WO1998015624A9 - Galectin 8, 9, 10 and 10sv - Google Patents

Galectin 8, 9, 10 and 10sv

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Publication number
WO1998015624A9
WO1998015624A9 PCT/US1997/018261 US9718261W WO9815624A9 WO 1998015624 A9 WO1998015624 A9 WO 1998015624A9 US 9718261 W US9718261 W US 9718261W WO 9815624 A9 WO9815624 A9 WO 9815624A9
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seq
gly
galectin
val
leu
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PCT/US1997/018261
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French (fr)
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WO1998015624A1 (en
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Priority to JP10517750A priority Critical patent/JP2001501831A/en
Priority to CA002268022A priority patent/CA2268022A1/en
Priority to EP97912689A priority patent/EP1012266A4/en
Publication of WO1998015624A1 publication Critical patent/WO1998015624A1/en
Publication of WO1998015624A9 publication Critical patent/WO1998015624A9/en

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  • the present invention relates to novel galectins. More specifically, isolated nucleic acid molecules are provided encoding human galectin 8, 9, 10, or 10SV. Galectin 8, 9, 10 and 10SV polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the same. The invention further relates to screening methods for identifying agonists and antagonists of galectin 8, 9, 10, or 10SV activity. Also provided are diagnostic methods for detecting cell growth disorders and therapeutic methods for cell growth disorders, including autoimmune diseases, cancer, and inflammatory diseases.
  • Lectins are proteins that bind to specific carbohydrate structures and can thus recognize particular glycoconjugates. Barondes et al, J. Biol. Chem.
  • Galectins are members of a family of ⁇ -galactoside-binding lectins with related amino acid sequences (For review see,
  • Galectin 1 (aka. L-14-1, L-14, RL-14.5, galaptin, MGBP, GBP, BHL, CHA, HBP, HPL, HLBP 14, rIML-1) is a homodimer with a subunit molecular mass of 14,500 which is abundant in smooth and skeletal muscle, and is present in many other cell types (Couraud et al, J. Biol. Chem. 264:1310-1316 (1989)).
  • Galectin 2 was originally found in hepatoma and is a homodimer with a subunit molecular weight of 14,650 (Gitt et al, J. Biol. Chem. 257. 10601-10606 (1992)).
  • Galectin 3 (aka. Mac-2, EPB,
  • CBP-35, CBP-30, and L-29 is abundant in activated macrophages and epithelial cells and is a monomer with an apparent molecular mass between 26,320 and 30,300 (Cherayil et al, Proc. Natl Acad. Sci. USA 87: 7324-7326 (1990)).
  • Galectin 4 has a molecular mass of 36,300 and contains two carbohydrate-binding domains within a single polypeptide chain (Oda et al, J. Biol. Chem. 268:5929- 5939 (1993)).
  • Galectins 5 and 6 are mentioned in Barondes et al, Cell 76:591- 598 (1994).
  • Human galectin 7 has a molecular mass of 15,073 and is found mainly in stratified squamous epithelium (Madsen et al, J. Biol Chem. 270(11):5823-5S29 (1995)).
  • Galectin 1 has been shown to either promote or inhibit cell adhesion depending upon the cell type in which it is present. Galectin 1 inhibits cell-matrix interactions in skeletal muscle (Cooper et al, J. Cell Biol. 115:1431- 1448 (1991)). In other cell types, galectin 1 promotes cell-matrix adhesion possibly by cross-linking cell surface and substrate glycoconjugates (Zhou et al, Arch. Biochem. Biophys. 300:6-11 (1993); Skrincosky et al, Cancer Res. 53:2661-2615 (1993)).
  • Galectin 1 also participates in regulating cell proliferation (Wells et al, Cell 64:91-91 (1991)) and some immune functions (Offner et al, J. Neuroimmunol. 25.177-184 (1990)). Galectin 1 has been shown to regulate the immune response by mediating apoptosis of T cells (Perillo et al, Nature 378: 736-739 (1995)).
  • Galectin 3 promotes the growth of cells cultured under restrictive culture conditions (Yang et al, Proc. Nat Acad. Sci. USA 93:6131-6142 (June 1996)). Galectin 3 expression in cells confers resistance to apoptosis which indicates that Galectin 3 could be a cell death suppressor which interferes in a common pathway of apoptosis. Id.
  • the present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the galectin 8, 9, 10, or 10SN polypeptide having the amino acid sequence is shown in FIGs. 1, 2A-2B, 3A-3B, and 4A-4B, respectively (SEQ ID ⁇ Os:2, 4, 6, and 8, respectively) or the amino acid sequence encoded by the cDNA clones deposited in bacterial hosts as ATCC Deposit Numbers 97732, 97733 and 97734 on September 24, 1996.
  • the present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of galectin 8, 9, 10, or 10SV polypeptides or peptides by recombinant techniques.
  • the invention further provides an isolated galectin 8, 9, 10, or 10SN polypeptide having an amino acid sequence encoded by a polynucleotide described herein.
  • the present invention also provides a screening method for identifying compounds capable of enhancing or inhibiting a cellular response induced by galectin 8, 9, 10, or 10SV, which involves contacting cells which express galectin 8, 9, 10, or 10SN with the candidate compound, assaying a cellular response, and comparing the cellular response to a standard cellular response, the standard being assayed when contact is made in absence of the candidate compound; whereby, an increased cellular response over the standard indicates that the compound is an agonist and a decreased cellular response over the standard indicates that the compound is an antagonist.
  • a screening assay for agonists and antagonists is provided which involves determining the effect a candidate compound has on galectin 8, 9, 10, or 10SV binding to the ⁇ -galactosidase sugar.
  • the method involves contacting the ⁇ -galactosidase sugar with a galectin 8, 9, 10, or 10SV polypeptide and a candidate compound and determining whether galectin 8, 9, 10, or 10SV binding to ⁇ -galactosidase sugar is increased or decreased due to the presence of the candidate compound.
  • the invention provides a diagnostic method useful during diagnosis disorder.
  • An additional aspect of the invention is related to a method for treating an individual in need of an increased level of galectin 8, 9, 10, or 10SN activity in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of an isolated galectin 8, 9, 10, or 10SV polypeptide of the invention or an agonist thereof.
  • a still further aspect of the invention is related to a method for treating an individual in need of a decreased level of galectin 8, 9, 10, or 10SV activity in the body comprising, administering to such an individual a composition comprising a therapeutically effective amount of a galectin 8, 9, 10, or 10SV antagonist.
  • FIG. 1 shows the nucleotide (SEQ ID ⁇ O:l) and deduced amino acid
  • SEQ ID NO:2 sequences of galectin 8.
  • the protein has a deduced molecular weight of about 36 kDa.
  • FIG. 2A-2B shows the nucleotide (SEQ ID NO:3) and deduced amino acid (SEQ ID NO:4) sequences of galectin 9.
  • the protein has a deduced molecular weight of about 34.7 kDa.
  • FIG. 3A-3B shows the nucleotide (SEQ ID NO:5) and deduced amino acid (SEQ ID NO:6) sequences of full length galectin 10.
  • the protein has a deduced molecular weight of about 35.7 kDa.
  • FIG. 4A-4B shows the nucleotide (SEQ ID NO:7) and deduced amino acid (SEQ ID NO:8) sequences of a galectin 10 splice variant (galectin 10SV).
  • the protein has a deduced molecular weight of about 22.4 kDa.
  • FIG. 5A-5E shows the regions of similarity between the amino acid sequences of the galectin 8, 9, and 10 proteins and human galectin 2 (SEQ ID NO:9), human galectin 3 (SEQ ID NO:10), rat galectin 4 (SEQ ID NO:l 1), rat galectin 5 (SEQ ID NO: 12), human galectin 7 (SEQ ID NO: 13), rat galectin 3 (SEQ ID NO: 14), rat galectin 8 (SEQ ID NO: 15), and human galectin 1 (SEQ ID NO:16).
  • FIG. 6 shows the regions of similarity between the amino acid sequences of the galectin 10SV protein and the rat RL30 protein (SEQ ID NO: 17).
  • FIG. 7 shows a homology comparison between the galectin 10 and galectin 10SV proteins.
  • FIGs. 8, 9, 10, and 11 show an analysis of the galectin 8, 9, 10, and 10SV amino acid sequence, respectively.
  • Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown.
  • SEQ ID NO:6 corresponds to the shown highly antigenic regions of the galectin 8, 9, 10, or 10SV protein, respectively.
  • the present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding a galectin 8, 9, 10, or 10SV polypeptide having the amino acid sequence shown in FIGs. 1, 2A-2B, 3A-3B, and 4A-4B, respectively (SEQ ID NOs:2, 4, 6, and 8, respectively), which was deterrnined by sequencing a cloned cDNA.
  • the galectin 8, 9, 10, and 10SV proteins of the present invention share sequence homology with other galectins and the rat RL30 protein (FIGs.
  • FIGs. 1, 2A-2B, and 4A-4B SEQ ID NO:l, 3, and 7, respectively
  • the nucleotide sequences shown in FIGs. 1, 2A-2B, and 4A-4B were obtained by sequencing the HSIAL77, HTPBR22, and HETAS87 clones, which were deposited on September 24, 1996 at the American Type Culture Collection, 12301 Park Lawn Drive, Rockville, Maryland 20852, and given accession numbers 97732, 97733 and 97734, respectively.
  • the deposited clones are contained in the pBluescript SK(-) plasmid (Stratagene, LaJolla, CA).
  • the nucleotide sequence shown in FIG. 3A-3B SEQ ID NO:5), which encodes the full-length galectin 10 protein, was obtained by sequencing a clone cD A obtained from a human endometrial tumor library.
  • nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer (such as the Model 373 from Applied Biosystems, Inc.), and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA sequence determined as above. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art.
  • a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion.
  • nucleic acid molecule of the present invention encoding a galectin 8, 9, 10, or 10SV, respectively
  • polypeptide may be obtained using standard cloning and screening procedures, such as those for cloning cDNAs using mRNA as starting material.
  • nucleic acid molecules described in FIGs. 1, 2A-2B, 3A-3B, and 4A-4B (SEQ ID NO:l, 3, 5, and 7, respectively) were discovered in cDNA libraries derived from human adult small intestine, human pancreatic tumor, human endometrial tumor and human endometrial tumor, respectively.
  • Galectin 8 (SEQ ID NO:l) appears to be mainly expressed in cells of the human colon and small intestine.
  • the determined nucleotide sequences of the galectin 8, 9, 10, and 10SV cDNAs of FIGs. 1, 2A-2B, 3A-3B, and 4A-4B, respectively (SEQ ID NOs:l, 3, 5, and 7) contain open reading frames encoding proteins of 323, 311, 317, and 200 amino acid residues, with an initiation codon at positions 52-54, 16-18, 118-
  • FIGs. 1 , 2A-2B, 3 A-3B, and 4A- 4B respectively (SEQ ID NOs:l, 3, 5, and 7), and a deduced molecular weight of about 36, 34.7, 35.7, and 22.4 kDa, respectively.
  • the galectin 8, 9, 10 and 10SV proteins shown in FIGs. 1, 2A-2B, 3A-3B, and 4A-4B respectively (SEQ ID NOs:2, 4, 6, and 8) share homology with other galectins (See, e.g., FIG. 5A-
  • the predicted galectin 8 and 9 polypeptides encoded by the deposited cDNAs comprise about 323 and 311 amino acids, but may be anywhere in the range of 300 - 333 amino acids.
  • the predicted galectin 10 polypeptide comprises about 317 amino acids, but may be anywhere in the range of 305 - 329 amino acids.
  • the predicted galectin 10SV polypeptide encoded by the deposited cDNA comprises about 200 amino acids, but may be anywhere in the range of 190 - 210 amino acids. Galectin 10SV is believed to be a splice variant of galectin 10.
  • splice variant refers to cDNA molecules produced from RNA molecules initially transcribed from the same genomic DNA sequence but which have undergone alternative RNA splicing.
  • Alternative RNA splicing occurs when a primary RNA transcript undergoes splicing, generally for the removal of introns, which results in the production of more than one mRNA molecule each of which may encode different amino acid sequences.
  • the term “splice variant” also refers to the proteins encoded by the above cDNA molecules.
  • nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced synthetically.
  • the DNA may be double-stranded or single-stranded.
  • Single-stranded DNA or RNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand.
  • isolated nucleic acid molecule(s) is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment
  • recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention.
  • Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution.
  • Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the present invention.
  • Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.
  • Isolated nucleic acid molecules of the present invention include DNA molecules comprising an open reading frame (ORF) shown in FIGs. 1, 2A-2B,
  • the invention provides nucleic acid molecules having nucleotide sequences related to extensive portions of SEQ ID NO:l which have been determined from the following related cDNA clones: HSIAL77R (SEQ ID NO:l).
  • HKCAA85R SEQ ID NO:21
  • HCNAI55R SEQ ID NO:22
  • HCNAI87R SEQ ID NO:
  • HCNAS74R SEQ ID NO:24
  • HCNAF43R SEQ ID NO:25
  • the invention provides nucleic acid molecules having nucleotide sequences related to extensive portions of SEQ ID NO: 3 which have been determined from the following related cDNA clones: HMSCP11R (SEQ ID NO:26), HMSEU32R (SEQ ID NO:27), HTPAO71R (SEQ ID NO:28), HJAAN54R (SEQ ID ⁇ O:29), HMSEU43R (SEQ ID NO:30), HILBP03R (SEQ ID NO:31), HTPCG81R (SEQ ID NO:32), HTBAA21R (SEQ ID NO:33), and HFXBU26R (SEQ ID NO:34).
  • HMSCP11R SEQ ID NO:26
  • HMSEU32R SEQ ID NO:27
  • HTPAO71R SEQ ID NO:28
  • HJAAN54R SEQ ID ⁇ O:29
  • HMSEU43R SEQ ID NO:30
  • HILBP03R SEQ ID NO:31
  • HTPCG81R SEQ ID NO
  • the invention provides nucleic acid molecules having nucleotide sequences related to extensive portions of SEQ ID NO: 5 which have been determined from the following related cDNA clones: HTNBX92R (SEQ ID NO:35), HLTAZ64RB (SEQ ID NO:36), HJBAI38R (SEQ ID NO:37), HETAS87R (SEQ ID NO:38), and HETAR45R (SEQ ID NO:39).
  • the invention provides nucleic acid molecules having nucleotide sequences related to extensive portions of SEQ ID NO:7 which have been determined from the following related cDNA clones: HTNBX92R (SEQ ID NO:35), HLTAZ64RB (SEQ ID NO:36), HBNAF37R (SEQ ID NO:40), and HETAS87R (SEQ ID NO:38).
  • the invention provides isolated nucleic acid molecules encoding the galectin 8, 9, 10 or 10SV polypeptide having an amino acid sequence encoded by the cDNA clone contained in the plasmid deposited as ATCC Deposit Nos. 97732, 97733 and 97734, respectively, on September 24, 1996.
  • nucleic acid molecules are provided encoding the full-length galectin 8, 9, 10, or 10SV polypeptide lacking the N-terminal methionine.
  • the invention further provides an isolated nucleic acid molecule having the nucleotide sequence shown in FIGs.
  • the present invention is further directed to fragments of the isolated nucleic acid molecules described herein.
  • a fragment of an isolated nucleic acid molecule having the nucleotide sequence of the deposited cDNA or the nucleotide sequence shown in FIGs. 1, 2A-2B, 3A-3B, or 4A-4B is intended fragments at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length which are useful as diagnostic probes and primers as discussed herein.
  • DNA fragments 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 100, 1050, 1100, or 1115 nt in length of the sequence shown in SEQ ID NO:l are also useful according to the present invention as are fragments corresponding to most, if not all, of the nucleotide sequence of the cDNA clone contained in the plasmid deposited as ATCC Deposit No. 97732 or as shown in SEQ ID NO: 1.
  • larger DNA fragments 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 100, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, or 1464 nt in length of the sequence shown in SEQ ID NO:5 are also useful according to the present invention as are fragments corresponding to most, if not all, of the nucleotide sequence of the cDNA molecule as shown in SEQ ID NO:5. Further, larger DNA fragments 50, 100, 150, 200, 250, 300, 350,
  • SEQ ID NO:7 are also useful according to the present invention as are fragments corresponding to most, if not all, of the nucleotide sequence of the cDNA clone contained in the plasmid deposited as ATCC Deposit No. 97734 or as shown in SEQ ID NO:7.
  • fragments at least 20 nt in length are intended fragments which include 20 or more contiguous bases from the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in SEQ ID NOs:l, 3, 5, or 7.
  • nucleic acid fragments of the present invention include nucleic acid molecules encoding epitope-bearing portions of the galectin 8, 9, 10, or 10SV protein.
  • nucleic acid fragments of the present invention include nucleic acid molecules encoding: a polypeptide comprising amino acid residues from about 55-101, 137-162, 180-193, 216-266 in FIG. 1 (SEQ ID NO:
  • the invention provides an isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent hybridization conditions to a portion of the polynucleotide in a nucleic acid molecule of the invention described above, for instance, a cDNA clone contained in ATCC Deposit Nos. 97732, 97733 and 97734.
  • stringent hybridization conditions is intended overnight incubation at 42°C in a solution comprising: 50% formamide, 5x SSC (150 mM NaCl, 15mM trisodium citrate), 50 raM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 g/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1 x SSC at about 65 °C.
  • a polynucleotide which hybridizes to a "portion" of a polynucleotide is intended a polynucleotide (either DNA or RNA) hybridizing to at least about 15 nucleotides (nt), and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably about 30-70 nt of the reference polynucleotide. These are useful as diagnostic probes and primers as discussed above and in more detail below.
  • a polynucleotide which hybridizes only to a poly A sequence such as the 3' terminal poly(A) tract of the galectin 8, 9, 10, or 10SV cDNA shown in FIGs.
  • nucleic acid molecules of the present invention which encode a galectin 8, 9, 10, or 10SN polypeptide may include, but are not limited to those encoding the amino acid sequence of the polypeptide, by itself; the coding sequence for the polypeptide and additional sequences, such as those encoding an amino acid leader or secretory sequence, such as a pre-, or pro- or prepro- protein sequence; the coding sequence of the polypeptide, with or without the aforementioned additional coding sequences, together with additional, non-coding sequences, including for example, but not limited to introns and non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing, including splicing and polyadenylation signals, for example - ribosome binding and stability of mRNA; an additional coding sequence which codes for additional amino acids, such as those which provide additional functionalities.
  • the sequence encoding the polypeptide may be fused to a marker sequence, such as a sequence encoding a peptide which facilitates purification of the fused polypeptide.
  • the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc.), among others, many of which are commercially available. As described in Gentz et al, Proc. Natl. Acad. Sci. USA ⁇ S6 ' :821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein.
  • the "HA” tag is another peptide useful for purification which corresponds to an epitope derived from the influenza hemagglutinin protein, which has been described by Wilson et al, Cell 37:161-11 (1984).
  • other such fusion proteins include the galectin 8, 9, 10, or 10SV fused to Fc at the N- or C -terminus.
  • the present invention further relates to variants of the nucleic acid molecules of the present invention, which encode portions, analogs or derivatives of the galectin 8, 9, 10, or 10SN protein. Variants may occur naturally, such as a natural allelic variant. By an "allelic variant" is intended one of several alternate forms of a gene occupying a given locus on a chromosome of an organism.
  • Non-naturally occurring variants may be produced using art-known mutagenesis techniques.
  • variants include those produced by nucleotide substitutions, deletions or additions which may involve one or more nucleotides.
  • the variants may be altered in coding regions, non-coding regions, or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the galectin 8, 9, 10, or 10SV protein or portions thereof. Also especially preferred in this regard are conservative substitutions.
  • nucleic acid molecules comprising a polynucleotide having a nucleotide sequence at least 95% identical, and more preferably at least 96%, 97%, 98% or 99% identical to (a) a nucleotide sequence encoding the galectin 8, 9, 10, or 10SV polypeptide having the amino acid sequence in FIGs. 1 , 2A-2B, 3 A-3B, or 4A-4B (SEQ ID NOs: 1 ,
  • nucleotide sequence encoding the polypeptide having the amino acid sequence in FIGs. 1, 2A-2B, 3A-3B, or 4A-4B (SEQ ID NOs:l, 3, 5, or 7), but lacking the N-terminal methionine;
  • a nucleotide sequence encoding the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit Nos. 97732, 97733 or 97734 on September 24, 1996; or (d) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), or (c).
  • nucleotide sequence having a nucleotide sequence at least, for example, 95%» “identical” to a reference nucleotide sequence encoding a galectin 8, 9, 10, or 10SV polypeptide is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the galectin 8, 9, 10, or 10SV polypeptide.
  • a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
  • These mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
  • nucleic acid molecule is at least 95%, 96%, 97%, 98% or 99% identical to, for instance, the nucleotide sequence shown in FIGs. 1 , 2A-2B, 3 A-3B, or 4A-4B (SEQ ID NOs: 1 , 3, 5, or
  • telomere sequence of the deposited cDNA clone can be determined conventionally using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711. Bestfit uses the local homology algorithm of Smith and Waterman, Advances in
  • the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.
  • the present application is directed to nucleic acid molecules at least 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence shown in FIGs. 1 ,
  • nucleic acid sequence of one of the deposited cDNAs irrespective of whether they encode a polypeptide having galectin 8, 9, 10, or 10SV activity. This is because even where a particular nucleic acid molecule does not encode a polypeptide having galectin 8, 9, 10, or 1 OSV activity, one of skill in the art would still know how to use the nucleic acid molecule, for instance, as a hybridization probe or a polymerase chain reaction (PCR) primer.
  • PCR polymerase chain reaction
  • nucleic acid molecules of the present invention that do not encode a polypeptide having galectin 8, 9, 10, or 10SV activity include, ter alia, (1) isolating the galectin 8, 9, 10, or 10SV gene or allelic variants thereof in a cDNA library; (2) in situ hybridization (e.g., "FISH") to metaphase chromosomal spreads to provide precise chromosomal location of the galectin 8, 9, 10, or 10SV gene, as described in Verma et al, Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York (1988); and (3) Northern Blot analysis for detecting galectin 8, 9, 10, or 10SV mRNA expression in specific tissues.
  • FISH in situ hybridization
  • nucleic acid molecules having sequences at least 95%o, 96%), 97%), 98%) or 99% identical to the nucleic acid sequence shown in FIGs. 1, 2A-2B, 3A-3B, or 4A-4B (SEQ ID NOs:l, 3, 5, or 7) or to the nucleic acid sequence of one of the deposited cDNAs which do, in fact, encode a polypeptide having galectin 8, 9, 10, or 10SV protein activity.
  • a polypeptide having galectin 8, 9, 10, or 10SV activity is intended polypeptides exhibiting activity similar, but not necessarily identical, to an activity of the galectin 8, 9, 10, or 10SV protein of the invention, as measured in a particular biological assay.
  • galectin 8, 9, 10, or 10SV protein activity can be measured using a lactose binding assay.
  • Lactose binding activity of the expressed galectin 8, 9, 10, or 10SV is assayed by immunodetection of in situ binding activity to asialofetuin (Sigma) immobilized on nitrocellulose (Amersham) (Madsen et al, J. Biol. Chem. 270(1 J.-5823-5829 (1995)). Thirty ⁇ g of asialofetuin dissolved in 3 ⁇ l of water is spotted on a 1-cm 2 strip of nitrocellulose.
  • nitrocellulose pieces are then placed in a 24- well tissue culture plate and incubated overnight in buffer B (58 mM Na 2 HPO 4 , 18 mM KH 2 PO 4 , 75 mM NaCl, 2 mM EDTA, and 3% BSA, pH7.2) with constant agitation at 22°C. Following incubation, the blocking medium is aspirated and the nitrocellulose pieces are washed three times in buffer A (58 mM Na ⁇ PO ⁇ 18 mM KH 2 PO 4 , 75 mM NaCl, 2 mM EDTA, 4 mM ⁇ - mercaptoethanol and 0.2%) BSA, pH7.2).
  • buffer B 58 mM Na 2 HPO 4 , 18 mM KH 2 PO 4 , 75 mM NaCl, 2 mM EDTA, 4 mM ⁇ - mercaptoethanol and 0.2%) BSA, pH7.2.
  • Cell extracts are prepared containing 1% BSA and either with or without 150 mM lactose (105 ⁇ l of primary extract, 15 ⁇ l of 10% BSA in buffer A and either 30 ⁇ l of 0.75 M lactose in buffer A or 30 ⁇ l of buffer A).
  • the immobilized asialofetuin is incubated with the extracts for 2 h and washed 5 times in buffer A.
  • the nitrocellulose pieces are then fixed in 2% formalin in PBS (58 mM Na ⁇ PO,,, 18 mM KH 2 PO 4 , 75 mM NaCl, 2 mM EDTA pH7.2) for 1 hour to prevent loss of bound galectin.
  • the pieces were incubated with rabbit anti-galectin 8, 9, 10, or 1 OSV polyclonal serum diluted 1 : 100 in PBS for 2 h at 22°C. The pieces are then washed in PBS and incubated with peroxidase-labeled goat anti-rabbit antibodies (DAKO). Following incubation for 2 h at 22°C, the pieces are washed in PBS and the substrate is added. Nitrocellulose pieces are incubated until the color develops and the reaction is stopped by washing in distilled water.
  • DAKO peroxidase-labeled goat anti-rabbit antibodies
  • nucleic acid molecules having a sequence at least 95%, 96%, 97%, 98%), or 99% identical to the nucleic acid sequence of the deposited cDNA or the nucleic acid sequence shown in FIGs. 1, 2A-2B, 3A-3B, or 4A-4B (SEQ ID NOs:l, 3, 5, or 7, respectively) will encode "a polypeptide having galectin 8, 9, 10, or 1 OSV protein activity.”
  • degenerate variants of these nucleotide sequences all encode the same polypeptide, this will be clear to the skilled artisan even without performing the above described comparison assay. It will be further recognized in the art that, for such nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a polypeptide having galectin 8, 9, 10, or
  • the present invention also relates to vectors which include the isolated DNA molecules of the present invention, host cells which are genetically engineered with the recombinant vectors, and the production of galectin 8, 9, 10, or 10SV polypeptides or fragments thereof by recombinant techniques.
  • the polynucleotides may be joined to a vector containing a selectable marker for propagation in a host.
  • a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
  • the DNA insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few.
  • an appropriate promoter such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few.
  • Other suitable promoters will be known to the skilled artisan.
  • the expression constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation.
  • the coding portion of the transcripts expressed by the constructs will preferably include a translation initiating at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.
  • the expression vectors will preferably include at least one selectable marker.
  • markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria.
  • Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS and Bowes melanoma cells; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.
  • vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNHl ⁇ a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.
  • preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.
  • Other suitable vectors will be readily apparent to the skilled artisan.
  • Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al, Basic Methods In Molecular Biology (1986).
  • the polypeptide may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art.
  • a preferred fusion protein comprises a heterologous region from immunoglobulin that is useful to solubilize proteins. For example, EP-A-O 464
  • fusion proteins comprising various portions of constant region of immunoglobin molecules together with another human protein or part thereof.
  • the Fc part in a fusion protein is thoroughly advantageous for use in therapy and diagnosis and thus results, for example, in improved pharmacokinetic properties (EP-A 0232262).
  • EP-A 0232262 it would be desirable to be able to delete the Fc part after the fusion protein has been expressed, detected and purified in the advantageous manner described. This is the case when Fc portion proves to be a hindrance to use in therapy and diagnosis, for example when the fusion protein is to be used as antigen for immunizations.
  • human proteins such as, hIL5-receptor has been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5.
  • Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5.
  • the galectin 8, 9, 10, or 1 OSV protein can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is employed for purification.
  • Polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells.
  • polypeptides of the present invention may be glycosylated or may be non-glycosylated.
  • polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.
  • the invention further provides an isolated galectin 8, 9, 10, or 10SV polypeptide having (1) the amino acid sequence encoded by one of the deposited cDNAs, (2) the amino acid sequence in FIGs. 1, 2A-2B, 3A-3B, or 4A-4B (SEQ ID NOs:2, 4, 6, or 8, respectively), or (3) the amino acid sequence of a peptide or polypeptide comprising a portion of the above polypeptides.
  • the invention further includes variations of the galectin 8, 9, 10, or 10SV polypeptide which show substantial galectin 8, 9, 10, or 10SV polypeptide activity or which include regions of galectin 8, 9, 10, or 10SV protein such as the protein portions discussed below.
  • Such mutants include deletions, insertions, inversions, repeats, and type substitutions.
  • guidance concerning which amino acid changes are likely to be phenotypically silent can be found in Bowie, J.U., et al, "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions," Science 247:1306-1310 (1990).
  • the fragment, derivative or analog of the polypeptide of SEQ ID As indicated above, guidance concerning which amino acid changes are likely to be phenotypically silent can be found in Bowie, J.U., et al, "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions," Science 247:1306-1310 (1990).
  • amino acid residues may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as an IgG Fc fusion region peptide or leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence.
  • a conserved or non-conserved amino acid residue preferably a conserved amino acid residue
  • substituted amino acid residue may or may not be one encoded by the genetic code
  • changes are preferably of a minor nature, such as conservative amino acid substitutions that do not significantly affect the folding or activity of the protein (see Table 1).
