WO2003051919A1 - Proteines secretees de type mucine - Google Patents

Proteines secretees de type mucine Download PDF

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Publication number
WO2003051919A1
WO2003051919A1 PCT/GB2002/005811 GB0205811W WO03051919A1 WO 2003051919 A1 WO2003051919 A1 WO 2003051919A1 GB 0205811 W GB0205811 W GB 0205811W WO 03051919 A1 WO03051919 A1 WO 03051919A1
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seq
polypeptide
nucleic acid
sequence
acid molecule
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PCT/GB2002/005811
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English (en)
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Mark Douglas Davies
Richard Joseph Fagan
Christopher Benjamin Phelps
Christine Power
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Ares Trading S.A.
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Priority to AU2002356306A priority Critical patent/AU2002356306A1/en
Publication of WO2003051919A1 publication Critical patent/WO2003051919A1/fr

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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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4727—Mucins, e.g. human intestinal mucin
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)

Definitions

  • This invention relates to novel proteins (herein termed INSP009, INSPOIO and INSPOl l), identified as secreted proteins (in particular as members of the cystine knot class of secreted proteins and furthermore as members of the mucin subfamily) and to the use of these proteins and nucleic acid sequences from the encoding genes in the diagnosis, prevention and treatment of disease.
  • novel proteins herein termed INSP009, INSPOIO and INSPOl l
  • bioinformatics tools increase in potency and in accuracy, these tools are rapidly replacing the conventional techniques of biochemical characterisation. Indeed, the advanced bioinformatics tools used in identifying the present invention are now capable of outputting results in which a high degree of confidence can be placed.
  • Various institutions and commercial organisations are examining sequence data as they become available and significant discoveries are being made on an on-going basis. However, there remains a continuing need to identify and characterise further genes and the polypeptides that they encode, as targets for research and for drug discovery.
  • Secreted proteins The ability for cells to make and secrete extracellular proteins is central to many biological processes. Enzymes, growth factors, extracellular matrix proteins and signalling molecules are all secreted by cells. This is through fusion of a secretory vesicle with the plasma membrane. In most cases, but not all, proteins are directed to the endoplasmic reticulum and into secretory vesicles by a signal peptide. Signal peptides are cis-acting sequences that affect the transport of polypeptide chains from the cytoplasm to a membrane bound compartment such as a secretory vesicle. Polypeptides that are targeted to the secretory vesicles are either secreted into the extracellular matrix or are retained in the plasma membrane.
  • polypeptides that are retained in the plasma membrane will have one or more transmembrane domains.
  • secreted proteins that play a central role in the functioning of a cell are cytokines, hormones, extracellular matrix proteins (adhesion molecules), proteases, and growth and differentiation factors.
  • Mucins are the main components of mucus.
  • Mucins are synthesized and secreted by specialized cells of the epithelium and in some case, by non mucin-secreting cells. Little was known about the structure of mucins until around a decade ago. This was principally due to the heavy glycosylation of mucins, which complicated their analysis. Mucins are now known to fall within the cystine-knot fold family (Moniaux N et al, (2001) Front Biosci. Oct 1;6:D1192-206).
  • mucins were thought to have the unique function of protecting and lubricating the epithelial surfaces.
  • the study of the mucin structure as well as the relationship between structure and function show that mucins also possess other important functions, such as in growth and differentiation (Simmons PJ et al, (2001) Ann NY Acad Sci Jun; 938:196-206) direct implication in the foetal development (Brandenberger R et /.,(2001) J Cell Biol, Jul 23;154(2):447-58), in epithelial renewal, differentiation and integrity (de Bolos C et al, (2001)Front Biosci, Oct l;6:D1256-63), in carcinogenesis (Hanaoka J et al, Cancer , Oct 15;92(8):2148-57, Silva F et al, Eur J Hum Genet Jul;9(7):548-52), and in reproduction (Gipson IK, (2001) Front Biosci, Oct l;6:D
  • dysregulation of mucin function is implicated in many diseases, including carcinogenisis (Paydas S et al, (2001) Leuk. Res. Mar; 25(3):221-5), reproductive health
  • cystine knot family The typical structure seen in cystine knot family is based on the presence of 6 cysteine residues creating 3 disulphide bonds. Two of the disulphide bonds create a 'ring-like' structure, which is penetrated by the third disulphide bond, (Sun et al. 1995). Cystine knot domains are often found with more than 6 cysteine residues. The extra cysteine residues are normally used to create further disulphide bonds within the cystine knot domain or interchain disulphide bonds, during dimerisation.
  • This cystine knot super family is divided into subfamilies, which include, the glycoprotein hormones (e.g. follicle stimulating hormone), the transforming growth factor beta (TGFBeta) proteins (e.g. bone morphogenentic protein 4), the platelet-derived growth factor-like (PDGF-like) proteins (e.g. platelet derived growth factor A), nerve growth factors (NGF) (e.g. brain-derived neurotrophic factor), the DAN family (eg. cerberus) and some members of the mucin family (e.g. MUC5AC).
  • glycoprotein hormones e.g. follicle stimulating hormone
  • TGFBeta transforming growth factor beta
  • PDGF-like proteins e.g. platelet derived growth factor A
  • nerve growth factors e.g. brain-derived neurotrophic factor
  • DAN family e.g. cerberus
  • mucin family e.g. MUC5AC
  • the mucin proteins function as major airway glycoproteins. Mucin protein sequences have been found to be secreted from cells found in respiratory, gastrointestinal and urogenital tracts (Eckhardt et al 1997). Mucins normally exist as multi-domain proteins. Domains other than cystine knots, identified in mucin sequences, but not found in all mucins, include von Willebrand factor D (N FD) and von Willebrand factor (NWFC). NWFD domains have been associated with binding of factor NIII and the multimerization (Jorieux et al. 2000). While NWFC domains have been associated with protein sequences, which are involved in maintaining homeostasis (Voorberg et al. 1991).
  • the invention is based on the discovery that the INSP009, INSPOIO, INSPOl l and INSP01 la proteins function as secreted proteins and moreover as secreted proteins of the cystine knot class. More specifically INSP009, INSPOIO, INSPOl l and INSPOl la belong to the mucin subfamily of cystine knot domain containing proteins. Not all mucin sequences contain cystine knot domains. This is illustrated by the fact that the protein sequence of tracheobronchial mucin (MUC5 AC) contains a cystine knot domain and the protein sequence of mucin 1 (MUC1) does not contain a cystine knot domain.
  • MUC5 AC protein sequence of tracheobronchial mucin
  • MUC1 protein sequence of mucin 1
  • the invention provides a polypeptide, which polypeptide:
  • (i) comprises an amino acid sequence as recited in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42,
  • (ii) is a fragment thereof which functions as a secreted protein or has an antigenic determinant in common with the polypeptides of (i); or (iii) is a functional equivalent of (i) or (ii).
  • a polypeptide according to part i) above comprises the sequence recited in SEQ ID NO: 180 and more preferably, consists of the sequence recited in SEQ ID NO: 180.
  • this embodiment of the invention provides a polypeptide comprising the sequence recited in SEQ ID NO: 186.
  • This polypeptide sequence will be referred to herein as the "INSP009 exons 24-26 consolidated polypeptide sequence" and contains the portion of the polypeptide in which the cystine knot domain is considered to reside.
  • the polypeptide having the sequence recited in SEQ ID NO:2 is encoded by exon 1 of the INSP009 protein and is referred to hereafter as "the INSP009 exon 1 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO:4 is encoded by exon 2 of the INSP009 protein and is referred to hereafter as "the INSP009 exon 2 polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NO: 6 is encoded by exon 3 of the INSP009 protein and is referred to hereafter as "the INSP009 exon 3 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 8 is encoded by exon 4 of the INSP009 protein and is referred to hereafter as "the INSP009 exon 4 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 10 is encoded by exon 5 of the INSP009 protein and is referred to hereafter as "the INSP009 exon 5 polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NO: 12 is encoded by exon 6 of the INSP009 protein and is referred to hereafter as "the INSP009 exon 6 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 14 is encoded by exon 7 of the INSP009 protein and is referred to hereafter as "the INSP009 exon 7 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 16 is encoded by exon 8 of the INSP009 protein and is referred to hereafter as "the INSP009 exon 8 polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NO: 18 is encoded by exon 9 of the INSP009 protein and is referred to hereafter as "the INSP009 exon 9 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO:20 is encoded by exon 10 of the INSP009 protein and is referred to hereafter as "the INSP009 exon 10 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO:22 is encoded by exon 11 of the INSP009 protein and is referred to hereafter as "the INSP009 exon 11 polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NO:24 is encoded by exon 12 of the INSP009 protein and is referred to hereafter as "the INSP009 exon 12 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO:26 is encoded by exon 13 of the INSP009 protein and is referred to hereafter as "the INSP009 exon 13 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO:28 is encoded by exon 14 of the INSP009 protein and is referred to hereafter as "the INSP009 exon 14 polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NO:30 is encoded by exon 15 of the INSP009 protein and is referred to hereafter as "the INSP009 exon 15 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO:32 is encoded by exon 16 of the INSP009 protein and is referred to hereafter as "the INSP009 exon 16 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO:34 is encoded by exon 17 of the INSP009 protein and is referred to hereafter as "the INSP009 exon 17 polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NO:36 is encoded by exon 18 of the INSP009 protein and is referred to hereafter as "the INSP009 exon 18 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 38 is encoded by exon 19 of the INSP009 protein and is referred to hereafter as "the INSP009 exon 19 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO:40 is encoded by exon 20 of the INSP009 protein and is referred to hereafter as "the INSP009 exon 20 polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NO:42 is encoded by exon 21 of the INSP009 protein and is referred to hereafter as "the INSP009 exon 21 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO:44 is encoded by exon 22 of the INSP009 protein and is referred to hereafter as "the INSP009 exon 22 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO:46 is encoded by exon 23 of the INSP009 protein and is referred to hereafter as "the INSP009 exon 23 polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NO:48 is encoded by exon 24 of the INSP009 protein and is referred to hereafter as "the INSP009 exon 24 polypeptide".
  • polypeptide having the sequence recited in SEQ ID NO:50 is encoded by exon 25 of the INSP009 protein and is referred to hereafter as "the INSP009 exon 25 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO:52 is encoded by exon 26 of the INSP009 protein and is referred to hereafter as "the INSP009 exon 26 polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NO: 180 is referred to hereafter as "the INSP009 polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NO: 186 is referred to hereafter as the "INSP009 exons 24-26 consolidated polypeptide sequence".
  • the invention provides a polypeptide, which polypeptide:
  • (i) comprises an amino acid sequence as recited in SEQ ID NO:54, SEQ ID NO:56,
  • (ii) is a fragment thereof which functions as a secreted protein or has an antigenic determinant in common with the polypeptides of (i); or
  • a polypeptide according to part i) above comprises the sequence recited in SEQ ID NO: 182 and more preferably, consists of the sequence recited in SEQ ID NO: 182.
  • Such a polypeptide includes the sequence recited in SEQ ID NO: 190, the cloning of which is reported herein in Example 2a.
  • the polypeptide having the sequence recited in SEQ ID NO:54 is referred to hereafter as "the INSPOIO exon 1 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO:56 is referred to hereafter as "the INSPOIO exon 2 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO:58 is referred to hereafter as "the INSPOIO exon 3 polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NO:60 is referred to hereafter as "the INSPOIO exon 4 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 62 is referred to hereafter as "the INSPOIO exon 5 polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NO:64 is referred to hereafter as "the INSPOIO exon 6 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO:66 is referred to hereafter as "the INSPOIO exon 7 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO:68 is referred to hereafter as "the INSPOIO exon 8 polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NO:70 is referred to hereafter as "the INSPOIO exon 9 polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NO: 72 is referred to hereafter as "the INSPOIO exon 10 polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NO:74 is referred to hereafter as "the INSPOIO exon 11 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO:76 is referred to hereafter as "the INSPOIO exon 12 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO:78 is referred to hereafter as "the INSPOIO exon 13 polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NO:80 is referred to hereafter as "the INSPOIO exon 14 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 82 is referred to hereafter as "the INSPOIO exon 15 polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NO:84 is referred to hereafter as "the INSPOIO exon 16 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO:86 is referred to hereafter as "the INSPOIO exon 17 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO:88 is referred to hereafter as "the INSPOIO exon 18 polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NO: 90 is referred to hereafter as "the INSPOIO exon 19 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO:92 is referred to hereafter as "the INSPOIO exon 20 polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NO:94 is referred to hereafter as "the INSPOIO exon 21 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO:96 is referred to hereafter as "the INSPOIO exon 22 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO:98 is referred to hereafter as "the INSPOIO exon 23 polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NO: 100 is referred to hereafter as "the INSPOIO exon 24 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 102 is referred to hereafter as "the INSPOIO exon 25 polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NO: 104 is referred to hereafter as "the INSPOIO exon 26 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 106 is referred to hereafter as "the INSPOIO exon 27 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 108 is referred to hereafter as "the INSPOIO exon 28 polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NOT 10 is referred to hereafter as "the INSPOIO exon 29 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NOT 12 is referred to hereafter as "the INSPOIO exon 30 polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NOT 14 is referred to hereafter as "the INSPOIO exon 31 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NOT 16 is referred to hereafter as "the INSPOIO exon 32 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NOT 18 is referred to hereafter as "the INSPOIO exon 33 polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NO:120 is referred to hereafter as "the INSPOIO exon 34 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 122 is referred to hereafter as "the INSPOIO exon 35 polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NO: 124 is referred to hereafter as "the INSPOIO exon 36 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 126 is referred to hereafter as "the INSPOIO exon 37 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO-.128 is referred to hereafter as "the INSPOIO exon 38 polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NO: 130 is referred to hereafter as "the INSPOIO exon 39 polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NO:132 is referred to hereafter as "the INSPOIO exon 40 polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NO: 134 is referred to hereafter as "the INSPOIO exon 41 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO:136 is referred to hereafter as "the INSPOIO exon 42 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 138 is referred to hereafter as "the INSPOIO exon 43 polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NO: 140 is referred to hereafter as "the INSPOIO exon 44 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 142 is referred to hereafter as "the INSPOIO exon 45 polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NO: 144 is referred to hereafter as "the INSPOIO exon 46 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 146 is referred to hereafter as "the INSPOIO exon 47 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 148 is referred to hereafter as "the INSPOIO exon 48 polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NO: 150 is referred to hereafter as "the INSPOIO exon 49 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NOT52 is referred to hereafter as "the INSPOIO exon 50 polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NOT 54 is referred to hereafter as "the INSPOIO exon 51 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NOT56 is referred to hereafter as "the INSPOIO exon 52 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 158 is referred to hereafter as "the INSPOIO exon 53 polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NO: 160 is referred to hereafter as "the INSPOIO exon 54 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 162 is referred to hereafter as "the INSPOIO exon 55 polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NOT64 is referred to hereafter as "the INSPOIO exon 56 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 182 is referred to hereafter as "the INSPOIO polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NO:190 is referred to herein as "the cloned INSPOIO partial polypeptide".
