AU717733B2 - Novel G-CSF receptor agonists - Google Patents

Description

NOVEL G-CSF RECEPTOR

AGONISTS

Field of the Invention The present invention relates to human G-CSF receptor agonists with activity on hematopoietic cell differentiation and expansion.

Background of the Invention The human blood-forming (hematopoietic) system replaces a variety of white blood cells (including neutrophils, macrophages, and basophils/mast cells), red blood cells (erythrocytes) and clot-forming cells (megakaryocytes/platelets). The hematopoietic systems of the average male has been estimated to produce on the order of 4.5 x 101 granulocytes and erythrocytes every year, which is equivalent to an annual replacement of total body weight (Dexter et al., BioEssays, 2;154-158, 1985).

It is believed that small amounts of certain hematopoietic growth factors account for the differentiation S, 0% of a small number of progenitor "stem cells" into the variety of blood cell lines, for the tremendous proliferation of those lines, and for the ultimate differentiation of mature blood cells from those lines.

Because the hematopoietic growth factors are present in extremely small amounts, the detection and identification of 25 these factors has relied upon an array of assays which as yet only distinguish among the different factors on the basis of stimulative effects on cultured cells under artificial conditions.

U.S. Patent 4,999,291 discloses DNA and methods for making G-CSF the disclosure of which is incorporated herein by reference in it entirety.

U.S. Patent 4,810,643 relates to DNA and methods of making G-CSF and Cys to Ser substitution variants of G-CSF.

Kuga et al. (Biochem. Biophys. Res. Comm. 159:103- 111, 1989) made a series of G-CSF variants to partially define the structure-function relationship. Kuga et al.

found that internal and C-terminal deletions abolished activity, while N-terminal deletions of up to 11 amino acids and amino acid substitutions at positions 1, 2 and 3 were active.

Watanabe et al. (Anal. Biochem. 195:38-44, 1991) made a var-iant..to st-udy G-CSF receptor binding in which amino acids 1 and 3 were changed to Tyr for radioiodination of the protein. Watanabe et al. found this Tyrl, Tyr 3 G-CSF variant to be active.

WO 95/27732 describes, but does not show that the molecule has biological activity, a circularly permuted G- CSF ligand with a breakpoint at positions 68/69 creating a 25 circularly permuted G-CSF ligand with a new N-terminus at the original position 69 of G-CSF and a new C-terminus at the original position 68 of G-CSF. WO 95/27732 also "discloses circularly permuted GM-CSF, IL-2 and IL-4.

Rearrangement of Protein Seauences In evolution, rearrangements of DNA sequences serve an important role in generating a diversity of protein structure and function. Gene duplication and exon shuffling provide an important mechanism to rapidly generate diversity WO 97/12977 PCT/US96/15935 3 and thereby provide organisms with a competitive advantage, especially since the basal mutation rate is low (Doolittle, Protein Science 1:191-200, 1992).

The development of recombinant DNA methods has made it possible to study the effects of sequence transposition on protein folding, structure and function. The approach used in creating new sequences resembles that of naturally occurring pairs of proteins that are related by linear reorganization of their amino acid sequences (Cunningham, et al., Proc. Natl. Acad. Sci. U.S.A. 76:3218-3222, 1979; Teather Erfle, J. Bacteriol. 172: 3837-3841, 1990; Schimming et al., Eur. J. Biochem. 204: 13-19, 1992; Yamiuchi and Minamikawa, FEBS Lett. 260:127-130, 1991: MacGregor et al., FEBS Lett. 378:263-266, 1996). The first in vitro application of this type of rearrangement to proteins was described by Goldenberg and Creighton Mol.

Biol. 165:407-413, 1983). A new N-terminus is selected at an internal site (breakpoint) of the original sequence, the new sequence having the same order of amino acids as the original from the breakpoint until it reaches an amino acid that is at or near the original C-terminus. At this point the new sequence is joined, either directly or through an additional portion of sequence (linker), to an amino acid that is at or near the original N-terminus, and the new sequence continues with the same sequence as the original until it reaches a point that is at or near the amino acid that was N-terminal to the breakpoint site of the original sequence, this residue forming the new C-terminus of the chain.

This approach has been applied to proteins which range in size from 58 to 462 amino acids (Goldenberg Creighton, J. Mol. Biol. 165:407-413, 1983; Li Coffino, Mol. Cell.

Biol. 13:2377-2383, 1993). The proteins examined have represented a broad range of structural classes, including proteins that contain predominantly a-helix (interleukin-4; WO 97/12977 PCTIUS96/15935 4 Kreitmnan et al., Cytokine 7:311-318, 1995), fsheet (interleukin-1; Horlick et al., Protein Eng. 5:427-431, 1992), or mixtures of the two (yeast phosphoribosyl anthranilate isomerase; Luger et al., Science 243:206-210, 1989). Broad categories of protein function are represented in these sequence reorganization studies: Enzymes T4 lysozyme dihydrofolate reductase ribonuclease Ti Zhang et al., Biochemistry 32:12311-12318 (1993); Zhang et al., Nature Struct. Biol. 1:434-438 (1995) Buchwalder et al., Biochemistry 31:1621-1630 (1994) Protasova et al., Prot. Eng. 7:1373-1377 (1995) Mullins et al., J. Am. Chem. Soc.

116:5529-5533 (1994); Garrett et al., Protein Science 5:204-211 (1996) Hahn et al., Proc. Natl. Acad. Sci.

U.S.A. 91:10417-10421 (1994) Yang Schachman, Proc. Natl. Acad.

Sci. U.S.A. 90:11980-11984 (1993) Luger et al., Science 243:206-210 (1989); Luger et al., Prot. Eng.

3:249-258 (1990) Lin et al., Protein Science 4:159- 166 (1995) Bacillus P-glucanse aspartate transcarbamoylase phosphoribosyl anthranilate isoinerase pepsin/pepsinogen WO 97/12977 WO 9712977PCTIUS96/15935 glyceraldehyde-3 phosphate dehydrogenase ornithine decarboxylase yeast phosphoglycerat e dehydrogenase Vignais et Protei-n Science 4:994-1000 (1995) Li Coffino, Mol. Cell. Biol.

13:2377-2383 (1993) Ritco-Vonsovici et Biochemistry 34:16543-16551 (1995) Enzyme Inhibitor basic pancreatic trypsin inhibitor Goldenberg Creighton, J. Moi.

Biol. 165:407-413 (1983) Cytokines interleuki-113 Horlick et Protein Eng. 5:427- 431 (1992) Kreitman et Cyvtokine 7:311- 318 (1995) interleukin-4 Tyrosine Kinase Recognition Domain a-spectrin SH3 domain Transmembrane Protein Viguera, et J.

Mol. Biol. 247:670-681 (1995) omp A Koebnik Krdmer, J. Mci. Biol.

250:617-626 (1995) WO 97/12977 PCTIUS96/15935 6 Chimeric Protein interleukin-4- Kreitman et al., Proc. Natl. Acad.

Pseudomonas Sci. U.S.A. 91:6889-6893 (1994).

exotoxin fusion molecule The results of these studies have been highly variable.

In many cases substantially lower activity, solubility or thermodynamic stability were observed coli dihydrofolate reductase, aspartate transcarbamoylase, phosphoribosyl anthranilate isomerase, glyceraldehyde-3-phosphate dehydrogenase, ornithine decarboxylase, omp A, yeast phosphoglycerate dehydrogenase). In other cases, the sequence rearranged protein appeared to have many nearly identical properties as its natural counterpart (basic pancreatic trypsin inhibitor, T4 lysozyme, ribonuclease Tl, Bacillus P-glucanase, interleukin-lp, aspectrin SH3 domain, pepsinogen, interleukin-4). In exceptional cases, an unexpected improvement over some properties of the natural sequence was observed, the solubility and refolding rate for rearranged o-spectrin SH3 domain sequences, and the receptor affinity and anti-tumor activity of transposed interleukin-4-Pseudomonas exotoxin fusion molecule (Kreitman et al., Proc. Natl. Acad. Sci. U.S.A. 91:6889-6893, 1994; Kreitman et al., Cancer Res. 55:3357-3363, 1995).

The primary motivation for these types of studies has been to study the role of short-range and long-range interactions in protein folding and stability. Sequence rearrangements of this type convert a subset of interactions that are long-range in the original sequence into shortrange interactions in the new sequence, and vice versa. The fact that many of these sequence rearrangements are able to attain a conformation with at least some activity is persuasive evidence that protein folding occurs by multiple WO 97/12977 PCT/US96/15935 7 folding pathways (Viguera, et al., J. Mol. Biol. 247:670- 681, 1995). In the case of the SH3 domain of aspectrin, choosing new termini at locations that corresponded to -hairpin turns resulted in proteins with slightly less stability, but which were nevertheless able to fold.

The positions of the internal breakpoints used in the studies cited here are found exclusively on the surface of proteins, and are distributed throughout the linear sequence without any obvious bias towards the ends or the middle (the variation in the relative distance from the original Nterminus to the breakpoint is ca. 10 to 80% of the total sequence length). The linkers connecting the original N- and C-termini in these studies have ranged from 0 to 9 residues.

In one case (Yang Schachman, Proc. Natl. Acad. Sci. U.S.A.

90:11980-11984, 1993), a portion of sequence has been deleted from the original C-terminal segment, and the connection made from the truncated C-terminus to the original N-terminus. Flexible hydrophilic residues such as Gly and Ser are frequently used in the linkers. Viguera, et al.(J. Mol. Biol. 247:670-681, 1995) compared joining the original N- and C- termini with 3- or 4-residue linkers; the 3-residue linker was less thermodynamically stable.

Protasova et al. (Protein Eng. 7:1373-1377, 1994) used 3- or linkers in connecting the original N-termini of E.

coli dihydrofolate reductase; only the 3-residue linker produced protein in good yield.

WO 97/12977 PCTIUS96/1 5935 8 Summary of the Invention The modified human G-CSF receptor agonists of the present invention can be represented by the Formula:

X

1 -(L)a-X 2 wherein; a is 0 or 1;

X

1 is a peptide comprising an amino acid sequence corresponding to the sequence of residues n+l through J;

X

2 is a peptide comprising an amino acid sequence corresponding to the sequence of residues 1 through n; n is an integer ranging from 1 to J-l; and L is a linker.

In the formula above the constituent amino acids residues of human G-CSF are numbered sequentially 1 through J from the amino to the carboxyl terminus. A pair of adjacent amino acids within this protein may be numbered n and n+l respectively where n is an integer ranging from 1 to J-l. The residue n+l becomes the new N-terminus of the new G-CSF receptor agonist and the residue n becomes the new Cterminus of the new G-CSF receptor agonist.

The present invention relates to novel G-CSF receptor agonists of the following formula: 1 Xaa Xaa Xaa Gly Pro Ala Ser Ser Leu Pro Gin Ser Xaa Leu Leu Xaa Xaa Xaa Glu Gin Val Xaa Lys Xaa Gin Gly Xaa Gly Ala Xaa Leu Gin Glu Xaa Leu Xaa Ala Thr Tyr Lys Leu Xaa Xaa Xaa Xaa Glu Xaa Xaa Val Xaa Gly His Ser Xaa Gly Ile Pro Trp Ala Pro Leu Ser Ser Xaa Pro Ser Xaa Ala Leu Xaa Leu Ala Gly Ser Xaa Leu Ser Gin Leu His Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu 100 Glu Leu Gly Pro Thr Leu Glu Gly Ile Ser Pro 110 Val Xaa Thr Leu Gin Xaa Asp Ala Asp Phe Ala Xaa Thr Ile Trp 120 Gin Xaa Met Glu 130 Pro Ala Leu Gin Pro Thr Xaa Leu Gly Met Ala 140 Phe Gin Gly Ala Met Pro Ala Ala Ser Ala Xaa Gin Xaa Xaa Ala 150 Gly-Gly-Val- Leu 160 Gin Xa- Phe -Leu Xaa Xaa Val Ala Ser-Xaa Leu 170 Xaa Ser Tyr Arg Val Leu Xaa Leu Ala Gin Pro (SEQ ID NO:1) 30 wherein

S

S

S

Xaa at Xaa at Xaa at 35 Xaa at Xaa at Xaa at Xaa at Xaa at Xaa at Xaa at Xaa at Xaa at Xaa at Xaa at Xaa at position position position position position position position position position position position position position position position 1 is Thr, Ser, Arg, 2 is Pro or Leu; Tyr or Gly; 3 is Leu, Arg, Tyr or Ser; Phe, Ser, His, Thr or Pro; Lys, Pro, Ser, Thr or His; Cys, Ser, Gly, Ala, Ile, Tyr or Arg; Leu, Thr, Pro, His, Ile or Cys; Arg, Tyr, Ser, Thr or Ala; Ile, Pro, Tyr or Leu; Asp, or Gly; Ala, Ile, Leu or Gly; Lys or Ser; Cys or Ser; Cys or Ser; His, Thr, Gly, Val, Lys, Trp, Ala, Arg, Cys, or Leu; Xaa at position 44 is Pro, Gly, Arg, Asp, Val, Ala, His, Trp, Gin, or Thr; Xaa at position 46 is Glu, Arg, Phe, Arg, Ile or Ala; Xaa at position 47 is Leu or Thr; Xaa at position 49 is Leu, Phe, Arg or Ser; i< Or Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa at position 50 at position 54 at position 64 at position 67 at position 70 at position 74 at position 104 at position 108 at position 115 at position 120 at position 123 at position 144 at position 146 at position 147 at position 156 at position 159 at position 162 at position 163 at position 169 at position 170 is is is is is is is is is is is is is is is is is is is is Leu, Ile, His, Pro or Tyr; Leu or His; Cys or Ser; Gin, Lys, Leu or Cys; Gin, Pro, Leu, Arg or Ser; Cys or Ser; Asp, Gly or Val; Leu, Ala, Val, Arg, Trp, Gin or SThr, His, Leu or Ala; Gin, Gly, Arg, Lys or His Glu, Arg, Phe or Thr Phe, His, Arg, Pro, Leu, Gin or Arg or Gin; Arg or Gin; His, Gly or Ser; Ser, Arg, Thr, Tyr, Val or Gly; Glu, Leu, Gly or Trp; Val, Gly, Arg or Ala; Arg, Ser, Leu, Arg or Cys; His, Arg or Ser; Gly; Glu; wherein optionally 1-11 amino acids from the N-terminus and from the C-terminus can be deleted; and wherein the N-terminus is joined to the C-terminus directly or through a linker capable of joining the N-terminus to the C-terminus and having new C- and N-termini at amino acids; a a a .5e 38-39 39-40 40-41 41-42 42-43 43-44 45-46 48-49 49-50 52-53 53-54 54-55 55-56 56-57 57-58 58-59 59-60 60-61 61-62 62-63 63-64 64-65 65-66 66-67 67-68 69-70 70-71 71-72 91-92 92-93 93-94 94-95 95-96 96-97 97-98 98-99 99-100 123-124 124-125 125-126 126-127 127-128 128-129 129-130 130-131 131-132 132-133 133-134 134-135 135-136 136-137 137-138 138-139 139-140 140-141 141-142 or 142-143.

The G-CSF receptor agonists of the present invention may contain amino acid substitutions, deletions and/or insertions and may also have amino acid deletions at either/or both the N- and C- termini.

The more preferred breakpoints at which new C-terminus and N-terminus can be made are; 38-39, 39-40, 40-41, 41-42, 48-49, 53-54, 54-55, 55-56, 56-57, 57-58, 58-59, 59-60, 61, 61-62, 62-63, 64-65, 65-66, 66-67, 67-68, 69-70, 96-97, 125-126, 126-127, 127-128, 128-129, 129-130, 130-131, 131-132, 132-133, 133-134, 134-135, 135-136, 136-137, 137- 138, 138-139, 139-140, 140-141 and 141-142.

The most preferred breakpoints at which new C-terminus and N-terminus can be made are; 38-39, 48-49, 96-97,.125- 12-6-, -132-13-3 -and 141-142.

A preferred embodiment of the present invention the linker joining the N-terminus to the C-terminus is a S: polypeptide selected from the group consisting of: GlyGlyGlySer (SEQ ID NO:2); GlyGlyGlySerGlyGlyGlySer (SEQ ID NO:61); GlyGlyGlySerGlyGlyGlySerGlyGlyGlySer (SEQ ID NO:62); 25 SerGlyGlySerGlyGlySer (SEQ ID NO:63); S( I GluPheGlyAsnMet (SEQ ID NO:64); GluPheGlyGlyAsnMet (SEQ ID GluPheGlyGlyAsnGlyGlyAsnMet (SEQ ID NO:66); and GlyGlySerAspMetAlaGly (SEQ ID NO:67).

The present invention also encompasses recombinant human G-CSF receptor agonists co-administered or sequentially with one or more additional colony stimulating factors (CSF) including, cytokines, lymphokines, interleukins, hematopoietic growth factors which include but WO 97/12977 PCT/US96/15935 12 are not limited to GM-CSF, c-mpl ligand (also known as TPO or MGDF), M-CSF, erythropoietin (EPO), IL-1, IL-4, IL-2, IL- 3, IL-5, IL 6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, LIF, flt3/flk2 ligand, human growth hormone, B-cell growth factor, B-cell differentiation factor, eosinophil differentiation factor and stem cell factor (SCF) also known as steel factor or c-kit ligand (herein collectively referred to as "colony stimulating factors" or These co-administered mixtures may be characterized by having the usual activity of both of the peptides or the mixture may be further characterized by having a biological or physiological activity greater than simply the additive function of the presence of the G-CSF receptor agonists or the second colony stimulating factor alone. The coadministration may also provide an enhanced effect on the activity or an activity different from that expected by the presence of the G-CSF ligand or the second colony stimulating factor. The co-administration may also have an improved activity profile which may include reduction of undesirable biological activities associated with native human G-CSF. In addition to the list above, IL-3 variants taught in WO 94/12639 and WO 94/12638 can be co-administered with the polypeptides of the present invention.

In addition, it is envisioned that in vitro uses would include the ability to stimulate bone marrow and blood cell activation and growth before the expanded cells are infused into patients WO 97/12977 PCTIUTS96/1 5935 13 Brief Description of the Figures Figure 1 schematically illustrates the sequence rearrangement of a protein. The N-terminus and the Cterminus of the native protein are joined through a linker, or joined directly. The protein is opened at a breakpoint creating a new N-terminus (new N) and a new Cterminus (new-C) resulting in a protein with a new linear amino acid sequence. A rearranged molecule may be synthesized de novo as linear molecule and not go through the steps of joining the original N-terminus and the Cterminus and opening of the protein at the breakpoint.

Figure 2 shows a schematic of Method I, for creating new proteins in which the original N-terminus and C-terminus of the native protein are joined with a linker and different N-terminus and C-terminus of the protein are created. In the example shown the sequence rearrangement results in a new gene encoding a protein with a new N-terminus created at amino acid 97 of the original protein, the original Cterminus 174) joined to the amino acid 11 1- are deleted) through a linker region and a new C-terminus created at amino acid 96 of the original sequence.

Figure 3 shows a schematic of Method II, for creating new proteins in which the original N-terminus and C-terminus of the native protein are joined without a linker and different N-terminus and C-terminus of the protein are created. In the example shown the sequence rearrangement results in a new gene encoding a protein with a new Nterminus created at amino acid 97 of the original protein, the original C-terminus 174) joined to the original Nterminus and a new C-terminus created at amino acid 96 of the original sequence.

WO 97/12977 PCT/US96/15935 14 Figure 4 shows a schematic of Method III, for creating new proteins in which the original N-terminus and C-terminus of the native protein are joined with a linker and different N-terminus and C-terminus of the protein are created. In the example shown the sequence rearrangement results in a new gene encoding a protein with a new N-terminus created at amino acid 97 of the original protein, the original Cterminus 174) joined to amino acid 1 through a linker region and a new C-terminus created at amino acid 96 of the original sequence.

WO 97/12977 PCTIUS96/15935 Detailed Description of the Invention Receptor agonists of the present invention may be useful in the treatment of diseases characterized by decreased levels of granulocytes of the hematopoietic system.

A G-CSF receptor agonist may be useful in the treatment or prevention of neutropenia. Many drugs may cause bone marrow suppression or hematopoietic deficiencies. Examples of such drugs are AZT, DDI, alkylating agents and antimetabolites used in chemotherapy, antibiotics such as chloramphenicol, penicillin, gancyclovir, daunomycin and sulfa drugs, phenothiazones, tranquilizers such as meprobamate, analgesics such as aminopyrine and dipyrone, anti-convulsants such as phenytoin or carbamazepine, antithyroids such as propylthiouracil and methimazole and diuretics. G-CSF receptor agonists may be useful in preventing or treating the bone marrow suppression or hematopoietic deficiencies which often occur in patients treated with these drugs.

Hematopoietic deficiencies may also occur as a result of viral, microbial or parasitic infections and as a result of treatment for renal disease or renal failure, e.g., dialysis. The present peptide may be useful in treating such hematopoietic deficiency.

Another aspect of the present invention provides plasmid DNA vectors for use in the method of expression of these novel G-CSF receptor agonists. These vectors contain the novel DNA sequences described above which code for the novel polypeptides of the invention. Appropriate vectors which can transform host cells capable of expressing the G- CSF receptor agonists include expression vectors comprising nucleotide sequences coding for the G-CSF receptor agonists joined to transcriptional and translational regulatory WO 97/12977 PCT/US96/15935 16 sequences which are selected according to the host cells used. Vectors incorporating modified sequences as described above are included in the present invention and are useful in the production of the modified G-CSF receptor agonist polypeptides. The vector employed in the method also contains selected regulatory sequences in operative association with the DNA coding sequences of the invention and capable of directing the replication and expression thereof in selected host cells.

As another aspect of the present invention, there is provided a novel method for producing the novel family of human G-CSF receptor agonists. The method of the present invention involves culturing suitable cells or cell line, which has been transformed with a vector containing a DNA sequence coding for expression of the novel G-CSF receptor agonist polypeptide. Suitable cells or cell lines may include various strains of bacteria such as E. coli, yeast, mammalian cells, or insect cells may be utilized as host cells in the method of the present invention.

Other aspects of the present invention are methods and therapeutic compositions for treating the conditions referred to above. Such compositions comprise a therapeutically effective amount of one or more of the G-CSF receptor agonists of the present invention in a mixture with a pharmaceutically acceptable carrier. This composition can be administered either parenterally, intravenously or subcutaneously. When administered, the therapeutic composition for use in this invention is preferably in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such a parenterally acceptable protein solution, having due regard to pH, isotonicity, stability and the like, is within the skill of the art.

WO 97/12977 PCT/US96/15935 17 The dosage regimen involved in a method for treating the above-described conditions will be determined by the attending physician considering various factors which modify the action of drugs, e.g. the condition, body weight, sex and diet of the patient, the severity of any infection, time of administration and other clinical factors. Generally, a daily regimen may be in the range of 0.5 150 gg/kg of nonglycosylated G-CSF receptor agonists protein per kilogram of body weight. Dosages would be adjusted relative to the activity of a given receptor agonist and it would not be unreasonable to note that dosage regimens may include doses as low as 0.1 microgram and as high as 1 milligram per kilogram of body weight per day. In addition, there may exist specific circumstances where dosages of G-CSF receptor agonist would be adjusted higher or lower than the range of 150 micrograms per kilogram of body weight. These include co-administration with other CSF or growth factors; co-administration with chemotherapeutic drugs and/or radiation; the use of glycosylated G-CSF receptor agonists; and various patient-related issues mentioned earlier in this section. As indicated above, the therapeutic method and compositions may also include co-administration with other human factors. A non-exclusive list of other appropriate hematopoietins, CSFs and interleukins for simultaneous or serial co-administration with the polypeptides of the present invention includes GM-CSF, c-mpl ligand (also known as TPO or MGDF), M-CSF, erythropoietin (EPO), IL-1, IL-4, IL-2, IL-3, IL-5, IL 6, IL-7, IL-8, IL-9, IL-10, IL-11, IL- 12, IL-13, IL-15, LIF, flt3/flk2 ligand, human growth hormone, B-cell growth factor, B-cell differentiation factor, eosinophil differentiation factor and stem cell factor (SCF) also known as steel factor or c-kit ligand (herein collectively referred to as "colony stimulating factors"), or combinations thereof. In addition to the list above, IL-3 variants taught in WO 94/12639 and WO 94/12638 WO 97/12977 PCT/US96/15935 18 can be co-administered with the polypeptides of the present invention.

The G-CSF receptor agonists of the present invention may be useful in the mobilization of hematopoietic progenitors and stem cells in peripheral blood. Peripheral blood derived progenitors have been shown to be effective in reconstituting patients in the setting of autologous marrow transplantation. Hematopoietic growth factors, including G- CSF and GM-CSF, have been shown to enhance the number of circulating progenitors and stem cells in the peripheral blood. This has simplified the procedure for peripheral stem cell collection and dramatically decreased the cost of the procedure by decreasing the number of pheresis required. The G-CSF receptor agonist of the present invention may be useful in mobilization of stem cells and further enhance the efficacy of peripheral stem cell transplantation.

The G-CSF receptor agonists of the present invention may also be useful in the ex vivo expansion of hematopoietic progenitors. Colony stimulating factors (CSFs), such as G- CSF, have been administered alone, co-administered with other CSFs, or in combination with bone marrow transplants subsequent to high dose chemotherapy to treat the neutropenia and which is often the result of such treatment.

However the period of severe neutropenia may not be totally eliminated. The myeloid lineage, which is comprised of monocytes (macrophages), granulocytes (including neutrophils) and megakaryocytes, is critical in preventing infections and bleeding which can be life-threatening.

Neutropenia may also be the result of disease, genetic disorders, drugs, toxins, radiation and many therapeutic treatments such as conventional oncology therapy.

Bone marrow transplants have been used to treat this patient population. However, several problems are associated WO 97/12977 PCT/US96/15935 19 with the use of bone marrow to reconstitute a compromised hematopoietic system including: 1) the number of stem cells in bone marrow or other tissues, such as spleen or peripheral blood, is limited, 2) Graft Versus Host Disease, 3) graft rejection and 4) possible contamination with tumor cells. Stem cells and progenitor cells make up a very small percentage of the nucleated cells in the bone marrow, spleen and peripheral blood. It is clear that a dose response exists such that a greater number of multipotential hematopoietic progenitors will enhance hematopoietic recovery. Therefore, the in vitro expansion of stem cells should enhance hematopoietic recovery and patient survival.

Bone marrow from an allogeneic donor has been used to provide bone marrow for transplant. However, Graft Versus Host Disease and graft rejection limit bone marrow transplantation even in recipients with HLA-matched sibling donors. An alternative to allogeneic bone marrow transplants is autologous bone marrow transplants. In autologous bone marrow transplants, some of the patient's own marrow is harvested prior to myeloablative therapy, e.g. high dose chemotherapy, and is transplanted back into the patient afterwards. Autologous transplants eliminate the risk of Graft Versus Host Disease and graft rejection. However, autologous bone marrow transplants still present problems in terms of the limited number of stems cells in the marrow and possible contamination with tumor cells. The limited number of multipotential hematopoietic progenitors may be overcome by ex-vivo expansion of the multipotential hematopoietic progenitors. In addition, stem cells can be specifically isolated based on the presence of specific surface antigens such as CD34+ in order to decrease tumor cell contamination of the marrow graft.

The following patents contain further details on separating stem cells, CD34+ cells, culturing the cells with WO 97/12977 PCT/S96/15935 hematopoietic factors, the use of the cells for the treatment of patients with hematopoietic disorders and the use of hematopoietic factors for cell expansion and gene therapy.

5,061,620 relates to compositions comprising human hematopoietic stem cells provided by separating the stem cells from dedicated cells.

5,199,942 describes a method for autologous hematopoietic cell transplantation comprising: obtaining hematopoietic progenitor cells from a patient; ex-vivo expansion of cells with a growth factor selected from the group consisting of IL-3, flt3 ligand, c-kit ligand, GM CSF, IL-1, GM-CSF/IL-3 fusion protein and combinations thereof; (3) administering cellular preparation to a patient.

5,240,856 relates to a cell separator that includes an apparatus for automatically controlling the cell separation process.

WO 91/16116 describes devices and methods for selectively isolating and separating target cells from a mixture of cells.

WO 91/18972 describes methods for in vitro culturing of bone marrow, by incubating suspension of bone marrow cells, using a hollow fiber bioreactor.

WO 92/18615 relates to a process for maintaining and expanding bone marrow cells, in a culture medium containing specific mixtures of cytokines, for use in transplants.

WO 93/08268 describes a method for selectively expanding stem cells, comprising the steps of separating CD34+ WO 97/12977 PCT[US96/15935 21 stem cells from other cells and incubating the separated cells in a selective medium, such that the stem cells are selectively expanded.

WO 93/18136 describes a process for in vitro support of mammalian cells derived from peripheral blood.

WO 93/18648 relates to a composition comprising human neutrophil precursor cells with a high content of myeloblasts and promyelocytes for treating genetic or acquired neutropenia.

WO 94/08039 describes a method of enrichment for human hematopoietic stem cells by selection for cells which express c-kit protein.

WO 94/11493 describes a stem cell population that are CD34+ and small in size, which are isolated using a counterflow elutriation method.

WO 94/27698 relates to a method combining immunoaffinity separation and continuous flow centrifugal separation for the selective separation of a nucleated heterogeneous cell population from a heterogeneous cell mixture.

WO 94/25848 describes a cell separation apparatus for collection and manipulation of target cells.

The long term culturing of highly enriched CD34+ precursors of hematopoietic progenitor cells from human bone marrow in cultures containing IL-la, IL-3, IL-6 or GM-CSF is discussed in Brandt et al Clin. Invest. 86:932-941, 1990).

WO 97/12977 PCT/US96/15935 22 One aspect of the present invention provides a method for selective ex-vivo expansion of stem cells. The term "stem cell" refers to the multipotential hematopoietic cells as well as early myeloid progenitor and precursors cells which can be isolated from bone marrow, spleen or peripheral blood. The term "expansion" refers to the proliferation and differentiation of the cells. The present invention provides a method for selective ex-vivo expansion of stem cells, comprising the steps of; separating stem cells from other cells, culturing the separated stem cells with a selective medium which contains a G-CSF receptor agonist and optionally a second colony stimulating factor, and (c) harvesting the cultured stems cells. Stem cells, as well as committed progenitor cells destined to become neutrophils, erythrocytes, platelets, etc., may be distinguished from most other cells by the presence or absence of particular progenitor marker antigens, such as CD34, that are present on the surface of these cells and/or by morphological characteristics. The phenotype for a highly enriched human stem cell fraction is reported as CD34+, Thy-l+ and lin-, but it is to be understood that the present invention is not limited to the expansion of this stem cell population. The CD34+ enriched human stem cell fraction can be separated by a number of reported methods, including affinity columns or beads, magnetic beads or flow cytometry using antibodies directed to surface antigens such as the CD34+. Further, physical separation methods such as counterflow elutriation may be used to enrich hematopoietic progenitors. The CD34+ progenitors are heterogeneous, and may be divided into several sub-populations characterized by the presence or absence of co-expression of different lineage associated cell surface associated molecules. The most immature progenitor cells do not express any known lineage associated markers, such as HLA-DR or CD38, but they may express CD90(thy-l). Other surface antigens such =s CD33, CD38, WO 97/12977 PCTIUS96/15935 23 CD41, CD71, HLA-DR or c-kit can also be used to selectively isolate hematopoietic progenitors. The separated cells can be incubated in selected medium in a culture flask, sterile bag or in hollow fibers. Various colony stimulating factors may be utilized in order to selectively expand cells.

Representative factors that have been utilized for ex-vivo expansion of bone marrow include, c-kit ligand, IL-3, G-CSF, GM-CSF, IL-1, IL-6, IL-11, flt-3 ligand or combinations thereof. The proliferation of the stem cells can be monitored by enumerating the number of stem cells and other cells, by standard techniques hemacytometer, CFU, LTCIC) or by flow cytometry prior and subsequent to incubation.

Several methods for ex-vivo expansion of stem cells have been reported utilizing a number of selection methods and expansion using various colony stimulating factors including c-kit ligand (Brandt et al., Blood 83:1507-1514, 1994; McKenna et al., Blood 86:3413-3420, 1995), IL-3 (Brandt et al., Blood 83:1507-1514, 1994; Sato et al., Blood 82:3600-3609, 1993), G-CSF (Sato et al., Blood 82:3600-3609, 1993), GM-CSF (Sato et al., Blood 82:3600-3609, 1993), IL-1 (Muench et al., Blood 81:3463-3473, 1993), IL-6 (Sato et al., Blood 82:3600-3609, 1993), IL-11 (Lemoli et al., Exp.

Hem. 21:1668-1672, 1993; Sato et al., Blood 82:3600-3609, 1993), flt-3 ligand (McKenna et al., Blood 86:3413 3420, 1995) and/or combinations thereof (Brandt et al., Blood 83:1507 1514, 1994; Haylock et al., Blood 80:1405-1412, 1992, Koller et al., Biotechnology 11:358-363, 1993; Lemoli et al., Exp. Hem. 21:1668-1672, 1993), McKenna et al., Blood 86:3413-3420, 1995; Muench et al., Blood 81:3463-3473, 1993; Patchen et al., Biotherapy 7:13-26, 1994; Sato et al., Blood 82:3600-3609, 1993; Smith et al., Exp. Hem. 21:870-877, 1993; Steen et al., Stem Cells 12:214-224, 1994; Tsujino et al., Exp. Hem. 21:1379-1386, 1993). Among the individual WO 97/12977 PCTIUS96/1 5935 24 colony stimulating factors, hIL-3 has been shown to be one of the most potent in expanding peripheral blood CD34+ cells (Sato et al., Blood 82:3600-3609, 1993; Kobayashi et al., Blood 73:1836-1841, 1989). However, no single factor has been shown to be as effective as the combination of multiple factors. The present invention provides methods for ex vivo expansion that utilize novel G-CSF receptor agonists.

Another aspect of the invention provides methods of sustaining and/or expanding hematopoietic precursor cells which includes inoculating the cells into a culture vessel which contains a culture medium that has been conditioned by exposure to a stromal cell line such as HS-5 (WO 96/02662, Roecklein and Torok-Strob, Blood 85:997-1105, 1995) that has been supplemented with a G-CSF receptor agonist of the present invention.

Another projected clinical use of growth factors has been in the in vitro activation of hematopoietic progenitors and stem cells for gene therapy. Due to the long life-span of hematopoietic progenitor cells and the distribution of their daughter cells throughout the entire body, hematopoietic progenitor cells are good candidates for ex vivo gene transfection. In order to have the gene of interest incorporated into the genome of the hematopoietic progenitor or stem cell one needs to stimulate cell division and DNA replication. Hematopoietic stem cells cycle at a very low frequency which means that growth factors may be useful to promote gene transduction and thereby enhance the clinical prospects for gene therapy. Potential applications of gene therapy (review Crystal, Science 270:404-410, 1995) include; 1) the treatment of many congenital metabolic disorders and immunodeficiencies (Kay and Woo, Trends Genet.

10:253-257, 1994), 2) neurological disorders (Friedmann, Trends Genet. 10:210-214, 1994), 3) cancer (Culver and WO 97/12977 PCT/US96/15935 Blaese, Trends Genet. 10:174-178, 1994) and 4) infectious diseases (Gilboa and Smith, Trends Genet. 10:139-144, 1994).

There are a variety of methods, known to those with skill in the art, for introducing genetic material into a host cell. A number of vectors, both viral and non-viral have been developed for transferring therapeutic genes into primary cells. Viral based vectors include; 1) replication deficient recombinant retrovirus (Boris-Lawrie and Temin, Curr. Opin. Genet. Dev. 3:102-109, 1993; Boris-Lawrie and Temin, Annal. New York Acad. Sci. 716:59-71, 1994; Miller, Current Top. Microbiol. Immunol. 158:1-24, 1992) and replication-deficient recombinant adenovirus (Berkner, BioTechniques 6:616-629, 1988; Berkner, Current Top.

Microbiol. Immunol. 158:39-66, 1992; Brody and Crystal, Annal. New York Acad. Sci. 716:90-103, 1994). Non-viral based vectors include protein/DNA complexes (Cristiano et al., PNAS USA. 90:2122-2126, 1993; Curiel et al., PNAS USA 88:8850-8854, 1991; Curiel, Annal. New York Acad. Sci.

716:36-58, 1994), electroporation and liposome mediated delivery such as cationic liposomes (Farhood et al., Annal.

New York Acad. Sci. 716:23-35, 1994).

The present invention provides an improvement to the existing methods of expanding hematopoietic cells, into which new genetic material has been introduced, in that it provides methods utilizing G-CSF receptor agonists that may have improved biological activity and/or physical properties.

Determination of the Linker The length of the amino acid sequence of the linker can be selected empirically or with guidance from structural information, or by using a combination of the two approaches.

WO 97/12977 PCTIUS96/15935 26 When no structural information is available, a small series of linkers can be prepared for testing using a design whose length is varied in order to span a range from 0 to A and whose sequence is chosen in order to be consistent with surface exposure (hydrophilicity, Hopp Woods, Mol.

Immunol. 20: 483-489, 1983; Kyte Doolittle, J. Mol. Biol.

157:105-132, 1982; solvent exposed surface area, Lee Richards, J. Mol. Biol. 55:379-400, 1971) and the ability to adopt the necessary conformation without deranging the configuration of the c-mpl receptor agonist (conformationally flexible; Karplus Schulz, Naturwissenschaften 72:212-213, (1985). Assuming an average of translation of 2.0 to 3.8 A per residue, this would mean the length to test would be between 0 to 30 residues, with 0 to 15 residues being the preferred range. Exemplary of such an empirical series would be to construct linkers using a cassette sequence such as Gly-Gly-Gly-Ser (SEQ ID NO:2) repeated n times, where n is 1, 2, 3 or 4. Those skilled in the art will recognize that there are many such sequences that vary in length or composition that can serve as linkers with the primary consideration being that they be neither excessively long nor short Sandhu, Critical Rev.

Biotech. 12: 437-462, 1992); if they are too long, entropy effects will likely destabilize the three-dimensional fold, and may also make folding kinetically impractical, and if they are too short, they will likely destabilize the molecule because of torsional or steric strain.

Those skilled in the analysis of protein structural information will recognize that using the distance between the chain ends, defined as the distance between the c-alpha carbons, can be used to define the length of the sequence to be used, or at least to limit the number of possibilities that must be tested in an empirical selection of linkers.