  • the number of amino acid substitutions a skilled artisan would make depends on many factors, including those described above and below. Generally speaking, the number of substitutions for any given galectin 8, 9, 10, or 10SV polypeptide or mutant thereof will not be more than 50, 40, 30, 20, 10, 5, or 3, depending on the objective.
  • Amino acids in a galectin 8, 9, 10, or 10SV protein of the present invention that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule. Sites that are critical for ligand binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al, J. Mol. Biol. 224:899-904 (1992) and de Vos et al, Science 255:306-312 (1992)).
  • the polypeptides of the present invention are preferably provided in an isolated form.
  • isolated polypeptide is intended a polypeptide removed from its native environment.
  • a polypeptide produced and/or contained within a recombinant host cell is considered isolated for purposes of the present invention.
  • polypeptides that have been purified, partially or substantially, from a recombinant host cell are polypeptides that have been purified, partially or substantially, from a recombinant host cell.
  • a recombinantly produced version of a galectin 8, 9, 10, or 10SV polypeptide can be substantially purified by the one-step method described in Smith and Johnson, Gene 57:31-40 (1988).
  • the polypeptides of the present invention include the polypeptides encoded by the deposited cDNAs; a polypeptide comprising amino acids about
  • polypeptides which are at least 95% identical, still more preferably at least 96%, 97%, 98%> or 99% identical to the polypeptides described above and also include portions of such polypeptides with at least 30 amino acids and more preferably at least 50 amino acids.
  • polypeptide having an amino acid sequence at least, for example,
  • identical to a reference amino acid sequence of a galectin 8, 9, 10, or 10SV polypeptide is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of the galectin 8, 9, 10, or 10SV polypeptide.
  • up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence.
  • alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence. As a practical matter, whether any particular polypeptide is at least 95%),
  • polypeptide of the present invention could be used as a molecular weight marker on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art.
  • the invention provides a peptide or polypeptide comprising an epitope-bearing portion of a polypeptide of the invention.
  • the epitope of this polypeptide portion is an immunogenic or antigenic epitope described herein.
  • An "immunogenic epitope” is defined as a part of a protein that elicits an antibody response when the whole protein is the immunogen.
  • a region of a protein molecule to which an antibody can bind is defined as an "antigenic epitope.”
  • the number of immunogenic epitopes of a protein generally is less than the number of antigenic epitopes. See, for instance, Geysen et al, Proc. Natl Acad. Sci.
  • Peptides capable of eliciting protein-reactive sera are frequently represented in the primary sequence of a protein, can be characterized by a set of simple chemical rules, and are confined neither to immunodominant regions of intact proteins (i.e., immunogenic epitopes) nor to the amino or carboxyl terminals.
  • Antigenic epitope-bearing peptides and polypeptides of the invention are therefore useful to raise antibodies, including monoclonal antibodies, that bind specifically to a polypeptide of the invention. See, for instance, Wilson et al, Cell 37:161-118 (1984) at 777.
  • Antigenic epitope-bearing peptides and polypeptides of the invention preferably contain a sequence of at least seven, more preferably at least nine and most preferably between about at least 15 to about 30 amino acids contained within the amino acid sequence of a polypeptide of the invention.
  • Non-limiting examples of antigenic polypeptides or peptides that can be used to generate galectin 8, 9, 10, or lOSV-specific antibodies include: a polypeptide comprising amino acid residues from about 55-101, 137-162, 180- 193, 216-266 in FIG. 1 (SEQ IDNO:2), 62-102, 226-259, 197-308 in FIG.
  • the epitope-bearing peptides and polypeptides of the invention may be produced by any conventional means Houghten, R. A. (1985) General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids. Proc. Natl. Acad. Sci. USA 52:5131-5135. This "Simultaneous Multiple Peptide Synthesis (SMPS)" process is further described in U.S. Patent No. 4,631,211 to Houghten et al. (1986).
  • SMPS Simultaneous Multiple Peptide Synthesis
  • galectin 8, 9, 10, or 10SV polypeptides of the present invention and the epitope-bearing fragments thereof described above can be combined with parts of the constant domain of immunoglobulins (IgG), resulting in chimeric polypeptides.
  • IgG immunoglobulins
  • These fusion proteins facilitate purification and show an increased half-life in vivo. This has been shown, e.g., for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins (EPA 394,827; Traunecker et al, Nature 331:84- 86 (1988)).
  • Fusion proteins that have a disulfide-linked dimeric structure due to the IgG part can also be more efficient in binding and neutralizing other molecules than the monomeric galectin 8, 9, 10, or 10SV protein or protein fragment alone (Fountoulakis et al, JBiochem 270:3958-3964 (1995)).
  • galectin 8, 9, 10, or 10SV protein express significantly altered (enhanced or decreased) levels of the galectin 8, 9, 10, or 10SV protein and mRNA encoding the galectin 8, 9, 10, or 10SV protein when compared to a corresponding "standard" mammal, i.e., a mammal of the same species not having the disease. Further, it is believed that altered levels of the galectin 8, 9, 10, or 10SV protein can be detected in certain body fluids (e.g., sera, plasma, urine, and spinal fluid) from mammals with the disease when compared to sera from mammals of the same species not having the disease.
  • body fluids e.g., sera, plasma, urine, and spinal fluid
  • the invention provides a diagnostic method useful during diagnosis, which involves assaying the expression level of the gene encoding the galectin 8, 9, 10, or 10SV protein in mammalian cells or body fluid and comparing the gene expression level with a standard galectin 8, 9, 10, or 10SV gene expression level, whereby an increase or decrease in the gene expression level over the standard is indicative of the disease.
  • the present invention is useful as a prognostic indicator, whereby patients exhibiting altered galectin 8, 9, 10, or 10SV gene expression will experience a worse clinical outcome relative to patients expressing the gene at a normal level.
  • test the expression level of the gene encoding the galectin 8, 9, 10, or 10SV protein is intended qualitatively or quantitatively measuring or estimating the level of the galectin 8, 9, 10, or 10SV protein or the level of the mRNA encoding the galectin 8, 9, 10, or 1 OSV protein in a first biological sample either directly (e.g., by determining or estimating absolute protein level or mRNA level) or relatively (e.g., by comparing to the galectin 8, 9, 10, or 10SV protein level or mRNA level in a second biological sample).
  • the galectin 8, 9, 10, or 1 OSV protein level or mRNA level in the first biological sample is measured or estimated and compared to a standard galectin 8, 9, 10, or 10SV protein level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the cancer.
  • a standard galectin 8, 9, 10, or 10SV protein level or mRNA level is known, it can be used repeatedly as a standard for comparison.
  • biological sample any biological sample obtained from an individual, cell line, tissue culture, or other source which contains galectin 8, 9, 10, or 10SV protein or mRNA.
  • Biological samples include mammalian body fluids (such as sera, plasma, urine, synovial fluid and spinal fluid) which contain secreted galectin 8, 9, 10, or 10SV protein, and ovarian, prostate, heart, placenta, pancreas liver, spleen, lung, breast and umbilical tissue.
  • the present invention is useful for detecting diseases in mammals (for example, cancer, autoimmune diseases, inflammatory diseases, asthma, and allergic diseases).
  • mammals for example, cancer, autoimmune diseases, inflammatory diseases, asthma, and allergic diseases.
  • the invention is useful during diagnosis of the of following types of cancers in mammals: melanoma, renal astrocytoma, Hodgkin disease, breast, ovarian, prostate, bone, liver, lung, pancreatic, and spleenic.
  • Preferred mammals include monkeys, apes, cats, dogs, cows, pigs, horses, rabbits and humans. Particularly preferred are humans.
  • Total cellular RNA can be isolated from a biological sample using the single-step guanidinium-thiocyanate-phenol-chloroform method described in Chomczynski and SacchL n ⁇ /. Biochem.
  • mRNA encoding the galectin 8, 9, 10, or 10SV protein are then assayed using any appropriate method. These include Northern blot analysis, (Harada et al. , Cell 55:303-312 (1990) SI nuclease mapping, (Fijita et al, Cell 49:351-361 (1987)) the polymerase chain reaction (PCR), reverse transcription in combination with the polymerase chain reaction (RT-PCR) (Makino et al, Technique 2:295-301 (1990), and reverse transcription in combination with the ligase chain reaction (RT-LCR). Assaying galectin 8, 9, 10, or 10SV protein levels in a biological sample can antibody-based techniques.
  • galectin 8, 9, 10, or 10SV protein expression in tissues can be studied with classical immunohistological methods. (Jalkanen, M., et al, J. Cell. Biol 101:916-985 (1985); Jalkanen, M., et al, J. Cell . Biol. 105:3081-3096 (1987)).
  • 10SV protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
  • immunoassays such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
  • Suitable labels are known in the art and include enzyme labels, such as, Glucose oxidase, and radioisotopes, such as iodine ( 125 1, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 112 In), and technetium ( 99m Tc), and fluorescent labels, such as fluorescein and rhodamine, and biotin.
  • enzyme labels such as, Glucose oxidase, and radioisotopes, such as iodine ( 125 1, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 112 In), and technetium ( 99m Tc)
  • fluorescent labels such as fluorescein and rhodamine, and biotin.
  • galectin 8, 9, 10, and 10SV share significant homology with other galectins. Galectin 1 induces apoptosis of T cells and T cell leukemia cell lines. Thus, it is believed by the inventors that galectin 8, 9, 10, and 10SV are active in modulating growth regulatory activities, immunomodulatory activity, cell-cell and cell-substrate interactions, and apoptosis.
  • galectin 8, 9, 10, or 10SV may be therapeutically valuable in the treatment of clinical manifestations of such cell regulatory disorders.
  • Disorders which can be treated include, but should not be limited to, autoimmune disease, cancer (preferably, melanoma, renal, astrocytoma, and Hodgkin disease), inflammatory disease, wound healing, arteriosclerosis, other heart diseases, microbe infection (virus, fungal, bacterial, and parasite), asthma, and allergic diseases.
  • galectin 8, 9, 10, and 10SV Given the activities modulated by galectin 8, 9, 10, and 10SV, it is readily apparent that a substantially altered (increased or decreased) level of expression of galectin 8, 9, 10, or 10SV in an individual compared to the standard or "normal” level produces pathological conditions such as those described above. It will also be appreciated by one of ordinary skill that the galectin 8, 9, 10, or 10SV protein of the invention will exert its modulating activities on any of its target cells. Therefore, it will be appreciated that conditions caused by a decrease in the standard or normal level of galectin 8, 9, 10, or 10SV activity in an individual, can be treated by administration of galectin 8, 9, 10, or 10SV protein or an agonist thereof.
  • the invention further provides a method of treating an individual in need of an increased level of galectin 8, 9, 10, or 10SV activity comprising administering to such an individual a pharmaceutical composition comprising an amount of an isolated galectin 8, 9, 10, or 10SV polypeptide of the invention or an agonist thereof to increase the galectin 8, 9, 10, or 10SV activity level in such an individual.
  • a still further aspect of the invention is related to a method for treating an individual in need of a decreased level of galectin 8, 9, 10, or 10SV activity in the body comprising, administering to such an individual a composition comprising a therapeutically effective amount of a galectin 8, 9, 10, or 10SV antagonist.
  • Preferred antagonists for use in the present invention are galectin 8, 9, 10, or lOSV-specific antibodies.
  • the invention further provides a method of treating an individual in need of an increased level of galectin 8, 9, 10, or 10SV activity comprising administering to such an individual a pharmaceutical composition comprising an effective amount of an isolated galectin 8, 9, 10, or 10SV polypeptide of the invention, particularly a mature form of the galectin 8, 9, 10, or 10SV, effective to increase the galectin 8, 9, 10, or 10SV activity level in such an individual.
  • the total pharmaceutically effective amount of galectin 8, 9, 10, or 10SV polypeptide administered parenterally per dose will be in the range of about 1 ⁇ g/kg/day to 10 mg/kg/day of patient body weight, although, as noted above, this will be subject to therapeutic discretion. More preferably, this dose is at least 0.01 mg/kg/day, and most preferably for humans between about 0.01 and 1 mg/kg/day for the hormone.
  • the galectin 8, 9, 10, or 10SV polypeptide is typically administered at a dose rate of about 1 ⁇ g/kg/hour to about 50 ⁇ g/kg/hour, either by 1-4 injections per day or by continuous subcutaneous infusions, for example, using a mini-pump.
  • An intravenous bag solution may also be employed.
  • compositions containing the galectin 8, 9, 10, or 10SV of the invention may be administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, drops or transdermal patch), bucally, or as an oral or nasal spray.
  • pharmaceutically acceptable carrier is meant a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • parenteral refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasteraal, subcutaneous and intraarticular injection and infusion.
  • the nucleic acid molecules of the present invention are also valuable for chromosome identification.
  • the sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome.
  • the mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease.
  • the cDNA herein disclosed is used to clone genomic DNA of a galectin 8, 9, 10, or 10SV protein gene. This can be accomplished using a variety of well known techniques and libraries, which generally are available commercially. The genomic DNA then is used for in situ chromosome mapping using well known techniques for this purpose.
  • sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis of the 3' untranslated region of the gene is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes.
  • Fluorescence in situ hybridization of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step.
  • This technique can be used with probes from the cDNA as short as 50 or 60 bp.
  • Verma et al Human Chromosomes: A Manual Of Basic Techniques, Pergamon Press, New York (1988).
  • the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, for example, in V.
  • Example 1 Expression and Purification of Galectin 8, 9, 10 and lOSVin E. coli
  • the DNA sequence encoding the galectin 9 protein in the deposited cDNA clone was amplified using PCR oligonucleotide primers specific to the amino terminal sequences of the galectin 9 protein and to vector sequences 3' to the gene. Additional nucleotides containing restriction sites to facilitate cloning are added to the 5' and 3' sequences.
  • the DNA sequence encoding the galectin 8 or 10SV protein in the deposited cDNA clone is amplified using PCR oligonucleotide primers specific to the nucleotide sequences encoding the amino terminal sequences of the galectin 8 or 10SV protein and to vector sequences 3' to the gene. Additional nucleotides containing restriction sites to facilitate cloning are added to the 5' and
  • the cDNA sequence encoding the galectin 10 protein is amplified from either a human endometrial tumor or human fetal heart cDNA library using PCR oligonucleotide primers specific to the nucleotide sequences encoding the amino terminal sequences of the galectin 10 protein and to vector sequences 3' to the gene. Additional nucleotides containing restriction sites to facilitate cloning are added to the 5' and 3' sequences.
  • the 5 ' galectin 8 oligonucleotide primer has the sequence 5' cgc ccATGg
  • CCTATGTCCCCGCACCG 3' (SEQ ID NO:41) containing the underlined Ncol restriction site and nucleotides 56 to 72 of the galectin 8 protein coding sequence in FIG. 1 (SEQ ID NO: 1).
  • the 3 ' galectin 8 primer has the sequence 5 ' cgc AAG CTT TTAGATC TGGACATAGGAC 3' (SEQ ID NO:42) containing the underlined Hindlll restriction site followed by nucleotides complementary to position 1005 to 1023 of the galectin 8 protein coding sequence in FIG. 1 (SEQ ID NO:l).
  • the 5' galectin 9 oligonucleotide primer has the sequence 5 'cgc ccATGg
  • CCTT CAGCGGTTCCCAG 3 ' (SEQ ID NO:43) containing the underlined Ncol restriction site and nucleotides 20 to 36 of the galectin 9 protein coding sequence in FIG. 2A-2B (SEQ ID NO:3).
  • the 3' galectin 9 primer has the sequence 5 'cgc AAG CTT CAGGGTT
  • the 5' galectin 10 and 10SV oligonucleotide primer has the sequence 5 'cgc CCATGc TGTTGTCCTTAAACAAC 3' (SEQ ID NO:45) containing the underlined Sphl restriction site and nucleotides 122-138 of the galectin 10 protein coding sequence in FIG. 3A-3B (SEQ ID NO:5).
  • the 3 ' galectin 10 primer has the sequence 5' cgc CTG CAG CACAGAA
  • GCCATTCTG 3' (SEQ ID NO:46) containing the underlined Pstl restriction site followed by nucleotides complementary to position 1105-1120 of the galectin 10 protein coding sequence in FIG. 3A-3B (SEQ ID NO:5).
  • the 3' galectin 10SV primer has the sequence 5' CGCCTGCAGCTA TGCAACTTTATAAAATATTCC 3 ' (SEQ ID NO:47) containing the underlined
  • restriction sites are convenient to restriction enzyme sites in the bacterial expression vector pQE60 (galectin 8 and 9) or pQE6 (galectin 10), which are used for bacterial expression in these examples. (Qiagen, Inc. 9259
  • pQE60 encodes ampicillin antibiotic resistance ("Amp r ”) and contains a bacterial origin of replication ("ori”), an IPTG inducible promoter, a ribosome binding site (“RBS”), a 6-His tag and restriction enzyme sites.
  • the amplified galectin 8, 9, 10, or 1 OSV DNA and the vector pQE60 or pQE6 both are digested with Ncol and Hindlll (for galectin 8 and 9) or Sphl and Pstl (for galectin 10) and the digested DNAs are then ligated together.
  • Insertion of the galectin 8, 9, 10, or 10SV protein DNA into the restricted pQE60 or pQE6 vector placed the galectin 8, 9, 10, or 10SV protein coding region downstream of and operably linked to the vector's IPTG-inducible promoter and in-frame with an initiating AUG appropriately positioned for translation of galectin 8, 9, 10, or 1 OSV protein.
  • E. coli strain M15/rep4 containing multiple copies of the plasmid pR ⁇ P4, which expresses lac repressor and confers kanamycin resistance ("Kan r "), is used in carrying out the example described herein.
  • This strain which is only one of many that are suitable for expressing galectin 8, 9, 10, or 10SV protein, is available commercially from
  • Transformants are identified by their ability to grow on LB plates in the presence of ampicillin and kanamycin. Plasmid DNA is isolated from resistant colonies and the identity of the cloned DNA confirmed by restriction analysis. Clones containing the desired constructs are grown overnight ("O/N") in liquid culture in LB media supplemented with both ampicillin (100 ⁇ g/ml) and kanamycin (25 ⁇ g/ml).
  • the O/N culture is used to inoculate a large culture, at a dilution of approximately 1 : 100 to 1 :250.
  • the cells are grown to an optical density at 600nm ("OD600") of between 0.4 and 0.6.
  • OD600 optical density at 600nm
  • IPTG IPTG
  • PBS 2X phosphate-buffered saline
  • the protein is purified by a further step of chromatography to remove endotoxin. Then, it is sterile filtered. The sterile filtered protein preparation is stored in 2X PBS at a concentration of 95 ⁇ /ml.
  • Example 2 Cloning and Expression of Galectin 8, 9, 10 and 10SV protein in a Baculovirus Expression System
  • the cDNA sequence encoding the full length galectin 8, 9, 10, or 10SV protein in the deposited clone is amplified using PCR oligonucleotide primers corresponding to the 5 ' and 3 ' sequences of the gene:
  • the 5' galectin 8 oligonucleotide primer has the sequence 5 'cgc CCC GGG GCCTATGTCCCCGCAC 3' (SEQ ID NO:48) containing the underlined Smal restriction site and nucleotides 55 to 70 of the galectin 8 protein coding sequence in FIG. 1 (SEQ ID NO:l).
  • the 3' galectin 8 primer has the sequence 5' cgc GGT ACC
  • TTAGATCTGG ACATAGGAC 3' (SEQ ID NO:49) containing the underlined Asp718 restriction site followed by nucleotides complementary to position 1005 to 1023 of the galectin 8 protein coding sequence in FIG. 1 (SEQ ID NO:l).
  • the 5' galectin 9 oligonucleotide primer has the sequence 5' cgc CCC GGG GCCTTCAGCGGTTCCCAG 3' (SEQ ID NO:50) containing the underlined Smal restriction site and nucleotides 19 to 36 of the galectin 9 protein coding sequence in FIG. 2A-2B (SEQ ID NO:3).
  • the 3' galectin 9 primer has the sequence 5' cgc GGT ACC
  • CAGGGTTGG AAAGGCTG 3' (SEQ ID NO:51) containing the underlined Asp718 restriction site followed by nucleotides complementary to position 1029 to 1045 of the galectin 9 protein coding sequence in FIG. 2A-2B (SEQ ID NO:3).
  • the 5' galectin 10 oligonucleotide primer has the sequence 5' cgc CCC
  • GGG TTGTCCTTAAACAACCTAC 3' (SEQ ID NO:52) containing the underlined Smal restriction site and nucleotides 124-142 of the galectin 10 protein coding sequence in FIG. 3A-3B (SEQ ID NO:5).
  • the 3 ' galectin 10 primer has the sequence 5 ' cgc GGT ACC CACA GAAGCCATTCTG 3' (SEQ ID NO:53) containing the underlined Asp718 restriction site followed by nucleotides complementary to position 1105-1120 of the galectin 10 protein coding sequence in FIG. 3A-3B (SEQ ID NO:5).
  • the 3' galectin 10SV primer has the sequence 5' CGCGGTACCCTA TGCAACTTTATAAAATATTCC 3 ' (SEQ ID NO:54) containing the underlined Asp718 restriction site followed by nucleotides complementary to the 3' end of the galectin 10SV protein coding sequence in FIG. 4A-4B (SEQ ID NO:7).
  • An efficient signal for initiation of translation in eukaryotic cells as described by Kozak, M., J Mol Biol. 196:941-950 (1987) is appropriately located in the vector portion of the construct.
  • the amplified fragment is isolated from a 1% agarose gel using a commercially available kit ("Geneclean,” BIO 101 Inc., La Jolla, Ca.). The fragment then is digested with Xbal and again is purified on a 1% agarose gel.
  • the vector pA2-GP is used to express the galectin 8, 9, 10, or 10SV protein in the baculovirus expression system, using standard methods, as described in Summers et al, A MANUAL OF METHODS FOR BACULOVIRUS VECTORS AND INSECT CELL CULTURE PROCEDURES, Texas
  • This expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by convenient restriction sites.
  • the signal peptide of AcMNPV gp67, including the N-terminal methionine, is located just upstream of a BamHI site.
  • the polyadenylation site of the simian virus 40 (“SV40") is used for efficient polyadenylation.
  • the beta-galactosidase gene from E. coli is inserted in the same orientation as the polyhedrin promoter and is followed by the polyadenylation signal of the polyhedrin gene.
  • the polyhedrin sequences are flanked at both sides by viral sequences for cell-mediated homologous recombination with wild-type viral DNA to generate viable virus that express the cloned polynucleotide.
  • baculovirus vectors could be used in place of pA2-GP, such as pAc373, pVL941 and pAcIMl provided, as those of skill readily will appreciate, that construction provides appropriately located signals for transcription, translation, trafficking and the like, such as an in-frame AUG and a signal peptide, as required.
  • pA2-GP such as pAc373, pVL941 and pAcIMl
  • the plasmid is digested with the restriction enzyme Smal and Asp718 and then is dephosphorylated using calf intestinal phosphatase, using routine procedures known in the art.
  • the DNA is then isolated from a 1% agarose gel using a commercially available kit ("Geneclean" BIO 101 Inc., La Jolla, Ca.). This vector DNA is designated herein "V2".
  • Fragment F2 and the dephosphorylated plasmid V2 are ligated together with T4 DNA ligase.
  • E coli HB 101 cells are transformed with ligation mix and spread on culture plates.
  • Bacteria are identified that contain the plasmid with the human galectin 8, 9, 10, or 10SV gene by digesting DNA from individual colonies using Xbal and then analyzing the digestion product by gel electrophoresis. The sequence of the cloned fragment is confirmed by DNA sequencing.
  • This plasmid is designated herein pBacgalectin 8, 9, 10, or 10SV. 5 ⁇ g of the plasmid pBacgalectin 8, 9, 10, or 10SV is co-transfected with
  • BaculoGoldTM baculovirus DNA a commercially available linearized baculovirus DNA
  • BaculoGoldTM virus DNA 5 ⁇ g of the plasmid pBacgalectin 8, 9, 10, or 10SV are mixed in a sterile well of a microtiter plate containing 50 ⁇ l of serum-free Grace's medium (Life Technologies Inc., Gaithersburg, MD).
  • plaque assay After four days the supernatant is collected and a plaque assay is performed, as described by Summers and Smith, cited above. An agarose gel with "Blue Gal” (Life Technologies Inc., Gaithersburg) is used to allow easy identification and isolation of gal-expressing clones, which produce blue-stained plaques. (A detailed description of a "plaque assay” of this type can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9-10).
  • the virus is added to the cells. After appropriate incubation, blue stained plaques are picked with the tip of an Eppendorf pipette. The agar containing the recombinant viruses is then resuspended in an Eppendorf tube containing 200 ⁇ l of Grace's medium. The agar is removed by a brief centrifugation and the supernatant containing the recombinant baculovirus is used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supernatants of these culture dishes are harvested and then they are stored at 4°C. A clone containing properly inserted hESSB I, II and III is identified by DNA analysis including restriction mapping and sequencing. This is designated herein as V-galectin 8, 9, 10, or 10SV.
  • Sf9 cells are grown in Grace's medium supplemented with 10% heat- inactivated FBS.
  • the cells are infected with the recombinant baculovirus V-galectin 8, 9, 10, or 10SV at a multiplicity of infection ("MOI") of about 2
  • vectors used for the transient expression of the galectin 8, 9, 10, or 10SV protein gene sequence in mammalian cells should carry the SV40 origin of replication. This allows the replication of the vector to high copy numbers in cells (e.g. COS cells) which express the T antigen required for the initiation of viral DNA synthesis. Any other mammalian cell line can also be utilized for this purpose.
  • a typical mammalian expression vector contains the promoter element, which mediates the initiation of transcription of mRNA, the protein coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription can be achieved with the early and late promoters from SV40, the long terminal repeats (LTRs) from Retroviruses, e.g. RSV, HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV).
  • LTRs long terminal repeats
  • cellular signals can also be used (e.g., human actin promoter).
  • Suitable expression vectors for use in practicing the present invention include, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109).
  • Mammalian host cells that could be used include, human HeLa, 283, H9 and Jurkart cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV1, African green monkey cells, quail QC1-3 cells, mouse L cells and Chinese hamster ovary cells.
  • the gene can be expressed in stable cell lines that contain the gene integrated into a chromosome.
  • a selectable marker such as dhfr, gpt, neomycin, hygromycin allows the identification and isolation of the transfected cells.
  • the transfected gene can also be amplified to express large amounts of the encoded protein.
  • the DHFR dihydrofolate reductase
  • GS glutamine synthase
  • Another useful selection marker is the enzyme glutamine synthase (GS) (Murphy et al, Biochem. J. 227:211-219 (1991); Bebbington et al, Bio/Technology 10:169-115 (1992)).
  • GS glutamine synthase
  • the mammalian cells are grown in selective medium and the cells with the highest resistance are selected.
  • These cell lines contain the amplified gene(s) integrated into a chromosome. Chinese hamster ovary (CHO) cells are often used for the production of proteins.
  • the expression vectors pCl and pC4 contain the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen et al, Molecular and Cellular Biology,
  • the vectors contain in addition the 3 ' intron, the polyadenylation and termination signal of the rat preproinsulin gene.
  • the expression plasmid, pgalectin 8, 9, 10, or 10SV HA is made by cloning a cDNA encoding galectin 8, 9, 10, or 10SV into the expression vector pcDNAI/Amp (which can be obtained from Invitrogen, Inc.).
  • the expression vector pcDNAI/amp contains: (1) an E coli origin of replication effective for propagation in E.
  • coli and other prokaryotic cells (2) an ampicillin resistance gene for selection of plasmid-containing prokaryotic cells; (3) an SV40 origin of replication for propagation in eukaryotic cells; (4) a CMV promoter, a polylinker, an SV40 intron, and a polyadenylation signal arranged so that a cDNA conveniently can be placed under expression control of the CMV promoter and operably linked to the SV40 intron and the polyadenylation signal by means of restriction sites in the polylinker.
  • a DNA fragment encoding the galectin 8, 9, 10, or 10SV protein and an HA tag fused in frame to its 3 ' end is cloned into the polylinker region of the vector so that recombinant protein expression is directed by the CMV promoter.
  • the HA tag corresponds to an epitope derived from the influenza hemagglutinin protein described by Wilson etal, Cell 57:767-778 (1984). The fusion of the HA tag to the target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope.
  • the plasmid construction strategy is as follows.
  • the galectin 8, 9, 10, or 10SV cDNA of the deposited clone is amplified using primers that contain convenient restriction sites, much as described above regarding the construction of expression vectors for expression of galectin 8, 9, 10, or 10SV in E. coli.
  • primers that contain convenient restriction sites, much as described above regarding the construction of expression vectors for expression of galectin 8, 9, 10, or 10SV in E. coli.
  • one of the primers contains a hemagglutinin tag ("HA tag") as described above.
  • Suitable primers include the following, which are used in this example.
  • the 5' galectin 8 primer has the sequence 5 'cgc CCC GGG gcc ate ATG GCCTATGTCCCCG 3' (S ⁇ Q ID NO:55) containing the underlined Smal restriction enzyme site followed by nucleotide sequence 52-67 of FIG. 1 (S ⁇ Q ID NO:l).
