CA2440846A1 - Protein cluster v - Google Patents

Abstract

The present invention relates to the identification of a human gene family expressed in metabolically relevant tissues. The genes encode a group polypeptides referred to as "Protein Cluster V" which are predicted to be useful in the diagnosis of metabolic diseases, such as obesity and diabetes, as well as in the identification of agents useful in the treatment of the said diseases.

Description

PROTEIN CLUSTER V
TECHNICAL FIELD
The present invention relates to the identification of a human gene family expressed in metabolically relevant tissues. The genes encode a group polypeptides referred to as "Protein Cluster V" which are predicted to be useful in the diagnosis of metabolic diseases, such as obesity and diabetes, as well as in the identification of agents useftil in the treatment of the said diseases.
BACKGROUND ART
Metabolic diseases are defined as any of the diseases or disorders that disrupt normal metabolism. They may arise from nutritional deficiencies; in comiection with diseases of the endocrine system, the liver, or the kidneys; or as a result of genetic defects.
Metabolic diseases are conditions caused by an abnormality in one or more of the chemical reactions essential to producing energy, to regenerating cellular constituents, or to eliminating unneeded products arising fiom these processes. Depending on which metabolic pathway is involved, a single defective chemical reaction may produce consequences that are narrow, involving a single body function, or broad, affecting many organs and systems.
One of the major hormones that influence metabolism is insulin, which is synthesized in the beta cells of the islets of Langerhans of the pancreas. Insulin primarily regulates the direction of metabolism, shifting many processes toward the storage of substrates and away from their degradation. Insulin acts to increase the transport of glucose and amino acids as well as 1<ey minerals such as potassium, magnesium, and phosphate from the blood into cells. It also regulates a variety of enzymatic reactions within the cells, all of which have a conunon overall direction, namely the synthesis of large molecules from small units. A deficiency in the action of insulin (diabetes mellitus) causes severe impairment in (i) the storage of glucose in the form of glycogen and the oxidation of glucose for energy; (ii) the synthesis and storage of fat from fatty acids and their precursors and the completion of fatty-acid oxidation; and (iii) the synthesis of proteins l.'com amino acids.
There are two varieties of diabetes. Type I is insulin-dependent diabetes mellitus (IDDM), for which insulin injection is required; it was formerly referred to as,juvenile onset diabetes. In this type, insulin is not secreted by the pancreas and hence must be taken by injection. Type II, non-insulin-dependent diabetes mellitus (NIDDM) may be controlled by dietary restriction. It derives ti~om insufficient pancreatic insulin secretion and tissue resistance to secreted insulin, which is complicated by subtle changes in the secretion of insulin by the beta cells. Despite their former classifications as juvenile or adult, either type can occur at any age; NIDDM, however, is the most common type, accounting for 90 percent of all diabetes. While the exact causes of diabetes remain obscure, it is evident that NIDDM is linked to heredity and obesity. There is clearly a genetic predisposition to NIDDM diabetes in those who become overweight or obese.
Obesity is usually defined in terms of the body mass index (BMI), i.e. weight (in kilograms) divided by the square of the height (il~ meters). Weight is regulated with great precision. Regulation of body weight is believed to occur not only in persons of normal weight but also among many obese persons, in whom obesity is attributed to an elevation in the set point around which weight is regulated. The determinants of obesity can be divided into genetic, environmental, and regulatory.
Recent discoveries have helped explain how genes may determine obesity and how they may influence the regulation of body weight. For example, mutations in the o%
gene have led to massive obesity in mice. Cloning the oh gene led to the identification of leptin, a protein coded by this gene; leptin is produced in adipose tissue cells and acts to control body fat. The existence of leptin supports the idea that body weight is regulated, because leptin serves as a signal between adipose tissue and the areas of the brain that control energy metabolism, which influences body weight.

Metabolic diseases like diabetes and obesity are clinically and genetically heterogeneous disorders. Recent advances in molecular genetics have led to the recognition of genes involved in IDDM and in some subtypes of NIDDM, 111Cllldlllg maturity-onset diabetes of the young (MODY) (Velho & Froguel (1997) Diabetes Metab. 23 Suppl 2:34-37). However, several IDDM susceptibility genes have not yet been identified, and very little is known about genes contributing to common forms of NIDDM. Studies of candidate genes and of genes mapped in animal models of IDDM
or NIDDM, as well as whole genome scaluung of diabetic families from different populations, should allow the identification of most diabetes susceptibility genes and of the molecular targets for new potential drugs. The identification of genes involved in metabolic disorders will thus contribute to the development of novel predictive and therapeutic approaches.
The (33-adrenergic receptor (AR) represents one of a number of potential anti-obesity drugs targets for which selective agonists have been developed. In rodents, (33-AR
mRNA is abundant in white adipose tissue (WA T) and brown adipose tissue (BAT). It has been demonstrated that mice lacking endogenous (33-adrenoreceptors have a slight increase in body fat, but otherwise appear normal (Susulic V.S., et al. (1995) J. Biol.
Chem. 270(49): 29483-29492). These mice are completely resistant to the specific (33-agonist CL-316,243, which has been shown to increase lipolysis, energy expenditure and affect insulin and leptin levels. When the (33-AR was ectopically expressed in white and brown adipose tissue or brown adipose tissue only, it was recently demonstrated that the anorectic and insulin secretagogue effects appeared to be mediated by white adipose tissue (Grujic D, et al. (1997) J Biol Chem. 272(28):17686-93). How these effects are mediated by (33-AR agonists remains poorly understood.
Lardizabal, K.D. et al. (J. Biol. Chem. 276: 38862-38869) and Cases, S. et al.
(:I. Biol.
Chem. 276: 38870-38876; both papers published 31 :Iuly 2001) disclose a new gene family, including members in fungi, plants and animals, which encode proteins corresponding to the "Cluster V" proteins according to the present invention.
The proteins were shown to have acyl CoA:diacylglycerol acyltransferase (DGAT; EC
2.3.1.20) function. The gene family is unrelated to the previously identified DGAT(l) family and was designated DGAT2. DGAT2 was shown to have high expression levels in liver and white adipose tissue, suggesting that it may play a signitica~it role in mammalian triglyceride metabolism.
DISCLOSURE OF THE INVENTION
According to the present invention, a family of genes and encoded homologous proteins (hereinafter referred to as "Protein Cluster V") has been identified.
Consequently, the present invention provides an isolated nucleic acid molecule selected from:
(a) nucleic acid molecules comprising a nucleotide sequence as shown in SEQ ID
NO:
3, 5, 7, 9, 11, 13, 15, 17, or 19.
(b) nucleic acid molecules comprising a nucleotide sequence capable of hybridizing, order stringent hybridization conditions, to a nucleotide sequence complementary to the polypeptide coding region of a nucleic acid molecule as defined in (a); and (c) nucleic acid molecules comprising a nucleic acid sequence which is degenerate as a result of the genetic code to a nucleotide seduence as defined in (a) or (b).
The nucleic acid molecules according to the present invention includes cDNA, chemically synthesized DNA, DNA isolated by PCR, genomic DNA, and combinations thereof.
RNA transcribed from DNA is also encompassed by the present invention.
The term "stringent hybridization conditions" is known in the art from standard protocols (e.g. Ausubel et al., supra) and could be understood as e.g.
hybridization to filter-bound DNA in 0.5 M NaHP04, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA
at +65°C, and washing in 0. IxSSC / 0.1 % SDS at +68°C.
In a preferred form of the invention, the said nucleic acid molecule has a nucleotide sequence identical with SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, or 19 of the Sequence Listing. However, the nucleic acid molecule according to the invention is not to be limited strictly to the sequence shown as SEQ ID NO: 3, 5, 7, 9, 1 1, 13, I5, 17, or 19.
Rather the invention encompasses nucleic acid molecules carrying modifications like substitutions, small deletions, insertions or inversions, which nevertheless encode proteins having substantially the features of the Protein Cluster V
pohypeptide according to the invention. Included in the invention are consequently nucleic acid molecules, the nucleotide sequence of which is at least 90% homologous, preferably at least 95%
homologous, with the nucleotide sequence shown as SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, or 19 in the Sequence Listing.
lncluded in the invention is also a nucleic acid molecule which nucleotide sequence is degenerate, because of the genetic code, to the nucleotide sequence shown as SEQ ID
NO: 3, 5, 7, 9, 11, 13, 15, 17, or 19. A sequential grouping of three nucleotides, a "codon", codes for one amino acid. Since there are 64 possible codons, but only 20 natural amino acids, most amino acids are coded for by more than one codon.
This natural "degeneracy", or "redundancy", of the genetic code is well known in the art. It will thus be appreciated that the nucleotide sequence shown in the Sequence Listing is only an example within a large but definite group of sequences which will encode the Protein Cluster V polypeptide.
The nucleic acid molecules according to the invention have numerous applications in techniques known to those skilled in the art of molecular biology. These techniques include their use as hybridization probes, for chromosome and gene mapping, in PCR
technologies, in the production of sense or antisense nucleic acids, in screening for new therapeutic molecules, etc.
More specifically, the sequence information provided by the invention makes possible large-scale expression of the encoded polypeptides by techniques well Known in the art.
Nucleic acid molecules of the invention also permit identification and isolation of nucleic acid molecules encoding related polypeptides, such as human allelic variants and species homologues, by well-known techniques including Southern and/or Northern hybridization, and PCR. Knowledge of the sequence of a human DNA also makes possible, through use of Southern hybridization or PCR, the identification of genomic DNA sequences encoding the proteins in Cluster V, expression control regulatory sequences such as promoters, operators, enhancers, repressors, and the like.
Nucleic -G-acid molecules of the invention are also useful in hybridization assays to detect the capacity of cells to express the proteins in Cluster V. Nucleic acid molecules of the invention may also provide a basis for diagnostic methods useful for identifying a genetic alterations) in a locus that underlies a disease state or states, which information is useful both for diagnosis and for selection of therapeutic strategies.
Tn a further aspect, the invention provides an isolated polypeptide encoded by the nucleic acid molecule as defined above. In a preferred form, the said polypeptide has an amino acid sequence according to SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18 or 20 of the Sequence Listing. However, the polypeptide according to the invention is not to be limited strictly to a polypeptide with an amino acid sequence identical with SEQ ID
NO: 4, 6, 8, 10, 12, 14, 16, 18 or 20 in the Sequence Listing. Rather the invention encompasses polypeptides carrying modifications like substitutions, small deletions, insertions or inversions, which polypeptides nevertheless have substantially the features of the Protein Cluster V polypeptide. Included in the invention are consequently polypeptides, the amino acid sequence of which is at least 90% homologous, preferably at least 95% homologous, with the amino acid sequence shown as SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18 or 20 in the Sequence Listing.
In a further aspect, the invention provides a vector harboring the nucleic acid molecule as defined above. The said vector can e.g. be a replicable expression vector, which carries and is capable of mediating the expression of a DNA molecule according to the invention. In the present context the term "replicable" means that the vector is able to replicate in a given type of host cell into which is has been introduced.
Examples of vectors are viruses such as bacteriophages, cosmids, plasmids and other recombination vectors. Nucleic acid molecules are inserted into vector genomes by methods well known in the art.
Included in the invention is also a cultured host cell harboring a vector according to the invention. Such a host cell can be a prokaryotic cell, a unicellular eulcaryotic cell or a cell derived from a multicellular organism. The host cell can thus e.g. be a bacterial cell such as an E. coli cell; a cell from yeast such as SuccharonZyce.s ceroi.viae or Pichi~~