  • the first 22 amino acids of the INSPOIO polypeptide form a signal peptide.
  • the INSPOIO polypeptide sequence without this postulated signal sequence is recited in SEQ ID NO: 188.
  • the polypeptide having the sequence recited in SEQ ID NO: 188 is referred to hereafter as "the INSPOIO mature polypeptide".
  • the polypeptides are included herein as aspects of the present invention.
  • the invention provides a polypeptide, which polypeptide:
  • (i) comprises an amino acid sequence as recited in SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176 and/or SEQ ID NOT78;
  • (ii) is a fragment thereof which functions as a secreted protein or has an antigenic determinant in common with the polypeptides of (i); or
  • a polypeptide according to part i) above comprises the sequence recited in SEQ ID NO: 184 and more preferably, consists of the sequence recited in SEQ ID NO: 184.
  • This embodiment also provides a polypeptide comprising the sequence recited in SEQ ID NOT92 (the INSPOlla cloned partial polypeptide sequence), or SEQ ID NO:194 (the INSP01 la full length polypeptide sequence).
  • the polypeptide having the sequence recited in SEQ ID NO: 166 is referred to hereafter as "the INSPOll exon 1 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 168 is referred to hereafter as "the INSPOll exon 2 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 170 is referred to hereafter as "the INSPOl l exon 3 polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NO: 172 is referred to hereafter as "the INSPOll exon 4 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO: 174 is referred to hereafter as "the INSPOll exon 5 polypeptide”.
  • polypeptide having the sequence recited in SEQ ID NO: 176 is referred to hereafter as "the INSPOIO exon 6 polypeptide".
  • the polypeptide having the sequence recited in SEQ ID NO 78 is referred to hereafter as "the INSPOl l exon 7 polypeptide”.
  • the polypeptide having the sequence recited in SEQ ID NO: 184 is referred to hereafter as "the INSP011 polypeptide".
  • This embodiment also provides a polypeptide comprising the sequence recited in SEQ ID NO.192 (the INSPOlla cloned partial polypeptide sequence), or SEQ ID NOT94 (the INSPOl la full length polypeptide sequence).
  • the INSPOl la cloned partial polypeptide sequence corresponds to a polypeptide sequence cloned from a pool of cDNA libraries (retina, bladder and thymus), confirming that this exists as a cDNA.
  • the INSPOlla full length polypeptide sequence is a fusion of the INSPOlla cloned partial polypeptide sequence and the contiguous portion of the INSP011 full length sequence.
  • polypeptides of the above aspects of the invention function as secreted proteins of the cystine knot class, and preferably of the mucin subfamily of proteins.
  • functions as a secreted protein we refer to polypeptides that comprise amino acid sequence or structural features that can be identified as conserved features within the polypeptides of the secreted protein family, such that the polypeptide' s interaction with ligand is not substantially affected detrimentally in comparison to the function of the full length wild type polypeptide.
  • cysteine residues in specific positions within the polypeptide that allow the formation of intra-domain disulphide bonds.
  • a polypeptide according to the first aspect of the invention functions as a member of the cystine knot fold cytokine superfamily, preferably as a member of the mucin subfamily.
  • cystine knot fold cytokine is well understood in the art and the skilled worker will readily be able to ascertain whether a polypeptide functions as a member of the cystine knot fold cytokine superfamily using one of a variety of assays known in the art.
  • mucin family are large secreted airway glycoproteins, which contain multiple domains.
  • a group of mucin sequences have been shown to contain a C-terminal cystine knot domain. This group includes MUC2, MUC5AC, MUC5B and MUC6.
  • the domain structure seen in MUC2, starting from the N-terminal, is made up of a signal peptide, followed by 3 von- Willebrand factor (VWF) domains. The D-domains are then followed a serine, threonine and proline rich region.
  • VWF von- Willebrand factor
  • the majority of oligosaccharides, which are associated with mucins are linked to hydroxyl groups of the serine and threonine residues, (Eckhardt et al (1997) J. Biol.
  • the serine, threonine and proline rich domain is followed by a tandem repeat made up of 100 tandem 23 residue repeats.
  • the C-terminus of the MUC2 is made up of a fourth D-domain and C- terminal cystine knot (Eckhardt et al. 1997).
  • the description defines a typical structure found in mucin protein sequences that contain a C-terminal cystine knot. Variations would be expected in the domain organisation between members of the mucin family. This variation can be the removal or the addition of the previously described domains. Further, this variation could be the inclusion of domains not previously described, (e.g. MUC5AC contains a VWFC domain).
  • the purified nucleic acid molecule which encodes a polypeptide of the first aspect of the invention.
  • the purified nucleic acid molecule has the nucleic acid sequence as recited in SEQ ID NOT (encoding the INSP009 exon 1 polypeptide), the nucleic acid sequence as recited in SEQ ID NO:3 (encoding the INSP009 exon 2 polypeptide), the nucleic acid sequence as recited in SEQ ID NO:5 (encoding the INSP009 exon 3 polypeptide), the nucleic acid sequence as recited in SEQ ID NO:7 (encoding INSP009 exon 4 polypeptide), the nucleic acid sequence as recited in SEQ ID NO:9 (encoding the INSP009 exon 5 polypeptide), the nucleic acid sequence as recited in SEQ ID NO:l l (encoding the INSP009 exon 6 polypeptide), the nucleic acid sequence as recited in SEQ ID NO:
  • the nucleic acid sequence according to this embodiment comprises, or more preferably, consists of the sequence recited in SEQ ID NO: 179 (encoding the INSP009 polypeptide).
  • the nucleic acid according to the embodiment comprises, or more preferably, consists of the sequence recited in SEQ ID NO: 185 (encoding the INSP009 exons 24-26 consolidated sequence).
  • a purified nucleic acid molecule that has the nucleic acid sequence as recited in SEQ ID NO:53 (encoding the
  • INSPOIO exon 1 polypeptide the nucleic acid sequence as recited in SEQ ID NO:55 (encoding the INSPOIO exon 2 polypeptide), the nucleic acid sequence as recited in SEQ ID NO:57 (encoding the INSPOIO exon 3 polypeptide), the nucleic acid sequence as recited in SEQ ID NO:59 (encoding the INSPOIO exon 4 polypeptide),the nucleic acid sequence as recited in SEQ ID NO:61 (encoding the INSPOIO exon 5 polypeptide), the nucleic acid sequence as recited in SEQ ID NO: 63 (encoding the INSPOIO exon 6 polypeptide), the nucleic acid sequence as recited in SEQ ID NO: 65 (encoding the INSPOIO exon 7 polypeptide), the nucleic acid sequence as recited in SEQ ID NO:67 (encoding the INSPOIO exon 8 polypeptide), the nucleic acid sequence as recited in SEQ ID NO:69 (en
  • the nucleic acid sequence according to this embodiment comprises, or more preferably, consists of the sequence recited in SEQ ID NOT81 (encoding the INSPOIO polypeptide), the sequence recited in SEQ ID NO: 187 (encoding the INSPOIO mature polypeptide) or the sequence recited in SEQ ID NO: 189 (encoding the INSPOIO cloned partial sequence).
  • a purified nucleic acid molecule that has the nucleic acid sequence as recited in SEQ ID NO: 165 (encoding the INSPOll exon 1 polypeptide), the nucleic acid sequence as recited in SEQ ID NOT67 (encoding the INSPOl 1 exon 2 polypeptide), the nucleic acid sequence as recited in SEQ ID NOT69 (encoding the INSPOl l exon 3 polypeptide), the nucleic acid sequence as recited in SEQ ID NO 71 (encoding the INSPOl l exon 4 polypeptide), the nucleic acid sequence as recited in SEQ ID NO: 173 (encoding the INSPOl l exon 5 polypeptide), the nucleic acid sequence as recited in SEQ ID NOT75 (encoding the INSPOl l exon 6 polypeptide) and/or the nucleic acid sequence as recited in SEQ ID NO: 177 (encoding the INSPOl
  • the nucleic acid sequence according to this embodiment comprises, or more preferably, consists of the sequence recited in SEQ ID NO: 183 (encoding the INSPOl l polypeptide), the sequence recited in SEQ ID NO: 191 (encoding the INSPOl la cloned partial polypeptide), or the sequence recited in SEQ ID NO: 193 (the INSPOl la full length nucleotide sequence).
  • the invention provides a purified nucleic acid molecule which hydridizes under high stringency conditions with a nucleic acid molecule of the second aspect of the invention.
  • the invention provides a vector, such as an expression vector, that contains a nucleic acid molecule of the second or third aspect of the invention.
  • the invention provides a host cell transformed with a vector of the fourth aspect of the invention.
  • the invention provides a ligand which binds specifically to, and which preferably inhibits the activity of (such as the activity as a mucin), a polypeptide of the first aspect of the invention.
  • the invention provides a compound that is effective to alter the expression of a natural gene which encodes a polypeptide of the first aspect of the invention or to regulate the activity of a polypeptide of the first aspect of the invention.
  • a compound of the seventh aspect of the invention may either increase (agonise) or decrease (antagonise) the level of expression of the gene or the activity of the polypeptide.
  • the identification of the function of the INSP009, INSPOIO, INSPOl 1 and INSPOl la polypeptides allows for the design of screening methods capable of identifying compounds that are effective in the treatment and/or diagnosis of disease.
  • the invention provides a polypeptide of the first aspect of the invention, or a nucleic acid molecule of the second or third aspect of the invention, or a vector of the fourth aspect of the invention, or a ligand of the fifth aspect of the invention, or a compound of the sixth aspect of the invention, for use in therapy or diagnosis.
  • These molecules may also be used in the manufacture of a medicament for the treatment of cell proliferative disorders, autoimmune/inflammatory disorders, cardiovascular disorders, neurological disorders, developmental disorders, metabolic disorders, infections and other pathological conditions.
  • the invention provides a method of diagnosing a disease in a patient, comprising assessing the level of expression of a natural gene encoding a polypeptide of the first aspect of the invention or the activity of a polypeptide of the first aspect of the invention in tissue from said patient and comparing said level of expression or activity to a control level, wherein a level that is different to said control level is indicative of disease.
  • a method will preferably be carried out in vitro.
  • Similar methods may be used for monitoring the therapeutic treatment of disease in a patient, wherein altering the level of expression or activity of a polypeptide or nucleic acid molecule over the period of time towards a control level is indicative of regression of disease.
  • a preferred method for detecting polypeptides of the first aspect of the invention comprises the steps of: (a) contacting a ligand, such as an antibody, of the sixth aspect of the invention with a biological sample under conditions suitable for the formation of a ligand-polypeptide complex; and (b) detecting said complex.
  • kits that are useful in these methods for diagnosing disease.
  • the invention provides for the use of a polypeptide of the first aspect of the invention as a secreted protein, preferably as a mucin.
  • the invention provides a pharmaceutical composition comprising a polypeptide of the first aspect of the invention, or a nucleic acid molecule of the second or third aspect of the invention, or a vector of the fourth aspect of the invention, or a host cell of the fifth aspect of the invention, or a ligand of the sixth aspect of the invention, or a compound of the seventh aspect of the invention, in conjunction with a pharmaceutically-acceptable carrier.
  • the present invention provides a polypeptide of the first aspect of the invention, or a nucleic acid molecule of the second or third aspect of the invention, or a vector of the fourth aspect of the invention, or a host cell of the fifth aspect of the invention, or a ligand of the sixth aspect of the invention, or a compound of the seventh aspect of the invention, for use in the manufacture of a medicament for the diagnosis or treatment of a disease, such as a disease relating to carcinogenesis, reproductive health, airway and gastrointestinal inflammation and infection.
  • a disease such as a disease relating to carcinogenesis, reproductive health, airway and gastrointestinal inflammation and infection.
  • the invention provides a method of treating a disease in a patient comprising administering to the patient a polypeptide of the first aspect of the invention, or a nucleic acid molecule of the second or third aspect of the invention, or a vector of the fourth aspect of the invention, or a host cell of the fifth aspect of the invention, or a ligand of the sixth aspect of the invention, or a compound of the seventh aspect of the invention.
  • the polypeptide, nucleic acid molecule, ligand or compound administered to the patient should be an agonist.
  • the polypeptide, nucleic acid molecule, ligand or compound administered to the patient should be an antagonist.
  • antagonists include antisense nucleic acid molecules, ribozymes and ligands, such as antibodies.
  • the invention provides transgenic or knockout non-human animals that have been transformed to express higher, lower or absent levels of a polypeptide of the first aspect of the invention.
  • Such transgenic animals are very useful models for the study of disease and may also be used in screening regimes for the identification of compounds that are effective in the treatment or diagnosis of such a disease.
  • polypeptide includes any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e. peptide isosteres. This term refers both to short chains (peptides and oligopeptides) and to longer chains (proteins).
  • the polypeptide of the present invention may be in the form of a mature protein or may be a pre-, pro- or prepro- protein that can be activated by cleavage of the pre-, pro- or prepro- portion to produce an active mature polypeptide.
  • the pre-, pro- or prepro- sequence may be a leader or secretory sequence or may be a sequence that is employed for purification of the mature polypeptide sequence.
  • the polypeptide of the first aspect of the invention may form part of a fusion protein.
  • the mature polypeptide may be fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol).
  • Polypeptides may contain amino acids other than the 20 gene-encoded amino acids, modified either by natural processes, such as by post-translational processing or by chemical modification techniques which are well known in the art.
  • modifications which may commonly be present in polypeptides of the present invention are glycosylation, lipid attachment, sulphation, gamma-carboxylation, for instance of glutamic acid residues, hydroxylation and ADP-ribosylation.
  • Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini.
  • blockage of the amino or carboxyl terminus in a polypeptide, or both, by a covalent modification is common in naturally-occurring and synthetic polypeptides and such modifications may be present in polypeptides of the present invention.
  • modifications that occur in a polypeptide often will be a function of how the polypeptide is made.
  • the nature and extent of the modifications in large part will be determined by the post-translational modification capacity of the particular host cell and the modification signals that are present in the amino acid sequence of the polypeptide in question. For instance, glycosylation patterns vary between different types of host cell.
  • polypeptides of the present invention can be prepared in any suitable manner.
  • Such polypeptides include isolated naturally-occurring polypeptides (for example purified from cell culture), recombinantly-produced polypeptides (including fusion proteins), synthetically-produced polypeptides or polypeptides that are produced by a combination of these methods.