  • the 3 ' galectin 8 primer has the sequence 5' cgc GGT ACC TTAGAT
  • CTGGACATAGGAC 3' (S ⁇ Q ID NO:56) containing the Asp718 restriction followed by nucleotides complementary to nucleotides 1005-1023 of the galectin 8 coding sequence set out in FIG. 1 (S ⁇ Q ID NO:l).
  • the 5 ' galectin 9 primer has the sequence 5 ' cgc CCC GGG gcc ate ATGGCCTTCAGCGGTTC 3 ' (S ⁇ Q ID NO:57) containing the underlined Smal restriction enzyme site followed by the nucleotide sequence of bases 16-32 of FIG. 2A-2B (S ⁇ Q ID NO:3).
  • the 3' galectin 9 primer has the sequence 5' cgc GGT ACC CAGGGTT GGAAAGGCTG 3' (S ⁇ Q ID NO:58) containing the Asp718 restriction followed by nucleotides complementary to nucleotides 1029-1045 of the galectin 9 coding sequence set out in FIG. 2A-2B (SEQ ID NO:3), including the stop codon.
  • the 5' galectin 10 and 10SV primer has the sequence 5' cgc CCC GGG gcc ate ATGATGTTGTCCTTAAAC 3' (SEQ ID NO:59) containing the underlined Smal restriction enzyme site followed by nucleotide sequence 118-135 of FIG. 3A-3B (SEQ ID NO:5).
  • the 3' galectin 10 primer has the sequence 5' cgc GGT ACC CACAG AAGCCATTCTG 3' (SEQ ID NO:60) containing the Asp718 restriction followed by nucleotides complementary to nucleotides 1105-1120 set out in FIG. 3A-3B (SEQ ID NO:5).
  • the 3' galectin 10SV primer has the sequence 5' CGCGGTACCCTA TGCAACTTTATAAAATATTCC 3' (SEQ ID NO: 54) containing the Asp718 restriction followed by nucleotides complementary to the 3' end of the galectin 10SV coding sequence set out in FIG. 4A-4B (SEQ ID NO:7).
  • the PCR amplified DNA fragment and the vector, pcDNAI/Amp are digested with Hindlll and Xhol and then ligated. The ligation mixture is transformed into E.
  • Plasmid DNA is isolated from resistant colonies and examined by restriction analysis and gel sizing for the presence of the galectin 8, 9, 10, or lOSV-encoding fragment.
  • COS cells are transfected with an expression vector, as described above, using DEAE- DEXTRAN, as described, for instance, in Sambrook et al, MOLECULAR
  • galectin 8, 9, 10, or 10SV HA fusion protein is detected by radiolabelling and immunoprecipitation, using methods described in, for example Harlow et al, ANTIBODIES: A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1988). To this end, two days after transfection, the cells are labeled by incubation in media containing 35 S-cysteine for 8 hours. The cells and the media are collected, and the cells are washed and the lysed with detergent-containing RIPA buffer:
  • Proteins are precipitated from the cell lysate and from the culture media using an HA-specific monoclonal antibody. The precipitated proteins then are analyzed by SDS-PAGE gels and autoradiography. An expression product of the expected size is seen in the cell lysate, which is not seen in negative controls.
  • Plasmid pCl is used for the expression of galectin 8, 9, 10, or 10SV protein.
  • Plasmid pCl is a derivative of the plasmid pSV2-dhfr [ATCC Accession No. 37146]. Both plasmids contain the mouse DHFR gene under control of the
  • SV40 early promoter Chinese hamster ovary- or other cells lacking dihydrofolate activity that are transfected with these plasmids can be selected by growing the cells in a selective medium (alpha minus MEM, Life Technologies) supplemented with the chemotherapeutic agent methotrexate.
  • a selective medium alpha minus MEM, Life Technologies
  • methotrexate The amplification of the DHFR genes in cells resistant to methotrexate (MTX) has been well documented (see, e.g., Alt, F.W., Kellems, R.M., Bertino, J.R., and Schimke, R.T., 1978, J. Biol. Chem. 255:1357-1370, Hamlin, J.L. and Ma, C. 1990, Biochem. etBiophys.
  • DHFR gene If a second gene is linked to the DHFR gene it is usually co- amplified and over-expressed. It is state of the art to develop cell lines carrying more than 1,000 copies of the genes. Subsequently, when the methotrexate is withdrawn, cell lines contain the amplified gene integrated into the chromosome(s).
  • Plasmid pCl contains for the expression of the gene of interest a strong promoter of the long terminal repeat (LTR) of the Rouse Sarcoma Virus (Cullen, et al, Molecular and Cellular Biology, March 1985:438-4470) plus a fragment isolated from the enhancer of the immediate early gene of human cytomegalovirus (CMV) (Boshart et al, Cell 47:521-530, 1985). Downstream of the promoter are the following single restriction enzyme cleavage sites that allow the integration of the genes: BamHI, PvuII, and Nrul.
  • LTR long terminal repeat
  • CMV cytomegalovirus
  • the plasmid contains translational stop codons in all three reading frames followed by the 3 ' intron and the polyadenylation site of the rat preproinsulin gene.
  • Other high efficient promoters can also be used for the expression, e.g. , the human ⁇ -actin promoter, the SV40 early or late promoters or the long terminal repeats from other retroviruses, e.g., HIV and HTLVI.
  • the polyadenylation of the mRNA other signals, e.g. , from the human growth hormone or globin genes can be used as well.
  • Stable cell lines carrying a gene of interest integrated into the chromosomes can also be selected upon co-transfection with a selectable marker such as gpt, G418 or hygromycin. It is advantageous to use more than one selectable marker in the beginning, e.g. , G418 plus methotrexate.
  • the plasmid pCl is digested with the restriction enzyme BamHI and then dephosphorylated using calf intestinal phosphates by procedures known in the art.
  • the vector is then isolated from a 1% agarose gel.
  • the DNA sequence encoding galectin 8, 9, or 10SV, ATCC Deposit Nos. 97732, 97733 and 97734, respectively, is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene.
  • the galectin 10 sequence is similarly amplified from a human endometrial tumor or human fetal heart cDNA library.
  • the 5' galectin 8 primer has the sequence 5' cgcCCCGGGgccatcATG GCCTATGTCCCCG 3' (SEQ ID NO:55) containing the underlined Smal restriction enzyme site followed by nucleotide sequence 52-67 of FIG. 1 (SEQ ID NO:l).
  • An efficient signal for initiation of translation in eukaryotic cells as described by Kozak, M., J. Mol Biol. 196:941-950 (1987) is appropriately located in the vector portion of the construct.
  • the 3' galectin 8 primer has the sequence 5' cgc GGT ACC TTAGAT CTGGACATAGGAC 3' (SEQ ID NO:56) containing the Asp718 restriction followed by nucleotides complementary to nucleotides 1005-1023 of the galectin 8 coding sequence set out in FIG. 1 (SEQ ID NO:l).
  • the 5' galectin 9 primer has the sequence 5' cgc CCC GGG gcc ate ATGGCCTTCAGCGGTTC 3 ' (SEQ ID NO:57) containing the underlined Smal restriction enzyme site followed by the nucleotide sequence of bases 16-32 of FIG. 2A-2B (SEQ ID NO:3). Inserted into an expression vector, as described below, the 5' end of the amplified fragment encoding human galectin 9 provides an efficient signal peptide. An efficient signal for initiation of translation in eukaryotic cells, as described by Kozak, M., J. Mol Biol 196:941-950 (1987) is appropriately located in the vector portion of the construct.
  • the 3 ' galectin 9 primer has the sequence 5 ' cgc GGT ACC CAGGGTT GGAAAGGCTG 3 ' (SEQ ID NO:58) containing the Asp718 restriction followed by nucleotides complementary to nucleotides 1029-1045 of the galectin 9 coding sequence set out in FIG. 2A-2B (SEQ ID NO:3), including the stop codon.
  • the 5' galectin 10 and 10SV primer has the sequence 5' cgc CCC GGG gcc ate ATGATGTTGTCCTTAAAC 3' (SEQ ID NO:59) containing the underlined Smal restriction enzyme site followed by nucleotide sequence 118-135 of FIG. 3A-3B (SEQ ID NO:5).
  • Inserted into an expression vector, as described below, the 5' end of the amplified fragment encoding human galectin 10 provides an efficient signal peptide.
  • An efficient signal for initiation of translation in eukaryotic cells, as described by Kozak, M., J. Mol. Biol. 196:947-950 (1987) is appropriately located in the vector portion of the construct.
  • the 3' galectin 10 primer has the sequence 5' cgcGGTACCCACAG AAGCCATTCTG 3' (SEQ ID NO:60) containing the Asp718 restriction followed by nucleotides complementary to nucleotides 1105-1120 set out in FIG. 3A-3B (SEQ ID NO:5).
  • the 3' galectin 10SV primer has the sequence 5' CGCGGTACCCTA
  • TGCAACTTTATAAAATATTCC 3' (SEQ ID NO:54) containing the Asp718 restriction followed by nucleotides complementary to the 3' end of the galectin 10SV coding sequence set out in FIG. 4A-4B (SEQ ID NO:7).
  • the amplified fragments are isolated from a 1%> agarose gel as described above and then digested with the endonucleases Smal and Asp718 and then purified again on a 1% agarose gel.
  • the isolated fragment and the dephosphorylated vector are then ligated with T4 DNA ligase.
  • E. coli HB101 cells are then transformed and bacteria identified that contained the plasmid pCl inserted in the correct orientation using the restriction enzyme Smal. The sequence of the inserted gene is confirmed by
  • Chinese hamster ovary cells lacking an active DHFR enzyme are used for transfection.
  • Five ⁇ g of the expression plasmid Cl are cotransfected with 0.5 ⁇ g of the plasmid pSVneo using the lipofecting method (Feigner et a , supra).
  • the plasmid pSV2-neo contains a dominant selectable marker, the gene neo from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including G418.
  • the cells are seeded in alpha minus MEM supplemented with 1 mg/ml G418.
  • the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) and cultivated from 10-14 days. After this period, single clones are trypsinized and then seeded in 6-well petri dishes using different concentrations of methotrexate (25 nM, 50 nM, 100 nM, 200 nM, 400 nM). Clones growing at the highest concentrations of methotrexate are then transferred to new 6-well plates containing even higher concentrations of methotrexate (500 nM, 1 ⁇ M, 2 ⁇ M, 5 ⁇ M). The same procedure is repeated until clones grow at a concentration of 100 ⁇ M.
  • the expression of the desired gene product is analyzed by Western blot analysis and SDS-PAGE.
  • Northern blot analysis is carried out to examine galectin 8, 9, 10, or 10SV gene expression in human tissues, using methods described by, among others, Sambrook et al, cited above.
  • a cDNA probe containing the entire nucleotide sequence of the galectin 8, 9, 10, or 10SV protein (SEQ ID NO:l, 3, 5, or 7, respectively) is labeled with 32 P using the re /primeTM DNA labeling system (Amersham Life Science), according to manufacturer's instructions. After labeling, the probe is purified using a CHROMA SPIN- 100TM column (Clontech Laboratories, Inc.), according to manufacturer's protocol number PT1200-1. The purified labeled probe is then used to examine various human tissues for galectin
  • MTN Multiple Tissue Northern
  • H human tissues
  • IM human immune system tissues
  • ADDRESSEE Sterne, Kessler, Goldstein, & Fox P.L.L.C.
  • ATC CAA GGA GTG GCC AGC GAG CAC ATG AAG CGG TTC TTC GTG AAC TTT 201 lie Gin Gly Val Ala Ser Glu His Met Lys Arg Phe Phe Val Asn Phe 35 40 45 50
  • CAGCCTTTCC AACCCTGCCT GGGATCTGGG CTTTAATGCA GAGGCCATGT CCTTGTCTGG 1088
  • CAACCCTTCA CCCCTCCTGG AAAGCAGGCC TGATGGCTTC CCACTGGCCT CCACCACCTG 1448
  • GAG TAC AAA CAC AGA TTT AAA GAG CTC AGC AGT ATT GAC ACG CTG GAA 1029 Glu Tyr Lys His Arg Phe Lys Glu Leu Ser Ser He Asp Thr Leu Glu 290 295 300
  • MOLECULE TYPE protein
  • SEQUENCE DESCRIPTION SEQ ID NO : 6 :
  • MOLECULE TYPE protein
  • AGCACCTCTT TGACTTTGCC CATCGNCTCT CGGCCTTCCA GAGGGTGGAC ANATTNGAAA 300
  • MOLECULE TYPE cDNA
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:22:
  • GGTCTTCATA GTCCTGGCTG AGCACTACAA GGTGGTGGTA AATGGAAATC CCTTCTATGA 180
  • MOLECULE TYPE cDNA
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:24:
  • CTGAGCACTA CAAGGTGGTG GTAAATGGAA ATCCCTTCTA TNAGTACGGG CACCGGCTTC 120
  • AATTCCGTTC TCTACTCCCG CCATCCCACC TATAATGTAC CCCCACCCCG CCTATCCAAT 60
  • MOLECULE TYPE cDNA
  • xi SEQUENCE DESCRIPTION: SEQ ID NO: 37:
  • AAGCCACTCT GCCCTCTCTC CTACTTTGGC TGACTCTTCA AGAATGCCAT TCAACAAGTA 60
  • GAAACACCAG TNTTTGGGGC CAGTNCCTCA NTTTCAATCC AGGTAACCTT TAANTGAAAC 60

Abstract

The present invention relates to novel galectin 8, 9, 10 and 10SV proteins which are members of the galectin superfamily. In particular, isolated nucleic acid molecules are provided encoding the human galectin 8, 9, 10 and 10SV proteins. Galectin 8, 9, 10 and 10SV polypeptides are also provided as are vectors, host cells and recombinant methods for producing the same. The invention further relates to screening methods for identifying agonists and antagonists of galectin 8, 9, 10 or 10SV activity. Also provided are diagnostic and therapeutic methods.

Description

Galectin 8, 9, 10 and 10SV
Background of the Invention
Field of the Invention
The present invention relates to novel galectins. More specifically, isolated nucleic acid molecules are provided encoding human galectin 8, 9, 10, or 10SV. Galectin 8, 9, 10 and 10SV polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the same. The invention further relates to screening methods for identifying agonists and antagonists of galectin 8, 9, 10, or 10SV activity. Also provided are diagnostic methods for detecting cell growth disorders and therapeutic methods for cell growth disorders, including autoimmune diseases, cancer, and inflammatory diseases.
Related Art
Lectins are proteins that bind to specific carbohydrate structures and can thus recognize particular glycoconjugates. Barondes et al, J. Biol. Chem.
269(33) .-20807-20810 (1994). Galectins are members of a family of β-galactoside-binding lectins with related amino acid sequences (For review see,
Barondes et al, Cell 76:591-59% (1994); Barondes et al, J. Biol. Chem.
269(33) .20807-20810 (August 1994)). Galectin 1 (aka. L-14-1, L-14, RL-14.5, galaptin, MGBP, GBP, BHL, CHA, HBP, HPL, HLBP 14, rIML-1) is a homodimer with a subunit molecular mass of 14,500 which is abundant in smooth and skeletal muscle, and is present in many other cell types (Couraud et al, J. Biol. Chem. 264:1310-1316 (1989)). Galectin 2 was originally found in hepatoma and is a homodimer with a subunit molecular weight of 14,650 (Gitt et al, J. Biol. Chem. 257. 10601-10606 (1992)). Galectin 3 (aka. Mac-2, EPB,
CBP-35, CBP-30, and L-29) is abundant in activated macrophages and epithelial cells and is a monomer with an apparent molecular mass between 26,320 and 30,300 (Cherayil et al, Proc. Natl Acad. Sci. USA 87: 7324-7326 (1990)). Galectin 4 has a molecular mass of 36,300 and contains two carbohydrate-binding domains within a single polypeptide chain (Oda et al, J. Biol. Chem. 268:5929- 5939 (1993)). Galectins 5 and 6 are mentioned in Barondes et al, Cell 76:591- 598 (1994). Human galectin 7 has a molecular mass of 15,073 and is found mainly in stratified squamous epithelium (Madsen et al, J. Biol Chem. 270(11):5823-5S29 (1995)).
Animal lectins, in general, often function in modulating cell-cell and cell- matrix interactions. Galectin 1 has been shown to either promote or inhibit cell adhesion depending upon the cell type in which it is present. Galectin 1 inhibits cell-matrix interactions in skeletal muscle (Cooper et al, J. Cell Biol. 115:1431- 1448 (1991)). In other cell types, galectin 1 promotes cell-matrix adhesion possibly by cross-linking cell surface and substrate glycoconjugates (Zhou et al, Arch. Biochem. Biophys. 300:6-11 (1993); Skrincosky et al, Cancer Res. 53:2661-2615 (1993)).
Galectin 1 also participates in regulating cell proliferation (Wells et al, Cell 64:91-91 (1991)) and some immune functions (Offner et al, J. Neuroimmunol. 25.177-184 (1990)). Galectin 1 has been shown to regulate the immune response by mediating apoptosis of T cells (Perillo et al, Nature 378: 736-739 (1995)).
Galectin 3 promotes the growth of cells cultured under restrictive culture conditions (Yang et al, Proc. Nat Acad. Sci. USA 93:6131-6142 (June 1996)). Galectin 3 expression in cells confers resistance to apoptosis which indicates that Galectin 3 could be a cell death suppressor which interferes in a common pathway of apoptosis. Id.
Accordingly, there is a need in the art for the identification of novel galectins which can serve as useful tools in the development of therapeutics and diagnostics for regulating immune response. Summary of the Invention
The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the galectin 8, 9, 10, or 10SN polypeptide having the amino acid sequence is shown in FIGs. 1, 2A-2B, 3A-3B, and 4A-4B, respectively (SEQ ID ΝOs:2, 4, 6, and 8, respectively) or the amino acid sequence encoded by the cDNA clones deposited in bacterial hosts as ATCC Deposit Numbers 97732, 97733 and 97734 on September 24, 1996.
The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of galectin 8, 9, 10, or 10SV polypeptides or peptides by recombinant techniques.
The invention further provides an isolated galectin 8, 9, 10, or 10SN polypeptide having an amino acid sequence encoded by a polynucleotide described herein.
The present invention also provides a screening method for identifying compounds capable of enhancing or inhibiting a cellular response induced by galectin 8, 9, 10, or 10SV, which involves contacting cells which express galectin 8, 9, 10, or 10SN with the candidate compound, assaying a cellular response, and comparing the cellular response to a standard cellular response, the standard being assayed when contact is made in absence of the candidate compound; whereby, an increased cellular response over the standard indicates that the compound is an agonist and a decreased cellular response over the standard indicates that the compound is an antagonist. In another aspect, a screening assay for agonists and antagonists is provided which involves determining the effect a candidate compound has on galectin 8, 9, 10, or 10SV binding to the β-galactosidase sugar. In particular, the method involves contacting the β-galactosidase sugar with a galectin 8, 9, 10, or 10SV polypeptide and a candidate compound and determining whether galectin 8, 9, 10, or 10SV binding to β-galactosidase sugar is increased or decreased due to the presence of the candidate compound.
The invention provides a diagnostic method useful during diagnosis disorder. An additional aspect of the invention is related to a method for treating an individual in need of an increased level of galectin 8, 9, 10, or 10SN activity in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of an isolated galectin 8, 9, 10, or 10SV polypeptide of the invention or an agonist thereof. A still further aspect of the invention is related to a method for treating an individual in need of a decreased level of galectin 8, 9, 10, or 10SV activity in the body comprising, administering to such an individual a composition comprising a therapeutically effective amount of a galectin 8, 9, 10, or 10SV antagonist.
Brief Description of the Figures
FIG. 1 shows the nucleotide (SEQ ID ΝO:l) and deduced amino acid
(SEQ ID NO:2) sequences of galectin 8. The protein has a deduced molecular weight of about 36 kDa.
FIG. 2A-2B shows the nucleotide (SEQ ID NO:3) and deduced amino acid (SEQ ID NO:4) sequences of galectin 9. The protein has a deduced molecular weight of about 34.7 kDa.
FIG. 3A-3B shows the nucleotide (SEQ ID NO:5) and deduced amino acid (SEQ ID NO:6) sequences of full length galectin 10. The protein has a deduced molecular weight of about 35.7 kDa.
FIG. 4A-4B shows the nucleotide (SEQ ID NO:7) and deduced amino acid (SEQ ID NO:8) sequences of a galectin 10 splice variant (galectin 10SV).
The protein has a deduced molecular weight of about 22.4 kDa.
FIG. 5A-5E shows the regions of similarity between the amino acid sequences of the galectin 8, 9, and 10 proteins and human galectin 2 (SEQ ID NO:9), human galectin 3 (SEQ ID NO:10), rat galectin 4 (SEQ ID NO:l 1), rat galectin 5 (SEQ ID NO: 12), human galectin 7 (SEQ ID NO: 13), rat galectin 3 (SEQ ID NO: 14), rat galectin 8 (SEQ ID NO: 15), and human galectin 1 (SEQ ID NO:16). FIG. 6 shows the regions of similarity between the amino acid sequences of the galectin 10SV protein and the rat RL30 protein (SEQ ID NO: 17).
FIG. 7 shows a homology comparison between the galectin 10 and galectin 10SV proteins.
FIGs. 8, 9, 10, and 11 show an analysis of the galectin 8, 9, 10, and 10SV amino acid sequence, respectively. Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown. In the "Antigenic Index - Jameson- Wolf graph, amino acid residues 55-101, 137-162, 180-193, 216-266 in FIG. 1 (SEQ ID NO:2), 62-102, 226-259, 197-308 in FIG. 2A-2B (SEQ ID NO:4), 25-77, 84-105, 129-140, 156-183, 195-215, and 241-257 in FIG. 3A-3B
(SEQ ID NO:6), and 25-77, 84-105, 129-140, and 156-183 in FIG. 4A-4B (SEQ ID NO:8) correspond to the shown highly antigenic regions of the galectin 8, 9, 10, or 10SV protein, respectively.
Detailed Description
The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding a galectin 8, 9, 10, or 10SV polypeptide having the amino acid sequence shown in FIGs. 1, 2A-2B, 3A-3B, and 4A-4B, respectively (SEQ ID NOs:2, 4, 6, and 8, respectively), which was deterrnined by sequencing a cloned cDNA. The galectin 8, 9, 10, and 10SV proteins of the present invention share sequence homology with other galectins and the rat RL30 protein (FIGs.
5A-5E and 6) (SEQ ID NOs:9-17). The nucleotide sequences shown in FIGs. 1, 2A-2B, and 4A-4B (SEQ ID NO:l, 3, and 7, respectively) were obtained by sequencing the HSIAL77, HTPBR22, and HETAS87 clones, which were deposited on September 24, 1996 at the American Type Culture Collection, 12301 Park Lawn Drive, Rockville, Maryland 20852, and given accession numbers 97732, 97733 and 97734, respectively. The deposited clones are contained in the pBluescript SK(-) plasmid (Stratagene, LaJolla, CA). The nucleotide sequence shown in FIG. 3A-3B (SEQ ID NO:5), which encodes the full-length galectin 10 protein, was obtained by sequencing a clone cD A obtained from a human endometrial tumor library.
Nucleic Acid Molecules
Unless otherwise indicated, all nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer (such as the Model 373 from Applied Biosystems, Inc.), and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA sequence determined as above. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art.
As is also known in the art, a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion.
Using the information provided herein, such as the nucleotide sequences in FIGs. 1, 2A-2B, 3A-3B, and 4A-4B a nucleic acid molecule of the present invention encoding a galectin 8, 9, 10, or 10SV, respectively, polypeptide may be obtained using standard cloning and screening procedures, such as those for cloning cDNAs using mRNA as starting material. Illustrative of the invention, the nucleic acid molecules described in FIGs. 1, 2A-2B, 3A-3B, and 4A-4B (SEQ ID NO:l, 3, 5, and 7, respectively) were discovered in cDNA libraries derived from human adult small intestine, human pancreatic tumor, human endometrial tumor and human endometrial tumor, respectively. These genes were also identified in cDNA libraries from the following tissues pancreas, colon, small intestine, brain, bone marrow, kidney, lung, spleen, and testes tissue. Galectin 8 (SEQ ID NO:l) appears to be mainly expressed in cells of the human colon and small intestine.
The determined nucleotide sequences of the galectin 8, 9, 10, and 10SV cDNAs of FIGs. 1, 2A-2B, 3A-3B, and 4A-4B, respectively (SEQ ID NOs:l, 3, 5, and 7) contain open reading frames encoding proteins of 323, 311, 317, and 200 amino acid residues, with an initiation codon at positions 52-54, 16-18, 118-
120, and 118-120 of the nucleotide sequences in FIGs. 1 , 2A-2B, 3 A-3B, and 4A- 4B, respectively (SEQ ID NOs:l, 3, 5, and 7), and a deduced molecular weight of about 36, 34.7, 35.7, and 22.4 kDa, respectively. The galectin 8, 9, 10 and 10SV proteins shown in FIGs. 1, 2A-2B, 3A-3B, and 4A-4B respectively (SEQ ID NOs:2, 4, 6, and 8) share homology with other galectins (See, e.g., FIG. 5A-
5E).
As one of ordinary skill would appreciate, due to the possibilities of sequencing errors discussed above, as well as the variability of processing sites for different known proteins, the predicted galectin 8 and 9 polypeptides encoded by the deposited cDNAs comprise about 323 and 311 amino acids, but may be anywhere in the range of 300 - 333 amino acids. Similarly, the predicted galectin 10 polypeptide comprises about 317 amino acids, but may be anywhere in the range of 305 - 329 amino acids. Further, the predicted galectin 10SV polypeptide encoded by the deposited cDNA comprises about 200 amino acids, but may be anywhere in the range of 190 - 210 amino acids. Galectin 10SV is believed to be a splice variant of galectin 10. As used herein the phrase "splice variant" refers to cDNA molecules produced from RNA molecules initially transcribed from the same genomic DNA sequence but which have undergone alternative RNA splicing. Alternative RNA splicing occurs when a primary RNA transcript undergoes splicing, generally for the removal of introns, which results in the production of more than one mRNA molecule each of which may encode different amino acid sequences. The term "splice variant" also refers to the proteins encoded by the above cDNA molecules.
As indicated, nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced synthetically. The DNA may be double-stranded or single-stranded. Single-stranded DNA or RNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand. By "isolated" nucleic acid molecule(s) is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment For example, recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution.
Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.
Isolated nucleic acid molecules of the present invention include DNA molecules comprising an open reading frame (ORF) shown in FIGs. 1, 2A-2B,
3A-3B, and 4A-4B, respectively (SEQ ID NOs:l, 3, 5, and 7); and DNA molecules which comprise a sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode the galectin 8, 9, 10, or 10SV protein. Of course, the genetic code is well known in the art. Thus, it would be routine for one skilled in the art to generate such degenerate variants.
In addition, the invention provides nucleic acid molecules having nucleotide sequences related to extensive portions of SEQ ID NO:l which have been determined from the following related cDNA clones: HSIAL77R (SEQ ID
NO: 18), HGBDK55R (SEQ ID NO: 19), HCNAH29R (SEQ ID NO:20),
HKCAA85R (SEQ ID NO:21), HCNAI55R (SEQ ID NO:22), HCNAI87R (SEQ
ID NO:23), HCNAS74R (SEQ ID NO:24) and HCNAF43R (SEQ ID NO:25).
In addition, the invention provides nucleic acid molecules having nucleotide sequences related to extensive portions of SEQ ID NO: 3 which have been determined from the following related cDNA clones: HMSCP11R (SEQ ID NO:26), HMSEU32R (SEQ ID NO:27), HTPAO71R (SEQ ID NO:28), HJAAN54R (SEQ ID ΝO:29), HMSEU43R (SEQ ID NO:30), HILBP03R (SEQ ID NO:31), HTPCG81R (SEQ ID NO:32), HTBAA21R (SEQ ID NO:33), and HFXBU26R (SEQ ID NO:34).
In addition, the invention provides nucleic acid molecules having nucleotide sequences related to extensive portions of SEQ ID NO: 5 which have been determined from the following related cDNA clones: HTNBX92R (SEQ ID NO:35), HLTAZ64RB (SEQ ID NO:36), HJBAI38R (SEQ ID NO:37), HETAS87R (SEQ ID NO:38), and HETAR45R (SEQ ID NO:39).
In addition, the invention provides nucleic acid molecules having nucleotide sequences related to extensive portions of SEQ ID NO:7 which have been determined from the following related cDNA clones: HTNBX92R (SEQ ID NO:35), HLTAZ64RB (SEQ ID NO:36), HBNAF37R (SEQ ID NO:40), and HETAS87R (SEQ ID NO:38).
In another aspect, the invention provides isolated nucleic acid molecules encoding the galectin 8, 9, 10 or 10SV polypeptide having an amino acid sequence encoded by the cDNA clone contained in the plasmid deposited as ATCC Deposit Nos. 97732, 97733 and 97734, respectively, on September 24, 1996. In a further embodiment, nucleic acid molecules are provided encoding the full-length galectin 8, 9, 10, or 10SV polypeptide lacking the N-terminal methionine. The invention further provides an isolated nucleic acid molecule having the nucleotide sequence shown in FIGs. 1, 2A-2B, 3A-3B, or 4A-4B (SEQ ID NOs:l, 3, 5, or 7) or the nucleotide sequence of the galectin 8, 9, or 10SV cDNA contained in the above-described deposited clones, or a nucleic acid molecule having a sequence complementary to one of the above sequences. Such isolated molecules, particularly DNA molecules, are useful as probes for gene mapping, by in situ hybridization with chromosomes, and for detecting expression of the galectin 8, 9, 10, or 10SV gene in human tissue, for instance, by Northern blot analysis.