pu,stori.s, or a mammalian cell. The methods employed to effect introduction of the vector into the host cell are standard methods well known to a person familiar with recombinant DNA methods.
In yet another aspect, the invention provides a process for production of a polypeptide, comprising culturing a host cell, according to the invention, under conditions whereby said polypeptide is produced, and recovering said polypeptide. The medium used to grow the cells may be any conventional medium suitable for the purpose. A
suitable vector may be any of the vectors described above, and an appropriate host cell may be any of the cell types listed above. The methods employed to construct the vector and effect introduction thereof into the host cell may be any methods known for such purpo-ses within the field of recombinant DNA. The recombinant polypeptide expressed by the cells may be secreted, i.e. exported tluough the cell membrane, dependent on the type of cell and the composition of the vector.
In a filrther aspect, the invention provides a method for identifying an agent capable of modulating a nucleic acid molecule according to the invention, comprising (i) providing a cell comprising the said nucleic acid molecule;
(ii) contacting said cell with a candidate agent; and (iii) monitoring said cell for an effect that is not present in the absence of said candidate agent.
For screening purposes, appropriate host cells can be transformed with a vector having a reporter gene under the control of the nucleic acid molecule according to this invention.
The expression of the reporter gene can be measured in the presence or absence o1' an agent with known activity (i.e. a standard agent) or putative activity (i.e. a "test agent"
or "candidate agent"). A change in the level of expression of the reporter gene in the presence of the test agent is compared with that effected by the standard agent. In this way, active agents are identified and their relative potency in this assay determined.
A transfection assay can be a particularly useful screening assay for identifying an effective agent. In a transfection assay, a nucleic acid contannng a gene such as a _g_ reporter gene that is operably linked to a nucleic acid molecule according to the invention, is transfected into the desired cell type. A test level of reporter gene expression is assayed in the presence of a candidate agent and compared to a control level of expression. An effective agent is identified as an agent that results in a test level of expression that is different than a control level of reporter gene expression, which is the level of expression determined in the absence of the agent. Methods l:or transfecting cells and a variety of convenient reporter genes are well known in the art (see, for example, Goeddel (ed.), Methods Enzymol., Vol. 185, San Diego: Academic Press, Inc.
(1990); see also Sambrook, sups°a).
Throughout this description the terms "standard protocols" and "standard procedures", when used in the context of molecular biology techniques, are to be understood as protocols and procedures found in an ordinary laboratory manual such as:
Current I?rotocols in Molecular Biology, editors F. Ausubel et al., :lohn Wiley and Sons, Inc.
1994, or Sambrook, J., Pritsch, E.F. and Maniatis, T., Molecular Cloning: A
laboratory manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
1989.
EXAMPLES
EXAMPLE 1: Identification of protein clusters A family of homologous proteins (hereinafter referred to as "frotein Cluster V") was identified by an "all-versus-all" BLAST procedure using all C'aenorhabdili.s elegan.s proteins in the Wormpep20 database release (http:llumo~..sanger.cac.ukLProjeccsl C=eleganslwormpeplindex.shtml). The Wormpep database contains the predicted proteins from the C. elegan.s genome sequencing project, carried out,jointly by the Sanger Centre in Cambridge, UK and the Genome Sequencing Center in St. Louis, USA. A number of 18,940 proteins were retrieved from Wormpep20. The proteins were used in a Smith-Waterman clustering procedure to group together proteins of similarity (Smith T.F. & Waterman M.S. (1981) Identification of common molec~.ilar .sZrh.seguences. J. Mot. Biol. 147(1 ): 195-197; Pearson WR. (1991 ) ,Searching protein ,sequence libraries: comparison of the .sen.s~itivily and selectiviy of the Smilh-Watermcrn and FASTA algorithms. Genomics 11: 635-650; Olsen et al. ( 1999) Optimizing ,Smuh-Walerman alignments. Pac Symp Biocomput.302-313). Completely annotated proteins were filtered out, whereby 10,130 proteins of uucnown function could be grouped into 1,800 clusters.
rChe obtained sequence clusters were compared to the Dr~osophilu melcmogaster proteins contained in the database Flybase (Berkeley Drosophila Genome Project;
http:llwww., fi~uitfly. org), and annotated clusters were removed. Non-amotated protein clusters, conserved in both C'. elegan.s and D. melanogaster, were saved to a worm/fly data set, which was used in a BLAST procedure (http:llww~~. nchi. nhn.
nih.govl EducationlBLASTinfolinformation3.hlml) against the Celera Human Genome Database (http:llu~~~w. celera. com). Overlapping fragments were assembled to, as close as possible, full-length proteins using the PHRAP software, developed at the University of Washington (http:llwww.genome. wu.shington. edul CWGClanaly,si.stool.slphrap.
htnZ). A
group of homologous proteins ("Protein Cluster V") with unknown function was chosen for further studies.
EST databases provided by the EMBL (hltp:ll~~wo~. emhl.
o~°glService,slindex. html) were used to check whether the human proteins in Cluster V were expressed, in order to identify putative pseudogenes. One putative pseudogene was identified and excluded.
EXAMPLE 2: Analyses of Protein Cluster V
(a) Alignment The human part of this protein family includes seven different 150-250 residue polypeptides shown as SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, I 8 and 20, encoded by the nucleic acid sequences shown as SEQ ID NO: l, 3, 5, 7, 9, 1 l, 13, 15, 17, alld 19. The amino acid sequence shown as SEQ ID NO: 2 was identified to correspond to a human 261 as sequence encoded by the gene "WUGSC: H DJ0747G18.5" (GenBank Accession No. AC004876). No fiunction has been associated with the said gene.
An alignment of the human polypeptides included in Protein Cluster V, using the ClustalW multiple alignment software (Thompson et al. (1994) Nucleic Acid Research 22: 4673-4680) is shown in Table I. The alignment showed a high degree of conservation over a 100 residues region in the protein (corresponding to positions 23-147 in SEQ ID NO: 2), indicating the presence of a novel domain.
(b) HMM PfanZ
A HMM-Pfam search was performed on the human family members. Pfam is a large collection of protein families and domains. Pfam contains multiple protein alignments and prof le-HMMs (Profile Hidden Markov Models) of these families. Profile-NMMs can be used to do sensitive database searching using statistical descriptions of a sequence family's consensus. Pfam is available on the WWW at hlth:lll?fanz.v~zz.sll.edu;
hltp:llwn~w.sanger.ac.uklSoftwaz°elPfam; and http:llwvu~.cgz°.ki..selPfam. The latest version (4.3) of Pfam contains 1815 families. These Pfam families match 63% of proteins in SWISS-PROT 37 and TrEMBL 9. For references to ffam, see Bateman et al.
(2000) The Pfam protein,familie.s dutabase. Nucleic Acids Res. 28:263-266;
SomZhammer et al. (1998) Pfam: Multiple Seguence Ali~lnment.v cznd HMM-Profile.v of I'rolcin Domains. Nucleic Acids Research, 26:322-325; Sonnhammer et al. (1997) Pfum: cz C'omprehensive Database ofProtein Domain Familie.s~ l3a.sed on Seed Alignments. Proteins 28: 405-420.
The HMM-Pfam search indicated that no previously known domains could be identified in I?rotein Cluster V.
(c) :>N1 HMM
The human proteins in Cluster V were analyzed using the TM-HMM tool available e.g.
at http: //www. cbs. dtu. dkls~erviceslTMHMM L 0. TM-HMM is a method to model and predict the location and orientation of alpha helices in membrane-spanning proteins (Som~hammer et al. (1998) A hidden Markov model, fur predicting lr~an.smem.hrcrne helices in protein seguences. ISMB 6:175-182). The results indicate that the human Cluster V proteins contain 3-4 transmembrane segments (d) Analysis of non-human orthologs T he Caenorhabditis elegan.s~ genome includes four genes, designated K07B 1.4 (GenBank Accession No. AF003384), F59A1.10 (GenBauc Accession No. 281557), Y53G8B.2 (GenBank Accession No. AC006804), and WOIAI 1.2 (GenBank Accession No. U64852) orthologous to the human Cluster V genes. The closest ancestor (K07B1.4) is on average 44% identical to the 10 human gene products. (See also:
G~rzome .seguence of~the nematode C'. elegan.s: a hlalfornz, for investigating biology; The C'. elegan.s Sequencing Consortium. Science (1998) 282:2012-2018. Published errata appear in Science (1999) 283:35; 283:2103; and 285:.1493.) The Dr°o.s~ophila melanogaster genome includes four genes orthologoLis to human Cluster V. The most closely related genes, designated "CG1942" (GenBank Accession No. AE003840 36) and gene: "CG1946" (GenBank Accession No. AL003840_37) are 39% identical to the human gene products. (See also Adams et al. (2000) The genome .seguence of Dro.sophila melanogaster; Science 287:2185-2195) is 42% identical to the human protein set.
The human proteins in Cluster V show 27% identity to two yeast proteins; S.
C'erevi.s~icre SCYOR245C_1 (GenBank Accession No. 275153) and S. pombe SfCC548_1 (GenBank Accession No. AL359685). The yeast proteins are of unknown function.

