggca ccccctccag gacattccct gccggcggcg ggcttcgacc aagtacttcc ctggtcgcca cacqaccat c ctccccgtcg gcgctgcggt ctcggcaagc gcggtggcac atctacccga ccgatcaccc tggaccatcc 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 WO 99/37660 WO 9937660PCT[US99/013 13 -89gcttcggtgc tggtcgagaa cccgatccac gct cqccggc tggagggccg cggctccccg cggctcccgg cgacgatccc gagcgtgacg acgacggacg gcaccgcccc gggtggtcgc caagcggatc gaagcgcatc cggcgaggtg gatcgcggtc gcacgcctac ggccggtct g catctcctcc cctgtgcgcq gtcqggcacc cgtgtggggg ctacgagatc gcgcgccttc cccggccttc gctggccgtc gtcggagaac ccagcacccg catcgtgctg ttcgcccgag cggcggcaga ccgccgccga gtgatggtca gcgaattcac gcgttcgaac ctcatcgggc gcgcttcccg ctgctcgtcc gcggt cgtgg ccgagcggcg accgtcgcca ggcatcgaga gtcgccgtga ctgttcgcga gcgtt ccgcg aagccgqagc tcctccggct cgcaagccca accatgttct ccggccaccg gtgatctcgc gccgcagcca acgqacggct gaggtgaagg ttcgtctgag gaccgtccct tgctgcgaac ccagctccgc tatagccatg ccgtagagca gcttacgatc gcgcactgcg tgttcgcgtc tgtccatcgc tcctgatgtt gcggcgacta gcgccctgct acctgggctc tcctgctgct ttccgacgta gactggtcct acggcgqcct gtgccgcgct agtccaagaa gcggcatcat acctcttcca agqtcgccga ccgcgctqgt tccccgagga acaccatcca ggccgcggct ctcgtccggg gctggcgaag ggacagatgc cgaaccccqc gaccggtgcg ctctgttgtg cagcgaccgg cgacccqctg gggcgtgtcg caccgtggtc cgaggt ggcc ggtcgactac cgcgatcccc cacgctgatg cgtcttcgtc ggacgacacc ggccggcttc caccggtqtc cgcggggaac cgcqctggcc caacggcgtc ggcggtcttc cctcttcctc cgccgcggag gcacacgctc cccggttcgc gccccgCCt c gccagaacgg atgggatctc cgt ccgcccg gccgaggagg tccaaactga ctgggtgaaa tcctccgtgg gcctaccact gcctcctacc accaccaacc gtcctgaccg ttcgtcgtcg aacctgcgcg gcgggcgtct atgcgtgccc gcgctgatct gaqgcgatct accctcgcga gccgcgaccg gcggtcggcg ggcgagggca gccgccaaca gacccgctgg aacgagatgc ccgagggcgg agccccccgc cgaggccgac cctcaggggg atccgcagcc ggtgqcgatt ccgacgtgcc cgctcctgcc cqtacgcgcc tcagcccgtg ggcagaacgt tcgggcccaa tcgcggtctc agcacaaggt gqgtcaggga tcatcatgat cgaccgcgga tcctcctcct ccaacggcgt tgatgggtct acgtgcggat cggactacgt gcttcctgtt ccgcgtacaa 3240 3300 3360 3420 .34 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 WO 99/37660 WO 9937660PCT/US99/01313 cqqcttcccg cacccgcggc gctcctggtc cgtcttcqtg ggccggcqag cttcggcgcc cggcgcctgg caagcactac gagcgacgac ccgccccacg gctcagcgtc cggcatcgcc catcgagtac ccccgagtac cctcaagqgc ggagtcctcc tgcggtgcgg ctcqtccacg ccgccatgca tctcggagcc acggcggcca tgcccggcga gggcggacgc tcgccgqccc gacgcctgaa aggaggccgg ctgctcggct gaccqcctgg gtcgtctacq tccttcacgc cgggaccagt ttcttcaccq gt cgcgctgc qaccgggtcg atggtgcgcc ctccgcgccc aacgtcqacc gtaccqctga gtcaagagcc gtggtcggcc cggctgctgt gaggccgcca cgcggaccgg tagactggac ggcagaaccg ggtctcgctg ctgcatcgcc gcgggtcgtg ggtcgagatc cggccgctgc gggcgaggtg ctgggacggc cgatcctcgc ccttctccaa gcgccgactc tcagccagat ccaagcgacg gcctcgtcct tcggcatgtg ccgaggagat cctcacgcgt tcgcctacqc cggccgagac aggtcctgga tgcgcaagga actggtacga tcacqccgg ggcgccgggc cccaccacca ggctgttgtc aagaagtcgc gtgggcgagg cgcacgtccq qcccgggtga ctggacccct ggcggctgcg gtcgccgagc acggtgatgc ccaggaccgc cggcat cgtq gacccggctg cggcatggtc ccacatgatg ggtggtggtC catcttcttc cgcggccccg tcactcqqtg caaqctgatg gaaggcgctg ctcgccgtac gtccccgcgc gcacctgctg cgtcatqgtc gcgcaaqcgc ccaggaccgt cctgtcatcc aggcggaaca agtacgaggt agggccaggt cggagggcga ccaaggaccg actggcagca agttgcagcg cggccgaggg tacctgccgc ctcctcgccg atccagctct cqccactgga cgctcccgcg ctgqcgacca gcgaccatga gaggaccccg gtcctgatct cgct ccgaca cqcgaggagt cqcgagatca gacgcggtct cacaaccaga acqtcggtcc caggactgga gaccgtacga ccccgttctc qcgagcggtc cgaggtcgqc gctgttcgtc ggagggtgcc catcgaagcc cgccaagccg cctggcgggt cqacaagctg gccagctgca gagccgccat acatcgtcgg accgcaacct cgatcaacgc agttcacgca cgqcgatccg aggaggcaca ccaagatcca gcctggaggc gggagcgccg cccggccggt cggtgatcat gcgccctgcg cgtaccagct gcgcgccggg aggactcctc tggagtcacc gcqqagccgg cccgtcgccc cggcacacgc cgcttcctgc ccctgcccct ggcgcccagc ctcaccccgg ccggccggcc 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 5520 5580 5640 5700 5760 5820 5880 5940 6000 6060 6120 6180 6240 6300 WO 99/37660 WO 9937660PCT/US99/01313 -91aggtcccgtc tgcgccgcca gcgt cagcga tcgcggcgac tccccatcgt gcqtccaccg gcgtcggctc cggtcatgca tcgqcctctt tcgagtccgg tccgcatcga acctcatcgt cgaccctg acctggggta tcccgatgac gacctqcatt gcgcacgacg cggtgccgcg tgacgctcgg ctctcgcgcc ggatcaggta atcagggcgc gcgttcagga agctcgccac gcgttgctgc aacgggatcg gtggcgcacg ccgctcccac actqqgcatc gggctcccag cgaactggac cgtccacggc cggcggcttc gggcctgctg cqccggcqcc caagcgcgcc gcagggcaag cctcgacccg cgcccgcagg cttccaggac tgcgcacgtt tttcttggct tagagcttgc atgtcggcgt gtgacgctat cgatgaagca gacggt cagg tqtcattggt qtctgtgcgg cagtccgaga ccccgctcgg tgtggagttt cgcgtgcagt gagatcgagc gagcgccgtg gaccgccagg cgcccggtct cqccccttcg tggcaggtcc ccccgcaagg ctggccgacc gtcgagqacg gtcgagtccg ccccgcqccg atcgcctacg ggggggtacc gagtgcgtqg ggatcaggaq ggaccgctcg gqtcggccct ttctgaaggg ggttctctct gaccgcaggqf qatggcqaag gcgccgcacc ggagccgata cagaccgggg cggaccggaa tcgcgqtgga cgatcgacca actggcccqg tcatcctgac cggtcatgcg tccgcgagcg acccgaaggc gcgacatggc gcgtcgggga cccgccacaa tcctgccccg gcgccggccq tggcctgcga gggtgcgqac cgattttqga cggcctgttg tgaaagccgc tctcctggcg tcgtcaccga gctccaggta t gcagggcqt gcgccgat ca ggcacggtct ctgtccggtg cagcaacgct cggcttggca cqccqacggt ctgcatgatc cat ggcgacg cccgcgcccc cgt cggggag cgccgacgac cgccgacacc cctggacctc ccagggagcg cctcgccgcc caccggcatc caagacggtc cccggccgcg gct gcgggtq gcccgccgca cgctcgacct ctgacctggc cgaaqgqaaa ctgqggaggc atcgtcgagg cgacqcccag caccqtcgac ggaagctcgg ccgggtgacc tgcacgatcg ggacgtgccc cgcgccggtc gcggcggaqg gtcgaggcga ggcgcccgcc aaggacggcg cgcacctacc ctggtcaccg tactgcggcg gtcctcggca ttcgaccgcg gacgaggtcg cagcacctct ctggcccggg ttcgatctgt aaggggctct gttctccaaa gttgcacgag ccgaaggtct agggccctgc gtgccctgac gtcgaggatc gqaacaggtt ctccgaccgc cttcgtgcaa gccggtact c gagcggtacg 6360 6420 6480 6540 6600 6660 6720 6780 6840 6900 6960 7020 7080 7140 7200 7260 7320 7380 7440 7500 7560 7620 7680 7740 7800 7860 WO 99/37660 WO 9937660PCTIUS99/01313 gctcctgggo gaaagggggt ccggotoogc cgcogaga cgatatcacc gccactgtoa cggtgtttcc gtgcogctc cggacctggo acaggcgagg actcggcggt got cggccga gcttcgaaga gcgcggaatq acggooaggt ggcacgttgg qcgggcgcgg ttggtgtgat tgcaccgcgt cggqtccgcg agtcatgaac cttcaqccgg gaggqtgccg cccggccagc cagcagacgg ttcggaggtg cacattgcac toogccoccc gctocgoca ctgtoccacc tggcctgcat ccgccatcga gttctccacg ctctgaac ctccgcogog gcggtoooto cacogoggac gcgagttatt ccgtgacgcg cctccgacga agccgtgtgc aaagggtggc cgacggtgaa gtcggtggct gtccgatcga gtcggctgag gggggt acgg atcgcgacca aagatccgta agaoctgcco ctggtgcogg ggcgtggagg accogcttc cccgggacct ccaacctgac aggocat ogg ctatgccaac cgacctcggg acttcagcgg atgtgaaogc gccagaatgc ggagccactg goggtgcggt ccggcacoog gttgtogtcg ggtgtaggcg gccttgCgg gcgcatgctg ctcggtgacg gtcccgccoq gttccgttgc ggagcctgoo cggccagcag ggacggtcag cgccgagttc acgtgcacgc tttcggcagc gtgtctgqgg gtcgatgggt tgcgcccac ccgacgtgag cgcacgcctc ctcggaaccg otcatccgc cgtccggagt ccacggccag ocggcccctg cctgtaaotc caggaggoac tgggaccaga gagagcgagt gtgaatgcgg ggacgcagca gcggtgcagt gcggtgctca gocggggooo ggggtgoggt tgtgcggcgg ggcgaagatc ggacaogtag ggtggtttog ttcgatgatc ccgtgcggcc ggtgoctggg gaaaggcggc gtcgocgacc accgaagtcc cacttgaccg actcgcqcac got gaacagt tgtqgtgotg ttcgagcogg cacotggtct cgaggggccg cgtotgcgtc aatgtcagcc gggtggggto cgtogtcctc gccgtgcgtt tgttctogaa tttctgtctt gcacggcgat tacagggctg gcccagattt gttgtccggc acgatgaagg gggatgtact gcgatccagg ggogogtcgg ogt gcgggOO acctttcagt ggctgatgaa tot caocggt aaggcaoogt Ogoogctgtg atgtggtggg gotgggottg aoaogcoooo gootaootgt ooottggoog atoaggogog otgogtgaoo ggogtgogog gaagttgagg gogoagoooo ogogccotgg ototoggttg ggogatgatg gagttgggot ogaaogogat ooagoogooa oggogoogtg tgaggtaoat tgaaogoqg cgga aggogg acagggcgcg 7920 7980 8040 8100 8160 8220 8280 8340 8400 8460 8520 8580 8640 8700 8760 8820 8880 8940 9000 9060 9120 9180 9240 9300 9360 9420 WO 99/37660 WO 9937660PCT/US99/01313 gttgccgagc caggatqccg caggacggcg tgtcagtgcg gctcatgatq ggtgacgcgg gcccaggqtg cgagcggagc ggtcgtgtcg gagcaccgtg qqgaccgatc gaagacggtc gtcggtgagc atggacgacg ctgtccgtgc gtagccgagc gacggggqcc ttcggcggcc ggcggggatg tccgaagccg ggtcgaggcg ggcgcggtct gtccgtqccc gatgactccg cacggcaaga caggacgagc aggcggacga caggccatgg aaggtcgccg qcggtgatcg aagatgttgt ttgccgatgc gccaggaacg agcaggacga cggtcgaggg qcgt cctgga qcqtcgagtg ttgccgtcct gcggtggcga acggtactgg tccgactcgg aqtcccgcgc cgtgcgacca ttgtccacgc aaggtgaacg tgaagtgccg ctggcgagga ggctcgtcga accccgaaca gcgtccgcga aqcggcacga acgacggtgg tggcggggac cgaagccgaa cgaggatgag ccttcgctgg agtcqacgcc cgtcqtcacc acagqagcag ggaccacggt cggagcggat cgccggcggc ccttggagct tgcccatgcg tgtcggcggg gggcqatctc tgqcqgcgcg tcagtaccac gggcggcgat ccccgggcca ccaccagcgc gggagacggc cgqact cggc gcagggtctc gcagcacgqt gaatcgcgcc gcgccatcag ccagcagcag cagqagcggg ctggaacagt gcccgcggag ggqctgggtg gagcgcgacg gaggacqgtc ggtcgcgacc gacgatggct gtcggcgttc ctgggctgcg gtaggggtcg ggggtctgcg tgcggggctg gccqggcccg gaagccgctg caggagtgcg gcggtgccag gaagacgctc catgacggcc cacgagcagg ggtgcggCgC gcggtagcgg catgatcgag gagagtctcc gggcgagcgg gactttgtca cggatgagga tcgcggatcg gagatgacgc cgcagttcct aggaagacga acqgt gaaga aqgtagagcg aggaccagca tgcgcggtct gattgtgtgg aggtcqagga acacggctga gagcgcaggt aattcctccc agggtgttga atcacggccg atqcctgcgc ctgcccagca acqaccagag gcaaacatgg acggcggcct gcggtgatga gcggtctqggg' gccacgcgca tagatcgcca ccgccgctga aggtgtccag gctqggt cat cgccggtgcg gcttgaaccg aqatgtacgc aggtggtgqc gggcaaggag qcacgatgag cgccgccgat cctgcttgaq tgacgtcgta cgtgatcggc tqtcctggga qaatgaggtg agctctcctg aggccacctt cgcgactgcg ggaggaccag cgaggtacgg cgagcacgat gcatcgcgcq tcagcgcgta aqctgaccgt tagccacgcc gcaggatgat agaccttcac 9480 9540 9600 9660 9720 9780 9840 9900 9960 10020 10080 10140 10200 10260 10320 10380 10440 10500 10560 10620 10680 10740 10800 10860 10920 10980 WO 99/37660 WO 9937660PCTIUS99/01313 ggtgtcggtg gcgatcccgq cqqggccqgt caccgcgacg ggacgcgagq ct ccatgggc gtcgggagcg aaggqccgag gccgcggacc atcacqggcc gacggattcc caccaccacg catcggttgc ctctaagttg gggagacggc gt ccgggagc qaagagggcg cgcaacagca cgcgaattcc qccgcccagg qgctggcqggg aatctgcaca cagcccgagc gaccaaggcq ct cgcggatg ccgtcgacag cagaccgctg cagtgcccga gacgttcgtg gcacgcacct acgatgacca gtcaccgtgg gtqatggcgg acgatgatcg ttcatggagt agggccgggg gcggcgcgct atcgggcagc cgtcaagcta acagcgtccg accgggcqca gtgccgaagc tctaccgcta ccgcctggat ctgccgccta ccgcgctcac tctcgctaca tgaccgtqgc acgatctgac acgacttgcc cgaagccccg gtggctcatg cgcccatcga gtgcgctcat cgtcgaccgc gctgagcacc agaccacgct cgaccgattg ccgqcgtcgc ccgagqcggc cgaccccggc tcttcgatcc cttcggcagg acacgccccc aqgtgaagcg gcggCtcgcg cctcacaqcc cttcaactcc cgacgcagtg cqtcagggcc gcgcgact cq cgaggcgctc ggtggtccaa aaccgtgtcc acatcacccg gcagaacgag cctcgtctca ttacgacgtc tggtcacccg attctqqaag gcggtcctgc cacgacatgg cgtggcgctc gtcctggccc gggcggcagg gaggaggccc caggaqacat aagtacggac tcggcctctc gagaaaatgc cagctgattg ggagcggttg atcgacgacc gagcaggcca aacctcgaac ctctccccgt gcccgqgtcg gcagtcqtcg gacttcgcgg tgacgcaccc ccggatcttq tccgatcttg cacgacccga gcctctgggg tcggcgggcc gact cgtccg qagaccaact tcgggtcggc ttcgcggcgt ggaagctggg ttggcqgagg gcgat cagga cgcagcagag agaacttagc gggcgtgggg ccaagattga acgcggccga ccgcgcgagc tgctcgaact tcgcggccga aggcagctcg cggcgcggga tgcgcgaact atgcgtgcat ccggagcgac cgtccaggcg agccgcaccg ccaccgggcg accagcgcgt gtcaccagcg cggcccggtc aggggctgcc cggccgcagc tggttccgga cggtgccctt cgcggacgag cgt tgt cct c cccaqaaqgc cgggggagtt gacagggtgt gtaggttggc agccggcagc ggagctcctg cgggatcgcc cgtcgtcacc gaccacaccc cggcacccac gcqggtgagg ggggcagcca ccgcagaatc gcgtcgactg gctcgcaggc gagcgtgacg qccgaccggt tcagtgcgtt cttttagggc 11040 11100 11160 11220 11280 11340 11400 11460 11520 11580 11640 11700 11760 11820 11880 11940 12000 12060 12120 12180 12240 12300 12360 12420 12480 12540 WO 99/37660 WO 9937660PCT/US99/0131 3 acgagactcg tccggcttcc gcaagcagga tcgatgacgt gagcggcgtg tgccgagcac gaacgtcttc tccgtcgccc cgcgtcttcc cccgacggac ctgggaccgg ttgtgggtct ctqcgtgtcg ggtcttggcg tcgcggagcq ccggcacccc atccaggtca cccacgtcct tcgcgggcga aaqcgctgac gtgccgtccq cagccgacgg tcatatcctc gcagcctcct atttcctcac ttgtcgaaga acggtggcgc cgacgacctg acttgccatc tctggccggt cgctacctcc agcgtgcgtg ggagccgagc gcaatcacgc tgcggttcaa ccgaggcccg cgctgctgat cggtggtcag gcgccgggcg tggtcgctgt catacggtgg ggcgaacagc gtcgccacag ccccggacac cgatccgctg tgctcgtgat tgccgacgag cgtaggcgtc tgagggtcac cgtgggctga cggcctcgac aggaggtggt qtgataccag gacacgaagc ggggccattc ctgatggaca agat cgacga acatqggcct tgtatcccac gctgatcgac cggcgccagc tcacgcgctg cgccctgt cc tgaggtggcg tctgggagcc caaactcatc cagaccggt c cgcacgtccc gcgcagaagc cacgaagtcc aagggcttcg cagccggtcg aggaccgccg gagctct ccc cttgaacagc tccgcgtccc gaggaggcgg gtggCccgtg gcaggcatca ttacggagcg gtgatgcccg gcgatgcgga gatcctggcc ccttgtgtcg cctgcacgag ggcaacaagc gccagtaccg ctcaccagca gccctggggg gcgccggccc ctgatcgccg gccgggcact ggaatcctcg cgtccggtcg ccggtgccgt ttggcctgcc aqgtcggctt gcggccttgc aaggtgtcgq t cggcccagc ccgtcggcct gccgcgtaac cgtgcctttt atgctcgtcg tgaccttatg ggctcgtggg ggaccgggcc qattctggac atcgtgcgca gcgatcgaac aagccgcggc gcaccgcctg tcccgcccca gccctctcct ttctcgccct ggtggqgctt gcgtactcaa gagagggcct qcqccgcgqc ggtcaggtcc ccaccqcgta acagcgggac cggcgtcgct tgccgtaccc gat cggggta tcttctggaa ctagttgctt cqtaggtgaa cggcgtcctt ggatgaggga gcqatgacac gagqqttgca gagctgaagq tacctgaagt cqgtcaacgg ggccccqgac actactgcac gttcgccatg cgggcgccgg cagcagcgca ggacacgqcg cgtgggcggg cgccgtgatc gtggtggtgt cggagcggtc gagccgtcag ctggccgccg gacqqgcacg ccggt cggcg cgtcgccatg gtcggcgacc ttcgtcccat tttcacctcc agacagcggg gtgagggtag gtagagcggg 12600 12660 12720 12780 12840 12900 12960 13020 13080 13140 13200 13260 13320 13380 13440 13500 13560 13620 13680 13740 13800 13860 13920 13980 14040* 14 100 WO 99/37660 PCT/US99/01313 -96tccacgqttc cgtcqtagac gtcgtaqgaa atccggtccc tgtctatcaq ccaggccgcc 14160 gcctgccgtg cgagtctccg gcgagcattt cgggcgagtg gcggcgccct acgacgccgt tacgtgqtcg tccggtgcgg tccaggaggg cggacgtcct catcgatagg ggct ccgcca gtcgagccgg accttgatgg gccgtggcga atgaggctcg tgcgtcggcc gaagtcttat ggtcggcacc tgcgcgggtc gccatgttcc aggtggcagt cacggccgga ggcgqcgctg cgcgtctgtc cgctctgcgc cgggggqaag aqt cgggggt tqtaccgagg acggtccggt agtcgacgat tcagtcggaa acgacggccc tggctgtcat tgcgtaggcc ctccgggctc agtcgtagcc tggtgccctc tcaccgtggg tcctcgcaaq aagtatgcgc gactgacacg ccctctcagc ccgcccacgg gtgttgcagg gcggtactcc tccctcatgg ccgctggccc gtgaaacggc ctccgtcacg ctgtctgagg gtcgtagaaq gttgqgcgag cccgtcgatc cgagcccgcc agtgacggtc cacqtcggaa tctgcgtccg gqaatcggtg cagggtcagc gccgqccggg ctqqtcgtcc tgtgatgacg atcgagacga aqgcgtcgct gtggaactgc aqccgttccg tctcactcct gcgtggccat gtttcccacg gaggcgctqt tgaccgtcgc ttgccgcggc cqaagccccg gcgacatcga tggagttcga agggagatct cggccgtccg ccggtcgtca cggt cgcgqq tctatcttct ctggagaagg aaqgagcagc agtgtctgga tcgaqtgacg gtcgggtaaa gacgcacgga acaggaattt tccttcggag tatgccccgt cctcgtctcc cgatgccatt gccaagtcgg tcctctcccc gagaaagtca catgagcgtg ggttgttgag gatcqctcgg tgcggtggga tcggccggcc tctcgccctt agcgcagqga gcttgaaggg cgtccatcga tgacccgttc tgatgtcatc tttcggctgc agacattgct tcggcggttc gcagcagacc tgtgcgcacg tcgtacccct cccgacggca tcgqcgagag ggtggtcctg ccctgtgcaa gaggtcgttc cttcagtcgg ctggtacggc ggcctgctgt gtacagqttt 14220 gttcagatcq 14280 tatccaggcc 14340 ggtgttctcg 14400 cqcgtaggag 14460 gtccqccggg 14520 gaacgtggcq 14580 ctcgatggtg 14640 gaacgagaac 14700 ccgcagccgg 14760 gtcgggaacg 14820 qtacagagtg 14880 cgtcqtcccg 14940 ggctgccagt 15000 aataggtctc 15060 gggtgqagag 15120 cttccggaac 15180 ggccgccaqg 15240 cggacccgct 15300 tgtcacctgt 15360 gtcttccgtc 15420 ccgtgctccg. 15480.
gaggtctggg 15540 cgcagccggc 15600 cggcgcagcc gggaacaccg ggtccgtcgt gcacgtcgcg gcqgacgacc cggagcacgc 15660 WO 99/37660 WO 9937660PCT/US99/01313 -97gggacccccg cacgagqgcq cggcaagacc caagaccggc gagcggcggc gttcaacctg cgcctacgcc gcagctcgtg gggcacctac cagcccgcac gctcatcgac ggactacgac cacggtgacc cggcctgatc ccagtcctac actgcgctcc ccggacggcc ctgctcctac gacctaccca gtcctccagc caacgqcccc caccgactac gtacctggag cgagtggctg cgacgqccgc cccgtcgcgc tccgagcttc qcgaaggcgc aactcccgtg gcgctggtcc accgggaacg ctqcgcgacq ctctacctgc ggcagcagcc tcccgcgacg agcgccgagq tccacctgga ttccgcggtc gacqcgctct ctcacgtcga aagqt cagcc ccqctgtggg tacggcacct tgcagctcca tgcagcagcc gtcqcgaacg cagacctacg gggaat ccgt cgtccggact ttcqacatgt agtcccccac cgcccgtgqc cggtccgtcc cgcgtgccgc agcagatcaa acgcctacta gctcgacgtc gcgccggcta tgcagaccgc tcaacgacgc agaacgcccg actcgtcqtg accaggtgcc acaacttcgc acgcgggacg ctctggccgg tcggtgtcgc gcgacctcca gcatcaccat tgcgcaacca acaacaacag ccggcgcgat cggcggccgg tccagatctg acggcgactt cgccgagacg cgcgagtaag ct cgaaqtcq cgcgtgcaac qqcgtccacg cgccttccgt ctacccgggc ctacgtgcac gatacgcgcc caacggcgag ctacatccac gtggatgctc cgcgttcgtg gaqcggccac tgaactcggc cggcctgctc cgagatgacc ggcacaactg caaggcgcag ggacgcctac caccatcgag gtacgggatc caaccaqccg gaacctcaac caacgccaac qagcacgtcg gacgcgctca acggacaagg gtctccgact accgactgcg gagtcgcaga aacgcctcca tactacaacg gggatcgacq acgctcgccg qtcgtcaagc aacgcggtca agtgccgtgc ctcgcgctqc cggttcctgc aactccagct gactactacg gcccgctccq cagatgacct ttccacaacg gtcgtggtct gacaccaaca cgcttcatcg cacgagtaca atcaccaccc cgtagggacg aggaggtgta cggtcgccgg tcaccagccg tcaacaccct tgacctcggt ccggtatgcc ccggcacgqt ccttcttcgc aggccgtcac gactqctggc acaacgtgta agtctgaccc tggqaacgga agcattccgc ccatcaagqg acaaggccaa tcctgacggt cgggcgagct tggtccgtga tcgactccag acggcqgcat Cctacgaggc cccactacct cgaccatctg 15720 15780 15840 15900 15960 16020 16080 16140 16200 16260 16320 16380 16440 16500 16560 16620 16680 16740 16800 16860 16920 16980 17040 17100 17160 gtgggtcgaa ggcttcgccg agtacgtctc ctactcctac cgcggcgtcc cctacaccga 17220 WO 99/37660 WO 9937660PCTIUS99/01313 -98ggccacgacc caqccacgac aaaccaccgc cgcccgcagc gctggcggcc ggcgcccacc gtccaccgac ctccacggcc gaccgtcacc cqgcggaggc ccaacgcggc ggccggcacc ctacagcacc caacaaccac cgccgtcagc ccaaggcacg gcggcggcag cgggcatccc cgcgccatgt gtcggcagcg caactcgcag gagcggctca gcctggatcg acggccctcg gtcgaccgca gcagcgaaca qaggcggggc accacgcgca gccqacatgg tacctgaccg tgcgcggccg gccgcgttca gccgacggca accaaccccg gacgacaagg ggcggcaccg aaccagtccg acccagctga agcggctggc accctgacca agcttcagcg accctcacga gggggagacc tacctcccag ggtccctcat gtgaccaqggg cggaccagca ccggcgtcgc accagaagca tcqaggcccg gcctgcggca agggcgccgc gtcgcacgta tctaccgctg acaccgtcct gcaccatcgq gcaactgcgq ccaccgccgt ccatcgcctc tcaagacgta gagccaccgc aatgcaacqg ccaccaccgg agatcaccac ccggcaccac tcaccaaccc gcgtcaccgt cggCCCgggq tccgtcgccc gaggcagagc catgtccgtc cacgtcacgg catctccacc aacagcgctg cggcggcaqg gtccgctcag actcgaccgc ccacggcctg cgcgctgagc gggctacctc cagccactac cacccgctac tggcgggggc ccaqggcctg ccgctcctgg cgggtcggcc caccgccacg gaccgacacc caactacgcc ctccggcggg gagctacacg gccggccggc gagttccgcc cggctctccc cggaccgaga gtgaagtcat ggtctcacct ctcagcgcct caggaggcac cgtgagcgcc ctgaccgtcg gctcaggcac atgtccgcag cagtccgcgg accctgttcg qccgtgcggt cgcgcgggaa gacaacgact accaacccgc aacgtcacct agctt cggcq ggqtcctaca aggacggtca cqggaactgg tacctgtacc acgggcgacg cagcgggcca gccaactaca tactgaccca cgccccgggc acacatcgcc tacccgcacg gcgcactcgc cgcaacaggc agcggcgcgt tgggttcccg ccgtcacccg ccgacacgac gactggccca tggtggtgca acaccacqta acatgctcqa actggaacgc ggtacacctg ccgqgaacca tcaccgacca acgqcaccac cggtcaagct ccgtcggcag gccaqaactg tctacgtccc cggacctgta cgggagccqg tcagcctgca cggctccgca ggcgtccggg cgcccgcaca caggcgacgc cacacccgcc cgcggccggc actgcggcag cttcgcagga gtcgacggcc caccgtcgtc ccgtatcgcc ggacaacaag 17280 17340 17400 17460 17520 17580 17640 17700 17760 17820 17880 17940 18000 18060 18120 18180 18240 18300 18360 18420 18480 18540 18600 18660 18720 18780 WO 99/37660 WO 9937660PCTIUS99/01313 qttcgtctgg tgggcgaagc cccttctact tacggcacga atgatgaccg tacggctacc gcgatcgtca acccgcatca acgaccggct cgcaccctga ggcgtctacg ggcatgatcc ggctaagcag ccgccgcacg tgggggcggg agaccgtacc gcgtcaccca catgatggtg gatgccgctg gcatggtggg cgagcatgtg agaaggcgcc ggtcggccaa acaccgggac ctgggtagtc ctccggtcgc acctgccacg ggaccctcqg gcggcggcca acgtccgctg ccggccactg agqqggtcgg gggtcgacga ccaactggga acacctgcgg ccggcatgac acggctcaac acgaaccgat cccgggcgga cgcggtcggc ggcgtggcac ggggagcgcc tttctggaag caccacctcg ccacagcgga ctgqacqgcg gaactcgtgg gggccagccc ggacacctcc ggttctgata gcaacccagg gtacacactg gggcaagacc cgatggcctc gtactcgtgc ctccagcgcc tgcggaggac tcacgtcgcc ccccqgctt c ctacgactac gcagatcacc ctggtccacc ggcccacqgc cagccgagca ccgtgagtac gtctttcccc gtcgtgcacg tttccggctc tgcggagcct acggccgacg tcccccgcga gccaccccca gtgacggcgg accccgtcgt agccggtgcg gctcggt ggt gcgacgt cga ctgaccgaga ctcacccccg gaggtcagca gaccccccgc ttcatggcgt agctcgtact gactacacct tggcagccgc gtcggccagt gagaccaacg gcatgcgacg atcctcagcg ctggcggacc gccgccccgq gtgctccgct ccgcagcgcg cgtt cgtggg gcgacagcag cctcgaccga qgggtttaac gacccttggc gcctgaatcc cogcggagac ccagtgggtg ccagcttcgg gctcgccggc cggtcaqatc cccagcacgc cctacgcgca tgcgctcggg acagcggcag gggaggtccc tcggctacta gcggctgggt acgtgtgcaa caacggtgtc ctcccggtga gggggccgaa gcggggt cac tcacatcacg ccgtccgcca gtggaacccg acgggcgggt ttgccagtgg atcccgccgc cagtactccq gatcatcgac cgcggcccca gaaccacgcg atcggcaggg agagtgtcga ctctaggaga ggggcaggct agtcgtggag tgcctccgaa cctgqccatc cagctactac cacctacagc cggcgactcc ctacccctcq gcagggctcc ctacccagqc cagcqgcagc cagcggatgc gctgctggcc aggacgtcga cccagcggac tcggccgatt gcgtatgcgc gcgcgtacgg cagacgagca cccaccgggc tgcagttggt caagcgtcgt tcctccgaca gtggccacga agaggcagcc tcgatgagtt tcacggagga 18840 18900 18960 19020 19080 19140 19200 19260 19320 19380 19440 19500 19560 19620 19680 19740 19800 19860 19920 19980 20040 20100 20160 20220 20280 20340 WO 99/37660 WO 9937660PCTIUS99/01313 -100cgtcgagcaa ccccggacqa gggcccggcc ggaagacccc gggtgtatcc qcacgcgcaa tccattatgc accagtagcc ccgggtgacc gcctgaagcg gtcgaagcga tggtggggag ctcctcacca cacgacatgg acggaggcgg cggggagtcg ccacaggagc gaccgggaac gcgatgtgag ctgagggcgt agaaacagcc cgcaccacgt ttttgttctc cggtgctcgt actcggacgc ggtcagcgac ggtqggtacc gcqgggaggc gacgacctgg ctcgccqagc gagcgccgca gtggcgcagc gccaggaqga cgagggttat gccgtggaaa caccqccatc ccggggccgc gqccggtcga acgggctgcg tcgtcgtcgc acgacgagtg agaccacttc gggctatcga gctggtcact ttcgtcctcc gatgacgctc tttgacctgg ggagtacgtg ct gcggcggg gtgtccggcc ctgagccgag cgctggcacc accggtgtgt cgcagccgtg gcgccqtagg agtccctcga gcagcccggc tcaagttcca aggcggtggg ggatgagccg tctcggacct gagqagcgac ggttcaccgc ggaagagctc gcacgcgggc ggtcctcgat cgggcgggga ccgcgggaag gtcggctccg ggctccccaa t cgggcggcc gtctgacgct ccttttctgc gcgattctgg cgcggcaagc ggcgggtggg atgcggttcg tgtgqaagaa tgatggcccc cgagattgt c cggtgagctc tgcggcgcag cgacgccccc cgggggcacc gaat ccggga gagcctctgg gcgtgttggt atcatgcctc gcccgccacc cgcttcagcg gggcccatga gacagccqggg ctcgccctga cgctgctggg gctgaggagg ggaagcggac cagtagccga ctggccgctt ggctgcctca agcctgctgg tcqtgcgggg ctgccttcgt ttcggcacca cctttgcgac gaggcgaagc ggagagcqcc cacgcccgcg ttgggtactc cqcgtcggct tcgctccggq cggcctgacg tatggcctgg ccgcagaggc acacctgcat gcgtctccac cggaactcgt ccgtcgagtt agctgccgcg tcqaggacct cggttctqcc cggaccacgg tactggcgag cccagggcag tcaagctgca ggccgacatc caaggggtt c caqttgggtt ttcagtgggt tggggtccgc aaggccctgg cgacgtgtcg agqatctccc ctgctgcqca atcgccggct atcgcacaca cgaacagagt ccttcggtcg ccggttgttc gactgcggaa cagcttcacc cgcgctgagc gacctccgag gacgctggtg gcctcggcag cagtctgata gctgcggacg cttcgaggca tctgtttccg gcgggcgtgc cagcgagccg tttccgatga tgacctgcgc #tcccgaaggc gcgagagggc a gga tga ccc ccgaggtt cg 20400 20460 20520 20580 20640 20700 20760 20820 20880 20940 21000 21060 21120 21180 21240 21300 21360 21420 21480 21540 21600 21660 21720 21780 21840 21900 WO 99/37660 WO 9937660PCT[US99/01313 -101ctcccacagc gaccacccgc cgaatgcgcc cgatcctgaa caccggcagc cgt cggaggg ccgtctgccc atcactgttc tagacattgg ggcagagacg gtcqaagcca gtggtcagg ggtgaaggcg agagcaqacg ctgagaggtt cgccatcagg ggtggtctcc gcggacacag gccctggtgc agcagacqat ggcggccgag gatcacgccg cgcggcccgc cgtcatcctg cccggccggc agggctcgcc gcctqctgga qaacgttggc cgccgcctqa gccctgcgcc ttcaccctgc tcgcggcttg ggggcttggg gagagtgatt gcagcagtgt aactgccggg ggatggtgca gccgccaccg tcggctgcgg caqgacgggc cgtgtcacca atcgcccggg ggtcaagaaa aaggccgqtg cqcatqgcac tcgtacggcc atgctcggta ctccgctcqq gcccgcgaac gaagaccagc ctggtggaca tgccaagacg ccacagcgaa atcagctcca acctgtccct ccgtgaagca tctaccgcag acqct caccg tcccggctgc acctctccgc gqttatgttt cagcagtacc ggacggcgac cagtgcagta acgcqcqcgc aacggggccg gcagtagagc cgcagttttg ccgcgat ccc caqtatcaqg cagggcccct qtctcgacga cgacccccqc aggqcggcca tcgtcgacca tccaagaagc aggccgaggg ggaagccctt cacaccccgc cgacctcctc caacaccgtc gctgtttcgc cacccagggc tat ccat cac ggcccgtagg cgqacacatg ctcctgtaac cgcaqttgca gggactgacg cccagcagcg cgggaggttc actgtcggac agtaccagag ggcccgggta cgcgcccccg gccggcagat ccatgcgttc cqacgattac tttcctgcgg ccgccgctac gggcactccc gcggcgtatc ctgaggccgc gccgcaacqa ccggt cggcq ggcaagggtg aagcgctacc qagqt ccagq cqqqcagaag tggaacggcg cccggctcac gaaatctqca ccaqaaggac ggacgqtgcq accggaccgg aagtcagtga qgcagtggtg agt ggcgctg qaaagaacgg ccgcaggaca gcaqcaggca ggt qtaggag cgcagagaqg cccgcctaca gccgtcggag tcccgctacc gt cgagqcgg aacgaggaac atctgccggc gaagaggcaa tccatgagca tcggcttgct cqcqcacccg agcagggctg gcgaccggcc acgtctgatc cccagcaccc tcggctggag cgcaqqgccc gtggtggagt gcggcccgca gcggtacctc gttccaagcc tcacaggtca aggaaccaag tcgatagtga qgtcgggtcc ttccttcggg tgcagatgac ccatggqgcg aagcccggct agttgcqcat cctgccgcat tgcagaggcg cggtcctgtg ctgtccgcac 21960 22020 22080 22140 22200 22260 22320 22380 22440 22500 22560 22620 22680 22740 22800 22860 22920 22980 23040 23100 23160 23220 23280 23340 23400 23460 WO 99/37660 WO 9937660PCTIUS99/01313 -102cgatgtgctg tggacgcgga gacaggacog ggcggcqgcg accagcgtgt cgggggcagt ctcaggaacg cacgqcgccg ttcgaacgag tgcggacgca gccaccgtcc ctcgcgtatg ccctgaacag agcgcaggta tcqtcttctg cggacgaatt ctcgcgggat cccgtatgtg tcctcacacg gaggcgccgt qcgttctgcc cgcctcttgc ggcggccgag gccgccgaca tccagtacgg ggcccggtcc accgqacggc gcgaaggtgt caggtgtcac catccacctg tgtaccgttc ggctggactg cgcgaccccg ccatctcatg agccgtctgg gtcccgccct ggccgaccat aagggctggc atcccgtgct ggcgacatcc gacgggcccg catcttggtg ggccgagatg cattcccctg tgcggtctct gcctggcgcg ccggcgcgtt gcgacctcgt caggcatgct cgtcqcggcg tqgctaggac agtgtcgtcq attcgtqttc agggacat cq atccaggctg cgccgcgag tcgcagtccq accacagcta tgtgccggta ggcgccaccc gcgtcgtcgc acggccaacq gggacctcct gtccgcgacc ctttagcgtg ttgggaacgg cgggctggaa ccggaaggct agccgtcggc gcggcgaggc gctgccggct gccgggtqga cgqccggcct ttgccatctc ctgccgcctc tcccgtcttc ggcgcggagc qgcggtgtga gttccgactt ccgcggtag agtgacccga ggccaactca cacgcgtggc tgaacgtgat acgactggct ccgtqtggcg ccagaggtaa ttccaagacc cagggctgtc ggcggtagcc caatgcggga caccgcagqc qacgtgcccg catggaacgg cggggccacg gttcacgaqa gagcaggtgg gaacggcttt cctcqctqtc actcgcgcat ttgccggagc gcggggcgca cggcqcacct ccaccacccg tgctcgacgt ttgaccaccg agcccgacca atgaccaagg cgacgtcccc cgccgccccg tcatgacggc agcgctgtcc gtgctgcagg ctccgtctgg gccgaaggga ggtcacgttt cqaggccgag aacgcagaca aaccagccgt cagggcgaag acaccgaggg tgaaggactg cactgtccca tactgacccc cgatcgggga gatgcggctg cgtcgatggc ggccggcgag tccgggttcc cgggggtgta qcctgtggcg tgaqqccgac cggcacatgc ggcactctg cccccgcggc gacagacttc atggtgagtt agtccgtcag ccgagggtcc ggcgccatgt ccggaccggt gcaatggccg acccqtaccg acgaaccccg aggccgcgcc aggagtcgcc gctcgctgaa gcggcggcag agcagcagga atgcggcatg 23520 23580 23640 23700 23760 23820 23880 23940 24000 24060 24120 24180 24240 24300 24360 24420 24480 24540 24600 24660 24720 24780 24840 24900 24960 tggtgagggc ccggccggcg tqccggtcgg gccctcacac tgttttggtg tcqggcgtt 25020 WO 99/37660 PTU9/11 PCT/US99/01313 -103t gt cgt gt og ggactgtcgc gccatggccc tggccgacct ggttcgggtt aagcgggcaa cgtctggcgg acgggcacca gacggtgaga tatcggqagc gcatcctcgt acctcqacgg gtgccgcgca tqcctcctgc cgtgqacttg ggtggcgccg ggtcggggcc ggtgacgagc gaggtagtcc gtggattccg ctgcgccaga taccccgctg catgatcacg gtggacatgg gtcagacgga tgcqcgcggg ttaggatttc gcttggcggg tgacgccctt tgacacagcg tt cagcggga gcccgagccg cgaagggctc tgtcccggac cgcagacgac tcatgtcgta atgggagagc gcagacgggc agtgcggggg cgcttcttgg tgggctccgg cggatcccgg gtgacgacqc ttgtqqtcgg cgtgccgcga gtgcgggtgc ttgagcaggt cggccgtcgc agtacgtggt caggtggggg qgcggcct cq gccggtgctg gtagtggcgt catcgattcc catgatgcct gagagcgagg t ccgaagggc gctgtcgtcg ggcggtgagc cagat got g ccagacctgt gcctacggag ggggttcctg tacgccgcgt ggcgttcagt tgatccggct cttccgacat tgccggactc tcggcgqgtc cgttggtcgc gctqcggctg cccgggtcag gtgcggcgat gctccatcgt gcgccgagca ggaagqgtgc aagtcgcggc cgtccgatga agcacgccgc cacaggcaqq acgcccaggg accgcgtcct gtgatgtcac cgggCctggg gcacgggcgg gqcccggcgg cgatcctgca atcaggacgg cgcqaggcg cgccggggcg gagggcctcg gagacggacc gcCggcgcgg gacgttgacg caccagcacg ggacttgccg tctgtcgacg ctcggtggtg qgtgacgqcg tggcggaagc ggcaggtgaa ggtcctggcg tcacggactc tcttggtcag agagttacgq cgct gccgtg oggcttcctc cgaggcggag tgaccaggcc cggccatcac cgtccatgac actgtcctgg tcctaggcgg gggcggcgtc gtgatgacga tcgcccgggc atgccgcggc gccgtgcggc acgaggtcga gtgacgtgcc ccgtgcaggg gcgtgcttgg gccgcgtgct ccggccgagg ccgctgcaac gcccagctgg ggtgatgacc gccggtgacc gtcgaacggg ggccggccgc ttcgtcgacc cctcgtggcc ccggtcggtg gacgcaccgg ggacagcgcc gtcggccacc cgtcaagggg ccgcgccagc ggccgcgtga ccgggatgcc tgacctgggt gctggttcgg cgccccggtg ggttgtcgac cggtcttgaa cggcggcccg tgtcgaggaa tgtcggcgcc ggtcgacgga 25080 25140 25200 25260 25320 25380 25440 25500 25560 25620 25680 25740 25800 25860 25920 25980 26040 26100 26160 26220 26280 26340 26400 26460 26520 gtgcacgacg gggtcgctga ggtagcggcg tacgagcagg tcgacgttgc ggtcgagggt 26580 WO 99/37660 WO 9937660PCT/US99/01313 -104ggcggaqaac cggcatgaag gttcaaccgg gacctcggcg cgtggccagc cgccagctca gccgqccgta cccqcggccc gaacggcacg atcgqcgaag cccctgaacg gcgcggcgcc ggtgcgagcg cacatatcaa gggccttgcc gqqqqtgctg ctgcaccttc aggtgaaatc ccagctacaa cttctqgccc ctgggcggcg gaagccgaag ccttcggggc cccgcccgga gcacttgaaa ccactgtgcg agcatgcqct cccatatcgg cagtcgccgc ccaccacgca cgcagcttca cgtgtcggta cgggccagca atgatgtcgc gtcacccctt ccctccgcag gcgccgggCC ggcgaaccga aagcggtcgt ggaatttccg agcggatctg tgggctcgat gaaggacgat actgcgggaa gtacgtgaca tgcgcgatgt atgttcgagt cccttttccg agtcgtacgg aaaagaccgg tttcgggaac gtgaagaacg ggccttcqg ccatctggtc qgt cgatqag gcgccgacgc cagagcgggc cgaggaccag gagccaggcc ggccggcgag gttggccqag cgggaagcgc qgcggccgta agcggctgcc tcgtgcgtgt aagcaatgaq gccgacagaa attcgaagcq ccgtgtgcgg acgcatqtag gt cggcgtca cgaagttcac agtgggcgaa cgttgaacca gatccgcacc aqatgtaaaa cacaactqca ccaccttgct acgcacctgg ggcctcgtcg gtccttgaga ctqcctgccg gtacggggtq ccccagcggc gaaggcgagg ggagttcggc cgcggccagc gggggtgatc accgccggag gccctttccg gcggttcata cagcacggag aatctgtqg tccactgcac taaacacacg ctggggcccg ggcqggaacg ct tct ggcc t cacat cagcg cttcacggta gcgcggacct gtgcttccag actgacatcg tattgcggca tcgagcagtg aggacggtga cgtcccggag atcgacatcc agcgcgt cgg tgccgaggct gtctttccgg agggtcgcgg agttccccgg gtccgqggga cggctgggtc gagtcggcac cggaaccttc aat cgcaaga gcacgtgcgc tgtagctatg ctgtccggag caccccgaag atgttctcgg tcgagcagct ccgccaccgt ccagcagcca cgtgtcgccg gggtactgag acagtagcac gagaatctat cggtgacctg cggagacctg tggcgacgac cgcccaccac tqacctgctg cggcccgccg aaccggtgcg cctggatcgg qcatgtcgag gggcgaactc cggccgqccg cgccatgacq ctcgatgcgg atggaccqgt tggaatgatt aaggatgcgg cgtcgtccgc gatgcgggcc ccgtcgggcc cgcggaagcc cctgctcgat tgtcatttct caatgatcac cccgaccgga gccacagcag ccgcatgctc 26640 26700 26760 26820 26880 26940 27000 27060 27120 27180 27240 27300 27360 27420 27480 27540 27600 27660 27720 27780 27840 27900 27960 28020 28080 28140 WO 99/37660 WO 9937660PCTIUS99/01313 -105cgatgaaaac gccgcgctcg cgcgggggca ccqgtcgggt gtcttgcctg gttctgcgcg cctcaggagg ttgccgccqt tccgtcaccc ttcgcgcggt tccgcgatcg Ccqgaa gt ggg ccctcgtact gccaggtcgt acttccacat atcacqtqcg cagctcttct acgagaatgt gcttcgtcga tcqgcggtct gttcgqccgg cccgccccgc ccggcctgct atcgttcacg tcccggccat t caaaccgcg cgtqat ccgg qacaggtccg gtacqtctcg gcaaccgqct cccgcttccq cgtgacccgg aggtgacgtc cgtatgccct ctgqgtcggc cgaacgtcgg agagcatctg ccgccagctc agtagatgtt gcagcggcgc agcgcgcgcc tcccgaacac cgagccgctc gatggtcggt cctcgatggt t cacatacac gggtagcgcc cggtgttctc ctaaaaccga agcgccaact cgcacagata tcccccacca gccatgacgc cgcgcggcga qtcggctcgg tqaacgcgcc ggaagccgag ctcgaagtat ggccaqttcc