The present invention is further directed to fragments of the isolated nucleic acid molecules described herein. By a fragment of an isolated nucleic acid molecule having the nucleotide sequence of the deposited cDNA or the nucleotide sequence shown in FIGs. 1, 2A-2B, 3A-3B, or 4A-4B (SEQ ID NO:l, 3, 5, or 7) is intended fragments at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length which are useful as diagnostic probes and primers as discussed herein. Of course larger DNA fragments 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 100, 1050, 1100, or 1115 nt in length of the sequence shown in SEQ ID NO:l are also useful according to the present invention as are fragments corresponding to most, if not all, of the nucleotide sequence of the cDNA clone contained in the plasmid deposited as ATCC Deposit No. 97732 or as shown in SEQ ID NO: 1. Similarly, larger DNA fragments 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 100, 1050, 1100, 1150, 1200, 1250, 1300, 1350,
1400, 1450, 1500, or 1525 nt in length of the sequence shown in SEQ ID NO:3 are also useful according to the present invention as are fragments corresponding to most, if not all, of the nucleotide sequence of the cDNA clone contained in the plasmid deposited as ATCC Deposit No. 97733 or as shown in SEQ ID NO:3. Similarly, larger DNA fragments 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 100, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, or 1464 nt in length of the sequence shown in SEQ ID NO:5 are also useful according to the present invention as are fragments corresponding to most, if not all, of the nucleotide sequence of the cDNA molecule as shown in SEQ ID NO:5. Further, larger DNA fragments 50, 100, 150, 200, 250, 300, 350,
400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 100, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, and 1908 nt in length of the sequence shown in SEQ ID NO:7 are also useful according to the present invention as are fragments corresponding to most, if not all, of the nucleotide sequence of the cDNA clone contained in the plasmid deposited as ATCC Deposit No. 97734 or as shown in SEQ ID NO:7. By a fragment at least 20 nt in length, for example, is intended fragments which include 20 or more contiguous bases from the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in SEQ ID NOs:l, 3, 5, or 7.
Preferred nucleic acid fragments of the present invention include nucleic acid molecules encoding epitope-bearing portions of the galectin 8, 9, 10, or 10SV protein. In particular, such nucleic acid fragments of the present invention include nucleic acid molecules encoding: a polypeptide comprising amino acid residues from about 55-101, 137-162, 180-193, 216-266 in FIG. 1 (SEQ ID
NO:2), 62-102, 226-259, 197-308 in FIG. 2A-2B (SEQ ID NO:4), 25-77, 84-105, 129-140, 156-183, 195-215, and 241-257 in FIG. 3A-3B (SEQ ID NO:6), and 25- 77, 84-105, 129-140, and 156-183 in FIG. 4A-4B (SEQ ID NO:8). The inventors have determined that the above polypeptide fragments are antigenic regions of the galectin 8, 9, 10, and 10SV proteins. Methods for determining other such epitope-bearing portions of the galectin 8, 9, 10, and 10SV proteins are described in detail below.
In another aspect, the invention provides an isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent hybridization conditions to a portion of the polynucleotide in a nucleic acid molecule of the invention described above, for instance, a cDNA clone contained in ATCC Deposit Nos. 97732, 97733 and 97734. By "stringent hybridization conditions" is intended overnight incubation at 42°C in a solution comprising: 50% formamide, 5x SSC (150 mM NaCl, 15mM trisodium citrate), 50 raM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 g/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1 x SSC at about 65 °C.
By a polynucleotide which hybridizes to a "portion" of a polynucleotide is intended a polynucleotide (either DNA or RNA) hybridizing to at least about 15 nucleotides (nt), and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably about 30-70 nt of the reference polynucleotide. These are useful as diagnostic probes and primers as discussed above and in more detail below.
By a portion of a polynucleotide of "at least 20 nt in length," for example, is intended 20 or more contiguous nucleotides from the nucleotide sequence of the reference polynucleotide (e.g. , the deposited cDNA or the nucleotide sequence as shown in FIGs. 1, 2A-2B, 3A-3B, or 4A-4B (SEQ ID NOs:l, 3, 5, or 7)). Of course, a polynucleotide which hybridizes only to a poly A sequence (such as the 3' terminal poly(A) tract of the galectin 8, 9, 10, or 10SV cDNA shown in FIGs. 1, 2A-2B, 3A-3B, or 4A-4B, respectively (SEQ ID NOs:l, 3, 5, or 7)), or to a complementary stretch of T (or U) resides, would not be included in a polynucleotide of the invention used to hybridize to a portion of a nucleic acid of the invention, since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone).
As indicated, nucleic acid molecules of the present invention which encode a galectin 8, 9, 10, or 10SN polypeptide may include, but are not limited to those encoding the amino acid sequence of the polypeptide, by itself; the coding sequence for the polypeptide and additional sequences, such as those encoding an amino acid leader or secretory sequence, such as a pre-, or pro- or prepro- protein sequence; the coding sequence of the polypeptide, with or without the aforementioned additional coding sequences, together with additional, non-coding sequences, including for example, but not limited to introns and non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing, including splicing and polyadenylation signals, for example - ribosome binding and stability of mRNA; an additional coding sequence which codes for additional amino acids, such as those which provide additional functionalities. Thus, the sequence encoding the polypeptide may be fused to a marker sequence, such as a sequence encoding a peptide which facilitates purification of the fused polypeptide. In certain preferred embodiments of this aspect of the invention, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc.), among others, many of which are commercially available. As described in Gentz et al, Proc. Natl. Acad. Sci. USA <S6':821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein.
The "HA" tag is another peptide useful for purification which corresponds to an epitope derived from the influenza hemagglutinin protein, which has been described by Wilson et al, Cell 37:161-11 (1984). As discussed below, other such fusion proteins include the galectin 8, 9, 10, or 10SV fused to Fc at the N- or C -terminus.
The present invention further relates to variants of the nucleic acid molecules of the present invention, which encode portions, analogs or derivatives of the galectin 8, 9, 10, or 10SN protein. Variants may occur naturally, such as a natural allelic variant. By an "allelic variant" is intended one of several alternate forms of a gene occupying a given locus on a chromosome of an organism.
Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985). Non-naturally occurring variants may be produced using art-known mutagenesis techniques.
Such variants include those produced by nucleotide substitutions, deletions or additions which may involve one or more nucleotides. The variants may be altered in coding regions, non-coding regions, or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the galectin 8, 9, 10, or 10SV protein or portions thereof. Also especially preferred in this regard are conservative substitutions.
Further embodiments of the invention include isolated nucleic acid molecules comprising a polynucleotide having a nucleotide sequence at least 95% identical, and more preferably at least 96%, 97%, 98% or 99% identical to (a) a nucleotide sequence encoding the galectin 8, 9, 10, or 10SV polypeptide having the amino acid sequence in FIGs. 1 , 2A-2B, 3 A-3B, or 4A-4B (SEQ ID NOs: 1 ,
3, 5, or 7); (b) a nucleotide sequence encoding the polypeptide having the amino acid sequence in FIGs. 1, 2A-2B, 3A-3B, or 4A-4B (SEQ ID NOs:l, 3, 5, or 7), but lacking the N-terminal methionine; (c) a nucleotide sequence encoding the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit Nos. 97732, 97733 or 97734 on September 24, 1996; or (d) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), or (c).
By a polynucleotide having a nucleotide sequence at least, for example, 95%» "identical" to a reference nucleotide sequence encoding a galectin 8, 9, 10, or 10SV polypeptide is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the galectin 8, 9, 10, or 10SV polypeptide. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
As a practical matter, whether any particular nucleic acid molecule is at least 95%, 96%, 97%, 98% or 99% identical to, for instance, the nucleotide sequence shown in FIGs. 1 , 2A-2B, 3 A-3B, or 4A-4B (SEQ ID NOs: 1 , 3, 5, or
7) or to the nucleotides sequence of the deposited cDNA clone can be determined conventionally using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711. Bestfit uses the local homology algorithm of Smith and Waterman, Advances in
Applied Mathematics 2:482-489 (1981)), to find the best segment of homology between two sequences. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.
The present application is directed to nucleic acid molecules at least 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence shown in FIGs. 1 ,
2A-2B, 3A-3B, or 4A-4B (SEQ ID NOs:l, 3, 5, or 7) or to the nucleic acid sequence of one of the deposited cDNAs, irrespective of whether they encode a polypeptide having galectin 8, 9, 10, or 10SV activity. This is because even where a particular nucleic acid molecule does not encode a polypeptide having galectin 8, 9, 10, or 1 OSV activity, one of skill in the art would still know how to use the nucleic acid molecule, for instance, as a hybridization probe or a polymerase chain reaction (PCR) primer. Uses of the nucleic acid molecules of the present invention that do not encode a polypeptide having galectin 8, 9, 10, or 10SV activity include, ter alia, (1) isolating the galectin 8, 9, 10, or 10SV gene or allelic variants thereof in a cDNA library; (2) in situ hybridization (e.g., "FISH") to metaphase chromosomal spreads to provide precise chromosomal location of the galectin 8, 9, 10, or 10SV gene, as described in Verma et al, Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York (1988); and (3) Northern Blot analysis for detecting galectin 8, 9, 10, or 10SV mRNA expression in specific tissues.
Preferred, however, are nucleic acid molecules having sequences at least 95%o, 96%), 97%), 98%) or 99% identical to the nucleic acid sequence shown in FIGs. 1, 2A-2B, 3A-3B, or 4A-4B (SEQ ID NOs:l, 3, 5, or 7) or to the nucleic acid sequence of one of the deposited cDNAs which do, in fact, encode a polypeptide having galectin 8, 9, 10, or 10SV protein activity. By "a polypeptide having galectin 8, 9, 10, or 10SV activity" is intended polypeptides exhibiting activity similar, but not necessarily identical, to an activity of the galectin 8, 9, 10, or 10SV protein of the invention, as measured in a particular biological assay. For example, galectin 8, 9, 10, or 10SV protein activity can be measured using a lactose binding assay.
Lactose binding activity of the expressed galectin 8, 9, 10, or 10SV is assayed by immunodetection of in situ binding activity to asialofetuin (Sigma) immobilized on nitrocellulose (Amersham) (Madsen et al, J. Biol. Chem. 270(1 J.-5823-5829 (1995)). Thirty μg of asialofetuin dissolved in 3 μl of water is spotted on a 1-cm2 strip of nitrocellulose. The nitrocellulose pieces are then placed in a 24- well tissue culture plate and incubated overnight in buffer B (58 mM Na2HPO4, 18 mM KH2PO4, 75 mM NaCl, 2 mM EDTA, and 3% BSA, pH7.2) with constant agitation at 22°C. Following incubation, the blocking medium is aspirated and the nitrocellulose pieces are washed three times in buffer A (58 mM Na^PO^ 18 mM KH2PO4, 75 mM NaCl, 2 mM EDTA, 4 mM β- mercaptoethanol and 0.2%) BSA, pH7.2). Cell extracts (preferably, COS cells) are prepared containing 1% BSA and either with or without 150 mM lactose (105 μl of primary extract, 15 μl of 10% BSA in buffer A and either 30 μl of 0.75 M lactose in buffer A or 30 μl of buffer A). The immobilized asialofetuin is incubated with the extracts for 2 h and washed 5 times in buffer A. The nitrocellulose pieces are then fixed in 2% formalin in PBS (58 mM Na^PO,,, 18 mM KH2PO4, 75 mM NaCl, 2 mM EDTA pH7.2) for 1 hour to prevent loss of bound galectin. Following extensive washing in PBS the pieces were incubated with rabbit anti-galectin 8, 9, 10, or 1 OSV polyclonal serum diluted 1 : 100 in PBS for 2 h at 22°C. The pieces are then washed in PBS and incubated with peroxidase-labeled goat anti-rabbit antibodies (DAKO). Following incubation for 2 h at 22°C, the pieces are washed in PBS and the substrate is added. Nitrocellulose pieces are incubated until the color develops and the reaction is stopped by washing in distilled water. Of course, due to the degeneracy of the genetic code, one of ordinary skill in the art will immediately recognize that a large number of the nucleic acid molecules having a sequence at least 95%, 96%, 97%, 98%), or 99% identical to the nucleic acid sequence of the deposited cDNA or the nucleic acid sequence shown in FIGs. 1, 2A-2B, 3A-3B, or 4A-4B (SEQ ID NOs:l, 3, 5, or 7, respectively) will encode "a polypeptide having galectin 8, 9, 10, or 1 OSV protein activity." In fact, since degenerate variants of these nucleotide sequences all encode the same polypeptide, this will be clear to the skilled artisan even without performing the above described comparison assay. It will be further recognized in the art that, for such nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a polypeptide having galectin 8, 9, 10, or
10SV protein activity. This is because the skilled artisan is fully aware of amino acid substitutions that are either less likely or not likely to significantly effect protein function (e.g., replacing one aliphatic amino acid with a second aliphatic amino acid). For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie, J. U. et al, "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions," Science 2^7.1306-1310 (1990), wherein the authors indicate that proteins are surprisingly tolerant of amino acid substitutions. Vectors and Host Cells
The present invention also relates to vectors which include the isolated DNA molecules of the present invention, host cells which are genetically engineered with the recombinant vectors, and the production of galectin 8, 9, 10, or 10SV polypeptides or fragments thereof by recombinant techniques.
The polynucleotides may be joined to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
The DNA insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan. The expression constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation. The coding portion of the transcripts expressed by the constructs will preferably include a translation initiating at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated. As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS and Bowes melanoma cells; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art. Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNHlόa, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to the skilled artisan.
Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al, Basic Methods In Molecular Biology (1986).
The polypeptide may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art. A preferred fusion protein comprises a heterologous region from immunoglobulin that is useful to solubilize proteins. For example, EP-A-O 464
533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobin molecules together with another human protein or part thereof. In many cases, the Fc part in a fusion protein is thoroughly advantageous for use in therapy and diagnosis and thus results, for example, in improved pharmacokinetic properties (EP-A 0232262). On the other hand, for some uses it would be desirable to be able to delete the Fc part after the fusion protein has been expressed, detected and purified in the advantageous manner described. This is the case when Fc portion proves to be a hindrance to use in therapy and diagnosis, for example when the fusion protein is to be used as antigen for immunizations. In drug discovery, for example, human proteins, such as, hIL5-receptor has been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. See, D. Bennett et al, Journal of Molecular Recognition, Vol. 8 52-58 (1995) and K. Johanson et al, The Journal of Biological Chemistry, Vol. 270, No. 16, pp 9459-9471 (1995).
The galectin 8, 9, 10, or 1 OSV protein can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is employed for purification. Polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes. Galectin 8, 9, and 10 Polypeptides and Fragments
The invention further provides an isolated galectin 8, 9, 10, or 10SV polypeptide having (1) the amino acid sequence encoded by one of the deposited cDNAs, (2) the amino acid sequence in FIGs. 1, 2A-2B, 3A-3B, or 4A-4B (SEQ ID NOs:2, 4, 6, or 8, respectively), or (3) the amino acid sequence of a peptide or polypeptide comprising a portion of the above polypeptides.
It will be recognized in the art that some amino acid sequences of the galectin 8, 9, 10, or 10SV polypeptide can be varied without significant effect of the structure or function of the protein. If such differences in sequence are contemplated, it should be remembered that there will be critical areas on the protein which determine activity.
Thus, the invention further includes variations of the galectin 8, 9, 10, or 10SV polypeptide which show substantial galectin 8, 9, 10, or 10SV polypeptide activity or which include regions of galectin 8, 9, 10, or 10SV protein such as the protein portions discussed below. Such mutants include deletions, insertions, inversions, repeats, and type substitutions. As indicated above, guidance concerning which amino acid changes are likely to be phenotypically silent can be found in Bowie, J.U., et al, "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions," Science 247:1306-1310 (1990). Thus, the fragment, derivative or analog of the polypeptide of SEQ ID
NOs:2, 4, 6, or 8, or that encoded by one of the deposited cDNAs, may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as an IgG Fc fusion region peptide or leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein. Of particular interest are substitutions of charged amino acids with another charged amino acid and with neutral or negatively charged amino acids. The latter results in proteins with reduced positive charge to improve the characteristics of a galectin 8, 9, 10, or 10SV protein. The prevention of aggregation is highly desirable. Aggregation of proteins not only results in a loss of activity but can also be problematic when preparing pharmaceutical formulations, because they can be immunogenic. (Pinckard et al, Clin. Exp. Immunol 2:331-340 (1967); Robbins etal, Diabetes 55:838-845 (1987); Cleland et al, Crit. Rev. Therapeutic Drug Carrier Systems 10:301-311 (1993)).
As indicated, changes are preferably of a minor nature, such as conservative amino acid substitutions that do not significantly affect the folding or activity of the protein (see Table 1).
TABLE 1. Conservative Amino Acid Substitutions.
Figure imgf000025_0001
Of course, the number of amino acid substitutions a skilled artisan would make depends on many factors, including those described above and below. Generally speaking, the number of substitutions for any given galectin 8, 9, 10, or 10SV polypeptide or mutant thereof will not be more than 50, 40, 30, 20, 10, 5, or 3, depending on the objective.
Amino acids in a galectin 8, 9, 10, or 10SV protein of the present invention that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule. Sites that are critical for ligand binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al, J. Mol. Biol. 224:899-904 (1992) and de Vos et al, Science 255:306-312 (1992)). The polypeptides of the present invention are preferably provided in an isolated form. By "isolated polypeptide" is intended a polypeptide removed from its native environment. Thus, a polypeptide produced and/or contained within a recombinant host cell is considered isolated for purposes of the present invention. Also intended as an "isolated polypeptide" are polypeptides that have been purified, partially or substantially, from a recombinant host cell. For example, a recombinantly produced version of a galectin 8, 9, 10, or 10SV polypeptide can be substantially purified by the one-step method described in Smith and Johnson, Gene 57:31-40 (1988). The polypeptides of the present invention include the polypeptides encoded by the deposited cDNAs; a polypeptide comprising amino acids about
1 to about 323 in SEQ ID NO:2, about 1 to about 311 in SEQ ID NO:4, about 1 to about 317 in SEQ ID NO:6, and about 1 to about 200 in SEQ ID NO:8; a polypeptide comprising amino acids about 2 to about 323 in SEQ ID NO:2, about 2 to about 311 in SEQ ID NO:4, about 2 to about 317 in SEQ ID NO:6 and about
2 to about 200 in SEQ ID NO: 8; as well as polypeptides which are at least 95% identical, still more preferably at least 96%, 97%, 98%> or 99% identical to the polypeptides described above and also include portions of such polypeptides with at least 30 amino acids and more preferably at least 50 amino acids. By a polypeptide having an amino acid sequence at least, for example,
95%> "identical" to a reference amino acid sequence of a galectin 8, 9, 10, or 10SV polypeptide is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of the galectin 8, 9, 10, or 10SV polypeptide. In other words, to obtain a polypeptide having an amino acid sequence at least 95%) identical to a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence. As a practical matter, whether any particular polypeptide is at least 95%),
96%, 97%o, 98% or 99%> identical to, for instance, the amino acid sequence shown in FIGs. 1, 2A-2B, 3A-3B, or 4A-4B (SEQ ID NOs:2, 4, 6, or 8, respectively) or to the amino acid sequence encoded by one of the deposited cDNA clones (ATCC Deposit Numbers 97732, 97733 and 97734) can be determined conventionally using known computer programs such the Bestfit program (Wisconsin Sequence
Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711). When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference amino acid sequence and that gaps in homology of up to 5% of the total number of amino acid residues in the reference sequence are allowed.
The polypeptide of the present invention could be used as a molecular weight marker on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art.
In another aspect, the invention provides a peptide or polypeptide comprising an epitope-bearing portion of a polypeptide of the invention. The epitope of this polypeptide portion is an immunogenic or antigenic epitope described herein. An "immunogenic epitope" is defined as a part of a protein that elicits an antibody response when the whole protein is the immunogen. On the other hand, a region of a protein molecule to which an antibody can bind is defined as an "antigenic epitope." The number of immunogenic epitopes of a protein generally is less than the number of antigenic epitopes. See, for instance, Geysen et al, Proc. Natl Acad. Sci. USA 81:3998- 4002 (1983). As to the selection of peptides or polypeptides bearing an antigenic epitope (i.e., that contain a region of a protein molecule to which an antibody can bind), it is well known in that art that relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked protein. See, for instance, Sutcliffe, J. G.,
Shinnick, T. M., Green, N. and Learner, R.A. (1983) Antibodies that react with predetermined sites on proteins. Science 219:660-666. Peptides capable of eliciting protein-reactive sera are frequently represented in the primary sequence of a protein, can be characterized by a set of simple chemical rules, and are confined neither to immunodominant regions of intact proteins (i.e., immunogenic epitopes) nor to the amino or carboxyl terminals.
Antigenic epitope-bearing peptides and polypeptides of the invention are therefore useful to raise antibodies, including monoclonal antibodies, that bind specifically to a polypeptide of the invention. See, for instance, Wilson et al, Cell 37:161-118 (1984) at 777.
Antigenic epitope-bearing peptides and polypeptides of the invention preferably contain a sequence of at least seven, more preferably at least nine and most preferably between about at least 15 to about 30 amino acids contained within the amino acid sequence of a polypeptide of the invention. Non-limiting examples of antigenic polypeptides or peptides that can be used to generate galectin 8, 9, 10, or lOSV-specific antibodies include: a polypeptide comprising amino acid residues from about 55-101, 137-162, 180- 193, 216-266 in FIG. 1 (SEQ IDNO:2), 62-102, 226-259, 197-308 in FIG. 2A-2B (SEQ IDNO:4), 25-77, 84-105, 129-140, 156-183, 195-215, and 241-257 in FIG. 3A-3B (SEQ ID NO:6), and 25-77, 84-105, 129-140, and 156-183 in FIG. 4A-4B
(SEQ ID NO:8), respectively. As indicated above, the inventors have determined that the above polypeptide fragments are antigenic regions of the galectin 8, 9, 10, or 1 OSV protein.
The epitope-bearing peptides and polypeptides of the invention may be produced by any conventional means Houghten, R. A. (1985) General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids. Proc. Natl. Acad. Sci. USA 52:5131-5135. This "Simultaneous Multiple Peptide Synthesis (SMPS)" process is further described in U.S. Patent No. 4,631,211 to Houghten et al. (1986).
As one of skill in the art will appreciate, galectin 8, 9, 10, or 10SV polypeptides of the present invention and the epitope-bearing fragments thereof described above can be combined with parts of the constant domain of immunoglobulins (IgG), resulting in chimeric polypeptides. These fusion proteins facilitate purification and show an increased half-life in vivo. This has been shown, e.g., for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins (EPA 394,827; Traunecker et al, Nature 331:84- 86 (1988)). Fusion proteins that have a disulfide-linked dimeric structure due to the IgG part can also be more efficient in binding and neutralizing other molecules than the monomeric galectin 8, 9, 10, or 10SV protein or protein fragment alone (Fountoulakis et al, JBiochem 270:3958-3964 (1995)).
Diagnosis and Prognosis
It is believed that certain tissues in mammals with certain diseases
(cancer, autoimmune diseases, inflammatory diseases, asthma, and allergic diseases) express significantly altered (enhanced or decreased) levels of the galectin 8, 9, 10, or 10SV protein and mRNA encoding the galectin 8, 9, 10, or 10SV protein when compared to a corresponding "standard" mammal, i.e., a mammal of the same species not having the disease. Further, it is believed that altered levels of the galectin 8, 9, 10, or 10SV protein can be detected in certain body fluids (e.g., sera, plasma, urine, and spinal fluid) from mammals with the disease when compared to sera from mammals of the same species not having the disease. Thus, the invention provides a diagnostic method useful during diagnosis, which involves assaying the expression level of the gene encoding the galectin 8, 9, 10, or 10SV protein in mammalian cells or body fluid and comparing the gene expression level with a standard galectin 8, 9, 10, or 10SV gene expression level, whereby an increase or decrease in the gene expression level over the standard is indicative of the disease.
Where a diagnosis has already been made according to conventional methods, the present invention is useful as a prognostic indicator, whereby patients exhibiting altered galectin 8, 9, 10, or 10SV gene expression will experience a worse clinical outcome relative to patients expressing the gene at a normal level.
By "assaying the expression level of the gene encoding the galectin 8, 9, 10, or 10SV protein" is intended qualitatively or quantitatively measuring or estimating the level of the galectin 8, 9, 10, or 10SV protein or the level of the mRNA encoding the galectin 8, 9, 10, or 1 OSV protein in a first biological sample either directly (e.g., by determining or estimating absolute protein level or mRNA level) or relatively (e.g., by comparing to the galectin 8, 9, 10, or 10SV protein level or mRNA level in a second biological sample).
Preferably, the galectin 8, 9, 10, or 1 OSV protein level or mRNA level in the first biological sample is measured or estimated and compared to a standard galectin 8, 9, 10, or 10SV protein level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the cancer. As will be appreciated in the art, once a standard galectin 8, 9, 10, or 10SV protein level or mRNA level is known, it can be used repeatedly as a standard for comparison.
By "biological sample" is intended any biological sample obtained from an individual, cell line, tissue culture, or other source which contains galectin 8, 9, 10, or 10SV protein or mRNA. Biological samples include mammalian body fluids (such as sera, plasma, urine, synovial fluid and spinal fluid) which contain secreted galectin 8, 9, 10, or 10SV protein, and ovarian, prostate, heart, placenta, pancreas liver, spleen, lung, breast and umbilical tissue.
The present invention is useful for detecting diseases in mammals (for example, cancer, autoimmune diseases, inflammatory diseases, asthma, and allergic diseases). In particular the invention is useful during diagnosis of the of following types of cancers in mammals: melanoma, renal astrocytoma, Hodgkin disease, breast, ovarian, prostate, bone, liver, lung, pancreatic, and spleenic. Preferred mammals include monkeys, apes, cats, dogs, cows, pigs, horses, rabbits and humans. Particularly preferred are humans. Total cellular RNA can be isolated from a biological sample using the single-step guanidinium-thiocyanate-phenol-chloroform method described in Chomczynski and SacchL nα/. Biochem. 752:156-159 (1987). Levels of mRNA encoding the galectin 8, 9, 10, or 10SV protein are then assayed using any appropriate method. These include Northern blot analysis, (Harada et al. , Cell 55:303-312 (1990) SI nuclease mapping, (Fijita et al, Cell 49:351-361 (1987)) the polymerase chain reaction (PCR), reverse transcription in combination with the polymerase chain reaction (RT-PCR) (Makino et al, Technique 2:295-301 (1990), and reverse transcription in combination with the ligase chain reaction (RT-LCR). Assaying galectin 8, 9, 10, or 10SV protein levels in a biological sample can antibody-based techniques. For example, galectin 8, 9, 10, or 10SV protein expression in tissues can be studied with classical immunohistological methods. (Jalkanen, M., et al, J. Cell. Biol 101:916-985 (1985); Jalkanen, M., et al, J. Cell . Biol. 105:3081-3096 (1987)). Other antibody-based methods useful for detecting galectin 8, 9, 10, or
10SV protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
Suitable labels are known in the art and include enzyme labels, such as, Glucose oxidase, and radioisotopes, such as iodine (1251, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99mTc), and fluorescent labels, such as fluorescein and rhodamine, and biotin.
Therapeutics
It is to be understood that although the following discussion is specifically directed to human patients, the teachings are also applicable to any animal that expresses galectin 8, 9, 10, or 10SV.
As noted above, galectin 8, 9, 10, and 10SV share significant homology with other galectins. Galectin 1 induces apoptosis of T cells and T cell leukemia cell lines. Thus, it is believed by the inventors that galectin 8, 9, 10, and 10SV are active in modulating growth regulatory activities, immunomodulatory activity, cell-cell and cell-substrate interactions, and apoptosis.
The ability of galectin 8, 9, 10, or 10SV to modulate growth regulatory activity may be therapeutically valuable in the treatment of clinical manifestations of such cell regulatory disorders. Disorders which can be treated include, but should not be limited to, autoimmune disease, cancer (preferably, melanoma, renal, astrocytoma, and Hodgkin disease), inflammatory disease, wound healing, arteriosclerosis, other heart diseases, microbe infection (virus, fungal, bacterial, and parasite), asthma, and allergic diseases.
Given the activities modulated by galectin 8, 9, 10, and 10SV, it is readily apparent that a substantially altered (increased or decreased) level of expression of galectin 8, 9, 10, or 10SV in an individual compared to the standard or "normal" level produces pathological conditions such as those described above. It will also be appreciated by one of ordinary skill that the galectin 8, 9, 10, or 10SV protein of the invention will exert its modulating activities on any of its target cells. Therefore, it will be appreciated that conditions caused by a decrease in the standard or normal level of galectin 8, 9, 10, or 10SV activity in an individual, can be treated by administration of galectin 8, 9, 10, or 10SV protein or an agonist thereof. Thus, the invention further provides a method of treating an individual in need of an increased level of galectin 8, 9, 10, or 10SV activity comprising administering to such an individual a pharmaceutical composition comprising an amount of an isolated galectin 8, 9, 10, or 10SV polypeptide of the invention or an agonist thereof to increase the galectin 8, 9, 10, or 10SV activity level in such an individual.