EXAMPLE 3: Expression analysis The tissue distribution of the 17L1111a11 genes was studied LlSlllg the Incyte LifeSeq't database (hltp:llwwm. incyte. conZ). The genes shown as SEQ ID NO: 1, 3, 5, 7, 9, 1 1, 13, 15, 17 were found to be expressed primarily in the following tissues:
SEQ ID NO: 1 and 3: Liver, digestive system SEQ ID NO: 7 and 9: Exocrine Glands, Comlective Tissue, Germ Cells SEQ ID NO: 11: Female genitalia, urinary tract SEQ ID NO: 17: Female genitalia, nervous system SEQ ID NO: 13 and 15: Digestive System SEQ ID NO: 5: Cardiovascular system Therefore, the said nucleic acid molecules and the encoded polypeptides shown are proposed to be useful for differential identification of the tissues or cell types present in a biological sample and for diagnosis of diseases and disorders related to the tissues where the genes are expressed.
EXAMPLE 4: Effect of (33-AR agonists on cluster V genes Microarrays consist of a highly ordered matrix of thousands of different DNA
sequences that can be used to measure DNA and RNA variation in applications that include gene expression profiling, comparative genomics and genotyping (For recent reviews, see e.g.: Harrington et al. (2000) Monitoring gene exhre.s.sion z~.s~ing DNA
I7ZlC1"oCIYYG'y.S. Curr. Opin. Microbiol. 3(3): 285-291; or Duggan et al.
(1999) Expre.s~.s~ion profiling using cDNA Microarrays. Nature Genetics Supplement 21:1.0-14).
In order to investigate the mechanisms whereby (33-AR agonists affect gene regulation in adipose tissue in vivo, a study was carried out using Alfymctrix GeneChip oligonucleotide arrays by comparing the transcript profiles of a large number oh genes in white adipose tissue derived from C57BL/6J mice treated with the (33-AR
agonist CL-316, 243, or from control mice injected with a saline solution.

PolyA+mRNAs were extracted from white adipose tissue from control and (33-AR
agonist treated mice respectively. They were reverse transcribed using a T7-tagged oligo-dT primer and double-stranded cDNAs were generated. These cDNAs were then amplified and labeled using In Vitro Transcription (1V1) with T7 RNA
polymerise and biotinylated nucleotides. The populations of cRNAs obtained after IV~T were purified and fiagmented by heat to produce a distribution of RNA fragment sizes from approximately 3 5 to 200 bases. Two Affymetrix Mu 19K and Mu 11 K sets oi~ 3 arrays (subA, subB and subC) and 2 arrays (subA and subB) respectively, were hybridized (using the recommended buffer) overnight at 45°C with the control or the treated denatured samples. The arrays were then washed and stained with R-phycoerythrin streptavidin with the help of an Affymetrix fluidics station. The cartridges were scanned using a 1-Iewlett-Packard confocal scanner and the images were analyzed with the GeneChip 3.1 software (Affymetrix).
The results indicate that the mouse gene (GenBank accession No. AA275948), orthologous to the worm gene I~59A1.10, is down-regulated by (33-AR agonist treatment. It is hypothesized that the human genes in Cluster V are similarly involved in metabolically important signaling pathways.
EXAMPLE 5: Multiple Tissue Northern blotting Multiple Tissue Northern blotting (MTN) is performed to make a more thorough analysis of the expression profiles of tine proteins in Cluster V. Multiple Tissue Northern (MTNTM) Blots (hltp:llnwww.clontecGa.comlnzln) are pre-made Northern blots featuring Premium Poly A+ RNA from a variety of different human, mouse, or rat tissues. MTN Blots can be used to analyze size and relative abundance of transcripts in different tissues. MTN Blots can also be used to investigate gene families and alternate splice forms and to assess cross species homology.

EXAMPLE 6: Identification of polypeptides binding to Protein Cluster V
In order to assay for proteins interacting with Protein Cluster V, the two-hybrid screening method can be used. T he two-hybrid method, first described by h fields &
Song (1989) Nature 340:245-247, is a yeast-based genetic assay to detect protein-protein interactions in vivo. The method enables not only identification of interacting proteins, but also results in the immediate availability of the cloned genes for these proteins.
The two-hybrid method can be used to determine il~two Known proteins (i.e.
proteins for which the corresponding genes have been previously cloned) interact.
Another important application of the two-hybrid method is to identify previously unknown proteins that interact with a target protein by screening a two-hybrid library. For reviews, see e.g.: Chien et al. (1991) The two-hybrid system: a method to identify and clone genes for proteins Chat interact with a p~°otein of interest.
Proc. Natl. Acad. Sci.
U.S.A. 88:9578-9582; Bartel PL, Fields (1995) Anulyzingprotean protein intercrctzons using two-hybrid system. Methods Enzymol. 254:241-263; or Wallach et al. ( 1998) The .yeast two-hybrid screening technigue and ilsw.re in the .study of protein protein interactions in apoptosi.r. Curr. Opin. Immunol. 10(2): 131-136. See also http: //www. clontech. comlnZatchmaker.
The two-hybrid method uses the restoration of transcriptional activation to indicate the interaction between two proteins. Central to this technique is the fact that many cukaryotic transcriptional activators consist of two physically discrete modular domains: the DNA-binding domain (DNA-BD) that binds to a specific promoter sequence and the activation domain (AD) that directs the RNA polymerase 11 complex to transcribe the gene downstream of the DNA binding site. The DNA-BD vector is used to generate a fusion of the DNA-BD and a bait protein X, and the AD
vector is used to generate a fusion of the AD and another protein Y. An entire library of hybrids with the AD can also be constructed to search for new or unknown proteins that interact with the bait protein. When interaction occurs between the bait protein X and a candidate protein Y, the two functional domains, responsible for DNA binding and activation, are tethered, resulting in functional restoration of transcriptional activation.
The two hybrids are cotransformed 111t0 a yeast host strain harboring reporter genes containing appropriate upstream binding sites; expression of the reporter genes then indicates interaction between a candidate protein and the target protein.
EXAMPLE 7: Full-length cloning of Cluster V genes The polymerase chain reaction (PCR), which is a well-lalown procedure for in vitro enzymatic amplification of a specific DNA segment, can be used for direct cloning of Protein Cluster V genes. Tissue cDNA can be amplified by PCR and cloned into an appropriate plasmid and sequenced. For reviews, see e.g. Hooft van Huijsduijnen (1998) PCR-assisted cDNA cloning.' a guided lour of~lhe minefield. Biotechnidues 24:390-392;
Lenstra (1995) The applications of the polyn~.ena.se chain reaction in the life .science.s.
Cellular & Molecular Biology 41:603-G14; or Rashtchian (1995) Novel n2ethods.fo~°
cloning and engineering genes using the polynZera.se chain i°eaction.
Current Opinion in Biotechnology 6:30-36. Various methods for generating suitable ends to facilitate the direct cloning of PCR products are given e.g. in Ausubel et al. .supra (section 15.7).
In an alternative approach to isolate a cDNA clone encoding a filll length protein of Protein Cluster V, a DNA fragment corresponding to a nucleotide sequence selected from the group consisting of SEQ ID NO: l, 3, 5, 7, 9, 1 l, 13, 15, 17 or 19, or a portion thereof, can be used as a probe for hybridization screening of a phage cDNA
library.
The DNA fragment is amplified by the polymerase chain reaction (PCR) method.
The primers are preferably 10 to 25 nucleotides in length and are determined by procedures well known to those skilled in the art. A lambda phage library containing cDNAs cloned into lambda phage-vectors is plated on agar plates with E. coli host cells, and grown.
1?hage plaques are transferred to nylon membranes, which are hybridized with a DNA
probe prepared as described above. Positive colonies are isolated from the plates.
flasmids containing cDNA are rescued from the isolated phages by standard methods.
Plasmid DNA is isolated from the clones. The size of the insert is determined by digesting the plasmid with appropriate restriction enzymes. The sequence of the entire insert is determined by automated sequencing of the plasmids.
EXAMPLE 8: Recombinant expression of proteins in eulcaryotic host cells To produce proteins of Cluster V, a polypeptide-encoding nucleic acid molecule is expressed in a suitable host cell using a suitable expression vector and standard genetic engineering techniques. For example, the polypeptide-encoding sequence is subcloned into a commercial expression vector and transfected into mammalian, e.g.
Chinese Hamster Ovary (CHO), cells using a standard transfection reagent. Cells stably expressing a protein are selected. Optionally, the protein may be purified lion the cells using standard chromatographic techniques. To facilitate purification, antisera is raised against one or more synthetic peptide sequences that correspond to portions of the amino acid sequence, and the antisera is used to affinity purify the protein.
CXAMPLE 9: Determination of gene function Methods are lmown in the art for elucidating the biological function or mode o.f action of individual genes. For instance, RNA interference (RNAi) offers a way of specifically and potently inactivating a cloned gene, and is proving a powerful tool for investigating gene function. For reviews, see e.g. Fire (1999) RNA-triggered gene silencing.
Trends in Genetics 15:358-363; or Kuwabara & Coulson (2000) RNAi ~~ro.s~~ect.r.
fo~° a genenul lechnigue,for determininggene,function. Parasitology Today 16:347-349. When double-stranded RNA (dsRNA) corresponding to a sense and antisense sequence of an endogenous mRNA is introduced into a cell, the cognate mRNA is degraded and the gene is silenced. This type ofposttranscriptional gene silencing (I'TGS) was first discovered in C. elegan.s (Fire et al., (1998) Nature 391:806-81 1 ). RNA
interference has recently been used for targeting nearly 90% of predicted genes on C'. elegan.s chromosome I (Fraser et al. (2000) Nature 408: 325-330) and 96% of predicted genes on C'. elegans chromosome III (Gonczy et al. (2000) Nature 408:331-336).

