WO1998040404A2 - Secreted proteins and polynucleotides encoding them - Google Patents

Secreted proteins and polynucleotides encoding them Download PDF

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Publication number
WO1998040404A2
WO1998040404A2 PCT/US1998/004601 US9804601W WO9840404A2 WO 1998040404 A2 WO1998040404 A2 WO 1998040404A2 US 9804601 W US9804601 W US 9804601W WO 9840404 A2 WO9840404 A2 WO 9840404A2
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WIPO (PCT)
Prior art keywords
amino acid
seq
polynucleotide
protein
sequence
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PCT/US1998/004601
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French (fr)
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WO1998040404A3 (en
Inventor
Kenneth Jacobs
John M. Mccoy
Edward R. Lavallie
Lisa A. Racie
David Merberg
Maurice Treacy
Vikki Spaulding
Michael J. Agostino
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Genetics Institute, Inc.
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Application filed by Genetics Institute, Inc. filed Critical Genetics Institute, Inc.
Priority to EP98911532A priority Critical patent/EP0970109A2/en
Priority to CA002283193A priority patent/CA2283193A1/en
Priority to AU65468/98A priority patent/AU6546898A/en
Priority to JP53968398A priority patent/JP2001514520A/en
Publication of WO1998040404A2 publication Critical patent/WO1998040404A2/en
Publication of WO1998040404A3 publication Critical patent/WO1998040404A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/04—Drugs for disorders of the muscular or neuromuscular system for myasthenia gravis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents

Definitions

  • the present invention provides novel polynucleotides and proteins encoded by such polynucleotides, along with therapeutic, diagnostic and research utilities for these polynucleotides and proteins.
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
  • (k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and (1) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i).
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:l from nucleotide 521 to nucleotide 1111; the nucleotide sequence of SEQ ID NO:l from nucleotide 536 to nucleotide 817; the nucleotide sequence of the full-length protein coding sequence of clone ax318_3 deposited under accession number ATCC 98353; or the nucleotide sequence of the mature protein coding sequence of clone ax318_3 deposited under accession number ATCC 98353.
  • the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone ax318_3 deposited under accession number ATCC 98353.
  • the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:2 from amino acid 4 to amino acid 99.
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
  • protein comprises the amino acid sequence of SEQ ID NO:2 or the amino acid sequence of SEQ ID NO:2 from amino acid 4 to amino acid 99.
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
  • polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i).
  • polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1
  • the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone bgl40_l deposited under accession number ATCC 98353.
  • the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:31 from amino acid 148 to amino acid 249.
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
  • fragments of the amino acid sequence of SEQ ID NO:4 comprising the amino acid sequence from amino acid 129 to amino acid 138 of SEQ ID NO:4;
  • fragments of the amino acid sequence of SEQ ID NO:31 comprising the amino acid sequence from amino acid 163 to amino acid 172 of SEQ ID NO:31;
  • amino acid sequence encoded by the cDNA insert of clone bgl40_l deposited under accession number ATCC 98353 the protein being substantially free from other mammalian proteins.
  • protein comprises the amino acid sequence of SEQ ID NO:4, the amino acid sequence of
  • SEQ ID NO:31 or the amino acid sequence of SEQ ID NO:31 from amino acid 148 to amino acid 249.
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
  • polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i).
  • polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1
  • the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone bg465_2 deposited under accession number ATCC 98353.
  • the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:6 from amino acid 260 to amino acid 343.
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
  • protein comprises the amino acid sequence of SEQ ID NO:6 or the amino acid sequence of SEQ ID NO:6 from amino acid 260 to amino acid 343.
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
  • a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 8 having biological activity, the fragment comprising the amino acid sequence from amino acid 85 to amino acid 94 of SEQ ID NO:8;
  • a polynucleotide which is an allelic variant of a polynucleotide of (a)-(h) above;
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:7 from nucleotide 48 to nucleotide 1055; the nucleotide sequence of SEQ ID NO:7 from nucleotide 216 to nucleotide 1055; the nucleotide sequence of SEQ ID NO:7 from nucleotide 494 to nucleotide 958; the nucleotide sequence of the full-length protein coding sequence of clone bk291_3 deposited under accession number ATCC 98353; or the nucleotide sequence of the mature protein coding sequence of clone bk291_3 deposited under accession number ATCC 98353.
  • the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone bk291_3 deposited under accession number ATCC 98353.
  • the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:8 from amino acid 188 to amino acid 306.
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
  • a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:10 having biological activity, the fragment comprising the amino acid sequence from amino acid 184 to amino acid 193 of SEQ ID NO:10;
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:9 from nucleotide 64 to nucleotide 1197; the nucleotide sequence of SEQ ID NO:9 from nucleotide 1 to nucleotide 828; the nucleotide sequence of the full-length protein coding sequence of clone bp537_4 deposited under accession number ATCC 98353; or the nucleotide sequence of the mature protein coding sequence of clone bp537_4 deposited under accession number ATCC 98353.
  • the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone bp537_4 deposited under accession number ATCC 98353.
  • the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:10 from amino acid 1 to amino acid 255.
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
  • protein comprises the amino acid sequence of SEQ ID NO:10 or the amino acid sequence of SEQ ID NO:10 from amino acid 1 to amino acid 255.
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
  • polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i).
  • polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1
  • the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone cs431_2 deposited under accession number ATCC 98353.
  • the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:12 from amino acid 150 to amino acid 310.
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:13 from nucleotide 29 to nucleotide 2227; the nucleotide sequence of SEQ ID NO:13 from nucleotide 1334 to nucleotide 2227; the nucleotide sequence of SEQ ID NO:13 from nucleotide 1 to nucleotide 746; the nucleotide sequence of the full-length protein coding sequence of clone cw976_l deposited under accession number ATCC 98353; or the nucleotide sequence of the mature protein coding sequence of clone cw976_l deposited under accession number ATCC 98353.
  • the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone cw976_l deposited under accession number ATCC 98353.
  • the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 14 from amino acid 1 to amino acid 239, or a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:14 from amino acid 119 to amino acid 733.
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
  • protein comprises the amino acid sequence of SEQ ID NO:14, the amino acid sequence of SEQ ID NO:14 from amino acid 1 to amino acid 239, or the amino acid sequence of SEQ ID NO:14 from amino acid 119 to amino acid 733.
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:15 from nucleotide 364 to nucleotide 777; the nucleotide sequence of SEQ ID NO:15 from nucleotide 1 to nucleotide 636; the nucleotide sequence of the full-length protein coding sequence of clone cwl233_3 deposited under accession number ATCC 98353; or the nucleotide sequence of the mature protein coding sequence of clone cwl233_3 deposited under accession number ATCC 98353.
  • the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone cwl233_3 deposited under accession number ATCC 98353.
  • the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:16 from amino acid 1 to amino acid
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of SEQ ID NO: 16;
  • protein comprises the amino acid sequence of SEQ ID NO:16 or the amino acid sequence of SEQ ID NO: 16 from amino acid 1 to amino acid 91.
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
  • polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i).
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:17 from nucleotide 619 to nucleotide 1434; the nucleotide sequence of SEQ ID NO:17 from nucleotide 520 to nucleotide 1323; the nucleotide sequence of the full-length protein coding sequence of clone dgl_l deposited under accession number ATCC 98353; or the nucleotide sequence of the mature protein coding sequence of clone dgl_l deposited under accession number ATCC 98353.
  • the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone dgl_l deposited under accession number ATCC 98353.
  • the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:18 from amino acid 1 to amino acid 235.
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
  • protein comprises the amino acid sequence of SEQ ID NO:18 or the amino acid sequence of SEQ ID NO: 18 from amino acid 1 to amino acid 235.
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID
  • nucleotide 2063 from nucleotide 2063 to nucleotide 2290;
  • a polynucleotide comprising the nucleotide sequence of SEQ ID NO:19 from nucleotide 2276 to nucleotide 2290;
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:19 from nucleotide 2063 to nucleotide 2290; the nucleotide sequence of SEQ ID NO:19 from nucleotide 2276 to nucleotide 2290; the nucleotide sequence of SEQ ID NO: 19 from nucleotide 2037 to nucleotide 2405; the nucleotide sequence of the full-length protein coding sequence of clone ep234_l deposited under accession number ATCC 98353; or the nucleotide sequence of the mature protein coding sequence of clone ep234_l deposited under accession number ATCC 98353.
  • the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone ep234_l deposited under accession number ATCC 98353.
  • the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:20 from amino acid 1 to amino acid 69.
  • Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:20 from amino acid 1 to amino acid 69.
  • Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of SEQ ID NO:20;
  • protein comprises the amino acid sequence of SEQ ID NO:20 or the amino acid sequence of SEQ ID NO:20 from amino acid 1 to amino acid 69.
  • the polynucleotide is operably linked to an expression control sequence.
  • the invention also provides a host cell, including bacterial, yeast, insect and mammalian cells, transformed with such polynucleotide compositions. Also provided by the present invention are organisms that have enhanced, reduced, or modified expression of the gene(s) corresponding to the polynucleotide sequences disclosed herein.
  • Processes are also provided for producing a protein, which comprise:
  • the protein produced according to such methods is also provided by the present invention.
  • Preferred embodiments include those in which the protein produced by such process is a mature form of the protein.
  • Protein compositions of the present invention may further comprise a pharmaceutically acceptable carrier.
  • Compositions comprising an antibody which specifically reacts with such protein are also provided by the present invention.
  • Methods are also provided for preventing, treating or ameliorating a medical condition which comprises administering to a mammalian subject a therapeutically effective amount of a composition comprising a protein of the present invention and a pharmaceutically acceptable carrier.
  • FIGS. IA and IB are schematic representations of the pED6 and pNOTs vectors, respectively, used for deposit of clones disclosed herein.
  • nucleotide and amino acid sequences are reported below for each clone and protein disclosed in the present application.
  • the nucleotide sequence of each clone can readily be determined by sequencing of the deposited clone in accordance with known methods. The predicted amino acid sequence (both full-length and mature) can then be determined from such nucleotide sequence.
  • the amino acid sequence of the protein encoded by a particular clone can also be determined by expression of the clone in a suitable host cell, collecting the protein and determining its sequence. For each disclosed protein applicants have identified what they have determined to be the reading frame best identifiable with sequence information available at the time of filing.
  • a "secreted” protein is one which, when expressed in a suitable host cell, is transported across or through a membrane, including transport as a result of signal sequences in its amino acid sequence.
  • "Secreted” proteins include without limitation proteins secreted wholly (e.g., soluble proteins) or partially (e.g. , receptors) from the cell in which they are expressed.
  • “Secreted” proteins also include without limitation proteins which are transported across the membrane of the endoplasmic reticulum.
  • ax318 3 A polynucleotide of the present invention has been identified as clone "ax318_3 "• ax318_3 was isolated from a human adult testes cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein.
  • ax318_3 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "ax318_3 protein").
  • nucleotide sequence of ax318_3 as presently determined is reported in SEQ ID NO:l. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the ax318_3 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:2.
  • the EcoRI/NotI restriction fragment obtainable from the deposit containing clone ax318_3 should be approximately 1200 bp.
  • ax318_3 demonstrated at least some similarity with sequences identified as AA255872 (zsl9a01.sl NCI_CGAP_GCB1 Homo sapiens cDNA clone), AA625610 (zv89h04.sl Soares NhHMPu SI Homo sapiens cDNA clone 766999 3' similar to WP:K10B2.1 CE02008 TRANSDUCIN BETA CHAIN), D86043 (Human mRNA for SHPS-1, complete eds), H23217 (ym52f03.sl Homo sapiens cDNA clone 518803'), U06701
  • SHPS-1 is a membrane glycoprotein that binds the SH2-domain-containing protein tyrosine phosphatase SHP-2 in response to mitogens and cell adhesion (Fujioka et al, 1996, Mol. Cell. Biol.
  • ax318_3 proteins and each similar protein or peptide may share at least some activity.
  • the TopPredll computer program predicts a putative transmembrane domain within the ax318_3 protein sequence, centered around amino acid 28 of SEQ ID NO:2; amino acids 15 to 27 of SEQ ID NO:2 are also a possible leader /signal sequence, with the predicted mature amino acid sequence beginning at amino acid 28.
  • bgl40_l A polynucleotide of the present invention has been identified as clone "bgl40_l".
  • bgl40_l was isolated from a human adult brain cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein.
  • bgl40_l is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "bgl40_l protein").
  • nucleotide sequence of bgl40_l as presently determined is reported in SEQ ID NO:3. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the bgl40_l protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:4. Another possible bgl40_l reading frame and predicted amino acid sequence, encoded by base pairs 641 to 1648 of SEQ ID NO:3, is reported in SEQ ID NO:31.
  • the EcoRI/NotI restriction fragment obtainable from the deposit containing clone bgl40_l should be approximately 2900 bp.
  • bgl40_l demonstrated at least some similarity with sequences identified as AA075629 (zm89a04.sl Stratagene ovarian cancer (#937219) Homo sapiens cDNA clone 545070 3'), AA113989 (zn27hl2.rl Stratagene neuroepithelium NT2RAMI 937234 Homo sapiens cDNA clone 548711 5'), N77135 (yz85hll.rl Homo sapiens cDNA clone 289893 5'), R11701 (yf49d02.rl Homo sapiens cDNA clone 25600 5'), R13178 (yf73e04.rl Homo sapiens cDNA clone 27855 5'), ar ⁇ d Y
  • the predicted amino acid sequence disclosed herein for bgl40_l was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol.
  • the predicted bgl40_l protein demonstrated at least some similarity to sequences identified as AF038969 (general transcription factor 2-1 [Homo sapiens]), U77948 (Bruton's tyrosine kinase-associated protein-135; BAP-135 [Homo sapiens]), and Y14946 (SPIN protein [Homo sapiens]). Based upon sequence similarity, bgl40_l proteins and each similar protein or peptide may share at least some activity.
  • a polynucleotide of the present invention has been identified as clone "bg465 . _2".
  • bg465_2 was isolated from a human adult brain cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein.
  • bg465_2 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "bg465_2 protein").
  • nucleotide sequence of bg465_2 as presently determined is reported in SEQ ID NO:5. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the bg465_2 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:6.
  • the EcoRI/NotI restriction fragment obtainable from the deposit containing clone bg465_2 should be approximately 2500 bp.
  • nucleotide sequence disclosed herein for bg465_2 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
  • bg465_2 demonstrated at least some similarity with sequences identified as AA133150 (zm25b07.rl Stratagene pancreas (#937208) Homo sapiens cDNA clone 526645 5' similar to WP:F07F6.4 CE01896 ZINC FINGER PROTEIN), AA144842 (mr69h01.rl Stratagene mouse testis (#937308) Mus musculus cDNA clone 602737 5'), AA484561 (nf06al2.sl NCI_CGAP_Lil Homo sapiens cDNA clone), D16939 (Human HepG2 3' region cDNA, clone hmd5d06), L24125 (Saccharomyces cerevisiae zinc finger protein (GCS1) gene, complete eds), R18422 (yg02f06.rl Homo sapiens cDNA clone 30950 5'), T325
  • the predicted amino acid sequence disclosed herein for bg465_2 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol.
  • the predicted bg465_2 protein demonstrated at least some similarity to sequences identified as U23486 (similar to S. cerevisiae zinc finger protein GCS1 (SP GCS1_YEAST) [Caenorhabditis elegans]). Based upon sequence similarity, bg465_2 proteins and each similar protein or peptide may share at least some activity.
  • a polynucleotide of the present invention has been identified as clone "bk291_3".
  • bk291_3 was isolated from a human adult retina cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein.
  • bk291_3 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "bk291_3 protein").
  • nucleotide sequence of bk291_3 as presently determined is reported in SEQ ID NO:7. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the bk291_3 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:8.
  • Amino acids 44 to 56 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 57, or are a transmembrane domain.
  • the EcoRI/NotI restriction fragment obtainable from the deposit containing clone bk291_3 should be approximately 1500 bp.
  • bk291_3 The nucleotide sequence disclosed herein for bk291_3 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. bk291_3 demonstrated at least some similarity with sequences identified as AA135915 (zll9f04.rl Soares pregnant uterus NbHPU Homo sapiens cDNA clone 502399 5'), AA156738 (zll9f04.sl Soares pregnant uterus NbHPU Homo sapiens cDNA clone 502399 3' similar to WP.T05E11.5 CE06364 YEAST YKK0 LIKE), AF017785 (Mus musculus MHC Class I H13a minor histocompatibility peptide (H13) mRNA, partial eds), N40134 (yw73al2.rl Homo sapiens cDNA clone 257854 5'
  • the predicted amino acid sequence disclosed herein for bk291_3 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol.
  • the predicted bk291_3 protein demonstrated at least some similarity to sequences identified as X71133 (YKL450 [Saccharomyces cerevisiae]), Z28100 (ORF YKLlOOc [Saccharomyces cerevisiae]), and Z68751 (T05E11.5 [Caenorhabditis elegans]). Based upon sequence similarity, bk291_3 proteins and each similar protein or peptide may share at least some activity.
  • the TopPredll computer program predicts six additional potential transmembrane domains within the bk291_3 protein sequence, centered around amino acids 70, 125, 170, 220, 260, and 280 of SEQ ID NO:8.
  • a polynucleotide of the present invention has been identified as clone "bp537_4".
  • bp537_4 was isolated from a human fetal kidney cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein.
  • bp537_4 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "bp537_4 protein").
  • nucleotide sequence of bp537_4 as presently determined is reported in SEQ ID NO:9. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the bp537_4 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:10.
  • the EcoRI/NotI restriction fragment obtainable from the deposit containing clone bp537_4 should be approximately 1500 bp.
  • bp537_4 The nucleotide sequence disclosed herein for bp537_4 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. bp537_4 demonstrated at least some similarity with sequences identified as AA007576 (zh99b06.rl Soares fetal liver spleen INFLS SI Homo sapiens cDNA clone 429395 5'), AA287563 (zs52g02.rl NCI_CGAP_GCB1 Homo sapiens cDNA 5'), R09093 (yf21h09.rl Homo sapiens cDNA clone 127553 5'), and T33088 (EST56631 Homo sapiens cDNA 5' end similar to None).
  • the predicted amino acid sequence disclosed herein for bp537_4 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol.
  • the predicted bp537_4 protein demonstrated at least some similarity to sequences identified as U34998 (Rad9 [Coprinus cinereus]). Based upon sequence similarity, bp537_4 proteins and each similar protein or peptide may share at least some activity.
  • cs431_2 A polynucleotide of the present invention has been identified as clone "cs431_2".
  • cs431_2 was isolated from a human fetal brain cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein.
  • cs431_2 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "cs431_2 protein").
  • nucleotide sequence of cs431_2 as presently determined is reported in SEQ ID NO:ll. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the cs431_2 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:12.
  • the EcoRI/NotI restriction fragment obtainable from the deposit containing clone cs431_2 should be approximately 2300 bp.
  • the nucleotide sequence disclosed herein for cs431_2 was searched against the
  • cs431_2 demonstrated at least some similarity with sequences identified as AA021033 (ze67hl2.sl Soares retina N2b4HR Homo sapiens cDNA clone 364103 3' similar to contains Alu repetitive element), H29349 (ym32c02.rl Homo sapiens cDNA clone 49839 5' similar to SP:KYES_XIPHE P27447 PROTO-ONCOGENE
  • TYROSINE-PROTELN KINASE YES L14577 (Homo sapiens cystathionine beta-synthase (CBS) mRNA, complete eds), Q87430 (Human cystathionine beta-synthase cDNA), and X92659 (H.sapiens intron 4 from p53 gene).
  • CBS Homo sapiens cystathionine beta-synthase
  • Q87430 Human cystathionine beta-synthase cDNA
  • X92659 H.sapiens intron 4 from p53 gene.
  • the predicted amino acid sequence disclosed herein for cs431_2 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol.
  • the predicted cs431_2 protein demonstrated at least some similarity to sequences identified as L19501 (cystathionine beta-synthase [Homo sapiens]), R71376 (Human cystathionine beta-synthase), U61167 (SH3 domain-containing protein SH3P18 [Homo sapiens]), and X82166 (cystathionine beta-synthase [Homo sapiens]). Based upon sequence similarity, cs431_2 proteins and each similar protein or peptide may share at least some activity.
  • the TopPredll computer program predicts a potential transmembrane domain within the cs431_2 protein sequence centered around amino acid 30 of SEQ ID NO:12. The nucleotide sequence of cs431_2 indicates that it may contain an Alu repetitive element.
  • cw976_l A polynucleotide of the present invention has been identified as clone "cw976_l".
  • cw976_l was isolated from a human fetal brain cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein.
  • cw976_l is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "cw976_l protein").
  • nucleotide sequence of cw976_l as presently determined is reported in SEQ ID NO:13. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the cw976_l protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:14.
  • Amino acids 423 to 435 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 436, or are a transmembrane domain.
  • the EcoRI/NotI restriction fragment obtainable from the deposit containing clone cw976_l should be approximately 3300 bp.
  • cw976_l demonstrated at least some similarity with sequences identified as AA280747 (zs96a09.rl NCI_CGAP_GCB1 Homo sapiens cDNA clone IMAGE:711448 5'), AA323946 (EST26798 Cerebellum II Homo sapiens cDNA 5' end), R13665 (yf60g06.rl Homo sapiens cDNA clone 26764 5'), R16892 (yf44d06.s2 Homo sapiens cDNA clone 1297073'), R85177 (yo43d07.rl Homo sapiens cDNA clone 1806855'), and R89648 (ym97c02.rl Homo sapiens cDNA clone 166850 5
  • cw976_l proteins and each similar protein or peptide may share at least some activity.
  • the TopPredll computer program predicts a potential transmembrane domain within the cw976_l protein sequence at the amino terminus of SEQ ID NO:14. Clone "cwl233 3"
  • a polynucleotide of the present invention has been identified as clone "cwl233_3 " ⁇ cwl233_3 was isolated from a human fetal brain cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein.
  • cwl233_3 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "cwl233_3 protein").
  • nucleotide sequence of cwl233_3 as presently determined is reported in SEQ ID NO:15. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the cwl233_3 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:16.
  • the EcoRI/NotI restriction fragment obtainable from the deposit containing clone cwl233_3 should be approximately 3000 bp.
  • the nucleotide sequence disclosed herein for cwl233_3 was searched against the
  • cwl233_3 demonstrated at least some similarity with sequences identified as R61600 (yhl6f08.rl Homo sapiens cDNA clone 378075'), R83929 (ypO ⁇ hll.sl Homo sapiens cDNA clone 186693 3' similar to contains Alu repetitive element;contains TARl repetitive element), R87877 (yo45h05.rl Homo sapiens cDNA clone 180921 5'), U14568 (***ALU WARNING Human Alu-Sb subfamily consensus sequence).
  • the predicted amino acid sequence disclosed herein for cwl233_3 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol.
  • the predicted cwl233_3 protein demonstrated at least some similarity to sequences identified as AB002317 (KIAA0319 [Homo sapiens]). Based upon sequence similarity, cwl233_3 proteins and each similar protein or peptide may share at least some activity.
  • the nucleotide sequence of cwl233_3 indicates that it may contain an Alu repetitive element.
  • dgl_l A polynucleotide of the present invention has been identified as clone "dgl_l”.
  • dgl_l was isolated from a human adult placenta cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein.
  • dgl_l is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "dgl_l protein").
  • nucleotide sequence of dgl_l as presently determined is reported in SEQ ID NO: 17. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the dgl_l protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:18.
  • the EcoRI/NotI restriction fragment obtainable from the deposit containing clone dgl_l should be approximately 1900 bp.
  • the nucleotide sequence disclosed herein for dgl_l was searched against the
  • dgl_l demonstrated at least some similarity with sequences identified as AA731281 (nw57f05.sl NCI_CGAP_GCB1 Homo sapiens cDNA clone LMAGE 1250721 similar to gb Y00345_cdsl POLYADENYLATE-BINDING PROTEIN (HUMAN)).
  • the predicted amino acid sequence disclosed herein for dgl_l was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol.
  • the predicted dgl_l protein demonstrated at least some similarity to sequences identified as M81878 (hyaluronidase [Clostridium perfringens]) and U28742 (similar to hyaluronoglucosaminidase (SPNAGH_CLOPE, P26831) [Caenorhabditis elegans]).
  • Hyaluronoglucosaminidase is a secreted protein found, for example, in bee venom.
  • dgl_l proteins and each similar protein or peptide may share at least some activity.
  • the TopPredll computer program predicts a potential transmembrane domain within the dgl_l protein sequence centered around amino acid 150 of SEQ ID NO:18.
  • ep234_l A polynucleotide of the present invention has been identified as clone "ep234_l".
  • ep234_l was isolated from a human adult placenta cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein.
  • ep234_l is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "ep234_l protein").
  • nucleotide sequence of ep234_l as presently determined is reported in SEQ ID NO:19. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the ep234_l protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:20. Amino acids 59 to 71 are a predicted leader /signal sequence, with the predicted mature amino acid sequence beginning at amino acid 72, or are a transmembrane domain.
  • the EcoRI/NotI restriction fragment obtainable from the deposit containing clone ep234_l should be approximately 2500 bp.
  • ep234_l demonstrated at least some similarity with sequences identified as AA044714 (zf54d09.rl Soares retina N2b4HR Homo sapiens cDNA clone 3807535'), AA454840 (zx79d09.sl Soares ovary tumor NbHOT Homo sapiens cDNA clone 809969 3'), AA470137 (zulleOl.sl Soares testis NHT Homo sapiens cDNA clone 731544 3'), H24852 (yl42cll.rl Homo sapiens cDNA clone 160916 5'), N64648 (yz87b06.sl Homo sapiens cDNA clone 290003 3"), R45788
  • ep234_l proteins and each similar protein or peptide may share at least some activity.
  • the TopPredll computer program predicts at least two potential transmembrane domains within the ep234_l protein sequence of SEQ ID NO:20.
  • Clones ax318_3, bgl40_l, bg465_2, bk291_3, bp537_4, cs431_2, cw976_l, cwl233_3, dgl_l, and ep234_l were deposited on March 11, 1997 with the American Type Culture Collection as an original deposit under the Budapest Treaty and were given the accession number ATCC 98353, from which each clone comprising a particular polynucleotide is obtainable. All restrictions on the availability to the public of the deposited material will be irrevocably removed upon the granting of the patent, except for the requirements specified in 37 C.F.R. ⁇ 1.808(b).
  • Each clone has been transfected into separate bacterial cells (E. coli) in this composite deposit. Each clone can be removed from the vector in which it was deposited by performing an EcoRI/NotI digestion (5' site, EcoRI; 3' site, NotI) to produce the appropriate fragment for such clone. Each clone was deposited in either the pED6 or pNOTs vector depicted in Fig. 1.
  • the pED6dpc2 vector (“pED6" was derived from pED ⁇ dpcl by insertion of a new polylinker to facilitate cDNA cloning (Kaufman et al., 1991, Nucleic Acids Res.
  • the pNOTs vector was derived from pMT2 (Kaufman et al, 1989, Mol. Cell. Biol. 9: 946-958) by deletion of the DHFR sequences, insertion of a new polylinker, and insertion of the M13 origin of replication in the Clal site.
  • the deposited clone can become "flipped" (i.e., in the reverse orientation) in the deposited isolate.
  • the cDNA insert can still be isolated by digestion with EcoRI and Notl. However, NotI will then produce the 5' site and EcoRI will produce the 3' site for placement of the cDNA in proper orientation for expression in a suitable vector.
  • the cDNA may also be expressed from the vectors in which they were deposited.
  • Bacterial cells containing a particular clone can be obtained from the composite deposit as follows: An oligonucleotide probe or probes should be designed to the sequence that is known for that particular clone. This sequence can be derived from the sequences provided herein, or from a combination of those sequences. The sequence of the oligonucleotide probe that was used to isolate each full-length clone is identified below, and should be most reliable in isolating the clone of interest.
  • the design of the oligonucleotide probe should preferably follow these parameters:
  • the oligonucleotide should preferably be labeled with g- 32 P ATP (specific activity 6000 Ci/mmole) and T4 polynucleotide kinase using commonly employed techniques for labeling oligonucleotides. Other labeling techniques can also be used. Unincorporated label should preferably be removed by gel filtration chromatography or other established methods. The amount of radioactivity incorporated into the probe should be quantitated by measurement in a scintillation counter. Preferably, specific activity of the resulting probe should be approximately 4e+6 dpm/pmole.
  • the bacterial culture containing the pool of full-length clones should preferably be thawed and 100 ⁇ l of the stock used to inoculate a sterile culture flask containing 25 ml of sterile L-broth containing ampicillin at 100 ⁇ g/ml.
  • the culture should preferably be grown to saturation at 37°C, and the saturated culture should preferably be diluted in fresh L-broth.
  • Aliquots of these dilutions should preferably be plated to determine the dilution and volume which will yield approximately 5000 distinct and well-separated colonies on solid bacteriological media containing L-broth containing ampicillin at 100 ⁇ g/ml and agar at 1.5% in a 150 mm pe ri dish when grown overnight at 37°C. Other known methods of obtaining distinct, well-separated colonies can also be employed.
  • Standard colony hybridization procedures should then be used to transfer the colonies to nitrocellulose filters and lyse, denature and bake them.
  • the filter is then preferably incubated at 65°C for 1 hour with gentle agitation in 6X SSC (20X stock is 175.3 g NaCl/liter, 88.2 g Na citrate /liter, adjusted to pH 7.0 with NaOH) containing 0.5% SDS, 100 ⁇ g/ml of yeast RNA, and 10 mM EDTA (approximately
  • the probe is then added to the hybridization mix at a concentration greater than or equal to le+6 dpm/mL.
  • the filter is then preferably incubated at 65°C with gentle agitation overnight.
  • the filter is then preferably washed in 500 mL of 2X SSC/0.5% SDS at room temperature without agitation, preferably followed by 500 mL of 2X SSC/0.1% SDS at room temperature with gentle shaking for 15 minutes.
  • a third wash with 0.1X SSC/0.5% SDS at 65°C for 30 minutes to 1 hour is optional.
  • the filter is then preferably dried and subjected to autoradiography for sufficient time to visualize the positives on the X-ray film.
  • Other known hybridization methods can also be employed.
  • the positive colonies are picked, grown in culture, and plasmid DNA isolated using standard procedures.
  • the clones can then be verified by restriction analysis, hybridization analysis, or DNA sequencing.
  • Fragments of the proteins of the present invention which are capable of exhibiting biological activity are also encompassed by the present invention.
  • Fragments of the protein may be in linear form or they may be cyclized using known methods, for example, as described in H.U. Saragovi, et al, Bio /Technology 10, 773-778 (1992) and in R.S. McDowell, et al, J. Amer. Chem. Soc. 114, 9245-9253 (1992), both of which are incorporated herein by reference.
  • Such fragments may be fused to carrier molecules such as immunoglobulins for many purposes, including increasing the valency of protein binding sites.
  • fragments of the protein may be fused through "linker" sequences to the Fc portion of an immunoglobulin.
  • a fusion could be to the Fc portion of an IgG molecule.
  • Other immunoglobulin isotypes may also be used to generate such fusions.
  • a protein - IgM fusion would generate a decavalent form of the protein of the invention.
  • the present invention also provides both full-length and mature forms of the disclosed proteins.
  • the full-length form of the such proteins is identified in the sequence listing by translation of the nucleotide sequence of each disclosed clone.
  • the mature form of such protein may be obtained by expression of the disclosed full-length polynucleotide (preferably those deposited with ATCC) in a suitable mammalian cell or other host cell.
  • sequence of the mature form of the protein may also be determinable from the amino acid sequence of the full-length form.
  • the present invention also provides genes corresponding to the polynucleotide sequences disclosed herein.
  • “Corresponding genes” are the regions of the genome that are transcribed to produce the mRNAs from which cDNA polynucleotide sequences are derived and may include contiguous regions of the genome necessary for the regulated expression of such genes. Corresponding genes may therefore include but are not limited to coding sequences, 5' and 3' untranslated regions, alternatively spliced exons, introns, promoters, enhancers, and silencer or suppressor elements. The corresponding genes can be isolated in accordance with known methods using the sequence information disclosed herein.
  • Such methods include the preparation of probes or primers from the disclosed sequence information for identification and /or amplification of genes in appropriate genomic libraries or other sources of genomic materials.
  • An "isolated gene” is a gene that has been separated from the adjacent coding sequences, if any, present in the genome of the organism from which the gene was isolated.
  • Organisms that have enhanced, reduced, or modified expression of the gene(s) corresponding to the polynucleotide sequences disclosed herein are provided.
  • the desired change in gene expression can be achieved through the use of antisense polynucleotides or ribozymes that bind and /or cleave the mRNA transcribed from the gene (Albert and Morris, 1994, Trends Pharmacol. Sci. 15(7): 250-254; Lavarosky et al., 1997, Biochem. Mol. Med. 62(1): 11-22; and Hampel, 1998, Prog. Nucleic Acid Res. Mol. Biol. 58: 1- 39; all of which are incorporated by reference herein).
  • Transgenic animals that have multiple copies of the gene(s) corresponding to the polynucleotide sequences disclosed herein, preferably produced by transformation of cells with genetic constructs that are stably maintained within the transformed cells and their progeny, are provided.
  • organisms are provided in which the gene(s) corresponding to the polynucleotide sequences disclosed herein have been partially or completely inactivated, through insertion of extraneous sequences into the corresponding gene(s) or through deletion of all or part of the corresponding gene(s).
  • Partial or complete gene inactivation can be accomplished through insertion, preferably followed by imprecise excision, of transposable elements (Plasterk, 1992, Bioessays 14(9): 629-633; Zwaal et al, 1993, Proc. Natl.
  • the present invention also provides for soluble forms of such protein.
  • the intracellular and transmembrane domains of the protein are deleted such that the protein is fully secreted from the cell in which it is expressed.
  • the intracellular and transmembrane domains of proteins of the invention can be identified in accordance with known techniques for determination of such domains from sequence information.
  • Proteins and protein fragments of the present invention include proteins with amino acid sequence lengths that are at least 25%(more preferably at least 50%, and most preferably at least 75%) of the length of a disclosed protein and have at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% or 95% identity) with that disclosed protein, where sequence identity is determined by comparing the amino acid sequences of the proteins when aligned so as to maximize overlap and identity while minimizing sequence gaps.
  • proteins and protein fragments that contain a segment preferably comprising 8 or more (more preferably 20 or more, most preferably 30 or more) contiguous amino acids that shares at least 75% sequence identity (more preferably, at least 85% identity; most preferably at least 95% identity) with any such segment of any of the disclosed proteins.
  • Species homologs of the disclosed polynucleotides and proteins are also provided by the present invention.
  • a "species homologue" is a protein or polynucleotide with a different species of origin from that of a given protein or polynucleotide, but with significant sequence similarity to the given protein or polynucleotide, as determined by those of skill in the art.
  • Species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from the desired species.
  • species homologs are those isolated from mammalian species.
  • the invention also encompasses allelic variants of the disclosed polynucleotides or proteins; that is, naturally-occurring alternative forms of the isolated polynucleotide which also encode proteins which are identical, homologous, or related to that encoded by the polynucleotides .
  • the invention also includes polynucleotides with sequences complementary to those of the polynucleotides disclosed herein.
  • the present invention also includes polynucleotides capable of hybridizing under reduced stringency conditions, more preferably stringent conditions, and most preferably highly stringent conditions, to polynucleotides described herein. Examples of stringency conditions are shown in the table below: highly stringent conditions are those that are at least as stringent as, for example, conditions A-F; stringent conditions are at least as stringent as, for example, conditions G-L; and reduced stringency conditions are at least as stringent as, for example, conditions M-R.
  • the hybrid length is that anticipated for the hybridized region(s) of the hybridizing polynucleotides.
  • the hybrid length is assumed to be that of the hybridizing polynucleotide.
  • the hybrid length can be determined by aligning the sequences of the polynucleotides and identifying the region or regions of optimal sequence complementarity.
  • SSPE (lxSSPE is 0 15M NaCl, lOmM NaH 2 P0 4 , and 1.25mM EDTA, pH 7.4) can be substituted for SSC (lxSSC is 0.15M NaCl and 15mM sodium citrate) in the hybridization and wash buffers, washes are performed for 15 minutes after hybridization is complete
  • T m melting temperature
  • each such hybridizing polynucleotide has a length that is at least 25%(more preferably at least 50%, and most preferably at least 75%) of the length of the polynucleotide of the present invention to which it hybridizes, and has at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% or 95% identity) with the polynucleotide of the present invention to which it hybridizes, where sequence identity is determined by comparing the sequences of the hybridizing polynucleotides when aligned so as to maximize overlap and identity while minimizing sequence gaps.
  • the isolated polynucleotide of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al, Nucleic Acids Res. 19, 4485-4490 (1991), in order to produce the protein recombinantly.
  • an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al, Nucleic Acids Res. 19, 4485-4490 (1991)
  • Many suitable expression control sequences are known in the art. General methods of expressing recombinant proteins are also known and are exemplified in R. Kaufman, Methods in Enzymology 185, 537-566 (1990).
  • operably linked means that the isolated polynucleotide of the invention and an expression control sequence are situated within a vector or cell in such a way that the protein is expressed by a host cell which has been transformed (transfected) with the ligated polynucleotide /expression control sequence.
  • Mammalian host cells include, for example, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.
  • monkey COS cells Chinese Hamster Ovary (CHO) cells
  • human kidney 293 cells human epidermal A431 cells
  • human Colo205 cells human Colo205 cells
  • CV-1 cells other transformed primate cell lines
  • normal diploid cells cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.
  • yeast eukaryotes
  • prokaryotes such as bacteria.
  • yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous proteins.
  • Potentially suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous proteins. If the protein is made in yeast or bacteria, it may be necessary to modify the protein produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain the functional protein. Such covalent attachments may be accomplished using known chemical or enzymatic methods.
  • the protein may also be produced by operably linking the isolated polynucleotide of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system.
  • suitable control sequences in one or more insect expression vectors, and employing an insect expression system.
  • Materials and methods for baculovirus /insect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego, California, U.S.A. (the MaxBac® kit), and such methods are well known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987), incorporated herein by reference.
  • an insect cell capable of expressing a polynucleotide of the present invention is "transformed.”
  • the protein of the invention may be prepared by culturing transformed host cells under culture conditions suitable to express the recombinant protein.
  • the resulting expressed protein may then be purified from such culture (i.e., from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography.
  • the purification of the protein may also include an affinity column containing agents which will bind to the protein; one or more column steps over such affinity resins as concanavalin A-agarose, heparin-toyopearl® or Cibacrom blue 3GA Sepharose®; one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography.
  • the protein of the invention may also be expressed in a form which will facilitate purification.
  • it may be expressed as a fusion protein, such as those of maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX). Kits for expression and purification of such fusion proteins are commercially available from New England BioLab (Beverly, MA), Pharmacia (Piscataway, NJ) and InVitrogen, respectively.
  • MBP maltose binding protein
  • GST glutathione-S-transferase
  • TRX thioredoxin
  • Kits for expression and purification of such fusion proteins are commercially available from New England BioLab (Beverly, MA), Pharmacia (Piscataway, NJ) and InVitrogen, respectively.
  • the protein can also be tagged with an epitope and subsequently purified by using a specific antibody directed to such epitope.
  • One such epitope (“Flag") is commercially available from Kod
  • RP- HPLC reverse-phase high performance liquid chromatography
  • hydrophobic RP-HPLC media e.g., silica gel having pendant methyl or other aliphatic groups
  • Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant protein.
  • the protein thus purified is substantially free of other mammalian proteins and is defined in accordance with the present invention as an "isolated protein.”
  • the protein of the invention may also be expressed as a product of transgenic animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells containing a nucleotide sequence encoding the protein.
  • the protein may also be produced by known conventional chemical synthesis. Methods for constructing the proteins of the present invention by synthetic means are known to those skilled in the art.
  • the synthetically-constructed protein sequences by virtue of sharing primary, secondary or tertiary structural and /or conformational characteristics with proteins may possess biological properties in common therewith, including protein activity. Thus, they may be employed as biologically active or immunological substitutes for natural, purified proteins in screening of therapeutic compounds and in immunological processes for the development of antibodies.
  • the proteins provided herein also include proteins characterized by amino acid sequences similar to those of purified proteins but into which modification are naturally provided or deliberately engineered.
  • modifications in the peptide or DNA sequences can be made by those skilled in the art using known techniques.
  • Modifications of interest in the protein sequences may include the alteration, substitution, replacement, insertion or deletion of a selected amino acid residue in the coding sequence.
  • one or more of the cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule.
  • Techniques for such alteration, substitution, replacement, insertion or deletion are well known to those skilled in the art (see, e.g., U.S. Patent No.4,518,584).
  • such alteration, substitution, replacement, insertion or deletion retains the desired activity of the protein.
  • polynucleotides and proteins of the present invention are expected to exhibit one or more of the uses or biological activities (including those associated with assays cited herein) identified below.
  • Uses or activities described for proteins of the present invention may be provided by administration or use of such proteins or by administration or use of polynucleotides encoding such proteins (such as, for example, in gene therapies or vectors suitable for introduction of DNA).
  • the polynucleotides provided by the present invention can be used by the research community for various purposes.
  • the polynucleotides can be used to express recombinant protein for analysis, characterization or therapeutic use; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in disease states); as molecular weight markers on Southern gels; as chromosome markers or tags (when labeled) to identify chromosomes or to map related gene positions; to compare with endogenous DNA sequences in patients to identify potential genetic disorders; as probes to hybridize and thus discover novel, related DNA sequences; as a source of information to derive PCR primers for genetic fingerprinting; as a probe to "subtract-out" known sequences in the process of discovering other novel polynucleotides; for selecting and making oligomers for attachment to a "gene chip” or other support, including for examination of expression patterns; to raise anti-protein antibodies using DNA immunization techniques;
  • the polynucleotide encodes a protein which binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction)
  • the polynucleotide can also be used in interaction trap assays (such as, for example, that described in Gyuris et al., Cell 75:791-803 (1993)) to identify polynucleotides encoding the other protein with which binding occurs or to identify inhibitors of the binding interaction.
  • the proteins provided by the present invention can similarly be used in assay to determine biological activity, including in a panel of multiple proteins for high- throughput screening; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its receptor) in biological fluids; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state); and, of course, to isolate correlative receptors or ligands.
  • the protein binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction)
  • the protein can be used to identify the other protein with which binding occurs or to identify inhibitors of the binding interaction. Proteins involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction.
  • Polynucleotides and proteins of the present invention can also be used as nutritional sources or supplements. Such uses include without limitation use as a protein or amino acid supplement, use as a carbon source, use as a nitrogen source and use as a source of carbohydrate.
  • the protein or polynucleotide of the invention can be added to the feed of a particular organism or can be administered as a separate solid or liquid preparation, such as in the form of powder, pills, solutions, suspensions or capsules.
  • the protein or polynucleotide of the invention can be added to the medium in or on which the microorganism is cultured. Cytokine and Cell Proliferation /Differentiation Activity
  • a protein of the present invention may exhibit cytokine, cell proliferation (either inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may induce production of other cytokines in certain cell populations.
  • cytokine cytokine
  • cell proliferation either inducing or inhibiting
  • cell differentiation either inducing or inhibiting
  • the activity of a protein of the present invention is evidenced by any one of a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+ (preB M+), 2E8, RB5, DAI, 123, T1165, HT2, CTLL2, TF-1, Mo7e and CMK.
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Assays for T-cell or thymocyte proliferation include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley- Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500, 1986; BertagnoUi et al., J. Immunol.
  • Assays for cytokine production and/or proliferation of spleen cells, lymph node cells or thymocytes include, without limitation, those described in: Polyclonal T cell stimulation, Kruisbeek, A.M. and Shevach, E.M. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and
  • Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include, without limitation, those described in: Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L.S. and Lipsky, P.E. In Current
  • Assays for T-cell clone responses to antigens include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their cellular receptors; Chapter 7, Immunologic studies in Humans); Weinberger et al., Proc. Natl. Acad. Sci.
  • a protein of the present invention may also exhibit immune stimulating or immune suppressing activity, including without limitation the activities for which assays are described herein.
  • a protein may be useful in the treatment of various immune deficiencies and disorders (including severe combined immunodeficiency (SOD)), e.g., in regulating (up or down) growth and proliferation of T and/or B lymphocytes, as well as effecting the cytolytic activity of NK cells and other cell populations.
  • SOD severe combined immunodeficiency
  • These immune deficiencies may be genetic or be caused by viral (e.g., HIV) as well as bacterial or fungal infections, or may result from autoimmune disorders.
  • infectious diseases causes by viral, bacterial, fungal or other infection may be treatable using a protein of the present invention, including infections by HIV, hepatitis viruses, herpesviruses, mycobacteria, Leishmania spp., malaria spp. and various fungal infections such as candidiasis.
  • a protein of the present invention may also be useful where a boost to the immune system generally may be desirable, i.e., in the treatment of cancer.
  • Autoimmune disorders which may be treated using a protein of the present invention include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye disease.
  • a protein of the present invention may also to be useful in the treatment of allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems.
  • Other conditions, in which immune suppression is desired may also be treatable using a protein of the present invention.
  • T cells may be inhibited by suppressing T cell responses or by inducing specific tolerance in T cells, or both.
  • Immunosuppression of T cell responses is generally an active, non-antigen-specific, process which requires continuous exposure of the T cells to the suppressive agent.
  • Tolerance which involves inducing non-responsiveness or anergy in T cells, is distinguishable from immunosuppression in that it is generally antigen-specific and persists after exposure to the tolerizing agent has ceased. Operationally, tolerance can be demonstrated by the lack of a T cell response upon reexposure to specific antigen in the absence of the tolerizing agent.
  • Down regulating or preventing one or more antigen functions (including without limitation B lymphocyte antigen functions (such as , for example, B7)), e.g., preventing high level lymphokine synthesis by activated T cells, will be useful in situations of tissue, skin and organ transplantation and in graft-versus-host disease (GVHD).
  • B lymphocyte antigen functions such as , for example, B7
  • GVHD graft-versus-host disease
  • blockage of T cell function should result in reduced tissue destruction in tissue transplantation.
  • rejection of the transplant is initiated through its recognition as foreign by T cells, followed by an immune reaction that destroys the transplant.
  • a molecule which inhibits or blocks interaction of a B7 lymphocyte antigen with its natural ligand(s) on immune cells such as a soluble, monomeric form of a peptide having B7-2 activity alone or in conjunction with a monomeric form of a peptide having an activity of another B lymphocyte antigen (e.g., B7- 1, B7-3) or blocking antibody
  • B7- 1, B7-3 or blocking antibody e.g., B7- 1, B7-3 or blocking antibody
  • Blocking B lymphocyte antigen function in this matter prevents cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant.
  • the lack of costimulation may also be sufficient to anergize the T cells, thereby inducing tolerance in a subject.
  • Induction of long-term tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of these blocking reagents.
  • To achieve sufficient immunosuppression or tolerance in a subject it may also be necessary to block the function of a combination of B lymphocyte antigens.
  • the efficacy of particular blocking reagents in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans.
  • Examples of appropriate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in vivo as described in Lenschow et al, Science 257:789-792 (1992) and Turka et al, Proc. Natl. Acad. Sci USA, 89:11102-11105 (1992).
  • murine models of GVHD see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 846-847) can be used to determine the effect of blocking B lymphocyte antigen function in vivo on the development of that disease.
  • Blocking antigen function may also be therapeutically useful for treating autoimmune diseases.
  • Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive against self tissue and which promote the production of cytokines and autoantibodies involved in the pathology of the diseases.
  • Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms.
  • Administration of reagents which block costimulation of T cells by disrupting recepto ⁇ ligand interactions of B lymphocyte antigens can be used to inhibit T cell activation and prevent production of autoantibodies or T cell-derived cytokines which may be involved in the disease process.
  • blocking reagents may induce antigen-specific tolerance of autoreactive T cells which could lead to long-term relief from the disease.
  • the efficacy of blocking reagents in preventing or alleviating autoimmune disorders can be determined using a number of well-characterized animal models of human autoimmune diseases. Examples include murine experimental autoimmune encephalitis, systemic lupus erythmatosis in MK /lprApr mice or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and murine experimental myasthenia gravis (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 840-856).
  • Upregulation of an antigen function (preferably a B lymphocyte antigen function), as a means of up regulating immune responses, may also be useful in therapy. Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response. For example, enhancing an immune response through stimulating B lymphocyte antigen function may be useful in cases of viral infection. In addition, systemic viral diseases such as influenza, the common cold, and encephalitis might be alleviated by the administration of stimulatory forms of B lymphocyte antigens systemically.
  • anti-viral immune responses may be enhanced in an infected patient by removing T cells from the patient, costimulating the T cells in vitro with viral antigen- pulsed APCs either expressing a peptide of the present invention or together with a stimulatory form of a soluble peptide of the present invention and reintroducing the in vitro activated T cells into the patient.
  • Another method of enhancing anti-viral immune responses would be to isolate infected cells from a patient, transfect them with a nucleic acid encoding a protein of the present invention as described herein such that the cells express all or a portion of the protein on their surface, and reintroduce the transfected cells into the patient.
  • the infected cells would now be capable of delivering a costimulatory signal to, and thereby activate, T cells in vivo.
  • up regulation or enhancement of antigen function may be useful in the induction of tumor immunity.
  • Tumor cells e.g., sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, carcinoma
  • a nucleic acid encoding at least one peptide of the present invention can be administered to a subject to overcome tumor-specific tolerance in the subject. If desired, the tumor cell can be transfected to express a combination of peptides.
  • tumor cells obtained from a patient can be transfected ex vivo with an expression vector directing the expression of a peptide having B7-2-like activity alone, or in conjunction with a peptide having B7-l-like activity and /or B7-3-like activity.
  • the transfected tumor cells are returned to the patient to result in expression of the peptides on the surface of the transfected cell.
  • gene therapy techniques can be used to target a tumor cell for transfection in vivo.
  • tumor cells which lack MHC class I or MHC class II molecules, or which fail to reexpress sufficient amounts of MHC class I or MHC class II molecules, can be transfected with nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class I chain protein and ⁇ 2 microglobulin protein or an MHC class II ⁇ chain protein and an MHC class II ⁇ chain protein to thereby express MHC class I or MHC class II proteins on the cell surface.
  • nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class I chain protein and ⁇ 2 microglobulin protein or an MHC class II ⁇ chain protein and an MHC class II ⁇ chain protein to thereby express MHC class I or MHC class II proteins on the cell surface.
  • a gene encoding an antisense construct which blocks expression of an MHC class II associated protein, such as the invariant chain can also be cotransfected with a DNA encoding a peptide having the activity of a B lymphocyte antigen to promote presentation of tumor associated antigens and induce tumor specific immunity.
  • a T cell mediated immune response in a human subject may be sufficient to overcome tumor-specific tolerance in the subject.
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1- 3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl. Acad. Sci. USA 78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J. Immunol. 135:1564-1572, 1985; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al.,
  • T-cell-dependent immunoglobulin responses and isotype switching (which will identify, among others, proteins that modulate T-cell dependent antibody responses and that affect Thl/Th2 profiles) include, without limitation, those described in: Maliszewski, J. Immunol. 144:3028-3033, 1990; and Assays for B cell function: In vitro antibody production, Mond, J.J. and Brunswick, M. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto. 1994.
  • MLR Mixed lymphocyte reaction
  • Dendritic cell-dependent assays (which will identify, among others, proteins expressed by dendritic cells that activate naive T-cells) include, without limitation, those described in: Guery et al., J. Immunol.
  • lymphocyte survival /apoptosis (which will identify, among others, proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis) include, without limitation, those described in: Darzynkiewicz et al., Cytometry 13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca et al., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991; Zacharchuk, Journal of Immunology 145:4037-4045, 1990; Zamai et al., Cytometry 14:891-897, 1993; Gorczyca et al., International Journal of Oncology 1:639-648, 1992.
  • Assays for proteins that influence early steps of T-cell commitment and development include, without limitation, those described in: Antica et al., Blood 84:111-117, 1994; Fine et al., Cellular Immunology 155:111-122, 1994; Galy et al., Blood 85:2770-2778, 1995; Toki et al., Proc. Nat. Acad Sci. USA 88:7548-7551, 1991.
  • a protein of the present invention may be useful in regulation of hematopoiesis and, consequently, in the treatment of myeloid or lymphoid cell deficiencies. Even marginal biological activity in support of colony forming cells or of factor-dependent cell lines indicates involvement in regulating hematopoiesis, e.g.
  • erythroid progenitor cells alone or in combination with other cytokines, thereby indicating utility, for example, in treating various anemias or for use in conjunction with irradiation /chemotherapy to stimulate the production of erythroid precursors and /or erythroid cells; in supporting the growth and proliferation of myeloid cells such as granulocytes and monocytes/macrophages (i.e., traditional CSF activity) useful, for example, in conjunction with chemotherapy to prevent or treat consequent myelo-suppression; in supporting the growth and proliferation of megakaryocytes and consequently of platelets thereby allowing prevention or treatment of various platelet disorders such as thrombocytopenia, and generally for use in place of or complimentary to platelet transfusions; and /or in supporting the growth and proliferation of hematopoietic stem cells which are capable of maturing to any and all of the above- mentioned hematopoietic cells and therefore find therapeutic utility in various stem cell disorders (such as those usually treated with
  • Assays for embryonic stem cell differentiation include, without limitation, those described in: Johansson et al. Cellular Biology 15:141-151, 1995; Keller et al., Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al., Blood
  • Assays for stem cell survival and differentiation include, without limitation, those described in: Methylcellulose colony forming assays, Freshney, M.G. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss, Inc., New York, NY. 1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992; Primitive hematopoietic colony forming cells with high proliferative potential, McNiece, I.K. and Briddell, R.A.
  • a protein of the present invention also may have utility in compositions used for bone, cartilage, tendon, ligament and /or nerve tissue growth or regeneration, as well as for wound healing and tissue repair and replacement, and in the treatment of burns, incisions and ulcers.
  • a protein of the present invention which induces cartilage and /or bone growth in circumstances where bone is not normally formed, has application in the healing of bone fractures and cartilage damage or defects in humans and other animals.
  • Such a preparation employing a protein of the invention may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints. De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma induced, or oncologic resection induced craniofacial defects, and also is useful in cosmetic plastic surgery.
  • a protein of this invention may also be used in the treatment of periodontal disease, and in other tooth repair processes. Such agents may provide an environment to attract bone-forming cells, stimulate growth of bone-forming cells or induce differentiation of progenitors of bone-forming cells.
  • a protein of the invention may also be useful in the treatment of osteoporosis or osteoarthritis, such as through stimulation of bone and/or cartilage repair or by blocking inflammation or processes of tissue destruction (collagenase activity, osteoclast activity, etc.) mediated by inflammatory processes.
  • tissue regeneration activity that may be attributable to the protein of the present invention is tendon /ligament formation.
  • a protein of the present invention which induces tendon /ligament-like tissue or other tissue formation in circumstances where such tissue is not normally formed, has application in the healing of tendon or ligament tears, deformities and other tendon or ligament defects in humans and other animals.
  • Such a preparation employing a tendon /ligament-like tissue inducing protein may have prophylactic use in preventing damage to tendon or ligament tissue, as well as use in the improved fixation of tendon or ligament to bone or other tissues, and in repairing defects to tendon or ligament tissue.
  • compositions of the present invention contributes to the repair of congenital, trauma induced, or other tendon or ligament defects of other origin, and is also useful in cosmetic plastic surgery for attachment or repair of tendons or ligaments.
  • the compositions of the present invention may provide an environment to attract tendon- or ligament-forming cells, stimulate growth of tendon- or ligament-forming cells, induce differentiation of progenitors of tendon- or ligament-forming cells, or induce growth of tendon /ligament cells or progenitors ex vivo for return in vivo to effect tissue repair.
  • the compositions of the invention may also be useful in the treatment of tendinitis, carpal runnel syndrome and other tendon or ligament defects.
  • the compositions may also include an appropriate matrix and /or sequestering agent as a carrier as is well known in the art.
  • the protein of the present invention may also be useful for proliferation of neural cells and for regeneration of nerve and brain tissue, i.e. for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders, which involve degeneration, death or trauma to neural cells or nerve tissue. More specifically, a protein may be used in the treatment of diseases of the peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome. Further conditions which may be treated in accordance with the present invention include mechanical and traumatic disorders, such as spinal cord disorders, head trauma and cerebrovascular diseases such as stroke. Peripheral neuropathies resulting from chemotherapy or other medical therapies may also be treatable using a protein of the invention.
  • Proteins of the invention may also be useful to promote better or faster closure of non-healing wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like. It is expected that a protein of the present invention may also exhibit activity for generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac) and vascular (including vascular endothelium) tissue, or for promoting the growth of cells comprising such tissues. Part of the desired effects may be by inhibition or modulation of fibrotic scarring to allow normal tissue to regenerate. A protein of the invention may also exhibit angiogenic activity.
  • organs including, for example, pancreas, liver, intestine, kidney, skin, endothelium
  • muscle smooth, skeletal or cardiac
  • vascular including vascular endothelium
  • a protein of the present invention may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and conditions resulting from systemic cytokine damage.
  • a protein of the present invention may also be useful for promoting or inhibiting differentiation of tissues described above from precursor tissues or cells; or for inhibiting the growth of tissues described above.
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Assays for tissue generation activity include, without limitation, those described in: International Patent Publication No. WO95/16035 (bone, cartilage, tendon); International Patent Publication No. W095/ 05846 (nerve, neuronal); International Patent Publication No. WO91/07491 (skin, endothelium ).
  • Assays for wound healing activity include, without limitation, those described in:
  • a protein of the present invention may also exhibit activin- or inhibin-related activities. Inhibins are characterized by their ability to inhibit the release of follicle stimulating hormone (FSH), while activins and are characterized by their ability to stimulate the release of follicle stimulating hormone (FSH).
  • FSH follicle stimulating hormone
  • a protein of the present invention alone or in heterodimers with a member of the inhibin ⁇ family, may be useful as a contraceptive based on the ability of inhibins to decrease fertility in female mammals and decrease spermatogenesis in male mammals. Administration of sufficient amounts of other inhibins can induce infertility in these mammals.
  • the protein of the invention may be useful as a fertility inducing therapeutic, based upon the ability of activin molecules in stimulating FSH release from cells of the anterior pituitary. See, for example, United States Patent 4,798,885.
  • a protein of the invention may also be useful for advancement of the onset of fertility in sexually immature mammals, so as to increase the lifetime reproductive performance of domestic animals such as cows, sheep and pigs.
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Assays for activin/inhibin activity include, without limitation, those described in: Vale et al., Endocrinology 91:562-572, 1972; Ling et al., Nature 321:779-782, 1986; Vale et al., Nature 321:776-779, 1986; Mason et al., Nature 318:659-663, 1985; Forage et al., Proc. Natl. Acad. Sci. USA 83:3091-3095, 1986.
  • a protein of the present invention may have chemotactic or chemokinetic activity (e.g., act as a chemokine) for mammalian cells, including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and /or endothelial cells.
  • Chemotactic and chemokinetic proteins can be used to mobilize or attract a desired cell population to a desired site of action.
  • Chemotactic or chemokinetic proteins provide particular advantages in treatment of wounds and other trauma to tissues, as well as in treatment of localized infections. For example, attraction of lymphocytes, monocytes or neutrophils to tumors or sites of infection may result in improved immune responses against the tumor or infecting agent.
  • a protein or peptide has chemotactic activity for a particular cell population if it can stimulate, directly or indirectly, the directed orientation or movement of such cell population.
  • the protein or peptide has the ability to directly stimulate directed movement of cells. Whether a particular protein has chemotactic activity for a population of cells can be readily determined by employing such protein or peptide in any known assay for cell chemotaxis.
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Assays for chemotactic activity consist of assays that measure the ability of a protein to induce the migration of cells across a membrane as well as the ability of a protein to induce the adhesion of one cell population to another cell population.
  • Suitable assays for movement and adhesion include, without limitation, those described in: Current Protocols in Immunology, Ed by J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W.Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest.
  • a protein of the invention may also exhibit hemostatic or thrombolytic activity. As a result, such a protein is expected to be useful in treatment of various coagulation disorders (including hereditary disorders, such as hemophilias) or to enhance coagulation and other hemostatic events in treating wounds resulting from trauma, surgery or other causes.
  • a protein of the invention may also be useful for dissolving or inhibiting formation of thromboses and for treatment and prevention of conditions resulting therefrom (such as, for example, infarction of cardiac and central nervous system vessels (e.g., stroke).
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Assay for hemostatic and thrombolytic activity include, without limitation, those described in: Linet et al., J. Clin. Pharmacol. 26:131-140, 1986; Burdick et al., Thrombosis Res.45:413-419, 1987; Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub, Prostaglandins 35:467-474, 1988.
  • a protein of the present invention may also demonstrate activity as receptors, receptor ligands or inhibitors or agonists of receptor/ligand interactions.
  • receptors and ligands include, without limitation, cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, receptors involved in cell-cell interactions and their ligands (including without limitation, cellular adhesion molecules (such as selectins, integrins and their ligands) and receptor/ligand pairs involved in antigen presentation, antigen recognition and development of cellular and humoral immune responses).
  • Receptors and ligands are also useful for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction.
  • a protein of the present invention (including, without limitation, fragments of receptors and ligands) may themselves be useful as inhibitors of receptor/ligand interactions.
  • Suitable assays for receptor-ligand activity include without limitation those described in:Current Protocols in Immunology, Ed by J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W.Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under static conditions 7.28.1-7.28.22), Takai et al, Proc. Natl. Acad. Sci. USA 84:6864-6868, 1987; Bierer et al., J. Exp. Med.
  • Anti-Inflammatory Activity Proteins of the present invention may also exhibit anti-inflammatory activity.
  • the anti-inflammatory activity may be achieved by providing a stimulus to cells involved in the inflammatory response, by inhibiting or promoting cell-cell interactions (such as, for example, cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the inflammatory process, inhibiting or promoting cell extravasation, or by stimulating or suppressing production of other factors which more directly inhibit or promote an inflammatory response.
  • Proteins exhibiting such activities can be used to treat inflammatory conditions including chronic or acute conditions), including without limitation inflammation associated with infection (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or resulting from over production of cytokines such as TNF or IL-1. Proteins of the invention may also be useful to treat anaphylaxis and hypersensitivity to an antigenic substance or material.
  • infection such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)
  • ischemia-reperfusion injury such as endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or resulting
  • Cadherins are calcium-dependent adhesion molecules that appear to play major roles during development, particularly in defining specific cell types. Loss or alteration of normal cadherin expression can lead to changes in cell adhesion properties linked to tumor growth and metastasis. Cadherin malfunction is also implicated in other human diseases, such as pemphigus vulgaris and pemphigus foliaceus (auto-immune blistering skin diseases), Crohn's disease, and some developmental abnormalities.
  • the cadherin superfamily includes well over forty members, each with a distinct pattern of expression. All members of the superfamily have in common conserved extracellular repeats (cadherin domains), but structural differences are found in other parts of the molecule.
  • the cadherin domains bind calcium to form their tertiary structure and thus calcium is required to mediate their adhesion. Only a few amino acids in the first cadherin domain provide the basis for homophilic adhesion; modification of this recognition site can change the specificity of a cadherin so that instead of recognizing only itself, the mutant molecule can now also bind to a different cadherin. In addition, some cadherins engage in heterophilic adhesion with other cadherins.
  • E-cadherin one member of the cadherin superfamily, is expressed in epithelial cell types. Pathologically, if E-cadherin expression is lost in a tumor, the malignant cells become invasive and the cancer metastasizes. Transfection of cancer cell lines with polynucleotides expressing E-cadherin has reversed cancer-associated changes by returning altered cell shapes to normal, restoring cells' adhesiveness to each other and to their substrate, decreasing the cell growth rate, and drastically reducing anchorage- independent cell growth. Thus, reintroducing E-cadherin expression reverts carcinomas to a less advanced stage. It is likely that other cadherins have the same invasion suppressor role in carcinomas derived from other tissue types.
  • proteins of the present invention with cadherin activity can be used to treat cancer.
  • Introducing such proteins or polynucleotides into cancer cells can reduce or eliminate the cancerous changes observed in these cells by providing normal cadherin expression.
  • Cancer cells have also been shown to express cadherins of a different tissue type than their origin, thus allowing these cells to invade and metastasize in a different tissue in the body.
  • Proteins of the present invention with cadherin activity, and polynucleotides of the present invention encoding such proteins can be substituted in these cells for the inappropriately expressed cadherins, restoring normal cell adhesive properties and reducing or eliminating the tendency of the cells to metastasize.
  • proteins of the present invention with cadherin activity can be used to generate antibodies recognizing and binding to cadherins.
  • Such antibodies can be used to block the adhesion of inappropriately expressed tumor-cell cadherins, preventing the cells from forming a tumor elsewhere.
  • Such an anti-cadherin antibody can also be used as a marker for the grade, pathological type, and prognosis of a cancer, i.e. the more progressed the cancer, the less cadherin expression there will be, and this decrease in cadherin expression can be detected by the use of a cadherin-binding antibody.
  • Fragments of proteins of the present invention with cadherin activity can also be used to block cadherin function by binding to cadherins and preventing them from binding in ways that produce undesirable effects. Additionally, fragments of proteins of the present invention with cadherin activity, preferably truncated soluble cadherin fragments which have been found to be stable in the circulation of cancer patients, and polynucleotides encoding such protein fragments, can be used to disturb proper cell-cell adhesion.
  • Assays for cadherin adhesive and invasive suppressor activity include, without limitation, those described in: Hortsch et al. J Biol Chem 270 (32): 18809-18817, 1995; Miyaki et al. Oncogene 11: 2547-2552, 1995; Ozawa et al. Cell 63: 1033-1038, 1990.
  • a protein of the invention may exhibit other anti- tumor activities.
  • a protein may inhibit tumor growth directly or indirectly (such as, for example, via
  • a protein may exhibit its tumor inhibitory activity by acting on tumor tissue or tumor precursor tissue, by inhibiting formation of tissues necessary to support tumor growth (such as, for example, by inhibiting angiogenesis), by causing production of other factors, agents or cell types which inhibit tumor growth, or by suppressing, eliminating or inhibiting factors, agents or cell types which promote tumor growth.
  • a protein of the invention may also exhibit one or more of the following additional activities or effects: inhibiting the growth, infection or function of, or killing, infectious agents, including, without limitation, bacteria, viruses, fungi and other parasites; effecting
  • bodily characteristics including, without limitation, height, weight, hair color, eye color, skin, fat to lean ratio or other tissue pigmentation, or organ or body part size or shape (such as, for example, breast augmentation or diminution, change in bone form or shape); effecting biorhythms or caricadic cycles or rhythms; effecting the fertility of male or female subjects; effecting the metabolism, catabolism, anabolism, processing, utilization, storage or elimination of dietary fat, lipid, protein, carbohydrate, vitamins, minerals, cofactors or other nutritional factors or component(s); effecting behavioral characteristics, including, without limitation, appetite, libido, stress, cognition (including cognitive disorders), depression (including depressive disorders) and violent behaviors; providing analgesic effects or other pain reducing effects; promoting differentiation and growth of embryonic stem cells in lineages other than hematopoietic lineages; hormonal or endocrine activity; in the case of enzymes, correcting deficiencies of the enzyme and treating deficiency-related diseases; treatment of hyperpro
  • a protein of the present invention may be used in a pharmaceutical composition when combined with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier may also contain (in addition to protein and a carrier) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art.
  • pharmaceutically acceptable means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s). The characteristics of the carrier will depend on the route of administration.
  • the pharmaceutical composition of the invention may also contain cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,
  • the pharmaceutical composition may further contain other agents which either enhance the activity of the protein or compliment its activity or use in treatment. Such additional factors and/or agents may be included in the pharmaceutical composition to produce a synergistic effect with protein of the invention, or to minimize side effects.
  • protein of the present invention may be included in formulations of the particular cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize side effects of the cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent.
  • a protein of the present invention may be active in mul timers (e.g., heterodimers or homodimers) or complexes with itself or other proteins.
  • pharmaceutical compositions of the invention may comprise a protein of the invention in such multimeric or complexed form.
  • the pharmaceutical composition of the invention may be in the form of a complex of the protein(s) of present invention along with protein or peptide antigens.
  • the protein and/or peptide antigen will deliver a stimulatory signal to both B and T lymphocytes.
  • B lymphocytes will respond to antigen through their surface immunoglobulin receptor.
  • T lymphocytes will respond to antigen through the T cell receptor (TCR) following presentation of the antigen by MHC proteins.
  • TCR T cell receptor
  • MHC and structurally related proteins including those encoded by class I and class II MHC genes on host cells will serve to present the peptide antigen(s) to T lymphocytes.
  • the antigen components could also be supplied as purified MHC-peptide complexes alone or with co-stimulatory molecules that can directly signal T cells.
  • the pharmaceutical composition of the invention may be in the form of a liposome in which protein of the present invention is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution.
  • amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution.
  • Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like.
  • the term "therapeutically effective amount” means the total amount of each active component of the pharmaceutical composition or method that is sufficient to show a meaningful patient benefit, i.e., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions.
  • a meaningful patient benefit i.e., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions.
  • the term refers to that ingredient alone.
  • a therapeutically effective amount of protein of the present invention is administered to a mammal having a condition to be treated.
  • Protein of the present invention may be administered in accordance with the method of the invention either alone or in combination with other therapies such as treatments employing cytokines, lymphokines or other hematopoietic factors.
  • protein of the present invention may be administered either simultaneously with the cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or sequentially. If administered sequentially, the attending physician will decide on the appropriate sequence of administering protein of the present invention in combination with cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors.
  • Administration of protein of the present invention used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection. Intravenous administration to the patient is preferred.
  • protein of the present invention When a therapeutically effective amount of protein of the present invention is administered orally, protein of the present invention will be in the form of a tablet, capsule, powder, solution or elixir.
  • the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant.
  • the tablet, capsule, and powder contain from about 5 to 95% protein of the present invention, and preferably from about 25 to 90% protein of the present invention.
  • a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added.
  • the liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol.
  • the pharmaceutical composition When administered in liquid form, contains from about 0.5 to 90% by weight of protein of the present invention, and preferably from about 1 to 50% protein of the present invention.
  • protein of the present invention When a therapeutically effective amount of protein of the present invention is administered by intravenous, cutaneous or subcutaneous injection, protein of the present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution.
  • parenterally acceptable protein solutions having due regard to pH, isotonicity, stability, and the like, is within the skill in the art.
  • a preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to protein of the present invention, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride
  • the pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art.
  • the amount of protein of the present invention in the pharmaceutical composition of the present invention will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the patient has undergone. Ultimately, the attending physician will decide the amount of protein of the present invention with which to treat each individual patient. Initially, the attending physician will administer low doses of protein of the present invention and observe the patient's response. Larger doses of protein of the present invention may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further.
  • compositions used to practice the method of the present invention should contain about 0.01 ⁇ g to about 100 mg (preferably about O.lng to about 10 mg, more preferably about 0.1 ⁇ g to about 1 mg) of protein of the present invention per kg body weight.
  • the duration of intravenous therapy using the pharmaceutical composition of the present invention will vary, depending on the severity of the disease being treated and the condition and potential idiosyncratic response of each individual patient. It is contemplated that the duration of each application of the protein of the present invention will be in the range of 12 to 24 hours of continuous intravenous administration.
  • Protein of the invention may also be used to immunize animals to obtain polyclonal and monoclonal antibodies which specifically react with the protein. Such antibodies may be obtained using either the entire protein or fragments thereof as an immunogen.
  • the peptide immunogens additionally may contain a cysteine residue at the carboxyl terminus, and are conjugated to a hapten such as keyhole limpet hemocyanin (KLH).
  • KLH keyhole limpet hemocyanin
  • Monoclonal antibodies binding to the protein of the invention may be useful diagnostic agents for the immunodetection of the protein.
  • Neutralizing monoclonal antibodies binding to the protein may also be useful therapeutics for both conditions associated with the protein and also in the treatment of some forms of cancer where abnormal expression of the protein is involved.
  • neutralizing monoclonal antibodies against the protein may be useful in detecting and preventing the metastatic spread of the cancerous cells, which may be mediated by the protein.
  • the therapeutic method includes administering the composition topically, systematically, or locally as an implant or device.
  • the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically acceptable form.
  • the composition may desirably be encapsulated or injected in a viscous form for delivery to the site of bone, cartilage or tissue damage.
  • Topical administration may be suitable for wound healing and tissue repair.
  • Therapeutically useful agents other than a protein of the invention which may also optionally be included in the composition as described above, may alternatively or additionally, be administered simultaneously or sequentially with the composition in the methods of the invention.
  • the composition would include a matrix capable of delivering the protein-containing composition to the site of bone and /or cartilage damage, providing a structure for the developing bone and cartilage and optimally capable of being resorbed into the body.
  • a matrix capable of delivering the protein-containing composition to the site of bone and /or cartilage damage, providing a structure for the developing bone and cartilage and optimally capable of being resorbed into the body.
  • Such matrices may be formed of materials presently in use for other implanted medical applications. The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties. The particular application of the compositions will define the appropriate formulation.
  • Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydrides.
  • Other potential materials are biodegradable and biologically well- defined, such as bone or dermal collagen.
  • Further matrices are comprised of pure proteins or extracellular matrix components.
  • Other potential matrices are nonbiodegradable and chemically defined, such as sintered hydroxapatite, bioglass, aluminates, or other ceramics.
  • Matrices may be comprised of combinations of any of the above mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalciumphosphate.
  • the bioceramics may be altered in composition, such as in calcium- aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradability.
  • a sequestering agent such as carboxymethyl cellulose or autologous blood clot
  • a preferred family of sequestering agents is cellulosic materials such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl- methylcellulose, and carboxymethylcellulose, the most preferred being cationic salts of carboxymethylcellulose (CMC).
  • sequestering agents include hyaluronic acid, sodium alginate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol).
  • the amount of sequestering agent useful herein is 0.5-20 wt%, preferably 1-10 wt% based on total formulation weight, which represents the amount necessary to prevent desorbtion of the protein from the polymer matrix and to provide appropriate handling of the composition, yet not so much that the progenitor cells are prevented from infiltrating the matrix, thereby providing the protein the opportunity to assist the osteogenic activity of the progenitor cells.
  • proteins of the invention may be combined with other agents beneficial to the treatment of the bone and /or cartilage defect, wound, or tissue in question.
  • agents include various growth factors such as epidermal growth factor (EGF), platelet derived growth factor (PDGF), transforming growth factors (TGF- ⁇ and
  • TGF- ⁇ TGF- ⁇
  • IGF insulin-like growth factor
  • the therapeutic compositions are also presently valuable for veterinary applications. Particularly domestic animals and thoroughbred horses, in addition to humans, are desired patients for such treatment with proteins of the present invention.
  • the dosage regimen of a protein-containing pharmaceutical composition to be used in tissue regeneration will be determined by the attending physician considering various factors which modify the action of the proteins, e.g., amount of tissue weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and diet, the severity of any infection, time of administration and other clinical factors.
  • the dosage may vary with the type of matrix used in the reconstitution and with inclusion of other proteins in the pharmaceutical composition.
  • IGF I insulin like growth factor I
  • the addition of other known growth factors, such as IGF I may also effect the dosage.
  • Progress can be monitored by periodic assessment of tissue/bone growth and/or repair, for example, X-rays, histomorphometric determinations and tetracycline labeling.
  • Polynucleotides of the present invention can also be used for gene therapy. Such polynucleotides can be introduced either in vivo or ex vivo into cells for expression in a mammalian subject. Polynucleotides of the invention may also be administered by other known methods for introduction of nucleic acid into a cell or organism (including, without limitation, in the form of viral vectors or naked DNA).
  • Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic purposes.
  • ACATGTGTTC CATGTGCAAC AAACGGAGAT GTCACAGACT GTATCAACTG GGGAGTCAAT 660
  • CATCTTGAGT TGCAGCGTAC CCGATACCTT ACCAAATGGA CCTGTCTTGT GGTTCAAGGG 720
  • AACAGGGCCA AACCGGAAAT TAATCTACAA TTTCAAACAA GGTAACTTTC CCAGAGTAAA 780
  • GACAATTCCA GATGATGATA ATGAAAGACT CTCGAAAGTT GAAAAAGCTA GACAGCTAAG 1260
  • CTTAGACTCT GCCGAGTTTA TCAAATTCAC
  • GGTCATTAGA CCATTTCCAG GACTTGTGAT 1500
  • AAAAAAAAAA AAAAA 2835 (2) INFORMATION FOR SEQ ID NO : 4 :
  • Lys Pro Glu Leu Val lie Ser Tyr Leu Pro Pro Gly Met Ala Ser Lys 180 185 190 lie Asn Thr Lys Ala Leu Gin Ser Pro Lys Arg Pro Arg Ser Pro Gly 195 200 205
  • GGCAAGCATA ACCTATGGAG TGTTCCTTTG CATTGATTGC TCAGGGTCCC ACCGGTCACT 240
  • AAAGAAACCA AATCAAGCTA AAAAAGGCCT TGGGGCCAAA
  • CTATTCAGAC AGACCTACTG CTCGCCGCAA GCCAGATTAT GAGCCAGTTG AAAATACAGA 1320
  • CTGTGTATAT CCTTGCTTTA TTTTCTTGGA ACCTTTGATT TCAACACTGA GGGCCTGGAG 1920
  • CAACACTCAC CACACACACT TCCTTCAAAA GACCAAAAGT GACTGGTGTC TCGTGTGACA 2100
  • AAAACTACCC CATCAAGGCT CTTTTCAGAT TGATGAATTT GTCCACACTG AAAGTTCTTC 1020
  • CAGTCTCAGC ATCACTGTGG AGCATGCCTT GGAGAGCTTC AGCTTCCTCA ATGAAAACGA 1140
  • AAAAGGCAGA CTTTTTGATA TTCAGAGCCT TGGAAGTCAA CACTGTCACA TGTCAGCTGA 1380
  • CTCTGTACCC AACGGGCAGA CCCCTCTGAA GGCCAGGAGC CCGCGGGAGG AGATCCTGTA 1860