A still further aspect of the invention is related to a method for treating an individual in need of a decreased level of galectin 8, 9, 10, or 10SV activity in the body comprising, administering to such an individual a composition comprising a therapeutically effective amount of a galectin 8, 9, 10, or 10SV antagonist. Preferred antagonists for use in the present invention are galectin 8, 9, 10, or lOSV-specific antibodies.
Modes of administration
It will be appreciated that conditions caused by a decrease in the standard or normal level of galectin 8, 9, 10, or 10SV activity in an individual, can be treated by administration of galectin 8, 9, 10, or 10SV protein or an agonist thereof. Thus, the invention further provides a method of treating an individual in need of an increased level of galectin 8, 9, 10, or 10SV activity comprising administering to such an individual a pharmaceutical composition comprising an effective amount of an isolated galectin 8, 9, 10, or 10SV polypeptide of the invention, particularly a mature form of the galectin 8, 9, 10, or 10SV, effective to increase the galectin 8, 9, 10, or 10SV activity level in such an individual.
As a general proposition, the total pharmaceutically effective amount of galectin 8, 9, 10, or 10SV polypeptide administered parenterally per dose will be in the range of about 1 μg/kg/day to 10 mg/kg/day of patient body weight, although, as noted above, this will be subject to therapeutic discretion. More preferably, this dose is at least 0.01 mg/kg/day, and most preferably for humans between about 0.01 and 1 mg/kg/day for the hormone. If given continuously, the galectin 8, 9, 10, or 10SV polypeptide is typically administered at a dose rate of about 1 μg/kg/hour to about 50 μg/kg/hour, either by 1-4 injections per day or by continuous subcutaneous infusions, for example, using a mini-pump. An intravenous bag solution may also be employed.
Pharmaceutical compositions containing the galectin 8, 9, 10, or 10SV of the invention may be administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, drops or transdermal patch), bucally, or as an oral or nasal spray. By "pharmaceutically acceptable carrier" is meant a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The term "parenteral" as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasteraal, subcutaneous and intraarticular injection and infusion.
Chromosome Assays
The nucleic acid molecules of the present invention are also valuable for chromosome identification. The sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome. The mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease. In certain preferred embodiments in this regard, the cDNA herein disclosed is used to clone genomic DNA of a galectin 8, 9, 10, or 10SV protein gene. This can be accomplished using a variety of well known techniques and libraries, which generally are available commercially. The genomic DNA then is used for in situ chromosome mapping using well known techniques for this purpose.
In addition, in some cases, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis of the 3' untranslated region of the gene is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes.
Fluorescence in situ hybridization ("FISH") of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step. This technique can be used with probes from the cDNA as short as 50 or 60 bp. For a review of this technique, see Verma et al, Human Chromosomes: A Manual Of Basic Techniques, Pergamon Press, New York (1988). Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, for example, in V. McKusick, Mendelian Inheritance In Man, available on-line through Johns Hopkins University, Welch Medical Library. The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes).
Next, it is necessary to determine the differences in the cDNA or genomic sequence between affected and unaffected individuals. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease.
Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended as limiting.
Examples Example 1: Expression and Purification of Galectin 8, 9, 10 and lOSVin E. coli
The DNA sequence encoding the galectin 9 protein in the deposited cDNA clone was amplified using PCR oligonucleotide primers specific to the amino terminal sequences of the galectin 9 protein and to vector sequences 3' to the gene. Additional nucleotides containing restriction sites to facilitate cloning are added to the 5' and 3' sequences.
The DNA sequence encoding the galectin 8 or 10SV protein in the deposited cDNA clone is amplified using PCR oligonucleotide primers specific to the nucleotide sequences encoding the amino terminal sequences of the galectin 8 or 10SV protein and to vector sequences 3' to the gene. Additional nucleotides containing restriction sites to facilitate cloning are added to the 5' and
3' sequences.
The cDNA sequence encoding the galectin 10 protein is amplified from either a human endometrial tumor or human fetal heart cDNA library using PCR oligonucleotide primers specific to the nucleotide sequences encoding the amino terminal sequences of the galectin 10 protein and to vector sequences 3' to the gene. Additional nucleotides containing restriction sites to facilitate cloning are added to the 5' and 3' sequences. The 5 ' galectin 8 oligonucleotide primer has the sequence 5' cgc ccATGg
CCTATGTCCCCGCACCG 3' (SEQ ID NO:41) containing the underlined Ncol restriction site and nucleotides 56 to 72 of the galectin 8 protein coding sequence in FIG. 1 (SEQ ID NO: 1).
The 3 ' galectin 8 primer has the sequence 5 ' cgc AAG CTT TTAGATC TGGACATAGGAC 3' (SEQ ID NO:42) containing the underlined Hindlll restriction site followed by nucleotides complementary to position 1005 to 1023 of the galectin 8 protein coding sequence in FIG. 1 (SEQ ID NO:l).
The 5' galectin 9 oligonucleotide primer has the sequence 5 'cgc ccATGg
CCTT CAGCGGTTCCCAG 3 ' (SEQ ID NO:43) containing the underlined Ncol restriction site and nucleotides 20 to 36 of the galectin 9 protein coding sequence in FIG. 2A-2B (SEQ ID NO:3).
The 3' galectin 9 primer has the sequence 5 'cgc AAG CTT CAGGGTT
GGAAAGGCTG (SEQ ID NO:44) containing the underlined Hindlll restriction site followed by nucleotides complementary to position 1029 to 1045 of the galectin 9 protein coding sequence in FIG. 2A-2B (SEQ ID NO:3). The 5' galectin 10 and 10SV oligonucleotide primer has the sequence 5 'cgc CCATGc TGTTGTCCTTAAACAAC 3' (SEQ ID NO:45) containing the underlined Sphl restriction site and nucleotides 122-138 of the galectin 10 protein coding sequence in FIG. 3A-3B (SEQ ID NO:5). The 3 ' galectin 10 primer has the sequence 5' cgc CTG CAG CACAGAA
GCCATTCTG 3' (SEQ ID NO:46) containing the underlined Pstl restriction site followed by nucleotides complementary to position 1105-1120 of the galectin 10 protein coding sequence in FIG. 3A-3B (SEQ ID NO:5).
The 3' galectin 10SV primer has the sequence 5' CGCCTGCAGCTA TGCAACTTTATAAAATATTCC 3 ' (SEQ ID NO:47) containing the underlined
Pstl restriction site followed by nucleotides complementary to 3' end of the galectin 10SV protein coding sequence in FIG. 4A-4B (SEQ ID NO:7).
The restriction sites are convenient to restriction enzyme sites in the bacterial expression vector pQE60 (galectin 8 and 9) or pQE6 (galectin 10), which are used for bacterial expression in these examples. (Qiagen, Inc. 9259
Eton Avenue, Chatsworth, CA, 91311). pQE60 encodes ampicillin antibiotic resistance ("Ampr") and contains a bacterial origin of replication ("ori"), an IPTG inducible promoter, a ribosome binding site ("RBS"), a 6-His tag and restriction enzyme sites. The amplified galectin 8, 9, 10, or 1 OSV DNA and the vector pQE60 or pQE6 both are digested with Ncol and Hindlll (for galectin 8 and 9) or Sphl and Pstl (for galectin 10) and the digested DNAs are then ligated together. Insertion of the galectin 8, 9, 10, or 10SV protein DNA into the restricted pQE60 or pQE6 vector placed the galectin 8, 9, 10, or 10SV protein coding region downstream of and operably linked to the vector's IPTG-inducible promoter and in-frame with an initiating AUG appropriately positioned for translation of galectin 8, 9, 10, or 1 OSV protein.
The ligation mixture is transformed into competent E. coli cells using standard procedures. Such procedures are described in Sambrook et al, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). E. coli strain M15/rep4, containing multiple copies of the plasmid pRΕP4, which expresses lac repressor and confers kanamycin resistance ("Kanr"), is used in carrying out the example described herein. This strain, which is only one of many that are suitable for expressing galectin 8, 9, 10, or 10SV protein, is available commercially from
Qiagen.
Transformants are identified by their ability to grow on LB plates in the presence of ampicillin and kanamycin. Plasmid DNA is isolated from resistant colonies and the identity of the cloned DNA confirmed by restriction analysis. Clones containing the desired constructs are grown overnight ("O/N") in liquid culture in LB media supplemented with both ampicillin (100 μg/ml) and kanamycin (25 μg/ml).
The O/N culture is used to inoculate a large culture, at a dilution of approximately 1 : 100 to 1 :250. The cells are grown to an optical density at 600nm ("OD600") of between 0.4 and 0.6. Isopropyl-B-D-thiogalactopyranoside
("IPTG") is then added to a final concentration of 1 mM to induce transcription from lac repressor sensitive promoters, by inactivating the lacl repressor. Cells subsequently are incubated further for 3 to 4 hours. Cells then are harvested by centrifugation and disrupted, by standard methods. Inclusion bodies are purified from the disrupted cells using routine collection techniques, and protein is solubilized from the inclusion bodies into 8M urea. The 8M urea solution containing the solubilized protein is passed over a PD-10 column in 2X phosphate-buffered saline ("PBS"), thereby removing the urea, exchanging the buffer and refolding the protein. The protein is purified by a further step of chromatography to remove endotoxin. Then, it is sterile filtered. The sterile filtered protein preparation is stored in 2X PBS at a concentration of 95 μ/ml. Example 2: Cloning and Expression of Galectin 8, 9, 10 and 10SV protein in a Baculovirus Expression System
The cDNA sequence encoding the full length galectin 8, 9, 10, or 10SV protein in the deposited clone is amplified using PCR oligonucleotide primers corresponding to the 5 ' and 3 ' sequences of the gene:
The 5' galectin 8 oligonucleotide primer has the sequence 5 'cgc CCC GGG GCCTATGTCCCCGCAC 3' (SEQ ID NO:48) containing the underlined Smal restriction site and nucleotides 55 to 70 of the galectin 8 protein coding sequence in FIG. 1 (SEQ ID NO:l). The 3' galectin 8 primer has the sequence 5' cgc GGT ACC
TTAGATCTGG ACATAGGAC 3' (SEQ ID NO:49) containing the underlined Asp718 restriction site followed by nucleotides complementary to position 1005 to 1023 of the galectin 8 protein coding sequence in FIG. 1 (SEQ ID NO:l).
The 5' galectin 9 oligonucleotide primer has the sequence 5' cgc CCC GGG GCCTTCAGCGGTTCCCAG 3' (SEQ ID NO:50) containing the underlined Smal restriction site and nucleotides 19 to 36 of the galectin 9 protein coding sequence in FIG. 2A-2B (SEQ ID NO:3).
The 3' galectin 9 primer has the sequence 5' cgc GGT ACC
CAGGGTTGG AAAGGCTG 3' (SEQ ID NO:51) containing the underlined Asp718 restriction site followed by nucleotides complementary to position 1029 to 1045 of the galectin 9 protein coding sequence in FIG. 2A-2B (SEQ ID NO:3).
The 5' galectin 10 oligonucleotide primer has the sequence 5' cgc CCC
GGG TTGTCCTTAAACAACCTAC 3' (SEQ ID NO:52) containing the underlined Smal restriction site and nucleotides 124-142 of the galectin 10 protein coding sequence in FIG. 3A-3B (SEQ ID NO:5).
The 3 ' galectin 10 primer has the sequence 5 ' cgc GGT ACC CACA GAAGCCATTCTG 3' (SEQ ID NO:53) containing the underlined Asp718 restriction site followed by nucleotides complementary to position 1105-1120 of the galectin 10 protein coding sequence in FIG. 3A-3B (SEQ ID NO:5). The 3' galectin 10SV primer has the sequence 5' CGCGGTACCCTA TGCAACTTTATAAAATATTCC 3 ' (SEQ ID NO:54) containing the underlined Asp718 restriction site followed by nucleotides complementary to the 3' end of the galectin 10SV protein coding sequence in FIG. 4A-4B (SEQ ID NO:7). An efficient signal for initiation of translation in eukaryotic cells, as described by Kozak, M., J Mol Biol. 196:941-950 (1987) is appropriately located in the vector portion of the construct.
The amplified fragment is isolated from a 1% agarose gel using a commercially available kit ("Geneclean," BIO 101 Inc., La Jolla, Ca.). The fragment then is digested with Xbal and again is purified on a 1% agarose gel.
This fragment is designated herein F2.
The vector pA2-GP is used to express the galectin 8, 9, 10, or 10SV protein in the baculovirus expression system, using standard methods, as described in Summers et al, A MANUAL OF METHODS FOR BACULOVIRUS VECTORS AND INSECT CELL CULTURE PROCEDURES, Texas
Agricultural Experimental Station Bulletin No. 1555 (1987). This expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by convenient restriction sites. The signal peptide of AcMNPV gp67, including the N-terminal methionine, is located just upstream of a BamHI site. The polyadenylation site of the simian virus 40 ("SV40") is used for efficient polyadenylation. For an easy selection of recombinant virus the beta-galactosidase gene from E. coli is inserted in the same orientation as the polyhedrin promoter and is followed by the polyadenylation signal of the polyhedrin gene. The polyhedrin sequences are flanked at both sides by viral sequences for cell-mediated homologous recombination with wild-type viral DNA to generate viable virus that express the cloned polynucleotide.
Many other baculovirus vectors could be used in place of pA2-GP, such as pAc373, pVL941 and pAcIMl provided, as those of skill readily will appreciate, that construction provides appropriately located signals for transcription, translation, trafficking and the like, such as an in-frame AUG and a signal peptide, as required. Such vectors are described in Luckow et al, Virology 170:31-39, among others.
The plasmid is digested with the restriction enzyme Smal and Asp718 and then is dephosphorylated using calf intestinal phosphatase, using routine procedures known in the art. The DNA is then isolated from a 1% agarose gel using a commercially available kit ("Geneclean" BIO 101 Inc., La Jolla, Ca.). This vector DNA is designated herein "V2".
Fragment F2 and the dephosphorylated plasmid V2 are ligated together with T4 DNA ligase. E coli HB 101 cells are transformed with ligation mix and spread on culture plates. Bacteria are identified that contain the plasmid with the human galectin 8, 9, 10, or 10SV gene by digesting DNA from individual colonies using Xbal and then analyzing the digestion product by gel electrophoresis. The sequence of the cloned fragment is confirmed by DNA sequencing. This plasmid is designated herein pBacgalectin 8, 9, 10, or 10SV. 5 μg of the plasmid pBacgalectin 8, 9, 10, or 10SV is co-transfected with
1.0 μg of a commercially available linearized baculovirus DNA ("BaculoGold™ baculovirus DNA", Pharmingen, San Diego, CA.), using the lipofection method described by Feigner et al, Proc. Natl. Acad. Sci. USA 84: 7413-7417 (1987). lμg of BaculoGold™ virus DNA and 5 μg of the plasmid pBacgalectin 8, 9, 10, or 10SV are mixed in a sterile well of a microtiter plate containing 50 μl of serum-free Grace's medium (Life Technologies Inc., Gaithersburg, MD). Afterwards 10 μl Lipofectin plus 90 μl Grace's medium are added, mixed and incubated for 15 minutes at room temperature. Then the transfection mixture is added drop- wise to Sf9 insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with 1 ml Grace's medium without serum. The plate is rocked back and forth to mix the newly added solution. The plate is then incubated for 5 hours at 27°C. After 5 hours the transfection solution is removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum is added. The plate is put back into an incubator and cultivation is continued at 27°C for four days. After four days the supernatant is collected and a plaque assay is performed, as described by Summers and Smith, cited above. An agarose gel with "Blue Gal" (Life Technologies Inc., Gaithersburg) is used to allow easy identification and isolation of gal-expressing clones, which produce blue-stained plaques. (A detailed description of a "plaque assay" of this type can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9-10).
Four days after serial dilution, the virus is added to the cells. After appropriate incubation, blue stained plaques are picked with the tip of an Eppendorf pipette. The agar containing the recombinant viruses is then resuspended in an Eppendorf tube containing 200 μl of Grace's medium. The agar is removed by a brief centrifugation and the supernatant containing the recombinant baculovirus is used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supernatants of these culture dishes are harvested and then they are stored at 4°C. A clone containing properly inserted hESSB I, II and III is identified by DNA analysis including restriction mapping and sequencing. This is designated herein as V-galectin 8, 9, 10, or 10SV.
Sf9 cells are grown in Grace's medium supplemented with 10% heat- inactivated FBS. The cells are infected with the recombinant baculovirus V-galectin 8, 9, 10, or 10SV at a multiplicity of infection ("MOI") of about 2
(about 1 to about 3). Six hours later the medium is removed and is replaced with SF900 II medium minus methionine and cysteine (available from Life Technologies Inc., Gaithersburg). 42 hours later, 5 μCi of 35S-methionine and 5 μCi 35S-cysteine (available from Amersham) are added. The cells are further incubated for 16 hours and then they are harvested by centrifugation, lysed and the labeled proteins are visualized by SDS-PAGE and autoradiography. Example 3: Cloning and Expression in Mammalian Cells
Most of the vectors used for the transient expression of the galectin 8, 9, 10, or 10SV protein gene sequence in mammalian cells should carry the SV40 origin of replication. This allows the replication of the vector to high copy numbers in cells (e.g. COS cells) which express the T antigen required for the initiation of viral DNA synthesis. Any other mammalian cell line can also be utilized for this purpose.
A typical mammalian expression vector contains the promoter element, which mediates the initiation of transcription of mRNA, the protein coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription can be achieved with the early and late promoters from SV40, the long terminal repeats (LTRs) from Retroviruses, e.g. RSV, HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV).
However, cellular signals can also be used (e.g., human actin promoter). Suitable expression vectors for use in practicing the present invention include, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109). Mammalian host cells that could be used include, human HeLa, 283, H9 and Jurkart cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV1, African green monkey cells, quail QC1-3 cells, mouse L cells and Chinese hamster ovary cells.
Alternatively, the gene can be expressed in stable cell lines that contain the gene integrated into a chromosome. The co-transfection with a selectable marker such as dhfr, gpt, neomycin, hygromycin allows the identification and isolation of the transfected cells.
The transfected gene can also be amplified to express large amounts of the encoded protein. The DHFR (dihydrofolate reductase) is a useful marker to develop cell lines that carry several hundred or even several thousand copies of the gene of interest. Another useful selection marker is the enzyme glutamine synthase (GS) (Murphy et al, Biochem. J. 227:211-219 (1991); Bebbington et al, Bio/Technology 10:169-115 (1992)). Using these markers, the mammalian cells are grown in selective medium and the cells with the highest resistance are selected. These cell lines contain the amplified gene(s) integrated into a chromosome. Chinese hamster ovary (CHO) cells are often used for the production of proteins.
The expression vectors pCl and pC4 contain the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen et al, Molecular and Cellular Biology,
438-4470 (March, 1985)) plus a fragment of the CMV-enhancer (Boshart et al, Cell 4i:521-530 (1985)). Multiple cloning sites, e.g., with the restriction enzyme cleavage sites BamHI, Xbal and Asp718, facilitate the cloning of the gene of interest. The vectors contain in addition the 3 ' intron, the polyadenylation and termination signal of the rat preproinsulin gene.
Example 3(a): Cloning and Expression in COS Cells
The expression plasmid, pgalectin 8, 9, 10, or 10SV HA, is made by cloning a cDNA encoding galectin 8, 9, 10, or 10SV into the expression vector pcDNAI/Amp (which can be obtained from Invitrogen, Inc.). The expression vector pcDNAI/amp contains: (1) an E coli origin of replication effective for propagation in E. coli and other prokaryotic cells; (2) an ampicillin resistance gene for selection of plasmid-containing prokaryotic cells; (3) an SV40 origin of replication for propagation in eukaryotic cells; (4) a CMV promoter, a polylinker, an SV40 intron, and a polyadenylation signal arranged so that a cDNA conveniently can be placed under expression control of the CMV promoter and operably linked to the SV40 intron and the polyadenylation signal by means of restriction sites in the polylinker. A DNA fragment encoding the galectin 8, 9, 10, or 10SV protein and an HA tag fused in frame to its 3 ' end is cloned into the polylinker region of the vector so that recombinant protein expression is directed by the CMV promoter. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein described by Wilson etal, Cell 57:767-778 (1984). The fusion of the HA tag to the target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope.
The plasmid construction strategy is as follows. The galectin 8, 9, 10, or 10SV cDNA of the deposited clone is amplified using primers that contain convenient restriction sites, much as described above regarding the construction of expression vectors for expression of galectin 8, 9, 10, or 10SV in E. coli. To facilitate detection, purification and characterization of the expressed galectin 8, 9, 10, or 10SV, one of the primers contains a hemagglutinin tag ("HA tag") as described above. Suitable primers include the following, which are used in this example.
The 5' galectin 8 primer has the sequence 5 'cgc CCC GGG gcc ate ATG GCCTATGTCCCCG 3' (SΕQ ID NO:55) containing the underlined Smal restriction enzyme site followed by nucleotide sequence 52-67 of FIG. 1 (SΕQ ID NO:l). The 3 ' galectin 8 primer has the sequence 5' cgc GGT ACC TTAGAT
CTGGACATAGGAC 3' (SΕQ ID NO:56) containing the Asp718 restriction followed by nucleotides complementary to nucleotides 1005-1023 of the galectin 8 coding sequence set out in FIG. 1 (SΕQ ID NO:l).
The 5 ' galectin 9 primer has the sequence 5 ' cgc CCC GGG gcc ate ATGGCCTTCAGCGGTTC 3 ' (SΕQ ID NO:57) containing the underlined Smal restriction enzyme site followed by the nucleotide sequence of bases 16-32 of FIG. 2A-2B (SΕQ ID NO:3).
The 3' galectin 9 primer has the sequence 5' cgc GGT ACC CAGGGTT GGAAAGGCTG 3' (SΕQ ID NO:58) containing the Asp718 restriction followed by nucleotides complementary to nucleotides 1029-1045 of the galectin 9 coding sequence set out in FIG. 2A-2B (SEQ ID NO:3), including the stop codon.
The 5' galectin 10 and 10SV primer has the sequence 5' cgc CCC GGG gcc ate ATGATGTTGTCCTTAAAC 3' (SEQ ID NO:59) containing the underlined Smal restriction enzyme site followed by nucleotide sequence 118-135 of FIG. 3A-3B (SEQ ID NO:5).
The 3' galectin 10 primer has the sequence 5' cgc GGT ACC CACAG AAGCCATTCTG 3' (SEQ ID NO:60) containing the Asp718 restriction followed by nucleotides complementary to nucleotides 1105-1120 set out in FIG. 3A-3B (SEQ ID NO:5).
The 3' galectin 10SV primer has the sequence 5' CGCGGTACCCTA TGCAACTTTATAAAATATTCC 3' (SEQ ID NO: 54) containing the Asp718 restriction followed by nucleotides complementary to the 3' end of the galectin 10SV coding sequence set out in FIG. 4A-4B (SEQ ID NO:7). The PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digested with Hindlll and Xhol and then ligated. The ligation mixture is transformed into E. coli strain SURE (available from Stratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla, CA 92037), and the transformed culture is plated on ampicillin media plates which then are incubated to allow growth of ampicillin resistant colonies. Plasmid DNA is isolated from resistant colonies and examined by restriction analysis and gel sizing for the presence of the galectin 8, 9, 10, or lOSV-encoding fragment.
For expression of recombinant galectin 8, 9, 10, or 10SV, COS cells are transfected with an expression vector, as described above, using DEAE- DEXTRAN, as described, for instance, in Sambrook et al, MOLECULAR
CLONING: A LABORATORY MANUAL, Cold Spring Laboratory Press, Cold Spring Harbor, New York (1989). Cells are incubated under conditions for expression of galectin 8, 9, 10, or 10SV by the vector.
Expression of the galectin 8, 9, 10, or 10SV HA fusion protein is detected by radiolabelling and immunoprecipitation, using methods described in, for example Harlow et al, ANTIBODIES: A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1988). To this end, two days after transfection, the cells are labeled by incubation in media containing 35S-cysteine for 8 hours. The cells and the media are collected, and the cells are washed and the lysed with detergent-containing RIPA buffer:
150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5, as described by Wilson et al. cited above. Proteins are precipitated from the cell lysate and from the culture media using an HA-specific monoclonal antibody. The precipitated proteins then are analyzed by SDS-PAGE gels and autoradiography. An expression product of the expected size is seen in the cell lysate, which is not seen in negative controls.
Example 3(b): Cloning and Expression in CHO Cells
The vector pCl is used for the expression of galectin 8, 9, 10, or 10SV protein. Plasmid pCl is a derivative of the plasmid pSV2-dhfr [ATCC Accession No. 37146]. Both plasmids contain the mouse DHFR gene under control of the
SV40 early promoter. Chinese hamster ovary- or other cells lacking dihydrofolate activity that are transfected with these plasmids can be selected by growing the cells in a selective medium (alpha minus MEM, Life Technologies) supplemented with the chemotherapeutic agent methotrexate. The amplification of the DHFR genes in cells resistant to methotrexate (MTX) has been well documented (see, e.g., Alt, F.W., Kellems, R.M., Bertino, J.R., and Schimke, R.T., 1978, J. Biol. Chem. 255:1357-1370, Hamlin, J.L. and Ma, C. 1990, Biochem. etBiophys. Acta, 1097:101-143, Page, M.J. and Sydenham, M.A., Biotechnology :64-68 (1991)). Cells grown in increasing concentrations of MTX develop resistance to the drug by overproducing the target enzyme, DHFR, as a result of amplification of the
DHFR gene. If a second gene is linked to the DHFR gene it is usually co- amplified and over-expressed. It is state of the art to develop cell lines carrying more than 1,000 copies of the genes. Subsequently, when the methotrexate is withdrawn, cell lines contain the amplified gene integrated into the chromosome(s).
Plasmid pCl contains for the expression of the gene of interest a strong promoter of the long terminal repeat (LTR) of the Rouse Sarcoma Virus (Cullen, et al, Molecular and Cellular Biology, March 1985:438-4470) plus a fragment isolated from the enhancer of the immediate early gene of human cytomegalovirus (CMV) (Boshart et al, Cell 47:521-530, 1985). Downstream of the promoter are the following single restriction enzyme cleavage sites that allow the integration of the genes: BamHI, PvuII, and Nrul. Behind these cloning sites the plasmid contains translational stop codons in all three reading frames followed by the 3 ' intron and the polyadenylation site of the rat preproinsulin gene. Other high efficient promoters can also be used for the expression, e.g. , the human β-actin promoter, the SV40 early or late promoters or the long terminal repeats from other retroviruses, e.g., HIV and HTLVI. For the polyadenylation of the mRNA other signals, e.g. , from the human growth hormone or globin genes can be used as well.
Stable cell lines carrying a gene of interest integrated into the chromosomes can also be selected upon co-transfection with a selectable marker such as gpt, G418 or hygromycin. It is advantageous to use more than one selectable marker in the beginning, e.g. , G418 plus methotrexate.
The plasmid pCl is digested with the restriction enzyme BamHI and then dephosphorylated using calf intestinal phosphates by procedures known in the art. The vector is then isolated from a 1% agarose gel.
The DNA sequence encoding galectin 8, 9, or 10SV, ATCC Deposit Nos. 97732, 97733 and 97734, respectively, is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene. The galectin 10 sequence is similarly amplified from a human endometrial tumor or human fetal heart cDNA library.
The 5' galectin 8 primer has the sequence 5' cgcCCCGGGgccatcATG GCCTATGTCCCCG 3' (SEQ ID NO:55) containing the underlined Smal restriction enzyme site followed by nucleotide sequence 52-67 of FIG. 1 (SEQ ID NO:l). Inserted into an expression vector, as described below, the 5' end of the amplified fragment encoding human galectin 8 provides an efficient signal peptide. An efficient signal for initiation of translation in eukaryotic cells, as described by Kozak, M., J. Mol Biol. 196:941-950 (1987) is appropriately located in the vector portion of the construct.
The 3' galectin 8 primer has the sequence 5' cgc GGT ACC TTAGAT CTGGACATAGGAC 3' (SEQ ID NO:56) containing the Asp718 restriction followed by nucleotides complementary to nucleotides 1005-1023 of the galectin 8 coding sequence set out in FIG. 1 (SEQ ID NO:l).
The 5' galectin 9 primer has the sequence 5' cgc CCC GGG gcc ate ATGGCCTTCAGCGGTTC 3 ' (SEQ ID NO:57) containing the underlined Smal restriction enzyme site followed by the nucleotide sequence of bases 16-32 of FIG. 2A-2B (SEQ ID NO:3). Inserted into an expression vector, as described below, the 5' end of the amplified fragment encoding human galectin 9 provides an efficient signal peptide. An efficient signal for initiation of translation in eukaryotic cells, as described by Kozak, M., J. Mol Biol 196:941-950 (1987) is appropriately located in the vector portion of the construct.
The 3 ' galectin 9 primer has the sequence 5 ' cgc GGT ACC CAGGGTT GGAAAGGCTG 3 ' (SEQ ID NO:58) containing the Asp718 restriction followed by nucleotides complementary to nucleotides 1029-1045 of the galectin 9 coding sequence set out in FIG. 2A-2B (SEQ ID NO:3), including the stop codon.