They will .lso recognize that it is sometimes the case that WO 97/12977 PCTIUS96/1 5935 27 the positions of the ends of the polypeptide chain are illdefined in structural models derived from x-ray diffraction or nuclear magnetic resonance spectroscopy data, and that when true, this situation will therefore need to be taken into account in order to properly estimate the length of the linker required. From those residues whose positions are well defined are selected two residues that are close in sequence to the chain ends, and the distance between their c-alpha carbons is used to calculate an approximate length for a linker between them. Using the calculated length as a guide, linkers with a range of number of residues (calculated using 2 to 3.8A per residue) are then selected.

These linkers may be composed of the original sequence, shortened or lengthened as necessary, and when lengthened the additional residues may be chosen to be flexible and hydrophilic as described above; or optionally the original sequence may be substituted for using a series of linkers, one example being the Gly-Gly-Gly-Ser (SEQ ID NO:2) cassette approach mentioned above; or optionally a combination of the original sequence and new sequence having the appropriate total length may be used.

Determination of the Amino and Carboxvl Termini of G-CSF Receptor Aaonists Sequences of G-CSF receptor agonists capable of folding to biologically active states can be prepared by appropriate selection of the beginning (amino terminus) and ending (carboxyl terminus) positions from within the original polypeptide chain while using the linker sequence as described above. Amino and carboxyl termini are selected from within a common stretch of sequence, referred to as a breakpoint region, using the guidelines described below. A novel amino acid sequence is thus generated by selecting WO 97/12977 PCTIUS96/15935 28 amino and carboxyl termini from within the same breakpoint region. In many cases the selection of the new termini will be such that the original position of the carboxyl terminus immediately preceded that of the amino terminus. However, those skilled in the art will recognize that selections of termini anywhere within the region may function, and that these will effectively lead to either deletions or additions to the amino or carboxyl portions of the new sequence.

It is a central tenet of molecular biology that the primary amino acid sequence of a protein dictates folding to the three-dimensional structure necessary for expression of its biological function. Methods are known to those skilled in the art to obtain and interpret three-dimensional structural information using x-ray diffraction of single protein crystals or nuclear magnetic resonance spectroscopy of protein solutions. Examples of structural information that are r.levant to the identification of breakpoint regions include the location and type of protein secondary structure (alpha and 3-10 helices, parallel and antiparallel beta sheets, chain reversals and turns, and loops; Kabsch Sander, Biopolymers 22: 2577-2637, 1983; the degree of solvent exposure of amino acid residues, the extent and type of interactions of residues with one another (Chothia, Ann. Rev. Biochem. 53:537-572; 1984) and the static and dynamic distribution of conformations along the polypeptide chain (Alber Mathews, Methods Enzymol. 154: 511-533, 1987). In some cases additional information is known about solvent exposure of residues; one example is a site of posttranslational attachment of carbohydrate which is necessarily on the surface of the protein. When experimental structural information is not available, or is not feasible to obtain, methods are also available to analyze the primary amino acid sequence in order to make predictions of protein tertiary and secondary structure, solvent accessibility and the occurrence of turns and loops.

WO 97/12977 PCT/US96/15935 29 Biochemical methods are also sometimes applicable for empirically determining surface exposure when direct structural methods are not feasible; for example, using the identification of sites of chain scission following limited proteolysis in order to infer surface exposure (Gentile Salvatore, Eur. J. Biochem. 218:603-621, 1993) Thus using either the experimentally derived structural informaticn or predictive methods Srinivisan Rose Proteins: Struct., Funct. Genetics, 22: 81-99, 1995) the parental amino acid sequence is inspected to classify regions according to whether or not they are integral to the maintenance of secondary and tertiary structure. The occurrence of sequences within regions that are known to be involved in periodic secondary structure (alpha and 3-10 helices, parallel and anti-parallel beta sheets) are regions that should be avoided. Similarly, regions of amino acid sequence that are observed or predicted to have a low degree of solvent exposure are more likely to be part of the socalled hydrophobic core of the protein and should also be avoided for selection of amino and carboxyl termini. In contrast, those regions that are known or predicted to be in surface turns or loops, and especially those regions that are known not to be required for biological activity, are the preferred sites for location of the extremes of the polypeptide chain. Continuous stretches of amino acid sequence that are preferred based on the above criteria are referred to as a breakpoint region.

WO 97/12977 WO 9712977PCTIUS96/15935 TABLE 1

OLIGONUCLEOTIDES

L-llstart.seq L-llstop. seq BlstartP. seq blstopP.seq 39 start. seq 38stop.Seq 97start .seq 96stop. Seq 126start .seq .Seq l33start.seq l32stop. seq 142start .seq l41stop .Seq 96for .Seq 96rev.Seq GCTCTGAGAG CCGCCAGAGC CGCCAGAGGG CTGCGCAAGG TGGCCTAGAA CGCG (SEQ ID NO:3) CAGCCCTCTG GCGGCTCTGG CGGCTCTCAG AGCTTCCTGC TCAAGTCTTT AGAG (SEQ ID NO:4) GGGCTCCGCA AGGTCGCG (SEQ ID ACACCATTGG GCCCTGCCAG C GATCGACCAT GGCTTACAAG C (SEQ ID NO:7) CGATCGAAGC TTATTAGGTG G (SEQ ID NO:8) GATCGACCAT GGCTCCCGAG T (SEQ ID NO:9) CGATCGAAGC TTATTAGGAT A (SEQ ID GATCGACCAT GGCTATGGCC C (SEQ ID NO:11) CGATCGAAGC TTATTATCCC A (SEQ ID NO:12) GATOGACCAT GGCTACCCAG G (SEQ ID NO:13) CGATCGAAGC TTATTAGGGC T (SEQ ID NO:14) GATCGACCAT GGCTTCTGCT TI (SEQ ID CGATCGAAGC TTATTAGGCG (SEQ ID NO:16) ATATCCATGC CTCCGGAACT C (SEQ ID NO:17) ACCTCCAGGA AGCTCTGCAGI (SEQ ID NO:18) (SEQ ID NO:6)

TGTGCCACC

CACACAGCT

TGGGTCCCA

TCCCTTCCA

CTGCCCTGC

.GTTCTTCCA

GTGCCATGC

~GCAGGGCAG

'TCCAGCGCC

LAGGCCGGCA

;GGTCCAACT

TGG

cc

TCTCCT

cc

GGGCCT

AG

TCTGCT

CG

GGGCCA

GG

TLGGCAC

CTG

WO 97/12977 WO 9712977PCTIUS96/1 5935 seq seq 132for.seq l32rev. seq 141for seq 141rev. seq 49start seq 48stop. seq 77start.seq 76stop.seq 82start-seq 8lstop seq S4start seq 83stop.seq 9lstart .seq TATATCCATG CCTATGGCTC CAGCTCTCCA ACCAACTCAA GGTGCAATGC CACCATTTGC ATCTG (SEQ ID NO:19) GATGGCTAGC AACCAGAACA CCACCTGCAC GACGTTGAAA AGCAGATGCA AATGCTGGCA TTG (SEQ ID TATATCCATG GCTACTCAAC GTGCTATGCC AGCTTTTGCT TCTGCTTTTC AACGTCG (SEQ ID NO:21) GCAGATGGCT AGCAACCAGA ACACCACCTC CACGACGTTG AAAACCAGAA GCAAAAGC (SEQ ID NO:22) CATCGCTTCT GCTTTTCAAC GTCGTCCAGG TGOTGTTCTG GTTC (SEQ ID NO:23) CTAGCAACCA GAACACCACC TGCACGACGT TGAAAAGCAG AAGC (SEQ ID NO:24) GATCGACCAT GGCTCTGCTC GGACACTCTC TG (SEQ ID NO:68) CGATCGAAGC TTATTACACC (SEQ ID NO:69) GATCGACCAT GGCTCAACTC (SEQ ID CGATCGAAGC TTATTAGCTC (SEQ ID NO:71) GATCGACCAT GGCTCTTTTC (SEQ ID NO:72) CGATCGAAGC TTATTAGCCG (SEQ ID NO:73) GATCGACCAT GGCTCTCTAC (SEQ ID NO:74) CGATCGAAGC TTATTAGAAA (SEQ ID GATCGACCAT GGCTGCCCTG (SEQ ID NO:76) CGATCGAAGC TTATTACTGC (SEQ ID NO:77) AGCTCCTCGG GGTGGC CATAGCGCCC TT AAGCAGCCTG CCAGCT CTCTACCAGG GG CTATGGAGTT GGCTCA CAGGGGCTCC TG AGGCCGCTAT GGAGTT GAAGGGATAT CC AGGAGCCCCT GGTAGA WO 97/12977 WO 9712977PCT[US96/15935 112 start. seq lllstop.seq ll7start .seq 1l6stop. seq ll9start .seq ll8stop. seq Z4849at.for Z4849at .rev GATCACCAT GGCTGACTTT (SEQ ID NO:78) CGATCGAAGC TTATTAGGCG (SEQ ID NO:79) GATOGACCAT GGCTATCTGG (SEQ ID CGATCGAAGC TTATTAGGTG (SEQ ID NO:81) GATCGACCAT GGCTCAGCAG (SEQ ID NO:82) CGATCGAAGC TTATTACCAG (SEQ ID NO:83) CATGGCTTTG TTAGGACATT TCCATGGGCT CCTCTGAGCT CAGAGGAGCC CATGGAATAC TCCTAACAAA GC (SEQ ID GOCACCACCA TO ACGTCCAGCT GCAGTG CAGCAGATGG

AA

GTGGCAAAGT CGGCGA ATGGAAGAAC TG ATGGTGGTGG CAAAGT

CTTTAGGTAT

(SEQ ID NO:84)

CTAAAGAATG

NO: WO 97/12977 WO 9712977PCTIUS96/15935 33 TABLE 2 DNA seauences pMON3485.Seq 1 51 101 151 201 251 301 351 401 451 501

ATCGCTTACA

TCTGGGCATC

AGCTGGCAGG

GGGCTCCTGC

GGACACACTG

AGATGGAAGA

ATGCCGGCCT

TGCTAGCCAT

ACCTTGCGCA

AAGTCTTTAG

GGAGAAGCTG

AGCTGTGCCA

CCCTGGGCTC

CTGCTTGAGC

AGGCCCTGGA

CAGCTGGACG

ACTGGGAATG

TCGCCTCTGC

CTGCAGAGCT

GCCCTCTGGC

AGCAAGTGAG

TGTGCCACCT

CCCCGAGGAG

CCCTGAGCTC

CAACTCCATA

AGGGATATCC

TCGCCGACTT

GCCCCTGCCC

TTTCCAGCGC

TCCTGGAGGT

GGCTCTGGCG

GAAGATCCAG

AATAA (SEQ

CTGGTGCTGC

CTGCCCCAGC

GCGGCCTTTT

CCCGAGTTGG

TGCCACCACC

TGCAGCCCAC

CGGGCAGGAG

GTCGTACCGC

GCTCTCAGAG

GGCGATGGCG

ID

TCGGACACTC

CAGGCCCTGC

CCTCTACCAG

GTCCCACCTT

ATCTGGCAGC

CCAGCCTGCC

GGGTCCTGGT

GTTCTACGCC

CTTCCTGCTC

CAGCGCTCCA

pMON3486.Seq 51 101 151 201 251 301 351 401 451 501

ATGGCTCCCG

CGACTTTGCC

CTCCCCTGCA

CAGCGCCGGG

GGAGG r7,TCG

CTGGCGGCTC

ATCCAGGGCG

GCTGTGCCAC

CCTGGGCTCC

TGCTTGAGCC

GGCCCTGGAA

AGTTGGGTCC

ACCACCATCT

GCCCACCCAG

CAGGAGGGGT

TACCGCCTTC

TCAGAGCTTC

ATGGCGCAGC

CCCGAGGAGC

CCTGAGCTCC

AACTCCATAG

GGGATATCCT

CACCTTGGAC

GGCAGCAGAT

GGTGCCATGC

CCTGGTTGCT

TACGCCACCT

CTGCTCA-AGT

GCTCCAGGAG

TGGTGCTGCT

TGCCCCACC

CGGCCTTTTC

AATAA (SEQ

ACACTGCAGC

GGAAGAACTG

CGGCCTTCGC

AGCCATCTGC

TGCGCAGCCC

CTTTAGAGCA

AAGCTGTGTG

CGGACACTCT

AGGCCCTGCA

CTCTACCAGC

ID NO:26)

TGGACGTCGC

GGAATGGCCC

CTCTGCTTTC

AGAGCTTCCT

TCTCGCGGCT

AGTGAGGAAG

CCACCTACAA

CTGGGCATCC

GCTGGCAGGC

GGCTCCTGCA

pMON3487.Seq 1 51 101 151 201 251 301 351 401 451 501

ATGGCTATGG

CGCCTCTGCT

TGCAGAGCTT

CCCTCTGGCG

GCAAGTGAGG

GTGCCACCTA

TC TCTGGGCA

GCAGCTGGCA

AGGGGCTCCT

TTGGACACAC

GCAGATGGAA

CCCCTGCCCT

TTCCAGCGCC

CCTGGAGGTG

GCTCTGGCGG

AAGATCCAGG

CAAGCTGTGC

TCCCCTGGGC

GGCTGCTTGA

GCAGGCCCTG

TGCAGCTGGA

GAACTGGGAT

GCAGCCCACC

GGGCAGGAGG

TCGTACCGCG

C TCTCAGAGC

GCGATGGCC

C AC CCC GAGG

TCCCCTGAGC

GCCAACTCCA

GAAGGGATAT

CGTCGCCGAC

AATAA (SEQ CAGGGTGCCA TCCCGGCCTT GGTCCTGGTT GCTAGCCATC TTCTACGCCA CCTTGCGCAG TTCCTGCTCA AGTCTTTAGA AGCGCTCCAG GAGAAGCTGT AGCTGGTGCT GCTCGGACAC TCCTGCCCCA GCCAGGCCCT TAGCGGCCTT TTCCTCTACC CCCCCGAGTT GGGTCCCACC TTTGCCACCA CCATCTGGCA ID NO:27) pMON3488.Seq WO 97/12977 WO 9712977PCT/US96/15935 1 51 101 151 201 251 301 351 401 451 501

ATGGCTACCC

GGCAGGAGGG

CC TAC CGC CT

TCTCAGAGCT

CGATCGCGCA

ACCCCGAGCA

CCCCTGAGCT

C CAAC TC CAT

AAGGCATATC

C TCC C CGAC T

GGCCCCTGCC

ACGGTGCCAT GCCGCCCTTC GTCCTCCTTG CTAGCCATCT TCTACGCCAC CTTGCGCAGC TCCTGCTCAA GTCTTTAGAG GCGCTCCAGG ACAAGCTCTG GCTGGTGCTG CTCGGACACT CCTGCCCCAG CCAGGCCCTG ACCGCCCTTT TCCTCTACCA CCCCGAGTTG GGTCCCACCT TTGCCACCAC CATCTCGCAG CTGCAGCCCT AATAA (SEQ CCCTCTCCTT TCCAGCCCCG CCAGAGCTTC CTGGACGTGT CCTCTCGCGG CTCTGGCGGC CAAGTCAGGA AGATCCAGGG TGCCACCTAC AAGCTGTGCC CTCTGGGCAT CCCCTGGGCT CAGCTGCCAG GCTCCTTGAG GGGGCTCCTG CAGCCCCTGG TGGACACACT CCAGCTGGAC CAGATCGAAC AACTGCAAT ID NO:28) pMON3489.Seq 1 51 101 151 201 251 301 351 401 451 501

ATGCCTTCTC

TCTGCAGAC

ACCCCTCTGG

GACCAAGTCA

GTCTCCCACC

ACTCTCTCGC

CTGCAGCTG

CCAGGGGCTC

CCTTGGACAC

CAGCAGATGG

TCCCATGCCG

CTTTCCAGCC

TTCCTCGAGG

CCCCTCTCGC

CGAACATCCA

TACAAGCTGT

CATCCCCTGG

CAGGCTGCTT

CTGCAGGCCC

ACTCCACCTG

AAGAACTCGG

GCCTTCGCCT

CCGGGCAGGA GGGGTCCTGG TGTCGTACCG CCTTCTACC GGCTCTCACA CCTTCCTGCT GGGCGATGGC CCAGCGCTCC GCCACCCCGA GGAGCTGGTG GCTCCCCTGA GCTCCTGCCC GACCCAACTC CATAGCGGCC TGGAAGGGAT ATCCCCCGAG GACGTCGCCG ACTTTGCCAC AATGGCCCCT GCCCTGCAGC AATAA (SEQ ID NO:29)

TTCCTAGCCA

CACCTTGCGC

CAAGTCTTTA

AGGAGAAGCT

CTGCTCGGAC

CAGCCAGGCC

TTTTCCTCTA

TTGGGTCCCA

CACCATCTGG

CCACC CAGGG pMON3490.seq 1 51 101 151 201 251 301 351 401 451 501

ATGGCTTACA

TCTGGGCATC

AGCTGGCAGG

GGGCTCCTC

GGACACACTG

AGATGGAAGA

ATGCC3GCCT

TGCTAGCCAT

ACCTTGCGCA

TTCCTGCTCA

AGCGCTCCAG

AGCTCTCCCA

CCCTGGGCTC

CTGCTTGAGC

CCCCCAGGAG

CCCTGAGCTC

CAACTCCATA

AGGCCCTGGA AGGGATATCC CAGCTGGACG TCCCCGACTT ACTGGGAATG GCCCCTGCCC TCGCCTCTGC TTTCCAGCGC CTGCAGAGCT TCCTGGAGGT GCCCACACCA TTGGGCCCTC AGTCTTTACA GCAAGTGACA GAGAACCTGT GTGCCACCTA

CTGGTGCTGC

CTGCCCCAGC

GCGGCCTTTT

CCCGAGTTGG

TGCCACCACC

TGCAGCCCAC

CGGGCAGGAG

CTCGTACCGC

CCAGCTCCCT

AAGATCCAGG

ATAA (SEQ

TCGGACACTC

CAGGCCCTGC

CCTCTACCAG

CTC C CAC CTT

ATCTGGCAGC

CCAGGGTGCC

GGGTCCTGGT

GTTCTACCC

GCCCCAGAGC

GCGATGGCGC

ID pMON3491.seq 1 51 101 151 201 251 301

ATGGCTCCCG

CGACTTTGCC

CTGCCCTGCA

CAGCGCCGGG

GGAGGTGTCG

CCCCTGCCAG

GTGAGAAAGA

ACTTCGGTCC

ACCACCATCT

GCCCACCCAG

CAGGAGGGGT

TACCGCCTTC

CTCCCTGCCC

TCCAGGGCGA

CACCTTGGAC ACACTGCAC GGCAGCAGAT GGAAGAACTG GGTGCCATGC CGGCCTTCGC CCTGGTTCCT AGCCATCTGC TACGCCACCT TGCGCAGCCC CACAGCTTCC TCCTCAAGTC TGGCCCAGCG CTCCAGGAGA

TGGACGTCC

GGAATGGCCC

CTCTGCTTTC

AGACCTTCCT

ACACCATTGC

TTTAGAGCAA

AGCTGTGTGC

WO 97/12977 WO 9712977PCTIUS96/1 5935 351 401 451 501 C AC CTAC AAG

TGGGCATCCC

CTGGCAGGCT

GCTCCTGCAG

C TO T CCAC C

CTGGGCTCCC

OCTTGAGCCA

GCCCTGGAAG

CCGAGGAGCT GGTGCTGCTC GGACACTCTC CTGAGCTCCT GCCCCAGCCA GGCCCTGCAG ACTCCATAGC GGCCTTTTCC TCTACCAGGG GGATATCCTA ATAA (SEQ ID NO:31) pMON3492.seq 1 51 101 151 201 251 301 351 401 451 501

ATGGCTATGG

CGCCTCTGCT

TGCAGAGCTT

CCCACACCAT

GTCTTTAGAC

AGAAGCTGTG

CTCGGACACT

CCAGGCCCTG

TCCTCTACCA

GGTCCCACCT

CATCTGGCAG

CCCCTGCCCT

TTC CAG CCCC

CCTGGAGGTG

TGGGCCCTGC

CAAGTGAGAA

TGCCACCTAC

CTCTGGGCAT

CAGCTGGCAG

GGGGCTCCTG

TGGACACACT

CAGATGGAAG

GCAGCC CAC C

GGGCAGGAGG

TCGTACCGCG

CAGCTCCCTG

AGATCCAGGG

AAGCTGTGCC

CCCCTGGGCT

GCTGCTTGAG

C AGGC C CTGG

GCAGCTGGAC

A.ACTGGGATA

CAGGGTGCCA TGCCGGCCTT GGTCCTGGTT GCTAGCCATC TTCTACGCCA CCTTGCGCAG CCCCAGAGCT TCCTGCTCAA CGATGGCGCA GCGCTCCAGG ACCCCGAGGA GCTGGTGCTG CCCCTGAGCT CCTGCCCCAG CCAACTCCAT AGCGGCCTTT AAGGGATATC CCCCGAGTTG GTCGCCGACT TTGCCACCAC ATAA (SEQ ID NO:32) pMON3493.seq 1 51 101 151 201 251 301 351 401 451 501

ATGGCTACCC

GGCAG' 7.!GGG

CGTACCGCGT

AGCTCCCTGC

GATCCAGGGC

AGCTGTGCCA

CCCTGGGCTC

CTGCTTGAGC

AGGCCCTGGA

CAGCTGGACG

ACTGGGAATG

AGGGTGCCAT

GTCCTGGTTG

TCTACGCCAC

CCCAGAGCTT

GATGGCGCAG

CCCCGAGGAG

CCCTGAGCTC

CAACTCCATA

AGGGATATCC

TCGCCGACTT

GCCCCTGCCC

GCCGGCCTTC

CTAGCCATCT

C TTGCGC AG C

CCTGCTCAAG

CGCTCCAGGA

CTGGTGCTGC

CTGCCCCAC

GCGGCCTTTT

CCCGAGTTGG

TGCCACCACC

T CAGC C CTA

GCCTCTGCTT

GCAGAGC TTC

CCACACCATT

TCTTTAGAGC

GAAGCTGTGT

TCGGACACTC

CAGGCCCTGC

CC TCTAC C A

GTCCCACCTT

ATCTGGCAGC

ATAA (SEQ

TCCAGCGCCG

CTGGAGGTGT

GGGCCCTGCC

AAGTGAGAAA

GCCACCTACA

TCTGGGCATC

AGCTGGCAGG

GGGCTCCTGC

GGACACACTG

AGATGGAAGA

ID NO:33) pMON3494.seq 1 51 101 151 201 251 301 351 401 451

ATGGCTTCTG

TCTGCAGAGC

AGCCCACACC

AAGTCTTTAG

GGAGAAGCTG

TGCTCGGACA

AG C CAGGCCC

TTTCCTCTAC

TGGGTCCCAC

ACCATCTGGC

CTTTCCAGCG CCGGGCACGA TTCCTGGAGG TGTCGTACCG ATTGGGCCCT GCCAGCTCCC AGCAAGTGAG AAAGATCCAG TGTGCCACCT ACAAGCTGTG CTCTCTGGGC ATCCCCTGGG TGCAGCTGGC AGGCTCCTTG CAGGGGCTCC TGCAGGCCCT CTTGGACACA CTGCAGCTGG AGCAGATGGA AGAACTGGGA

GGGGTCCTGG

CGTTCTACGC

TGCCCCAGAG

GGCGATGGCG

CCACCCCGAG

CTCCCCTGAG

AGCCAACTCC

GGAAGGGATA

ACGTCGCCGA

ATGGCCCCTG

ATAA (SEQ

TTGCTAGCCA

CACCTTGCGC

CTTCCTGCTC

CAGCGCTCCA

GAGCTGGTGC

CTCCTGCCCC

ATAGCGGCCT

TCCCCCGAGT

CTTTGCCACC

CCC TGCAGCC ID NO:34) 501 CACCCAGGGT GCCATGCCGG CCTTCGCCTA pMON2 5181. seq WO 97/12977 WO 9712977PCTIUS96/1 5935 1 51 101 151 201 251 301 351 401 451 501

ATGGCTCCGG

CCACTTTGCC

CTGCCCTGCA

C AGCCC GGG

GGACGTGTCG

GCCCTGCCAC

GTGAGAAAGA

C AC CTAC AAG

TGGGCATCCC

CTGGCACGCT

GCTCCTGCAG

AACTCGGTCC

ACCACCATCT

GCCCACCCAG

CAGGAGGCGT

TACCGCCTTC

CTCCCTCCCC

TCCAGGGCGA

CTGTGCCACC

CTGGGCTCCC

CCTTGAGCCA

GCCCTGGAAC

AACTCTCGAC

GCCAGCAGAT

CCTGCCATC

CCTGGTTGCT

TACCCCACCT

CAGAGCTTCC

TGGCCC AC

CCCAGGAGCT

CTGACCTCCT

ACTCCATAC

CCATATCCTA

ACACTGCAC

CCAACAACTC

CCCCCTTCC

ACCCATCTC

TCCCACCCC

TCCTCAACTC

CTCCAGCACA

CCTCCTCCTC

CCCCCACCCA

CCCC-TTTTCC

A (SEQ ID

TCCACCTCC

CCAATCCCCC

CTCTCCTTTC

ACACCTTCCT

ACACCATTCC

TTTACACCAA

ACCTCTCTC

CCACACTCTC

GCCCTCCAG

TCTACCAGCC

pMON25182.seq 1 51 101 151 201 251 301 351 401 451 501

ATCCCTATCC

TCCATCTCCT

TCCACACCTT

CC CACAC CAT

CTCTTTACAC

AGAACCTCTC

CTCCCACACT

C CAGCCCC TC

TCCTCTACCA

CCTCCCACCT

CATCTCCCAC

CTCCACCTCT

TTTCA.ACCTC

CCTCGACCTC

TCCCCCCTC

CAAGTGAGAA

TGCCACCTAC

CTCTGGCCAT

CACCTCCCAG

CCCCC TCCTC

TCCACACACT

CACATCCAAC

CCAACCAACT

CTCCACCTCC

TCGTACCCC

CACCTCCCTC

ACATCCACCC

AAGCTCTCC

CCCCTGGCCT

CCTCCTTCAC

C AGGC CC TGC

CCACCTCCAC

AACTCCCATA

CAACCTCCAA

TCTTCTGCTT

TTCTACCCCA

CCCCAGACCT

CCATGCCCA

ACCCCCACCA

CCCCTCACCT

CCAACTCCAT

AACCCATATC

CTCCCCGACT

TCCCACCATT

CCTACCCATC

CCTTCCCAC

TCCTGCTCAA

CCCTCCACC

CCTCGTCCTC

CCTCCCCCAC

ACCCCCTTT

CCCCCACTTC

TTCCCACCAC

A (SEQ ID NO:36) pMON25183.seg 1 51 101 151 201 251 301 351 401 451 501

ATCCCTACTC

TCCACCTCGT

CCTACCGCGT

ACCTCCCTC

CATCCACCCC

ACCTCTCCCA

CCCTCCCCTC

CTCCTTCAC

ACCCCCTGCA

CACCTCCACC

ACTCCCAATC

AACCTGCTAT

CTTCTGCTTC

TCTACCCCAC

CCCAGACCTT

CATGCGCAG

CCCCGAGGAG

CCCTGACCTC

CAACTCCATA

AGGCATATCC

TCCCCACTT

CCCCCTCCCC

CCCACCTTTT

CTACCCATCT

CTTCCCAGC

CCTCCTCAAC

CCCTCCACCA

C TGC TCC TOC

CTGCCCCAGC

CCCCTTTT

CCCCACTTCC

TCCCACCACC

TGCACCCCTA

CCTTCTCCTT TTCAACCTCC CCACACCTTC CTCCACCTGT CCACACCATT CCCCCCTGCC TCTTTACACC AACTCACAAA CAACCTGTCT CCCACCTACA TCCCACACTC TCTGGCCATC CAGCCCCTCC ACCTCCCACG CCTCTACCAC GCCCTCCTGC CTCCCACCTT CCACACACTC ATCTCCCACC ACATCCAAGA A (SEQ ID NO:37) pMON25184.seq 1 51 101 151 201 251

ATCCCTTCTC

TCTCCAGAC

ACCCACAC C

AACTCTTTAC

CCACAACCTC

TCCTCGGACA

CTTTTCAACC

TTCC TCCAGC

ATTCGCCCT

ACCAACTCAC

TCTCCCACCT

CTCTCTCCCC

TCCTCCAGGT

TCTCCTACCC

CCACTCC C

AAAGATCCAC

ACAACCTCTC

ATCCCCTGCC

CCTCTTCTCC

CGTTCTACC

TCCCCCAGAC

GCCATGCC

CCACCCCGAC

CTCCCCTCAC

TTCCTACCCA

CACCTTCC

CTTCCTCCTC

C ACCCTCCA

CACCTCCTC

CTCCTCCCCC

WO 97/12977 WO 9712977PCTIUS96/1 5935 301 351 401 451 501 AGCC"br-GCCC TTTCCfC0TAO

TGGGTCCCAC

ACCATCTGGC

CACCCAGGGT

TGCAGCTGGC

CAGGGGCTCC

CTTGGACACA

AGCAGATGGA

GCCATGCCGG

AGGCTGCTTG

TGCAGGCCCT

CTGCAGCTGG

AGAACTGGGA

CCTTCGCCTA

AGCCAACTCC ATAGCGGCCT GGAAGGGATA TCCCCCGAGT ACGTCGCCGA CTTTGCCACC ATGGCCCCTG CCCTGCAGCC A (SEQ ID NO:38) pMON25185.seq 1 51 101 151 201 251 301 351 401 451 501

ATGGCTCCGG

CGACTTTCC

CTGCCCTCCA

CAGCGCCGGG

GGAGGTGTCG

CTGGCGGCTC

ATCCAGGGCG

GCTGTGCCAC

CCTGGGCTCC

TGCTTGAGCC

GGCCCTGGAA

AACTGGGTCC

ACCACCATCT

GCCC ACCC AG

CAGGAGGGGT

TACCGCGTTC

TCAGAGCTTC

ATGGCGCAGC

CCCGAGGAGC

CCTGAGCTCC

AACTCCATAG

GGGATATCCT

AACTCTGGAC

GGCACCACAT

GGTGCCATGC

CCTGGTTGCT

TACGCCACCT

CTGCTCAAGT

GCTCCAGGAG

TGGTGCTGCT

TGCCCCAGCC

CGGCCTTTTC

AA (SEQ ID

ACACTGCAGC

GGAAGAACTG

CGGCCTTCGC

AGCCATCTGC

TGCGCAGCCC

CTTTAGAGCA

AAGCTGTGTG

CGGACACTCT

AGGCCCTGCA

CTCTACCAGG

NO: 9)

TGGACGTCGC

GGAATGGCCC

CTCTGCTTTC

AGAGCTTCCT

TCTGGCGGCT

AGTGAGAAAG

CCACCTACA-A

CTGGGCATCC

GCTGGCAGGC

GGCTCCTGCA

pMON25186.seq 1 51 101 151 201 251 301 351 401 451 501

ATGGCTATGG

TGCATCTGCT

TGCAGAGCTT

CCCTCTGGCG

GCAAGTGAGA

GTGCCACCTA

TC TCTGGGCA

GCAGCTGGCA

AGGGGCTCCT

TTGGACACAC

GCAGATGGAA

CTCCAGCTCT

TTTCAACGTC

CCTGGAGGTG

GCTCTGGCGG

AAGATCCAGG

CAAGCTGTGC

TCCCCTGGGC

GGCTGCTTGA

GCAGGCCCTG

TGCAGCTGGA

GAACTGGGAT

GCAACCAACT

GTGCAGGTGG

TCGTACCGCG

CTCTCAGAGC

GCGATGGCGC

CACCCCGAGG

TCCCCTGAGC

GCCAACTCCA

GAAGGGATAT

CGTCGCCGAC

AA (SEQ ID

CAAGGTGCAA

TGTTCTGGTT

TTCTACGCCA

TTCCTGCTCA

AGCGCTCCAG

AGCTGGTGCT

TCCTGCCCCA

TAGCGGCCTT

CCCCCGAGTT

TTTGCCACCA

NO:

TGCCAGCATT

GCTAGCCATC

CCTTGCGCAG

AGTCTTTAGA

GAGAAGCTGT

GCTCGGACAC

CCCAGGC CC T

TTCCTCTACC

GGGTCCCACC

CCATCTGGCA

pMON25187.seq 1 51 101 151 201 251 301 351 401 451 501

ATGGCTACTC

TGCAGGTGGT

CGTACCGCGT

TCTCAGAGCT

CGATGGCGCA

ACCCCGAGGA

C CCC T'-AGC T

CCAACTCCAT

AAGGGATATC

GTCGCCGACT

GC CCC CTGC C

AAGGTCTAT

GTTCTGGTTG

TCTACGCCAC

TCCTGCTCAA

GCGCTCCAGG

GCTGGTGCTG

CC TG C CCCAG

AGCGGCCTTT

CCCCGAGTTG

TTGCCACCAC

C TGCAG CCC T

GCCAGCTTTT

CTAGCCATCT

CTTGCGCAGC

GTCTTTAGAG

AGAAGCTGTG

CTCGGACACT

C CAGGC CC TG

TCCTCTACCA

GGTC C CACC T

CATCTGGCAG

GCTTCTGCTT

GCAGAGCTTC

CCTCTGGCGG

CAAGTGAGAA

TGCCACCTAC

CTCTGGGCAT

CAGCTGGCAG

GGGGCTCCTG

TGGACACACT

CAGATGGA.AG

TTCAACGTCG

CTGGAGGTGT

CTCTGGCGGC

AGATCCAGGG

AAGCTGTGCC

CCCCTGGGCT

GCTGCTTGAG

CAGGCCCTGG

GCAGCTGGAC

AACTGGGAAT

AA (SEQ ID NO:41) WO 97/12977 WO 9712977PCTIUS96/1 5935 pMON2 5188. seq 1 51 101 151 2021 251 301 351 401 451 501

ATGGCTTCTG

TCTGCAGAGC

AGCCCTCTG

GAGCAAGTGA

GTGTGCCACC

ACTCTCTGGG

C TGCAGC TGG CCAGG,.- ;CTC CC TTGGAC AC

CAGCAGATGG

TGCCATGCCG

CTTTTCAACG

TTCCTGGAGG

CGGCTCTGGC

GAAAGATCCA

TACAAGCTGT

C ATC CCC TGG

CAGGCTGCTT

CTGCAGGCCC

ACTGCAGCTG

AAGAACTGGG

GCCTTCGCCT

TCGTGCAGGT

TGTCGTACCG

GGCTCTCAGA

GGGCGATGGC

GCCACCCCGA

GCTCCCCTGA

GAGCCAAC TC

TGGAAGGGAT

GACGTCGCCG

A.ATGGCCCCT

AA (SEQ ID

GGTGTTCTGG

CGTTCTACGC

GCTTCCTGCT

GCAGCGCTCC

GGAGCTGGTG

GCTCCTGCCC

CATAGCGGCC

ATCCCCCGAG

ACTTTGCCAC

GCCCTGCAGC

NO:42)

TTGCTAGCCA

CACCTTGCGC

CAAGTCTTTA

AGGAGAAGCT

CTGCTCGGAC

C AGC CAGGC C

TTTTCCTCTA

TTGGGTCCCA

CACCATCTGG

CCACCCAGGG

pMON3460 .seq 1 ATGGCTCTGC TCGGACACTC TCTGGGCATC CCCTGGGCTC CCCTGAGCTC 51 CTGCCCCAGC CAGGCCCTGC AGCTGGCAGG CTGCTTGAGC CAACTCCATA 101 151 201 251 301 351 401 451 501

GCGGCCTTTT

CCCGAGTTGG

TGCCACCACC

TGCAGCCCAC

CGGGCAGGAG

GTCGTACCGC

CCAGCTCCCT

AAGATCCAGG

CAAGCTGTGC

C CTC TAC CAG

GTCCCACCTT

ATCTGGCAGC

CCAGGGTGCC

GGGTCCTGGT

CTTCTACGCC

GCCCCAGAGC

GCGATGGCGC

CACCCCGAGG

GGGCTCCTGC

GGACACACTG

AGATGGAAGA

ATGCCGGCCT

TGCTAGCCAT

AC CTTGCGCCA

TTCCTGCTCA

AGCGCTCCAG

AGCTGGTGTA

AGGCCCTGGA AGGGATATCC CAGCTGGACG TCGCCGACTT ACTGGGAATG GCCCCTGCCC TCGCCTCTGC TTTCCAGCGC CTGCAGAGCT TCCTGGAGGT GCCCACACCA TTGGGCCCTG AGTCTTTAGA GCAAGTGAGA GAGAAGCTGT GTGCCACCTA ATAA (SEQ ID NO:86) pMON3461 .seq 1 51 101 151 201 251 301 351 401 451 501 ATGGC'rCAAC

CCTGGAAGGG

TGGACGTCGC

GGAATGGCCC

CTCTGCTTTC

AGAGCTTCCT

ACACCATTGG

TTTAGAGCAA

AGCTGTGTGC

GGACACTCTC

GGCCCTGCAG

TCCATAGCGG

ATATOC C CC G

CGACTTTCC

CTGCCCTGCA

CAGCGCCGGG

GGAGGTGTCG

GCCCTGCCAG

GTGAGAAAGA

CACCTACAAG

TGGGCATCCC

CTGGCAGGCT

CCTTTTCCTC

AGTTGGGTCC

ACCACCATCT

GCCCACCCAG

CAGGAGGGGT

TACCGCGTTC

CTCCCTGCCC

TCCAGGGCGA

CTGTGCCACC

CTGGGCTCCC

GCTTGAGCTA

TACCAGGGGC

CACCTTGGAC

GGCAGCAGAT

GGTGCCATGC

CCTGGTTGCT

TAC GC CACC T

CAGAGCTTCC

TGGCGCAGCG

CCGAGGAGCT

CTGAGCTCCT

TCCTGCAGGC

ACACTGCAGC

GGAAGAACTG

CGGCCTTCGC

AGCCATCTGC

TGCGCAGCCC

TGCTCAAGTC

CTCCAGGAGA

GGTGCTGCTC

CCC CCAGC CA ATAA (SEQ ID NO:87) pMON3462 .seq 1 51 101 151 201

ATGGCTCTTT

CCCCGAGTTG

TTGCCACCAC

CTGCAGCCCA

CCGGGCAGGA

TCCTCTACCA

GGTCCCACCT

CATCTGGCAG

CCCAGGGTGC

GGGGTCCTGG

GGGGCTCCTG

TGGACACACT

CAGATGGAAG

CATGCCGGCC

TTGCTAGCCA

CAGGCCCTGG AAGGGATATC GCAGCTGGAC GTCGCCGACT A.ACTGGGAAT GGCCCCTGCC TTC,,;CCTCTG CTTTCCAGCG TCTGCAGAGC TTCCTGGAGG WO 97/12977 WO 9712977PCTIUS96/15935 251 301 351 401 451 501

