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 II” 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:
TECHNICAL FIELD  
         [0001]    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 II” 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.  
         BACKGROUND ART  
         [0002]    Metabolic diseases are defined as any of the diseases or disorders that disrupt normal metabolism. They may arise from nutritional deficiencies; in connection 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 from 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.  
           [0003]    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 key 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 common 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 from amino acids.  
           [0004]    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 from 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.  
           [0005]    Obesity is usually defined in terms of the body mass index (BMI), i.e. weight (in kilograms) divided by the square of the height (in 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.  
           [0006]    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 ob gene have led to massive obesity in mice. Cloning the ob 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.  
           [0007]    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, including maturity-onset diabetes of the young (MODY) (Velho &amp; 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 scanning 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.  
         DESCRIPTION OF THE INVENTION  
         [0008]    According to the present invention, a family of genes and encoded homologous proteins (hereinafter referred to as “Protein Cluster II”) has been identified. Consequently, the present invention provides an isolated nucleic acid molecule selected from:  
           [0009]    (a) nucleic acid molecules comprising a nucleotide sequence as shown in SEQ ID NO: 1, or 3;  
           [0010]    (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  
           [0011]    (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).  
           [0012]    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.  
           [0013]    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 NaHPO 4 , 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at +65° C., and washing in 0.1×SSC/0.1% SDS at +68° C.  
           [0014]    In a preferred form of the invention, the said nucleic acid molecule has a nucleotide sequence identical with SEQ ID NOS: 1 or 3 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 NOS: 1 or 3. 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 II polypeptide 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 NOS: 1 or 3 in the Sequence Listing.  
           [0015]    Included 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: 1 or 3. 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 II polypeptide.  
           [0016]    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.  
           [0017]    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 II, expression control regulatory sequences such as promoters, operators, enhancers, repressors, and the like. Nucleic acid molecules of the invention are also useful in hybridization assays to detect the capacity of cells to express the proteins in Cluster II. Nucleic acid molecules of the invention may also provide a basis for diagnostic methods useful for identifying a genetic alteration(s) in a locus that underlies a disease state or states, which information is useful both for diagnosis and for selection of therapeutic strategies.  
           [0018]    In 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: 2 or 4 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: 2 or 4 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 II 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: 2 or 4 in the Sequence Listing.  
           [0019]    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.  
           [0020]    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 eukaryotic 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 a yeast such as  Saccharomyces cervisiae  or  Pichia pastoris , 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.  
           [0021]    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 purposes within the field of recombinant DNA. The recombinant polypeptide expressed by the cells may be secreted, i.e. exported through the cell membrane, dependent on the type of cell and the composition of the vector.  
           [0022]    In a further aspect, the invention provides a method for identifying an agent capable of modulating a nucleic acid molecule according to the invention, comprising  
           [0023]    (i) providing a cell comprising the said nucleic acid molecule;  
           [0024]    (ii) contacting said cell with a candidate agent; and  
           [0025]    (iii) monitoring said cell for an effect that is not present in the absence of said candidate agent.  
           [0026]    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 of 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.  
           [0027]    A transfection assay can be a particularly useful screening assay for identifying an effective agent. In a transfection assay, a nucleic acid containing a gene such as a 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 for 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, supra).  
           [0028]    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 Protocols in Molecular Biology, editors F. Ausubel et al., John Wiley and Sons, Inc. 1994, or Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A laboratory manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 1989.  
           [0029]    Additional features of the invention will be apparent from the following Examples. Examples 1 to 3 are actual, while Examples 4 to 9 are prophetic.  
       