TAB Ll:, I
Alignment of.polypeptides in Protein Cluster V
SEQ 2 ____________________________________________________________ ~F 4 ____________________________________________________________ ~' .. Q
SEQ 8 ____________________________________________________________ ~'EQ_10 ____________________________________________________________ SEQ_12 _______________________________-__________________-_____-___ 14 ____________________________________________________________ SEQ_ SEQ_20 MVNGKSITSLQSNKNLAAIHGPKYLCGNFGPRWQAFSLGTKLDPMEVFPKLLPSKVPVAQ 60 SEQ 16 ____________________________________________________________ SEQ 18 ____________________________________________________________ SEQ 6 ____________________________________________________________ SEQ_2 ____________________________________________________________ SEQ 4 ____________________________________________________________ SEQ_8 ____________________________________________________________ SEQ_10 ____________________________________________________________ SEQ_12 _______________________________-____________________-_______ SEQ_14 ____________________________________-____-______________-___ SEQ_20 TLAPYSAPCFQRLWWSAAKVKAPSHNAKQGPKMDGQLVKTHDLSPKHNYIIANHPHGILS 120 SEQ_16 _____________________________________________________RPGGSEG 7 SEQ 18 ________________________________-_____________-_____________ r~E~ ~ ____________________________-___--__________________________ pEQ2 -------EAPLFSRCLAFHPPFILLNTPKLVKTAELPPDRNYVLGAHPHGIMCTGFLCNF53 _ 4 LGTLLGWRAPLFSRCLAFHPPFILLNTPKLVKTAELPPDRNYVLGAI-IPHGIMCTGFLCNF60 SEQ

SEQ_10 _________________________________________-__________________ SEQ12 ____________________________________-_______________________ _ 7.9 _________________________________________________________NLF3 SEQ

'_~EQ20 FGVFINFATEATGIARIFPSITPFVGTLERIFWIPIVREYVMSMGVCPVSSSALKYLLTQ180 _ 16 RFPKVTPVSGRVRAGTQAPPWLSRLPSLQLVKTAELDPSRNYIAGFHPHGVLAVGAFANL67 SEQ

_ 18 -----------------w w SDYVPLKLLKTHDICPSRNYILVCHPHGLFAHGWFGHF38 SEQ

_ 6 ---------------------'----CSEIFASLRLPR---IMAHSKQPSHFQSLMLLQW31 SEQ -SEQ_ STESNGFSQLFPGLRPWLAVLAG-----LFYLPVYRDYIMSFGASLVPVYSFGENDIFRL115 SEQ_ NGSGNAIIIVVGGAAESLSSMPGKNAVTLRNRKGFVKLALRHGADLVPIYSFGENEVYKQ118 ~EQ_ -----------------------------RNRKGFVKLALRHGADLVPIYSFGENEVYKQ31 SEQ_ --------------KESLDAHPGKFTLFIRQRKGFVKIALTHGASLVPVVSFGENELFKQ46 SEQ_ EAHKLKFNIIVGGAQEALDARPGSFTLLLRNRKGFVRLALTHGAPLVXIFSFGENDLFDQ63 SEQ_ KGSGNAVVIVVGGAAEALLCRPGASTLFLKQRKGFVKMALQTGAYLVPSYSFGENEVFNQ240 SEQ_ CTESTGFSSIFPGIRPHLMMLTL-----WFRAPFFRDYIMSAGLVTSEKESAAHILNRKG122 ,'~EQ_ PLSYLAIFWILQPLFVYLLFTSLWPLPVLYFAWLFLDWKTPERGGRRSAWVRNWCVWTHI91 _18_ r~'ABLIJ I (continued) SEQ_2 SQPQLG-------QAVVI----MVGGAEALYSVPGEHCLTLQKRKGFVRLALRHGASLVP153 _ 10 VIFEEGSWGRWVQKKFQ----KYIGFAPCIFHGRGLFSSDTWGLVPYSKPITTVGGGKIQ87 SEQ_ SEQ_12 TDNPEGSWIRTVQNKLQ----KIMGFALPLFHARGVFQYN-FGLMTYRKAIHTVVGRPIP101 SEQ_14 IPNSSGSWLRYIQNRLQ----KIMG-----------------------------------84 SEQ_20 ETFPEGTWLRLFQKTFQDTFKKILGLNFCTFHGRG-FTRGSWGFLPFNRPITTVVGEPLP299 SEQ_16 GGNLLGIIVG--------------GAQEALDARPGSFTLLLRNRKGFVRLALTHG-----163 SEQ_18 TGNMVIVVIG--------------GLAECRYSLPGSSTLVLKNRSGFVRMALQHGVPLIP139 SEQ_2 VYS---FGENDIFRLKAFATGSWQHWCQLTFKKL-MGFSPCIFWVAV 196 SEQ_4 VPQRLHPTEEEVNHYHALYMTDLEQLFEEHKESCGVPASTCLTFI-- 216 _ 10 S----RSKKRKINXX-------------QNDSCYSL----------- 106 SEQ

_ 12 VRQTLNPTQEQIEELHQTYMEELRKLFEEHKGKYGIPEHETLVLK-- 146 SEQ_ SEQ_14 _______________________________________________ SEQ_20 IPRIKRPNQKTVDKYHALYISALRKLFDQHKVEYGLPETQELTIT-- 394 ='EQ16 _______________________________________________ SEQ_18 AYAFGETDL--____________________________________ 198 SEQ6 _______________________________________________ SEQUENCE LISTING
<110> Pharmacia AB
<7.20> Protein Cluster V
<130> 00907 <160> 20 <7.70> PatentIn version 3.0 <210> 1 <211> 593 <212> DNA
<213> human <220>
<221> CDS
<222> (3)..(593) <400> 1 tg gag gCC CCt Ctt ttC agC Cgg tgt Ctt gCC ttC Cdt CCt CCC ttC 47 Glu Ala Pro Leu Phe Ser Arg Cys Leu Ala Phe His Pro Pro Phe atc ctg ctc aac acc ccg aag ctg gtg aaa aca gca gag ctg CCC ccg 95 Ile Leu Leu Asn Thr Pro Lys Leu Val. Lys Thr Ala Glu Leu Pro Pro gatcggaactac gtgctgggc gcccaccct catgggatc atgtgtaca 143 AspArgAsnTyr ValLeuGly AlaHisPro HisGlyIle MetCysThr ggcttcctctgt aatttctcc accgagagc catggcttc tcccagctc 191 GlyPheLeuCys AsnPheSer ThrGluSer HisGlyPhe SerGlnLeu ttcccggggete cggccctgg ttatccgtg ctggetgge etettetac 239 PheProGlyLeu ArgProTrp LeuSerVal LeuAlaGly LeuPheTyr ctcccggtctat cgcgactac atcatgtcc tttggactc tgtccggtg 287 LeuProValTyr ArgAspTyr IleMetSer PheGlyLeu CysProVal agccgccagagc ctggacttc atcctgtcc cagccccag ctcgggcag 335 SerArgGlnSer LeuAspPhe IleLeuSer GlnProGln LeuGlyGln gccgtggtcatc atggtgggg ggtgcgcac gaggccctg tattcagtc 383 AlaValValIle MetValGly GlyAlaHis GluAlaLeu TyrSerVal cccggggagcac tgccttacg ctccagaag cgcaaaggc ttcgtgcgc 431 ProGlyGluHis CysLeuThr LeuGlnLys ArgLysGly PheValArg ctggcg ctgaggcac ggggcgtcc ctggtgccc gtgtactcc tttggg 479 LeuAla LeuArgHis GlyAlaSer LeuValPro ValTyrSer PheGly gagaat gacatcttt agacttaag gettttgcc acaggctcc tggcag 527 GluAsn AspIlePhe ArgLeuLys AlaPheAla ThrGlySer TrpGln cattgg tgccagctc accttcaag aagctcatg ggcttCtCt ccttgc 575 HisTrp CysGlnLeu ThrPheLys LysLeuMet GlyPheSer ProCys atcttc tgggtcgcg gtc 593 IlePhe TrpValAla Val <210> 2 <211> 197 <212> PRT

