EPO PRIMARY RESPONSE GENE 1, EPRG1

Abstract: EPRG1 polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing EPRG1 polypeptides and polynucleotides in therapy, and diagnostic assays for such.

Description: EPO PRIMARY RESPONSE GENE 1, EPRGl Field of the Invention This invention relates to newly identified polypeptides and polynucleotides encoding such polypeptides, to their use in therapy and in identifying compounds which may be agonists, antagonists and/or inhibitors which are potentially useful in therapy, and to production of such polypeptides and polynucleotides Background of the Invention The drug discovery process is currently undergoing a fundamental revolution as it embraces 'functional genomics', that is, high throughput genome- or gene-based biology This approach is rapidly superceding earlier approaches based on 'positional cloning' A phenotype, that is a biological function or genetic disease, would be identified and this would then be tracked back to the responsible gene, based on its genetic map position Functional genomics relies heavily on the various tools of bioinformatics to identify gene sequences of potential interest from the many molecular biology databases now available There is a continuing need to identify and characterise further genes and their related polypeptides/proteins, as targets for drug discovery EPRGl (SOCS-3) was isolated from a human hematopoietic cell line which requires EPO for proliferation The expression of EPRG 1 is induced by EPO, and the presence of EPO is required for maintenance of its expression EPRGl is involved in the proliferation of EPO- dependent cells and may be important in the growth and development of erythroid and other hematopoietic lineages Expression of EPRGl is induced to a high level in human bone marrow treated with G-CSF, and a substantial level of EPRGl is also expressed in fetal liver, peripheral blood leukocytes, lung, and in human bone marrow stimulated with EPO Furthermore, expression of EPRGl is induced by EPO and by TPO in the presence of cycloheximide, indicative of a primary genetic response As a primary response gene, expression of EPRGl is a direct result of activation of signalling pathways following binding of EPO or of TPO to their cognate receptors These data indicate that EPRGl is useful in preventing, ameliorating or correcting dysfunctions or diseases, including, but not limited to, cytopenias, including anemia, neutropenia, and thrombocytopenia (including those caused by genetic factors, myelosuppressive cancer chemotherapy, radiotherapy or accidental irradiation), polycythemia, cancer, metabolic or genetic growth deficiencies; obesity; infection via immunostimulation; infectious disease or cancer via adjuvant enhancement of vaccine therapy; AIDS; drug-induced anemias; autoimmune diseases, such as rheumatoid arthritis, diabetes, multiple sclerosis; and inflammatory diseases, such as asthma and allergies. Summary of the Invention The present invention relates to EPRGl, in particular EPRGl polypeptides and EPRGl polynucleotides, recombinant materials and methods for their production. In another aspect, the invention relates to methods for using such polypeptides and polynucleotides, including the treatment or prophylaxis of cytopenias, including anemia, neutropenia, and thrombocytopenia (including those caused by genetic factors, myelosuppressive cancer chemotherapy, radiotherapy or accidental irradiation); polycythemia; cancer; metabolic or genetic growth deficiencies; obesity; infection via immunostimulation; infectious disease or cancer via adjuvant enhancement of vaccine therapy ; AIDS; drug-induced anemias; autoimmune diseases, such as rheumatoid arthritis, diabetes, multiple sclerosis; and inflammatory diseases, such as asthma and allergies, hereinafter referred to as "the Diseases", amongst others. In a further aspect, the invention relates to methods for identifying agonists and antagonists/inhibitors using the materials provided by the invention, and treating conditions associated with EPRGl imbalance with the identified compounds. In a still further aspect, the invention relates to diagnostic assays for detecting diseases associated with inappropriate EPRGl activity or levels. Description of the Invention In a first aspect, the present invention relates to EPRGl polypeptides. Such peptides include isolated polypeptides comprising an amino acid sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% identity, to that of SEQ ID NO:2 over the entire length of SEQ ID NO:2. Such polypeptides include those comprising the amino acid of SEQ ID NO:2. Further peptides of the present invention include isolated polypeptides in which the amino acid sequence has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% identity, to the amino acid sequence of SEQ ID NO:2 over the entire length of SEQ ID NO:2. Such polypeptides include the polypeptide of SEQ ID NO:2. Further peptides of the present invention include isolated polypeptides encoded by a polynucleotide comprising the sequence contained in SEQ ID NO 1 EPRGl polypeptides of the present invention also include isolated polypeptides comprising an amino acid sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% identity, to that of SEQ ID NO 4 over the entire length of SEQ ID NO 4 Such polypeptides include those composing the amino acid of SEQ ID NO 4 Further peptides of the present invention include isolated polypeptides