    
    
     EXAMPLES cl Example 1  
       [0030]    Identification of Protein Clusters  
         [0031]    A family of homologous proteins (hereinafter referred to as “Protein Cluster II”) was identified by an “all-versus-all” BLAST procedure using all  Caenorhabditis elegans  proteins in the Wormpep20 database release (http://www.sanger.ac.uk/Projects/ C—elegans /wormpep/index.shtml). The Wormpep database contains the predicted proteins from the  C. elegans  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. &amp; Waterman M. S. (1981)  Identification of common molecular subsequences . J. Mol. Biol. 147(1): 195-197; Pearson W R. (1991)  Searching protein sequence libraries: comparison of the sensitivity and selectivity of the Smith - Waterman and FASTA algorithms . Genomics 11: 635-650; Olsen et al. (1999)  Optimizing Smith - Waterman alignments . Pac Symp Biocomput.302-313). Completely annotated proteins were filtered out, whereby 10,130 proteins of unknown function could be grouped into 1,800 clusters.  
         [0032]    The obtained sequence clusters were compared to the  Drosophila melanogaster  proteins contained in the database Flybase (Berkeley Drosophila Genome Project; http://www.fruitfly.org), and annotated clusters were removed. Non-annotated protein clusters, conserved in both  C. elegans  and  D. melanogaster , were saved to a worn/fly data set, which was used in a BLAST procedure (http://www.ncbi.nlm.nih.gov/Education/BLASTinfo/information3.html) against the Celera Human Genome Database (http://www.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://www.genome.washington.edu/UWGC/analysistools/phrap.htm). A group of homologous proteins (“Protein Cluster II”) with unknown function was chosen for further studies.  
         [0033]    EST databases provided by the EMBL (http://www.embl.org/Services/index.html) were used to check whether the human proteins in Cluster II were expressed, in order to identify putative pseudogenes. One putative pseudogene was identified and excluded.  
       Example 2  
       [0034]    Analyses of Protein Cluster II  
         [0035]    (a) Alignment  
         [0036]    The human part of Protein Cluster II comprises polypeptides encoded by the nucleic acid sequences shown as SEQ ID NOS: 1, 3 and 5. The sequence shown as SEQ ID NO: 5 was identified as the gene annotated as  Homo sapiens  core1 UDP-galactose:N-acetylgalactosamine-alpha-R beta 1,3-galactosyltransferase (C1GALT1; GenBank Accession No. AF155582; see also International Patent Application No. WO 99/65712.)  
         [0037]    An alignment of the human polypeptides included in Protein Cluster II (SEQ ID NOS: 2, 4, and 6), 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 in a distinct part of the protein cluster II, indicating the presence of a novel domain (see positions marked with stars in Table I).  
         [0038]    The sequence shown as SEQ ID NO: 6 is identical to positions 53-363 of the polypeptide encoded by the C1GALT1 gene mentioned above.  
         [0039]    (b) HMM-Pfam  
         [0040]    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 profile-HMMs (Profile Hidden Markov Models) of these families. Profile-HMMs can be used to do sensitive database searching using statistical descriptions of a sequence family&#39;s consensus. Pfam is available on the WWW at http://pfam.wustl.edu; http://www.sanger.ac.uk/Software/Pfam; and http://www.cgr.ki.se/Pfam. 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 Pfam, see Bateman et al. (2000)  The Pfam protein families database . Nucleic Acids Res. 28:263-266; Sonnhammer et al. (1998)  Pfam: Multiple Sequence Alignments and HMM - Profiles of Protein Domains . Nucleic Acids Research, 26:322-325; Sonnhammer et al. (1997)  Pfam: a Comprehensive Database of Protein Domain Families Based on Seed Alignments . Proteins 28:405-420.  
         [0041]    The HMM-Pfam search indicated that no previously known domains could be identified in Protein Cluster II, with exception for a weak homology to Galactosyltransferase (Pfam Accession No. PF01762; see also Kolbinger et al. (1998) J. Biol. Chem. 273: 433-440).  
         [0042]    A Pfam-B search revealed identity to the Pfam-B 7357 domain (Pfam Accession No. PB007357). Pfam-B domains are generated automatically from an alignment taken from the database ProDom 2000.1 (http://www.lunix.toulouse.inra.fr/prodom) subtracting sequence segments already covered by Pfam-A. The ProDom database has been designed as tool to help analyze domain arrangements of proteins and protein families (Corpet et al. (1999) Nucleic Acid Research 27: 263-267). Pfam-B domains are curated manually at the Sanger Centre, UK, to become Pfam-A domains.  