Abstract

Novel polynucleotides and the proteins encoded thereby are disclosed.

Description

SECRETED PROTEINS AND POLYNUCLEOTIDES ENCODING THEM
This application is a continuation-in-part of Ser. No. 60/XXX,XXX (converted to a provisional application from non-provisional application Ser. No. 08/815,381), filed March 11, 1997, which is incorporated by reference herein.
FIELD OF THE INVENTION The present invention provides novel polynucleotides and proteins encoded by such polynucleotides, along with therapeutic, diagnostic and research utilities for these polynucleotides and proteins.
BACKGROUND OF THE INVENTION Technology aimed at the discovery of protein factors (including e.g., cytokines, such as lymphokines, interferons, CSFs and interleukins) has matured rapidly over the past decade. The now routine hybridization cloning and expression cloning techniques clone novel polynucleotides "directly" in the sense that they rely on information directly related to the discovered protein (i.e., partial DNA/amino acid sequence of the protein in the case of hybridization cloning; activity of the protein in the case of expression cloning). More recent "indirect" cloning techniques such as signal sequence cloning, which isolates DNA sequences based on the presence of a now well-recognized secretory leader sequence motif, as well as various PCR-based or low stringency hybridization cloning techniques, have advanced the state of the art by making available large numbers of DNA/amino acid sequences for proteins that are known to have biological activity by virtue of their secreted nature in the case of leader sequence cloning, or by virtue of the cell or tissue source in the case of PCR-based techniques. It is to these proteins and the polynucleotides encoding them that the present invention is directed. SUMMARY OF THE INVENTION In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:l;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:l from nucleotide 521 to nucleotide 1111;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:l from nucleotide 536 to nucleotide 817; (d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone ax318_3 deposited under accession number ATCC 98353;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone ax318_3 deposited under accession number ATCC 98353; (f ) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone ax318_3 deposited under accession number ATCC 98353;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone ax318_3 deposited under accession number ATCC 98353; (h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:2;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:2 having biological activity, the fragment comprising the amino acid sequence from amino acid 93 to amino acid 102 of SEQ ID NO:2;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and (1) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:l from nucleotide 521 to nucleotide 1111; the nucleotide sequence of SEQ ID NO:l from nucleotide 536 to nucleotide 817; the nucleotide sequence of the full-length protein coding sequence of clone ax318_3 deposited under accession number ATCC 98353; or the nucleotide sequence of the mature protein coding sequence of clone ax318_3 deposited under accession number ATCC 98353. In other preferred embodiments, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone ax318_3 deposited under accession number ATCC 98353. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:2 from amino acid 4 to amino acid 99.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID NO.1. In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:2;
(b) the amino acid sequence of SEQ ID NO:2 from amino acid 4 to amino acid 99;
(c) fragments of the amino acid sequence of SEQ ID NO:2 comprising the amino acid sequence from amino acid 93 to amino acid 102 of SEQ ID NO:2; and
(d) the amino acid sequence encoded by the cDNA insert of clone ax318_3 deposited under accession number ATCC 98353; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:2 or the amino acid sequence of SEQ ID NO:2 from amino acid 4 to amino acid 99.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:3;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:3 from nucleotide 61 to nucleotide 864; (c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:3 from nucleotide 826 to nucleotide 1386;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone bgl40_l deposited under accession number ATCC 98353; (e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone bgl40_l deposited under accession number ATCC 98353;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone bgl40_l deposited under accession number ATCC 98353;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone bgl40_l deposited under accession number ATCC 98353;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:4; (i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:4 having biological activity, the fragment comprising the amino acid sequence from amino acid 129 to amino acid 138 of SEQ ID NO:4;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i). Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
NO:3 from nucleotide 61 to nucleotide 864; the nucleotide sequence of SEQ ID NO:3 from nucleotide 826 to nucleotide 1386; the nucleotide sequence of the full-length protein coding sequence of clone bgl40_l deposited under accession number ATCC 98353; or the nucleotide sequence of the mature protein coding sequence of clone bgl40_l deposited under accession number ATCC 98353. In other preferred embodiments, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone bgl40_l deposited under accession number ATCC 98353. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:31 from amino acid 148 to amino acid 249.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:3. In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:4; (b) the amino acid sequence of SEQ ID NO:31;
(c) the amino acid sequence of SEQ ID NO:31 from amino acid 148 to amino acid 249;
(d) fragments of the amino acid sequence of SEQ ID NO:4 comprising the amino acid sequence from amino acid 129 to amino acid 138 of SEQ ID NO:4; (e) fragments of the amino acid sequence of SEQ ID NO:31comprising the amino acid sequence from amino acid 163 to amino acid 172 of SEQ ID NO:31; and
(f) the amino acid sequence encoded by the cDNA insert of clone bgl40_l deposited under accession number ATCC 98353; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:4, the amino acid sequence of
SEQ ID NO:31, or the amino acid sequence of SEQ ID NO:31 from amino acid 148 to amino acid 249.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:5;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:5 from nucleotide 77 to nucleotide 1624; (c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:5 from nucleotide 390 to nucleotide 789;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone bg465_2 deposited under accession number ATCC 98353; (e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone bg465_2 deposited under accession number ATCC 98353; (f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone bg465_2 deposited under accession number ATCC 98353; (g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone bg465_2 deposited under accession number ATCC 98353; (h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:6; (i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:6 having biological activity, the fragment comprising the amino acid sequence from amino acid 253 to amino acid 262 of SEQ ID NO:6;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i). Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
NO:5 from nucleotide 77 to nucleotide 1624; the nucleotide sequence of SEQ ID NO:5 from nucleotide 390 to nucleotide 789; the nucleotide sequence of the full-length protein coding sequence of clone bg465_2 deposited under accession number ATCC 98353; or the nucleotide sequence of the mature protein coding sequence of clone bg465_2 deposited under accession number ATCC 98353. In other preferred embodiments, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone bg465_2 deposited under accession number ATCC 98353. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:6 from amino acid 260 to amino acid 343.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:5.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:6;
(b) the amino acid sequence of SEQ ID NO:6 from amino acid 260 to amino acid 343; (c) fragments of the amino acid sequence of SEQ ID NO:6 comprising the amino acid sequence from amino acid 253 to amino acid 262 of SEQ ID NO:6; and
(d) the amino acid sequence encoded by the cDNA insert of clone bg465_2 deposited under accession number ATCC 98353; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:6 or the amino acid sequence of SEQ ID NO:6 from amino acid 260 to amino acid 343.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:7;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:7 from nucleotide 48 to nucleotide 1055; (c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:7 from nucleotide 216 to nucleotide 1055;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:7 from nucleotide 494 to nucleotide 958;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone bk291_3 deposited under accession number ATCC 98353;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone bk291_3 deposited under accession number ATCC 98353;
(g) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone bk291_3 deposited under accession number
ATCC 98353;
(h) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone bk291_3 deposited under accession number ATCC 98353;
(i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:8;
(j) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 8 having biological activity, the fragment comprising the amino acid sequence from amino acid 85 to amino acid 94 of SEQ ID NO:8; (k) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(h) above;
(1) a polynucleotide which encodes a species homologue of the protein of (i) or (j) above ; and (m) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(j).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:7 from nucleotide 48 to nucleotide 1055; the nucleotide sequence of SEQ ID NO:7 from nucleotide 216 to nucleotide 1055; the nucleotide sequence of SEQ ID NO:7 from nucleotide 494 to nucleotide 958; the nucleotide sequence of the full-length protein coding sequence of clone bk291_3 deposited under accession number ATCC 98353; or the nucleotide sequence of the mature protein coding sequence of clone bk291_3 deposited under accession number ATCC 98353. In other preferred embodiments, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone bk291_3 deposited under accession number ATCC 98353. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:8 from amino acid 188 to amino acid 306.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:7.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:8; (b) the amino acid sequence of SEQ ID NO:8 from amino acid 188 to amino acid 306;
(c) fragments of the amino acid sequence of SEQ ID NO:8 comprising the amino acid sequence from amino acid 85 to amino acid 94 of SEQ ID NO:8; and
(d) the amino acid sequence encoded by the cDNA insert of clone bk291_3 deposited under accession number ATCC 98353; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:8 or the amino acid sequence of SEQ ID NO:8 from amino acid 188 to amino acid 306. In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9; (b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:9 from nucleotide 64 to nucleotide 1197;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9 from nucleotide 1 to nucleotide 828;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone bp537_4 deposited under accession number ATCC 98353;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone bp537_4 deposited under accession number ATCC 98353;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone bp537_4 deposited under accession number
ATCC 98353;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone bp537_4 deposited under accession number ATCC 98353;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:10;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:10 having biological activity, the fragment comprising the amino acid sequence from amino acid 184 to amino acid 193 of SEQ ID NO:10; (j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:9 from nucleotide 64 to nucleotide 1197; the nucleotide sequence of SEQ ID NO:9 from nucleotide 1 to nucleotide 828; the nucleotide sequence of the full-length protein coding sequence of clone bp537_4 deposited under accession number ATCC 98353; or the nucleotide sequence of the mature protein coding sequence of clone bp537_4 deposited under accession number ATCC 98353. In other preferred embodiments, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone bp537_4 deposited under accession number ATCC 98353. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:10 from amino acid 1 to amino acid 255.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:9. In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO: 10;
(b) the amino acid sequence of SEQ ID NO: 10 from amino acid 1 to amino acid 255;
(c) fragments of the amino acid sequence of SEQ ID NO:10 comprising the amino acid sequence from amino acid 184 to amino acid 193 of SEQ ID NO:10; and
(d) the amino acid sequence encoded by the cDNA insert of clone bp537_4 deposited under accession number ATCC 98353; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:10 or the amino acid sequence of SEQ ID NO:10 from amino acid 1 to amino acid 255.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:ll;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:ll from nucleotide 581 to nucleotide 1534; (c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:ll from nucleotide 928 to nucleotide 1510;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone cs431_2 deposited under accession number ATCC 98353; (e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone cs431_2 deposited under accession number ATCC 98353;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone cs431_2 deposited under accession number ATCC 98353;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone cs431_2 deposited under accession number ATCC 98353;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:12; (i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:12 having biological activity, the fragment comprising the amino acid sequence from amino acid 154 to amino acid 163 of SEQ ID NO: 12;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i). Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
NO:ll from nucleotide 581 to nucleotide 1534; the nucleotide sequence of SEQ ID NO:ll from nucleotide 928 to nucleotide 1510; the nucleotide sequence of the full-length protein coding sequence of clone cs431_2 deposited under accession number ATCC 98353; or the nucleotide sequence of the mature protein coding sequence of clone cs431_2 deposited under accession number ATCC 98353. In other preferred embodiments, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone cs431_2 deposited under accession number ATCC 98353. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:12 from amino acid 150 to amino acid 310.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:ll. In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:12; (b) the amino acid sequence of SEQ ID NO:12 from amino acid 150 to amino acid 310;
(c) fragments of the amino acid sequence of SEQ ID NO: 12 comprising the amino acid sequence from amino acid 154 to amino acid 163 of SEQ ID NO:12; and (d) the amino acid sequence encoded by the cDNA insert of clone cs431_2 deposited under accession number ATCC 98353; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:12 or the amino acid sequence of SEQ ID NO:12 from amino acid 150 to amino acid 310. In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:13;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:13 from nucleotide 29 to nucleotide 2227;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 13 from nucleotide 1334 to nucleotide 2227;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:13 from nucleotide 1 to nucleotide 746; (e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone cw976_l deposited under accession number ATCC 98353;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone cw976_l deposited under accession number ATCC 98353; (g) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone cw976_l deposited under accession number ATCC 98353;
(h) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone cw976_l deposited under accession number ATCC 98353; (i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:14;
(j) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 14 having biological activity, the fragment comprising the amino acid sequence from amino acid 361 to amino acid 370 of
SEQ ID NO:14;
(k) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(h) above;
(1) a polynucleotide which encodes a species homologue of the protein of (i) or (j) above ; and
(m) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(j).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:13 from nucleotide 29 to nucleotide 2227; the nucleotide sequence of SEQ ID NO:13 from nucleotide 1334 to nucleotide 2227; the nucleotide sequence of SEQ ID NO:13 from nucleotide 1 to nucleotide 746; the nucleotide sequence of the full-length protein coding sequence of clone cw976_l deposited under accession number ATCC 98353; or the nucleotide sequence of the mature protein coding sequence of clone cw976_l deposited under accession number ATCC 98353. In other preferred embodiments, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone cw976_l deposited under accession number ATCC 98353. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 14 from amino acid 1 to amino acid 239, or a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:14 from amino acid 119 to amino acid 733.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:13.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:14;
(b) the amino acid sequence of SEQ ID NO: 14 from amino acid 1 to amino acid 239; (c) the amino acid sequence of SEQ ID NO:14 from amino acid 119 to amino acid 733;
(d) fragments of the amino acid sequence of SEQ ID NO: 14 comprising the amino acid sequence from amino acid 361 to amino acid 370 of SEQ ID NO:14; and
(e) the amino acid sequence encoded by the cDNA insert of clone cw976_l deposited under accession number ATCC 98353; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:14, the amino acid sequence of SEQ ID NO:14 from amino acid 1 to amino acid 239, or the amino acid sequence of SEQ ID NO:14 from amino acid 119 to amino acid 733.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:15;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 15 from nucleotide 364 to nucleotide 777;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:15 from nucleotide 1 to nucleotide 636; (d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone cwl233_3 deposited under accession number ATCC 98353;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone cwl233_3 deposited under accession number ATCC 98353; (f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone cwl233_3 deposited under accession number
ATCC 98353;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone cwl233_3 deposited under accession number ATCC 98353; (h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:16;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 16 having biological activity, the fragment comprising the amino acid sequence from amino acid 64 to amino acid 73 of SEQ ID NO: 16;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above; (k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:15 from nucleotide 364 to nucleotide 777; the nucleotide sequence of SEQ ID NO:15 from nucleotide 1 to nucleotide 636; the nucleotide sequence of the full-length protein coding sequence of clone cwl233_3 deposited under accession number ATCC 98353; or the nucleotide sequence of the mature protein coding sequence of clone cwl233_3 deposited under accession number ATCC 98353. In other preferred embodiments, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone cwl233_3 deposited under accession number ATCC 98353. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:16 from amino acid 1 to amino acid
91. Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID NO:15.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of SEQ ID NO: 16;
(b) the amino acid sequence of SEQ ID NO: 16 from amino acid 1 to amino acid 91;
(c) fragments of the amino acid sequence of SEQ ID NO:16 comprising the amino acid sequence from amino acid 64 to amino acid 73 of SEQ ID NO: 16; and
(d) the amino acid sequence encoded by the cDNA insert of clone cwl233_3 deposited under accession number ATCC 98353; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:16 or the amino acid sequence of SEQ ID NO: 16 from amino acid 1 to amino acid 91.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:17;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:17 from nucleotide 619 to nucleotide 1434; (c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:17 from nucleotide 520 to nucleotide 1323;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone dgl_l deposited under accession number ATCC 98353; (e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone dgl_l deposited under accession number ATCC 98353;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone dgl_l deposited under accession number ATCC 98353; (g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone dgl_l deposited under accession number ATCC 98353;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:18;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:18 having biological activity, the fragment comprising the amino acid sequence from amino acid 131 to amino acid 140 of SEQ ID NO:18;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above; (k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i). Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:17 from nucleotide 619 to nucleotide 1434; the nucleotide sequence of SEQ ID NO:17 from nucleotide 520 to nucleotide 1323; the nucleotide sequence of the full-length protein coding sequence of clone dgl_l deposited under accession number ATCC 98353; or the nucleotide sequence of the mature protein coding sequence of clone dgl_l deposited under accession number ATCC 98353. In other preferred embodiments, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone dgl_l deposited under accession number ATCC 98353. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:18 from amino acid 1 to amino acid 235.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:17.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO: 18;
(b) the amino acid sequence of SEQ ID NO:18 from amino acid 1 to amino acid 235; (c) fragments of the amino acid sequence of SEQ ID NO: 18 comprising the amino acid sequence from amino acid 131 to amino acid 140 of SEQ ID NO:18; and
(d) the amino acid sequence encoded by the cDNA insert of clone dgl_l deposited under accession number ATCC 98353; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:18 or the amino acid sequence of SEQ ID NO: 18 from amino acid 1 to amino acid 235.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:19;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:19 from nucleotide 2063 to nucleotide 2290; (c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:19 from nucleotide 2276 to nucleotide 2290;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:19 from nucleotide 2037 to nucleotide 2405; (e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone ep234_l deposited under accession number ATCC 98353;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone ep234_l deposited under accession number ATCC 98353; (g) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone ep234_l deposited under accession number ATCC 98353;
(h) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone ep234_l deposited under accession number ATCC 98353; (i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:20;
(j) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:20 having biological activity, the fragment comprising the amino acid sequence from amino acid 33 to amino acid 42 of SEQ ID NO:20;
(k) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(h) above;
(1) a polynucleotide which encodes a species homologue of the protein of (i) or (j) above ; and (m) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(j).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:19 from nucleotide 2063 to nucleotide 2290; the nucleotide sequence of SEQ ID NO:19 from nucleotide 2276 to nucleotide 2290; the nucleotide sequence of SEQ ID NO: 19 from nucleotide 2037 to nucleotide 2405; the nucleotide sequence of the full-length protein coding sequence of clone ep234_l deposited under accession number ATCC 98353; or the nucleotide sequence of the mature protein coding sequence of clone ep234_l deposited under accession number ATCC 98353. In other preferred embodiments, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone ep234_l deposited under accession number ATCC 98353. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:20 from amino acid 1 to amino acid 69. Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID NO:19.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of SEQ ID NO:20;
(b) the amino acid sequence of SEQ ID NO:20 from amino acid 1 to amino acid 69;
(c) fragments of the amino acid sequence of SEQ ID NO:20 comprising the amino acid sequence from amino acid 33 to amino acid 42 of SEQ ID NO:20; and
(d) the amino acid sequence encoded by the cDNA insert of clone ep234_l deposited under accession number ATCC 98353; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:20 or the amino acid sequence of SEQ ID NO:20 from amino acid 1 to amino acid 69.
In certain preferred embodiments, the polynucleotide is operably linked to an expression control sequence. The invention also provides a host cell, including bacterial, yeast, insect and mammalian cells, transformed with such polynucleotide compositions. Also provided by the present invention are organisms that have enhanced, reduced, or modified expression of the gene(s) corresponding to the polynucleotide sequences disclosed herein.
Processes are also provided for producing a protein, which comprise:
(a) growing a culture of the host cell transformed with such polynucleotide compositions in a suitable culture medium; and (b) purifying the protein from the culture.
The protein produced according to such methods is also provided by the present invention. Preferred embodiments include those in which the protein produced by such process is a mature form of the protein. Protein compositions of the present invention may further comprise a pharmaceutically acceptable carrier. Compositions comprising an antibody which specifically reacts with such protein are also provided by the present invention.
Methods are also provided for preventing, treating or ameliorating a medical condition which comprises administering to a mammalian subject a therapeutically effective amount of a composition comprising a protein of the present invention and a pharmaceutically acceptable carrier.
BRIEF DESCRIPTION OF THE DRAWINGS Figures IA and IB are schematic representations of the pED6 and pNOTs vectors, respectively, used for deposit of clones disclosed herein.
DETAILED DESCRIPTION
ISOLATED PROTEINS AND POLYNUCLEOTIDES
Nucleotide and amino acid sequences, as presently determined, are reported below for each clone and protein disclosed in the present application. The nucleotide sequence of each clone can readily be determined by sequencing of the deposited clone in accordance with known methods. The predicted amino acid sequence (both full-length and mature) can then be determined from such nucleotide sequence. The amino acid sequence of the protein encoded by a particular clone can also be determined by expression of the clone in a suitable host cell, collecting the protein and determining its sequence. For each disclosed protein applicants have identified what they have determined to be the reading frame best identifiable with sequence information available at the time of filing.
As used herein a "secreted" protein is one which, when expressed in a suitable host cell, is transported across or through a membrane, including transport as a result of signal sequences in its amino acid sequence. "Secreted" proteins include without limitation proteins secreted wholly (e.g., soluble proteins) or partially (e.g. , receptors) from the cell in which they are expressed. "Secreted" proteins also include without limitation proteins which are transported across the membrane of the endoplasmic reticulum.
Clone "ax318 3" A polynucleotide of the present invention has been identified as clone "ax318_3 "• ax318_3 was isolated from a human adult testes cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. ax318_3 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "ax318_3 protein").
The nucleotide sequence of ax318_3 as presently determined is reported in SEQ ID NO:l. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the ax318_3 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:2.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone ax318_3 should be approximately 1200 bp.
The nucleotide sequence disclosed herein for ax318_3 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. ax318_3 demonstrated at least some similarity with sequences identified as AA255872 (zsl9a01.sl NCI_CGAP_GCB1 Homo sapiens cDNA clone), AA625610 (zv89h04.sl Soares NhHMPu SI Homo sapiens cDNA clone 766999 3' similar to WP:K10B2.1 CE02008 TRANSDUCIN BETA CHAIN), D86043 (Human mRNA for SHPS-1, complete eds), H23217 (ym52f03.sl Homo sapiens cDNA clone 518803'), U06701
(Human clone CCA53 mRNA containing CCA trinucleotide repeat), and W05822 (za90b02.rl Soares fetal lung NbHL19W Homo sapiens cDNA clone 299787 5'). The predicted amino acid sequence disclosed herein for ax318_3 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted ax318_3 protein demonstrated at least some similarity to sequences identified as D85183 (SHPS-1 [Rattus rattus]), D86043 (SHPS-1 [Homo sapiens]), R85852 (WD-40 domain-containing beta-transducin protein), Y10376 (SIRP-betal [Homo sapiens]), and Z79757 (F55B12.3 [Caenorhabditis elegans]). SHPS-1 is a membrane glycoprotein that binds the SH2-domain-containing protein tyrosine phosphatase SHP-2 in response to mitogens and cell adhesion (Fujioka et al, 1996, Mol. Cell. Biol. 16(12): 6887-6899, incorporated by reference herein). Based upon sequence similarity, ax318_3 proteins and each similar protein or peptide may share at least some activity. The TopPredll computer program predicts a putative transmembrane domain within the ax318_3 protein sequence, centered around amino acid 28 of SEQ ID NO:2; amino acids 15 to 27 of SEQ ID NO:2 are also a possible leader /signal sequence, with the predicted mature amino acid sequence beginning at amino acid 28.
Clone "bg!40 1" A polynucleotide of the present invention has been identified as clone "bgl40_l". bgl40_l was isolated from a human adult brain cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. bgl40_l is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "bgl40_l protein").
The nucleotide sequence of bgl40_l as presently determined is reported in SEQ ID NO:3. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the bgl40_l protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:4. Another possible bgl40_l reading frame and predicted amino acid sequence, encoded by base pairs 641 to 1648 of SEQ ID NO:3, is reported in SEQ ID NO:31. A frameshift in the nucleotide sequence of SEQ ID NO:3, caused, for example, by a single base pair deletion near position 825 of SEQ ID NO:3, could join the open reading frame encoding SEQ ID NO:4 with that encoding SEQ ID NO.31.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone bgl40_l should be approximately 2900 bp.
The nucleotide sequence disclosed herein for bgl40_l was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. bgl40_l demonstrated at least some similarity with sequences identified as AA075629 (zm89a04.sl Stratagene ovarian cancer (#937219) Homo sapiens cDNA clone 545070 3'), AA113989 (zn27hl2.rl Stratagene neuroepithelium NT2RAMI 937234 Homo sapiens cDNA clone 548711 5'), N77135 (yz85hll.rl Homo sapiens cDNA clone 289893 5'), R11701 (yf49d02.rl Homo sapiens cDNA clone 25600 5'), R13178 (yf73e04.rl Homo sapiens cDNA clone 27855 5'), ar^d Y14946 (Homo sapiens mRNA for
SPIN protein). The predicted amino acid sequence disclosed herein for bgl40_l was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted bgl40_l protein demonstrated at least some similarity to sequences identified as AF038969 (general transcription factor 2-1 [Homo sapiens]), U77948 (Bruton's tyrosine kinase-associated protein-135; BAP-135 [Homo sapiens]), and Y14946 (SPIN protein [Homo sapiens]). Based upon sequence similarity, bgl40_l proteins and each similar protein or peptide may share at least some activity.
Clone "bg465 2"
A polynucleotide of the present invention has been identified as clone "bg465._2". bg465_2 was isolated from a human adult brain cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. bg465_2 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "bg465_2 protein").
The nucleotide sequence of bg465_2 as presently determined is reported in SEQ ID NO:5. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the bg465_2 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:6.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone bg465_2 should be approximately 2500 bp.
The nucleotide sequence disclosed herein for bg465_2 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. bg465_2 demonstrated at least some similarity with sequences identified as AA133150 (zm25b07.rl Stratagene pancreas (#937208) Homo sapiens cDNA clone 526645 5' similar to WP:F07F6.4 CE01896 ZINC FINGER PROTEIN), AA144842 (mr69h01.rl Stratagene mouse testis (#937308) Mus musculus cDNA clone 602737 5'), AA484561 (nf06al2.sl NCI_CGAP_Lil Homo sapiens cDNA clone), D16939 (Human HepG2 3' region cDNA, clone hmd5d06), L24125 (Saccharomyces cerevisiae zinc finger protein (GCS1) gene, complete eds), R18422 (yg02f06.rl Homo sapiens cDNA clone 30950 5'), T32543 (EST50558 Homo sapiens cDNA 5' end similar to None), W88569 (zh70bl2.sl Soares fetal liver spleen 1NFLS SI Homo sapiens cDNA clone 417407 3'), Z74274 (S.cerevisiae chromosome IV reading frame ORF YDL226c), and Z82199 (Human DNA sequence *** SEQUENCING IN PROGRESS *** from clone 316D5; HTGS phase 1). The predicted amino acid sequence disclosed herein for bg465_2 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted bg465_2 protein demonstrated at least some similarity to sequences identified as U23486 (similar to S. cerevisiae zinc finger protein GCS1 (SP GCS1_YEAST) [Caenorhabditis elegans]). Based upon sequence similarity, bg465_2 proteins and each similar protein or peptide may share at least some activity.