ggccagcgcc ctcttgggcg ctccgaacgc ctqagccagc ctcgcggtaq qtcqgagcgg cagctccag cgaggtggcc gtactcccgg gggaactgcq ctcggtcgtc ggtagcgccg* cgccacgagg agtggtctgc cagaacacgt acgtcctcgc ttgaccttcg cgctctccgc cggccatgct gcactgcggg gctggttggt gcagccccgg gatcagcgqt gcccagqcat ccgctggagg gcgaccgtac atcgcgtcac aggaagcccg ccgaacgcga cggcgqttgg at cagcagcg atccgcaqgc agaaggtgct tcgatccgct attccqgtqc cggccgacct gcccgcccca gcgat ccgtc ctggtgatgt caccttttat actgagcgtt cgcgacgcca tactqcacgg cggggcgtag ggaggggccg cagccgtggc ctgccttcta agtcgtcagg ccggccqqct tggacttgcc gggccaggta gccagttctc gggacaccgc ggcggatcgg agtgcgcggt gaagcagcag cgtcqgcgag cgccgcccca cgacgagggc cgcccgctgc cgctggcccg gttccacagc ctgccagcgg cctcgttgct gtggggggta ttcaqcgtgg tatatcgcat gtcgcaccgc cggacgcctg gttgccagac aqgtgatgtg ccaggccgac ggaggcttcc gccgtccacg gttccaccgc gtgcggqgac ctcactcaca gaccgccgtg ggcgttcttc accgtcggtg cgccgccgtq cggtgtctcc caggtcgttg qcggacctgg ggtgacgagt ggcccgggtg Ctgagct cgt acccttcgga catgtcatcc ccgcgcatca gccaccgatc 28200 28260 28320 28380 28440 28500 28560 28620 28680 28740 28800 28860 28920 28980 29040 29100 29160 29220 29280 29340 29400 29460 29520 29580 29640 gggtgacgcc ggtcaggtcg gggtaggggc cgcaacgcac aaqgctcgcg tgcacgacat 29700 WO 99/37660 WO 9937660PCT/US99/01313 -106qgccaccgcg gagaccaqct agacgcgttc ggaccatcgq cacggtaagc cggccgaccg ggcacgggca gtgacgggct caggttgatc gatgctgagc gagcacgttg cgagcgcagg ctgaccggtc ctccaggagg gatctcggtc ggacaqqgac ccgctgqagg gagcggtt cq gcqct gct gc gaccttggtg cttcagcggc gaagaccagg gaagacctgg cgacaaggtg cgcgacggtg ctcqacgacc cgcatgatct cacccgggac agcagcccac gct cggcacc ggtgaggtgt gcatgccgag tgggcggcac t cgggt caga tcgccgtccg tcgtactcga acqgcaccgt cccacggtgg tcacccagtt ttgcagcggc gqtgtgccca atggcctcga tccttgagcc t ccaggagca tgtccgccgg aggacgtcgt agtgcgaggt ccgagattgc tcgccgacac gtggacttgc aagttgacgt agct caggca cccagcggga acccqgtccg atcccaggag gggagtgcac cgaggtccag cgggcagccc ccccaccccg cctgtgcggg tgtgccqgac atcgcgctcc cgtgcggqgc cggacgaccc cgacctgtac cqatgaagcc cctgctggag cctggtcgtq agacgcgggc gcacgccggg agagctggtg cgatgaggcg tgtcggcgac ggccttcggg ggatqgtgcc cgcagcccga cctccaggac t gct tct tcc gcccagccgt acaccgcaca ccgtctaccg agttqatcag atgggcggcg accqgtgtgc cagcacctga gaacagcagg ggggctctcg ggtgtacgag gtcggcgcgg cgcgqagcag ggctccgccc qgcgacctcg gtgtccgtgc ggtgacgtac ctggtcgcgc ggagtagacg ggggtagcgg ccgttgct cg qgtcatgtgt qqgcaccgtg cgagt cgggg cgggccgagc cgcggtcccg ccttcaggag cccqggcagc cgatcaagta tcccaggaac taactggcaa qcqgtggtqc cgaqcggcgg agtcggtcag caqtcgtccg gcatggacgc cactqctcga tcggtgagcg gcgccggcca tcggtggcgc ggagtggcgg atgaacacgg acggtggtga atcttcgcgt atgcctcggg tcgcgcaggt cccttggtga ggccagagcg ctgcgccggg gtctccagac agagtgaaga tggaaggact gaqaccggcg CCcagccgct gt cqacct cc CCCtgctccg cqagctcgtg ccccagcggt acccggcggc gaccggccgc ggcggatgat gcacgtcgcc tcctcgcccg tgatgcgttc ccctcaagcq cgacqcgccc gagtctcgta cgatgcqgtc tgccgacctc ccaggttgga cgagggcgac gaqccatgtc ccttgcggag cgtacgactg tgccgtcgaa ccgcgacgca actccccgtc tcttgatgtt agqccggcga 29760 29820 29880 29940 30000 30060 30120 30180 30240 30300 30360 30420 30480 30540 30600 30660 30720 30780 30840 30900 30960 31020 31080 31140 31200 31260 WO 99/37660PCUS9031 PCT/US99/01313 -107cgacggcggt gggtccacag tggcgggggc cgggcacgag cgcgtgcggc acgccgacgg ccgcqtcgcc ccgggaccgc agcggcgtgc aqgccacggt ggaagttgtc acgcgcgcag cgaaggcgaa tqccgcgcac gggcgatggt tgatcqagtc cgcccaggat cgtagagcat ccgcgccgac cgcagaacag gtgagtacag cggccgggcc gccagcgcag tcgccatcgg tgtcqagqac tgaaggcgcc gacqqcgatc atcgatggcg gtactcgcqq gagacggagc gttgt cgaqt gagtgcccgg gccgggtatc gtgcggtgac cacqagqacg gatgctgttg caqggtgagc gatgagcgcg gcccagccac cgacacgaac ggtgagcagc gatgccctgc gagcgggatg cggcgqgcgc gctgcgggCC gtaggcgatg gttccagggc gtagacgacg cacctgcccg caccgcgatg gatcgaaccg gccggccgCg tggagggtgg gtggtgccga atcatctggg atgatccqg tcggctccga atqccgaagg caggtgaagg tgcgctgtcg accaccgtgc acgatcgact aggtcgaacg agcatcqqca ttccaqgcac cgctgggcgt atqaaggtgg tcgtaqagga gacagggtct agcatcggca gcgttct cca gggacctggg acccagccga atccacccga aacagqcqgq qcgagcgcga aagcccgccg tacaggggct cgaqggcggc tgacctgtga tccagatgag acaccgtgcg gctgcatggt cgatgatcag cccagaacag tctccaggag cgaacaqqgt cggtgtaggg ggttcaccag qcagtgcgcc cqatqtcgag gccccagcag acgccqcgcc agttggaqaa cgatcgccgt ccacgaccga gagaggtgtc cgacqqcct c agccctcgcg aggccaggac cggcggggaa acacggtcqt acqtgaacag ggttgcggac cacggagccg ctcggctccg caqqaacgaq ccaccagtcg cgccggqag cgcgaagagc gccgatgccg acgggcgaag cacggccacc ggcgtagtcg cggagtgagc qaagacgacq caggtcggag ccgcgtctgg cagcagjgccg gagggtgatg gccgaagatc qcqcaggacg ggaggcqgcc gacgaacgcc caccgcgccg gagcgggqag gcgggtgcq3 caqgacggcg gt qggtgtag cgactgqtag gtctcaccct gcgaggaaca gcgagcatcg gcgccggcga atcgcgccgt gtgccgcgca acgatcaggc cggaagtcgg gcccccacga aagatcagac agcgaggtga tacagaccga cgggtcgcct aacacgacca tagtccggat ccgqcgqqct agcaqacccq cgcagaqgcc aqcgcgttgg ataccgggca gtcaggaagc atgtagatgg tccagcagaa aagaggag tt cgaqaggq aggatcggta 31320 31380 31440 31500 31560 31620 31680 31740 31800 31860 31920 31980 32040 32100 32160 32220 32280 32340 32400 32460 32520 32580 32640 32700 32760 32820 WO 99/37660 WO 9937660PCT/US99/01313 -108cgacgggggc cacgtccggc tgcgggacgg ggggt gggga gcgcgacgta ggtccgcgcc ccggccggta ggaagt ccag cgcgcggcat gctcgqcqgc ggccagttcc gggccggacg gaaggtgaat ctccagggqc ggctatcacc gccggcgccg cgccgcggct atggacgatg cgccgatcag acagcgacga gaccgtcgac gccgacgcgc cgacctgccc cggccggtgt tggcacccgc ggccgtttcg gaggacgagc gccgaacagg cgcgqgtggc caggggcgtt cqcctcccgc gaqcctgcqc cggcaccagg gaagagacg gacgacgqtg gqgtatcqcg tggcgcgagt agggacaggg tcctcctctc agtacaccca gcccgcactc tagttgcggc tcgtggacgg cggccgggtc gacgacgccg ccccqgtgac cgcgccgcct cgccgggcac acctggtgcc acgccccgaa t ccgqcggcg agtggaccgc acggcggtga cgccggtact gccgggqgtg gggcgggcgg acgcgct ccq cgcatgtcct ccgccctcgg gccgcgtt cg ccctccgcgt acgccatcac ggaggcgcag cqacggggtt cggtgaaggc tqccgat cag ccagcagcgc ccgcgaccac gccccatccg ccttcgaggq tgcaacggac ttccgatgac gctctgggtc cgtcgtccac acatgacagg gctcaccgcc gcaggagcag ggacgCggaa cgaggaacqt gggqcgtgc tctggatggc gggcggctca gcacccgcac gcagactgtc cqaccgccgc ggtgcggggc agtagctcca gtacggcagg cctgagacgq cgtqccqacg gtgggccgat gttgttgcgg ct gt gcct tg gacqagatcg caqttgcgtg cctggccgcc gcgaggacag gcgcacgccc accctcccgc gtcgtcqcca tcacacctgt gacgcgcgcg ccccggaacc cgt cacggcg cagccagtgg cagctcgccc cat gacgact gccggccgcc gqgccgqtac catggcgtcc ctgcccttcg ggtcttcacg ccccagcagt atcccgccgt accgtggcgc cacaggtcqg gcggtgt cqc tggatgcgct gcqgcccagt tggcccgcgg ccccgatggc gggtcgttcc cgtcagcttc ccgccgcccg tccgcctgcg ccgacccccg ctcggcacqc ccacccgccc ggtgacqcat tccggaaacc atggtgacct gcgcgcggga ccgtacgaac ttctcccagc agatggacgc tggcgcacgt gcggagagca acgqacagcg cccccgctgt gccgcaggta ggggt gcgt C cgaggccqtc cctcctcgaa cgaaggactc cgcggctgqa cgcggtaggt cgccctgcgc tgacccgagg qtccgcggca aacccgagct agatcagatc cggcaccgct ccccgqcgaa ggacgcaccc ctcgtcggcc ccgqaaggtg 32880 32940 33000 33060 33120 33180 33240 33300 33360 33420 33480 33540 33600 33660 33720 33780 33840 33900 33960 34020 34080 34140 34200 34260 34320 34380 WO 99/37660PC/S9O31 PCTIUS99/01313 -109accggcctgc tcggccacgt acgaggcggg tcgqt ccagc cggacaccca cgcgcggcga t cqgacgcqq tcaagactga gcgaaggcgc acggacacgg cggqcgagat tcgtcgtcqt cqcggtaggc gtgcagtttg tgccggcgga gagcttgagc cctcgttctg ccggcgaagg cagcttcagg gtcggtcaqa gcgaagagga gtggtcgccc atcacccgga gcgctcttgg caggcggaga ctcctcgtgq gtgtcttgaa gcaggaaggg cggcataacc ccggagqacg ggtaggtgcc gatccagcat agcgagttcg aaaccqgctc cgggaccgag aggagaaggt gagcgtgccg tcatcatcgt gaaaagtcqg ccgggtcgcc ggcacgcctt gatgcagcgc ggtgagagca ccaggacctg cccqacgtag cctgttcgaa tgcactccac actcctcgga ctccttgggc ccgcgtagaa ccatcgtcac tcaggaccgt gccgagccgt acgatcaccg ctgcccgcgc caggcgttcc gagttcatgg ctcaggcacg gccggggcgg ccaacccggc taggccggcc cqccacgtcg accactgagc gccgtttcct cggcggggcc tgaggactt c ctgacgtgcc cgccggaagt gtcctgcctt tcccacggag ggggaggggc gtggaacgcc gagggactgc gagcgggtcg gcgcaggcgc ggcgtcgtgt gacgtcqqgt gccggtgqct tcgtacaagq cgcgccgaga qcctcgggag ccggccatcg gtacggggcc ctgtccgcgc ccgtcgccgg gqcggaacgg aggtcggccc gtgagataqq gactgaccta tcctqgtgag cqtggcccga cggctggaqq tccaccggca cgagcgcctc gtcgagtgat tggctcatcc gactcggaac ttqatgaagc gcqgtgaggg gcgttgtcga ttggagagga gcgtccgagc gtgtccgagt ccggaggcga cqatcqcgcc tqcgctcggt cggcgcaqac ccaccagggt agaacggccc ccagctcgac gccaggtcat ccggggagct agtcctccgc tqgctgctcg ccgggtcgtc cgcggtggtc tagtcgtagc ccaatgccaa ccactcagtt aatgcatcga gcggcgttcg acctccccct ccggcgtccc agtcccgggg cgt cctcggc agt cgggcgg tttcgqtgaa ggtggtqgcc tgagcaqqag tggtgtccac qqtgttcgcc gagagcgcgg ggqcctgatc gccgtcqacc cggctcggtc cacqtcggtg ctgacggccc gaacatgtcg gtccgggtcg accgaaccgt qagtgcgggg gaagggtgqc ccttgtcacc agcgctctcg caggcagatt gcqgcgactt gaccgtcacc cctcgqccca gctcgcgaag ttgggcgacg ttcgcgtgaa gt agagcgag ccctgcctgg cqgtgttccg ggaggaaa gacgt cggcg 34440 34500 34560 34620 34680 34740 34800 34860 34920 34980 35040 35100 35160 35220 35280 35340 35400 35460 35520 35580 35640 35700 35760 35820 35880 35940 WO 99/37660 WO 9937660PCTIUS99/01313 -110tcggttgccg tcgacgcqqt gtcaggtcgc acccgttggg aggcggatgg ttgtcccatc accqcgtctc atgttgaggt atcqggaagg cctgggacat gtgaagtcga cgacctggac tggacggcga t cggccccgq acgacctcat t caccagccc aggagaccga tcatcggcag gqttcgtctc ggctcgacac cczqgccgccc tqctcgcccg agcaggtgta agtggatgcc t gcccggcct acgaatgacg acagcaggtc cqgtgtgttc agqcgaaggc cgqcggcgag ccagggttcg tcatggtgtc tacgctatcg gcccaacccc tcgtgccttt cqtgggcggt ggaqgagacc gggggacgac cctggaccac cgatctcacc agccggtgcc ggaccggctg ccgcctgacc cgcggaccgc ggtgctggac cctgcacaac gaccacgctq cctcactccc t ccqcactgc cqccgacgcc gaccctggac acgagtcgat cggcccggtg cgcgacgccg gtgcacccgc ccggcgtgcc tccgaagtcc gttccccagt gtcatggtcg qgtgcggacg gcccagaagc catgtcgagg gqctggcqcc ggcggcggcC ccqaggctgg cgcct caagg gttqccgact cgcgagctct gaggagacco gagggccggg gagcagaccc gtgctggaga cggatcaacg gtcggaaagc ccgaagtcac cagccqagca cggatcgagg gagcgccgat aggtagcggg gaggcgaagt tggtgtccgc gagaqgaaga Cggccggcgg cgccgtttcg ctcacgaacg aggtctggac qgagccggga agggcgaqac tgacccgtgt tgcgtcacga aatggtccaa agaaccgcgg cgcctttcga acccgcagcc gggcgctgat cggacgtgac tggtgatccc ccggacgcct gacgcgcctg cgccggtcac tgatgcgcgc gcgccgaggt atgccgaacg gagccgatga agccgttgtt cqcgqatcga ggt cgcggat cct ccgcgcc ccgtgatgac tggatcgqgt gtcgttcacg cgtcggcgcg ccgtcgcctg qcttctggag gcggcggttc ggccgactac gcact ccgcc accaqgggag cttgctccgg gaacccgaac cggctgttcg gccacgtgqc cgacgcgacc ggggctgctc tgtttcggcc cgcgctggtg cgacgcctgg gacccttgcg ggagtcgctg gacccgacgg gacgagtacg ggcaggatcc ctcgtcgcgg gaggtccgcq gacgcggtgg ggtgccgtgc gtcaatgatg gtcatcctca ttccccgggg gccctcgcqc ctcggcacca ctggtcgaqg tacgactggt tacctgatcc gcggacgccc tcgctgcqca tcactggtgt ggcccggczg ggcaacaaac ttcctcgtcc cgcccggaqc gtgcaggtcg cgacccqagc cagctgaacc cgcctgcggt gatccqacgg 36000 36060 36120 36180 36240 36300 36360 36420 36480 36540 36600 36660 36720 36780 36840 36900 36960 37020 37080 37140 37200 37260 37320 37380 37440 37500 WO 99/37660 WO 9937660PCTIUS99/01313
-III-
gtcggcgtCC cgcgacggtc aggtgtacag ggccggactg cgccccagca acttgtcgcc cgctgtggca cgaggcagcg agtcgccgcc tgttgaacag cggtgacgcc aqqccggggc agttctgcag ggaagccgac tggcctgggt cggacgggtc gttgcaggtt cgtcccactg tcgcctggcg aggccagggt cgqoggagct gcggcgagca agtactgtcc ctggatcagg gacgccqacg qctgtagatc gtagaccctg gtcccacaqc gcccqaaccg gttgagtgcg caacggcgtc gaggcagcgc gccctggatg tgtggtgcgg gcagtcgatc cttggcccag ggcggcgcgc ggagtcccgc qacgatcttg cagcgcgctg gtggccgcgc gaagttgaac gaccggtcgc ggtcaggtgc gtgccgttgg acggacccgt tggaccttgg agctcgcccg tggagctggg acgccct tga tcgaqgacgg tgctccgacc gagacggcca tccaqctcgg aagttgctgt ggcacqccgc gtccagttct gcggtgcgga cgcgct cccq cccttgtagt ccqctgaggc acctgcttgc tggccccgct gaaggtcacc gggtccagcg ccgtgccqtt cggaqttgag aqccgttgcc cqtcagtqgc tgccgtcgqa tctgtccccc cggtgtacqc gccagqagtc ggcaqtcgtt tgatggccac tgtaggggct gctgcttgaa cgacgttqta aggcgacctc aactgccqtc gggccatcac tctgcatcca cqttctgcac 9c99 gcccgcacgg ttggttgctg cccgacggcq gcgccacttc ggtgcctqcg qqcccactgc ggtgctqgcg gtccgcqgggg ggcCttcttg gctgtcgcgc ggtcacgttg gtcgacgccg gccgctgttg gtcccgcacc gtcgttgtag gatccagtcg qgcgaaggcc gccgttgatg gccgggctgc cgcccagttg cggcgaccct ccgttggagc tcgaggcaga tggttgtcgC gcgtccaggc tggttgqtgc tcgggcacgt qgct ccgagq ctgccgtcgt acaccccaga gcgtaggt cg agggcggcga aagtgcgact atgttgtaca cagagcttgg ttgccgctgc tcgttcacga tggtcgatca tgggagtgcc tagacgcggt 37560 37620 37680 37740 37800 37860 37920 37980 38040 38100 38160 38220 38280 38340 38400 38460 38520 38580 38640 38700 38734 <210> 31 <211> 3331 <212> DNA <213> Unknown <220> <223> Description of Unknown Organisn:Unknown WO 99/37660 PT1S90 1 PCT/US99/01313 -112- <400> 31 tcggatctcc ggtggttctc accagtatta tgccatggtt t ta cqgtgt c cggacgcggc tgatgaatgg tggtggtgcc tgctggactc tcttggcgga agctcttgcc cgctgctgcc cgct qcagga tgccgctgca tggcggactt tgccqctgct ctacggtgga tgccgctgct aggaggaggc agcttctaga gagtaatgga tcaaattgac atctgcttca cggaggtggt tgctgcagga ccacacaaca accaccctcg ggcttcaatt tatgatccat qgagggggcg ggcggcggtg gactgggatg ggaggtggtg qgacttggat cttggcgqtg ctcggtggag gccgggggtg ggaggcgcag ggaggcgcag ggaggcctcg gctgctgccg cgaggaggta gcagctgccg ggaggcgctg caaatgagtg gcctctgcaa gatttgaagq gcatctgcat aacgqaggag gccqgaggtg tagatagaqg tgggctt tag cacagtatat gggcatgtgg gaggcgcctg gagattggga atgacggtgg ctggtgctgg tgggcqgagg gagacgattt ctggtggagc gagttggtgg gtagacttgg gtggacttgg gaggtcttgg ccgctgctqc gacgcggtcg ccgcaqccgc gtgctqccgc gtataaggga aagcctctgd atgtcttaaa ctgcatcagc gaqgcggcgg gacttgqagg atatccgcct ctcagcacta gctgqgagga ttgcgtttcg gggagctggc atatgactat aatqggagct tgctggagca tctcgqaggt atttgattta tggaggtqct agctgctgcc aqgagctgct aggactcggt tggcctcgga cggagqtgga aggaagagga tggtggtggc tgccgctgca cgcattagga taaagcatca ggatcttgca ttcagctgga tqgagctgga tggaggcqga gggttcacaa agtttcgtt ctaaqacttt gcatggagca ggtgctqgag gatgacgaca ggcgccggag ggcgcaggag ggacttggcg gatttcqatg gctgctgctg gcagccgcag gctgcagccg ggcggacttg ggatatggag ggactcggtg gqccgcagac ggaggaggtg gccgctgctg gacattaaag gcagtagcaa ggtctattga ggtggaggcg gctctagctg ggcggagctt tgaagtt act gtaattacag tctgtatgcc gtgcaggtct gagccgacgg gcgatgacga gtggtgctgg caggagcaqg gacttggagg atcttggtgc ctgcagctqc ccgctqctgc cagccgctgc gaggactcgg gatctgctgc gtgttggttt gtqctgctgc gaggtggtgg catctgcttc accttctcaq gcacaaaatc aaagct cagc gtggtggtaa ctgctctcgc tagccgctgc 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 actaqctgct gctggtgcag gtqgaggagg ttttggtgga cttggaggac taggcggtct 1560 PCTIUS99/01 313 WO 99/37660 -113tggtggggga tctqccqcag ctgctgcagc aagaqcactt tgctgctgct cggaqt aggt tgctgccgct agqacgtgga tgctgctgct tatgtacggt aaatggagqt tggaggtggc tggcggatca cgatgatgac tccgccattt tagtttgatt attagttqac atatqttcag agaaggqctt gctgcagctg ctcggtqgag gctgcagccg gqcgatggcq gctggaggat gacggtgctq ggcggatctg ggtgatctg gqcggcggtg tgtgatgaat gactcatttt gaataaatta ctgtaaactt tattgtggtz gagaaqaca :tgctggag ;attcggag ::cgctggatt acggtaacg ctgctgctga atggacctga gtggtggcg gtggatcagg gaaacaatgg atggtaaccc tcttagttct t gttt t cga t tttcatggag aagcacctgt caaaatcttc ataatatcct acctgatctt attgagaagt aatgataacl aatqttctc' caagtatca, taacaatgc attaatgca cgctgccgct aatgcgtgga tggatgga aggatttggt tggtgqaggt agctagtgct tgttgccgct tttcgataat atccggcgga cgqtggcgc atggqgaaat tattaqaag ctatgtttta ataaattttt ttataatcta accgcaaaca -tacactctca *gaacatgaac -tcactgaaaz -tttctataaw -tacattaatl Sctaactcacl a ggtttcaca, c tcataaata c cattattgc gctgcatcag ggtggatccg qgtggaatqg ggtggatcat ggacgaagag qtagctgcaq gccgctgctg ggattcggtg ggtggatccg ggatctgqtg aacggcaaca gggtaaatta tacttcacct tttaaattaa aggaacaaaa caatcacctc cctacacact Itttgtqttga itttacgacaa iaaacaatcgq :aaggccccaa a cctctcatgc c ataaaactai a ttttctaaai qtggtggagq 1620 ctgctqccgc 1680 gtggaqgatt 1740 cagcagcaqc 1800 gtaqaggtag 1860 ccgccqccqc 1920 caqccqcagc 1980 gtggaaacgg 2040 gtgqcgqatc 2100 qttcaqqcgg 2160 ataaatatga 2220 tttqacatta 2280 taqattgttt 2340 attaaacttt 2400 aacatacata 2460 tatacatgta 2520 cgcacacaqt 2580 taaaaagttc 2640 gtattgaaaa 2700 aacaatgact 2760 ktaatctctct 2820 1tggaaattca 2880 -cttcatggca 2940 a ctaacaaaat 3000 a caaagcatac 3060 tacaaaatca gtaatgctga cctcttacat agaaaaatct tagcagaaag ggaatgacaa attttcaaac catactatac gaccatgctg aggtcagtct acacagcact cccctacatc aaaacgggaa ggatgaaaat ttttttttca taaattacca ggtatttgtc gtaacaaatt actggatatt ggcagacaaa atgttqttat tggatacctt tccattctat ctagacactt 3120 WO 99/37660 PCTIUS99/01313 -114gctttccaca agtcatcata aataaatccc ccctatccca aatgtcaatg gaatgcccca 3180 acccttcccc cataatttta aaacctagaa taaattaaaa catctatagt tcgtcatgat 3240 catctttctt atcatcctct tcttcttcct cctcctcctt cttcttcttc ctcctcctca 3300 ggttcttggc tgcctgctcc ttccttgcca a 3331 <210> 32 <211> 5224 <212> DNA <213> Unknown <220> <223> Description of Unknown Orqanism:Ulklowf <400> 32 ggatcccctq ctcgacqccg gcggcccggt acacctccat cgtgcggacg ttgttcccgc gcccgcgaqg gccggcccag gaaccctgtg gcqtgatqtc agcgcggtgC cggcacgccc ttaccgttcc cccggaccgt gtggacgqag gaacacggac gaccgtgtcc cgcccactcg gccggggacc gacgatcgtg tccaggaaga qtccccctca gtgccggcgt gacgctccac cagcatgtgc cqcaccccga 120 gtcgacagqc ccggcccgat gccggtttcg acacttcacc ctcacggcc ggtgcgcctc ccgtgttcga aagcggaggt acatcagcga agtqggagag tccgtcccct agacggccgz ccacgagcga tccgccgacg acqaqgaccq 180 cggctcttgc gccggggtca cgcttaaccc gacqcaccqt aatgacggtC cggttcccqg cctggacgcc ccagctgtgc gttcatcttt tgcatgcccc gctgcgttcg catgtgacgc tctqccgctg gtgcgggtcg tacgagcgga cagtcgctgq z:tcctccagc 240 gcgaggctgg 300 cgcaggqgca 360 gtcggccgcc 420 tgtccacgqt 480 tgctgatgct 540 tgtccgaccg 600 agaaccccac 660 gggccaacag 720 cccactcqat 780 cggcgqcccg 840 ccgtcaccta 900 ggacatccgc gacgggacgc ggtcgccgcc ccagtggctc cgaggaacac gcgctactcg gtgcCggggC atcaccagcg ctggagatgg gtgctqgCg cqcaccggag ttcctcgcct ggagccctac ggagcgggcc gtccqcgqca qtccggcacg gggcgcgctg cagcccggc cgcgcatcgg cgacaacgtg caccgtcaag cccqacagq tcaccgaqgt cgtqaaqatc ctctccgcct acacctcqcc 960 cgaggtgcgc gtqgacgaca ccacgcggct cgtgcgcacc tccctcttcc tgtacggcaa 1020 PCT/US99/01 313 WO 99/37660 -115ccgggtcgtc cgggcgatcg agqtgcgggg cgacctgcag gccgccctgc gccacqtggc 1080 ccgtcgagga agccctcacc ccgcacatcg aacaggaccg 1140 ccggcagcc ggacctcacc ccaccacgtg cccggcccac caccgtggqc cgtcgtacgg cctgcacgag cgccgacggc gcctctgaca tgaccyacat ggcgcqgg gcttcatgq actccgaggz cgcatcccci gaggtgcgcg gacccgcggt gtgtcCgggC cccggcgCCg gacccgggca ctcgtcgaca ccggacgccc cccccggacg gaccgtcagt gacagaacag Icqgcgacgtc fcgtggccgtc gttcgtgtaC gcgcaccgg( ccgcccggct qcgggacgg gaccggcgct qtccggtcgt cggcqgqgC tcgccgaggc accqgggcgct cgccggcctc caggcacgca cgggccctgc qtacggccc'gtcaccjccc Scagggcctg( gttcttCacc cggcgacacg cgcccgqctg cgcctgcaCC accqgaqgcg cgggcgqctg gccgacgttc ctgacccccc tcgtcgcctt tcacgcccac Igagaacgcat cgggCCgCqC cagcggtgcg ctqgCgCqgc gtcttcacc gcgcCCgggg ctggacgccg ctcgcgcacg gct cgcccga cgacgacgtc gaccgcggg ctccgagcac tgccggccgt cgctgtacta agggcgaggc gcgacgacct ccgtcctggc ccgcgctcgg cccggatgcg cctcagggag ccgcccaacc gcgaccaqg taccacccgt gacgacgtgc 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 gcgccttcga ggtggacctg tcatccccaa ggacgtgcgc caccgcttcc 1920 gcaacaccgg cgacgtcgag gcgcgcctcg cggacctcgq catgagccgc ggcccggttc gcacgtcgac cqqgt cgt cq tgggacctgc accgaggaga tcaccggcat tggccggCgg tcttccacct ccgacgagac aggcgtcgtc gcgtacggcg qggtccgctg cgcgCCggCC qccCCgggCg acgcgccgga gccCCCCggC ggggtggtgt gcatcggcgc tctccctgtt cgtccgcgca 2040 2100 2160 2220 cgacccggcg cgcctgcgct cgcagatcgc cgccgaqtgc gacttcgacc cggcctggac caccqccgag qggggccgtc qgtcagcgag ccgcgccttc cccggtgcag gacgagacg gcggtCCgCg ctcggcacgg qgcggctcgc gcccccagca accgtctcca tccggcggtg acgcgggcct cqgtCggCg gctgggacqt cqctcgcct c ccggctgcac cgaccggtac ggacaccacg caccacccgc ggaccaccgq caccgtcgcc gtccgggctg gtgcagttcg cgcgaggacc ctggagcacg cgggccgagc gagaccttcg gacgcggtgg cgctgqtCgC cctggcgcat actacgtCct cgcacctgca qcgcgca qq ggtacgccta 2280 2340 2400 2460 2520 2580 PCT/US99/01313 WO 99/37660 -116ccacgccatc atcgccgatc cccggagcac cggcgcggtg ctgcgagctc ggagggcctg cacggacatc gaccgcggcg caagtcgatg cctcgccctg gtgcgacctg ggtgggcagc gctgagatga ggcatcggtg atcgaccggg gacccggccg gcgatggcgg tcgccgtact cgcgagctgc gcctggttct tgcggcgtg gcggtacggc tactcgatg( gcctaccgti ttcgtcctg, gccgagggcc accatggcqt gcctcccgcc ctcgtgctqg gccggctacg gagatggcgc gactacgtca qt caaqcggt gtgggccact gcgcaccagg gactacqtgc ggcttcggcg gccaccccg ccgagacqtt agggctgcq ccctgatcge ccacccagct cqgtgggCgt agaaactgtS.
acgcggcga( jtqgccgccg.
gcggcaccg, j tctqccagc ccttcacct g agqaggagg gtgccgacgt gcttcgacgc ccttcgacgc aggagctgqa ccaccttcgq gggccatcga acgcgcacgg cgctgggcqa cgctcggCgC tggtgCCqCC cgcgcgaggc gcttccagtc gcgagccgtc ctggaagacg ccacctgcc ggacacctac cqccctcgaC ggtgacggcC I gggccaggg( 3 caccggcca( a cgaggccgq, ccaccgtcgt t gggttaccc gtgcctggcg catcaaqgg ccgccgcaac gcacgcccgg caacqcccac caccgcgctg ctccggcacc gcacgcgtac gatcggctcg cacggccaac acgcgagcgg cgcggtcgtg gtcaccgqac *gccgtggacg ctgaagatcg ctcgtccaga -gacgcccgqc -gcgggctccc -tcgaagtacc.
SatCtccatc( c ggcctggac( c gccggcqgg g gagctcagc gcgcctcgg acctcgccca qggttcgtga gcccqgqCgg cacatgaccg gacatggccc cagcagaacg cggaccccga atcgaggtcg tacqaqacac gagctgcgca ctgaccggac tcqgagtggt gcaccagcag ccggccaggt ccgaccgctt tctcccgcgc igcqgcggCga Itcggcccctz gcqgcggctt 3 ccctcgcgci a ccgagqccc( c gcaqcgccg.
c ccgccgagg actcgccgat 2 gcaacgacga 2 tgggcgaggg 2 cggacgtcta 2 ggctcacccg 2 gcctqgacgg2 accggcacga tgagctcgattcgcctgcqt cggaccccga gcgtgctgtc cggagaggag qqcaccccac cctggcccgg ccccqacttc cacccacttc cgacatcgac qttcggccag iccagtcgatc :caagggcccc a cgccgcgCtg :gctggCCCCg a cccqggccgg g cgggqcqatg g ggcgacgqtg 640 700 :760 820 ~880 ?940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 cgccgcctgc gggttcgtq cgcggcacgc gagcgcggcg ccgacgcgc gccggccacg cggccacgtt caccggcgcc tcccgctgqg aggagtccag ggccgqcctg 4140 WO 99/37660 PCTIUS99/01313 -117gcgcacgcga tcggcacgqc gctgqgcqcg gccggctgcc gtccqcagqa cgtggacgtc 4200 gtqttcgccg a gacgcgctcg cgggcgttct gagctqatcc aacaactcg acaggtgtct ccgacatqac agcqcaccgc tcgacgqctt agcacggcgt cgcaqgtgaa atcgtcatcg ccgcqgctgt cgcttccggc ~cgccctcgg C Icccgcacgc jcgCggccgC cgcccacccc gtcccgcccg cgctcgtCCt cacgtcccct cgaacgcgtg gggcgtgcac cggcctqgac gggactgccg cgccaccctc ccgcgccgct tcagcgagta tcaccatgca ~gtgccggag gccgaccggg ccgaggccct ~cqgcgggtc c gtgctcgac :catgtgctc gccgcgcacc gcgcagggqc tcgggCgCg ggcacccag qtggaaccgc tccctgggcc gagcaggtgg aggacggcgg gqacctggt c cgcctcCgCC cccggacgac ccgtcaccg ccccqaaggc ~tggcgaccg 3acgtctgcc gcgctggtgC gccgcgccgt gcacccgagt tgaccttcqa ctgacctgcg tgctgggcat agcgctgcaa tgtgacatgg tgggacatga gagatcctgg cgctgctcgC acgacctgga tcagccgcgq tccccgagta caaggagctc ggaactgtcc ggcgcggqCC cgtggccgag gacgcccgcg ccgggcacac ccaacgacgt agcgcgaggg jgccctggcc4 gggcatcggc catggagcac zctggtggtC actcatgggc agtaccccga aaccacatga gccctqatga gaggagggct ctggagaaga gagttcctcg cgagaacgag cgagaactgg cgaccgcgtc qgtctccgaa cggccccttc 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5224 gaggccggg tgtggagctg cgcgtcgccg accgcgcctc cctgacggtc cgcgcccacc gcgtqgagac cagttcatgg atcc acatccagtg gqtgtacgag cagacgcccg agggcgtgct gatgcgctgg <210> 33 <211> 30601 <212> DNA <213> Unknown <220> <223> Description of Unknown Organism:UlklOWf <400> 33 gatcttagac cttattcact tgatacqtgt aataqttatt acgatagtat gtttttqqcc gattcctccg cgtcttcttt cgacgacgtg qagqtggagg caaaagcqaa gtagttgtgg 120 PCT[US99/01313 WO 99/37660 -118aagaataaga cgctccatgc aagcggaaqa ttttgttgtg taatctctga ttttgcggcg ttcaatgatg gagtqgtgga gcaagaagac gcactgaaca ccaatctcca gcaatttctt ccgaccaccg acaagtagct acaatttctt aacatqatct ggctgtccat tactctgggC ctgaaatgal attcatcaa( ctgatccac, gaagatggc cagattgtt cagaacact ttacagcac attatgatta agacgtttgc cqtcgtattt ataatgagat gcactcgata ttgaactccc ttctaagaac catagttctt gaagtgcaag atcgtacatt ataatccagg gcacttgttg t cttccqcac ggacactctt gttgatactt *cggcacttgz *ggtccggtts -ccatcacaa( -attttcgtt, aatagttcc a agtgactga a agtttgact gcgqtatat g aaacqtatt t ccaatcaqt tcatgattat caggagacga gaattcgaag cggcatgqag tcgtcttgtt ccgccacacg ttacgtgagt cattgcattt atacactgcg agtaaattct atccaatatg gtctcttttg tgacgcatgt cccattccgc ccttatccaE igaagacgacc.
Scccatcggai Statcttttt( aagattaaa.
t tcagatcca g caaggqgac a gtttctggc g cattcggtt t atggtattc.
tattcaaatt cgggtqaag atgataatga gcatttcaca tacgaccgga atacagaaca ccatgqagat atcaaacatc acgacgccat aaatctgaaa ttcttctccc cacatcacaa aacatttcta cattttccag iatgattgcae I ttgaqtaccz tggtggtacc acaagtcttt ai gcaaactga gagtacacg.
aatgacttg a ttttqqtat g tacgttccc a caacagCaa tc at al a t t
C
rt t1 t.
C
g ctctattg ttacqtaccg 1 ~taggaagc gaagaagcgg 2 ;tcattqat gctgatgatg 3 :ctctttqt tcqcgaqgct 3 jgcacgtct tcacaccaga 4 :ggaattat tattagaaqc 4 tctgccaag aattatttqt 5 tqgttttc tggtttcatc ccccaccac aaqaaqcaga ttatatatc ccacttttga ttggacaqg gttcaagtcg caacatctt tcaaaatcgt tttgttgaa catgagttcc atgaacaat cacgaagg *actcatcag gaagatcaCg :taccacaag ttqctgaaca :cgqcttgaa gtttttgaag :ttaggcgtg gacgagtatt acgtactcga tcacaaaaat aacacttcta gtctgaattc tttccaagat gtacatggca tccacaaaaa gaagcatcaa.
atgaccgatt ccacaagata tctggagtat ttqaataaac 00 300 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 tttctccaaa atgggCaagt gcctaaatta catgtctqat 1620 gagaagcagg ccgatcaqat gcagtaccac aaagtgacat atcgacttca gttccatttc 1680 WO 99/37660 PCTIUS99/01313 -119caqaaacaca accattctcc tttccatttt cqtctcgata gccaccaagt atgtatgtgt catcacaact tgttacattc tgaaactctt aagtttatat aaggcaacga ctgtacacca acaagcatgt ttcccgatcg tcgagagcac ccgttctgtc gacgattcga atcatcaacg tttcaqaaca aactq-.caat gacgatcacc tggcactaca aaatctaatg cttgttttg tgtccagaca aagtatccgt gaacagccgc catgaacata cagaaccaga cttggaaatg ataaagattt aaaaacactt cctttctgcc ccaaaggttc tgaagtccat atcggatggt ctccattttt actagacaca aa a atat gt a tttgagaaca cattaatttt aactctccac agtcatgtac aatgtcactt atattcgaga accgttaagt tcatgggtat tgtatagaaa gqatatcaat cttgctcatt cttqacgacc :ttggacatg tgttccggta tctccacact tcattacaat gctgcatcca tctgaccaat gcctctttct caatatgact tctccatagt tttgaattac acgcaagaaa tcttcagtga gtttaccttg ttttgacaat tgcgattgct aaatacaatg tcatatgaga ctaatcggac acagtgtctt aggaattcaLc tgataacaca ctcacaaaqa atttgcagaa aaatgttcct atccatcctc attctctatg gtactgatct tagctgagca gttcttgtgt aacattgagc gcccataaac gttctctgcc tccgatgcat tccattttct gatcacatag ctcgttgacg tccatctgta tagaacaatc ttctatcatg gtccacgaca cactggatat tgtcqggtgg cagttccatt ggaatgcaat cattggcggc agggtgagtg taattgtcat ccagctggaa ttgacaacct icaagaaaca c icatggtttt itgacgatcg gtcagaccagE ctgtgctgga atgacgcatt ccatcttgqa gcacaatgacI ttttccattc tccagagcag atgttcqgat tttctgtcca aatataaaaa tatattgcac actttgacga ggctctatca tggccgaatt atgactacca tatccgttca ttgttggcga acggagaqct atgaaaaaag cttctttcat tcttcattac tacaggaaga ~tacactgag 1740 ;taattatct 1800 ;tacaattag 1860 ictccgaact 1920 :cacatctgg 1980 :tqactccac 2040 catqgaattc 2100 :catcaactc 2160 gatccgcaaq 2220 tcaatgtaac 2280 gctqgagttt 2340 gatccacatt 2400 tttattatag 2460 attcgtgttg 2520 tgagtcqaca 2580 cattctgtcc 2640 acagcatttg 2700 acagaaagaa 2760 ataattgcat 2820 gccagtgata 2880 qaaggttaaa 2940 taaatgttcc 3000 attattatat 3060 ttcgttataa 3120 atcatcccca 3180 ccgcaaacac cacatttgtc tcttcggaqa gttgaatgaa gttgatgatc acagcctgaa 3240 WO 99/37660 PCTIUS99/01313 -120aatccactta ctccagctac ctcgaagcag tt t tca at tt acaaatatcg atagaacgct ttggtgcaac attcgcatat ttccaatatc atgggcattc caccatttcg tagccagtgt ataaccggct gaaaagcctt atttaagaqt cctcgttttc ttcagactat cttaatcctt tcaccccatt tgatgacaga actcaccatg tcgacagccc tttattgttg ttgtgtatta agqtctaaag ggcctaaaag acttcaggtg aaactatctc gatcagaagc acttctgaac ttgatatcat tttgaacacc gtccgtcaat actaaatatc cttcttgatt atctggcttc tcttttgaaa tctccatttc gcagatccac tttggaaccc aattcagaac catgaccgat taatatqqga ctggaaqttt attctataaa gaagcggccc actccatacc tcgccgattg ttcqtttggC accaccacaa tttggtaag caagttttta cgtcacaagq cgtaqaatqt ttaaagaatz i tacaaactt( atcctcgta( Ek atcgttcga( a acqttqgtg t ggaacttqc g gaatcgaaa tcagatcaat tatcacatg tgagccgaca agtgagtatt attqaacttc aacgttctcg aaatgtcatt ttttaaagat ttttataaaq gaaagcccag ttgatqttac aaaatatttt agtttaactc gtacgactgc gattcggggg tgagtttctt fagttccqtcg itgcacaccaa itatttaggca 4cactgttgat j taacgacgtt a aattgacaat g cattccagct c cattatgtg a aaccttatti a cacatgacc, gttacctgct tgttccatca atacaqcttg cgttgaagcg acggtatggt ttgaccaaca ttaatattga gcgttttcta cttggaagct aaaagcagag atttgatttt gattgaatat actttgggct attctcccca ctagacgaac ttgtgattaa gcccatggca agacggcqqc aactaaccat gagcatcgta cgaccggctc taaatagaat qaacatggcc a accqgcatgl aattcatgac ggcatacaag 3300 acaactttat 3360 caacqttcat 3420 acaccttqqa 3480 tqaqtatccc 3540 cagtactttc 3600 taggaaagct 3660 tcaatttaaq 3720 aqgtaaatct 3780 acggacaaac 3840 agtgtttcca 3900 attatttqct 3960 gtcacaatct 4020 tgatcgccaq 4080 acaggctcca 4140 tttctgagat 4200 tatqctqaqt 4260 atgtcggctg 4320 ataagggcac 4380 tttctttcct 4440 atcgaataga 4500 caaaatttta 4560 accaactcca 4620 qaaactgcgt 4680 aqgtagagca 4740 3 caattgtgaa 4800 taaaatatca atgtttcatc aactcagaag ggttcatcac cattgagttt atagctatct tqgatgagtg tttatcaaat ttttcgttcg ctqgtccaa gtacaaaga( gacctcgatl tactcgaqgg ttatattcc t t aqtgct t tcatcatga WO 99/37660 WO 9937660PCTIUS99/01313 -121tgcagoactc tccaagttca cgtcaaaagg ctgctgccat agcgttttcc ggattggtga agatcttccg atcccaagag tttoataaaa agctccactt aatggggatg tatctgtttc aatccattqt goaagtccaa attgcaacgt ctacagaaat gt tt toa aa a cgatagatgg tgatattogt ttggcagctc ttatttaaac tgaootatct caagactaat attgattcaa cataacataq aacagcctgq cttctatgat gtgtatcqca catgatqttg cttgaagtgt cgatcaactt aggcaactgt acatttttgt ttcgggatct atttttttac ttgttgtcat aaaaggtatt ttttcagatg cagcccgtga gatocattat gcaaaattct gatgggatca tottttttct atgcatccgc ttttcccgga ctgcgcaatc ctctgtotqt tottgtgtct agcgccagtt ct tgt totga aagaatctag tgoaoaaott ttttoottgt gaoaotoqaa ttgttgagog gaoaaoaaog ggagaagaga atoogcoaoo tgotttgogt tgtatootto tgaototato ggtaaotggt tttatataao aaaggtgctg ootttgotot atagtttotg goagaatotg aoatttttta otaotoaaaa cgtaogoaao ttttoootgo otoooaooao tototgaoot gaaaaaaata oataaaoooa aoaaatttca ttttgcatat gatogacaaa toatotggat t totgagt ga aoattgatag gtgagaaogt aoaaggaott totataatta otattaatot aaaaactgtt tooagtagag oatottaaoo aaatcooagt tttaottoqt tgtgtgttag agatottttg oagaaotago agtogttttt aoaaaaoogo agctototot otaaatoato ttaoaogtcg qtgtgtooaa aaaaaaogaa gaagttctta oaoaoattgt 4860 tatotttaog 4920 oattgtaata 4980 ttogtggtoo 5040 atgcaogaag 5100 agtottoaag 5160 oaaoatagtg 5220 aatoottttg 5280 tqoaooooag 5340 oaaotatgaa 5400 otttttttao 5460 gttoaaatot 5520 aatttoaatg 5580 taaotaooat 5640 qgtgttttaa 5700 ggaaooggaa 5760 aggtaaaott 5820 aaaatgattg 5880 ttgogoqagq 5940 ototoaottt 6000 atoagoatca 6060 actagggaaa 6120 atgagoagtt 6180 otaggoggoa 6240 oocaataaoat 6300 goattogaco taqotatagg tgggotatta aaaaatattg toagattgaa tttotttgtg atgtaoaoaa ttotaotooa toaoagtooo agototoaag togaatttga aaagagatot aoaoaoaagt ggaoooaoga oaoaacaooo tgataacttc atotogaaaa tttgtootot aacgtacqtt taaatoagtt oooaooaagt oagaoaotog aoogtttgtt ggataatcta ottoooaotg atottgooao gtggoaaoog togtotoogt otoqttgaoa otggtogagt 6360 WO 99/37660 PCT/US99/0131 3 -122taagatggtc ggcgacttgc tctacctgtt aagcattccg cgtcttccga ggtagataca aatataagac aaattaatac ttgattccat acaagcatgt gacatcacgt gacgtgtata gaacattatt agcacatctg aaaacaaaag aggcagacga ccgatggagc agaaaaaaga cttccaaaaa tgatcttttc gtggaatgac tagaatgcae tccgatttgC acaccgttaE ctttaaatt( aaacgatttg tccgtcgtgc ccgtttccac tactgaaaaa gtcgcataaa atcgggatct catcgqgcg qactqttata ctgtgcaaat ccacagtctc tttccacgac ccggcgtctt tcacttcaaa atgagaattc ctqataaatt cgaggagtat gcataccaac gatggaaaga ctacctgctc ataatctcc fcacttgcaat igcaatttttE -gcagaataa agtctgagcc( -ataaaattc, aagtqcattg tctagatgtt taataattgg ggcttttatt ttgacgatgc gttcgagaca aaatttgaat ataaaattga gaacgatgta tggcaactcc gtccataaga gacgczcatcg actttttgga tgagatcgtg aaaataatta gagagcggcg *agctccgtga *agcagaaqct gaggtgaaq gagtttgtgt agggaacagE iaatttgtttt a taaaaactt(.