in which the amino acid sequence has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% identity, to the amino acid sequence of SEQ ID NO 4 over the entire length of SEQ ID NO 4 Such polypeptides include the polypeptide of SEQ ID NO 4 Further peptides of the present invention include isolated polypeptides encoded by a polynucleotide comprising the sequence contained in SEQ ID NO 3 Polypeptides of the present invention are believed to be members of the SOCS family of polypeptides They are therefore of interest because genes in this family modulate activation of signal transduction pathways from cell growth and differentiation factors These properties are hereinafter referred to as "EPRGl activity" or "EPRGl polypeptide activity" or "biological activity of EPRGl " Also included amongst these activities are antigenic and immunogenic activities of said EPRGl polypeptides, in particular the antigenic and immunogenic activities of the polypeptide of SEQ ID NO 2 or 4 Preferably, a polypeptide of the present invention exhibits at least one biological activity of EPRGl The polypeptides of the present invention may be in the form of the ' mature' protein or may be a part of a larger protein such as a fusion protein It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro- sequences, sequences which aid in purification such as multiple histidine residues, or an additional sequence for stability during recombinant production The present invention also includes include variants of the aforementioned polypeptides, that is polypeptides that vary from the referents by conservative ammo acid substitutions, whereby a residue is substituted by another with like characteristics Typical such substitutions are among Ala, Val, Leu and He, among Ser and Thr, among the acidic residues Asp and Glu among Asn and Gin, and among the basic residues Lys and Arg, or aromatic residues Phe and Tyr Particularly preferred are vaπants in which several, 5-10, 1-5, 1-3, 1-2 or 1 amino acids are substituted, deleted, or added in any combination Polypeptides of the present invention can be prepared in any suitable manner Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods Means for preparing such polypeptides are well understood in the art In a further aspect, the present invention relates to EPRGl polynucleotides Such polynucleotides include isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, to the amino acid sequence of SEQ ID NO 2, over the entire length of SEQ ID NO 2 In this regard, polypeptides which have at least 97% identity are highly preferred, whilst those with at least 98-99% identity are more highly preferred, and those with at least 99% identity are most highly preferred Such polynucleotides include a polynucleotide comprising the nucleotide sequence contained in SEQ ID NO 1 encoding the polypeptide of SEQ ID NO 2 Further polynucleotides of the present invention include isolated polynucleotides comprising a nucleotide sequence that has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, to a nucleotide sequence encoding a polypeptide of SEQ ID NO 2, over the entire coding region In this regard, polynucleotides which have at least 97% identity are highly preferred, whilst those with at least 98-99% identity are more highly preferred, and those with at least 99% identity are most highly preferred Further polynucleotides of the present invention include isolated polynucleotides comprising a nucleotide sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, to SEQ ID NO 1 over the entire length of SEQ ID NO 1 In this regard, polynucleotides which have at least 97% identity are highly preferred, whilst those with at least 98-99% ldentiy are more highly preferred, and those with at least 99% identity are most highly preferred Such polynucleotides include a polynucleotide comprising the polynucleotide of SEQ ID NO 1 as well as the polynucleotide of SEQ ID NO 1 The invention also provides polynucleotides which are complementary to all the above described polynucleotides The nucleotide sequence of SEQ ID NO 1 is a cDNA sequence and comprises a polypeptide encoding sequence (nucleotide 28 to 705) encoding a polypeptide of 225 amino acids, the polypeptide of SEQ ID NO 2 The nucleotide sequence encoding the polypeptide of SEQ ID NO 2 may be identical to the polypeptide encoding sequence contained in SEQ ID NO 1 or it may be a sequence other than the one contained in SEQ ID NO 1 , which, as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO 2 Preferred polypeptides and polynucleotides of the present invention are expected to have, inter alia, similar biological functions/properties to their homologous polypeptides and polynucleotides Furthermore, preferred polypeptides and polynucleotides of the present invention have at least one EPRGl activity The present invention also relates to partial or other polynucleotide and polypeptide sequences which were first identified prior to the determination of the corresponding full length sequences of SEQ ID NO 1 and SEQ ID NO 2 Accordingly, in a further aspect, the present invention provides for an isolated polynucleotide (a) comprising a nucleotide sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at least 97-997c identity to SEQ ID NO 5 over the entire length of SEQ ID NO 5, (b) comprising a nucleotide sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at least 97-99% identity, to SEQ ID NO 5 over the entire length of SEQ ID NO 