  • the functionally-equivalent polypeptides of the first aspect of the invention may be polypeptides that are homologous to the INSP009, INSPOIO, INSPOll and INSPOlla polypeptides.
  • Two polypeptides are said to be "homologous", as the term is used herein, if the sequence of one of the polypeptides has a high enough degree of identity or similarity to the sequence of the other polypeptide. "Identity” indicates that at any particular position in the aligned sequences, the amino acid residue is identical between the sequences.
  • Similarity indicates that, at any particular position in the aligned sequences, the amino acid residue is of a similar type between the sequences.
  • Homologous polypeptides therefore include natural biological variants (for example, allelic variants or geographical variations within the species from which the polypeptides are derived) and mutants (such as mutants containing amino acid substitutions, insertions or deletions) of the INSP009, INSPOIO, INSPOl l and INSPOlla polypeptides.
  • Such mutants may include polypeptides in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code.
  • Typical such substitutions are among Ala, Val, Leu and lie; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gin; among the basic residues Lys and Arg; or among the aromatic residues Phe and Tyr.
  • Particularly preferred are variants in which several, i.e. between 5 and 10, 1 and 5, 1 and 3, 1 and 2 or just 1 amino acids are substituted, deleted or added in any combination.
  • silent substitutions, additions and deletions which do not alter the properties and activities of the protein. Also especially preferred in this regard are conservative substitutions.
  • Such mutants also include polypeptides in which one or more of the amino acid residues includes a substituent group.
  • identity between two polypeptides is considered to be an indication of functional equivalence.
  • functionally equivalent polypeptides of the first aspect of the invention have a degree of sequence identity with the INSP009, INSPOIO, INSPOl l and INSPOl la polypeptides, or with active fragments thereof, of greater than 30%. More preferred polypeptides have degrees of identity of greater than 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99%, respectively.
  • the functionally-equivalent polypeptides of the first aspect of the invention may also be polypeptides which have been identified using one or more techniques of structural alignment.
  • the Inpharmatica Genome Threader technology that forms one aspect of the search tools used to generate the Biopendium search database may be used (see co-pending United Kingdom patent application GB0006153.1) to identify polypeptides of presently-unknown function which, while having low sequence identity as compared to the INSP009, INSPOIO, INSPOl l and INSPOl la polypeptides, are predicted to have activity as secreted molecules, by virtue of sharing significant structural homology with the INSP009, INSPOIO, INSPOll and INSPOl la polypeptide sequences.
  • significant structural homology is meant that the Inpharmatica Genome Threader predicts two proteins to share structural homology with a certainty of 10% and above.
  • polypeptides of the first aspect of the invention also include fragments of the INSP009, INSPOIO, INSPOll and INSPOlla polypeptides and fragments of the functional equivalents of the INSP009, INSPOIO, INSPOl l and INSPOlla polypeptides, provided that those fragments retain activity as secreted proteins or have an antigenic determinant in common with the INSP009, INSPOIO, P SP011 and INSPOl la polypeptides.
  • fragment refers to a polypeptide having an amino acid sequence that is the same as part, but not all, of the amino acid sequence of the INSP009, INSPO 10, INSPO 11 and INSPO 11 a polypeptides or one of its functional equivalents.
  • the fragments should comprise at least n consecutive amino acids from the sequence and, depending on the particular sequence, n preferably is 7 or more (for example, 8, 10, 12, 14, 16, 18, 20 or more). Small fragments may form an antigenic determinant.
  • Fragments of the full length INSP009, INSPOIO, INSPOll and INSPOl la polypeptides may consist of combinations of 2 or 3 of neighbouring exon sequences in the INSP009, INSPOIO, INSPOl 1 or INSPOl la polypeptide sequences, respectively.
  • such combinations include exons 1 and 2, 2 and 3 or 1, 2 and 3 - in the case of the INSPOl l protein, the combination of exons 5, 6 and 7 is a preferred fragment.
  • Such fragments are included in the present invention.
  • Such fragments may be "free-standing", i.e. not part of or fused to other amino acids or polypeptides, or they may be comprised within a larger polypeptide of which they form a part or region.
  • the fragment of the invention When comprised within a larger polypeptide, the fragment of the invention most preferably forms a single continuous region. For instance, certain preferred embodiments relate to a fragment having a pre- and/or pro- polypeptide region fused to the amino terminus of the fragment and/or an additional region fused to the carboxyl terminus of the fragment. However, several fragments may be comprised within a single larger polypeptide.
  • the polypeptides of the present invention or their immunogenic fragments can be used to generate ligands, such as polyclonal or monoclonal antibodies, that are immunospecific for the polypeptides.
  • ligands such as polyclonal or monoclonal antibodies
  • Such antibodies may be employed to isolate or to identify clones expressing the polypeptides of the invention or to purify the polypeptides by affinity chromatography.
  • the antibodies may also be employed as diagnostic or therapeutic aids, amongst other applications, as will be apparent to the skilled reader.
  • immunospecific means that the antibodies have substantially greater affinity for the polypeptides of the invention than their affinity for other related polypeptides in the prior art.
  • antibody refers to intact molecules as well as to fragments thereof, such as Fab, F(ab')2 and Fv, which are capable of binding to the antigenic determinant in question. Such antibodies thus bind to the polypeptides of the first aspect of the invention.
  • a selected mammal such as a mouse, rabbit, goat or horse
  • a polypeptide of the first aspect of the invention may be immunised with a polypeptide of the first aspect of the invention.
  • the polypeptide used to immunise the animal can be derived by recombinant DNA technology or can be synthesized chemically.
  • the polypeptide can be conjugated to a carrier protein.
  • Commonly used carriers to which the polypeptides may be chemically coupled include bovine serum albumin, thyroglobulin and keyhole limpet haemocyanin.
  • the coupled polypeptide is then used to immunise the animal. Serum from the immunised animal is collected and treated according to known procedures, for example by immunoaffinity chromatography.
  • Monoclonal antibodies to the polypeptides of the first aspect of the invention can also be readily produced by one skilled in the art.
  • the general methodology for making monoclonal antibodies using hybridoma technology is well known (see, for example, Kohler, G. and Milstein, C, Nature 256: 495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Cole et al, 77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985).
  • Panels of monoclonal antibodies produced against the polypeptides of the first aspect of the invention can be screened for various properties, i.e., for isotype, epitope, affinity, etc. Monoclonal antibodies are particularly useful in purification of the individual polypeptides against which they are directed. Alternatively, genes encoding the monoclonal antibodies of interest may be isolated from hybridomas, for instance by PCR techniques known in the art, and cloned and expressed in appropriate vectors.
  • Chimeric antibodies in which non-human variable regions are joined or fused to human constant regions (see, for example, Liu et al, Proc. Natl. Acad. Sci. USA, 84, 3439 (1987)), may also be of use.
  • the antibody may be modified to make it less immunogenic in an individual, for example by humanisation (see Jones et al, Nature, 321, 522 (1986); Verhoeyen et al, Science, 239, 1534 (1988); Kabat et al, J. Immunol., 147, 1709 (1991); Queen et al, Proc. Natl Acad. Sci. USA, 86, 10029 (1989); Gorman et al, Proc. Natl Acad. Sci.
  • humanised antibody refers to antibody molecules in which the CDR amino acids and selected other amino acids in the variable domains of the heavy and/or light chains of a non-human donor antibody have been substituted in place of the equivalent amino acids in a human antibody.
  • the humanised antibody thus closely resembles a human antibody but has the binding ability of the donor antibody.
  • the antibody may be a "bispecific" antibody, that is an antibody having two different antigen binding domains, each domain being directed against a different epitope.
  • Phage display technology may be utilised to select genes which encode antibodies with binding activities towards the polypeptides of the invention either from repertoires of PCR amplified V-genes of lymphocytes from humans screened for possessing the relevant antibodies, or from naive libraries (McCafferty, J. et al., (1990), Nature 348, 552-554; Marks, J. et al, (1992) Biotechnology 10, 779-783).
  • the affinity of these antibodies can also be improved by chain shuffling (Clackson, T. et al, (1991) Nature 352, 624-628).
  • Antibodies generated by the above techniques have additional utility in that they may be employed as reagents in immunoassays, radioimmunoassays (RIA) or enzyme-linked immunosorbent assays (ELISA).
  • the antibodies can be labelled with an analytically-detectable reagent such as a radioisotope, a fluorescent molecule or an enzyme.
  • Preferred nucleic acid molecules of the second and third aspects of the invention are those which encode the polypeptide sequences recited in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NOT4, SEQ ID NO:16, SEQ ID NOT8, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, S
  • SEQ ID NOT06 SEQ ID NOT08, SEQ ID NOT 10, SEQ ID NO 112, SEQ ID NO: 114, SEQ ID NOT 16, SEQ ID NOT 18, SEQ ID NOT20, SEQ ID NO 122, SEQ ID NOT24, SEQ ID NOT26, SEQ ID NOT28, SEQ ID NOT30, SEQ ID NO 132, SEQ ID NO: 134, SEQ ID NO:136, SEQ ID NOT38, SEQ ID NOT40, SEQ ID NO 142, SEQ ID NO: 144, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NOT52, SEQ ID NOT54, SEQ ID NO:156, SEQ ID NOT58, SEQ ID NOT60, SEQ ID NOT62, SEQ ID NOT64, SEQ ID NOT66, SEQ ID NOT68, SEQ ID NO:170, SEQ ID NOT72, SEQ ID NOT74, SEQ ID NOT76, SEQ ID NOT78, SEQ ID NO:180, SEQ ID NOT82, SEQ ID NOT84, S
  • nucleic acid molecules may be used in the methods and applications described herein.
  • the nucleic acid molecules of the invention preferably comprise at least n consecutive nucleotides from the sequences disclosed herein where, depending on the particular sequence, n is 10 or more (for example, 12, 14, 15, 18, 20, 25, 30, 35, 40 or more).
  • nucleic acid molecules of the invention also include sequences that are complementary to nucleic acid molecules described above (for example, for antisense or probing purposes).
  • Nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance cDNA, synthetic DNA or genomic DNA. Such nucleic acid molecules may be obtained by cloning, by chemical synthetic techniques or by a combination thereof. The nucleic acid molecules can be prepared, for example, by chemical synthesis using techniques such as solid phase phosphoramidite chemical synthesis, from genomic or cDNA libraries or by separation from an organism. RNA molecules may generally be generated by the in vitro or in vivo transcription of DNA sequences.
  • the nucleic acid molecules may be double-stranded or single-stranded.
  • Single-stranded DNA may be the coding strand, also known as the sense strand, or it may be the non- coding strand, also referred to as the anti-sense strand.
  • the term "nucleic acid molecule” also includes analogues of DNA and RNA, such as those containing modified backbones,and peptide nucleic acids (PNA).
  • PNA peptide nucleic acids
  • PNA refers to an antisense molecule or an anti-gene agent which comprises an oligonucleotide of at least five nucleotides in length linked to a peptide backbone of amino acid residues, which preferably ends in lysine.
  • PNAs may be pegylated to extend their lifespan in a cell, where they preferentially bind complementary single stranded DNA and RNA and stop transcript elongation (Nielsen, P.E. et al (1993) Anticancer Drug Des. 8:53-63).
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:2 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NOT.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:4 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:3.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:6 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:5.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 8 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:7.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 10 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 9.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 12 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NOT 1.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 14 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 13.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 16 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 15.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 18 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 17.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:20 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 19.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:22 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:21.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:24 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:23.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:26 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:25.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:28 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:27.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:30 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:29.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 32 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:31.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:34 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:33.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:36 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:35.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:38 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:37.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:40 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:39.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:42 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:41.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:44 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:43.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:46 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:45.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:48 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:47.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:50 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:49.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 52 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:51.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 54 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:53.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:56 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:55.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 58 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:57.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:60 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:59.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:62 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:61.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 64 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 63.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:66 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:65.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:68 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:67.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:70 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 69.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 72 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:71.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:74 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:73.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:76 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:75.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:78 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:77.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:80 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:79.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 82 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:81.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:84 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:83.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:86 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:85.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:88 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:87.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:90 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 89.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 92 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:91.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 94 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 93.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 96 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:95.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:98 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:97.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 100 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:99.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 102 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 101.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 104 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 103.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 106 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 105.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 108 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 107.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NOT 10 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 109.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NOT 12 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NOT 11.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NOT 14 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NOT 13.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NOT 16 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 115.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NOT 18 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NOT 17.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 120 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 119.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 122 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NOT2L
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 124 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 123.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 126 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 125.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 128 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 127.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 130 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NOT 29.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 132 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 131.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 134 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 133.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 136 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 135.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 138 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 137.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 140 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 139.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 142 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 141.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 144 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 143.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 146 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 145.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 148 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 147.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 150 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NOT49.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 152 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NOT51.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 154 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NOT 53.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 156 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 155.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 158 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NOT 57.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 160 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 159.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 162 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 161.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 164 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 163.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 166 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 165.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 168 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 167.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 170 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 169.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NOT 72 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 171.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 174 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NOT 73.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 176 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 175.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 178 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 177.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NOT 80 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 179.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 182 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NOT81.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 184 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 183.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 186 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 185.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 188 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 187.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 190 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NOT 89.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 192 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 191.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO: 193 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 194.
  • SEQ ID NO:2 SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NOT0, SEQ ID NOT2, SEQ ID NOT4, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66,
  • nucleic acid molecules that encode the polypeptide of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NOT0, SEQ ID NOT2, SEQ ID NOT4, SEQ ID NOT6, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ
  • SEQ ID NO 110 SEQ ID NO T 12, SEQ ID NOT 14, SEQ ID NOT16, SEQ ID NO 118 SEQ ID NO 120, SEQ ID NO :122, SEQ ID NOT24, SEQ ID NOT26, SEQ ID NO 128 SEQ ID NO 130, SEQ ID NO T32, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO 138 SEQ ID NO 140, SEQ ID NO T42, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO 148 SEQ ID NO 150, SEQ ID NO T52, SEQ ID NOT54, SEQ ID NOT56, SEQ ID NO 158 SEQ ID NO 160, SEQ ID NO T62, SEQ ID NOT64, SEQ ID NOT66, SEQ ID NO 168 SEQ ID NO 170, SEQ ID NO T72, SEQ ID NOT74, SEQ ID NOT76, SEQ ID NO 178 SEQ ID NO 180, SEQ ID NO T82, SEQ ID NOT84, SEQ ID NOT86, SEQ ID NO
  • nucleic acid molecules of the second and third aspects of the invention may also encode the fragments or the functional equivalents of the polypeptides and fragments of the first aspect of the invention.
  • a nucleic acid molecule may be a naturally- occurring variant such as a naturally-occurring allelic variant, or the molecule may be a variant that is not known to occur naturally.
  • non-naturally occurring variants of the nucleic acid molecule may be made by mutagenesis techniques, including those applied to nucleic acid molecules, cells or organisms.