acctttcgc caattttaai gatcgaactt taaagtgtca ctcaaccgta caccaaaatt tgtgctqatq ttccacctct tggaaaagtg aatctcatac gacggcat ca ttggagtcga atctcttcgg actgtgatgc ttgttacctg ccactccctg gataaaaacg acgacgggct tgatqattat ccgataaagt gaagtcgtct *tttcgtttag i catcacaga *aaaatcactt 3actqtaatat tttttttgtt a aacatctaaE tcgqacgaga qagaaagtga tggattccgc cgaacttata tacaccttta aacctcctc gqaacactt ttgttatgtg tctgatcgta cgtcgccqat acgatgtgat actggcacac agtacttqgc ctgaaaggaa aacattgcaa gcagcagatg gattgtgtgg tcqtccgtct gattgaactg tcgaattaaa ctgaaaaatt *attctgacgc *tgggaaaatt gacgaaaaaa gtcaattcct :gttgcctat 6420 :tacaaagtt 6480 :gggtaqtgc 6540 caaaccaatc 6600 acgtqtqcac 6660 tccgagtcca 6720 ctggaattga 6780 aqtccggtat 6840 atcgtaaqtq 6900 ctgttqacgt 6960 ggctgtcgat 7020 ctqaaactta 7080 cctqqagaac 7140 catttgqtta 7200 cgcataaacg 7260 gaatqagccq 7320 aqagcagcaa 7380 cttctgaaac 7440 ctactgcttc 7500 attgtagatt 7560 aaaaattatc 7620 catcttcttt 7680 tcgaaaaaaa 7740 accaaacaag 7800 cccaataatg 7860 catttgtata tgaacaaaag tctgttgacc ataaqtcgtt atattactac aagcaattgg 7920 WO 99/37660 PCTIUS99/0 1313 -123tcatcaacaa acctcataaa aatcatttt gaacgggagc aatttatata aactctgtgt 7980 gctcttttgc ctcttgacga ggtaggagct gatgaaggtg aagaagaaga aaatagtaat gatgtgtatc ccacccccga cctactaatg aatgacutggt gtctcttgtg caaggtaagg tttgaaaaat gccctaattt ccttcttaaa aagaacaggg caaaatggaa aat cat cacE aatctcagtE gggtggttcE atgttgct gttatctggl tctttttctt aatctgtaaa ctcaagctgc tcataaaaca aaaaggagat aacaatgaac tgagqggatc ctagatqatt atctagtcaa gattgaaaaa acgtattttt agtgttttqa tagacaccct ctgctcggtc tcaaaaqctt Iagcaagcatt caactcactc icaggcacatc.
icacacacac iatgctgatgl i tttcaatcc t aatggtqaa atttcttagt ttgttcgtca taatagtcct ctgataagaa ttagagaaga gcagagtctt caatgtgtaq qgaagcaatt gattcttctg taaaacggtt tcttccaggc attttgaata catgggaaaa gggtagaatg aaggtcatct tacaacaaaa zccactcgagz 3 gaacttctg a aaaatcaaa g gagagagag, t aaatttatc' 9 ttttataaa tgtcttctag t tttttgattt a atagtaaagt a tcatcatqat t gaaacaagaa t ccatgttgga tgatggtagt gatgatagat ataagaqaaa tatgaagtcg acgttcgcgt taaaaattta attataqgggc tctaatctaa ctgaaacgtg Laagtaaacaa iacgttttgaa 3 gacacaaaac a aagacggaaa Sgagaaacttc t aaacaattct c aaaacaagac tccgccacc a taaqatttg t ctaaaaaca c :aggttgqtg c :aaaaatgca ;aaggaacag aaacacttga gtagagccaa aacaagga gggtgtacta tattcacgat aaagaaatta gaggagaggo atcctacctc cagttgacaa aaattgcatt ggggagagga aatacaaact ttaggagcag aaaaaaagaa aaaaatgctg aaacaattct ctttcgctg 8040 ttggctctc 8100 aaagaagca 8160 agagaaaag 8220 aaataaaaa 8280 facccatgtt 8340 jagggaactt 8400 igaattggga 8460 igaacatgaa 8520 aagatgcaag 8580 :ttatgcaaa 8640 aagttagaca 8700 ggtgagaggc 8760 atgtttggct 8820 gttcaatggt 8880 tgtcgcagtt 8940 agaagaggaa 9000 gggtgccgtg 9060 atgtggtaaa 9120 gtttagattt 9180 gttttggaag 9240 tgagatctta 9300 tattgattgt 9360 tttaactttt 9420 gatgatgatg 9480 aaaatcttag cgactacaac aatatttagg tattttttaa tggaaaaaag tgacttggga tagagcacat aattgaacag ttcgtagaca ca at ttt tt g aaagggaaaa tacttttaaa cgattggtcc tcagttatat aacatgtgaa PCTIUS99/01313 WO 99/37660 -124tcaacaagtt ttggatcqga gccaaaaaag aaacaaaaca aagaggtgca cccagccagt gatcaagaqa aacgqaqgaa ggagagcata gtcgagcqqt atggcaccca ttttttgcaa tat accgqga caatttattt attatctaaa gactatacct gcaacaactt aattcttttc cgcaqactat aaaaggacga ttcttttgag cggcgggaag ccgtggaaca cggcgcgttt tgtctaagaa tggttatttc ctgccgttct cgattaatca ttatcaatat cgtagagttg caaagtaaaa tgataagaga aaaaatggga acaaaaagtg tgatcatqat tggaaaacta ttgtaaaatt taaqcgcctt tcctatattt taaaaatgtc ttgccaaaaa aattgqaatt caqtttaagt acgaaattaa tacattcact aatgacccgc acaagagaaz tgatagatgz tttttggta acctgatta( I gtttacgat( tttgtttatg acctagaaaa tgagtaaaaq ttgaagactt cctaggggca aagaaggatg qattgagagt acaaagttga gagtgatcct tttatactag attattgcac acaaaagaat ttcqgccaat tgtcgatttt ttacattttc *aagataataa fgacataggat *ctcgaaactc aactgtcaac ictgaaaaaat attttccggtt a gaaaaaataz j aaacgtgta( g tctgaaaaai ttcataccat ttttgattat tgttggaggc gaaqtgatat ggaagagtgt actgacgcaa ggcaagaaga gaaaattgga ccaaaaaatt ttctgtgaca catgttatat cccctttgtt aaaaacaact cggaaaaatg ccgaaatgat aagtttactg aaaagcaatg taataatatt tcccttttct jatgagttaqt agagtaaaac ttcaacaage a attgatagtc j taattgaagE a acatagatat gatggqaaac 9540 ttttctqgca 9600 ggagtcttag 9660 aaacttagga 9720 gctctttgcg 9780 ttgaaacatg 9840 agcaagagaa 9900 aatctcgaac 9960 attttacatg 10020 taatttaaag 10080 tgttaatttt 10140 cattctqaca 10200 aaaattaaga 10260 cttggttgcc 10320 atgaaagttt 10380 taatttttcc 10440 caaacatagc 10500 agtctaactt 10560 gccatcaact 10620 ttcaagattc 10680 taataataat 10740 aaaacaacaa 10800 atacggtatg 10860 iagaaaagttg 10920 -tatggtaaga 10980 aaatacaaqa aatttatcaa aatttgtcaa gaatgatgca aaaatatgaa tatgaaatct tctatagaac accgtactca tctgaaacag accacataaa tttccgagaa actattactt aatttgagaa aaatttagtc gctaattttg qaagagtttt gatcccgact actaaataaa cttttgactc caataatcta ttgacatgca atcctcatac ctaaagactt gttgaat cgz tcaagcatag aaaaaatqga aaaatacaaq aaaatagaga ctagagattg cataggtttt 11040 WO 99/37660 WO 9937660PCTIUS99/01313 -125gcggtggcga tctctattta agaccgagtg gtactacgag cqacatattt caaacaactt caacactatt t t tt gt t tg aattgtagga tttaactaat aaqcttgtaa catttattta tctacaatta acaagggcaa gacctctttg ttcgtggcct aacaaccggg tgtgaatcgg gacgcggaag tgctccaaat aacgatagtg gaacttttca ttggaagaac tatataccga tttgaaaaaa ataacttgca aaccgcacac ctgtccagaa atgagagccg aagcacacac aattttcgaa tcagttttqc gcgtatcaaa caaacattta tacaaatagc acaaaaacac aatttaaata tatatcttqg aatcagttgt agagtacatt gattgaaaaa cgagaatagc acgaatggat atgaaatgtg aacagqaatg gtgagagata cgcaggtgct aggqgtttcq tttcaggata ttattqaaac aaatcacaat acccgggtat atttttgtct qaatgaagaa cagttttgca acatacgaaa ctaaagttta ggaagaaaat aatttttcga aaaqaactca tacagaacaa agaaactttg tggatacagt caaatcaaat tttttcatcc aggaagagtt gcgaacgaat aaattggcgg tactgaatca atgattttag atcaaggggt atcaggtagg tgatttctgt gaaagggttc acatcgctca attttaatcg tttttcaatt t tcat tct aa gtgttatctc tatgggggaa aaactttttc actqttqagt gagggtctgt tacagcgatt tttgccaaaa aaaaacattt ttagaatata aaactcgaaa acaaacaata cgtatccctt tcccgqacta tatgatgaca tgaqgtttag aattaattat aaaatgaaag aataacctaa acatcttata aagagacgta gcttttgcat gagatttcgc agcttgtttg tttcttacta atttagacca tttttttcat taattttacg ctctcggtgt 11100 aagtgcgcgg gggcaataat taaaacctaa gcgtgcattt ttttcgaata ttcaqactaa ttagatgttc aaatagagtt tttttatatc taatcataga acccactcat ttagtttaac ggaaaaaagc gacccccgga ccqtaagagt aaagaagaga atqcaacaaa gqqqaaaaat gaataggaac gtgttccgat atqtgagctt ttagatttca ttagtaaagt aactaattat ttggaaccac gaaaattgag ccgccgqcga aaaattgttt ttqaattttc tttctgaaaa gttttggtgg gaaaccgtac gtcaaacatg aaaattgaaa tcaaataqtt attcqatgag ttccacttga tatgtaaaat aaatgaagaa gtgaattgga tgaaaatacg acqacgtaaa gcactttttg aggaaacggt ggaatttttg tggaagaatt gacttcaaag ttaatcacag ggtacagaaa tagtttttga 11160 11220 11280 11340 11400 11460 11520 11580 11640 11700 11760 11820 11880 11940 12000 12060 12120 12180 12240 12300 12360 12420 12480 12540 12600 WO 99/37660 WO 9937660PCTIUS99/01313 -126aatagaaact cagatctaag tact taggca acgtgaqgaa gatttctgtg gcgggaatcg acgttccgat gcgtgacatt cagcacacat gatagtatcc ttctacatca atatttttga gtttaaaacc gttcagaact agagaggttt acagatggtc ggtcaactgg caaatatcta aatcactcag tcttactatt tattcaaaaa cgaaacattc atttcagtca agtcaaaaca aqcaaaatca gtctttcaaa cgttaggatt ttcagtctaa cagggcatcc t caaggaqaa ctcattcctt ccattgccga ttttaaaatg tgttaatatq tttgaagact ttaatgctta aaaaatcata tttttggaag tctgaccttc tgtctcgggt atgactatta ggatttcatt ttttaggttt ctaaattgaa agcaaaaatg agtagatcaa tgagcaaacc gctctacctt agqttttgga cacaatagga aattctggaa atttaaattc cttcacatat ttagaatcgc ttctaacttt aaaggaaaag gttcggatga actttttcaa gaaaaaaatg qaaaaagcat tttttacaaa atcatatcca cttagtgatt gatgataact tacagaaaaa caagccatga ttgtagttga cataaaatat cttggaattg gtttqttatt ttgggaaaac aaaaccaqac a a ga aat tg~c ctttttttct aaattgttcg atataaaatt ctgcgacttt cagaagagtt tat cataact aaatttgacc tttgtccccg aacaggaagc gctcactgtt gagacagaga aaagaggaag gattacttca aacaatggtt agtttagttt atatgcaaaa tttttgtgat acattacctc agacatgaga agagaaaaat cataagaaaa tttcaaacat gtttttgaca gtgagaaaaa caattattca acctaattta gtctaggaat actaaccatt cgtagaagaa tttaqtgcaa ataacaaaac atcagtattt atcacacttt acaaaatgat gaaaagtagg tgcaacattg gagagagaaa atgatgaaaa aaattgtaca taagcaagct taagttttga caatttttaa tccgaaaaag tatgaatttt tagggtgtaa gatatctcaa ggtacgttta ttttaggaaa tattgtagat tccaagagac tattcctact tcatcccaca tataaagggc atgaaagtta aagctttttt ttatccattc agacaggtgt tgttgcacat aaaacaaatt gacaaaaatg agaagctctt gcgttqgtgc gagaaggaaa aatgaactct ctaatcccca ttaaaaaatt tttcaaaaat attcaaggac attaaagtag ttttcatctc aacgt t cga tggatttcat tqactgtcta tattttcttg tttagagaag aaaagaatgg attcaatata tatattccga cataattata aaaaccaatc tttggtcgaa aacgcaagtt acaagtaaaa 12660 12720 12780 12840 12900 12960 13020 13080 13140 13200 13260 13320 13380 13440 13500 13560 13620 13680 13740 13800 13860 13920 13980 14040 14100 14160 WO 99/37660 WO 9937660PCTIUS99/01313 -127gaaaaataca ctttctcgaa acatatggca qaaactgtaa tcgaactttc cgtaagagaa aaaaaaaccc aatcgtatag ctgcaaqcgc cccccagatc tttagaacct tgtatcatct tgattaatag tcacagtgat cattttggat gagacaacgt tccgacagaa gaacacagtt tgctcgaaaa ttatgcttta gctttcaaga aaaaaagagt gaagatacga cagttgcgaa catggtcagg catctttttt agttttatcg acccagatgc agtgttttgg gtatgacgac aaaactttta agctcaaaat ccaaaatgcg atot tggt to tcctgaagag actttctttt atccttcttc tttctctttt gtgacatgaa tccaqatatg gttttgataa gcagaataat cgaataacat ttgttgaggg gaacagaatt caaatgacat ctagtttcaa ataaagtgag aatgagtttg aaagctgtga atgactccaa aactagtttt taaaaactga cqtacgaagt cgcaaattgt ttaaccacac agctctctcg ttttcaaact agccccatga acgacatgaa aagaaacatc ttcttqcttc ttcttctttt ttttttgtta ctcttgtcac agcaactcca tgttggaaca gtctaccagc aaagatcaaq gaccaaatca tagaagttqa ccaatctaaa atttgtcgat aaggaaqaaa aaqactgcac aaatgataca ctgggatatt tacaatatga tacgaatcta aaqcagcagc a a aga tt tto aaaatcaaat catctaccgt ttttctgaat acctgacaac gaqoaccgcg ttttttccag atctgatttg tgatacttcg tctattcatt ttttcaattt atqaqgtgtt aataaattga tggtttcagt aaaaactgtt taattaatga tgaacaatat gcatctggta tcaatggatc gtgtgaaaaa ttttcgagcc ttgtgtcggc cagttgtaaa gaaattgttc gatacacctg caactaaaca cgtgtgggta ttcaaaagat agtcttctaa gacagaatca cagacaagtc ggggcacacg gaccactggc tgtctgcgtc acatcagaac cattcacttt caacttctta gagtagaaac aacaaacaag ggcatattgt tqggagcaga ctaaatttta ttttactttt atctgaaatt aagtgtgtaa agaagaacgt tcgatggtca tctcgtagag gaacacaatt tgtgcgaaaa ttaaaaaagg t t ttg agta a actagaattt cttaaaatqt taacaacagt gttgtataca ga aat tgta a agagcaacta cagtagtgct gtctgatctc aacatcgaca t cat t tagt t aatcttaaac ctaaatataa acttgaaata accacgaacg caaacaatca acgaagaaag agattaacaa tgtcaaaaca ttgatccaat gaaacgtgca gtcacttggt aagaaagcca -gatatttttg atatgacttc 14220 14280 14340 14400 14460 14520 14580 14640 14700 14760 14820 14880 14940 15000 15060 15120 15180 15240 15300 15360 15420 15480 15540 15600 15660 15720 WO 99/37660 WO 9937660PCTIUS99/01313 -128ttccttgttg tcagcagaat aaaatttctt gagcacaaaa aaaaaactac gaggcggca tacatacaag agttgtggca aaaattcaca cggacgcgga tctcatcatt acactattct ttgttgataa catttttcca tattactttt ggaaaaattg agcaggagaa caaacaatgg aatgtattag agtttgtctg cagtgagaaa atgtgaagtg gactatttgt gaatttataa aattttqaac agqtgttttt ccgaagtgta gtgttcttac caaaatgttc cacctgccga aaaataacca acaagcgtcc tttctaaaat atgaaatgtt aagaaatgtt tattacacat tttttccaaa taaaattaat aataqtgcca ccaaaaaaca ttgacatgaa aacgqcgtat gcggaccqaa actgttgtat aataggtttc aagtttatga aacacgtcat aattacgtaa ggaatgaaac ttaccggaac ctgcaaaaca tcacagaact tttccctgga attttgttgt ctacgtcttc cgcctctaaa atacaaattg tcatgacgcc ttaacttaca ttaaaaatct acaatctgtg tcagtcacac gtttattcta gtataatagt aacatcctta ctgaaatggt attttcaatg tqcaaaaatc gaattcagaa aaaaataaag actacctgtt cqqgaagtqt tttggaacgt taaaat cagt aattgggggq aaaaatatct taagcacaaa ttaaacaaca aattccagta aataataatg tatqcacatt atagttataa agtagggcga ttgttcattt aaatttaaaa aaatattgag aatgaagacg aataaacgtt aagttcaata ttagaaaata tgttacgcag ccataaaacc atgtaatgta ggtgtqcgga gcttttaaaa cqqagqcggt gttqaactca ccatcaaata taaaatgggt tttattaagc qttttttttt ttaaattatt ataatggagt aatactcata aatacctaat tattaaaatt atttaqgtca ctgcgcatga tggagaggtg agaatgtgtt aaagtcacaa ttttaaataa aacaatgaaa cggacactac ttttagtttg ttttqaaaca ttatccaacc tattcaaatg aaacaaagaa ggaggagaag taagaacggc agagagtcaa aaaaggtgtg actttcaaaa ggcaccgcaa ttattaaact ccaattttcg aagatttgag t t ttgt t tta atttgaatga gtaaaaaatc gcattaatta cagtttcatg aatcaggtag ggcqgttgga tgctttqaat caataataaa atgatttttg aagtgaggaa cacacatttc attattttgg tgaaactttt acatttttct gatatgagaa aaatattgcg gaaaaggaag gactttcaqa agctttcaga aaaaagtqaa tttgacttat aatgttcaca aacccttccg attttttttt tgatgtttga aaatatcaat aaacagtaga 15780 15840 15900 15960 16020 16080 16140 16200 16260 16320 16380 16440 16500 16560 16620 16680 16740 16800 16860 16920 16980 17040 17100 17160 17220 1'7280 WO 99/37660 WO 9937660PCT/US99/01313 -129tcccacaata aaaacaaaaa aaattgggca gtgttcctag taaaacqaac aatgggcacc t ggct tct ag gaagagaaat gaggaacgga aagttatggt ggagatttca aaaacagtca atgacacaca tatagaaaga tagaaaataa gaacttgcaa tataagtctt tcaagaagtg ttatcaaaat aaaaccacct tctttgtcga tgctagggtg aattattcaa gagacaatga gctgacattg ttttgaaaac gaaaaaaaca atatggtgaa t gga at gaa a agcggaagaa tatcatcatc ttcaagaaac atgagaaaat aacqttatct atctttgaaa ttcgaagaac atcacagqaa atatcacaga gagggtqaac caatttttga atttttagaa gcagtatttc ttcaggaaat tttagaggtc agtgtatggt tatgcggqtg tcaaaccaaa attctcgtaa tcgaaaqtta gaacatatca ttatctatat aataatttgt gaaatacgqt ctaacagaaq aagatctagt tcacttgttt gaaaaaaaga qatgatgata ctgatcgcca gaaaagaaaa ctaacccaat caggtgtctq caaaatgatc aggagataga acaacaaaga ttatcatata ttaaatttaa tttaggacat taqtacacat aaagtacatg atggtgtttc tttatttcaa tttaatgaat acaaaaaaaa aaaagtggta atatatatat cagttgataa agttcgtcqc ctatacggta ggtcacttcc atttgaacag gcaataacgg ataataataa tctcattatt caggaaatta ttgaactcac tcttttcaaa aatgaggttg gaatcaaaat aataatttag aaaataacaa cagttcattt caaattttta ctacccaaaa agaagtgacg tgtgagcagt cccattcttg cactcagtaa ctgaggatta agtgaaagtg atatatataa tttttagata actgtcagac tataatatta gatttctcag tctcgacttt aacagaaaat gttagaagag attatgagac tacaqaacac tcccacttcc atgtatacgt cagaaaagaa ttgcatagat tggcatataa tgtttctata cttggtaatc tttattttct agaagacttt tgtctttggg aacaggaaat cttcaaaaaa ctgtaacgtt aacgttattt aaacgaaaag ttacqaaaaa tgagttgcca taattttcaa gqaacacaat ctgacttttg ttccaattgt tcagaaagtg ggttatcgat acaaagatgt acacaatttc tcttgtctat tttccgaata tgcaaaaaaa aaatatgcaa tatagcgatq ttttatgccc tttattttta aaatctactt qgagctctca cagctggcca tctggacacc cccccaacta ttttttttca ggtatctaca tgcaacattt 17340 17400 17460 17520 17580 17640 17700 17760 17820 17880 17940 18000 18060 18120 18180 18240 18300 18360 18420 18480 18540 18600 18660 18720 18780 gaaattgaga gtagaaaaga tcattgaagc agaaatatgg aagtgaattg aaagccgtgg 18840 WO 99/37660 WO 9937660PCTIUS99/01313 -130cgccaaaacg acacaaagac tttcttagtt aatggaaaca actaacaaag tttctctaaa ttcgcattac qagaaatcat ttttgagtaa aaaatactag tcaattaaag ctagcacaqg taaacacat c aqtgtgtgac ttttgcataa tat qtqt at t agcaaattag attgtacaat caacaacgtt ttttttaaat caaagtttaa ctaacttcaa tttacgtgcc tcttcaaaaa tccaataaaa aattaaagat acggt cagqc aacgtgaaaa agctgaqaat tgtctagact gttacaaagc ataataacac tgaqcacaaa taaaaacagt gactaagtat taaaaatgag gaatcaattt gagcattgga atatctatca aaatataccg atactaaata atcaaccatc gagacgcaga aagacaggta catttatttt tcagatttct ttatttgagc aaaatcctga tgcctacggt tcttccttaa tactcttatg a a a a attat gccattgaga attaqtttgg agtttacatt tcagataaat tattaggaaa cattaatttg acttqqaaaa acaatttcag gcactcacaa catttcgaaa tataataqtt agatataaaa aaccatcact taatattaag attgaaagaa aacagttttt ttgagaaaaa gcatctctca ttagccttca cattcatgta gcagaaaatt ataaatacca ctattttcta aaaatctcca caaaaacccc qatagacagg aaattaatga agaagataaa gattttcggg ttgtagaatt aatacqgaat ct atat t tgc agtttaaaaa acagaaataa cttttctgag agcaatatat ctaggcaatc acataaagat tccatcaaat ttcaaatggt acgcagagaa gattaaaacc atgagaaaat aagaacctat ttatgatatc caagtaaagt tgaatggaaa attcttttcc atttttttaa aaaaaatcaa ccattcattt aagctgattt gagttcggaa ggttgaaaca 18900 aaatgtctgg aaaacgcaga tatatttgta gtatttttga tatatatgct aaaaggaaac atctttcagt tgttccaaaa catacaacta ccacttttag aaaggtgata ccacagattt gqaaaagacg tgtaagagaa atggagaagc aatgaaagta tgtcaagcag tcattggttc tgttaattgg tttcagaaaa aagtatgatt tgataaaatt gttcaggaaa tgcagaaaaa attagatcaa agatgqacga atgttagaaa gcaaaagcac aaatttttga atatagtatg ttgttttctg gaaagctttt atgtttaaag cacaaacatt attcaatttt aaagatccat atcacaaatc cagacaaagc aaatatacaa gatatacagg cggaaqqgtt tttaaacatt tataattgga ttattatgcc ctgattcatg ctcgagcctg ttagagtaga actaaagtac qacaaacaag tgaatcqact 18960 19020 19080 19140 19200 19260 19320 19380 19440 19500 19560 19620 19680 19740 19800 19860 19920 19980 20040 20100 20160 20220 20280 20340 20400 WO 99/37660 WO 9937660PCTJUS99O 1313 -131tttagttttt aaagatcaaa aaaatgtgcg tcggctactg tgtttccaaa taaagtaaaq ataaattaga tccaaaagtt cctaaaaatg caccgtctct ttaaaaagag ttccaatttt tggaaaaatc ctataaaaaa aaatttttat ggattgcatt gagatccaga aaaaaagatt ttctggtcac tcccatttac tttcgaaata ttcccaagag gacaaaaatq aaattctgga attttttagt tgaatacact cttgaactct agttataatt gtacatgaga ataatttcct aattgtqggc aagaaaatga ttattaaaga caagtcctaa gcggacagct tatcatcatg aqagtgtgag taaaatgtat gtttttqgat ctttgtqttc gaatgtttta taaaagtgca agctcqaaaa ttagcccaat tttgaaggaa tatttcttgt ttgcggtttt aacttgatga qaaatgactg aaacttttta gatggagaag acgtqttttt a at t tga aat gaaaatcaac cagtagtagt tttttactqa gtggtttatg cagaggagaa taatagttac gaatcttgct gtcgggacac ccatttttcg aaataagagt ttaaaagagt tattttttga acttttttat ttgtaaaatc ttagcagaaa acatccaaat caactaaaaa acactaatcc tgtcttcaaa cgttaaaacc ccttcatcat aatcggaaag aaatttaaga tacgtgataa atttatggta agtattcgaa aaattgatag agtaqccata taatttgatq aattggtcaa attattttcg ggtttaaaat attttgcaaa tatccaagaa aattttaaac acacatggaa taccgttcca ggaatatttt cccgttttta ataaaaaggt tgtattccta ttcttccaag aaaactctcg caaactgaga aagtcttaga ttttttgagt caaaattagt agcaaqaaaa agggcaacqa gqttaaggtg gaatatagca tgagaaaatt tgtttgtata gtacgtattg tcatttcgta gacatqaatt tttgctttgg aaataggtga aaaaaagcat ttcgtgataa tcagactttg aatgcaagat tttttgatta tgaatgcgct tttttatttt gcgaaa cat c tggaatgttt aaagttgatg tttttttcat accgaacatg attqtgcaaa agattgaggg gqtcattgaa tcgaaaaaaa taagaaacag gaacactagt cttaaagaac gaaaatatac qgattaaatt gtggctccac attttatttg aaaqgaattg aaattgcaaa taaaaaaata gagcttttgg aegggtgaaq ataaagaaaa ttgaatttgt gctttataag ttcaattttc tacaactttc taaattgcct tttgtgcaac ttccaaaaat atttcacatt gattaaacca aaatagaatg tccattagaa tgtttgatca aaqt at t tgg qaaattaggg gcacacgttt gtttttaata 20460 20520 20580 20640 20700 20760 20820 20880 20940 21000 21060 21120 21180 21240 21300 21360 21420 21480 21540 21600 21660 21720 21780 21840 21900 21960 WO 99/37660 WO 9937660PCT/US99/01313 -132caaactgaaa taaaaatacq gaaatgtaat tttttttttt gaattttcac gtcttaaagt tgtcctatgt aaattcaagt gcaataaaaa gttactcccg aqtgaattta tgcacagcca caaaaactct ttgaattgca ttacatacaa cacaacaata cttcqgagca tatgtgtgtt acctttttct gccatagaca aatacaacqa ttaggaggag attgctcata catacttaca tttgtttaaa gaaggcacgt qattgtgaat qattgaacag cttttcaggt atcaaacggt tttcgactat aqccttttct a ga a agttat gtcagctact tagtacggat cagaqaaata tattqttaca gaaagtcaca ttgattccca atatacacac tcgttccctt tcttgtgctt taggtgttgt tatgcttctt aggcggcgac tcacaacaac ttggagtttc aaattgtgat acctttattc agaaaattca gtaaatgcca tgaaaattta gaaaqtgcag aaagaattat agtgattctc aatttaaqta acacttgaaa ttagtcttgt gtttqattta acagagtgaa attataaaac cttggagtta tcaagaaatt agagaaatag aaaaaagagt ttatctctaa gctccttgta ttgaacaaat cttgttttqt atttttgaaa aacacaacga tacaatcatg accatgaaga tqacttgaat attaggaaat cccggaaatt agagaatcat aaatatcaga acagaaatcg tttatgcgtt atatgtttgt aatqataatt gtqccaagca aaaacttaga aactagtqat ttaaaatttt caaattctgc ccaacaaaaa tagtaaaagt qtagacctct ttctctgcgt cggcggtaca aagtttttgg gcacattttg aaattatatt aatgaaaaca aaatgtttat ccgaaaaact ttcagttttt aagcaataaa agaaaaaata tqcaaaaqta ttaccgtccc taataaattt gaaagaatcc gggtgatatg caaaaatcat tttatttgaa ataaacacat ctactggtgt tgtactttag tagcagctca aaaccatcta acacattaga gacccctatc tccttctaac ctccataagc gtttcctccc ccatccacct gctcttgctt agtactqtta tqagatcaaa ctagttatta ctatqcaact ggtgtttgca ttttggcatg agatttctca cacaaatgaa aacagaaacc aaaacaaaag gcctgaaact tatttacgct catggtgctg aatggtattt aqtctattag gcctgtgcaa atcggctgat gtcttcaggc ggattqaatg tgccaactca cattcctccg ccaaccaaca tttgagagct tctgctccct ccttctcaaa gtctgctcga tatagtactt agacaaattg taaaactgaa gcatactgca gttattctat tatttcgata aactgaaaat tcatttttca ggaaataaca agagttatga 22020 22080 22140 22200 22260 22320 22380 22440 22500 22560 22620 22680 22740 22800 22860 22920 22980 23040 23100 23160 23220 23280 23340 23400 23460 23520 WO 99/37660 WO 9937660PCT/US99/01 313 -133cacattgcag tggtcatact gaaaatggaq gctttggttt caagtgaaag tattgaatat ctggaagatt aaaattacaa ttttctttta tttaaaaata ttgataggaa acaactctca attttatgaa aagagttata cgtttgagca gaaacgggga aaaacaacag ttgtttttat gaatggtagc gggagataat gactatcaaa ttatqatagt tataagatta gtgttttaca ggaagttgg agacaaagac aacgqtctct tacaaaaaaa gtctttcqCg aaaagtttcg gaaaccattg actqatatgt catttctgat aaaataaata caataaagqt acaactttat aactatttta tttgaaagta tacggctctt actcatqgag gaagactgta attgaaatac aacgatggtc tccgtacgca acccaatttc ggaaatgacg actacaataa tgqgatttac catcaaaata tctcatgtag tgaagaaaag attcctaact gtggaaaata acagagttta gattcatttt caaacaaata agagcaaaaa gaatactgga tcccttqtgt ctcatcttca aatgtaaacg ggtatttttc acattttggg ataaatatga ggtagttttt accctaattt ggaagagcac gatatgcgat tcaggctcac cagaaacttt aaatagtgtt tgatgaaatg tactaatqga tggtttcaag aaactaaaat gaaacgaaca aataggqgcc cggaggtatt ggaggaggtg actaaaaatt acttcttctt aacaatgaac caacaatcaa aaaataatta ctttgaccaa tcgattcagt ttccgagcgt ttaaagtctt aactcggcaa aaataaatta gacgagaaaa ctggtttcac acgtcataaa gagttttggt ttgactcatc ttttgaggta tgatttgaag gaacqqaqqa gaaaagttca ttcaaatgtc ttgaaggata aaatctttga agaatagaag ccaaaaactg ctgcaaaaac aaattaagtq ttttccactt attgtgttga ttgaaataaa gtctctataa aactttattt gqtgttttaa gtagttttct aaactgaaac aagctctgca tcqaagtttt ttacatgttq tagaaaatca aattagqqga tcgaaccggc tcggtgggaa tttttgatcg agacaatgaa aaactqgtat ggattcttga aaacatctgg aagtaaaaca acacctaqat gtcattttga ttccaatatt tccttaqaca 23580 agaaaatgaa 23640 ttcgttgcaa 23700 gaagacaagc 23760 gagtaaagtt 23820 ttqgtaccgc 23880 ttttcagttc 23940 actcctacgt 24000 ctqaaaattt 24060 cgaaacattt 24120 qqcttgccga 24180 tttttttgat 24240 cataaatttc 24300 aaaaatcaaq 24360 tcaacqatcc 24420 tttqtcccaa 24480 caatttttat 24540 tgggtgggtg 24600 agaaacaagt 24660 aaatatcgtt 24720 agattttaca 24780 aagaaaaacg 24840 gcataatata 24900 aacttcaaac 24960 caaagtgaac 25020 qaagaatgac 25080 WO 99/37660 WO 9937660PCT/US99/01313 -134actatttgat atcgcaaaat ataattaatt gttttcttta agttcggcgt caaatatttt gtattattct tgtttagaac tacatqagtt ctatgagaga ctcccactga ggcatcttcc tgcatcctta aaatgttttc aattaccacg cacttaacta acgaacgtta attcaaacga tccttggatt aaccaccacc aaaaaacttg ctagttatct gtacttcagt aacttttatt ttttatgcat tttataagat tttatatcat aacattatta aaaaagtggc cttgatgaqt ataaaaatgc tqatacttga ttccgttctc actttgagtt tttttgtcga taattccatc agccagagtq aaatttatta gttgaaqqaa catgttqcat tttataacct cgtttaaaca tgaaacagta tcagatacta ttctgataca ttggtttcca attacagatt tctqttctag tcatttttat cttcaqtttc ccatctctcc ctctctcact tqccacgtca tcatcatcat gcatcaaaat attcgatatc ttttctatqc gttattattt tqqtggaaaa cctgaaaatg actgtcatta atctttctat tggtccaaat qactcaaaaa cggatttgtt qataactcac atatgaagtg ttgaaaaatt tgtatttcaa atattaatat aattaccacc cacattttga agqatgtttt ctcattttgq caaattaata tctaattttt tttctcgcta ttaaattagc atcacatgqc ttttttctta ggaaatttat cattcattca aaacatqcct aaaaaccttc qqacccgttt ttttatattt ataaacagtt tttttctgaa aatactaaaa aaattaacaa tgattatttt cggttttttt acctaattat tcttattaat atgtttttca atttgttttg tttaaatctq ttqttaatac agcggccaca taaattgtat gccatattta ttgcaagtac tattttaaac aaaaatacac attttccccg atttttttaa aaacttgaaa ttcctcaagt atacctccaa agacacctag attcgacttc atcttttqtc taattttccg tacatttctt cactcttctt gttcactaaa tctcaattca caatgccacc ttctcagagc ccattcgaaa acgaaaa'atg cttctcaatt ccacttcata tttatttctt tgttttcact atattttatc ttcaaqttac atcagcaatt tatqattcaa ttgattcttc aattcgtcga cgqgaaatat catcaagaaa tgaaactcaa attgagctta aaatttcagt aagtttagga ttttttttca gataattgqg ccgggcttac atcagagtat tcaaacattg taqagattcg aaatgcgaca ctgtcaaaaa agttcccaaa aattgtcgaa cacgattgga gtgttttttt aacaaatccc aaatcaataa tttggattaa ttcaacttgt aaaaaaaaac tatcttcaaa caattagcgt 25140 25200 25260 25320 25380 25440 25500 25560 25620 25680 25740 25800 25860 25920 25980 26040 26100 26160 26220 26280 26340 26400 26460 26520 26580 26640 WO 99/37660 WO 9937660PCTIUS99/01313 -135gctttaaaac aactatqctc attctaaagt ttccattatt aaagacataa cgtttccatt tgggagattc accccagtct aggacatcca gttaccactt ttatggdgat aacatttaat catatgaaga atgtcaggtt aaaattatca taatgcataa cgatttcctc caccacgagt gcttaggcga aattaaccqg tttactgttt acgcggtaaa gaaaacattg gacacttatc tgtaagacca ttctaaattt tactaggcct agaatttggt aagattaqtc taacaaccaa gcaaaagctg ccctcgtttg ataatgtgaa ttaactgtga tatttagaag caagaaccat tttgagatgg ttttatagct gcttactgga aactgcattt tttgaaaatg aaaccgggcc gacgctggaa tgaaggatat cttttcggat aaattctaaa ctattttggc atgtatccat gacaaattqq aaagaatgtt tcaqttagac ttgaatgttq ttgttggcaa aatgtttgaa atctattttc gactqjagcaa gatcaaagct taaagtacaa aactaaatgt atgcagaaca taagttcaaa actccgattt ttttaacagg ttaacatcat gagctttaca ccaatgatac atcaaactta aaattcatga aatcccaatt gatattttag ttctccatca acattaatca atttatttca ttttcaaaaa atacaataac cagaaaattc atcgatctct aaagtttttt cgtcttttca aatgttttgg cattccattt acattttcct tqggcaaatc ttggtaatct taagatttgg tccaaagtca tcagaaatcc tcgcgacatt tttaacaaat ccatatcaat agtttctacg gtcat cagcc aaacaqattt tcttccaaaa acgcctctga aatataatgt ggcagacaat tqaqaatggt gcattctcca tttcccatct agaaataatt gaaatcttgg tcatggatgg ttgttcagat cattttggca gcaagcatat ttccattttt gttttaattt ctattcaaaq taaqqcttaa tcatcaattg ttgcttttac ccaatccatt gttagttgtt taatttqgtt gggatcattt tgtatttgaa tcgcttcgat aacgcatttg ctctgctqaa atttacacga caaaatttca tgaaqcagca atcacttttt actccacaga ccaccacaag gaagaatctS aaatgctcgt tattcatatg tttcaataaa caaaactata agttattcca cacctatttt tcatatatga tattttccaa ttaattattg aaaaggaaaa gagatcacac ttttcttgta tcagtccaat tcttttttaa gttaqtatac aaaacgggac tcggaaaatg cattgttqqc attgttttac atatttgaaa acqgataaac aaaqcagaag ttcaaaataa atctcttaaa ttattccaaa atgcattctg caagaaaatg at att catt t cttttaagaa 26700 26760 26820 26880 26940 27000 27060 27120 27180 27240 27300 27360 27420 27480 27540 27600 27660 27720 27780 27840 27900 27960 28020 28080 28140 28200 WO 99/37660 WO 9937660PCTIUS99/01313 -136aagaataatt tacattcqaa tgttgtcatt ttatttagca cacaaaaatt ttcaaattct tgctcgcgtc aaccaaaaac ttttcagatc aggttgaaqq taaaatcaaa tttgatttat gagttaccat attatacaaa atcgattttc ggtttggaca tccattggaa ccttcgagag ccagcacaqa caaaattttc at tcggt at c tcgattccat ggagtataga aatttgaaaa ctatagcctt caatttctgq ttaaattcta aaagctcaat tgaagttctg tacaacatga gqaacacttg cagtaaaact gctgaagtat tcagataaag acttctatca agcatcaccg ttaaacaccg tctgattttc tqcgtatgcg tttgtatttg tcatttcttg tatacgqaaq tatgatcggg ttaaaagttc ttctatactg tcaccgtaat cgtt ctgaac catqgtacac acccaqttct ggttggaatt ctcgtgacag acgcaatatc taattcctga aagcaaaacg gagtgcgatg atttgaccaa cgaagcgaat qaacgttttt tttctagagt agcataactt gcaqtattct gaaatagagg aaaatatgaa cgccgctgat agttcgaact aaaacggaca tgttttttcc aatgttcgag aagttcaaag acgaactgct aaattgcttg gaatctgaca acgtggtaaa ttcccaacac tggaacttct tttgacgttc tccaaagtcg gcgatgaatt atttccaact accacgaaat aaaagtctct ctcgaaataa tcaaqacttt tttatqctct atgtttaata ttataacgca tcttttttcc atgactgctt acatattcac ctgctctgac ggacgccqat acatacactc tqgcgttcag acttqaatct tagctgttgg tcactcattg ttgtgttqtt atgqctggtg acctcagcaa atgactccat ggagccaacc tctaactttt gatatcttta atttgaagtq atgtttatca aacagtatta cacctcggac ggttaagact tcgaagttat ccaaatcatt aaactaatat ttttcagaac aaagacacca attgttcttc taacctgaac ttttgaaacc tcttctttct cttcttccgc gttcaaaagg tttttggttg atcctggagc tgaccctttc ttactcaaaa ggacgttagc ctttcattcc aagcgaaaac atctaatagg tgtgaggatt ct tgt acat t aatqaagata tttcccaaag aaaaaaaaga tttctgtaat gaaaattttt taaacaagct ttaatatggc gtaaatgaaa tcttcaagct aqaactgaaa tttttctgaa agctttcaqq gggacttgga gaataaacga cgaattgctc tgqagcaact ctcaatattc ttgatcaaat ctcttcggca gaatagtgaa ttcaaatgqc cgqataaggt atcagtcttt aactctgaaa catccgatag cccgtagaga ttcaagacca 28260 28320 28380 28440 28500 28560 28620 28680 28740 28800 28860 28920 28980 29040 29100 29160 29220 29280 29340 29400 29460 29520 29580 29640 29700 29760 WO 99/37660 WO 9937660PCTIUS99/01313 -13 7agaccagctt tttagatatg cttagtaaaa aaggttcctg tcatcaatct ttgccagctt cctctccaaa cccacggttc atgaaaataa gatgatagta tgttattgtq agcaatcgtt tggctctccc gaagaaccat aatccaaagc taaaattaat tcctgcagcc ggcttcgtgc gattgcaqct tgctccttct acgaccaatt qtttactcac attgagaaga ctctcgaatt ttacgtacgt acttttgqat cgtatgtttc tccattgtca cagttaatta acaccataaa atqatttctt tttttgacgg cctaatttct ggacggatga t ta at t tqta tctgcgatac acataatgac cctcaattac cagtaaatcg ttggaaattg gcctaaaata attaacatac aagtgacgac tgatctgctc tatttcctat tgattaatgt ttacagtttc agatcacccc tagaaaacca gaatctgaaa tccaacatag aactagaaaa gcaatgaacc atttacataa caactccatt attcggatgt ttttgtcaaa gcgaatttct cacgtcagaa tgggccctaa agcaacaggt ttttttatca taatattctt acagagagaa tcgcctcqtt aatgttcttc gacatttttt atgaccatca ttgaactttc gattccaact cccaattgaa tgttqctttt aagcaaacaa tctttagaac actgcaaact aaaaattatg caaatttcct gaqtgaqcaa 29820 29880 29940 30000 30060 30120 30180 30240 30300 30360 30420 30480 30540 ctgtttcata tcctcacgtq gcaataagcc atggtaccag gqttcttttg caagtacttg 30600 C 30601 <210> 34 <211> 8009 <212> DNA <213> Unknown <220> <223> Description of Unknown Organisra:Unknown <400> 34 ggatccttgg ccacgccatq ggcgatqaaa ttgaccgcgt cgtaacgqgt tcctgcagga agaaggccgc gttcgattcc cgttccgcaa agatcgcgac gcccccgtca cctcggtccg gcccgagctt tgcacatgga tcgcggcgcg cgctggaagg cgqcqgtcgg cacggcttcc qagccttcga cttcggtgat agatcatcgt cgatgaactc ggtcagggtq qtgcgcaact cgccaagatc gcgcgcatca cgcggctqgc qtcgggctcg tcgatcagcg cgacgagaaq catgtcctgc aaggacattc ctggatcacc cagcgtcgag gacgccgcag atqttcgagc 120 180 240 300 360 WO 99/37660 WO 9937660PCT/US99/0 1313 -13 8gtcgccagtt agcgtggtcg catgtgatta acctctcgcc cagcgcgcga gagcggcgcg caaaaaqqqa cagccgcaaq gctgagcgcg gggcggcagc gagacgcccg cagataccag catccccaga ggcatcggcc cgcgatgtga ggtcagcacc ggcggtgtcg gagggtcatg caacctgcaa gggcgcacgg tccggcatgg tatctgggcc tggcccgccg accgggatga gcgctgatgg ccqaaccggg catgtttgcg aaaacgaagg agtttcggtg gctctqttgc aagtgctttc gcatccccgg ttcgttgccc gccgtcaccc aaccggccgt gcggccagac cagccccagc gcgatatggc tgcagcacgt gcggcccCCg tcggcgtggt ttcagcaccg tqccagagcc gccttttgcg tgcatctcga cgcaaaaggc ccgggctgac gggcgcggcg gagaqcccaa tcgaccggct aggaatgctg tcgggctttg cgtgttggcc catgcggcgc gatttcgccg aaaactgacc agaaccqgtc gcaggccgag gttcctcgcc ggtccatccg tcgcggcggt qggtcagcgt tcatcaggct cgagcgtatg cgcccgggqc ccaccttggc gatgggtgat gggccgcctc aggcgtaatt catctttcgc cgtgctcgac gat ccgcgac ggt ggcgggc ggtgatcggg tgtctcggtq ggtggtgctg gcgcacgctg ggcgccgCgC agcgccagtg tcctttcctc cgcttcaagg agcgcggcgg cttgcgcgcc cgcggcgcgc caaagtcacc gtcqtgcagc gtattcatgc gtcgaacatc gtcqccggaa gccgtcggcc caccggatga cccggtcgcc caggatgtgg gggacatcg gtcggtcagg tatcctgacc ctgcgtgatt aaggtggtcg tacggcttcg ctgatgcccg ctctgtgccg cacgggcttg ggccccggCg gaaaccacgc cggcgtgaat gggtctcccg cccggacgcg gctttcgcgc cgcgccgcgg aacgcagcgt ggcaaactgq cgaccgttcc cccgaacaga tgcgcggggg aagagcagcc gcgatcacct tcqagcggcg tgcgccagcg tgcagctgcc accaccacca caggqgatcg cagcttcgcc tctactaccg aactctggcc cggtgccgct cgcagcaggg aaaccagctq aagtctccga ggcgggcgct ccttcggctt tgcttgctcg atactggcct tgttttttcc qatccgcagg tgaagacggc catcggcgcg gttccccggc ccggttccag cccgggtttc tcccctcgaa ccgccccgg gcgcggcctc gcagccggtg cctcgacccc agcgccggtc cggtat cggt gggtcagttc caaggaaggc cacccaattg ggacacccag gttgcgcccc cgtgatgccc gccgctggag cgaccccgat gttcatcgtg tggccgcccc 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 WO 99/37660PCUS/033 PCTfUS99/01313 -13 9tatacgatgg gcggcgqcgc tgggaacggc gtgatcaagc ctctcgatcc cgcaagaatc acgcagcggg ccagcctgat cqcaaccccg tgacggaacg tgtccgaacc acct cgcgca ccgccttggc aggagcaggg ccaacggtct agggcctggc ccaccgcgct gcaagaccgt agctgggttc ccatgaaaga agcagatcaa t cggtgacgg aattccccgg t gat et t gg tcqtggacgt cgatcgacgg gccagctcga tgtacattcc tgggcacaag aggtgcattc ttgaaggggc gcccggCCgg gcgggcttcg gcggattcct aatgcggcgt attggttccg agcttcgatt ggaggccaag cqgttcggc ggacgcgatc gaaactgatg cgccgcgat c gagcgcggcg caaactgaac gcgcatgatc ggtcggataa gaacttcggg gatcgcgcgc cggcatccgc gtcggaccgc tccggcgggc *caagggcccg ggcgcaggca gccctcgcag ggcggcgggC ggtgcgccgc gcccaagccg aaaagcccqt accctttgcg gccgccaaqa gcgagatcat gtqtccgcgt tcqcagoca ggcat cgacg gagctgcgtg gccgcggtgg gcgacgaagc gccgaagcca caggccgaga gtcgccgtcg gacaccacgg atgggcatcc caggatgccc gtgcacgggc gggatggcgc gacatcaagg gaaggcctgc atcgtggcga cgacgggtgg cggcggttct tatctggcgg tcggggcttg gtggtcgc ttccgcggca ctaacactcc tggttgcgag tttgggcgcc gcggaggcaa ttgccgggcq cgctctcggc acctgatttc ctgcgaagat gccqtctgtt aggqcgagat agctggcggc atgaaagcct tcaaagccaa aagcagctga aggtcgccga tcgacaacgt tgaaccttga aagqcqacac tgggccgcgt aagagcgtcc ggtttgcccc ggctgcgctc cgggggtggt gcgcggcggt ggatgtgagc cttcgccctg gtgccggtqc ggtcttgatg gaggggggcc aagcatcqga ttatgccacg cgacgtggac ctcgccggtc ggqcctgtcg cgcgctgccg gaccgoggat gacgctggcg catcggtggc actcgcttcc gatttctgcg agtgggccgc ccaggcgqgc agtcgacaac cgtcaagcgc cgtggacggc Icat egccgac cgaacgtcag 1980 ctccgagatg 2040 gatgcttgaq 2100 gcgcaagccg 2160 cgcccgcggc 2220 cggcggggaa 2280 agaaaatgtg 2340 ctctgctaga 2400 tctgaatcgg 2460 aqggtgqacg 2520 gccatcttcg 2580 gcgctgacgg 2640 tacacccgcg 2700 gcgccqcttg 2760 cagctgctca 2820 gtcacctcgg 2880 aaacagacgg 2940 atgatcgtca 3000 cttcagaacg 3060 atcctcaagg 3120 gtgctctcgg 3180 gagatggtcg 3240 gtcgqgatcg 3300 accaacgcca 3360 cttggcaacc 3420 gtcaaagccc 3480 WO 99/37660 WO 9937660PCT/US99/01313 -140cgggcat cat acgcgatgat gcaagaccgc cgqgcaacaa agctgqtgaa ccgcttcgga aatacttccg ccgtggccta cgqgcgacgt atttcggttc cgqccttcat aactgttcta gttcgtcggc cgcagtatcg aaaagctqct cgctgaccaa ccgttccggt cccgcaagqa agaagaagct ggacagggag tcgcaagatc aagcgccgaa cqgtgcggtg gqtccatctc tatcgcgaaa gatcctcacc tccgcggaaa cccgatcggc gatcgcgctc gctgcactqc gaagctcgaa cccggcgccg cgacaacggc tcgtcagatg gttctatctg gggctcqctg cccgaccaac ccagggcatc ccagaccaac cgaaatggcc gaaccgcggt cgccgaaatc gaaggaagtc cgtgcttgag caaagacgcg atgcccagcc acgaaagcga gctgcccggc ggctcgaccg ctcgtcatca ctcgcqaaqg gtcggcaaga tcggtgcatg cgcqgccagc gacaccattc ttctatgtcg gaagccgqcg atgcaqttcc atgcacqcgc tcgctgctgc cactcgcqcc accgcgctgc gtgatctcga cgcccggccg tcgatgaagt gcctttgcgc gcccgtctga gtggcggtga ggccggttcg tggctcacca atcgccgagt ttaaggacct tgcagatqgt cctatqccga acggtgcgcc tgacgqgcga cgaaqqcgat aaggtcgcga aqccgatggc gcgagctgat tgaaccagaa ccatcgggca cgatggaata ttgcccccta tgatcatcta tgcgccgtcc tgctggaacg cggtcatcga tcaccgacgg tgaacaccqg cggttgccgg agttcggttc ccgagctgat t ct ttgcggg agaaaqgcct aggaagaccc tcgccaaqac caagaaccgg cgcqqcggcg gcggatgaac qcgcctactt gcgcgqgctt ggaactgctg cgcgctgcgt gaccggcct c cat cggcgac gtcgtacaac gaagcqctcg caccaccgtc ttcggcgacc tgatgacctc gccggggcgt ttcggcgaaa aacccagggc tcagatcttc tctctcggtg tccggtgaaa cqaccttgac gaaacagccg caccaacggc gctggcctat caagatcaag cttcgcttga atcgtgagtq aacatt cgcc gccgtgatgt qcgcacgg tgcggcggct gcccagggca cgtgatctqg aagtcggtcg cgtcagaccg gacgccaacc accgtggcgc gtcgccgcga qcgatgqcgg tcgaaqcaag gaagcctatc ctgaacgagg ggcgacgtgt ctggaaaccq tcgcgcgtcg ctggagcttg gccgcgacgc caatattcgc ttcctcgatg ctgcgctcga ggcgacgccg cggcctgaaa tcaagaacac gcgcccaqga cgagccttgc gctccgacaa tcaacgccaa agacggtgaa gccagtatta 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 WO 99/37660 WO 9937660PCT/US99/01313 -141catcgatcac ttcgcaaaac ctcggtcttc gttcgaaacc aatcctgacc aaacaacgcg gggtgacatq caaagagctc gagaatggca cgaagacggc cgttctcgaa gaccgagggt ggtgggcaac cggtgacgtg gcagtcgacg ctattccaaq gatcatggaa cgttggcgaa tatcaacctc acccccggg cgaccagtcg cggttcqgaa gctgqccacc cacctcggtt qtcctttgcc ctacccggcc atcgacctga atcatcqacc cagagcgtga gatgcggcct gcgctgctgc tccttcaacg atcgatcgct atcgaaatca agcaaaggca ctcccggcga gtggcgcagc ctcgtgcgcg gccaccctgg tcgaaagccg gaaagccaga ggcggcaaga ctgatcaaca cggacccqtg gagaagctcq gcccgtgccc ggcaccgacq gtgtcggcgc gacatggqcg caggccatct cacctcgacg gtcgacccgc gcqacgtgaa gcttcgaggc tcagccaggt cggcctcggc cgcgtgcggt gggcgcagat tgaccatcqa tctcgggcqc aagtgaccca ttctqaacgc acctgggcga gcgcggccgt gccgcatcct aagcccgggc tcctcgtcac tcggtctctt acatcqcgaa agggcaacga aagaatcgaa gcgtqgcgct tgctgttctt tccttggccq cgctgcaaga acgttccggc ccacqaccgt tcgactccac gaaactgagc gggcqaatac gccqaccgcc qgtttacqac ggccacggcg gtcgqccatg gtataaccgc cqaggcgctc ggtcatcggc ccttgaaacc gaacaccgtc gtccgacacc gaacgtcatc gatccaccag cggcatcaag cggcgqcgcc agtgcactcg cctttaccac agtggcgctg qaccggcctg cgtcgacaac tatcccctcq acgcatcacc cgacgacctt tctqtcgcqt ctcgcggatc tacccggtgg gatgtggcga aagcaggtga tacgaaccgg atctttgccg gacaacgcca tcgcgtcagg tgacggaacc gccgtcgtcg accaacaacg cgcaccatcq ggqcgcccga ggcgagccgg cccgcgcccg gtgatcgacc ggtgtgggca ggcttctcgg gaqatgatcg gtctacggcc accctggcgg atcttccgct gccgtgggct tcgaccaaag accgacccgg gcgatctcgg cttqacccgc cgcagaagat cgatcttctt t cccggcgca gcgatcagga cgctqctgga cccgcaacgc ccgccatcac ggagatagaa acgtgcagtt gcaagcgcct cgatggacgc t caccgtt cc tggacgaacg atttcgcggc tgctcgcccc agaccgttct tgttcgcqg aatcgggcgt agatgaacga aacagttccg tcacccaggc accagccgac ccggttcgat ccccggccac aactcgqgat aaqtcgtcqg 5100 5160 5220 5280 5340 5400 5460 5520 5580 5640 5700 5760 5820 5880 5940 6000 6060 6120 6180 6240 6300 6360 6420 6480 6540 6600