5, (c) comprising the polynucleotide of SEQ ID NO 5, (d) the polynucleotide of SEQ ID NO 5 , or (e) a polynucleotide which is complementary to a polynucleotide of (a), (b), (c) or (d) In a further aspect, the present invention provides for an isolated polynucleotide (a) comprising a nucleotide sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at least 97-99% identity to SEQ ID NO over the entire length of SEQ ID NO 3, (b) comprising the polynucleotide of SEQ ID NO 3, (c) comprising a nucleotide sequence encoding a polypeptide which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at least 97-99% identity, to the amino acid sequence of SEQ ID NO 4, over the entire length of SEQ ID NO 4, (d) the polynucleotide of SEQ ID NO 3, or (e) a polynucleotide which is complementary to a polynucleotide of (a), (b), (c), or (d) The present invention further provides for a polypeptide which (a) comprises an amino acid sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% identity, to that of SEQ ID NO 4 over the entire length of SEQ ID NO 4, (b) has an amino acid sequence which is at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% identity, to the amino acid sequence of SEQ ID NO 4 over the entire length of SEQ ID NO 4, (c) comprises the amino acid of SEQ ID NO 4, and (d) is the polypeptide of SEQ ID NO 4, as well as polypeptides encoded by a polynucleotide comprising the sequence contained in SEQ ID NO 3 The nucleotide sequence of SEQ ID NO 5 is derived from EST (Expressed Sequence Tag) sequences It is recognised by those skilled in the art that there will inevitably be some nucleotide sequence reading errors in EST sequences (see Adams, M D et al. Nature 377 (supp) 3, 1995) Accordingly, the nucleotide sequence of SEQ ID NO 5 is therefore subject to the same inherent limitations in sequence accuracy Polynucleotides of the present invention may be obtained, using standard cloning and screening techniques, from a cDNA library derived from mRNA in cells of human bone marrow and hematopoietic cells, using the expressed sequence tag (EST) analysis (Adams, M D , et al Science ( 1991 ) 252 1651 - 1656, Adams, M D et al , Nature, ( 1992) 355 632-634, Adams, M O , et al , Nature ( 1995) 377 Supp 3- 174) Polynucleotides of the invention can also be obtained from natural sources such as genomic DNA libraries or can be synthesized using well known and commercially available techniques When polynucleotides of the present invention are used for the recombinant production of polypeptides of the present invention, the polynucleotide may include the coding sequence for the mature polypeptide, by itself; or the coding sequence for the mature polypeptide in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence, or other fusion peptide portions. For example, a marker sequence which facilitates purification of the fused polypeptide can be encoded. In certain preferred embodiments of this aspect of the invention, the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz et al, Proc Natl Acad Sci USA (1989) 86:821-824, or is an HA tag. The polynucleotide may also contain non-coding 5' and 3' sequences, such as transcribed, non-translated sequences, splicing and polyadenylation signals, ribosome binding sites and sequences that stabilize mRNA. Further embodiments of the present invention include polynucleotides encoding polypeptide variants which comprise the amino acid sequence of SEQ ID NO:2 or 4 and in which several, for instance from 5 to 10, 1 to 5, 1 to 3, 1 to 2 or 1, amino acid residues are substituted, deleted or added, in any combination. Polynucleotides which are identical or sufficiently identical to a nucleotide sequence contained in SEQ ID NO: 1 , 3 or 5 may be used as hybridization probes for cDNA and genomic DNA or as primers for a nucleic acid amplification (PCR) reaction, to isolate full-length cDNAs and genomic clones encoding polypeptides of the present invention and to isolate cDNA and genomic clones of other genes (including genes encoding homologs and orthologs from species other than human) that have a high sequence similarity to SEQ ID NO: 1 , 3 or 5. Typically these nucleotide sequences are 70% identical, preferably 80% identical, more preferably 90% identical, most preferably 95% identical to that of the referent. The probes or primers will generally comprise at least 15 nucleotides, preferably, at least 30 nucleotides and may have at least 50 nucleotides. Particularly preferred probes will have between 30 and 50 nucleotides. A polynucleotide encoding a polypeptide of the present invention, including homologs and orthologs from species other than human, may be obtained by a process which comprises the steps of screening an appropriate library under stringent hybridization conditions with a labeled probe having the sequence of SEQ ID NO: 1 , 3 or 5, or a fragment thereof; and isolating full- length cDNA and genomic clones containing said polynucleotide sequence. Such hybridization techniques are well known to the skilled artisan. Preferred stringent hybridization conditions include overnight incubation at 42°C in a solution comprising: 50% formamide, 5xSSC ( 150mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10 % dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm DNA; followed by washing the filters in 0. lx SSC at about 65°C. Thus the present invention also includes polynucleotides obtainable by screening an appropπate library under stingent