TGTCGTACCG

GCCAGCTCCC

AAAGATCCAG

ACAAGCTGTG

ATCCCCTGGG

AGGCTGCTTG

CGTTCTACGC

TGCCCCAGAG

GGCGATGGCG

CCACCCCGAG

CTCCCCTGAG

AGCCAACTCC

CACCTTGCGC

CTTCCTGCTC

CAGCGCTCCA

GAGCTGGTGC

CTCCTGCCCC

ATAGCGGCTA

AGCCCACACC ATTGGGCCCT AAGTCTTTAG AGCAAGTGAG GGAGAAGCTG TGTGCCACCT TGCTCGGACA CTCTCTGGGC AGCCAGGCCC TGCAGCTGGC ATAA (SEQ ID NO:88) pMON3463.seq 1 51 101 151 201 251 301 351 401 451 501

ATGGCTCTCT

GTTGGGTCCC

CCACCATCTG

CCC AC CCAGG

AGGAGGGGTC

ACCGCGTTCT

TCCCTGCCCC

CCAGGGCGAT

TGTGC"..CCC

TGGGCTCCCC

CTTGAGCCAA

ACCAGGGGCT

ACCTTGGACA

GCAGCAGATG

GTGCCATGCC

CTGGTTGCTA

ACGCCACCTT

AGAGCTTCCT

GGCGCAGCGC

CGAGGAGCTG

TGAGCTCCTG

CTCCATAGCG

CCTGCAGGCC

CACTGCAGCT

GAAGAAC TGG

GGCCTTCGCC

GCCATCTGCA

GCGCAGCCCA

GCTCAAGTCT

TCCAGGAGAA

GTGCTGCTCG

CCCCAGCCAG

GCCTTTTCTA

CTGGAAGGGA TATCCCCCGA GGACGTCGCC GACTTTGCCA GAATGGCCCC TGCCCTGCAG TCTGCTTTCC AGCGCCGGGC GAGCTTCCTG GAGGTGTCGT CACCATTGGG CCCTGCCAGC TTAGAGCAAG TGAGAAAGAT GCTGTGTGCC ACCTACAAGC GACACTCTCT GGGCATCCCC GCCCTGCAGC TGGCAGGCTG ATAA (SEQ ID NO:89) pMON3464 .seq 1 51 101 151 201 251 301 351 401 451 501

ATGGCTGCCC

ACTGCAGCTG

AAGAACTGGG

GCCTTCGCCT

CCATCTGCAG

C GCAG C CCAC

CTCAAGTCTT

CCAGGAGAAG

TGCTGCTCGG

CCCAGCCAGG

CCTTTTCCTC

TGGAAGGGAT

GACGTCGCCG

AATGCC CCC T

CTGCTTTCCA

AGCTTCCTGG

ACCATTGGGC

TAGAGCAAGT

CTGTGTGCCA

ACACTCTCTG

CCC TGCAGCT

TACCAGGGGC

ATCCCCCGAG

ACTTTGCCAC

GCCCTGCAGC

GCGCCGGGCA

AGGTGTCGTA

CCTGCCAGCT

GAGAAAGATC

CCTACAAGCT

GGCATCCCCT

GGCAGGCTGC

TCCTGCAGTA

TTGGGTCCCA

CACCATCTGG

CCACCCAGGG

GGAGGGGTCC

CCGCGTTCTA

CCCTGCCCCA

CAGGGCGATG

GTGCCACCCC

GGGCTCCCCT

TTGAGCCAAC

CCTTGGACAC

CAGCAGATGG

TGCCATGCCG

TGGTTGC TAG

CGCCACCTTG

GAGCTTCCTG

GCGCAGCGCT

GAGGAGCTGG

GAGCTCCTGC

TCCATAGCGG

ATAA (SEQ ID pMON3465.seq 1 51 101 151 201 251 301 351 401 451 501

ATGGCTGACT

GGCCC 'TGCC

CTTTCCAGCG

TTCCTGGAGG

ATTGGGCCCT

AGCAAGTGAG

TGTGCCACCT

CTCTCTGGGC

TGCAGCTGGC

CAGGGGCTCC

CTTGGACACA

TTGCCACCAC

CTGCACCCCA

CCGGGCAGGA

TGTCGTACCG

GCCAGCTCCC

AAAGATCCAG

ACAAGCTGTG

ATCCCCTGGG

AGGCTGCTTG

TGCAGGCCCT

CTGCAGCTGG

CATCTGGCAG

CCCAGGGTGC

GGGGTCCTGG

CGTTCTACGC

TGCCCCAGAG

GGCGATGGCG

CCACCCCGAG

CTCCCCTGAG

AG CC AAC TC C

GGAAGGGATA

AC GTCCC CTA

CAGATGGAAG

CATGCCGCC

TTGCTACCCA

CACCTTCCC

CTTCCTCCTC

CAGCCCTCCA

GACCTGGTGC

CTCCTGCCCC

ATACCGCCCT

TCCCCCGAGT

A.ACTGCGAAT

TTCCCCTCTC

TCTGCAGAGC

ACC CCAC AC C

AACTCTTTAG

GGACAACCTG

TGCTCGGACA

AGCCAGGCCC

TTTCCTCTAC

TGGGTCCCAC

ATAA (SEQ ID NO:91) WO 97/12977 WO 9712977PCTIUS96/1 5935 pMON 3466.seq 1 51 101 151 201 251 301 351 401 451 501 ATGGCTATCT GGCAGCAGAT GCCCACCCAG GGTGCCATGC CAGGAGGGGT CCTGGTTGCT TACCCG'TTC TACGCCACCT CTCCCTCCCC CAGAGCTTCC TCCAGGGCGA TGGCGCAGCG CTGTGCCACC CCGACGACCT CTGGGCTCCC CTGAGCTCCT GCTTGAGCCA ACTCCATAGC GCCCTGGAAG GGATATCCCC CCTGGACGTC GCCGACTTTG GGAAGAACTG GGAATGGCCC CTGCCCTGCA CGGCCTTCGC CTCTGCTTTC CAGCGCCGGG AGCCATCTGC AGAGCTTCCT GGAGGTGTCG TGCGCAGCCC ACACCATTGG GCCCTGCCAG TGCTCAAGTC TTTAGAGCAA GTGAGAAAGA CTCCAGGAGA AGCTGTGTGC CACCTACAAG GGTGCTGCTC GGACACTCTC TGGGCATCCC GCCCCAGCCA GGCCCTGCAG CTGGCAGGCT GGCCTTTTCC TCTACCAGGG GCTCCTGCAG CGAGTTGGGT CCCACCTTGG ACACACTGCA CCACCACCTA ATAA (SEQ ID NO:92) pMON3467.seq 1 51 101 151 201 251 301 351 401 451 501 ATGGCTCAGC AGATGGAAGA ACTGGGAATG GCCCCTGCCC TGCAGCCCAC CCAGGGTGCC ATGCCGGCCT TCGCCTCTGC TTTCCAGCGC CGGGCAGGAG GGGTCCTGGT TGCTAGCCAT CTGCAGAGCT TCCTGGAGGT GTCGTACCGC GTTCTACGCC ACCTTGCGCA GCCCACACCA TTGGGCCCTG CCAGCTCCCT GCCCCAGAGC TTCCTGCTCA AGTCTTTAGA GCAAGTGAGA AAGATCCAGG GCGATGGCGC AGCGCTCCAG GAGAAGCTGT GTGCCACCTA CAAGCTGTGC CACCCCGAGG AGCTGGTGCT GCTCGGACAC TCTCTGGGCA TCCCCTGGGC TCCCCTGAGC TCCTGCCCCA GCCAGGCCCT GCAGCTGGCA GGCTGCTTGA GCCAACTCCA TAGCGGCCTT TTCCTCTACC AGGGGCTCCT GCAGGCCCTG GAAGGGATAT CCCCCGAGTT GGGTCCCACC TTGGACACAC TGCAGCTGGA CGTCG 7CGAC TTTGCCACCA CCATCTGGTA ATAA (SEQ ID NO:93) pMON3499 .seq 1 51 101 151 201 251 301 351 401 451 501 ATGGCTTTGT TAGGACATTC TTTAGCTATT CCATGGGCTC CTGCCCCAGC CAGGCCCTGC AGCTGGCAGG CTGCTTGAGC GCGGCCTTTT CCTCTACCAG GGGCTCCTGC AGGCCCTGGA CCCGAGTTGG GTCCCACCTT GGACACACTG CAGCTGGACG TGCCACCACC ATCTGGCAGC AGATGGAAGA ACTGGGAATG TGCAGCCCAC CCAGGGTGCC ATGCCGGCCT TCGCCTCTGC CGGGCAGGAG GGGTCCTGGT TGCTAGCCAT CTGCAGAGCT GTCGTACCGC CTTCTACGCC ACCTTGCGCA GCCCACACCA CCAGCTCCCT GCCCCAGAGC TTCCTGCTCA AGTCTTTAGA AAGATCCAGG GCGATGGCGC AGCGCTCCAG GAGAAGCTGT CAAGCTGTGC CACCCCGAGG AGCTGGTGTA ATAA (SEQ

CTCTGAGCTC

CAACTCCATA

AGGGATATCC

TCGCCGACTT

GCCCCTGCCC

TTTCCAGCGC

TCCTGGAGGT

TTGGGCCCTG

GCAAGTGAGA

GTGCCACCTA

ID NO:94) WO 97/12977 PCTIUS96/1 5935 41 TABLE 3 PROTEIN SEOUENCES pMON3485.Pep Tyr Leu Leu Leu Leu Ala Ala Phe Ser Pro Leu Glu Lys Leu Cys Gly Ile Pro Gin Leu Ala Tyr Gin Gly Gly Pro Thr Thr Thr Ile Leu Gin Pro Gin Arg Arg Phe Leu Glu Ser Glv Gly Glu Gin Vai Lys Leu Cys His Trp Gly Leu Leu Trp Thr Ala Val Ser Arg Ala Pro Ala Cys Leu Asp Gin Gin Gly Ser Gly Lys Thr Glu Glu Leu Val Leu Leu Pro Leu Ser Ser Cys Pro Leu Ser Gin Leu His Ser Gin Ala Leu Giu Giy Ile Thr Leu Gin Leu Asp Vai Gin Met Giu Giu Leu Gly Gly Ala Met Pro Ala Phe Gly Val Leu Val Ala Ser Tyr Arg Val Leu Arg His Gly Ser Gin Ser Phe Leu Ile Gin Gly Asp Giy Ala (SEQ ID NO:43) Gly His Ser Ser Gin Ala Gly Leu Phe Ser Pro Glu Ala Asp Phe Met Ala Pro Ala Ser Ala His Leu Gin Leu Ala Gin Leu Lys Ser Ala Leu Gin pMON3486.Pep Pro Asp Ala Ser Leu Ala Lys Leu Glu Leu Ser Ala Glu Leu Phe Ala Pro Ala Ala Phe Gin Ser Gin Pro Ser Leu Gin Glu Leu Val Ser Ser Gin Leu Leu Glu Gly Thr Leu Gin Phe Ser Glu Lys Leu Cys His Gly Pro Thr Gin Arg Leu Gly Gin Leu Leu Pro Ser Ile Thr Leu Asp Thr Leu Ile Trp Gin Gin Met Pro Thr Gin Giy Ala Arg Ala Gly Giy Vai Glu Val Ser Tyr Arg Gly Ser Gly Giy Ser Vai Arg Lys Ile Gin Cys Ala Thr Tyr Lys Gly His Ser Leu Gly Ser Gin Ala Leu Gin Gly Leu Phe Leu Tyr Ser (SEQ ID NO:44) Gin Glu Met Leu Val Gin Gly Leu Ile Leu Gin Leu Glu Pro Vai Leu Ser Asp Cys Pro Ala Gly Asp Val Leu Gly Ala Phe Ala Ser Arg His Phe Leu Gly Ala His Pro Trp Ala Gly Cys Leu Leu Ala Met Ala His Leu Leu Ala Glu Pro Leu Gin pMON3487.Pep Met Ala Pr.) Ala Ser Ala His Leu Gin Leu Ala Gin Leu Lys Ser Ala Leu Gin Glu Giu Leu Pro Leu Ser Leu Ser Gin Gin Ala Leu Gin Met Glu Ala Phe Ser Pro Leu Glu Val Ser Leu Glu Glu Leu Gin Phe Ser Glu Lys Leu Cys His Gly Leu Gin Arg Leu Gly Gin Leu Leu Pro Ser Ile Gly Pro Thr Gin Gly Ala Met Arg Ala Gly Gly Val Leu Glu Val Ser Tyr Arg Val Gly Ser Gly Gly Ser Gin Vai Arg Lys Ile Gin Gly Cys Ala Thr Tyr Lys Leu Gly His Ser Leu Gly Ile Ser Gin Ala Leu Gin Leu Gly Leu Phe Leu Tyr Gin Ser Pro Giu Leu Gly Pro (SEQ ID Pro Ala Val Ala Leu Arg Ser Phe Asp Gly Cys His Pro Trp Ala Gly Gly Leu Thr Leu Phe Ser His Leu Ala Pro Ala Cys Leu Asp pMON3488.Pep WO 97/12977 PCT/US96/15935 Thr Gin Gly Ala Gly Gly Val Ser Tyr Ser Gly Gly Arg Lys Ile Ala Thr Tyr His Ser Leu Gin Ala Leu Leu Phe Leu Pro Glu Leu Asp Phe Ala Ala Pro Ala Ala Val Arg Ser Gin Lys Gly Gin Tyr Gly Thr Leu Met Pro Leu Val Val Leu Gin Ser Gly Asp Leu Cys Ile Pro Leu Ala Gin Gly Pro Thr Thr Ile Gin Pro Ala Phe Ala Ser Arg His Phe Leu Gly Ala His Pro Trp Ala Gly Cys Leu Leu Leu Asp Trp Gin Ala Ser Ala Phe His Leu Gin Ser Leu Ala Gin Pro Leu Lys Ser Leu Ala Leu Gin Glu Glu Glu Leu Val Pro Leu Ser Ser Leu Ser Gin Leu Gin Ala Leu Glu Thr Leu Gin Leu Gin Met Giu Glu Gin Arg Phe Leu Ser Gly Glu Gin Lys Leu Leu Leu Cys Pro His Ser Gly Ile Asp Val Leu Gly Arg Glu Gly Val Cys Gly Ser Gly Ser Ala Met (SEQ ID NO:46) pMON3489.Pep Ser Ala Leu Gin Ala Gin Lys Ser Leu Gin Glu Leu Leu Ser Ser Gin Ala Leu Leu Gin Met Glu Ala Met Phe Gin Arg Arg Ser Phe Leu Glu Pro Ser Gly Gly Leu Giu Gin Val Glu Lys Leu Cys Val Leu Leu Gly Ser Cys Pro Ser His Ser Gly Glu Gly Ile Ser Leu Asp Val Ala Glu Leu Gly Met Pro Ala Phe Ala Ala Val Ser Arg Ala His Gin Leu Pro Asp Ala Gly Ser Gly Lys Thr Ser Ala Phe Glu Phe Pro Gly Tyr Gly Ile Tyr Leu Leu Leu Leu Ala Ala Va1 Arg Ser Gin Lys Gly Gin Tyr Gly Thr Leu Leu Val Val Leu Gin Ser Gly Asp Leu Cys Ile Pro Leu Ala Gin Gly Pro Thr Thr Ile Gin Pro Ala Arg Phe Gly His Trp Gly Leu Leu Trp Thr Ser His His Leu Leu Leu Ala Ala Pro Glu Ala Pro Cys Leu Leu Gin Asp Thr Gin Gin Gin Gly (SEQ ID NO:47) pMON3490.Pep Tyr Lys Leu Gly Leu Gin Leu Tyr Leu Gly Ala Thr Ala Leu Phe Gin Ser Phe Pro Thr Leu Lys Ala Leu Leu Cys Ile Pro Leu Ala Gin Gly Pro Thr Thr Ile Gin Pro Arg Arg Leu Glu Pro Leu Ser Leu Gin Glu His Trp Gly Leu Leu Trp Thr Ala Val Gly Glu Lys Pro Ala Cys Leu Asp Gin Gin Gly Ser Pro Gin Leu Glu Glu Pro Leu Leu Ser Gin Ala Thr Leu Gin Met Gly Ala Gly Val Tyr Arg Ala Ser Val Arg Cys Ala Leu Ser Gin Leu Gin Glu Met Leu Val Ser Lys Thr Val Leu Ser Cys Leu His Glu Gly Leu Asp Glu Leu Pro Ala Val Ala Leu Arg Leu Pro Ile Gin (SEQ ID Leu Gly Pro Ser Ser Gly Ile Ser Vai Ala Gly Met Phe Ala Ser His His Leu Gin Ser Gly Asp NO: 48) His Ser Gin Ala Leu Phe Pro Glu Asp Phe Ala Pro Ser Ala Leu Gin Ala Gin Phe Leu Gly Ala pMON3491.Pep Pro Glu Leu Gly Pro Thr Leu Asp Thr Asp Phe Ala Thr Thr Ile Trp Gin Gin Ala Pro Ala Leu Gin Pro Thr Gin Gly Leu Gin Leu Asp Val Ala Met Glu Giu Leu Gly Met Ala Met Pro Ala Phe Ala WO 97/12977 PCT/US96/15935 Ser Leu Ala Phe Gly His Trp Gly Leu Ala Phe Gin Ser Gin Pro Leu Leu Ala Ala Pro Glu Ala Pro Cys Leu Leu Gin Gin Phe Thr Lys Leu Glu Leu Ser Ala Arg Leu Pro Ser Gin Leu Ser Gin Leu Arg Glu Leu Leu Glu Val Ser Leu Glu Ala Val Gly Glu Lys Leu Cys His Gly Gly Ser Pro Gin Leu Leu Pro Ser Ile Gly Tyr Ala Val Cys Gly Ser Gly Ser Val Leu Val Ala Arg Val Leu Arg Ser Ser Leu Pro Arg Lys Ile Gin Ala Thr Tyr Lys His Ser Leu Gly Gin Ala Leu Gin Leu Phe Leu Tyr (SEQ ID NO:49) Ser His His Leu Gin Ser Gly Asp Leu Cys Ile Pro Leu Ala Gin Gly pMON3492.Pep Met Ala Ala Ser His Leu Leu Ala Ser Phe Asp Gly Cys His Pro Trp Ala Gly Gly Leu Thr Leu Ile Trp Pro Ala Ala Phe Gin Ser Gin Pro Leu Leu Ala Ala Pro Glu Ala Pro Cys Leu Leu Gin Asp Thr Gin Gin Leu Gin Phe Thr Lys Leu Glu Leu Ser Ala Leu Met Gin Arg Leu Pro Ser Gin Leu Ser Gin Leu Gin Glu Pro Arg Glu Leu Leu Glu Val Ser Leu Glu Leu Glu Thr Ala Val Gly Glu Lys Leu Cys His Gly Asp Leu Gin Gly Ser Pro Gin Leu Leu Pro Ser Ile Val Gly Gly Gly Tyr Ala Val Cys Gly Ser Gly Ser Ala Ala Met Val Leu Arg Val Ser Ser Arg Lys Ala Thr His Ser Gin Ala Leu Phe Pro Glu Asp Phe Pro Ala Val Ala Leu Arg Leu Pro Ile Gin Tyr Lys Leu Gly Leu Gin Leu Tyr Leu Gly Ala Thr Phe Ser His Gin Gly Leu Ile Leu Gin Pro Thr (SEQ ID pMON3493.Pep Thr Ala Val Gly Glu Lys Leu Cys His Gly Asp Leu Gin Gly Ser Pro Gin Leu Leu Pro Ser Ile Val Gly Gly Ala Gly Vai Tyr Arg Ala Ser Val Arg Cys Ala Gly His Ser Gin Gly Leu Ser Pro Ala Asp Met Ala Met Leu Val Ser Lys Thr Ser Ala Phe Glu Phe Pro Pro Ala Val Ala Leu Arg Leu Pro Ile Gin Tyr Lys Leu Gly Leu Gin Leu Tyr Leu Gly Ala Thr Ala Leu Phe Ala Ser His His Leu Gin Ser Gly Asp Leu Cys Ile Pro Leu Ala Gin Gly Pro Thr Thr Ile Gin Pro Ser Ala Leu Gin Ala Gin Phe Leu Gly Ala His Pro Trp Ala Gly Cys Leu Leu Leu Asp Trp Gin (SEQ ID Phe Gin Arg Ser Phe Leu Pro Thr Pro Leu Lys Ser Ala Leu Gin Glu Giu Leu Pro Leu Ser Leu Ser Gin Gin Ala Leu Thr Leu Gin Gin Met Glu NO: 51) Arg Glu Leu Leu Glu Val Ser Leu Glu Leu Glu pMON3494.Pep Ser Ala Phe Gin Leu Gin Ser Phe Ala Gin Pro Thr Phe Leu Leu Lys Gly Ala Ala Leu His Pro Giu Glu Trp Ala Pro Leu Arg Arg Ala Leu Giu Val Pro Leu Gly Ser Leu Glu Gin Giu Lys Leu Val Leu Ser Ser Cys Gly Gly Ser Tyr Pro Ala Gin Val Leu Cys Leu Gly Pro Ser Val Leu Arg Val Ser Ser Arg Lys Ala Thr His Ser Gin Ala Vai Ala Leu Arg Leu Pro Ile Gin Tyr Lys Leu Gly Leu Gin Ser His His Leu Gin Ser Gly Asp Leu Cys Ile Pro Leu Ala WO 97/12977 PCT/US96/1 5935 Gly Cys Leu Ser Leu Leu Gin Ala Leu Asp Thr Leu Trp Gin Gin Met Thr Gin Gly Ala Gin Leu Gin Glu Met Leu Glu Leu Glu Pro His Gly Asp Leu Ala Ser Ile Vai Gly Phe Gly Ser Ala Met Ala Leu Phe Pro Glu Asp Phe Ala Pro (SEQ ID Leu Tyr Gin Gly Leu Gly Pro Thr Ala Thr Thr Ile Ala Leu Gin Pro NO: 52) pMON25181.pep Pro Glu Asp Phe Ala Pro Ser Ala Leu Gin Ala Gin Phe Leu Gly Ala His Pro Trp Ala Gly Cys Leu Leu Leu Gly Ala Thr Ala Leu Phe Gin Ser Phe Pro Thr Leu Lys Ala Leu Glu Glu Pro Leu Leu Ser Gin Ala Pro Thr Gin Arg Leu Pro Ser Gin Leu Ser Gin Leu Thr Ile Pro Arg Glu Leu Leu Glu Val Ser Leu Glu Leu Trp Thr Ala Va1 Gly Glu Lys Leu Cys His Gly Asp Gin Gin Gly Ser Pro Gin Leu Leu Pro Ser Ile Thr Gin Gly Gly Tyr Ala Val cys Gly Ser Gly Ser Leu Gin Met Glu Ala Met Val Leu Arg Val Ser Ser Arg Lys Ala Thr His Ser Gin Ala Leu Phe (SEQ ID Leu Asp Val Glu Leu Gly Pro Ala Phe Val Ala Ser Leu Arg His Leu Pro Gin Ile Gin Gly Tyr Lys Leu Leu Gly Ile Leu Gin Leu Leu Tyr Gin NO: 53) Ala Met Ala His Leu Ser Asp Cys Pro Ala Gly pMON25182.pep Met Ala His Leu Ser Asp Cys Pro Ala Gly Thr Ile Ala Ser Leu Ala Phe Gly His Trp Gly Leu Leu Trp Pro Ala Ala Phe Gin Ser Gin Pro Leu Leu Ala Ala Pro Glu Ala Pro Cys Leu Leu Gin Asp Thr Gin Gin Leu Gin Phe Thr Lys Leu Glu Leu Ser Ala Leu Met Gin Pro Arg Arg Leu Glu Pro Leu Ser Leu Gin Glu Leu Val Ser Ser Gin Leu Leu Glu Gin Leu Glu Glu Thr Ala Val Gly Glu Lys Leu Cys His Gly Asp Leu Gin Gly Ala Gly Gly Val Ser Tyr Arg Pro Ala Ser Gin Val Arg Leu Cys Ala Leu Gly His Pro Ser Gin Ser Gly Leu Ile Ser Pro Val Ala Asp Gly (SEQ ID Met Pro Ala Leu Val Ala Val Leu Arg Ser Leu Pro Lys Ile Gin Thr Tyr Lys Ser Leu Gly Ala Leu Gin Phe Leu Tyr Glu Leu Gly Phe Ala Thr NO: 54) Phe Ser His Gin Gly Leu Ile Leu Gin Pro Thr pMON25183.pep Thr Ala Val Gly Glu Lys Leu Cys His Gly Gin Gly Ser Pro Gin Leu Leu Pro Ser Ile Gly Ala Gly Val Ty, Arg Ala Ser Vai Arg Cys Ala Gly His Ser Gin Gly Leu Ser Pro Met Leu Val Ser Lys Thr Ser Ala Phe Glu Pro Ala Val Ala Leu Arg, Leu Pro Ile Gin Tyr Lys Leu Gly Leu Gin Leu Tyr Leu Gly Phe Ala Ser Ser His Leu His Leu Ala Gin Ser Phe Gly Asp Gly Leu Cys His Ile Pro Trp Leu Ala Gly Gin Giy Leu Pro Thr Leu Ala Gin Gin Leu Ala Pro Ala Cys Leu Asp Phe Gin Arg Ser Phe Leu Pro Thr Pro Leu Lys Ser Ala Leu Gin Glu Giu Leu Pro Leu Ser Leu Ser Gin Gin Ala Leu Thr Leu Gin Arg Glu Leu Leu Glu Val Ser Leu Glu Leu WO 97/12977 PCTIUS96/15935 Asp Val Ala Asp Phe Ala Thr Thr Ile Trp, Gin Gin Met Glu Giu Leu Gly Met Ala Pro Ala Leu Gin Pro (SEQ ID pMON25184.pep Ser Leu Ala Phe Gly His Trp Gly Leu Leu Trp Thr Ala Gin Gin Leu Ala Pro Ala Cys Leu Asp Gin Gln Phe Gin Sei Phe Pro Thr Leu Lys Ala Leu Giu Giu Pro Leu Leu Ser Gin Ala Thr Leu Gin Met Gly Ala Arg Arg Ala Leu Giu Val Pro Leu Gly Ser Leu Giu Gin Giu Lys Leu Val Leu Ser Ser Cys Gin Leu His Leu Glu Gly Gin Leu Asp Giu Giu Leu Met Pro Ala Giy Ser Pro Gin Leu Leu Pro Ser Ile Val Gly Phe Giy Ala Val1 Cy s Gly Ser Giy Ser Aila Met Ala Val Leu Arg Vai Ser Ser Arg Lys Aia Thr His Ser Gin Ala Leu Phe Pro Giu Asp Phe Ala Pro (SEQ ID Vai Ala Leu Arg Leu Pro Ile Gin Tyr Lys Leu Gly Leu Gin Leu T~yr Leu Giy Ala Thr Ala Leu NO: 5 6) Ser His His Leu Gin Ser Gly Asp Leu Cys Ile Pro Leu Ala Gin Giy Pro Thr Thr Ile Gin Pro pMON25185 .pep Pro Asp Ala Ser Leu Ala Lys Leu Giu Leu Ser Ala Giu Phe Pro Ala Gin Gin Ser Gin Leu Ser Gin Leu Leu Ala Ala Phe Ser Pro Leu Giu Val Ser Leu Giu Gly Thr Leu Gin Phe Ser Giu Lys Leu Cys His G ly Pro Thr Gin Arg Leu G ly Gin Leu Leu Pro Ser Ile Thr Ile Pro Arg Giu Gly Val Cys Gly Ser Gly Ser Leu Asp Trp, Gin Thr Gin Ala Gly Val Ser Ser Gly Arg Lys Ala Thr His Ser Gin Ala Leu Phe (SEQ ID Thr Leu Gin Met Gly Ala Gly Val Tyr Arg Gly Ser Ile Gin Tyr Lys Leu Gly Leu Gin Leu Tyr NO: 57) Gin Giu Met L ,u Val Gin G ly Leu Ile Leu Gin Leu Giu Pro Val Leu Ser Asp Cys Pro Ala G ly Asp Leu Ala Ala Arg Phe Gly His Trp Gly Leu Val1 Gly Phe Ser His Leu Ala Pro Ala Cys Leu Ala Met Ala His Leu Leu Ala Giu Pro Leu Gin pMON25186 .pep Met Ala His Leu Leu Ala Leu Ser Gin Leu Gin Giu Ala Pro Ser Ala Leu Gin Ala Gin Lys Ser Leu Gin Val Leu Ser Cys Leu His Glu Gly Leu Asp Giu Leu Ala Phe Ser Pro Leu Giu Leu Pro Ser Ile Val Gly Leu Gin Gin Arg Phe Leu Ser Gly Giu Gin Lys Ala Gly His Ser Gin Gly Leu Ser Pro Ala Asp (SEQ ID Pro Thr Arg Ala Giu Val Gly Ser Val Arg Thr Tyr Ser Leu Ala Leu Phe Leu Giu Leu Phe Ala NO: 5 8) Gin Giy Gly Gly Ser Tyr Gly Gly Lys Ile Lys Leu Gly Ile Gin Leu Tyr Gin Giy Pro Thr Thr Ala Met Val Leu Arg Val Ser Gin Gin Gly Cys His Pro Trp Ala Gly Gly Leu Thr Leu Ile Trp Pro Ala Phe Val Ala Ser Leu Arg His Ser Phe Leu Asp Gly Ala Pro Glu Giu Ala Pro Leu Cys Leu Ser Leu Gin Ala Asp Thr Leu Gin Gin Met WO 97/12977 PCT/US96/1 5935 pMON25187.pep Thr Gin Ala Gly Val Ser Ser Gly Arg Lys Ala Thr His Ser Gin Ala Leu Phe Pro Glu Asp Phe Ala Pro Gly Ala Gly Val Tyr Arg Gly Ser Ile Gin Tyr Lys Leu Gly Leu Gin Leu Tyr Leu Gly Ala Thr Ala Leu Met Leu Val Gin Gly Leu Ile Leu Gin Pro Thr Gin Pro Val Leu Ser Asp Cys Pro Ala Gly Thr Ile Pro Ala Phe Ala Ser Arg His Phe Leu Gly Ala His Pro Trp Ala Gly Cys Leu Leu Leu Asp Trp Gin (SEQ ID Ala Ser Ala Phe Gin Arg Arg His Leu Gin Ser Phe Leu Glu Leu Ala Gin Pro Ser Gly Gly Leu Lys Ser Leu Giu Gin Val Ala Leu Gin Glu Lys Leu Cys Glu Glu Leu Val Leu Leu Gly Pro Leu Ser Ser Cys Pro Ser Leu Ser Gin Leu His Ser Gly Gin Ala Leu Giu Gly Ile Ser Thr Leu Gin Leu Asp Val Ala Gin Met Glu Giu Leu Gly Met NO:59) pMON25188.pep Ser Ala Phe Leu Gin Ser Ala Gin Pro Lys Ser Leu Leu Gin Glu Glu Leu Val Leu Ser Ser Ser Gin Leu Ala Leu Glu Leu Gin Leu Met Giu Glu Ala Met Pro Gin Phe Ser Glu Lys Leu Cys His Gly Asp Leu Ala Arg Leu Gly Gin Leu Leu Pro Ser Ile Val Gly Phe Arg Glu Gly Val Cys Gly Ser Gly Ser Ala Met Ala Ala Gly Val Ser Ser Gly Arg Lys Ala Thr His Ser Gin Ala Leu Phe Pro Glu Asp Phe Ala Pro (SEQ ID Gly Val Tyr Arg Gly Ser Ile Gin Tyr Lys Leu Gly Leu Gin Leu Tyr Leu Gly Ala Thr Ala Leu NO: Leu Val Val Leu Gin Ser Gly Asp Leu Cys Ile Pro Leu Ala Gin Gly Pro Thr Thr Ile Gin Pro Ala Arg Phe Gly His Trp Gly Leu Leu Trp Thr Ser His His Leu Leu Leu Ala Ala Pro Glu Ala Pro Cys Leu Leu Gin Asp Thr Gin Gin Gin Gly pMON3460.Pep Leu Leu Cys Pro His Ser Gly Ile Asp Val Leu Gly Ala Phe Ala Ser Arg His Pro Gin Gin Gly Lys Leu Gly His Ser Gin Gly Leu Ser Pro Ala Asp Met Ala Aia Ser His Leu Leu Ala Ser Phe Asp Gly Cys His Ser Ala Phe Glu Phe Pro Ala Gin Gin Leu Ala Pro Leu Leu Leu Leu Ala Ala Phe Ser Pro Leu Ala Glu Gly Gin Tyr Gly Thr Leu Gin Phe Thr Lys Leu Glu Ile Pro Leu Ala Gin Gly Pro Thr Thr Ile Gin Pro Arg Arg Leu Glu Pro Leu Ser Leu Gin Glu Leu Val Trp Ala Gly Cys Leu Leu Leu Asp Trp Gin Thr Gin Ala Gly Val Ser Gly Pro Glu Gin Lys Leu (SEQ ID Pro Leu Leu Ser Gin Ala Thr Leu Gin Met Gly Ala Gly Val Tyr Arg Ala Ser Val Arg Cys Ala NO: Ser Ser Gin Leu Leu Glu Gin Leu Glu Glu Met Pro Leu Val Val Leu Ser Leu Lys Ile Thr Tyr pMON3461.Pep Gin Leu His Ser Leu Giu Gly Ile Gin Leu Asp Val Gly Leu Ser Pro Ala Asp Phe Leu Tyr Glu Leu Gly Phe Ala Thr Gin Gly Leu Leu Gin Ala Pro Thr Leu Asp Thr Leu Thr Ile Trp Gin Gin Met WO 97/12977 PCT/US96/1 5935 Glu Giu Leu Gly Met Met Pro Ala Phe Ala Leu Val Ala Ser His Val Leu Arg His Leu Ser Leu Pro Gin Ser Lys Ile Gin Gly Asp Thr Tyr Lys Leu Cys Ser Leu Gly Ile Pro Ala Leu Gin Leu Ala Ala Ser Leu Ala Phe Gly His Trp Gly Pro Ala Gin Gin Leu Ala Pro Ala Cys Ala Phe Ser Pro Leu Ala Glu Pro Leu Leu Gin Phe Thr Lys Leu Glu Leu Ser Gin Pro Arg Arg Leu Glu Pro Leu Ser Leu Gin Glu Leu Vai Ser Ser (SEQ ID Thr Gin Ala Gly Val Ser Gly Pro Glu Gin Lys Leu Leu Leu Cys Pro NO: 96) Gly Gly Tyr Ala Va1 Cys Gly Ser Ala Val Arg Ser Arg Ala His Gin 3462.Pep Leu Pro Asp Ala Ser Leu Ala Phe Gly His Trp Gly Phe Glu Phe Pro Ala Gin Gin Leu Ala Pro Ala Cys Leu Leu Ala Ala Phe Ser Pro Leu Ala Glu Pro Leu Tyr Gly Thr Leu Gin Phe Thr Lys Leu Glu Leu Ser Gin Pro Thr Gin Arg Leu Pro Ser Gin Leu Ser Gin Gly Thr Ile Pro Arg Glu Leu Leu Glu Val Ser Leu Leu Leu Trp Thr Ala Val Gly Glu Lys Leu Cys His Leu Asp Gin Gin Gly Ser Pro Gin Leu Leu Pro Ser Gin Thr Gin Gly Gly Tyr Ala Val Cys Gly Ser Gly Ala Leu Leu Gin Met Glu Ala Met Val Leu Arg Val Ser Ser Arg Lys Ala Ti'r His Ser Gin Ala (SEQ ID Glu Gly Leu Asp Glu Leu Pro Ala Val Ala Leu Arg Leu Pro Ile Gin Tyr Lys Leu Gly Leu Gin NO: 97) Ile Ser Val Ala Gly Met Phe Ala Ser His His Leu Gin Ser Gly Asp Leu Cys Ile Pro Leu Ala 3463.Pep Leu Leu Ala Ala Phe Ser Pro Leu Ala Glu Pro Leu Tyr Gin Gly Pro Thr Thr Leu Gin Gin Arg Phe Leu Thr Pro Lys Ser Leu Gin Glu Leu Leu Ser Ser Gin Gly Thr Ile Pro Arg Glu Leu Leu Glu Val Ser Leu Leu Leu Trp Thr Ala Vai Gly Glu Lys Leu Cys His Leu Asp Gin Gin Gly Ser Pro Gin Leu Leu Pro Ser Gin Thr Gin Gly Gly Tyr Ala Val Cys Gly Ser Gly Ala Leu Met Ala Va1 Arg Ser Arg Ala His Gin Leu Leu Gin Glu Met Leu Va1 Ser Lys Thr Ser Ala Phe Glu Gly Leu Asp Glu Leu Pro Ala Val Ala Leu Arg Leu Pro Ile Gin Tyr Lys Leu Gly Leu Gin (SEQ ID Ile Ser Pro Glu Val Ala Asp Phe Gly Met Ala Pro Phe Ala Ser Ala Ser His Leu Gin His Leu Ala Gin Gin Ser Phe Leu Gly Asp Gly Ala Leu Cys His Pro Ile Pro Trp Ala Leu Ala Gly Cys NO: 98) 3464.Pep Ala Leu Glu Gly Ile Leu Gin Leu Asp Val Met Giu GI.i Leu Gly Ala Met Pro Ala Phe Val Leu Val Ala Ser Arg Val Leu Arg His Ser Ser Leu Pro Gin Ser Pro Glu Ala Asp Phe Met Ala Pro Ala Ser Ala His Leu Gin Leu Ala Gin Ser Phe Leu Leu Gly Pro Thr Leu Asp Ala Thr Thr Ile Trp Gin Ala Leu Gin Pro Thr Gin Phe Gin Arg Arg Ala Gly Ser Phe Leu Giu Val Ser Pro Thr Pro Leu Gly Pro Leu Lys Ser Leu Giu Gin Thr Gin Gly Gly Tyr Ala Val WO 97/12977 PCTIUS96/1 5935 Arg Lys Ile Ala Thr Tyr His Ser Leu Gin Ala Leu Leu Phe Leu Gin Lys Gly Gin Tyr Gly Leu Ile Leu Gin Asp Cys Pro Ala Gly Gly His Trp Gly Leu Ala Pro Ala Cys Leu Ala Glu Pro Leu Gin Leu Gin Glu Leu Leu Ser Ser Gin (SEQ ID Glu Lys Leu Cys Val Leu Leu Gly Ser Cys Pro Ser Leu His Ser Gly NO: 99) 3465.Pep Asp Ala Ser Leu Ala Phe Gly His Trp Gly Leu Leu Phe Ala Pro Ala Ala Phe Gin Se 7 Gin Pro Leu Leu Ala Ala Pro Glu Ala Pro Cys Leu Leu Gin Asp Thr Thr Leu Gin Phe Thr Lys Leu Glu Leu Ser Ala Leu Thr Gin Arg Leu Pro Ser Gin Leu Ser Gin Leu Gin Ile Pro Arg Glu Leu Leu Glu Val Ser Leu Glu Leu Trp Thr Ala Val Gly Glu Lys Leu Cys His Gly Asp Gin Gin Gly Ser Pro Gin Leu Leu Pro Ser Ile Val Gin Gly Gly Tyr Ala Val Cys Gly Ser Gly Ser Ala Met Glu Ala Met Val Leu Arg Val Ser Ser Arg Lys Ala Thr His Ser Gin Ala Leu Phe Pro Glu (SEQ ID Glu Leu Pro Ala Val Ala Leu Arg Leu Pro Ile Gin Tyr Lys Leu Gly Leu Gin Leu Tyr Leu Gly NO: 100) Gly Met Phe Ala Ser His His Leu Gin Ser Gly Asp Leu Cys Ile Pro Leu Ala Gin Gly Pro Thr 3466.Pep Ile Trp Pro Thr Arg Ala Glu Val Leu Gly Leu Glu Glu Lys Val Leu Ser Cys Leu His Glu Gly Leu Asp Gin Gin Gly Ser Pro Gin Leu Leu Pro Ser Ile Val Gin Gly Gly Tyr Ala Val Cys Gly Ser Gly Ser Ala Met Ala Val Arg Ser Arg Ala His Gin Leu Pro Asp Glu Glu Met Pro Leu Val Val Leu Ser Leu Lys Ile Thr Tyr Ser Leu Ala Leu Phe Leu Glu Leu Phe Ala Leu Gly Met Ala Pro Ala Phe Ala Ser Ala Ala Ser His Leu Gin Arg His Leu Ala Gin Pro Gin Ser Phe Leu Gin Gly Asp Gly Ala Lys Leu Cys His Pro Gly Ile Pro Trp Ala Gin Leu Ala Gly Cys Tyr Gin Gly Leu Leu Gly Pro Thr Leu Asp Ala Phe Ser Pro Leu Ala Glu Pro Leu Gin Thr Leu Gin Phe Thr Lys Leu Glu Leu Ser Ala Leu Gin Arg Leu Pro Ser Gin Leu Ser Gin Leu Gin Thr Thr (SEQ ID NO:101) 3467.Pep Gin Gin Gin Gly Gly Gly Ser Tyr Pro Ala Gin Val Leu Cys Leu Gly Pro Ser Ser Gly Ile Ser Met Ala Val Arg Ser Arg Ala His Gin Leu Pro Glu Met Leu Val Ser Lys Thr Ser Ala Phe Glu Glu Leu Gly Pro Ala Phe Val Ala Ser Leu Arg His Leu Pro Gin Ile Gin Gly Tyr Lys Leu Leu Gly Ile Leu Gin Leu Leu Tyr Gin Leu Gly Pro Met Ala Pro Ala Ser Ala His Leu Gin Leu Ala Gin Ser Phe Leu Asp Gly Ala Cys His Pro Pro Trp Ala Ala Gly Cys Gly Leu Leu Thr Leu Asp Ala Phe Ser Pro Leu Ala Glu Pro Leu Gin Thr Leu Gin Phe Thr Lys Leu Glu Leu Ser Ala Leu Gin Pro Thr Arg Arg Ala Leu Giu Val Pro Leu Gly Ser Leu Glu Gin Giu Lys Leu Val Leu Ser Ser Cys Gin Leu His Leu Glu Gly Gin Leu Asp WO 97/12977 PCT/US96/15935 49 Val Ala As,) Phe Ala Thr Thr Ile Trp (SEQ ID NO:102) 3499 .Pep Leu Leu Gly Cys Pro Ser His Ser Gly Giy Ile Ser Asp Val Ala Leu Gly Met Ala Phe Ala Ala Ser His Arg His Leu Pro Gln Ser Gin Gly Asp Lys Leu Cys His Gin Leu Pro Asp Ala Ser Leu Ala Phe Gly His Ser Ala Phe Giu Phe Pro Ala Gin Gin Leu Ala Pro Leu Gly Leu Gin Leu Tyr Leu Gly Ala Thr Ala Leu Phe Gin Ser Phe Pro Thr Leu Lys Ala Leu Glu Giu Ile Leu Gin Pro Thr Gin Arg Leu Pro Ser Gin Leu Pro Trp Ala Pro Leu Ser Ala Gly Cys Leu Ser Gin Gly Leu Leu Gin Ala Leu Thr Leu Asp Thr Leu Gin Ile Trp Gin Gin Met Giu Pro Thr Gin Gly Ala Met Arg Ala Gly Giy Vai Leu Glu Val Ser Tyr Arg Val Leu Gly Pro Ala Ser Ser Leu Glu Gin Val Arg Lys Giu Lys Leu Cys Ala Thr Val (SEQ ID NO:103) Ser Leu Giu Leu Giu Pro Val Leu Leu Ile 'Tyr WO 97/12977 PCT/US96/15935 Materials and Methods Recombinant DNA methods Unless noted otherwise, all specialty chemicals were obtained from Sigma Co., (St. Louis, MO). Restriction endonuclea-es and T4 DNA ligase were obtained from New England Biolabs (Beverly, MA) or Boehringer Mannheim (Indianapolis, IN).