         [0043]    (c) TM-HMM  
         [0044]    The human proteins in Cluster II were analyzed using the TM-HMM tool available e.g. at http://www.cbs.dtu.dk/services/TMHMM-1.0. TM-HMM is a method to model and predict the location and orientation of alpha helices in membrane-spanning proteins (Sonnhammer et al. (1998)  A hidden Markov model for predicting transmembrane helices in protein sequences . ISMB 6:175-182). No transmembrane regions were identified.  
         [0045]    (d) Analysis of non-human orthologs  
         [0046]    The  Caenorhabditis elegans  genome includes eight genes encoding proteins within Protein Cluster II, of which the closest ancestor in evolution, a sequence included the  C. elegans  cosmid C38H2.2 (GenBank Accession No. Z35461) and annotated as UDP-galactose:N-acetylgalactosamine-alpha-R beta 1,3-galactosyltransferase mRNA (GenBank Accession No. AF269063) is 55%, 54%, and 42% identical to the three identified human proteins shown as SEQ ID NOS: 2, 4 and 6, respectively. (See also:  Genome sequence of the nematode C. elegans: a platform for investigating biology ; The  C. elegans  Sequencing Consortium. Science (1998) 282:2012-2018. Published errata appear in Science (1999) 283:35; 283:2103; and 285:1493.)  
         [0047]    The  Drosophila melanogaster  genome comprises 10 genes belonging to Protein Cluster II, of which the closest relative “CG9520” (GenBank Accession No. AE003623; see also Adams et al. (2000) The genome sequence of Drosophila melanogaster ; Science 287:2185-2195) is 42% identical to the human protein set.  
         [0048]    No counterparts to Protein Cluster II in  Saccharomyces cerevisiae  were identified.  
       Example 3  
       [0049]    Expression Analysis  
         [0050]    The tissue distribution of the human genes was studied using the Incyte LifeSeq® database (http://www.incyte.com). The nucleic acid molecules shown as SEQ ID NO: 1, 3 and 5 were found to be expressed primarily in germ cells and in the nervous system. Therefore, the said nucleic acid molecules shown as SEQ ID NO: 1, 3 and 5 and the polypeptides shown as SEQ ID NO: 2, 4 and 6 are proposed to be useful for differential identification of the tissue(s) or cell types(s) present in a biological sample and for diagnosis of diseases and disorders, including disorders of the central nervous system.  
       Example 4  
       [0051]    Multiple Tissue Northern Blotting  
         [0052]    Multiple Tissue Northern blotting (MTN) is performed to make a more thorough analysis of the expression profiles of the proteins in Cluster II. Multiple Tissue Northern (MTN™) Blots (http://www.clontech.com/mtn) 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 5  
       [0053]    Expressing Profiling Using Microarrays  
         [0054]    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 expression using DNA microarrays . Curr. Opin. Microbiol. 3(3): 285-291; or Duggan et al. (1999)  Expression profiling using cDNA Microarrays . Nature Genetics Supplement 21:10-14).  
         [0055]    The expression pattern of the proteins in Cluster II can be analyzed using GeneChip® expression arrays (http://www.affymetrix.com/products/app_exp.html). Briefly, mRNAs are extracted from various tissues. They are reverse transcribed using a T7-tagged oligo-dT primer and double-stranded cDNAs are generated. These cDNAs are then amplified and labeled using In Vitro Transcription (IVT) with T7 RNA polymerase and biotinylated nucleotides. The populations of cRNAs obtained are purified and fragmented by heat to produce a distribution of RNA fragment sizes from approximately 35 to 200 bases. GeneChip® expression arrays are hybridized with the samples. The arrays are washed and stained. The cartridges are scanned using a confocal scanner and the images are analyzed with the GeneChip 3.1 software (Affymetrix).  
       Example 6  
       [0056]    Identification of Polypeptides Binding to Protein Cluster II  
         [0057]    In order to assay for proteins interacting with Protein Cluster II, the two-hybrid screening method can be used. The two-hybrid method, first described by Fields &amp; 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.  
         [0058]    The two-hybrid method can be used to determine if 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 that interact with a protein of interest . Proc. Natl. Acad. Sci. U.S.A. 88:9578-9582; Bartel P L, Fields (1995)  Analyzing protein - protein interactions using two - hybrid system . Methods Enzymol. 254:241-263; or Wallach et al. (1998)  The yeast two - hybrid screening technique and its use in the study of protein-protein interactions in apoptosis . Curr. Opin. Immunol. 10(2): 131-136. See also http://www.clontech.com/matchmaker.  