hybridization conditions with a labeled probe having the sequence of SEQ ID NO 1 , 3 or 5, or a fragment thereof The skilled artisan will appreciate that, in many cases, an isolated cDNA sequence will be incomplete, in that the region coding for the polypeptide is cut short at the 5' end of the cDNA This is a consequence of reverse transcπptase, an enzyme with inherently low 'processivity' (a measure of the ability of the enzyme to remain attached to the template during the polymerisation reaction), failing to complete a DNA copy of the mRNA template duπng 1st strand cDNA synthesis There are several methods available and well known to those skilled in the art to obtain full-length cDNAs, or extend short cDNAs, for example those based on the method of Rapid Amplification of cDNA ends (RACE) (see, for example, Frohman et al , PNAS USA 85, 8998-9002, 1988) Recent modifications of the technique, exemplified by the Marathon™' technology (Clontech Laboratories Inc ) for example, have significantly simplified the search for longer cDNAs In the Marathon™ technology, cDNAs have been prepared from mRNA extracted from a chosen tissue and an 'adaptor' sequence ligated onto each end Nucleic acid amplification (PCR) is then carried out to amplify the 'missing' 5' end of the cDNA using a combination of gene specific and adaptor specific ohgonucleotide primers The PCR reaction is then repeated using nested' primers, that is, primers designed to anneal within the amplified product (typically an adaptor specific primer that anneals further 3' in the adaptor sequence and a gene specific primer that anneals further 5' in the known gene sequence) The products of this reaction can then be analysed by DNA sequencing and a full-length cDNA constructed either by joining the product directly to the existing cDNA to give a complete sequence, or carrying out a separate full-length PCR using the new sequence information for the design of the 5' primer Recombinant polypeptides of the present invention may be prepared by processes well known in the art from genetically engineered host cells comprising expression systems Accordingly, in a further aspect, the present invention relates to expression systems which comprise a polynucleotide or polynucleotides of the present invention, to host cells which are genetically engineered with such expression sytems and to the production of polypeptides of the invention by recombinant techniques Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention For recombinant production, host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention Introduction of polynucleotides into host cells can be effected by methods descπbed in many standard laboratory manuals, such as Davis et al , Basic Methods in Molecular Biology (1986) and Sambrook et al , Molecular Cloning A Laboratory Manual, 2nd Ed , Cold Spπng Harbor Laboratory Press, Cold Spπng Harbor, N Y (1989) Preferred such methods include, for instance, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, micromjection, cationic hpid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection Representative examples of appropπate hosts include bacterial cells, such as streptococci, staphylococci, E coli, Streptomyces and Bacillus subtilis cells, fungal cells, such as yeast cells and Aspergillus cells, insect cells such as Drosophda S2 and Spodoptera Sf9 cells, animal cells such as CHO, COS, HeLa, C127, 3T3. BHK, HEK 293 and Bowes melanoma cells, and plant cells A great variety of expression systems can be used, for instance, chromosomal, episomal and virus-derived systems, e g , vectors derived from bacterial plasmids, from bactenophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bactenophage genetic elements, such as cosmids and phagemids The expression systems may contain control regions that regulate as well as engender expression Generally, any system or vector which is able to maintain, propagate or express a polynucleotide to produce a polypeptide in a host may be used The appropriate nucleotide sequence may be inserted into an expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al , MOLECULAR CLONING, A LABORATORY MANUAL (supra) Appropriate secretion signals may be incorporated into the desired polypeptide to allow secretion of the translated protein into the lumen of the endoplasmic reticulum, the peπplasmic space or the extracellular environment These signals may be endogenous to the polypeptide or they may be heterologous signals If a polypeptide of the present invention is to be expressed for use in screening assays, it is generally preferred that the polypeptide be produced at the surface of the cell In this event, the cells may be harvested prior to use in the screening assay If the polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify the polypeptide If produced intracellularly, the cells must first be lysed before the polypeptide is recovered Polypeptides of the present invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography Most preferably, high performance liquid chromatography is employed for purification Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification This invention also relates to the use of polynucleotides of the present invention as diagnostic reagents Detection of a mutated form of the gene characterised by the polynucleotide of SEQ ID NO 1 , 3 or 5 which is associated with a dysfunction will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, or