'N
WO 99/37660 WO 9937660PCTIUS99/01313 -142cgaagagcac gcaqgacatc qgcccgcgcc caccggctcg ggttgcgqgc tqacgtqatc taccatgcag gqtccgtctt cctctcgctg cgtgaccggc tctgacccg agtcgcggac ggaagcgctt cggtcatcgc gaagqtgcaq atcggcgtgg tggacgggcg gagatcccgg cgcatggaca tggggcgaca aatgacgacg cgatggtgcg ctgqcagctc tatcaggtcg atcgccatcc cggaagatcc gacggcgtgc gaatacgacc gcgaaagccc ttcgatctcg cccggcgtgg cgtcccggca ggcttcgccg gcggaactga gccgcgcatc ggctcgcaca gccggggccc gcagccggag cccagggatc agggcccgcg cggtgcaqgt tttccgccga cccgcgtcgc actgqgacgt cgcgactttc gtggtgatcc cccgtgacgt tcggcatgga agcgcttcct aggttccgct acctgccgga agcgcctcgc tqtcgccgga aaggcqatct tcctgaccgt aggtttcqggg ccgccgcggt cgtcggtggc tcaatctctg ttqctttttg tgagcgacag gctqgtgctg cgaattgcgc gtcgctgtcg catggtgacg ccgcgtccgc ggcctgatcc ccatccaacg tgtgggggac ccaagggatq cgaactgtcg qtcgcagcc cgaaqacacc agcggccttc cgctgcggcg acggcggctt qacggcgatg ggtcagcgcc cgagaaggt g tcatgccgag cgatgccgcc acgggacat c cttttgtctt gaacggatga ttttcggatt aggctggtgg atgtgggaca gccqagggct gcgqactggc cgcccggctt aggcccgccc ctgcaacgct gaagaaqaca ttcgacgtgg atcaagtcgt tacatggtcg taagggggaa gcctccgttg cqggccatg gcgggcacgg accgttctgg atgctggccg gcqaagatgc ccgccggata gccgcgccgc aqaagttttc atctggacgg tgttcgacga tcgaccagaa tcgtgatcgt tggcgatcgg gactttccgc gtgcaacaag acaagtcgct agctgacqgt cgaaagtctt t caaqgcggt gcggcatcga ccatggccga ccgcgagcga cgcccgtcat ccqaatacgc ccgagcgcgg agqccaagaa tggccgacat tctcgggccc atattagcgt ctcgacccqq cggcgtgatg agccttccgc gcacaatccg ctttcgcacc Cggctgcqcc CCccccgcqc atgccccccg 6660 6720 6780 6840 6900 6960 7020 7080 7140 7200 7260 7320 7380 7440 7500 7560 7620 7680 7740 7800 7860 7920 gaaatatccg gtcgccgaac itcaacgccct 7980 gatcgagacc qtgtggcccg ggcctcgag <210> <211> 9810 8009 WO 99/37660 WO 9937660PCT/US99/01313 -143- <212> DNA <213> Unknown <220> <223> Description of Unknown Organism:Unknown <400> gatatcqggc gtggccgtcc catcggcatt ggcgtgaccc gagaccggcc tactgcctcg cttgcggccc gogccogato qtgatcgagq cgctt caccg ccgcaccggc gccaagtgcg atgccgcgcg gggagctcgg ggctgatcca tcctccggcg atcccctgcc agcggccgat tgacctcgat atggctcggc actatccgat cgctaaagcc ggctgggaca ttgtcatttt tcatcgcaga ttctgggccg t gcccgaqac tqcgggccat ccacgtcgag tcttcgaggg tgttcctgct ataccgagcc gcggtagcca ggttcgacag agaccctgac cgcgggctgg cacctcgcgt cgtccggctc cttcgggttg ctccatcgcc ggtqqtggcc ggtctgcgaa ctcctactac gccgatgccg cgtgcagtcg gatgggcgag cgattqcgac actggaccgg qagcttcccg ctt ccaggt c gctcggggcc cacgccggcc acgctgcttc cgccgcggcc cggcqtgcgc tttcgcgaac cttcgaccgt atgatcqacc ctctattatg cagcgggcgc gagcaccqgq aagctggccc cccaccgccg ttcggcaccg cagcacaaga gatgtgatct gtcatcqgcg qtgctgCggC gacgcgccgc ggqgttctgg gcgggcgtgc gctqttcagg gaggaacgtg gcggagaccg cggctcacgc accgcgagcc gagatggqcg qcaggcctcg cgat ccc ccg ttctacgaga ggcccgagag tcgcaggcct agcggctggt tctcgggctg gcgtggcgcc ccgccgaggt gccggtcgct tcgtgtcgct tccgcatcgc aggatgcggc ttgcgcgcaa tcctgaagga ttgattcgga gggtcgacga aggtcgtgca cccggctgct tccgcgcgct gctcgctgga aggtggcgcg tcgcgcccga cggccgggat aggatgcgcc ccctgcccgg cgagccgacg gactcaggat gagccgggcc cctgcatctg gagtctcggg ggagcgggtg gcgcgccacc caacggaaat cgaccqtgtg ggagcgggag tgccgatgtg cgggttcatc agttctgqcc ggccttcgtg gcqccagttc gtcagccttc gtcggtgccc gatcacgggc cggcaagccc acgctgcctc gcaggtctgg cgatgccctg cctgcgccgg cccctcgacg cagat cgcgc atctccgaac gaagccgcgc tccgaggtgg ctgcgctcqg gtcgagqaga atttctgcgg cqtgcgccgc ctqtttcatg accttcgtcq acgcccgagg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 WO 99/37660 WO 9937660PCT/US99/01313 -144agctgaccga cggagggtcg tttccgaqqa acgcaggctt cacttctgtc tcggtgagta gatgaccgca cgccggcgcc ggggctcatg cgaggaaaac cgacgtgctg gagcctgaac cggcctgacc catgtatcgc ggacttcgtg ctqcctgcgc cagctacggc gaaggccacg gaaaaacggc cgacgcgacg ggtgggcttc ctggaacctc ctgggcgacc gcctccgggg gaagatgacg gccttacgtc gatgcaggat ctacctcgaa tcggacggtg aagggccgt ctgaagccgc attattcgcc agatttcgcg qaaaccatca tccgagttca gtgctgcgcc accatcggca gacctgccgc gtcgacggcg aaqgacctga aaggaagcgg ggcaaggccg gcgcgctggt ctgaccgact ttcaacgaga gtgqccgcct gcgctcgccc gcgatcccgg tcgaaqgact acgcggacgt ctcgacagca ggtgtgcagt ctgggcgccg accaqcatca gtcgccatcg cttttcaacg cgaaaqgcgc gcacgaaata ccctgatqggg ccgtggcgac acgcgcagca agaaggtcac cctacqaggt cggagtatga agctctatgc tggaqaaggc cgcagaagat gctggggcga t cgacgaqaa atctcgacat acctcgcqct ccttcgtgac ccqataccgg cgggctcgca atgccgagct cgctctacga tcaacqcggc tcgtgqcaat tcggagaggt atcagcgggt ccggtggcag gcctgatcac ggcaaaaagt atgctcaccg cgcgtgcgcc tgtcaacaac ccccgacatc qaccgacatc tccgatctgg tgccgacgac cgcgcccttc ggggctgacc gaccqacaag qaacatggcc ctggcagccg gatgacgaac gttccagcag caaccccgag caagggcaag gaaggtcgat ggtggcctcg gaacccggaa tgacccgacc ccccgagttc ggcgggatgg tgcgggcgtc gcqcaagctc cgacgagttc atttgacagq tgcaggccag qtggctgcgc ggcgacatga accgtcgagt gccaccaagg ggca agcagq atcct gcccg tacggcgaga atgcccgacg gatgccgagg ttcct cagcg cagttcgacg tacggcccgc ggcaagtgcg gaat ccacgg cgggccaact gccgccaagc aaggaaggct tat cagaagg cacccggcgg cagggcat cg gtcttcgact Cgtgtcgaac gcggcgctgc acggcgcagg ctggcacccc ggaggatact tctcgtccgc tccgcatgca gggtgacgct gcgggcagtt gctggctcgt cgatccgcaa gct cgatgat ccccacctq tctacggcat ccatggccaa gcgaggcctg ccggcgcctc gcatgtggat tggccgacaa qgctcggggc agttcatcgc gggccaacgt tgccgttcgc tcgatccggt gcaccgccgt 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 WO 99/37660 WO 9937660PCT/U599/01313 -145gggccaqcag ggcccagcag gcgggccggc ggcctttccg cqcgtctgat cgatgacgct tcaccggctg ccaacacgat tcgcgctcct ctcccttctt ccgtgaacgg tgtcgcaqgc ccacgctgat agatgqacgg gtgccatcac tcctcqtcac atgcgcagtc tqgtgctcgc cctgacatgt acgatagcct gaaggagacg tacgccgatg tcgctgqgct ttcgtqccgg ccggcggtgg acgcggatcg ttctcggcag ttcacgacgc cctgagcggc ccccatcagc gatttccccg ctacttcagc ggacaattac cctcctcgtg gctcgaccag cgtcatgccc gatgttcgcc gccgctggcc ccttctgacg cgcctcggcg cgtggtggtg gacgaacqgt gctcctgaat caatatcgtg cacgccgcac tcctcatctt ccat caaggc tgcaggaacg cqaccctcgt gccqqcggac gcgtgctcat gcctcgtgat ccctcgcggg gcgaaatgac cggcccqcac cccgaggcct gccgtgatcc ttcctgcqct tattacttcc gtcggcgtcc cccttctqggg accgtctcqg catatcqccc tcqatcatcg gcgct ccagt ctcgaccggt ctgatccaga ggacccggca tacqacgtgg gcgatcttcc ctcaacccgc ctt cccgat c gcccttcgcc gtcgaactac ggcgctcgcc gaaggacgtg cccgctctat cgtgctcacg ctcgatgtcg ccgcgcgggc cgcttgccgc ccatggcgac tcctgttcct acaacctcct tqaccgatcc ttctcatcac ggcagggcat cgctggtctg gcgggctcgg gcatcgtggc cgctcgaccg tcatccacat ccatcttcct ccgcctcgac gggqcgqgtc tgatgcgcat cgcacgctga ctctggacqg atgctcttct gcccgccact atcgcgatcc ctgatgtgga ctgatcttcc ctcatcaacc gccgagcagg tacatcaagt ttccgqccgt ccagcattca gtggatgatc catgccgggg qqccttct CC cgtggtgggc cgtgcgcgtg gaagaacatg ccttcqccg ctggcagtqq cgagcagatg caccqtgccg tctgggcgtc caacatcacc ggccggcggc gat cqgcaag tcgtcacgct tgctgatgag cggactggac tcatgaattc ccgccgcctg tqctgtcgac gcgacacggg tgccgatcgt cgCttcaggc gagcccttcc atccgccqga aagactgcgg gtgccqctgt atggagagct gcggccctga ggggtcctgc ctggtqatcg ttcatgaacc ttcgacttcc ctgcccttcg gaqgcggccg cacctgacgc ttCgCcqaga tacctcgtct atcgtcgccq aatctggacg cgccgcctgg cttcaaatcg CCtgcaatcc ggtggtgatc qqccatqqcc caagatgatg ccttctccrac ggtctgqatg 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 WO 99/37660 WO 9937660PCT/US99/01313 -146ctctacacct gcgacgctcg tcqacqctqc accacctcgc ctcttctacg ggctggttca ccacatgggc cccctcgctc ctgcggcaaa gatcatgatc ggtgtttcag gctgcggatg gatcctgaac gcagcgggtg gccgctctcg gcaccaqtcg catgqccgac caccctctac gaacctgatc cgaacatatc acatctcggc gcggacgggc cgacaagatc agaccgccct tgcgtgaagg cggccgagat acttcaagga qctccgaqat ttctgaacgt qggcggcCCC ccaaactgtc gccagaaaca aagataaccc gacctcgaca tccacgctcc gacgggcgcg agctacgcgc gcgaagatqg ctcaccaact gccatcgggc aacctcgatg ctcgagacca aagatcgtgg cgcaatccg qaggggcccg gacctgtcgc tcggacacgt ggagagttcg caccqcttcg catcaccggc cgcqcgcgtg cggccccgcg gatcccgggc cctctatatc qatcctcgcc gctcacqcag gqcggcct cg actcgtccgc tgcgcaacgt tcgaggatgg tgcgcctgat acqccaccga tctatccgca agccacagga atctcgaccq qcgccatcgt cggcgctgcg cgatqatcta tgctgaacgc cgaacctctt aggccgccaa gcqaggcggg t cctqcatgt gcgtccatca qcgccgacgg tcggcgcgcg gccatcgccq gcctgcgcca gaqatcctcg ctcacgccga tqqaacgagg ttcatcgcga accatggcca ggcctgacct ccagaagcgg cgagttcgtc cgcgggcctc gatgccgccc catgacggtg gatcqagcgg ccgccccggc gcgcgagccg ggtcaacatq tqtcacccac gggccggatc cgtggcgggc gcacggcgcc ggcgtgggag qcatgtcgcg cqgcgaccgg aaaggcgctc gcataggccg acatcaacct tcgccctcga aggcgqcgcg tggccgtgcc ccttctggac gctattccag tcgcgccgat tcggcgcggt ttcggtgagg gtcttcgtcg gaggatgtgt gcgaagcgcg aagaagaaca cgcgtgtcga cagctctcgg gcggcctt cc cqgctcgaga gatcagqtcg gagcaggtgg ttcatcggca accaccatcg ggcqaggt cg gggatgccca gtctggctca tgacatgcgg cgccttcgcc cgaggcagcc cgtgaccgat gatggacggg gqgcatcgcc gctgcagctg ccccgagggc cctgatcctt gaagtgagga cggtcgtcat gcccctcggq cgqacggcca gcctcgccat tcgccttccc acgcggccaa gcgggcaacg tgttcgacga tcaccgagct aggccatgac gct cgcccct gcccgaagat gqatccgccc gcgtctcgga ccct caccgt cgccgcaggc ctcgacggca gaggcctatg cgcgccaccg caggccaqca 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 5520 5580 5640 5700 5760 5820 5880 5940 6000 6060 WO 99/37660 WO 9937660PCTIUS99/01313 -147tcgaccgctg atgcggccct tqttcqcgat tcgcgggcgg aggcgctqgt cgggqctgaa acggcgagca gcgagaagaa acctgaccgg cctacaacgt tcgaacgcgc gaccgcaagq catcaggcgg ctcgqcgcgg aatctctcga atggtgggct ccgcgcatcc ggcgccttcg gcgaccgcct cccttcaccg gtggtgggcc gccttcccga ctggcgcagg tgqgtgatcg ttcaccccgc gcggtgatcg cgtggccqag cttcgatctg caacgtctcg ccggqgcggC gggcgtctat cctcatccgc ctgggacqg gcgtcaggtc catqgcqatc ggacggcqgc cccctat ccg cgctcactcc tctatctcga gcgtccgccc cggtqatcga tcctgccggt gcatcgtctc atccgacgca tcggcgcgat tgatgtcctg tggccgagct acggcatggt gcttcggcct aqgatcattt atgtccatgc cctatccgtc cttctcgacc gcgcccatcg gqcacgctct aagatcatca tgcgcgacca cacgggatca gtggatgcga ggcgcggcgg ttcctcgcca aactqgatga aggtaat ccc cggcatcgtc cgatctcttc gaccgatgcg gctcgatccg cgaqgccgac gctgaccgtg tcccgatatc cctcgccgcc cgacaacctc ctacqacgcc cqaccqcatc cgccgatccg ccccgccgga ctacgaggcg ggcgctcatg gctgqggcag tcgaqatcac tcatqatgca acatggcaag aggccgccgt acgtcaatgc agttcgccga tgcccttcgg cgcccgaggc gctgaggccc atqacccgct catatcggcg gcgctgggcg cggatgcgcg gcgggccacc aatgcgqccc accgagggcg gtggccgatg ctccgcgccc cccggcaacg gagcttgcgg acccccgcca gtgcccgtca cgccccgcgc atgaagatcc gacatccagc catcgacatc ccgcqaqagc gqcggtgqca ccaggccggc catctcgctc catcgccccg ctacgagaac ccgcatggqc cgactacatc aaggcccgqc ccgtcacccg tcggcaactt agggccacga aggctctggc gggcccggca tgatcgaggc gctattatgt cqqcccatcc gccgcgacgc gccatgtcac gctgggtgaa ccggcccgca cctgcgagcc tcgagaaggt gcatcctgaa tcgtgcacgc ctcgtgaaca tacgaccggc cgggcgatga cqccgcgqcg acccagagcg ggcgtggtgg ctgccccgcg cgcgccgagg qtggcccaga cctccccccg tccctcctat ccaccgggcg ctgggccatc cgcgcaggac ggtgggqgcg catgtcgqat cgatqcctcg tgcgcggccc gggggttaca ccgcaacgcc ggcgcaggtg cgaqcgcgaa gttccggcag gggcgtqacc cgggqgccat ggccatggcc 6120 6180 6240 6300 6360 6420 6480 6540 6600 6660 6720 6780 6840 6900 6960 7020 7080 7140 7200 7260 7320 7380 7440 7500 7560 7620 WO 99/37660 WO 9937660PCTIUS99/01313 -148catccqctga ccgcccgtgc aaccccgaga aagttcatcg gtgctgctct gtcgaqccga acgccggccg ctggcggccg ctgcggcgct gcccctgccc ctcggtaaca ccgggcgagg cccgttagca acacggggca cggaaggcac ggcttgcgga ctcagcgcgc ggatcgtcgt cgcgccggcg gaaatcgctc caccgacggc ggccgccgac ctggggcaag cqgcatcaac gcagcacaac ccggcgcgag tcgcggcctt ccgacaccag tcgccgacac tgccgtcqct cggcgctctg acgatccgaa agtggctggc agttcqcggc tcctcgcggc cttgcggcgc ccatggtatc cgccggactt cagtggtagt cgcgatcctc ctgcccgctt aagccgtgcg aqccggcgga tcattcatca cttgcccagt qacacgatct aacttccaga gccgtgaccg gatcccacat gcctggcact atcgtgqcct atcaacaCCg cctgcacaag cat ccccgac gacgcqcaqg qcgcgacaat gtgccgctac ctggacggcg gatgaccgaa agccct cgag ctgatccgca accgtgaggc gcgcaagaat gtcaaqgcqg gcagcgctct cctccgcatc gtgcgccgcg cctcggttct cagaagccgg ccaaggccqc ccgcgcccaa tcggcgqcgc tccaggtctt agggcaccgt t cgcqctggc accatgqcgg tcccqqgcgg tgqccgacat gt cgaggt cg tat cttaccc ctctgcctcg ctggcggcgg tgcttcqgca ctgcaggacc gtctacggcg gcggtctggc gggcccagcc gaaacgaccg gccggcgcct cggccctcgg tgtaaaqcga ggcgctcgcc ccctttcctc ggacaqccgc cccgccacgt cgtggqaqggg ggtcacctgg cgaagtgctg ctcggcgggc cgaatgctgc cgcqgccgtg cggqat cgac caacaccggc gcagggcctg aggagatcct tgatcgagag acggttcgaa qcacggtgcc cgacggactc gggcgcggcg atctgqcgca gcgacggggc qct cqgagca ggccaccccg ctgccgaacg gtagagagqg aqqtcgttcg cccggtctgg gcttcccaag cccttqcggt ccacaaggga gccgccgcga aggctcgcct tcgaagatgc gagctggtgc cacacggtcg cccttctcqc ctctacaacg gtgcagatgg aagatgcggg gccccatqtc ccgcttctcg ccggcagccg gaaggggctq ggqcgttgtg qgcgaaggag gaacgatctt cgaggcggtq ccgaaqcgga gggccaccgc ggcccggct g cgggcgtggc ttcaaatcgg actgcctctt cgtctgtcac gtaatctgcc ggaatgccat gcgccctcqc cctccttccc tctccgaggc cgggcctgca getactatta tgtcggcgcg atttcctcgc gcqgctggtt tcggcggctt 7680 7740 7800 7860 7920 7980 8040 8100 8160 8220 8280 8340 8400 8460 8520 8580 8640 8700 8760 8820 8880 8940 9000 9060 9120 9180 WO 99/37660 WO 9937660PCTIUS99/01313 -149tgcggggaag ctatccgqcg cgaqaagctc cggcccgacc tcagtcgctg ctggaagaac cagccccgag agcctcgaac ct a cacc tgg cgccggcaaq gtcttttc;at gtgatggagc ctggagaagg ggcttcttca gtccatttca ctgcgcaccg ccgacggcga atcctgcagg cccgccttca gcgcagatcg ctctgagccc gggcgacagq gt ctgqgcgt ggacqatcqa aggtggcgcc tgttcaacaa cctgccacgc tcaagtcgct cctgtttcga agaagatctg ccgaatacaa gagcgccgg gqCCgqcgCg cgtgccgcag cgcgaccgaa ctactactac gagcgcctac ggccgatgcg ggtggcgcag ggccgcqaac ggactcgatc ctacgacacc cgaaagagga cagatcgcgg tgggtcggcc tatCCCgct qaggggctqa aacatgctcc gqaacccagc gaggtctatq aaggccttcc ttcatgatg ccccggagcc gcggcgacat cctatgacga qqtgggaagg ccc cqgcct a agctctacga tcagqccctt ccgagatgga gctccgagca tgcagcagaa gcgttccggg 9240 9300 9360 9420 9480 9540 9600 9660 9720 9780 9810 <210> 36 <211> 1886 <212> DNA <213> Unknown <220> <223> Description of Unknown Organism:Unknown <400> 36 tgagtgtcta cttcaatgtg gcaqctgtgt gctaattggt tccactctgc gcgatgtcgg atctcctcca ttgatggcag gctgagccgg aagccgctct ttttttttCq accaaccatc gtgccactgg cggcacaqgg tgcctggaag aggccacaac tttggcttca ctgtaacqca agggggcgga tctccacccc gqttttttta ccaaggctct gCtgttggtg tatttccaca cgcgcctqcc aaacaattca actggctggt acqcaaggaq gcccctgaag ggcaaattcc agtgtgaatc aattcaacgt ttggtgCttt aaggcgctgt aaacaccagc ttcaaacagt gtggccgcca atcgccgtct cggccttttc ttcagcaaca acatggttag ttgggtgtgg actccccctc atccggcagt tgcatgtttg cattatttgq ctttctttgc tCtccqCCtC cqtctcctgc tcttccaggc gagcagttgt gggcccgctg atcgcaaacg qcctgtgcct qagggcacat gtacaatgcc atttggaqct gggtcaggct tgccaaacct gcct ccatcg 120 180 240 300 360 420 480 540 600 WO 99/37660 WO 9937660PCT/US99/01313 -150ctgcgcacgg aactggccct gtcgacttcc agotccaccc ttcggcaagg caggaggtgc atgaacgaqq gacgagaagc atcgacctcg tcgaagttga aagqaqgtga gtgaagcagg aaggggcgcg gacctggcgg ctgaacctgg gacaggaaaq aacgaggacg gagacagcgg ccacctcccc tgccatcaac ccaggttgct accgtgatgt actccaccta ccaacaacgc tgggggacag tcagcaacct qcttcggcaa tgtcgtgggc acatcaagta tcggtgtgat tcctcgtcag agtacgagtc aggagctgaa cccqaaagtt agaaccaqtt acatgggcaa ccccggccgg ccgcggctga aggatgtgga aggggacgga cccggcctcc ggccgtcctt ggcctggctg gcgccttttc tggccgagqc actccqcaac aagccctgga cggcatgggc aggccggqgg cgaccgccgg cccgcagctg gt cgaggcag catcgacacc cgagaagaag ggtgtcccat cctgqagggc cgtggagatc ggcggacgcc ggaggcgctg ggaggagggg gggggagggc ggtgccaacc ctgccggggg gagcttaatg cccgcagct c gccttc ccgcgctcga ccccagtcgg aacccgttca ctaggcctgg ttccccgtg cgggcgctgq cggctggtgc gaggcgctgg gaccccccgg aagaaggaca aagatcggcc ggccaccgca ggccgcgcgg gtgcccaaga aaggtgattg gagggcqacg gaggaggaag cgcagctgat ccggcaccat ctcccgccct ctcccctccc ccagcttoac tgattgacat cqcggctgCg gqctgggcaa aggaggaggt oggaccccga gggccgtgcc agctggcgga tggccaagct gccacaagaa agcacgacta tcaagqtgtc tgatgaagcg agctcggcac oggagcggag acctcgactt aggccgagga cgccgatccg ccgtcgttgc ccgttqgccc cgactgacac cgacatcagc cgggggagga ggggtccccg gggcaagggc qgaggaggag cgccccgccg gggcggccgg ggcggaagac agtcaattac ggggaaggtg cgacgtccgc gatggagttc ctt ccagaac ccggctgatc ggcagagcgc cgtggacgag gct ggaggag cgggggacag ggt gcggcgc gcggcggtcg agtgtggatg 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1886 <210> 37 <211> 3015 <c212> DNA <213> Unknown <220> WO 99/37660 WO 9937660PCTfUS99/01 313 -151- <223> Description of Unknown Organism:Unknown <400> 37 ccgct cat ct cagatacagc aqcqgccgtg cggtgcqcgc qtgcggtgag cagccgctac ctcctctgtc ctttctgcqc aacgaccgaa cgacaacccg cgccatccag ccgcaccaac tcttgacaat ctggttcaag cgcctcgcag qtcgtacaac gt tcggt ga c gctqctccag tgtgaaggca cgatgaagcc ccgcatggtc tcagtacagc ctgctggctc cgcggtcgac cagcgaggqt ccaqgcctcc accatggcct tcggtcgacg agcgcactca ctgaccggcq aacaacctct gtgcacacac ggcctcttCC acgctgctca gcgctgcacg agcgctgcca tgcgtcctcg gccaccaatc tatgtcttca aaccttgcgc atgttccagt cagggcqaga gacaccgcag gccgacgqca gggaacaagt gactactcgc aacatggatt taccgccacg caccagtgct accaccacca ctgagtgcgt ctaggctcgt cgcgcttcgt tcgacgacaa agggtgtcqa cattggtgga gctccaccca aggacqacta gcactgacgc ccgccctcaa agqacgcatg acatgtacga tcaaggcggt gctggaacca aqacggtgct acagcgctca cgacqatgcg atgccagtgg cctatgtctt acctcgcaca gccccgccgt gcccqcgcac tttgccctag accccqggcc gcagcagcga acccgagagc ccgtgtqctg ggtgcgcatg cacqacggag gatggtccgt gagccctctc cacccagcgc cttctacccg ggtqccgtCC cccggtgatc ggtcgagggc cgtggccgCC gtatcccggc cacgaqcaaq ggccaacatc cgacacaacg tccgcccttc ctqgtacgtg ccaggagtct caccggcatc ggctgacqgt tatcgcggac caaggcatgc cgacgttacg gggtaccgcc qgcaagtaqa gcgqccgcca gtgcaggtgg atttgtggca gctatcggcg ttcccgtcga accatccaga gtggtgtact gtggtgggcc gatqagcacc ctgtgcgcg gccttcgagg cttcaggagg ctcgatctca aacgcccacc gtgact ccct gcggttacca cgcctcatcc ggtat caagg tqcccgctga cactgcgcca aacaagccgg ccggacagct.
aaccccacca accagcagca gaaaggaaca tgctggttgC tacaccqcca ccctgtaccc agtttgcccg cgcggtacaa gcgcgaccgc cgaccaacag gtagctggct tcagctgqga actacaacgc ccgccgqgcg tgaacgccgc cgcagggctc gcctctctcC tggctgtcac tcttcgtgqa gcggcaaccc catactgcat at agct t ccg tgacacagac tqccgtcgcg acattctctc gcagcagcag gcgacgttac 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 WO 99/37660 WO 9937660PCT/US99/01313 -152caccaccagc cqagggtacc caccagcagc cgctaccacc cagcagcgag cgccaccagc cagcgacgct cagcagcagc cgttaccacc cagcagcagc qggtaccacc cagcagcgac taccaccagc cagcgagggt caccaccagc cgacgctacc caccagcagc cgagggtacc cccttccttc ccacattgcc ccgaaagcac ttgtgcatgt cacaaccgct ctttgtgggc ccatcgctct cgcgtccctc agcagcgagg gccaccaqca agcgagggta accagtagca qgtaccgcca agcagcgacg accaccagca agcgagggta accagcagca gagggtaccg agcagcaqca gctaccacca agcagcgagq accaccagca agcgacgcta accaccagca agcgacgcta accaccagca aaaaagcccg qggacatca gatgagtaca ttgaatggag cgtttcaccc tctctctggg tacctqtggg tgcag qtaccgccac gcagcgacgc ccaccagcaq gcgagggtac ccagcagcag ctaccaccac qcagcgaggg ccaccagcag gcagcgaggg ccaccaccaq gcgacgctac gcagcaqcaq qtaccgccac gcagcagcga ccaccagcag gcgacgttac ccaccaccag gcagcagcag cgaactggag tccgccgcgt gccgqcaccg aggaggcttc ccgtcatctt tgtgggcgct ctattttttt cagcagcagc taccaccaqc cagcagcgac cgccaccagc cgacgttacc cagcagcagc taccgccacc cagcagcgac taccgccacc cagcgacgct caccagcagc cgaggqtacc caccagcagc cgctaccacc cagcagcgag caccaccagc cagcgacgtt cagcagcaaa cccgcgcgtt gacgaacggt ccaacaqtag tgtacagcgt ttcattttga gacgctacca ccagcagcag 1560 agcagcagcg gctaccacca agcagcgacg accaccagca gagqgtacca accagcagcg gctaccacca agcagcagcg acca ccaqca agcgagggta accagcagca gacgctacca agcagcagcg ggtaccacca agcagcagcg accaccacca agcacaagtt ctctcgccgg gttacgatcg cacaacggca acattgtttc tctccgtcgt acgctaccac qcagcagcga ctaccaccac gcgaggqtac ccaqcagcag acgctaccac gcagcagcga acqctaccac gcagcagcga ccgccaccac gaagtgacgc ccagcagcag agggtaccgc gcaqcagcag aqqgtaccgc qcagcagcag catcgqatgt aaaggggccg gtgcgggtgt tgtaactctt gagaaggtat ctcatactgc gtcgttgacg tccctctctq 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3015 tgtgcgtqtg tcgctgtaaa aattatqqtt tattattact WO 99/37660 PTU9/11 PCT/US99/01313 -153- <210> 38 <211> 38186 <212> DNA <213> Unknown <220> <223> Description of Unknown Organism:Unknown <400> 38 gatccttcct tccctgatct tgtaaattca gggccatgtc aggatgtcag attgagcgcc cctagggatg gacgaggacg tgtagcacct gggcaggacg t aca ccc gt t catttgatga atgcataaaa actccagaac cccgtcccca caaagtcaaa gcactttcag ccaacatggt cgcctgtaat ggcggttgca gcctcttccg ctgcctccat tccgtcgttg tgcagagacc cctgtctttc gactgcqtgc gcqgccgtga gggaggcgac ggctcgggca ggcctgacac gctcccagga ctacqgtttc tgcggtqgct attctcatqq gcccctgqca tctttaaacc agqccgagac gaagccccgt cccagctact gtgagccgag gcgtctgggg ctgctcacag gatcagggcc ctatttccac tgggagatgt cctgccgtgt tcacgcaggc atcagcaagg gaggccccga tagggacctc ggcacacact cattcattca tttagtatta gcccaaaagg acccacgcta cacatatggc ggqcagatca ctccactaaa ctggaggctg attqtgccac ct ccaggcgc ccttctcccc cacccggttc ataaggtcct agttcaaccc gcttgaagqt aqcagagagc ccgtgtqtga ggagggctg gggCCCCggg gcggttcgct tttgtagaga acagaqtqct agacctgggg cttagtcatt taggcgtggt cctqagqtca aatacaaaat agacaggaqa tgcactccag ccttggcttg tgtgtgtctc ctcgtggcct attcacagga acaacacaca cccaccctca agct ctggga qccaggcagg gaggagctgg aatgcctctq tcgccaagaa tataacactc gcacccgata tgttagtcac atttaggtgt ggct cacgcc ggagttcgag tagccgggcg atcqcttgaa cctggacaac cagattqatc tgtctcttct cgccttaact accggqggtc tcaaacagtt ggaagcgggg agcqgggagg gtgtccccgg gcgaggaggc ggggggcgtg tqtttaattg agaccacaaa ccacagcctc cagctcactc ttaggagttg tgtaatccca accagcctgg tggtggtggg cccaggaggc agagcgagac 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 tccgtctcaa aaaaaaaaaa agtaccaaaa agtgccccag gtcataaggg cacagctcga 1260 WO 99/37660 WO 9937660PCTIUS99/01313 -154tagctggtcc tgccqtcctt tcatctccag aatgtagctt atggagtctc cctcctcctc gcacccgcca qttggccaqq agtgctqgga tttttttttt atcacggctc ccggtagaga cgatcctccc gcctcctttc gtcgccaata tggcggcaqc gggtccgaga cagcggaccc ggtggcctcc caagctcaag gaagaagatg gatgaagctg gggagggggc ccgqqgatgg ccggggatgg ct aaaqggaa agaaqctgcc acccttggtt tccactgqtg gctctgtcac ccgggttcaa ccacacccag ctggtctcga ttacaggtgt ttgagtcagg actgcagcct cgggqqtctt qcctcggcct caccgctctt cggtgtcaaa agctcggttc ggtgagccgg agccctgcct atgccgctgt tt cgaggtct tacttcatcc tccgatggct acgcgacccc ggatggggat ggatggqgat cgtggtgtaa tttgctaagg ttcatgttga tgtgtgtgtg ccgggctcca gagactcqtg ctaatttttt act cctgaca gagccaccac gtcttgttct cqacttcctg accgtgttgc ccgcaaatgc gtctacagcc cggcgaaggq cggctacggg tcccagaggc cccccctgca tcgccaacgc tccagccgqq agcacggcqt cctacttcgq cgcggtgtgc ggggatgggg 99g9CC9gqq ccaccacaca ggaattgccc tttttcaaaa tgtgtgtgtg gtqcagttgt cctcagcctc gtatttttag tcaggcqatc qcccggcctt qcgctcaggc caccaccacg ccaggctgqt tgagatcaca gcccctcctq qcactgagcg gctgagcccg agggcgaqg ggagatcgtc cgaccccaac tgactacatc ggtcagcgtg gggtgagctt agagccaggg ccggggatgg atggggatgg gaacgaagct tgacttccca atcacctgat tgtgtgtgtg gtgatcttgg ccgagtagct tagagatggq cacccacctt atttttcccc tggagggcag cctggctgtt ctagaactcc cgcgtgagcc qtccgattgt ttttttcttt tctctcagac gqaagcggga aacttcaact ttcgtcacqg atccqcgaag ctcactaagg gaggggggcg ggCCggggcc ggatggggat ggccggggat ggaacqttcc caccattgat aqtctgaccg tqagagagag ttcactgtaa gggattacag gtttcaccat ggcctcccag cattttcttt tggtgtgggg tttttttttt tgggctcaag cccgcacccg attggcagat ctcccgtcct qaggaaactg gtgqggt ccg qccggaagct ccatgctgac gcaccatcgg qcaacaaqqa cgcctggagg gqggCCqgqg ggggatqgg ggqgCCgggg 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 atgggqccgg ggatggggcc ggggccggca ccagggaqag cctgqgtggg aagcgcccac 2820 WO 99/37660 WO 9937660PCT/US99/01313 gctggccaag tcgtccagca ccgcacggcg cttcaacgag cgaccqcctg gggggagggg gtggctgtgg gccatggcag ttgggtctaq cccaagggag tgtgtgctgg tggactggtg tgggcttccc cggggaggaa agaggcctag cagacacaca agccatggag cgtgtccagg cggcagagcc gtgggaatgg gggagctccc aacccgtt cc acaggctgtg cataatcagc tgtacacagc qtgcagaggc acccccccct agcgtgcggg gtgctqgagg gaccgcat cg cgtggccaag catcaggggc caggggcgtq aqgctgat tt ggaatgcaca gggtgcaggg atgaaatcac tgagttggga gatgtgttcg agtccagaga qggcccaqtg ggttctaggt agggagacgg ctqatgactg gaatgaagac tggtgtacag tgagtatcag tacaqqcagg gtatacaggt aggcattaaa cgggccgtgt gcgcgccacg ctgacaccta agtaccccat gtgagcgggc gcatcaggag acggttgggg gct gqggcag tct ga cct at gggtgtttgc gacagacccq acacgactgg atgcagagtg tggagtggac gccagggagg ggcggcaggg gggcggagqa tgacctqgcc gatgtaggcg catggctgaa taggaaqctc gtgcttaggt tgttttcggt ggtacacatg tacctgttta gcctgggcgg tgcagagatc ctgccqcctc gatgcgqcgc cgggggcqt g agtggcttgg cagagacgtg gggcagcggc tqtcctactt ggttgtgccg ggggcccact gctgtgtgcc gagaccaggg acagcacatc cctggaggag agagtttaga cctggtcaga tctcaggqggg ccagagagat acgcaggaca tccacaactt tataacttct atgcctgtgg catacttggt ctgccaggcg ggagggccgc ggcgcccgcc 2880 tqcctgctca tattcgctga gccttcgaga gCCggggcgg acagtggcag gccaagqcat tggccgctcc cagccaqagg aatgctcggt caga ctccca agcagggcag taagqqatgc ccaaggccct gttggggaag ctaaatcagg ttgtatccgc gcagt ctctq qgcggctcat ggtgcgacgg gctctataca gtatacagca cacactggag gcacagtgat cggtqqctca cccggggccg gcgtggacaa cggtgqccat gtgccctggc ggggaagggc caggagtgtg taggacccct caqcctgttt gagcacctgc gggaggctta gtggggccgg catgtggaaa gaggtggaaa aaggggaggc agcatcaggg caaggcgggc gggcagggag gctgctgttc agtggtgtca gtgagtatgc ggtgct caga gcagtcatta acctgctcca cqcctgtagt 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 cccagcactt tcggaggcca aggtgggtgg atcacgaggt caggagattg agaccatcct 4380 WO 99/37660 WO 9937660PCTIUS99/01 313 -156ggctaacatg ggtggcgggc ccgggaggtg gagcaagact cttcatgcat tccatactat catgcttgct tqcccgctgt aagaagaatt aaccaggaqa gccgagctg tcggccatcg ccgctcgtgg cccgcacctg qccagccccc gggcccgccc ccctcgctgc acaccgcgct gactcggcct ctctcgtcca ggccgtcatc qccatagacg ccctccgcgc cccggcggcg ctgtaagaca gtgaaacccc qcctgtagtc gaccttgcag ccgtctcaga gctgqctgtg aggccagaga ctacacggca acgcagcagq ccatcctcct acgccatcat gt cagcqcqt ccacgctgca ggccgctggc ccgccgcctc gggcaccgcg tgcccgcgcg ctcacggcgc tggggcccac cacccggcgc act tgt gacc cagaccaaag agacgtaggt ccccggccgt gcctcgcgtg gqgacggggc gtctctacta ccagctactc tgggccgaqa aacaaagcaa catagcaggt tqctacatat agcactgtgt cgctccctqt gcacaaggtg ccaggagatc gggcctcttc gcagqcggcg gctcggct cg acccgggccc gacctcgccc ccqcctgagc ccccqqcccc gcccgctgcc cgccgqcggc ctcgccgacc ccatgccatt agccgtagtt cccccctcat cgagggggct ggcccagtg aaaaaaaaat gggaggatga tcgcgccact aacaaaagcc gctcagcctg gtqcttattg gcgcaccc gcacacgcta cagcatgacc gtcaagtacg ccgccgccgc gccatgagct ccgcgcctcg ccgccccccg tacggcggcc cgcqcgtcgc gcggcctcca cgggccgccg ctggaccccc gccccgCggg gcgctgcccc ggacggacg cgccccgcgc cccttcacct ctgaqaggag acaaaaaatt ggcaggagaa gcactccagc ctgctttctq tatatggcag tatacagtag ggtgcagagt acgccccctc tcaactcgqq accgcqagat cgccgccgcc tctgcccqca tgcgccgccc ccagcccccc tgcccgccgc gcccactgtc cacgcccggc cgcccagccc aggactccgc cccaggcggg ggccgccagt gcagggccgg ccacccccat cggtgcctca ccggctgtgg agccgtgt tggtgtgaac ccgggcgaca tatgcaggtg gtactcaata gtggtaaatg aggtgctcgg tcccgcaggc cgtattcaac ggtgcagcag gcaggtcacc ggtggcgcgg gcccccgggg gggcgcgccc cccccttgct cgcctcgcaq cagcagctcc ggaccgcagg gcgctcgcgc cC9cggcg ccgcccagaa cggggcagcc cgcccctgcc gttcccccag agccccgccc 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 5520 5580 5640 5700 5760 5820 5880 gccccccacc ctctaggtgg cccccgtccq aqgaqqatcq ttttctaagt qcaatacttq 5940 WO 99/37660 WO 9937660PCTIUS99/01313 -157qcccgccggc gcgcgcgtcc agaggcaggc atgtactqac ccacaccccc tgcccgagac agagctttat gcttcacctq cgggaaagcc acctgqcttq gggcctgggg aatccaggta ctggacaggt tgggcaggag caacgcccca ctqgttcatg ggtcaggccc acccagtgac ccaccctcct aggtatcagt ccccacqctg gccaccaccc acctccacgt gggcagcqcc ctacattcgg act ctgccca tt cccgctgc cccggtgacc ctggctgcgc gaqccgaggC attccgcgca ccgcagggcg ttacacactg ctccaggtcg ccgccctggc qqcaccgcct ttggaagcag gttggggtca cctgcaggat tcagaggatc gtgtgggggc acggagacat ttcctgtggc cagcatcctg qcagqcctcg acagggggag ctgtgggagc cgcatcctga qctccagatc catgctcggc ggcacacagc qtccctgccc ccccatcgcg tcgggqagca agggcgcggg agcagtgccc ataaacgaca tgcacccttc acaaggctca aaggcccctq ccgcagtt cq gcagtqtctc ggtggggtga gggagcgcct gggctcgctg cccaggggtg cccacccatg cagctgcgtg agagcqgag gccctgcgct ccccacccct gaaatgttcc ctcagctccg gcattcccag taaccacaca tctccctgtc ctcagggcct ccagctagac ctcacgcaat gcaccccqcc ggggaggctg ccaccgtggc gcattggcgc ctgaagacag cggggtgtca cggaggaagc agccaccctt cttcagctgg ggctgaggcc tactcagagc tgcaaqcgga atcaggcagg ggtggactga agtccagtaa actcctgaag cagccccctc cgccccgccc cagaagcctc agggcqgcta ctcccgtggc tcgcggtgcc gggccacagg ctacacagqc ccagcctt gc aaccggcccg tccctccagc gggt cccgcc cccccacgcc caagcctggc tggctcctgg gctgaagaag aqaqcgqacg gcgaggctgc ctggcctcca aggttttggg agaaccgctt caaactqctc ctctgggcac ggctcaqact cagtcgtgca ggaggggagc cacttgaggt ctccccaaac ctcgccccac cgaggtcccc cggtagattc aagaaatgcc aggggagctg cccacagaca cagctgtgcc gcccccgtcc actggcaccg gccgtgatga ccattaaccc cgcgtgtgat gggtggcaaa taggtgqaac gcgtgggtgg ccacccgcct agatcttctg gtgggggggg gaccaggaat ccggcacacc cacccctacc acggqggcac cagagacgaa tcacagggcc ccagggaagc atcctgggtt ccctgccgcc tcctgccagg tqctggaacg cagcaggaag ccaggaccac ca gcaga I:cc ctgctagcca 6000 6060 6120 6180 6240 6300 6360 6420 6480 6540 6600 6660 6720 6780 6840 6900 6960 7020 7080 7140 7200 7260 7320 7380 7440 7500 WO 99/37660 WO 9937660PCTIUS99/01313 -158gaaqacgccc tgcagggcgg ccgttcttct gcccagggac caggccccag gctctgaatt gcccagcacg ggtggcacac tacgctgggg ggatggggtq acgcccaqgg tcggtcagcc cccgtccact gcccgqgtcc gaggcctccc gcccgttcac agcaggqgggg ctaacaggca gqqcacaccc ctggcgcacc gccatagcgc gatgaaacct cacctccacc ggccaccaag ggtgagggca ctgggaggcg tgaqcatcat gcttacgtgt acccctaact cccggqcCCC ccacctccta gtggtggtac tqqcgcgqga cactggtaca gtacactgac gtgtqaccca agtgctgtgg tgcttaggga ccgagaacat acacgaactc gccctactcc caggggaatg gccagtqqt c gtctgagttt tggggaaagt gtgaccccgt tccagcacct tgcacggcgg cacccatqaa cctggggccg agcggcaccc gtgggaggag gttqqgcttt ggccgctgtc ccactcaccg tttgctaaaa attgagqtgg tggggtggca ctgggacgct cttgaccttg ctccaccaca qacacaggcc gtcctggCCg ctcctgtaqa ctgcagaggg cccctatttc gggcctggcg tgggggagca taaatggcaq cgctcagctc acaccaccgt gtgccacccg gtgggccggg gcccccgccc agactcaggg acgccgtccg tccagcgagt ctgaggacgg tccaccgtgg gctgatqtct aggggaaqg tggtacactq cactggggcq gttacactgg gagtccaggc gagcccatgt gtctcagggc agcggggaca ctcttgaaga cgggcagcag agagccactg cccacgcagt gccagggctc tgaaaccaac