Transformation of E. coli strains E. coli strains, such as DH50C (Life Technologies, Gaithersburg, MD) and TG1 (Amersham Corp., Arlington Heights, IL) are used for transformation of ligation reactions and are the source of plasmid DNA for transfecting mammalian cells. E. coli strains, such as MON105 and JM101, can be used for expressing the G-CSF receptor agonist of the present invention in the cytoplasm or periplasmic space.

MON105 ATCC#55204: lamda-,IN(rrnD, rrE)1, rpoD+, rpoH358 phi80dlacZdeltaM15, delta(lacZYA-argF)U169, deoR, recAl, endAl, hsdRl7(rk-,mk+), phoA, supE441amda-, thi-l, gyrA96, relAl TG1: delta(lac-pro), supE, thi-1, hsdD5/F'(traD36, proA+B+, lacIq, DH5Yc T Subcloning efficiency cells are purchased as competent cells and are ready for transformation using the manufacturer's protocol, while both E. coli strains TG1 and MON105 are rendered competent to take up DNA using a CaCl2 method. Typically, 20 to 50 mL of cells -re grown in LB medium Bacto-tryptone, 0.5% Bacto-yeast extract, 150 mM WO 97/12977 PCTIUS96/1 5935 51 NaCI) to a density of approximately 1.0 optical density unit at 600 nanometers (OD600) as measured by a Baush Lomb Spectronic spectrophotometer (Rochester, NY). The cells are collected by centrifugation and resuspended in one-fifth culture volume of CaCl2 solution (50 mM CaCl2, 10 mM Tris- Cl, pH7.4) and are held at 4°C for 30 minutes. The cells are again collected by centrifugation and resuspended in one-tenth culture volume of CaCl2 solution. Ligated DNA is added to 0.2mL of these cells, and the samples are held at 4'C for 1 hour. The samples are shifted to 42'C for two minutes and ImL of LB is added prior to shaking the samples at 37'C for one hour. Cells from these samples are spread on plates ;LB medium plus 1.5% Bacto-agar) containing either ampicillin (100 micrograms/mL, ug/mL) when selecting for ampicillin-resistant transformants, or spectinomycin ug/mL) when selecting for spectinomycin-resistant transformants. The plates are incubated overnight at 37'C.

Single colonies are picked, grown in LB supplemented with appropriate antibiotic for 6-16 hours at 37'C with shaking.

Colonies are picked and inoculated into LB plus appropriate antibiotic (100 ug/mL ampicillin or 75 ug/mL spectinomycin) and are grown at 37 0 C while shaking. Before harvesting the cultures, 1 ul of cells are analyzed by PCR for the presence of a G-CSF gene. The PCR is carried out using a combination of primers that anneal to the G-CSF gene and/or vector. After the PCR is complete, loading dye is added to the sample followed by electrophoresis as described earlier. A gene has been ligated to the vector when a PCR product of the expected size is observed.

Methods for creation of genes with new N-terminus/C-terminus Method I. Creation of genes with new N-terminus/C-terminus which contain a linker region.

WO 97/12977 PCT/US96/15935 52 Genes with new N-terminus/C-terminus which contain a linker region separating the original C-terminus and Nterminus can be made essentially following the method described in L. S. Mullins, et al J. Am. Chem. Soc. 116, 5529-5533 '1994). Multiple steps of polymerase chain reaction (PCR) amplifications are used to rearrange the DNA sequence encoding the primary amino acid sequence of the protein. The steps are illustrated in Figure 2.

In the first step, the primer set ("new start" and "linker start") is used to create and amplify, from the original gene sequence, the DNA fragment ("Fragment Start") that contains the sequence encoding the new N-terminal portion of the new protein followed by the linker that connects the C-terminal and N-terminal ends of the original protein. In the second step, the primer set ("new stop" and "linker stop") is used to create and amplify, from the original gene sequence, the DNA fragment ("Fragment Stop") that encodes the same linker as used above, followed by the new C-terminal portion of the new protein. The "new start" and "new stop" primers are designed to include the appropriate restriction enzyme recognition sites which allow cloning of the new gene into expression plasmids. Typical PCR conditions are one cycle 95 0 C melting for two minutes; 25 cycles 94 0 C denaturation for one minute, 50 0 C annealing for one minute and 72 0 C extension for one minute; plus one cycle 72 0 C extension for seven minutes. A Perkin Elmer GeneAmp PCR Core Reagents kit is used. A 100 ul reaction contains 100 pmole of each primer and one ug of template DNA; and lx PCR buffer, 200 uM dGTP, 200 uM dATP, 200 uM dTTP, 200 uM dCTP, 2.5 units AmpliTaq DNA polymerase and 2 mM MgCl 2 PCR reactions are performed in a Model 480 DNA thermal cycler (Perkin Elmer Corporation, Norwalk, CT).

WO 97/12977 PCTIUS96/1 5935 53 "Fragment Start" and "Fragment Stop", which have complementary sequence in the linker region and the coding sequence for the two amino acids on both sides of the linker, are joined together in a third PCR step to make the full-length gene encoding the new protein. The DNA fragments "Fragment Start" and "Fragment Stop" are resolved on a 1% TAE gel, stained with ethidium bromide and isolated using a Qiaex Gel Extraction kit (Qiagen). These fragments are combined in equimolar quantities, heated at 700C for ten minutes and slow cooled to allow annealing through their shared sequence in "linker start" and "linker stop". In the third PCR step, primers "new start" and "new stop" are added to the annealed fragments to create and amplify the fulllength new N-terminus/C-terminus gene. Typical PCR conditions are one cycle 95 0 C melting for two minutes; cycles 94C denaturation for one minute, 600C annealing for one minute and 720C extension for one minute; plus one cycle 720C extension for seven minutes. A Perkin Elmer GeneAmp PCR Core Reagents kit is used. A 100 ul reaction contains 100 pmole of each primer and approximately 0.5 ug of DNA; and Ix PCR buffer, 200 uM dGTP, 200 uM dATP, 200 uM dTTP, 200 uM dCTP, 2.5 units AmpliTaq DNA polymerase and 2 mM MgC12. PCR reactions are purified using a Wizard PCR Preps kit (Promega).

Method II. Creation of genes with new N-terminus/C-terminus without a linker region.

New N-terminus/C-terminus genes without a linker joining the original N-terminus and C-terminus can be made using two steps of PCR amplification and a blunt end ligation. The steps are illustrated in Figure 3. In the first step, the primer set ("new start" and "P-bl start") is used to create and amplify, from the original gene sequence, the DNA fragment ("Fragment Start") that contains the WO 97/12977 PCTIUS96/15935 54 sequence encoding the new N-terminal portion of the new protein. In the second step, the primer set ("new stop" and "P-bl stop") is used to create and amplify, from the original gene sequence, the DNA fragment ("Fragment Stop") that contains the sequence encoding the new C-terminal portion of the new protein. The "new start" and "new stop" primers are designed to include appropriate restriction sites which allow cloning of the new gene into expression vectors. Typical PCR conditions are one cycle 95 0 C melting for two minutes; 25 cycles 94 0 C denaturation for one minute, 0 C annealing for 45 seconds and 72 0 C extension for seconds. Deep Vent polymerase (New England Biolabs) is used to reduce the occurrence of overhangs in conditions recommended by the manufacturer. The "P-bl start" and "P-bl stop" primers are phosphorylated at the 5' end to aid in the subsequent blunt end ligation of "Fragment Start" and "Fragment Stop" to each other. A 100 ul ieaction contained 150 pmole of each primer and one ug of template DNA; and Ix Vent buffer (New England Biolabs), 300 uM dGTP, 300 uM dATP, 300 uM dTTP, 300 uM dCTP, and 1 unit Deep Vent polymerase.

PCR reactions are performed in a Model 480 DNA thermal cycler (Perkin Elmer Corporation, Norwalk, CT). PCR reaction products are purified using a Wizard PCR Preps kit (Promega).

The primers are designed to include appropriate restriction enzyme recognition sites which allow for the cloning of the new gene into expression vectors. Typically "Fragment Start" is designed to create a NcoI restriction site and "Fragment Stop" is designed to create a HindIII restriction site. Restriction digest reactions are purified using a Magic DNA Clean-up System kit (Promega). Fragments Start and Stop are resolved on a 1% TAE gel, stained with ethidium bromide and isolated using a Qiaex Gel Extraction kit (Qiagen). These fragments are combined with and WO 97/12977 PCT/US96/15935 annealed to the ends of the 3800 base pair NcoI/HindIII vector fragment of pMON3934 by heating at 50 0 C for ten minutes and allowed to slow cool. The three fragments are ligated together using T4 DNA ligase (Boehringer Mannheim).

The result is a plasmid containing the full-length new Nterminus/C-terminus gene. A portion of the ligation reaction is used to transform E. coli strain DH5acells (Life Technologies, Gaithersburg, MD). Plasmid DNA is purified and sequence confirmed as below.

Method III. Creation of new N-terminus/C-terminus genes by tandem-duplication method New N-terminus/C-terminus genes can be made based on the method described in R. A. Horlick, et al Protein Eng.

5:427-431 (1992). Polymerase chain reaction (PCR) amplification of the new N-terminus/C-terminus genes is performed using a tandemly duplicated template DNA. The steps are illustrated in Figure 4.

The tandemly-duplicated template DNA is created by cloning and contains two copies of the gene separated by DNA sequence encoding a linker connecting the original C- and Nterminal ends of the two copies of the gene. Specific primer sets are used to create and amplify a full-length new N terminus/C-terminus gene from the tandemly-duplicated template DNA. These primers are designed to include appropriate restriction sites which allow for the cloning of the new gene into expression vectors. Typical PCR conditions are one cycle 95 0 C melting for two minutes; 25 cycles 94°C denaturation for one minute, 50 0 C annealing for one minute and 72 0 C extension for one minute; plus one cycle 72°C extension for seven minutes. A Perkin Elmer GeneAmp PCR Core Reagents kit (Perkin Elmer Corporation, Norwalk, CT) is used. A 100 ul reaction contains 100 pmole of each primer WO 97/12977 PCT/US96/15935 56 and one ug of template DNA; and Ix PCR buffer, 200 uM dGTP, 200 uM dATP, 200 uM dTTP, 200 uM dCTP, 2.5 units AmpliTaq DNA polymerase and 2 mM MgCl 2 PCR reactions are performed in a Model 480 DNA thermal cycler (Perkin Elmer Corporation, Norwalk, CT). PCR reactions are purified using a Wizard PCR Preps kit (Promega).

DNA isolation and characterization Plasmid DNA can be isolated by a number of different methods and using commercially available kits known to those skilled in the art. A few such methods are shown herein.

Plasmid DNA is isolated using the Promega Wizard T Miniprep kit (Madison, WI), the Qiagen QIAwell Plasmid isolation kits (Chatsworth, CA) or Qiagen Plasmid Midi kit. These kits follow the same general procedure for plasmid DNA isolation.

Briefly, c..lls are pelleted by centrifugation (5000 x g), plasmid DNA released with sequential NaOH/acid treatment, and cellular debris is removed by centrifugation (10000 x The supernatant (containing the plasmid DNA) is loaded onto a column containing a DNA-binding resin, the column is washed, and plasmid DNA eluted with TE. After screening for the colonies with the plasmid of interest, the E. coli cells are inoculated into 50-100 mLs of LB plus appropriate antibiotic for overnight growth at 37 0 C in an air incubator while shaking. The purified plasmid DNA is used for DNA sequencing, further restriction enzyme digestion, additional subcloning of DNA fragments and transfection into mammalian, E. coli or other cells.

Seauence confirmation.

Purified plasmid DNA is resuspended in dH20 and quantitated by measuring the absorbance at 260/280 nm in a Bausch and Lomb Spectronic 601 UV spectrometer. DNA samples are sequenced using ABI PRISM T M DyeDeoxy

T

terminator WO 97/12977 PCT/US96/15935 57 sequencing chemistry (Applied Biosystems Division of Perkin Elmer Corporation, Lincoln City, CA) kits (Part Number 401388 or 402078) according to the manufacturers suggested protocol usually modified by the addition of 5% DMSO to the sequencing mixture. Sequencing reactions are performed in a Model 480 DNA thermal cycler (Perkin Elmer Corporation, Norwalk, CT) following the recommended amplification conditions. Samples are purified to remove excess dye terminators with Centri-Sep T spin columns (Princeton Separations, Adelphia, NJ) and lyophilized. Fluorescent dye labeled sequencing reactions are resuspended in deionized formamide, and sequenced on denaturing 4.75% polyacrylamide- 8M urea gels using an ABI Model 373A automated DNA sequencer. Overlapping DNA sequence fragments are analyzed and assembled into master DNA contigs using Sequencher v2.1 DNA analysis software (Gene Codes Corporation, Ann Arbor,

MI).

Expression of G-CSF receptor aaonists in mammalian cells Mammalian Cell Transfection/Production of Conditioned Media The BHK-21 cell line can be obtained from the ATCC (Rockville, MD). The cells are cultured in Dulbecco's modified Eagle media (DMEM/high-glucose), supplemented to 2mM (mM) L-glutamine and 10% fetal bovine serum (FBS). This formulation is designated BHK growth media. Selective media is BHK growth media supplemented with 453 units/mL hygromycin B (Calbiochem, San Diego, CA). The BHK-21 cell line was previously stably transfected with the HSV transactivating protein VP16, which transactivates the IE110 promoter found on the plasmid pMON3359 (See Hippenmeyer et al., Bio/Technology, pp.1037-1041, 1993). The VP16 protein drives expression of genes inserted behind the IE110 promoter. BHK-21 cells expressing the transactivating WO 97/12977 PCT/US96/15935 58 protein VP'6 are designated BHK-VP16. The plasmid pMON1118 (See Highkin et al., Poultry Sci., 70: 970-981, 1991) expresses the hygromycin resistance gene from the promoter. A similar plasmid is available from ATCC, pSV2hph.

BHK-VP16 cells are seeded into a 60 millimeter (mm) tissue culture dish at 3 X 105 cells per dish 24 hours prior to transfection. Cells are transfected for 16 hours in 3 mL of "OPTIMEM"T (Gibco-BRL, Gaithersburg, MD) containing ug of plasmid DNA containing the gene of interest, 3 ug hygromycin resistance plasmid, pMON1118, and 80 ug of Gibco- BRL "LIPOFECTAMINE"T per dish. The media is subsequently aspirated and replaced with 3 mL of growth media. At 48 hours post-transfection, media from each dish is collected and assayed for activity (transient conditioned media). The cells are removed from the dish by trypsin-EDTA, diluted 1:10 and transferred to 100 mm tissue culture dishes containing 10 mL of selective media. After approximately 7 days in selective media, resistant cells grow into colonies several millimeters in diameter. The colonies are removed from the dish with filter paper (cut to approximately the same size as the colonies and soaked in trypsin/EDTA) and transferred to individual wells of a 24 well plate containing 1 mL of selective media. After the clones are grown to confluence, the conditioned media is re-assayed, and positive clones are expanded into growth media.

Expression of G-CSF receptor agonists in E. coli E. coli strain MON105 or JM101 harboring the plasmid of interest are grown at 37 0 C in M9 plus casamino acids medium with shaking in a air incubator Model G25 from New Brunswick Scientific (Edison, New Jersey). Growth is monitored at OD600 until it reaches a value of 1, at which time nalidixic acid (10 milligrams/mL) in 0.1 N NaOH is added to a final WO 97/12977 PCTIUS96/1 5935 59 concentration of 50 |lg/mL. The cultures are then shaken at 37 0 C for three to four additional hours. A high degree of aeration is maintained throughout culture period in order to achieve maximal production of the desired gene product. The cells are examined under a light microsccpe for the presence of inclusion bodies One mL aliquots of the culture are removed for analysis of protein content by boiling the pelleted cells, treating them with reducing buffer and electrophoresis via SDS-PAGE (see Maniatis et al. Molecular Cloning: A Laboratory Manual, 1982). The culture is centrifuged (5000 x g) to pellet the cells.

Inclusion Body preparation. Extraction. Refolding, Dialysis, DEAE Chromatoaraphv. and Characterization of the G-CSF receptor aaonists which accumulate as inclusion bodies in E coli.

Isolation of Inclusion Bodies: The cell pellet from a 330 mL E. coll culture is resuspended in 15 mL of sonication buffer (10 mM 2-amino-2- (hydroxymethyl) 1,3-propanediol hydrochloride (Tris-HCl), pH 1 mM ethylenediaminetetraacetic acid (EDTA)). These resuspended cells are sonicated using the microtip probe of a Sonicator Cell Disrupter (Model W-375, Heat Systems- Ultrasonics, Inc., Farmingdale, New York). Three rounds of sonication in sonication buffer followed by centrifugation are employed to disrupt the cells and wash the inclusion bodies The first round of sonication is a 3 minute burst followed by a 1 minute burst, and the final two rounds of sonication are for 1 minute each.

Extraction and refolding of proteins from inclusion body pellets: WO 97/12977 PCT/US96/15935 Foll-owing the final centrifugation step, the IB pellet is resuspended in 10 mL of 50 mM Tris-HC1, pH 9.5, 8 M urea and 5 mM dithiothreitol (DTT) and stirred at room temperature for approximately 45 minutes to allow for denaturation of the expressed protein.

The extraction solution is transferred to a beaker containing 70 mL of 5mM Tris-HC1, pH 9.5 and 2.3 M urea and gently stirred while exposed to air at 40C for 18 to 48 hours to allow the proteins to refold. Refolding is monitored by analysis on a Vydac (Hesperia, Ca.) C18 reversed phase high pressure liquid chromatography (RP-HPLC) column (0.46x25 cm). A linear gradient of 40% to acetonitrile, containing 0.1% trifluoroacetic acid (TFA), is employed to monitor the refold. This gradient is developed over 30 minutes at a flow rate of 1.5 mL per minute.

Denatured proteins generally elute later in the gradient than the refolded proteins.

Purification: Following the refold, contaminating E. coli proteins are removed by acid precipitation. The pH of the refold solution is titrated to between pH 5.0 and pH 5.2 using acetic acid (HOAc). This solution is stirred at for 2 hours and then centrifuged for 20 minutes at 12,000 x g to pellet any insoluble protein.

The supernatant from the acid precipitation step is dialyzed using a Spectra/Por 3 membrane with a molecular weight cut off (MWCO) of 3,500 daltons. The dialysis is against 2 changes of 4 liters (a 50-fold excess) of Tris-HC1, oH 8.0 for a total of 18 hours. Dialysis lowers the sample conductivity and removes urea prior to DEAE chromatography. The sample is then centrifuged (20 minutes at 12,000 x g) to pellet any insoluble protein following dialysis.

WO 97/12977 PCT/US96/15935 61 A Bio-Rad Bio-Scale DEAE2 column (7 x 52 mm) is used for ion exchange chromatography. The column is equilibrated in a buffer containing 10mM Tris-HCl, pH 8.0. The protein is eluted using a 0-to-500 mM sodium chloride (NaCI) gradient, in equilibration buffer, over 45 column volumes. A flow rate of 1 mL per minute is used throughout the run. Column fractions (2 mL per fraction) are collected across the gradient and analyzed by RP HPLC on a Vydac (Hesperia, Ca.) C18 column (0.46 x 25 cm). A linear gradient of 40% to acetonitrile, containing 0.1% trifluoroacetic acid (TFA), is employed. This gradient is developed over 30 minutes at a flow rate of 1.5 mL per minute. Pooled fractions are then dialyzed against 2 changes of 4 liters (50-to-500-fold excess) of 10 mM ammonium acetate (NH 4 Ac), pH 4.0 for a total of 18 hours. Dialysis is performed using a Spectra/Por 3 membrane with a MWCO of 3,500 daltons.

Finally, the sample is sterile filtered using a 0.22pm syringe filter (lStar LB syringe filter, Costar, Cambridge, and stored at 4 0

C.

In some cases the folded proteins can be affinity purified using affinity reagents such as mAbs or receptor subunits attached to a suitable matrix. Alternatively, (or in addition) purification can be accomplished using any of a variety of chromatographic methods such as: ion exchange, gel filtration or hydrophobic chromatography or reversed phase HPLC.

These and other protein purification methods are described in detail in Methods in Enzymology, Volume 182 'Guide to Protein Purification' edited by Murray Deutscher, Academic Press, San Diego, CA (1990).

Protein Characterization:

I',

62 The purified protein is analyzed by RP-HPLC, electrospray mass spectrometry, and SDS-PAGE. The protein quantitation is done by amino acid composition, RP-HPLC, and Bradford protein determination. In some cases tryptic peptide mapping is performed in conjunction with electrospray mass spectrometry to confirm the identity of the protein.

AML Proliferation Assay The factor-dependent cell line AML 193 was obtained from the American Type Culture Collection (ATCC, 10801 University Blvd, Manassas, VA, 20110-2209. This cell line, established from a patient with acute myelogenous leukemia, is a growth factor dependent cell line which displayed enhanced growth in GM-CSF supplemented medium (Lange, et al., Blood 70: 192, 1987; Valtieri, et al., J. Immunol. 138:4042, 1987). The ability of AML 193 cells to proliferate in the presence of human IL-3 has also been documented. (Santoli, et al., J. Immunol.

139: 348, 1987). A cell line variant was used, AML 193 1.3, which was adapted for long term growth in IL-3 by washing o: out the growth factors and starving the cytokine dependent AML 193 cells for growth factors for 24 hours. The cells are then replated at 1x10 5 cells/well in a 24 well plate in media containing 100 U/mL IL-3. It took approximately 2 months for the cells to grow rapidly in IL-3. These cells are maintained as -AML 193 1.3 thereafter by supplementing tissue culture medium (see below) with human IL-3.

AML 193 1.3 cells are washed 6 times in cold Hanks balanced salt solution (HBSS, Gibco, Grand Island, NY) by centrifuging cell suspensions at 250 x g for 10 minutes followed by decantation of the supernatant. Pelleted cells are resuspended in HBSS and the procedure is repeated until six wash cycles are completed. Cells washed six times by t'S 'tis procedure are resuspended in tissue culture medium at a degnsity ranging from 2 x 105 to 5 x 105 viable cells/mL.

WO 97/12977 PCT/US96/15935 63 This medium is prepared by supplementing Iscove's modified Dulbecco's Medium (IMDM, Hazelton, Lenexa, KS) with albumin, transferrin, lipids and 2-mercaptoethanol. Bovine albumin (Boehringer-Mannheim, Indianapolis, IN) is added at 500 ptg/mL; human transferrin (Boehringer-Mannheim, Indianapolis, IN) is added at 100 pg/mL; soybean lipid (Boehringer- Mannheim, Indianapolis, IN) is added at 50 pg/mL; and 2mercaptoethanol (Sigma, St. Louis, MO) is added at 5 x 10-

M.

Serial dilutions of G-CSF receptor agonist proteins are made in triplicate series in tissue culture medium supplemented as stated above in 96 well Costar 3596 tissue culture plates. Each well contained 50 .l of medium containing G-CSF receptor agonist proteins once serial dilutions are completed. Control wells contained tissue culture medium alone (negative control). AML 193 1.3 cell suspensions prepared as above are added to each well by pipetting 50 .l (2.5 x 104 cells) into each well. Tissue culture plates are incubated at 37 0 C with 5% C02 in humidified air for 3 days. On day 3, 0.5 (pCi 3 H-thymidine (2 Ci/mM, New England Nuclear, Boston, MA) is added in 50 .1 of tissue culture medium. Cultures are incubated at 37 0

C

with 5% C02 in humidified air for 18-24 hours. Cellular DNA is harvested onto glass filter mats (Pharmacia LKB, Gaithersburg, MD) using a TOMTEC cell harvester (TOMTEC, Orange, CT) which utilized a water wash cycle followed by a ethanol wash cycle. Filter mats are allowed to air dry and then placed into sample bags to which scintillation fluid (Scintiverse II, Fisher Scientific, St. Louis, MO or BetaPlate Scintillation Fluid, Pharmacia LKB, Gaithersburg, MD) is added. Beta emissions of samples from individual tissue culture wells are counted in a LKB BetaPlate model 1205 scintillation counter (Pharmacia LKB, Gaithersburg, MD) and data is expressed as counts per minute of 3 H-thymidine incorporated into cells from each tissue culture well.

WO 97/12977 PCT/US96/15935 64 Activity of each G-CSF receptor agonist proteins preparation is quantitated by measuring cell proliferation 3 H-thymidine incorporation) induced by graded concentrations of G-CSF receptor agonist. Typically, concentration ranges from 0.05 pM 105 pM are quantitated in these assays. Activity is determined by measuring the dose of G-CSF receptor agonist protein which provides 50% of maximal proliferation (EC50 x (maximum average counts per minute of 3 H-thymidine incorporated per well among triplicate cultures of all concentrations of G-CSF receptor agonists tested background proliferation measured by 3 H-thymidine incorporation observed in triplicate cultures lacking any factor). This EC50 value is also equivalent to 1 unit of bioactivity. Every assay is performed with native interleukin-3 and G-CSF as reference standards so that relative activity levels could be assigned.

Typically, the G-CSF receptor agonist proteins were tested in a concentration range of 2000 pM to 0.06 pM titrated in serial 2 fold dilutions.

Activity for each sample was determined by the concentration which gave 50% of the maximal response by fitting a four-parameter logistic model to the data. It was observed that the upper plateau (maximal response) for the sample and the standard with which it was compared did not differ. Therefore relative potency calculation for each sample was determined from EC50 estimations for the sample and the standard as indicated above.

Other in vitro cell based proliferation assays Other in vitro cell based proliferation assays, known to those skilled in the art, may also be useful to determine the activity of the G-CSF receptor agonists in a similar manner as described in the AML 193.1.3 cell proliferation assay.

WO 97/12977 PCTIUS96/1 5935 Transfected cell lines: Cell lines, such as BHK or the murine pro B cell line Baf/3, can be transfected with a colony stimulating factor receptor, uch as the human G-CSF receptor which the cell line does not have. These transfected cell lines can be used to determine the activity of the ligand of which the receptor has been transfected.

EXAMPLE 1 Construction of DMON3485 The new N-terminus/C-terminus gene in pMON3485 was created using Method I as described in Materials and Methods. Fragment Start was created and amplified from G- CSF Ser 17 sequence in pMON13037 using the primer set, 39 start (SEQ ID NO:7) and L-ll start (SEQ ID NO:3). Fragment Stop was created and amplified from G-CSF Ser 17 sequence in the plasmid, pMON13037 (WO 95/21254), using the primer set, 38 stop (SEQ ID NO:8) and L-ll stop (SEQ ID NO:4). The full-length new N terminus/C-terminus G-CSF Ser 17 gene was created and amplified from the annealed Fragments Start and Stop using the primers 39 start (SEQ ID NO:7) and 38 stop (SEQ ID NO:8).

The resulting DNA fragment which contains the new gene was digested with restriction endonucleases NcoI and HindIII and purified using a Magic DNA Clean-up System kit (Promega, Madison, WI). The plasmid, pMON3934 (derivative of pMON3359), was digested with restriction endonucleases HindIII and NcoI, resulting in an approximately 3800 base pair vector fragment, and gel-purified. The purified restriction fragments were combined and ligated using T4 DNA ligase. A portion of the ligation reaction was used to WO 97/12977 PCT/US96/15935 66 transform E. coli strain DH5a cells (Life Technologies, Gaithersburg, MD). Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert. The resulting plasmid was designated pMON3485.

BHK cells were transfected with the plasmid, pMON3485, for protein expression and bioassay.

The plasmid, pMON3485 containing the gene sequence of (SEQ ID NO:25), Tyr Lys Leu Cys Leu Gly Ile Pro Leu Gin Leu Ala Leu Tyr Gln Gly Leu Gly Pro Thr Ala Thr Thr Ile Ala Leu Gin Pro Phe Gin Arg Arg Ser Phe Leu Glu Pro Ser Gly Gly Leu Glu Gin Val Glu Lys Leu Cys encodes the following amino acid sequence: His Trp Gly Leu Leu Trp Thr Ala Val Ser Arg Ala Pro Ala Cys Leu Asp Gin Gin Gly Ser Gly Lys Thr Glu Glu Pro Leu Leu Ser Gin Ala Thr Leu Gin Met Gly Ala Gly Val Tyr Arg Gly Ser Ile Gin (SEQ ID Leu Val Leu Ser Ser Cys Gin Leu His Leu Glu Gly Gin Leu Asp Glu Glu Leu Met Pro Ala Leu Val Ala Val Leu Arg Gin Ser Phe Gly Asp Gly NO:43) Leu Gly His Ser Pro Ser Gin Ala Ser Gly Leu Phe Ile Ser Pro Glu Val Ala Asp Phe Gly Met Ala Pro Phe Ala Ser Ala Ser His Leu Gin His Leu Ala Gin Leu Leu Lys Ser Ala Ala Leu Gin EXAMPLE 2 Construction of DMON3486 The new N-terminus/C-terminus gene in pMON3486 was created using Method I as described in Materials and Methods. Fragment Start was created and amplified from G- CSF Ser 17 sequence in the plasmid, pMON13037, using the primer set, 97 start (SEQ ID NO:9) and L-ll start (SEQ ID NO:3). Fragment Stop was created and amplified from G-CSF Ser 17 sequence in pMON13037 using the primer set, 96 stop WO 97/12977 PCT/US96/15935 67 (SEQ ID NO:10) and L-ll stop (SEQ ID NO:4). The full-length new N terminus/C-terminus G-CSF Ser 17 gene was created and amplified from the annealed Fragments Start and Stop using the primers 97 start (SEQ ID NO:9) and 96 stop (SEQ ID The resulting DNA fragment which contains the new gene was digested with restriction endonucleases NcoI and HindIII and gel-purified using a Magic DNA Clean-up System kit. The plasmid, pMON3934, was digested with restriction endonucleases HindIII and NcoI, resulting in an approximately 3800 base pair vector fragment, and gelpurified. The purified restriction fragments were combined and ligated using T4 DNA ligase. A portion of the ligation reaction was used to transform E. coli strain DH5a cells.

Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert. The resulting plasmid was designated pMON3486.

BHK cells were transfected with the plasmid, pMON3486, for protein expression and bioassay.

The plasmid, pMON3486 containing the gene sequence of (SEQ ID NO:26), encodes the following amino acid sequence: Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gin Gin Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gin Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gin Pro Ser Gly Gly Ser Gly Gly Ser Gin Ser Phe Leu Leu Lys Ser Leu Glu Gin Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gin Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gin Ala Leu Gin Leu Ala Gly Cys Leu WO 97/12977 PCT/US96/15935 68 Ser Gin Leu His Ser Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly Ile Ser (SEQ ID NO:44) EXAMPLE 3 Construction of pMON3487 The new N-terminus/C-terminus gene in pMON3487 was created using Method I as described in Materials and Methods. Fragment Start was created and amplified from G- CSF Ser 17 sequence in the plasmid, pMON13037, using the primer set, 126 start (SEQ ID NO:11) and L-ll start (SEQ ID NO:3). Fragment Stop was created and amplified from G-CSF Ser 17 sequence in pMON13037 using the primer set, 125 stop (SEQ ID NO:12) and L-11 stop (SEQ ID NO:4). The full-length new N terminus/C-terminus G-CSF Ser 1 7 gene was created and amplified from the annealed Fragments Start and Stop using the primers 126 start (SEQ ID NO:11) and 125 stop (SEQ ID NO:12).

The resulting DNA fragment which contains the new gene was digested with restriction endonucleases NcoI and HindIII and purified using a Magic DNA Clean-up System kit. The plasmid, pMON3934, was digested with restriction endonucleases HindIII and NcoI, resulting in an approximately 3800 base pair vector fragment, and gelpurified. The purified restriction fragments were combined and ligated using T4 DNA ligase. A portion of the ligation reaction was used to transform E. coli strain DH5a cells.

Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert. The resulting plasmid was designated pMON3487.

BHK cells were transfected with the plasmid, pMON3487, for protein expression and bioassay.