susceptibility to a disease, which results from under-expression, over-expression or altered expression of the gene Individuals carrying mutations in the gene may be detected at the DNA level by a variety of techniques The present invention also contemplates a method of detecting the presence of or absence of variations in an EPRG 1 polynucleotide in an individual from that of SEQ ID NOS 1 , 3 or 5, comprising the step of comparing an EPRG l polynucleotide sequence contained in a sample obtained from the individual with that of SEQ ID NOS 1 , 3, or 5, respectively Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy material The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR or other amplification techniques prior to analysis RNA or cDNA may also be used in similar fashion Deletions and insertions can be detected by a change in size of the amplified product in compaπson to the normal genotype Point mutations can be identified by hybridizing amplified DNA to labeled EPRG 1 nucleotide sequences Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures DNA sequence differences may also be detected by alterations in electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing (ee, e g , Myers et al , Science ( 1985) 230 1242) Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and S 1 protection or the chemical cleavage method (see Cotton et al , Proc Natl Acad Sci USA ( 1985) 85 4397-4401) In another embodiment, an array of o gonucleotides probes comprising EPRGl nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e g , genetic mutations Array technology methods are well known and have general applicability and can be used to address a variety of questions m molecular genetics including gene expression, genetic linkage, and genetic variability (see for example M Chee et al , Science, Vol 274, pp 610-613 (1996)) The diagnostic assays offer a process for diagnosing or determining a susceptibility to the Diseases through detection of mutation in the EPRGl gene by the methods described In addition, such diseases may be diagnosed by methods comprising determining from a sample derived from a subject an abnormally decreased or increased level of polypeptide or mRNA Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, nucleic acid amplification, for instance PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods Assay techniques that can be used to determine levels of a protein, such as a polypeptide of the present invention, in a sample derived from a host are well-known to those of skill in the art Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays Thus in another aspect, the present invention relates to a diagonostic kit which comprises (a) a polynucleotide of the present invention, preferably the nucleotide sequence of SEQ ID NO 1 , 3 or 5, or a fragment thereof , (b) a nucleotide sequence complementary to that of (a), (c) a polypeptide of the present invention, preferably the polypeptide of SEQ ID NO 2 or 4, or a fragment thereof, or (d) an antibody to a polypeptide of the present invention, preferably to the polypeptide of SEQ ID NO 2 or 4 It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component Such a kit will be of use in diagnosing a disease or suspectability to a disease, particularly cytopenias, including anemia, neutropenia, and thrombocytopenia (including those caused by genetic factors, myelosuppressive cancer chemotherapy, radiotherapy or accidental irradiation), polycythemia, cancer, metabolic or genetic growth deficiencies, obesity, infection via immunostimulation, infectious disease or cancer via adjuvant enhancement of vaccine therapy, AIDS, drug-induced anemias, autoimmune diseases, such as rheumatoid arthritis, diabetes, multiple sclerosis, and inflammatory diseases, such as asthma and allergies, amongst others The nucleotide sequences of the present invention are also valuable for chromosome identification The sequence is specifically targeted to, and can hybπdize with, a particular location on an individual human chromosome The mapping of relevant sequences to chromosomes according to the present invention is an important first step in correlating those sequences with gene associated disease Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data Such data are found in, for example, V McKusick, Mendehan Inheritance in Man (available on-line through Johns Hopkins University Welch Medical Library) The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheπtance of physically adjacent genes) The differences in the cDNA or genomic sequence between affected and unaffected individuals can also be determined If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease The polypeptides of the invention or their fragments or analogs thereof, or cells expressing them, can also be used as immunogens to produce antibodies lmmunospecific for polypeptides of the present invention The term "lmmunospecific" means that the antibodies have substantially greater affinity for the polypeptides of the invention than their affinity for other related polypeptides in the prior art Antibodies generated against polypeptides of the present invention may be obtained by administering the polypeptides or epitope-beaπng fragments, analogs or cells to an animal, preferably a non-human animal, using routine protocols For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used Examples include the hybπdoma technique (Kohler, G and Milstein, C , Nature (1975) 256 495-497), the tπoma technique, the human B-cell hybπdoma technique (Kozbor et al , Immunology Today (1983) 4 72) and the EBV-hybπdoma technique (Cole et al , MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp 77-96, Alan R Liss, Inc , 1985) Techniques for the production of single chain antibodies, such as those described in U S Patent No 4,946,778, can also be adapted to produce single chain antibodies to polypeptides of this invention Also, transgenic mice, or other organisms, including other mammals, may be used to express humanized antibodies The above-described antibodies may be employed to isolate or to identify clones expressing the polypeptide or to purify the polypeptides by affinity chromatography Antibodies against polypeptides of the present invention may also be employed to treat the Diseases, amongst others In a further aspect, the present invention relates to genetically engineered soluble fusion proteins comprising a polypeptide of the present invention, or a fragment thereof, and various portions of the constant regions of heavy or light chains of immunoglobulins of various subclasses (IgG, IgM, IgA, IgE) Preferred as an immunoglobulin is the constant part of the heavy chain of human IgG, particularly IgGl , where fusion takes place at the hinge region In a particular embodiment, the Fc part can be removed simply by incorporation of a cleavage sequence which can be cleaved with blood clotting factor Xa Furthermore, this invention relates to processes for the preparation of these fusion proteins by genetic engineering, and to the use thereof for drug screening, diagnosis and therapy A further aspect of the invention also relates to polynucleotides encoding such fusion proteins Examples of fusion protein technology can be found in International Patent Application Nos W094/29458 and W094/22914 Another aspect of the invention relates to a method for inducing an immunological response in a mammal which comprises inoculating the mammal with a polypeptide of the present invention, adequate to produce antibody and/or T cell immune response to protect said animal from the Diseases hereinbefore mentioned, amongst others Yet another aspect of the invention relates to a method of inducing immunological response in a mammal which comprises, delivering a polypeptide of the present invention via a vector directing expression of the polynucleotide and coding for the polypeptide in vivo in order to induce such an immunological response to produce antibody to protect said animal from diseases A further aspect of the invention relates to an immunological/vaccine formulation (composition) which, when introduced into a mammalian host, induces an immunological response in that mammal to a polypeptide of the present invention wherein the composition comprises a polypeptide or polynucleotide of the present invention The vaccine formulation may further comprise a suitable carrier Since a polypeptide may be broken down in the stomach, it is preferably administered parenterally (for instance, subcutaneous, intramuscular, intravenous, or intradermal injection) Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteπostats and solutes which render the formulation instonic with the blood of the recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dπed condition requiring only the addition of the sterile liquid carrier immediately prior to use The vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known in the art The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation Polypeptides of the present invention are responsible for many biological functions, including many disease states, in particular the Diseases hereinbefore mentioned It is therefore desirous to devise screening methods to identify compounds which stimulate or which inhibit the function of the polypeptide Accordingly, in a further aspect, the present invention provides for a method of screening compounds to identify those which stimulate or which inhibit the function of the polypeptide In general, agonists or antagonists may be employed for therapeutic and prophylactic purposes for such Diseases as hereinbefore mentioned Compounds may be identified from a variety of sources, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures Such agonists, antagonists or inhibitors so-identified may be natural or modified substrates, ligands, receptors, enzymes, etc , as the case may be, of the polypeptide, or may be structural or functional mimetics thereof (see Coligan et al , Current Protocols in Immunology 1(2) Chapter 5 ( 1991 )) The screening method may simply measure the binding of a candidate compound to the polypeptide, or to cells or membranes bearing the polypeptide, or a fusion protein thereof by means of a label directly or indirectly associated with the candidate compound Alternatively, the screening method may involve competition with a labeled competitor Further, these screening methods may test whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide, using detection systems appropriate to the cells bearing the polypeptide Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed Constitutively active polpypeptides may be employed in screening methods for inverse agonists or inhibitors, in the absence of an agonist or inhibitor, by testing whether the candidate compound results in inhibition of activation of the polypeptide Further the screening methods may simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide of the present invention, to form a mixture, measunng EPRGl activity in the mixture, and comparing the EPRGl activity of the mixture to a standard Fusion proteins, such as those made from Fc portion and EPRGl polypeptide, as hereinbefore described, can also be used for high-throughput screening assays to identify antagonists for the polypeptide of the present invention (see D Bennett et al , J Mol Recognition, 8 52-58 ( 1995), and K Johanson et al , J Biol Chem, 270(16) 9459-9471 (1995)) The interaction of the SH2 domain with a phosphotyrosine peptide is highly specific for a given SH2 domain and its particular peptide substrate The formation of this complex can be used as the basis to configure an in vitro high-throughput screen using ELISA or other suitable means for detection of the association The polynucleotides, polypeptides and antibodies to the polypeptide of the present invention may also be used to configure screening methods for detecting the effect of added compounds on the production of mRNA and polypeptide in cells For example, an ELISA assay may be constructed for measuring secreted or cell associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known in the art This can be used to discover agents which may inhibit or enhance the production of polypeptide (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues The polypeptide may be used to identify membrane bound or soluble receptors, if any, through standard receptor binding techniques known in the art These include, but are not limited to, ligand binding and crosslinking assays in which the polypeptide is labeled with a radioactive isotope (for instance, '-^1), chemically modified (for instance, biotinylated), or fused to a peptide sequence suitable for detection or purification, and incubated with a source of the putative receptor (cells, cell membranes, cell supernatants, tissue extracts, bodily fluids) Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy These screening methods may also be used to identify agonists and antagonists of the polypeptide which compete with the binding of the polypeptide to its receptors, if any Standard methods for conducting such assays are well understood in the art Examples of potential polypeptide antagonists include antibodies or, in some cases, o gonucleotides or proteins which are closely related to the ligands, substrates, receptors, enzymes, etc , as the case may be, of the polypeptide, e g , a fragment of the ligands, substrates, receptors, enzymes, etc , or small molecules which bind to the polypeptide of the present invention but do not elicit a response, so that the activity of the polypeptide is prevented Included in the definition of antagonists are anti-sense ohgonucleotides (ASO) compπsing the sequences of SEQ ID NOS 6 and 7 (anti-sense) gaCCATGGCGCACGGAGccA (SEQ ID NO 6) (anti-sense) gcTGTGGGTGACCATGGcgC (SEQ ID NO 7) Lower case nucleotide base indicates 3'-phosphorothιoate linkage Thus, in another aspect, the present invention relates to a screening kit for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, etc for polypeptides of the present invention, or compounds which decrease or enhance the production of such polypeptides, which comprises (a) a polypeptide of the present invention, (b) a recombinant cell expressing a polypeptide of the present invention, (c) a cell membrane expressing a polypeptide of the present invention, or (d) antibody to a polypeptide of the present invention, which polypeptide is preferably that of SEQ ID NO 2 or 4 It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component It will be readily appreciated by the skilled artisan that a polypeptide of the present invention may also be used in a method for the structure-based design of an agonist, antagonist or inhibitor of the polypeptide, by (a) determining in the first instance the three dimensional structure of the polypeptide (b) deducing the three-dimensional structure for the likely reactive or binding sιte(s) of an agonist, antagonist or inhibitor, (c) synthesing candidate compounds that are predicted to bind to or react with the deduced binding or reactive site, and (d) testing whether the candidate compounds are indeed agonists antagonists or inhibitors It will be further appreciated that this will normally be an interative process In a further aspect, the present invention provides methods of treating or preventing abnormal conditions such as, for instance, cytopenias, including anemia, neutropenia, and thrombocytopenia (including those caused by genetic factors, myelosuppressive cancer chemotherapy, radiotherapy or accidental irradiation), polycythemia, cancer, metabolic or genetic growth deficiencies, obesity, infection via immunostimulation, infectious disease or cancer via adjuvant enhancement of vaccine therapy, AIDS, drug-induced anemias, autoimmune diseases, such as rheumatoid arthritis, diabetes, multiple sclerosis, and inflammatory diseases, such as asthma and allergies, related to either an excess of, or an under-expression of, EPRGl polypeptide activity If the activity of the polypeptide is in excess, several approaches are available One approach compπses administering to a subject m need thereof an inhibitor compound (antagonist) as heieinabove described, optionally in combination with a pharmaceutically acceptable carrier, in an amount effective to inhibit the function of the