  • variants in this regard are variants that differ from the aforementioned nucleic acid molecules by nucleotide substitutions, deletions or insertions.
  • the substitutions, deletions or insertions may involve one or more nucleotides.
  • the variants may be altered in coding or non-coding regions or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or insertions.
  • the nucleic acid molecules of the invention can also be engineered, using methods generally known in the art, for a variety of reasons, including modifying the cloning, processing, and/or expression of the gene product (the polypeptide).
  • DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides are included as techniques which may be used to engineer the nucleotide sequences.
  • Site-directed mutagenesis may be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations and so forth.
  • Nucleic acid molecules which encode a polypeptide of the first aspect of the invention may be ligated to a heterologous sequence so that the combined nucleic acid molecule encodes a fusion protein.
  • Such combined nucleic acid molecules are included within the second or third aspects of the invention.
  • a fusion protein that can be recognised by a commercially-available antibody.
  • a fusion protein may also be engineered to contain a cleavage site located between the sequence of the polypeptide of the invention and the sequence of a heterologous protein so that the polypeptide may be cleaved and purified away from the heterologous protein.
  • the nucleic acid molecules of the invention also include antisense molecules that are partially complementary to nucleic acid molecules encoding polypeptides of the present invention and that therefore hybridize to the encoding nucleic acid molecules (hybridization).
  • antisense molecules such as oligonucleotides, can be designed to recognise, specifically bind to and prevent transcription of a target nucleic acid encoding a polypeptide of the invention, as will be known by those of ordinary skill in the art (see, for example, Cohen, J.S., Trends in Pharm. Sci., 10, 435 (1989), Okano, J. Neurochem. 56, 560 (1991); O'Connor, J. Neurochem 56, 560 (1991); Lee et al, Nucleic Acids Res 6, 3073 (1979); Cooney et al, Science 241, 456 (1988); Dervan et al, Science 251, 1360 (1991).
  • hybridization refers to the association of two nucleic acid molecules with one another by hydrogen bonding. Typically, one molecule will be fixed to a solid support and the other will be free in solution. Then, the two molecules may be placed in contact with one another under conditions that favour hydrogen bonding. Factors that affect this bonding include: the type and volume of solvent; reaction temperature; time of hybridization; agitation; agents to block the non-specific attachment of the liquid phase molecule to the solid support (Denhardt's reagent or BLOTTO); the concentration of the molecules; use of compounds to increase the rate of association of molecules (dextran sulphate or polyethylene glycol); and the stringency of the washing conditions following hybridization (see Sambrook et al. [supra]).
  • the inhibition of hybridization of a completely complementary molecule to a target molecule may be examined using a hybridization assay, as known in the art (see, for example, Sambrook et al. [supra]).
  • a substantially homologous molecule will then compete for and inhibit the binding of a completely homologous molecule to the target molecule under various conditions of stringency, as taught in Wahl, G.M. and S.L. Berger (1987; Methods Enzymol. 152:399-407) and Kimmel, A.R. (1987; Methods Enzymol. 152:507-511).
  • Stringency refers to conditions in a hybridization reaction that favour the association of very similar molecules over association of molecules that differ.
  • High stringency hybridisation conditions are defined as overnight incubation at 42°C in a solution comprising 50% formamide, 5XSSC (150mM NaCl, 15mM trisodium citrate), 50mM sodium phosphate (pH7.6), 5x Denhardts solution, 10%» dextran sulphate, and 20 microgram/ml denatured, sheared salmon sperm DNA, followed by washing the filters in OTX SSC at approximately 65(C.
  • Low stringency conditions involve the hybridisation reaction being carried out at 35(C (see Sambrook et al. [supra]).
  • the conditions used for hybridization are those of high stringency.
  • Preferred embodiments of this aspect of the invention are nucleic acid molecules that are at least 70% identical over their entire length to a nucleic acid molecule encoding the INSP009 polypeptide or fragments thereof (SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO:12, SEQ ID NOT4, SEQ ID NOT6, SEQ ID NOT8, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50 and SEQ ID NO:52, SEQ ID NOT80, and SEQ ID NOT86), and nucleic acid molecules that are substantially complementary to such nucleic acid molecules.
  • a nucleic acid molecule according to this aspect of the invention comprises a region that is at least 80% identical over its entire length to any one of the above-listed nucleic acid molecules or a nucleic acid molecule that is complementary thereto.
  • nucleic acid molecules at least 90%, preferably at least 95%, more preferably at least 98% or 99% identical over their entire length to the same are particularly preferred.
  • Preferred embodiments in this respect are nucleic acid molecules that encode polypeptides which retain substantially the same biological function or activity as the INSP009 polypeptides.
  • nucleic acid molecules that are at least 70% identical over their entire length to a nucleic acid molecule encoding the INSPOIO polypeptide or fragments thereof (SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NOT04, SEQ ID NOT06, SEQ ID NOT08, SEQ ID NOT 10, SEQ ID NOT 12,
  • a nucleic acid molecule according to this aspect of the invention comprises a region that is at least 80% identical over its entire length to any one of the above-listed nucleic acid molecules or to a nucleic acid molecule that is complementary thereto.
  • nucleic acid molecules at least 90%, preferably at least 95%, more preferably at least 98% or 99% identical over their entire length to the same are particularly preferred.
  • Preferred embodiments in this respect are nucleic acid molecules that encode polypeptides which retain substantially the same biological function or activity as the INSPOIO polypeptides.
  • nucleic acid molecules that are at least 70% identical over their entire length to a nucleic acid molecule encoding the INSPOll polypeptide or fragments thereof (SEQ ID NOT66, SEQ ID NOT68, SEQ ID NOT70, SEQ ID NOT72, SEQ ID NOT74, SEQ ID NOT76 and SEQ ID NOT78, SEQ ID NOT84) or the INSPOlla polypeptide or fragments thereof (SEQ ID NO: 192 and SEQ ID NO: 194), and nucleic acid molecules that are substantially complementary to such nucleic acid molecules.
  • a nucleic acid molecule according to this aspect of the invention comprises a region that is at least 80% identical over its entire length to any one of the above-listed nucleic acid molecules or to a nucleic acid molecule that is complementary thereto.
  • nucleic acid molecules at least 90%, preferably at least 95%, more preferably at least 98% or 99% identical over their entire length to the same are particularly preferred.
  • Preferred embodiments in this respect are nucleic acid molecules that encode polypeptides which retain substantially the same biological function or activity as the INSPOl 1 or INSPOl la polypeptides.
  • the invention also provides a process for detecting a nucleic acid molecule of the invention, comprising the steps of: (a) contacting a nucleic probe according to the invention with a biological sample under hybridizing conditions to form duplexes; and (b) detecting any such duplexes that are formed.
  • a nucleic acid molecule as described above may be used as a hybridization probe for RNA, cDNA or genomic DNA, in order to isolate full-length cDNAs and genomic clones encoding the INSP009, INSPOIO, INSPOll and INSPOlla polypeptides and to isolate cDNA and genomic clones of homologous or orthologous genes that have a high sequence similarity to the gene encoding this polypeptide.
  • the following techniques among others known in the art, may be utilised and are discussed below for purposes of illustration.
  • DNA sequencing and analysis are well known and are generally available in the art and may, indeed, be used to practice many of the embodiments of the invention discussed herein. Such methods may employ such enzymes as the Klenow fragment of DNA polymerase I, Sequenase (US Biochemical Corp, Cleveland, OH), Taq polymerase (Perkin Elmer), thermostable T7 polymerase (Amersham, Chicago, IL), or combinations of polymerases and proof-reading exonucleases such as those found in the ELONGASE Amplification System marketed by Gibco/BRL (Gaithersburg, MD).
  • Klenow fragment of DNA polymerase I Sequenase
  • Sequenase US Biochemical Corp, Cleveland, OH
  • Taq polymerase Perkin Elmer
  • thermostable T7 polymerase Amersham, Chicago, IL
  • combinations of polymerases and proof-reading exonucleases such as those found in the ELONGASE Amplification System marketed by Gibco/BRL (Gaithersburg, MD).
  • the sequencing process may be automated using machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno, NV), the Peltier Thermal Cycler (PTC200; MJ Research, Watertown, MA) and the ABI Catalyst and 373 and 377 DNA Sequencers (Perkin Elmer).
  • machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno, NV), the Peltier Thermal Cycler (PTC200; MJ Research, Watertown, MA) and the ABI Catalyst and 373 and 377 DNA Sequencers (Perkin Elmer).
  • One method for isolating a nucleic acid molecule encoding a polypeptide with an equivalent function to that of the INSP009, INSPOIO, INSPOll and INSPOl la polypeptides is to probe a genomic or cDNA library with a natural or artificially-designed probe using standard procedures that are recognised in the art (see, for example, "Current Protocols in Molecular Biology", Ausubel et al. (eds). Greene Publishing Association and John Wiley Interscience, New York, 1989,1992).
  • Probes comprising at least 15, preferably at least 30, and more preferably at least 50, contiguous bases that correspond to, or are complementary to, nucleic acid sequences from the appropriate encoding genes (which for INSP009 are SEQ ID NOT, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NOT1, SEQ ID NOT3, SEQ ID NOT5, SEQ ID NOT7, SEQ ID NOT9, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49 and SEQ ID NO:51, SEQ ID NO.T79, and SEQ ID NOT85; for INSPOIO, these are SEQ ID NO:53, SEQ ID NO:55
  • Such probes may be labelled with an analytically-detectable reagent to facilitate their identification.
  • Useful reagents include, but are not limited to, radioisotopes, fluorescent dyes and enzymes that are capable of catalysing the formation of a detectable product.
  • the ordinarily skilled artisan will be capable of isolating complementary copies of genomic DNA, cDNA or RNA polynucleotides encoding proteins of interest from human, mammalian or other animal sources and screening such sources for related sequences, for example, for additional members of the family, type and/or subtype.
  • isolated cDNA sequences will be incomplete, in that the region encoding the polypeptide will be cut short, normally at the 5' end.
  • Several methods are available to obtain full length cDNAs, or to extend short cDNAs. Such sequences may be extended utilising a partial nucleotide sequence and employing various methods known in the art to detect upstream sequences such as promoters and regulatory elements. For example, one method which may be employed is based on the method of Rapid Amplification of cDNA Ends (RACE; see, for example, Frohman et al, PNAS USA 85, 8998-9002, 1988).
  • RACE Rapid Amplification of cDNA Ends
  • Another method which may be used is capture PCR which involves PCR amplification of DNA fragments adjacent a known sequence in human and yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods Applic, 1, 111-119). Another method which may be used to retrieve unknown sequences is that of Parker, J.D. et al. (1991); Nucleic Acids Res. 19:3055- 3060). Additionally, one may use PCR, nested primers, and PromoterFinderTM libraries to walk genomic DNA (Clontech, Palo Alto, CA). This process avoids the need to screen libraries and is useful in finding intron/exon junctions.
  • libraries that have been size-selected to include larger cDNAs.
  • random-primed libraries are preferable, in that they will contain more sequences that contain the 5' regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA.
  • Genomic libraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
  • the nucleic acid molecules of the present invention may be used for chromosome localisation.
  • a nucleic acid molecule is specifically targeted to, and can hybridize with, a particular location on an individual human chromosome.
  • the mapping of relevant sequences to chromosomes according to the present invention is an important step in the confirmatory correlation of those sequences with the gene-associated disease. Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found in, for example, V. McKusick, Mendelian Inheritance in Man (available on-line through Johns Hopkins University Welch Medical Library).
  • the relationships between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes). This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the disease or syndrome has been crudely localised by genetic linkage to a particular genomic region, any sequences mapping to that area may represent associated or regulatory genes for further investigation.
  • the nucleic acid molecule may also be used to detect differences in the chromosomal location due to translocation, inversion, etc. among normal, carrier, or affected individuals.
  • the nucleic acid molecules of the present invention are also valuable for tissue localisation.
  • Such techniques allow the determination of expression patterns of the polypeptide in tissues by detection of the mRNAs that encode them.
  • These techniques include in situ hybridization techniques and nucleotide amplification techniques, such as PCR. Results from these studies provide an indication of the normal functions of the polypeptide in the organism.
  • comparative studies of the normal expression pattern of mRNAs with that of mRNAs encoded by a mutant gene provide valuable insights into the role of mutant polypeptides in disease. Such inappropriate expression may be of a temporal, spatial or quantitative nature.
  • Gene silencing approaches may also be undertaken to down-regulate endogenous expression of a gene encoding a polypeptide of the invention.
  • RNA interference (Elbashir, SM et al., Nature 2001, 411, 494-498) is one method of sequence specific post- transcriptional gene silencing that may be employed. Short dsRNA oligonucleotides are synthesised in vitro and introduced into a cell. The sequence specific binding of these dsRNA oligonucleotides triggers the degradation of target mRNA, reducing or ablating target protein expression.
  • the vectors of the present invention comprise nucleic acid molecules of the invention and may be cloning or expression vectors.
  • the host cells of the invention which may be transformed, transfected or transduced with the vectors of the invention may be prokaryotic or eukaryotic.
  • polypeptides of the invention may be prepared in recombinant form by expression of their encoding nucleic acid molecules in vectors contained within a host cell.
  • Such expression methods are well known to those of skill in the art and many are described in detail by Sambrook et al. (supra) and Fernandez & Hoeffler (1998, eds. "Gene expression systems. Using nature for the art of expression”. Academic Press, San Diego, London, Boston, New York, Sydney, Tokyo, Toronto).
  • any system or vector that is suitable to maintain, propagate or express nucleic acid molecules to produce a polypeptide in the required host may be used.
  • nucleotide sequence may be inserted into an expression system by any of a variety of well-known and routine techniques, such as, for example, those described in Sambrook et al, (supra).
  • the encoding gene can be placed under the control of a control element such as a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator, so that the DNA sequence encoding the desired polypeptide is transcribed into RNA in the transformed host cell.
  • suitable expression systems include, for example, chromosomal, episomal and virus-derived systems, including, for example, vectors derived from: bacterial plasmids, bacteriophage, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses such as baculoviruses, papova viruses such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, or combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, including cosmids and phagemids.
  • Human artificial chromosomes may also be employed to deliver larger fragments of DNA than can be contained and expressed in a plasmid.
  • Particularly suitable expression systems include microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (for example, baculovirus); plant cell systems transformed with virus expression vectors (for example, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (for example, Ti or pBR322 plasmids); or animal cell systems.
  • Cell-free translation systems can also be employed to produce the polypeptides of the invention.
  • nucleic acid molecules encoding a polypeptide of the present invention into host cells can be effected by methods described in many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology (1986) and Sambrook et al, [supra]. Particularly suitable methods include calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid- mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection (see Sambrook et al, 1989 [supra]; Ausubel et al., 1991 [supra]; Spector, Goldman & Leinwald, 1998).