ccggaggcgc catcaccgtc catgccccgc ggcqcqqgt C cagccccacc ctcacattgc gagacgccca ggatgaaqtt aacaggtgcc ctgctctcca cttgtg gccggtgagt qggtagtggc gtggtacact gatggtggca gatagggctg gggagatgag aggqgctca gqacagqacg cctggcgtac gtgcaggtcc aggcccaagg cagcaagaaa ctgctgggag gtgttcqcag ttctcaatct tgcttcacca tgggcgtcct agccccgcta acatcctcag ccccacgccg cctgtagcag gggcgggaag ggtqggcg gacccccggc tgtaggtgat cccacccgag acgctaggat gggqtggtgg cactggqag gatgacgggg gcggqcact c ggccggggat tacctggatg gagatagtga tcaggggctg cctagggcct cgcccaagcc gctgggtcgq cgcgacatgc gcaggcgccc ccttgcgggt gcctacggaa gcagcccagg gacaggccaa agatgccccc 7560 7620 7680 7740 7800 7860 7920 7980 8040 8100 8160 8220 8280 8340 8400 8460 8520 8580 8640 8700 8760 8820 8880 8940 9000 gggcagcagg gcacacccta cctgcgcqgc cacgccgctg tacacgtcct gcggcacatc 9060 WO 99/37660 WO 9937660PCTfUS99/01313 9cgaqqgctcc ctgcaggccg agagqcagag gcacccccca gqctcccgca accctcaagt tggctcacct gcgttcqcca cactttcggc gcccgctggg tagtgaacac gccacggatg gccgggggaq atgcaqqgga gcaggacggg aggggaggg gacgggcagg gggtgccggq gagtccagga cgcttcttca ccgagcgggc ttgaagtqcq aagacqcqgt ctgtgcatct cqqgcgggcg ggggccggca aggttgacgg ttqcaagago acgtgtggga gccaagtgca cact ctaqga cgagccccag gatggacggg cctccataca cctgcgqga aggctgtgtt qggacgcatg gaggatggga gagggtgqac ggggggaacg cagggggcgc gaggaggaag gqcgcgggg agggcqga tgtcatccat accccgtgag ggccctgggc gcgggcaggg cccgcaggtg cccgggccac cggcgctgtg cgcctagctg aggcggcgcc cgtcctgaqg ccccaaacca ccggagcccc ccacctccaq gcccagtgca gaggtgggag gcaggccagc caqcggatgg gcqgggaggt tgggcgagag gctgcgggtg agaggacctg tgggqaacgc ggggaggag acgggcaggg gcgccggggg atqcgggggc cacctcctcc attgaccagg gaactccagc gtaqqtgcgq ctgcgccagc cttctggcag cggcaggtgg ggggcagccc cacgctgtcg aaggggcgc ccccccaggt cgcacgctcc agaataccac cactgcacgg acataggcgg gt ctgccagg ggggcagtga ggqaaatggg acggggcqgt qaccgggctg ggggcgccqg gggggccctg agcgqgcggg ggcgccaggg agggcqcggg cccgccccta gcaaaggcca tgqatcttga agggcccgcg ccgcggaagt gagaqgcgqt tgcgccagct gcctcgggcg ttggcctgga cggcccagag aaacgggaga cgagccgttc cgggagagac gagcgaaggt cctcgggggc cagggtcgga gttcctccgc gqcccgggcc gaggagacgc ggctggatga aaacaagcgt gggaggaagc gggcagggga ggacgtgggg gagggggagg ggcgccggqg ccgtcaaggg ggcgcttccg gccaatccag ccacgtcgct ccatgttgtg acagcqcctc cacggcgcag gctggggcgc agagctgcag cagcataatg tggaagctag ctagggccgt caggagccat gaaatctcac cagacccagc ggcqcgcaca gcccatccac cgatccctga acacccgtga gttctccata gtccqgagct agctcggcgg gaa~ggagaa qcgccagggg ggaggaqgaa gagggcgcgg ttggtccgcg cagcqgctcc gccgtqcggg gcccaggtgg cggcagccag cgcccgcagg ctcggccttg ctcqqagggc caccaggtcc 9120 9180 9240 9300 9360 9420 9480 9540 9600 9660 9720 9780 9840 9900 9960 10020 10080 10140 10200 10260 10320 10380 10440 10500 10560 10620 WO 99/37660 WO 9937660PCTIUS99/01313 -160agcacgcgcc ccat gcagcq accgtgcgca ggcagcqgggg gccttctcca acaggcagac ggtgagag ccacctccag aggtactgac cctggtcctc cgttgcggaa tgtccaggct gctccatctg cctcccagta gctgggcacc catctgtcag ccagggaggg ctcacctcgg tgcacctggc agctcccggg ggaaggcagc gcacctgcag gtgagaaccg ggcacagtgc gaacgggccc ccqcagctcc cqtt qacqg cggtgqgcgg tcagcttggt ggcaaaqcat gcagctgcac gggtcagggc qccggct ccc aaacggccgg ggctgcgctg ccagcgggat ggaatacacg gcatggcatc cactggcagq ctgccgcggc ggggcccctg cccaagcata agagcgccca cgtcggaggc cactgacccg ccagggtggt cgtgagtgcc gagcatccgc gccctcqtac tttctcccat ggtgagcccc ctgcagagac ccaggcgcag gcaggtttcc ggggctgagc gggtacatcc gtaggccggg cccggtgctg cggccaacaa acacctgcat agatcaaggc gccccccagc tgqtagagca tgcgtagcct qgccagggct aggcagggct agctagatgc cagatgaaca cccaccactq cagcaagcag ctgcctctqc gaaggactca tgcccgcccc accacctcgc acctcgcgct tgqtcccgca gtgqcagctg ctcatggccc ttgcccaqtt agcagctcct aggaaaqcac accgcctcaa tqcggcttca gggctttcct gaaaccagtg ggacacccat tccgcccaaa tcaggagggc cggggacaag gcaccagcat gctcccgcaq cgggcacct q cccaccgccc gactgaagct gccccagcca aggtacttcc accacgtgcc ccttgggcgg gcccqgggac gctcgtccag ctaqgcggt t gtcttcag gtgggaccca tcaaggtccc gggtgagggc ggtgctgcgt cagagtgcgg aqqt cagcgt gqatgccgat gttcccaccc tggcctccca ggtgtgtccc ggcgccaacc actgcctggc ccgagaggaa ctccgccagc ggcctcgggc taggacaqg qtgcctgacg tgggtgcaaa ggaggagagg tgtagtggtt ggctgaaagt gcagcgcctg ccggccgcgc caggcacagg cttggtctcc qgtcttccgc ggct cacagg tgctqtgtgt qtccagtgcg ggcgccttcc cgatgtccag gggctccgcg ctggggtgcg cttaaacttg cgaacagaag gagtcctggg ccatqgggtc acctgctgga cgatgcggct agcttgccca gcccccagct cggtcagggc cccggtgggg cccggctqct gggtgcgagc ctqcagcgcc gcgcqcactc caggacct ge ggaqgaagac aaggggtaaa cgcagcgccc tgcgggggat acgqgggt ca tccgggtagc 10680 10740 10800 10860 10920 10980 11040 11100 11160 11220 11280 11340 11400 11460 11520 11580 11640 11700 11760 11820 11880 11940 12000 12060 12120 12180 WO 99/37660 WO 9937660PCT/US99/01313 -161tcctcacccc cccagctgca tccctgagcg aattcaaccg ctgcggcgga ccctgcggcg gcaacgtggg gcttctcaaa gcccatcctg cccaggaccc gctatcgctg aaaggccttg ggtggcaqgq ccagatgttg tagatgaatt cataaaagct gcccaacgtc catgactcag gggagatgcc agcagactca cttttagggt ttacaccttt tgcgtgcacc gcggcccccg tctgggtcct ggcctgccct gtgtgcqcag gcagccaggc catacacgga cacggqaccc gacacgggac cttctccacg gaggcactgc cagggatggg cgagacagca acgcggggaa accctcctgg gcggtgatgc gctgttttct aaaaataaca tgtacacaaa ccataaacgg ccctqaccca aaacacaaaa tccacagatg gatggaacat aagagggttt tggcagcccc accacctcgt cccatcttct ctgccggctt aagcacaggc tgagttccta gctcagcccc cggctcagcc cccggctcag gacaccacgc agggtcttca ggtaqtgagg tggqtgcacg aggcgggctg cctcgacccc tgggagctgg cctcaaactc actaaagcct cgttcacaqc acgaacagac qgt cgcctcc qgtctcgcag gggaggggat tctagaatta tatgqcagqt actcaaacgc ccccctgagc cagaggagga cagcqtqcct caagacccaa ggccctgcat tgcggcggac cgtgcggtgg cccctaccgc acaccctgct agggcaggtg ttggqggct t cqtttctgcg cgggtaaagt tctagaagg cagagcctgg agaactgtat agcgctttga tgcatgactc gggcacggcg cggaggcacc tgtgtgqtcc ggggagtgac gacaaccgtg gaattacacc actgctctcc ccacaccctc agggaggaat gacgcagcca cctcgggacc gaccagacca acgcgacccc acacgcgacc gtgcttgacc ggccagctcc cagcttcggg gccagagggc tctcctgcaa cagtgccagc acactgggca ggagaccgtt gaatgtgacc gaat caaaga gcagtcgata cggccatcca atgaggacgt catttctatg ggggatggg actacactaa tcagtaacaq tgt caccoca aggqccaaqa tcagggccca agagcaaaag ccacaagttt gggcatgagc ggctcagccc ccggctcagc tccttgcttg atgtqgagct taggacacac gacctgccct gttgctggtg agtgcaaacc ccgtgcaggg cactgcaccc catccagaaa cgcacgtcca agtagaaaca cgcaccggag cacgctcagt gaatgtccag acgaggcttc aatcgctgaa acgaqcccac ccctctctct ccctcccagc gcccaggtga 12240 12300 12360 12420 12480 12540 12600 12660 12720 12780 12840 12900 12960 13020 13080 13140 13200 13260 13320 13380 13440 13500 1356-0 13620 13680 gccctgqgca ccggggaggc ccattggtct gagctgaggc tccaqgaacc cccaaagggc 13740 WO 99/37660 WO 9937660PCT/US99/01313 -162agctataagg gagcagcttg caccttgtgc gaagagtgcc gcggcctqct tcgtgaccac cccagccccc ggtgggggcc cccatgcact accatqaata qggtctgggg taqccaccag atgcatcccc ccaggtggga ttgtcaattg tgccttccat qgtttagtga gatgtttcac agtggcacaa ctcagcctcc attcttagca gtgatccgcc ggccatcacc tcagctgcat ttcgaacttc gt ctcgctct actgaagtct gaggtgttga acggccttca tqcagcttca cgaggqacgq ctcctgctag agcccaggca ctcccgacac ggagggcagc acacatatac ggatggccca ctcagcaggg ctgaggttct ctgtgggagg tgtggatctt ttcatcgatt cagcttcttc tttttttccc tctcagctgg tgagtagctg gatacggqgt agcctcagcc tttccgaata cccaggactt ataqtttact gtcgcccagg gccggggccc ccqggggCgg gaacagtggc gcccctcctg gccagcccca cctgcaggtc ccgtcccaga cacctacctt cgcataggac cagctccttg acctccacat gacagggtca gacacacaag ttcacgttcc ttcaaaggac tgttttaatc ttctagtttc cccaagatgg qccgggtt ct ggattacaqq ttcaccatgc tcccaaagtg taggcatttt ctgacaggtg tcttctttgg ctggagtgca acgtgggctc cagctqcggc ccgatcctcc gctcatctgt cgctqggctt tcggtgtqgc tcttaaaacc tcgatggtcc agcaggtccg aaggcaccct cctccctgct ccaggcagga ctccacctgc tctgggaggc tgacttggct tttgtaactt ctaagqtgaa agtcttgctc ctgcctccca cacacgccac tggccaggct ctgggatgac gtgactataa gtgttttcat aaattttatt gtqgcgcaat accttggcat gggaggctga tcagacagca tccagacacc ccacagaccc tqt caggccc ctgggaggga cggcgtcctg qagtcaagcc gggagaccaa ccctggagac qtggccagct agaggcagcc agcttgttaa tgactqttct cctctcatct aggtgacgta tgttgcccag ggttccagca cacaccagct ggtctcgaac aggtgtgcac attaccctgco tttcattatc taattatttt ctcagctcac acacgtccct aggtgqgctt gaacggcggt tgtggtgcag cagqgaacc tctgggggtc catggt gggg gtcctgcctc ggcatccttc gccagggtga cccttctctg gggcagaccg gcatggcccg acctccagat ctgctgtttc acttgcttta tttggtctga gctggagtgc cttctcctgc aattttttgt tcctgacatc caccgcgccc gagcactgtg tccaagtgtt tttagataga tgtcaacctc 13800 13860 13920 13980 14040 14100 14160 14220 14280 14340 14400 14460 14520 14580 14640 14700 14760 14820 14880 14940 15000 15060 15120 15180 15240 15300 WO 99/37660 WO 9937660PCT/US99/01313 -163cqcct cccgg acatgccacc ttggccaggc gtgctgggat caattttttt agcactttgg caacatagtg gcgtggtggc tgggcgtggt ttgaacgcag gagacagaaa acatatatat cctgacatcc gtaactgtaa tcctgcagct agttttgggg gctggagtgc tcctcctgcc aattttttgt ctcctgacct ccactgtgcc tggccacaca ttcagtagca actggatggt cacttaggga caacatggca gttcaaccga acacccaqct tggtctccaa tacaggtgtg ttttttcatt gaggccaaga aaaccccatc tcatgcctgt ggcacatgcc a aa g cgg aa a ctttgctgtc atatatataa ctgttctgag cagactattc gaagacatqc catttttttt aqtggctcga tcagcctccc atatttttag cgtgatccgc cggccggttt tagtccttaa cacacagctg gctggttaga ggctgaggcg aaagcccgtc ttctcctgcc aattattttq ctcctgacct agccaccacg taatttttqg caqgaggat c tccactaaaa gctccctcca tgtaatctca ttgcqgtaag gacagacttg aataacatat caatttctcc caaagggaaa tgtgacatga tttttttttt tctcggctca aaqtaqctgg tagagacggg ccgcctcagc tggggcagtt attgaaatat ttggcagatg atacgtggcc ggcggattac tctactaaaa tcagcctcct tatttttaqt cagqqqatcc cccagccatg ccaggcacag acaaggtcag atacaaaaaa ctaaaaatac gctactggga ccqggatctc gagactctgt atatataatt tttgacccag aaaattccct ggtggccaca tttgaqacgg ctgcaagctc gactgcaggc gtttcactgt ctcccaaagt tctaaacaac tcagttcttc cggaaaattc gggcgCggtC ctgaggtcag atacaaaaat gaqtagctgg agagatggg acccgcctcg tatagcttaa tqqctcatgc gagtttaaga aaaaaaaaaa aaaaaaaaaa gcctqgggca accactgcac cttaaaatat tttttcttgt tggctgctta tacatcctcc caccagagac agtctcgctc tgcctcccag gcccgccacc gttagccagg gctgqgatta ctctgtatgg cctttcacca ccaacatcat gctcacqcct gagtttgaga tgqccgggcg gactacaggc tttcgccctg gcctcccaaa atatcccctg ctgtaacccc ccagcctggc aattagctgg aaaaattagc ggagaatcac tccaqcctgg acacacacac attcattttt agagtggcct caccccatag caqagacatq tgtcgcccag gttcactcca acacccggct atggtctcat caggcgtgag tagacctcac gcttcaagtg agaaagttct gtaatcccag ccaqcccggc tqgtggtqag 15360 15420 15480 15540 15600 15660 15720 15780 15840 15900 15960 16020 16080 16140 16200 16260 16320 16380 16440 16500 16560 16620 16680 16740 16800 16860 WO 99/37660 WO 9937660PCTIUS99/01313 -164tccctgtaat ggaggctgta tctatctcaa ctgaaacccc cgcctgtaat tggccacccc ttcggqtctg ctgtgcaagc acagtgtggg acgaccctca cagcacgccg acctgatcct acgaattgqt caaatattaa gggaqaaagc aaccaagagt ctcctcggga acacggtgaa acctgtaact gaggtggcca t cccagaaaa tccagatqaa cggcgccagt cacctcagga gagggctctg gagccccgag cccagccact gtgagccgag aaaaaaaaaa acctctacta cccagctgct qgaacgctgg cagacatctt cgccaggttt *ctct ctgccc tcctcagtcc cctccacctc gaatttcgct cttaatattq gagaagcttt tgggaacgtt tccgaggaga ggctcagggg acccccgtct ccagctactt tgagccgaga caaataaata ccacaacgaa gaacacccac aaccaqaaaa caggctgccc aaaqaacgga caqgaggctg atcatggcac aaaaaaaaga aaaataaaaa cqqgaggctg gagaggcagg gattccagac gggacacttt agggaatcca tcccggagtc gcggtaagag qtttgatgcg cgatatctgt tactgttgtt tqctgctttg aactttggcc ggcagatcac ctactcaaaa aggagqctga tcgcaccacc aacaaacaaa gat gggt ggg atttcacaca aggagcaaag cccgacgaqq aggaaatgct cgqcaqggag tgcaccctag taqaagcaat ttaaaacaat agct cqcagt aggggcccct ttctgggctc cgtaggggcc gggggct tgt tggcgccccc cactgcggac ttaataaaga attttaatat tctcaaatta aaagggtgtg ggatacggtg gaggtcagga tacaaaaatt ggcaggataa gcactccaac aagaaaacg atacatctaa gaaggatctc tcaaccccaa ccaaaaqcac caaggcatta aattattqaa cctgggcaac gccttagcct tatccggqggg ccagcgacat gctaqagcct gtgctaagag gatcccaaaa gaccttggag tgcagcaag ttcaccgcaa agcacatcaa aatagtatcc gggctgaagg taaacaacac qctcacgcct gtttgagacc aatcqggggt tcacttgaac ctagtaacag caagggaaat agctgtgctc agcacagcag caccaaagcc ccctgcccag gcagaatttc cccaggaggc agacagagag ggctaacatg tggtggcaca ccaggactgc ctggagagac tgcgtttctg gcgccctgtt gggaaaatac aqgaaccagg gactggcccc qttctctacc catgtttacc atcatggggg cctccaaaac gtaatctcag agcttggcca qgtggcgggc ctgggaggtg ggagagtatg tggaaaatac agagggaatg cccgaccttc tcatcctgac acagattcac tccctacttt 16920 16980 17040 17100 17160 17220 17280 17340 17400 17460 17520 17580 17640 17700 17760 17820 17880 17940 18000 18060 18120 18180 18240 18300 18360 18420 WO 99/37660 WO 9937660PCT[US99/01313 -165tttggtcatt ttattaaaat aatgagggca cacctgccgc cttctgccgc gagcaggcag cgagtgctgc cagcagccgg caggcacccg cgccttgaac catctgccgg ggtgqcatcc gccacattct agcctgcagc gaggqacccc gcaggtggct ttcaaaattt aaaaaacaaa agttcctgtc gcccaqccaa tggccgtqgt cacttqaaga cccgacgggg gccagctgct gccatctgct tccccgctct gtccgcttat ttqqccccca gggagccgcg tqccgcaccc ctccccattc gagctcgctg gagaqt caca ggacttgaac tccaacgacc gcatcacggc ggacgaccag aggccaggag cctgctccac cctcccaggg tgctcaggag gqaatggcat ccttctccaq tctggacctt ccacatccac gcgcctggag gagcacccgt ggacccaagg tccagacaca agccctaaca agaccctcca ctcagtacct ccctctcact ccctqgctca tggagctgcc ctaqctcgca caatcaatgc gccgggcatg cgtgatttgt ctgggggcta agggcaggtg gttgtacatg cagqtggtgg cctctgctgc atcgaggctc gctctcgqg ccggggctcc ctgcagcttc tatctttgcc cctgggtggc cctgttcacc cactgtgagg ctctctggac cgtgaattcc cqcaccccac agctgccacc ggtgcacagg cctctgggct gtctagctca accatggcct acacacacac gggaaaacgc ctaagtgttc gcgqctcacg atttgaaaag agagagaaaa gcccggctcc tccagcgtga gcgaggqgca tgacctgaga agcttcccag gcctggcgcg acct gcaqgc gccttcaacc cagcggccac tggagqctac accacctccg gqcagaaggc cctqagccag tcatacttgc cacctccqca gaaacctgca cccagaaccc tgcctccaga qqqcctctgc gcaaqttcgc tcctaggctc ggctcctgct tgtatctctt tcgqtcatcc cctaaaagaa gtccagtcta cggctgcact gcagcttccg gctggtcagt gctgggcctg gggcctcctg tgcagtcctc gcctggtcaq gctgcatacg agggcaccgg catctccact acacgctctc gaggacatga gccaggaggt caacaacgtt cctccccacc gcaccacgtc ggggtctgtg atctatccag acagactccc accccgcct c ccagtgtcta cqtgccgcct taaagaagcc cagcactttg 18480 18540 18600 18660 18720 18780 18840 18900 18960 19020 19080 19140 19200 19260 19320 19380 19440 19500 19560 19620 19680 19740 19800 19860 19920 19980 gtaaqqtctg cccgctgctt cgagcctcac tccccggtca acaactccct gtggcccccg aggaccctga agcaagaccc caccggtgga caggttgcat ttgttggaaa agaactcaqc ctggccgctc ccgtccacct cctcccctgc accgcccact ccccagctgg cgctgaqcca ttttgccaac tctattgctg WO 99/37660 WO 9937660PCTfLUS99/01 313 -166ggaggccgaq gtgaaacccc tagtaccaqc tgcagtqact taaaaaaaaa actccggtg gtgatcaggc aagggggggg taggaggccg qtgaaacccc tagttccagc tgcagtgagc caagaaaaaa caaaactcag ttttgaqatg ccgcaacctc cgattttcct gctaattttt actcatgacc gccaccgcac qtgctgggag gcccacctcg gttttttccc acaggatgtg ccacaccacc ccaagtttcg gcaggaagat gtctctatta tacttgqgag caagatagcg agactgttga ctgaggtgaa caccgtgctg gggacccagg aggcaggcg gtccctacta tactcgggag caagatcgca aaaattgctg gagatacttt qaatttccct caccttccgq gcctcagcct gtatttttag tcttgatccg caggcctcgg tacaggcgtg gccacccaaa tttataaagg ggacagaagc agccaggcca cagtcatcca cacctaaqgt gaaatccaaa gctgaggcag ccattgaact aataagccgg aqaatcacct cagcctgaga tgtccagatg atcacgaggt aaaatacqaa gttgaqgcag ccattgcact aaataaaaag tagqattaac cgttgcccag gttcaagcga cctgagtagc tagaqacagt cccgcctcgg acccttgacc agccaccgca agtgctggga ttctcccgcc accggcggga qgacgagggc gtctccatta caqqagtt cg aaattagctg gagaattgct ccagcctggq gtacaqqgcc aagcctagga gacagagcag tggtggctca caggagatca aaattaaccg gagaattgct ccagcctagc acaagcgtga agttgagagc gctagagtqc ttctcctqtc tggcactqca gtttcaccat cctcccaaag tcttgatccg ccaggcctcq ttacagqcgt tctcccttcc agcaagcaca ctgccaccgc ggcgcctacc agaccagcct ggcgtggtgg tgagcctggg caacaqaaca gcgcacctgt gttcctggct gaccctgtct cgcctgtaat agaccatcct qgcqtggtqq tgaactcggg aacagattga tgtccgcctt tttgttttgt aatggcatga tcagtctccc ggcgttcacc qttggccagg tgctgggatt cccaccttgg a accc ccga c gagccaccgc cggctgccta gggaagctcc tggagcctgg ccccagagcc ggccaacatg catgtgtcta aggcagagqt ataatccatc ggt cccagct gctgtgagcc caaaaaaaaa cccagcactt ggctaacacg tgcgcgcctg aggcggaqgc gaatccgtct cagagtgctc tttgttttgt tctcggctca cgqttcaag accatgccca ctggtcttga acaggcgtga ccacccaaaa ctcttgatcc acctggccag atggacgcag cacctccctc gctgtccctc aagccaggac 20040 20100 20160 20220 20280 20340 20400 20460 20520 20580 20640 20700 20760 20820 20880 20940 21000 21060 21120 21180 21240 21300 21360 21420 21480 21540 WO 99/37660 WO 9937660PCTIUS99/01 313 -167agctgagtca gggggggggg cacctqgtg aaggacaggg gqacccgag tcccggcagc ccagcaqct c cggacqagct cggctgagcg gaagggcaaa gaaactaaga gagagaggqt cacctggggt gcctcagtcq acgcccaggt gcccacgccg ggagtcccgg agcaaagtgc tgcgcccgcg accccagccg Cgctctgatt tggcaactqg ctgtttttct tggcgtgatc ggcttcccaa tttcaaggca gttcagggtt tctctccaga ggttctgggg cccatgagcg aggtgqagac agggagcgga cacgtggccc cctcctgggg qggccgggcg ggagccctaa cagcgaaggt tcctcgcact caggagqctg aaaagcgggc gcaaagaggc tggcccccgg gcggccgcaa cgacattacg ccgcctgccg gaagtagact ggttcctcct gaggcactct ttct t tt t tg atagctcaqt agcgctggga tactcaitcta cacatCCtgg cataatcttg agggggaatt qtgcaaqaga cgccct cagc ctctcctctc cagtccttcc ccqcagacqc tgtgggcggg acgcagcgga ggaagggccc cgaqqtgcag cataaqaaac aagggcgagt aaaggtcaaa gctgcctggc ggccgtaggq cacgccgctc ggagttgtgg cccagqatgc gctgttttta caggctgttt gttttgtttt gcagCttcga ttgcagacgc ataacgagga ctctgcacat qqcctcacct acgagagctc tgtttcagca ctgtctcaga ccqggcgcc tgcggtcttg caccggcggt ggcctccata aaCtctCgga cgccgcggcg caggtcaaag cacgagtcaa gqaaagcggg gcgccaaagg cgtctccctt ctcgtttqag caggccaccc tttcatggtc agcggaggcg aagggagggg tcccgaggac gqacgcgttg actgctgggg cgccaccgtg cagcatctgc gtggccttgq atgtccctgg Caggaaggag acgccgtctg atctgagcct gtggqgtcg gtcttgctcc ccctgcggga aaggcagaag gcacgqctt aacacgggcg gttaagaqcc aggtcagact gccgggt cgg ccccqgccgc tqtgttacct ccccgccgct caccqgcccg gacggaggct cgaaggcatg gcgggacaga ctcaaatccg tggcccagqc taaagagatc CCcggctttt aatttagaga CgCgggcc aaagtgggag cctgctcagc ggcgtgtcct ttgccttttc cgctcaccct tgggggctgg aagacgagag ccgaagggtc aagttggaaa cggaaccgcc ctaaacacca gcacggagga tgggctgcgc gcgqgagga tctttgccgg ccgcggcccc cqcctgcgca gcgaagggaa cgccggtqga gctgttgccq gacttttttt tggagtgcag ctcgcccctc tttttttttt ttcctgtccg 21600 21660 21720 21780 21840 21900 21960 22020 22080 22140 22200 22260 22320 22380 22440 22500 22560 22620 22680 22740 22800 22860 22920 22980 23040 23100 WO 99/37660 WO 9937660PCT/US99/01313 -168caaccttcat ggaggaagct tcagggaggc tttaccctcc tcacctgcta cctggtctcc qaaggtgtgc tgtggqqcag ggctaatatc ctttgggagg tacgcaagca ccgggggt gq gcttgaaccc ggqtgacaga gcctcaggcc ggagttccag aaaagaaatt ggcaqgagga qtactccagc cctgaqgcag ggagcaaaga tcagctcaag acgaggactc ctqctgccga gqcagcaccc cagacctcac tgctccaacg gaggctcaga tgcaaggaga aatccccaag agtggggtga gcgtgatcct ccacctqtgt taggcttggg ccagttgcaa ctgaggcqggg tgcctggcaa tggcgggcac gggaggcgga gcgagaacct tgtaatccca acaagccttg agccaggcat ctgcttgagc ctgggttgac gaacaagcgt tgtagctggg ctatcctcca gacctcactc cgcctgcgtg tccctcacct ccactactgg acaacttttg gaggqccacc gaaagcccca ccctcaggg ggt caaggga gcct aqgagg cgtt cagccg aagggtgaga aggggagctg cggatcacct catggcaaaa ctgtaatccc ggttgcagtg gtctcaaaaa gcactttgg ggcaaccgag ggtggtgcac ccaggaggtt agaacaggac ggacagagqa gctcagttaq tccccaaacc tggaagggcc agcgcactga gcctcagtcc ggtcacctgc qgtaagagtc aaqttgctgg gtccgcgaqa caggagccga atgaaataaa agcagggcgg gggcaqcagg ataggaatat gtttggtggc aaggtcaqag ccccgtctct agctact cgg agccaagatc aaaaaaagtg aggctgcggc atactgagac acctgtqgtc tagactgcag cctgtctcaa gcaatttqag ggggcctgac ctggcacttc tggcagcctc tgccaccggc acctcagggt ctggccctga attaggatgc aagacacagc ctcccagcct atggagcggc cctcgqagcc gagctttaga ccagaggcgg ctgggggtaa tcaggcctgt ttcgagacca agtaaaaata gaggctgagg tcgccactgc caaagggagq gggaggatcg ccagtctcca ccagatactc tgagctgaga aacaaaacaa cagagtgggg cacacggggg agtttcccca cttacagcac tgggaatgtt gccccagcgg atcagccagg cgtctatcat acgtgcgacc ccagcttcag aqgcttggat tagagcctgc atgqaacct g gagcqcctgc ctgtqttcca cat cccagca gcttggcaaa caaaaattat caggagaatc actccaqcct tcagttcagt cttgagccca ccaaaggaaa qggaggctga tggcgccact gtgcaaaggc ctggggagag ctcgqgggcc tcagcccaga attccagacg ttcqacagac gctgtgacct cctggtgtgc 23160 23220 23280 23340 23400 23460 23520 23580 23640 23700 23760 23820 23880 23940 24000 24060 24120 24180 24240 24300 24360 24420 24480 24540 24600 24660 WO 99/37660 WO 9937660PCT[US99/01313 -169caagacctac aaccgacttc qatcccagca agcctggcca gtggcctgtg tgagaggtgg tagccaaacc cctgtggccc aqgcagagqc agaccctgtc gaaacagctc dccaccattt tggctggtca at cccctgag taaaaattca ggaggctgag ctcgccgctg aaat acaaca aaaagtggga acgggaggc atggtgaaac gtaatcccag ttqcagggag ctaaaaacaa atggaagggg cttaaagcaa agacaccccc tgaactattt ctttgggagg acatgqtgaa cctgtaatcc aggttacagt ccatcactac catctacttg tgcagtgagc tcaaaaaaaa attgcccaaa attttaacta tggtgqctca gtcaggagtt aaaattagcc gtaagagaaa cactccagcc ttaattattt aaccatatat gaggcgggcq cctcatctcc ctattcagga ccgagatcac aacaaaacaa atqgcccagg cgttcatttt tgcacccctg caccattoct ccgaggcggg accccgtctc cagctactca gagccaagat aaataataca ggaggctgag tgtgattgaq aagaacataa ttccagcctc catctgtctc tgcctgtaat cgagactggc ggacqtggta tgcttgaatc tgaacgacag ttcttgctta ctggccgggc qatcccctga aataaaaata ggctgaggca accactgccc aacccaacca tccctgqagc cttacatttc caggctggca tatgcgttag cggatcacgt tactaaaaat ggaqccaag cgcaccactg aaaaaatttt gtgggaagat ccactgcact tctggqtttt gcaactctgt caccactcct tccaqcactt ctggccaacg gtgqqtggtg caagagactg agcgagactc agttttacga gtggtggctc qqccgggagc acaaaaatta gaagaatcac tccggcctgg agcaaacccc ccctqtqaca tggaaatgaa gagccaqaaa tcttttcttt gaggtcagga acgaaaatta ggaggaaaat cactccagcc gttggctgtg gtagaattgc ccagcctggg ggaataagac agccgccacc gtattaagta tgggaggctg tgqtgaaacc cctgtaatcc aqgttgcagjt cgtctcaaaa agagacttaa ccgcctgtca tggagaccag gccgggcatg ttgaacccgg gcqacagagc acagagtcga aattaccaca aagt ccaaaa cttqggtgga tat t tga tga gtttgagacc gccgggcatg cacttgaacc ttgggcaatg atggtgcctg ttgagccagg cgacagagcg agcagtttct accccccaqc aatgcaatat aggcaggcag ctgtctccac cagctacttg qagctqagat ataaattaat tatcaccatc tcccagcact cctggctaac gtgggtgcct gaggcggagg qgagactctgt gaatcgctaq aactcggtgc tcagqactgc 24720 24780 24840 24900 24960 25020 25080 25140 25200 25260 25320 25380 25440 25500 25560 25620 25680 25740 25800 25860 25920 25980 26040 26100 26160 26220 WO 99/37660 WO 9937660PCT/US99/01313 -170ggggctgaag tgcctctccc ctcggtctct tccqtattqt tcatggtgtc tcagggtggg aatacaaagc gacagagtct acctccgcct caggcgtgaa cat gctcagt aaatgtccac ggcagagggg actagacagt gtgcagtggc ggtcaggagt aaaaattagc aggagaatcg ctccagtctg ctgaactgta cattaaagaa atcatttctc gggaagacct gcgccctctg actggcccat tgggaagcca tcaaggtgtg agcttttgaa gtggtcgcac gaccgcgttt aatatattga gacttacctt gtgcqccagc cgctctgtcg cctgqgttgt aaggagcaag gaaagacgcc aacaggccca atgagtgaca cgtggtggtt tcatgcctgt ttgagaccag tgggtgtqgt cttgaaccag ggtgacagag cagtqtaaqt actcagacgc acaaggccct qggctcctgc cccagcgtgc tttacagagg qgccaggctg tggaggcctc gqctccaggt tgcagcctcc aggatqcccc gactcttttt tgggccaacc ccagccctga cccaggctg atagattctc got ctgcccc agacacagaa tccacagagg gctgatgggg gcacaactct aatcccagca cctggccaac ggcaggtgcc ggaggcagag ccagactccg gggtgaattg ctggggtggg cagatcaccc tggcqagaag tccgtggagg cggaaccgaa gacctcaqcc gctccctcca gtgcttggc tcgtctgcct aggacaatct ccaaataagg acagaggcta tcggtgtttg agggcagtgg ctgcctcagc agccacagcg ggacacacag caggaagggg cttcttcttg acgaggtact ctttgggaqg atggtaaaac tgtaatccca tttgcagtga tot caaagaa tgtggtatat gccagtctca ttccgcggtg gcggtggaca gtctcattgt gcttggggtg atggggacc qaggccctgq tgcggooaca gtgtgaaatc tctccatatc caaatgtcac caaagaggaa ttgttgttgt catgatct ca ctcctgaqta cggatgcacc tgtgtgatcc atgtgtgggt cggtgatgga aaaatcactg cagaagoaqg tctgtctcta gctactcagg gcogagatog ataataataa gagtgatgtt ccgctgtggg gggggcggac tttcttcagt cttcctccag ttggccacag ctgtccotga ggctccttoc tcactcccgt tootcctgtc gttcagatct attctaggqa gacaccactc ttttgtttga gctcattgca qctgggatta ttgaggatgt Oct ttatatg gccgggggct atcttctgga aactggctgg tagatcacga ctaaaaatac aggctgaggc caccactgca aataaaatca tccgaggtgt tcccatcccc actctaagaa qtctggtgcc acgtctcttt gtctagtg gactqtggca 26280 26340 26400 26460 26520 26580 26640 26700 26760 26820 26880 26940 27000 27060 27120 27180 27240 27300 27360 27420 27480 27540 27600 27660 27720 27780 WO 99/37660 WO 9937660PCT[US99101313 -171cctgccacac gccaccaaga gcaggacagg gggctgtggc gccgcccctc gtgagggtca ggggccagcc agcaagtgtg ccggccaccg acgtccccag tttcqggaaa ctggtcccac ctccgcctct tgtctcttga aggtctctgc ggggactcgg agcgcggagt attggccggc ccattggccg cccgccgcgc tgccgggtca ccggacgccq ggcgacgtgc ggcggCCgCg ggggcCtggg tgcgcccgcg cctctgtgtg aggcatccca ctggtcgcca cgcagggcca tcaggcctcg ctgtgctcac gccttgctgg ggtgcgaatg acaggtctac tcgtcacctg ctacaatttg ggctgacgca gcccctctct ctgccgccgt ggcccgcgtc ctgactcacg cccgcacqgc gccctagcct gcgtccccgc gaaggcaqag tgcgccgccg cgggaacccc gctggcqgcg tgcagggcac gt ggggaggc ggggagtccc acccgcctaa gcgtccagcc tgtcttcagc ggactgggct gtttccccag cttttggggc gtggttcccc gggccgcccc gtggctatcc cccacccacc cggggagaga cgcggcagga gtctctgtgt ctttctgtct t caca ctcc c gacacgcccc cgcgcccctq ggtcccgccc cccagcgaac ccgcqgacgc cctgtggctq gagcgcgtcg cctcttctcc ccgctqgcgc qqcgggtgac ggaggct cct gccaggaaga agaccggtta ctggtgctat gctgggagct ttgtcaatgc ccagcgcatg gtggggcctg attcctgccg tccctcctgc cgcgcattcc agtcccacqa cctcccgtgt ctctctcgtc ctgtctccct gcccccgcaa gcgagacaaa tgcacctggc aagtggaccc ccggccccgc ccgggaqcga qgcctggcct cggggaccgc tccactcact cacggccaqg ggcaacqcgg ctgtgcagcc gagggtcagg tccctccaga ttaaaggtgg gtgtccccac ctccacttgq gggcaggcag gtatggct cctccgacgt ccacctacct cacacccttg gggcatgccc ccatctctgt tctcccggtc ccqggtctct cccgaggtcc caacaaacgc ccccgccccc cgcccccgcc ccccgaggcg cgagcgcgca ggctgctgct gcagctaccc tcttcctgcg acagtgagtg ccgccgtctt tcggcctcag agatgcctga gggctccccg gtgccacctg agcggaggtc ctgtgagtct aqgaaggqtg ctaagggaqg gccccgccag gcccaaacac tgggcctggc cgqagcctgg ccccaagcat atcttccttg gtctccctcc tagcccgccc gcggaggccg gagacgt ccc ccgaggcacc ccccattggc gcgaaccggg ggcgcgggcg gcacctggaq cgtqgatccc cggggcggcg cacggtgacc tttccgtggt 27840 27900 27960 28020 28080 28140 28200 28260 28320 28380 28440 28500 28560 28620 28680 28740 28800 28860 28920 28980 29040 29100 29160 29220 29280 29340 PCT/US99/0 13 1 3 Wo 99/37660 -172ctgtgagatg ggtgcaq CCt gcctggtggg agggttgcac tgtt cggcggaccc ggctcagggg cagagaa gcg tccgtgqgt acaa acccatctqc aqqtgqgaaa ctgaggctcc agaggggctg 999c cggaagcggc ggggggctga cctccggact cctgacatca caga ctgagtcggg gccccctctg cttCttccca gacaccccat ctgc ggcacacaga agggatqgga cctgcccagg gtcacactga cag~ ccccacaggg cccaqgacgt cacgqagcgg gcgtctctqt ccc actgaggtag gccctcagtg tttgtqgaat gtcaggaqca aga caggqgatgt gggtacctgg aggccagggg agtcggtgtc ccc ggqaag9gg cccggqcccg ctggctgccq cctgaatcac cac accccctgtc cttcccaccg ctttcatctg ggcgccaagg ccc acgagggCgg acaggggact gctgggCCg gtccatccat gg gtggcctcgg gccggggcag agqcctggct ccgctgcctg ac ctctccaagc. ctcggtttCCcc~agctggac ggtgatgggg 9t ~cttccct tcgtgcattc gctggtcact aatcgggcac ct catgggggac ccagtggtga cagagacgcc ~ccctcctg g9 cgcagcagtt agacacgtga acaagggcgc aggtgggtgc a ccgggtgcag tggctcacgt cgqtaatccc agcactttgg 9 acgaqgtcag gagatcgaga ccatcccggc taacacggtg a atacaaaaat tagccgggtg tggtggcggg cgcctgtagt
I
aggcaggaga atgacgtgaa gccgggaggt ggagcttgca tgccc-tccac cctgggcgac agagCgagac tccgtctcaa cagtgaatga cgtgaacaag ggtgcaggtg ggtgcgcaga gaggcacctt gccagqggag gggaggtgca gggcgaggaa cacagacgcc ccagaacaac cacctcaaag acgttcctgt gggaggctgc cccgaggagc tttcactttg acagggagct aaagcg aaggctgcag 2940 ccctgt gggtggggCC 2946 aggccc agctgcatgg 2952 atccag tcagggctgc 2958 ;caggtg aggacaagga 296~ jggtggc qgagctgjggt 297' cagggtc tgccgagcac 297 ggagagg ctgggcacag 298 gccgggc ggggggcact 298 :catcagg gcaggtaatc 29~ -tcattag gccgca~gtg 301 cgggcatg gccaggcggg ctgqaaca gtctctgcct gagggcta gctgagggct :tgtgggtg ctgtgCtCCg 3( jgctcccag agcagaggcg 31 cagaacagt gaacggttgg 3 aggccgagg cgggcaqatc. 3 .aaccccgtc tctaccaaaa3 ~ccagctact cgggaggctg gtgagctgag atcgcgccac aaaaaaaaaa aaaaaaagaa acagtgaacg gcggtqttgg ggggccaggg gagatcgtga gtgtcctgga aggtcgggct ggccgggcac gcagqaact 0 0 0 00 00 060 020 1080 )040 030600 020 080 3040 3000 PCTIUS99/01 3 1 3 Wo 99/37660 -173gtacacccag ctgacaaagc ggcagacacc cagg gtggcggctg qccccagggt ccttgctgga caaC ctcacccagg gcta~CCaca gacgatggcg tcc caactggccc ctctgcaacg tgggctgctg agc tcggcgctca agcatgtctg tggtgtccca taa gccagcccgg cctccagtcc agagtcccag tcc qqcagctgag ctqtgagggc cqqqctgqqg gct gccgggctgg gggctccata tggqgtggtg tgl tatggggtgg tgtgagctqt gagggccgg9 ctc qtgagggccg gqctgggggg tccctggggt qg tctctggggt ggtgtgagct gtgaggccg gq ctgtgagggc cgggctgggg gctccatatg gg ggtccctg gggtggtgtq agctgtgagg gc agctctgaqg qccqgctgg ggggtctctg qc gggctccata tggggtggtg tgagctctqa g( ctqgtcgctg gctcattgac agttatcagt g ccacatcca ggaacccttt cccaaccttc c tccctctggg tgtgtggqag ~cccccqccg a gagctqcagg gacagcgctg acctggccct c ccccagctcg tgggggccgt cctgcttccc cacatgctgg ggggggctcc tggtgtcagt gtggtcgqqa gggqactcgt ggtcccgggg ccctgcacga agcacagcgg acagcagcgg gacccccgcc tcccccaggc atcctggaga tcaaagcagt gtcctcaggc ttctacgtgg cggtgagtgc cgggcagggc tggqgqcggc ccgggq ;acactt a aatet g gggcttg accaccg ggagtcc .ccatatg kgctgtgc tgtgagc ctggqqg gtggtgt :cgggctg ~gtggtgt 9gCCqqg( gtctggq tcgtggt tacacac ~ccacqggj gttccct( cacgqct gtctcCt tgctggg tccgctC ccatga gggag tgagcgagtg tgg cagctcaggg t99 gctctgccac tcc gtaggjacagc t99 gtgtcccact cta cagtgactgt c9 gggtggtgtg agc agggccgggct 99 ccatatgggt gg t gtagggccg2 gg c tccatatggg t g agctqtgag c jg gggctccCtg gc .q agctgtgagg g( ct ggggctccc t tg gccctqCCC
C
gt tgctgCCCCC
C
.ac acagatgctg c ;qt cctcatcgat ctg cctgctCCtt ctg gqqatcctg ggt atctgtctg rqqt gagccagcaa .tqt acacgtqgqc accg ccqgggCgccg ggtg gggagggtqg ~tgagga 309( ggcaggg 310 caqgcct 310~ catacag 311 ggccact 312 ~tgggccg 312 ~tgtgagg 313 3ggctcca 31: tgtgagct 31 ctgqgggg 31 ggtgtgag 31 :cgggctgg 31 ;gtggtgtg 31 ccggqctgg 3~ ggggtgctg 3~ ttctgactc 3 tgacgtccg 3 :tcttgggct 3 -tctgcactc3 gCtCctCCCt agtgtccgtc gtcctgaggg ggccctCCCC gtcgtggtca otct a cgqgt gccggcctca 00 560 440 100 1860 120 1740 3100 3160 32280 240 32400 3260 PCTIUS99/01 3 1 3 Wo 99/37660 -174cccccgccC0 cggccacaac gct gqacagq ccacttcctg ctgctgt ctgagtgccc cagcctqagg ccaacagggc agqcagggc( agcggaggg( gaagccggt caggtggga cagggtcct gctgtggCC tggggaagC ttgatatg acgtaqta ggtgtCtg ccaacgcc ctcggat( qgcaga4 gctgtCC cagcC ctccagc acaccct tccccg( cagcgactct acaccgtgga ctgcaggttcC9cgq acctacgcct cacagcgCtg gcgccgCCgC ggcc aggggggqqc cccqqccagg cgqccggacg cgg( ~ccqtcctgg tctcctgagg ccctgagagg ccg qqcgagqggc ccggccacgc ttgttcttcc ccc accgtgtgcg ggcgctgtgg acacagccca gga gggtggtggc caccaagcag gttcaatcct gag gcctctcggg ctgaaggacg cagacgtcga aac -cqgcagaggc agqgctcgg ggtgggqagc acc g aggggagg qccggggaga cggtgacaga cg ccgtcccggg aatctgcaaa tacaacgcct tg ggagatggag gagcatccag ggtggggggt cc acgaCaggag gcgggaCagt gagagccaga ga g tgaagqggag gaagggccct aagctgaggg ac ;c gqqaacggtt ccgcactgga gcccccggga g( ag cccagtqqga cccagtttgg actctggcct c ag ccatcaactt tgtggtCttt tqttacagca g aa actgttCtca tgacaaaata agCCtcagat c tc ggtgttcctc cgatcccccg ggaagggcgg
E
cc ccgggaaggg cagaggCCga cgcctcggtg ggc tgagggcagg agccgtgCtg ggtgCagggc tgc gggacgcaga qaggCtggc cgtcggtgtg ctc ctgaCatCCt gactccgctg ggacttctqc .ctc: tcagagaaaa ggtcagagcc aagagcccca .ggg cctgtctccc cttctgtgtg tggggcgaca ggga cggccagggc tccatcccca gccgccqcct agcgca :agccca -ggtacc 3 cggctc tgCgggC gccctCC ttgggga ;gt cgagc jttggga( ccgcaqa ogaggac agggccc igagatgi -gtgggt( ggaccgg cagaacc acgtcgg :cCccggg ~ggccga( ctcctca aggcctg ggggCC ctacagc cagcct( gcagca tccaca tcgaaqagaa 3252 tgttcctggC 3258 acctgtccgc 326' cccaaggtgc 327' tctgtaagcg 327 aggggggtCC 328 cctcgaggac 328 jgggacgtccc 32~ g tgggggCagg 33( a caccagcctC 33 a aaggcacctg 33 C aqtgtcctca 33 ;g gatgggccgc 33 gc ctCCagatgc 3~ cc tgctcctgcc 3~ gc cagaaaataa 3 raa atatgCacac 3 ;ga aggcggagg3 cgC ctcggtgttC 2 gat cccccgggaa ggg gcttCatgCc ccc cacctgtgCC ~cct gggagtcaaa ggag ccaggcagtg tcgc cctggtgaag taaa tacaggaaga :0 000 060 120 180 ~240 3300 3360 3420 3480 3540 ~3600 33660 33-720 3 378 0 33840 33900 33960 34020 PCTIUS99/01 313 WO 99/37660 -175ctgggccgag agggaggtc ccccagggga acagggctgc gccagtcgga gaaataaaac agcaggccgg tggatcacga ctaaaaatac gaggctgag cgccactgc~ aagaaacag ctccgcaca caagcccaa agaggagc'g taagacaac gqqcggat( cttaccag' ggccactt ccggtgtC tcctgctc ccggcagc cctcaccI acacaga aggaaaa actcggg gcacttgctg ggaggtgctg aqc~ gtgCCCg9cc cgagagcttc tgc caccaggcgg cctctgggga cat acccaggatg tccccaggtt 99c ggqcgtCgCC acttgagttg tca ttgctggcac cccacgcagg aac gcgcgggggc tcacqcctgt cat ggtcaggaga tcaaggccat ct~ -aaaaaattag ccgggcgtgg t9 cggggaatcg cttgaatCCg 99 a ctccagcctg ggcgacagag c9 g tcaqcagttq tttctttgtt tc t cccagggcag tgaaatccCg 9t a cctggggaCC cccgttgtcc t g cctqgggggca cagaaccagc g ~c ccaaactgtt gcaaactgtg c 3g ggagagggc gctqcaggcg
C
gg ccctgtCCtg tcgtgcagca 9 ct tactgctgtc tgacctcgag ct ctgggcccag cgcccccgag :ct ctggccactc cacgcgaggg zat ctctgacaaa ggccctccag :gc ccagccctcc tctccagctc acc cgagccttgt ctccctcaac cgq ctcggccggg gqactgtgac gcc taaaacaCtt gttctctcag agcctga cac( tcaccct gca ctttggC tgg agtgtga ggg .ctgggaqj ctq ~gtaacat gtc -cccagca ctt cggtcaac at gcgggCqC ct aggcggag gt agactccg tc :taaaaCag ac :tcacacag ac caggcagtg a( gctccccag q ttccgctta c :gaggggccc g ~gctcctq99 a zaatgcggcc t gctcatgCCt aagtcccagc ttccgagtct caactccaac gcctcctgac ccggagcagg tccggagatc ggaaaac gacagaa catggag gagatCg rcacaggt ;cctcgaa :tgggagg ggtgaaac gtaattc( tgcagtg .tcaaaaa jcgtqgaa ~ccctcag jgtggggi aaatCg~c gjaagcac 1 gggacgc cagggaa :cacagcc jggtggqcj ctcacag( ccaggtc( tcccaag tcaaaac cggccca aaggac ccttctgga 340 gcgagcccca 341 tgggtggagg 342 gcccacgttg 342 caccacagct 34: gaaacgqgtc 341 ccgaggcggg 34 cccgtgtcta 34 -aqctacttga 34 a qctgagatcg 34 a aaaaaaaaaa 34~ t gggcqtacag 3' c agcttattcg 3.