<213> human <400> 2 GluAlaPro LeuPhe SerArgCys LeuAlaPhe HisProPro PheTle 7. 5 1.0 15 LeuLeuAsn ThrPro LysLeuVal LysThrAla GluLeuPro ProAsp ArgAsnTyr ValLeu GlyAlaHis ProHisGly IleMetCys ThrGly PheLeuCys AsnPhe SerThrGlu SerHisGly PheSerGln LeuPhe 5p 55 60 ProGlyLeu ArgPro TrpLeuSer ValLeuAla GlyLeuPhe TyrLeu ProValTyr ArgAsp TyrIleMet SerPheGly LeuCysPro ValSer ArgGlnSer LeuAsp PheIleLeu SerGlnPro GlnLeuGly GlnAla ValValIle MetVal GlyGlyAla HisGluAla LeuTyrSer ValPro G'lyGluHis CysLeu ThrLeuGln LysArgLys GlyPheVal ArgLeu AlaLeuArg HisGly AlaSerLeu ValProVal TyrSerPhe GlyGlu AsnAspIle PheArg LeuLysAla PheAlaThr GlySerTrp GlnHis TrpCysGln LeuThr PheLysLys LeuMetGly PheSerPro CysIle -J_ Phe Trp Val Ala Val <210> 3 <211> 822 <212> DNA
<213> human <220>
<221> CDS
<222> (93)..(740) <400> 3 aaaaaaaaac ctgggccctt aaccctatcc taagaacctt taactcggaa ctctgctggg 60 gtggcccttg accctatcct aagaaccttt as ctc gga act ctg ttg ggg tgg 113 Leu Gly Thr Leu Leu Gly Trp agggcccctctt ttcagccgg tgtcttgcc ttccatcct cccttcatc 161 ArgAlaProLeu PheSerArg CysLeuAla PheHisPro ProPheIle ctgctcaacacc ccgaagctg gtgaaaaca gcagagctg cccccggat 209 LeuLeuAsnThr ProLysLeu ValLysThr AlaGluLeu ProProAsp cggaactacgtg ctgggcgcc caccctcat gggatcatg tgtacaggc 257 ArgAsnTyrVal LeuGlyAla HisProHis GlyIleMet CysThrGly ttcctctgtaat ttctccacc gagagcaat ggcttctcc cagctcttc 305 PheLeuCysAsn PheSerThr GluSerAsn GlyPheSer GlnLeuPhe ccggggctcegg ccctggtta gccgtgctg getggecte ttctacctc 353 ProGlyLeuArg ProTrpLeu AlaValLeu AlaGlyLeu PheTyrLeu ccggtctatcgc gactacatc atgtccttt ggggcgtcc ctggtgccc 901 ProValTyrArg AspTyrIle MetSerPhe GlyAlaSer LeuValPro gtgtactccttt ggggagaat gacatcttt agacttaag gettttgcc 449 ValTyrSerPhe GlyGluAsn AspIlePhe ArgLeuLys AlaPheAla acaggctcctgg cagcattgg tgccagctc accttcaag aagctcatg 497 ThrGlySerTrp GlnHisTrp CysGlnLeu ThrPheLys LysLeuMet ggcttctctcct tgcatcttc tggggtcgc ggtctcttc tcagccacc 545 GlyPheSerPro CysIlePhe TrpGlyArg GlyLeuPhe SerAlaThr tcctggggcctg ctgcccttt getgtgccc atcaccact gtggtgggc 593 SerTrpGlyLeu LeuProPhe AlaValPro IleThrThr ValValGly cgcccc atccccgtc ccccagcgc ctccacccc accgaggag gaagtc 641 ArgPro IleProVal ProGlnArg LeuHisPro ThrGluGlu GluVal aatcac tatcacgcc ctctacatg acggacctg gagcagctc ttcgag 689 AsnHis TyrHisAla LeuTyrMet ThrAspLeu GluGlnLeu PheGlu gagcac aaggaaagc tgtggggtc cccgettcc acctgcctc accttc 737 GluHis LysGluSer CysGlyVal ProAlaSer ThrCysLeu ThrPhe atctaggcctggc cgcggccttt ggcactgaga 790 cgctgagccc ctgagcccaa Ile cctccaccca ctgtggactc catgcctcca at 822 <210>

<211> 16 <212> RT
P

<213> uman h <400>

LeuGly ThrLeu LeuGlyTrpArg AlaProLeu PheSerArg CysLeu AlaPhe HisPro ProPheIleLeu LeuAsnThr ProLysLeu ValLys ThrAla GluLeu ProProAspArg AsnTyrVal LeuGlyAla HisPro HisGly IleMet CysThrGlyPhe LeuCysAsn PheSerThr GluSer AsnGly PheSer GlnLeuPhePro GlyLeuArg ProTrpLeu AlaVal LeuAla GlyLeu PheTyrLeuPro ValTyrArg AspTyrIle MetSer PheGly AlaSer LeuValProVal TyrSerPhe GlyGluAsn AspIle PheArg LeuLys AlaPheAlaThr GlySerTrp GlnHisTrp CysGln LeuThr PheLys LysLeuMetGly PheSerPro CysIlePhe TrpGly ArgGly LeuPhe SerAlaThrSer TrpGlyLeu LeuProPhe AlaVal ProIle ThrThr ValValGlyArg ProIlePro ValProGln ArgLeu HisPro ThrGlu GluGluValAsn HisTyrHis AlaLeuTyr MetThr Asp Leu Glu Gln Leu Phe Glu Glu His Lys Glu Ser Cys Gly Val Pro Ala Ser Thr Cys Leu Thr Phe Ile <210> 5 <211> 392 <212> DNA
<213> human <220>

<221>CDS

<222>(3)..(392) <400>5 ac tct atcttt tccctc aggctc agaatcatg 47 tgt gag gcc ccg get Cys Ser IlePhe SerLeu ArgLeuPro IleMet Glu Ala Arg Ala cat aag cagcctagt cacttccag agtctgatg cttctgcag tgg 95 tcc His Lys GlnProSer HisPheGln SerLeuMet LeuLeuGln Trp Ser cct agc taccttgcc atcttttgg atcttgcag ccattgttc gtc 143 ttg Pro Ser TyrLeuAla IlePheTrp IleLeuGln ProLeuPhe Val Leu tac ctg tttacatcc ttgtggccg ctaccagtg ctttacttt gcc 191 ctg Tyr Leu PheThrSer LeuTrpPro LeuProVal LeuTyrPhe Ala Leu tgg ttc ctggactgg aagacccca gagcgaggt ggcaggcgt tcg 239 ttg Trp Phe LeuAspTrp LysThrPro GluArgGly GlyArgArg Ser Leu gcc gta aggaactgg tgtgtctgg acccacatc agggactat ttc 287 tgg Ala Val ArgAsnTrp CysValTrp ThrHisIle ArgAspTyr Phe Trp ccc acg atcctgaag acaaaggac ctatcacct gagcacaac tac 335 att Pro Thr IleLeuLys ThrLysAsp LeuSerPro GluHisAsn Tyr Ile ctc ggg gttcacccc atgggcctc ctgaccttt ggcgccttc tgc 383 atg Leu Gly ValHisPro MetGlyLeu LeuThrPhe GlyAlaPhe Cys Met aac tgc 392 ttc Asn Cys Phe <210>6 <211>130 <212>PRT