Clone "bk291 3"
A polynucleotide of the present invention has been identified as clone "bk291_3". bk291_3 was isolated from a human adult retina cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. bk291_3 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "bk291_3 protein").
The nucleotide sequence of bk291_3 as presently determined is reported in SEQ ID NO:7. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the bk291_3 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:8. Amino acids 44 to 56 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 57, or are a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone bk291_3 should be approximately 1500 bp.
The nucleotide sequence disclosed herein for bk291_3 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. bk291_3 demonstrated at least some similarity with sequences identified as AA135915 (zll9f04.rl Soares pregnant uterus NbHPU Homo sapiens cDNA clone 502399 5'), AA156738 (zll9f04.sl Soares pregnant uterus NbHPU Homo sapiens cDNA clone 502399 3' similar to WP.T05E11.5 CE06364 YEAST YKK0 LIKE), AF017785 (Mus musculus MHC Class I H13a minor histocompatibility peptide (H13) mRNA, partial eds), N40134 (yw73al2.rl Homo sapiens cDNA clone 257854 5' similar to SW:YKK0_YEAST P34248 HYPOTHETICAL 67.5 KD PROTEIN IN APE1/LAP4-CWP1 INTERGENIC REGION), N42726 (yylldOl.rl Homo sapiens cDNA clone 2709135' similar to SW:YKK0_YEAST P34248 HYPOTHETICAL 67.5 KD PROTEIN LN APE1 /LAP4-CWP1 INTERGENIC REGION), R67144 (yi31h04.rl Homo sapiens cDNA clone 1408875' similar to SP:YKK0_YEAST P34248 HYPOTHETICAL 67.5 KD PROTEIN LN APE1/LAP4-MBR1 INTERGENIC), R78822 (yi90b05.rl Homo sapiens cDNA clone 146481 5'), R79317 (yi90b05.sl Homo sapiens cDNA clone 146481 3'), T23944 (Human gene signature HUMGS05889), and W04243 (za58hl0.rl Soares fetal liver spleen INFLS Homo sapiens cDNA clone). The predicted amino acid sequence disclosed herein for bk291_3 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted bk291_3 protein demonstrated at least some similarity to sequences identified as X71133 (YKL450 [Saccharomyces cerevisiae]), Z28100 (ORF YKLlOOc [Saccharomyces cerevisiae]), and Z68751 (T05E11.5 [Caenorhabditis elegans]). Based upon sequence similarity, bk291_3 proteins and each similar protein or peptide may share at least some activity. The TopPredll computer program predicts six additional potential transmembrane domains within the bk291_3 protein sequence, centered around amino acids 70, 125, 170, 220, 260, and 280 of SEQ ID NO:8.
Clone "bp537 4"
A polynucleotide of the present invention has been identified as clone "bp537_4". bp537_4 was isolated from a human fetal kidney cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. bp537_4 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "bp537_4 protein").
The nucleotide sequence of bp537_4 as presently determined is reported in SEQ ID NO:9. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the bp537_4 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:10. The EcoRI/NotI restriction fragment obtainable from the deposit containing clone bp537_4 should be approximately 1500 bp.
The nucleotide sequence disclosed herein for bp537_4 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. bp537_4 demonstrated at least some similarity with sequences identified as AA007576 (zh99b06.rl Soares fetal liver spleen INFLS SI Homo sapiens cDNA clone 429395 5'), AA287563 (zs52g02.rl NCI_CGAP_GCB1 Homo sapiens cDNA 5'), R09093 (yf21h09.rl Homo sapiens cDNA clone 127553 5'), and T33088 (EST56631 Homo sapiens cDNA 5' end similar to None). The predicted amino acid sequence disclosed herein for bp537_4 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted bp537_4 protein demonstrated at least some similarity to sequences identified as U34998 (Rad9 [Coprinus cinereus]). Based upon sequence similarity, bp537_4 proteins and each similar protein or peptide may share at least some activity.
Clone "cs431 2"
A polynucleotide of the present invention has been identified as clone "cs431_2". cs431_2 was isolated from a human fetal brain cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. cs431_2 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "cs431_2 protein").
The nucleotide sequence of cs431_2 as presently determined is reported in SEQ ID NO:ll. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the cs431_2 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:12.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone cs431_2 should be approximately 2300 bp. The nucleotide sequence disclosed herein for cs431_2 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. cs431_2 demonstrated at least some similarity with sequences identified as AA021033 (ze67hl2.sl Soares retina N2b4HR Homo sapiens cDNA clone 364103 3' similar to contains Alu repetitive element), H29349 (ym32c02.rl Homo sapiens cDNA clone 49839 5' similar to SP:KYES_XIPHE P27447 PROTO-ONCOGENE
TYROSINE-PROTELN KINASE YES), L14577 (Homo sapiens cystathionine beta-synthase (CBS) mRNA, complete eds), Q87430 (Human cystathionine beta-synthase cDNA), and X92659 (H.sapiens intron 4 from p53 gene). The predicted amino acid sequence disclosed herein for cs431_2 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted cs431_2 protein demonstrated at least some similarity to sequences identified as L19501 (cystathionine beta-synthase [Homo sapiens]), R71376 (Human cystathionine beta-synthase), U61167 (SH3 domain-containing protein SH3P18 [Homo sapiens]), and X82166 (cystathionine beta-synthase [Homo sapiens]). Based upon sequence similarity, cs431_2 proteins and each similar protein or peptide may share at least some activity. The TopPredll computer program predicts a potential transmembrane domain within the cs431_2 protein sequence centered around amino acid 30 of SEQ ID NO:12. The nucleotide sequence of cs431_2 indicates that it may contain an Alu repetitive element.
Clone "cw976 1"
A polynucleotide of the present invention has been identified as clone "cw976_l". cw976_l was isolated from a human fetal brain cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. cw976_l is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "cw976_l protein").
The nucleotide sequence of cw976_l as presently determined is reported in SEQ ID NO:13. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the cw976_l protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:14. Amino acids 423 to 435 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 436, or are a transmembrane domain. The EcoRI/NotI restriction fragment obtainable from the deposit containing clone cw976_l should be approximately 3300 bp.
The nucleotide sequence disclosed herein for cw976_l was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. cw976_l demonstrated at least some similarity with sequences identified as AA280747 (zs96a09.rl NCI_CGAP_GCB1 Homo sapiens cDNA clone IMAGE:711448 5'), AA323946 (EST26798 Cerebellum II Homo sapiens cDNA 5' end), R13665 (yf60g06.rl Homo sapiens cDNA clone 26764 5'), R16892 (yf44d06.s2 Homo sapiens cDNA clone 1297073'), R85177 (yo43d07.rl Homo sapiens cDNA clone 1806855'), and R89648 (ym97c02.rl Homo sapiens cDNA clone 166850 5'). Based upon sequence similarity, cw976_l proteins and each similar protein or peptide may share at least some activity. The TopPredll computer program predicts a potential transmembrane domain within the cw976_l protein sequence at the amino terminus of SEQ ID NO:14. Clone "cwl233 3"
A polynucleotide of the present invention has been identified as clone "cwl233_3 "■ cwl233_3 was isolated from a human fetal brain cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. cwl233_3 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "cwl233_3 protein").
The nucleotide sequence of cwl233_3 as presently determined is reported in SEQ ID NO:15. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the cwl233_3 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:16.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone cwl233_3 should be approximately 3000 bp. The nucleotide sequence disclosed herein for cwl233_3 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. cwl233_3 demonstrated at least some similarity with sequences identified as R61600 (yhl6f08.rl Homo sapiens cDNA clone 378075'), R83929 (ypOόhll.sl Homo sapiens cDNA clone 186693 3' similar to contains Alu repetitive element;contains TARl repetitive element), R87877 (yo45h05.rl Homo sapiens cDNA clone 180921 5'), U14568 (***ALU WARNING Human Alu-Sb subfamily consensus sequence). The predicted amino acid sequence disclosed herein for cwl233_3 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted cwl233_3 protein demonstrated at least some similarity to sequences identified as AB002317 (KIAA0319 [Homo sapiens]). Based upon sequence similarity, cwl233_3 proteins and each similar protein or peptide may share at least some activity. The nucleotide sequence of cwl233_3 indicates that it may contain an Alu repetitive element.
Clone "dgl 1"
A polynucleotide of the present invention has been identified as clone "dgl_l". dgl_l was isolated from a human adult placenta cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. dgl_l is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "dgl_l protein").
The nucleotide sequence of dgl_l as presently determined is reported in SEQ ID NO: 17. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the dgl_l protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:18.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone dgl_l should be approximately 1900 bp. The nucleotide sequence disclosed herein for dgl_l was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. dgl_l demonstrated at least some similarity with sequences identified as AA731281 (nw57f05.sl NCI_CGAP_GCB1 Homo sapiens cDNA clone LMAGE 1250721 similar to gb Y00345_cdsl POLYADENYLATE-BINDING PROTEIN (HUMAN)). The predicted amino acid sequence disclosed herein for dgl_l was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted dgl_l protein demonstrated at least some similarity to sequences identified as M81878 (hyaluronidase [Clostridium perfringens]) and U28742 (similar to hyaluronoglucosaminidase (SPNAGH_CLOPE, P26831) [Caenorhabditis elegans]). Hyaluronoglucosaminidase is a secreted protein found, for example, in bee venom. Based upon sequence similarity, dgl_l proteins and each similar protein or peptide may share at least some activity. The TopPredll computer program predicts a potential transmembrane domain within the dgl_l protein sequence centered around amino acid 150 of SEQ ID NO:18.
Clone "ep234 1"
A polynucleotide of the present invention has been identified as clone "ep234_l". ep234_l was isolated from a human adult placenta cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. ep234_l is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "ep234_l protein"). The nucleotide sequence of ep234_l as presently determined is reported in SEQ ID NO:19. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the ep234_l protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:20. Amino acids 59 to 71 are a predicted leader /signal sequence, with the predicted mature amino acid sequence beginning at amino acid 72, or are a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone ep234_l should be approximately 2500 bp.
The nucleotide sequence disclosed herein for ep234_l was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. ep234_l demonstrated at least some similarity with sequences identified as AA044714 (zf54d09.rl Soares retina N2b4HR Homo sapiens cDNA clone 3807535'), AA454840 (zx79d09.sl Soares ovary tumor NbHOT Homo sapiens cDNA clone 809969 3'), AA470137 (zulleOl.sl Soares testis NHT Homo sapiens cDNA clone 731544 3'), H24852 (yl42cll.rl Homo sapiens cDNA clone 160916 5'), N64648 (yz87b06.sl Homo sapiens cDNA clone 290003 3"), R45788 (Ha662-f Homo sapiens cDNA clone a662-f), T70546 (ydl5b07.sl Homo sapiens cDNA clone 108277 3'), W73481 (zd54e01.sl Soares fetal heart NbHH19W Homo sapiens cDNA clone 344472 3'), and W73553 (zd54e01.rl Soares fetal heart). Based upon sequence similarity, ep234_l proteins and each similar protein or peptide may share at least some activity. The TopPredll computer program predicts at least two potential transmembrane domains within the ep234_l protein sequence of SEQ ID NO:20.
Deposit of Clones Clones ax318_3, bgl40_l, bg465_2, bk291_3, bp537_4, cs431_2, cw976_l, cwl233_3, dgl_l, and ep234_l were deposited on March 11, 1997 with the American Type Culture Collection as an original deposit under the Budapest Treaty and were given the accession number ATCC 98353, from which each clone comprising a particular polynucleotide is obtainable. All restrictions on the availability to the public of the deposited material will be irrevocably removed upon the granting of the patent, except for the requirements specified in 37 C.F.R. § 1.808(b).
Each clone has been transfected into separate bacterial cells (E. coli) in this composite deposit. Each clone can be removed from the vector in which it was deposited by performing an EcoRI/NotI digestion (5' site, EcoRI; 3' site, NotI) to produce the appropriate fragment for such clone. Each clone was deposited in either the pED6 or pNOTs vector depicted in Fig. 1. The pED6dpc2 vector ("pED6") was derived from pEDόdpcl by insertion of a new polylinker to facilitate cDNA cloning (Kaufman et al., 1991, Nucleic Acids Res. 19: 4485-4490); the pNOTs vector was derived from pMT2 (Kaufman et al, 1989, Mol. Cell. Biol. 9: 946-958) by deletion of the DHFR sequences, insertion of a new polylinker, and insertion of the M13 origin of replication in the Clal site. In some instances, the deposited clone can become "flipped" (i.e., in the reverse orientation) in the deposited isolate. In such instances, the cDNA insert can still be isolated by digestion with EcoRI and Notl. However, NotI will then produce the 5' site and EcoRI will produce the 3' site for placement of the cDNA in proper orientation for expression in a suitable vector. The cDNA may also be expressed from the vectors in which they were deposited.
Bacterial cells containing a particular clone can be obtained from the composite deposit as follows: An oligonucleotide probe or probes should be designed to the sequence that is known for that particular clone. This sequence can be derived from the sequences provided herein, or from a combination of those sequences. The sequence of the oligonucleotide probe that was used to isolate each full-length clone is identified below, and should be most reliable in isolating the clone of interest.
Clone Probe Sequence ax318_3 SEQ ID NO:21 bgl40_l SEQ ID NO:22 bg465_2 SEQ ID NO:23, SEQ ID NO:32 bk291_3 SEQ ID NO:24 bp537_4 SEQ ID NO:25 cs431_2 SEQ ID NO:26 cw976_l SEQ ID NO:27 cwl233_3 SEQ ID NO:28 dgl_l SEQ ID NO:29 ep234_l SEQ ID NO:30
In the sequences listed above which include an N at position 2, that position is occupied in preferred probes /primers by a biotinylated phosphoaramidite residue rather than a nucleotide (such as , for example, that produced by use of biotin phosphoramidite (1- dimethoxytrityloxy-2-(N-biotinyl-4-aminobutyl)-propyl-3-0-(2-cyanoethyl)-(N,N- diisopropyl)-phosphoramadite) (Glen Research, cat. no. 10-1953)).
The design of the oligonucleotide probe should preferably follow these parameters:
(a) It should be designed to an area of the sequence which has the fewest ambiguous bases ("N's"), if any;
(b) It should be designed to have a Tm of approx. 80 ° C (assuming 2° for each A or T and 4 degrees for each G or C). The oligonucleotide should preferably be labeled with g-32P ATP (specific activity 6000 Ci/mmole) and T4 polynucleotide kinase using commonly employed techniques for labeling oligonucleotides. Other labeling techniques can also be used. Unincorporated label should preferably be removed by gel filtration chromatography or other established methods. The amount of radioactivity incorporated into the probe should be quantitated by measurement in a scintillation counter. Preferably, specific activity of the resulting probe should be approximately 4e+6 dpm/pmole.
The bacterial culture containing the pool of full-length clones should preferably be thawed and 100 μl of the stock used to inoculate a sterile culture flask containing 25 ml of sterile L-broth containing ampicillin at 100 μg/ml. The culture should preferably be grown to saturation at 37°C, and the saturated culture should preferably be diluted in fresh L-broth. Aliquots of these dilutions should preferably be plated to determine the dilution and volume which will yield approximately 5000 distinct and well-separated colonies on solid bacteriological media containing L-broth containing ampicillin at 100 μg/ml and agar at 1.5% in a 150 mm pe ri dish when grown overnight at 37°C. Other known methods of obtaining distinct, well-separated colonies can also be employed.
Standard colony hybridization procedures should then be used to transfer the colonies to nitrocellulose filters and lyse, denature and bake them.
The filter is then preferably incubated at 65°C for 1 hour with gentle agitation in 6X SSC (20X stock is 175.3 g NaCl/liter, 88.2 g Na citrate /liter, adjusted to pH 7.0 with NaOH) containing 0.5% SDS, 100 μg/ml of yeast RNA, and 10 mM EDTA (approximately
10 mL per 150 mm filter). Preferably, the probe is then added to the hybridization mix at a concentration greater than or equal to le+6 dpm/mL. The filter is then preferably incubated at 65°C with gentle agitation overnight. The filter is then preferably washed in 500 mL of 2X SSC/0.5% SDS at room temperature without agitation, preferably followed by 500 mL of 2X SSC/0.1% SDS at room temperature with gentle shaking for 15 minutes. A third wash with 0.1X SSC/0.5% SDS at 65°C for 30 minutes to 1 hour is optional. The filter is then preferably dried and subjected to autoradiography for sufficient time to visualize the positives on the X-ray film. Other known hybridization methods can also be employed.
The positive colonies are picked, grown in culture, and plasmid DNA isolated using standard procedures. The clones can then be verified by restriction analysis, hybridization analysis, or DNA sequencing.
Fragments of the proteins of the present invention which are capable of exhibiting biological activity are also encompassed by the present invention. Fragments of the protein may be in linear form or they may be cyclized using known methods, for example, as described in H.U. Saragovi, et al, Bio /Technology 10, 773-778 (1992) and in R.S. McDowell, et al, J. Amer. Chem. Soc. 114, 9245-9253 (1992), both of which are incorporated herein by reference. Such fragments may be fused to carrier molecules such as immunoglobulins for many purposes, including increasing the valency of protein binding sites. For example, fragments of the protein may be fused through "linker" sequences to the Fc portion of an immunoglobulin. For a bivalent form of the protein, such a fusion could be to the Fc portion of an IgG molecule. Other immunoglobulin isotypes may also be used to generate such fusions. For example, a protein - IgM fusion would generate a decavalent form of the protein of the invention.
The present invention also provides both full-length and mature forms of the disclosed proteins. The full-length form of the such proteins is identified in the sequence listing by translation of the nucleotide sequence of each disclosed clone. The mature form of such protein may be obtained by expression of the disclosed full-length polynucleotide (preferably those deposited with ATCC) in a suitable mammalian cell or other host cell.
The sequence of the mature form of the protein may also be determinable from the amino acid sequence of the full-length form.
The present invention also provides genes corresponding to the polynucleotide sequences disclosed herein. "Corresponding genes" are the regions of the genome that are transcribed to produce the mRNAs from which cDNA polynucleotide sequences are derived and may include contiguous regions of the genome necessary for the regulated expression of such genes. Corresponding genes may therefore include but are not limited to coding sequences, 5' and 3' untranslated regions, alternatively spliced exons, introns, promoters, enhancers, and silencer or suppressor elements. The corresponding genes can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include the preparation of probes or primers from the disclosed sequence information for identification and /or amplification of genes in appropriate genomic libraries or other sources of genomic materials. An "isolated gene" is a gene that has been separated from the adjacent coding sequences, if any, present in the genome of the organism from which the gene was isolated.
Organisms that have enhanced, reduced, or modified expression of the gene(s) corresponding to the polynucleotide sequences disclosed herein are provided. The desired change in gene expression can be achieved through the use of antisense polynucleotides or ribozymes that bind and /or cleave the mRNA transcribed from the gene (Albert and Morris, 1994, Trends Pharmacol. Sci. 15(7): 250-254; Lavarosky et al., 1997, Biochem. Mol. Med. 62(1): 11-22; and Hampel, 1998, Prog. Nucleic Acid Res. Mol. Biol. 58: 1- 39; all of which are incorporated by reference herein). Transgenic animals that have multiple copies of the gene(s) corresponding to the polynucleotide sequences disclosed herein, preferably produced by transformation of cells with genetic constructs that are stably maintained within the transformed cells and their progeny, are provided. Transgenic animals that have modified genetic control regions that increase or reduce gene expression levels, or that change temporal or spatial patterns of gene expression, are also provided (see European Patent No. 0 649 464 Bl, incorporated by reference herein). In addition, organisms are provided in which the gene(s) corresponding to the polynucleotide sequences disclosed herein have been partially or completely inactivated, through insertion of extraneous sequences into the corresponding gene(s) or through deletion of all or part of the corresponding gene(s). Partial or complete gene inactivation can be accomplished through insertion, preferably followed by imprecise excision, of transposable elements (Plasterk, 1992, Bioessays 14(9): 629-633; Zwaal et al, 1993, Proc. Natl.
Acad. Sci. USA 90(16): 7431-7435; Clark et al, 1994, Proc. Natl. Acad. Sci. USA 91(2): 719-722; all of which are incorporated by reference herein), or through homologous recombination, preferably detected by positive/negative genetic selection strategies (Mansour et al, 1988, Nature 336: 348-352; U.S. Patent Nos. 5,464,764; 5,487,992; 5,627,059; 5,631,153; 5,614, 396; 5,616,491; and 5,679,523; all of which are incorporated by reference herein). These organisms with altered gene expression are preferably eukaryotes and more preferably are mammals. Such organisms are useful for the development of non-human models for the study of disorders involving the corresponding gene(s), and for the development of assay systems for the identification of molecules that interact with the protein product(s) of the corresponding gene(s).
Where the protein of the present invention is membrane-bound (e.g., is a receptor), the present invention also provides for soluble forms of such protein. In such forms part or all of the intracellular and transmembrane domains of the protein are deleted such that the protein is fully secreted from the cell in which it is expressed. The intracellular and transmembrane domains of proteins of the invention can be identified in accordance with known techniques for determination of such domains from sequence information.
Proteins and protein fragments of the present invention include proteins with amino acid sequence lengths that are at least 25%(more preferably at least 50%, and most preferably at least 75%) of the length of a disclosed protein and have at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% or 95% identity) with that disclosed protein, where sequence identity is determined by comparing the amino acid sequences of the proteins when aligned so as to maximize overlap and identity while minimizing sequence gaps. Also included in the present invention are proteins and protein fragments that contain a segment preferably comprising 8 or more (more preferably 20 or more, most preferably 30 or more) contiguous amino acids that shares at least 75% sequence identity (more preferably, at least 85% identity; most preferably at least 95% identity) with any such segment of any of the disclosed proteins. Species homologs of the disclosed polynucleotides and proteins are also provided by the present invention. As used herein, a "species homologue" is a protein or polynucleotide with a different species of origin from that of a given protein or polynucleotide, but with significant sequence similarity to the given protein or polynucleotide, as determined by those of skill in the art. Species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from the desired species. Preferably, species homologs are those isolated from mammalian species.
The invention also encompasses allelic variants of the disclosed polynucleotides or proteins; that is, naturally-occurring alternative forms of the isolated polynucleotide which also encode proteins which are identical, homologous, or related to that encoded by the polynucleotides .
The invention also includes polynucleotides with sequences complementary to those of the polynucleotides disclosed herein. The present invention also includes polynucleotides capable of hybridizing under reduced stringency conditions, more preferably stringent conditions, and most preferably highly stringent conditions, to polynucleotides described herein. Examples of stringency conditions are shown in the table below: highly stringent conditions are those that are at least as stringent as, for example, conditions A-F; stringent conditions are at least as stringent as, for example, conditions G-L; and reduced stringency conditions are at least as stringent as, for example, conditions M-R.
Figure imgf000038_0001
*: The hybrid length is that anticipated for the hybridized region(s) of the hybridizing polynucleotides. When hybridizing a polynucleotide to a target polynucleotide of unknown sequence, the hybrid length is assumed to be that of the hybridizing polynucleotide. When polynucleotides of known sequence are hybridized, the hybrid length can be determined by aligning the sequences of the polynucleotides and identifying the region or regions of optimal sequence complementarity.
+: SSPE (lxSSPE is 0 15M NaCl, lOmM NaH2P04, and 1.25mM EDTA, pH 7.4) can be substituted for SSC (lxSSC is 0.15M NaCl and 15mM sodium citrate) in the hybridization and wash buffers, washes are performed for 15 minutes after hybridization is complete
*TB - TR- The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5-10°C less than the melting temperature (Tm) of the hybrid, where Tm ιs determined according to the following equations. For hybrids less than 18 base pairs in length, Tm(°C) = 2(# of A + T bases) + 4(# of G + C bases) For hybrids between 18 and 49 base pairs in length, Tm(°C) = 81.5 + 16.6(log10[Na+]) + 0.41(%G+C) - (600/N), where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridization buffer ([Na+] for lxSSC = 0.165 M)
Additional examples of stringency conditions for polynucleotide hybridization are provided in Sambrook, J., E.F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, chapters 9 and 11, and Current Protocols in Molecular Biology, 1995, F.M. Ausubel et al, eds.,
John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, incorporated herein by reference.
Preferably, each such hybridizing polynucleotide has a length that is at least 25%(more preferably at least 50%, and most preferably at least 75%) of the length of the polynucleotide of the present invention to which it hybridizes, and has at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% or 95% identity) with the polynucleotide of the present invention to which it hybridizes, where sequence identity is determined by comparing the sequences of the hybridizing polynucleotides when aligned so as to maximize overlap and identity while minimizing sequence gaps.
The isolated polynucleotide of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al, Nucleic Acids Res. 19, 4485-4490 (1991), in order to produce the protein recombinantly. Many suitable expression control sequences are known in the art. General methods of expressing recombinant proteins are also known and are exemplified in R. Kaufman, Methods in Enzymology 185, 537-566 (1990). As defined herein "operably linked" means that the isolated polynucleotide of the invention and an expression control sequence are situated within a vector or cell in such a way that the protein is expressed by a host cell which has been transformed (transfected) with the ligated polynucleotide /expression control sequence.
A number of types of cells may act as suitable host cells for expression of the protein. Mammalian host cells include, for example, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.
Alternatively, it may be possible to produce the protein in lower eukaryotes such as yeast or in prokaryotes such as bacteria. Potentially suitable yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous proteins. Potentially suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous proteins. If the protein is made in yeast or bacteria, it may be necessary to modify the protein produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain the functional protein. Such covalent attachments may be accomplished using known chemical or enzymatic methods.
The protein may also be produced by operably linking the isolated polynucleotide of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system. Materials and methods for baculovirus /insect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego, California, U.S.A. (the MaxBac® kit), and such methods are well known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987), incorporated herein by reference. As used herein, an insect cell capable of expressing a polynucleotide of the present invention is "transformed."
The protein of the invention may be prepared by culturing transformed host cells under culture conditions suitable to express the recombinant protein. The resulting expressed protein may then be purified from such culture (i.e., from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography. The purification of the protein may also include an affinity column containing agents which will bind to the protein; one or more column steps over such affinity resins as concanavalin A-agarose, heparin-toyopearl® or Cibacrom blue 3GA Sepharose®; one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography.
Alternatively, the protein of the invention may also be expressed in a form which will facilitate purification. For example, it may be expressed as a fusion protein, such as those of maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX). Kits for expression and purification of such fusion proteins are commercially available from New England BioLab (Beverly, MA), Pharmacia (Piscataway, NJ) and InVitrogen, respectively. The protein can also be tagged with an epitope and subsequently purified by using a specific antibody directed to such epitope. One such epitope ("Flag") is commercially available from Kodak (New Haven, CT).
Finally, one or more reverse-phase high performance liquid chromatography (RP- HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify the protein. Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant protein. The protein thus purified is substantially free of other mammalian proteins and is defined in accordance with the present invention as an "isolated protein."
The protein of the invention may also be expressed as a product of transgenic animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells containing a nucleotide sequence encoding the protein.
The protein may also be produced by known conventional chemical synthesis. Methods for constructing the proteins of the present invention by synthetic means are known to those skilled in the art. The synthetically-constructed protein sequences, by virtue of sharing primary, secondary or tertiary structural and /or conformational characteristics with proteins may possess biological properties in common therewith, including protein activity. Thus, they may be employed as biologically active or immunological substitutes for natural, purified proteins in screening of therapeutic compounds and in immunological processes for the development of antibodies.
The proteins provided herein also include proteins characterized by amino acid sequences similar to those of purified proteins but into which modification are naturally provided or deliberately engineered. For example, modifications in the peptide or DNA sequences can be made by those skilled in the art using known techniques. Modifications of interest in the protein sequences may include the alteration, substitution, replacement, insertion or deletion of a selected amino acid residue in the coding sequence. For example, one or more of the cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule. Techniques for such alteration, substitution, replacement, insertion or deletion are well known to those skilled in the art (see, e.g., U.S. Patent No.4,518,584). Preferably, such alteration, substitution, replacement, insertion or deletion retains the desired activity of the protein.
Other fragments and derivatives of the sequences of proteins which would be expected to retain protein activity in whole or in part and may thus be useful for screening or other immunological methodologies may also be easily made by those skilled in the art given the disclosures herein. Such modifications are believed to be encompassed by the present invention.