;c cccccaaca 3 ,a gcagtgaaaa 3 c ctgagcggca 3 ag gggtgccggc 3 ga caccaggggc cc caccagggtg ;cg aaccaatCgg 3ca gjjgcacaCC cg ccgctgcaag aac caccacqgac tcc atcttccaac 9CC tgtCqcgcag ;atc cgaggtaacc :00 320 380 440 500 560 620 ~680 4740 4800 4860 4920 .4980 ~5040 M510 35160 35220 35280 35340 35400 35460 35520 35580 PCTIUS99/0131 3 Wo 99/37660 -176tccctacctc ggtgtCCtcc aggagccgag tccgcgggtc ccctggcggc cacagtcatg cggccacgag gagggcccgc acgcgcgacc ccagaqgcg agaaggtgcc ctgcgcacgg gacgcagaca cgtcgcggga ttggagaaca ggtaggggcg gagggacacg ggtggtcctg cttggatccc aggatggtgq tgagcccgca gqtccccacc gcatccagca accttagci gctgagqtgt gaccgaagci tttctqqttt taaatttca ctcctqgggct taacttcgg agqaggCgca gacccctgg ttqqcctttt cctgacgCz agtagtgtat tttaaataS.
ggagcagttt tgttcttgi gggtcgtgga ggcggccg tgcagcactg qqggagcc gtcttccgcc acaaacca gcagtaataa tttaaaat agacagagga cggggag aaqccagggg qccggtgl gctgaccagc caaqcgt atgcaacctc gtcttagggc acc ctqgggttga gatgtggacg ccc gaagtcccaa cgcttctctc 99c cacgcacgac cccagagggc 99c gccaccagga gggcccgcca C99 tccggacagg tgggatccqa gtt cacagtgaag agtgtggtgc ag~ tgqgggcacc gcctCCCacg c9 ctggaggctc ctctccgtta gc Igatcatcagc aactgagatg aa ccttgccagc tgtgcccqag ct Sgtggggtcc gcttttcaga ac g tqggqcagag gcgctgqqcc ci c gacccatga cactgccacc ci 9 agagcagttt tgctagCCgg c a gacaggaccg gactctqcag a tc gggcgCagaa ggaaagccac a 3c tttcaagata cacatatttt t aa ttttgctggt catcctcatg ag ggggaggctg ggggcccacg gc tgaaccccgt gcttcagqcC tg catqgCgCCg acgtgagctc :,ta taaaaatctt tccaccgctg 3ct ggggacgccc cagaggggac ggc cgggCCCqqg tcctgcCCtg ggc gccgCCgggg cacccagtct gggtacg ttacctcgtg 35640 tcagggc tggcactctg 35700 tccgcaa ccccagaggg 35760 .caccagg agggcccgcc 35820 cgttgCg gcagcagccc 35880 :acctggc caagggggct 35940 agcggagg gcgggagtct 36000 caggagcc gtctaccgtg 36060 tgtctcca tcgtctgatt 36120 cccactgc cccggccccc 36180 .ggctgcac cccgggccag 36240 gqggccagg aacccgcgtg 36300 :ggcgcttt aacgctggtg 36360 tgctacctc gccagcagcc 36420 cctgggtgc caagccctgc 36480 gcccgacca gcCtcccagc 36540 .gcaccggct tctctttgta 36600 :tcctttaaa aaagtctgtt 36660 3tcccgagCC cccctactC~c 36720 tggcccgtcc tggcggcacc 36780 tgggggagcc gctgggcccc 36840 aaacgtccgt ttatttcaaa 36900 aacgtttoaga ggqtgqaggtt 36960 catgtggccc acgccttccc 37020 gaacaggcgg gacctgcagc 37080 gtgggtgcCg tgtggcgctg 37140 PCTIUS99/013 13 WO 99/37660 -177gctgaqqgtg qgtgggaaag 9c gcttqtgqCq ttctcgttgC tg gctcacccca gtgaggCCag gg gcagacgggg cagctgtcqt qc ttagagqgg gagggccgcg gt ccagccaggg cacgatgcag c( gctcacccaq cgcgtagtcg ti tqtgcagctg cggtcacaqc g ccacagcctc agccggaggc c tcccccgggc tgccagaggg 9 aaatgctcga ggccgggtgc gqcaggagga tcacctgagg tgtctctact gaaaatacaa tactcgggag gctgagctct gcctgqattt tctctttatc tgaaagacaa gaggcgaqaq cgcgagqctg caggacctgc ttggagcccg cccggggtcg cccgtgCt ggcgagct gtt cqt :tttgtggg :cggggtgg ::gtcgtqjga ccttgcaca ggcgtgggi gcccctga~ acctggac~ ;gtggctca :caggagtt gtatgagcc catacctac tgccggt9g tgccggga aaaagcac gagato ttcccgacgg ccgacgtggg ot ggaggaggaC gatgaCgacg ag ggccgtgttC tgtcccgtga gc gacagaggCa ggqjacgggag a~ gggggcgggt gggcggggca ci acaggtggtt gcagggcagc t cagggcactc gagcccggag c ggcctgccga gccttcaagg g ;cccagcgagg ggagaaaagc c gaaccctct 0 ctcccagccc2 gcctgtcatc ccagcacttt c gagacctgcc tgaccaacat a ggcgtggcgg cgggtgCCtg ~g tgctcctcag tgacggggac 3g gggcctgtgt tttcaaactg gc tcctcggggg CCC99CCC99 cg aggccgcgtt tgtcctgggc cacgagtt 37 gaggagaa 37 aqgggcagg 3tcacctgct 3 gccgcacac 3 ctgcgggag 3 caggctact 3 :gtgtgtgtg3 ccttcaggg ~gaggccga ggtgaaaccc taattcccac gqtggggaqg attgaggagc ggctctgaaa cctgggCCC 200 260 p320 1380 744 0 7500 7560 7620 ~7680 -7 74 0 37800 37860 37920 3-7980 38040 38100 38160 38186 <210> 39 <211> 720 <212> DNA <213> Unknown <220>oworaimUnon <223> Descriptionl Of UnknwnOaimUkow <400> 39 cgccggcgct tgacctgact ttcatgaatc gaaaaqgaaa tcctCtatga acgcactgca tcgcatcggc gccqgaacgc tactggccgt gt tgctcgct tttggcctga ccggctgC99 120 ggagaaggag gaggttcagc agtcgctcga .gccggtggct tttcacgact ctgacgagtg 180 PCTIUS99/0131 3 WO 99/37660 -178tcacgtgtgc gcggggagtq aaaccgtctg gccggatgac caaaggcgcc tgccggcatg ggaggctgca cgcacacgca gcacaatgat ggcatgatca aagaaatttt ctcgatgcca ggtcatctga atgggcgcgt cacggcggtc agcatgcagc ggccactqag gggtatcagc tcactgactt gttccaccgc agctct acgt tcgacgcaac cgcttgcaag gtgtgctgcg acggcggcat caqcagtggt gtctggcaac ccCCggCCCC cgaaatgctt ccacgacatg cagcgcctac ctttgccgag cttcgaggaa gcacgaccat ctgaacagca tcctgatcat aagggccagg ggttgqtggC qggcgcagcg tatgtggtcg gagcaggacg atcgatcagg gcgccaaacg cacacaagaa tcttctgatc cggtcgaaaa 240 tqcaqccqga 300 tttgggaaaa 360 gtacgtcact 420 ccaaggcgct 480 cgctgctgca 540 gtgcacataa 600 atcgaggtaa 660 gtcgtcatgc 720 <210> <211> 127 <212> DNA <213> Unknown <220> <223> Description of Unknown Organism:iUnknown <220> <221> UNSURE <222> (9) <223> May be any nucleic acid <220> <221> UNSURE <222> (101) <223> May be any nucleic acid <220> <221> UNSURE <222> (119) <223> May be any nucleic acid <400> 40 gcggccqcnc ggcgctggct gctgtgcgga ggccacgqcq ggccgcqaqc cgcctcgtcc tcgccctcct gccctgggtg cggccccccg ggtcccggcg ncccactcgc cccggcgtnc 120 ccgcgct 127 <210> 41 <211> 6858 <212> DNA PCT/US99/O1 313 WO 99/37660 -179- <213> Unknown <220> <223> Description of Unknown Orqanism:Unknown <400> 41 actcgccaag tgatcgaccg gcccctgag qcacaggcgg tccatcctgt cgcqcccctc ggcgggcggC caaggctcgc aggagtcgcg tgctcggcgg aggacgccat gcacccgcaa cggtcatcct gcgagctgcg ctcatggcca aagcagggca aaccgcgtga ctgcaggtcg aagaagccga accgtgaagg agaccgagaa tgcaggagca tcaacatggg acctcaccgc agttcaagct -cttgtgcgt 3tgccggggg cgttctcgac ccgcctcggg ccatcgtggg gctcaaggtc ttccggacgc ccccggcggC qcagtcccgc gaagaccgac tgacaagaag agatcaagaa aggtcctgcg ccaagcacct tcgaggcccc cccgtgtggg tccgcttcgC ggtcaccccc gttccagtac cgtcggcgac catcaccggc gcgtgaggg( taqactctga ccgcgcacac ctccccgacg ggaggtaa ggagaaccgg gccgacaaca cgctacgcag aacgtcaaga cqtcccgacg ggcgagcccc ttcatgaaga ggacgacctc cgtgttcCCq gcgcqgcqqC qatccacatc ctaccgcttc caaggcctcj cgtctgaag ggqaacgtci Igccgccaag, cagaaqccg atgcccatc gccgcgacgc tat t cqtgcg ogat cccqga ctgtctgaac gcgtcccggg cgcgacqcta ccggtgcgaa gcatcggcga aggcgcacgt gctcgtacat gtggcacgcg tcgtqtcgct gtgcagqtca acggatgagc caggacaaca tcgaacgtgg gagaccgtcg 3gggaaggagc a ccaagtaccc a tgcaggtgcc g actccaagat a tgatcaccaz g gcacgcacg( aqagggcgcc ccctctcqtt tccgcctcgg acgcgtcgc atgcccgttc ggagagacaa ggaaatcctg caccatcgtc cgt caaggcc caagttcgac catcttcggc cqccccggag tcagtggcaa gcgtgctcgt acggttccac ccgtggtgga agaaggacgg tgtgatgacc cgaggaqatc gggcctcgtC catcgacgac i ggcccgCaat.
:caccctccgi ccgtgcactg gqcaggacca 120 ccctgccctg 180 ggggqacgac 240 aaccgttccg 300 gtgatccagc 360 accatccgtg 420 gccaccgtga 480 qtggtggtcc 540 gagaacgcgg 600 cccgtgggtc 660 gtqatctgac 720 ggacaagggc 780 cgagggcgtg 840 cgagggcgqc 900 cccggagacc 960 cgtgacqaag 1020 gaggtgcagc 1080 cgcggacgcc 1140 aaggtcgtcg 1200 gccgtcaccg 1260 tccatcgcgc 1320 iggcgatcqca 1380 WO 99/37660 WO 9937660PCTIUS99/01313 tgtgggagtt gcctqtccga ccgtgttcca tqgtgacgac cgttcaagac ggaaaccacg gctgacccgt gaagctgaag cgagqaggag tgagcgtgcg gtccacgaac tqqtctcctc ctacgtctgg tctcccqatc gaagggcccc caccctcgag cctgcacggt ctccaagcag cgagttcgac cacggtggag gaccgccgcc cgcgggcgag ggcaagggca ggcaccacgt gtcgtqgacg cgtgcgctcg cctggaccqc ccgccagttc cgagatcgat cgccaagaac cgaccagtaa gcgagaaagg ctgcgcaacg actcgcgtcg gccgaggtcg atcgccggcc ctgccccacg acgaaccagc tgacgggcaa accatccccg aagggcccca gaggggcagg ctgacccgta ctcgaqgtcg ctgggctact qgtaacaggg aacatccgca cagatccgcc agttcaacgc ccgtcccccg acacgcagag agggtgacaa ctggtcacgc gacggcaacg caqgacaaqa gacqacgaqg gacctccacg gcgtgaagca caaactcggc ccqagat cct qcaagaagct tgcgccgcat tgctgggcqg aqgccgccaa qacggaagaa ccggcgt cga aqqgtcaqct tcaccgtggc ccct cat cag tcggcaccgg cccaccccgt tcaccgtcgc agctgcgccg gcaaggccgg ccqcacccgc cctcgtcgtc ccgcacqat c g a cgcc aa g -180tgccgctgcc gcaactacac tcqaccgcgt gccgcgcgct ccacaggtcc catgaccatg ctaccacgac caaggccgag gaccatcqac ctccaagccg cctcggcatc gaaggctggc aggctgaact tgtgaccatc cgagcactcg ccgccccgac caacatggtc ctaccqcgtg cccggtgaag cggtatcgac ccccgacccg aaagaagtga cgccacctcc aaccqctctg gcgtacgcct qccaagcgcg gcgcatccgt cttcggcctg gcgcggcatg gct caaggcg tccaccggtg accgatcccg accqtgtcca ggctacatcc ctgaagttcg ggcctgcgcg gccatcctgt qtgggcggag gacatgtctc gacggcgacc ctgcccacgc gacgagcgtq gagggcgtga caggccaaqg gtgtcccagg aagcaqcagc tacaagggca tgtctactct gggtqcgcaa cacggcacat ccaccatgga tgggcgagct gacttccqcg tccgagcaga gacatcaccg ctgggcttcc aaccggtggc tcgcaqacat tgccgtcctc aggactggcg gcccgcagcg tgtacqcqaa ccacctcctc aagtcctcgc gaatcqgacg gcgtctccgt ccatcacggc agtcccgctc ccaacgqctt gccaggacct gcatcacctt aggtcggcga agqgcqtcta gaagqtgaag gcqqatctcc gttcgtgcag qgccgacgtg cgtcgccgag 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 WO 99/37660 PTU9/11 PCTIUS99/01313 cgtgccaaqg gggcgcgtcg acatcaacca ggtcgggctc cggccgcggt cgcccaggac ccaaggtcgg ccgtcqgcgt aggaggccaa tgcagggtga tgatcgccqg ccaagtccat agaagctgaa cccccatgc gtgcgtqacg tcatcaccca cgctgggcct gcatggtcaa gacgccatca gtgggtcgcg gcccqttacc ccqaagctcc aaqctctccg qgcctcgtcc gtgcaggtga cggccggcat cqqccgtggc gaaggacact gcgagccaga ggccqgacgg gacgaggaag ccgccgcttc cggctacggc gaagtccttc ggacgccgcc cqgt ccgqtq gggctccgtg gagcccccag gatgctgcgc cgtgtttgaq gacccgcqgc gaagcggat c caccgtgccg aggtccacga gtgaggcgtc aggtccgtgc gcggcttcaa cgcacttccc gtcgtggcca aggcgaacgc cgaggccgcg cgacggtgCg caggtgaccg ccaccgagcg cgatcgtcgt gaccaqttcc tccttcaccg aaggcqaaqg ttccgcgtcc qqCgtCgtgC cgcgccgtgc aacgcgatca gccgtcgccg accatggaga tccactcgca gtcaCqggct ggccaccagg cacctggtgt cctgcgtccg gaagggcaag gggcttcgag gaacccgttc cgaqqgcqgt gcccgtgaag cttctctgcg -181gtcttcgacc cgagagggtq agagcaccga cgccaccggt qgcgccgtc tcqagcgcgt ccctcgtqgt aqgt ccccgc cccqcgtcgg tgctccgccc tcgagtgcgc acatcgtgcg cccgccgcgg acgatcgcgc agaacatcca ccaagcaqaa tcacccgcaa ccqtggagqa gcccccggtg accgccggtc ggcgqtcagc cgcacggaqt gaggtcaccg gtgctgggca t ccgccgtgg ggqcgqgcaa ggctgcagct qaCCaccgtc ggccgcgqcg ggacgaccga cgt gqqcatc ggtgggtgac tgcqatccag ctccaccatc ggcctccccg cggCatccac cggcacggtg caaggccctg ccagaaqagc qccctcggac ccatcqggac gqccgacgcg ggtcaacaat ccaagaccgc gcggcaccaa tgcccctgca accaggtcgt tggacgcgct cgggggagat aqaagatcca caagtaccac gtgaccqaga tcqaqaccg gtcgcgacgg accgtcgtgg aaccqcgtct ggcgacggca aagqccgtgg ccacctgg ggtaccgcgg gacgtgctct qagggcct ca gacgagatcg gcgaagqcag gccaccctgg accctgcgct gtgacggtcg ggctgacaac caaqacccgc gggcaccaag gatgcgtctg gaacctgqac cgtctccaag caccgcggcc ggccgCCggc 3000 3060 3120 3180 3240 3300 3360 3420 3480 354-0 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 gggtccaccg agaccctctg acacgccgac ccatcqaccg agggCCCtgg ccggagcagc 4500 WO 99/37660 WO 9937660PCT/US99/01313 cgct cgggcc cgtctcgtgc tcgtgctcaa tcacgctcgg actacccggc tcgtgaacat tgccgtacat agcagctcca tgaccctcgc ccctgctcgg tggtcatcgc agaacggcgt cggccggtct ggatcggcct cggtccagta tcccggtcaa tgctcccggg ggatcacgtg acttcctgct agatctcgga ocaccgagcg acctcggtat acttcccgct agcaggtcag cccgatcccg tcatgggccc aggccctggt occogoccog ggccatcgcc gctcatcgcc ggtgcagoag gttct cgggc cacggcgtcg coaggagogo cctcgccctg atgcagcctg cctgaccaoc gggoaacggc cgqccaggtg gctcaccatg ogooaagcgg ggtgaacatg catcctcatc gctgaqccgg catcatcggc caacatgaag ttacctgcag cgtggccatg cggcggcacg cgcacagatg oaaccgtc tcccggttcc ccgt cogtgt cggaacggcg cggatcgtoc gtctatcgga tgcctggcag ggggcgct cc atcatcgtgc cgcaggggoc ctgcaggcga cogotgct go ggctgtctca at gt cct go gtccagacgc ctggccat cg cagatcggct gccaacgtca cagttcaaca tacttcggct ttoaogtact cgctacggcg tacgtcatca atcccgctga tccatcctca gagcagcgoc cgtatcgaca ggcaagggca -182agactogoac cacgccccac ggacgcctga tgggcgactt cgggcaacgc t gcaggtgt 0 agctgctgcg aggcgacgct ccacgatggc gcgacggcto tcgtcatgtg toatottoac agggctggcg tcttcgtgga oacggaccgt tcccggtcat cgoogcagga ccggtgacca tctacgtgtc gottcatccc googcatoac tcgccttogo tcatggtggg actacgaggg gtqaggaaca cccaggccac agcogocccg aggaccagcc cctgttgcgg cgtgccggcc coagggcggc tgtcttcgcg cgtggtgatc gacgcagtac ctcgctggcc cat cotcacg gtt cggggag otocatcgcg cgtgttcgcg ggagtcgcag gggogggtog ottogcotoc cggcagtgcg cccggtgtac catcaogttc ggcgtccgcg gttcgtggtg ggtgatcggc cgtcggoctc cctgctgcgc cacgatgacc coggatcgc gtgtggccgc gcaggaggac aagatcgcct accggcgtgg ctgtactcct ctgggcatca cogogottcg acccgctacc cgoaocgggg gtgctgctcg oggatoacog gcaggcttcc atcgtcatgg cgooggatc agcacotaca tccgtgctga ccggccccgt atggccctgt aacccggtgg coggooggoc ggggccct ct accagccaga cagacogt ga tgagccccga cgcatgctgc gacaagctgg 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 5520 5580 5640 5700 5760 5820 5880 5940 6000 6060 WO 99/37660 WO 9937660PCT/US99/01313 -183ggatcgtccc tcggcgtcga accgcatggt gctacccgcg agtcgctgtc tcaagcgtgc tggacctgta tcgcccgcgt tgtactccgt gaccccgtga cagcgcgcgg ttcaccaccg tccaacttcc gtcgtgcacg gatctccacc ggccaagag cgccgaccgc cacgaagggc cgccgtcgtc cgagatcqag ccaccgcgag qgacggcacc gcgctccgcc tcggccgccg gggtggtcct cggaqctgga tgggctacta gcatcccc ggtgacatct tacatcgaca atcgcccaqq caggtcgagq gagctggagg qgccgcgcgg accgagtccg ggccaqatcg acgggctccc ctcgctcgag gtccgaggca cgccgtgttc cgacttcccg tccgccacaa acggcgactt ccgacgcgga cgctggacgC tgcccgacga acgacaccca tcatccagga acgacqtcac tgcccgtgat ctcaagaccg ctggacgccg gcggtggtgc gcctcgatct cgtgaagtcg cgtccccgat gcacggcttc catgctcgcc ggagctcgtg ggaggtcatc gtacgtggag cgagcgcctg ccagccgggc ccccccaqct cgctggccgg tggccgaacg qcacctcggt atgacgccgc gagqt cacga ctgctgqacg gaggccggcc gagcgcctgc gagcaccgcc cgcggcatcg ctgcaggccg gcqgagtcct gctqgccatg cgcgccgggc cggtgcgacc caacgaqgag 6120 6180 6240 6300 6360 6420 6480 6540 6600 6660 6720 6780 6840 6858 <210> <211> <212> <213> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> 42 578
DNA
Homo sapiens
UNSURE
May be any nucleic acid
UNSURE
(23) May be any nucleic acid
UNSURE
(31) May be any nucleic acid
UNSURE
(48) May be any nucleic acid WO 99/37660 PCT/US99/01313 -184- <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223>
UNSURE
(211) May be
UNSURE
(292) May be
UNSURE
(308) May be
UNSURE
(350) May be
UNSURE
(384) May be
UNSURE
(477) May be
UNSURE
(507) May be
UNSURE
(529) May be
UNSURE
(549) May be
UNSURE
(551) May be
UNSURE
(558) May be any nucleic acid any nucleic acid any nucleic acid any nucleic acid any nucleic acid any nucleic acid any nucleic acid any nucleic acid any nucleic acid any nucleic acid any nucleic acid <400> 42 ttctngtcta tggcagagat ggncaggttg ncgttgagca ggtactgncc atcagccgtc WO 99/37660 WO 9937660PCTIUS99/01313 -185ttcagcgcca atattagtgg agggacccgg agcttccgag cagatggnca ttggaactcg aacccagcat ggtgggcatt tcccccccng ggtagttccc caccagctgg agaccttcct gcgaqt ccac gtgtttctgg ctcctccccc tcttqgggga attcttctca ntggaatnat atcgttctgc gatggtgaca gcaggagttg cacatggtca cccacacagg qggntcqgga cccactgcag caattgntgc tqgtacttgg acacccgggt atgtcattgt nctttqcccc cagccggcct gtgccatcaa ggaacaactt gaggattccc tccctgaagg gtctccga ggctccgctg agccataatt cacacacccc tgacacactg tcaccttqgn gcagggqtcc cgtccatgtc ttttcccgnc cttcacgtca ggtggggqtg gcatttgtcc gncacqqaca ctcgaacact cgggggqac aagtgtnatt aaggggggat 120 180 240 300 360 420 480 540 578 <210> 43 <211> 305 <212> DNA <213> Homo sapiens <220> <221> UNSURE <222> (128) <223> May be any nucleic acid <220> <221> UNSURE <222> (146) <223> May be any nucleic acid <400> 43 catttaagtt tgctagtcct ttgcaaacag actgacgctg aqtgtcctgt ctqaqtcaat aagtgcactt ttacctttta acctatgccc tctacttqaa cccgagcaag gtccagtcca ctggacangt tqatgatagg qtctgncgcc ccataccctc tcctcttccc ccttaggaat ttgtgcagta ctggaggggt tgcgqcaatg ggaqqcctgg gtgggccgtq ctgccttgat atggccaagg gacccagtca ccacagtqga gacccttgtc tgcacctcag taccgcatgt ccagg 120 180 240 300 305 <210> 44 <211> 333 <212> DNA <213> Homo sapiens WO 99/37660 WO 9937660PCT/US99/01313 -186- <220> <221> <222> <223>
UNSURE
(82) May be <220> <221> UNSUF <222> (255) <223> May h <220> <221> UNSUF <222> (275) <223> May h <220> <221> UNSUF <222> (299) <223> May h <220> <221> UNSUF <222> (313) <223> May h <220> <221> UNSUF <222> (324) <223> May h <400> 44 ggcacaggtg ccgctgctcc ccggctggca tgcaacaaqq gtgatttcag agacaaggtc any nucleic acid any nucleic acid any nucleic acid any nucleic acid any nucleic acid any nucleic acid actttagcat acgcacagtg gaggcgaact ctctggaaac ggggncaggg ttncattgtg gcagagcagc gntgctgggg gtagagtgca ccqagqatgc gccattttgt gtgnatgggt aaagagagag caaccaccaa catcatccaq gactqgtctg agtgctctag cactgcgggg qggacccctc cgqtgcaggc ctctgccacc caagccctgg cagaaccagc tgtqccccct gctcngggac atgcggtaat ggaggttqnc t cc 120 180 240 300 333 <210> <211> 102 <212> DNA <213> Unknown <220> <223> Description of Unknown Orqanism:Unknown <400> WO 99/37660 PCT/US99/01313 -187- <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223>
UNSURE
(64) May be
UNSURE
(69) May be
UNSURE
(71) May be
UNSURE
(72) May be any nucleic acid any nucleic acid any nucleic acid any nucleic acid gcagcagcag cgcagcgcag agagagcagc agcagcagca gcagcagcag cagagcagat cntnctggna nnaaaaaatc gcggcagcag ctgctctagc ag <210> 46 <211> 123 <212> DNA <213> Unknown <220> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> Description of Unknown Organism:Unknown
UNSURE
(9) May be
UNSURE
(51) May be
UNSURE
(52) May be
UNSURE
(57) May be any nucleic acid any nucleic acid any nucleic acid any nucleic acid WO 99/37660 PCT/US99/01313 -188- <221> UNSURE <222> (67) <223> May be any nucleic acid <220> <221> UNSURE <222> (123) <223> May be any nucleic acid <400> 46 caggcaagnc ggcacgtagg agcagcagca gcagcagcag cagcagtaac nnagtcnacg agggggngcc cgggacccaa ggcgcccgaa cagagaggcg gagcacaatc cactggtcgg 120 cgn 123 <210> 47 <211> 109 <212> DNA <213> Unknown <220> <223> Description of Unknown Organism:Unknown <220> <221> UNSURE <222> (87) <223> May be any nucleic acid <220> <221> UNSURE <222> <223> May be any nucleic acid <220> <221> UNSURE <222> (102) <223> May be any nucleic acid <220> <221> UNSURE <222> (106) <223> May be any nucleic acid <220> <221> UNSURE <222> (107) <223> May be any nucleic acid <400> 47 ggcacgcagg agcagcagca gcagcagcag cagcagcagc agagagagag cagcagagag agagagcagc agagcagagc agagcanagt agagnagagc anagcnnac 109 WO 99/37660 PCT/US99/01313 -189- <210> <211> <212> <213> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> 48 293
DNA
Homo sapiens
UNSURE
(86) May be
UNSURE
(166) May be
UNSURE
(185) May be
UNSURE
(209) May be
UNSURE
(214) May be
UNSURE
(219) May be
UNSURE
(234) May be
UNSURE
(290) May be any nucleic acid any nucleic acid any nucleic acid any nucleic acid any nucleic acid any nucleic acid any nucleic acid any nucleic acid <400> 48 ggcacgaggg ggaaactgct ccgcgcgcgc cggggaggag gaaccgcccg gtcctttagg gtccgggccc ggccgggcat ggattnaatg cctgagcccg ggtcccgctg tcttctgctt 120 cttcccttgc tgctgctgct gctgctgctg ctgccggccc cggagntggg cccgagccag 180 gccgnagctg aggagaacga cttgggttng cctncccana aaatgggaag gganttgggg 240 WO 99/37660 PCT/US99/01313 -190ttaatcgaag tcattgggac cattttaaaa ggggcttcct ggattatagn ctt <210> 49 <211> 506 <212> DNA <213> Homo sapiens 293 <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223>
UNSURE
(283) May be
UNSURE
(342) May be
UNSURE
(356) May be
UNSURE
(362) May be
UNSURE
(364) May be
UNSURE
(368) May be
UNSURE
(429) May be
UNSURE
(454) May be
UNSURE
(461) May be any nucleic acid any nucleic acid any nucleic acid any nucleic acid any nucleic acid any nucleic acid any nucleic acid any nucleic acid any nucleic acid <400> 49 aattcggcac gagcacccgg ccactgcagt cttctgccct gctggacagc agcagcagca WO 99/37660 PTU9/11 PCTIUS99/01313 -191gcagcagcag cttctacctc agctgtcaga tgatgtcctc gattatatct tntnaacntt gggggtttnc ttcggcttcc cagcagcagc ctttqaqccc aatctttgat agaagtgttt acaacctgga tgactgacag ttgggacttt ttaacgaaag agcagcaaca atcaaggcag cccacacgag gcccctctgc cgagagtgaa ggacatgcct tcaaccgacc ttttgg gtaacagcag accccacag agtgcatgag tttcgtcttt ggtgtttgtg tttttggttg ctanagagtt cagttcgtcc tgttttggaa ctcqgagctq ctncaccccc anctcttttg ggacccaqat nagagcaaan ggacccaacc ctccccaaag ctgqaqgagt gggagaccac atgtgnctgt tttttgactt aggttggttt 120 180 240 300 360 420 480 506 <210> <211> 419 <212> DNA <213> Homo sapiens <220> <221> UNSURE <222> (137) <223> May be any nucleic acid <220> <221> UNSURE <222> (221) <223> May be any nucleic acid <220> <221> UNSURE <222> (259) <223> May be any nucleic acid <220> <221> UNSURE <222> (327) <223> May be any nucleic acid <220> <221> UNSURE <222> (385) <223> May be any nucleic acid <220> <221> UNSURE <222> (389) <22.3> May be any nucleic acid <220> <221> UNSURE <222> (416) WO 99/37660 PTU9/11 PCT/US99/01313 -192- <223> May be any nucleic acid <220> <221> UNSURE <222> (418) <223> May be any nucleic acid <400> tttaagcacc tgcatgctct ggggtggtgg ttgctctgtg tgttgtggtg gaactcactt ggcccaga aaaacttgtg tgtctccctt ttatgancag ctgaatggct ggctgctgnt cctcttcctc tggagccttq ttttaatgat aatggagaga cagtctgtca cagtggccct gctgctgctg ctqctgncac ctccntgant gttggatgga gtgtgacact cagctcagcg acaaagcgga ctgctgctgc ctgtcttttq tatgataggc aatctttcct gcttagcact aqqtgaagcc ntcagctctt tgct gccctt gcttgtggga ccctcggtct aaatgtgtca tggatggctt tqtqggcgtt ggt gqct tt C gcctctaaaa ttggagtcat cttttntnc 120 180 240 300 360 419 <210> <211> <212> <213> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> 51 495
DNA
Saccharomyces cerevisiae
UNSURE
(177) May be
UNSURE
(322) May be
UNSURE
(328) May be
UNSURE
(342) May be
UNSURE
(368) May be any nucleic acid any nucleic acid any nucleic acid any nucleic acid any nucleic acid WO 99/37660 PCT/US99/01313 -193- <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223>
UNSURE
(371) May be any nucleic acid
UNSURE
(375) May be any nucleic acid
UNSURE
(380) May be any nucleic acid
UNSURE
(386) May be any nucleic acid
UNSURE
(396) May be any nucleic acid
UNSURE
(404) May be any nucleic acid
UNSURE
(423) May be any nucleic acid
UNSURE
(426) May be any nucleic acid
UNSURE
(436) May be any nucleic acid
UNSURE
(443) May be any nucleic acid
UNSURE
(456) May be any nucleic acid <220> <221> UNSURE <222> (460) L WO 99/37660 WO 9937660PCT/US99/01313 -194- <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> May be any nucleic acid
UNSURE
(467) May be
UNSURE
(468) May be
UNSURE
(471) May be
UNSURE
(474) May be any nucleic acid any nucleic acid any nucleic acid any nucleic acid <400> 51 aattcggcac tccatctggg cagaggtgca agtccctggg gtctgctctt ccggagttcc tgggaaantt ttnacnaaaa ttttttcggg gagcaaagtt ggacacagtg gctqaaggaq actcccacct ccaccacctc ttccttgcct naggnagttn attttnaaca tccgg ctgcgctcca gactctgatc gaatctqctg ccgagccagc ttgcacaaag tnaggggntt aaggantacc gtntaaattg ttgtgggcat agttcaagcg ctgctgctgc tcccaccccc cccagtcctc ttcagcaagt aggttnttgc ggtttnttgn caaacgacac ggaggaggat tgctgctgcc agcatgactg cggcccagaa tnagttcctt catnctttcc ccctttnngg gtcaaagccc ttctactaca gcagacnccc gcctgcctct catcctgggc gggtcctttt agatccaagt nggntgtttt 120 180 240 300 360 420 480 495 <210> <211> <212> <213> <220> <223> 52 81
DNA
Unknown Description of Unknown Organism:Unknown <220> <221> UNSURE <222> WO 99/37660 PCT/US99/01313 -195- <223> May be any nucleic acid <220> <221> UNSURE <222> (67) <223> May be any nucleic acid <220> <221> UNSURE <222> (71) <223> May be any nucleic acid <220> <221> UNSURE <222> <223> May be any nucleic acid <400> 52 ggcacgcagg agcagagcag cagcagcaga gagagcagca gcagcagcag cagcagcaga gagananata natanatata t <210> <211> <212> <213> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> 53 305
DNA
Homo sapiens
UNSURE
(11) May be
UNSURE
(62) May be
UNSURE
(81) May be
UNSURE
(256) May be
UNSURE
(289) May be any nucleic acid any nucleic acid any nucleic acid any nucleic acid any nucleic acid <400> 53 aggcacttga nttgaaaatg gaaaacccta ctgctggtgg tgctgcggtg atgaggccta WO 99/37660 WO 9937660PCTIUS99/01313 -196tnatgcagcc ccagggtttt nttaatgctc aaatgqtcgc ccaacgcagc agagagctqc 120 taaqtcatca cttccgacaa cagagqtgg ctataatgat qcagcagcag cagcagcagc 180 aacagcagca gcagcagcag cagcagcagc aacagcaaca gcaacagcaa cagcagcaac 240 agcagcaaac ccaggncttc agcccacctc ctaatgtgac tgcttcccnc agcatggatg 300 ggctt 305 <210> 54 <211> 307 <212> DNA <213> Hepatitis C virus <220> <221> UNSURE <222> (212) <223> May be any nucle <400> 54 tgggqtgtga aqctccggtg ccccctcagc agtctttctq ic acid ctggtgcggc gggggactgc ggggccagcc tcagtttaaa tcgttgccct ccacactgcg agactctgga qqqcgatctg ttcctgggag atttggggqt aqtctccaat ctgtccctg cggcggcctt gncaggagta ttctagaatg agtgcacata cagcaqcagc agcagcagca qcagcaagca gcagcagcag gaggtctgga ctcatcttgt aaaatacctt cagcagc agataaccga gacccgaagc caaacggtaq <210> <211> <212> <213> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> 88
DNA
Unknown
UNSURE
(6) May be any nucleic acid
UNSURE
(7) May be any nucleic acid
UNSURE
WO 99/37660 PCT/US99/01313 -197- <222> (78) <223> May be any nucleic acid <220> <221> UNSURE <222> (83) <223> May be any nucleic acid <220> <221> UNSURE <222> (87) <223> <220> <223> May be any nucleic acid Description of Unknown Organism:Unknown <400> ggacanngac tactctctct ctctctctct ctctctctgc tgctgctgct gtgctgctgc tgctgctgct gctgccgntg tgngcana <210> 56 <211> 346 <212> DNA <213> Unknown <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223>
UNSURE
(278) May be
UNSURE
(288) May be
UNSURE
(299) May be
UNSURE
(313) May be
UNSURE
(342) May be any nucleic acid any nucleic acid any nucleic acid any nucleic acid any nucleic acid <220> <223> Description of Unknown Organism:Unknown WO 99/37660 PTU9/11 PCTfUS99/01313 -198- <400> 56 qgcacagccc ctgatactct gagggaaaaa cggtcacaag tctcagcatg agatgctgag aactqgtgat agtggggctg aaaaaccggc gaaaatgqat ggcttccagg acntcgtgac gctgctgctg gaagggtggt agccactgct tcagtttgca gcagcqattg tccagqataa ctgctgctgc tcctattcgc gaatgttggg tctctccctc aggagacntt gaaacattaa tgccqccgcc gcctctattg accatcgcca accaqagaca ttcggaqgct gcatccgact ctttaaacag cttctccqggg accaaggngc accacacant cngggg <210> <211> <212> <213> <220> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> 57 496
DNA
Unknown Description of Unknown Organisrn:Unknown
UNSURE
(11) May be
UNSURE
(78) May be
UNSURE
(195) May be
UNSURE
(197) May be
UNSURE
(286) May be
UNSURE
(291) May be
UNSURE
(293) May be any nucleic acid any nucleic acid any nucleic acid any nucleic acid any nucleic acid any nucleic acid any nucleic acid WO 99/37660 PCT/US99/01313 -199- <220> <221> UNSURE <222> (315) <223> May be <220> <221> UNSURE <222> (328) <223> May be <220> <221> UNSURE <222> (329) <223> May be <220> <221> UNSURE <222> (344) <223> May be <220> <221> UNSURE <222> (346) <223> May be <220> <221> UNSURE <222> (352) <223> May be <220> <221> UNSURE <222> (354) <223> May be <220> <221> UNSURE <222> (358) <223> May be <220> <221> UNSURE <222> (366) <223> May be <220> <221> UNSURE <222> (399) <223> May be <220> <221> UNSURE <222> (406) <223> May be <220> <221> UNSURE <222> (410) <223> May be any nucleic acid any nucleic acid any nucleic acid any nucleic acid any nucleic acid any nucleic acid any nucleic acid any nucleic acid any nucleic acid any nucleic acid any nucleic acid any nucleic acid WO 99/37660 PCT/US99/01313 -200- <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223>
UNSURE
(418) May be
UNSURE
(420) May be
UNSURE
(435) May be
UNSURE
(443) May be
UNSURE
(453) May be
UNSURE
(454) May be
UNSURE
(459) May be
UNSURE
(471) May be
UNSURE
(473) May be
UNSURE
(474) May be
UNSURE
(481) May be any nucleic acid any nucleic acid any nucleic acid any nucleic acid any nucleic acid any nucleic acid any nucleic acid any nucleic acid any nucleic acid any nucleic acid any nucleic acid WO 99/37660 WO 9937660PCTIUS99/0131 3 1- <400> 57 qaattcggca ccctgtgtat gtcaatgggt aagaaacgga ttaccagcag tcccaccacc gatttriggca tacaattacc ntttgaagtc naggtgcaca tctqacglct qqctggagat aagtflgfagc cagcagcagc aagtnccaca ttgtgggttg atttncggtt catttg qatgtqqtgg gagaaaggag gctcatggtc cccccacacc agcagcagca atttgggnna cttgcatgga gtnaaggtta atggggaggg catttcatcc tatccccgga acaggagcct gcagcagcat aaaccaaggt aggacattng aanntccgnc ccgcacggga gggcggagac ccaacaaqca gggactgcca ccccantgct tgtngnagac gttgtnggtn attcagaagg cagaagttct caaggagatc gaatcagaag agttgggctg ntnggaaagt gngntttnqg ccttggangn ntnnaaggtg 120 180 240 300 360 420 480 496 <210> <211> <212> <213> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> 58 268
DNA
Drosophila sp.