<213>human -G-<400> 6 Cys Ser Glu Ile Phe Ala Ser Leu Arg Leu Pro Arg Ile Met Ala His Ser Lys Gln Pro Ser His Phe Gln Ser Leu Met Leu Leu Gln Trp Pro Leu Ser Tyr Leu Ala Ile Phe Trp Ile Leu Gln Pro Leu Phe Val Tyr Leu Leu Phe Thr Ser Leu Trp Pro Leu Pro Val Leu Tyr Phe Ala Trp Leu Phe Leu Asp Trp Lys Thr Pro Glu Arg Gly Gly Arg Arg Ser Ala Trp Val Arg Asn Trp Cys Val Trp Thr His Ile Arg Asp Tyr Phe Pro g5 90 95 Ile Thr Ile Leu Lys Thr Lys Asp Leu Ser Pro Glu His Asn Tyr Leu Met Gly Val His Pro Met Gly Leu Leu Thr Phe Gly Ala Phe Cys Asn Phe Cys <210> 7 <211> 2519 <212> DNA
<213> human <220>
<221> CDS
<222> (714)..(1373) <400> 7 gccgcctctg ctggggtcta ggctgtttct ctcgcgccac cactggccgc cggccgcagc 60 tccaggtgtc ctagccgccc agcctcgacg ccgtcccggg acccctgtgc tctgcgcgaa 120 gccctggccc cgggggccgg ggcatgggcc aggggcgcgg ggtgaagcgg cttcccgcgg 180 ggccgtgact gggcgggctt cagccatgaa gaccctcata gccgcctact ccggggtcct 240 gcgcggcgagcgtcaggccgaggctgaccggagccagcgctctcacggaggacctgcgct300 gtcgcgcgaggggtctgggagatggggcactggatccagcatcctctccgccctccagga360 cctcttctctgtcacctggctcaataggtccaaggtggaaaagcagctacaggtcatctc420 agtgctccagtgggtcctgtccttccttgtactgggagtggcctgcagtgccatcctcat480 gtacatattctgcactgattgctggctcatcgctgtgctctacttcacttggctggtgtt540 tgactggaacacacccaagaaaggtggcaggaggtcacagtgggtccgaaactgggctgt600 gtggcgctactttcgagactactttcccatccagctggtgaagacacacaacctgctgac660 caccaggaac ecatggtatc atgggcctgg getgce tatatetttg gataccacec Ala ttctgcaacttc agcacagag gccacagaagtg agcaag aagttccca 764 PheCysAsnPhe SerThrGlu AlaThrGluVal SerLys LysPhePro ggcatacggcct tacctgget acactggcaggc aacttc cgaatgcct 812 GlyIleArgPro TyrLeuAla ThrLeuAlaGly AsnPhe ArgMetPro gt ttgagggag tacctgatg tctggaggtatc tgccct gtcagccgg 860 g ValLeuArgGlu TyrLeuMet SerGlyGlyIle CysPro ValSerArg gacaccatagac tatttgett tcaaagaatggg agtggc aatgetatc 908 Asp'I'hrIleAsp TyrLeuLeu SerLysAsnGly SerGly AsnAlaIle atcatcgtggtc gggggtgcg getgagtctctg agctcc atgcetggc 956 IleIleValVal GlyGlyAla AlaGluSerLeu SerSer MetProGly aagaatgcagtc accctgcgg aaccgcaagggc tttgtg aaactggcc 1004 LysAsnAlaVal ThrLeuArg AsnArgLysGly PheVal LysLeuAla ctgcgtcatgga getgacctg gttcccatctac tccttt ggagagaat 1052 LeuArgHisGly AlaAspLeu ValProIleTyr SerPhe GlyGluAsn gaagtgtacaag caggtgatc ttcgaggagggc tcctgg ggccgatgg 1100 GluValTyrLys GlnValIle PheGluGluGly SerTrp GlyArgTrp 115 120 7.25 gtccagaagaag ttccagaaa tacattggtttc gcccca tgcatcttc 1148 ValGlnLysLys PheGlnLys TyrIleGlyPhe AlaPro CysIlePhe catggtcgaggc ctcttctcc tccgacacctgg gggctg gtgccctac 1196 HisGlyArgGly LeuPheSer SerAspThrTrp GlyLeu ValProTyr tccaagcccatc accactgtt gtgggagagccc atcacc atccccaag 1244 SerLysProIle ThrThrVal ValGlyGluPro IleThr IleProLys ctggagcaccca acccagcaa gacatcgacctg taccac accatgtac 1292 LeuGluHisPro ThrGlnGln AspIleAspLeu TyrHis ThrMetTyr atggaggccctg gtgaagctc ttcgacaagcac aagacc aagttcggc 1340 MetGluAlaLeu ValLysLeu PheAspLysHis LysThr LysPheGly ctcccggagact gaggtcctg gaggtgaactga gccagccttc ggggccaatt1393 LeuProGluThr GluValLeu GluValAsn _g_ ccctggaggaaccagctgcaaatcacttttttgctctgtaaatttggaagtgtcatgggt1453 gtctgtgggttatttaaaagaaattataacaattttgctaaaccattacaatgttaggtc1513 ttttttaagaaggaaaaagtcagtatttcaagttctttcacttccagcttgccctgttct1573 aggtggtggctaaatctgggcctaatctgggtggctcagctaacctctcttcttcccttc1633 ca gaagtgacaaaggaaactcagtcttcttggggaagaaggattgccattagtgacttgg1693 accagttagatgattcactttttgcccctagggatgagaggcgaaagccacttctcatac1753 aagcccctttattgccactaccccacgctcgtctagtcctgaaactgcaggaccagtttc1813 t ctgccaaggggaggagttggagagcacagttgccccgttgtgtgagggcagtagtaggc1873 atctggaatgctccagtttgatctcccttctgccacccctacctcacccctagtcactca1933 tatcggagcctggactggcctccaggatgaggatgggggtggcaatgacaccctgcaggg1993 gaaaggactgccccccatgcaccattgcagggaggatgccgccaccatgagctaggtgga2053 gtaactggtttttcttgggtggctgatgacatggatgcagcacagactcagccttggcct2113 ggagcacatgcttactggtggcctcagtttaccttccccagatcctagattctggatgtg2173 aggaagagatccctcttcagaaggggcctggccttctgagcagcagattagttccaaagc2233 aggtggcccccgaacccaagcctcacttttctgtgccttcctgagggggttgggccgggg2293 aggaaacccaaccctctcctgtgtgttctgttatctcttgatgagatcattgcaccatgt2353 cagacttttgtatatgccttgaaaataaatgaaagtgagacatggtgcaatgatctcatc2413 aagagataacagaacagacaggagagggttgggttatctcttgatgagatcattgcacca2473 tgtcagacttttgtatatgccttgaaaataaatgaaagtgagaatc 2519 <210> 8 <211> 219 <212> PRT
<213> human <400> 8 Ala Phe Cys Asn Phe Ser Thr Glu Ala Thr Glu Val Ser Lys Lys Phe Pro Gly Ile Arg Pro Tyr Leu Ala Thr Leu Ala Gly Asn Phe Arg Met Pro Val Leu Arg Glu Tyr Leu Met Ser Gly Gly Ile Cys Pro Val Ser Rrg Asp Thr Ile Asp Tyr Leu Leu Ser Lys Asn Gly Ser Gly Asn Ala Ile Ile Ile Val Val Gly Gly Ala Ala Glu Ser Leu Ser Ser Met Pro Gly Lys Asn Ala Val Thr Leu Arg Asn Arg Lys Gly Phe Val Lys Leu Ala Leu Arg His Gly Ala Asp Leu Val Pro Ile Tyr Ser Phe Gly Glu Asn Glu Val Tyr Lys Gln Val Ile Phe Glu Glu Gly Ser Trp Gly Arg 'rrp Val Gln Lys Lys Phe Gln Lys Tyr Ile Gly Phe Ala Pro Cys Ile Phe His Gly Arg Gly Leu Phe Ser Ser Asp Thr Trp Gly Leu Val Pro Tyr Ser Lys Pro Ile Thr Thr Val Val Gly Glu Pro Ile Thr Ile Pro Lys Leu Glu His Pro Thr Gln Gln Asp Ile Asp Leu Tyr His Thr Met Tyr Met Glu Ala Leu Val Lys Leu Phe Asp Lys His Lys Thr Lys Phe Gly Leu Pro Glu Thr Glu Val Leu Glu Val Asn <210> 9 <211> 685 <212> DNA
<213> human <220>
<221> misc_feature <222> ( ) . . ( ) <223> n = A, C. G or T
<220>
<221> CDS
<222> (2)..(322) <400> 9 g 49 cgg aac cgc aag gge ttt gtg aaa etg gcc ctg cgt cat gga get gac Arg e a s y Ala Asn Val Leu Gl Asp Arg Lys Arg Lys Leu Hi Gly Al Ph ctggtt cccatctac tcctttgga gagaat gaagtgtac aagcag gtg 97 LeuVal ProIleTyr SerPheGly GluAsn GluValTyr LysGln Val atcttc gaggagggc tcctggggc cgatgg gtccagaag aagttc cag 145 IlePhe GluGluGly SerTrpGly ArgTrp ValGlnLys LysPhe Gln aaatac attggtttc gccccatgc atcttc catggtcga ggcctc ttc 193 LysTyr IleGlyPhe AlaProCys IlePhe HisGlyArg GlyLeu Phe tcc tcc acc tgg ggg ctg ccc tac aag ccc acc act 241 gac gtg tcc atc Ser Ser Thr Trp Gly Leu Pro Tyr Lys Pro Thr Thr Asp Val Ser Ile gtt ggt gga aaa att cag agg agt aaa agg atc aac 289 ggt tct aaa aag Val Gly Gly Lys Ile Gln Arg Ser Lys Arg Ile Asn Gly Ser Lys Lys atn ntg aat gac tca tgc tca tta aagcaattgctggagatgnt342 cag tat tag Xaa Xaa Asn Asp Ser Cys Ser Leu Gln Tyr atcattgtggatcacggaag tcttcatggaagaggtggcatttgagctgggccttcactg402 aagcggtgaatcggcgtcct gggtgcctggcacaccttgtagctcagcttactagctagt462 ggagtgcgaaggggcgtgta cttgtcggttggagctggtcatgaaagagctcgtgggact522 gcccgacggttctcaggtcc cagtgcatcctgcgtggtggctctctgctgaaccataaag582 cattccttttcaatccctgc acgctcacgccgggaaaagactgcacaaggggctccaagg642 cagacaagcgatcgccaccc agctggcttccgagggtccccgc 685 <210> 10 <211> 106 <212> PRT

<213> human <220>

<221> misc_feature <222> ( ( ) ) .
.

<223> n G
= or A, T
C.

<900> 10 llrg Arg Lys GlyPheValLys LeuAlaLeu ArgHisGly AlaAsp Asn Leu Pro Ile TyrSerPheGly GluAsnGlu ValTyrLys GlnVal Val Ile Glu Glu GlySerTrpGly ArgTrpVal GlnLysLys PheGln Phe Lys Ile Gly PheAlaProCys IlePheHis GlyArgGly LeuPhe Tyr Ser Asp Thr TrpGlyLeuVal ProTyrSer LysProIle ThrThr Ser Val Gly Gly LysIleGlnSer ArgSerLys LysArgLys IleAsn Gly Xaa Gln Asn AspSerCysTyr SerLeu Xaa <210> 11 <211> 474 <212> DNA
<213> human <220>

<221>CDS

<222>(2)..(492) <400>11 a g c c cgc 49 aaa tt act gaa ctg tca ttc ctg at gat get cat cct gga aa Lys 1u a s s e e Arg G Ser Hi Pro Ph Thr Leu Gly Leu Asp Ly Phe Al Il cag aaa ggattt gttaaaatt getttg acccatggcgcc tctctg 97 cgg Gln Lys GlyPhe ValLysIle AlaLeu ThrHisGlyAla SerLeu Arg gtc gtg gtttct tttggtgaa aatgaa ctgtttaaacaa actgac 145 cca Val Val ValSer PheGlyGlu AsnGlu LeuPheLysGln ThrAsp Pro aac gaa ggatca tggattaga actgtt cagaataaactg cagaag 193 cct Asn Glu GlySer TrpIleArg ThrVal GlnAsnLysLeu GlnLys Pro atc ggg tttget ttgcccctg tttcat gccaggggagtt tttcag 241 atg Ile Gly PheAla LeuProLeu PheHis AlaArgGlyVal PheGln Met tac ttt ggccta atgacctat aggaaa gccatccacact gttgtt 289 aat 'Lyr Phe GlyLeu MetThrTyr ArgLys AlaIleHisThr ValVal Asn ggc ccg atccct gttcgtcag actctg aacccgacccag gagcag 337 r_gc Gly Pro IlePro ValArgGln ThrLeu AsnProThrGln GluGln Arg att gag ttacat cagacctat atggag gaacttaggaaa ttgttt 385 gag Ile Glu LeuHis GlnThrTyr MetGlu GluLeuArgLys LeuPhe Glu gag cac aaagga aagtatggc attcca gagcacgagact cttgtt 433 gaa Glu His LysGly LysTyrGly IlePro GluHisGluThr LeuVal Glu tta tga cttgactata gc 479 aaa aaaaaaaaaa aaaagcggcc Leu Lys <210>12 <211>146 <212>PRT