  • expression systems may either be transient (for example, episomal) or permanent (chromosomal integration) according to the needs of the system.
  • the encoding nucleic acid molecule may or may not include a sequence encoding a control sequence, such as a signal peptide or leader sequence, as desired, for example, for secretion of the translated polypeptide into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment.
  • signals may be endogenous to the polypeptide or they may be heterologous signals.
  • Leader sequences can be removed by the bacterial host in post-translational processing.
  • regulatory sequences that allow for regulation of the expression of the polypeptide relative to the growth of the host cell.
  • regulatory sequences are those which cause the expression of a gene to be increased or decreased in response to a chemical or physical stimulus, including the presence of a regulatory compound or to various temperature or metabolic conditions.
  • Regulatory sequences are those non-translated regions of the vector, such as enhancers, promoters and 5' and 3' untranslated regions. These interact with host cellular proteins to carry out transcription and translation. Such regulatory sequences may vary in their strength and specificity. Depending on the vector system and host utilised, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used.
  • inducible promoters such as the hybrid lacZ promoter of the Bluescript phagemid (Stratagene, LaJolla, CA) or pSportlTM plasmid (Gibco BRL) and the like may be used.
  • the baculovirus polyhedrin promoter may be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (for example, heat shock, RUBISCO and storage protein genes) or from plant viruses (for example, viral promoters or leader sequences) may be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of the sequence, vectors based on SV40 or EBV may be used with an appropriate selectable marker.
  • An expression vector is constructed so that the particular nucleic acid coding sequence is located in the vector with the appropriate regulatory sequences, the positioning and orientation of the coding sequence with respect to the regulatory sequences being such that the coding sequence is transcribed under the "control" of the regulatory sequences, i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence.
  • control i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence.
  • control sequences and other regulatory sequences may be ligated to the nucleic acid coding sequence prior to insertion into a vector.
  • the coding sequence can be cloned directly into an expression vector that already contains the control sequences and an appropriate restriction site.
  • cell lines which stably express the polypeptide of interest may be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media.
  • the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells that successfully express the introduced sequences.
  • Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the cell type.
  • Mammalian cell lines available as hosts for expression are known in the art and include many immortalised cell lines available from the American Type Culture Collection (ATCC) including, but not limited to, Chinese hamster ovary (CHO), HeLa, baby hamster kidney (BHK), monkey kidney (COS), C127, 3T3, BHK, HEK 293, Bowes melanoma and human hepatocellular carcinoma (for example Hep G2) cells and a number of other cell lines.
  • ATCC American Type Culture Collection
  • baculovirus/insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego CA (the "MaxBac” kit). These techniques are generally known to those skilled in the art and are described fully in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987).
  • Particularly suitable host cells for use in this system include insect cells such as Drosophila S2 and Spodoptera Sf9 cells.
  • plant cell culture and whole plant genetic expression systems known in the art. Examples of suitable plant cellular genetic expression systems include those described in US 5,693,506; US 5,659,122; and US 5,608,143.
  • yeast cells for example, S. cerevisiae
  • Aspergillus cells examples include yeast cells (for example, S. cerevisiae) and Aspergillus cells.
  • any number of selection systems are known in the art that may be used to recover transformed cell lines. Examples include the herpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et al (1980) Cell 22:817-23) genes that can be employed in tk " or aprt* cells, respectively. Also, antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection; for example, dihydrofolate reductase (DHFR) that confers resistance to methotrexate (Wigler, M. et al (1980) Proc. Natl. Acad.
  • DHFR dihydrofolate reductase
  • npt which confers resistance to the aminoglycosides neomycin and G-418 (Colbere-Garapin, F. et al. (1981) J. Mol. Biol. 150:1-14) and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively. Additional selectable genes have been described, examples of which will be clear to those of skill in the art.
  • marker gene expression suggests that the gene of interest is also present, its presence and expression may need to be confirmed.
  • transformed cells containing the appropriate sequences can be identified by the absence of marker gene function.
  • a marker gene can be placed in tandem with a sequence encoding a polypeptide of the invention under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
  • host cells that contain a nucleic acid sequence encoding a polypeptide of the invention and which express said polypeptide may be identified by a variety of procedures known to those of skill in the art.
  • DNA-DNA or DNA-RNA hybridizations include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassays, for example, fluorescence activated cell sorting (FACS) or immunoassay techniques (such as the enzyme-linked immunosorbent assay [ELISA] and radioimmunoassay [RIA]), that include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein (see Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS Press, St Paul, MN) and Maddox, D.E. et al. (1983) J. Exp. Med, 158, 1211-1216).
  • FACS fluorescence activated cell sorting
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • Means for producing labelled hybridization or PCR probes for detecting sequences related to nucleic acid molecules encoding polypeptides of the present invention include oligolabelling, nick translation, end-labelling or PCR amplification using a labelled polynucleotide.
  • sequences encoding the polypeptide of the invention may be cloned into a vector for the production of an mRNA probe.
  • RNA polymerase such as T7, T3 or SP6 and labelled nucleotides. These procedures may be conducted using a variety of commercially available kits (Pharmacia & Upjohn, (Kalamazoo, MI); Promega (Madison WI); and U.S. Biochemical Corp., Cleveland, OH)).
  • Suitable reporter molecules or labels include radionuclides, enzymes and fluorescent, chemiluminescent or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
  • Nucleic acid molecules according to the present invention may also be used to create transgenic animals, particularly rodent animals. Such transgenic animals form a further aspect of the present invention. This may be done locally by modification of somatic cells, or by germ line therapy to incorporate heritable modifications. Such transgenic animals may be particularly useful in the generation of animal models for drug molecules effective as modulators of the polypeptides of the present invention.
  • the polypeptide can be recovered and purified from recombinant cell cultures by well- known methods including ammonium sulphate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. High performance liquid chromatography is particularly useful for purification. Well known techniques for refolding proteins may be employed to regenerate an active conformation when the polypeptide is denatured during isolation and or purification.
  • Specialised vector constructions may also be used to facilitate purification of proteins, as desired, by joining sequences encoding the polypeptides of the invention to a nucleotide sequence encoding a polypeptide domain that will facilitate purification of soluble proteins.
  • purification-facilitating domains include metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilised metals, protein A domains that allow purification on immobilised immunoglobulm, and the domain utilised in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, WA).
  • cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen, San Diego, CA) between the purification domain and the polypeptide of the invention may be used to facilitate purification.
  • One such expression vector provides for expression of a fusion protein containing the polypeptide of the invention fused to several histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification by IMAC (immobilised metal ion affinity chromatography as described in Porath, J. et al. (1992), Prot. Exp. Purif.
  • the polypeptide is to be expressed for use in screening assays, generally it is preferred that it be produced at the surface of the host cell in which it is expressed. In this event, the host cells may be harvested prior to use in the screening assay, for example using techniques such as fluorescence activated cell sorting (FACS) or immunoaffinity techniques. If the polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify the expressed polypeptide. If polypeptide is produced intracellularly, the cells must first be lysed before the polypeptide is recovered.
  • FACS fluorescence activated cell sorting
  • the polypeptide of the invention can be used to screen libraries of compounds in any of a variety of drug screening techniques. Such compounds may activate (agonise) or inhibit (antagonise) the level of expression of the gene or the activity of the polypeptide of the invention and form a further aspect of the present invention. Preferred compounds are effective to alter the expression of a natural gene which encodes a polypeptide of the first aspect of the invention or to regulate the activity of a polypeptide of the first aspect of the invention.
  • Agonist or antagonist compounds may be isolated from, for example, cells, cell-free preparations, chemical libraries or natural product mixtures. These agonists or antagonists may be natural or modified substrates, ligands, enzymes, receptors or structural or functional mimetics. For a suitable review of such screening techniques, see Coligan et al., Current Protocols in Immunology l(2):Chapter 5 (1991).
  • Compounds that are most likely to be good antagonists are molecules that bind to the polypeptide of the invention without inducing the biological effects of the polypeptide upon binding to it.
  • Potential antagonists include small organic molecules, peptides, polypeptides and antibodies that bind to the polypeptide of the invention and thereby inhibit or extinguish its activity. In this fashion, binding of the polypeptide to normal cellular binding molecules may be inhibited, such that the normal biological activity of the polypeptide is prevented.
  • the polypeptide of the invention that is employed in such a screening technique may be free in solution, affixed to a solid support, borne on a cell surface or located intracellularly.
  • screening procedures may involve using appropriate cells or cell membranes that express the polypeptide that are contacted with a test compound to observe binding, or stimulation or inhibition of a functional response.
  • the functional response of the cells contacted with the test compound is then compared with control cells that were not contacted with the test compound.
  • Such an assay may assess whether the test compound results in a signal generated by activation of the polypeptide, using an appropriate detection system.
  • Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist in the presence of the test compound is observed.
  • a preferred method for identifying an agonist or antagonist compound of a polypeptide of the present invention comprises:
  • the polypeptide being associated with a second component capable of providing a detectable signal in response to the binding of a compound to the polypeptide, with a compound to be screened under conditions to permit binding to the polypeptide; and (b) determining whether the compound binds to and activates or inhibits the polypeptide by measuring the level of a signal generated from the interaction of the compound with the polypeptide.
  • a further preferred method for identifying an agonist or antagonist of a polypeptide of the invention comprises:
  • the general methods that are described above may further comprise conducting the identification of agonist or antagonist in the presence of labelled or unlabelled ligand for the polypeptide.
  • the method for identifying an agonist or antagonist of a polypeptide of the present invention comprises: determining the inhibition of binding of a ligand to cells which have a polypeptide of the invention on the surface thereof, or to cell membranes containing such a polypeptide, in the presence of a candidate compound under conditions to permit binding to the polypeptide, and determining the amount of ligand bound to the polypeptide.
  • a compound capable of causing reduction of binding of a ligand is considered to be an agonist or antagonist.
  • the ligand is labelled.
  • a method of screening for a polypeptide antagonist or agonist compound comprises the steps of:
  • step (c) adding a candidate compound to a mixture of labelled ligand and the whole cell or the cell membrane of step (a) and allowing the mixture to attain equilibrium;
  • step (d) measuring the amount of labelled ligand bound to the whole cell or the cell membrane after step (c);
  • step (e) comparing the difference in the labelled ligand bound in step (b) and (d), such that the compound which causes the reduction in binding in step (d) is considered to be an agonist or antagonist.
  • polypeptides may be found to modulate a variety of physiological and pathological processes in a dose-dependent manner in the above-described assays.
  • the "functional equivalents" of the polypeptides of the invention include polypeptides that exhibit any of the same modulatory activities in the above-described assays in a dose- dependent manner.
  • the degree of dose-dependent activity need not be identical to that of the polypeptides of the invention, preferably the "functional equivalents" will exhibit substantially similar dose-dependence in a given activity assay compared to the polypeptides of the invention.
  • simple binding assays may be used, in which the adherence of a test compound to a surface bearing the polypeptide is detected by means of a label directly or indirectly associated with the test compound or in an assay involving competition with a labelled competitor.
  • competitive drug screening assays may be used, in which neutralising antibodies that are capable of binding the polypeptide specifically compete with a test compound for binding. In this manner, the antibodies can be used to detect the presence of any test compound that possesses specific binding affinity for the polypeptide. Assays may also be designed to detect the effect of added test compounds on the production of mRNA encoding the polypeptide in cells.
  • an ELISA may be constructed that measures secreted or cell-associated levels of polypeptide using monoclonal or polyclonal antibodies by standard methods known in the art, and this can be used to search for compounds that may inhibit or enhance the production of the polypeptide from suitably manipulated cells or tissues. The formation of binding complexes between the polypeptide and the compound being tested may then be measured.
  • Assay methods that are also included within the terms of the present invention are those that involve the use of the genes and polypeptides of the invention in overexpression or ablation assays. Such assays involve the manipulation of levels of these genes/polypeptides in cells and assessment of the impact of this manipulation event on the physiology of the manipulated cells. For example, such experiments reveal details of signaling and metabolic pathways in which the particular genes/polypeptides are implicated, generate information regarding the identities of polypeptides with which the studied polypeptides interact and provide clues as to methods by which related genes and proteins are regulated.
  • Another technique for drug screening which may be used provides for high throughput screening of compounds having suitable binding affinity to the polypeptide of interest (see International patent application WO84/03564).
  • This method large numbers of different small test compounds are synthesised on a solid substrate, which may then be reacted with the polypeptide of the invention and washed.
  • One way of immobilising the polypeptide is to use non-neutralising antibodies. Bound polypeptide may then be detected using methods that are well known in the art. Purified polypeptide can also be coated directly onto plates for use in the aforementioned drug screening techniques.
  • the polypeptide of the invention may be used to identify membrane-bound or soluble receptors, through standard receptor binding techniques that are known in the art, such as ligand binding and crosslinking assays in which the polypeptide is labelled with a radioactive isotope, is chemically modified, or is fused to a peptide sequence that facilitates its detection or purification, and incubated with a source of the putative receptor (for example, a composition of cells, cell membranes, cell supernatants, tissue extracts, or bodily fluids).
  • a source of the putative receptor for example, a composition of cells, cell membranes, cell supernatants, tissue extracts, or bodily fluids.
  • the efficacy of binding may be measured using biophysical techniques such as surface plasmon resonance and spectroscopy.
  • Binding assays may be used for the purification and cloning of the receptor, but may also identify agonists and antagonists of the polypeptide, that compete with the binding of the polypeptide to its receptor. Standard methods for conducting screening assays are well understood in the art.
  • the invention also includes a screening kit useful in the methods for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, that are described above.
  • the invention includes the agonists, antagonists, ligands, receptors, substrates and enzymes, and other compounds which modulate the activity or antigenicity of the polypeptide of the invention discovered by the methods that are described above.
  • compositions comprising a polypeptide, nucleic acid, ligand or compound of the invention in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier may be suitable as therapeutic or diagnostic reagents, as vaccines, or as other immunogenic compositions, as outlined in detail below.
  • a composition containing a polypeptide, nucleic acid, ligand or compound [X] is "substantially free of impurities [herein, Y] when at least 85% by weight of the total X+Y in the composition is X.
  • X comprises at least about 90% by weight of the total of X+Y in the composition, more preferably at least about 95%, 98% or even 99%) by weight.
  • compositions should preferably comprise a therapeutically effective amount of the polypeptide, nucleic acid molecule, ligand, or compound of the invention.
  • therapeutically effective amount refers to an amount of a therapeutic agent needed to treat, ameliorate, or prevent a targeted disease or condition, or to exhibit a detectable therapeutic or preventative effect.