UNSURE
(16) May be any nucleic acid
UNSURE
(51) May be any nucleic acid
UNSURE
May be any nucleic acid
UNSURE
(202) May be any nucleic acid <400> 58 aacacttatc cttqanagct ctgtttggga agc agqacaa aqctacatgt naggaaactn tqgagcctcc gcagactctc caccaqcagc agcagcagca gcagcagcag caagagaaqc 120 ttccaattag gcagggggtt gtacgctccc tgtcctatga ggaacccaga agacactcac 180 cccccattga gaagcagctc tntccagcca ttcagaaact catggtcaqg agcgcagacc 240 WO 99/37660 WO 9937660PCT/US99/01313 -202tccacccatt gtcagaqctg cctgaaaa 268 <210> 59 <211> 471 <212> DNA <213> Homo sapiens <220> <221> UNSURE <222> (249) <223> May be any nucleic acid <220> <221> UNSURE <222> (386) <223> May be any nucleic acid <220> <221> UNSURE <222> (449) <223> May be any nucleic acid <400> 59 tcqacccacq cqtccgctga qgaacaq agacatgctg ctgctgctgc tgctgct( agacaqacaa tatggggatg gttactt ggqcctgtgt gtccatgtgc cctgctc tgacccaqnt catggctact ggttccg ggccacaaac aacccagaca qagaagt tggggacatt tqgagcaacg actgcnc ggggtcatat ttctttcggc tagagag acg gcc gct ctt 9gc gca cct ang ttccctggcg cctgctctgg gcaagtgcag ctcctacccc aggagacaga ggcagagacc gagcatcaga aagcatgaaa gccctggcgc gggacaaagg gagctggtga caggatggct ccataccaag cagggccgat gacgccagga tggagttaca cttcaaaccc ggatggaggg cggtgcagga ggactgactc acgct ccagt tccaactcct agagggataa 120 180 240 300 360 420 471 <210> <211> <212> <213> <220> <223> <220> <221> <222> <223> 379
DNA
Unknown Description of Unknown Orqanism:Unknown
UNSURE
(2) May be any nucleic acid WO 99/37660 PTU9/11 PCTIUS99/01313 -203- <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223>
UNSURE
(14) May be
UNSURE
(31) May be
UNSURE
(135) May be
UNSURE
(315) May be
UNSURE
(332) May be
UNSURE
(349) May be
UNSURE
(357) May be
UNSURE
(374) May be any nucleic acid any nucleic acid any nucleic acid any nucleic acid any nucleic acid any nucleic acid any nucleic acid any nucleic acid <400> anttcggcan gttcaggagg cgccaccacc ggaatagaga ttcttcagag atcttttctt aggnaaggga catggaqctg gtagncagca cagccagaga qqgacqqgtg tttcntaaaa gagggtgacc ngcatcccaa ctagatttca gtggagtgaa acaaccatga ggcctcggca gccaccgcca ccaccgccgc 120 gcagcagcag caqcagcagc aagagttaac tctgacttag 180 gaaatqtqat caatgaagga gacatctgga qtgtgcgtqc 240 atgggcaqat ttggaaaaag caccgcagat tgggaacctt 300 ttgttqttat gnaaatttgg gtttttccng taacttntta 360 WO 99/37660PCIS9133 PCTIUS99/01313 -204aaaacttaaa agtngqttt <210> <211> <212> <213> <220> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> 61 255
DNA
Unknown Description of Unknown Organism:Unknown
UNSURE
(121) May be
UNSURE
(183) May be
[INSURE
(254) May be
UNSURE
(255) May be any nucleic acid any nucleic acid any nucleic acid any nucleic acid <400> 61 aattccgaca atttcagcat nactgccagg ggnatggcat aatgttcttt atggaaagca ctcttaqcct tqcaqtqqtc tacattttta aggaaccaat tctttattac ccggcacqct gtgtcctttq tcagagttca agtttatggt 120 gtcagacagt ccatttqctg ctgctgctgc tgctqctqct ttctcgaact 180 tagggaagct qctqtctgag tgttagggaa tgtcttggct aagtaaagcc 240 cctnn 255 <210> 62 <211> 5289 <212> DNA <213> Unknown <220> <223> Description of Unknown Organism:Unknown <400> 62 cgagctctcc cagccgcagc ctccgaatcc acqgcctcca ccccgcgcct ctccagcgct WO 99/37660 WO 9937660PCTIUS99/01313 -205ctatcccgtc tccttgctgg ctgttgcatg gacatctttg cctgaccctt qacaagaagt gcctccctga cactctggcc ctgaccgtgc caqtggaaga gagaagatgg gccaqoatog ctgcagaatg tqctccagct cctgccatcc atotcatgtg accacgctgc ctgaggaggc aoggtggaca tcctgtccca tgctggcoca tctgtgacct agatgcgagg tttaaacagg ggagacggtg aagccatgtg gctgcgccct gcacaaatag cctgcggctc aactcaccgq ctagcccagc tccaagacct ggcaaatgaa aggtcttoag agqggaaqca goatca coot agaatgcgga ocaggotoog taaggtttgt otaooagtgt goaoogaota acgagotgto aggaoagtat coooaototg gctgoaotga toatgcootg gogaototgo gtggoaatgg gotoototgt atggoggctg tgatcaoaag agggoaaaqo tgt ogooggo otcoaooatg caacogoatt agotgooogc tttccgcato agtggatgot gaagaoccgg cgtgatotcc goatgtggtg gtttgtgcag gotggatgto oattgooaaa otttggaaoo otttgtcaoo oattggccao oagcatggtc oogoaaagtg ctaooaoaac gtgtogotgo ctooaatgoo agaogatggo aatcoagoag gcagacgcgg gagooaotgg gatooggcto ccgggagacc cooggoogct gggotggoot ooagagtctq aagcggtotg gaggatgcoa gtgogggogg ggtaccotgo aatggoaagq toggtggaag gaggacaggg coatocaga ggaggtgtoa aoaooagaag ottgaoaaoa aagaoaaagq otggagotca acogaagaga ggagtgoagt oagaaotcaq aoagttoogg tggtoooogt ogtggocgct aootgcoaoa tooooatggt tgcaaotcoo aaagcctgcc goat ocgogt ggggactogg gqggagaoaa ggogoogact acotgat coo agaaaggttt tggctgtgga cgggoaooot aagoaotoot oocagctgta goatott cac aogaoaattt acatootcag aogtggtgaa aoctgoaago ggggtotacg aoaaagagot acaggaotgg ttacoatotg atggagaatg ggtctgagtg ootgogaoag tooaggagtg oatottgotc ocagcocca agaaagactc cogcaoaggo tgtootgcto oagtgtgttt ggtgaagggo oootgtgoct ootoctootg goggaaagao ggaootgago ggogaotggo oatogaotgt oagggaootg ooagggggtg gaaoaaaggc tgggtooago oatotgtggo caooatogtg ggccaaogag ogaogagtgg caaaaaagtg otgocoaogg gaootcttgo cctoaaoaao tgaoaagaga cgtaaoat gt gatgaatggq ctgccooatc 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 WO 99/37660 WO 9937660PCTIUS99/01313 -206aatgqaggct gtacagaaac gttggtgatg ctqtccaatc tgtggtgcct tgcaaagaag gaccccqgct cgqggcqtgq gacqggacac cccatgtacc gacacagacc cactgcagaa gatggaatcg gacaactgtc gaccgctgtg ggqgaaggag aactgccagt cagtgtgaca attggagaca gacaactgtc qatgcctgtg ctggtgccca gatgattttg atcagtgaga cagaatgacc tgtgaccctg ggggaccttg gtagccggct tgacagaaaa cctqctttgc gtcccccagg tccctgatgc acaactgcct aacatgccac acgactqcaa qctgcgagtq tggacggctg aggataattg gcgatgcctg cattccatta acaactqccc acgcctgtgc atgtctacaa actgccccct cctqtgacaa cctacgtgcc accatgatga atcctgacca accaggacaa ctgatttccg ctaactgggt gacttgctgt gtcaccatgg ctgcaacaac ccagatctgc tqqtgtccag ctatagtgga ctgcttcaac gccctgccca cgccaacaag caagaacgcc caaqcctggc gcccaatgag ccccaacctt cgatgatgac caacccagcc ctacaaccac agctgacatt tgtggaccag ggaacacaat caatcaggat caacgccaac cgacaatgat gaaggactct ggtgccagac ccgattccag tgtacgccat aggttatgac gacatctgtt cccaaacccc aacaagcagg tgtaccagct gatggagtcg cacaatggag ccgcgcttca caggtatgca aagtgcaact tacgccggca gacctgctgt cccaactcag gatgacaatg cagtacgact aacccagacc gatggggaca aaagacactg ccaqaccagc attgatgaag caggctgacc ggcattcctg gatgqtgatq attgatgaca atgattcctc cagggtaaag gaatttaacg ctgtcacctg agtttggagg actgtcccat accctgatgg agtgcaaaga agcacaggtg ctggctcgca agccccgaaa acctgqgcca acggcatcat gcgtggccaa ggcaggaaga ataagattcc atgacaqaga aggctgacac gtatcctcaa acatggacqg tcgactctga acggccacca atgacaagga atgaccqqga gtcgaggtga tctgtcccga tagatcccaa aactcgtcca ccgtggactt tggaggaqggg caaggactgc tqacggatgc cagctggaag cgttgatgag tqagaacaca gccctttggc cccctgcaca ctacagcgac ctgcggggaq cgcaacttac ctatgacaag agatgacagg tgacgtggga agataacaat tgaacgggac gqttggtgat ctcggaccgc qaacaatctg tggcaaaggc caactgcagg tqcttgcaaa aaatqttgat agggacatcc gactgtcaac cagtggcacc 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 WO 99/37660PC/S/013 PCTIUS99/01313 -207ttcttcatca agcagccgct cccacgaggg cctggcgaqc acactqtggc ctgagccaca atggctgact gtcttctctc tcatcaaact accatqgqct catgagtgtg gaccctgcat ttggcttttg tgcttttgtc ttttcaggca cctactccct cccaagagat attttcagqg taacccacat gacttcctcc gqagttaaac gttgtattcc agcgagcacc aggcacaaaa gttttccagt acacogagag tctatgttgt ctcaggggta acctgcggaa atgaccctcg ggccaaagac caggacccat aagaaatgqt gttqatcaaa tagaaaaccc gactcctaga tcagcctctg caaaagcaaa agagcagaat tgtgagagaa ctggaatggg gattgtatga gattgaaaga aaataggcac cagatttcca cgacacaagg agatgtagct aggctgtctc ttatcaacta tgtctctttt qgatgacgac gatgtggaag ctctggactt tgccctgtgg tcacattggc aggtttcatc ctatgacaaa gttcttctcc agactgatca coaggat cgc acatgtgact actgagaaga agcatccaca gcgactgtga gggaggactc ggcgggtgg aqaaaatatg ctattgctg ttaaatagga ccccttaaca caqggccagc tctgcagatg ccaagggacg agacattcct ggactgtagt tatgccggct caaqtcactc tccgtgaaqg cacacaggaa tggaaagatt agagtggtaa acctatgctg gacctgaaat taaaccaatg gcctcgctgc tqcctcaaga atcttccaaq tgctttqgtt ggccagctct actagaattg qgggccaaag gaggaactgt atttcatgat gcagggaagg catcacctgt tgctgcagct tagcagcaaa gcagccatgc tctttctctt ttttagtgtt ttgtgtttgg agtcctactg ttgtaaactc acacctctgq tcactgccta tgtatqaagg gtgggaggct atqaatgcag ctggtattgc ctgcctttgc aaatgcaatt gaqacaaaca ggaaggtgcc qagcagtgga acaaacaaaa cccaaagggg tacatttttg gctgaccggt aaggaaaaga agtgaccaga tqqttctatt ataagaatac ttgtattttt tttttcctga ttaaacaaac ctaccagtcc ggacaccaac caccacgggg ccaggtqcgc cagatggcat gaagaaaatc aggcttgttc aqactcctaa accttctgga tctctgcttg ttccaaatca atgactttgg tgtcccactc ctccaaaatq ccagccctga aggatgcata gtactaaatc gttagctgat ctggcttctg acagggagt c gaaattgtca ccaccatctc atggttagaa acatcatgga actttacaat 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 gtaaactatt tattttttac ttattctggg ggatctgtct gaaagactat tcatggaaca 4740 WO 99/37660 PTU9/11 PCT/US99/01313 -208ggaagaagcg gtaaatacag tttgagagca cagcatagca cttttttttc gaaagccaaq tgaaaagcat aaacttgtat aacagaaaga aaaaaaaaa taaggactat attatttatt ggtagttgag agctgctctt ccaagtqgaa aggccattaa ctacatttat acaaatatta gtttaagtgt ccatatcatc aactctqttc atcgatcaqc ca cct tgt gc ttacttqqtt tactgtctta tatttttgcc cctcatttgt ctaacaaact tttgctacaa tacctqqaat aaatctttca ttagactgga gtccatttgc tcccatccct tttttccaaa tgtgtgactg taaagctact gtcattatga ctagttt cat caggaatqgc tgatttggaa aagtgttttt tgtgcctatt agaaaaaaat aqtaaagaat gtagtaccta ctgtaagatt atggaaagtg acaaggaaac ttcttttttc agtttgcaaa tccaqggaga gacaaaggtg tttqqqatca aaaaaaaaaa 4800 4860 4920 4980 5040 5100 5160 5220 5280 5289 <210> 63 <211> 2053 <212> DNA <213> Unknown <220> <223> Description of Unknown Organism:Unknown <400> 63 gaattccggc ccacggcctc gccccggcgc tggggctggc ttccagagtc gcaacggtac tcgaggatgc ctgtgcgggc ggqgtaccct ccaatggcaa ggccgctgag caccccgcgc tgcatccgcg ctggggactc tgggggagac tgggcgccga caacctgatc ggagaaaggt gctggctgtg qgcggqcacc agcccaccct ctctccagcg tccgcacagg ggtgtcctgc aacagtgtgt ctggtgaagg ccccctgtgc ttcctcctcc gagcggaaag ctggacctga ggcgagctct ctctatcccg ctccttgact tcctgttgca ttgacatctt gccctgaccc ctgacaagaa tggcctccct accactctgg qcctgaccgt cccagccgca tcgctgcgcc gggcacaaat tgcctgcggc tgaactcacc ttctagccca gttccaagac gaggcaaatg ccaggtcttc gcaqqqgaag gcctccgaat cttgtcgccg aqctccacca tccaaccgca qqagctgccc gctttccgca ctagtgqatg aagaagaccc aqcgtgatct caqcat ct gg 120 180 240 300 360 420 480 540 600 tgtcggtgga agaagcactc ctggcgactg gccaqtqqaa gagcatcacc ctqtttgtgc 660 WO 99/37660 WO 9937660PCTIUS99/01313 -209aggaggacag tccccatcca aaggaggtgt ccacaccaga cccttgacaa acaagacaaa tcctggagct tgaccgaaga acggagtgca gccagaactc ccacagttcc gctggtcccc agctggccgc gacctgccac gtccccatgg ctgcaactcc caaagcctgc ggacatctgt ccccacaccc caacaagcag gtgtaccagc agatggagtc ccacaatgga accgcccgga <210> 64 <211> 4339 ggcccagctg gaqcatcttc caacgacaat agacatcctc caacgtggtg ggacctgcaa caggggtcta gaacaaagag gtacaggact agttaccatc ggatqgagaa gtqgtctgag tcctgcgaca atccaggagt tcatcttgct cccagccccc cagaaagact tctgtcacct cagtttggag gactgtccca taccctgatq gagtgcaaag qagcacaggt att tacatcqact accagggacc tt ccagggg aggaacaaag aatgggtcca gccatctgtg cgcaccatcg ctggccaacg ggcgacgagt tgcaaaaaag tgctqcccac tggacctctt gcctcaacaa gt gaca agag ccgtaacatg agatgaatgg cctgccccat gtggaggagg gcaaggactg t tgacggat g gcagctggaa acgttgatga gtgagaaqat tggccagcat tcctgcagaa gctgctccag gccctgccat gcatctcatg tgaccacgct agctqaggag ggacggtgga tqtcctgtcc ggtgctggcc gctctgtgac cagatgcgag atttaaacag tggagacggt gaagccatgt caatggaggc gqtacagaaa cattggtgat cctgtccaat gtgtqgtgcc gt gcaaagaa ggagaatgcg cqccaggctc tgtaaqgttt ctctaccagt ccgcaccgac tgacgagctg gcaggacagt gcccccactc cagctgcact cat catgccc cagcgactct ctgtggcaat ggctcctctg gatggcggct qtgatcacaa gagggcaaag tggggacctt cqtagccggc gtgacagaaa ccctqctttg tgtcccccag gtccctgatg gagctggatg cgcattgcca gtctttggaa gtctttgtca tacattggcc tccagcatgg atccgcaaag tgctaccaca gagtgtcgct tqctccaatg gcagacqacg ggaatccagc tgcagacgcg ggagccactg ggatccggct cccgggagac ggtcaccatg tctgcaacaa accagatctg ctggtgtcca gctatagtqg cctgcttcaa 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2053 gtgagaacac agaccccggc tacaactgcc tgccctgccc WO 99/37660 WO 9937660PCTJUS99/01313 -2 <212> DNA <213> Unknown <220> <223> Description of Unknown Organism:Unknown <400> 64 agccactgcc tccagttctg gcgtccttgt caggctccgt gttcctgttq gtttgacatc gggccaaqat gccggatgac ott gqct tcc agacaacact gaqcctgagc tggccagtgg ctgtgataag tctggccagc ggtgctgcag aggctgctcc cagccctgct tggcctctcc cgtgaccact tgagctgaag gtggactgta ggtqtcctgt tggagtcagc ccacggtct c ccttcacttc gttgqcaca catatgtgtg tttgaactca ctatccaqcc aagttccaag ttgaggcaga ggccagatct ct gccaggga aagagcatca atqqagagcg gttgccaggc aatgtgaggt aqct ctacca atccgcacca tgtgatgaac ctgcaggaca cggcctcccc gacagttgca cccatcatgc cagcctcatc t cccgqcgca acctttqcca aaggctccac gaagcaaccg ttqgaqgtgc ccgccttccg acctactgqa tgaagaagac tcagtgtggt agcaacaagt cgctgtttgt cggagctgga tccgagttgc ttgtctttgg acgtccttct actacatcgg tatccagcat gcatccgaaa tctgctttca caqagtgtca cctgctccaa qgacttctgc ccggcagt ct cctctccggg catggagctc cattccagag acqaaqqqgc cattgagaat cgctgtgtgg ccgqqqcaca ctccaacggc ggtgtcagtg tcaagaggac tgtacccatc aaagggagat aaccacccca tacccttgac ccacaaaaca gqtcctggaa agtgacggaa caatqgagtc ctgccagaac cgccacagtt aggcaatcgc cagcgtcttc ttactgagcc ctgcggggac tctgggggag cccggtcgcc gccaacctga gccgacaaaq ctcctqgctg aaagctggca gaggaagctc cgggctcaac cagagcatct qtcaatgaca gaagacattc aacaacgtgg aaggacctcc ctgaagggcc gagaacagag cagtacaagas tcggttacca cctgatqgtg gaagctgcta accgqactca ccggtgcaca taggtgtcct ataacggtgt gactggtgaa tccccgctgt gct tcat ct t tggaacggaa ccctggacct t cctqgccac tctacataga tcaccaggga attttcaggg tcagqaacaa tgaacggttc aagctatctg tgcgcaccat agctggtcag acaacgagga tctqcaaaaa aatgctgccc 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 acqgtgctgg cccagcgact ctgctqacga tgqctggtct ccctggtctg agtggacctc 1380 WO 99/37660 PTU9/11 PCTIUS99/01313 -211ctqctctgcc caacagatgc aagatttaaa ctgtggtgac cgggaagccc aattaatgga aggagtgcag ctgtqttgqc atgcctgtcc gaaatgtggt tgagtgcaaa cacagatcct cggccgaggt cacgqacggg cgaccccatg agaggacaca ctaccactgc caaggacggg cagggacaac gggagaccgc aaacggggag agacaactgc agatcagtgt cctcataggg cctggacaac aggagatgcc acatgtggca gaggqctctt caggatggtg ggtgtgatca tgtgaagqtg ggctggggtc agacgcagcc gatgtgacag aatccctgct gcgtgtcctc gaagtgcctg ggctacaact gtcgaacatg acgcatgact taccqctgtg gacctggacg aaaaaqgaca attggcqatg tgtccattcc tgtgacaact ggcgatgcct cagtacgttt gacaactqcc gacacttgtg tgtccctatg tgtgaccatg atqgaattca cggtacagac gctggagtca caaggatccg aagcccggga cctggtcacc qactctgtaa aaaatcaagt ttgctgqtgc ctggctacag atgcttgctt gcctgccctg ccatqqccaa gcaacaagaa agtgcaagcc gctggcctaa actgccccaa cctgcgatga attacaaccc gcccctacaa gtgctgtgga acaacgtgqa ccctggaaca acaacaatca tgcctaacgc acgatgacaa gcaacgtggt gaggacctgc ctggtctcca tctctgcaac gaccaaagcc atgggacatc caaccccaca ttgcaacaag caagtgtact tggaaatqqc caatcacaac cccaccacga caaacagqtg cgctaagtgc cgqctatgca tgaaaacctq ccttcccaac tqacgat gac agcccagtat ccacaaccct catcgat gga ccagagggac caatccagac ggacatcgat caaccaggcc tgacggcatc cgttcctqtg cacattcagg tggtcgtcct tcccccagcc tgcaagaaag tgctctgtca ccccagtttg caggactgcc agctaccctg atccaqtgca ggagaacatc ttcactggct tgcaaaccgc aactacctgg ggcaatggca gtgtgtgtgg tcggggcagg aacgacaaga qactatgaca qaccaagcag gatggaatcc acggacatgg cagctggact gaggatqgcc gaccatgata cctgatqaca acagcctcaa agtqtgacaa gttctgtqac cccagatgaa acgcctqccc cctgtggagg gaggcaaaga caattgatgg atggtagctq aagacgtcqa ggtqcaagaa cacagccctt gaaacccctg gtcactacag tcatctgcgg ccaacgcaac aagactatga tccctgatga gagatgatgt acacagacaa tcaatgaacg atggggttgg ctgactcaga atcagaacaa aagatggcaa gagacaactg 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 WO 99/37660 WO 9937660PCTIUS99/0 1313 -212caggctggtg caaagacgac tgacatcagt ctcccaaaat aaactgtgac taccttcttc gtccagcagc caaccccaca cggccctggc gcgcaccctg gcgtctc-aqc aatcatggct gttcqtcttc ctaatcatca aqaqaaaccc catagtgtgg agtcagcact gggtttgctt cactctgcct gaagcatttt aacctctgac gtacccaaga tcaggggaca atcttccaca <210> <211> 186 cccaatcctq tttgaccatg gaaaccgatt gaccctaact cctggacttg atcaacaccg cgcttctacg aqggctcagg gagcacctgc tggcatgacc cacaqgccaa gactcgggac tctcaggaaa qctgccaatc cccacccctg acttgtaaag cggcctttaa ttgggaaagc tactcacaga caggcatgtc ataggtcctg gatgactgta gacagacttg caggcactt accagaagga acaatqtgcc tccgacgatt gggttgtccg ctgtaggtta aqagagatga ttgtgatgtg gatactcagq ggaatgcact ctcqccacat agaccggtta ccatctatga tggtqttctt ataaccaqcg ccgggatcgc cctgagacct cgaatgagaa caaagcgcct qccagaactt aaagaaaqga agatgtgggg tgaagaaaat ctgcatttcc ctctqatggt agatattgat ccagatgatt ccatcaqggc tgatgagttt tqactacgct gaaacaagtc cctgtctgta gtggcacaca cggctggaaa tatcagagtg caaaacctac ctcagacatg ctggcaatgc ctttcctcgc gcctcaagaa tgcatcttcc atttacttcc cttcgaggcc aaaatgactc aggcaggagc gtggaggagc gcatgctgct gatggccgag gacatctgtc cctctagatc aaagaactcg aatgctgtgq gqcttggtat acccagtcct aaggttgtga qgaaacaccc gatttcactg gtgatgtatg gccgqcggta aaatacgagt accttctaaa cttccttgcc aatgcagttt aaqaccatga cactaggaag acctctgagc actagaactc caaagctcta tgttcggtac ggtgagagct gtgacgcctg ctgagaattt ccaaaggaac tccagactgt acttcagcqg tcggctacca actgggacac act ccaccac ctggccaggt cqtacagatg aaggaaagaa gactaggcct gtcgaqattc aacaagggct tctcttcttg tcgaacccag agagttcctt qtgcccgctc agcacacaca accgccaaac gggagggcat taaatcattt gattgaccca 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4339 WO 99/37660 PCT/US99/01313 -213- <212> DNA <213> Unknown <220> <223> Description of Unknown Organisrn:Unknown <400> gcacagttaa tggagqctgg ggtccctgqt caccatggga catctgctct gtcacctgtq qaggaggaqt gcagagacgc agccgactct gtaacaaccc cacaccccag tttqqaggca 120 aagactgtgt tggcgatgtg acagaaaatc aaqtttgcaa caagcaggac tgcccaattg 180 gtaagc 186 <210> 66 <211> 5774 <212> DNA <213> Unknown <220> <223> Description of Unknown Organism:Unknown <400> 66 gtcactttgg tggtaatgaa ggtgact cca tcacaaaaag cacgqcattt tggggctgtt taacaaccat taactgtgct ctgtgatggt ggaaagaggt agctgtagaa ggaaggcctt aatgagacgc ggatgactct ttgatagcag gaaatggtgc qt cagcacaa aggtcagcga ggaaaagatt gtggagtggc caaccaggaa tatgttggtg ctggctggaa aaacagatqg caggctccca gatgatctag cgcagacacg gtggtccgtt ccgctctggt aaatagattt atctagaagg gggacgtgtc ttcatctqcg atgagacatc gtgctacgta acatcgtgga tgataaaaaq aggaagaaaa tagacatgtc gtactgttta cgggagaaaa t ccatgqcaa agaggttagq accaataaag acgctatctc ttccaaccct actaaagccc tctggtgcct tagaatccgg cattccagga tgataatgaa aggaaggatt caaaqacttc tggcaacatc cqattacaat agagcacgtc acttcagctg agatatagag tcccatactc gagcagttgt aacactcaac gggaatataa aaaacagagc acctctgttg gagtatttca catgttgtct cact acagag caccagcagc atcgaggtac caaaactacc atggacaagc agtatgagct tttctgcgag tctttaacat tagtagctcc ccgatcccat ctttgcaqac ccatcagcaa ttqaaccct t acaagagatc agtcggacct tgaatgaaac tgctgggagt tcctgaccct 120 180 240 300 360 420 480 540 600 660 720 780 840 WO 99/37660 PTU9/11 PCTIUS99/01313 -2 14aatgaacatt cctggtgcgc cocat ccaga caaccactct tqgaatgcaa qaatcatgag gggaatggag catqgctccc aqaactgaaa ttggcctaaa t ga t t ttggt gctgtggtgt tgatgggact tgctaatcag tcggacatgt caatggtggt atgccaaaaa tgaataccag atgccacctt gcatgatgga tgtgaaagtg ctgtggagga gaagcttggg agaagacgag tttaaatqgc ggattataac gtgaatgaaa atgataatgc agcttggaga gaacaccatg ggatatgctc gatggttttt catgatggac ttggtacaag agatatatcc ctcccagaac gttggctata aqccatcctg gaatgtgctg caaaaacaag ggaactggtg caggattgtc cactttgaqg aataccaaac tactgtcagt acqcactgtt ggctgtgata gataattccc taccttaaga gct tct cct C aaaggggagg attgaagatg tttaccatqa tgggatatgc atgtgtgtcg accatgcaat cagtcaccg catctgcttt aaggcaacag cagcattcca attcctatga ttcctggaat aaatgtgcac ataatcccta ctggaaaatg atqgcaattg ttcqtttcag ctggtgttaa acttcagagc accactggtt ccaaggagac cttacaaaga aagaaattgg actgccgaac tqtttgatat atattCttgc aagccaagt c acattgaaag tgagtccctc aaagtccatc ctgggcgtcc ttttttaacc catgtgtcat tgtagtagcc gtgtqgtgat tcgttaccac ctgtctcctt caattattct cgcgttccqa cttttgtaag gtgctataag ggggtcatgg aacacgccag ttttgagtac acagcagtgt gccatatgaa tggagatgtt tccatatagc ttctaataag cgtgaagggg accccctggg tattaaqaac gcggacCttc tcttcacacc gqaqtgcata agcctcatag caacagcaaa aggcaagact ccagtgagaa catgaaacgg gagactgcta tggtcccgat gatgaccctt atqgatgagc acctttgacc actaaaaagg ggtcattgca actaaatttg tgcaataatc cagctttgta cagcagcgaa catcctgacc gcttacatga atatgtgtgc gttgaggata acatttacca gctagacatg caggctacag atagatcttg gatggacctt taaatgtggt aaagggqaaa gatctgatct ttggacctgc gt tgt accct gccatgtgtt tgggaagtgt gcagtggtca ttgatcatga aatgtcgttt catgtaaaca gacctccact tgtgqaagaa gctcctgttc ccatgcccat acacagaaga actcccactt ccaagaaaag aacaactggt gaggagagtg agtgtggtgt gaactcccag tgttaatcca gccattatat gtgtggagtg tacatgatcc 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 WO 99/37660 WO 9937660PCTIUS99/01313 -2 tgttattgtt catcatccat agatactttt tttccagtac cttctgtgaq tacacatcca tgqctatcag tqtgcacagc gccctgccct aggaacggag tgagtcggtc qtccatattc gttatgttgt agctgctgaa gatgcctaca tagcatctct tgatggtgct ggtcaccgta ggctgctgct ctcaaagaaa aagatgagaa ttcccatqgt tctgtaattq gttctgcatt agaaaaatat caattgtgca ttgattatac gaagactctg gagtgggctt actaaatatg gccaacaaaa ctctgggtag cttcgcactg aaatactgca qcacaqtgga gtgaggcagg agagcctgtc tgtcaaatgg qagtcctgca actcatgatg tctttggttc tcaqtgggag aatttacgcc ccatcctccc tccttctttq gatggaaaga agtgaaccaa gcatatgctt gaagaacaga gactcattta qttgctaaga tatcagctga ctcaagaaaa tacctacaat tgaagagctg gatgccgtaq agccgaaacc cagaaqaatg tacqctgcct tgqgtgaccg aaacaggacc tcctctgcag aactgcctcc aaqtgttggc gcaagcgcag atgtcatctc cttatcattc gtccaaatgc agaggagtgc cacccaccaa caqccagtga tcattgacaa a a a ggct agac gtttaaagtg aaqtgctggc ccagaattca ttgtgttggt ctttttgttt tgatacccgc caacagcaac gtctcaggtt gaaaagtgat tattagacga ggaacactgc tcagccactc t cccqagagc ctggagtgag gqctgqqgac ttgtaatgat acgatactgc tagcaccctg taaccctaqt agagacccct atatgctgct tcaqcaagca gagggtccac ttcaatagqt cagacgtccg aaccagagga gaaatctcta tcactttcta ttggaagaaa cqctctctga gttttagaaa tctagcctga aatgtcatcc tccaaaccct aataaaatgg atgtgcaata accaaaacct cttqatggca cqccggccct tgttcagtga cactqtgatg gaaccatgtt tccataccag ccaccaccat gacct cccta gcaaagaaga ttcaggccaa ggaagtaaga ctcagttcag gcttcttctc acaagatcat aaacctggac tagatcqtca gttgctttca tcaccaaaga agcagaaaag agttacagta catataagta aggaagaatt gtggtggagg tccatcgcag ttcaagagtq gtgqaagttc ccaaccgctc gtaacagaqt cctgcggtga gtgaaaagcc tgggagacaa gttataacaa accttctaga qatctctaqt tgtctttgag acagtaaacc ctgtgagact cttcacaaat aggcaaqaac ccaccttaga aacctctctc gctcatttta tcctcctttt ttattacaaa ggactggaac aaaattaaaa 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 WO 99/37660 WO 9937660PCTIUS99/013 13 -216agagatacca gacttgtatt gtaaatactg tttactatat cattctatag ctgacaatat aagaaacaca aatttaaagg atqaactcqt ggtgtccttt aaacag,-tat ttqtatgttg agctttggqg ttccatatgg taataattta agtagatgta ttttttttct ttggtaaact aagcaaacta atcttgcatt aaattgtatg tttttgaaag gcataatgat gtagatgaat taaactaaca agaactttga atggtttaca cctatttatc tatattgcaa tacatttctg gttaattttc aaagggtttt aaagctatag attggtgtct gcaagacaaa gtttttgtgt actcaatgta gtagctgaga ctttgaaggc ttcatgttca tgtcagtagt aagtacttgt tgatagtatt agagagacat ctacagccaa tttagtagtt gactcagcta agataagtqc tttgaggtca taacctctca gcttaaacta aggtcaaatt ctttaacaag tatattagaa aaatcagtat gatgttctag aaagcagagt gttgaatcaa atatacagat tagtacactt actgaaaccc gtgtgtgttc ctgtaataat aaaatatcat aggttcaaga aatgttcaca caccttgctc acatagaatt tttatgtact aattagagat ctcaaaagct gatattaagt gctgttcaat atcatgaatt gtacctgagc aagcacagaa ttgactctat aqgcaaattc aaattttgga atattgtatg tattttaaga gtgcctgtcq attacaaaag caatgtgata atcagcttac gtggtcacag tctttccagg ttttttaatt cgcaaaggaa cagtctagaa aagcatatgt accacaatgc acagcaaagc cagaactgaa aaatatttac gcatgctttg aaaactgaac tgatgacttg gaaattgtga acatgtacat tgtctaaaaa tgtgcaagaa acctctaaag cagatagtaa tat ggaacaa agcaaatttg qatgtgttct ttgagtattg agaagttaga cgtaaattat ct at tgcct t ggatcaacqa acctcagtaq ttcgcattgt aaagttttgg ttgatatttg cgatggttga atctgactgc cagaaatgct gatatttatt actaatatca ttctgtgcat aaatttgatg tttcccttca attagaaaca gagaggagat tatattatac cttttgcatt aattgctgct aacaatccct agaattctta cagctgttgt caaatgattg ccttgtttga attacagcta agaaaaagaa ctatacttat atagtaaata gcaccgttga tgacagatac gataacttat agtatagtag gatatttatt aataatgtgc ctctccaggg aaaagttgat attacatatt agagaccttt ttatgcaaac aggaaacatt tttttaaaag agtgatatca aaactaaaat ttaatcgttg actttgt-gca aagccttaag 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 5520 WO 99/37660 PCTIUS99/01313 -2 17tctttttttt ttcctaaaaa ttcccataqa ataaaattct ctctagttta cttgtgtgtg 5580 catacatctc atccacaggg qaaqataaag atggtcacac aaacagtttc cataaagatg 5640 tacatattca ttatacttct gacctttggg ctttcttttc tactaagcta aaaattcctt 5700 tttatcaaag tgtacactac tgatqctgtt tgttgtactg agagcacgta ccaataaaaa 5760 tgttaacaaa atat 5774 <210> 67 <211> 5535 <212> DNA <213> Unknown <220> <223> Description of Unknown Organism:Unknown <400> 67 ggactttaga cccttgcccc gtcaagacct aggatgaggq ctgctgctgc cccgaqccgt gtqttcccgg tacactctct cgcacctacc tt cqacgagq aagcagttcc gccggqccgc aaccccaqcc aqt cgcagct gcgggcggcc gtqgctggtq agccgttgct gcctccctgc ggtccgcgcc gccaggccgc tgctgggacg gcqccacgct ccaacqcctc acatgaaggt agttcgactc tqctqcggct tgcagatqcg cgggccccac gtgccgcctg cgcacccctg ctgccgcggg gccctgaaaa gccctctctg ccccaccggg tgcctgccca cgccccgggc ccgcgcgcgg ggtqcaggga gcgctgctcc ggccaaggcg cttcctcgag ctgcgacccc qcgccagcag cgacgacttc ccagatgctq cgggatcatg acccctggcc ctgcctcacc tcacctgaag ccggccctqc gcccgcggaa cccgtctgga gcggccgccg aagttcttcg tgqacgctac cccgtgccct tccacgcgca tccgcacccc ccgccccaqc tccgtggagt tgccgctggC caqaccccct ccccgcgqgq agcctgaccc cggggccctc ccgccgccgg ccccggcggc tcct cgc ccc gagcaqacgc qctacttctc gcaacccgga gcagcggccc cctacctggg tggccttcct acgacgggct acctggtqgt tggacqcqtg' gcgcctgcct atgtctqctt aggaccgggg tcccatccca accctggcat cccgcgagct gctgctactg ggggccc9gg cgcggccgcc cccqcggcgc cgqccgcgtg cgtggagagc gcaggccagc ccggccccgg gggqaaccgc tctggccggt gggcggcgag gagagatgcg cgggcacggc 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 WO 99/37660 WO 9937660PCTIUS99/01313 -2 18gccacaggcg ggcctooaga gagggggtgc aocagctcoc gacgagctgo tggtcoogt ttctgcgtgt tgcaacaact ctotgctcca cagtttgggg ctgtgoootg tcogccagot gggggtgcgg ccagtggatg gctggcagoc cagggoccoc atctgtgatg gctgotgtcc gaggaaggca agaaaoatoo gagggggt ct agtggggacc qcgtactaca ttggcagagg gagctgttcc agggatgcat qotggaagct cgcggacgcg tgqaggaggg ggagccagto agoagtttgg qgagcgtgtg octoctocta ctgccgtgtg gcacctgtgg gcaacccctg gccgggcagt gct cccaggg aqtgooaggg gcaagtggca agcgacggga aggatgagta aggacaaott ggtgtcocg tcgcctactg agatgatgac oggaggtcat t gct gt cca o gooccacoc aqaatoggga ggctggtgga accaggtgac gtggtccctg cacctgcctg tcgccagtgc octgcggtcc gttcoagcc ctccagcacc cagcacgcag oocagtqcat ccgtggcttt tgaggqccct ggatggaaac ccgacagcag ccactgggtg qgcotgggog gcgtgtctgc ccggcagtgc tggtgctgtg oaacgccaca ggagcccccc ccgggagcac ccagacactg catcqatgtc tggggacgta caagtgggag ggactttgtg agacaaOctg tggggcgaat ooogcogg aaccgcgagg acagatgcoo ccocagaccg tgcqgcgagg tgoagcggac gtqotggg cqggatcgca gagaagcaaa tggaatgagt cgcacgcgtg gagacoogag tcatggggca tctgggccct ggcacccagc atctggaagg gqactcatcc acctacat cc ctggccaagg gtggagatct ctgaggaaca cagaactttg gaggcccagc gacgtcatcg gttctcagca gcacgcggga gcgt ggaggg cct gcggcoc ggcggcgcga gtgacccagc got ggcagac cct gcgcga atgagtggtc cgcgcacctg ccaagttctg ggt cgagctg aatgcaacgg actgcttcct gttgcagcgt tot tcggggg ggtqtcccga agaccccagc tgcgacggtg gotgtgtttc ctcagcgagg ctcaggacgg tgacagagat tocagatcct tggcgggoccc gott oogoat tooataagot otgoggggga oggcggotgo ogotgggogo ggagctgggg agoogaggag ccgcacgcgo gcagoggotg gcot ggago oaggoooooo oaaoattgoo gagogootgo gcottootao gcagoagtgo oaogtgtggg agoagootgo gooooatgag gggagaggtg tgagctggao oattgaotao gotgootggg gacoagotac tttooggaga tagoaaoctg oaaogooaag gaaggacctg coagooago 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 WO 99/37660 PTU9/11 PCTIUS99/01313 -2 19qgagccactg acatcagctt ccccatgaag ggctggcggg gtgccagagg gatqaagcat gccctgcaga ccgcctcgct accaaccaga cagctcqggc cgctgcctct aacatggaga ctcaccctgc cgttctgtca atcgggcaga ttcttcttcc gtgacgggcc ctccctgcac atgaactact gctqccgttg aaagacggca tgcgtggtgc cgccqct ccg atcgtcatqg gttgaccggc cagctcatqa gacatcgcgg gaggaggaga ccggccaacg acagggtcac ccgtgtttgt ggaacacgac ccctgcgcac cctgtatcct cctggtcgtg gtgaccgqct aggcgactct tcatgctggt tcctcatcaa cccagacccg tgtcctcctt acctccggaa tggttgtggc gctggctctc tgctggtgaa tcacggacaa tgccgctgct ccctcttcca tgcactgtat aggaggaggg ccgacttcga cctgccgcac agctgaagct tgtccaagct tgtgtccaag ggtgqgcacc cgtcctgaat acccttqgag gtgggatgag gcgcggctgc ctccaccttc gccgtcggtg catcatctac cttctgcctg caacaaggtg ctgctgggtg ccgcctcatc cat tt ctgt g cctggagggg catggtcatt gaagctgaag ggcgctgacc gatcctcttc cctccgtaga caacggggac gaaggacgtg tgccaccatc ggcccatgcc gcacctgcac aqtgtcttct gtgctctaca tctaaggtga atcgagtttg acggatgtac cgcacggtgc gccatcttag acgctcatcg gtgtccgtqt tccatcatct atgtgcacgc ctcaccgagg cgcaagcqct gqattcacca ggactgctct qggatcctgg gagcgggcag tggatqtcgg gctgtcttcg gaggtccagg tcagggggct gatctggcct acggqcacac aaggggccgc ggctcacccc ccacgggtga ccacggggct gqaacctggg tctccgtqac cccacatgta cctcctcctc ccctcqacqc cccagctcag tgggctgtgg ggaqgtacat cctccaatgc tggtggccgc cctggcagtc tcct ctgcct aggccaaagg atgccttcgt tgttcaacaa gggcctccct ctgtgctcgc act cgctgga acgctgtgaa ccttccagaa gtagatcagt tgaagcggcc ccaccaattt gctatcccgg ctgggccaag gacagaggc cagcttcctg tgtgaaaccc taatggcacc cgcccccccg cctccggacg cgccgacgcg cgtgtcctct tcgctcagag cctcatcctc cttcctgcac ctacatggcc gggctggggg gtacagcacc gggacctgcc gctcgtgtcc gtggagctcc cgtcaccgac gggcttcgtc atgccgtgtg cggccacgcc gctgaacaag gtctctgccc caacagcctg cgggcccctg 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 WO 99/37660 PTU9/11 PCT/US99/01313 -220cccgacttcc qtcggtgacg gctctggaca aaggaggagc ct caqcacgg aqcggcgggc ccacctcccc ggggatcccg gagaatgtcg ctgqactttg aaccggaagc ccggaaaagc acgcccacgt agcgtqgagt ctccagaccg gctgctccgc cccgcacccc accagagoca gaaggtgcct ctgtggaccg ctgcggagga ccctcgggaa gtggagggca gaagaagcag aaaacccaaa ccaaccactc ggqacatctt cgagctacgt ccaagtacag cccccgaggc cccccqaggc agcagcccct gggaqcctgc ccaccttgtc agaagatcat tgcagcacgc agcagacgcc gggtgaaqaa gggagaggtc aggtct gage ccgctcctgc ggcctcaggg gatgcaggac cagactccgc tggacaggcc gctgcctgct gcagccagct cagccctccg gggggaatct aaaaa actgaccctc caagaaqctg qatcctgccc catccacatt cagcctcccc accccctgcc gcccccaccq cgcccatccg tgtgagctcc gcacacccgg agcggagaag caacaagagg ggaqctggag gggcgccacg gggtgggCgg cgcagacggg cgctcagacg aggaggcggc cctcctcqggg cagcgcggec tggccCggCC ccccacacct accctcatgg attttttctc aagagggaca gactcggagc acggccacgg gaccaqatgc gcccgcagcc cagcccccac cccaatctgq ggacccagca ctggagcggc aagcggcacc gacaaggaqg ccctgggaga ccgctgcagc atcccgctgg cggccacqca cacagacacg gcqgccaggc ccggccagcg ccgaggccca agcgt cccag ggcctggcac tccagggccc cccccaggqg tccttttctt aggcgcccaa tqagccgqgc ccacgctgcg cgcagacccg cgccctcccg cgcctccgcc agccggcacc cggggcccag ggaagtcgcq aagacatgtt tgctggggcc gcctccggaa cgtcgccgct tgggccagga ctgggccacg ctcgcggqca acagggcccg ggcacagggc qcgggcagat ggtacccgcc cttttttaa taggcccctc caggactgag ttcttcaata gtcctccttc ccaggagaag gcccaagccc cctcatccac ccagcccccc cccaccgcca ccccagcctg caccaaqaac gtatgcagaa ccaggacctg ggacagcaag agcccacggg ggagcttcgc catcatcgac gaggaggqat gcgggccagg cagtqctggg accagaggec gggcggacgg tgaqctcctg acacccccat ctagacccag tcccctccag aaaagaatta 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 5520 5535 WO 99/37660 WO 9937660PCT/US99/01313 -221- <210> 68 <211> 398 <212> DNA <213> Unknown <220> <223> Description of Unknown Organism:Unknown <400> 68 cggggcaacc cgctggagtq gacgggccag gtgacggtgc gcaagaagcq tccaagttcc agacgctcga gctcgagaaq gagttcctct tcaacgcgta cagaagcgct gggagctqgc gcgcaacctc aacctcaccg aqcqccaggt ttccagaacc ggcgcatgaa gaacaagaag aacagccaqc gccaqgcqqc agcagcaaca gcagcagcag caqcagcagc aacagcagca agcggccgcc cggccgccgc caacggccac caqqqccacc aagcgcacca ccacgcgccc ccgtcgcagc cctcaagcac caccagtgac ccgtagcg <210> 69 <211> 8670 <212> DNA <213> Unknown <220> <223> Description of Unknown Organism:Unknown caagccctac cgtcagcaag caagatctqg cagcaqcagc gqcggggcgt cccaacqgcg 120 180 240 300 360 398 <400> 69 cccggqtgcg gcgcgacacq ggacatagcg gcgtgcgtgc agqggtaaga gcctgcatgt ggcgggcccc gcgctcgccc qaggagccgc gaagagqcgt qtgtcgtgtg gcgcccgacc gccaaggtga gtgcgtgcgg gtgcatacac atccaggtqq agcggcagcg cgacctcagc ccgtgtacgc gggggcgaga tggggctggg gccgccagct gggcagggtt tgttgcgtgt atgcatqcga cagcgtggcg ccgccagtgg caccgcggcc gtgcgagcaa gagcaaaggg cgccatgttc ggacgaccgg ttgcgtgcgt gtatttgaac ccggtggcct acgagcacgt cagcggcgcc gcqct cacct acaggtqcqt actaqggaaa ctggacatgc atgatggggg gcttgattgt tgtgttttgt tacaaatcaa gqcttcgagg agcggctcgg tcacgcggqt aagcgacgtg cgcacagcca tgagggccaa cggacccggg gcgtgtqcqt gtatgtactt caacacgtac gcccaggcac caccgcagqc gaaccccggc t gggcagcgc aatacggtat WO 99/37660 WO 9937660PCT/US99/01313 -222gcgggcaacg aattgctqggg actgcagtag qgcccatgcc ccgccgccct ccgcttcctc ccgcagcgaa agcgcgtgtt gaactgtgta tgccccacgt gcacgcacgt ctatgcccgc cacgagtgcg ggaggacggc gggccttgta gtacggcttg ttgccccgct agtgcgagaa ccgcagqgggg gtattgcacq aggcgctgca gttgagttgg agaaggcgtg atttttgtta ttgcgcatgc ttgtgtatcg aggcgatggc cgactaccgt tgccgttgcc tcgcgcactc ct cccccgcc aggctgggtg ctgctggtgt tctcagggcc tgtggagtag tgcagagt ca cctccgcact acacgctccc aggaggatga ctgggactgg cgtgactgcg tgggcatgca ccctgcacac cgcgcagcag agtgtgttca cgtacagcat gcgagttgtg cqtttggagg accgttttac caagcgacga acacgtgacc gttggcactt cctggaaatc agtctactga agccgcgctg cgagcaccca ttcacttctt cacgtgtgcg gcccggtgag tgtgtgggac cgtgqcgtgt gctgacaacg cccaacaaat gcgccctcag ggcgqcggc gcatcacag gttctgcatg gtgccttgac actgcagccq ctcaacaggc tattgtqgtt tggtgactgg gggcatgcgt tgtqggacga gagctcgtat atgcgtgqq aaatttatgc ataacaattg gcagggccct tgcattgcac ctgccctttg gagcacccag aattaatcgt cqacgcctgc tcgacgaqqa accgaaacgt gcggccggac acacgtgcgc tqacagcgac qtgtcgggcc tgcggcgqcg tgggtggcag gctagtggct qtqaggctgc gcttcgggcg tgctggggta gtttggqccg tcaggtgtaa catgcgcaga cgtggggttt gatgtagcat ttttggatgg tcaacgacgt gctcgtcaaa tttgaaatcg tacttgtatt gctcccttcc aagccgttgc gaatgtaatc acggagggtg ggaggtgcaa ggtaaaaqag cqacaaggca cttcctgtca acgtgcgcct agaccacaqa ccggcggggc gctggcaggg cacgcgctgc gctgccttgc ctacttcacc caaggcgctg tqggcgcggg gcggccggca cggcccctgg gtgccgtcaa ggattgaata caggggtttc gaccattgct ttgacgcqag tgtaaggcgc actgtatcct caatccaaat gtgcgctccg cccccccccc tggtggatgc qggggatacc acccgcccgc gcttgtggac ttgcccgtgc tcctataagc ccggttggtc gccgcggcga actcacgcat gcacgttcac tgctgccgcc gcgggctacg tgagagcgcg ctgcatgtgc gtgcgccgcg acgacaaggc agcacagaac atgacatgtg aqtcgcccga ttatacatac gctgcacttc 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 WO 99/37660PCJS9/11 PCTIUS99/01313 -223gatcctgaaa atcaaggggc gctggactcg caattgtgga cgtctcagcg gccgcaccga ggcagctgct gacggacccg tgaggacatc cgcqggtttg gggcccaggg cggccatccc cacccacata tcttgccaag caaggtgcgc agaggcgcgg gcgtctqagg gcgggcgqgc tttgcgcacg cacacactc tcccctggcc ggaaggcggg ggcgcctcac acgaccaggt ggtgggtggg aaccgaaccc gccccagttc ctctaagtgc tgcacttgtc agccgctgac tcatggccca gtctgcgcgg gcgcctqctg ctcatggaga aaggtqcggc gtggccaggg taacaccaga caccgccctc cccacacacc gccggcattc acacccggca gctgagaggg ggggctgaga gggcgggCgg ccacagccca ccacacccac cccgcctgct tgcctttgtg ggagctggtg gagggtgggt tgggtgggtg aagqggtqq aacaagtcgg ctcgcqgcaa gacgccgtcq gaattgtccg ggtgcggacg acqtctcgct acaacactaa agttcaacga acagggaggg cagggaggga ccgtgccgtq cccaccagca cacgcattcc tggcacatga gcagggcg gqctgagagg tggtggtggg gcgacgtgta catgacaccg gcqtgcgcgC ccccccatcc atcaaggcg ggggcggtg gggtgggggt ggt ggqt ggq cgtagggcg atagccaaat cccagcgcaa gcaccgcaat catqtgacat ttgqgactqc gacqtttcqc gaagctgtgg qtcgctgccc ggqcqagtqg agacgtgcgg tcgcgtgccc atgacacgta ccaacagggc cgaggccgtg tgggtgggtg ggggtcagcq ggagctgcgq cgtgagtagc ccgcaaggcc gcctccttcc gqccgccccg gtgacgagga gcqgcaaqct ggggtgqgtg ggtttgagat tqqgaggggg ggccccgctc gtgtgctcgc cgaaagacgc tgcaggctcq act tgcacga gttqaatgca ggcggtcgct tttgacaagc tggggtgggg qgct aggcaa ggcttqctgc cacattcaca agccaggcgt accattgtgg ggtggggtgg aqgcgcaggc gtgctggggc cgctgaccgg cgccgcgccc cctcaataca cctgcaggtg cat ccacacg gcacaccggc ggtgggtggq accggtacca ggagtgcgga gctctccagc 2220 gttgcggtga 2280 gtgcqtcgag 2340 cgtccccqct 2400 atgtgatgqg 2460 tctcgcaata 2520 tcacqggcaa 2580 gcctgtgqgc 2640 ctgggqqqga 2700 gaggctgcqa 2760 ccaccttgcc 2820 cactccccca 2880 acgccaaggc 2940 aggggctggc 3000 ggqgcagaq 3060 tcagggggag 3120 tgctgcggtg 3180 gcgctgqgcc 3240 cacccacgtt 3300 cgcgcctcct 3360 gctqaggagt 3420 gccaacgagc 3480 cqctcgcqca 3540 tgggtgggtg 3600 ggccaaacta 3660 agccgagg 3720 WO 99/37660 PTU9/11 PCT/US99/01313 -224caggagtaag gcagtcqgtg gggcggcggc agccccgtcc cgagqcactt aaacccaact ttcttaacta acttaatgtg ttgccaccga gcgagctgat gtgagggggc aqgggagggg tgagggcgat acctaaacaa gctgcctgct cacgcacctt gqcctggcag qctgggctcg aggggagaag agagggtggg agacgggagg ctggagtggg qgccggggcg agqagcacga acacacacac acgccccccc ggcgggagga gcggcgqtgg qgggCgggC gccactgccg cacagatgcg ttcccttccc accatgaatg tgtaaccccc ctaccggctg qcgcgtggcg aggqaggggq cagaggaggg agaaagttgc qggagctgga cccccacctc cagaatgcca cgcgacgaca qqtgggtgag ggggggggag qctgggtggg agggatgcag ccgacgagtg tgtqcacqac cgtgggacac acacacacac ccctacacac gggqqccgga cgggtqtgcc cgggggtgqg ccaccaccac aagcaaccac gccttaccta cccccccccg cttacacaca tgqctggtgg qcqgaccgct ggaggqqgag agggggagga gtattgtcqg gcgaggccaa ctcccccatc tgactqtgcg tgcggctgcg gqagggqagg acgaggaggg tggacggagt gggcgaaqgt tgcggacggq aggggcttgc qcatagatac acacacacaa acgccccctc ggagaaqcag gttgtgcaqt agacttgtcc cgccgccgcc accgttctcc accatgaccc gctgtacctg cacacacacc gtcaggtgga ccgaggcaga ggggaggtgc gggggcggct taaactcaaa atctgaacgt tcgccccccc ct ggagcca c ggacctqctq ggaggggagg tqqaagggtg gcactggtag ggggaggagg gcaggcggca gcgtctgcga gccgcattga acacaaacac cccaggcgcc ggacgaaqtc qqctqtggag aqaccccgtg gtagccacca acatgaacag gctacccccc gctacgactt cct ccccgcc ggt gatgcqq ggtggagqtg tcatgccggt qagtgcggga ggactagacq gacatcqccc ccccacacac tggctgatga ccgcgggtg ggggcagggc aggagggata ggaggggagq acggggatta ttgtgggggc? caacacacac acacaaacac ctggccggca gatgacaggc gccatgtgca gccctcttcc cccctcacgt ctaccctccc ccccctttat cacttcgtaa cctccaaagq tccgaggtqg ctcatgccgg gagggtagg gaggcaggga aagagaacaa gcctcctccc acacaqgctt gccacgccgc ccacactgcc gagggagagg ggtggaggct gqgtacattg aggcgtgqag aacggcgggg acacagggac acacacacac aaacacacac acccctttct 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 WO 99/37660PCUS9O13 PCTfUS99/01313 -225ggtggaccgc catggacgcg qaggaggggg aggagggggc qaggqggagg qagggggttg gtactgcctg tccccacacg aggatataca cacgggcgcg aacacact cc cgcgactttg tgggcggagg gcctacgcca atcaggtgcg tgaaggcgta tagccaggaa gttgggtaaa agcaggcagg aacacgtgac agggctgaga gctgcctcca cctcctcctq cggtqctcaa gagggaggga cagggacagg cagttcatcg gtgaggggaq ggagggggtt ctggaggagg attgggaggg ctgctggtgt gggccccgcg cgggccgctg cacacacaca ggctttcgtt atct t tct ca tgatcgagac acctcatcat ccggctcctc gqagggatgg gggggtggat cagtagtaga gcggtacagg cggggcgagg acggagatgc ggcgcgtgtg cctcctccac ctgcaggggc gggcacgccc gggagagcga gacagggaca ccaaggagtt gaggaggggg aactttgaag qqgggaggag ggctggggga aaagggcctg ctgcagagqt ccqctgcccg cacacacaca tcgtttttta cacaaacaca ggt gt tt gcg ctacagctcc gctcatgccg ggtgggggtg ggggtgqqtt gcagacgcgt atgtactgag ggcgtggggg accacacaaa gaggggt gcg cgcctcctgc aagggcggtc accacataca gggaqaqgga gggacaggga qggtttcqqc aggagggcgg cgtaaggaaa gaggqtggct gggtgcccgc taggcactga gt cqcgcagt ccacaccctt cacacacaca acacaaacac cacgcacaca gccagcctgc qgccccttcg cagaagaaga ggqgggttac acattcatqa tgtagtgtgt gactgcagac cgggggttac gaqggacgtg gggttgggcq cacctccacc qtgtgcagg acaaggactt gggagaggga caggqacagg ggcggcqtgt gggggggcag cagtcgggag ggagggqgct agctggggga gagcactgtg gtggcggcgc gccactttgt cacacacaaa acactccccc cacatgcgca tgtgcgtgca gctacgtgca accccgacgc attcatggtt acatttaaga gggtttgggt cgaaggagcg tggcaccgtg gggagtggca gggggctgtt tcccccactg caacctgatq ccaqgcgaga gggagaggga ggcaggggca gccccaactc gaggggggag gaggggggga gggqgaggag ggtggggagg gggagccggg atcccccgca gtgctttcct cacaaacaca tgtgctcctc ggtgtctgac cctgtcgcgc gtgcagcgac cctggagctc agttaagaag aqtgaaggcg gggagggatg ggggaggggg ccqggtaagc ggcqggggcc tcatgatacc cccctccccg ggcgtcatgg gagcgagagc gacagaggqa ggggcagggg 5340 5400 5460 5520 5580 5640 5700 5760 5820 5880 5940 6000 6060 6120 6180 6240 6300 6360 6420 6480 6540 6600 6660 6720 6780 6840 WO 99/37660 WO 9937660PCTIUS99/01313 -226caggggcagg tagqggcqct gggagcqgac tgggcttact ttgtgcagga gcatcgccac agcgtagccg agcgggagcg aggaagggaa ggggtcgtgt gagcctgcac ctcttgcgcc cctgacctcc gacttggccg tgcggccggg tcgaacgttg catatgtata gcaccacgcc ttcgacctca gcggtgagcg tagcctgggg gcagccgagc atgctggtgt ccttactgtg ccacaacccc cgcaccaaca ggcaggggag gcactgaggc atgcgccgca gacaattcat gtgttgggag cggcgtgctg agcagggctg ggagcagcag ggaqqagcag ccttggccqc ccacgctccg caccccctta tcgccaaacc agtacctggt cgggagggcg tgccgcgcct acaaactgac accacagcgg ccqtggacga gcggcgcgga gggagcgtgt gcacgtggtg gctgctaggt gccatgtgaa gcaccaccca acacgacgca gccccccggg cgcgaggcgg aacacagacg cattgtqcgc ctgctgtttq tccaccctgc gagcagcagc gaggggtggt gagcaggaqg atgggcqgag cgggtcccta gtgcatcctg tcccccgccc gcgcaaggqc tgagggcgtg gtttcacaat caccaatcat cgccgccgtg cctcaagacc gcagcagcaq gggaggaacg ctttgcccca ggttcacacc cgcatcctc gctcctcaat tgtgtttgcc ggcqgcgqgc gcqqgaggca ggttgagaaa a tat gtgt gt acacggtgga ggatcaaqcc cgggcagcag tgggaagcgg aagaggagga gcggggaggc ccgtcttgcg tacccctctt ccaggcctgt gtgccgttcc ggtggggcat gtcgggccac gcacqctcac aagatggccg atccacccgc caqcagcagc gcgggggagg ttccatgcca tacgtgtgaa atgtcctcgt ccagtgcaag acgtgcgcgc ccgggqcatg 99g9gc999g gcacaacgac atqtgtatgt cacggtgcac cgaccgcatg tagcagcagg tgggggtagg ggaagggcgg gggqaggagg cctaaccccg tccaaacatc ccgccgacat ggqagacaca gcccggggtt agggtatgca acgctctccc aggaccgcgg tcttcaccga agcagcagca ggaggcgggg gcagggtgac tttgtgctgg gattgcgccc qaaaggaaat acacacgcgc aggtcagaca ggcgqggggc tggaacgcag gtgtgtttgt gacgtggtgc aaggccggtg gcaggggagc gtgggagcgg t gqqgggtgq cggggaagca tqcgcctagc cttgcaactc gctcgccacg ccaccacagg gtgagaqcta gtttcctctc acacacacgc ctgcacgctg cgacgtggcg gcagcagcag ggtgtcgttt acacctgacc cgtqcgcaca ggcacattqc gcacgcccgc aggtgtggga 6900 6960 7020 7080 7140 7200 7260 7320 7380 7440 7500 7560 7620 7680 7740 7800 7860 7920 7980 8040 8100 8160 8220 8280 8340 8400 WO 99/37660 WO 9937660PCTIUS99/013 13 -227cttcaaccgc agcgccgaga tgcgcgacac ggagggcggc accagcaagc gctcggtqct 8460 gqagcaggtg cagaagatgc gcacotacot ggcggcggag ggacagcact gagcgggtcg 8520 ggggaggggq ggcgggtgtg tatgtgtgtg tgtqtgcgtg tgtaagtctc gqtqgagggg 8580 tggtcctcta tatgqcggcg ggqccacagg gggacgggtg tgacaqagtt acggccggag 8640 ccagcggagt cccgggatgg attaaggatc 8670 <210> <211> 745 <212> DNA <213> Unknown <220> <223> Description of Unknown Organism:Unknown <400> atgagatggc ggct ccgccg accgcggccc gtgtgctact gtggtgatcg gcggcggcgg qgcccacggg ccctcttggC ctggtgaagg ctcaccgtgc gaggacagca gccgccttCC agccgggtgc gacgcgcccc cccgctcgtC tggcgccggg cgtccccgCC aqggaaaggt cggcgqqcga cgctggggcc ccaccgcccc tgcaccaggt qcctgggqac ggtacatctt gagcctcttt tgtgcaaqcq gcgccgctcc gccgccgctg ggcggcggcc.