The 5' galectin 10 and 10SV primer has the sequence 5' cgc CCC GGG gcc ate ATGATGTTGTCCTTAAAC 3' (SEQ ID NO:59) containing the underlined Smal restriction enzyme site followed by nucleotide sequence 118-135 of FIG. 3A-3B (SEQ ID NO:5). Inserted into an expression vector, as described below, the 5' end of the amplified fragment encoding human galectin 10 provides an efficient signal peptide. An efficient signal for initiation of translation in eukaryotic cells, as described by Kozak, M., J. Mol. Biol. 196:947-950 (1987) is appropriately located in the vector portion of the construct. The 3' galectin 10 primer has the sequence 5' cgcGGTACCCACAG AAGCCATTCTG 3' (SEQ ID NO:60) containing the Asp718 restriction followed by nucleotides complementary to nucleotides 1105-1120 set out in FIG. 3A-3B (SEQ ID NO:5). The 3' galectin 10SV primer has the sequence 5' CGCGGTACCCTA
TGCAACTTTATAAAATATTCC 3' (SEQ ID NO:54) containing the Asp718 restriction followed by nucleotides complementary to the 3' end of the galectin 10SV coding sequence set out in FIG. 4A-4B (SEQ ID NO:7).
The amplified fragments are isolated from a 1%> agarose gel as described above and then digested with the endonucleases Smal and Asp718 and then purified again on a 1% agarose gel.
The isolated fragment and the dephosphorylated vector are then ligated with T4 DNA ligase. E. coli HB101 cells are then transformed and bacteria identified that contained the plasmid pCl inserted in the correct orientation using the restriction enzyme Smal. The sequence of the inserted gene is confirmed by
DNA sequencing.
Transfection of CHO-DHFR-cells
Chinese hamster ovary cells lacking an active DHFR enzyme are used for transfection. Five μg of the expression plasmid Cl are cotransfected with 0.5 μg of the plasmid pSVneo using the lipofecting method (Feigner et a , supra). The plasmid pSV2-neo contains a dominant selectable marker, the gene neo from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including G418. The cells are seeded in alpha minus MEM supplemented with 1 mg/ml G418. After 2 days, the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) and cultivated from 10-14 days. After this period, single clones are trypsinized and then seeded in 6-well petri dishes using different concentrations of methotrexate (25 nM, 50 nM, 100 nM, 200 nM, 400 nM). Clones growing at the highest concentrations of methotrexate are then transferred to new 6-well plates containing even higher concentrations of methotrexate (500 nM, 1 μM, 2 μM, 5 μM). The same procedure is repeated until clones grow at a concentration of 100 μM.
The expression of the desired gene product is analyzed by Western blot analysis and SDS-PAGE.
Example 4: Tissue distribution of protein expression
Northern blot analysis is carried out to examine galectin 8, 9, 10, or 10SV gene expression in human tissues, using methods described by, among others, Sambrook et al, cited above. A cDNA probe containing the entire nucleotide sequence of the galectin 8, 9, 10, or 10SV protein (SEQ ID NO:l, 3, 5, or 7, respectively) is labeled with 32P using the re /prime™ DNA labeling system (Amersham Life Science), according to manufacturer's instructions. After labeling, the probe is purified using a CHROMA SPIN- 100™ column (Clontech Laboratories, Inc.), according to manufacturer's protocol number PT1200-1. The purified labeled probe is then used to examine various human tissues for galectin
8, 9, 10, or 1 OSV mRNA.
Multiple Tissue Northern (MTN) blots containing various human tissues (H) or human immune system tissues (IM) are obtained from Clontech and are examined with labeled probe using ExpressHyb™ hybridization solution (Clontech) according to manufacturer's protocol number PT1190- 1. Following hybridization and washing, the blots are mounted and exposed to film at -70 °C overnight, and films developed according to standard procedures.
It will be clear that the invention may be practiced otherwise than as particularly described in the foregoing description and examples. Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the appended claims. The entire disclosure of all publications (including patents, patent applications, journal articles, laboratory manuals, books, or other documents) cited herein are hereby incorporated by reference.
SEQUΈNCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Human Genome Sciences, Inc. 9410 Key West Avenue Rockville, MD 20850 United States of America APPLICANTS/INVENTORS: Ni, Jian
Gentz, Reiner L. Ruben, Steven M.
(ii) TITLE OF INVENTION: Galectin 8, 9, 10 and 10SV
(iii) NUMBER OF SEQUENCES: 60
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Sterne, Kessler, Goldstein, & Fox P.L.L.C.
(B) STREET: 1100 New York Ave . , Suite 600
(C) CITY: Washington
(D) STATE: D.C.
(E) COUNTRY: USA
(F) ZIP: 20005-3934
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: To be assigned
(B) FILING DATE: Herewith
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/028,093
(B) FILING DATE: 09-OCT-1996
(viii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: PCT/US96/16565
(B) FILING DATE: 09-OCT-1996
(ix) ATTORNEY/AGENT INFORMATION:
(A) NAME: Steffe, Eric K.
(B) REGISTRATION NUMBER: 36,688
(C) REFERENCE/DOCKET NUMBER: 1488.056PC01/EKS/SGW
(x) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 202-371-2600
(B) TELEFAX: 202-371-2540
(2) INFORMATION FOR SEQ ID NO : 1 : (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1138 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : double
(D) TOPOLOGY: both
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 52..1020
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 :
TTCGGCACGA GAGCTCTTCT CACAGGACCA GCCACTAGCG CACCTCGAGC G ATG GCC 57
Met Ala
1
TAT GTC CCC GCA CCG GGC TAC CAG CCC ACC TAC AAC CCG ACG CTG CCT 105 Tyr Val Pro Ala Pro Gly Tyr Gin Pro Thr Tyr Asn Pro Thr Leu Pro 5 10 15
TAC TAC CAG CCC ATC CCG GGC GGG CTC AAC GTG GGA ATG TCT GTT TAC 153 Tyr Tyr Gin Pro lie Pro Gly Gly Leu Asn Val Gly Met Ser Val Tyr 20 25 30
ATC CAA GGA GTG GCC AGC GAG CAC ATG AAG CGG TTC TTC GTG AAC TTT 201 lie Gin Gly Val Ala Ser Glu His Met Lys Arg Phe Phe Val Asn Phe 35 40 45 50
GTG GTT GGG CAG GAT CCG GGC TCA GAC GTC GCC TTC CAC TTC AAT CCG 249 Val Val Gly Gin Asp Pro Gly Ser Asp Val Ala Phe His Phe Asn Pro 55 60 65
CGG TTT GAC GGC TGG GAC AAG GTG GTC TTC AAC ACG TTG CAG GGC GGG 297 Arg Phe Asp Gly Trp Asp Lys Val Val Phe Asn Thr Leu Gin Gly Gly 70 75 80
AAG TGG GGC AGC GAG GAG AGG AAG AGG AGC ATG CCC TTC AAA AAG GGT 345 Lys Trp Gly Ser Glu Glu Arg Lys Arg Ser Met Pro Phe Lys Lys Gly 85 90 95
GCC GCC TTT GAG CTG GTC TTC ATA GTC CTG GCT GAG CAC TAC AAG GTG 393 Ala Ala Phe Glu Leu Val Phe He Val Leu Ala Glu His Tyr Lys Val 100 105 110
GTG GTA AAT GGA AAT CCC TTC TAT GAG TAC GGG CAC CGG CTT CCC CTA 441 Val Val Asn Gly Asn Pro Phe Tyr Glu Tyr Gly His Arg Leu Pro Leu 115 120 125 130
CAG ATG GTC ACC CAC CTG CAA GTG GAT GGG GAT CTG CAA CTT CAA TCA 489 Gin Met Val Thr His Leu Gin Val Asp Gly Asp Leu Gin Leu Gin Ser 135 140 145
ATC AAC TTC ATC GGA GGC CAG CCC CTC CGG CCC CAG GGA CCC CCG ATG 537 He Asn Phe He Gly Gly Gin Pro Leu Arg Pro Gin Gly Pro Pro Met 150 155 160
ATG CCA CCT TAC CCT GGT CCC GGA CAT TGC CAT CAA CAG CTG AAC AGC 585 Met Pro Pro Tyr Pro Gly Pro Gly His Cys His Gin Gin Leu Asn Ser 165 170 175
CTG CCC ACC ATG GAA GGA CCC CCA ACC TTC AAC CCG CCT GTG CCA TAT 633 Leu Pro Thr Met Glu Gly Pro Pro Thr Phe Asn Pro Pro Val Pro Tyr 180 185 190
TTC GGG AGG CTG CAA GGA GGG CTC ACA GCT CGA AGA ACC ATC ATC ATC 681 Phe Gly Arg Leu Gin Gly Gly Leu Thr Ala Arg Arg Thr He He He 195 200 205 210
AAG GGC TAT GTG CCT CCC ACA GGC AAG AGC TTT GCT ATC AAC TTC AAG 729 Lys Gly Tyr Val Pro Pro Thr Gly Lys Ser Phe Ala He Asn Phe Lys 215 220 225
GTG GGC TCC TCA GGG GAC ATA GCT CTG CAC ATT AAT CCC CGC ATG GGC 777 Val Gly Ser Ser Gly Asp He Ala Leu His He Asn Pro Arg Met Gly 230 235 240
AAC GGT ACC GTG GTC CGG AAC AGC CTT CTG AAT GGC TCG TGG GGA TCC 825 Asn Gly Thr Val Val Arg Asn Ser Leu Leu Asn Gly Ser Trp Gly Ser 245 250 255
GAG GAG AAG AAG ATC ACC CAC AAC CCA TTT GGT CCC GGA CAG TTC TTT 873 Glu Glu Lys Lys He Thr His Asn Pro Phe Gly Pro Gly Gin Phe Phe 260 265 270
GAT CTG TCC ATT CGC TGT GGC TTG GAT CGC TTC AAG GTT TAC GCC AAT 921 Asp Leu Ser He Arg Cys Gly Leu Asp Arg Phe Lys Val Tyr Ala Asn 275 280 285 290
GGC CAG CAC CTC TTT GAC TTT GCC CAT CGC CTC TCG GCC TTC CAG AGG 969 Gly Gin His Leu Phe Asp Phe Ala His Arg Leu Ser Ala Phe Gin Arg 295 300 305
GTG GAC ACA TTG GAA ATC CAG GGT GAT GTC ACC TTG TCC TAT GTC CAG 1017 Val Asp Thr Leu Glu He Gin Gly Asp Val Thr Leu Ser Tyr Val Gin 310 315 320
ATC TAATCTATTC CTGGGGCCAT AACTCATGGG AAAACAGAAT TATCCCCTAG 1070
He
GACTCCTTTC TAAGCCCCTA ATAAAATGTC TGAGGGTGTC TCATGAAAAA AAAAAAAAAA 1130 AAAAAAAA 1138 (2) INFORMATION FOR SEQ ID NO : 2 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 323 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Ala Tyr Val Pro Ala Pro Gly Tyr Gin Pro Thr Tyr Asn Pro Thr 1 5 10 15
Leu Pro Tyr Tyr Gin Pro He Pro Gly Gly Leu Asn Val Gly Met Ser 20 25 30
Val Tyr He Gin Gly Val Ala Ser Glu His Met Lys Arg Phe Phe Val 35 40 45
Asn Phe Val Val Gly Gin Asp Pro Gly Ser Asp Val Ala Phe His Phe 50 55 60
Asn Pro Arg Phe Asp Gly Trp Asp Lys Val Val Phe Asn Thr Leu Gin 65 70 75 80
Gly Gly Lys Trp Gly Ser Glu Glu Arg Lys Arg Ser Met Pro Phe Lys 85 90 95
Lys Gly Ala Ala Phe Glu Leu Val Phe He Val Leu Ala Glu His Tyr 100 105 110
Lys Val Val Val Asn Gly Asn Pro Phe Tyr Glu Tyr Gly His Arg Leu 115 120 125
Pro Leu Gin Met Val Thr His Leu Gin Val Asp Gly Asp Leu Gin Leu 130 135 140
Gin Ser He Asn Phe He Gly Gly Gin Pro Leu Arg Pro Gin Gly Pro 145 150 155 160
Pro Met Met Pro Pro Tyr Pro Gly Pro Gly His Cys His Gin Gin Leu 165 170 175
Asn Ser Leu Pro Thr Met Glu Gly Pro Pro Thr Phe Asn Pro Pro Val 180 185 190
Pro Tyr Phe Gly Arg Leu Gin Gly Gly Leu Thr Ala Arg Arg Thr He 195 200 205
He He Lys Gly Tyr Val Pro Pro Thr Gly Lys Ser Phe Ala He Asn 210 215 220
Phe Lys Val Gly Ser Ser Gly Asp He Ala Leu His He Asn Pro Arg 225 230 235 240 Met Gly Asn Gly Thr Val Val Arg Asn Ser Leu Leu Asn Gly Ser Trp 245 250 255
Gly Ser Glu Glu Lys Lys He Thr His Asn Pro Phe Gly Pro Gly Gin 260 265 270
Phe Phe Asp Leu Ser He Arg Cys Gly Leu Asp Arg Phe Lys Val Tyr 275 280 285
Ala Asn Gly Gin His Leu Phe Asp Phe Ala His Arg Leu Ser Ala Phe 290 295 300
Gin Arg Val Asp Thr Leu Glu He Gin Gly Asp Val Thr Leu Ser Tyr 305 310 315 320
Val Gin He
(2) INFORMATION FOR SEQ ID NO : 3 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1545 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : double
(D) TOPOLOGY: both
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 16..948
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
AGAGGCGGCG GAGAG ATG GCC TTC AGC GGT TCC CAG GCT CCC TAC CTG AGT 51 Met Ala Phe Ser Gly Ser Gin Ala Pro Tyr Leu Ser 1 5 10
CCA GCT GTC CCC TTT TCT GGG ACT ATT CAA GGA GGT CTC CAG GAC GGA 99 Pro Ala Val Pro Phe Ser Gly Thr He Gin Gly Gly Leu Gin Asp Gly 15 20 25
CTT CAG ATC ACT GTC AAT GGG ACC GTT CTC AGC TCC AGT GGA ACC AGG 147 Leu Gin He Thr Val Asn Gly Thr Val Leu Ser Ser Ser Gly Thr Arg 30 35 40
TTT GCT GTG AAC TTT CAG ACT GGC TTC AGT GGA AAT GAC ATT GCC TTC 195 Phe Ala Val Asn Phe Gin Thr Gly Phe Ser Gly Asn Asp He Ala Phe 45 50 55 60
CAC TTC AAC CCT CGG TTT GAA GAT GGA GGG TAC GTG GTG TGC AAC ACG 243 His Phe Asn Pro Arg Phe Glu Asp Gly Gly Tyr Val Val Cys Asn Thr 65 70 75 AGG CAG AAC GGA AGC TGG GGG CCC GAG GAG AGG AAG ACA CAC ATG CCT 291 Arg Gin Asn Gly Ser Trp Gly Pro Glu Glu Arg Lys Thr His Met Pro 80 85 90
TTC CAG AAG GGG ATG CCC TTT GAC CTC TGC TTC CTG GTG CAG AGC TCA 339 Phe Gin Lys Gly Met Pro Phe Asp Leu Cys Phe Leu Val Gin Ser Ser 95 100 105
GAT TTC AAG GTG ATG GTG AAC GGG ATC CTC TTC GTG CAG TAC TTC CAC 387 Asp Phe Lys Val Met Val Asn Gly He Leu Phe Val Gin Tyr Phe His 110 115 120
CGC GTG CCC TTC CAC CGT GTG GAC ACC ATC TCC GTC AAT GGC TCT GTG 435 Arg Val Pro Phe His Arg Val Asp Thr He Ser Val Asn Gly Ser Val 125 130 135 140
CAG CTG TCC TAC ATC AGC TTC CAG ACC CAG ACA GTC ATC CAC ACA GTG 483 Gin Leu Ser Tyr He Ser Phe Gin Thr Gin Thr Val He His Thr Val 145 150 155
CAG AGC GCC CCT GGA CAG ATG TTC TCT ACT CCC GCC ATC CCA CCT ATG 531 Gin Ser Ala Pro Gly Gin Met Phe Ser Thr Pro Ala He Pro Pro Met 160 165 170
ATG TAC CCC CAC CCC GCC TAT CCG ATG CCT TTC ATC ACC ACC ATT CTG 579 Met Tyr Pro His Pro Ala Tyr Pro Met Pro Phe He Thr Thr He Leu 175 180 185
GGA GGG CTG TAC CCA TCC AAG TCC ATC CTC CTG TCA GGC ACT GTC CTG 627 Gly Gly Leu Tyr Pro Ser Lys Ser He Leu Leu Ser Gly Thr Val Leu 190 195 200
CCC AGT GCT CAG AGG TTC CAC ATC AAC CTG TGC TCT GGG AAC CAC ATC 675 Pro Ser Ala Gin Arg Phe His He Asn Leu Cys Ser Gly Asn His He 205 210 215 220
GCC TTC CAC CTG AAC CCC CGT TTT GAT GAG AAT GCT GTG GTC CGC AAC 723 Ala Phe His Leu Asn Pro Arg Phe Asp Glu Asn Ala Val Val Arg Asn 225 230 235
ACC CAG ATC GAC AAC TCC TGG GGG TCT GAG GAG CGA AGT CTG CCC CGA 771 Thr Gin He Asp Asn Ser Trp Gly Ser Glu Glu Arg Ser Leu Pro Arg 240 245 250
AAA ATG CCC TTC GTC CGT GGC CAG AGC TTC TCA GTG TGG ATC TTG TGT 819 Lys Met Pro Phe Val Arg Gly Gin Ser Phe Ser Val Trp He Leu Cys 255 260 265
GAA GCT CAC TGC CTC AAG GTG GCC GTG GAT GGT CAG CAC CTG TTT GAA 867 Glu Ala His Cys Leu Lys Val Ala Val Asp Gly Gin His Leu Phe Glu 270 275 280
TAC TAC CAT CGC CTG AGG AAC CTG CCC ACC ATC AAC AGA CTG GAA GTG 915 Tyr Tyr His Arg Leu Arg Asn Leu Pro Thr He Asn Arg Leu Glu Val 285 290 295 300 GGG GGC GAC ATC CAG CTG ACC CAT GTG CAG ACA TAGGCGGCTT CCTGGCCCTG 968 Gly Gly Asp He Gin Leu Thr His Val Gin Thr 305 310
GGGCCGGGGG CTGGGGTGTG GGGCAGTCTG GGTCCTCTCA TCATCCCCAC TTCCCAGGCC 1028
CAGCCTTTCC AACCCTGCCT GGGATCTGGG CTTTAATGCA GAGGCCATGT CCTTGTCTGG 1088
TCCTGCTTCT GGCTACAGCC ACCCTGGAAC GGAGAAGGCA GCTGACGGGG ATTGCCTTCC 1148
TCAGCCGCAG CAGCACCTGG GGCTCCAGCT GCTGGAAATC CTACCATCCC AGGAGGCAGG 1208
CACAGCCAGG GAGAGGGGAG GAGTGGGCAG TGAAGATGAA GCCCCATGCT CAGTCCCCTC 1268
CCATCCCCCA CGCAGCTCCA CCCCAGTCCC AAGCCACCAG CTGTCTGCTC CTGGTGGGAG 1328
GTGGCCTCCT CAGCCCCTCC TCTCTGACCT TTAACCTCAC TCTCACCTTG CACCGTGCAC 1388
CAACCCTTCA CCCCTCCTGG AAAGCAGGCC TGATGGCTTC CCACTGGCCT CCACCACCTG 1448
ACCAGAGTGT TCTCTTCAGA GGACTGGCTC CTTTCCCAGT GTCCTTAAAA TAAAGAAATG 1508
AAAATGCTTG TTGGCAAAAA AAAAAAAAAA AAAAAAA 1545
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 311 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 :
Met Ala Phe Ser Gly Ser Gin Ala Pro Tyr Leu Ser Pro Ala Val Pro 1 5 10 15
Phe Ser Gly Thr He Gin Gly Gly Leu Gin Asp Gly Leu Gin He Thr 20 25 30
Val Asn Gly Thr Val Leu Ser Ser Ser Gly Thr Arg Phe Ala Val Asn 35 40 45
Phe Gin Thr Gly Phe Ser Gly Asn Asp He Ala Phe His Phe Asn Pro 50 55 60
Arg Phe Glu Asp Gly Gly Tyr Val Val Cys Asn Thr Arg Gin Asn Gly 65 70 75 80
Ser Trp Gly Pro Glu Glu Arg Lys Thr His Met Pro Phe Gin Lys Gly 85 90 95
Met Pro Phe Asp Leu Cys Phe Leu Val Gin Ser Ser Asp Phe Lys Val 100 105 110
Met Val Asn Gly He Leu Phe Val Gin Tyr Phe His Arg Val Pro Phe 115 120 125
His Arg Val Asp Thr He Ser Val Asn Gly Ser Val Gin Leu Ser Tyr 130 135 140
He Ser Phe Gin Thr Gin Thr Val He His Thr Val Gin Ser Ala Pro 145 150 155 160
Gly Gin Met Phe Ser Thr Pro Ala He Pro Pro Met Met Tyr Pro His 165 170 175
Pro Ala Tyr Pro Met Pro Phe He Thr Thr He Leu Gly Gly Leu Tyr 180 185 190
Pro Ser Lys Ser He Leu Leu Ser Gly Thr Val Leu Pro Ser Ala Gin 195 200 205
Arg Phe His He Asn Leu Cys Ser Gly Asn His He Ala Phe His Leu 210 215 220
Asn Pro Arg Phe Asp Glu Asn Ala Val Val Arg Asn Thr Gin He Asp 225 230 235 240
Asn Ser Trp Gly Ser Glu Glu Arg Ser Leu Pro Arg Lys Met Pro Phe 245 250 255
Val Arg Gly Gin Ser Phe Ser Val Trp He Leu Cys Glu Ala His Cys 260 265 270
Leu Lys Val Ala Val Asp Gly Gin His Leu Phe Glu Tyr Tyr His Arg 275 280 285
Leu Arg Asn Leu Pro Thr He Asn Arg Leu Glu Val Gly Gly Asp He 290 295 300
Gin Leu Thr His Val Gin Thr 305 310
(2) INFORMATION FOR SEQ ID NO : 5 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1479 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : double
(D) TOPOLOGY: both
(ii) MOLECULE TYPE: CDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 118..1068 (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 5 :
ACACCAGTCT TTGGGGCCAG TGCCTCAGTT TCAATCCAGG TAACCTTTAA ATGAAACTTG 60
CCTAAAATCT TAGGTCATAC ACAGAAGAGA CTCCAATCGA CAAGAAGCTG GAAAAGA 117
ATG ATG TTG TCC TTA AAC AAC CTA CAG AAT ATC ATC TAT AAC CCG GTA 165 Met Met Leu Ser Leu Asn Asn Leu Gin Asn He He Tyr Asn Pro Val 1 5 10 15
ATC CCG TTT GTT GGC ACC ATT CCT GAT CAG CTG GAT CCT GGA ACT TTG 213 He Pro Phe Val Gly Thr He Pro Asp Gin Leu Asp Pro Gly Thr Leu 20 25 30
ATT GTG ATA CGT GGG CAT GTT CCT AGT GAC GCA GAC AGA TTC CAG GTG 261 He Val He Arg Gly His Val Pro Ser Asp Ala Asp Arg Phe Gin Val 35 40 45
GAT CTG CAG AAT GGC AGC AGT GTG AAA CCT CGA GCC GAT GTG GCC TTT 309 Asp Leu Gin Asn Gly Ser Ser Val Lys Pro Arg Ala Asp Val Ala Phe 50 55 60
CAT TTC AAT CCT CGT TTC AAA AGG GCC GGC TGC ATT GTT TGC AAT ACT 357 His Phe Asn Pro Arg Phe Lys Arg Ala Gly Cys He Val Cys Asn Thr 65 70 75 80
TTG ATA AAT GAA AAA TGG GGA CGG GAA GAG ATC ACC TAT GAC ACG CCT 405 Leu He Asn Glu Lys Trp Gly Arg Glu Glu He Thr Tyr Asp Thr Pro 85 90 95
TTC AAA AGA GAA AAG TCT TTT GAG ATC GTG ATT ATG GTG CTA AAG GAC 453 Phe Lys Arg Glu Lys Ser Phe Glu He Val He Met Val Leu Lys Asp 100 105 110
AAA TTC CAG GTG GCT GTA AAT GGA AAA CAT ACT CTG CTC TAT GGC CAC 501 Lys Phe Gin Val Ala Val Asn Gly Lys His Thr Leu Leu Tyr Gly His 115 120 125
AGG ATC GGC CCA GAG AAA ATA GAC ACT CTG GGC ATT TAT GGC AAA GTG 549 Arg He Gly Pro Glu Lys He Asp Thr Leu Gly He Tyr Gly Lys Val 130 135 140
AAT ATT CAC TCA ATT GGT TTT AGC TTC AGC TCG GAC TTA CAA AGT ACC 597 Asn He His Ser He Gly Phe Ser Phe Ser Ser Asp Leu Gin Ser Thr 145 150 155 160
CAA GCA TCT AGT CTG GAA CTG ACA GAG ATA GTT AGA GAA AAT GTT CCA 645 Gin Ala Ser Ser Leu Glu Leu Thr Glu He Val Arg Glu Asn Val Pro 165 170 175
AAG TCT GGC ACG CCC CAG CTT AGC CTG CCA TTC GCT GCA AGG TTG AAC 693 Lys Ser Gly Thr Pro Gin Leu Ser Leu Pro Phe Ala Ala Arg Leu Asn 180 185 190 ACC CCC ATG GGC CCT GGA CGA ACT GTC GTC GTT AAA GGA GAA GTG AAT 741 Thr Pro Met Gly Pro Gly Arg Thr Val Val Val Lys Gly Glu Val Asn 195 200 205
GCA AAT GCC AAA AGC TTT AAT GTT GAC CTA CTA GCA GGA AAA TCA AAG 789 Ala Asn Ala Lys Ser Phe Asn Val Asp Leu Leu Ala Gly Lys Ser Lys 210 215 220
GAT ATT GCT CTA CAC TTG AAC CCA CGC CTG AAT ATT AAA GCA TTT GTG 837 Asp He Ala Leu His Leu Asn Pro Arg Leu Asn He Lys Ala Phe Val 225 230 235 240
AGA AAT TCT TTT CTT CAA GAG TCC TGG GGA GAA GAA GAG AGA AAT ATT 885 Arg Asn Ser Phe Leu Gin Glu Ser Trp Gly Glu Glu Glu Arg Asn He 245 250 255
ACC GCT TTC CCA TTT AGT CCT GGG ATG TAC TTT GAG ATG ATA ATT TAT 933 Thr Ala Phe Pro Phe Ser Pro Gly Met Tyr Phe Glu Met He He Tyr 260 265 270
TGT GAT GTT AGA GAA TTC AAG GTT GCA GTA AAT GGC GTA CAC AGC CTG 981 Cys Asp Val Arg Glu Phe Lys Val Ala Val Asn Gly Val His Ser Leu 275 280 285
GAG TAC AAA CAC AGA TTT AAA GAG CTC AGC AGT ATT GAC ACG CTG GAA 1029 Glu Tyr Lys His Arg Phe Lys Glu Leu Ser Ser He Asp Thr Leu Glu 290 295 300
ATT AAT GGA GAC ATC CAC TTA CTG GAA GTA AGG AGC TGG TAGCCTACCT 1078 He Asn Gly Asp He His Leu Leu Glu Val Arg Ser Trp 305 310 315
ACACAGCTGC TACAAAAACC AAAATACAGA ATGGCTTCTG TGATACTGGC CTTGCTGAAA 1138
CGCATCTCAC TGTCATTCTA TTGTTTATAT TGTTAAAATG AGCTTGTGCA CCATTAGGTC 1198
CTGCTGGGTG TTCTCAGTCC TTGCCATGAA GTATGGTGGT GTCTAGCACT GAATGGGGAA 1258
ACTGGGGGCA GCAACACTTA TAGCCAGTTA AAGCCACTCT GCCCTCTCTC CTACTTTGGC 1318
TGACTCTTCA AGAATGCCAT TCAACAAGTA TTTATGGAGT CCTACTATAT ACAGTAGCTA 1378
ACATGTATTG AGCACAGATT TTTTTGGTAA ACCTGTGAGG GCTAGGGTAT ATCCTTGGGA 1438
ACAAACCAGA ATGTCCTGTC CCTTGAAAAA AAAAAAAAAA A 1479
(2) INFORMATION FOR SEQ ID NO : 6 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 317 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 6 :
Met Met Leu Ser Leu Asn Asn Leu Gin Asn He He Tyr Asn Pro Val 1 5 10 15
He Pro Phe Val Gly Thr He Pro Asp Gin Leu Asp Pro Gly Thr Leu 20 25 30
He Val He Arg Gly His Val Pro Ser Asp Ala Asp Arg Phe Gin Val 35 40 45
Asp Leu Gin Asn Gly Ser Ser Val Lys Pro Arg Ala Asp Val Ala Phe 50 55 60
His Phe Asn Pro Arg Phe Lys Arg Ala Gly Cys He Val Cys Asn Thr 65 70 75 80
Leu He Asn Glu Lys Trp Gly Arg Glu Glu He Thr Tyr Asp Thr Pro 85 90 95
Phe Lys Arg Glu Lys Ser Phe Glu He Val He Met Val Leu Lys Asp 100 105 110
Lys Phe Gin Val Ala Val Asn Gly Lys His Thr Leu Leu Tyr Gly His 115 120 125
Arg He Gly Pro Glu Lys He Asp Thr Leu Gly He Tyr Gly Lys Val 130 135 140
Asn He His Ser He Gly Phe Ser Phe Ser Ser Asp Leu Gin Ser Thr 145 150 155 160
Gin Ala Ser Ser Leu Glu Leu Thr Glu He Val Arg Glu Asn Val Pro 165 170 175
Lys Ser Gly Thr Pro Gin Leu Ser Leu Pro Phe Ala Ala Arg Leu Asn 180 185 190
Thr Pro Met Gly Pro Gly Arg Thr Val Val Val Lys Gly Glu Val Asn 195 200 205
Ala Asn Ala Lys Ser Phe Asn Val Asp Leu Leu Ala Gly Lys Ser Lys 210 215 220
Asp He Ala Leu His Leu Asn Pro Arg Leu Asn He Lys Ala Phe Val 225 230 235 240
Arg Asn Ser Phe Leu Gin Glu Ser Trp Gly Glu Glu Glu Arg Asn He 245 250 255