         [0059]    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 eukaryotic 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 II 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 into 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  
       [0060]    Full-length Cloning of Cluster II Genes  
         [0061]    The polymerase chain reaction (PCR), which is a well known procedure for in vitro enzymatic amplification of a specific DNA segment, can be used for direct cloning of Protein Cluster II 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 tour of the minefield . Biotechniques 24:390-392; Lenstra (1995)  The applications of the polymerase chain reaction in the life sciences . Cellular &amp; Molecular Biology 41:603-614; or Rashtchian (1995)  Novel methods for cloning and engineering genes using the polymerase chain reaction . 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).  
         [0062]    In an alternative approach to isolate a cDNA clone encoding a full length protein of Protein Cluster II, a DNA fragment corresponding to a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, 3, 5 or 7, 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. Phage plaques are transferred to nylon membranes, which are hybridized with a DNA probe prepared as described above. Positive colonies are isolated from the plates. Plasmids 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  
       [0063]    Recombinant Expression of Proteins in Eukaryotic Host Cells  
         [0064]    To produce proteins of Cluster II, 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 from 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.  
       Example 9  
       [0065]    Determination of Gene Function  
         [0066]    Methods are known in the art for elucidating the biological function or mode of 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 &amp; Coulson (2000)  RNAi - prospects for a general technique for determining gene 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 of posttranscriptional gene silencing (PTGS) was first discovered in  C. elegans  (Fire et al., (1998) Nature 391:806-811). RNA interference has recently been used for targeting nearly 90% of predicted genes on  C. elegans  chromosome I (Fraser et al. (2000) Nature 408: 325-330) and 96% of predicted genes on  C. elegans  chromosome III (Gönczy et al. (2000) Nature 408:331-336).  
                                 TABLE I                       Alignment of polypeptides in Protein Cluster II:       “*” = identical or conserved residues in all       sequences in the alignment.                                SEQ_ID_NO_4   ------------------------------------------------------------               SEQ_ID_NO_6   ------------------------------------------------------------       SEQ_ID_NO_2   MTENSLSEMASKSWLNFLTFLYGSAIGFILFSQLLSILLGEEGDTQTNVLHNDPHARHSD   60               SEQ_ID_NO_4   -----------------------NTGVTDKLYQKMKILCWIMTGPQNLEKKIRRIRDTWA   37       SEQ_ID_NO_6   DNGQNHLEGQMNFNADSSQHKDENTDIAENLYQKVRILCWVMTGPQNLEKKAKHVKATWA   60       SEQ_ID_NO_2   DNGQNHLGGQMNFNADSSQRKDENTEIAENLYXQVKILCWVMTGSQNLQKKAKHVKATWA   60                                  **     **    **** *** *** **      ***               SEQ_ID_NO_4   QGCNKALFMSSKENKDFSTVGLHTKEDRNQLSWKIVKAFLYAHDHYLEYMDWFMKADDDI   97       SEQ_ID_NO_6   QRCNKVLFMSSEENKDFPAVGLKTKEGRDQLYWKTIKAFQYVHEHYLEDADWFLKADDDT   120       SEQ_ID_NO_2   QRCLKVFFMSSEENKDFRAVGLKTKAGRDELYWKTINLF---------------------   159           * * *  **** *****  *** **  *  * **    *                                    SEQ_ID_NO_4   CIYITLDNLKWLLTNYNPDESTYFGKRFKHCRKQDYNTGGAGYVLSKE------------   145       SEQ_ID_NO_6   --YVILDNLRWLLSKYDPEEPIYFGRRFKPYVKQGYMSGGAGYVLSKEALKRFVDAFKTD   178       SEQ_ID_NO_2   ------------------------------------------------------------               SEQ_ID_NO_4   ------------------------------------------------------------       SEQ_ID_NO_6   KCTHSSSIEDLALGRCMEIMNVEAGDSRDTIGKETFHPFVPEHHLIKGYLPRTFWYWNYN   238       SEQ_ID_NO_2   ------------------------------------------------------------               SEQ_ID_NO_4   ------------------------------------------------------------       SEQ_ID_NO_6   YYPPVEGPGCCSDLAVSFHYVDSTTMYELEYLVYHLRPYGYLYRYQPTLPERILKEISQA   298       SEQ_ID_NO_2   ------------------------------------------------------------               SEQ_ID_NO_4   -------------       SEQ_ID_NO_6   NKNEDTKVKLGNP  311       SEQ_ID_NO_2   -------------                  
 