cagcgtgqga gcacccgcag ggcaggggcg gcccgccgag ggtgcccagc gtqqgcggtg ctgggqccac cttcatggag cccccctctg gtgcg gggcgtcccg ccgctgctgC ggcaacgagg tcggtgcagg cggcggcagc tggggcqgcg gagccgctgc gccggcgagc aaagccgggg cccqccttCC cccgacgcca gagacgggcc gcccccgggc cactactgct cggctcccgC agctagctca agggggcact atcgcgagcc tcgccgccaa ccggggagga gcttgaagaa cctcctgcgg acagcaccag ggaacctcaa ccagcgcccc gctgctggg ggggqcctcg gcgcgccgcg cgacaggaag gccagccgcg cqggaccgtg ggcgccctat gqactcgctg qaggctcaag ccgcqcgccg gaaggaggtC 120 180 240 300 360 420 480 540 600 660 720 745 <210> 71 <211> 1986 <212> DNA <213> Unknown WO 99/37660 WO 9937660PCTIUS99/01 313 -228- <220> <223> Description of Unknown Organism:Unknown <400> 71 gaattccttt ctgtggttcc cccccaataa ggggaaggaa agcccgcacg qgcccccggg ccactactgc gcgqctcccg gagctagctc cagggggcac gatcgcgagc ctcgccgcca cccggggagg ggcttgaaga ccctcctqcg aacagcacca cggaacctca ttgaaagaga accagttctg cgaaaaaaca attaacaaag ggaaatqaca accactggga ggaggggagt tttttttttt atccacttct ataaataaaa aagggaggca cacctcgcac cccagcgccc tgctgctggg cgggggcctc aqcgcgccgc tcgacaggaa cgccagccgc acgggaccgt aqgcgcccta aqgactcgct ggaggctcaa gccgcgcgcc agaaggaggt tgaaaagcca aatactcctc aaccacaaaa catcactggc gtgcctctgc caagccatct gcttcatggt ttttttcttt tccccctcct ggagqagggc gcgcgagaag catgagatg cggctccgcc gaccgcggcc ggtgtgctac ggtggtgatc qgcggcggcg gggcccacgg gccctcttgg tctggtgaag gctcaccgtg qgaggacagc ggccgccttc cagccgggtg ggaatcggct tctcagattc tatcaagata tgattctgga caatatcacc tgtaaaatgt gaaagacctt ttttttttqc cctcccataa aaggggggag agccgggcag cgacgcgcc qcccgctcqt ctggcgccqg tcgtccccqc gagggaaagg gcqgcgggcg gcqct gggqc cccaccqccc gtgcaccagg cgcctgggga aggtacatct cgagcctctt ctgtgcaagc gcaggttcca aagtggttca caaaaaaagc gagtatatgt atcgtggaat gcggaqaagg tcaaacccct ccttatacct acaactctcc gaqgaggagt agtccgaacc cgcgccgctc cgccgccgct gqcggcgqc ccagcgtggg tgcacccgca aggcaggggc cgcccgccga cggtgcccag tgtgggcggt cctggqcca tcttcatgga tcccccctct ggtgcgcctt aactaqtcct agaatqggaa cagqaagtc gcaaagtgat caaacgctac agaaaacttt cgagatactt cttcgccttt tacccctgca qgtgctgcga gacagocaga cggqcgtccc gccgctgctg cggcaacgag atcqgtgcag gcggcgqcag gtggggcqgc ggagccgctg cgccggcgag gaaagccggg ccccgccttc gcccgacgcc ggagacgggc gcctccccaa tcggtgtgaa tgaattgaat agaacttcgc cagcaaatta atctacatcc ct gt gt aa t gtgcaagtgc 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 WO 99/37660 WO 9937660PCT/US99/01313 -229ccaaatgagt tccactccct tgctgcatct actgcctctg gcgactagtt atattgaatg ggcatttcaa ttctttatac tgaaatgatg aaaag ttactggtqa ttctgtctct cccctcagat cctqtcgcat gqctctgaqa atgtgataca agtctcactt aatgaccaca gtaagttaat tcgctgccaa gcctgaatag tccacctaqa gagaacatta tactaatagg aattgatagt ttattgataa tcctgaaaag t t tgat tcag aactacgtaa gagcatgctc gctagatgtg acaaaagcaa tgtgtgaggc caatatcaag aataaaaatc ggtgttgcta aatgtgttat tggccaqctt agttgqtgct tcttaccaga ttgtattact tccggatgtt cagtgaaata attctactga agctgtaacc ttgtcacaaa ctacaqtacg gctttcttgt tctaatattg tcctctgttc tctggaattg tgataataaa acagtccatc qatatgcact taaacataat 1500 1560 1620 1680 1740 1800 1860 1920 1980 1986 <210> <211> <212> <213> <220> <223> <220> <221> <222> <223> <220> <221> <222> <223> 72 2003
DNA
Unknown Description of Unknown Organism:Unknown
UNSURE
(31) May be any nucleic acid UNS URE (32) May be any nucleic acid <400> 72 ggaattcctt tttctgtqgt gcacccccaa cgagqggaag agaagcccqc cccggccccc tttttttttt tccatccact taaataaata gaaaagggag acqcacctcq gggcccagcg tttttttctt tcttccccct aaaggaggag gcagcgcgag caccatgaga ccccgqctcC nntttttttt cctcctccca ggcaaggggq aagagccggg tggcgacgcg qccqcccgct tgcccttata taaacaactc gaggaqgagg cagagtccga ccccgcgccg cgtcgccgcc cctcttcgcc tcctacccct agtggtgctg accqacagcc ctccgqgcgt gctgccgctg 120 180 240 300 360 WO 99/37660PC/S9013 PCT/US99/01313 -230ctgccactac gaggcggctc cagqagctag cagcaggggg ggcgatcgcg ctgctcgccg gagcccqqqg gggggcttga ttcccctcct gccaacagca ggccggaac caattgaaag gaa acc agt t aatcgaaaaa cgcattaaca ttaggaaatg tccaccactg aatggagggg tgcccaaatg acgtccactc tgttgctgca ttgactgcct ttcqcgacta ttgatattga aaaggcattt atcttcttta tgctgctgct ccgcqgggggC ctcagcgcgc cactcgacag agccgccagc ccaacgggac aggaggcgcc agaaggactc gcqggaggct ccagccgcgc tcaagaaqga agatqaaaag ctgaatactc acaaaccaca aagcatcact acagtgcctc ggacaagcca agtgcttcat agtttactgg cctttctgtc tctcccctca ctgcctgtcg gttggctctg atgatgtgat caaagtctca tacaatgacc ggggaccqcg ctcggtgt gc cgcggtggtg gaaggcgqcg cgcgqgccca cgtgccctct ctatctggtg gctqctcacc caaggaggac gccggccgcc ggtcagccgg ccaggaatcg ctctctcaga aaatatcaag ggctgattct tgccaatatc tcttgtaaaa ggtgaaagac tgatcgctgc tctgcctgaa gattccacct catgagaaca agatactaat acaaattgat cttttattga acatcctgaa gccctqgcgC tactcgtccc atcgagggaa gcggcggcgq cgggcgctgg tggcccaccg aaggtgcacc gtgcgcctgg agcaggtaca ttccgagcct gtgctgtgca gctgcaggtt ttcaagtggt atacaaaaaa ggagagtata accatcqtgg tgtgcggaga ctttcaaacc caaaactacg taggagcatg agagctagat ttaacaaaag aggtgtgtga aqtcaatatc taaaataaaa aagggtgttg Cgggq9gc cgcccagcgt aggtgcaccc gcgaggcagg ggccgcccgc ccc cggt gcc agqt gtgggc ggacctgggg tcttcttcat ctttcccccc agcggtgcgc ccaaactagt tcaagaatgg agccagggaa tgtgcaaagt aatcaaacgc aggagaaaac cct cgagata taatggccag ctcagttggt gtgtcttacc caattgtatt ggctccggat aaqcagtgaa atcattctac ctaagctgta ggccggcaac gggatcggtg gcagcggcgg ggcgtggggc cgagqagccg cagcgccggc ggtgaaagcc ccaccccgcc ggqacccgac tctggagacg cttgcctccc ccttcggtqt gaatqaattg gtcagaactt gatcagcaaa tacatctaca tttctgtqtg cttgtgcaaq cttctacagt gctgctttct agatctaata acttcctctg gtttctggaa atatgataat tgaacagtcc accgatatgc 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 WO 99/37660 WO 9937660PCTIUS99/01313 -231acttgaaatg atgqtaagtt aattttgatt cagaatgtgt tatttgtcac aaataaacat 1980 aataaaagga aaaaaaaaaa aaa 2003 <210> <211> <212> <213> <220> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> 73 957
DNA
Unknown Description of Unknown Orqanism:Unknown
UNSURE
(809) May be any nucleic acid
UNSURE
(810) May be any nucleic acid
UNSURE
(811) May be any nucleic acid <400> 73 t ctcgcccca caaaatggca agatttgcct ctqctgcagc ttttqttgca cgctgcggag gctcgagaag cttgaggagc gcggggtttg ccaggagaaq actttttccc gacctcttca gcggaqggcg aaactgcaag gggaagctgg gggcggctgc caggacctcg agcaaactgg ctgctggtcg ctggcccagg ccgcgctccg gcggactcgt agagggcccc aaggggcccg cctqctgcct ccagcaqcag agcagagcct ccctcgaggc agagcagcaa cctacagttc cagcagcagc gggcqgcgtc ccgccccgcc cgagctggag gcgggtgggg cagcaqcagc cgaggccggc cctcctcgag agacacggtg tttcctgcgg agcagcagca gtcggcgctg cccggcactg ggttttgtgc gcggagcagc agctgctgcg aaqcagggcg ggggcccgcg9 ctgcgcagca ggctaccagg gcagcagcag ttqctgcaqc cctggacttg agcagctgag tggcgcgctg cgctgctgca cggagtgcct ttgcagcaac ttccccacac gggcagcagc 120 180 240 300 360 420 480 540 600 ggaggtct ctgggctacg gggcccctqc tgctqcttac ggccagcaqc agcaqcccaq 660 PCTIUS99/01313 WO 99/37660 cagctacgg cagcagcagc ggcgccgctg actccacag gcgccagctg -232g cgccccccg cctccagcca gcagccctcC ggcttcttct ggtagccctg 720 agcagcagca gcagcagcag cagcgcgggc ggcacgcg qcgqggccgq 780 cagcaacagc agcagccgnn ncgqctagcg ccgcggagca ctcgcaggga 840 cagcgggaga gcagcaqgga cqagaagcag gtcatqtagc gcaggcagca 900 cagcagcagc agcagcagca gcagcagcag cagcagctcc tgcaccg 957 <210> <211> <212> <213> <220> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> 74 957
DNA
Unknown Description of Unknown Organism:Ulklowf
UNSURE
(809) May be
UNSURE
(810) May be
UNSURE
(811) May be any nucleic acid any nucleic acid any nucleic acid <400> 74 tctcgcccca caaaatggca agatttgcct ctgctgcagc ttttgttgca cqctgcggag gct cqaqaag cttgaggagc gcggggtttg actttttccc gacctcttca gcggagggcg aaactgcaag gggaagctg gggcggctgc caggacctcg agcaaactgg ctgctggtcg ccgcgctccq gcggactcgt agagggcccc aaggggcccg cctgctqcct ccagcaqcag agcagagcct ccctcgaggc agaqcagcaa cagcagcagc agcagcagca gggcgqcqtc qtcggcgctg ccgccccgcc cccggcactq cgagctggag ggttttqtqc gcgggtqgqg gcggaqcagc cagcagcagc agctgctgcg cgaggccggc aagcagggcg cctcctcgag gqqgcccgcg agacacgqtq ctgcgcagca gcagcagcag ttgctgcagc cctggacttg agcaqctqag tggcgcgctg cgctgctqca cqgagtgcct ttqcagcaac ttccccacac 120 180 240 300 360 420 480 540 WO 99/37660 PTU9/11 PCT/US99/01313 -233ccaggagaag ggggaggt ct cagctacggg caqcaqcagc ggcgccgctg actccacagg qcgccagctg ctggcccagg ctgggctacg gcgccccCcg agcagcagca cagcaacaqc cagcgqgaga cagcagcagc cctacagttc gqgcccctgc cctccagcca gcagcagcag agcagccgnn gcagcaggga agcagcagca tttcctccgq tgctgcttac gcagccctcc cagcgcgggc ncggctagcg cgagaaqcag gcagcagcag ggctaccagg qgccagcagc gqcttcttct ggcagccgcq ccgcggagca gtcatgtagc cagcagctcc gggcagcagc agcagcccag ggtagccctg gcggggccgg ctcgcaggga gcaggcagca tgcaccg 600 660 720 780 840 900 957 210> <211> <212> <213> <220> <223> <220> <221> <222> <223> <220> <221> <222> <~223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> 1089
DNA
Unknown Description of Unknown Organism:Unknown
UNSURE
(376) May be UNS URE (377) May be
UNSURE
(847) May be
UNSURE
(848) May be
UNSURE
(849) May be
UNSURE
(850) May be any nucleic acid any nucleic acid any nucleic acid any nucleic acid any nucleic acid any nucleic acid r- I PCTIUS99/01313 WO 99/37660 -234- <400> qaattccctc cgcagcagca ggcgtcgtcq cccgcccccg ctggagggtt gtggqggCgg agcagcagct gccggcaagc ctcgaggggg acggtgctgc ctgcggggct gcttacggcc ccctccgqct ggcggcggca ctagcgnnnn agcaggtcta cagcagcagc caactcttcg gcagcagcag gcgctgttgc gcactgcctg ttctqcagca agcagctggc gctgcnngct agggcgcgga cccgcgttgc gcagcattcc accagggggc agcagcagca tcttctggta gccgcggCgg gagcactcgc tgtagcgcag agctcctgca cqactctctc cagcagcaaa tgcagcagat gacttgctgc gctgagtttt gcgctgcgct gctgcagctc gtgcctcttg aqcaacgcg ccacacccag agcagcgggg gcccagcagc gccctgcagc ggCCgqggcg agggaactcc gcagcagcgc ccgcagcgtt tctctcgccc atgqcagacc ttgcctgcgq tgcagcaaac gttgcaggga gcggaqggc gaqaagcag aggagcagca ggtttgctgc gagaagctgg aqgtctctgg tacqgggcgc agcagcagca ccgctgcagc acaggcagcq cagctgcagc caactttttc tcttcagcqg agggcgagag tqcaagaagg agctggcctg ggctgcccag acctcgagca aactggccct tggtcgagag ct caggocta gctacggggc cccccqcctc gcagcagcag aacagcagca ggagagcagc agcagcagca ccccgcgCc actcgtgggc ggccccccgc qgcccgcgag ctgcctgcgg cagcagcagc qagcctcgag cgaggccct c cagcaaagac cagttctttc ccctgctgct cagccagcag cagcagcagc gccgCggcqg agggacgaga gcagcagCag agctctgagg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 gtgtcattta ttacgttggc cctcggCgca acggaattc qccaaCgCgC ctcaggtatc tttcagactc ttttctctaa ggtcttccag 1080 1089 <210> 76 <211> 1985 <212> DNA <213> Unknown <220> <223> Description of Unknown Organism:Unklowf <400> 76 cgccgagctt tcggcacctc tgccggqtgg taccgagcct tcccggcc ccctcctctc PCT/US99/01313 WO 99/37660 ctcccaccgg ctctcccggc gtggcggCgg tgctactact cctcatagaa gaaatatata tatatcaaat catatattta ctctcaactg gctqttaatt tacgttaatg tatattgata cgtgtaatga agggatattg atagatgatg tatcaaccta ggttgtttca aacttaccat tggacatatg gattactttg aaccagaace atgttacctc gacagtggac aagcggatac agaaatgtg cctgcccttc cgaqccgCgq tggccgggga acgacggtga tccgcatgac ggccccataa ttctacggtc atqttqgaga gcggttcagt gggctggagq atattgtgct tagatattca cggtatcatt gtgctggaaa agtcatatgg gtgctgtggt atctaacagt tactgatgct agactgcagt agtattttgc ctccagaatz atgcacctg jatgaagatg( Icttgtgatgi I ctgatcata.
cccgcgggac cggcagcagc qcccatggcg tattqqaaat ccataacttg agccactgcc aataagacca agattgtcca tgctggagct attacatcat tgccatcctt tcatggtgat ccataaatat aggcaaatac gcagatattt attacagtgt caaaggtcat tggaggaggt tgcccttgat accagacttc Itatggaaaac 3 tgtccagatc 3 agaagatccE a agaattctc a gaaaggaqc -23 tatcgccccc 2 agcagcagca tacagtcaaq tattattatg ctqttaaatt qaagaaatga gataacatgt gcgtttgatg qtgaagttaa gctaagaaat gaattactaa ggtgttgaag ggggaatact tatgctgtca aagcctatta ggtgcagact gctaaatgtg ggctacacaa tqtgagattc aaactgcata Iataaaacagc Icaagctattc igacaagagaa igattctgagg iaagaaagcta kcgtttccct c ;cagcaggaq g aggcqgcaa a 4acagggtca t caaaatatca ctgagtatag gactctttga accgacaaca acgaagcatc agtatcatca aagcttttta ttcctggcac attttccaat tctcaaaggt cattatctgg tagaagttgt tccgtaatgt ccaatgagtt ttagtccttc gtttgtttga cagaagatgc tttctattcg atgaaggaga gaattgaaga ~agccctttt 120 ~aggagcccg 180 iaaaaaagtc 240 :cccatgaag 300 ~agaaaaatg 360 agtgatgag 420 :aagcagatg 480 jttttgtcag 540 gactgatatg 600 aggattctgt 660 gagagtctta 720 tacaacagat 780 aggagacttg 840 gtgtqatggt 900 gatggagatg 960 tqatagactg 1020 aaaaactttt 1080 tgctcgatgt 1140 gccatataat 1200 aaacatgaca 1260 aaatttqcgc 1320 tgttcatgaa 1380 agcatcagac 1440 aggaggtcga 1500 agataagaaa 1560 gaaacagagg acaaaaaaac agacgttaag gaagaagata aatccaagga caacagtggt 1620 PCTIUS99/01313 WO 99/37660 gaaaaaacag tctcaccaat gaagacttct actttttcgt aaatttcttt gtcaaaaaaa aaaag ataccaaagg ttcagaaaat ggcttcattt ttttgttttt tctccaccat ctgatctatt aaccaaatca cattaaaaag tat act act t ctgggcaagt gctttatgtg aaagaagtaa -236gaacagct ca aaaatattga tggcatggac tttattgtqa atagtattta ttggcctttC gcaacccctg aaggaaaatg tgtatttatt gattttctaa aaattgatgt tgagctgaaa aatttgacag ttttcttttt ttcaaatggg ttatgaagca gagttattat aaaaaaaaaa 1680 1740 1800 1860 1920 1980 1985 <210> 77 <211> 476 <212> DNA <213> Unknown <220> <223> Description of Unknown Organism:Unknown <400> 77 ccaccctcct ccccctcccc cggccacttc gctaacttqg tggctgttgt cctgtagatc cgagcaccag ccggcgcttc agccccccct ccagcagcct aaaatgagcg acgtgagccc ggtggtggct gcgcaacagc agcagcaaca caacagcagc agcagcagca qcaacagcag cagcagcagc agqaqggcg gcggcggcaq cgqcggctgc ggcggcqqca gctgcagtgc cccqgttgcg gacaaccgca ccatggtgga gatcatcgcc qaccacccgg ccgaactcgt agccccaact tcctgtgctc ggtgctgccc tcgcactgqc gctgcaacaa gtggccttca aggtaagagg ctaccccgcc ccccgccccc qgccgggagc <210> 78 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:DNA Primer <400> 78 gcattttgqa tccgcctttt catg gatqcgtatt gcagcccggc gcagcagcag ggcggcggct gccgccccac ccgcaccgac gaccctgccc ggcgqa 120 180 240 300 360 420 476 WO99/37660 PCT/US99/01313 WO 99/37660 -237- <210> 79 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:DNA Primer <400> 79 gttgtgtgct gcagattgtt cc 22 <210> <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:DNA Primer <400> gaaaaatggg gatccgaggt g 21 <210> 81 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:DNA Primer <400> 81 gcaggagaat tccgtccatg <210> 82 <211> <212> PRT <213> Homo sapiens <220> <221> UNSURE <222> (3) <223> Can be any amino acid <400> 82 Trp Ser Xaa Trp Ser 1 PCT/US99/01313 WO 99/37660 -23 8- <210> 83 <211> 6 <212> PRT <213> Homo sapiens <400> 83 Cys Ser Val Thr Cys Gly <210> 84 <211> <212> PRT <213> Homo sapiens <220> <221> UNSURE <222> (4) <223> Can be any amino acid <400> 84 Gly Cys Gin Xaa Arg 1 <210> <211> <212> <213> <400> 733
DNA
Homo sapiens gggatccgga gcccaaatct tctgacaaaa ctcacacatq cccaccgtgc ccaqcacctg aattcgaggg tctcccggac tcaagttcaa aggagcagta ggctgaatgg agaaaaccat catcccggga atccaagcga ccacgcctcc tgcaccgtca tcctgaggtc ctggtacgtg caacagcacq caaqgagtac ctccaaagcc tgagctgacc catcgccgtg cgtgctggac gtcttcctct acatgcgtqg gacggcgtg taccqtgtg aagtgcaagg aaaqggcagc aagaaccagg gagtgggaga tccgacggct tccccccaaa tggtqgacgt aggtgcataa tcagcgtcct tctccaacaa cccgagaacc tcagcctgac gcaatgggca ccttcttcct acccaaggac aagccacgaa tgccaaqaca caccqtcctg agccctccca acaggtgtac ctgcctggtc gccqqaqaac ctacagcaag accctcatga 120 qaccctgaqg 180 aagccgcggg 240 caccaggact 300 acccccatcg 360 accctgcccc 420 aaaggcttct 480 aactacaaga 540 ctcaccgtgg 600 acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat qaggctctgc 660 WO 99/37660 PCT/US99/01313 -239acaaccacta cacgcagaag agcctctccc tgtctccggg taaatgagtg cgacggccgc 720 gactctagag gat 733 <210> 86 <211> 86 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:DNA Primer <400> 86 gcgcctcgag atttccccga aatctagatt tccccgaaat gatttccccg aaatgatttc cccgaaatat ctgccatctc aattag 86 <210> 87 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:DNA Primer <400> 87 gcggcaagct ttttgcaaag cctaggc 27 <210> 88 <211> 271 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:PCR Fragment <400> 88 ctcgagattt ccccgaaatc tagatttccc cgaaatgatt tccccgaaat gatttccccg aaatatctgc catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc 120 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat 180 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt 240 ttttggaggc ctaggctttt gcaaaaagct t 271 <210> 89 WO 99/37660 PCT/US99/01313 -240- <211> 32 <212> DNA <213> Homo sapiens <400> 89 gcgctcgagg gatgacagcg atagaacccc gg 32 <210> <211> 31 <212> DNA <213> Homo sapiens <400> gcgaagcttc gcgactcccc ggatccgcct c 31 <210> 91 <211> 12 <212> DNA <213> Homo sapiens <400> 91 ggggactttc cc 12 <210> 92 <211> 73 <212> DNA <213> Homo sapiens <400> 92 gcggcctcga ggggactttc ccggggactt tccggggact ttccgggact ttccatcctg ccatctcaat tag 73 <210> 93 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:PCR Fragment <400> 93 gcggcaagct ttttgcaaag cctaggc 27

Claims (23)

  1. 4.Sep. 2003 13:39 WRAY&ASSOCIATES No.3724 P. 4 180 What Is Claimed Is: 1. An isolated nucleic acid molecule comprising a polynucleotide selected from the group consisting of: a polynucleotide encoding a polypeptide comprising amino acids 1 to 950 in SEQ ID NO:2; a polynucleotide encoding a polypeptide comprising amino acids 2 to 950 in SEQ ID NO:2; a polynucleotide encoding a polypeptide comprising amino acids 29 to 950 in SEQ ID NO:2; a polynucleotide encoding a polypeptide comprising amino acids 30 to 950 in SEQ ID NO:2; a polynucleotide comprising a nucleotide sequence encoding the METH1 polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 209581; a polynucleotide comprising a nucleotide sequence encoding the mature METH1 polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 209581; a polynucleotide encoding a polypeptide comprising amino acids 1 to 890 in SEQ ID NO:4; a polynucleotide encoding a polypeptide comprising amino acids 2 to 890 in SEQ ID NO:4; a polynucleotide encoding a polypeptide comprising amino acids 24 to 890 in SEQ ID NO:4; a polynucleotide encoding a polypeptide comprising amino acids 112 to 890 in SEQ ID NO:4; a polynucleotide comprising a nucleotide sequence encoding the METH2 polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 209582; a polynucleotide variant created by altering a polynucleotide of or wherein: said altering includes a nucleotide insertion, deletion, or substitution, or any combination thereof, and COMS ID No: SMBI-00404055 Received by IP Australia: Time 15:00 Date 2003-09-04 4.Sep. 2003 13:39 WRAY&ASSOCIATES No.3724 P. 181 (ii) the number of alterations is equal to or less than 5% of the total number of nucleotides present in the unaltered polynucleotide; a polynucleotide encoding amino acids 235 to 459 in SEQ ID NO:2; a polynucleotide encoding amino acids 460 to 544 in SEQ ID NO:2; a polynucleotide encoding amino acids 545 to 598 in SEQ ID NO:2; a polynucleotide encoding amino acids 841 to 894 in SEQ ID NO:2; a polynucleotide encoding amino acids 895 to 934 in SEQ ID NO:2; a polynucleotide encoding amino acids 536 to 613 in SEQ ID NO:2; a polynucleotide encoding amino acids 214 to 439 in SEQ ID NO:4; a polynucleotide encoding amino acids 440 to 529 in SEQ ID NO:4; apolynucleotide encoding amino acids 530 to 583 in SEQ ID NO:4; a polynucleotide encoding amino acids 837 to 890 in SEQ ID NO:4; a polynucleotide encoding amino acids 280 to 606 in SEQ ID NO:4; a polynucleotide encoding amino acids 529 to 548 in SEQ ID NO:4; and a nucleotide sequence complementary to any of the nucleotide sequences in or 2. An isolated nucleic acid molecule, comprising a polynucleotide selected from the group consisting of 50 contiguous nucleotides of the coding region of SEQ ID NO: 1, provided that said nucleotide sequence is not any one of SEQ ID Nos:14-41, or any subfragment thereof; and .a nucleotide sequence complementary to the nucleotide sequence in 44 4 *eo COMS ID No: SMBI-00404055 Received by IP Australia: Time 15:00 Date 2003-09-04 4.Sep. 2003 13:40 WRAY&ASSOCIATES No.3724 P. 6 182 3. An isolated nucleic acid molecule, comprising a polynucleotide selected from the group consisting of: 50 contiguous nucleotides of the coding region of SEQ ID NO:3, provided that said nucleotide sequence is not SEQ ID Nos: 19-22, 24, 42-77, or any subfragment thereof; and a nucleotide sequence complementary to the nucleotide sequence in 4. A method for making a recombinant vector comprising inserting an isolated nucleic acid molecule of claim 1 into a vector in operable linkage to a promoter. A recombinant vector produced by the method of claim 4.
  2. 6. A method of making a recombinant host cell comprising introducing the recombinant vector of claim 5 into a host cell.
  3. 7. A recombinant host cell produced by the method of claim 6.
  4. 8. A recombinant method for producing a METH1 or METH2 polypeptide, comprising culturing the recombinant host cell of claim 7 under conditions such that said polypeptide is expressed and recovering said polypeptide.
  5. 9. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of: 9 amino acids 1 to 950 in SEQ ID NO:2; amino acids 2 to 950 in SEQ ID NO:2; amino acids 29 to 950 in SEQ ID NO:2; amino acids 30 to 950 in SEQ ID NO:2; the amino acid sequence of the METH1 polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 209581; 9 9 *oo *~e *w *oo *e COMS ID No: SMBI-00404055 Received by IP Australia: Time 15:00 Date 2003-09-04 4.Sep. 2003 13:40 WRAY&ASSOCIATES No.3724 P. 7 183 the amino acid sequence of the mature METH1 polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 209581; amino acids I to 890 in SEQ ID NO:4; amino acids 2 to 890 in SEQ ID NO:4; amino acids 24 to 890 in SEQ ID NO:4; amino acids 112 to 890 in SEQ ID NO:4; an amino acid sequence of the mature METH2 polypeptide having the amino acid sequence encoded by the METH2 cDNA clone contained in ATCC Deposit No. 209582; the amino acid sequence of a polypeptide variant created by altering a polypeptide of or wherein: said altering includes an amino acid insertion, deletion, or substitution, or any combination thereof; and (ii) the number of alterations is equal to or less than 5% of the total number of amino acids present in the unaltered amino acid sequence; amino acids 235 to 459 in SEQ ID NO:2; amino acids 460 to 544 in SEQ ID NO:2; amino acids 545 to 598 in SEQ ID NO:2; S* amino acids 841 to 894 in SEQ ID NO:2; amino acids 895 to 934 in SEQ ID NO:2; amino acids 536 to 613 in SEQ ID NO:2; amino acids 214 to 439 in SEQ ID NO:4; amino acids 440 to 529 in SEQ ID NO:4; *494 o9*9 **o COMS ID No: SMBI-00404055 Received by IP Australia: Time 15:00 Date 2003-09-04 4.Sep. 2003 13:40 WRAY&ASSOCIATES No.3724 P. 8 184 amino acids 530 to 583 in SEQ ID NO:4; amino acids 837 to 890 in SEQ ID NO:4; amino acids 280 to 606 in SEQ ID NO:4; amino acids 529 to 548 in SEQ ID NO:4; The isolated polypeptide of claim 9, which is produced in a recombinant host cell.
  6. 11. The isolated polypeptide of claim 10, wherein said recombinant host cell is mammalian.
  7. 12. An isolated nucleic acid molecule comprising a polynucleotide encoding a METHI or METH2 polypeptide wherein, except for one to fifty conservative amino acid substitutions, said polypeptide has a sequence selected from the group consisting of: amino acids from about 1 to about 950 in SEQ ID NO:2; amino acids from about 2 to about 950 in SEQ ID NO:2; amino acids from about 29 to about 950 in SEQ ID NO:2; amino acids from about 30 to about 950 in SEQ ID NO:2; the amino acid sequence of the METH1 polypeptide as encoded by the cDNA clone contained in ATCC Deposit No. 209581; o the amino acid sequence of the mature METH 1 polypeptide as encoded by the cDNA clone contained in ATCC Deposit No. 209581; amino acids from about 1 to about 890 in SEQ ID NO:4; amino acids from about 2 to about 890 in SEQ ID NO:4; amino acids from about 24 to 890 in SEQ ID NO:4; amino acids from about 112 to about 890 in SEQ ID NO:4; and the amino acid sequence of the METH2 polypeptide as encoded by the cDNA clone contained in ATCC Deposit No. 209582. 9** COMS ID No: SMBI-00404055 Received by IP Australia: Time 15:00 Date 2003-09-04 4.Sep. 2003 13:40 WRAY&ASSOCIATES No.3724 P. 9 185
  8. 13. An isolated polypeptide wherein, except for one to fifty conservative amino acid substitutions, said polypeptide has a sequence selected from the group consisting of: amino acids from about 1 to about 950 in SEQ ID NO:2; amino acids from about 2 to about 950 in SEQ ID NO;2; amino acids from about 29 to about 950 in SEQ ID NO:2; amino acids from about 30 to about 950 in SEQ ID NO:2; the amino acid sequence of the METHI polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 209581; the amino acid sequence of the mature METH1 polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 209581; amino acids from about 1 to about 890 in SEQ ID NO:4; amino acids from about 2 to about 890 in SEQ ID NO:4; amino acids from about 24 to about 890 in SEQ ID NO:4; amino acids from about 112 to about 890 in SEQ ID NO:4; the amino acid sequence of the METH2 polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 209582;
  9. 14. An isolated nucleic acid molecule comprising a polynucleotide at least 95% identical to a polynucleotide selected from the group consisting of: a polynucleotide encoding a polypeptide comprising amino acids I to 950 in SEQ ID NO:2; a polynucleotide encoding a polypeptide comprising amino acids 2 to 950 in S SEQ ID NO:2; a polynucleotide encoding a polypeptide comprising amino acids 29 to 950 in SEQ ID NO:2; COMS ID No: SMBI-00404055 Received by IP Australia: Time 15:00 Date 2003-09-04 4.Sep. 2003 13:40 WRAY&ASSOCIATES No.3724 P. 186 a polynucleotide encoding a polypeptide comprising amino acids 30 to 950 in SEQ ID NO:2; a polynucleotide comprising a nucleotide sequence encoding the METH1 polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 209581; a polynucleotide comprising a nucleotide sequence encoding the mature METHI polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 209581; a polynucleotide encoding a polypeptide comprising amino acids 1 to 890 in SEQ ID NO:4; a polynucleotide encoding a polypeptide comprising amino acids 2 to 890 in SEQ ID NO:4; a polynucleotide encoding a polypeptide comprising amino acids 24 to 890 in SEQ ID NO:4; a polynucleotide encoding a polypeptide comprising amino acids 112 to 890 in SEQ ID NO:4; a polynucleotide comprising a nucleotide sequence encoding the METH2 polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 209582; a nucleotide sequence complementary to any of the nucleotide sequences in or wherein said identity is calculated using the FASTDB computer program, with the parameters: Matrix=Unitary, k-tuple=4, Mismatch Penalty=-, Joining Randomization Group Length=0, Cutoff Score=l, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the subject nucleotide sequence, whichever is shorter. An isolated polypeptide comprising a polypeptide having 95% identity to a polypeptide having an amino acid sequence selected from the group consisting of: amino acids from about 1 to about 950 in SEQ ID NO:2; amino acids from about 2 to about 950 in SEQ ID NO:2; amino acids from about 29 to about 950 in SEQ ID NO:2; amino acids from about 30 to about 950 in SEQ ID NO:2; the amino acid sequence of the METH1 polypeptide having the amino acid COMS ID No: SMBI-00404055 Received by IP Australia: Time 15:00 Date 2003-09-04 4.Sep. 2003 13:41 WRAY&ASSOCIATES No.3724 P. 11 187 sequence encoded by the cDNA clone contained in ATCC Deposit No. 209581; the amino acid sequence of the mature METH1 polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 209581; amino acids from about 1 to about 890 in SEQ ID NO:4; amino acids from about 2 to about 890 in SEQ ID NO:4; amino acids from about 24 to about 890 in SEQ ID NO:4; amino acids from about 112 to about 890 in SEQ ID NO:4; the amino acid sequence of the METH2 polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 209582; and wherein said identity is calculated using the FASTDB computer program, with the parameters: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=l, Joining Randomization Group Length=0, Cutoff Score=l, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the subject amino acid sequence, whichever is shorter.
  10. 16. A method for inhibiting angiogenesis in an individual, comprising administering an effective amount of a polypeptide according to any one of claims 9, 13 and 15 to said individual.
  11. 17. An isolated antibody of the polypeptide of any one of claims 9, 13 and 9
  12. 18. A method of identifying antagonists or agonists of the polypeptide of any one of claims 9,13 and 15 comprising: producing cells which express said polypeptide either as a secreted protein or on the cell membrane; contacting the polypeptide produced in step with a test sample potentially containing an antagonist or agonist; and identifying an antagonist or agonist by observing binding and stimulation or inhibition of activity of said protein. *19. e nucleic acid molecule of claim which is DNA or RNA.
  13. 19. The nucleic acid molecule of claim 1 which is DNA or RNA. 9*9 COMS ID No: SMBI-00404055 Received by IP Australia: Time 15:00 Date 2003-09-04 4.Sep. 2003 13:41 WRAY&ASSOCIATES No.3724 P. 12 188 A polynucleotide that is capable of hybridizing under stringent conditions to any one of the polynucleotides specified in claim 1(a) to wherein said polynucleotide capable of hybridizing under stringent conditions: does not hybridize under stringent conditions to a nucleic acid molecule having a nucleotide sequence of only A residues or of only T residues; (ii) is not any one of SEQ ID NO:14 to 77 or any subfragment thereof; and (iii) is at least 95% identical to any one of the polynucleotides specified in claim l(a) to 1
  14. 21. An in vitro method of diagnosing cancer in a subject comprising the step of determining the expression level of the nucleic acid molecules of claims 1 or 19.
  15. 22. An in vitro method of diagnosing cancer in a subject comprising the step of determining the protein level of the polypeptide of any one of claims 9, 13 and
  16. 23. A pharmaceutical composition comprising the nucleic acid molecules of any one of claims I and 19, the polypeptide of any one of claims 9, 13 and 15 or the antibody of claim 17.
  17. 24. A method for stimulating angiogenesis in an individual, comprising administering an effective amount of an antagonist of the polypeptide of any one of claims 9, 13 and 15 to said individual.
  18. 25. A method according to claim 24 wherein the antagonist is selected from the group comprising: an antibody, an oligonucleotide, a protein and a small molecule.
  19. 26. A method for inhibiting angiogenesis in an individual, comprising administering an effective amount of an agonist of the polypeptide of any one of claims 9, 13 and to said individual.
  20. 27. The antibody of claim 17, which is selected from the group consisting of: a humanized antibody; a single chain antibody; a polyclonal antibody; a monoclonal antibody; and 4*49 COMS ID No: SMBI-00404055 Received by IP Australia: Time 15:00 Date 2003-09-04 4.Sep. 2003 13:41 WRAY&ASSOCIAIES No.3724 P. 13 189 a Fab fragment.
  21. 28. A nucleic acid according to claim 1 substantially as herein before described with reference to any on~e of' the examples.
  22. 29. A method according to any one of claims 4, 6 or 8 substantially as herein before described with reference to any one of the examples. A recombinant vector according to claim 5 substantially as herein before described with reference to any one of the examples.
  23. 31. A recombinant host cell according to claim 7 substantially as herein before described with reference to any one of the examples. Dated this FOURTH day of SEPTEMBER 2003. Jfluian Genorne Sciences. Jnc. and Beth Israel Deaconess Medical Center Applicants Wray.& Associates Perth, Western Australia Patent Attorneys for the Applicants COMS ID No: SMBI-00404055 Received by IP Australia: Time 15:00 Date 2003-09-04
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WO1999007850A1 (en) 1997-08-06 1999-02-18 Millennium Biotherapeutics, Inc. Tango-71, tango-73, tango-74, tango-76, and tango-83 nucleic acid molecules and polypeptides
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US6649377B1 (en) * 1999-05-10 2003-11-18 Syntex (U.S.A.) Llc Human aggrecanase and nucleic acid compositions encoding the same
ATE444971T1 (en) * 1999-06-09 2009-10-15 Genentech Inc COMPOSITIONS AND METHODS FOR TUMOR TREATMENT
US6395889B1 (en) * 1999-09-09 2002-05-28 Millennium Pharmaceuticals, Inc. Nucleic acid molecules encoding human protease homologs
EP1892250A3 (en) * 2000-01-31 2008-09-17 Munin Corporation CYR61 compositions and methods
WO2001074852A2 (en) * 2000-03-31 2001-10-11 Bayer Corporation Protein having activity as an angiogenesis modulator
JP2002330761A (en) * 2000-04-26 2002-11-19 Pfizer Prod Inc Adamts polypeptide, nucleic acid encoding the same and use thereof
US20020099012A1 (en) * 2000-06-29 2002-07-25 Tongtong Wang Compositions and methods for the therapy and diagnosis of lung cancer
AU2002258626B2 (en) 2001-04-10 2007-01-18 Agensys, Inc. Nucleid acid and corresponding protein entitled 158P3D2 useful in treatment and detection of cancer
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