  • the therapeutically effective dose can be estimated initially either in cell culture assays, for example, of neoplastic cells, or in animal models, usually mice, rabbits, dogs, or pigs. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • an effective amount for a human subject will depend upon the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. This amount can be determined by routine experimentation and is within the judgement of the clinician. Generally, an effective dose will be from 0.01 mg/kg to 50 mg/kg, preferably 0.05 mg/kg to 10 mg/kg. Compositions may be administered individually to a patient or may be administered in combination with other agents, drugs or hormones.
  • a pharmaceutical composition may also contain a pharmaceutically acceptable carrier, for administration of a therapeutic agent.
  • a pharmaceutically acceptable carrier for administration of a therapeutic agent.
  • Such carriers include antibodies and other polypeptides, genes and other therapeutic agents such as liposomes, provided that the carrier does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity.
  • Suitable carriers may be large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles.
  • Pharmaceutically acceptable salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulphates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulphates, and the like
  • organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • compositions of therapeutic compositions may additionally contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such compositions. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient. Once formulated, the compositions of the invention can be administered directly to the subject.
  • the subjects to be treated can be animals; in particular, human subjects can be treated.
  • compositions utilised in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra- arterial, intrameduUary, intrathecal, intraventricular, transdermal or transcutaneous applications (for example, see WO98/20734), subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal or rectal means.
  • Gene guns or hyposprays may also be used to administer the pharmaceutical compositions of the invention.
  • the therapeutic compositions may be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.
  • Direct delivery of the compositions will generally be accomplished by injection, subcutaneously, intraperitoneally, intravenously or intramuscularly, or delivered to the interstitial space of a tissue.
  • the compositions can also be administered into a lesion. Dosage treatment may be a single dose schedule or a multiple dose schedule.
  • One approach comprises administering to a subject an inhibitor compound (antagonist) as described above, along with a pharmaceutically acceptable carrier in an amount effective to inhibit the function of the polypeptide, such as by blocking the binding of ligands, substrates, enzymes, receptors, or by inhibiting a second signal, and thereby alleviating the abnormal condition.
  • an inhibitor compound as described above
  • a pharmaceutically acceptable carrier in an amount effective to inhibit the function of the polypeptide, such as by blocking the binding of ligands, substrates, enzymes, receptors, or by inhibiting a second signal, and thereby alleviating the abnormal condition.
  • antagonists are antibodies.
  • such antibodies are chimeric and/or humanised to minimise their immunogenicity, as described previously.
  • polypeptide that retain binding affinity for the ligand, substrate, enzyme, receptor, in question, may be administered.
  • polypeptide may be administered in the form of fragments that retain the relevant portions.
  • expression of the gene encoding the polypeptide can be inhibited using expression blocking techniques, such as the use of antisense nucleic acid molecules (as described above), either internally generated or separately administered.
  • Modifications of gene expression can be obtained by designing complementary sequences or antisense molecules (DNA, RNA, or PNA) to the control, 5' or regulatory regions (signal sequence, promoters, enhancers and introns) of the gene encoding the polypeptide.
  • inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules.
  • the complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
  • Such oligonucleotides may be administered or may be generated in situ from expression in vivo.
  • Ribozymes are catalytically active RNAs that can be natural or synthetic (see for example Usman, N, et al, Curr. Opin. Struct. Biol (1996) 6(4), 527-33). Synthetic ribozymes can be designed to specifically cleave mRNAs at selected positions thereby preventing translation of the mRNAs into functional polypeptide. Ribozymes may be synthesised with a natural ribose phosphate backbone and natural bases, as normally found in RNA molecules. Alternatively the ribozymes may be synthesised with non-natural backbones, for example, 2'-O-methyl RNA, to provide protection from ribonuclease degradation and may contain modified bases.
  • RNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of non-traditional bases such as inosine, queosine and butosine, as well as acetyl-, methyl-, thio- and similarly modified forms of adenine, cytidine, guanine, thymine and uridine which are not as easily recognised by endogenous endonucleases.
  • One approach comprises administering to a subject a therapeutically effective amount of a compound that activates the polypeptide, i.e. an agonist as described above, to alleviate the abnormal condition.
  • a therapeutic amount of the polypeptide in combination with a suitable pharmaceutical carrier may be administered to restore the relevant physiological balance of polypeptide.
  • Gene therapy may be employed to effect the endogenous production of the polypeptide by the relevant cells in the subject. Gene therapy is used to treat permanently the inappropriate production of the polypeptide by replacing a defective gene with a corrected therapeutic gene.
  • Gene therapy of the present invention can occur in vivo or ex vivo.
  • Ex vivo gene therapy requires the isolation and purification of patient cells, the introduction of a therapeutic gene and introduction of the genetically altered cells back into the patient.
  • in vivo gene therapy does not require isolation and purification of a patient's cells.
  • the therapeutic gene is typically "packaged" for administration to a patient.
  • Gene delivery vehicles may be non-viral, such as liposomes, or replication-deficient viruses, such as adenovirus as described by Berkner, K.L., in Curr. Top. Microbiol. Immunol., 158, 39-66 (1992) or adeno-associated virus (AAV) vectors as described by Muzyczka, N., in Curr. Top. Microbiol. Immunol, 158, 97-129 (1992) and U.S. Patent No. 5,252,479.
  • a nucleic acid molecule encoding a polypeptide of the invention may be engineered for expression in a replication-defective retroviral vector.
  • This expression construct may then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA encoding the polypeptide, such that the packaging cell now produces infectious viral particles containing the gene of interest.
  • These producer cells may be administered to a subject for engineering cells in vivo and expression of the polypeptide in vivo (see Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeutic Approaches, (and references cited therein) in Human Molecular Genetics (1996), T Strachan and A P Read, BIOS Scientific Publishers Ltd).
  • Another approach is the administration of "naked DNA" in which the therapeutic gene is directly injected into the bloodstream or muscle tissue.
  • the invention provides that they can be used in vaccines to raise antibodies against the disease causing agent.
  • Vaccines according to the invention may either be prophylactic (i.e. to prevent infection) or therapeutic (i.e. to treat disease after infection).
  • Such vaccines comprise immunising antigen(s), immunogen(s), polypeptide(s), protein(s) or nucleic acid, usually in combination with pharmaceutically-acceptable carriers as described above, which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Additionally, these carriers may function as immunostimulating agents ("adjuvants").
  • the antigen or immunogen may be conjugated to a bacterial toxoid, such as a toxoid from diphtheria, tetanus, cholera, H. pylori, and other pathogens.
  • a bacterial toxoid such as a toxoid from diphtheria, tetanus, cholera, H. pylori, and other pathogens.
  • polypeptides may be broken down in the stomach, vaccines comprising polypeptides are preferably administered parenterally (for instance, subcutaneous, intramuscular, intravenous, or intradermal injection).
  • Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents.
  • the vaccine formulations of the invention may be presented in unit-dose or multi-dose containers.
  • sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use.
  • the dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.
  • This invention also relates to the use of nucleic acid molecules according to the present invention as diagnostic reagents. Detection of a mutated form of the gene characterised by the nucleic acid molecules of the invention which is associated with a dysfunction will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, or susceptibility to a disease, which results from under-expression, over-expression or altered spatial or temporal expression of the gene. Individuals carrying mutations in the gene may be detected at the DNA level by a variety of techniques.
  • Nucleic acid molecules for diagnosis may be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy material.
  • the genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR, ligase chain reaction (LCR), strand displacement amplification (SDA), or other amplification techniques (see Saiki et al., Nature, 324, 163-166 (1986); Bej, et al., Crit. Rev. Biochem. Molec. Biol, 26, 301-334 (1991); Birkenmeyer et al, J. Virol. Meth., 35, 117-126 (1991); Van Brunt, J., Bio/Technology, 8, 291-294 (1990)) prior to analysis.
  • LCR ligase chain reaction
  • SDA strand displacement amplification
  • this aspect of the invention provides a method of diagnosing a disease in a patient, comprising assessing the level of expression of a natural gene encoding a polypeptide according to the invention and comparing said level of expression to a control level, wherein a level that is different to said control level is indicative of disease.
  • the method may comprise the steps of: a) contacting a sample of tissue from the patient with a nucleic acid probe under stringent conditions that allow the formation of a hybrid complex between a nucleic acid molecule of the invention and the probe; b) contacting a control sample with said probe under the same conditions used in step a); c) and detecting the presence of hybrid complexes in said samples; wherein detection of levels of the hybrid complex in the patient sample that differ from levels of the hybrid complex in the control sample is indicative of disease.
  • a further aspect of the invention comprises a diagnostic method comprising the steps of: a) obtaining a tissue sample from a patient being tested for disease; b) isolating a nucleic acid molecule according to the invention from said tissue sample; and c) diagnosing the patient for disease by detecting the presence of a mutation in the nucleic acid molecule which is associated with disease.
  • an amplification step for example using PCR, may be included.
  • Deletions and insertions can be detected by a change in the size of the amplified product in comparison to the normal genotype.
  • Point mutations can be identified by hybridizing amplified DNA to labelled RNA of the invention or alternatively, labelled antisense DNA sequences of the invention. Perfectly-matched sequences can be distinguished from mismatched duplexes by RNase digestion or by assessing differences in melting temperatures.
  • the presence or absence of the mutation in the patient may be detected by contacting DNA with a nucleic acid probe that hybridises to the DNA under stringent conditions to form a hybrid double-stranded molecule, the hybrid double-stranded molecule having an unhybridised portion of the nucleic acid probe strand at any portion corresponding to a mutation associated with disease; and detecting the presence or absence of an unhybridised portion of the probe strand as an indication of the presence or absence of a disease-associated mutation in the corresponding portion of the DNA strand.
  • Such diagnostics are particularly useful for prenatal and even neonatal testing.
  • Point mutations and other sequence differences between the reference gene and "mutant" genes can be identified by other well-known techniques, such as direct DNA sequencing or single-strand conformational polymorphism, (see Orita et al, Genomics, 5, 874-879 (1989)).
  • a sequencing primer may be used with double-stranded PCR product or a single-stranded template molecule generated by a modified PCR.
  • the sequence determination is performed by conventional procedures with radiolabelled nucleotides or by automatic sequencing procedures with fluorescent-tags.
  • Cloned DNA segments may also be used as probes to detect specific DNA segments. The sensitivity of this method is greatly enhanced when combined with PCR.
  • point mutations and other sequence variations, such as polymorphisms can be detected as described above, for example, through the use of allele-specific oligonucleotides for PCR amplification of sequences that differ by single nucleotides.
  • DNA sequence differences may also be detected by alterations in the electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing (for example, Myers et al, Science (1985) 230:1242). Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and SI protection or the chemical cleavage method (see Cotton et al, Proc. Natl Acad. Sci. USA (1985) 85: 4397-4401).
  • mutations such as microdeletions, aneuploidies, translocations, inversions, can also be detected by in situ analysis (see, for example, Keller et al, DNA Probes, 2nd Ed., Stockton Press, New York, N.Y., USA (1993)), that is, DNA or RNA sequences in cells can be analysed for mutations without need for their isolation and/or immobilisation onto a membrane.
  • Fluorescence in situ hybridization is presently the most commonly applied method and numerous reviews of FISH have appeared (see, for example, Trachuck et al., Science, 250, 559-562 (1990), and Trask et al, Trends, Genet., 7, 149-154 (1991)).
  • an array of oligonucleotide probes comprising a nucleic acid molecule according to the invention can be constructed to conduct efficient screening of genetic variants, mutations and polymorphisms.
  • Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability (see for example: M.Chee et al., Science (1996), Vol 274, pp 610-613).
  • the array is prepared and used according to the methods described in PCT application WO95/11995 (Chee et al); Lockhart, D. J. et al. (1996) Nat. Biotech. 14: 1675-1680); and Schena, M. et al. (1996) Proc. Natl. Acad. Sci. 93: 10614-10619).
  • Oligonucleotide pairs may range from two to over one million.
  • the oligomers are synthesized at designated areas on a substrate using a light-directed chemical process.
  • the substrate may be paper, nylon or other type of membrane, filter, chip, glass slide or any other suitable solid support.
  • an oligonucleotide may be synthesized on the surface of the substrate by using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application W095/251116 (Baldeschweiler et al .
  • a "gridded" array analogous to a dot (or slot) blot may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedures.
  • An array such as those described above, may be produced by hand or by using available devices (slot blot or dot blot apparatus), materials (any suitable solid support), and machines (including robotic instruments), and may contain 8, 24, 96, 384, 1536 or 6144 oligonucleotides, or any other number between two and over one million which lends itself to the efficient use of commercially-available instrumentation.
  • diseases may be diagnosed by methods comprising determining, from a sample derived from a subject, an abnormally decreased or increased level of polypeptide or mRNA.
  • Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, nucleic acid amplification, for instance PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods.
  • nucleic acid amplification for instance PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods.
  • Assay techniques that can be used to determine levels of a polypeptide of the present invention in a sample derived from a host are well-known to those of skill in the art and are discussed in some detail above (including radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays).
  • This aspect of the invention provides a diagnostic method which comprises the steps of: (a) contacting a ligand as described above with a biological sample under conditions suitable for the formation of a ligand- polypeptide complex; and (b) detecting said complex.
  • Protocols such as ELISA, RIA, and FACS for measuring polypeptide levels may additionally provide a basis for diagnosing altered or abnormal levels of polypeptide expression.
  • Normal or standard values for polypeptide expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably humans, with antibody to the polypeptide under conditions suitable for complex formation The amount of standard complex formation may be quantified by various methods, such as by photometric means.
  • Antibodies which specifically bind to a polypeptide of the invention may be used for the diagnosis of conditions or diseases characterised by expression of the polypeptide, or in assays to monitor patients being treated with the polypeptides, nucleic acid molecules, ligands and other compounds of the invention.
  • Antibodies useful for diagnostic purposes may be prepared in the same manner as those described above for therapeutics. Diagnostic assays for the polypeptide include methods that utilise the antibody and a label to detect the polypeptide in human body fluids or extracts of cells or tissues.
  • the antibodies may be used with or without modification, and may be labelled by joining them, either covalently or non-covalently, with a reporter molecule.
  • a wide variety of reporter molecules known in the art may be used, several of which are described above.
  • Diagnostic assays may be used to distinguish between absence, presence, and excess expression of polypeptide and to monitor regulation of polypeptide levels during therapeutic intervention. Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials or in monitoring the treatment of an individual patient.
  • a diagnostic kit of the present invention may comprise:
  • a diagnostic kit may comprise a first container containing a nucleic acid probe that hybridises under stringent conditions with a nucleic acid molecule according to the invention; a second container containing primers useful for amplifying the nucleic acid molecule; and instructions for using the probe and primers for facilitating the diagnosis of disease.
  • the kit may further comprise a third container holding an agent for digesting unhybridised RNA.