WO 97/12977 PCTIUS96/1 5935 69 The plasmid, pMON3487 containing the gene sequence of (SEQ ID NO:27), encodes the following amino acid sequence: Met Ala Ala Ser His Leu Leu Ala Leu Lys Ala Leu Glu Glu Pro Leu Leu Ser Gln Ala Thr Leu Pro Ala Gin Gin Ser Gin Leu Ser Gin Leu Gin Ala Phe Ser Pro Leu Glu Val Ser Leu Glu Leu Leu Gin Phe Ser Glu Lys Leu Cys His Gly Asp Gin Arg Leu Gly Gin Leu Leu Pro Ser Ile Val Pro Thr Arg Ala Glu Val Gly Ser Val Arg Cys Ala Gly His Ser Gin Gly Leu Ser Pro Ala Asp (SEQ ID Gin Gly Gly Gly Ser Tyr Gly Gly Lys Ile Thr Tyr Ser Leu Ala Leu Phe Leu Glu Leu Phe Ala Ala Val Arg Ser Gin Lys Gly Gin Tyr Gly Thr Met Pro Leu Val Val Leu Gin Ser Gly Asp Leu Cys Ile Pro Leu Ala Gin Gly Pro Thr Thr Ile Ala Phe Ala Ser Arg His Phe Leu Gly Ala His Pro Trp Ala Gly Cys Leu Leu Leu Asp Trp Gin Gin Met Glu Glu Leu Gly EXAMPLE 4 Construction of PMON3488 The new N-terminus/C-terminus gene in pMON3488 was created using Method I as described in Materials and Methods. Fragment Start was created and amplified from G- CSF Ser 17 sequence in the plasmid, pMON13037, using the primer set, 133 start (SEQ ID NO:13) and L-ll start (SEQ ID NO:3). Fragment Stop was created and amplified from G-CSF Ser 17 sequence in the plasmid, pMON13037 using the primer set, 132 stop (SEQ ID NO:14) and L-ll stop (SEQ ID NO:4).

The full-length new N terminus/C-terminus G-CSF Ser 17 gene was created and amplified from the annealed Fragments Start and Stop using the primers 133 start (SEQ ID NO:13) and 132 stop (SEQ ID NO:14).

The resulting DNA fragment which contains the new gene was digested with restriction endonucleases NcoI and HindIII and purified using a Magic DNA Clean-up System kit. The WO 97/12977 PCTIUS96/15935 plasmid, pMON3934, was digested with restriction endonucleases HindIII and NcoI, resulting in an approximately 3800 base pair vector fragment, and gelpurified. The purified restriction fragments were combined and ligated using T4 DNA ligase. A portion of the ligation reaction was used to transform E. coli strain Transformaznt bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert. The resulting plasmid was designated pMON3488.

BHK cells were transfected with the plasmid, pMON3488, for protein expression and bioassay.

The plasmid, pMON3488 containing the gene sequence of (SEQ ID NO:28), encodes the following amino acid sequence: Thr Ala Val Ser Arg Ala His Gln Leu Pro Asp Gin Gly Ser Gly Lys Thr Ser Ala Phe Glu Phe Gly Ala Gly Val Tyr Arg Gly Ser Ile Gln Tyr Lys Leu Gly Leu Gln Leu Tyr Leu Gly Ala Thr Met Leu Val Gin Gly Leu Ile Leu Gin Pro Thr Pro Val Leu Ser Asp Cys Pro Ala Gly Thr Ile Ala Phe Ala Ser Arg His Phe Leu Gly Ala His Pro Trp Ala Gly Cys Leu Leu Leu Asp Trp Gin Ala His Leu Leu Ala Glu Pro Leu Gin Thr Gin Ser Leu Ala Lys Leu Glu Leu Ser Ala Leu Met Ala Phe Gin Gin Ser Phe Gin Pro Ser Ser Leu Glu Gin Glu Lys Leu Val Leu Ser Ser Cys Gin Leu His Leu Glu Gly Gin Leu Asp Glu Glu Leu Arg Leu Gly Gin Leu Leu Pro Ser Ile Val Gly Arg Glu Gly Val Cys Gly Ser Gly Ser Ala Met Ala Pro Ala Leu Gin Pro (SEQ ID NO:46) EXAMPLE Construction of PMON3489 The new N-terminus/C-terminus gene in pMON3489 was created using Method I as described in Materials and WO 97/12977 PCT/US96/15935 71 Methods. Fragment Start was created and amplified from G- CSF Ser 17 sequence in the plasmid, pMON13037, using the primer set, 142 start (SEQ ID NO:15) and L-ll start (SEQ ID NO:3). Fragment Stop was created and amplified from G-CSF Ser 17 sequence in pMON13037 using the primer set, 141 stop (SEQ ID NO:16) and L-11 stop (SEQ ID NO:4). The full-length new N terminus/C-terminus G-CSF Ser 17 gene was created and amplified from the annealed Fragments Start and Stop using the primers 142 start (SEQ ID NO:15) and 141 stop (SEQ ID NO:16).

The resulting DNA fragment which contains the new gene was digested with restriction endonucleases NcoI and HindIII and purified using a Magic DNA Clean-up System kit. The plasmid, pMON3934, was digested with restriction endonucleases HindIII and NcoI, resulting in an approximately 3800 base pair vector fragment, and gelpurified. The purified restriction fragments were combined and ligated using T4 DNA ligase. A portion of the ligation reaction was used to transform E. coli strain Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert. The resulting plasmid was designated pMON3489.

BHK cells were transfected with the plasmid, pMON3489, for protein expression and bioassay.

The plasmid, pMON3489 containing the gene sequence of (SEQ ID NO:29), encodes the following amino acid sequence: Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro Ser Gly Gly Ser Gly Gly Ser Gln Ser Phe Leu Leu Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro WO 97/12977 PCT/US96/1 5935 72 Leu Ser Ser Cys Pro Ser Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His Ser Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Gl,1 Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gin Gin Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala (SEQ ID NO:47) EXAMPLE 6 Construction of DMON3490 The new N-terminus/C-terminus gene in pMON3490 was created using Method II as described in Materials and Methods. Fragment Start was created and amplified from G- CSF sequence in the plasmid, pMON13037, using the primer set, 39 start (SEQ ID NO:7) and P-bl start (SEQ ID Fragment Stop was created and amplified from G-CSF Ser 17 sequence in pMON13037 using the primer set, 38 stop (SEQ ID NO:8) and P-bl stop (SEQ ID NO:6). Fragment Start was digested with restriction endonuclease NcoI, and Fragment Stop was digested with restriction endonuclease HindIII.

After purification, the digested Fragments Start and Stop were combined with and ligated to the approximately 3800 base pair NcoI-HindIII vector fragment of pMON3934.

Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert. The resulting plasmid was designated pMON3490.

BHK cells were transfected with the plasmid, pMON3490, for protein expression and bioassay.

The plasmid, pMON3490 containing the gene sequence of (SEQ ID NO:30), encodes the following amino acid sequence: WO 97/12977 PCT/US96/15935 73 Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His Ser Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gin Gin Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gin Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gin Pro Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gin Ser Phe Leu Leu Lys Ser Leu Glu Gin Val Arg Lys Ile Gin Gly Asp Gly Ala Ala Leu Gin Glu Lys Leu Cys Ala Thr (SEQ ID NO:48) EXAMPLE 7 Construction of DMON3491 The new N-terminus/C-terminus gene in pMON3491 was created using Method II as described in Materials and Methods. Fragment Start was created and amplified from G- CSF sequence in the plasmid, pMON13037, using the primer set, 97 start (SEQ ID NO:9) and P-bl start (SEQ ID Fragment Stop was created and amplified from G-CSF Ser 17 sequence in pMON13037 using the primer set, 96 stop (SEQ ID NO:10) and P-bl stop (SEQ ID NO:6). Fragment Start was digested with restriction endonuclease NcoI, and Fragment Stop was digested with restriction endonuclease HindIII.

After purification, the digested Fragments Start and Stop were combined with and ligated to the approximately 3800 base pair NcoI-HindIII vector fragment of pMON3934. A portion of the ligation reaction was used to transform E.

coli strain DH5a cells. Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert. The resulting plasmid was designated pMON3491.

WO 97/12977 PCT/US96/15935 74 BHK cells were transfected with the plasmid, pMON3491, for protein expression and bioassay.

The.plasmid, pMON3491 containing the gene sequence of (SEQ ID NO:31), encodes the following amino acid sequence: Pro Asp Ala Ser Leu Ala Phe Gly His Trp Gly Leu Glu Phe Pro Ala Gin Gin Leu Ala Pro Ala Cys Leu Leu Gly Ala Thr Ala Leu Phe Gin Ser Phe Pro Thr Leu Lys Ala Leu Glu Glu Pro Leu Le-. Ser Gin Ala Pro Thr Gin Arg Leu Pro Ser Gin Leu Ser Gin Leu Thr Ile Pro Arg Glu Leu Leu Glu Val Ser Leu Glu Leu Trp Thr Ala Val Gly Glu Lys Leu Cys His Gly Asp Thr Gin Gin Gin Gly Gly Gly Ser Tyr Pro Ala Gin Val Leu Cys Leu Gly Pro Ser Ser Gly Ile Ser Leu Gin Met Glu Ala Met Val Leu Arg Val Ser Ser Arg Lys Ala Thr His Ser Gin Ala Leu Phe (SEQ ID Leu Asp Glu Leu Pro Ala Val Ala Leu Arg Leu Pro Ile Gin Tyr Lys Leu Gly Leu Gin Leu Tyr NO:49) Val Ala Gly Met Phe Ala Ser His His Leu Gin Ser Gly Asp Leu Cys Ile Pro Leu Ala Gin Gly EXAMPLE 8 Construction of pMON3492 The new N-terminus/C-terminus gene in pMON3492 was created using Method II as described in Materials and Methods. Fragment Start was created and amplified from G- CSF sequence in the plasmid, pMON13037, using the primer set, 126 start (SEQ ID NO:11) and P-bl start (SEQ ID Fragment Stop was created and amplified from G-CSF Ser 17 sequence in pMON13037 using the primer set, 125 stop (SEQ ID NO:12) and P-bl stop (SEQ ID NO:6). Fragment Start was digested w-th restriction endonuclease NcoI, and Fragment Stop was digested with restriction endonuclease HindIII.

After purification, the digested Fragments Start and Stop were combined with and ligated to the approximately 3800 WO 97/12977 PCTIUS96/1 5935 base pair NcoI-HindIII vector fragment of pMON3934. A portion of the ligation reaction was used to transform E.

coli strain DH5a cells. Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert. The resulting plasmid was designated pMON3492.

BHK cells were transfected with the plasmid, pMON3492, for protein expression and bioassay.

.0 The plasmid, pMON3492 containing the gene sequence of (SEQ ID NO:32), encodes the following amino acid sequence: Met Ala His Leu Ser Asp Cys Pro Ala Gly Thr Ala Ser Leu Ala Phe Gly His Trp Gly Leu Leu Pro Ala Ala Phe Gin Ser Gin Pro Leu Leu Ala Ala Pro Glu Ala Pro Cys Leu Leu Gin Asp Thr Leu Gln Phe Thr Lys Leu Glu Leu Ser Ala Leu Gin Arg Leu Pro Ser Gin Leu Ser Gin Leu Gin Pro Thr Arg Ala Glu Val Leu Gly Leu Glu Glu Lys Val Leu Ser Cys Leu His Glu Gly Leu Asp Gin Gly Ser Pro Gin Leu Leu Pro Ser Ile Val Gly Ala Gly Val Tyr Arg Ala Ser Val Arg Cys Ala Gly His Ser Gin Gly Leu Ser Pro Ala Asp (SEQ ID Met Pro Ala Phe Leu Val Ala Ser Val Leu Arg His Ser Leu Pro Gin Lys Ile Gin Gly Thr Tyr Lys Leu Ser Leu Gly Ile Ala Leu Gin Leu Phe Leu Tyr Gin Glu Leu Gly Pro Phe Ala Thr Thr Ile Trp Gin Gin Met Glu Glu Leu Gly EXAMPLE 9 Construction of PMON3493 The new N-terminus/C-terminus gene in pMON3493 was created using Method II as described in Materials and Methods. Fragment Start was created and amplified from G- CSF sequence in the plasmid, pMON13037, using the primer set, 133 start (SEQ ID NO:13) and P-bl start (SEQ ID WO 97/12977 PCT/US96/15935 76 Fragment Stop was created and amplified from G-CSF Ser 17 sequence in pMON13037 using the primer set, 132 stop (SEQ ID NO:14) and P-bl stop (SEQ ID NO:6). Fragment Start was digested with restriction endonuclease NcoI, and Fragment Stop was digested with restriction endonuclease HindIII.

After purification, the digested Fragments Start and Stop were combined with and ligated to the approximately 3800 base pair NcoI-HindIII vector fragment of pMON3934. A portion of the ligation reaction was used to transform E.

coli strain DH5a cells. Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert. The resulting plasmid was designated pMON3493.

BHK cells were transfected with the plasmid, pMON3493, for prote-i expression and bioassay.

The plasmid, pMON3493 containing the gene sequence of (SEQ ID NO:33), encodes the following amino acid sequence: Thr Ala Val Gly Glu Lys Leu Cys His Gly Asp Gin Gly Ser Pro Gin Leu Leu Pro Ser Ile Val Gly Gly Tyr Ala Val Cys Gly Ser Gly Ser Ala Ala Met Val Leu Arg Val Ser Ser Arg Lys Ala Thr His Ser Gin Ala Leu Phe Pro Glu Asp Phe Pro Ala Val Ala Leu Arg Leu Pro Ile Gin Tyr Lys Leu Gly Leu Gin Leu Tyr Leu Gly Ala Thr Phe Ala Ser Ser His Leu His Leu Ala Gin Ser Phe Gly Asp Gly Leu Cys His Ile Pro Trp Leu Ala Gly Gin Gly Leu Pro Thr Leu Thr Ile Trp Ala Gin Gin Leu Ala Pro Ala Cys Leu Asp Gin Phe Ser Pro Leu Ala Glu Pro Leu Gin Thr Gin Gin Arg Phe Leu Thr Pro Lys Ser Leu Gin Glu Leu Leu Ser Ser Gin Ala Leu Leu Gin Met Glu Arg Glu Leu Leu Glu Val Ser Leu Glu Leu Glu Leu Gly Met Ala Pro Ala Leu Gin Pro (SEQ ID NO:51) EXAMPLE Construction of DMON3494 WO 97/12977 PCTIUS96/1 5935 77 The new N-terminus/C-terminus gene in pMON3494 was created using Method II as described in Materials and Methods. Fragment Start was created and amplified from G- CSF sequence in the plasmid, pMON13037, using the primer set, 142 start (SEQ ID NO:15) and P-bl start (SEQ ID Fragment Stop was created and amplified from G-CSF Ser 17 sequence i, pMON13037 using the primer set, 141 stop (SEQ ID NO:16) and P-bl stop (SEQ ID NO:6). Fragment Start was digested with restriction endonuclease NcoI, and Fragment Stop was digested with restriction endonuclease HindIII.

After purification, the digested Fragments Start and Stop were combined with and ligated to the approximately 3800 base pair NcoI-HindIII vector fragment of pMON3934. A portion of the ligation reaction was used to transform E.

coli strain DH5a cells. Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the correct insert. The resulting plasmid was designated pMON3494.

BHK cells were transfected with the plasmid, pMON3494, for protein expression and bioassay.

The piasmid, pMON3494 containing the gene sequence of (SEQ ID NO:34), encodes the following amino acid sequence: Ser Leu Ala Phe Gly His Trp Gly Leu Leu Ala Gin Gin Leu Ala Pro Ala Cys Leu Asp Phe Gin Ser Phe Pro Thr Leu Lys Ala Leu Glu Glu Pro Leu Leu Ser Gin Ala Thr Leu Arg Arg Leu Glu Pro Leu Ser Leu Gin Glu Leu Val Ser Ser Gin Leu Leu Glu Gin Leu Ala Val Gly Glu Lys Leu Cys His Gly Asp Gly Gly Ser Tyr Pro Ala Gin Val Leu Cys Leu Gly Pro Ser Ser Gly Ile Ser Val Ala Val Arg Ser Arg Ala His Gin Leu Pro Asp Leu Val Ser Lys Thr Ser Ala Phe Glu Phe Val Ala Leu Arg Leu Pro Ile Gin Tyr Lys Leu Gly Leu Gin Leu Tyr Leu Gly Ala Thr Ser His His Leu Gin Ser Gly Asp Leu Cys Ile Pro Leu Ala Gin Gly Pro Thr Thr Ile WO 97/12977 PCT/US96/15935 78 Trp Gin Gin Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala (SEQ ID NO:52) Examples 11-20 The genes encoding the G-CSF receptor agonists of Examples 1-10 were excised from the BHK vectors as a NcoI/HindIII fragment and ligated with the 3630 base pair NcoI/HindIII vector fragment of pMON2341 (WO 94/12638). The resulting plasmids (Examples 11-20) are indicated in Table 4. The plasmids were transformed into E. coli strain JM101 cells and expression of the G-CSF receptor agonist protein was evaluated. The proteins expressed are the same as those expressed in the parental BHK expression vector except the proteins were immediately preceded by a Methionine-Alanine dipeptide and the Methionine is processed off by methionine aminopeptidase. Overnight growths of cells (20 Klett units) were inoculated in 10mL of minimal M9 medium supplemented with vitamin B1 and trace minerals and incubated with shaking at 37 0 C until initial Klett readings of -120 units were obtained. At 120 Klett units 50uL of 10mg/mL nalidixic acid was added. Four hours post-induction, a 1ml aliquot was removed for protein expression analysis by SDS-PAGE. Cells were also examined using light microscopy for the presence of inclusion bodies. Only pMON3450 and pMON3455 had significant expression levels of the G-CSF receptor agonist protein. In an effort to improve expression levels of G-CSF receptor agonists, the 5' end of the genes were reengineered to incorporate AT-rich codons and E. coli preferred codons between the unique NcoI and NheI restriction endonuclease recognition sites (Examples 21-28).

WO 97/12977 PCT/US96/15935 79 TABLE 4 E. coli expression plasmids Example Example Example Example Example Example Example Example Example Example Example Resulting E. coli expression plasmid pMON# pMON3450 pMON3455 pMON3451 pMON3456 pMON3452 pMON3457 pMON3453 pMON3458 pMON3454 pMON3459 Breakpoint 38/39 38/39 96/97 96/97 125/126 125/126 132/133 132/133 141/142 141/142 Parental Linker BHK plasmid pMON# zero Al-10 zero Al-10 zero Al-10 zero Al-10 zero Al-10 pMON3490 pMON3485 pMON3491 pMON3486 pMON3492 pMON3487 pMON3493 pMON3488 pMON3494 pMON3489 Example 21 Construction of PMON25184 The complementary pair of synthetic oligomers, 141for.seq (SEQ ID NO:23) and 141rev.seq (SEQ ID NO:24), (Midland Certified Reagent Co., Midland TX) were annealed by heating 2ug of each synthetic oligomer in a 20ul reaction mixture containing 20mM Tris-HC1 10mM MgCl 2 and NaC1, at 80 0 C for 5 minutes, and allowing the mixture to slowly cool to ambient temperature (approximately minutes). When properly annealed the oligomers create an NcoI site at the 5' end and a NheI site at the 3' end.

Approximately 15 ng of the annealed oligomer pair was ligated with the gel-purified 4120 base pair NcoI/NheI WO 97/12977 PCTIUS96/15935 vector fragment of pMON3454 (-molar ratio of 10:1). The resulting gene, had seven codon changes at the 5' end of the gene. The ligation reaction was used to transform E. coli strain DH5a and the desired codon changes were confirmed by DNA sequence analysis. The resulting plasmid was designated pMON25184. Plasmid, pMON25184 containing the gene sequence of (SEQ ID NO:38), DNA was retransformed into E. coli strain JM101 cells for protein expression. The protein expressed is the same as that expressed from pMON3454.

Example 22 Construction of pMON25188 The complementary pair of synthetic oligomers, 141for.seq (SEQ ID NO:23) and 141rev.seq (SEQ ID NO:24), (Midland Certified Reagent Co., Midland TX) were annealed by heating 2ug of each synthetic oligomer in a 20ul reaction mixture containing 20mM Tris-HCl 10mM MgC12 and 50mM NaCl, at 80 0 C for 5 minutes, and allowing the mixture to slowly cool to ambient temperature (approximately minutes). When properly annealed the oligomers create an NcoI site at the 5' end and a NheI site at the 3' end.

Approximately 15ng of the annealed oligomer pair was ligated with the 4110 base pair NcoI/NheI gel-purified pMON3459 (-molar ratio of 10:1). The ligation mixture was used to transform E. coli strain DH5a and the desired codon changes were confirmed by DNA sequence analysis. The resulting plasmid was designated pMON25188. The resulting gene, had seven codon changes at the 5' end of the gene. Plasmid, pMON25188 containing the gene sequence of (SEQ ID NO:42), DNA was retransformed into E. coli strain JM101 cells for protein expression. The protein expressed is the same as that expressed from pMON3459.

WO 97/12977 PCT/US96/15935 81 Example 23 Construction of DMON25183 pMON25183 was constructed using an overlapping PCR primer method. The synthetic oligomers, 132for.seq (SEQ ID NO:321 and 132rev.seq (SEQ ID NO:22), encode the NcoI and NheI restriction recognition sequence, respectively.

Amplified DNA was generated by the DNA polymerase chain amplification method using the PCR Optimizer Kit (Invitrogen). The PCR reactions were performed using the manufacturer's recommended conditions using 5X buffer B (300mM Tris-HCl pH8.5, 75 mM (NH4)2S0 4 10mM MgCl 2 for seven cycles consisting of 94C for 65 0 C for and 72 0 C for followed by 20 cycles of 94 0 C for and 72 0

C

for and a final cycle of 7 minutes at 72 0 C using a Perkin Elmer Model 480 DNA thermal cycler (Perkin Elmer).

The reaction product was desalted using Centri-Sep spin columns (Princeton Separations) following the manufacturer's recommended protocol, digested with NcoI/NheI, and gel purified from TAE-agarose gels using Gene Clean (Bio 101) and the DNA product was eluted in dH 2 0 The purified PCR product was ligated with the 4090 base pair NcoI/NheI pMON3453 vector fragment. Positive clones containing the AT-rich replacement insert were identified as described in Example 21. The resulting plasmid was designated pMON25183.

The resulting gene, had 14 codon changes at the 5' end of the gene. Plasmid, pMON25183 containing the gene sequence of (SEQ ID NO:37), DNA was retransformed into E. coli strain JM101 cells for protein expression. The protein expressed is the same as that expressed from pMON3453.

Example 24 Construction of pMON25187 WO 97/12977 PCT/US96/15935 82 pMON25187 was constructed using an overlapping PCR primer method. The synthetic oligomers, 132for.seq (SEQ ID NO:21) and 132rev.seq (SEQ ID NO:22), encode the NcoI and NheI restriction recognition sequence, respectively.

Amplified DNA was generated by the DNA polymerase chain amplification method using the PCR Optimizer Kit (Invitrogen). The PCR reactions were performed using the manufacturer's recommended conditions, in 5X buffer B for seven cycles consisting of 94 0 C for 65 0 C for and 72 0 C for followed by 20 cycles of 94 0 C for and 72 0

C

for and a final cycle of 7 minutes at 72 0 C using a Perkin Elmer Model 480 DNA thermal cycler (Perkin Elmer).

The reaction product was desalted using Centri-Sep spin columns (P-inceton Separations) following the manufacturer's recommended protocol, digested with NcoI/NheI, and gel purified from TAE-agarose gels using Gene Clean (Bio 101) and the DNA product was eluted in dH 2 0. The purified PCR product was ligated with the 4080 base pair NcoI/NheI pMON3458 vector fragment. Positive clones containing the AT-rich replacement insert were identified as described in Example 21. The resulting plasmid was designated pMON25187.

The resulting gene, had 14 codon changes at the 5' end of the gene. Plasmid, pMON25187 containing the gene sequence of (SEQ ID NO:41), DNA was retransformed into E. coli strain JM101 cells for protein expression. The protein expressed is the same as that expressed from pMON3458.

Example Construction of pMON25182 pMON25182 was constructed using the overlapping PCR primer approach described in Example 23. The synthetic oligomer primers 125for.seq (SEQ ID NO:19) and 125rev.seq WO 97/12977 PCTIUS96/1 5935 83 (SEQ ID NO:20) were used in the PCR reaction. The PCR reaction conditions were identical to those used in Example 23 except the annealing temperature for the first seven cycles was 60 0 C. The purified PCR product was ligated with 4070 base pair NcoI/NheI pMON3452 vector fragment.

Positive clones containing the AT-rich replacement insert were identified as described in Example 21. The resulting plasmid was designated pMON25182. The resulting gene, had 19 codon changes at the 5' end of the gene. Plasmid, pMON25182 containing the gene sequence of (SEQ ID NO:36), DNA was retransformed into E. coli strain JM101 cells for protein expression. The protein expressed is the same as that expressed from pMON3452.

Example 26 Construction of MON25186 pMON25186 was constructed using the overlapping PCR primer approach described in Example 23. The synthetic oligomer primers 125for.seq (SEQ ID NO:19) and 125rev.seq (SEQ ID NO:20) were used in the PCR reaction. The PCR reaction conditions were identical to those used in Example 23 except the annealing temperature for the first seven cycles was 60 0 C. The purified PCR product was ligated with the 4060 base pair NcoI/NheI pMON3457 vector fragment.

Positive clones containing the AT-rich replacement insert were identified as described in Example 21. The resulting plasmid was designated pMON25186. The resulting gene, had 19 codon changes at the 5' end of the gene. Plasmid, pMON25186 containing the gene sequence of (SEQ ID DNA was retransformed into E. coli strain JM101 cells for protein expression. The protein expressed is the same as that expressed from pMON3457.

WO 97/12977 PCT/US96/15935 84 Examples 27 Construction of pMON25181 pMON25181 was constructed using PCR to amplify a DNA fragment from pMON3451 as the template using the oligomers 96for.seq (SEQ ID NO:17) and 96rev.seq (SEQ ID NO:18). The oligomer 96for.seq was designed to create six codon changes.

The PCR reaction conditions were the same as described in Example 25, except 10ng of pMON3451 plasmid DNA was added.

The purified PCR product was ligated with the 3980 base pair NcoI/NheI pMON3451 vector fragment. Positive clones containing the AT-rich replacement insert were identified as described in Example 21. The resulting plasmid was designated pMON25181. The resulting gene, had 6 codon changes at the 5' end of the gene. Plasmid, pMON25181 containing the gene sequence of (SEQ ID NO:35), DNA was retransformed into E. coli strain JM101 cells for protein expression. The protein expressed is the same as that expressed from pMON3451.

Examples 28 Construction of pMON25185 pMON25185 was constructed using PCR to amplify a DNA fragment from pMON3451 as the template using the oligomers 96for.seq (SEQ ID NO:17) and 96rev.seq (SEQ ID NO:18). The oligomer 9697for.seq was designed to create six codon changes. The PCR reaction conditions were the same as described in Example 25, except 10ng of pMON3456 plasmid DNA was added. The purified PCR product was ligated with the 3970 base pair NcoI/NheI pMON3456 vector fragment. Positive clones containing the AT-rich replacement insert were identified as described in Example 21. The resulting WO 97/12977 PCT/US96/15935 plasmid was designated pMON25185. The resulting gene, had 6 codon changes at the 5' end of the gene. Plasmid, pMON25185 containing the gene sequence of (SEQ ID NO:39), DNA was retransformed into E. coli strain JM101 cells for protein expression. The protein expressed is the same as that expressed from pMON3456.

EXAMPLE 29 The G-CSF amino acid substitution variants of the present invention were made using PCR mutagenesis techniques as described in WO 94/12639 and WO 94/12638. These and other variants amino acid substitutions, insertions or deletions and N-terminal or C-terminal extensions) could also be made, by one skilled in the art, using a variety of other methods including synthetic gene assembly or sitedirected mutagenesis (see Taylor et al., Nucl. Acids Res., 13:7864-8785, 1985; Kunkel et al., Proc. Natl. Acad. Sci.

USA, 82:488-492, 1985; Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, WO 94/12639 and WO 94/12638). These substitutions can be made one at a time or in combination with other amino acid substitutions, and/or deletions, and/or insertions and/or extensions. After sequence verification of the changes, the plasmid DNA can be transfected into an appropriate mammalian cell, insect cell or bacterial strain such as E. coli for production. Known variants of G-CSF, which are active, include substitutions at positions 1 (Thr to Ser, Arg or Gly, 2 (Pro to Leu), 3 (Leu to Arg or Ser) and 17 (Cys to Ser) and deletions of amino acids 1-11 (Kuga et al. Biochemicla and Biophysical Research Comm. 159:103-111, 1989). It is understood that these G-CSF amino acid substitution variants could serve as the template sequence for the rearrangement of the amino acid sequence as described in the other examples.

WO 97/12977 PCT/US96/15935 86 Bioactivity determination of G-CSF amino acid substitution variants.

The G-CSF amino acid substitution variants were assayed in the Baf/3 cell line, transfected with the human G-CSF receptor, proliferation assay to determine their bioactivity relative to native G-CSF. The G-CSF variants tested and their relative bioactivity are shown in Table 5. A indicates that the activity was comparable to native G-CSF and indicates that the activity was significantly decreased or not detected.

WO 97/12977 WO 9712977PCTIUS96/15935 CELL PROLIFERATION ACTIVTTY OF Q-CSF ARIANT-q INT BAF/-1 QFIL I LINE TRANSFECTED WITH THE HUMAN G-CSF RECEPTOR -aa positicr native aa mutant aa activity Phe Phe Phe Phe Lys Lys Ly s Lys Leu Leu Leu Leu Leu Glu Giu Giu Giu Glu Giu Giu Arg Arg Arg Arg Arg Ile Ile Ile Ser His Thr Pro Pro Ser Thr His Pro Thr His Cys Ile Ala Thr Arg Pro Leu Gly Ser Tyr Ser Ala Val Thr Pro *Leu Tvr WO 97/12977 WO 9712977PCTIUS96/1 5935 TABLE 5 cont.

aa position native aa mutant aa activity* Asp Ala Ala Lys His His His His His His His His His Pro Pro Pro Pro Pro Pro Pro Pro Pro Giu Glu Giu Giu Leu Leu Leu Leu Gly Ile Leu Ser G ly Thr Val1 Lys Trp Ala Arg Cys Leu Arg Asp Vai Ala His Gin Trp Gly Thr Ala Arg Phe Ile Thr Phe Arg Ser WO 97/12977 WO 9712977PCT[US96/15935 TABLE 5 cont.

aa position native aa mutant aa activity s0 51 51 54 67 67 67 67 104 104 108 108 108 108 108 108 115 115 115 115 120 120 120 120 Leu Leu Gly Gly Leu Gin Gin Gin Gin Gin Gin Gin Gin Asp Asp Leu Leu Leu Leu Leu Leu Thr Thr Thr Thr Gin Gin Gin Gin His Pro Ser Met His Lys Leu Cys Lys Pro Leu Arg Ser Gly Val1 Ala Val1 Arg Gly Trp Gin His Leu Ala Ile G ly Arg Lys His WO 97/12977PCIS/155 PCT[US96/15935 TABLE 5 cont.

I aa position native aa mutant aa act ivitv 123 123 123 144 144 144 144 144 146 147 156 156 156 159 159 159 159 159 162 162 162 163 163 163 165 169 169 169 170 170 Glu Glu Glu Phe Phe Phe Phe Phe Arg Arg His His His Ser Ser Ser Ser Ser Glu Glu Glu Val1 Val1 Val1 Tyr Ser Ser S er His His Arg Phe Thr His Arg Pro Leu Glu Gin Gin Asp Ser Gly Arg Thr Tyr Val1 Gly Gly Trp Leu Arg Ala Gly Cys Leu Cys Arg Arg Ser not determined WO 97/12977 PCT/US96/15935 EXAMLPLE 30-37 Examples 30-37 were made in a similar manner as described in Example 6 using the plasmid pMON13037 as the template and the oligonucleotide primers indicated in Table 6. The resulting gene and the designated plasmid pMON and the protein encoded are indicated in Table 6.

WO 97/12977 WO 9712977PCT[US96/15935 92 TABLE 6 Example breakpoint primers resulting gene resulting protein 48/49 49start (SEQ ID NO:68) pMON3460 (SEQ ID 48stop (SEQ ID NO:86) (SEQ ID NO:69) 31 76/77 77start (SEQ ID NO:70) pMON3461 (SEQ ID NO:96) 76stop (SEQ ID NO:87) (SEQ ID NO:71) 32 81/82 82start (SEQ ID N0:72) pMON3462 (SEQ ID NO:97) 81stop (SEQ ID NO:88) (SEQ ID NO:73) 33 83/84 84start (SEQ ID NO:74) pMON3463 (SEQ ID NO:98) 83stop (SEQ ID NO:88) (SEQ ID 34 90/91 91start (SEQ ID NO:76) pMON3464 (SEQ ID NO:99) (SEQ ID N0:89) (SEQ ID NO:77) 111/112 112start pMON3465 (SEQ ID NO:78) (SEQ ID NO:90) (SEQ ID NO:100) ills top (SEQ ID NO:79) 36 116/117 ll7start (SEQ ID NO:80) pMON3466 (SEQ ID NO:l0l) ll6stop (SEQ ID N0:91) (SEQ ID N0:81) 37 118/119 ll9start (SEQ ID NO:82) pMON3467 (SEQ ID NO:102) 118stop (SEQ ID NO:92) (SEQ ID NO:83) The G-CSF receptor agonist genes in pMON364O, pMON3461, pMON3462, pMON3463, pMON3464, pMON3465, pMON3466 and pMON3467 were transferred to an E. copu expression vector, pMON2341, as an NcoI/HindIII restriction fragment, resulting WO 97/12977 PCT/US96/15935 93 in the plasmids pMON3468, pMON3469, pMON3470, pMON3471, pMON3472, pMON3473, pMON3474 and pMON3498 respectively.

EXAMPLE 38 The plasmid, pMON3468, resulted in low expression levels in E. coli of the desired G-CSF receptor agonist. The end of the gene was redesigned to use codon selection that was AT rich to increase expression levels. The oligonucleotides, Z4849AT.for (SEQ ID NO:84) and Z4849AT.rev (SEQ ID NO:85), were used to re-engineer the gene. The resulting plasmid, pMON3499, containing the gene (SEQ ID NO:94) encodes the G-CSF receptor agonist of (SEQ ID NO:103).

EXAMPLE 39 The G-CSF receptor agonists were assayed in the Baf/3 cell line, transfected with the human G-CSF receptor, (Baf/3-G-CSF) proliferation assay to determine their bioactivity relative to native G-CSF. The activity of the receptor agonists is shown in Table 7.

WO 97/12977 WO 9712977PCTIUS96/15935 TABLE 7 G-CSF receptor agonist activity in Baf proliferation assay /3-G-CSF cell pMON# breakpoint Expression E. coli EC50 (pM) refold native G-cSF 60 pM pMON25182 125/126 38 pM pMON25183 132/133 58 pM pMON25184 141/142 70 pM pMON25186 125/126 92 pM pMON25187 132/133 83 pM pMON25188 141/142 41 pM pMON3450 38/39 '121 pM pMON3455 38/39 102 pM pMON3499 48/49 137 pM pMON3470 81/82 no activity pMON3473 111/112 Additional techniques for the construction of the variant genes, recombinant protein expression protein purification, protein characterization, biological activity determination can be found in WO 94/12639, WO 94/12638, WO 95/20976, WO 95/21197, WO 95/20977, WO 95/21254 which are hereby incorporated by reference in their entirety.

All references, patents or applications cited herein are incorporated by reference in their entirety as if written herein.

Various other examples will be apparent to the person skilled in the art after reading the present disclosure without departing from the spirit and scope of the invention. It is intended that all such other examples be included within the scope of the appended claims.

20

C

25 With reference to the use of the word(s) "comprise" or "comprises" or "comprising" in the foregoing description and/or in the following claims, we note that unless the context requires otherwise, those words are used on the basis and clear understanding that they are to be interpreted inclusively, rather than exclusively, and that we intend each of those words to be so interpreted in construing the foregoing description and/or the following claims.

SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: ZURFLUH, LINDA L MCWHERTER, CHARLES A MCKEARN, JOHN P KLEIN, BARBARA K FENG, YIQING BRAFORD-GOLDBERG, SARAH R LEE, STEPHEN C (ii) TITLE OF INVENTION: G-CSF RECEPTOR AGONISTS (iii) NUMBER OF SEQUENCES: 129 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: S. CHRISTOPHER BAUER, MONSANTO/G. D. SEARLE CO., PATENT DEPARTMENT CENTRAL STREET: P.O. BOX 5110 CITY: CHICAGO STATE: ILLINOIS COUNTRY: USA ZIP: 60680 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: AU 73900/96 FILING DATE: 04-OCT-1996 i CLASSIFICATION: (vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: WO PCT/US 96/15935 FILING DATE: 04-OCT-1996 O:oo a:* S*(vii) PRIOR APPLICATION

DATA:

APPLICATION NUMBER: US 60/004,382 FILING DATE: 05-OCT-1995 a (viii) ATTORNEY/AGENT INFORMATION: NAME: BAUER, S. CHRISTOPHER REFERENCE/DOCKET NUMBER: 2907AU o* a a (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: 636-737-6257 TELEFAX: 636-737-5452 00 a* INFORMATION FOR SEQ ID NO:l: SEQUENCE CHARACTERISTICS: LENGTH: 174 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 1 [R OTHER INFORMATION: /note= "Xaa at position 1 is Thr, Ser, 9Arg, Tyr or Gly;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 2 OTHER INFORMATION: /note= Leu;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 3 OTHER INFORMATION: /note= Arg, Tyr or Ser;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 13 OTHER INFORMATION: /note= Ser, His, Thr or Pro;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 16 OTHER INFORMATION: /note= Pro, Ser, thr or His;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 17 OTHER INFORMATION: /note= Ser, Gly, Ala, Ile, Tyr or Arg;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 18 OTHER INFORMATION: /note= Thr, Pro, His, Ile or Cys;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 22 OTHER INFORMATION: /note= Tyr, Ser, Thr or Ala;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 24 OTHER INFORMATION: /note= Pro, Tyr or Leu;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 27 OTHER INFORMATION: /note= or Gly;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION: OTHER INFORMATION: /note= Ile, Leu or Gly;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 34 OTHER INFORMATION: /note= or Ser;" "Xaa at position 2 is Pro or "Xaa at position 3 is Leu, "Xaa at position 13 is Phe, "Xaa at position 16 is Lys, "Xaa at position 17 is Cys, "Xaa at position 18 is Leu, "Xaa at position 22 is Arg, "Xaa at position 24 is Ile, "Xaa at position 27 is Asp, "Xaa at position 30 is Ala, "Xaa at position 34 is Lys

C

C

Ce

C

C

C

C

C

C

C

C

(ix) FEATURE: NAME/KEY: Modified-site LOCATION: 36 OTHER INFORMATION: /note= Ser;" "Xaa at position 36 is Cys (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 42 OTHER INFORMATION: /note= "Xaa at position 42 is Cys or Ser;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 43 OTHER INFORMATION: /note= Thr, Gly, Val, Lys, Trp, Ala, Arg, Cys (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 44 OTHER INFORMATION: /note= Gly, Arg, Asp, Val, Ala, His, Trp, Gin (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 46 OTHER INFORMATION: /note= Arg, Phe, Arg, Ile or Ala;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 47 OTHER INFORMATION: /note= or Thr;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 49 OTHER INFORMATION: /note= Phe, Arg or Ser;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION: OTHER INFORMATION: /note= Ile, His, Pro or Tyr;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 54 OTHER INFORMATION: /note= or His;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 64 OTHER INFORMATION: /note= or Ser;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 67 OTHER INFORMATION: /note= Lys, Leu or Cys;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION: OTHER INFORMATION: /note= Pro, Leu, Arg or Ser;" "Xaa at position 43 is His, or Leu;" "Xaa at position 44 is Pro, or Thr;" "Xaa at position 46 is Glu, "Xaa at position 47 is Leu "Xaa at position 49 is Leu, "Xaa at position 50 is Leu, "Xaa at position 54 is Leu "Xaa at position 64 is Cys "Xaa at position 67 is Gin, "Xaa at position 70 is Gin, fees 0 6 0 o 6*@e *6.0 6 00.9 0..

of66 6 9 6 6 6 6 6 6 6 (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 74 OTHER INFORMATION: /note= Ser;" "Xaa at position 74 is Cys (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 104 OTHER INFORMATION: /note= "Xaa at position 104 is Asp, Gly or Val;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 108 OTHER INFORMATION: /note= "Xaa at position 108 is Leu, Ala, Val, Arg, Trp, Gin or Gly;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 115 OTHER INFORMATION: /note= "Xaa at position 115 is Thr, His, Leu or'Ala;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 120 OTHER INFORMATION: /note= "Xaa at position 120 is Gin, Gly, Arg, Lys or His" (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 123 OTHER INFORMATION: /note= "Xaa at position 123 is Glu, Arg, Phe or Thr" (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 144 OTHER INFORMATION: /note= "Xaa at position 144 is Phe, His, Arg, Pro, Leu, Gin or Glu;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 146 OTHER INFORMATION: /note= "Xaa at position 146 is Arg or Gin;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 147 OTHER INFORMATION: /note= "Xaa at position 147 is Arg or Gin;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 156 OTHER INFORMATION: /note= "Xaa at position 156 is His, Gly or Ser;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 159 OTHER INFORMATION: /note= "Xaa at position 159 is Ser, Arg, Thr, Tyr, Val or Gly;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 162 OTHER INFORMATION: /note= "Xaa at position 162 is Glu, Leu, Gly or Trp;" t 0 0000 0 0, 60 0.

S

40 0 0

S

4, *0 0 *r 0 (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 163 OTHER INFORMATION: /note= y, Arg or Ala;" "Xaa at position 163 is Val, 100 (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 169 OTHER INFORMATION: /note= "Xaa at position 169 is Arg, Ser, Leu, Arg or Cys;" (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 170 OTHER INFORMATION: /note= "Xaa at position 170 is His, Arg or Ser;" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: Xaa Xaa Xaa Gly Pro Ala Ser Ser Leu Xaa Glu Xaa Pro Gly Pro Phe Ala Gln 145 Xaa Lys Xaa Thr Tyr Lys Xaa Gly Ile Xaa Leu Ala 70 Gin Gly Leu Thr Leu Xaa Trp Gin Xaa 120 Gin Gly Ala 135 Gly Val Leu 150 Arg Val Leu Pro Gin Gly Xaa Xaa Xaa Trp Ala Xaa Leu Gin Ala Leu Gin Glu Xaa Pro Ala Ala Ser 155 Xaa Leu 170 Ser Gly Xaa Pro Ser Leu Xaa Leu Phe 140 Xaa Ala Xaa Ala Glu Leu Gin Glu Asp Gly 125 Ala Leu Gin 0 o* 0 00 0 0.0.

0 0S 0 Leu Xaa Xaa Ser INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 4 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (2) (P '1 0 a-- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Gly Gly Gly Ser 1 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 54 base pairs TYPE: nucleic acid STRANDEDNESS: single 101 TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GCTCTGAGAG CCGCCAGAGC CGCCAGAGGG CTGCGCAAGG TGGCGTAGAA CGCG 54 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 54 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: CAGCCCTCTG GCGGCTCTGG CGGCTCTCAG AGCTTCCTGC TCAAGTCTTT AGAG 54 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID GGGCTGCGCA AGGTGGCG 18 S(2) INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: ACACCATTGG GCCCTGCCAG C 21 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs SC(B) TYPE: nucleic acid STRANDEDNESS: single 102 TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: GATCGACCAT GGCTTACAAG CTGTGCCACC CC 32 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: CGATCGAAGC TTATTAGGTG GCACACAGCT TCTCCT 36 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid S(C) STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid S DESCRIPTION: /desc "DNA (synthetic)" *o 0* (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: GATCGACCAT GGCTCCCGAG TTGGGTCCCA CC 32 o, INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear o (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID CGATCGAAGC TTATTAGGAT ATCCCTTCCA GGGCCT 36 INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single 103 TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: GATCGACCAT GGCTATGGCC CCTGCCCTGC AG 32 INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: CGATCGAAGC TTATTATCCC AGTTCTTCCA TCTGCT 36 INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: GATCGACCAT GGCTACCCAG GGTGCCATGC CG 32 INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: CGATCGAAGC TTATTAGGGC TGCAGGGCAG GGGCCA 36 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid S STRANDEDNESS: single 104 TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID GATCGACCAT GGCTTCTGCT TTCCAGCGCC GG 32 INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: CGATCGAAGC TTATTAGGCG AAGGCCGGCA TGGCAC 36 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: ATATCCATGG CTCCGGAACT GGGTCCAACT CTG 33 INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: ACCTCCAGGA AGCTCTGCAG ATGG 24 INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 65 base pairs TYPE: nucleic acid S(C) STRANDEDNESS: single 105 TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: TATATCCATG GCTATGGCTC CAGCTCTGCA ACCAACTCAA GGTGCAATGC CAGCATTTGC ATCTG INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 63 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID GATGGCTAGC AACCAGAACA CCACCTGCAC GACGTTGAAA AGCAGATGCA AATGCTGGCA TTG 63 INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: o LENGTH: 57 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" o* (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: TATATCCATG GCTACTCAAG GTGCTATGCC AGCTTTTGCT TCTGCTTTTC AACGTCG 57 INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 58 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" SEQUENCE DESCRIPTION: SEQ ID NO:22: GCAGATGGCT AGCAACCAGA ACACCACCTG CACGACGTTG AAAAGCAGAA GCAAAAGC 58 \STP INFORMATION FOR SEQ ID NO:23: 106 SEQUENCE CHARACTERISTICS: LENGTH: 44 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: CATGGCTTCT GCTTTTCAAC GTCGTGCAGG TGGTGTTCTG GTTG INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 44 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: CTAGCAACCA GAACACCACC TGCACGACGT TGAAAAGCAG AAGC INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 525 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID ATGGCTTACA AGCTGTGCCA CCCCGAGGAG CTGGTGCTGC TCGGACACTC TCTGGGCATC 0 0 0000 0 0S a 0* 0 0 000

CCCTGGGCTC

CAACTCCATA

CCCGAGTTGG

ATCTGGCAGC

ATGCCGGCCT

CTGCAGAGCT

GGCTCTGGCG

CCCTGAGCTC

GCGGCCTTTT

GTCCCACCTT

AGATGGAAGA

TCGCCTCTGC

TCCTGGAGGT

GCTCTCAGAG

CTGCCCCAGC

CCTCTACCAG

GGACACACTG

ACTGGGAATG

TTTCCAGCGC

GTCGTACCGC

CTTCCTGCTC

CAGGCCCTGC AGCTGGCAGG GGGCTCCTGC AGGCCCTGGA CAGCTGGACG TCGCCGACTT GCCCCTGCCC TGCAGCCCAC CGGGCAGGAG GGGTCCTGGT GTTCTACGCC ACCTTGCGCA AAGTCTTTAG AGCAAGTGAG

CTGCTTGAGC

AGGGATATCC

TGCCACCACC

CCAGGGTGCC

TGCTAGCCAT

GCCCTCTGGC

GAAGATCCAG

GGCGATGGCG CAGCGCTCCA GGAGAAGCTG TGTGCCACCT AATAA (2) r~ r s c i INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: LENGTH: 525 base pairs 107 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: ATGGCTCCCG AGTTGGGTCC CACCTTGGAC ACACTGCAGC TGGACGTCGC CGACTTTGCC

ACCACCATCT

GGTGCCATGC

AGCCATCTGC

TCTGGCGGCT

ATCCAGGGCG

CCCGAGGAGC

TGCCCCAGCC

GGCAGCAGAT

CGGCCTTCGC

AGAGCTTCCT

CTGGCGGCTC

ATGGCGCAGC

TGGTGCTGCT

AGGCCCTGCA

GGAAGAACTG

CTCTGCTTTC

GGAGGTGTCG

TCAGAGCTTC

GCTCCAGGAG

CGGACACTCT

GCTGGCAGGC

GGAATGGCCC

CAGCGCCGGG

TACCGCGTTC

CTGCTCAAGT

AAGCTGTGTG

CTGGGCATCC

TGCTTGAGCC

CTGCCCTGCA

CAGGAGGGGT

TACGCCACCT

CTTTAGAGCA

CCACCTACAA

CCTGGGCTCC

AACTCCATAG

GCCCACCCAG

CCTGGTTGCT

TGCGCAGCCC

AGTGAGGAAG

GCTGTGCCAC

CCTGAGCTCC

CGGCCTTTTC

CTCTACCAGG GGCTCCTGCA GGCCCTGGAA GGGATATCCT AATAA INFORMATION FOR SEQ ID NO:27: SEQUENCE CHARACTERISTICS: LENGTH: 525 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" p p p p a p.

p 0 p *0 p (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: ATGGCTATGG CCCCTGCCCT GCAGCCCACC CAGGGTGCCA TGCCGGCCTT TTCCAGCGCC GGGCAGGAGG GGTCCTGGTT GCTAGCCATC TGCAGAGCTT TCGTACCGCG TTCTACGCCA CCTTGCGCAG CCCTCTGGCG GCTCTGGCGG TTCCTGCTCA AGTCTTTAGA GCAAGTGAGG AAGATCCAGG GCGATGGCGC GAGAAGCTGT GTGCCACCTA CAAGCTGTGC CACCCCGAGG AGCTGGTGCT TCTCTGGGCA TCCCCTGGGC TCCCCTGAGC TCCTGCCCCA GCCAGGCCCT GGCTGCTTGA GCCAACTCCA TAGCGGCCTT TTCCTCTACC AGGGGCTCCT GAAGGGATAT CCCCCGAGTT GGGTCCCACC TTGGACACAC TGCAGCTGGA TTTGCCACCA CCATCTGGCA GCAGATGGAA GAACTGGGAT AATAA INFORMATION FOR SEQ ID NO:28 SEQUENCE CHARACTERISTICS: LENGTH: 525 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear

CGCCTCTGCT

CCTGGAGGTG

CTCTCAGAGC

AGCGCTCCAG

GCTCGGACAC

GCAGCTGGCA

GCAGGCCCTG

CGTCGCCGAC

108 (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: ATGGCTACCC AGGGTGCCAT GCCGGCCTTC GCCTCTGCTT TCCAGCGCCG GGCAGGAGGG

GTCCTGGTTG

CTTGCGCAGC

CAAGTGAGGA

AAGCTGTGCC

CCCCTGAGCT

AGCGGCCTTT

GGTCCCACCT

CTAGCCATCT

CCTCTGGCGG

AGATCCAGGG

ACCCCGAGGA

CCTGCCCCAG

TCCTCTACCA

TGGACACACT

GCAGAGCTTC

CTCTGGCGGC

CGATGGCGCA

GCTGGTGCTG

CCAGGCCCTG

GGGGCTCCTG

GCAGCTGGAC

CTGGAGGTGT

TCTCAGAGCT

GCGCTCCAGG

CTCGGACACT

CAGCTGGCAG

CAGGCCCTGG

GTCGCCGACT

CGTACCGCGT

TCCTGCTCAA

AGAAGCTGTG

CTCTGGGCAT

GCTGCTTGAG

AAGGGATATC

TTGCCACCAC

TCTACGCCAC

GTCTTTAGAG

TGCCACCTAC

CCCCTGGGCT

CCAACTCCAT

CCCCGAGTTG

CATCTGGCAG

CAGATGGAAG AACTGGGAAT GGCCCCTGCC CTGCAGCCCT AATAA INFORMATION FOR SEQ ID NO:29: SEQUENCE CHARACTERISTICS: LENGTH: 525 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE-TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" .r 4

*W

S

S

S

S.

S

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: ATGGCTTCTG CTTTCCAGCG CCGGGCAGGA GGGGTCCTGG TTGCTAGCCA TTCCTGGAGG TGTCGTACCG CGTTCTACGC CACCTTGCGC AGCCCTCTGG GGCTCTCAGA GCTTCCTGCT CAAGTCTTTA GAGCAAGTGA GGAAGATCCA GCAGCGCTCC AGGAGAAGCT GTGTGCCACC TACAAGCTGT GCCACCCCGA CTGCTCGGAC ACTCTCTGGG CATCCCCTGG GCTCCCCTGA GCTCCTGCCC CTGCAGCTGG CAGGCTGCTT GAGCCAACTC CATAGCGGCC TTTTCCTCTA CTGCAGGCCC TGGAAGGGAT ATCCCCCGAG TTGGGTCCCA CCTTGGACAC GACGTCGCCG ACTTTGCCAC CACCATCTGG CAGCAGATGG AAGAACTGGG GCCCTGCAGC CCACCCAGGG TGCCATGCCG GCCTTCGCCT AATAA INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 534 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)"

TCTGCAGAGC

CGGCTCTGGC

GGGCGATGGC

GGAGCTGGTG

CAGCCAGGCC

CCAGGGGCTC

ACTGCAGCTG

AATGGCCCCT

109 (xi) SEQUENCE DESCRIPTION: SEQ ID ATGGCTTACA AGCTGTGCCA CCCCGAGGAG CTGGTGCTGC TCGGACACTC TCTGGGCATC

CCCTGGGCTC

CAACTCCATA

CCCGAGTTGG

ATCTGGCAGC

ATGCCGGCCT

CTGCAGAGCT

TTGGGCCCTG

CCCTGAGCTC

GCGGCCTTTT

GTCCCACCTT

AGATGGAAGA

TCGCCTCTGC

TCCTGGAGGT

CCAGCTCCCT

CTGCCCCAGC

CCTCTACCAG

GGACACACTG

ACTGGGAATG

TTTCCAGCGC

GTCGTACCGC

GCCCCAGAGC

CAGGCCCTGC

GGGCTCCTGC

CAGCTGGACG

GCCCCTGCCC

CGGGCAGGAG

GTTCTACGCC

TTCCTGCTCA

GAGAAGCTGT

AGCTGGCAGG CTGCTTGAGC AGGCCCTGGA AGGGATATCC TCGCCGACTT TGCCACCACC TGCAGCCCAC CCAGGGTGCC GGGTCCTGGT TGCTAGCCAT ACCTTGCGCA GCCCACACCA AGTCTTTAGA GCAAGTGAGA GTGCCACCTA ATAA AAGATCCAGG GCGATGGCGC AGCGCTCCAG INFORMATION FOR SEQ ID NO:31: SEQUENCE CHARACTERISTICS: LENGTH: 534 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" 4 0* 9*

S

S

0' 0 90 *0 0 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: ATGGCTCCCG AGTTGGGTCC CACCTTGGAC ACACTGCAGC TGGACGTCGC CGACTTTGCC ACCACCATCT GGCAGCAGAT GGAAGAACTG GGAATGGCCC CTGCCCTGCA GCCCACCCAG GGTGCCATGC CGGCCTTCGC CTCTGCTTTC CAGCGCCGGG CAGGAGGGGT CCTGGTTGCT AGCCATCTGC AGAGCTTCCT GGAGGTGTCG TACCGCGTTC TACGCCACCT TGCGCAGCCC ACACCATTGG GCCCTGCCAG CTCCCTGCCC CAGAGCTTCC TGCTCAAGTC TTTAGAGCAA GTGAGAAAGA TCCAGGGCGA TGGCGCAGCG CTCCAGGAGA AGCTGTGTGC CACCTACAAG CTGTGCCACC CCGAGGAGCT GGTGCTGCTC GGACACTCTC TGGGCATCCC CTGGGCTCCC CTGAGCTCCT GCCCCAGCCA GGCCCTGCAG CTGGCAGGCT GCTTGAGCCA ACTCCATAGC GGCCTTTTCC TCTACCAGGG GCTCCTGCAG GCCCTGGAAG GGATATCCTA ATAA INFORMATION FOR SEQ ID NO:32: SEQUENCE CHARACTERISTICS: LENGTH: 534 base pairs TYPE: nucleic acid STRANDEDNESS; single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (Synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: 110 ATGGCTATGG CCCCTGCCCT GCAGCCCACC CAGGGTGCCA TGCCGGCCTT TTCCAGCGCC GGGCAGGAGG GGTCCTGGTT GCTAGCCATC TGCAGAGCTT TCGTACCGCG TTCTACGCCA CCTTGCGCAG CCCACACCAT TGGGCCCTGC CCCCAGAGCT TCCTGCTCAA GTCTTTAGAG CAAGTGAGAA AGATCCAGGG GCGCTCCAGG AGAAGCTGTG TGCCACCTAC AAGCTGTGCC ACCCCGAGGA CTCGGACACT CTCTGGGCAT CCCCTGGGCT CCCCTGAGCT CCTGCCCCAG CAGCTGGCAG GCTGCTTGAG CCAACTCCAT AGCGGCCTTT TCCTCTACCA CAGGCCCTGG AAGGGATATC CCCCGAGTTG GGTCCCACCT TGGACACACT GTCGCCGACT TTGCCACCAC CATCTGGCAG CAGATGGAAG AACTGGGATA INFORMATION FOR SEQ ID NO:33: SEQUENCE CHARACTERISTICS: LENGTH: 534 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)"

CGCCTCTGCT

CCTGGAGGTG

CAGCTCCCTG

CGATGGCGCA

GCTGGTGCTG

CCAGGCCCTG

GGGGCTCCTG

GCAGCTGGAC

ATAA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: ATGGCTACCC AGGGTGCCAT GCCGGCCTTC GCCTCTGCTT TCCAGCGCCG GGCAGGAGGG

GTCCTGGTTG

CTTGCGCAGC

TCTTTAGAGC

GCCACCTACA

CCCTGGGCTC

CAACTCCATA

CCCGAGTTGG

CTAGCCATCT

CCACACCATT

AAGTGAGAAA

AGCTGTGCCA

CCCTGAGCTC

GCGGCCTTTT

GTCCCACCTT

GCAGAGCTTC

GGGCCCTGCC

GATCCAGGGC

CCCCGAGGAG

CTGCCCCAGC

CCTCTACCAG

GGACACACTG

CTGGAGGTGT

AGCTCCCTGC

GATGGCGCAG

CTGGTGCTGC

CAGGCCCTGC

GGGCTCCTGC

CAGCTGGACG

CGTACCGCGT

CCCAGAGCTT

CGCTCCAGGA

TCGGACACTC

AGCTGGCAGG

AGGCCCTGGA

TCGCCGACTT

TCTACGCCAC

CCTGCTCAAG

GAAGCTGTGT

TCTGGGCATC

CTGCTTGAGC

AGGGATATCC

TGCCACCACC

r r a i cc

J

h a ATCTGGCAGC AGATGGAAGA ACTGGGAATG GCCCCTGCCC TGCAGCCCTA ATAA INFORMATION FOR SEQ ID NO:34: SEQUENCE CHARACTERISTICS: LENGTH: 534 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)' (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: ATGGCTTCTG CTTTCCAGCG CCGGGCAGGA GGGGTCCTGG TTGCTAGCCA TCTGCAGAGC TTCCTGGAGG TGTCGTACCG CGTTCTACGC CACCTTGCGC AGCCCACACC ATTGGGCCCT 111 GCCAGCTCCC TGCCCCAGAG CTTCCTGCTC AAGTCTTTAG AGCAAGTGAG AAAGATCCAG GGCGATGGCG CAGCGCTCCA GGAGAAGCTG TGTGCCACCT ACAAGCTGTG CCACCCCGAG GAGCTGGTGC TGCTCGGACA CTCTCTGGGC ATCCCCTGGG CTCCCCTGAG CTCCTGCCCC AGCCAGGCCC TGCAGCTGGC AGGCTGCTTG AGCCAACTCC ATAGCGGCCT TTTCCTCTAC CAGGGGCTCC TGCAGGCCCT GGAAGGGATA TCCCCCGAGT TGGGTCCCAC CTTGGACACA CTGCAGCTGG ACGTCGCCGA CTTTGCCACC ACCATCTGGC AGCAGATGGA AGAACTGGGA ATGGCCCCTG CCCTGCAGCC CACCCAGGGT GCCATGCCGG CCTTCGCCTA ATAA INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 531 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID ATGGCTCCGG AACTGGGTCC AACTCTGGAC ACACTGCAGC TGGACGTCGC ACCACCATCT GGCAGCAGAT GGAAGAACTG GGAATGGCCC CTGCCCTGCA GGTGCCATGC CGGCCTTCGC CTCTGCTTTC CAGCGCCGGG CAGGAGGGGT AGCCATCTGC AGAGCTTCCT GGAGGTGTCG TACCGCGTTC TACGCCACCT ACACCATTGG GCCCTGCCAG CTCCCTGCCC CAGAGCTTCC TGCTCAAGTC GTGAGAAAGA TCCAGGGCGA TGGCGCAGCG CTCCAGGAGA AGCTGTGTGC CTGTGCCACC CCGAGGAGCT GGTGCTGCTC GGACACTCTC TGGGCATCCC CTGAGCTCCT GCCCCAGCCA GGCCCTGCAG CTGGCAGGCT GCTTGAGCCA GGCCTTTTCC TCTACCAGGG GCTCCTGCAG GCCCTGGAAG GGATATCCTA INFORMATION FOR SEQ ID NO:36: SEQUENCE CHARACTERISTICS: LENGTH: 531 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)"

CGACTTTGCC

GCCCACCCAG

CCTGGTTGCT

TGCGCAGCCC

TTTAGAGCAA

CACCTACAAG

CTGGGCTCCC

ACTCCATAGC

A

5555 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: ATGGCTATGG CTCCAGCTCT GCAACCAACT CAAGGTGCAA TGCCAGCATT TGCATCTGCT TTTCAACGTC GTGCAGGTGG TGTTCTGGTT GCTAGCCATC TGCAGAGCTT CCTGGAGGTG TCGTACCGCG TTCTACGCCA CCTTGCGCAG CCCACACCAT TGGGCCCTGC CAGCTCCCTG 0 11",CCCCAGAGCT TCCTGCTCAA GTCTTTAGAG CAAGTGAGAA AGATCCAGGG COATGGCGCA

<U

7- C 112 GCGCTCCAGG AGAAGCTGTG TGCCACCTAC AAGCTGTGCC ACCCCGAGGA GCTGGTGCTG CTCGGACACT CTCTGGGCAT CCCCTGGGCT CCCCTGAGCT CCTGCCCCAG CCAGGCCCTG CAGCTGGCAG GCTGCTTGAG CCAACTCCAT AGCGGCCTTT TCCTCTACCA GGGGCTCCTG CAGGCCCTGG AAGGGATATC CCCCGAGTTG GGTCCCACCT TGGACACACT GCAGCTGGAC GTCGCCGACT TTGCCACCAC CATCTGGCAG CAGATGGAAG AACTGGGATA A INFORMATION FOR SEQ ID NO:37: i) SEQUENCE CHARACTERISTICS: LENGTH: 531 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: ATGGCTACTC AAGGTGCTAT GCCAGCTTTT GCTTCTGCTT TTCAACGTCG GTTCTGGTTG CTAGCCATCT GCAGAGCTTC CTGGAGGTGT CGTACCGCGT CTTGCGCAGC CCACACCATT GGGCCCTGCC AGCTCCCTGC CCCAGAGCTT TCTTTAGAGC AAGTGAGAAA GATCCAGGGC GATGGCGCAG CGCTCCAGGA GCCACCTACA AGCTGTGCCA CCCCGAGGAG CTGGTGCTGC TCGGACACTC CCCTGGGCTC CCCTGAGCTC CTGCCCCAGC CAGGCCCTGC AGCTGGCAGG CAACTCCATA GCGGCCTTTT CCTCTACCAG GGGCTCCTGC AGGCCCTGGA CCCGAGTTGG GTCCCACCTT GGACACACTG CAGCTGGACG TCGCCGACTT ATCTGGCAGC AGATGGAAGA ACTGGGAATG GCCCCTGCCC TGCAGCCCTA INFORMATION FOR SEQ ID NO:38: SEQUENCE CHARACTERISTICS: LENGTH: 531 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" c

I

E

D

TGCAGGTGGT

TCTACGCCAC

CCTGCTCAAG

GAAGCTGTGT

TCTGGGCATC

CTGCTTGAGC

AGGGATATCC

TGCCACCACC

A

TCTGCAGAGC

ATTGGGCCCT

AAAGATCCAG

CCACCCCGAG

CTCCTGCCCC

TTTCCTCTAC

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: ATGGCTTCTG CTTTTCAACG TCGTGCAGGT GGTGTTCTGG TTGCTAGCCA TTCCTGGAGG TGTCGTACCG CGTTCTACGC CACCTTGCGC AGCCCACACC GCCAGCTCCC TGCCCCAGAG CTTCCTGCTC AAGTCTTTAG AGCAAGTGAG GGCGATGGCG CAGCGCTCCA GGAGAAGCTG TGTGCCACCT ACAAGCTGTG GAGCTGGTGC TGCTCGGACA CTCTCTGGGC ATCCCCTGGG CTCCCCTGAG AGCCAGGCCC TGCAGCTGGC AGGCTGCTTG AGCCAACTCC ATAGCGGCCT.

113 CAGGGGCTCC TGCAGGCCCT GGAAGGGATA TCCCCCGAGT TGGGTCCCAC CTTGGACACA CTGCAGCTGG ACGTCGCCGA CTTTGCCACC ACCATCTGGC AGCAGATGGA AGAACTGGGA ATGGCCCCTG CCCTGCAGCC CACCCAGGGT GCCATGCCGG CCTTCGCCTA A INFORMATION FOR SEQ ID NO:39: SEQUENCE CHARACTERISTICS: LENGTH: 522 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: Idesc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: ATGGCTCCGG AACTGGGTCC AACTCTGGAC ACACTGCAGC TGGACGTCGC ACCACCATCT GGCAGCAGAT GGAAGAACTG GGAATGGCCC CTGCCCTGCA GGTGCCATGC CGGCCTTCGC CTCTGCTTTC CAGCGCCGGG CAGGAGGGGT AGCCATCTGC AGAGCTTCCT GGAGGTGTCG TACCGCGTTC TACGCCACCT TCTGGCGGCT CTGGCGGCTC TCAGAGCTTC CTGCTCAAGT CTTTAGAGCA ATCCAGGGCG ATGGCGCAGC GCTCCAGGAG AAGCTGTGTG CCACCTACAA CCCGAGGAGC TGGTGCTGCT CGGACACTCT CTGGGCATCC CCTGGGCTCC TGCCCCAGCC AGGCCCTGCA GCTGGCAGGC TGCTTGAGCC AACTCCATAG CTCTACCAGG GGCTCCTGCA GGCCCTGGAA GGGATATCCT AA INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 522 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" q *0

CGACTTTGCC

GCCCACCCAG

CCTGGTTGCT

TGCGCAGCCC

AGTGAGAAAG

GCTGTGCCAC

CCTGAGCTCC

CGGCCTTTTC

TGCATCTGCT

CCTGGAGGTG

CTCTCAGAGC

AGCGCTCCAG

GCTCGGACAC

GCAGCTGGCA

GCAGGCCCTG

CGTCGCCGAC

120 180 240 300 360 420 480 522 120 180 240 300 360 420 480 0 (xi) SEQUENCE DESCRIPTION: SEQ ID ATGGCTATGG CTCCAGCTCT GCAACCAACT CAAGGTGCAA TTTCAACGTC GTGCAGGTGG TGTTCTGGTT GCTAGCCATC e TCGTACCGCG TTCTACGCCA CCTTGCGCAG CCCTCTGGCG TTCCTGCTCA AGTCTTTAGA GCAAGTGAGA AAGATCCAGG GAGAAGCTGT GTGCCACCTA CAAGCTGTGC CACCCCGAGG TCTCTGGGCA TCCCCTGGGC TCCCCTGAGC TCCTGCCCCA GGCTGCTTGA GCCAACTCCA TAGCGGCCTT TTCCTCTACC GAAGGGATAT CCCCCGAGTT GGGTCCCACC TTGGACACAC id,/ Kio" 1

TGCCAGCATT

TGCAGAGCTT

GCTCTGGCGG

GCGATGGCGC

AGCTGGTGCT

GCCAGGCCCT

AGGGGCTCCT

TGCAGCTGGA

114 TTTGCCACCA CCATCTGGCA GCAGATGGAA GAACTGGGAT AA INFORMATION FOR SEQ ID NO:41: SEQUENCE CHARACTERISTICS: LENGTH: 522 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:

ATGGCTACTC

GTTCTGGTTG

CTTGCGCAGC

CAAGTGAGAA

AAGCTGTGCC

CCCCTGAGCT

AGCGGCCTTT

GGTCCCACCT

CAGATGGAAG

AAGGTGCTAT

CTAGCCATCT

CCTCTGGCGG

AGATCCAGGG

ACCCCGAGGA

CCTGCCCCAG

TCCTCTACCA

TGGACACACT

AACTGGGAAT

GCCAGCTTTT

GCAGAGCTTC

CTCTGGCGGC

CGATGGCGCA

GCTGGTGCTG

CCAGGCCCTG

GGGGCTCCTG

GCAGCTGGAC

GGCCCCTGCC

GCTTCTGCTT

CTGGAGGTGT

TCTCAGAGCT

GCGCTCCAGG

CTCGGACACT

CAGCTGGCAG

CAGGCCCTGG

GTCGCCGACT

CTGCAGCCCT

TTCAACGTCG

CGTACCGCGT

TCCTGCTCAA

AGAAGCTGTG

CTCTGGGCAT

GCTGCTTGAG

AAGGGATATC

TTGCCACCAC

AA

TGCAGGTGGT

TCTACGCCAC

GTCTTTAGAG

TGCCACCTAC

CCCCTGGGCT

CCAACTCCAT

CCCCGAGTTG

CATCTGGCAG

INFORMATION FOR SEQ ID NO:42: SEQUENCE CHARACTERISTICS: LENGTH: 522 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: *S

ATGGCTTCTG

TTCCTGGAGG

GGCTCTCAGA

GCAGCGCTCC

CTGCTCGGAC

CTGCAGCTGG

CTGCAGGCCC

GACGTCGCCG

GCCCTGCAGC

CTTTTCAACG

TGTCGTACCG

GCTTCCTGCT

AGGAGAAGCT

ACTCTCTGGG

CAGGCTGCTT

TGGAAGGGAT

ACTTTGCCAC

CCACCCAGGG

TCGTGCAGGT

CGTTCTACGC

CAAGTCTTTA

GTGTGCCACC

CATCCCCTGG

GAGCCAACTC

ATCCCCCGAG

CACCATCTGG

TGCCATGCCG

GGTGTTCTGG TTGCTAGCCA CACCTTGCGC AGCCCTCTGG

TCTGCAGAGC

CGGCTCTGGC

GAGCAAGTGA GAAAGATCCA GGGCGATGGC TACAAGCTGT GC( GCTCCCCTGA GC' CATAGCGGCC TT TTGGGTCCCA CC' CAGCAGATGG AA( GCCTTCGCCT AA 'ACCCCGA GGAGCTGGTG rCCTGCCC CAGCCAGGCC rTCCTCTA

TGGACAC

GAACTGGG

CCAGGGGCTC

ACTGCAGCTG

AATGGCCCCT

115 SEQUENCE CHARACTERISTICS: LENGTH: 171 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu 1 5 10 Gly Leu Gly Leu Gin Gly Val Val Ser 145 Ile Ala Leu Asp Gin Ala Leu Leu 130 Phe Pro Trp Gly Cys Leu Gin Thr Leu Met Glu Met Pro 100 Val Ala 115 Arg His Leu Leu Ser Leu Gly Val Met Ser Gin 120 Pro Glu Pro Gly Pro Phe Ala Gin Leu Gly Arg 155 a a a Gly Ala Ala Leu Gin Glu Lys Leu Cys Ala Thr 165 170 INFORMATION FOR SEQ ID NO:44: SEQUENCE CHARACTERISTICS: LENGTH: 171 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) Pro 1 Phe Ala SEQUENCE DESCRIPTION: SEQ ID NO:44: Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala Asp 5 10 Ala Thr Thr Ile Trp Gin Gin Met Glu Glu Leu Gly Met Ala Pro 25 Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Phe 40 Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gin Ser Phe 50 55 >13 ?t2 /-ii

"IA

Gin 116 Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gin Pro Ser Gly 70 75 Gly Ser Gly Gly Ser Gin Ser Phe Leu Leu Lys Ser Leu Glu Gin Val 90 Arg Lys Ile Gin Gly Asp Gly Ala Ala Leu Gin Glu Lys Leu Cys Ala 100 105 110 Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser 115 120 125 Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gin Ala Leu 130 135 140 Gin Leu Ala Gly Cys Leu Ser Gin Leu His Ser Gly Leu Phe Leu Tyr 145 150 155 160 Gin Gly Leu Leu Gin Ala Leu Glu Gly Ile Ser 165 170 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 171 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gin Arg Arg Ala 1 5 10 Gly Gly Val Leu Val Ala Ser His Leu Gin Ser Phe Leu Glu Val Ser 25 Tyr Arg Val Leu Arg His Leu Ala Gin Pro Ser Gly Gly Ser Gly Gly 0 35 40 SSer Gin Ser Phe Leu Leu Lys Ser Leu Glu Gin Val Arg Lys Ile Gin 50 55 Gly Asp Gly Ala Ala Leu Gin Glu Lys Leu Cys Ala Thr Tyr Lys Leu 65 70 75 Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro 85 90 **Trp Ala Pro Leu Ser Ser Cys Pro Ser Gin Ala Leu Gin Leu Ala Gly 100 105 110 Cys Leu Ser Gin Leu His Ser Gly Leu Phe Leu Tyr Gin Gly Leu Leu 115 120 125 SGin Ala Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr 130 135 140 Leu Gin Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gin Gin Met 145 150 155 160 Glu Glu Leu Gly Met Ala Pro Ala Leu Gin Pro 165 170 y INFORMATION FOR SEQ ID NO:46: <-oV 117 SEQUENCE CHARACTERISTICS: LENGTH: 118 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: TGGAATAAAA AAGAGAGAAG GAAAAGGATA GAAGAAGGGG GGGGAAGGGA GAAAAGGCAA TTCGGAGGTA ACGAAGAAGC GGTGGGAAGG GGTATGAAAA AAATTTGGTG GGTAAAAG INFORMATION FOR SEQ ID NOf47: SEQUENCE CHARACTERISTICS: LENGTH: 171 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu

S

Phe Leu Gly Gly Val Arg Ala Thr Ser Leu Leu Gln 100 Tyr Gln 115 Pro Thr Ile Trp Leu Arg Phe Leu Ala Ala Glu Glu Leu Ser Gln Leu Glu Gly Asp Val 140 Gly Met 155 Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala 165 170 INFORMATION FOR SEQ ID NO:48: SEQUENCE CHARACTERISTICS: LENGTH: 174 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear 118 (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Ser Gin Leu Phe Glu Leu Ala Thr Leu Gin Arg Arg Glu Val 125 Gly Pro 140 Gin Val Cys Ala 9

A

9 9.9.

9* 9 A 9..