USES AND BIOLOGICAL ACTIVITY The polynucleotides and proteins of the present invention are expected to exhibit one or more of the uses or biological activities (including those associated with assays cited herein) identified below. Uses or activities described for proteins of the present invention may be provided by administration or use of such proteins or by administration or use of polynucleotides encoding such proteins (such as, for example, in gene therapies or vectors suitable for introduction of DNA).
Research Uses and Utilities
The polynucleotides provided by the present invention can be used by the research community for various purposes. The polynucleotides can be used to express recombinant protein for analysis, characterization or therapeutic use; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in disease states); as molecular weight markers on Southern gels; as chromosome markers or tags (when labeled) to identify chromosomes or to map related gene positions; to compare with endogenous DNA sequences in patients to identify potential genetic disorders; as probes to hybridize and thus discover novel, related DNA sequences; as a source of information to derive PCR primers for genetic fingerprinting; as a probe to "subtract-out" known sequences in the process of discovering other novel polynucleotides; for selecting and making oligomers for attachment to a "gene chip" or other support, including for examination of expression patterns; to raise anti-protein antibodies using DNA immunization techniques; and as an antigen to raise anti-DNA antibodies or elicit another immune response. Where the polynucleotide encodes a protein which binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction), the polynucleotide can also be used in interaction trap assays (such as, for example, that described in Gyuris et al., Cell 75:791-803 (1993)) to identify polynucleotides encoding the other protein with which binding occurs or to identify inhibitors of the binding interaction.
The proteins provided by the present invention can similarly be used in assay to determine biological activity, including in a panel of multiple proteins for high- throughput screening; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its receptor) in biological fluids; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state); and, of course, to isolate correlative receptors or ligands. Where the protein binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction), the protein can be used to identify the other protein with which binding occurs or to identify inhibitors of the binding interaction. Proteins involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction.
Any or all of these research utilities are capable of being developed into reagent grade or kit format for commercialization as research products.
Methods for performing the uses listed above are well known to those skilled in the art. References disclosing such methods include without limitation "Molecular
Cloning: A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E.F. Fritsch and T. Maniatis eds., 1989, and "Methods in Enzymology: Guide to Molecular Cloning Techniques", Academic Press, Berger, S.L. and A.R. Kimmel eds., 1987.
Nutritional Uses
Polynucleotides and proteins of the present invention can also be used as nutritional sources or supplements. Such uses include without limitation use as a protein or amino acid supplement, use as a carbon source, use as a nitrogen source and use as a source of carbohydrate. In such cases the protein or polynucleotide of the invention can be added to the feed of a particular organism or can be administered as a separate solid or liquid preparation, such as in the form of powder, pills, solutions, suspensions or capsules. In the case of microorganisms, the protein or polynucleotide of the invention can be added to the medium in or on which the microorganism is cultured. Cytokine and Cell Proliferation /Differentiation Activity
A protein of the present invention may exhibit cytokine, cell proliferation (either inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may induce production of other cytokines in certain cell populations. Many protein factors discovered to date, including all known cytokines, have exhibited activity in one or more factor dependent cell proliferation assays, and hence the assays serve as a convenient confirmation of cytokine activity. The activity of a protein of the present invention is evidenced by any one of a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+ (preB M+), 2E8, RB5, DAI, 123, T1165, HT2, CTLL2, TF-1, Mo7e and CMK.
The activity of a protein of the invention may, among other means, be measured by the following methods:
Assays for T-cell or thymocyte proliferation include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley- Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500, 1986; BertagnoUi et al., J. Immunol. 145:1706-1712, 1990; BertagnoUi et al., Cellular Immunology 133:327-341, 1991; BertagnoUi, et al., J. Immunol. 149:3778-3783, 1992; Bowman et al., J. Immunol. 152: 1756-1761, 1994.
Assays for cytokine production and/or proliferation of spleen cells, lymph node cells or thymocytes include, without limitation, those described in: Polyclonal T cell stimulation, Kruisbeek, A.M. and Shevach, E.M. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and
Measurement of mouse and human Interferon γ, Schreiber, R.D. In Current Protocols in
Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John Wiley and Sons, Toronto. 1994.
Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include, without limitation, those described in: Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L.S. and Lipsky, P.E. In Current
Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons, Toronto. 1991; deVries et al., J. Exp. Med. 173:1205-1211, 1991; Moreau et al., Nature 336:690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci. U.S.A. 80:2931-2938, 1983; Measurement of mouse and human interleukin 6 - Nordan, R. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto. 1991; Smith et al, Proc. Natl. Acad. Sci. U.S.A. 83:1857-1861, 1986; Measurement of human Interleukin 11 - Bennett, F., Giannotti, J., Clark, S.C. and Turner, K. J. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.15.1 John Wiley and Sons, Toronto. 1991; Measurement of mouse and human Interleukin 9 - Ciarletta, A., Giannotti, J., Clark, S.C. and Turner, K.J. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.13.1, John Wiley and Sons, Toronto. 1991.
Assays for T-cell clone responses to antigens (which will identify, among others, proteins that affect APC-T cell interactions as well as direct T-cell effects by measuring proliferation and cytokine production) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their cellular receptors; Chapter 7, Immunologic studies in Humans); Weinberger et al., Proc. Natl. Acad. Sci. USA 77:6091-6095, 1980; Weinberger et al., Eur. J. Immun. 11:405-411, 1981; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988.
Immune Stimulating or Suppressing Activity A protein of the present invention may also exhibit immune stimulating or immune suppressing activity, including without limitation the activities for which assays are described herein. A protein may be useful in the treatment of various immune deficiencies and disorders (including severe combined immunodeficiency (SOD)), e.g., in regulating (up or down) growth and proliferation of T and/or B lymphocytes, as well as effecting the cytolytic activity of NK cells and other cell populations. These immune deficiencies may be genetic or be caused by viral (e.g., HIV) as well as bacterial or fungal infections, or may result from autoimmune disorders. More specifically, infectious diseases causes by viral, bacterial, fungal or other infection may be treatable using a protein of the present invention, including infections by HIV, hepatitis viruses, herpesviruses, mycobacteria, Leishmania spp., malaria spp. and various fungal infections such as candidiasis. Of course, in this regard, a protein of the present invention may also be useful where a boost to the immune system generally may be desirable, i.e., in the treatment of cancer. Autoimmune disorders which may be treated using a protein of the present invention include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye disease. Such a protein of the present invention may also to be useful in the treatment of allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems. Other conditions, in which immune suppression is desired (including, for example, organ transplantation), may also be treatable using a protein of the present invention.
Using the proteins of the invention it may also be possible to immune responses, in a number of ways. Down regulation may be in the form of inhibiting or blocking an immune response already in progress or may involve preventing the induction of an immune response. The functions of activated T cells may be inhibited by suppressing T cell responses or by inducing specific tolerance in T cells, or both. Immunosuppression of T cell responses is generally an active, non-antigen-specific, process which requires continuous exposure of the T cells to the suppressive agent. Tolerance, which involves inducing non-responsiveness or anergy in T cells, is distinguishable from immunosuppression in that it is generally antigen-specific and persists after exposure to the tolerizing agent has ceased. Operationally, tolerance can be demonstrated by the lack of a T cell response upon reexposure to specific antigen in the absence of the tolerizing agent.
Down regulating or preventing one or more antigen functions (including without limitation B lymphocyte antigen functions (such as , for example, B7)), e.g., preventing high level lymphokine synthesis by activated T cells, will be useful in situations of tissue, skin and organ transplantation and in graft-versus-host disease (GVHD). For example, blockage of T cell function should result in reduced tissue destruction in tissue transplantation. Typically, in tissue transplants, rejection of the transplant is initiated through its recognition as foreign by T cells, followed by an immune reaction that destroys the transplant. The administration of a molecule which inhibits or blocks interaction of a B7 lymphocyte antigen with its natural ligand(s) on immune cells (such as a soluble, monomeric form of a peptide having B7-2 activity alone or in conjunction with a monomeric form of a peptide having an activity of another B lymphocyte antigen (e.g., B7- 1, B7-3) or blocking antibody), prior to transplantation can lead to the binding of the molecule to the natural ligand (s) on the immune cells without transmitting the corresponding costimulatory signal. Blocking B lymphocyte antigen function in this matter prevents cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant. Moreover, the lack of costimulation may also be sufficient to anergize the T cells, thereby inducing tolerance in a subject. Induction of long-term tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of these blocking reagents. To achieve sufficient immunosuppression or tolerance in a subject, it may also be necessary to block the function of a combination of B lymphocyte antigens. The efficacy of particular blocking reagents in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans. Examples of appropriate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in vivo as described in Lenschow et al, Science 257:789-792 (1992) and Turka et al, Proc. Natl. Acad. Sci USA, 89:11102-11105 (1992). In addition, murine models of GVHD (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 846-847) can be used to determine the effect of blocking B lymphocyte antigen function in vivo on the development of that disease. Blocking antigen function may also be therapeutically useful for treating autoimmune diseases. Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive against self tissue and which promote the production of cytokines and autoantibodies involved in the pathology of the diseases. Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms. Administration of reagents which block costimulation of T cells by disrupting receptoπligand interactions of B lymphocyte antigens can be used to inhibit T cell activation and prevent production of autoantibodies or T cell-derived cytokines which may be involved in the disease process. Additionally, blocking reagents may induce antigen-specific tolerance of autoreactive T cells which could lead to long-term relief from the disease. The efficacy of blocking reagents in preventing or alleviating autoimmune disorders can be determined using a number of well-characterized animal models of human autoimmune diseases. Examples include murine experimental autoimmune encephalitis, systemic lupus erythmatosis in MK /lprApr mice or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and murine experimental myasthenia gravis (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 840-856).
Upregulation of an antigen function (preferably a B lymphocyte antigen function), as a means of up regulating immune responses, may also be useful in therapy. Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response. For example, enhancing an immune response through stimulating B lymphocyte antigen function may be useful in cases of viral infection. In addition, systemic viral diseases such as influenza, the common cold, and encephalitis might be alleviated by the administration of stimulatory forms of B lymphocyte antigens systemically.
Alternatively, anti-viral immune responses may be enhanced in an infected patient by removing T cells from the patient, costimulating the T cells in vitro with viral antigen- pulsed APCs either expressing a peptide of the present invention or together with a stimulatory form of a soluble peptide of the present invention and reintroducing the in vitro activated T cells into the patient. Another method of enhancing anti-viral immune responses would be to isolate infected cells from a patient, transfect them with a nucleic acid encoding a protein of the present invention as described herein such that the cells express all or a portion of the protein on their surface, and reintroduce the transfected cells into the patient. The infected cells would now be capable of delivering a costimulatory signal to, and thereby activate, T cells in vivo.
In another application, up regulation or enhancement of antigen function (preferably B lymphocyte antigen function) may be useful in the induction of tumor immunity. Tumor cells (e.g., sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, carcinoma) transfected with a nucleic acid encoding at least one peptide of the present invention can be administered to a subject to overcome tumor-specific tolerance in the subject. If desired, the tumor cell can be transfected to express a combination of peptides. For example, tumor cells obtained from a patient can be transfected ex vivo with an expression vector directing the expression of a peptide having B7-2-like activity alone, or in conjunction with a peptide having B7-l-like activity and /or B7-3-like activity. The transfected tumor cells are returned to the patient to result in expression of the peptides on the surface of the transfected cell. Alternatively, gene therapy techniques can be used to target a tumor cell for transfection in vivo.
The presence of the peptide of the present invention having the activity of a B lymphocyte antigen(s) on the surface of the tumor cell provides the necessary costimulation signal to T cells to induce a T cell mediated immune response against the transfected tumor cells. In addition, tumor cells which lack MHC class I or MHC class II molecules, or which fail to reexpress sufficient amounts of MHC class I or MHC class II molecules, can be transfected with nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class I chain protein and β2 microglobulin protein or an MHC class II α chain protein and an MHC class II β chain protein to thereby express MHC class I or MHC class II proteins on the cell surface. Expression of the appropriate class I or class II MHC in conjunction with a peptide having the activity of a B lymphocyte antigen (e.g., B7-1, B7-2, B7-3) induces a T cell mediated immune response against the transfected tumor cell. Optionally, a gene encoding an antisense construct which blocks expression of an MHC class II associated protein, such as the invariant chain, can also be cotransfected with a DNA encoding a peptide having the activity of a B lymphocyte antigen to promote presentation of tumor associated antigens and induce tumor specific immunity. Thus, the induction of a T cell mediated immune response in a human subject may be sufficient to overcome tumor-specific tolerance in the subject.
The activity of a protein of the invention may, among other means, be measured by the following methods:
Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1- 3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl. Acad. Sci. USA 78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J. Immunol. 135:1564-1572, 1985; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al.,
J. Immunol. 140:508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA 78:2488-2492, 1981; Herrmann et al, J. Immunol. 128:1968-1974, 1982; Handa et al., J. Immunol. 135:1564-1572, 1985; Takai et al., J. Immunol. 137:3494-3500, 1986; Bowmanet al., J. Virology 61:1992-1998; Takai et al., J. Immunol. 140:508-512, 1988; BertagnoUi et al., Cellular Immunology 133:327-341, 1991; Brown et al., J. Immunol. 153:3079-3092, 1994.
Assays for T-cell-dependent immunoglobulin responses and isotype switching (which will identify, among others, proteins that modulate T-cell dependent antibody responses and that affect Thl/Th2 profiles) include, without limitation, those described in: Maliszewski, J. Immunol. 144:3028-3033, 1990; and Assays for B cell function: In vitro antibody production, Mond, J.J. and Brunswick, M. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto. 1994.
Mixed lymphocyte reaction (MLR) assays (which will identify, among others, proteins that generate predominantly Thl and CTL responses) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley- Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988; BertagnoUi et al., J. Immunol. 149:3778-3783, 1992. Dendritic cell-dependent assays (which will identify, among others, proteins expressed by dendritic cells that activate naive T-cells) include, without limitation, those described in: Guery et al., J. Immunol. 134:536-544, 1995; Inaba et al., Journal of Experimental Medicine 173:549-559, 1991; Macatonia et al., Journal of Immunology 154:5071-5079, 1995; Porgador et al., Journal of Experimental Medicine 182:255-260, 1995; Nair et al., Journal of Virology 67:4062-4069, 1993; Huang et al., Science 264:961-965, 1994; Macatonia et al., Journal of Experimental Medicine 169:1255-1264, 1989; Bhardwaj et al., Journal of Clinical Investigation 94:797-807, 1994; and Inaba et al., Journal of Experimental Medicine 172:631-640, 1990.
Assays for lymphocyte survival /apoptosis (which will identify, among others, proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis) include, without limitation, those described in: Darzynkiewicz et al., Cytometry 13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca et al., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991; Zacharchuk, Journal of Immunology 145:4037-4045, 1990; Zamai et al., Cytometry 14:891-897, 1993; Gorczyca et al., International Journal of Oncology 1:639-648, 1992.
Assays for proteins that influence early steps of T-cell commitment and development include, without limitation, those described in: Antica et al., Blood 84:111-117, 1994; Fine et al., Cellular Immunology 155:111-122, 1994; Galy et al., Blood 85:2770-2778, 1995; Toki et al., Proc. Nat. Acad Sci. USA 88:7548-7551, 1991.
Hematopoiesis Regulating Activity
A protein of the present invention may be useful in regulation of hematopoiesis and, consequently, in the treatment of myeloid or lymphoid cell deficiencies. Even marginal biological activity in support of colony forming cells or of factor-dependent cell lines indicates involvement in regulating hematopoiesis, e.g. in supporting the growth and proliferation of erythroid progenitor cells alone or in combination with other cytokines, thereby indicating utility, for example, in treating various anemias or for use in conjunction with irradiation /chemotherapy to stimulate the production of erythroid precursors and /or erythroid cells; in supporting the growth and proliferation of myeloid cells such as granulocytes and monocytes/macrophages (i.e., traditional CSF activity) useful, for example, in conjunction with chemotherapy to prevent or treat consequent myelo-suppression; in supporting the growth and proliferation of megakaryocytes and consequently of platelets thereby allowing prevention or treatment of various platelet disorders such as thrombocytopenia, and generally for use in place of or complimentary to platelet transfusions; and /or in supporting the growth and proliferation of hematopoietic stem cells which are capable of maturing to any and all of the above- mentioned hematopoietic cells and therefore find therapeutic utility in various stem cell disorders (such as those usually treated with transplantation, including, without limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well as in repopulating the stem cell compartment post irradiation/chemotherapy, either in-vivo or ex-vivo (i.e., in conjunction with bone marrow transplantation or with peripheral progenitor cell transplantation (homologous or heterologous)) as normal cells or genetically manipulated for gene therapy. The activity of a protein of the invention may, among other means, be measured by the following methods:
Suitable assays for proliferation and differentiation of various hematopoietic lines are cited above.
Assays for embryonic stem cell differentiation (which will identify, among others, proteins that influence embryonic differentiation hematopoiesis) include, without limitation, those described in: Johansson et al. Cellular Biology 15:141-151, 1995; Keller et al., Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al., Blood
81:2903-2915, 1993.
Assays for stem cell survival and differentiation (which will identify, among others, proteins that regulate lympho-hematopoiesis) include, without limitation, those described in: Methylcellulose colony forming assays, Freshney, M.G. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss, Inc., New York, NY. 1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992; Primitive hematopoietic colony forming cells with high proliferative potential, McNiece, I.K. and Briddell, R.A. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 23-39, Wiley-Liss, Inc., New York, NY. 1994; Neben et al., Experimental Hematology 22:353-359, 1994; Cobblestone area forming cell assay, Ploemacher, R.E. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 1-21, Wiley-Liss, Inc., New York, NY. 1994; Long term bone marrow cultures in the presence of stromal cells, Spooncer, E., Dexter, M. and Allen, T. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 163-179, Wiley-Liss, Inc., New York, NY. 1994; Long term culture initiating cell assay, Sutherland, H.J. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 139-162, Wiley-Liss, Inc., New York, NY. 1994.
Tissue Growth Activity
A protein of the present invention also may have utility in compositions used for bone, cartilage, tendon, ligament and /or nerve tissue growth or regeneration, as well as for wound healing and tissue repair and replacement, and in the treatment of burns, incisions and ulcers.
A protein of the present invention, which induces cartilage and /or bone growth in circumstances where bone is not normally formed, has application in the healing of bone fractures and cartilage damage or defects in humans and other animals. Such a preparation employing a protein of the invention may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints. De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma induced, or oncologic resection induced craniofacial defects, and also is useful in cosmetic plastic surgery.
A protein of this invention may also be used in the treatment of periodontal disease, and in other tooth repair processes. Such agents may provide an environment to attract bone-forming cells, stimulate growth of bone-forming cells or induce differentiation of progenitors of bone-forming cells. A protein of the invention may also be useful in the treatment of osteoporosis or osteoarthritis, such as through stimulation of bone and/or cartilage repair or by blocking inflammation or processes of tissue destruction (collagenase activity, osteoclast activity, etc.) mediated by inflammatory processes.
Another category of tissue regeneration activity that may be attributable to the protein of the present invention is tendon /ligament formation. A protein of the present invention, which induces tendon /ligament-like tissue or other tissue formation in circumstances where such tissue is not normally formed, has application in the healing of tendon or ligament tears, deformities and other tendon or ligament defects in humans and other animals. Such a preparation employing a tendon /ligament-like tissue inducing protein may have prophylactic use in preventing damage to tendon or ligament tissue, as well as use in the improved fixation of tendon or ligament to bone or other tissues, and in repairing defects to tendon or ligament tissue. De novo tendon/ligament-like tissue formation induced by a composition of the present invention contributes to the repair of congenital, trauma induced, or other tendon or ligament defects of other origin, and is also useful in cosmetic plastic surgery for attachment or repair of tendons or ligaments. The compositions of the present invention may provide an environment to attract tendon- or ligament-forming cells, stimulate growth of tendon- or ligament-forming cells, induce differentiation of progenitors of tendon- or ligament-forming cells, or induce growth of tendon /ligament cells or progenitors ex vivo for return in vivo to effect tissue repair. The compositions of the invention may also be useful in the treatment of tendinitis, carpal runnel syndrome and other tendon or ligament defects. The compositions may also include an appropriate matrix and /or sequestering agent as a carrier as is well known in the art.
The protein of the present invention may also be useful for proliferation of neural cells and for regeneration of nerve and brain tissue, i.e. for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders, which involve degeneration, death or trauma to neural cells or nerve tissue. More specifically, a protein may be used in the treatment of diseases of the peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome. Further conditions which may be treated in accordance with the present invention include mechanical and traumatic disorders, such as spinal cord disorders, head trauma and cerebrovascular diseases such as stroke. Peripheral neuropathies resulting from chemotherapy or other medical therapies may also be treatable using a protein of the invention.
Proteins of the invention may also be useful to promote better or faster closure of non-healing wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like. It is expected that a protein of the present invention may also exhibit activity for generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac) and vascular (including vascular endothelium) tissue, or for promoting the growth of cells comprising such tissues. Part of the desired effects may be by inhibition or modulation of fibrotic scarring to allow normal tissue to regenerate. A protein of the invention may also exhibit angiogenic activity.
A protein of the present invention may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and conditions resulting from systemic cytokine damage.
A protein of the present invention may also be useful for promoting or inhibiting differentiation of tissues described above from precursor tissues or cells; or for inhibiting the growth of tissues described above.
The activity of a protein of the invention may, among other means, be measured by the following methods:
Assays for tissue generation activity include, without limitation, those described in: International Patent Publication No. WO95/16035 (bone, cartilage, tendon); International Patent Publication No. W095/ 05846 (nerve, neuronal); International Patent Publication No. WO91/07491 (skin, endothelium ). Assays for wound healing activity include, without limitation, those described in:
Winter, Epidermal Wound Healing, pps. 71-112 (Maibach, HI and Rovee, DT, eds.), Year Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J. Invest. Dermatol 71:382-84 (1978).
Activin/ Inhibin Activity
A protein of the present invention may also exhibit activin- or inhibin-related activities. Inhibins are characterized by their ability to inhibit the release of follicle stimulating hormone (FSH), while activins and are characterized by their ability to stimulate the release of follicle stimulating hormone (FSH). Thus, a protein of the present invention, alone or in heterodimers with a member of the inhibin α family, may be useful as a contraceptive based on the ability of inhibins to decrease fertility in female mammals and decrease spermatogenesis in male mammals. Administration of sufficient amounts of other inhibins can induce infertility in these mammals. Alternatively, the protein of the invention, as a homodimer or as a heterodimer with other protein subunits of the inhibin- β group, may be useful as a fertility inducing therapeutic, based upon the ability of activin molecules in stimulating FSH release from cells of the anterior pituitary. See, for example, United States Patent 4,798,885. A protein of the invention may also be useful for advancement of the onset of fertility in sexually immature mammals, so as to increase the lifetime reproductive performance of domestic animals such as cows, sheep and pigs.
The activity of a protein of the invention may, among other means, be measured by the following methods:
Assays for activin/inhibin activity include, without limitation, those described in: Vale et al., Endocrinology 91:562-572, 1972; Ling et al., Nature 321:779-782, 1986; Vale et al., Nature 321:776-779, 1986; Mason et al., Nature 318:659-663, 1985; Forage et al., Proc. Natl. Acad. Sci. USA 83:3091-3095, 1986.
Chemotactic/Chemokinetic Activity
A protein of the present invention may have chemotactic or chemokinetic activity (e.g., act as a chemokine) for mammalian cells, including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and /or endothelial cells. Chemotactic and chemokinetic proteins can be used to mobilize or attract a desired cell population to a desired site of action. Chemotactic or chemokinetic proteins provide particular advantages in treatment of wounds and other trauma to tissues, as well as in treatment of localized infections. For example, attraction of lymphocytes, monocytes or neutrophils to tumors or sites of infection may result in improved immune responses against the tumor or infecting agent.
A protein or peptide has chemotactic activity for a particular cell population if it can stimulate, directly or indirectly, the directed orientation or movement of such cell population. Preferably, the protein or peptide has the ability to directly stimulate directed movement of cells. Whether a particular protein has chemotactic activity for a population of cells can be readily determined by employing such protein or peptide in any known assay for cell chemotaxis.
The activity of a protein of the invention may, among other means, be measured by the following methods:
Assays for chemotactic activity (which will identify proteins that induce or prevent chemotaxis) consist of assays that measure the ability of a protein to induce the migration of cells across a membrane as well as the ability of a protein to induce the adhesion of one cell population to another cell population. Suitable assays for movement and adhesion include, without limitation, those described in: Current Protocols in Immunology, Ed by J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W.Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest. 95:1370-1376, 1995; Lind et al. APMIS 103:140-146, 1995; Muller et al Eur. J. Immunol. 25: 1744-1748; Gruber et al. J. of Immunol. 152:5860-5867, 1994; Johnston et al. J. of Immunol. 153: 1762-1768, 1994.
Hemostatic and Thrombolytic Activity
A protein of the invention may also exhibit hemostatic or thrombolytic activity. As a result, such a protein is expected to be useful in treatment of various coagulation disorders (including hereditary disorders, such as hemophilias) or to enhance coagulation and other hemostatic events in treating wounds resulting from trauma, surgery or other causes. A protein of the invention may also be useful for dissolving or inhibiting formation of thromboses and for treatment and prevention of conditions resulting therefrom (such as, for example, infarction of cardiac and central nervous system vessels (e.g., stroke).
The activity of a protein of the invention may, among other means, be measured by the following methods:
Assay for hemostatic and thrombolytic activity include, without limitation, those described in: Linet et al., J. Clin. Pharmacol. 26:131-140, 1986; Burdick et al., Thrombosis Res.45:413-419, 1987; Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub, Prostaglandins 35:467-474, 1988.
Receptor/Ligand Activity A protein of the present invention may also demonstrate activity as receptors, receptor ligands or inhibitors or agonists of receptor/ligand interactions. Examples of such receptors and ligands include, without limitation, cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, receptors involved in cell-cell interactions and their ligands (including without limitation, cellular adhesion molecules (such as selectins, integrins and their ligands) and receptor/ligand pairs involved in antigen presentation, antigen recognition and development of cellular and humoral immune responses). Receptors and ligands are also useful for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction. A protein of the present invention (including, without limitation, fragments of receptors and ligands) may themselves be useful as inhibitors of receptor/ligand interactions.