         [0067]    [0067] 
     
       
       
         1 
         
           
             6  
           
           
             1  
             299  
             DNA  
             HUMAN  
             
               CDS  
               (21)..(299)  
                 
             
           
            1 

gcaagaggga gccacggccg atg aca gaa aat tca ctt tcc gag atg gcc tct     53 
                      Met Thr Glu Asn Ser Leu Ser Glu Met Ala Ser 
                      1               5                   10 

aaa tcc tgg ctg aat ttt tta acc ttc ctc tat gga tcg gca ata ggg      101 
Lys Ser Trp Leu Asn Phe Leu Thr Phe Leu Tyr Gly Ser Ala Ile Gly 
            15                  20                  25 

ttt att tta ttt tct cag cta ctt agt att ttg ttg gga gaa gag ggt      149 
Phe Ile Leu Phe Ser Gln Leu Leu Ser Ile Leu Leu Gly Glu Glu Gly 
        30                  35                  40 

gac acc cag act aat gtt ctt cat aat gat cct cat gcg agg cat tca      197 
Asp Thr Gln Thr Asn Val Leu His Asn Asp Pro His Ala Arg His Ser 
    45                  50                  55 

gat gat aat gga cag aat cat cta gga gga caa atg aac ttc aat gca      245 
Asp Asp Asn Gly Gln Asn His Leu Gly Gly Gln Met Asn Phe Asn Ala 
60                  65                  70                  75 

gat tct agc caa cgt aaa gat gag aac aca gaa atc gct gaa aac ctc      293 
Asp Ser Ser Gln Arg Lys Asp Glu Asn Thr Glu Ile Ala Glu Asn Leu 
                80                  85                  90 

tat tag                                                              299 
Tyr 

 
           
             2  
             92  
             PRT  
             HUMAN  
           
            2 

Met Thr Glu Asn Ser Leu Ser Glu Met Ala Ser Lys Ser Trp Leu Asn 
1               5                   10                  15 

Phe Leu Thr Phe Leu Tyr Gly Ser Ala Ile Gly Phe Ile Leu Phe Ser 
            20                  25                  30 

Gln Leu Leu Ser Ile Leu Leu Gly Glu Glu Gly Asp Thr Gln Thr Asn 
        35                  40                  45 

Val Leu His Asn Asp Pro His Ala Arg His Ser Asp Asp Asn Gly Gln 
    50                  55                  60 

Asn His Leu Gly Gly Gln Met Asn Phe Asn Ala Asp Ser Ser Gln Arg 
65                  70                  75                  80 

Lys Asp Glu Asn Thr Glu Ile Ala Glu Asn Leu Tyr 
                85                  90 

 
           
             3  
             489  
             DNA  
             HUMAN  
             
               CDS  
               (55)..(489)  
                 
             
           
            3 

catctaaaaa gactgatgaa gttgattgca aatgctagtc atcataaata ccag aac       57 
                                                            Asn 
                                                            1 

aca ggt gtc act gac aaa ctc tat caa aag atg aaa att ctt tgc tgg      105 
Thr Gly Val Thr Asp Lys Leu Tyr Gln Lys Met Lys Ile Leu Cys Trp 
            5                   10                  15 

att atg aca gga cct caa aat cta gaa aaa aag atc aga cgc atc aga      153 
Ile Met Thr Gly Pro Gln Asn Leu Glu Lys Lys Ile Arg Arg Ile Arg 
        20                  25                  30 