999 Gin Gly Asp Gly Ala Ala Leu Gin 165 INFORMATION FOR SEQ ID NO:49: SEQUENCE CHARACTERISTICS: LENGTH: 174 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) Pro 1 Phe Ala Gin Leu Leu SEQUENCE DESCRIPTION: SEQ ID NO:49: Glu Leu Gly Pro Thr Leu Asp Thr Leu 5 Ala Thr Thr Ile Trp Gin Gin Met Glu Leu Gin Pro Thr Gin Gly Ala Met Pro Arg Arg Ala Gly Gly Val Leu Val Ala Glu Val Ser Tyr Arg Val Leu Arg His Gly Pro Ala Ser Ser Leu Pro Gin Ser 119 Glu Gin Val Arg Lys Ile Gin Gly I 100 3 Leu Cys Ala Thr Tyr Lys Leu Cys 1 115 120 Gly His Ser Leu Gly Ile Pro Trp 1 130 135 Gin Ala Leu Gin Leu Ala Gly Cys I 145 150 Phe Leu Tyr Gin Gly Leu Leu Gin 1 165 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 174 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein Gly Ala Ala Leu Gin Glu Lys 110 Pro Glu Glu Leu Val Leu Leu 125 Pro Leu Ser Ser Cys Pro Ser 140 Ser Gin Leu His Ser Gly Leu 155 160 Leu Glu Gly Ile Ser 170 (xi) SEQUENCE DESCRIPTION: SEQ ID Met Ala Pro Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala

S

S

S

S. Si .5S Ser Ala Phe Gin Arg Arg Ala Gly Gin Ser Phe Leu Glu Val Ser Tyr Pro Thr Pro Leu Gly Pro Ala Ser 55 Lys Ser Leu Glu Gin Val Arg Lys 65 70 Gin Glu Lys Leu Cys Ala Thr Tyr Val Leu Leu Gly His Ser Leu Gly 100 Cys Pro Ser Gin Ala Leu Gin Leu 115 120 Ser Gly Leu Phe Leu Tyr Gin Gly 130 135 Ser Pro Glu Leu Gly Pro Thr Leu 145 150 Asp Phe Ala Thr Thr Ile Trp Gin 165 INFORMATION FOR SEQ ID NO:51: Val Leu Val Leu Leu Pro Gin Gly Leu Cys Pro Trp Gly Cys Leu Gin Thr Leu 155 Met. Glu 170 Val Arg Gin Asp His Ala Leu Ala 140 Gin Glu -3 nc SEQUENCE CHARACTERISTICS: LENGTH: 174 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear 120 (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51: Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gin Arg Arg Ala Gly Tyr Ser Lys Tyr Gly Leu Gly Leu 145 Gin Ser Pro Thr Lys Ser Gin Glu Val Leu Cys Pro Ser Gly Ser Pro Asp Phe 155 Pro Ala 170 0 a 00.: 9 p p .p p Gin Gin Met Glu Glu Leu Gly Met 1 165 INFORMATION FOR SEQ ID NO:52: SEQUENCE CHARACTERISTICS: LENGTH: 174 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) Ser 1 Gin Pro Lys Gin Val SEQUENCE DESCRIPTION: SEQ ID NO:52: Ala Phe Gin Arg Arg Ala Gly Gly Val 5 Ser Phe Leu Glu Val Ser Tyr Arg Val 20 Thr Pro Leu Gly Pro Ala Ser Ser Leu Ser Leu Glu Gin Val Arg Lys Ile Gin Glu Lys Leu Cys Ala Thr Tyr Lys Leu Leu Leu Gly His Ser Leu Gly Ile Pro Cys Pro Ser Gin Ala Leu Gin Leu Ala 100 105 Ser Gly Leu Phe Leu Tyr Gin Gly Leu 115 120 Ser Pro Glu Leu Gly Pro Thr Leu Asp 130 135 Asp Phe Ala Thr Thr Ile Trp Gin Gin 145 150 Pro Ala Leu Gin Pro Thr Gin Gly Ala 165 INFORMATION FOR SEQ ID NO:53: SEQUENCE CHARACTERISTICS: LENGTH: 174 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein Gly Cys Leu Ser Gin Leu His 110 Leu Gin Ala Leu Glu Gly Ile 125 Thr Leu Gin Leu Asp Val Ala 140 Met Glu Glu Leu Gly Met Ala 155 160 Met Pro Ala Phe Ala 170 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53: Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala Asp 1 5 10 0 a.

a a a.

*0 a Phe Ala Gin Leu 65 Leu Glu Leu Gly Gin 145 Ala Leu Arg Glu Gly Gin Cys His 130 Ala Trp Gin Gly Arg 70 Ser Ile Lys Ile Ala 150 Glu Glu Pro Ala Ala Ser His Leu Ser Phe Gly Ala Pro Glu Pro Leu Ser Gin 155 Leu Glu 170 Phe Leu Tyr Gin Leu Leu Gin INFORMATION FOR SEQ ID NO:54: SEQUENCE CHARACTERISTICS: LENGTH: 174 amino acids TYPE: amino acid STRANDEDNESS: single S(D) TOPOLOGY: linear 122 (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54: Met Ala Pro Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala Gly Arg Ser Ile Lys Ile 105 Al a Leu Asp Gin Leu Leu Pro Gly Cys Trp Cys Gin Leu 155 Glu Asp Phe Ala Thr Thr Ile Trp Gin 165 *got 000 :9.

000 *4 0 00 0 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 174 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) Thr Gly Tyr Ser Lys Tyr SEQUENCE DESCRIPTION: SEQ ID Gin Gly Ala Met Pro Ala Phe Ala Ser 5 Gly Val Leu Val Ala Ser His Leu Gin 20 Arg Val Leu Arg His Leu Ala Gin Pro Ser Leu Pro Gin Ser Phe Leu Leu Lys Ile Gin Giy Asp Gly Ala Ala Leu Gln Lys Leu Cys His Pro Giu Glu Leu Val 123 Gly Ile Pro Trp Ala Pro Leu Ser E 100 3 Leu Ala Gly Cys Leu Ser Gin Leu 1 115 120 Gly Leu Leu Gin Ala Leu Glu Gly 130 135 Leu Asp Thr Leu Gin Leu Asp Val 1 145 150 Gin Gin Met Glu Glu Leu Gly Met I 165 INFORMATION FOR SEQ ID NO:56: SEQUENCE CHARACTERISTICS: LENGTH: 174 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein Cys Pro Ser Gin Ala 110 Ser Gly Leu Phe Leu 125 Ser Pro Glu Leu Gly 140 Asp Phe Ala Thr Thr 155 Pro Ala Leu Gin Pro 170 Leu Gin Tyr Gin Pro Thr Ile Trp 160 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56: Ser Ala Phe Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu 1 5 10 o n 0 0 050 0000 0000 9000 0* .500 0 0 0

S

5 *50* *055

S

S

S

CO

Gin Ser Phe Leu Glu Val Ser Tyr Pro Thr Pro Leu Gly Pro Ala Ser 40 Lys Ser Leu Glu Gin Val Arg Lys 55 Gin Glu Lys Leu Cys Ala Thr Tyr 65 Val Leu Leu Gly His Ser Leu Gly 85 Cys Pro Ser Gin Ala Leu Gin Leu 100 Ser Gly Leu Phe Leu Tyr Gin Gly 115 120 Ser Pro Glu Leu Gly Pro Thr Leu 130 135 Asp Phe Ala Thr Thr Ile Trp Gin 145 150 Pro Ala Leu Gin Pro Thr Gin Gly Arg Val Ser Leu Ile Gin Lys Leu Ile Pro Ala Gly 105 Leu Leu Asp Thr Gin Met Ala Met 170 Arg His Gin Ser Asp Gly His Pro Ala Pro Leu Ser Ala Leu 125 Gin Leu 140 Glu Leu Ala Phe INFORMATION FOR SEQ ID NO:57: SEQUENCE CHARACTERISTICS: LENGTH: 171 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear 124 (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57: Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala Asp 1 5 10 Phe Ala Gin Leu Gly Arg Thr Leu Gin 145 Ala Thr Leu Gin Arg Arg Glu Val Ser Gly Lys Ile Tyr Lys 115 Gly Ile 130 Leu Ala Trp Gin Gly Arg 70 Gin Asp His Ala Leu 150 Gin Gly Val Val Ser Gly Pro Pro 135 Ser Gin Ala Leu Leu Phe Ala Glu 120 Leu Gin Met Glu Met Pro Val Ala Arg His Leu Leu Ala Leu 105 Glu Leu Ser Ser Leu His Glu Ala Ser Leu Lys Gin Val Cys Ser 155 Ala Ala Ser Ser Gin Cys His Ala Leu r r r o Gin Gly Leu Leu Gin Ala Leu Glu Gly 165 INFORMATION FOR SEQ ID NO:58: SEQUENCE CHARACTERISTICS: LENGTH: 169 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein Ile Ser 170 (xi) Met 1 Ser Gin Pro Glu Ala SEQUENCE DESCRIPTION: SEQ ID NO:58: Ala Pro Ala Leu Gin Pro Thr Gin Gly 5 10 Ala Phe Gin Arg Arg Ala Gly Gly Val Ser Phe Leu Glu Val Ser Tyr Arg Val Ser Gly Gly Ser Gly Gly Ser Gin Ser Gin Val Arg Lys Ile Gin Gly Asp Gly 70 Thr Tyr Lys Leu Cys His Pro Glu Glu Phe Ala His Leu Ala Gin Ser Leu Glu Lys Gly His

I,

125 Ser Leu Gly Ile Pro Trp Ala 100 Leu Gin Leu Ala Gly Cys Leu 115 Tyr Gin Gly Leu Leu Gin Ala 130 135 Pro Thr Leu Asp Thr Leu Gin 145 150 Ile Trp Gin Gin Met Glu Glu 165 INFORMATION FOR SEQ ID NO:59: SEQUENCE CHARACTERISTICS LENGTH: 171 amino z TYPE: amino acid STRANDEDNESS: sing] TOPOLOGY: linear (ii) MOLECULE TYPE: protein 90 Pro Leu Ser Ser Cys Pro Ser Gin Ala 105 110 Ser Gin Leu His Ser Gly Leu Phe Leu 120 125 Leu Glu Gly Ile Ser Pro Glu Leu Gly 140 Leu Asp Val Ala Asp Phe Ala Thr Thr 155 160 Leu Gly (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59: Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gin Arg Arg Ala 9 Gly Tyr Ser Gly Cys Trp Cys Gin Leu 145 Gly Arg Gin Asp His Ala Leu Ala 130 Gin Val Arg Leu Ala Glu 85 Ser Leu Gly Val Ser His Leu Leu Ala Gln Lys Ser Leu Gin Glu Lys Val Leu Leu Cys Pro Ser 105 Ser Gly Leu 120 Ser Pro Glu 135 Asp Phe Ala Gin Pro Glu Leu Gly 90 Gin Phe Leu Thr Val Gly Ile Lys Ile Ala Leu Asp Gin Glu Glu Leu Gly Met Ala Pro Ala Leu Gin Pro 165 170 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 171 amino acids TYPE: amino acid STRANDEDNESS: single I TOPOLOGY: linear 126 (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu i C 10 Gin Pro Glu Leu Gly Gin Phe Leu Ser Ser Gin Cys His Ala Leu Gly 130 Glu Ser Lys Tyr Gly Leu Gly Leu Val Ser Tyr Gly Gly Ser Ile Gin Gly Lys Leu Cys 70 Ile Pro Trp Ala Gly Cys Leu Leu Gin 120 Asp Thr Leu 135 Gin Met Glu 150 Val Leu Ser Phe Gly Ala Pro Glu Pro Leu Ser Gin Leu Glu Leu Asp Leu Gly 155 9 9 9*S9 99** 99 9 9 9 9 9 99 9 Thr Thr Ile Trp Gin Gin Pro Thr Gin Ala Met Pro Ala Phe Ala INFORMATION FOR SEQ ID NO:61: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61: Gly Gly Gly Ser Gly Gly Gly Ser 1 INFORMATION FOR SEQ ID NO:62: SEQUENCE CHARACTERISTICS: LENGTH: 12 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

L

X 127 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62: Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser 1 5 INFORMATION FOR SEQ ID NO:63: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63: Ser Gly Gly Ser Gly Gly Ser 1 INFORMATION FOR SEQ ID NO:64: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide OA(xi) SEQUENCE DESCRIPTION: SEQ ID NO:64: O Glu Phe Gly Asn Met 1 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide ee (xi) SEQUENCE DESCRIPTION: SEQ ID Glu Phe Gly Gly Asn Met 1 INFORMATION FOR SEQ ID NO:66: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 21I\ O 128 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66: Glu Phe Gly Gly Asn Gly Gly Asn Met INFORMATION FOR SEQ ID NO:67: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67: Gly Gly Ser Asp Met Ala Gly 1 INFORMATION FOR SEQ ID NO:68: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68: GATCGACCAT GGCTCTGCTC GGACACTCTC TG INFORMATION FOR SEQ ID NO:69: SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" 0* 0* 0 00 0 0 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69: CGATCGAAGC TTATTACACC AGCTCCTCGG GGTGGC INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid 9 DESCRIPTION: /desc "DNA (synthetic)" i(K OK" i~ 129 (xi) SEQUENCE DESCRIPTION: SEQ ID GATCGACCAT GGCTCAACTC CATAGCGGCC TT 32 INFORMATION FOR SEQ ID NO:71: SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71: CGATCGAAGC TTATTAGCTC AAGCAGCCTG CCAGCT 36 INFORMATION FOR SEQ ID NO:72: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72: GATCGACCAT GGCTCTTTTC CTCTACCAGG GG 32 9.9 INFORMATION FOR SEQ ID NO:73: SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear **go (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73: CGATCGAAGC TTATTAGCCG CTATGGAGTT GGCTCA 36 INFORMATION FOR SEQ ID NO:74: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" PC i 130 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74: GATCGACCAT GGCTCTCTAC CAGGGGCTCC TG 32 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID CGATCGAAGC TTATTAGAAA AGGCCGCTAT GGAGTT 36 INFORMATION FOR SEQ ID NO:76: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76: GATCGACCAT GGCTGCCCTG GAAGGGATAT CC 32 INFORMATION FOR SEQ ID NO:77: S(i) SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid o STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77: CGATCGAAGC TTATTACTGC AGGAGCCCCT GGTAGA 36 INFORMATION FOR SEQ ID NO:78: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" I-L 131 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78: GATCGACCAT GGCTGACTTT GCCACCACCA TC 32 INFORMATION FOR SEQ ID NO:79: SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79: CGATCGAAGC TTATTAGGCG ACGTCCAGCT GCAGTG 36 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" 9** (xi) SEQUENCE DESCRIPTION: SEQ ID GATCGACCAT GGCTATCTGG CAGCAGATGG AA 32 .9 INFORMATION FOR SEQ ID NO:81: SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid S(C) STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81: CGATCGAAGC TTATTAGGTG GTGGCAAAGT CGGCGA 36 INFORMATION FOR SEQ ID NO:82: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" 71J 132 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82: GATCGACCAT GGCTCAGCAG ATGGAAGAAC TG 32 INFORMATION FOR SEQ ID NO:83: SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single.

TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:83: CGATCGAAGC TTATTACCAG ATGGTGGTGG CAAAGT 36 INFORMATION FOR SEQ ID NO:84: SEQUENCE CHARACTERISTICS: LENGTH: 50 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:84: CATGGCTTTG TTAGGACATT CTTTAGGTAT TCCATGGGCT CCTCTGAGCT INFORMATION FOR SEQ ID o SEQUENCE CHARACTERISTICS: LENGTH: 40 base pairs c o(B) TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID CAGAGGAGCC CATGGAATAC CTAAAGAATG TCCTAACAAA INFORMATION FOR SEQ ID NO:86: SEQUENCE CHARACTERISTICS: LENGTH: 534 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" 133 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:86: ATGGCTCTGC TCGGACACTC TCTGGGCATC CCCTGGGCTC CCCTGAGCTC CTGCCCCAGC CAGGCCCTGC AGCTGGCAGG CTGCTTGAGC CAACTCCATA GCGGCCTTTT CCTCTACCAG GGGCTCCTGC AGGCCCTGGA AGGGATATCC CCCGAGTTGG GTCCCACCTT GGACACACTG CAGCTGGACG TCGCCGACTT TGCCACCACC ATCTGGCAGC AGATGGAAGA ACTGGGAATG GCCCCTGCCC TGCAGCCCAC CCAGGGTGCC ATGCCGGCCT TCGCCTCTGC TTTCCAGCGC CGGGCAGGAG GGGTCCTGGT TGCTAGCCAT CTGCAGAGCT TCCTGGAGGT GTCGTACCGC GTTCTACGCC ACCTTGCGCA GCCCACACCA TTGGGCCCTG CCAGCTCCCT GCCCCAGAGC TTCCTGCTCA AGTCTTTAGA GCAAGTGAGA AAGATCCAGG GCGATGGCGC AGCGCTCCAG GAGAAGCTGT GTGCCACCTA CAAGCTGTGC CACCCCGAGG AGCTGGTGTA ATAA INFORMATION FOR SEQ ID NO:87: SEQUENCE CHARACTERISTICS: LENGTH: 534 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)' (xi) SEQUENCE DESCRIPTION: SEQ ID NO:87: ATGGCTCAAC TCCATAGCGG CCTTTTCCTC TACCAGGGGC TCCTGCAGGC CCTGGAAGGG 0 *e 0 0 **so foe.

.01.0 0*

U

ATATCCCCCG

ACCACCATCT

GGTGCCATGC

AGCCATCTGC

ACACCATTG

GTGAGAAAGA

CTGTGCCACC

AGTTGGGTCC

GGCAGCAGAT

CGGCCTTCGC

AGAGCTTCCT

GCCCTGCCAG

TCCAGGGCGA

CC GAGGAGCT

CACCTTGGAC

GGAAGAACTG

CTCTGCTTTC

GGAGGTGTC6

CTCCCTGCCC

TGGCGCAGCG

GGTGCTGCTC

ACACTGCAGC

GGAATGGCCC

CAGCGCCGGG

TACCGCGTTC

CAGAGCTTCC

CTCCAGGAGA

GGACACTCTC

CTGGCAGGCT

TGGACGTCGC

CTGCCCTGCA

CAGGAGGGGT

TACGCCACCT

TGCTCAAGTC

AGCTGTGTGC

TGGGCATCCC

GCTTGAGCTA

CGACTTTGCC

GCCCACCCAG

CCTGGTTGCT

TGCGCAGCCC

TTTAGAGCAA

CACCTACAAG

CTGGGCTCCC

ATAA

CTGAGCTCCT GCCCCAGCCA GGCCCTGCAG INFORMATION FOR SEQ ID NO:88: SEQUENCE CHARACTERISTICS: LENGTH: 534 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc ="DNA (synthetic)" (xi) SEUNEDESCRIPTION: SEQ ID NO:88: a A:TGGCTCTTT TCCTCTACCA GGGGCTCCTG CAGGCCCTGG AAGGGATATC CCCCGAGTTG r" 134 GGTCCCACCT TGGACACACT GCAGCTGGAC GTCGCCGACT TTGCCACC1 CAGATGGAAG AACTGGGAAT GGCCCCTGCC CTGCAGCCCA CCCAGGGTC TTCGCCTCTG CTTTCCAGCG CCGGGCAGGA GGGGTCCTGG TTGCTAGCC TTCCTGGAGG TGTCGTACCG CGTTCTACGC CACCTTGCGC AGCCCACAC GCCAGCTCCC TGCCCCAGAG CTTCCTGCTC AAGTCTTTAG AGCAAGTGI GGCGATGGCG CAGCGCTCCA GGAGAAGCTG TGTGCCACCT ACAAGCTG GAGCTGGTGC TGCTCGGACA CTCTCTGGGC ATCCCCTGGG CTCCCCTG AGCCAGGCCC TGCAGCTGGC AGGCTGCTTG AGCCAACTCC ATAGCGGC INFORMATION FOR SEQ ID NO:89: SEQUENCE CHARACTERISTICS: LENGTH: 534 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)"

CC

AA

CC

GG

TAA

CATCTGGCAG

CATGCCGGCC

TCTGCAGAGC

ATTGGGCCCT

AAAGATCCAG

CCACCCCGAG

CTCCTGCCCC

ATAA

120 180 240 300 360 420 480 534 *9 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:89: ATGGCTCTCT ACCAGGGGCT CCTGCAGGCC CTGGAAGGGA TATCCCCCGA ACCTTGGACA CACTGCAGCT GGACGTCGCC GACTTTGCCA CCACCATCTG GAAGAACTGG GAATGGCCCC TGCCCTGCAG CCCACCCAGG GTGCCATGCC TCTGCTTTCC AGCGCCGGGC AGGAGGGGTC CTGGTTGCTA GCCATCTGCA GAGGTGTCGT ACCGCGTTCT ACGCCACCTT GCGCAGCCCA CACCATTGGG TCCCTGCCCC AGAGCTTCCT GCTCAAGTCT TTAGAGCAAG TGAGAAAGAT GGCGCAGCGC TCCAGGAGAA GCTGTGTGCC ACCTACAAGC TGTGCCACCC GTGCTGCTCG GACACTCTCT GGGCATCCCC TGGGCTCCCC TGAGCTCCTG GCCCTGCAGC TGGCAGGCTG CTTGAGCCAA CTCCATAGCG GCCTTTTCTA INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 534 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)"

GTTGGGTCCC

GCAGCAGATG

GGCCTTCGCC

GAGCTTCCTG

CCCTGCCAGC

CCAGGGCGAT

CGAGGAGCTG

CCCCAGCCAG

ATAA

(xi) SEQUENCE DESCRIPTION: SEQ ID ATGGCTGCCC TGGAAGGGAT ATCCCCCGAG TTGGGTCCCA CCTTGGACAC ACTGCAGCTG GACGTCGCCG ACTTTGCCAC CACCATCTGG CAGCAGATGG AAGAACTGGG AATGGCCCCT GCCCTGCAGC CCACCCAGGG TGCCATGCCG GCCTTCGCCT CTGCTTTCCA GCGCCGGGCA 135 GGAGGGGTCC TGGTTGCTAG CCATCTGCAG AGCTTCCTGG AGGTGTCGTA CGCCACCTTG CGCAGCCCAC ACCATTGGGC CCTGCCAGCT CCCTGCCCCA CTCAAGTCTT TAGAGCAAGT GAGAAAGATC CAGGGCGATG GCGCAGCGCT CTGTGTGCCA CCTACAAGCT GTGCCACCCC GAGGAGCTGG TGCTGCTCGG GGCATCCCCT GGGCTCCCCT GAGCTCCTGC CCCAGCCAGG CCCTGCAGCT TTGAGCCAAC TCCATAGCGG CCTTTTCCTC TACCAGGGGC TCCTGCAGTA INFORMATION FOR SEQ ID NO:91: SEQUENCE CHARACTERISTICS: LENGTH: 534 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)"

CCGCGTTCTA

GAGCTTCCTG

CCAGGAGAAG

ACACTCTCTG

GGCAGGCTGC

ATAA

9@ e 0*00 04

U

S

*0 04 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:91: ATGGCTGACT TTGCCACCAC CATCTGGCAG CAGATGGAAG AACTGGGAAT CTGCAGCCCA CCCAGGGTGC CATGCCGGCC TTCGCCTCTG CTTTCCAGCG GGGGTCCTGG TTGCTAGCCA TCTGCAGAGC TTCCTGGAGG TGTCGTACCG CACCTTGCGC AGCCCACACC ATTGGGCCCT GCCAGCTCCC TGCCCCAGAG AAGTCTTTAG AGCAAGTGAG AAAGATCCAG GGCGATGGCG CAGCGCTCCA TGTGCCACCT ACAAGCTGTG CCACCCCGAG GAGCTGGTGC TGCTCGGACA ATCCCCTGGG CTCCCCTGAG CTCCTGCCCC AGCCAGGCCC TGCAGCTGGC AGCCAACTCC ATAGCGGCCT TTTCCTCTAC CAGGGGCTCC TGCAGGCCCT TCCCCCGAGT TGGGTCCCAC CTTGGACACA CTGCAGCTGG ACGTCGCCTA INFORMATION FOR SEQ ID NO:92: SEQUENCE CHARACTERISTICS: LENGTH: 534 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: Idesc "DNA (synthetic)"

GGCCCCTGCC

CCGGGCAGGA

CGTTCTACGC

CTTCCTGCTC

GGAGAAGCTG

CTCTCTGGGC

AGGCTGCTTG

GGAAGGGATA

ATAA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:92: ATGGCTATCT GGCAGCAGAT GGAAGAACTG GGAATGGCCC CTGCCCTGCA GCCCACCCAG GGTGCCATGC CGGCCTTCGC CTCTGCTTTC CAGCGCCGGG CAGGAGGGGT CCTGGTTGCT AGCCATCTGC AGAGCTTCCT GGAGGTGTCG TACCGCGTTC TACGCCACCT TGCGCAGCCC ACACCATTGG GCCCTGCCAG CTCCCTGCCC CAGAGCTTCC TGCTCAAGTC TTTAGAGCAA GTGAGAAAGA TCCAGGGCGA TGGCGCAGCG CTCCAGGAGA AGCTGTGTGC CACCTACAAG

("I

>K7- C'> 136 CTGTGCCACC CCGAGGAGCT GGTGCTGCTC GGACACTCTC TGGGCATCCC CTGGGCTCCC CTGAGCTCCT GCCCCAGCCA GGCCCTGCAG CTGGCAGGCT GCTTGAGCCA ACTCCATAGC GGCCTTTTCC TCTACCAGGG GCTCCTGCAG GCCCTGGAAG GGATATCCCC CGAGTTGGGT CCCACCTTGG ACACACTGCA GCTGGACGTC GCCGACTTTG CCACCACCTA ATAA INFORMATION FOR SEQ ID NO:93: SEQUENCE CHARACTERISTICS: LENGTH: 534 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:93:

ATGGCTCAGC

ATGCCGGCCT

CTGCAGAGCT

TTGGGCCCTG

AAGATCCAGG

CACCCCGAGG

TCCTGCCCCA

TTCCTCTACC

TTGGACACAC

AGATGGAAGA

TCGCCTCTGC

TCCTGGAGGT

CCAGCTCCCT

GCGATGGCGC

AGCTGGTGCT

GCCAGGCCCT

AGGGGCTCCT

TGCAGCTGGA

ACTGGGAATG

TTTCCAGCGC

GTCGTACCGC

GCCCCAGAGC

AGCGCTCCAG

GCTCGGACAC

GCAGCTGGCA

GCAGGCCCTG

CGTCGCCGAC

GCCCCTGCCC

CGGGCAGGAG

GTTCTACGCC

TTCCTGCTCA

GAGA.AGCTGT

TCTCTGGGCA

GGCTGCTTGA

GAAGGGATAT

TTTGCCACCA

TGCAGCCCAC CCAGGGTGCC GGGTCCTGGT TGCTAGCCAT

S

9*

C

S

e g.

S. S

S.

C

ACCTTGCGCA

AGTCTTTAGA

GTGCCACCTA

TCCCCTGGGC

GCCAACTCCA

CCCCCGAGTT

CCATCTGGTA

GCCCACACCA

GCAAGTGAGA

CAAGCTGTGC

TCCCCTGAGC

TAGCGGCCTT

GGGTCCCACC

ATAA

INFORMATION FOR SEQ ID NO:94: SEQUENCE CHARACTERISTICS: LENGTH: 534 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:94: ATGGCTTTGT TAGGACATTC TTTAGGTATT CCATGGGCTC CTCTGAGCTC CAGGCCCTGC AGCTGGCAGG CTGCTTGAGC CAACTCCATA GCGGCCTTTT GGGCTCCTGC AGGCCCTGGA AGGGATATCC CCCGAGTTGG GTCCCACCTT CAGCTGGACG TCGCCGACTT TGCCACCACC ATCTGGCAGC AGATGGAAGA GCCCCTGCCC TGCAGCCCAC CCAGGGTGCC ATGCCGGCCT TCGCCTCTGC \,CGGGCAGGAG GGGTCCTGGT TGCTAGCCAT CTGCAGAGCT TCCTGGAGGT .GTTCTACGCC ACCTTGCGCA GCCCACACCA TTGGGCCCTG CCAGCTCCCT 4 ti

CTGCCCCAGC

CCTCTACCAG

GGACACACTG

ACTGGGAATG

TTTCCAGCGC

GTCGTACCGC

GCCCCAGAGC

137 TTCCTGCTCA AGTCTTTAGA GCAAGTGAGA AAGATCCAGG GCGATGGCGC! AGCGCTCCAG GAGAAGCTGT GTGCCACCTA CAAGCTGTGC CACCCCGAGG AGCTGGTGTA ATAA INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 174 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys

S

Se S S

C

S

*55*

S.

S

.5

S

Leu Tyr Pro I le Thr Gly Tyr Ser Lys Tyr 165 Leu Ala Gly Leu Leu Asp Gln Gin Gly Ala Val Leu Val Leu 120 Leu Pro 135 Gln Gly Gly Leu Thr Met Met Val1 105 Arg Gln Asp His Gln Glu Asp Gly Ala Leu Gln 125 Leu Leu Leu Leu Cys Ala Thr Lys Leu Cys INFORMATION FOR SEQ ID NO:96: SEQUENCE CHARACTERISTICS: LENGTH: 174 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein 1~ (xi) SEQUENCE DESCRIPTION: SEQ ID NO:96: Gin Leu His Ser Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu 1 5 10 Glu Gly Ile Ser Pro Glu Leu Gly Pro.Thr Leu Asp Thr Leu Gin Leu 138 Asp Val Ala Asp Phe Ala Thr Thr 40 Gly Met Ala Pro Ala Leu Gln Pro 55 Ala Ser Ala Phe Gln Arg Arg Ala 70 Leu Gln Ser Phe Leu Glu Val Ser Gln Pro Thr Pro Leu Gly Pro Ala 100 Leu Lys Ser Leu Glu Gln Val Arg 115 120 Leu Gln Glu Lys Leu Cys Ala Thr 130 135 Leu Val Leu Leu Gly His Ser Leu i 145 150 Ser Cys Pro Ser Gln Ala Leu Gln 165 INFORMATION FOR SEQ ID NO:97: SEQUENCE CHARACTERISTICS: LENGTH: 174 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein Gin Gly Val Val Leu Gin Leu Pro 155 Gly Leu Leu Leu Phe His 75 Ala Leu Ala Glu Ser i *fl.

Gin Ala Leu Leu Pro Gly Cys 140 Trp Cys Glu Asp Gly Ala Leu Gln Leu Leu Leu Ser Met Met Val Arg Gin Asp 125 His Ala Leu Gly Val Met Ser Gin Pro Lys Gin 125 Val Cys (xi) Leu 1 Glu Ala Leu Arg Glu Gly Gin Cys His SEQUENCE DESCRIPTION: SEQ ID NO:97: Phe Leu Tyr Gin Gly Leu Leu Gin Ala 5 10 Leu Gly Pro Thr Leu Asp Thr Leu Gin 25 Thr Thr Ile Trp Gin Gin Met Glu Glu 35 40 Gin Pro Thr Gin Gly Ala Met Pro Ala 50 55 Arg Ala Gly Gly Val Leu Val Ala Ser 70 Val Ser Tyr Arg Val Leu Arg His Leu 90 Pro Ala Ser Ser Leu Pro Gin Ser Phe 100 105 Val Arg Lys Ile Gin Gly Asp Gly Ala 115 120 Ala Thr Tyr Lys Leu Cys His Pro Glu 130 135 Ser Leu Gly Ile Pro Trp Ala Pro Leu Ile Ser Pro Ala Asp Phe Ala Pro Ala Ala Phe Gin Ser Phe Leu Thr Pro Leu Ser Leu Glu 110 Glu Lys Leu Leu Leu Gly Pro Ser Gin

-C,

139 145 150 155 160 Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His Ser Gly 165 170 INFORMATION FOR SEQ ID NO:98: SEQUENCE CHARACTERISTICS: LENGTH: 174 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:98: Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly Ile Ser Pro Glu Leu a

S

S

S

Gin Leu Ala Gly Leu Ser Gin S(2) INFORMATION FOR SEQ ID NO:99: SEQUENCE CHARACTERISTICS: LENGTH: 174 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:99: S Ala Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu S1 5 10 Gln Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gin Gin Met Glu x 140 Glu Leu Gly Met Ala Pro Ala Leu 40 Ala Phe Ala Ser Ala Phe Gin Arg 55 Ser His Leu Gin Ser Phe Leu Glu 70 Leu Ala Gin Pro Thr Pro Leu Gly Phe Leu Leu Lys Ser Leu Glu Gn 100 Ala Ala Leu Gin Glu Lys Leu Cys 115 120 Glu Glu Leu Val Leu Leu Gly His 130 135 Leu Ser Ser Cys Pro Ser Gin Ala 145 150 Gin Leu His Ser Gly Leu Phe Leu 165 INFORMATION FOR SEQ ID NO:100: SEQUENCE CHARACTERISTICS: LENGTH: 174 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein Gin Arg Val Pro Val 105 Ala Ser Leu Tyr Pro Ala Ser Ala 90 Arg Thr Leu Gin Gin 170 Thr Gly Tyr 75 Ser Lys Tyr Gly Leu 155 Gly Glu Pro Ala His Ser 75 Gly Pro Pro Ser Leu Gin Gly Arg Ser Ile Lys Ile 140 Ala Leu Glu Ala Ser Leu Phe Ala Glu Leu Gin 140 Glu Gly Ala Val Leu Val Leu Leu Pro Gln Gly 110 Leu Cys 125 Pro Trp Gly Cys Leu Gin Leu Gly Phe Ala His Leu Ala Gin Leu Leu Ala Leu Glu Leu 110 Ser Ser 125 Leu His Gly Ile Met Val Arg Gin Asp His Ala Leu Met Ser Gin Pro Lys Gln Val Cys Ser Ser Pro Ala His Ser Gly Pro Pro Ser 160 Ala Ala Ser Thr Ser Glu Leu Pro Gly Pro .u (xi) Asp 1 Pro Phe Phe Pro Leu Lys Leu Ser Leu SEQUENCE DESCRIPTION: SEQ ID NO:100: Phe Ala Thr Thr Ile Trp Gin Gin Met 5 10 Ala Leu Gln Pro Thr Gin Gly Ala Met 20 25 Gln Arg Arg Ala Gly Gly Val Leu Val 40 Leu Glu Val Ser Tyr Arg Val Leu Arg 55 Leu Gly Pro Ala Ser Ser Leu Pro Gin 70 Glu Gln Val Arg Lys Ile Gin Gly Asp 90 Leu Cys Ala Thr Tyr Lys Leu Cys His 100 105 Gly His Ser Leu Gly Ile Pro Trp Ala 115 120 Gin Ala Leu Gin Leu Ala Gly Cys Leu 130 135 Phe Leu Tyr Gln Gly Leu Leu Gln Ala 141 145 150 155 160 Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala 165 170 INFORMATION FOR SEQ ID NO:101: SEQUENCE CHARACTERISTICS: LENGTH: 174 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:101: Ile Trp Gin Gin Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gin Pro 1 5 10 Thr Gin Gly Ala Met Pro Gly Tyr Ser Lys Tyr Gly Leu Gly 145 Gly Val Arg Val Ser Leu Ile Gin Lys Leu Ile Pro 115 Ala Gly 130 Leu Leu Ala His Ser 70 Gly Pro Pro Ser Leu 150 Phe His 40 Ala Leu Ala Glu Ser 120 Leu Gly Ala Leu Gin Leu Leu Leu 105 Ser His Ile

U

U

9 9*9 Ser Gin Pro Lys Gln Val Cys Ser Ser Asp 170 Ala Phe Ser Phe Thr Pro Ser Leu 75 Glu Lys Leu Leu Pro Ser Gly Leu 140 Pro Glu 155 Gin Leu Leu Glu Leu Gly Gin 125 Phe Leu Ala Ser Ala Arg Thr Leu Gin Gin Thr 160 Leu Asp Thr Leu Gin Leu Asp Val Ala 165 INFORMATION FOR SEQ ID NO:102: SEQUENCE CHARACTERISTICS: LENGTH: 174 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein Phe Ala Thr Thr

S

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:102: Gin Gin Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gin Pro Thr Gin 1 5 10 Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gin Arg Arg Ala Gly Gly 142 Val Leu Val Ala Ser His Leu Gin 40 Val Leu Arg His Leu Ala Gin Pro 55 Leu Pro Gin Ser Phe Leu Leu Lys 70 Gin Gly Asp Gly Ala Ala Leu Gin Leu Cys His Pro Glu Glu Leu Val 100 Pro Trp Ala Pro Leu Ser Ser Cys 115 120 Gly Cys Leu Ser Gin Leu His Ser 130 135 Leu Gin Ala Leu Glu Gly Ile Ser 1 145 150 Thr Leu Gin Leu Asp Val Ala Asp I 165 INFORMATION FOR SEQ ID NO:103: SEQUENCE CHARACTERISTICS: LENGTH: 174 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein Phe Leu Pro Leu Leu Glu 75 Lys Leu Leu Gly Ser Gin Leu Phe Glu Leu 155 Ala Thr 170 Val Ser Pro Ala Val Arg Ala Thr Ser Leu 110 Leu Gin 125 Tyr Gin Pro Thr Ile Trp Arg Ser Ile Lys Ile Ala Leu Asp 160 SS (xi) Leu 1 Pro Gly Pro r Phe Ala Gin Leu Leu ,Glu SEQUENCE DESCRIPTION: SEQ ID NO:103: Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro 5 10 Ser Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser 25 Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu 40 Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu 50 55 Ala Thr Thr Ile Trp Gin Gin Met Glu Glu Leu 70 Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe 90 Arg Arg Ala Gly Gly Val Leu Val Ala Ser His 100 105 Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala 115 120 Gly Pro Ala Ser Ser Leu Pro Gin Ser Phe Leu 130 135 140 Gin Val Arg Lys Ile Gin Gly Asp Gly Ala Ala Leu Gin Glu Asp Gly Ala Leu Gin 125 Leu Leu Ser Ser Leu His Gly Ile Val Ala Met Ala Ser Ala Gin Ser 110 Pro Thr Lys Ser Gin Glu 143 145 150 155 Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val 165 170 INFORMATION FOR SEQ ID NO:104: SEQUENCE CHARACTERISTICS: LENGTH: 531 base pairs TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "synthetic" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:104: CAGAGCTTCC TGCTCAAGTC TTTAGAGCAA GTGAGGAAGA TCCAGGGCGA CTCCAGGAGA AGCTGTGTGC CACCTACAAG CTGTGCCACC CCGAGGAGCT GGACACTCTC TGGGCATCCC CTGGGCTCCC CTGAGCTCCT GCCCCAGCCA CTGGCAGGCT GCTTGAGCCA ACTCCATAGC GGCCTTTTCC TCTACCAGGG GCCCTGGAAG GGATATCCCC CGAGTTGGGT CCCACCTTGG ACACACTGCA GCCGACTTTG CCACCACCAT CTGGCAGCAG ATGGAAGAAC TGGGAATGGC CAGCCCACCC AGGGTGCCAT GCCGGCCTTC GCCTCTGCTT TCCAGCGCCG GTCCTGGTTG CTAGCCATCT GCAGAGCTTC CTGGAGGTGT CGTACCGCGT CTTGCGCAGC CCGACATGGC TACACCATTA GGCCCTGCCA GCTCCCTGCC INFORMATION FOR SEQ ID NO:105: SEQUENCE CHARACTERISTICS: LENGTH: 531 base pairs TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc synthetic" (xi) SEQUENCE DESCRIPTION: SEQ ID GAACTGGGAA TGGCCCCTGC CCTGCAGCCC ACCCAGGGTG CCATGCCGGC GCTTTCCAGC GCCGGGCAGG AGGGGTCCTG GTTGCTAGCC ATCTGCAGAG GTGTCGTACC GCGTTCTACG CCACCTTGCG CAGCCCGACA TGGCTACACC GCCAGCTCCC TGCCCCAGAG CTTCCTGCTC AAGTCTTTAG AGCAAGTGAG GGCGATGGCG CAGCGCTCCA GGAGAAGCTG TGTGCCACCT ACAAGCTGTG GAGCTGGTGC TGCTCGGACA CTCTCTGGGC ATCCCCTGGG CTCCCCTGG AGCCAGGCCC TGCAGCTGGC AGGCTGCTTG AGCCAACTCC ATAGCGGCCT CAGGGGCTCC TGCAGGCCCT GGAAGGGATA TCCCCCGAGT TGGGTCCCAC CTGCAGCTGG ACGTCGCCGA CTTTGCCACC ACCATCTGGC AGCAGATGGA 1 -i: c