The activity of a protein of the invention may, among other means, be measured by the following methods: Suitable assays for receptor-ligand activity include without limitation those described in:Current Protocols in Immunology, Ed by J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W.Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under static conditions 7.28.1-7.28.22), Takai et al, Proc. Natl. Acad. Sci. USA 84:6864-6868, 1987; Bierer et al., J. Exp. Med. 168:1145-1156, 1988; Rosenstein et al, J. Exp. Med. 169:149-160 1989; Stoltenborg et al., J. Immunol. Methods 175:59-68, 1994; Stitt et al., Cell 80:661-670, 1995.
Anti-Inflammatory Activity Proteins of the present invention may also exhibit anti-inflammatory activity. The anti-inflammatory activity may be achieved by providing a stimulus to cells involved in the inflammatory response, by inhibiting or promoting cell-cell interactions (such as, for example, cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the inflammatory process, inhibiting or promoting cell extravasation, or by stimulating or suppressing production of other factors which more directly inhibit or promote an inflammatory response. Proteins exhibiting such activities can be used to treat inflammatory conditions including chronic or acute conditions), including without limitation inflammation associated with infection (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or resulting from over production of cytokines such as TNF or IL-1. Proteins of the invention may also be useful to treat anaphylaxis and hypersensitivity to an antigenic substance or material.
Cadherin/ Tumor Invasion Suppressor Activity
Cadherins are calcium-dependent adhesion molecules that appear to play major roles during development, particularly in defining specific cell types. Loss or alteration of normal cadherin expression can lead to changes in cell adhesion properties linked to tumor growth and metastasis. Cadherin malfunction is also implicated in other human diseases, such as pemphigus vulgaris and pemphigus foliaceus (auto-immune blistering skin diseases), Crohn's disease, and some developmental abnormalities.
The cadherin superfamily includes well over forty members, each with a distinct pattern of expression. All members of the superfamily have in common conserved extracellular repeats (cadherin domains), but structural differences are found in other parts of the molecule. The cadherin domains bind calcium to form their tertiary structure and thus calcium is required to mediate their adhesion. Only a few amino acids in the first cadherin domain provide the basis for homophilic adhesion; modification of this recognition site can change the specificity of a cadherin so that instead of recognizing only itself, the mutant molecule can now also bind to a different cadherin. In addition, some cadherins engage in heterophilic adhesion with other cadherins.
E-cadherin, one member of the cadherin superfamily, is expressed in epithelial cell types. Pathologically, if E-cadherin expression is lost in a tumor, the malignant cells become invasive and the cancer metastasizes. Transfection of cancer cell lines with polynucleotides expressing E-cadherin has reversed cancer-associated changes by returning altered cell shapes to normal, restoring cells' adhesiveness to each other and to their substrate, decreasing the cell growth rate, and drastically reducing anchorage- independent cell growth. Thus, reintroducing E-cadherin expression reverts carcinomas to a less advanced stage. It is likely that other cadherins have the same invasion suppressor role in carcinomas derived from other tissue types. Therefore, proteins of the present invention with cadherin activity, and polynucleotides of the present invention encoding such proteins, can be used to treat cancer. Introducing such proteins or polynucleotides into cancer cells can reduce or eliminate the cancerous changes observed in these cells by providing normal cadherin expression. Cancer cells have also been shown to express cadherins of a different tissue type than their origin, thus allowing these cells to invade and metastasize in a different tissue in the body. Proteins of the present invention with cadherin activity, and polynucleotides of the present invention encoding such proteins, can be substituted in these cells for the inappropriately expressed cadherins, restoring normal cell adhesive properties and reducing or eliminating the tendency of the cells to metastasize.
Additionally, proteins of the present invention with cadherin activity, and polynucleotides of the present invention encoding such proteins, can used to generate antibodies recognizing and binding to cadherins. Such antibodies can be used to block the adhesion of inappropriately expressed tumor-cell cadherins, preventing the cells from forming a tumor elsewhere. Such an anti-cadherin antibody can also be used as a marker for the grade, pathological type, and prognosis of a cancer, i.e. the more progressed the cancer, the less cadherin expression there will be, and this decrease in cadherin expression can be detected by the use of a cadherin-binding antibody. Fragments of proteins of the present invention with cadherin activity, preferably a polypeptide comprising a decapeptide of the cadherin recognition site, and polynucleotides of the present invention encoding such protein fragments, can also be used to block cadherin function by binding to cadherins and preventing them from binding in ways that produce undesirable effects. Additionally, fragments of proteins of the present invention with cadherin activity, preferably truncated soluble cadherin fragments which have been found to be stable in the circulation of cancer patients, and polynucleotides encoding such protein fragments, can be used to disturb proper cell-cell adhesion.
Assays for cadherin adhesive and invasive suppressor activity include, without limitation, those described in: Hortsch et al. J Biol Chem 270 (32): 18809-18817, 1995; Miyaki et al. Oncogene 11: 2547-2552, 1995; Ozawa et al. Cell 63: 1033-1038, 1990.
Tumor Inhibition Activity
In addition to the activities described above for immunological treatment or prevention of tumors, a protein of the invention may exhibit other anti- tumor activities. A protein may inhibit tumor growth directly or indirectly (such as, for example, via
ADCC). A protein may exhibit its tumor inhibitory activity by acting on tumor tissue or tumor precursor tissue, by inhibiting formation of tissues necessary to support tumor growth (such as, for example, by inhibiting angiogenesis), by causing production of other factors, agents or cell types which inhibit tumor growth, or by suppressing, eliminating or inhibiting factors, agents or cell types which promote tumor growth.
Other Activities
A protein of the invention may also exhibit one or more of the following additional activities or effects: inhibiting the growth, infection or function of, or killing, infectious agents, including, without limitation, bacteria, viruses, fungi and other parasites; effecting
(suppressing or enhancing) bodily characteristics, including, without limitation, height, weight, hair color, eye color, skin, fat to lean ratio or other tissue pigmentation, or organ or body part size or shape (such as, for example, breast augmentation or diminution, change in bone form or shape); effecting biorhythms or caricadic cycles or rhythms; effecting the fertility of male or female subjects; effecting the metabolism, catabolism, anabolism, processing, utilization, storage or elimination of dietary fat, lipid, protein, carbohydrate, vitamins, minerals, cofactors or other nutritional factors or component(s); effecting behavioral characteristics, including, without limitation, appetite, libido, stress, cognition (including cognitive disorders), depression (including depressive disorders) and violent behaviors; providing analgesic effects or other pain reducing effects; promoting differentiation and growth of embryonic stem cells in lineages other than hematopoietic lineages; hormonal or endocrine activity; in the case of enzymes, correcting deficiencies of the enzyme and treating deficiency-related diseases; treatment of hyperproliferative disorders (such as, for example, psoriasis); immunoglobulin-like activity (such as, for example, the ability to bind antigens or complement); and the ability to act as an antigen in a vaccine composition to raise an immune response against such protein or another material or entity which is cross-reactive with such protein.
ADMINISTRATION AND DOSING
A protein of the present invention (from whatever source derived, including without limitation from recombinant and non-recombinant sources) may be used in a pharmaceutical composition when combined with a pharmaceutically acceptable carrier. Such a composition may also contain (in addition to protein and a carrier) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s). The characteristics of the carrier will depend on the route of administration. The pharmaceutical composition of the invention may also contain cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,
IL-12, IL-13, IL-14, IL-15, IFN, TNFO, TNF1, TNF2, G-CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin. The pharmaceutical composition may further contain other agents which either enhance the activity of the protein or compliment its activity or use in treatment. Such additional factors and/or agents may be included in the pharmaceutical composition to produce a synergistic effect with protein of the invention, or to minimize side effects. Conversely, protein of the present invention may be included in formulations of the particular cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize side effects of the cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent.
A protein of the present invention may be active in mul timers (e.g., heterodimers or homodimers) or complexes with itself or other proteins. As a result, pharmaceutical compositions of the invention may comprise a protein of the invention in such multimeric or complexed form.
The pharmaceutical composition of the invention may be in the form of a complex of the protein(s) of present invention along with protein or peptide antigens. The protein and/or peptide antigen will deliver a stimulatory signal to both B and T lymphocytes. B lymphocytes will respond to antigen through their surface immunoglobulin receptor. T lymphocytes will respond to antigen through the T cell receptor (TCR) following presentation of the antigen by MHC proteins. MHC and structurally related proteins including those encoded by class I and class II MHC genes on host cells will serve to present the peptide antigen(s) to T lymphocytes. The antigen components could also be supplied as purified MHC-peptide complexes alone or with co-stimulatory molecules that can directly signal T cells. Alternatively antibodies able to bind surface immunolgobulin and other molecules on B cells as well as antibodies able to bind the TCR and other molecules on T cells can be combined with the pharmaceutical composition of the invention. The pharmaceutical composition of the invention may be in the form of a liposome in which protein of the present invention is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Patent No. 4,235,871; U.S. Patent No. 4,501,728; U.S. Patent No. 4,837,028; and U.S. Patent No. 4,737,323, all of which are incorporated herein by reference. As used herein, the term "therapeutically effective amount" means the total amount of each active component of the pharmaceutical composition or method that is sufficient to show a meaningful patient benefit, i.e., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously. In practicing the method of treatment or use of the present invention, a therapeutically effective amount of protein of the present invention is administered to a mammal having a condition to be treated. Protein of the present invention may be administered in accordance with the method of the invention either alone or in combination with other therapies such as treatments employing cytokines, lymphokines or other hematopoietic factors. When co-administered with one or more cytokines, lymphokines or other hematopoietic factors, protein of the present invention may be administered either simultaneously with the cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or sequentially. If administered sequentially, the attending physician will decide on the appropriate sequence of administering protein of the present invention in combination with cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors.
Administration of protein of the present invention used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection. Intravenous administration to the patient is preferred.
When a therapeutically effective amount of protein of the present invention is administered orally, protein of the present invention will be in the form of a tablet, capsule, powder, solution or elixir. When administered in tablet form, the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant. The tablet, capsule, and powder contain from about 5 to 95% protein of the present invention, and preferably from about 25 to 90% protein of the present invention. When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added. The liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the pharmaceutical composition contains from about 0.5 to 90% by weight of protein of the present invention, and preferably from about 1 to 50% protein of the present invention.
When a therapeutically effective amount of protein of the present invention is administered by intravenous, cutaneous or subcutaneous injection, protein of the present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable protein solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art. A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to protein of the present invention, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride
Injection, Lactated Ringer's Injection, or other vehicle as known in the art. The pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art.
The amount of protein of the present invention in the pharmaceutical composition of the present invention will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the patient has undergone. Ultimately, the attending physician will decide the amount of protein of the present invention with which to treat each individual patient. Initially, the attending physician will administer low doses of protein of the present invention and observe the patient's response. Larger doses of protein of the present invention may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further. It is contemplated that the various pharmaceutical compositions used to practice the method of the present invention should contain about 0.01 μg to about 100 mg (preferably about O.lng to about 10 mg, more preferably about 0.1 μg to about 1 mg) of protein of the present invention per kg body weight.
The duration of intravenous therapy using the pharmaceutical composition of the present invention will vary, depending on the severity of the disease being treated and the condition and potential idiosyncratic response of each individual patient. It is contemplated that the duration of each application of the protein of the present invention will be in the range of 12 to 24 hours of continuous intravenous administration.
Ultimately the attending physician will decide on the appropriate duration of intravenous therapy using the pharmaceutical composition of the present invention.
Protein of the invention may also be used to immunize animals to obtain polyclonal and monoclonal antibodies which specifically react with the protein. Such antibodies may be obtained using either the entire protein or fragments thereof as an immunogen. The peptide immunogens additionally may contain a cysteine residue at the carboxyl terminus, and are conjugated to a hapten such as keyhole limpet hemocyanin (KLH). Methods for synthesizing such peptides are known in the art, for example, as in R.P. Merrifield, J. Amer.Chem.Soc. 85, 2149-2154 (1963); J.L. Krstenansky, et al, FEBS Lett. 211, 10 (1987). Monoclonal antibodies binding to the protein of the invention may be useful diagnostic agents for the immunodetection of the protein. Neutralizing monoclonal antibodies binding to the protein may also be useful therapeutics for both conditions associated with the protein and also in the treatment of some forms of cancer where abnormal expression of the protein is involved. In the case of cancerous cells or leukemic cells, neutralizing monoclonal antibodies against the protein may be useful in detecting and preventing the metastatic spread of the cancerous cells, which may be mediated by the protein.
For compositions of the present invention which are useful for bone, cartilage, tendon or ligament regeneration, the therapeutic method includes administering the composition topically, systematically, or locally as an implant or device. When administered, the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically acceptable form. Further, the composition may desirably be encapsulated or injected in a viscous form for delivery to the site of bone, cartilage or tissue damage. Topical administration may be suitable for wound healing and tissue repair. Therapeutically useful agents other than a protein of the invention which may also optionally be included in the composition as described above, may alternatively or additionally, be administered simultaneously or sequentially with the composition in the methods of the invention. Preferably for bone and /or cartilage formation, the composition would include a matrix capable of delivering the protein-containing composition to the site of bone and /or cartilage damage, providing a structure for the developing bone and cartilage and optimally capable of being resorbed into the body. Such matrices may be formed of materials presently in use for other implanted medical applications. The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties. The particular application of the compositions will define the appropriate formulation. Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydrides. Other potential materials are biodegradable and biologically well- defined, such as bone or dermal collagen. Further matrices are comprised of pure proteins or extracellular matrix components. Other potential matrices are nonbiodegradable and chemically defined, such as sintered hydroxapatite, bioglass, aluminates, or other ceramics. Matrices may be comprised of combinations of any of the above mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalciumphosphate. The bioceramics may be altered in composition, such as in calcium- aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradability. Presently preferred is a 50:50 (mole weight) copolymer of lactic acid and glycolic acid in the form of porous particles having diameters ranging from 150 to 800 microns. In some applications, it will be useful to utilize a sequestering agent, such as carboxymethyl cellulose or autologous blood clot, to prevent the protein compositions from disassociating from the matrix. A preferred family of sequestering agents is cellulosic materials such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl- methylcellulose, and carboxymethylcellulose, the most preferred being cationic salts of carboxymethylcellulose (CMC). Other preferred sequestering agents include hyaluronic acid, sodium alginate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol). The amount of sequestering agent useful herein is 0.5-20 wt%, preferably 1-10 wt% based on total formulation weight, which represents the amount necessary to prevent desorbtion of the protein from the polymer matrix and to provide appropriate handling of the composition, yet not so much that the progenitor cells are prevented from infiltrating the matrix, thereby providing the protein the opportunity to assist the osteogenic activity of the progenitor cells.
In further compositions, proteins of the invention may be combined with other agents beneficial to the treatment of the bone and /or cartilage defect, wound, or tissue in question. These agents include various growth factors such as epidermal growth factor (EGF), platelet derived growth factor (PDGF), transforming growth factors (TGF-α and
TGF-β), and insulin-like growth factor (IGF).
The therapeutic compositions are also presently valuable for veterinary applications. Particularly domestic animals and thoroughbred horses, in addition to humans, are desired patients for such treatment with proteins of the present invention. The dosage regimen of a protein-containing pharmaceutical composition to be used in tissue regeneration will be determined by the attending physician considering various factors which modify the action of the proteins, e.g., amount of tissue weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and diet, the severity of any infection, time of administration and other clinical factors. The dosage may vary with the type of matrix used in the reconstitution and with inclusion of other proteins in the pharmaceutical composition. For example, the addition of other known growth factors, such as IGF I (insulin like growth factor I), to the final composition, may also effect the dosage. Progress can be monitored by periodic assessment of tissue/bone growth and/or repair, for example, X-rays, histomorphometric determinations and tetracycline labeling.
Polynucleotides of the present invention can also be used for gene therapy. Such polynucleotides can be introduced either in vivo or ex vivo into cells for expression in a mammalian subject. Polynucleotides of the invention may also be administered by other known methods for introduction of nucleic acid into a cell or organism (including, without limitation, in the form of viral vectors or naked DNA).
Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic purposes.
Patent and literature references cited herein are incorporated by reference as if fully set forth.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Jacobs, Kenneth McCoy, John M. LaVallie, Edward R. Racie, Lisa A. Merberg, David Treacy, Maurice Spaulding, Vikki Agostino, Michael
(ii) TITLE OF INVENTION: SECRETED PROTEINS AND POLYNUCLEOTIDES ENCODING THEM
(iii) NUMBER OF SEQUENCES: 32
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Genetics Institute, Inc.
(B) STREET: 87 CambridgePark Drive
(C) CITY: Cambridge
(D) STATE: MA
(E) COUNTRY: U.S.A.
(F) ZIP: 02140
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentin Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Sprunger, Suzanne A.
(B) REGISTRATION NUMBER: 41,323
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (617) 498-8284
(B) TELEFAX: (617) 876-5851
(2) INFORMATION FOR SEQ ID NO : 1 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1328 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 :
CGTTAACAAG TGGAATGGAA CTCAAAGACA ATATTCTTGT CTCTGGGAAT GCAGATTCTA 60
CAGTTAAAAT CTGGGATATC AAAACAGGAC AGTGTTTACA AACATTGCAA GGTCCCAACA 120
AGCATCAGAG TGCTGTGACC TGTTTACAGT TCAACAAGAA CTTTGTAATT ACCAGCTCAG 180
ATGATGGAAC TGTAAAACTA TGGGACTTGA AAACGGGTGA ATTTATTCGA AACCTAGTCA 240
CATTGGAGAG TGGGGGGAGT GGGGGAGTTG TGTGGCGGAT CACAGCCTCA AACACAAAGC 300
TGGTGTGTGC AGTTGGGAGT CGGAATGGGA CTGAAGAAAC CAAGCTGCTG GTGCTGGACT 360
TTGATGTGGA CATGAAGTGA AGAGCAGAAA AGATGAATTT GTCCAATTGT GTAGACGATA 420
TACTCCCTGC CCTTCCCCCT GCAAAAAGAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 480
AAAAAAAAAA AAAAAAGCGC AAGCTCCAGG TTTCACCACA ATGCCCATCC CTGCCTCCCC 540
ACTCCACCCA CCTCTGCCTT CCTTACTGCT GTATCTGCTG CTTGAACTGG CAGGAGTCAC 600
ACATGTGTTC CATGTGCAAC AAACGGAGAT GTCACAGACT GTATCAACTG GGGAGTCAAT 660
CATCTTGAGT TGCAGCGTAC CCGATACCTT ACCAAATGGA CCTGTCTTGT GGTTCAAGGG 720
AACAGGGCCA AACCGGAAAT TAATCTACAA TTTCAAACAA GGTAACTTTC CCAGAGTAAA 780
AGAGATTGGA GACACCACCA AGCCTGGCAA CACAGACTTT TCCACCCGCA TCCGTGAAAT 840
CTCTCTTGCT GATGCTGGCA CCTATTACTG CGTGAAGTTC ATAAAAGGAA GAGCTATCAA 900
GGAGTACCAA TCAGGTCGGG GCACTCAGGT GTTTGTTACT GAGCAGAATC CAAGACCTCC 960
CAAGAACAGA CCTGCAGGCA GAGCAGGCTC CAGGGCCCAC CATGATGCCC ATACCTGCCT 1020
CTCGGCCCTG CCTGAGAGAA ACAGCACAAA CTATTTCGTC CAACCCTGCT GCTGCCTCCG 1080
GCTGCTGGGA CTCACAGGCT TGCTGTCAAA ATAATCCAAA CAGGGAAGGA ACGTACAAGT 1140
AAATAACAAA AGCCCCCATA CTCTTCTGAC TCCCTGGAGA CAGCTACTTT TTAGGAGTTT 1200
CATTTGCCTT CTTCAAGAGA GCTTTCTTCC ACTGACATAA AATGCCAGCT TGATCGTACA 1260
ATAAATCTGT CTATTTACCT GGGTCCAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 1320
AAAAAAAA 1328
(2) INFORMATION FOR SEQ ID NO : 2 : (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 197 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2 :
Met Pro lie Pro Ala Ser Pro Leu His Pro Pro Leu Pro Ser Leu Leu 1 5 10 15
Leu Tyr Leu Leu Leu Glu Leu Ala Gly Val Thr His Val Phe His Val 20 25 30
Gin Gin Thr Glu Met Ser Gin Thr Val Ser Thr Gly Glu Ser lie lie 35 40 45
Leu Ser Cys Ser Val Pro Asp Thr Leu Pro Asn Gly Pro Val Leu Trp 50 55 60
Phe Lys Gly Thr Gly Pro Asn Arg Lys Leu lie Tyr Asn Phe Lys Gin 65 70 75 80
Gly Asn Phe Pro Arg Val Lys Glu lie Gly Asp Thr Thr Lys Pro Gly 85 90 95
Asn Thr Asp Phe Ser Thr Arg lie Arg Glu lie Ser Leu Ala Asp Ala 100 105 110
Gly Thr Tyr Tyr Cys Val Lys Phe lie Lys Gly Arg Ala lie Lys Glu 115 120 125
Tyr Gin Ser Gly Arg Gly Thr Gin Val Phe Val Thr Glu Gin Asn Pro 130 135 140
Arg Pro Pro Lys Asn Arg Pro Ala Gly Arg Ala Gly Ser Arg Ala His 145 150 155 160
His Asp Ala His Thr Cys Leu Ser Ala Leu Pro Glu Arg Asn Ser Thr 165 170 175
Asn Tyr Phe Val Gin Pro Cys Cys Cys Leu Arg Leu Leu Gly Leu Thr 180 185 190
Gly Leu Leu Ser Lys 195
(2) INFORMATION FOR SEQ ID NO : 3 :
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2835 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 :
GCTTGATAAG CCAGCTTCAG GAGTAAAGGA AGAATGGTAT GCCAGAATCA CTAAATTAAG 60
ATGGTGGATC AGCTTTTCTG CAAAAAATTT GCGGAAGCCT TGGGGAGCAC TGAAGCCAAG 120
GCTGTACCGT ACCAAAAATT TGAGGCACAC CCGAATGATC TGTACGTGGA AGGACTGCCA 180
GAAAACATTC CTTTCCGAAG TCCCTCATGG TATGGAATCC CAAGGCTGGA AAAAATCATT 240
CAAGTGGGCA ATCGAATTAA ATTTGTTATT AAAAGACCAG AACTTCTGAC TCACAGTACC 300
ACTGAAGTTA CTCAGCCAAG AACGAATACA CCAGTCAAAG AAGATTGGAA TGTCAGAATT 360
ACCAAGCTAC GGAAGCAAGT GGAAGAGATT TTTAATTTGA AATTTGCTCA AGCTCTTGGA 420
CTCACCGAGG CAGTAAAAGT ACCATATCCT GTGTTTGAAT CAAACCCGGA GTTCTTGTAT 480
GTGGAAGGCT TGCCAGAGGG GATTCCCTTC CGAAGCCCTA CCTGGTTTGG AATTCCACGA 540
CTTGAAAGGA TCGTCCACGG GAGTAATAAA ATCAAGTTCG TTGTTAAAAA ACCTGAACTA 600
GTTATTTCCT ACTTGCCTCC TGGGATGGCT AGTAAAATAA ACACTAAAGC TTTGCAGTCC 660
CCCAAAAGAC CACGAAGTCC TGGGAGTAAT TCAAAGGTTC CTGAAATTGA GGTCACCGTG 720
GAAGGCCYTA ATAACAACAA TCCTCAAACC TCAGCTGTTC GAACCCCGAC CCAGACTAAC 780
GGTTCTAACG TTCCCTTCAA GCCACGAGGG AGAGAGTTTT CCTTTGGCCT GGAATGCCAA 840
AATCACGGAC CTAAAACAGA AAGTTGAAAA TCTCTTCAAT GAGAAATGTG GGGAAGCTCT 900
TGGCCTTAAA CAAGCTGTGA AGGTGCCGTT CGCGTTATTT GAGTCTTTCC CGGAAGACTT 960
TTATGTGGAA GGCTTACCTG AGGGTGTGCC ATTCCGAAGA CCATCGACTT TTGGCATTCC 1020
GAGGCTGGAG AAGATACTCA GAAACAAAGC CAAAATTAAG TTCATCATTA AAAAGCCCGA 1080
AATGTTTGAG ACGGCGATTA AGGAGAGCAC CTCCTCTAAG AGCCCTCCCA GAAAAATAAA 1140
TTCATCACCC AATGTTAATA CTACTGCATC AGGTGTTGAA GACCTTAACA TCATTCAGGT 1200
GACAATTCCA GATGATGATA ATGAAAGACT CTCGAAAGTT GAAAAAGCTA GACAGCTAAG 1260
AGAACAAGTG AATGACCTCT TTAGTCGGAA ATTTGGTGAA GCTATTGGTA TGGGTTTTCC 1320 TGTGAAAGTT CCCTACAGGA AAATCACAAT TAACCCTGGC TGTGTGGTGG TTGATGGCAT 1380
GCCCCCGGGG GTGTCCTTCA AAGCCCCCAG CTACCTGGAA ATCAGCTCCA TGAGAAGGAT 1440
CTTAGACTCT GCCGAGTTTA TCAAATTCAC GGTCATTAGA CCATTTCCAG GACTTGTGAT 1500
TAATAACCAG CTGGTTGATC AGAGTGAGTC AAAAGGCCCC GTGATACAAG AATCAGCTGA 1560
ACCAAGCCAG TTGGAAGTTC CAGCCACAGA AGAAATAAAA GAGACTGATG GAAGCTCTCA 1620
GATCAAGCAA GAACCAGACC CCACGTGGTA GACCTCTTCC CTCCTAGGCT TAAAGTATCA 1680
GTGGTTGAGA AGAGCTTTTC GGACCTGTTA CTACCCCAAG CTGTGTAATA TACTTGTATA 1740
ACAGAAATAC CTTCTATACA AACCTTTTTT TCTACTTTTA GATAGAAATG TCTACTTTTT 1800
CAGCAGTTCT GTGAATTAAA GAGCAGAGTG ACTGTGGGTC TGGAATGGCT GGTGTACTTG 1860
GGAATGTACT ATCAGGATTT TACAGCAATG CTGGGAAATG ACAGGGAAAA TGACAGGAAT 1920
GAATCTCACC AGATTTTTTA TGTACTCAGC AGAGCCTTGA GTTACGGTGT TTATTTTCCA 1980
ATCAAGTGAA GATATCTCCT ACTTCTCCTA CTGGAACATC TCAGCTTCTG CAGTGAAGAA 2040
AAATTCCTGT GATAGTTCAG TTCTTTAGTT TTTCTATTTG AAAAAAAAAA ATCATTTAAA 2100
TGATCCTTTG TTCACGGCTC TCCTTAATGA CTGAGTGAAC AGTTCCTATC TGTATATTTG 2160
ACTAAACCTT TTCCTAAGCT ATCTCTCATG GTTCCTATGT TTTTTTATCA TAATTAAAAG 2220
CAAAACCATT TGGATCACCT AACAGTCAGA GGTCAGTATC TCAGCGTGTG AATTATAGAG 2280
GAAATACAGA GAGAACCTCT TCCACTTTTA CTTTTCGTCC AAATAAAATG CATGGTGTAC 2340
CAGAAGTTGA AGATCGGGTT GAGGATTGGG GCTAGCTCGA TGACACTAAG GCCCCAACAT 2400
CGCGGGACCT GCTGTGGCGC GGATTCTTAG GAACGCTGTT CTAGCCGGCC CCCTCTCCAG 2460
GGGTCGCCGT GGCCGGCATT ATTTCCTAGT TCTTCTTGTA ACCCTGAGGT GCCAGCGCGG 2520
GGAGTGAGGA GGGGTCAGGG GGCTAAGGAT GCAACCTCTG ACGTTCTGCG CCTTCCTAGG 2580
AGAGTCTTAC ATGTGTTGAG ATTTCACAAG CAATGCGAGT TGTAAAATAC CAGCTCTACA 2640
AGAAGCTAGG CTCTGTGACG GCATAGTTTT CAGTAGCTTT ATCACAATAT TCACAATGGA 2700
GAATTATATG ACATGGTAGC AGAAATAGGC CCTTTTATGT GTTGCTTCTA TTTTACCTCA 2760
AATTGTAGAT ATAGGGTAAT CAATAAAATC CATCCATGCC TTTCAAAAAA AAAAAAAAAA 2820
AAAAAAAAAA AAAAA 2835 (2) INFORMATION FOR SEQ ID NO : 4 :
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 268 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 :
Met Val Asp Gin Leu Phe Cys Lys Lys Phe Ala Glu Ala Leu Gly Ser 1 5 10 15
Thr Glu Ala Lys Ala Val Pro Tyr Gin Lys Phe Glu Ala His Pro Asn 20 25 30
Asp Leu Tyr Val Glu Gly Leu Pro Glu Asn lie Pro Phe Arg Ser Pro 35 40 45
Ser Trp Tyr Gly lie Pro Arg Leu Glu Lys lie lie Gin Val Gly Asn 50 55 60
Arg lie Lys Phe Val lie Lys Arg Pro Glu Leu Leu Thr His Ser Thr 65 70 75 80
Thr Glu Val Thr Gin Pro Arg Thr Asn Thr Pro Val Lys Glu Asp Trp 85 90 95
Asn Val Arg lie Thr Lys Leu Arg Lys Gin Val Glu Glu lie Phe Asn 100 105 110
Leu Lys Phe Ala Gin Ala Leu Gly Leu Thr Glu Ala Val Lys Val Pro 115 120 125
Tyr Pro Val Phe Glu Ser Asn Pro Glu Phe Leu Tyr Val Glu Gly Leu 130 135 140
Pro Glu Gly lie Pro Phe Arg Ser Pro Thr Trp Phe Gly lie Pro Arg 145 150 155 160
Leu Glu Arg lie Val His Gly Ser Asn Lys lie Lys Phe Val Val Lys 165 170 175
Lys Pro Glu Leu Val lie Ser Tyr Leu Pro Pro Gly Met Ala Ser Lys 180 185 190 lie Asn Thr Lys Ala Leu Gin Ser Pro Lys Arg Pro Arg Ser Pro Gly 195 200 205
Ser Asn Ser Lys Val Pro Glu He Glu Val Thr Val Glu Gly Xaa Asn 210 215 220
Asn Asn Asn Pro Gin Thr Ser Ala Val Arg Thr Pro Thr Gin Thr Asn 225 230 235 240
Gly Ser Asn Val Pro Phe Lys Pro Arg Gly Arg Glu Phe Ser Phe Gly 245 250 255
Leu Glu Cys Gin Asn His Gly Pro Lys Thr Glu Ser 260 265
(2) INFORMATION FOR SEQ ID NO : 5 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2705 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 5 :
TAGGCCATGA AGGCCGCGCT TTTCGTCGAC TCTTACCGGT TGGCTGGGCC AGCTGCGCCG 60
CGGCTCACAG CTGACGATGG GGGACCCCAG CAAGCAGGAC ATCTTGACCA TCTTCAAGCG 120
CCTCCGCTCG GTGCCCACTA ACAAGGTGTG TTTTGATTGT GGTGCCAAAA ATCCCAGCTG 180
GGCAAGCATA ACCTATGGAG TGTTCCTTTG CATTGATTGC TCAGGGTCCC ACCGGTCACT 240
TGGTGTTCAC TTGAGTTTTA TTCGATCTAC AGAGTTGGAT TCCAACTGGT CATGGTTTCA 300
GTTGCGATGC ATGCAAGTCG GAGGAAACGC TAGTGCATCT TCCTTTTTTC ATCAACATGG 360
GTGTTCCACC AATGACACCA ATGCCAAGTA CAACAGTCGT GCTGCTCAGC TCTATAGGGA 420
GAAAATCAAA TCGCTCGCCT CTCAAGCAAC ACGGAAGCAT GGCACTGATC TGTGGCTTGA 480
TAGTTGTGTG GTTCCACCTT TGTCCCCTCC ACCAAAGGAG GAAGATTTTT TTGCCTCTCA 540
CGTTTCTCCT GAGGTGAGTG ACACAGCGTG GGCATCAGCA ATAGCAGAAC CATCTTCTTT 600
AACATCAAGG CCTGTGGAAA CCACTTTGGA AAATAATGAA GGTGGACAAG AGCAAGGACC 660
AAGTGTGGAA GGTCTTAATG TACCAACAAA GGCTACTTTA GAGGTATCCT CTATCATAAA 720
AAAGAAACCA AATCAAGCTA AAAAAGGCCT TGGGGCCAAA AAAGGAAGTT TGGGAGCTCA 780
GAAACTGGCA AACACATGCT TTAATGAAAT TGAAAAACAA GCTCAAGCTG CGGATAAAAT 840
GAAGGAGCAG AAAGACCTGG CCAAGGTGGT ATCTAAAGAA GAATCAATTG TTTCATCATT 900
ACGATTAGCC TATAAGGATC TTGAAATTCA AATGAAGAAA GACGAAAAGA TGAACATTAG 960 TGGCAAAAAA AATGTTGACT CAGACAGACT CGGCATGGGA TTTGGAAATT GCAGAAGTGT 1020
TATTTCACAT TCAGTGACTT CAGATATGCA GACCATAGAG CAGGAATCAC CCATTATGGC 1080
AAAACCAAGA AAAAAGTATA ATGATGACAG TGACGATTCA TATTTTACTT CCAGCTCAAG 1140
TTACTTTGAC GAGCCAGTGG AGTTAAGGAG CAGTTCTTTC TCTAGCTGGG ATGACAGTTC 1200
AGATTCCTAT TGGAAAAAAG AGACCAGCAA AGATACTGAA ACAGTTCTGA AAACCACAGG 1260
CTATTCAGAC AGACCTACTG CTCGCCGCAA GCCAGATTAT GAGCCAGTTG AAAATACAGA 1320
TGAGGCCCAG AAGAAGTTTG GCAATGTCAA GGCCATTTCA TCAGATATGT ATTTTGGAAG 1380
ACAATCCCAG GCTGATTATG AGACCAGGGC CCGCCTAGAG AGGCTGTCGG CAAGTTCCTC 1440
CATAAGCTCG GCTGATCTGT TCGAGGAGCC GAGGAAGCAG CCAGCAGGGA ACTACAGCCT 1500
GTCCAGTGTG CTGCCCAACG CCCCCGACAT GGCGCAGTTC AAGCAGGGAG TGAGATCGGT 1560
TGCTGGAAAA CTCTCCGTCT TTGCTAATGG AGTCGTGACT TCAATTCAGG ATCGCTACGG 1620
TTCTTAATAC TGAAGTCATG ATGTGTATTT CCTGGAGAAA TTCCTCTTTA AATGAACAAG 1680
TAACCACATC TCAGGCGGCA GTGAAGTCCA GATAGTTTTG CAGATTGTTT TGCTACTTTT 1740
TCATATGGTA TATGTTTCTG ATTTTTAATA TTTCTTTTGA GAAATTCTGA GTTCTGATGT 1800
AGGAGCTTTC CTGTGATTTC TGTTTCACGT TCCTTCCTGT CACACCCTCC TTTGGCGTCT 1860
CTGTGTATAT CCTTGCTTTA TTTTCTTGGA ACCTTTGATT TCAACACTGA GGGCCTGGAG 1920
ACCTCGGCTC CTCCTGCTCC TGAACCAGGA GGCTTCATGT GGGGGAGGAG GAGAGGTCTC 1980
CATGTGACAC ATGGGCTCAG GGCTGCCAGA ATCAGCGGAT GCTGGATGGG CCTGCAGAAA 2040
CAACACTCAC CACACACACT TCCTTCAAAA GACCAAAAGT GACTGGTGTC TCGTGTGACA 2100
GATTGCTTCA TTTATGTTTC TACATAGTAA GGTGACTGCC AAATAATATT TGAAGTCATC 2160
TGTCTCTTTG TAAATTATTT TATATGACCT ATAAATTTAA AAATGTTTTT CAGTGAGTGC 2220
TTTTAACAAA CTTAAGCTTC TGCCCTGCCA AGGGAATTAA TGTTATCTTG TGAAAGGTGT 2280
TGCTGTTTGA ATTGATGAGA AATGGAAGAT GAGAACTCCC TAAGAGTTCT CATAATAAAT 2340
CATCTCATCA CAAATCAATA CGGTATACAG AGTTAAAGTG GAATGAGGTA AGAAGATACA 2400
GCTACAGAAA ATAGTTGCGT GTATGGGAGA ACAGTCATTG TAATTGGGTA GTTTTGTTAA 2460
TAAATATTTT TAAATCTTGC TTTTCAGAAA TTACCGAATG TGTATAAACA AATAAAGAAA 2520
AATAATTTAG CTGTGTTTTA GACAGCATTA GAATATATTG TTCAGCACAG TAAAATATAT 2580
TTGAAATTTG ATAAGCCAAA AATGTGGTTT TGAATGAATA TTTTGTGAAT CTTTCTTAAA 2640 AGCTCAAATT TGTAGACTTC TAAATAGAAT AAACACTTGC AGCAGATGGA AAAAAAAAAA 2700 AAAAA 2705
(2) INFORMATION FOR SEQ ID NO : 6 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 516 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 6 :
Met Gly Asp Pro Ser Lys Gin Asp He Leu Thr He Phe Lys Arg Leu 1 5 10 15
Arg Ser Val Pro Thr Asn Lys Val Cys Phe Asp Cys Gly Ala Lys Asn 20 25 30
Pro Ser Trp Ala Ser He Thr Tyr Gly Val Phe Leu Cys He Asp Cys 35 40 45
Ser Gly Ser His Arg Ser Leu Gly Val His Leu Ser Phe He Arg Ser 50 55 60
Thr Glu Leu Asp Ser Asn Trp Ser Trp Phe Gin Leu Arg Cys Met Gin 65 70 75 80