gat aca tgg gcc cag ggt tgc aat aaa gcg ttg ttt atg agc tca aaa      201 
Asp Thr Trp Ala Gln Gly Cys Asn Lys Ala Leu Phe Met Ser Ser Lys 
    35                  40                  45 

gaa aat aaa gac ttc tct act gtg gga tta cac acc aaa gaa gac aga      249 
Glu Asn Lys Asp Phe Ser Thr Val Gly Leu His Thr Lys Glu Asp Arg 
50                  55                  60                  65 

aac caa ctg tcc tgg aaa ata gtt aaa gct ttt cta tat gct cat gac      297 
Asn Gln Leu Ser Trp Lys Ile Val Lys Ala Phe Leu Tyr Ala His Asp 
                70                  75                  80 

cat tat ctg gaa tac atg gat tgg ttc atg aaa gca gat gat gat ata      345 
His Tyr Leu Glu Tyr Met Asp Trp Phe Met Lys Ala Asp Asp Asp Ile 
            85                  90                  95 

tgt ata tat atc aca ttg gac aac ttg aaa tgg ctt ctc aca aac tat      393 
Cys Ile Tyr Ile Thr Leu Asp Asn Leu Lys Trp Leu Leu Thr Asn Tyr 
        100                 105                 110 

aac cct gat gaa tcc act tac ttt ggg aaa aga ttt aag cac tgc aga      441 
Asn Pro Asp Glu Ser Thr Tyr Phe Gly Lys Arg Phe Lys His Cys Arg 
    115                 120                 125 

aaa cag gac tac atg act gga gga gca gga tat gta ctg agc aaa gaa      489 
Lys Gln Asp Tyr Met Thr Gly Gly Ala Gly Tyr Val Leu Ser Lys Glu 
130                 135                 140                 145 

 
           
             4  
             145  
             PRT  
             HUMAN  
           
            4 

Asn Thr Gly Val Thr Asp Lys Leu Tyr Gln Lys Met Lys Ile Leu Cys 
1               5                   10                  15 

Trp Ile Met Thr Gly Pro Gln Asn Leu Glu Lys Lys Ile Arg Arg Ile 
            20                  25                  30 

Arg Asp Thr Trp Ala Gln Gly Cys Asn Lys Ala Leu Phe Met Ser Ser 
        35                  40                  45 

Lys Glu Asn Lys Asp Phe Ser Thr Val Gly Leu His Thr Lys Glu Asp 
    50                  55                  60 

Arg Asn Gln Leu Ser Trp Lys Ile Val Lys Ala Phe Leu Tyr Ala His 
65                  70                  75                  80 

Asp His Tyr Leu Glu Tyr Met Asp Trp Phe Met Lys Ala Asp Asp Asp 
                85                  90                  95 

Ile Cys Ile Tyr Ile Thr Leu Asp Asn Leu Lys Trp Leu Leu Thr Asn 
            100                 105                 110 

Tyr Asn Pro Asp Glu Ser Thr Tyr Phe Gly Lys Arg Phe Lys His Cys 
        115                 120                 125 

Arg Lys Gln Asp Tyr Met Thr Gly Gly Ala Gly Tyr Val Leu Ser Lys 
    130                 135                 140 

Glu 
145 

 
           
             5  
             1560  
             DNA  
             HUMAN  
             
               CDS  
               (2)..(934)  
                 
             
           
            5 

a gat aat gga cag aat cat cta gaa gga caa atg aac ttc aat gca gat     49 
  Asp Asn Gly Gln Asn His Leu Glu Gly Gln Met Asn Phe Asn Ala Asp 
  1               5                   10                  15 

tct agc caa cat aaa gat gag aac aca gac att gct gaa aac ctc tat       97 
Ser Ser Gln His Lys Asp Glu Asn Thr Asp Ile Ala Glu Asn Leu Tyr 
            20                  25                  30 

cag aaa gtt aga att ctt tgc tgg gtt atg acc ggc cct caa aac cta      145 
Gln Lys Val Arg Ile Leu Cys Trp Val Met Thr Gly Pro Gln Asn Leu 
        35                  40                  45 