  • a diagnostic kit may comprise an array of nucleic acid molecules, at least one of which may be a nucleic acid molecule according to the invention.
  • a diagnostic kit may comprise one or more antibodies that bind to a polypeptide according to the invention; and a reagent useful for the detection of a binding reaction between the antibody and the polypeptide.
  • kits will be of use in diagnosing a disease or susceptibility to disease, particularly cell proliferative disorders, autoimmune/inflammatory disorders, cardiovascular disorders, neurological disorders, developmental disorders, metabolic disorders, infections and other pathological conditions.
  • Figure 1 Results from BLAST against NCBI non-redundant database using combined SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NOT2, SEQ ID NOT4, SEQ ID NO:16, SEQ ID NOT8, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50 and SEQ ID NO:52 polypeptide sequence.
  • Figure 2 Alignment generated by BLAST between combined SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50 and SEQ ID NO:52 polypeptide sequence and the closet related sequence, AAC72492T, submaxillary mucin (Bos Taurus).
  • Figure 3 Results from BLAST against NCBI non-redundant database using combined SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO
  • SEQ ID NO: 104 SEQ ID NO 106, SEQ ID NO 108, SEQ ID NOT 10, SEQ ID NO 112, SEQ ID NOT 14, SEQ ID NO 116, SEQ ID NO 118, SEQ ID NO: 120, SEQ ID NO 122, SEQ ID NOT24, SEQ ID NO 126, SEQ ID NO 128, SEQ ID NO: 130, SEQ ID NO 132, SEQ ID NO: 134, SEQ ID NO 136, SEQ ID NO 138, SEQ ID NOT40, SEQ ID NO 142, SEQ ID NO:144, SEQ ID NO 146, SEQ ID NO 148, SEQ ID NOT50, SEQ ID NO 152, SEQ ID NOT 54, SEQ ID NO 156, SEQ ID NO T58, SEQ ID NOT60, SEQ ID NO: 162 and SEQ ID NO: 164 polypeptide sequence.
  • Figure 4 Alignment generated by BLAST between combined SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NOT 04, SEQ ID NOT06, SEQ ID NOT08, SEQ ID NOT 10, SEQ ID NOT 12, SEQ ID NOT 14, SEQ ID NOT16, SEQ ID NOT18, SEQ ID NOT20, SEQ ID NOT22, SEQ ID NOT24, SEQ ID
  • Figure 5 Results from BLAST against NCBI non-redundant database using combined SEQ ID NOT66, SEQ ID NOT68, SEQ ID NOT70, SEQ ID NOT72, SEQ ID NOT74, SEQ ID NOT76 and SEQ ID NOT78 polypeptide sequence.
  • Figure 6 Alignment generated by BLAST between combined SEQ ID NOT 66, SEQ ID NOT68, SEQ ID NOT70, SEQ ID NO:172, SEQ ID NOT74, SEQ ID NOT76 and SEQ ID NOT78 polypeptide sequence and the closet related sequence, NP_038652T (AAB96561T) otogelin (Mus musculus).
  • Figure 7 Alignment of INSPO 10 against NP_038652.1
  • the mouse orthologue Figure 8 SigP output showing the presence of a signal peptide in INSPOIO
  • Figure 9 INSPOIO Predicted sequence with translation, showing cloned region
  • Figure 10 INSPOIO cloned sequence with translation
  • Figure 11 Map of PCRII-TOPO-INSP010 partial
  • Figure 12 Full length virtual cDNA encoding INSPOl la
  • Figure 13 INSPOl la cloned sequence with translation
  • Figure 14 Clustal(W) alignment of cloned nucleotide sequence of INSPOlla against the predicted sequence of INSPOl 1
  • Figure 15 Pairwise alignment of amino acid sequence of cloned INSPOl la compared to the predicted sequence of INSPOl 1
  • Figure 16 Alignment of cloned INSPOl la sequence with genomic DNA
  • the top ten matches include sequences annotated as submaxillary mucin, (Bos taurus) (Jiang, W., Woitach, J. T., Gupta, D. and Bhavanandan,V.P., Biochem. Biophys. Res. Commun. (1998) 251 (2), 550-556).
  • Bos taurus submaxillary mucin
  • polypeptide sequence derived from combining SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NOT00, SEQ ID NOT02, SEQ ID NOT04, SEQ ID NOT06, SEQ ID
  • the top ten matches include sequences annotated as otogelin, Mus musculus, (Cohen-Salmon,M., El-Amraoui,A., Leibovici,M. and Petit,C, Proc. Natl. Acad. Sci. U.S.A. (1997) 94 (26), 14450-14455).
  • polypeptide sequence derived from combining SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NOT00, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NOT06, SEQ ID NOT08, SEQ ID NOT 10, SEQ ID NOT 12, SEQ ID NOT 14, SEQ ID NOT 16, SEQ ID NOT 18, SEQ ID NOT20, SEQ ID NO:122, SEQ ID NO: 124, SEQ ID NOT
  • Example 2a Summary of INSPOIO cloning
  • Human cDNA libraries (in bacteriophage lambda ( ⁇ ) vectors) were purchased from Stratagene or Clontech or prepared at the Serono Pharmaceutical Research Institute in ⁇ ZAP or ⁇ GT10 vectors according to the manufacturer's protocol (Stratagene). Bacteriophage ⁇ DNA was prepared from small scale cultures of infected E. coli host strain using the Wizard Lambda Preps DNA purification system according to the manufacturer's instructions (Promega, Corporation, Madison WL). The list of libraries and host strains used is shown in Table I.
  • a partial cDNA encoding INSPOIO ( Figure 9) was obtained as a PCR amplification product of 150 bp ( Figure 10) using gene specific cloning primers (04211-CPl and 04211-CP2, Figure 9 and Table II).
  • PCR products were visualized on 0.8 % agarose gels in 1 X TAE buffer (Invitrogen) and PCR products migrating at the predicted molecular mass were purified from the gel using the Wizard PCR Preps DNA Purification System (Promega). PCR products eluted in 50 ⁇ l of sterile water were either subcloned directly or stored at — 20°C.
  • Pairs of PCR primers having a length of between 18 and 25 bases were designed for amplifying the full length sequence of the virtual cDNA using Primer Designer Software (Scientific & Educational Software, PO Box 72045, Durham, NC 27722-2045, USA). PCR primers were optimized to have a Tm close to 55 + 10 °C and a GC content of 40- 60%. Primers were selected which had high selectivity for the target sequence INSPOIO (little or no none specific priming to other templates).
  • PCR products were subcloned into the topoisomerase I modified cloning vector (pCR II TOPO) using the TOPO TA cloning kit purchased from the Invitrogen Corporation (cat.
  • TOP 10 cells was thawed on ice and 2 ⁇ l of TOPO reaction was added. The mixture was incubated for 15 min on ice and then heat shocked by incubation at 42 °C for exactly 30 s. Samples were returned to ice and 250 ⁇ l of warm SOC media (room temperature) was added. Samples were incubated with shaking (220 rpm) for 1 h at 37 °C. The transformation mixture was then plated on L-broth (LB) plates containing ampicillin (100 ⁇ g/ml) and incubated overnight at 37 °C. Ampicillin resistant colonies containing cDNA inserts were identified by colony PCR.
  • LB L-broth
  • Colonies were inoculated into 50 ⁇ l sterile water using a sterile toothpick. A 10 ⁇ l aliquot of the inoculum was then subjected to PCR in a total reaction volume of 20 ⁇ l as described above, except the primers pairs used were SP6 and T7.
  • the cycling conditions were as follows: 94 °C, 2 min; 30 cycles of 94 °C, 30 sec, 47 °C, 30 sec and 72 °C for 1 min); 1 cycle, 72 °C, 7 min. Samples were then maintained at 4 °C (holding cycle) before further analysis. PCR reaction products were analyzed on 1 % agarose gels in 1 X TAE buffer.
  • Colonies which gave the expected PCR product size (150 bp cDNA + 187 bp due to the multiple cloning site or MCS) were grown up overnight at 37 °C in 5 ml L-Broth (LB) containing ampicillin (50 ⁇ g /ml), with shaking at 220 rpm at 37 C. 2a.6 Plasmid DNA preparation and Sequencing
  • Miniprep plasmid DNA was prepared from 5 ml cultures using a Qiaprep Turbo 9600 robotic system (Qiagen) or Wizard Plus SN Minipreps kit (Promega cat. no. 1460) according to the manufacturer's instructions. Plasmid D ⁇ A was eluted in 100 ⁇ l of sterile water. The D ⁇ A concentration was measured using an Eppendorf BO photometer. Plasmid D ⁇ A (200-500 ng) was subjected to D ⁇ A sequencing with T7 primer and SP6 primer using the BigDyeTerminator system (Applied Biosystems cat. no. 4390246) according to the manufacturer's instructions.
  • PCR products obtained with 04211-CPl and 04211-CP2 and migrating at the correct size (150 bp) were identified in the fetal kidney cDNA library (library 12).
  • the sequence of the PCR product cloned in pCRII-TOPO vector is shown in figure 10, and the plasmid map (plasmid ID. 13317) is in figure 11.
  • polypeptide sequence derived from combining SEQ ID NOT66, SEQ ID NOT68, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176 and SEQ ID NOT78, which represents the translation of consecutive exons from INSPOl 1 was used as a BLAST query against the NCBI non-redundant Sequence database.
  • the top ten matches include sequences annotated as otogelin, (Mus musculus), (Cohen-Salmon, M., El- Amraoui, A., Leibovici, M. and Petit, C, Proc. Natl. Acad. Sci. U.S.A. (1997) 94 (26), 14450-14455).
  • Human cDNA libraries (in bacteriophage lambda ( ⁇ ) vectors) were purchased from Stratagene or Clontech or prepared at the Serono Pharmaceutical Research Institute in ⁇ ZAP or ⁇ GT10 vectors according to the manufacturer's protocol (Stratagene). Bacteriophage ⁇ DNA was prepared from small scale cultures of infected E.coli host strain using the Wizard Lambda Preps DNA purification system according to the manufacturer's instructions (Promega, Corporation, Madison WL) The list of libraries and host strains used is shown in Table I.
  • PCR products were visualized on 0.8 % agarose gels in 1 X TAE buffer (Invitrogen) and PCR products migrating at the predicted molecular mass were purified from the gel using the Wizard PCR Preps DNA Purification System (Promega). PCR products eluted in 50 ⁇ l of sterile water were either subcloned directly or stored at -20 °C.
  • Pairs of PCR primers having a length of between 18 and 25 bases were designed for amplifying the full length sequence of the virtual cDNA using Primer Designer Software
  • PCR primers were optimized to have a Tm close to 55 + 10 °C and a GC content of 40- 60%. Primers were selected which had high selectivity for the target sequence INSPOl la (little or no none specific priming to other templates).
  • PCR products were subcloned into the topoisomerase I modified cloning vector (pCR II TOPO) using the TOPO TA cloning kit purchased from the Invitrogen Corporation (cat.
  • Colonies were inoculated into 50 ⁇ l sterile water using a sterile toothpick. A 10 ⁇ l aliquot of the inoculum was then subjected to PCR in a total reaction volume of 20 ⁇ l as described above, except the primers pairs used were SP6 and T7.
  • the cycling conditions were as follows: 94 °C, 2 min; 30 cycles of 94 °C, 30 sec, 47 °C, 30 sec and 72 °C for 1 min; 1 cycle, 72 °C, 7 min. Samples were then maintained at 4 °C (holding cycle) before further analysis.
  • PCR reaction products were analyzed on 1 % agarose gels in 1 X TA ⁇ buffer. Colonies which gave the expected PCR product size (436 bp cDNA +187 bp due to the multiple cloning site or MCS) were grown up overnight at 37 °C in 5 ml L-Broth (LB) containing ampicillin (100 ⁇ g /ml), with shaking at 220 rpm at 37 °C. 3a.6 Plasmid DNA preparation and Sequencing
  • Miniprep plasmid DNA was prepared from 5 ml cultures using a Qiaprep Turbo 9600 robotic system (Qiagen) or Wizard Plus SN Minipreps kit (Promega cat. no. 1460) according to the manufacturer's instructions. Plasmid D ⁇ A was eluted in 100 ⁇ l of sterile water. The D ⁇ A concentration was measured using an Eppendorf BO photometer. Plasmid D ⁇ A (200-500 ng) was subjected to D ⁇ A sequencing with T7 primer and SP6 primer using the BigDyeTerminator system (Applied Biosystems cat. no. 4390246) according to the manufacturer's instructions. Sequencing reactions were purified using Dye-Ex columns (Qiagen) or Montage SEQ 96 cleanup plates (Millipore cat. no. LSKS09624) then analysed on an Applied Biosystems 3700 sequencer.
  • Qiaprep Turbo 9600 robotic system Qiagen
  • Wizard Plus SN Minipreps kit Promega cat. no
  • PCR products obtained with CP1 and CP2 and migrating at the correct size (436 bp) were identified in a pool of the following libraries: U373 cells, retina, bladder and thymus.
  • the sequence of the cloned PCR fragment and the map of plasmid pCRII-TOPO-INSPOlla are shown in figures 13 and 17 respectively.
  • the cloned sequence contains 3 nucleotide substitutions G-A, T-G and T-G and a 3 base pair insertion (AAG) compared to the prediction (Figure 14) resulting in the following amino acid changes: N40S, W41E and insertion of Gly at position 42 ( Figure 15). These changes are consistent with the sequence of genomic D ⁇ A (100% identity) ( Figure 16).
  • the sequence of the cloned fragment spans 4 exons.
  • amino acids encoded by exon-exon junctions the amino acid will be assigned to the more 5' exon.