Val Gly Gly Asn Ala Ser Ala Ser Ser Phe Phe His Gin His Gly Cys 85 90 95
Ser Thr Asn Asp Thr Asn Ala Lys Tyr Asn Ser Arg Ala Ala Gin Leu 100 105 110
Tyr Arg Glu Lys He Lys Ser Leu Ala Ser Gin Ala Thr Arg Lys His 115 120 125
Gly Thr Asp Leu Trp Leu Asp Ser Cys Val Val Pro Pro Leu Ser Pro 130 135 140
Pro Pro Lys Glu Glu Asp Phe Phe Ala Ser His Val Ser Pro Glu Val 145 150 155 160
Ser Asp Thr Ala Trp Ala Ser Ala He Ala Glu Pro Ser Ser Leu Thr 165 170 175
Ser Arg Pro Val Glu Thr Thr Leu Glu Asn Asn Glu Gly Gly Gin Glu 180 185 190 Gin Gly Pro Ser Val Glu Gly Leu Asn Val Pro Thr Lys Ala Thr Leu 195 200 205
Glu Val Ser Ser He He Lys Lys Lys Pro Asn Gin Ala Lys Lys Gly 210 215 220
Leu Gly Ala Lys Lys Gly Ser Leu Gly Ala Gin Lys Leu Ala Asn Thr 225 230 235 240
Cys Phe Asn Glu He Glu Lys Gin Ala Gin Ala Ala Asp Lys Met Lys 245 250 255
Glu Gin Lys Asp Leu Ala Lys Val Val Ser Lys Glu Glu Ser He Val 260 265 270
Ser Ser Leu Arg Leu Ala Tyr Lys Asp Leu Glu He Gin Met Lys Lys 275 280 285
Asp Glu Lys Met Asn He Ser Gly Lys Lys Asn Val Asp Ser Asp Arg 290 295 300
Leu Gly Met Gly Phe Gly Asn Cys Arg Ser Val He Ser His Ser Val 305 310 315 320
Thr Ser Asp Met Gin Thr He Glu Gin Glu Ser Pro He Met Ala Lys 325 330 335
Pro Arg Lys Lys Tyr Asn Asp Asp Ser Asp Asp Ser Tyr Phe Thr Ser 340 345 350
Ser Ser Ser Tyr Phe Asp Glu Pro Val Glu Leu Arg Ser Ser Ser Phe 355 360 365
Ser Ser Trp Asp Asp Ser Ser Asp Ser Tyr Trp Lys Lys Glu Thr Ser 370 375 380
Lys Asp Thr Glu Thr Val Leu Lys Thr Thr Gly Tyr Ser Asp Arg Pro 385 390 395 400
Thr Ala Arg Arg Lys Pro Asp Tyr Glu Pro Val Glu Asn Thr Asp Glu 405 410 415
Ala Gin Lys Lys Phe Gly Asn Val Lys Ala He Ser Ser Asp Met Tyr 420 425 430
Phe Gly Arg Gin Ser Gin Ala Asp Tyr Glu Thr Arg Ala Arg Leu Glu 435 440 445
Arg Leu Ser Ala Ser Ser Ser He Ser Ser Ala Asp Leu Phe Glu Glu 450 455 460
Pro Arg Lys Gin Pro Ala Gly Asn Tyr Ser Leu Ser Ser Val Leu Pro 465 470 475 480
Asn Ala Pro Asp Met Ala Gin Phe Lys Gin Gly Val Arg Ser Val Ala 485 490 495
Gly Lys Leu Ser Val Phe Ala Asn Gly Val Val Thr Ser He Gin Asp 500 505 510
Arg Tyr Gly Ser 515
(2) INFORMATION FOR SEQ ID NO : 7 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1414 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 7 :
CTTCCACGCC CGAGGGCATC GCGCTGGCCT ACGGCAGCCT CCTGCTCATG GCGCTGCTGC 60
CCATCTTCTT CGGCGCCCTG CGCTCCGTAC GCTGCGCCCG CGGCAAGAAT GCTTCAGACA 120
TGCCTGAAAC AATCACCAGC CGGGATGCCG CCCGCTTCCC CATCATCGCC AGCTGCACAC 180
TCTTGGGGCT CTACCTCTTT TTCAAAATAT TCTCCCAGGA GTACATCAAC CTCCTGCTGT 240
CCATGTATTT CTTCGTGCTG GGAATCCTGG CCCTGTCCCA CACCATCAGC CCCTTCATGA 300
ATAAGTTTTT TCCAGCCAGC TTTCCAAATC GACAGTACCA GCTGCTCTTC ACACAGGGTT 360
CTGGGGAAAA CAAGGAAGAG ATCATCAATT ATGAATTTGA CACCAAGGAC CTGGTGTGCC 420
TGGGCCTGAG CAGCATCGTT GGCGTCTGGT ACCTGCTGAG GAAGCACTGG ATTGCCAACA 480
ACCTTTTTGG CCTGGCCTTC TCCCTTAATG GAGTAGAGCT CCTGCACCTC AACAATGTCA 540
GCACTGGCTG CATCCTGCTG GGCGGACTCT TCATCTACGA TGTCTTCTGG GTATTTGGCA 600
CCAATGTGAT GGTGACAGTG GCCAAGTCCT TCGAGGCACC AATAAAATTG GTGTTTCCCC 660
AGGATCTGCT GGAGAAAGGC CTCGAAGCAA ACAACTTTGC CATGCTGGGA CTTGGAGATG 720
TCGTCATTCC AGGGATCTTC ATTGCCTTGC TGCTGCGCTT TGACATCAGC TTGAAGAAGA 780
ATACCCACAC CTACTTCTAC ACCAGCTTTG CAGCCTACAT CTTCGGCCTG GGCCTTACCA 840
TCTTCATCAT GCACATCTTC AAGCATGCTC AGCCTGCCCT CCTATACCTG GTCCCCGCCT 900
GCATCGGTTT TCCTGTCCTG GTGGCGCTGG CCAAGGGAGA AGTGACAGAG ATGTTCAGTT 960 ATGAGGAGTC AAATCCTAAG GATCCAGCGG CAGTGACAGA ATCCAAAGAG GGAACAGAGG 1020
CATCAGCATC GAAGGGGCTG GAGAAGAAAG AGAAATGATG CAGCTGGTGC CCGAGCCTCT 1080
CAGGGCCAGA CCAGACAGAT GGGGGCTGGG CCCACACAGG CGTGCACCGG TAGAGGGCAC 1140
AGGAGGCCAA GGGCAGCTCC AGGACAGGGC AGGGGGCAGC AGGATACCTC CAGCCAGGCC 1200
TCTGTGGCCT CTGTTTCCTT CTCCCTTTCT TGGCCCTCCT CTGCTCCTCC CCACACCCTG 1260
CAGGCAAAAG AAACCCCCAG CTTCCCCCCT CCCCGGGAGC CAGGTGGGAA AAGTGGGTGT 1320
GATTTTTAGA TTTTGTATTG TGGACTGATT TTGCCTCACA TTAAAAACTC ATCCCATGGC 1380
CAGGGCGGGC CACTGTAAAA AAAAAAAAAA AAAA 1414 (2) INFORMATION FOR SEQ ID NO : 8 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 336 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 8 :
Met Ala Leu Leu Pro He Phe Phe Gly Ala Leu Arg Ser Val Arg Cys 1 5 10 15
Ala Arg Gly Lys Asn Ala Ser Asp Met Pro Glu Thr He Thr Ser Arg 20 25 30
Asp Ala Ala Arg Phe Pro He He Ala Ser Cys Thr Leu Leu Gly Leu 35 40 45
Tyr Leu Phe Phe Lys He Phe Ser Gin Glu Tyr He Asn Leu Leu Leu 50 55 60
Ser Met Tyr Phe Phe Val Leu Gly He Leu Ala Leu Ser His Thr He 65 70 75 80
Ser Pro Phe Met Asn Lys Phe Phe Pro Ala Ser Phe Pro Asn Arg Gin 85 90 95
Tyr Gin Leu Leu Phe Thr Gin Gly Ser Gly Glu Asn Lys Glu Glu He 100 105 110
He Asn Tyr Glu Phe Asp Thr Lys Asp Leu Val Cys Leu Gly Leu Ser 115 120 125 Ser He Val Gly Val Trp Tyr Leu Leu Arg Lys His Trp He Ala Asn 130 135 140
Asn Leu Phe Gly Leu Ala Phe Ser Leu Asn Gly Val Glu Leu Leu His 145 150 155 160
Leu Asn Asn Val Ser Thr Gly Cys He Leu Leu Gly Gly Leu Phe He 165 170 175
Tyr Asp Val Phe Trp Val Phe Gly Thr Asn Val Met Val Thr Val Ala 180 185 190
Lys Ser Phe Glu Ala Pro He Lys Leu Val Phe Pro Gin Asp Leu Leu 195 200 205
Glu Lys Gly Leu Glu Ala Asn Asn Phe Ala Met Leu Gly Leu Gly Asp 210 215 220
Val Val He Pro Gly He Phe He Ala Leu Leu Leu Arg Phe Asp He 225 230 235 240
Ser Leu Lys Lys Asn Thr His Thr Tyr Phe Tyr Thr Ser Phe Ala Ala 245 250 255
Tyr He Phe Gly Leu Gly Leu Thr He Phe He Met His He Phe Lys 260 265 270
His Ala Gin Pro Ala Leu Leu Tyr Leu Val Pro Ala Cys He Gly Phe 275 280 285
Pro Val Leu Val Ala Leu Ala Lys Gly Glu Val Thr Glu Met Phe Ser 290 295 300
Tyr Glu Glu Ser Asn Pro Lys Asp Pro Ala Ala Val Thr Glu Ser Lys 305 310 315 320
Glu Gly Thr Glu Ala Ser Ala Ser Lys Gly Leu Glu Lys Lys Glu Lys 325 330 335
(2) INFORMATION FOR SEQ ID NO : 9 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1583 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 9 : AGACGAGCCT TTCTTATTTC TTTACTCAAC CTCTTTGATG ACACAGCAAA AACAGACGTG 60
ACTATGCTCT TGTATATAGC AGACAATCTA GCCTGTTTTC CATACCAGAC ACAGGAAGAG 120
CCGTTGTTTA TAATGCATCA TATAGACATT ACACTCTCAG TTTCTGGTAG TAACCTACTG 180
CAGTCATTCA AGGAGTCTAT GGTAAAGGAC AAAAGGAAAG AGAGAAAATC ATCACCTAGT 240
AAGGAAAATG AGTCAAGCGA CAGTGAAGAA GAAGTTTCCA GGCCTCGGAA GTCACGGAAA 300
CGTGTAGATT CAGATTCAGA TTCAGATTCA GAAGACGATA TAAATTCAGT GATGAAATGT 360
TTGCCAGAAA ATTCAGCTCC TTTAATCGAA TTTGCAAATG TGTCCCAGGG TATTTTATTA 420
CTTCTCATGT TAAAACAACA TTTGAAGAAT CTTTGTGGAT TTTCTGATAG TAAAATTCAG 480
AAGTACTCTC CATCTGAATC TGCAAAAGTA TATGATAAAG CGATAAACCG AAAAACAGGA 540
GTTCATTTTC ATCCAAAACA AACACTGGAC TTCCTGCGGA GTGACATGGC TAATTCCAAA 600
ATCACAGAAG AGGTGAAAAG GAGTATAGTA AAACAGTATC TAGATTTCAA ACTTCTCATG 660
GAACATCTGG ACCCTGATGA AGAAGAAGAA GAAGGGGAGG TTTCAGCTAG CACAAATGCT 720
CGGAACAAAG CAATTACCTC ACTGCTTGGA GGAGGCAGCC CTAAAAATAA TACAGCAGCA 780
GAGACAGAAG ATGATGAAAG TGATGGGGAG GATAGAGGAG GAGGCACTTC AGGGTCATTG 840
AGAAGGTCAA AACGAAATTC AGACTCTACG GAGTTGGCAG CACAGATGAA TGAAAGTGTT 900
GACGTCATGG ATGTCATCGC TATTTGCTGT CCAAAGTACA AAGATCGACC ACAAATTGCA 960
AGAGTAGTGC AGAAAACCAG CAGTGGCTTC AGTGTTCAGT GGATGGCAGG CTCCTACAGT 1020
GGCTCCTGGA CTGAGGCTAA GCGCCGTGAT GGCCGCAAAC TGGTGCCTTG GGTAGACACT 1080
ATTAAAGAGT CAGACATTAT TTACAAAAAA ATTGCTCTAA CGAGTGCTAA TAAGCTGACT 1140
AATAAAGTTG TTCAGACTTT ACGATCCCTG TATGCCGCCA AGGATGGGAC TTCCAGCTAA 1200
TGAATTTGTA CATGCAGCCA AATTTACAGG AATTTTTTTA AAAGGCAGAA AAACTTGAAA 1260
TACCAACATT CTGGCAAAAA AAAATCAGTT TTATGAAGAG TAAGTGGAAC CTGGGATGCA 1320
GGAACAAAAG AAGGAAATGT TGGGCAAACA TTTTTGTGGG AGCTCCCTTC GCTGTTGTGC 1380
AGCAGAAACA GATTCTCAGT TCATTTTTAC TCCCACTGTA TTATAGTTTA ACAAAAATTG 1440
TTTATATCTT GGAAAAAAAA ACTTTCTGTT TAAAAAAAAT AAACAAGTGA ATGTTGGAAA 1500
TTAGTCTGTT AATGTTCTTA ATAAAGTGTT CTTGGAGTTT TAAAAAAAAA AAAAAAAAAA 1560
AAAAAAAAAA AAAAAAAAAA AAA 1583 (2) INFORMATION FOR SEQ ID NO: 10: ( i ) SEQUENCE CHARACTERISTICS :
( A) LENGTH : 378 amino acids
( B ) TYPE : amino ac id
( C ) STRANDEDNESS :
( D) TOPOLOGY : linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
Met Leu Leu Tyr He Ala Asp Asn Leu Ala Cys Phe Pro Tyr Gin Thr 1 5 10 15
Gin Glu Glu Pro Leu Phe He Met His His He Asp He Thr Leu Ser 20 25 30
Val Ser Gly Ser Asn Leu Leu Gin Ser Phe Lys Glu Ser Met Val Lys 35 40 45
Asp Lys Arg Lys Glu Arg Lys Ser Ser Pro Ser Lys Glu Asn Glu Ser 50 55 60
Ser Asp Ser Glu Glu Glu Val Ser Arg Pro Arg Lys Ser Arg Lys Arg 65 70 75 80
Val Asp Ser Asp Ser Asp Ser Asp Ser Glu Asp Asp He Asn Ser Val 85 90 95
Met Lys Cys Leu Pro Glu Asn Ser Ala Pro Leu He Glu Phe Ala Asn 100 105 110
Val Ser Gin Gly He Leu Leu Leu Leu Met Leu Lys Gin His Leu Lys 115 120 125
Asn Leu Cys Gly Phe Ser Asp Ser Lys He Gin Lys Tyr Ser Pro Ser 130 135 140
Glu Ser Ala Lys Val Tyr Asp Lys Ala He Asn Arg Lys Thr Gly Val 145 150 155 160
His Phe His Pro Lys Gin Thr Leu Asp Phe Leu Arg Ser Asp Met Ala 165 170 175
Asn Ser Lys He Thr Glu Glu Val Lys Arg Ser He Val Lys Gin Tyr 180 185 190
Leu Asp Phe Lys Leu Leu Met Glu His Leu Asp Pro Asp Glu Glu Glu 195 200 205
Glu Glu Gly Glu Val Ser Ala Ser Thr Asn Ala Arg Asn Lys Ala He 210 215 220 Thr Ser Leu Leu Gly Gly Gly Ser Pro Lys Asn Asn Thr Ala Ala Glu 225 230 235 240
Thr Glu Asp Asp Glu Ser Asp Gly Glu Asp Arg Gly Gly Gly Thr Ser 245 250 255
Gly Ser Leu Arg Arg Ser Lys Arg Asn Ser Asp Ser Thr Glu Leu Ala 260 265 270
Ala Gin Met Asn Glu Ser Val Asp Val Met Asp Val He Ala He Cys 275 280 285
Cys Pro Lys Tyr Lys Asp Arg Pro Gin He Ala Arg Val Val Gin Lys 290 295 300
Thr Ser Ser Gly Phe Ser Val Gin Trp Met Ala Gly Ser Tyr Ser Gly 305 310 315 320
Ser Trp Thr Glu Ala Lys Arg Arg Asp Gly Arg Lys Leu Val Pro Trp 325 330 335
Val Asp Thr He Lys Glu Ser Asp He He Tyr Lys Lys He Ala Leu 340 345 350
Thr Ser Ala Asn Lys Leu Thr Asn Lys Val Val Gin Thr Leu Arg Ser 355 360 365
Leu Tyr Ala Ala Lys Asp Gly Thr Ser Ser 370 375
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2353 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
TCCCGTCGCG CTCAGCATCC TCAATCATCC GCAGGCTGAT GCGGTCCTTC ACGCTCCCGC 60
CCGCGTTGAA GAACTCACAC TTGGCCAAGA GCTCACACTT CAGGCCGAAC TTCTTCCCAA 120
TCTTGTTGAT TCTGACCATA GGGGTGTCCC CGATTTTCTT CAGAATATCT GGCAAGATTT 180
TTGGAGATTT TGCCGGGGCA GTGTGGTGAT GTGGGGACTC GGAGGCAGGC CGGCCCAGCT 240
GCCAGGTGCA CCTGCTCGGA GCATCGGGCC GGATCCACAG GGGCTCCTTG GCTTCCTTAT 300 CCTCTGGGGA CCCCTTCTCC AGGCTCCCCT TCGCCGAGTG TGGCCCTGAG CGGTGGGGGC 360
AGCCTGTGGG CCCCACTTCT GCCTGGGGGG TCTCAGAAGG CATGCTGGGA CCGGACAGAT 420
GGTGCTGACA GTTCAGCACC GCTCAAACTC TGTAAGGAGA CGTGACAACA GAATCTGTCA 480
TGGGATCCTG GAATAGAAAA GGGCAGTGAG GAGAAAACTC TGCAAATCTG ACTCAAGCGC 540
GGACTTTGGT TAGGCCTCTT TGGACAGTAC TTTGAGAATT ATGGATCTCC GGATATTAGT 600
AGCAAGGAAG TTTGTAGGAG CAGCAAATTA TCGGGAGAAG ATTCATAGTA CTTTGACTCC 660
ATGTGGGACT TTTCTGTTTG CTGGAAGTGA GGATGGTATA GTGTATGTTT GGAACCCAGA 720
AACAGGAGAA CAAGTAGCCA TGTATTCTGA CTTGCCATTC AAGTCACCCA TTCGAGACAT 780
TTCTTATCAT CCATTTGAAA ATATGGTTGC ATTCTGTGCA TTTGGGCAAA ATGAGCCAAT 840
TCTTCTGTAT ATTTACGATT TCCATGTTGC CCAGCAGGAG GCTGAAATGT TCAAACGCTA 900
CAATGGAACA TTTCCATTAC CTGGAATACA CCAAAGTCAA GATGCCCTAT GTACCTGTCC 960
AAAACTACCC CATCAAGGCT CTTTTCAGAT TGATGAATTT GTCCACACTG AAAGTTCTTC 1020
AACGAAGATG CAGCTAGTAA AACAGAGGCT TGAAACTGTC ACAGAGGTGA TACGTTCCTG 1080
TGCTGCAAAA GTCAACAAAA ATCTCTCATT TACTTCACCA CCAGCAGTTT CCTCACAACA 1140
GTCTAAGTTA AAGCAGTCAA ACATGCTGAC CGCTCAAGAG ATTCTACATC AGTTTGGTTT 1200
CACTCAGACC GGGATTATCA GCATAGAAAG AAAGCCTTGT AACCATCAGG TAGATACAGC 1260
ACCAACGGTA GTGGCTCTTT ATGACTACAC AGCGAATCGA TCAGATGAAC TAACCATCCA 1320
TCGCGGAGAC ATTATCCGAG TGTTTTTCAA AGATAATGAA GACTGGTGGT ATGGCAGCAT 1380
AGGAAAGGGA CAGGAAGGTT ATTTTCCAGC TAATCATGTG GCTAGTGAAA CACTGTATCA 1440
AGAACTGCCT CCTGAGATAA AGGAGCGATC CCCTCCTTTA AGCCCTGAGG AAAAAACTAA 1500
AATAGAAAAA TCTCCAGCTC CTCAAAAGGT AAAATAAAAC AAAACAGCTC ACAGAGACAC 1560
CTCCTTCTTC CCCCTGCTTC TGCCTCCATG AGTAACTACC TATATGACGT GCTGCTGCTG 1620
AGACCAAGGA ATGAGTGAGT AAAGGTGTTT GGAAGTCAAA TATTGGTCCT AGTTAAGTAA 1680
GTGTCCTTTT CAAGTAAGTG GTAAACTTTG TAATGTGGAC CCCCTTCTAA CTTAGGATTC 1740
ATATAATTTG AACACAGGCT AAGCTGCCTC TGCATCATTA AAAAGGATTT GGGTATGCCA 1800
GTGATAGGAA TACACAGTAG AAAAGATGGC AGGGACCATG TTTAACATAC CTAAGACAAA 1860
CTTTCTGAGG ATATCACAGC ATCCATTTCA CATGAACAGA CTTAGCAGAA TTGGCAAGAC 1920
CTGCACATGG CAACATATTA TTCATTTTTC AGGATAACAA AAAATAATTA AAAGTAACAT 1980 TGGCTTAACA CTGGTATATA GTTCAGTATG ACTTCTCTCC CCTCAACCTC TGGAATAGGT 2040
TCAAATTGAT GAGAATTTCT GATTAGAGCC CTTAATGTTG AGTTTTTTGA AAAGTTTTAT 2100
CAAGTTTCAT ATATACCTAT ATTGATGGTA AGTTGCTGGT CTTGCCATGG GCAAAGAGAG 2160
AAAAATGATA YTGAGACCTT GTAAAGAATA GYTGGACATG GAGGCGCACA CCTGTAATCC 2220
CAGCAAYTCA GAAGGCTGAG GCGGGAGGAT TGCTTGAGCC CAGGAGTTCA AGGCTGCAGT 2280
GAGCTACGAT CATGCCACTG TACTCCAGCC TGAGCAACAA AGCATGACCC AATCTCTTAA 2340
AAAAAAAAAA AAA 2353 (2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 318 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 12 :
Met Asp Leu Arg He Leu Val Ala Arg Lys Phe Val Gly Ala Ala Asn 1 5 10 15
Tyr Arg Glu Lys He His Ser Thr Leu Thr Pro Cys Gly Thr Phe Leu 20 25 30
Phe Ala Gly Ser Glu Asp Gly He Val Tyr Val Trp Asn Pro Glu Thr 35 40 45
Gly Glu Gin Val Ala Met Tyr Ser Asp Leu Pro Phe Lys Ser Pro He 50 55 60
Arg Asp He Ser Tyr His Pro Phe Glu Asn Met Val Ala Phe Cys Ala- 65 70 75 80
Phe Gly Gin Asn Glu Pro He Leu Leu Tyr He Tyr Asp Phe His Val 85 90 95
Ala Gin Gin Glu Ala Glu Met Phe Lys Arg Tyr Asn Gly Thr Phe Pro 100 105 110
Leu Pro Gly He His Gin Ser Gin Asp Ala Leu Cys Thr Cys Pro Lys 115 120 125
Leu Pro His Gin Gly Ser Phe Gin He Asp Glu Phe Val His Thr Glu 130 135 140 Ser Ser Ser Thr Lys Met Gin Leu Val Lys Gin Arg Leu Glu Thr Val 145 150 155 160
Thr Glu Val He Arg Ser Cys Ala Ala Lys Val Asn Lys Asn Leu Ser 165 170 175
Phe Thr Ser Pro Pro Ala Val Ser Ser Gin Gin Ser Lys Leu Lys Gin 180 185 190
Ser Asn Met Leu Thr Ala Gin Glu He Leu His Gin Phe Gly Phe Thr 195 200 205
Gin Thr Gly He He Ser He Glu Arg Lys Pro Cys Asn His Gin Val 210 215 220
Asp Thr Ala Pro Thr Val Val Ala Leu Tyr Asp Tyr Thr Ala Asn Arg 225 230 235 240
Ser Asp Glu Leu Thr He His Arg Gly Asp He He Arg Val Phe Phe 245 250 255
Lys Asp Asn Glu Asp Trp Trp Tyr Gly Ser He Gly Lys Gly Gin Glu 260 265 270
Gly Tyr Phe Pro Ala Asn His Val Ala Ser Glu Thr Leu Tyr Gin Glu 275 280 285
Leu Pro Pro Glu He Lys Glu Arg Ser Pro Pro Leu Ser Pro Glu Glu 290 295 300
Lys Thr Lys He Glu Lys Ser Pro Ala Pro Gin Lys Val Lys 305 310 315
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2567 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
AGCCCACCTA CACACCCAGC TCCCACCCAT GGAGAGCACC CCAGTCCTGT TCCTCCTGCC 60
CTGGACCCTG GCCAACTCTG CCACAAGCTC TACCCTCGGT ACAACAGGCT CTGTCCCCAC 120
ATCTACAGAC CCTGCCCCAT CTGCACACCT AGACTCAGTT CATAAGTCCA CAGACTCTGG 180
CCCTTCAGAA CTGCCAGGCC CCACTCACAC CACTACAGGC TCTACCTATA GTGCCATTAC 240 CACTACCCAC AGTGCTCCAA GCCCCCTCAC TCACACTACT ACAGGCTCCA CCCACAAGCC 300
CATAATCTCT ACCCTTACTA CTACAGGCCC TACCCTCAAT ATCATAGGCC CAGTCCAGAC 360
TACCACAAGC CCCACCCACA CTATGCCAAG CCCTACCCAT ACCACAGCAA GCCCCACTCA 420
TACTTCCACA AGCCCCACCC ATACCCCCAC AAGTCCCACC CACAAAACCA GTATGTCACC 480
TCCCACCACT ACAAGTCCTA CCCCCAGTGG TATGGGCCTA GTCCAGACTG CCACAAGTCC 540
CACCCATCCT ACCACAAGCC CCACCCATCC CACCACAAGC CCCATCCTTA TAAATGTAAG 600
CCCTTCCACT TCTCTAGAAC TTGCTACCCT CTCCAGCCCC TCCAAACACT CAGACCCCAC 660
CCTCCCAGGC ACTGACTCCC TTCCCTGTAG TCCCCCAGTC TCCAATTCCT ACACTCAGGC 720
AGACCCTATG GCCCCCAGAA CTCCCCACCC AAGTCCTGCC CATTCCAGTA GGAAACCCCT 780
CACAAGCCCT GCCCCAGATC CCTCAGAGTC TACGGTTCAG AGTCTAAGCC CCACTCCCTC 840
ACCCCCAACC CCTGCACCCC AGCATTCAGA CCTTTGCCTG GCCATGGCTG TCCAGACCCC 900
AGTCCCAACG GCAGCCGGAG GGTCTGGGGA CAGGAGCCTG GAGGAGGCAC TGGGGGCCCT 960
AATGGCTGCC CTGGATGACT ACCGTGGCCA GTTTCCTGAG CTGCAGGGCC TGGAGCAGGA 1020
GGTGACCCGC CTAGAAAGTC TGCTCATGCA GAGACAAGGT CTGACTCGCA GCCGGGCCTC 1080
CAGTCTCAGC ATCACTGTGG AGCATGCCTT GGAGAGCTTC AGCTTCCTCA ATGAAAACGA 1140
AGATGAAGAC AATGATGTTC CTGGGGACAG GCCTCCAAGC AGCCCGGAGG CTGGGGCTGA 1200
GGACAGCATA GACTCACCCA GTGCCCGCCC CCTCAGCACG GGGTGTCCAG CTCTGGATGC 1260
TGCCTTGGTC CGGCACCTGT ACCACTGCAG TCGCCTCCTG CTGAAACTGG GCACATTTGG 1320
GCCCCTGCGC TGCCAGGAGG CATGGGCCCT GGAGCGGCTG CTGCGGGAAG CCCGAGTACT 1380
GGAGGCAGTA TGCGAGTTCA GCAGGCGGTG GGAGATCCCG GCCAGCTCTG CCCAGGAAGT 1440
GGTGCAGTTC TCGGCCTCTC GGCCTGGCTT CCTGACCTTC TGGGACCAGT GCACAGAGAG 1500
ACTCAGCTGC TTCCTCTGCC CGGTGGAGCG GGTGCTTCTC ACCTTCTGCA ACCAGTATGG 1560
TGCCCGCCTC TCCCTGCGCC AGCCAGGCTT GGCTGAGGCT GTGTGTGTGA AGTTCCTGGA 1620
GGATGCCCTG GGGCAGAAGC TGCCCAGAAG GCCCCAGCCA GGGCCTGGAG AGCAGCTCAC 1680
AGTCTTCCAG TTCTGGAGTT TTGTGGAAAC CTTGGACAGC CCCACCATGG AGGCCTACGT 1740
GACTGAGACC GCTGAGGAGG TGCTACTGGT GCGGAATCTG AACTCGGATG ATCAGGCTGT 1800
TGTGCTGAAG GCCCTGAGAT TGGCGCCCGA GGGGCGTCTG CGAAGGGACG GGCTGCGGGC 1860
CCTCAGCTCC CTGCTCGTCC ATGGCAACAA CAAGGTCATG GCTGCTGTCA GCACCCAGCT 1920 CCGGAGCCTG TCACTGGGCC CTACCTTCCG GGAGAGGGCC CTCCTGTGCT TCCTGGACCA 1980
GCTGGAGGAT GAGGACGTGC AGACTCGAGT GGCTGGCTGC CTGGCCCTAG GCTGCATCAA 2040
GGCTCCCGAG GGCATTGAGC CCCTGGTGTA CCTCTGCCAA ACTGACACAG AAGCTGTGAG 2100
GGAAGCTGCC CGGCAAAGCC TACAGCAGTG TGGAGAAGAG GGACAGTCTG CCCATCGACG 2160
GCTGGAGGAG TCCCTGGACG CCCTGCCCCG CATCTTTGGG CCTGGCAGCA TGGCCAGCAC 2220
AGCATTCTAA ACTATTCACC CATGGGTTCC TGGTGCCCCT TTCCCCCCAC TTTCAGGGCT 2280
CACCAGGCAC TGGCAGGGAG GGTAAGGGCT GGCTCCAGAT ACCCCTCCCC CACAGATTCC 2340
TAGCAATGAA AATCTAATAT ATTCTTCTGT TGCCCCTGGG GTTGGAGAGT CAGTGCCTGC 2400
AGTCAAGTGC CTCCCAGCCT CGGCTCAGCA CATCCCTTGC CACAAATCAG TGTCTGGGGC 2460
TTGGCCACCC TGCCGCTGCC CAGCCACATC CCTTGGTTTT GTATTTTATT TACAGAGTTT 2520
TACAGAAAAT AAAAAAGCAA AATGTCTTTC CTAAAAAAAA AAAAAAA 2567 (2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 733 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
Met Glu Ser Thr Pro Val Leu Phe Leu Leu Pro Trp Thr Leu Ala Asn
1 5 10 15
Ser Ala Thr Ser Ser Thr Leu Gly Thr Thr Gly Ser Val Pro Thr Ser 20 25 30
Thr Asp Pro Ala Pro Ser Ala His Leu Asp Ser Val His Lys Ser Thr 35 40 45
Asp Ser Gly Pro Ser Glu Leu Pro Gly Pro Thr His Thr Thr Thr Gly 50 55 60
Ser Thr Tyr Ser Ala He Thr Thr Thr His Ser Ala Pro Ser Pro Leu 65 70 75 80
Thr His Thr Thr Thr Gly Ser Thr His Lys Pro He He Ser Thr Leu
85 90 95 Thr Thr Thr Gly Pro Thr Leu Asn He He Gly Pro Val Gin Thr Thr 100 105 110
Thr Ser Pro Thr His Thr Met Pro Ser Pro Thr His Thr Thr Ala Ser 115 120 125
Pro Thr His Thr Ser Thr Ser Pro Thr His Thr Pro Thr Ser Pro Thr 130 135 140
His Lys Thr Ser Met Ser Pro Pro Thr Thr Thr Ser Pro Thr Pro Ser 145 150 155 160
Gly Met Gly Leu Val Gin Thr Ala Thr Ser Pro Thr His Pro Thr Thr 165 170 175
Ser Pro Thr His Pro Thr Thr Ser Pro He Leu He Asn Val Ser Pro 180 185 190
Ser Thr Ser Leu Glu Leu Ala Thr Leu Ser Ser Pro Ser Lys His Ser 195 200 205
Asp Pro Thr Leu Pro Gly Thr Asp Ser Leu Pro Cys Ser Pro Pro Val 210 215 220
Ser Asn Ser Tyr Thr Gin Ala Asp Pro Met Ala Pro Arg Thr Pro His 225 230 235 240
Pro Ser Pro Ala His Ser Ser Arg Lys Pro Leu Thr Ser Pro Ala Pro 245 250 255
Asp Pro Ser Glu Ser Thr Val Gin Ser Leu Ser Pro Thr Pro Ser Pro 260 265 270
Pro Thr Pro Ala Pro Gin His Ser Asp Leu Cys Leu Ala Met Ala Val 275 280 285
Gin Thr Pro Val Pro Thr Ala Ala Gly Gly Ser Gly Asp Arg Ser Leu 290 295 300
Glu Glu Ala Leu Gly Ala Leu Met Ala Ala Leu Asp Asp Tyr Arg Gly 305 310 315 320
Gin Phe Pro Glu Leu Gin Gly Leu Glu Gin Glu Val Thr Arg Leu Glu 325 330 335
Ser Leu Leu Met Gin Arg Gin Gly Leu Thr Arg Ser Arg Ala Ser Ser 340 345 350
Leu Ser He Thr Val Glu His Ala Leu Glu Ser Phe Ser Phe Leu Asn 355 360 365
Glu Asn Glu Asp Glu Asp Asn Asp Val Pro Gly Asp Arg Pro Pro Ser 370 375 380
Ser Pro Glu Ala Gly Ala Glu Asp Ser He Asp Ser Pro Ser Ala Arg 385 390 395 400
Pro Leu Ser Thr Gly Cys Pro Ala Leu Asp Ala Ala Leu Val Arg His 405 410 415
Leu Tyr His Cys Ser Arg Leu Leu Leu Lys Leu Gly Thr Phe Gly Pro 420 425 430
Leu Arg Cys Gin Glu Ala Trp Ala Leu Glu Arg Leu Leu Arg Glu Ala 435 440 445
Arg Val Leu Glu Ala Val Cys Glu Phe Ser Arg Arg Trp Glu He Pro 450 455 460
Ala Ser Ser Ala Gin Glu Val Val Gin Phe Ser Ala Ser Arg Pro Gly 465 470 475 480
Phe Leu Thr Phe Trp Asp Gin Cys Thr Glu Arg Leu Ser Cys Phe Leu 485 490 495
Cys Pro Val Glu Arg Val Leu Leu Thr Phe Cys Asn Gin Tyr Gly Ala 500 505 510
Arg Leu Ser Leu Arg Gin Pro Gly Leu Ala Glu Ala Val Cys Val Lys 515 520 525
Phe Leu Glu Asp Ala Leu Gly Gin Lys Leu Pro Arg Arg Pro Gin Pro 530 535 540
Gly Pro Gly Glu Gin Leu Thr Val Phe Gin Phe Trp Ser Phe Val Glu 545 550 555 560
Thr Leu Asp Ser Pro Thr Met Glu Ala Tyr Val Thr Glu Thr Ala Glu 565 570 575
Glu Val Leu Leu Val Arg Asn Leu Asn Ser Asp Asp Gin Ala Val Val 580 585 590
Leu Lys Ala Leu Arg Leu Ala Pro Glu Gly Arg Leu Arg Arg Asp Gly 595 600 605
Leu Arg Ala Leu Ser Ser Leu Leu Val His Gly Asn Asn Lys Val Met 610 615 620
Ala Ala Val Ser Thr Gin Leu Arg Ser Leu Ser Leu Gly Pro Thr Phe 625 630 635 640
Arg Glu Arg Ala Leu Leu Cys Phe Leu Asp Gin Leu Glu Asp Glu Asp 645 650 655
Val Gin Thr Arg Val Ala Gly Cys Leu Ala Leu Gly Cys He Lys Ala 660 665 670
Pro Glu Gly He Glu Pro Leu Val Tyr Leu Cys Gin Thr Asp Thr Glu 675 680 685 Ala Val Arg Glu Ala Ala Arg Gin Ser Leu Gin Gin Cys Gly Glu Glu 690 695 700
Gly Gin Ser Ala His Arg Arg Leu Glu Glu Ser Leu Asp Ala Leu Pro 705 710 715 720
Arg He Phe Gly Pro Gly Ser Met Ala Ser Thr Ala Phe 725 730
(2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2501 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
GGCAGTCAAA GCACTGATGA TGATAAAATC GTTCAGTACC ATTGGGAAGA ACTTAAGGGG 60
CCTCTAAGAG AAGAGAAGAT TTCTGAAGAT ACAGCCATAT TAAAACTAAG TAAACTCGTC 120
CCTGGGAACT ACACTTTCAG CTTGACTGTA GTAGACTCTG ATGGAGCTAC CAACTCTACT 180
ACTGCAAACC TGACAGTGAA CAAAGCTGTG GATTACCCCC CTGTGGCCAA CGCAGGCCCC 240
AACCAAGTGA TCACCCTGCC CCAAAACTCC ATCACCCTCT TTGGGAACCA GAGCACTGAT 300
GATCATGGCA TCACCAGCTA TGAGTGGTCA CTCAGCCCAA GCAGCAAAGG GAAAGTGGTG 360
GAGATGCAGG GTGTTAGAAC ACCAACCTTA CAGCTCTCTG CGATGCAAGA AGGAGACTAC 420
ACTTACCAGC TCACAGTGAC TGACACAATA GGACAGCAGG CCACTGCTCA AGTGACTGTT 480
ATTGTGCAAC CTGAAAACAA TAAGCCTCCT CAGGCAGATG CAGGCCCAGA TAAAGAGCTG 540
ACCCTTCCTG TGGATAGCAC AACCCTGGAT GGCAGCAAGA GCTCAGATGA TCAGAAAATT 600
ATCTCATATC TCTGGGAAAA AACACAGGGA CCTGATGGGG TGCAGCTCGA GAATGCTAAC 660
AGCAGTGTTG CTACTGTGAC TGGGCTGCAA GTGGGGACCT ATGTGTTCAC CTTGACTGTC 720
AAAGATGAGA GGAACCTGCA AAGCCAGAGC TCTGTGAATG TCATTGTCAA AGAAGAATAA 780
ACAAACCACC TATAGCCAAG AT ACTGGGA ATGTGGTGAT TACCCTACCC ACGAGCACAG 840
CAGAGCTGGA TGGCTCTAAG TCCTCAGATG ACAAGGGAAT AGTCAGCTAC CTCTGGACTC 900
GAGATGAGGG GAGCCCAGCA GCAGGGGAGG TGTTAAATCA CTCTGACCAT CACCCTATCC 960 TTTTTCTTTC AAACCTGGTT GAGGGAACCT ACACTTTTCA CCTGAAAGTG ACCGATGCAA 1020
AGGGTGAGAG TGACACAGAC CGGACCACTG TGGAGGTGAA ACCTGATCCC AGGAAAAACA 1080
ACCTGGTGGA GATCATCTTG GATATCAACG TCAGTCAGCT AACTGAGAGG CTGAAGGGGA 1140
TGTTCATCCG CCAGATTGGG GTCCTCCTGG GGGTGCTGGA TTCCGACATC ATTGTGCAAA 1200
AGATTCAGCC GTACACGGAG CAGAGCACCA AAATGGTATT TTTTGTTCAA AACGAGCCTC 1260
CCCACCAGAT CTTCAAAGGC CATGAGGTGG CAGCGATGCT CAAGAGTGAG CTGCGGAAGC 1320
AAAAGGCAGA CTTTTTGATA TTCAGAGCCT TGGAAGTCAA CACTGTCACA TGTCAGCTGA 1380
ACTGTTCCGA CCATGGCTAC TGTGACTCGT TCACCAAACG CTGTATCTGT GACCCTTTTT 1440
GGATGGAGAA TTTCATCAAG GTGCAGCTGA GGGATGGAGA CAGCAACTGT GAGTGGAGCG 1500
TGTTATATGT TATCATTGCT ACCTTTGTCA TTGTTGTTGC CTTGGGAATC CTGTCTTGGA 1560
CTGTGATCTG TTGTTGTAAG AGGCAAAAAG GAAAACCCAA GAGGAAAAGC AAGTACAAGA 1620
TCTGGATGCC ACGGATCAGG AAAGCTTGGA GCTGAAGCCA ACCTCCCGAG CAGGCATCAA 1680
ACAGAAAGGC CTTTTGCTAA GTAGCAGCCT GATGCACTCC GAGTCAGAGC TGGACAGCGA 1740
TGATGCCATC TTTACATGGC CAGACCGAGA GAAGGGCAAA CTCCTGCATG GTCAGAATGG 1800
CTCTGTACCC AACGGGCAGA CCCCTCTGAA GGCCAGGAGC CCGCGGGAGG AGATCCTGTA 1860
GCCACCTGGT CTGTCTCCTC AGGGCAGGGC CCAGCACACT GCCCGGCCAG TCCTCCTACC 1920
TCCCGAGTCT GCGGGCAGCT GCTGTCCCAG CATCTGCTGG TCATTTCGCC CTGACAGTCC 1980
CAACCAGAAC CCCTGGGACT TGAATCCAGA GACGTCCTTC AGGAACCCCT CAACGAAGCT 2040
GTGAATGAAG AGGTTTCCTC TTTAAACCTG TCTGGTGGGC CCCCAGATAT CCTCACCTCA 2100
GGGCCTCCTT TTTTTGCAAA CTCCTCCCCT CCCCCGAGGG CAGACCCAGC CAGCTGCTAA 2160
GCTCTGCAGC TCCCCAGTGG ACAGTGTCAT TGTGCCCAGA GTGCTGCAAG GTGAGGCCTG 2220
CTGTGCTGCC CGCACACCTG AGTGCAAAAC CAAGCACTGT GGGCATGGTG TTTCCCTCTC 2280
TGGGGTAGAG TACGCCCTCT CGCTGGGCAA AGAGGAAGTG GCACCCCTCC CCTCACCACA 2340
GATGCTGAGA TGGTAGCATA GAAATGATGG CCGGGCGCGG TGGCTMACGC CTGTAATCCC 2400
AGCACTTTGG GAGGCCGAGG CGGGCGGATC ATGAGGTCAG GAGATCAAGA CCACCCTGGC 2460
TAACACGGTG AAACCCCATC TCTACTAAAA AAAAAAAAAA A 2501 (2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 138 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
Met Gin Gly Val Arg Thr Pro Thr Leu Gin Leu Ser Ala Met Gin Glu 1 5 10 15
Gly Asp Tyr Thr Tyr Gin Leu Thr Val Thr Asp Thr He Gly Gin Gin 20 25 30
Ala Thr Ala Gin Val Thr Val He Val Gin Pro Glu Asn Asn Lys Pro 35 40 45
Pro Gin Ala Asp Ala Gly Pro Asp Lys Glu Leu Thr Leu Pro Val Asp 50 55 60
Ser Thr Thr Leu Asp Gly Ser Lys Ser Ser Asp Asp Gin Lys He He 65 70 75 80
Ser Tyr Leu Trp Glu Lys Thr Gin Gly Pro Asp Gly Val Gin Leu Glu 85 90 95
Asn Ala Asn Ser Ser Val Ala Thr Val Thr Gly Leu Gin Val Gly Thr 100 105 110
Tyr Val Phe Thr Leu Thr Val Lys Asp Glu Arg Asn Leu Gin Ser Gin 115 120 125
Ser Ser Val Asn Val He Val Lys Glu Glu 130 135
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1820 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: GGCAGCAGCA GAGGGAGAGC TCGGGGCTTG GAGGGGAAAC AGCGGAAGAC CTAAGATTAT 60 CGGGAGGGCA GCAGAGGCAG AGAACGAGGA CAGGACCCTT GGCCGTCTTC TTCCAGGGAA 120
CGAGAGGTCA CAGCCTCGCT CTCCGCTTAG GCTTCTGGCG CCCCAGCTTA AAGCCGAGGC 180
TGCGGCTGAC AAAGGGCTCG CGCCGGTGCC GCCGCCCTTC TCATCCGGGC ATTCGGGTCC 240
CTGCGGAGAG GGAGGGGGAA GGGCAGAGGG GGAGGGGAAG GAGCCSGAGG GGCSCACACT 300
TGGAGCTGAA GCCCTCTCCA GGGCTCCGGG CCGGTGCCCC AACGGACAGA GGTCGAGGAG 360
GACCCGCAGA GGTGGCAGCG GCCGGGGGCA GGAGGATGGT GCAGAAGGAG AGTCAAGCGA 420
CGTTGGAGGA GCGGGAGAGC GAGCTCAGTT CCAACCCTGC CGCCTCTGCG GGGGCATCGC 480
TGGAGCCGCC GGCAGCTCCG GCACCCGGAG AAGACAACCC CGCCGGGGCT GGGGGAGCGG 540
CGGTGGCCGG GGCTGCAGGA GGGGCTCGGC GGTTTCCTCT GCGGTGTGGT GGAAGGATTT 600
TATGGAAGAC CTTGGGTTAT GGAACAGAGA AAAGAACTCT TTAGAAGGCT CCAGAAATGG 660
GAATTAAATA CATACTTGTA TGCCCCAAAA GATGACTACA AACATAGGAT GTTTTGGCGA 720
GAGATGTATT CAGTGGAGGA AGCTGAGCAA CTTATGACTC TCATCTCTGC TGCACGAGAA 780
TATGAGATAG AGTTCATCTA TGCGATCTCA CCTGGATTGG ATATCACTTT TTCTAACCCC 840
AAGGAAGTAT CCACATTGAA ACGTAAATTG GACCAGGTTT CTCAGTTTGG GTGCAGATCA 900
TTTGCTTTGC TTTTTGATGA TATAGACCAT AATATGTGTG CAGCAGACAA AGAGGTATTC 960
AGTTCTTTTG CTCATGCCCA AGTCTCCATC ACAAATGAAA TCTATCAGTA CCTAGGAGAG 1020
CCAGAAACTT TCCTCTTCTG TCCCACAGAA TACTGTGGCA CTTTCTGTTA TCCAAATGTG 1080
TCTCAGTCTC CATATTTAAG GACTGTGGGT GAAAAGCTTC TACCTGGAAT TGAAGTGCTT 1140
TGGACAGGTC CCAAAGTTGT TTCTAAAGAA ATTCC GTAG AGTCCATCGA AGAGGTTTCT 1200
AAGATTATTA AGAGAGCTCC AGTAATCTGG GATAACATTC ATGCTAATGA TTATGATCAG 1260
AAGAGACTGT TTCTGGGCCC GTACAAAGGA AGATCCACAG AACTCATCCC ACGGTTAAAA 1320
GGAGTCCTCA CTAATCCAAA TTGTGAATTT GAAGCCAACT ACGTTGCTAT CCACACCCTT 1380
GCCACCTGGT ACAAATCAAA CATGAATGGG AGTGAGAAAA GATGTAGTGA TGACTGACAG 1440
TGAAGATAGT ACTGTGTCCA TCCAGATAAA ATTAGAAAAT GAAGGCAGTG ATGAAGATAT 1500
TGAAACTGAT GTACTCTATA GTCCACAGAT GGCTCTAAAG CTAGCATTAA CAGAATGGTT 1560
GCAAGAGTTT GGTGTGCYTC ATCAATACAG CAGTAGGCAA GTTGCACACA GTGGAGCTAA 1620
AGCAAGTGTA GTTGATGGGA CTCCTTTAGT TGCAGCACCC TCTTTAAATG CCACAACCGT 1680
AGTAACAACA GTTTATCAGG AGCCCATTAT GAGCCAGGGA GCAGCCTTGA GTGGTGAGCC 1740 TACTACTCTG ACCAAGGAAG AAGAAAAGAA ACAGCCTGAT GAAGAACCCA TGGACATGGT 1800 GGTGGAAAAA AAAAAAAAAA 1820
(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 272 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
Met Glu Gin Arg Lys Glu Leu Phe Arg Arg Leu Gin Lys Trp Glu Leu 1 5 10 15
Asn Thr Tyr Leu Tyr Ala Pro Lys Asp Asp Tyr Lys His Arg Met Phe 20 25 30
Trp Arg Glu Met Tyr Ser Val Glu Glu Ala Glu Gin Leu Met Thr Leu 35 40 45
He Ser Ala Ala Arg Glu Tyr Glu He Glu Phe He Tyr Ala He Ser 50 55 60
Pro Gly Leu Asp He Thr Phe Ser Asn Pro Lys Glu Val Ser Thr Leu 65 70 75 80
Lys Arg Lys Leu Asp Gin Val Ser Gin Phe Gly Cys Arg Ser Phe Ala 85 90 95
Leu Leu Phe Asp Asp He Asp His Asn Met Cys Ala Ala Asp Lys Glu 100 105 110
Val Phe Ser Ser Phe Ala His Ala Gin Val Ser He Thr Asn Glu He 115 120 125
Tyr Gin Tyr Leu Gly Glu Pro Glu Thr Phe Leu Phe Cys Pro Thr Glu 130 135 140
Tyr Cys Gly Thr Phe Cys Tyr Pro Asn Val Ser Gin Ser Pro Tyr Leu 145 150 155 160
Arg Thr Val Gly Glu Lys Leu Leu Pro Gly He Glu Val Leu Trp Thr 165 170 175
Gly Pro Lys Val Val Ser Lys Glu He Pro Val Glu Ser He Glu Glu 180 185 190 Val Ser Lys He He Lys Arg Ala Pro Val He Trp Asp Asn He His 195 200 205
Ala Asn Asp Tyr Asp Gin Lys Arg Leu Phe Leu Gly Pro Tyr Lys Gly 210 215 220
Arg Ser Thr Glu Leu He Pro Arg Leu Lys Gly Val Leu Thr Asn Pro 225 230 235 240
Asn Cys Glu Phe Glu Ala Asn Tyr Val Ala He His Thr Leu Ala Thr 245 250 255
Trp Tyr Lys Ser Asn Met Asn Gly Ser Glu Lys Arg Cys Ser Asp Asp 260 265 270
(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2405 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
TATCCATTAC GTCGACTAAT ACGTACATAA GAATTCAATC GGGAGACAAT GTAATAACCC 60
AAACACTGGG TATTCATATT TGACACATGG GCAAACTTGC CAGTGGAATG GAATTGTGAC 120
CTGACAGAGA AGGGAAGGCA GGCTGACGAA GGTGATCGAA TGGGAGAACA GTGCTGTGGT 180
GATCATGAGA ATGAGGCTTT TCTGTAGCAT GTAAACCAAA CCGGACCCTT GGCAGTTCGT 240
CGTCCCTCAG TTCTCCAGAT GCTATTTTTT GCAGGTTCTA CCAAGTGCTT GTTGATTACC 300
CTAGTTGTAA TTATCTAGGG AAGAGATGAA TGTAAGTGAG AGTGCAGAGC ACTGGGGAGG 360
GTGACAGTGA AATGCAATTA GAGGCAGCAA GAGAGTCCTA GTCTGTTCTC ACATAACCGG 420
ACTTGAACGC TCCAGTGCGA GCAGAGTGCT GGGGGTGGAT TCCACTGCCG AACCACGGCA 480
GCTTTGCTTT ACTCTTCAGC ATGGGGGTGG TAACTAGCTG CACAGCAAGT TATGAAATGG 540
AAAGCAAGCT TAACAGCTGT AATCTCATCG GATACCCTGG AGCAAATGCC TTGGGATTGC 600
CTGAAGTGAA GTGTTTAGCA TCCACCAAAT AGTTGAGTTT CTAAGATGGG CCATGCGGGA 660
TCCCTGCCAC ACGGGTGTGG GGGCAGCGCG CTCCTCCCTG CCTCGGCTGC TGTGTGTCTT 720 AGTCTTCATG TTTCCCCTTG GCGTCACCTC TGGTCCTGTC CATCTTGTGC ACATGTGTCC 780
CTGAGGGCGA GTCACACCTG TGTCTTGAGT CTGCTGTAAG GTGTTCAGTC TACCTCAAGG 840
GGTCATCATG GTAAGCTCTA TCCCACGGCA TCCTCCGTCC ATCACCACAC ACACTGGAAT 900
GTATCCCCCC CGCCCTGCCC CTTCGATAAC CTCCTGACCT TGTCTTTCCT ATAATCACCA 960
CAAACTCCTA GTGCAAATTG GATGCTGCTT TAAATGTGAA AACAATTGTT CAAAAGCTAT 1020
AAAACCTGAA TGAAAGCTGA AGCTGAATTT ATAAGCCTTG TTGCATATGA GCCAAAAGTG 1080
CAAAAAGCTC TATATAATGA ACTAACCTGC CACTCGTATA AATATAAATA TATATAAATA 1140
TATTAAAATC AGACTGTTCT ACAAAATTGT GTATTTGTAT TTTTGTGTAC GTACAATTTT 1200
CTGCTAAGCA GAAGGAGGAT GTAGTATAGG ATGCTGTGAG AAGAGATTTG GTTTAATCCT 1260
ATTTCTTTGT TACTTATTTT TGTTAACATC AGATGCCTGT CTTTGTCTTA TATGTGTATG 1320
ACACTGTTTG GAAAGCTGCA GTATCTCTCT TCCTGAGGTC CAGAGGCCAT TAAAGAATAA 1380
ACATGGGGCT TAGAGGTGTC TTGGCCACCA AACACAGGTA AGCCAACAGA GTTCGGCTCC 1440
CGTACGTGGT GTGGTGGACC TGGGGCCTGG AGCCACTTGT ACTCCTGGGG TGGGACTGAG 1500
GGAGGGAAAG GGCTCCCCCA CTTCAGGCTG GTCACTGTAT GTTGACTTCC CTCTGGGAAG 1560
CAAAATTCCA CTTGTAAAAA TCAGCTTCCA AGACAAAAGA TGATCCGATG TCACTTCTGG 1620
GTCACCAGGA AGGGACACAG GATCTCTCTG AACTTTGGAA CAGACCCTCA TATTCTGGGG 1680
CCAGAAGTTT GCCCAGAAAG CAGCAGGTGG CTCTGCCTGG CTGTAGAGCC CAGCTCATTG 1740
GCTGTCCCTG GGCTTGGTCT CCCTCTTCCG AGTAGTTGCT GCCTTTCTTC AGATCAGGTT 1800
ACCACAATGC CTCCCCGCTG CTGACGCTTC ATCCCCCACA CCTCCAGCCC CAGTTACCTG 1860
GAGCTTCTCA GAACCCACTT TGCCGGTGCT AAAACACAAG AGGGGGTGAA AGTGGCTGCC 1920
AGTAATGGCC AGAAACCAAC CACCAGAGGC CAGGCTGAAA GACAAGCTCC GGGTGTCCAG 1980
GGGCTGACGG GCCAACCATG TGGCAGGTCC CAGGCCCCAC CCACTGCGCC ATCCGCCTCT 2040
GAGCTCCACA GTGGTCCCAC TAATGGGAAC CTCCTCTAGG GAGAGTGATA CTGCACCTTC 2100
ACCCGTAGGA CTCATATTTA TAACAATGTG TAATGGCTGT AGCAAAAAGC CCTTGTTTCT 2160
AGATGTAAAT GGTCAAAGAA ACAAGCGCTC TATTGTTTTG AATAAAATAG TTCAAATGAG 2220
TCCTGTATCA TTGTATCTCC TATTCTGGAT TAGTGCCTTT TGGACAGTAG ACTGTTCTGT 2280
AATTAAAATG TAGTATACTG CTTTTTTGTA CAGTTTTGTT TTAATAAAAC TTTTTTTTAA 2340
TTTGTGTTTA TTTTAGTATT GTACCTATTA GAGAATAAAA TGTATAACTG AAAAAAAAAA 2400 AAAAA 2405
( 2 ) INFORMATION FOR SEQ ID NO : 20 :
( i ) SEQUENCE CHARACTERISTICS :
(A) LENGTH : 76 amino acids
( B ) TYPE : amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:
Met Gly Thr Ser Ser Arg Glu Ser Asp Thr Ala Pro Ser Pro Val Gly 1 5 10 15
Leu He Phe He Thr Met Cys Asn Gly Cys Ser Lys Lys Pro Leu Phe 20 25 30
Leu Asp Val Asn Gly Gin Arg Asn Lys Arg Ser He Val Leu Asn Lys 35 40 45
He Val Gin Met Ser Pro Val Ser Leu Tyr Leu Leu Phe Trp He Ser 50 55 60
Ala Phe Trp Thr Val Asp Cys Ser Val He Lys Met 65 70 75
(2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: CNAGCAGCAGA TACAGCAGTA AGGAAGGC 29
(2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: GNGCTCTCCTT AATCGCCGTC TCAAACAT 29
(2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: CNTTGGCCAGG TCTTCCTGCT CCTTCATT 29
(2) INFORMATION FOR SEQ ID NO: 24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: ANGCTGCAAAG CTGGTGTAGA AGTAGGTG 29
(2) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: ANTTAGCCATG TCACTCCGCA GGAAGTCC 29
(2) INFORMATION FOR SEQ ID NO: 26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: TNTAGTCATAA AGAGCCACTA CCGTTGGT 29
(2) INFORMATION FOR SEQ ID NO: 27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: TNGTATGGGTA GGGCTTGGCA TAGTGTGG 29
(2) INFORMATION FOR SEQ ID NO: 28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: TNTCAGGTTTG CAGTAGTAGA GTTGGTAG 29
(2) INFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: CNTCTTCGATG GACTCTACTG GAATTTCT 29
(2) INFORMATION FOR SEQ ID NO: 30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30: TNAGCCTGGCC TCTGGTGGTT GGTTTCTG 29
(2) INFORMATION FOR SEQ ID NO: 31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 336 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:
Thr Leu Lys Leu Cys Ser Pro Pro Lys Asp His Glu Val Leu Gly Val 1 5 10 15
He Gin Arg Phe Leu Lys Leu Arg Ser Pro Trp Lys Ala Leu He Thr 20 25 30
Thr He Leu Lys Pro Gin Leu Phe Glu Pro Arg Pro Arg Leu Thr Val 35 40 45
Leu Thr Phe Pro Ser Ser His Glu Gly Glu Ser Phe Pro Leu Ala Trp 50 55 60
Asn Ala Lys He Thr Asp Leu Lys Gin Lys Val Glu Asn Leu Phe Asn 65 70 75 80
Glu Lys Cys Gly Glu Ala Leu Gly Leu Lys Gin Ala Val Lys Val Pro 85 90 95
Phe Ala Leu Phe Glu Ser Phe Pro Glu Asp Phe Tyr Val Glu Gly Leu 100 105 110
Pro Glu Gly Val Pro Phe Arg Arg Pro Ser Thr Phe Gly He Pro Arg 115 120 125
Leu Glu Lys He Leu Arg Asn Lys Ala Lys He Lys Phe He He Lys 130 135 140
Lys Pro Glu Met Phe Glu Thr Ala He Lys Glu Ser Thr Ser Ser Lys 145 150 155 160
Ser Pro Pro Arg Lys He Asn Ser Ser Pro Asn Val Asn Thr Thr Ala 165 170 175
Ser Gly Val Glu Asp Leu Asn He He Gin Val Thr He Pro Asp Asp 180 185 190
Asp Asn Glu Arg Leu Ser Lys Val Glu Lys Ala Arg Gin Leu Arg Glu 195 200 205
Gin Val Asn Asp Leu Phe Ser Arg Lys Phe Gly Glu Ala He Gly Met 210 215 220
Gly Phe Pro Val Lys Val Pro Tyr Arg Lys He Thr He Asn Pro Gly 225 230 235 240
Cys Val Val Val Asp Gly Met Pro Pro Gly Val Ser Phe Lys Ala Pro 245 250 255
Ser Tyr Leu Glu He Ser Ser Met Arg Arg He Leu Asp Ser Ala Glu 260 265 270 Phe He Lys Phe Thr Val He Arg Pro Phe Pro Gly Leu Val He Asn 275 280 285
Asn Gin Leu Val Asp Gin Ser Glu Ser Lys Gly Pro Val He Gin Glu 290 295 300
Ser Ala Glu Pro Ser Gin Leu Glu Val Pro Ala Thr Glu Glu He Lys 305 310 315 320
Glu Thr Asp Gly Ser Ser Gin He Lys Gin Glu Pro Asp Pro Thr Trp 325 330 335
(2) INFORMATION FOR SEQ ID NO: 32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide'
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: ANCTTGGCCA GGTCTTCCTG CTCCTTCAT 29