<213>human <400> 12 Lys Glu Ser Leu Asp Ala His Pro Gly Lys Phe Thr Leu Phe Ile Arg Gln Arg Lys Gly Phe Val Lys Ile Ala Leu Thr His Gly Ala Ser Leu Val Pro Val Val Ser Phe Gly Glu Asn Glu Leu Phe Lys Gln Thr Asp Asn Pro Glu Gly Ser Trp Ile Arg Thr Val Gln Asn Lys Leu Gln Lys Ile Met Gly Phe Ala Leu Pro Leu Phe His Ala Arg Gly Val Phe Gln Tyr Asn Phe Gly Leu Met Thr Tyr Arg Lys Ala Ile His Thr Val Val g5 90 95 Gly Arg Pro Ile Pro Val Arg Gln Thr Leu Asn Pro Thr Gln Glu Gln Ile Glu Glu Leu His Gln Thr Tyr Met Glu Glu Leu Arg Lys Leu Phe Glu Glu His Lys Gly Lys Tyr Gly Ile Pro Glu His Glu Thr Leu Val Leu Lys <210> 13 <211> 254 <212> DNA

<213> human <220>

<221> misc_feature <222> ( ) . . ( ) <223> n = G or A, C, T

<220>

<221> CDS

<222> (3)..(254) <400> 13 gc aac ctc gcc ctt aagttc attgta ggg 47 ttc gag cac aac aaa atc Asn Leu Phe Ala is LysPhe IleIleVal Gly Glu H Lys Asn Leu ggt gcc cag gccctg gatgccagg cctgga tccttcacgctg tta 95 gag Gly Ala Gln AlaLeu AspAlaArg ProGly SerPheThrLeu Leu Glu ctg cgg aac aagggc ttcgtcagg ctcgcc ctgacacacggg gca 143 cga Leu Arg Asn LysGly PheValArg LeuAla LeuThrHisGly Ala Arg ccc ctg gtt atcttC tCCttcggg gagaat gacctatttgac cag 191 nta Pro Leu Val IlePhe SerPheGly GluAsn AspLeuPheAsp Gln Xaa att ccc aac tctggc tcctggtta cgctat atccagaatcgg ttg 239 tct Ile Pro Asn SerGly SerTrpLeu ArgTyr IleGlnAsnArg Leu Ser cag aag atc atg ggc 254 Gln Lys Ile Met Gly <210> 14 <211> 84 <212> PRT
<213> human <220>

<221> _feature misc <222> ( ( ) ) .
.

<223> n A, G or = C, T

<400> 14 Asn PheGlu AlaHisLys LeuLysPhe AsnIleIle ValGlyGly Leu Ala GluAla LeuAspAla ArgProGly SerPheThr LeuLeuLeu Gln Arg ArgLys GlyPheVal ArgLeuAla LeuThrHis GlyAlaPro Asn Leu XaaIle PheSerPhe GlyGluAsn AspLeuPhe AspGlnIle Val Pro SerSer GlySerTrp LeuArgTyr IleGlnAsn ArgLeuGln Asn Lys Ile Met Gly <210> 15 <211> 887 <212> DNA
<213> human <220>
<221> CDS
<222> (314)..(805) <400> 15 ggctgtttca gcatggcggt gcctccatgt ggccttttgg tgtcttcatg ttatatcctg 60 tccaggtggt gttggtataa ataattctag gcaccatcat acctgagttt ctcagtagcc 120 ctaggaggta gcagggacag gtccaaatac tctattgcca ctttacaaat gaagagcctg 180 taggagaggg aagcaatttg tcccaagcca gcatcaagtc tgtggcacag ccagcaccat 240 aatatctcca ggtgctgtca cataccatat ctgaatcttc gtaagaaccc agggtggtca 300 gacatatgga tga aga cct gga ggc tca gag ggg agg ttt ccc aag gtc 349 Arg Pro Gly Gly Ser Glu Gly Arg Phe Pro Lys Val acacca gtgagtggc agagtcagg getggtaca caggccccg ccctgg 397 ThrPro ValSerGly ArgValArg AlaGlyThr GlnAlaPro ProTrp ctcagc aggttgceg tccctgcag ctggtcaag actgetgag ctggac 995 LeuSer ArgLeuPro SerLeuGln LeuValLys ThrAlaGlu LeuAsp ccctct cggaactac attgcgggc ttccacccc catggagtc ctggca 493 ProSer ArgAsnTyr IleAlaGly PheHisPro HisGlyVal LeuAla gtcgga -gcctttgcc aacctgtgc actgagagc acaggcttc tcttcg 541 ValGly AlaPheAla AsnLeuCys ThrGluSer ThrGlyPhe SerSer atcttc cccggtatc cgcccccat ctgatgatg ctgaccttg tggttc 589 IlePhe ProGlyIle ArgProHis LeuMetMet LeuThrLeu TrpPhe g0 85 90 cgggcc cccttcttc agagattac atcatgtct gcagggttg gtcaca 637 ArgAla ProPhePhe ArgAspTyr IleMetSer AlaGlyLeu ValThr tcagaa aaggagagt getgetcac attctgaac aggaagggt ggcgga 685 SerGlu LysGluSer AlaAlaHis IleLeuAsn ArgLysGly GlyGly aacttg ctgggcatc attgtaggg ggtgcccag gaggccctg gatgcc 733 AsnLeu LeuGlyIle IleValGly GlyAlaGln G1_uAlaLeu AspAla aggcct ggatccttc acgctgtta ctgcggaac cgaaagggc ttcgtc 781 ArgPro GlySerPhe ThrLeuLeu LeuArgAsn ArgLysGly PheVal aggctc gccctgaca cacgggtat caagcctctg ggaagagcac 835 tctgggttca ArgLeu AlaLeuThr HisGlyTyr gttggcaatt ggcaagcgat ctttattttg gtgggaagat ggcagagacg as 887 <210> 16 <211> 164 <212> PRT
<213> human <400> 16 Arg Pro Gly Gly Ser Glu Gly Arg Phe Pro Lys Val Thr Pro Val Ser Gly Arg Val Arg Ala Gly Thr Gln Ala Pro Pro Trp Leu Ser Arg Leu Pro Ser Leu Gln Leu Val Lys Thr Ala Glu Leu Asp Pro Ser Arg Asn Tyr Ile Ala Gly Phe His Pro His Gly Val Leu Ala Val Gly Ala Phe Ala Asn Leu Cys Thr Glu Ser Thr Gly Phe Ser Ser Ile Phe Pro Gly Ile Arg Pro His Leu Met Met Leu Thr Leu Trp Phe Arg Ala Pro Phe Phe Arg Asp Tyr Ile Met Ser Ala Gly Leu Val Thr Ser Glu Lys Glu Ser Ala Ala His Ile Leu Asn Arg Lys Gly Gly Gly Asn Leu Leu Gly I1e Ile Val Gly Gly Ala Gln Glu Ala Leu Asp Ala Arg Pro Gly Ser Phe Thr Leu Leu Leu Arg Asn Arg Lys Gly Phe Val Arg Leu Ala Leu Thr His Gly Tyr <210> 17 <211> 446 <212> DNA

<213> human <220>

<221> CDS

<222> (1)..(444) <400> 17 agc tat gtccctctcaag cttctgaagact catgacatc tgcccc 48 gat Ser Tyr ValProLeuLys LeuLeuLysThr HisAspIle CysPro Asp agc aac tacatcctcgtc tgccaccctcat gggctcttt gcccat 96 cgc Ser Asn TyrIleLeuVal CysHisProHis GlyLeuPhe AlaHis Arg gga ttt ggccactttgcc acagaggcctca ggcttctcc aagata 144 tgg Gly Phe GlyHisPheAla ThrGluAlaSer GlyPheSer LysIle Trp ttt ggc atcaccccttac atactcacactg ggagccttt ttctgg 192 cct Phe Gly IleThrProTyr IleLeuThrLeu GlyAlaPhe PheTrp Pro atg ttc ctcagagaatat gtaatgtctaca ggggcctgc tctgtg 240 cct Met Phe LeuArgGluTyr ValMetSerThr GlyAlaCys SerVal Pro agt tcc tccattgacttt ctgctgactcat aaaggcaca ggcaac 288 cga Ser Ser SerIleAspPhe LeuLeuThrHis LysGlyThr GlyAsn Arg atg att gtggtgattggt ggactggetgag tgcagatac agcctg 336 gtc Met Ile ValValIleGly GlyLeuAlaGlu CysArgTyr SerLeu Val cca ggt tct tct acc ctg gtg ttg aag aac cgg tct ggc ttt gtg cgc 384 Pro Gly Ser Ser Thr Leu Val Leu Lys Asn Arg Ser Gly Phe Val Arg atg gcc ctt cag cat ggg gtg cct cta ata cct gcc tat gcc ttt ggg 432 Met Ala Leu Gln His Gly Val Pro Leu Ile Pro Ala Tyr Ala Phe Gly gag acg gac ctc to 496 Glu Thr Asp Leu <210> 18 <211> 148 <212> PRT
<213> human <400> 18 Ser Asp Tyr Val Pro Leu Lys Leu Leu Lys Thr His Asp Ile Cys Pro Ser Arg Asn Tyr Ile Leu Val Cys His Pro His Gly Leu Phe Ala His Gly Trp Phe Gly His Phe Ala Thr Glu Ala Ser Gly Phe Ser Lys Ile Phe Pro Gly Ile Thr Pro Tyr Ile Leu Thr Leu Gly Ala Phe Phe Trp Met Pro Phe Leu Arg Glu Tyr Val Met Ser Thr Gly Ala Cys Ser Val Ser Arg Ser Ser Ile Asp Phe Leu Leu Thr His Lys Gly Thr Gly Asn Met Val Ile Val Val Ile Gly Gly Leu Ala Glu Cys Arg Tyr Ser Leu Pro Gly Ser Ser Thr Leu Val Leu Lys Asn Arg Ser Gly Phe Val Arg Met Ala Leu Gln His Gly Val Pro Leu Ile Pro Ala Tyr Ala Phe Gly Glu Thr Asp Leu <210> 19 <211> 1670 <212> DNA
<213> human <220>
<221> misc_feature <222> ()..() _17_ <223> A, C, T
n = G or <220>