Thr Ala Phe Pro Phe Ser Pro Gly Met Tyr Phe Glu Met He He Tyr 260 265 270
Cys Asp Val Arg Glu Phe Lys Val Ala Val Asn Gly Val His Ser Leu 275 280 285 Glu Tyr Lys His Arg Phe Lys Glu Leu Ser Ser He Asp Thr Leu Glu 290 295 300
He Asn Gly Asp He His Leu Leu Glu Val Arg Ser Trp 305 310 315
(2) INFORMATION FOR SEQ ID NO : 7 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1936 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : single
(D) TOPOLOGY: linear
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 118..717
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 7 :
ACACCAGTCT TTGGGGCCAG TGCCTCAGTT TCAATCCAGG TAACCTTTAA ATGAAACTTG 60
CCTAAAATCT TAGGTCATAC ACAGAAGAGA CTCCAATCGA CAAGAAGCTG GAAAAGA 117
ATG ATG TTG TCC TTA AAC AAC CTA CAG AAT ATC ATC TAT AAC CCG GTA 165 Met Met Leu Ser Leu Asn Asn Leu Gin Asn He He Tyr Asn Pro Val 1 5 10 15
ATC CCG TTT GTT GGC ACC ATT CCT GAT CAG CTG GAT CCT GGA ACT TTG 213 He Pro Phe Val Gly Thr He Pro Asp Gin Leu Asp Pro Gly Thr Leu 20 25 30
ATT GTG ATA CGT GGG CAT GTT CCT AGT GAC GCA GAC AGA TTC CAG GTG 261 He Val He Arg Gly His Val Pro Ser Asp Ala Asp Arg Phe Gin Val 35 40 45
GAT CTG CAG AAT GGC AGC AGC ATG AAA CCT CGA GCC GAT GTG GCC TTT 309 Asp Leu Gin Asn Gly Ser Ser Met Lys Pro Arg Ala Asp Val Ala Phe 50 55 60
CAT TTC AAT CCT CGT TTC AAA AGG GCC GGC TGC ATT GTT TGC AAT ACT 357 His Phe Asn Pro Arg Phe Lys Arg Ala Gly Cys He Val Cys Asn Thr 65 70 75 80
TTG ATA AAT GAA AAA TGG GGA CGG GAA GAG ATC ACC TAT GAC ACG CCT 405 Leu He Asn Glu Lys Trp Gly Arg Glu Glu He Thr Tyr Asp Thr Pro 85 90 95
TTC AAA AGA GAA AAG TCT TTT GAG ATC GTG ATT ATG GTG CTG AAG GAC 453 Phe Lys Arg Glu Lys Ser Phe Glu He Val He Met Val Leu Lys Asp 100 105 110 AAA TTC CAG GTG GCT GTA AAT GGA AAA CAT ACT CTG CTC TAT GGC CAC 501 Lys Phe Gin Val Ala Val Asn Gly Lys His Thr Leu Leu Tyr Gly His 115 120 125
AGG ATC GGC CCA GAG AAA ATA GAC ACT CTG GGC ATT TAT GGC AAA GTG 549 Arg He Gly Pro Glu Lys He Asp Thr Leu Gly He Tyr Gly Lys Val 130 135 140
AAT ATT CAC TCA ATT GGT TTT AGC TTC AGC TCG GAC TTA CAA AGT ACC 597 Asn He His Ser He Gly Phe Ser Phe Ser Ser Asp Leu Gin Ser Thr 145 150 155 160
CAA GCA TCT AGT CTG GAA CTG ACA GAG ATA AGT AGA GAA AAT GTT CCA 645 Gin Ala Ser Ser Leu Glu Leu Thr Glu He Ser Arg Glu Asn Val Pro 165 170 175
AAG TCT GGC ACG CCC CAG CTT GTG AGT ATT TTT GCC TGG GTT ATT TCA 693 Lys Ser Gly Thr Pro Gin Leu Val Ser He Phe Ala Trp Val He Ser 180 185 190
TGT GGA ATA TTT TAT AAA GTT GCA TAGAAAATGA ACAGTTTAAA CCGTGGAGGG 747 Cys Gly He Phe Tyr Lys Val Ala 195 200
CAGCTTCATT CATTCCATTC CTTACTGTAG AACTGTTTCC CTACAGCCTA GTAATAGAGG 807
AGGAGACATT TCTAAAATCG CACCCAGAAC TGTCTACACC AAGAGCAAAG ATTCGACTGT 867
CAATCACACT TTGACTTGCA CCAAAATACC ACCTATGAAC TATGTGTCAA AGGGTTTGAA 927
GAGCACCAAA TTTTCTTAAC TCTATATAAA AATTAAGTTG TAATGAGCTG TTACGAGTAA 987
CCTGTATCCA CAATAGAGGC CCAAAGCAGC CCCCTCTGCA TTTGTGTGCC GTCCCTGGAC 1047
GGATTCGAGA GTCAACCAGG CCTGCCTCTG AGCCATTTCT GTGTATTTCC TCAGCACCTC 1107
CCTGCTTGGC TGCTTCCCCT TCAGGCAGAA CACAGTACTG CCTCAGACCC CAGGCACAGG 1167
GGGCCTTCCT GGCGTGTTTC ACTCATACAG AGGGCATCGG GTCCCACCCT GTCACTCATT 1227
TCATCGTCTA AAATGTAATC ATGTGTGTTT GCTTCGAGCC AGGGACAGTG CTGCTGCAGG 1287
GGACCCAGCT GGGACCAAGG CAGACTGTCT CTCCCCTCCT GGGATTTACA GGGTCATGGC 1347
TCTGAAACAT TCCGTAGTGT TCTTTGGACA CGAGTTTTCC CTGGAGATCG CTTTCTGCAG 1407
GCTCTTGGTC CTGACTGTGG CTTCTTTTCA GAGGCTGCCA TTTCGCTGCA AGGTTGAACA 1467
CCCCCATGGG CCCTGGACGA ACTGTCGTCG TTAAAGGAGA AGTGAATGCA AATGCCAAAA 1527
GCTTTAATGT TGACCTACTA GCAGGAAAAT CAAAGGATAT TGCTCTACAC TTGAACCCAC 1587
GCCTGAATAT TAAAGCATTT GTAAGAAATT CTTTTCTTCA GGAGTCCTGG GGAGAAGAAG 1647
AGAGAAATAT TACCTCTTTC CCATTTAGTC CTGGGATGTA CTTTGAGATG ATAATTTATT 1707 GTGATGTTAG AGAATTCAAG GTTGCAGTAA ATGGCGTACA CAGCCTGGAG TACAAACACA 1767
GATTTAAAGA GCTCAGCAGT ATTGACACGC TGGAAATTAA TGGAGACATC CACTTACTGG 1827
AAGTAAGGAG CTGGTAGCCT ACCTACACAG CTGCTACAAA AACCAAAATA CAGAATGGCT 1887
TCTGTGATAC TGGCCTTGCT GAAACGCAAA AAAAAAAAAA AAAAAAAAA 1936
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 200 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 8 :
Met Met Leu Ser Leu Asn Asn Leu Gin Asn He He Tyr Asn Pro Val 1 5 10 15
He Pro Phe Val Gly Thr He Pro Asp Gin Leu Asp Pro Gly Thr Leu 20 25 30
He Val He Arg Gly His Val Pro Ser Asp Ala Asp Arg Phe Gin Val 35 40 45
Asp Leu Gin Asn Gly Ser Ser Met Lys Pro Arg Ala Asp Val Ala Phe 50 55 60
His Phe Asn Pro Arg Phe Lys Arg Ala Gly Cys He Val Cys Asn Thr 65 70 75 80
Leu He Asn Glu Lys Trp Gly Arg Glu Glu He Thr Tyr Asp Thr Pro 85 90 95
Phe Lys Arg Glu Lys Ser Phe Glu He Val He Met Val Leu Lys Asp 100 105 110
Lys Phe Gin Val Ala Val Asn Gly Lys His Thr Leu Leu Tyr Gly His 115 120 125
Arg He Gly Pro Glu Lys He Asp Thr Leu Gly He Tyr Gly Lys Val 130 135 140
Asn He His Ser He Gly Phe Ser Phe Ser Ser Asp Leu Gin Ser Thr 145 150 155 160
Gin Ala Ser Ser Leu Glu Leu Thr Glu He Ser Arg Glu Asn Val Pro 165 170 175
Lys Ser Gly Thr Pro Gin Leu Val Ser He Phe Ala Trp Val He Ser 180 185 190 Cys Gly He Phe Tyr Lys Val Ala 195 200
(2) INFORMATION FOR SEQ ID NO : 9 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 132 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS : not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
Met Thr Gly Glu Leu Glu Val Lys Asn Met Asp Met Lys Pro Gly Ser 1 5 10 15
Thr Leu Lys He Thr Gly Ser He Ala Asp Gly Thr Asp Gly Phe Val 20 25 30
He Asn Leu Gly Gin Gly Thr Asp Lys Leu Asn Leu His Phe Asn Pro 35 40 45
Arg Phe Ser Glu Ser Thr He Val Cys Asn Ser Leu Asp Gly Ser Asn 50 55 60
Trp Gly Gin Glu Gin Arg Glu Asp His Leu Cys Phe Ser Pro Gly Ser 65 70 75 80
Glu Val Lys Phe Thr Val Thr Phe Glu Ser Asp Lys Phe Lys Val Lys 85 90 95
Leu Pro Asp Gly His Glu Leu Thr Phe Pro Asn Arg Leu Gly His Ser 100 105 110
His Leu Ser Tyr Leu Ser Val Arg Gly Gly Phe Asn Met Ser Ser Phe 115 120 125
Lys Leu Lys Glu 130
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 250 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
Met Ala Asp Asn Phe Ser Leu His Asp Ala Leu Ser Gly Ser Gly Asn 1 5 10 15
Pro Asn Pro Gin Gly Trp Pro Gly Ala Trp Gly Asn Gin Pro Ala Gly 20 25 30
Ala Gly Gly Tyr Pro Gly Ala Ser Tyr Pro Gly Ala Tyr Pro Gly Gin 35 40 45
Ala Pro Pro Gly Ala Tyr Pro Gly Gin Ala Pro Pro Gly Ala Tyr His 50 55 60
Gly Ala Pro Gly Ala Tyr Pro Gly Ala Pro Ala Pro Gly Val Tyr Pro 65 70 75 80
Gly Pro Pro Ser Gly Pro Gly Ala Tyr Pro Ser Ser Gly Gin Pro Ser 85 90 95
Ala Pro Gly Ala Tyr Pro Ala Thr Gly Pro Tyr Gly Ala Pro Ala Gly 100 105 110
Pro Leu He Val Pro Tyr Asn Leu Pro Leu Pro Gly Gly Val Val Pro 115 120 125
Arg Met Leu He Thr He Leu Gly Thr Val Lys Pro Asn Ala Asn Arg 130 135 140
He Ala Leu Asp Phe Gin Arg Gly Asn Asp Val Ala Phe His Phe Asn 145 150 155 160
Pro Arg Phe Asn Glu Asn Asn Arg Arg Val He Val Cys Asn Thr Lys 165 170 175
Leu Asp Asn Asn Trp Gly Arg Glu Glu Arg Gin Ser Val Phe Pro Phe 180 185 190
Glu Ser Gly Lys Pro Phe Lys He Gin Val Leu Val Glu Pro Asp His 195 200 205
Phe Lys Val Ala Val Asn Asp Ala His Leu Leu Gin Tyr Asn His Arg 210 215 220
Val Lys Lys Leu Asn Glu He Ser Lys Leu Gly He Ser Gly Asp He 225 230 235 240
Asp Leu Thr Ser Ala Ser Tyr Thr Met He 245 250
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 324 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
Met Ala Tyr Val Pro Ala Pro Gly Tyr Gin Pro Thr Tyr Asn Pro Thr 1 5 10 15
Leu Pro Tyr Lys Arg Pro He Pro Gly Gly Leu Ser Val Gly Met Ser 20 25 30
He Tyr He Gin Gly He Ala Lys Asp Asn Met Arg Arg Phe His Val 35 40 45
Asn Phe Ala Val Gly Gin Asp Glu Gly Ala Asp He Ala Phe His Phe 50 55 60
Asn Pro Arg Phe Asp Gly Trp Asp Lys Val Val Phe Asn Thr Met Gin 65 70 75 80
Ser Gly Gin Trp Gly Lys Glu Glu Lys Lys Lys Ser Met Pro Phe Gin 85 90 95
Lys Gly His His Phe Glu Leu Val Phe Met Val Met Ser Glu His Tyr 100 105 110
Lys Val Val Val Asn Gly Thr Pro Phe Tyr Glu Tyr Gly His Arg Leu 115 120 125
Pro Leu Gin Met Val Thr His Leu Gin Val Asp Gly Asp Leu Glu Leu 130 135 140
Gin Ser He Asn Phe Leu Gly Gly Gin Pro Ala Ala Ser Gin Tyr Pro 145 150 155 160
Gly Thr Met Thr He Pro Ala Tyr Pro Ser Ala Gly Tyr Asn Pro Pro 165 170 175
Gin Met Asn Ser Leu Pro Val Met Ala Gly Pro Pro He Phe Asn Pro 180 185 190
Pro Val Pro Tyr Val Gly Thr Leu Gin Gly Gly Leu Thr Ala Arg Arg 195 200 205
Thr He He He Lys Gly Tyr Val Leu Pro Thr Ala Lys Asn Leu He 210 215 220
He Asn Phe Lys Val Gly Ser Thr Gly Asp He Ala Phe His Met Asn 225 230 235 240
Pro Arg He Gly Asp Cys Val Val Arg Asn Ser Tyr Met Asn Gly Ser 245 250 255
Trp Gly Ser Glu Glu Arg Lys He Pro Tyr Asn Pro Phe Gly Ala Gly 260 265 270
Gin Phe Phe Asp Leu Ser He Arg Cys Gly Thr Asp Arg Phe Lys Val 275 280 285
Phe Ala Asn Gly Gin His Leu Phe Asp Phe Ser His Arg Phe Gin Ala 290 295 300
Phe Gin Arg Val Asp Met Leu Glu He Lys Gly Asp He Thr Leu Ser 305 310 315 320
Tyr Val Gin He
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 145 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
Met Ser Ser Phe Ser Thr Gin Thr Pro Tyr Pro Asn Leu Ala Val Pro 1 5 10 15
Phe Phe Thr Ser He Pro Asn Gly Leu Tyr Pro Ser Lys Ser He Val 20 25 30
He Ser Gly Val Val Leu Ser Asp Ala Lys Arg Phe Gin He Asn Leu 35 40 45
Arg Cys Gly Gly Asp He Ala Phe His Leu Asn Pro Arg Phe Asp Glu 50 55 60
Asn Ala Val Val Arg Asn Thr Gin He Asn Asn Ser Trp Gly Pro Glu 65 70 75 80
Glu Arg Ser Leu Pro Gly Ser Met Pro Phe Ser Arg Gly Gin Arg Phe 85 90 95
Ser Val Trp He Leu Cys Glu Gly His Cys Phe Lys Val Ala Val Asp 100 105 110 Gly Gin His He Cys Glu Tyr Ser His Arg Leu Met Asn Leu Pro Asp 115 120 125
He Asn Thr Leu Glu Val Ala Gly Asp He Gin Leu Thr His Val Glu 130 135 140
Thr 145
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 136 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Met Ser Asn Val Pro His Lys Ser Ser Leu Pro Glu Gly He Arg Pro 1 5 10 15
Gly Thr Val Leu Arg He Arg Gly Leu Val Pro Pro Asn Ala Ser Arg 20 25 30
Phe His Val Asn Leu Leu Cys Gly Glu Glu Gin Gly Ser Asp Ala Ala 35 40 45
Leu His Phe Asn Pro Arg Leu Asp Thr Ser Glu Val Val Phe Asn Ser 50 55 60
Lys Glu Gin Gly Ser Trp Gly Arg Glu Glu Arg Gly Pro Gly Val Pro 65 70 75 80
Phe Gin Arg Gly Gin Pro Phe Glu Val Leu He He Ala Ser Asp Asp 85 90 95
Gly Phe Lys Ala Val Val Gly Asp Ala Gin Tyr His His Phe Arg His 100 105 110
Arg Leu Pro Leu Ala Arg Val Arg Leu Val Glu Val Gly Gly Asp Val 115 120 125
Gin Leu Asp Ser Val Arg He Phe 130 135
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 262 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 14 :
Met Ala Asp Gly Phe Ser Leu Asn Asp Ala Leu Ala Gly Ser Gly Asn 1 5 10 15
Pro Asn Pro Gin Gly Trp Pro Gly Ala Trp Gly Asn Gin Pro Gly Ala 20 25 30
Gly Gly Tyr Pro Gly Ala Ser Tyr Pro Gly Ala Tyr Pro Gly Gin Ala 35 40 45
Pro Pro Gly Gly Tyr Pro Gly Gin Ala Pro Pro Ser Ala Tyr Pro Gly 50 55 60
Pro Thr Gly Pro Ser Ala Tyr Pro Gly Pro Thr Ala Pro Gly Ala Tyr 65 70 75 80
Pro Gly Pro Thr Ala Pro Gly Ala Phe Pro Gly Gin Pro Gly Gly Pro 85 90 95
Gly Ala Tyr Pro Ser Ala Pro Gly Ala Tyr Pro Ser Ala Pro Gly Ala 100 105 110
Tyr Pro Ala Thr Gly Pro Phe Gly Ala Pro Thr Gly Pro Leu Thr Val 115 120 125
Pro Tyr Asp Met Pro Leu Pro Gly Gly Val Met Pro Arg Met Leu He 130 135 140
Thr He He Gly Thr Val Lys Pro Asn Ala Asn Ser He Thr Leu Asn 145 150 155 160
Phe Lys Lys Gly Asn Asp He Ala Phe His Phe Asn Pro Arg Phe Asn 165 170 175
Glu Asn Asn Arg Arg Val He Val Cys Asn Thr Lys Gin Asp Asn Asn 180 185 190
Trp Gly Arg Glu Glu Arg Gin Ser Ala Phe Pro Phe Glu Ser Gly Lys 195 200 205
Pro Phe Lys He Gin Val Leu Val Glu Ala Asp His Phe Lys Val Ala 210 215 220
Val Asn Asp Val His Leu Leu Gin Tyr Asn His Arg Met Lys Asn Leu 225 230 235 240 Arg Glu He Ser Gin Leu Gly He He Gly Asp He Thr Leu Thr Ser 245 250 255
Ala Ser His Ala Met He 260
(2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 316 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
Met Leu Ser Leu Ser Asn Leu Gin Asn He He Tyr Asn Pro Thr He 1 5 10 15
Pro Tyr Val Ser Thr He Thr Glu Gin Leu Lys Pro Gly Ser Leu He 20 25 30
Val He Arg Gly His Val Pro Lys Asp Ser Glu Arg Phe Gin Val Asp 35 40 45
Phe Gin His Gly Asn Ser Leu Lys Pro Arg Ala Asp Val Ala Phe His 50 55 60
Phe Asn Pro Arg Phe Lys Arg Ser Asn Cys He Val Cys Asn Thr Leu 65 70 75 80
Thr Asn Glu Lys Trp Gly Trp Glu Glu He Thr His Asp Met Pro Phe 85 90 95
Arg Lys Glu Lys Ser Phe Glu He Val He Met Val Leu Lys Asn Lys 100 105 110
Phe His Val Ala Val Asn Gly Lys His He Leu Leu Tyr Ala His Arg 115 120 125
He Asn Pro Glu Lys He Asp Thr Leu Gly He Phe Gly Lys Val Asn 130 135 140
He His Ser He Gly Phe Arg Phe Ser Ser Asp Leu Gin Ser Met Glu 145 150 155 160
Thr Ser Thr Leu Gly Leu Thr Gin He Ser Lys Glu Asn He Gin Lys 165 170 175
Ser Gly Lys Leu His Leu Ser Leu Pro Phe Glu Ala Arg Leu Asn Ala 180 185 190
Ser Met Gly Pro Gly Arg Thr Val Val Val Lys Gly Glu Val Asn Thr 195 200 205
Asn Ala Thr Ser Phe Asn Val Asp Leu Val Ala Gly Arg Ser Arg Asp 210 215 220
He Ala Leu His Leu Asn Pro Arg Leu Asn Val Lys Ala Phe Val Arg 225 230 235 240
Asn Ser Phe Leu Gin Asp Ala Trp Gly Glu Glu Glu Arg Asn He Thr 245 250 255
Cys Phe Pro Phe Ser Ser Gly Met Tyr Phe Glu Met He He Tyr Cys 260 265 270
Asp Val Arg Glu Phe Lys Val Ala Val Asn Gly Val His Ser Leu Glu 275 280 285
Tyr Lys His Arg Phe Lys Asp Leu Ser Ser He Asp Thr Leu Ala Val 290 295 300
Asp Gly Asp He Arg Leu Leu Asp Val Arg Ser Trp 305 310 315
(2) INFORMATION FOR SEQ ID NO : 16 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 135 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
Met Ala Cys Gly Leu Val Ala Ser Asn Leu Asn Leu Lys Pro Gly Glu 1 5 10 15
Cys Leu Arg Val Arg Gly Glu Val Ala Pro Asp Ala Lys Ser Phe Val 20 25 30
Leu Asn Leu Gly Lys Asp Ser Asn Asn Leu Cys Leu His Phe Asn Pro 35 40 45
Arg Phe Asn Ala His Gly Asp Ala Asn Thr He Val Cys Asn Ser Lys 50 55 60
Asp Gly Gly Ala Trp Gly Thr Glu Gin Arg Glu Ala Val Phe Pro Phe 65 70 75 80 Gln Pro Gly Ser Val Ala Glu Val Cys He Thr Phe Asp Gin Ala Asn 85 90 95
Leu Thr Val Lys Leu Pro Asp Gly Tyr Glu Phe Lys Phe Pro Asn Arg 100 105 110
Leu Asn Leu Glu Ala He Asn Tyr Met Ala Ala Asp Gly Asp Phe Lys 115 120 125
He Lys Cys Val Ala Phe Asp 130 135
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 316 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
Met Leu Ser Leu Ser Asn Leu Gin Asn He He Tyr Asn Pro Thr He 1 5 10 15
Pro Tyr Val Ser Thr He Thr Glu Gin Leu Lys Pro Gly Ser Leu He 20 25 30
Val He Arg Gly His Val Pro Lys Asp Ser Glu Arg Phe Gin Val Asp 35 40 45
Phe Gin His Gly Asn Ser Leu Lys Pro Arg Ala Asp Val Ala Phe His 50 55 60
Phe Asn Pro Arg Phe Lys Arg Ser Asn Cys He Val Cys Asn Thr Leu 65 70 75 80
Thr Asn Glu Lys Trp Gly Trp Glu Glu He Thr His Asp Met Pro Phe 85 90 95
Arg Lys Glu Lys Ser Phe Glu He Val He Met Val Leu Lys Asn Lys 100 105 110
Phe His Val Ala Val Asn Gly Lys His He Leu Leu Tyr Ala His Arg 115 120 125
He Asn Pro Glu Lys He Asp Thr Leu Gly He Phe Gly Lys Val Asn 130 135 140
He His Ser He Gly Phe Arg Phe Ser Ser Asp Leu Gin Ser Met Glu 145 150 155 160
Thr Ser Thr Leu Gly Leu Thr Gin He Ser Lys Glu Asn He Gin Lys 165 170 175
Ser Gly Lys Leu His Leu Ser Leu Pro Phe Glu Ala Arg Leu Asn Ala 180 185 190
Ser Met Gly Pro Gly Arg Thr Val Val Val Lys Gly Glu Val Asn Thr 195 200 205
Asn Ala Thr Ser Phe Asn Val Asp Leu Val Ala Gly Arg Ser Arg Asp 210 215 220
He Ala Leu His Leu Asn Pro Arg Leu Asn Val Lys Ala Phe Val Arg 225 230 235 240
Asn Ser Phe Leu Gin Asp Ala Trp Gly Glu Glu Glu Arg Asn He Thr 245 250 255
Cys Phe Pro Phe Ser Ser Gly Met Tyr Phe Glu Met He He Tyr Cys 260 265 270
Asp Val Arg Glu Phe Lys Val Ala Val Asn Gly Val His Ser Leu Glu 275 280 285
Tyr Lys His Arg Phe Lys Asp Leu Ser Ser He Asp Thr Leu Ala Val 290 295 300
Asp Gly Asp He Arg Leu Leu Asp Val Arg Ser Trp 305 310 315
(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 499 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
AATTCGGCAC GAGAGCTCTT NTCACAGGAC CAGCCACTAG CGCANCTCGA GCGATGGCCT 60
ATGTCCCCGC ACCGGGCTAC CAGCCCACCT ACAACCCGAC GCTGCCTTAC TACCAGCCCA 120
TCCCGGGCGG GCTCAACGTG GGAATGTCTG TTTACATCCA AGGAGTGGCC AGCGAGCACA 180
TGAAGCGGTT CTTCGTGAAC TTTGTGGTTG GGCAGGATCC GGGCTCAGAC GTCGCCTTCC 240 ACTTCAATCC GCGGTTTGAC GGCTGGGACA AGGTGGTCTT CAACACGTTG CAGGGCGGGA 300
AGTGGGGCAG CGAGGAGAGG AAGAGGAGCA TGCCCTTCAA AAAGGGTGCC GCCTTTGAGC 360
TTGGTCTTCA TAGTCCTNGG TTGAGCACTA CAAGGTNGTN GTAAATGGAA TCCCTCTATG 420
ANTAGGGGAC CGNTTTCCCT ANAATTGTAA CCANCTNNAA TTGATGGGNN TCAATTAATN 480
ATCAATTATT GGNGGCANC 499 (2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 391 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
AGTGGATGGG GATCTGCAAC TTCAATCAAT CAACTTCATC GGAGGCCAGC CCCTCCGGCC 60
CCAGGGACCC CCGATGATGC CACCTTACCC TGGTCCCGGA CATTGCCATC AACAGCTGAA 120
CAGCCTGCCC ACCATGGAAG GACCCCCAAC CTTCAACCCG CCTGTGCCAT ATTTNGGGAG 180
GCTGCAAGGA GGGCTCACAG CTCGAAGAAC CATCATCATC AAGGGCTATG TGCCTCCCAC 240
AGGCAAGAGC TTTGCTATCA ACTTCAAGGT GGGCTCCTCA GGGGACATAG CTCTGCACAT 300
TAATCCCCGC ATGGGCAACG GTACCGTGGT CCGGAACAGC CTTCTTGAAT GGTTCGTGGG 360
GTTNCGAGGA GAAGAAGNTC ACCCACAACC C 391 (2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 423 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20: CCGGCCCCAG GGACCCCCGA TGATGCCACC TTACCCTGGT CCCGGACATT GCCATCAACA 60 GCTGAACAGC CTGCCCACCA TGGAAGGACC CCCAACCTTC AACCCGCCTG TGCCATATTT 120
CGGGAGGCTG CAAGGAGGGC TCACAGCTCG AAGAACCATC ATCATCAAGG GCTATGTGCC 180
TCCCACAGGC AAGAGCTTTG CTATCAACTT CAAGGTGGGC TCCTCAGGGG ACATAGCTCT 240
GCACATTAAT CCCCGCATGG GCAACGGTAC CGTGGTCCGG AACAGNCTTC TGAATGGCTC 300
GTGGGGATNC GAGGAGAAGG AAGGTCANCC ACAANCCATT TTGTNCCGGA CANTTTTTTT 360
NATCTGTCCA NTTGGTTGTG GTTTGGATCG TTTCAAGGTT TAAGGCAATG GCCAGAACTT 420
TTT 423 (2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 434 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
AATTCGGCAC GAGCACAGGC AAGAGCTTTG CTATCAACTT CAAGGTGGGC TCCTCAGGGG 60
ACATAGCTCT GCACATTAAT CCCCGCATGG GCAACGGTAC CGTGGTCCGG AACAGCCTTC 120
TGAATGGCTC GTGGGGATCC GAGGAGAAGA AGATCACCCA CAACCCATTT GGTCCCGGAC 180
AGTTCTTTGA TCTGTCCATT CGCTGTGGCT TGGATCGCTT CAAGGTTTAC GGCAATGGCC 240
AGCACCTCTT TGACTTTGCC CATCGNCTCT CGGCCTTCCA GAGGGTGGAC ANATTNGAAA 300
TCCAGGGTGA TGTCAACTTG TCCTATGTCC AGATCTAATC TTATTCCTGG GGCCATAATT 360
CATGGGAAAC AGATTATNCN CTAGGGTTCT TTTTTAGGCC CTAATAAAAT GTCTTAGGGG 420
GGTAAAAAAA AAAA 434 (2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 354 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
CTTCAATCCG CGGTTTGACG GCTGGGACAA GGTGGTCTTC AACACGTTGC AGGGCGGGAA 60
GTGGGGCAGC GAGGAGAGGA AGAGGAGCAT GCCCTTCAAA AAGGGTGCCG CCTTTAAGCT 120
GGTCTTCATA GTCCTGGCTG AGCACTACAA GGTGGTGGTA AATGGAAATC CCTTCTATGA 180
GTACGGGCAC CGGCTTCCCC TACAGATGGT CACCCACCTG CAAGTGGATG GGGATCTNCA 240
ACTTCAATCA ATCAACTTCA TCGGGAGGNC AGCCCNTCCG GCCCCAGGGA CCCCCGATGA 300
TGCCACCTTA CCCTGGTNCC GGACATTGGC CATCAGCAGT TGAACAGCTG TCCA 354 (2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 329 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
GTGGTCCGGA ACAGCCTTCT GAATGGCTCG TGGGGATCCG AGGAGAAGAA GATCACCCAC 60
AACCCATTTG GTCCCGGACA GTTCTTTGAT CTGTCCATTC GCTGTGGCTT GGATCGCTTC 120
AAGGTTTACG CCAATGGCCA GCACCTCTTT GACTTTGCCC ATCGCCTCTC GGCCTTCCAG 180
AGGGTGGACA CATTGGAAAT CCAGGGTGAT GTCACCTTGT CCTATGTCCA GATCTAATCT 240
ATTNCTGGGG CCATAACTCA TGGGAAAACA GAATTATCCC CTAGGACTCC TTTCTAAAGC 300
CCNCTAATAA AAANGTCTGA GGGTGTCTC 329 (2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 229 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
GCGGGCTCAA CGTGGGAATG TCTGTTTACA TCCAAGGAGT GGCCAGCGAG CACATGAAGC 60
GGTTCTTCGT GAACTTTGTG GTTGGGCAGG ATCCGGGCTC AGACGTCGCC TTCCACTTCA 120
ATCCGCGGTT TGACGGCTGG GACAAGGTGG TCTTCAACAC GTTGCAGGGC GGGAAGTGGG 180
GCAGCNAGGA GAGGAAGAGG AGCATGCCCT TCAAAAAGGG TGCCGCCTT 229 (2) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 194 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
GGAAGAGGAG CATGCCCTTC AAAAAGGGTG CCGCCTTTAA CCTGGTNTTC ATAGTCCTGG 60
CTGAGCACTA CAAGGTGGTG GTAAATGGAA ATCCCTTCTA TNAGTACGGG CACCGGCTTC 120
CCCTACAGAT GGTCACCCAC CTGCAAGTGG ATGGGGATCT GCAACTTCAT TCATTCAACT 180
TCATCGGAGG CCAG 194 (2) INFORMATION FOR SEQ ID NO: 26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 499 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:
AATTCCGTTC TCTACTCCCG CCATCCCACC TATAATGTAC CCCCACCCCG CCTATCCAAT 60
GCCTTTAATC ACCACCATTC TGGGAGGGCT GTACCCATCC AAGTCCATCC TCCTGTAAGG 120
CACTTGCCTG CCCAGTGCTC ANAGGTTCCA CATCAACCTG TGCTCTGGGA AACCACATCG 180
CCTTCCACCT GNAACCCCCG TTTTGAATGA GAATGCTGTG GTCCGCAACA CCCAGATNGA 240 CAACTCCTGG GGGTCTGAGG AGCGAAGTGT GCCCCGAAAA ATGCCCTTGG TNCGTGGCCA 300
GAGGTTNTNA GGTGGATCTT GTGTGAAGTT CAATGNGTNC AAGTGGGCCT GGATGGTNAG 360
NANTGTTTGN ATNATTANNC TGGGNTTGNG GNAACTGNGC AANNTTNAAC AGATNGNAGT 420
TGGGGGGGNG ANANTCAGNT GNACCGTTTT GNAGNNATAG GGGGNTTTNT TGGCCTTGGG 480
GGGGGGGGTT GGGGTTTTG 499 (2) INFORMATION FOR SEQ ID NO: 27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 376 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:
CTTTTGCCAA CAAGCATTTT NATTTCTTTA TTTTAAGGAC ACTGGGAAAG GAGCCAGTCC 60
CCTGAAGAGA ACACTCTGGT CAGGTGGTGG AGGCCAGTGG GAAGCCATCA GGCCTGCTTT 120
CCAGGAGGGG TGAAGGGTTG GTGCACGGTG CAAGGTGAGA GTGAAGGTTA AAGGTCAGAG 180