Claims

What is claimed is:
1. An isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:l;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:l from nucleotide 521 to nucleotide 1111;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:l from nucleotide 536 to nucleotide 817;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone ax318_3 deposited under accession number ATCC 98353;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone ax318_3 deposited under accession number ATCC 98353;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone ax318_3 deposited under accession number ATCC 98353;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone ax318_3 deposited under accession number ATCC 98353;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:2;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:2 having biological activity, the fragment comprising the amino acid sequence from amino acid 93 to amino acid 102 of SEQ ID NO:2;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i).
2. The polynucleotide of claim 1 wherein said polynucleotide is operably linked to at least one expression control sequence.
3. A host cell transformed with the polynucleotide of claim 2.
4. The host cell of claim 3, wherein said cell is a mammalian cell.
5. A process for producing a protein encoded by the polynucleotide of claim 2, which process comprises:
(a) growing a culture of the host cell of claim 3 in a suitable culture medium; and
(b) purifying said protein from the culture.
6. A protein produced according to the process of claim 5.
7. The protein of claim 6 comprising a mature protein.
8. A protein comprising an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:2;
(b) the amino acid sequence of SEQ ID NO:2 from amino acid 4 to amino acid 99;
(c) fragments of the amino acid sequence of SEQ ID NO:2 comprising the amino acid sequence from amino acid 93 to amino acid 102 of SEQ ID NO:2; and
(d) the amino acid sequence encoded by the cDNA insert of clone ax318_3 deposited under accession number ATCC 98353; the protein being substantially free from other mammalian proteins.
9. The protein of claim 8, wherein said protein comprises the amino acid sequence of SEQ ID NO:2.
10. The protein of claim 8, wherein said protein comprises the amino acid sequence of SEQ ID NO:2 from amino acid 4 to amino acid 99.
11. A composition comprising the protein of claim 8 and a pharmaceutically acceptable carrier.
12. A method for preventing, treating or ameliorating a medical condition which comprises administering to a mammalian subject a therapeutically effective amount of a composition of claim 11.
13. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:l.
14. An isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:3;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:3 from nucleotide 61 to nucleotide 864;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:3 from nucleotide 826 to nucleotide 1386;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone bgl40_l deposited under accession number ATCC 98353;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone bgl40_l deposited under accession number ATCC 98353;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone bgl40_l deposited under accession number ATCC 98353;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone bgl40_l deposited under accession number ATCC 98353;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:4;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:4 having biological activity, the fragment comprising the amino acid sequence from amino acid 129 to amino acid 138 of SEQ ID NO:4;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and (1) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i).
15. A protein comprising an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:4;
(b) the amino acid sequence of SEQ ID NO:31;
(c) the amino acid sequence of SEQ ID NO:31 from amino acid 148 to amino acid 249;
(d) fragments of the amino acid sequence of SEQ ID NO:4 comprising the amino acid sequence from amino acid 129 to amino acid 138 of SEQ ID NO:4;
(e) fragments of the amino acid sequence of SEQ ID NO:31comprising the amino acid sequence from amino acid 163 to amino acid 172 of SEQ ID NO:31; and
(f) the amino acid sequence encoded by the cDNA insert of clone bgl40_l deposited under accession number ATCC 98353; the protein being substantially free from other mammalian proteins.
16. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:3.
17. An isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:5;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:5 from nucleotide 77 to nucleotide 1624;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:5 from nucleotide 390 to nucleotide 789;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone bg465_2 deposited under accession number ATCC 98353;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone bg465_2 deposited under accession number ATCC 98353; (f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone bg465_2 deposited under accession number ATCC 98353;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone bg465_2 deposited under accession number ATCC 98353;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:6;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:6 having biological activity, the fragment comprising the amino acid sequence from amino acid 253 to amino acid 262 of SEQ ID NO:6;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i).
18. A protein comprising an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:6;
(b) the amino acid sequence of SEQ ID NO:6 from amino acid 260 to amino acid 343;
(c) fragments of the amino acid sequence of SEQ ID NO:6 comprising the amino acid sequence from amino acid 253 to amino acid 262 of SEQ ID NO:6; and
(d) the amino acid sequence encoded by the cDNA insert of clone bg465_2 deposited under accession number ATCC 98353; the protein being substantially free from other mammalian proteins.
19. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:5.
20. An isolated polynucleotide selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:7;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:7 from nucleotide 48 to nucleotide 1055;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:7 from nucleotide 216 to nucleotide 1055;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:7 from nucleotide 494 to nucleotide 958;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone bk291_3 deposited under accession number ATCC 98353;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone bk291_3 deposited under accession number ATCC 98353;
(g) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone bk291_3 deposited under accession number ATCC 98353;
(h) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone bk291_3 deposited under accession number ATCC 98353;
(i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:8;
(j) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:8 having biological activity, the fragment comprising the amino acid sequence from amino acid 85 to amino acid 94 of SEQ ID NO:8;
(k) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(h) above;
(1) a polynucleotide which encodes a species homologue of the protein of (i) or (j) above ; and
(m) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(j).
21. A protein comprising an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:8; (b) the amino acid sequence of SEQ ID NO:8 from amino acid 188 to amino acid 306;
(c) fragments of the amino acid sequence of SEQ ID NO:8 comprising the amino acid sequence from amino acid 85 to amino acid 94 of SEQ ID NO:8; and
(d) the amino acid sequence encoded by the cDNA insert of clone bk291_3 deposited under accession number ATCC 98353; the protein being substantially free from other mammalian proteins.
22. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:7.
23. An isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9 from nucleotide 64 to nucleotide 1197;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9 from nucleotide 1 to nucleotide 828;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone bp537_4 deposited under accession number ATCC 98353;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone bp537_4 deposited under accession number ATCC 98353;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone bp537_4 deposited under accession number ATCC 98353;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone bp537_4 deposited under accession number ATCC 98353;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:10;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:10 having biological activity, the fragment comprising the amino acid sequence from amino acid 184 to amino acid 193 of SEQ ID NO:10; (j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i).
24. A protein comprising an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:10;
(b) the amino acid sequence of SEQ ID NO:10 from amino acid 1 to amino acid 255;
(c) fragments of the amino acid sequence of SEQ ID NO: 10 comprising the amino acid sequence from amino acid 184 to amino acid 193 of SEQ ID NO:10; and
(d) the amino acid sequence encoded by the cDNA insert of clone bp537_4 deposited under accession number ATCC 98353; the protein being substantially free from other mammalian proteins.
25. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:9.
26. An isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:ll;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:ll from nucleotide 581 to nucleotide 1534;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:ll from nucleotide 928 to nucleotide 1510;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone cs431_2 deposited under accession number ATCC 98353;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone cs431_2 deposited under accession number ATCC 98353; (f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone cs431_2 deposited under accession number ATCC 98353;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone cs431_2 deposited under accession number ATCC 98353;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:12;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:12 having biological activity, the fragment comprising the amino acid sequence from amino acid 154 to amino acid 163 of SEQ ID NO:12;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i).
27. A protein comprising an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO: 12;
(b) the amino acid sequence of SEQ ID NO:12 from amino acid 150 to amino acid 310;
(c) fragments of the amino acid sequence of SEQ ID NO:12 comprising the amino acid sequence from amino acid 154 to amino acid 163 of SEQ ID NO:12; and
(d) the amino acid sequence encoded by the cDNA insert of clone cs431_2 deposited under accession number ATCC 98353; the protein being substantially free from other mammalian proteins.
28. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:ll.
29. An isolated polynucleotide selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:13;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:13 from nucleotide 29 to nucleotide 2227;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:13 from nucleotide 1334 to nucleotide 2227;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:13 from nucleotide 1 to nucleotide 746;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone cw976_l deposited under accession number ATCC 98353;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone cw976_l deposited under accession number ATCC 98353;
(g) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone cw976_l deposited under accession number ATCC 98353;
(h) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone cw976_l deposited under accession number ATCC 98353;
(i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:14;
(j) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 14 having biological activity, the fragment comprising the amino acid sequence from amino acid 361 to amino acid 370 of SEQ ID NO:14;
(k) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(h) above;
(1) a polynucleotide which encodes a species homologue of the protein of (i) or (j) above ; and
(m) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(j).
30. A protein comprising an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO: 14; (b) the amino acid sequence of SEQ ID NO: 14 from amino acid 1 to amino acid 239;
(c) the amino acid sequence of SEQ ID NO:14 from amino acid 119 to amino acid 733;
(d) fragments of the amino acid sequence of SEQ ID NO: 14 comprising the amino acid sequence from amino acid 361 to amino acid 370 of SEQ ID NO:14; and
(e) the amino acid sequence encoded by the cDNA insert of clone cw976_l deposited under accession number ATCC 98353; the protein being substantially free from other mammalian proteins.
31. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:13.
32. An isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:15;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:15 from nucleotide 364 to nucleotide 777;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:15 from nucleotide 1 to nucleotide 636;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone cwl233_3 deposited under accession number ATCC 98353;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone cwl233_3 deposited under accession number ATCC 98353;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone cwl233_3 deposited under accession number ATCC 98353;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone cwl233_3 deposited under accession number ATCC 98353;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:16;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:16 having biological activity, the fragment comprising the amino acid sequence from amino acid 64 to amino acid 73 of SEQ ID NO:16;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i).
33. A protein comprising an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO: 16;
(b) the amino acid sequence of SEQ ID NO: 16 from amino acid 1 to amino acid 91;
(c) fragments of the amino acid sequence of SEQ ID NO: 16 comprising the amino acid sequence from amino acid 64 to amino acid 73 of SEQ ID NO:16; and
(d) the amino acid sequence encoded by the cDNA insert of clone cwl233_3 deposited under accession number ATCC 98353; the protein being substantially free from other mammalian proteins.
34. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:15.
35. An isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:17;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 17 from nucleotide 619 to nucleotide 1434;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:17 from nucleotide 520 to nucleotide 1323;
(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone dgl_l deposited under accession number ATCC 98353; (e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone dgl_l deposited under accession number ATCC 98353;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone dgl_l deposited under accession number ATCC 98353;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone dgl_l deposited under accession number ATCC 98353;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 18;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:18 having biological activity, the fragment comprising the amino acid sequence from amino acid 131 to amino acid 140 of SEQ ID NO:18;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i).
36. A protein comprising an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:18;
(b) the amino acid sequence of SEQ ID NO: 18 from amino acid 1 to amino acid 235;
(c) fragments of the amino acid sequence of SEQ ID NO: 18 comprising the amino acid sequence from amino acid 131 to amino acid 140 of SEQ ID NO:18; and
(d) the amino acid sequence encoded by the cDNA insert of clone dgl_l deposited under accession number ATCC 98353; the protein being substantially free from other mammalian proteins.
37. An isolated gene corresponding to the cDNA sequence of SEQ ID NO:17.
38. An isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:19;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:19 from nucleotide 2063 to nucleotide 2290;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:19 from nucleotide 2276 to nucleotide 2290;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:19 from nucleotide 2037 to nucleotide 2405;
(e) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone ep234_l deposited under accession number ATCC 98353;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone ep234_l deposited under accession number ATCC 98353;
(g) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone ep234_l deposited under accession number ATCC 98353;
(h) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone ep234_l deposited under accession number ATCC 98353;
(i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:20;
(j) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:20 having biological activity, the fragment comprising the amino acid sequence from amino acid 33 to amino acid 42 of SEQ ID NO:20;
(k) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(h) above;
(1) a polynucleotide which encodes a species homologue of the protein of (i) or (j) above ; and
(m) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(j).
39. A protein comprising an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of SEQ ID NO:20;
(b) the amino acid sequence of SEQ ID NO:20 from amino acid 1 to amino acid 69;
(c) fragments of the amino acid sequence of SEQ ID NO:20 comprising the amino acid sequence from amino acid 33 to amino acid 42 of SEQ ID NO:20; and
(d) the amino acid sequence encoded by the cDNA insert of clone ep234_l deposited under accession number ATCC 98353; the protein being substantially free from other mammalian proteins.
40. An isolated gene corresponding to the cDNA sequence of SEQ ID NO: 19.
PCT/US1998/004601 1997-03-11 1998-03-09 Secreted proteins and polynucleotides encoding them WO1998040404A2 (en)

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EP98911532A EP0970109A2 (en) 1997-03-11 1998-03-09 Secreted proteins and polynucleotides encoding them
CA002283193A CA2283193A1 (en) 1997-03-11 1998-03-09 Secreted proteins and polynucleotides encoding them
AU65468/98A AU6546898A (en) 1997-03-11 1998-03-09 Secreted proteins and polynucleotides encoding them
JP53968398A JP2001514520A (en) 1997-03-11 1998-03-09 Secreted proteins and polynucleotides encoding them

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US81538197A 1997-03-11 1997-03-11
US08/815,381 1997-03-11
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US09/036,321 1998-03-06

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WO2000043505A2 (en) * 1999-01-22 2000-07-27 Memorec Medical Molecular Research Cologne Stoffel Gmbh Protease with two aspartate radicals in the catalytically active structure
US6833240B2 (en) 2000-03-08 2004-12-21 Mcgill University Very low density lipoprotein receptor polymorphisms and uses therefor
EP1683867A1 (en) 1999-06-18 2006-07-26 Research Foundation Of State University Of New York Groups of Borrelia burgdorferi and Borrelia afzelii that cause lyme disease in humans
US7094543B2 (en) 2000-09-27 2006-08-22 Massachusetts Institute Of Technology Methods for detecting rare polymorphic variants in genomic DNA sequences
WO2012068463A2 (en) 2010-11-18 2012-05-24 Beth Israel Deaconess Medicall Center, Inc. Methods of treating obesity by inhibiting nicotinamide n-methyl transferase (nnmt)
CN112930359A (en) * 2018-10-25 2021-06-08 味之素株式会社 Secretory production method of protein

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000043505A2 (en) * 1999-01-22 2000-07-27 Memorec Medical Molecular Research Cologne Stoffel Gmbh Protease with two aspartate radicals in the catalytically active structure
WO2000043505A3 (en) * 1999-01-22 2001-11-29 Memorec Medical Molecular Res Protease with two aspartate radicals in the catalytically active structure
EP1683867A1 (en) 1999-06-18 2006-07-26 Research Foundation Of State University Of New York Groups of Borrelia burgdorferi and Borrelia afzelii that cause lyme disease in humans
US6833240B2 (en) 2000-03-08 2004-12-21 Mcgill University Very low density lipoprotein receptor polymorphisms and uses therefor
US7094543B2 (en) 2000-09-27 2006-08-22 Massachusetts Institute Of Technology Methods for detecting rare polymorphic variants in genomic DNA sequences
WO2012068463A2 (en) 2010-11-18 2012-05-24 Beth Israel Deaconess Medicall Center, Inc. Methods of treating obesity by inhibiting nicotinamide n-methyl transferase (nnmt)
CN112930359A (en) * 2018-10-25 2021-06-08 味之素株式会社 Secretory production method of protein

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CA2283193A1 (en) 1998-09-17
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EP0970109A2 (en) 2000-01-12
AU6546898A (en) 1998-09-29

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