TGGCGCAGCG

GGTGCTGCTC

GGCCCTGCAG

GCTCCTGCAG

GCTGGACGTC

CCCTGCCCTG

GGCAGGAGGG

TCTACGCCAC

C

CTTCGCCTCT

CTTCCTGGAG

ATTAGGCCCT

GAAGATCCAG

CCACCCCGAG

CTCCTGCCCC

TTTCCTCTAC

CTTGGACACA

A

144 INFORMATION FOR SEQ ID NO:106: SEQUENCE CHARACTERISTICS: LENGTH: 531 base pairs TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY; unknown (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "synthetic" 0* 9 9 9* (xi) SEQUENCE DESCRIPTION: SEQ ID NO:106: GGAATGGCCC CTGCCCTGCA GCCCACCCAG GGTGCCATGC CGGCCTTCGC CAGCGCCGGG CAGGAGGGGT CCTGGTTGCT AGCCATCTGC AGAGCTTCCT TACCGCGTTC TACGCCACCT TGCGCAGCCC GACATGGCTA CACCATTAGG TCCCTGCCCC AGAGCTTCCT GCTCAAGTCT TTAGAGCAAG TGAGGAAGAT GGCGCAGCGC TCCAGGAGAA GCTGTGTGCC ACCTACAAGC TGTGCCACCC GTGCTGCTCG GACACTCTCT GGGCATCCCC TGGGCTCCCC TGAGCTCCTG GCCCTGCAGC TGGCAGGCTG CTTGAGCCAA CTCCATAGCG GCCTTTTCCT CTCCTGCAGG CCCTGGAAGG GATATCCCCC GAGTTGGGTC CCACCTTGGA CTGGACGTCG CCGACTTTGC CACCACCATC TGGCAGCAGA TGGAAGAACT INFORMATION FOR SEQ ID NO:107: SEQUENCE CHARACTERISTICS: LENGTH: 531 base pairs TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "synthetic" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:107: TTCCTGCTCA AGTCTTTAGA GCAAGTGAGG AAGATCCAGG GCGATGGCGC GAGAAGCTGT GTGCCACCTA CAAGCTGTGC CACCCCGAGG AGCTGGTGCT TCTCTGGGCA TCCCCTGGGC TCCCCTGAGC TCCTGCCCCA GCCAGGCCCT GGCTGCTTGA GCCAACTCCA TAGCGGCCTT TTCCTCTACC AGGGGCTCCT GAAGGGATAT CCCCCGAGTT GGGTCCCACC TTGGACACAC TGCAGCTGGA TTTGCCACCA CCATCTGGCA GCAGATGGAA GAACTGGGAA TGGCCCCTGC ACCCAGGGTG CCATGCCGGC CTTCGCCTCT GCTTTCCAGC GCCGGGCAGG GTTGCTAGCC ATCTGCAGAG CTTCCTGGAG GTGTCGTACC GCGTTCTACG CAGCCCGACA TGGCTACACC ATTAGGCCCT GCCAGCTCCC TGCCCCAGAG INFORMATION FOR SEQ ID NO:l08: SEQUENCE CHARACTERISTICS: bt

CTCTGCTTTC

GGAGGTGTCG

CCCTGCCAGC

CCAGGGCGAT

CGAGGAGCTG

CCCCAGCCAG

CTACCAGGGG

CACACTGCAG

G

AGCGCTCCAG

GCTCGGACAC

GCAGCTGGCA

GCAGGCCCTG

CGTCGCCGAC

CCTGCAGCCC

AGGGGTCCTG

:CACCTTGCG

120 180 240 300 360 420 480 531 120 180 240 300 360 420 480 531

NU

145 LENGTH: 531 base pairs TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "synthetic" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1Q8: AGCTTCCTGG AGGTGTCGTA CCGCGTTCTA CGCCACCTTG CGCAGCCCGA CATGGCTACA

CCATTAGGCC

AGGAAGATCC

TGCCACCCCG

AGCTCCTGCC

CTTTTCCTCT

ACCTTGGACA

GAAGAACTGG

CTGCCAGCTC

AGGGCGATGG

AGGAGCTGGT

CCAGCCAGGC

ACCAGGGGCT

CACTGCAGCT

GAATGGCCCC

CCTGCCCCAG

CGCAGCGCTC

GCTGCTCGGA

CCTGCAGCTG

CCTGCAGGCC

GGACGTCGCC

TGCCCTGCAG

AGCTTCCTGC

CAGGAGAAGC

CACTCTCTGG

GCAGGCTGCT

CTGGAAGGGA

GACTTTGCCA

CCCACCCAGG

CTGGTTGCTA

TCAAGTCTTT

TGTGTGCCAC

GCATCCCCTG

TGAGCCAACT

TATCCCCCGA

CCACCATCTG

GTGCCATGCC

GCCATCTGCA

AGAGCAAGTG

CTACAAGCTG

GGCTCCCCTG

CCATAGCGGC

GTTGGGTCCC

GCAGCAGATG

GGCCTTCGCC

G TCTGCTTTCC AGCGCCGGGC AGGAGGGGTC INFORMATION FOR SEQ ID NO:109: SEQUENCE CHARACTERISTICS: LENGTH: 531 base pairs TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE; other nucleic acid DESCRIPTION: /desc "synthetic" 4 4 .4

S

S

S

S.

4 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l09: AGCTTCCTGG AGGTGTCGTA CCGCGTTCTA CGCCACCTTG C CCATTAGGCC CTGCCAGCTC CCTGCCCCAG AGCTTCCTGC T AGGAAGATCC AGGGCGATGG CGCAGCGCTC CAGGAGAAGC T TGCCACCCCG AGGAGCTGGT GCTGCTCGGA CACTCTCTGG G AGCTCCTGCC CCAGCCAGGC CCTGCAGCTG GCAGGCTGCT T CTTTTCCTCT ACCAGGGGCT CCTGCAGGCC CTGGAAGGGA T.

ACCTTGGACA CACTGCAGCT GGACGTCGCC GACTTTGCCA C GAAGAACTGG GAATGGCCCC TGCCCTGCAG CCCACCCAGG G TCTGCTTTCC AGCGCCGGGC AGGAGGGGTC CTGGTTGCTA G INFORMATION FOR SEQ ID NO:110: SEQUENCE CHARACTERISTICS: LENGTH: 531 base pairs TYPE: nucleic acid STRANOEDNESS: unknown TOPOLOGY: unknown

GCAGCCCGA

CAAGTCTTT

GTGTGCCAC

CATCCCCTG

GAGCCAACT

ATCCCCCGA

CACCATCTG

TGCCATGCC

CCATCTGCA

CATGGCTACA

AGAGCAAGTG

CTACAAGCTG

GGCTCCCCTG

CCATAGCGGC

GTTGGGTCCC

GCAGCAGATG

GGCCTTCGCC

G

V.

1 V 146 (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "synthetic" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:110: TTAGGCCCTG CCAGCTCCCT G2CCCAGAGC TTCCTGCTCA AGTCTTTAGA GCAAGTGAGG AAGATCCAGG GCGATGGCGC AGCGCTCCAG GAGAAGCTGT GTGCCACCTA CAAGCTGTGC CACCCCGAGG AGCTGGTGCT GCTCGGACAC TCTCTGGGCA TCCCCTGGGC TCCCCTGAGC TCCTGCCCCA GCCAGGCCCT GCAGCTGGCA GGCTGCTTGA GCCAACTCCA TAGCGGCCTT TTCCTCTACC AGGGGCTCCT GCAGGCCCTG GAAGGGATAT CCCCCGAGTT GGGTCCCACC TTGGACACAC TGCAGCTGGA CGTCGCCGAC TTTGCCACCA CCATCTGGCA GCAGATGGAA GAACTGGGAA TGGCCCCTGC CCTGCAGCCC ACCCAGGGTG CCATGCCGGC CTTCGCCTCT GCTTTCCAGC GCCGGGCAGG AGGGGTCCTG GTTGCTAGCC ATCTGCAGAG CTTCCTGGAG GTGTCGTACC GCGTTCTACG CCACCTTGCG CAGCCCGACA TGGCTACACC A INFORMATION FOR SEQ ID NO:lll: SEQUENCE CHARACTERISTICS: LENGTH: 531 base pairs TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "synthetic" 9 9 9 9 0 *999 9999 9 9 99 9 9.

99 0 (xi) SEQUENCE DESCRIPTION:.SEQ ID NO:lll: CTGCTCGGAC ACTCTCTGGG CATCCCCTGG GCTCCCCTGA GCTCCTGCCC CAGCCAGGCC

CTGCAGCTGG

CTGCAGGCCC

GACGTCGCCG

GCCCTGCAGC

GGAGGGGTCC

CGCCACCTTG

AGCTTCCTGC

CAGGCTGCTT

TGGAAGGGAT

ACTTTGCCAC

CCACCCAGGG

TGGTTGCTAG

CGCAGCCCGA

TCAAGTCTTT

GAGCCAACTC

ATCCCCCGAG

CACCATCTGG

TGCCATGCCG

CCATCTGCAG

CATGGCTACA

AGAGCAAGTG

CATAGCGGCC TTTTCCTCTA TTGGGTCCCA CCTTGGACAC CAGCAGATGG AAGAACTGGG GCCTTCGCCT CTGCTTTCCA AGCTTCCTGG AGGTGTCGTA CCATTAGGCC CTGCCAGCTC AGGAAGATCC AGGGCGATGG

CCAGGGGCTC

ACTGCAGCTG

AATGGCCCCT

GCGCCGGGCA

CCGCGTTCTA

CCTGCCCCAG

CGCAGCGCTC

CAGGAGAAGC TGTGTGCCAC CTACAAGCTG TGCCACCCCG AGGAGCTGGT G INFORMATION FOR SEQ ID NO:112: SEQUENCE CHARACTERISTICS: LENGTH: 531 base pairs TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc ="synthetic" L1 531 147 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:112: CCCCTGAGCT CCTGCCCCAG CCAGGCCCTG CAGCTGGCAG GCTGCTTGAG AGCGGCCTTT TCCTCTACCA GGGGCTCCTG CAGGCCCTGG AAGGGATATC GGTCCCACCT TGGACACACT GCAGCTGGAC GTCGCCGACT TTGCCACCAC CAGATGGAAG AACTGGGAAT GGCCCCTGCC CTGCAGCCCA CCCAGGGTGC TTCGCCTCTG CTTTCCAGCG CCGGGCAGGA GGGGTCCTGG TTGCTAGCCA TTCCTGGAGG TGTCGTACCG CGTTCTACGC CACCTTGCGC AGCCCGACAT TTAGGCCCTG CCAGCTCCCT GCCCCAGAGC TTCCTGCTCA AGTCTTTAGA AAGATCCAGG GCGATGGCGC AGCGCTCCAG GAGAAGCTGT GTGCCACCTA CACCCCGAGG AGCTGGTGCT GCTCGGACAC TCTCTGGGCA TCCCCTGGGC INFORMATION FOR SEQ ID NO:l13: SEQUENCE CHARACTERISTICS: LENGTH: 531 base pairs TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "synthetic"

CCAACTCCAT

CCCCGAGTTG

CATCTGGCAG

CATGCCGGCC

TCTGCAGAGC

GGCTACACCA

GCAAGTGAGG

CAAGCTGTGC

T

see* 0 so*.

S

0

S

5.

S

S

9* *9 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1l3: CAGGCCCTGC AGCTGGCAGG CTGCTTGAGC CAACTCCATA GCGGCCTTTT GGGCTCCTGC AGGCCCTGGA AGGGATATCC CCCGAGTTGG GTCCCACCTT CAGCTGGACG TCGCCGACTT TGCCACCACC ATCTGGCAGC AGATGGAAGA GCCCCTGCCC TGCAGCCCAC CCAGGGTGCC ATGCCGGCCT TCGCCTCTGC CGGGCAGGAG GGGTCCTGGT TGCTAGCCAT CTGCAGAGCT TCCTGGAGGT GTTCTACGCC ACCTTGCGCA GCCCGACATG GCTACACCAT TAGGCCCTGC CCCCAGAGCT TCCTGCTCAA GTCTTTAGAG CAAGTGAGGA AGATCCAGGG GCGCTCCAGG AGAAGCTGTG TGCCACCTAC AAGCTGTGCC ACCCCGAGGA CTCGGACACT CTCTGGGCAT CCCCTGGGCT CCCCTGAGCT CCTGCCCCAG INFORMATION FOR SEQ ID NO:114: SEQUENCE CHARACTERISTICS: LENGTH: 531 base pairs TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "synthetic"

CCTCTACCAG

GGACACACTG

ACTGGGAATG

TTTCCAGCGC

GTCGTACCGC

CAGCTCCCTG

CGATGGCGCA

GCTGGTGCTG

C

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:114:

I'

148 CTGCAGCTGG CAGGCTGCTT GAGCCAACTC CATAGCGGCC TTTTCCTCTA CTGCAGGCCC TGGAAGGGAT ATCCCCCGAG TTGGGTCCCA CCTTGGACAC GACGTCGCCG ACTTTGCCAC CACCATCTGG CAGCAGATGG AAGAACTGGG GCCCTGCAGC CCACCCAGGG TGCCATGCCG GCCTTCGCCT CTGCTTTCCA.

GGAGGGGTCC TGGTTGCTAG CCATCTGCAG AGCTTCCTGG AGGTGTCGTA.

CGCCACCTTG CGCAGCCCGA CATGGCTACA CCATTAGGCC CTGCCAGCTC AGCTTCCTGC TCAAGTCTTT AGAGCAAGTG AGGAAGATCC AGGGCGATGG CAGGAGAAGC TGTGTGCCAC CTACAAGCTG TGCCACCCCG AGGAGCTGGT CACTCTCTGG GCATCCCCTG GGCTCCCCTG AGCTCCTGCC CCAGCCAGGC INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 531 base pairs TYPE: nucleic acid STRAN~DEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "synthetic"

CCAGGGGCTC

ACTGCAGCTG

AATGGCCCCT

GCGCCGGGCA

CCGCGTTCTA

CCTGCCCCAG,

CGCAGCGCTC

GCTGCTCGGA

C

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:iis: CTGGCAGGCT GCTTGAGCCA ACTCCATAGC GGCCTTTTCC TCTACCAGGG GCTCCTGCAG 0 0

S.

00 0

GCCCTGGAAG

GCCGACTTTG

CAGCCCACCC

GTCCTGGTTG

CTTGCGCAGC

CTGCTCAAGT

AAGCTGTGTG

GGATATCCCC

CCACCACCAT

AGGOTGCCAT

CTAGCCATCT

CCGACATGGC

CTTTAGAGCA

CCACCTACAA

CGAGTTGGGT

CTGGCAGCAG,

GCCGGCCTTC

GCAGAGCTTC

TACACCATTA

AGTGAGGAAG

GCTGTGCCAC

CCCACCTTGG

ATGGAAGAAC

GCCTCTGCTT

CTGGAGGTGT

GGCCCTGCCA

ATCCAGGGCG

CCCGAGGAGC

ACACACTGCA

TGGGA.ATGGC

TCCAGCGCCG

CGTACCGCGT

GCTCCCTGCC

ATGGCGCAGC

TGGTGCTGCT

GCTGGACGTC

CCCTGCCCTG

GGCAGGAGGG

TCTACGCCAC

CCAGAGCTTC

GCTCCAGGAG

CGGACACTCT

CTGGGCATCC CCTGGGCTCC CCTGAGCTCC TGCCCCAGCC AGGCCCTGCA G INFORMATION FOR SEQ ID NO:iiG: SEQUENCE CHARACTERISTICS: LENGTH: 177 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:116: Gin Ser Phe Leu Leu Lys Ser Leu Giu Gin Val Arg Lys Ile Gin Gly 1 5 10 'Asp Giy Ala Ala Leu Gin Giu Lys Leu Cys Ala Thr Tyr Lys Leu Cys

N

149 His Pro Giu Giu Leu Val Leu Leu( Ala Pro Leu Ser Ser Cys Pro Ser( Leu Ser Gin Leu His Ser Gly Leu 1 Ala Leu Giu Gly Ile Ser Pro Glu 1 Gin Leu Asp Val Ala Asp Phe Ala 100 Giu Leu Gly Met Ala Pro Ala Leu 115 120 Ala Phe Ala Ser Ala Phe Gin Arg 130 135 Ser His Leu Gin Ser Phe Leu Giu 145 150 Leu Ala Gin Pro Asp Met Ala Thr 165 Pro INFORMA.TION FOR SEQ ID NO:117: SEQUENCE CHARACTERISTICS: LENGTH; 177 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide Ile Pro Ala Gly Leu Leu Asp Thr Gin Met 110 Ala Met Leu Val Leu Arg Ser Ser 175 a a a a a a a.

a (xi) Giu 1 Al a Ser Leu Pro Gly Cys SEQUENCE DESCRIPTION: SEQ ID NO:117: Leu Gly Met Ala Pro Ala Leu Gin Pro 5 Phe Ala Ser Ala Phe Gin Arg Arg Ala 20 His Leu Gin Ser Phe Leu Giu Val Ser Ala Gin Pro Asp Met Ala Thr Pro Leu 50 Gin Ser Phe Leu Leu LYS Ser Leu Glu Asp Gly Ala Ala Leu Gin Giu Lys Leu His Pro Giu Giu Leu Val Leu Leu Gly 100 105 Ala Pro Leu Ser Ser Cys Pro Ser Gin 115 120 Leu Ser Gin Leu His Ser Gly Leu Phe Xi 7,

C

Cys 150 130 135 140 Gin Ala Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr 145 150 155 160 Leu Gin Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gin Gin Met 165 170 175 Glu INFORMATION FOR SEQ ID NO:118: SEQUENCE CHARACTERISTICS: LENGTH: 177 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:118: Gly Met Ala Pro Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe 1 5 10 SSer Phe Leu Leu Lys Ser Leu Glu 65 Gly Ala Ala Leu Gin Glu Lys Leu Pro Glu Glu Leu Val Leu Leu Gly I i 100 Pro Leu Ser Ser Cys Pro Ser Gin j 115 120 Ser Gin Leu His Ser Gly Leu Phe I 130 135 e Leu Glu Gly Ile Ser Pro Glu Leu 145 150 Leu Asp Val Ala Asp Phe Ala Thr 165 Leu INFORMATION FOR SEQ ID NO:119: SEQUENCE CHARACTERISTICS: LENGTH: 177 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown V (ii) MOLECULE TYPE: peptide 5 151 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:ii9:' Phe Leu Leu Lys Ser Leu Giu Gin Val Arg Lys Ile Gin Giy Asp Giy Ala Ala Glu Giu Leu Ser Gin Leu Giu Gly Asp Vai Gly Met Aia Ser 130 Leu Gin 145 Gin Pro Thr Leu Gin Gin Thr Trp Gin Gly Arg Pro 170 His Ala Leu Ala Gin Giu Ala Ser Leu Pro 175 9 *i e' 9 9 9* 9* i *99* 9 *i Ser INFORMATION FOR SEQ ID NO:120: SEQUENCE CHARACTERISTICS: LENGTH: 177 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) Ser Asp Leu Ala Glu Ser SEQUENCE DESCRIPTION: SEQ ID NO:120: Phe Leu Giu Val Ser Tyr Arg Val Leu 5 Met Ala Thr Pro Leu Gly Pro Ala Ser Leu Lys Ser Leu Giu Gin Val Arg Lys Leu Gin Giu Lys Leu Cys Ala Thr Tyr Leu Val Leu Leu Gly His Ser Leu Gly Ser Cys Pro Ser Gin Ala Leu Gin Leu 152 Leu His Ser Gly Leu Phe Leu Tyr 100 Gly Ile Ser Pro Giu Leu Gly Pro 115 120 Val Ala Asp Phe Ala Thr Thr Ile 130 135 Met Ala Pro Ala Leu Gln Pro Thr 145 150 Ser Ala Phe Gin Arg Arg Ala Gly 165 Gin INFORMATION FOR SEQ ID NO:121: SEQUENCE CHARACTERISTICS: LENGTH: 177 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide 90 Gly Leu Leu Gin Ala 110 Leu Asp Thr Leu Gin 125 Gin Gin Met Giu Giu 140 Gly Ala Met Pro Ala 155 Val Leu Val Ala Ser 170 Leu Giu Leu Asp Leu Gly Phe Ala 160 His Leu 175 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:121: Glu Gin Val Arg Lys Ile Gin Gly Asp Gly Ala Ala Leu Gin Glu Lys b i.* 4

S..

Leu Gly Gin Phe Leu Thr Gin Arg Val 145 Pro (2) V

V~N

INFORMATION FOR. SEQ ID NO:122: 153 i)SEQUENCE CHARACTERISTICS: LENGTH: 177 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0U.22: Gly Pro Ala Ser Ser Leu Pro Gin Ser Phe Leu Leu Lys Ser Leu 10 p inC..

n.m m *mm.

*.m m mm em m,

U

mm in .m.

me..

C

9 mm.m *mmm m m m Cm m.

mm Cm m mm i m m~m in inmmm INFORMATION FOR SEQ ID NO:123: SEQUENCE CHARACTERISTICS: LENGTH: 177 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) Leu Pro 'Gly SEQUENCE DESCRIPTION: SEQ ID NO:123: Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys 10 Ser Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His Ser 25 Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Giu Gly Ile Ser 154 40 Pro Glu Leu Gly Pro Thr Leu Asp 55 Phe Ala Thr Thr Ile Trp Gin Gin 70 Ala Leu Gin Pro Thr Gin Gly Ala Gin Arg Arg Ala Gly Gly Val Leu 100 Leu Glu Val Ser Tyr Arg Val Leu 115 120 Ala Thr Pro Leu Gly Pro Ala Ser 130 135 Lys Ser Leu Glu Gin Val Arg Lys 145 150 Gin Glu Lys Leu Cys Ala Thr Tyr 165 Val INFORMATION FOR SEQ ID NO:124: SEQUENCE CHARACTERISTICS: LENGTH: 177 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide 0 9 e a r 0 9.

i a '0 *0

U

Leu Asp Leu Gly Phe Ala His Leu Ala Gin 125 Gin Ser 140 Asp Gly His Pro Leu Ala Gly Leu Leu Asp Gin Gin Gly Ala Val Leu Val Leu Ala Ser 125 Arg Lys 140 Thr Tyr Ala Asp Ala Pro Ala Phe Ser Phe Asp Met Leu Leu Ala Leu 160 Glu Leu 175 Cys Leu Gin Ala Leu Gin Glu Glu Pro Ala Ala Ser His Leu Leu Pro Gin Gly Leu Cys (xi) Pro 1 Ser Leu Leu Leu Phe His Ala Gin Asp SEQUENCE DESCRIPTION: SEQ ID NO:124: Leu Ser Ser Cys Pro Ser Gin Ala Leu 5 10 Gin Leu His Ser Gly Leu Phe Leu Tyr Glu Gly Ile Ser Pro Glu Leu Gly Pro 40 Asp Val Ala Asp Phe Ala Thr Thr Ile 50 55 Gly Met Ala Pro Ala Leu Gin Pro Thr 70 Ala Ser Ala Phe Gin Arg Arg Ala Gly 90 Leu Gin Ser Phe Leu Glu Val Ser Tyr 100 105 Gin Pro Asp Met Ala Thr Pro Leu Gly 115 120 Ser Phe Leu Leu Lys Ser Leu Glu Gin 130 135 Gly Ala Ala Leu Gin Glu Lys Leu Cys 2 N 155 145 150 155 160 His Pro Giu Giu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp 165 170 175 Ala INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 177 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His Ser Gly Leu

U-

U

U

:s

U.

U,

US

U

-U

U

S

U.

U

i Ser INFORMATION FOR SEQ ID NO:i26: SEQUENCE CHARACTERISTICS: LENGTH: 177 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide N 156 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:126: Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His Ser Gly Leu Phe Leu 1 5 10 Glu Gly Asp Val Gly Met Ala Ser Leu Gin Gin Pro 105 Ser Phe Gly Ala Pro Glu Pro Leu 170 ::f a a a.

ah Ala INFORMATION FOR SEQ ID NO:127: SEQUENCE CHARACTERISTICS: LENGTH: 177 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) Leu 1 Gly Leu Gin Gly Val Val SEQUENCE DESCRIPTION: SEQ ID NO:127: Ala Gly Cys Leu Ser Gin Leu His Ser 5 Leu Leu Gin Ala Leu Glu Gly Ile Ser 20 Asp Thr Leu Gin Leu Asp Val Ala Asp Gin Met Glu Glu Leu Gly Met Ala Pro Ala Met Pro Ala Phe Ala Ser Ala Phe Leu Val Ala Ser His Leu Gin Ser Phe 1 Leu Arg His Leu Ala Gin Pro Asp Met LI. 157 100 105 Ala Ser Ser Leu Pro Gin Ser Phe Leu 115 120 Arg Lys Ile Gin Gly Asp Gly Ala Ala 130 135 Thr Tyr Lys Leu Cys His Pro Glu Glu 145 150 Leu Gly Ile Pro Trp Ala Pro Leu Ser 165 Gin INFORMATION FOR SEQ ID NO:128: SEQUENCE CHARACTERISTICS: LENGTH: 177 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide 110 Leu Lys Ser Leu Glu Gin Val 125 Leu Gin Glu Lys Leu Cys Ala 140 Leu Val Leu Leu Gly His Ser 155 160 Ser Cys Pro Ser Gin Ala Leu 170 175 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:128: His Leu Ala Gin Pro Asp Met Ala Thr Pro Leu Gly Pro Ala Ser Ser 4. 9 9 *c4 4 4c 4 a.

S.

Leu Gin Leu Pro 65 Gly Leu Thr Met Met 145 Val Leu Glu Lys Leu Leu Gly Ser Gin Leu Phe Glu Leu Ala Thr Leu Gin Arg Arg 155 Glu Val 170 INFORMATION FOR SEQ ID NO:129: S(i) SEQUENCE CHARACTERISTICS: LENGTH: 531 base pairs 158 TYPE: nucleic acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "synthetic" (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:129: CACCTTGCGC AGCCCGACAT GGCTACACCA TTAGGCCCTG, CCAGCTCCCT TTCCTGCTCA AGTCTTTAGA GCAAGTGAGG AAGATCCAGG GCGATGGCGC GAGAAGCTGT GTGCCACCTA CAAGCTGTGC CACCCCGAGG AGCTGGTGCT TCTCTGGGCA TCCCCTGGGC TCCCCTGAGC TCCTGCCCCA GCCAGGCCCT GGCTGCTTGA GCCAACTCCA TAGCGGCCTT TTCCTCTACC AGGGGCTCCT GAAGGGATAT CCCCCGAGTT GGGTCCCACC TTGGACACAC TGCAGCTGGA TTTGCCACCA CCATCTGGCA GCAGATGGAA GAACTGCGAA TGGCCCCTGC ACCCAGGGTG CCATGCCGGC CTTCGCCTCT GCTTTCCAGC GCCGGGCAGG GTTGCTAGCC ATCTGCAGAG CTTCCTGGAG GTGTCGTACC GCGTTCTACG

GCCCCAGAGC

AGCGCTCCAG

GCTCGGACAC

GCAGCTGGCA

GCAGGCCCTG

CGTCGCCGAC

CCTGCAGCCC

AGGGGTCCTG

C

C. *e

C

a

C

9~ *ea.

C C ~j1

NZ~

Claims (10)

1. A human G-CSF receptor agonist polypeptide, comprising a modified G-CSF amino acid sequence comprising a sequence selected from the group consisting of: the sequence of SEQ ID NO:1; wherein t...2 :2 ,2 St Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa at position at position at position at position at position at position Arg; at position at position at position at position at position at position at position at position at position is is is is is Sis is is is is is is is is is 3 Phe, Lys, Cys, Ser, Pro, Ser, His, Ser, Gly, Thr or Pro; Thr or His; Ala, Ile, Tyr or Thr, Ser, Arg, Tyr or Gly; Pro or Leu; Leu, Arg, Tyr or Ser; Leu, Thr, Pro, His, Ile or Cys; Arg, Tyr, Ser, Thr or Ala; Ile, Pro, Tyr or Leu; Asp, or Gly; Ala, Ile, Leu or Gly; Lys or Ser; Cys; Cys; His, Thr, Gly, Val, Lys, Trp, Ala, Arg, Cys, or Leu; Xaa at position 44 is Pro, Gly, Arg, Asp, Val, Ala, His, Trp, Gin, or Thr; Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa at position at position at position at position at position at position at position at position at position at position at position Gly; at position at position at position at position 46 is Glu, Arg, Phe, Arg, Ile or Ala; 47 is Leu or Thr; 49 is Leu, Phe, Arg or Ser; 50 is Leu, Ile, His, Pro or Tyr; 54 is Leu or His; 64 is Cys; 67 is Gin, Lys, Leu or Cys; 70 is Gin, Pro, Leu, Arg or Ser; 74 is Cys; 104 is Asp, Gly or Val; 108 is Leu, Ala, Val, Arg, Trp, Gin or A 45 -i 01; 115 120 123 144 Thr, His, Leu or Ala; Gin, Gly, Arg, Lys or His Glu, Arg, Phe or Thr Phe, His, Arg, Pro, Leu, Gin or Glu; 160 Xaa at position 146 is Xaa at position 147 is Xaa at position 156 is Xaa at position 159 is Xaa at position 162 is Xaa at position 163 is Xaa at position 169 is Xaa at position 170 is residues to Arg or Gin; Arg or Gin; His, Gly or Ser; Ser, Arg, Thr, Tyr, Val or Gly; Glu, Leu, Gly or Trp; Val, Gly, Arg or Ala; Arg, Ser, Leu, Arg or Cys; His, Arg or Ser;

12-174 of SEQ ID NO:1 according i 0@ 0* 25 residues 1-169 of SEQ ID NO:1 according to residues 12-169 of SEQ ID NO:1 according to and a polypeptide comprising the sequence according to or and an N- terminal methionine residue, alanine residue or methionine-alanine di-peptide wherein said methionine residue, alanine residue or methionine-alanine di-peptide immediately preceding said sequence according to or a- sq ft f wherein the N-terminus is joined to the C-terminus directly or through a linker and wherein a new C-terminus and N- terminus are created between the amino acid reside pairs of SEQ ID NO:1 selected from the group consisting of:

38-39, 39-40, 40-41, 41-42,

48-49, 49-50, 52-53, 53-54, 35 57-58, 58-59, 59-60, 60-61, S' 64-65, 65-66, 66-67, 67-68, -0 1

91-92, 92-93, 93-94, 94-95, V 42-43, 54-55, 61-62, 69-70,

95-96, 43-44, 45-46, 55-56, 56-57, 62-63, 63-64, 70-71, 71-72,

96-97, 97-98, 161

98-99, 99-100, 123-124, 124-125, 125-126, 126-127,

127-128, 128-129, 129-130, 130-131, 131-132, 132- 133, 133-134, 134-135, 135-136, 136-137, 137-138,

138-139, 139-140, 140-141, 141-142 and 142-143. 2. The G-CSF receptor agonist polypeptide, as recited in claim 1, wherein said linker is selected from the group consisting of; (SEQ ID NO:2), (SEQ ID NO:61), (SEQ ID NO:62), (SEQ ID NO:63), (SEQ ID NO:64), (SEQ ID NO:65), (SEQ ID NO:66), and (SEQ ID NO:67). 3. The G-CSF receptor agonist polypeptide of claim 1, selected from the group consisting of: (SEQ ID NO:48), (SEQ ID NO:49), (SEQ ID NO:50), (SEQ ID NO:51) and (SEQ ID NO:52). 20 4. A nucleic acid molecule comprising a DNA sequence S encoding the G-CSF receptor agonist polypeptide of claim .1. A nucleic acid molecule comprising a DNA sequence encoding the G-CSF receptor agonist polypeptide of claim 2. 6. A nucleic acid molecule comprising a DNA sequence encoding the G-CSF receptor agonist polypeptide of claim 3. 7. The nucleic acid molecule of claim 6 selected from group consisting of: Li 0 162 (SEQ ID NO:30), (SEQ ID NO:31), (SEQ ID NO:32), (SEQ ID NO:33), (SEQ ID NO:34), (SEQ ID NO:35), (SEQ ID NO:36), (SEQ ID NO:37), (SEQ ID NO:38), (SEQ ID NO:39), (SEQ ID and (SEQ ID NO:42). 8. A method of producing a G-CSF receptor agonist polypeptide comprising: growing under suitable nutrient conditions, a host cell transformed or transfected with a replicable vector comprising said nucleic acid molecule of claim 4, 5, 6, or 7 in a manner allowing expression of said G-CSF receptor agonist polypeptide and recovering said G- CSF receptor agonist polypeptide. 15 a a a a.. a a a a 9. The G-CSF receptor agonist polypeptide of any one of claims 1 to 3 when prepared by the method of claim 8. 10. A composition comprising the G-CSF receptor agonist polypeptide according to claim 1, 2, 3 or 9 and a pharmaceutically acceptable carrier. 11. A composition comprising: a G-CSF receptor agonist polypeptide according to claim 1, 2, 3 or 9; a colony stimulating factor; and a pharmaceutically acceptable carrier. 12. A composition comprising: a G-CSF receptor agonist polypeptide according to claim 1, 2, 3 or 9; a colony stimulating factor selected from the.group consisting of: -1) r S41 GM-CSF, c-mpl ligand, M-CSF, erythropoietin, IL-1, IL-4, IL-2, IL-3, IL-5, IL 6, IL-7, IL-8, IL-9, IL-10, IL-11, IL- 12, IL-13, IL-15, LIF, flt3 ligand, human growth hormone, B-cell growth factor, B-cell differentiation factor, eosinophil differentiation factor and stem cell factor; and a pharmaceutically acceptable carrier. 13. A method of stimulating the production of hematopoietic cells in a patient comprising the step of administering said G-CSF receptor agonist polypeptide of claim 1, 2, 3 or 9 to said patient. 15 S. 20 0 9* 50 25 14. A method of stimulating the production of hematopoietic cells in a patient comprising the step of administering said composition of claim 10 to said patient. 15. A method of stimulating the production of hematopoietic cells in a patient comprising the step of administering said composition of claim 11 or 12 to said patient. 16. A method for selective ex vivo expansion of stem cells, comprising the steps of: separating stem cells from other cells; culturing said separated stem cells with a selected culture medium comprising the polypeptide of claim 1, 2, 3 or 9; and harvesting said cultured cells. 17. A method for selective ex vivo expansion of stem ,'..cells, comprising the steps of: '0 separating stem cells from other cells; 164 culturing said separated stem cells with a selected culture medium comprising the composition of claim and harvesting said cultured cells. 18. A method for selective ex vivo expansion of stem cells, comprising the steps of: separating stem cells from other cells; culturing said separated stem cells with a selected culture medium comprising the composition of claim 11 or 12; harvesting said cultured cells. 9 0i i 9 V V V V. *9 V V 19. A method for treatment of a patient having a 15 hematopoietic disorder, comprising the steps of: removing stem cells from said patient; separating stem cells from other cells; culturing said separated stem cells with a selected culture medium comprising the polypeptide of claim 1, 2, 3 or 9; harvesting said cultured cells; and transplanting said cultured cells into said patient. A method for treatment of a patient having a hematopoietic disorder, comprising the steps of: removing stem cells from said patient; separating stem cells from other cells; culturing said separated stem cells with a selected culture medium comprising the composition of claim harvesting said cultured cells; and 2 4 165 transplanting said cultured cells into said patient. 21. A method for treatment of a patient having a hematopoietic disorder, comprising the steps of: removing stem cells; separating stem cells from other cells; culturing said separated stem cells with a selected culture medium comprising the composition of claim 11 or 12; harvesting said cultured cells; and transplanting said cultured cells into said patient. removing stem cells from a patient; S 20 culturing said separated stem cells with a selected culture medium comprising the hematopoietic protein of claim 1, 2, 3 or 9; introducing DNA into said cultured cells; harvesting said transduced cells; and 25 transplanting said transduced cells into said 6* patient. 23. A method of human gene therapy, comprising the steps of: removing stem cells from a patient; separating said stem cells from other cells; '25 culturing said separated stem cells with a SIL-selected media comprising the composition of claim U e (oefp ,(rtm 166 introducing DNA into said cultured cells; harvesting said transduced cells; and transplanting said transduced cells into said patient. 24. A method of human gene therapy, comprising the steps of: removing stem cells from a patient; separating said stem cells from other cells; culturing said separated stem cells with a selected media comprising the composition of claim 11 or 12; 1 S S (d) (e) (f) introducing DNA into said cultured cells; harvesting said transduced cells; and transplanting said transduced cells into said patient. S 25. isolated S 26. isolated S. 27. isolated 28. isolated 0- 29. isolated C2.. V .1:TJ A method of claim 19 wherein said stem cells are from peripheral blood. A method of claim 20 wherein said stem cells are from peripheral blood. A method of claim 21 wherein said stem cells are from peripheral blood. A method of claim 22 wherein said stem cells are from peripheral blood. A method of claim 23 wherein said stem cells are from peripheral blood. 167 A method of claim 24 wherein said stem cells are isolated from peripheral blood. 31. The use of the G-CSF receptor agonist polypeptide of any one of claims 1-3 or 9 for preparing medicament for increasing hematopoietic cell production in a mammal in need thereof. DATED this 4th day of February 2000 G. D. SEARLE CO., By its Patent Attorneys, F -WELLINGTON CO. Os.. 0 0 0000 *000 0 0 @000 00 @0 S. 0e 0* @0 0 0 0 00 6 00S 0 0 S @500 @000 0 0 00 0 00 60 0 S 00@0 0 0006 (Bruce Well BA! 2016 .jTh ~-0 C