  • SEQ ID NO:2 (INSP009 Protein sequence exon 1) 1 SDIMGESSRT TILSGSSNTE
  • SEQ ID NO:4 (INSP009 Protein sequence exon 2)
  • SEQ ID NO:5 (INSP009 Nucleotide sequence exon 3) 1 GATCTATGAC CACTGCACTG GGGAGCCAAC TCTCCAGCAG TCAAACAG
  • SEQ ID NO:6 (INSP009 Protein sequence exon 3)
  • SEQ ID NO:10 (INSP009 Protein sequence exon 5)
  • SEQ ID NO:l 1 (INSP009 Nucleotide sequence exon 6) 1 AAGCTACCAG TGGCACATCT GAGAGGCCAA ACCCTGGAAG TGAAATAG
  • SEQ ID NO: 12 (INSP009 Protein sequence exon 6)
  • SEQ ID NO:14 (INSP009 Protein sequence exon 7) 1 TTGIVSGTTV APGSSNTE
  • SEQ ID NO: 15 (INSP009 Nucleotide sequence exon 8) 1 AAGCCACAAC TTCTTTGGGC AATGGTGGAA CCACTGAGGC TGGAAGTAAA 51 ATAG
  • SEQ ID NO:16 (INSP009 Protein sequence exon 8)
  • SEQ ID NO: 19 (INSP009 Nucleotide sequence exon 10)
  • SEQ ID NO:20 (INSP009 Protein sequence exon 10)
  • SEQ ID NO:25 (INSP009 Nucleotide sequence exon 13) 1 AGGCCACAAC TTTTAAAGGT GATGTTGGAA CCACTGAAGC AGAAATTTCA
  • SEQ ID NO:32 (INSP009 Protein sequence exon 16) 1 PSKEASDKTT APGPPTTG
  • SEQ ID NO:36 (INSP009 Protein sequence exon 18) 1 TTWATREVE TENKTE
  • SEQ ID NO:41 (INSP009 Nucleotide sequence exon 21) 1 CCTGGAGACA TATGGACTGC CAATTGCCAC AGAGGTACCT GTACTGATGC 51 AAAGACTATA GACTGTAAAC CTGAGGAGTG CCCTTCTCCA CCCACATGCA 101 AAACCGGCGA GAAGCTTGTG AAGTTCCAAT CTAATGACAC CTGCTGTGAA 151 ATCGGATACT GTG SEQ IDNO:42 (INSP009 Proteinsequence exon 21)
  • SEQ IDNO:43 (INSP009Nucleotide sequence exon22) 1 AACCAAGAAC ATGTTTATTT AACAATACTG ACTATGAG
  • SEQ ID NO:49 (INSP009 Nucleotide sequence exon 25) 1 GTAAGAATAA TTGCAGATCT TCCCTTGTAA ATGTGACTGT TATCTACAGT
  • SEQ ID NO:50 (INSP009 Protein sequence exon 25) 1 KNNCRSSLVN VTVIYSGCKK RVQ AKCTGE CEKTAK
  • SEQ ID NO:52 (INSP009 Protein sequence exon 26) 1 YNYD1 LLEH SCLCCREENY ELRDIVLDCP DGSTIPYQYK HITTCSCLDI 51 CQ YTTFMYS
  • SEQ ID NO:54 (INSPOIO Protein sequence exon 1) 1 GVLASA C LLCV LP GE QAAESLRVQR LA
  • SEQ ID NO:56 (INSPOIO Protein sequence exon 2)
  • SEQ ID NO:57 (INSPOIO Nucleotide sequence exon 3) 1 CAGCAGCCAC CAGGAGGCGA CCCTTGCCAT GGGGGACAAG GCTACAGTCG
  • SEQ ID NO:58 (INSPOIO Protein sequence exon 3)
  • SEQ ID NO:59 (INSPOIO Nucleotide sequence exon 4) 1 CAGGCTGAAG CCCCAGACTC CGTGGCCATG TCTTCCTGGG AAAGGCGGCT 51 CCATCGGGCC AAGTGTGCAC CATCCT SEQ ID NO:60 (INSPOIO Protein sequence exon 4)
  • SEQ ID NO:61 (INSPOIO Nucleotide sequence exon 5)
  • SEQ ID NO:62 (INSPOIO Protein sequence exon 5) 1 LFSCFNGGEC VHPAFCDCRR FNATGPRCQM V SEQ ID NO:63 (INSPOIO Nucleotide sequence exon 6)
  • SEQ ID NO:64 (INSPOIO Protein sequence exon 6)
  • SEQ ID NO:67 (INSPOIO Nucleotide sequence exon 8) 1 GGTCCAACTG CCACATGTCA TGGGGAGCGC GCGTCTGCAG CAGCTTGCCG 51 GCTATGTCAT CGTGCGGCAT CAGTCAGCCT TCACACTGGC CTGGGATGGT
  • SEQ ID NO:69 (INSPOIO Nucleotide sequence exon 9)
  • SEQ ID NO:70 (INSPOIO Protein sequence exon 9)
  • SEQ ID NO:71 (INSPOIO Nucleotide sequence exon 10) 1 GGCGTGTACG AGCAGTGTGA GGCTCTACTG CGGCCCCCCT TTGACGCCTG
  • SEQ ID NO:72 (INSPOIO Protein sequence exon 10) 1 GVYEQCEALL RPPFDACHAY VSPLPFTASC TSDLCQ
  • SEQ ID NO:77 (INSPOIONucleotide sequence exon 13) 1 GGCTCATCTT CGAGGATGGG GGCTGCGTGG CACCAGCTGA GTGTCCCTGT 51 GAGTTTCACG GGACTCTGTA CCCACCTGGC TCTGTGGTGA AGGAAGACTG 101 CAATACTTG
  • SEQ ID NO:89 (INSPOIO Nucleotide sequence exon 19) 1 CCTCCTACTC AGTGCAGGCC TGCAGCGTGC TCACGGGGGA GATGTTTGCG
  • SEQ ID NO:90 (INSPOIO Protein sequence exon 19) 1 SYSVQACSVL TGEMFAPCSA FLSPVPYFEQ CRRDACRCGQ PCLCATLAHY 51 AHLCRRHGLP VDFRARLPAC A SEQ ID NO:91 (INSPOIO Nucleotide sequence exon 20)
  • SEQ IDNO:105 (INSPOIONucleotide sequence exon 27) 1 AGTCCAGAGA GCTTCCTGGA TGACAAGCAG GAGGTCCACA CATGGCGAGT 51 GGGATTTTTC ACACTGGTGC ATTTCCCACA GGAGCACATC ACCCTCTTGT 101 GGGACCAGAG AACCACAGTG CACGTCCAGG CTGGGCCTCA GTGGCAG
  • SEQ ID NO:106 (INSPOIO Protein sequence exon 27) 1 SPESFLDDKQ EVHT RVGFF TLVHFPQEHI TLLWDQRTTV HVQAGPQ Q
  • SEQ ID NO:l 13 (INSPOIO Nucleotide sequence exon 31) 1 CGTATGACTG TGACTTCTTT AACAAAG
  • SEQ ID NO:l 16 (INSPOIO Protein sequence exon 32) 1 LGKGPYQLSS LAAGGALVGM KAVGDDIVLV RTEDVAPADI VSFLLTAALY 51 KAKAHD
  • SEQ ID NO:l 17 (INSPOIO Nucleotide sequence exon 33) 1 ACCCAGATGT GGTGTCCCTG GAGGCAGCAG ACAGACCCAA CTTCTTCCTT 51 CACGTCACAG CCAACGGGTC TCTGGAGCTG GCTAAGTGGC AGGGCCGTGA 101 CACCTTCCAA CAGCATGCCT CCTTCTTGCT GCACCGGGGG ACACGGCAGG 151 CAGGCCTGGT GGCCCTGGAG TCCCTGGCCA AGCCCAGCTC CTTCCTCTAT
  • SEQ ID NO:l 18 (INSPOIO Protein sequence exon 33) 1 PDWSLEAAD RPNFFLHVTA NGSLELAKWQ GRDTFQQHAS FLLHRGTRQA
  • SEQ IDNO:125 (INSPOIONucleotide sequence exon 37) 1 CCAATCGCCG AGCAGGACTG CGTCCGCCAC ATCTGCCTGG AGGGCCAGCT 51 GATTCGCGTG AATCAGTCCC AGCACTGTCC CCAGGGTGCT GCTCCCCCTC 101 GCTGTGGGAT CCTGGGCCTC GCCGTGCGGG TGGGTGGGGA CCGCTGCTGC
  • SEQ ID NO:127 (INSPOIO Nucleotide sequence exon 38) 1 GCCGGTGCTC AATCTTCCCT GACCTGAGCT TCGTGACCTT CGATGGGAGC 51 CACGTAGCTC TGTTCAAGGA GGCCATCTAC ATCCTCAGCC AGAGCCCAGA 101 TGAAATGCTC ACCGTCCATG TACTGGACTG CAAAAGTGCC AACCTG
  • SEQ ID NO:128 (INSPOIO Protein sequence exon 38) 1 RCSIFPDLSF VTFDGSHVAL FKEA1YILSQ SPDEMLTVHV LDCKSANL SEQ ID NO:129 (INSPOIO Nucleotide sequence exon 39)
  • SEQ ID NO:138 (INSPOIO Protein sequence exon 43) 1 FLCSSDSTYQ ACVTACEPPK TCQDGILGPL DPEHCQVLGE GCVCSEGTIL 51 HRRHSALCIP EAKCA
  • SEQ ID NO-.141 (INSPOIO Nucleotide sequence exon 45) 1 TGTGTAACCA GACTCTGTGT GAGGGTCTCG CCCCCACATG CCGCCCAGGC 51 CACCGCCTCC TCACCCACTT CCAGGAGGAC TCCTGCTGCC CCAGCTACAG 101 CTGTG
  • SEQ ID NO:142 (INSPOIO Protein sequence exon 45) 1 CNQTLCEGLA PTCRPGHRLL THFQEDSCCP SYSCE
  • SEQ ID NO:152 (INSPOIO Protein sequence exon 50)
  • VLCDIHCEA SEQ IDNO:155 (INSPOIO Nucleotide sequence exon 52)
  • SEQ ID NO:164 (INSPOIO Protein sequence exon 56) 1 VNLVSCDGRC PSASIYNYNI NTYARFCKCC REVGLQRRSV QLFCATNATW 51 VPYTVQEPTD CACQWS
  • SEQ ID NO:165 (INSPOl1 Nucleotide sequence exon 1) 1 CATAACTTCC AGAGTGATTG TGGATGCATA CAGTATCTCT GTG
  • SEQ ID NO:167 (INSPOl 1 Nucleotide sequence exon 2) 1 AAAAGGATGA TGTGTGTGTA TTTCAAGAAG TATCAGTATT GAATCCTGGA 51 CAATCCATGA TAAAGTATTT GGAAGAAGAC TTTTGTTATG CTATAGAGTG 101 TCTGGAAGAA AAAGATAACC ATACGGGCTT TCACACTCTG AATTTTACAC 151 TGGTGAATTG TTCAAAAAAA TGTGATGTT SEQ ID NO:168 (INSPOl 1 Protein sequence exon 2)
  • SEQ ID NO:169 (INSPOl 1 Nucleotide sequence exon 3) 1 CACCAGGTAT ATACTCCATC CCCAAGTGAT TATGGTTGTT GTGGTACCTG 51 CAAAAATGTA TCCTGCAAAT TTCACATGGA AAATGGAACA TCAGTTGTAT 101 ACGCG
  • SEQ IDNO:170 (INSPOl1 Protein sequence exon3) 1 HQVYTPSPSD YGCCGTCKNV SCKFHMENGT SWYA
  • SEQ IDNO:171 (INSPOll Nucleotide sequence exon 4)
  • SEQ ID NO:172 (INSPOl 1 Protein sequence exon 4)
  • SEQ ID NO:174 (INSPOl 1 Protein sequence exon 5)
  • SEQ ID NO:175 (INSPOll Nucleotide sequence exon 6)
  • SEQ IDNO:177 (INSPOll Nucleotide sequence exon 7) 1 ATAAATGTTG CATCTTGTGA CGGCAAATGC CCATCAGCTA CCATATATAA 51 CATCAATATT GAAAGTCACC TAAGATTCTG CAAGTGTTGT CGTGAAAATG 101 GAGTACGAAA CTTGTCTGTG CCTCTGTATT GCTCAGGAAA TGGCACTGAA 151 ATTATGTACA CTCTCCAGGA ACCCATAGAC TGTACGTGCC AGTGGAAT SEQ ID NO: 178 (INSPOl 1 Protein sequence exon 7)
  • SEQ IDNO:180 (INSP009 Protein sequence) 1 SDIMGESSRT TILSGSSNTE ATNSIEETGT SGTGFKTGSM TTALGSQLSS 51 SQTGTTGVAL STTVAPGSSS TEATTSTGVH RTTWGQKTE ATSGTSERPN 101 PGSEIGTTGI VSGTTVAPGS SNTEATTSLG NGGTTEAGSK IVTTGITTGT 151 TIVPGSFNTK ATTSTDVGVA TGVGMATGIT N1ISGRSQPT GSKTGYTVTG 201 SGTTALPGGF RTEATTFKGD VGTTEAEISS GNTPGSTGVT SSQEGEITMM 251 DAGTTWSSG ITGIPETSIS GPSKEASDKT TAPGPPTTGS TLVTAGVPTR 301 PQVSQPETTV VATREVETEN KTECLASLPP APVCHGPLGE EKSPGDIWTA 351 NCHRGTCTDA KTIDCKPEEC PSPPTCKTGE KLVKFQSNDT CCEIGY
  • SEQ ID NO:182 (INSPOIO Protein sequence) 1 GVLASALC LLCV LPWGE QAAESLRVQR LAAAPVL GS AEPQPEPAGQ
  • SEQ ID NO:l 83 (INSPOl 1 Nucleotide sequence)
  • SEQ ID NO: 186 (INSP009 exons 24-26 consolidated polypeptide sequence)
  • SEQ ID NO: 187 (INSPOIO mature nucleotide sequence)
  • ACATGTCAGT ACATCCTGGC CAAGAGCCGC TCTTCGGGCA CCTTCACCGT GACATTGCAG
  • SEQ ID NO: 189 (INSPOIO cloned partial nucleotide sequence)
  • SEQ ID NO: 190 (INSPOIO cloned partial polypeptide sequence) 1 CPSASIYNYN INTYARFCKC CREVGLQRRS VQLFCATNAT WVPYTVQEPT
  • SEQ ID NO: 191 (INSPOlla cloned partial nucleotide sequence)
  • SEQ ID NO: 192 (INSPOlla cloned partial polypeptide sequence)
  • SEQ ID NO: 193 (INSPOlla full length nucleotide sequence)
  • SEQ ID NO: 194 (INSPOlla full length polypeptide sequence)

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Abstract

L'invention concerne des nouvelles protéines, appelées INSP009, INSP010, INSP011, identifiées ici comme des membres de la sous-famille mucine de protéines contenant des domaines de noeuds de la cystine ainsi que l'utilisation desdites protéines et des séquences d'acide nucléique provenant des gènes de codage dans le diagnostic, la prévention et le traitement de maladies.
PCT/GB2002/005811 2001-12-19 2002-12-19 Proteines secretees de type mucine WO2003051919A1 (fr)

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Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
COHEN-SALMON MARTINE ET AL: "Otogelin: A glycoprotein specific to the acellular membranes of the inner ear.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES, vol. 94, no. 26, Q8WWQ5, 23 December 1997 (1997-12-23), Dec. 23, 1997, pages 14450 - 14455, XP002237800, ISSN: 0027-8424, Retrieved from the Internet <URL:TREMBL> *
DATABASE EMBL [online] 1 June 1998 (1998-06-01), retrieved from TREMBL Database accession no. O55225 *
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