gag aaa aag gcc aaa cac gtc aaa gct act tgg gcc cag cgt tgt aac      193 
Glu Lys Lys Ala Lys His Val Lys Ala Thr Trp Ala Gln Arg Cys Asn 
    50                  55                  60 

aaa gtg ttg ttt atg agt tca gaa gaa aat aaa gac ttc cct gct gtg      241 
Lys Val Leu Phe Met Ser Ser Glu Glu Asn Lys Asp Phe Pro Ala Val 
65                  70                  75                  80 

gga ctg aaa acc aaa gaa ggc aga gat caa cta tac tgg aaa aca att      289 
Gly Leu Lys Thr Lys Glu Gly Arg Asp Gln Leu Tyr Trp Lys Thr Ile 
                85                  90                  95 

aaa gct ttt cag tat gtt cat gaa cat tat tta caa gat gct gat tgg      337 
Lys Ala Phe Gln Tyr Val His Glu His Tyr Leu Gln Asp Ala Asp Trp 
            100                 105                 110 

ttt ttg aaa gca gat gat gac acg tat gtc ata cta gac aat ttg agg      385 
Phe Leu Lys Ala Asp Asp Asp Thr Tyr Val Ile Leu Asp Asn Leu Arg 
        115                 120                 125 

tgg ctt ctt tca aaa tac gac cct gaa gaa ccc att tac ttt ggg aga      433 
Trp Leu Leu Ser Lys Tyr Asp Pro Glu Glu Pro Ile Tyr Phe Gly Arg 
    130                 135                 140 

aga ttt aag cct tat gta aag cag ggc tac atg agt gga gga gca gga      481 
Arg Phe Lys Pro Tyr Val Lys Gln Gly Tyr Met Ser Gly Gly Ala Gly 
145                 150                 155                 160 

tat gta cta agc aaa gaa gcc ttg aaa aga ttt gtt gat gca ttt aaa      529 
Tyr Val Leu Ser Lys Glu Ala Leu Lys Arg Phe Val Asp Ala Phe Lys 
                165                 170                 175 

aca gac aag tgt aca cat agt tcc tcc att gaa gac tta gca ctg ggg      577 
Thr Asp Lys Cys Thr His Ser Ser Ser Ile Glu Asp Leu Ala Leu Gly 
            180                 185                 190 

aga tgc atg gaa att atg aat gta gaa gca gga gat tcc aga gat acc      625 
Arg Cys Met Glu Ile Met Asn Val Glu Ala Gly Asp Ser Arg Asp Thr 
        195                 200                 205 

att gga aaa gaa act ttt cat ccc ttt gtg cca gaa cac cat tta att      673 
Ile Gly Lys Glu Thr Phe His Pro Phe Val Pro Glu His His Leu Ile 
    210                 215                 220 

aaa ggt tat cta cct aga acg ttt tgg tac tgg aat tac aac tat tat      721 
Lys Gly Tyr Leu Pro Arg Thr Phe Trp Tyr Trp Asn Tyr Asn Tyr Tyr 
225                 230                 235                 240 

cct cct gta gag ggt cct ggt tgc tgc tct gat ctt gca gtt tct ttt      769 
Pro Pro Val Glu Gly Pro Gly Cys Cys Ser Asp Leu Ala Val Ser Phe 
                245                 250                 255 

cac tat gtt gat tct aca acc atg tat gag tta gaa tac ctc gtt tat      817 
His Tyr Val Asp Ser Thr Thr Met Tyr Glu Leu Glu Tyr Leu Val Tyr 
            260                 265                 270 

cat ctt cgt cca tat ggt tat tta tac aga tat caa cct acc tta cct      865 
His Leu Arg Pro Tyr Gly Tyr Leu Tyr Arg Tyr Gln Pro Thr Leu Pro 
        275                 280                 285 

gaa cgt ata cta aag gaa att agt caa gca aac aaa aat gaa gat aca      913 
Glu Arg Ile Leu Lys Glu Ile Ser Gln Ala Asn Lys Asn Glu Asp Thr 
    290                 295                 300 

aaa gtg aag tta gga aat cct tgaaagaaaa tcatgaatga acaaaggtaa         964 
Lys Val Lys Leu Gly Asn Pro 
305                 310 