AGGAGGGGCT GAGGAGGCCA CCTTCCACCA GGAGCAGACA GCTGGTGGCT TGGGAACTGG 240
GGTGGAGCTG CGTGGGGGAT GGGAAGGGGA CTGAGCATGG GGCTTCATCT TNCACTGCCC 300
ACTCCTGCCC TCTTCCCTGG CTGTGCCTGC CTNCCTGGGA TGGTAGGGTT TCCANCANTT 360
GGAGGCCCCA NGTGCT 376 (2) INFORMATION FOR SEQ ID NO: 28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 282 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: TTCAGATCAC TGTCAATGGG ACCGTTCTCA GCTCCAGTGG AACCAGGTTT NCTGTGAACT 60 TTCAGACTGG CTTCAGTGGA AATAACATTG CCTTCCACTT CAACCCTCGG TTTGAAGATG 120
GAGGGTACGT GGTGTGCACA GNAGGCAGAA CGGAAGCTGG GGGCCCGAGG AGAGGAAGAC 180
ACACATGCCT TTCCAGAAGG GGATGCCCTT TAACCTCTGC TTCCTGGTGC AGAGCTCAGA 240
TTTCAAGGTG ATGGTGAACG GGATCCTCTT CGTGCAGTAC TT 282 (2) INFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 274 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:
GTGCAGAGCG CCCCTGGACA GATGTNCTCT ACTCCCGCCA TCCCACCTAT GATGTACCCC 60
CACCCCGCCT ATCCGATGCC TTTNAACACC ACCATTCTGG GAGGGCTGTA CCCATCCAAG 120
ATCCATCCTC CTGTCAGGCA CTGTCCTGCC CAGTGCTCAG AGGTTCCACA TCAACCTGTG 180
CTCTGGGAAC CACATCGCCT TCCACCTGAA CCCCCGTTTT GATGAGAATG CTGTGGTCCG 240
CAACACCCAG ATCGACAAAT TCCTGGGGGG TCTT 274 (2) INFORMATION FOR SEQ ID NO: 30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 342 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:
CTTTTGCCAA CAAGCATTTT NATTTCTTTA TTTTAAGGAC ACTGGGAAAG GAGCCAGTCC 60
CCTGAAGAGA ACACTCTGGT CAGGTGGTGG AGGCCAGTGG GAAGCCATCA GGCCTGCTTT 120
CCAGGAGGGG TGAAGGGTTG GTGCACGGTG CAAGGTGAGA GTNAAGGTTA AAGGTCAGAG 180
AGGAGGGGCT GAGGAGGCCA CCTTCCACCA GGAGCAGACA GCTGGTGGCT TGGGAACTGG 240 GGTGGGAGCT GTCGTNGGGG GATGGNAAGG GGACTGAGCC ATGGGGGCTT TCATCTTNCA 300 CTGCCCACTC CTGCCCTTTT CCCTGGTTTG TGNCTGNCCT TC 342
(2) INFORMATION FOR SEQ ID NO: 31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 246 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:
CCTGCTTCTG GCTACAGCCA CCNTGGAACG GAGAAGGCAG CTGACGGGGA TTGCCTTCNT 60
CAGCCGCAGC AGCACCTGGG GCTCCAGCTG CTGGAATCNT ACCATCCCAG GAGGCAGGCA 120
CAGCCAGGGA GAGGGGAGGA GTGGGCAGTG AAGATNAAGC CCCATGCTCA GTCCCCTCCC 180
ATCCCCCACG CAGCTCCACC CCAGTTCCAA GNCACCAGCT GTCTGCTCCT GGTGGGAGGT 240
GGCCTC 246 (2) INFORMATION FOR SEQ ID NO: 32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 228 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: GGCANAGCAG AGGTGTGGAT CTTNTNTAAA GCTCACTGCC TCAAGGTGGC CGTGGATGGT 60 CAGCACCTGT TTAAATACTA CCATCGCCTG AGGAACCTGC CCACCATCAA CAGACTGGGA 120 GTGGGGGGCG AACATCCAGC TGACCCATGT GCAGACATAG GCGGCTTCCT GGCCCTGGGG 180 CGGGGGCTNA GNTTTGGGGN AGTCTGGGTC CTNTAATNAT CCNCANTT 228
(2) INFORMATION FOR SEQ ID NO: 33: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 161 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: TTCCCTCTAC AAAGGACTTC CTAGTGGGTG TNAAAGGCAG CGGTGGCCAC ANAGGCGGCG 60 GAGAGATGGC CTTCAGCGGT TCCCAGGCTC CCTACCTGAG TCCAGCTGTC CCCTTTTTTG 120 GGACTATTCA AGGAGGTCTC CAGGACGGAC TTCAGATCAC T 161
(2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 306 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
CTCTGTGCAG CTGTCCTACA TCAGCTTCCA GGNNAGACTG TCCACCTGGC ACCGGTNCCA 60
GGGGCGGGGA ATGCGGGGNG NAGCGTAGTT GATACTGAAG NCNCTGATGG GTGGGGCNNA 120
AGNCANATCT CCTNACCCAG GTCACTCTGG GGGACAACCT CTGGCTTCCC TGTCCCAGTA 180
CCTGGCTGNC NACTTCTCCT CTGTGAACTC TGANCCCTCC TTCTGTGTTT ACTGTCTCTG 240
TCCGGAACAA CTGCCTTGGT CTCCCAGANT GCTCAGGTGA CCCTTTNTTN TTTCNACCCT 300
TCAATT 306 (2) INFORMATION FOR SEQ ID NO: 35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 449 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:
CTCATACAGA GGGCATCGGG TCCCACCCTG TCACTCATTT CATCGTCTAA AATGTAATCA 60
TGAGTGTTTG CTTCGAGCCA GGGACAGTNC TGCTGCAGGG GACCCAGCTG GGACCAAGGC 120
AGACTGTCTC TCCCCTCCTG GGATTTACAG GGTCATGGCT CTGAAACATT CTGTAGTGTT 180
CTTTGAACAC GAGTTTTCCC TGGAGATCGC TTTCTGCAGG CCTCTTGGTC CTGACTGTGG 240
CTTCTTTTCA GAGCCTGCCA TTCGCTGCAA GGTTGAACAN CCCCATGGGC CCTGGGACGA 300
ACTGTCGTCG TTAAAAGGAG AAGTGAATGC AAATGNCCAA AAAGCTTTTA ATGTTTGACC 360
TACTAGCAGG AAATCAAAGG GTATTGCNTC TTACAATTGN ACCCAGGCTG AATATTAAAG 420
CATTTTAAAG AATTCTTTTT CTTCAGGAG 449 (2) INFORMATION FOR SEQ ID NO: 36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 265 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:
TTCAATCCTC GTTTCAAAAG GGCCGGCTGC ATTGTTTGCA ATACTTTNAT AAATGAAAAA 60
TGGGGACGGG AAGAGATCAC CTATGACACG CCTTTCAAAA GAGAAAAGTC TTTTNAGATC 120
GTAATTATGG TGCTGAAGGA CAAATTCCAG GTGGCTGTAA ATGGAAAACA TACTCTGCTC 180
TATGGCCACA GGATCGGCCC AGAGAAAATA GACACTCTGG GCATTTATGG CAAAGTGAAT 240
ATTCACTCAA TTGGTTTTAG CTTCA 265 (2) INFORMATION FOR SEQ ID NO: 37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 353 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:
AAGCCACTCT GCCCTCTCTC CTACTTTGGC TGACTCTTCA AGAATGCCAT TCAACAAGTA 60
TTTATGGAGT ACCTACTATA ATACAGTAGC TAACATGTAT TGAGCACAGA TTTTTTTTGG 120
TAAAACTGTG AGGAGCTAGG ATATATACTT GGTGAAACAA ACCAGTATGT TCCCTGTTCT 180
CTTGAGCTTC GACTCTTCTG TGCTCTATTG CTGCGCACTG CTTTTTCTAC AGGCATTACA 240
TCAACTCCTA AGGGGTCCTC TGGGGATTAG TTAAGCAGCT ATTTAAATCA CCCGAAGGAC 300
ACTTAATTTA CAGATGACAC AANTCCTTTC CCCAGTGATT CAACTGTTCA TAA 353
(2) INFORMATION FOR SEQ ID NO: 38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 234 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: CDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:
GAAACACCAG TNTTTGGGGC CAGTNCCTCA NTTTCAATCC AGGTAACCTT TAANTGAAAC 60
TTGCCTAAAA TNTTAGGTCA TACACAGAAG AGACTCCAAT CGACAAGAAG CTGGAAAAGA 120
ATGATGTTGT CCTTAAACAA CCTACAGANT ATCATCTATA ACCCGGTAAT CCCGTTTNTT 180
GGCACCATTC CTGATCAGCT GGATCCTGGA ACTTTGATTG TAATACGTGG GCAT 234 (2) INFORMATION FOR SEQ ID NO: 39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 344 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39: ACACGCTGGA AATTAATGGA GACATCCACT TACTGGAAGT AAGGNGNTGG TAGCCTACCT 60
ACACAGCTGC TACAAAAACC AAAATACAGA ATGGCTTCTG TGATACTGGC CTTGCTGAAA 120
CGCATCTCAC TGTCATTCTA TTGTTTATAT TGTTAAAATG AGCTTGTGCA CCATTAGGTC 180
CTGCTGGGTG TTCTCAGTCC TTGCCATGAA GTATGGTGGT GTCTAGCACT GAATGGGGAA 240
ACTGGGGGCA GCAACACTTA TAGCCAGTTA AAGCCACTCT GCCCTCTCTC CTACTTTGGG 300
CTGACTCTTC AAGAATGCCA TTCAACAAGT ATTTATGGGG TACC 344 (2) INFORMATION FOR SEQ ID NO: 40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 502 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:
AATTCGGCAN AGCTTCAAAC CTTTGAGACA TAGTTCATAG GTGGTATTTT GGTGCAAGTC 60
AAAGTGTGAT NGACAGTCGA ATNTTTGCTC TTGGTGTAGA CAGTTCTGGG TGCGATTTTA 120
GAAATGTCTG CTCCTCTATT ACTAGGCTGT NGGGAAACAG TTCTACAGTA AGGAATGGAA 180
TGANATGAAG CTGCCCTCCA CGGTTTAAAC TGTTCATTTT CTATGCAACT TTATAAAATA 240
TTCCACATGA ANTAACCCAG GCAAAAATAC TTCACAGGCT GGGGGGCGTG GCCAGANCTT 300
TGGGAACCTA TTGGGAAAAG GAAACCAAAN CACANCAATG TTTAGAAGGG GGAAGGATTT 360
TTAGTTTATN AATNTGAAGT NTTGGGNNGT TGCTGAGGCT GAGGCCTGGG CCGGNGGCTT 420
GGGGATTGTT TCCNGGTTNC CACTCTGGTG NGGNNTTNCC NGGGCAGTTG GGTGNTTTTA 480
TGACGGGATT GGTATTGTGT TG 502 (2) INFORMATION FOR SEQ ID NO: 41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41: CGCCCATGGC CTATGTCCCC GCACCG 26
(2) INFORMATION FOR SEQ ID NO: 42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42: CGCAAGCTTT TAGATCTGGA CATAGGAC 28
(2) INFORMATION FOR SEQ ID NO: 43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: CGCCCATGGC CTTCAGCGGT TCCCAG 26
(2) INFORMATION FOR SEQ ID NO: 44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44: CGCAAGCTTC AGGGTTGGAA AGGCTG 26
(2) INFORMATION FOR SEQ ID NO: 45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45: CGCCCATGCT GTTGTCCTTA AACAAC 26
(2) INFORMATION FOR SEQ ID NO: 46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46: CGCCTGCAGC ACAGAAGCCA TTCTG 25
(2) INFORMATION FOR SEQ ID NO: 47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47: CGCCTGCAGC TATGCAACTT TATAAAATAT TCC 33
(2) INFORMATION FOR SEQ ID NO: 48: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: CGCCCCGGGG CCTATGTCCC CGCAC 25
(2) INFORMATION FOR SEQ ID NO: 49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49: CGCGGTACCT TAGATCTGGA CATAGGAC 28
(2) INFORMATION FOR SEQ ID NO: 50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50: CGCCCCGGGG CCTTCAGCGG TTCCCAG 27
(2) INFORMATION FOR SEQ ID NO: 51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51: CGCGGTACCC AGGGTTGGAA AGGCTG 26
(2) INFORMATION FOR SEQ ID NO: 52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52: CGCCCCGGGT TGTCCTTAAA CAACCTAC 28
(2) INFORMATION FOR SEQ ID NO: 53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53: CGCGGTACCC ACAGAAGCCA TTCTG 25
(2) INFORMATION FOR SEQ ID NO:54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 54 : CGCGGTACCC TATGCAACTT TATAAAATAT TCC 33
(2) INFORMATION FOR SEQ ID NO: 55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55: CGCCCCGGGG CCATCATGGC CTATGTCCCC G 31
(2) INFORMATION FOR SEQ ID NO: 56:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:56: CGCGGTACCT TAGATCTGGA CATAGGAC 28
(2) INFORMATION FOR SEQ ID NO: 57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:57: CGCCCCGGGG CCATCATGGC CTTCAGCGGT TC 32
(2) INFORMATION FOR SEQ ID NO: 58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:58: CGCGGTACCC AGGGTTGGAA AGGCTG 26
(2) INFORMATION FOR SEQ ID NO: 59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59: CGCCCCGGGG CCATCATGAT GTTGTCCTTA AAC 33
(2) INFORMATION FOR SEQ ID NO: 60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:60: CGCGGTACCC ACAGAAGCCA TTCTG 25 INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCT Rule I3bis)
A. The indications made below relate to the microorganism referred to in the description on page , line
B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet [ vl
Name of depositary institution
American Type Culture Collection
Address of depositary institution (including postal code and country)
12301 Parklav Drive Rockville, Maryland 20852 United States of America
Date of deposit Accession Number
September 24, 1996 ATCC 97732
C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet Q
DNA Plasmid , 93442
D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not for all designated States)
E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable)
The indications listed below will be submitted to the International Bureau later (specify the general nature of the indications e.g., "Accession Number of Deposit")
For International Bureau use only j j This sheet was received by the International Bureau on:
Λullioπ/.cd υl l iccr
Figure imgf000094_0001
INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCT Rule I3bis)
A. The indications made below relate to the microorganism referred to in the description on page 3 , line 6
B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet IX
Name of depositary institution
American Type Culture Collection
Address of depositary institution (including postal code and country)
12301 Parklan Drive Rockville, Maryland 20852 United States of America
Date of deposit Accession Number
September 24 , 1996 ATCC 97733
C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet | |
DNA Plasmid, 91715
D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not for all designated States)
E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable)
The indications listed below will be submitted to the International Bureau later (specify the general natureof theinώcations e.g., "Accession Number of Deposit")
For receiving Office use only For International Bureau use only
Q This sheet was received with the international application j j This sheet was received by the International Bureau on:
Λuthoπ/ed officer Λullioπ/cti ol ficcr
Figure imgf000095_0001
l onn l'C "l /KO/ n4 (July | Q<>? ) INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCT Rule I3bis)
A. The indications made below relate to the microorganism referred to in the description on page , line
B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet | |
Name of depositary institution
American Type Culture Collection
Address of depositary institution (includin postal code and country)
12301 Parklawn Drive Rockvile, Maryland 20852 United States of America
Date of deposit Accession Number
September 24 , 1996 ATCC 97734
C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet Q
DNA Plasmid, 221441
D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not for all designated States)
E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable)
The indications listed below will be submitted to the International Bureau later (specify the general nature of theindicationse.g., "Accession Number of Deposit")
For International Bureau use only
[ j This sheet was received by the International Bureau on:
Authorized officer
Figure imgf000096_0001

Claims

What Is Claimed Is:
1. An isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence at least 95% identical to a sequence selected from the group consisting of: (a) a nucleotide sequence encoding a polypeptide comprising amino acids from about 1 to about 323 in SEQ ID NO:2, from about 1 to about 311 in SEQ ID NO:4, from about 1 to about 317 in SEQ ID NO:6, or from about
1 to about 200 in SEQ ID NO:8;
(b) a nucleotide sequence encoding a polypeptide comprising amino acids from about 2 to about 323 in SEQ ID NO:2, from about 2 to about
311 in SEQ ID NO:4, from about 2 to about 317 in SEQ ID NO:6, or from about
2 to about 200 in SEQ ID NO:8;
(c) a nucleotide sequence encoding a polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97732, 97733 or 97734;
(d) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), or (c).
2. An isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide having a nucleotide sequence identical to a nucleotide sequence in (a), (b), (c), or
(d) of claim 1 wherein said polynucleotide which hybridizes does not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence consisting of only A residues or of only T residues.
3. An isolated nucleic acid fragment of the polynucleotide of claim 1 , wherein said fragment is selected from the group consisting of:
(a) a nucleotide sequence comprising at least 520 contiguous nucleotides of SEQ ID NO: 1 ; (b) a nucleotide sequence comprising at least 460 contiguous nucleotides of SEQ ID NO:3; and
(c) a nucleotide sequence complementary to any of the nucleotide sequences in (a) or (b).
4. A method for making a recombinant vector comprising inserting an isolated nucleic acid molecule of claim 1 into a vector.
5. A recombinant vector produced by the method of claim 4.
6. A method of making a recombinant host cell comprising introducing the recombinant vector of claim 5 into a host cell.
7. A recombinant host cell produced by the method of claim 6.
8. A recombinant method for producing a galectin 8, 9, 10 or 10SV polypeptide, comprising culturing the recombinant host cell of claim 7 under conditions such that said polypeptide is expressed and recovering said polypeptide.
9. An isolated galectin 8, 9, 10, or 10SN polypeptide having an amino acid sequence at least 95% identical to a sequence selected from the group consisting of:
(a) amino acids from about 1 to about 323 in SEQ ID ΝO:2, from about 1 to about 311 in SEQ ID NO:4, from about 1 to about 317 in SEQ ID NO:6, or from about 1 to about 200 in SEQ ID NO:8;
(b) amino acids from about 2 to about 323 in SEQ ID NO:2, from about 2 to about 311 in SEQ ID NO:4, from about 2 to about 317 in SEQ ID NO:6, or from about 2 to about 200 in SEQ ID NO:8; (c) the amino acid sequence of the galectin 8, 9, 10, or 10SN polypeptide having the amino acid sequence encoded by the cDΝA clone contained in ATCC Deposit No. 97732, 97733 or 97734; and
(d) the amino acid sequence of an epitope-bearing portion of any one of the polypeptides of (a), (b), or (c).
10. An isolated antibody that binds specifically to a galectin 8, 9, 10, or 1 OSV polypeptide of claim 9.
11. An isolated nucleic acid molecule comprising a polynucleotide encoding a galectin 8, 9, 10, or 10SV polypeptide wherein, except for at least one conservative amino acid substitution, said polypeptide has a sequence selected from the group consisting of:
(a) a nucleotide sequence encoding a polypeptide comprising amino acids from about 1 to about 323 in SEQ ID NO:2, from about 1 to about 311 in SEQ ID NO:4, from about 1 to about 317 in SEQ ID NO:6, or from about 1 to about 200 in SEQ ID NO:8;
(b) a nucleotide sequence encoding a polypeptide comprising amino acids from about 2 to about 323 in SEQ ID NO:2, from about 2 to about 311 in SEQ ID NO:4, from about 2 to about 317 in SEQ ID NO:6, or from about 2 to about 200 in SEQ ID NO:8; (c) a nucleotide sequence encoding a polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97732, 97733 or 97734;
(d) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), or (c).
12. An isolated galectin 8, 9, 10, or 1 OS V polypeptide wherein, except for at least one conservative amino acid substitution, said polypeptide has a sequence selected from the group consisting of: (a) amino acids from about 1 to about 323 in SEQ ID NO:2, from about 1 to about 311 in SEQ ID NO:4, from about 1 to about 317 in SEQ ID NO:6, or from about 1 to about 200 in SEQ ID NO:8;
(b) amino acids from about 2 to about 323 in SEQ ID NO:2, from about 2 to about 311 in SEQ ID NO:4, from about 2 to about 317 in SEQ
ID NO:6, or from about 2 to about 200 in SEQ ID NO:8;
(c) the amino acid sequence of the galectin 8, 9, 10, or 10SV polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97732, 97733 or 97734; and (d) the amino acid sequence of an epitope-bearing portion of any one of the polypeptides of (a), (b), or (c).
13. A method of detecting a galectin 8, 9, 10, or 10SN polypeptide in a sample, comprising: a) contacting said sample with an antibody according to claim 10, under conditions such that immunocomplexes form, and b) detecting the presence of said antibody bound to said polypeptide.
14. A method of treatment of a cell growth disorder in a mammal, comprising administering a therapeutically effective amount of the polypeptide of claim 9 to said mammal.
15. The method of claim 14, wherein said disorder is selected from the group consisting of cancer, autoimmune diseases, inflammatory diseases, asthma, and allergic diseases.
PCT/US1997/018261 1996-10-09 1997-10-09 Galectin 8, 9, 10 and 10sv WO1998015624A1 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
JP10517750A JP2001501831A (en) 1996-10-09 1997-10-09 Galectin 8, Galectin 9, Galectin 10, and Galectin 10 SV
CA002268022A CA2268022A1 (en) 1996-10-09 1997-10-09 Galectin 8, 9, 10 and 10sv
EP97912689A EP1012266A4 (en) 1996-10-09 1997-10-09 Galectin 8, 9, 10 and 10sv

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US2809396P 1996-10-09 1996-10-09
USPCT/US96/16565 1996-10-09
US60/028,093 1996-10-09
US9616565 1996-10-09

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US6953658B2 (en) 2000-03-09 2005-10-11 Diadexus, Inc. Method of diagnosing, monitoring, staging, imaging and treating gastrointestinal cancer
CA2427523A1 (en) * 2000-11-01 2003-04-30 Galpharma Co., Ltd. Predicting agent for metastasis
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US20060009378A1 (en) 2002-11-14 2006-01-12 Itshak Golan Novel galectin sequences and compositions and methods utilizing same for treating or diagnosing arthritis and other chronic inflammatory diseases
AU2003279511A1 (en) * 2002-11-14 2004-06-03 Medical Research Fund At The Tel Aviv Sourasky Medical Center Novel galectin sequences and compositions and methods utilizing same for treating or diagnosing arthritis and other chronic inflammatory diseases
CA2514108A1 (en) * 2003-01-24 2004-08-05 Galpharma Co., Ltd. Drugs containing galectin 9
CN1795206A (en) * 2003-04-28 2006-06-28 株式会社嘉尔药物 Galectin 9-inducing factors
DE10333406A1 (en) * 2003-07-15 2005-02-10 Protagen Ag T-regulatory cells containing galectins for the therapy and diagnosis of diseases
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TW200539890A (en) 2004-03-12 2005-12-16 Brigham & Womens Hospital Methods of modulating immune responses by modulating tim-1, tim-2 and tim-4 function
JP4792390B2 (en) * 2004-03-29 2011-10-12 株式会社ガルファーマ Novel galectin-9 variant protein and use thereof
CN1989245A (en) * 2004-06-14 2007-06-27 株式会社嘉尔药物 Novel galectin 8 modification protein and use thereof
CN1332030C (en) * 2005-04-28 2007-08-15 武汉大学 Polypeptide, its coding sequence and preparation method and application of fungus galactose agglutinin protein activity
JP2007131540A (en) * 2005-11-08 2007-05-31 Galpharma Co Ltd Antiallergic agent, immunosuppressive agent and antitumor agent based on cd44 function inhibiting factor
CN102205111B (en) * 2011-05-19 2013-01-23 武汉大学 Application of agrocybe aegerita galactose agglutinin in preparation of anti-HIV (Human Immunodeficiency Virus) infection medicament
WO2014171018A1 (en) * 2013-04-20 2014-10-23 株式会社ガルファーマ Method for diagnosing disease(s), method for treating or preventing disease(s), kit for diagnosing disease(s), and drug for treating or preventing disease(s)
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