<221>
CDS

<222>
(635)..(1666) <400>

gggaagagaatatcgtttttcttgcaaaatacacgctaaaaactatttagaagcaaaagg60 ttgtaatctctgtgatgtattctcaaatacaaacatatatgtatatacttacatttttac120 atttaaagataaatcaaacgtaaaatgttgacaatgggtagatgtagatgaagattaaac180 aagactttattaaaataatcttgttttttcaaaataaaaagtttaattaaaaaacctcca240 tcaagagtttttgtagcaataaacaagctgattcaaaaatttatatagaaaaacaaagaa300 actacaaataattaaaacaattttgagaacgaataaagttaaaggaattataccatctga360 ttttgagacttagca-taagactagagcaatcaagacagtgatgtatttgtgaaggaatag420 atatattgatccacagaacagaaaagagtcaagaaataaacacatgaatatggtcaattg480 atttttgacaaagatgaaaaagcaattccatggaggatgaataagtgcttttcaaggaac540 ggtgtaggaaaatttgatgtccatatgtggcaaaatgaatcttgacccaaacttcaggct600 ctataaaaattaactcaagtatgacatcaacaag atg aag tcc 655 gtg aat atc ggg Met Val Lys Ser Asn Gly Ile acatctctccag agcaacaag aatctggca gccatccat ggaccaaag 703 ThrSerLeuGln SerAsnLys AsnLeuAla AlaIleHis GlyProLys tacctttgtggg aattttgga cccaggtgg caggcgttc agcttgggt 751 TyrLeuCysGly AsnPheGly ProArgTrp GlnAlaPhe SerLeuGly acgaaactggac cctatggaa gtatttccg aaattactt cccagtaaa 799 ThrLysLeuAsp ProMetGlu ValPhePro LysLeuLeu ProSerLys gtccctgttgcc cagaccctt getccctac tcagetcca tgttttcag 847 ValProValAla GlnThrLeu AlaProTyr SerAlaPro CysPheGln aggctttggtgg tcagcagcg aaggtcaag gccccgagt cataatgca 895 ArgLeuTrpTrp SerAlaAla LysValLys AlaProSer HisAsnAla aagcaagggccc aagatggat gggcagctg gtgaagact catgatctt 943 LysGlnGlyPro LysMetAsp GlyGlnLeu ValLysThr HisAspLeu tctcccaaacac aactacatc attgccaat cacccccat ggcattctc 991 SerProLysHis AsnTyrIle IleAlaAsn HisProHis GlyIleLeu tcttttggtgtc ttcatcaac tttgccact gaggccactggc attget 1039 SerPheGlyVal PheIleAsn PheAlaThr GluAlaThrGly IleAla cggattttccca tccatcact ccctttgta gggaccttagaa aggata 1087 ArgIlePhePro SerIleThr ProPheVal GlyThrLeuGlu ArgIle ttttggatccca attgtgcga gaatatgtg atgtcaatgggt gtgtgc 1135 PheTrpIlePro IleValArg GluTyrVal MetSerMetGly ValCys cctgtgagtagc tcagccttg aagtacttg ctgacccagaaa ggctca 1183 P.roValSerSer SerAlaLeu LysTyrLeu LeuThrGlnLys GlySer ggcaatgccgtg gttattgtg gtgggtgga getgetgaaget etcttg 1231 GlyAsnAlaVal ValIleVal ValGlyGly AlaAlaGluAla LeuLeu tgccgaccagga gcctccact ctcttcctc aagcagcgtaaa ggtttt 1279 CysArgProGly AlaSerThr LeuPheLeu LysGlnArgLys GlyPhe gtgaagatggca ctgcaaaca ggggcatac cttgtcccttca tattcc 1327 ValLysMetAla LeuGlnThr GlyAlaTyr LeuValProSer TyrSer tttggtgagaac gaagttttc aatcaggag accttccctgag ggcacg 1375 PheGlyGluAsn GluValPhe AsnGlnGlu ThrPheProGlu GlyThr tggttaaggttg ttccaaaaa accttccag gacacattcaaa aaaatc 1423 TrpLeuArgLeu PheGlnLys ThrPheGln AspThrPheLys LysIle ctgggactaaat ttctgtacc ttccatggc cggggcttcact cgcgga 1471 LeuGlyLeuAsn PheCysThr PheHisGly ArgGlyPheThr ArgGly tcctggggcttc ctgcctttc aatcggccc attaccactgtt gttggg 1519 SerTrpGlyPhe LeuProPhe AsnArgPro IleThrThrVal ValGly gaaccccttcca attcccagg attaagagg ccaaaccagaag acagta 1567 C~7_uProLeuPro IleProArg IleLysArg ProAsnGlnLys ThrVal gacaagtatcac gcactctac atcagtgcc ctgcgcaagctc tttgac 1615 AspLysTyrHis AlaLeuTyr IleSerAla LeuArgLysLeu PheAsp caacacaaagtt gaatatggc ctccctgag acccaagagctg acaatt 1663 GlnHisLysVal GluTyrGly LeuProGlu ThrGlnGluLeu ThrIle acantaa 1670 Thr <210> 20 <211> 344 <212> PRT
<213> human <220>
<221> misc_feature <222> ()..() <223> n = A, C, G or T
<400> 20 Met Val Asn Gly Lys Ser Ile Thr Ser Leu Gln Ser Asn Lys Asn Leu Ala Ala Ile His Gly Pro Lys Tyr Leu Cys Gly Asn Phe Gly Pro Arg Tr_p Gln Ala Phe Ser Leu Gly Thr Lys Leu Asp Pro Met Glu Val Phe Pro Lys Leu Leu Pro Ser Lys Val Pro Val Ala Gln Thr Leu Ala Pro Tyr Ser Ala Pro Cys Phe Gln Arg Leu Trp Trp Ser Ala Ala Lys Val ~'5 70 75 80 Lys Ala Pro Ser His Asn Ala Lys Gln Gly Pro Lys Met Asp Gly Gln Leu Val Lys Thr His Asp Leu Ser Pro Lys f-lis Asn Tyr Ile Ile Ala Asn His Pro His Gly Ile Leu Ser Phe Gly Val Phe Ile Asn Phe Ala ')hr Glu Ala Thr Gly Ile Ala Arg Ile Phe Pro Ser Ile Thr Pro Phe Val Gly Thr Leu Glu Arg Ile Phe Trp Ile Pro Ile Val Arg Glu Tyr Val Met Ser Met Gly Val Cys Pro Val Ser Ser Ser Ala Leu Lys Tyr Leu Leu Thr Gln Lys Gly Ser Gly Asn Ala Val Val Ile Val Val Gly Gly Ala Ala Glu Ala Leu Leu Cys Arg Pro Gly Ala Ser Thr Leu Phe Leu Lys Gln Arg Lys Gly Phe Val Lys Met Ala Leu Gln Thr Gly Ala Tyr Leu Val Pro Ser Tyr Ser Phe Gly Glu Asn Glu Val Phe Asn Gln Glu Thr Phe Pro Glu Gly Thr Trp Leu Arg Leu Phe Gln Lys Thr Phe Gln Asp Thr Phe Lys Lys Ile Leu Gly Leu Asn Phe Cys Thr Phe His Gly Arg Gly Phe Thr Arg Gly Ser Trp Gly Phe Leu Pro Phe Asn Arg Pro Ile Thr Thr Val Val Gly Glu Pro Leu Pro Ile Pro Arg Ile Lys Arg Pro Asn Gln Lys Thr Val Asp Lys Tyr His Ala Leu Tyr Ile Ser Ala Leu Arg Lys Leu Phe Asp Gln His Lys Val Glu Tyr Gly Leu Pro Glu Thr Gln Glu Leu Thr Ile Thr

Claims (8)

1. An isolated nucleic acid molecule selected from:

(a) nucleic acid molecules comprising a nucleotide sequence as shown in SEQ ID
NO: 3, 5, 7, 9, 11, 13, 15, 17, or 19;

(b) nucleic acid molecules comprising a nucleotide sequence capable of hybridizing, under stringent hybridization conditions, to a nucleotide sequence complementary to the polypeptide coding region of a nucleic acid molecule as defined in (a); and (c) nucleic acid molecules comprising a nucleic acid sequence which is degenerate as a result of the genetic code to a nucleotide sequence as defined in (a) or (b).

2. An isolated polypeptide encoded by the nucleic acid molecule according to claim 1.

3. The isolated polypeptide according to claim 2 having an amino acid sequence shown as SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18 or 20 in the Sequence Listing

4. A vector harboring the nucleic acid molecule according to claim 1.

5. A replicable expression vector which carries and is capable of mediating the expression of a nucleotide sequence according to claim 1.

6. A cultured host cell harboring a vector according to claim 4 or 5.

7. A process for production of a polypeptide, comprising culturing a host cell according to claim 6 under conditions whereby said polypeptide is produced, and recovering said polypeptide.

8. A method for identifying an agent capable of modulating a nucleic acid molecule according to claim 1, comprising (i) providing a cell comprising the said nucleic acid molecule;

(ii) contacting said cell with a candidate agent; and (iii) monitoring said cell for an effect that is not present in the absence of said candidate agent.