tatgtctagc actgcactga aaaaggactt ctgcatttct gacatagaac actggaatcc   1024 

cagtgaggaa ttctaagtga acattcctta tagaaacctt tcacatgaat gactataaac   1084 

tgaagcttta aatgagctgt gaagtgtgtt aaaatgtgtt ttgatacagt aatatataaa   1144 

tatgtctata tatatgagga acttgtgttt tttaaatggt ggccaggtag aggaactaga   1204 

aaagagattt tgttgcctgt tttctgacca tctgtgttat tgtcactgag aaactaaaat   1264 

agtaaattta ctaaaactac actgcaccat gttagtaata aacagatctg ccttaaagaa   1324 

aagaaaattt tagaaagaaa tattgttgct cagtgttgtt aatatagctc aagaattgag   1384 

tttatatttg cagtatgcta taaatgatac ccccctacca cacccacaca cacagttttt   1444 

gtctaatgaa aatgttgctg tgattattta taattggtag tatttcttcc agaagaagct   1504 

aaaataagac tggcacttac cctgaagtgc attaataaaa ccacacttta aaatta       1560 

 
           
             6  
             311  
             PRT  
             HUMAN  
           
            6 

Asp Asn Gly Gln Asn His Leu Glu Gly Gln Met Asn Phe Asn Ala Asp 
1               5                   10                  15 

Ser Ser Gln His Lys Asp Glu Asn Thr Asp Ile Ala Glu Asn Leu Tyr 
            20                  25                  30 

Gln Lys Val Arg Ile Leu Cys Trp Val Met Thr Gly Pro Gln Asn Leu 
        35                  40                  45 

Glu Lys Lys Ala Lys His Val Lys Ala Thr Trp Ala Gln Arg Cys Asn 
    50                  55                  60 

Lys Val Leu Phe Met Ser Ser Glu Glu Asn Lys Asp Phe Pro Ala Val 
65                  70                  75                  80 

Gly Leu Lys Thr Lys Glu Gly Arg Asp Gln Leu Tyr Trp Lys Thr Ile 
                85                  90                  95 

Lys Ala Phe Gln Tyr Val His Glu His Tyr Leu Gln Asp Ala Asp Trp 
            100                 105                 110 

Phe Leu Lys Ala Asp Asp Asp Thr Tyr Val Ile Leu Asp Asn Leu Arg 
        115                 120                 125 

Trp Leu Leu Ser Lys Tyr Asp Pro Glu Glu Pro Ile Tyr Phe Gly Arg 
    130                 135                 140 

Arg Phe Lys Pro Tyr Val Lys Gln Gly Tyr Met Ser Gly Gly Ala Gly 
145                 150                 155                 160 

Tyr Val Leu Ser Lys Glu Ala Leu Lys Arg Phe Val Asp Ala Phe Lys 
                165                 170                 175 

Thr Asp Lys Cys Thr His Ser Ser Ser Ile Glu Asp Leu Ala Leu Gly 
            180                 185                 190 

Arg Cys Met Glu Ile Met Asn Val Glu Ala Gly Asp Ser Arg Asp Thr 
        195                 200                 205 

Ile Gly Lys Glu Thr Phe His Pro Phe Val Pro Glu His His Leu Ile 
    210                 215                 220 

Lys Gly Tyr Leu Pro Arg Thr Phe Trp Tyr Trp Asn Tyr Asn Tyr Tyr 
225                 230                 235                 240 

Pro Pro Val Glu Gly Pro Gly Cys Cys Ser Asp Leu Ala Val Ser Phe 
                245                 250                 255 

His Tyr Val Asp Ser Thr Thr Met Tyr Glu Leu Glu Tyr Leu Val Tyr 
            260                 265                 270 

His Leu Arg Pro Tyr Gly Tyr Leu Tyr Arg Tyr Gln Pro Thr Leu Pro 
        275                 280                 285 

Glu Arg Ile Leu Lys Glu Ile Ser Gln Ala Asn Lys Asn Glu Asp Thr 
    290                 295                 300 

Lys Val Lys Leu Gly Asn Pro 
305                 310