polypeptide, such as, for example, by blocking the binding of ligands, substrates, receptors, enzymes, etc , or by inhibiting a second signal, and thereby alleviating the abnormal condition In another approach, soluble forms of the polypeptides still capable of binding the ligand, substrate, enzymes, receptors, etc in competition with endogenous polypeptide may be administered Typical examples of such competitors include fragments of the EPRGl polypeptide In still another approach, expression of the gene encoding endogenous EPRGl polypeptide can be inhibited using expression blocking techniques Known such techniques involve the use of antisense sequences, such as those comprising the sequence of SEQ ID NO 6 or 7, either internally generated or separately administered (see, for example, O'Connor, J Neurochem ( 1991 ) 56 560 in Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL ( 1988)) Alternatively, a gene therapy-based approach can be used to deliver an expression cassette for production of an anti-sense message directly in vivo The anti-sense message can be optimized for effectiveness by size and location, and avoid toxicity issues associated with artificial ohgonucleotides In yet another approach, ohgonucleotides which form triple helices with the gene can be supplied (see, for example, Lee et al , Nucleic Acids Res ( 1979) 6 3073, Cooney et al , Science ( 1988) 241 456, Dervan et al , Science ( 1991 ) 251 1360) These oligomers can be administered per se or the relevant oligomers can be expressed in vivo EPRGl is a negative regulator that acts as a feedback inhibitor Inhibition of EPRGl function enhances the growth and differentiation of hematopoietic cells Consequently, ex vivo treatment of hematopoietic progenitors / stem cells from bone marrow, cord blood or blood following stem cell mobilization protocols is expected to improve the effectiveness of hematopoietic recovery Treatment would consist of incubation of inhibitors specific for EPRG 1 , such as anti-sense ohgonucleotides (ASO), with hematopoietic cells for a peπod of time under conditions in which inhibitor (e.g ohgonucleotide) uptake can occur EPRG-1 inhibitor treated hematopoietic cells could be directly transplanted into a patient needing a stem cell transplant or cultured ex vivo in expansion medium containing one or more hematopoietic growth factors (such as G-CSF, EPO, TPO, IL- 11 , IL-3, PEG-rHuMGDF (polyethylene glycol-conjugated recombinant human megakaryocyte growth and development factor), FLT-3, NESP (novel erythropoiesis stimulating protein), NEUPOGEN SD, etc ) to increase the number of progenitor / stem cells before transplantation. In view of the above, one particular envisioned utility of the inhibitor is the in vivo administration for the treatment of cytopenia The cytopenia condition may be genetic or due to myelosuppressive cancer chemotherapy, radiotherapy or accidental irradiation In vivo administration of an inhibitor of EPRG- 1 may be effective alone or optimally in combination with cytokine therapy (l e G-CSF, PEG-rHuMGDF , EPO, TPO, IL-1 1 , IL-3, FLT-3, NESP, NEUPOGEN SD, etc ) In one preferred aspect, the present invention relates to the EPRGl anti-sense ohgonucleotides Such anti-sense ohgonucleotides preferably include chemically synthesized phosphorothioate linked ohgonucleotides which contain nucleotide sequences which effectively hybridize with SOCS-3 mRNA and prevent SOCS-3 protein production, and any other ohgonucleotides which can hybridize to SOCS-3 mRNA that have other backbone modifications made to confer nuclease resistance Examples of such chemically synthesized phosphorothioate linked ohgonucleotides are the sequences comprising the sequence of SEQ ID NO 6 or 7, In another preferred aspect of the invention relates to a method for increasing the effectiveness and engraftment of hematopoietic stem cell transplantation by treating human hematopoietic progenitor / stem cells with an inhibitor of EPRG l , comprising (a) mixing a EPRGl inhibitor with a solution containing hematopoietic progenitor and stem cells, ex vivo, followed by transplantation of the hematopoietic cells into a patient in need thereof, or (b) mixing a EPRGl inhibitor with a solution containing hematopoietic cells in an ex vivo stem cell expansion culture system followed by transplantation of the hematopoietic cells into the patient, or (c) administering EPRGl inhibitor in vivo as an adjunct to hematopoietic stem cell transplantation or during the treatment of cytopenia (e g chemotherapy-induced neutropenia, anemia, thrombocytopenia) The preferred EPRGl inhibitor is an antisense ohgonucleotide, and in particular the sequence comprising the sequence of SEQ ID NO 6 or 7 Yet in another aspect the instant invention relates to a method for increasing the efficiency of gene therapy transfer into hematopoietic stem cells comprising (a) isolating hematopoietic stem cells or modified stem cell lines and incubating with EPRGl inhibitor before transfection with plasmid vectors containing any corrective gene of interest useful in treating a hereditary genetic diseases (e g ADA deficiency gene therapy) Preferred EPRG 1 inhibitor is an antisense ohgonucleotide, and in particular the sequence comprising the sequence of SEQ ID NO 6 or 7 For treating abnormal conditions related to an under-expression of EPRGl and its activity, several approaches are also available One approach comprises administering to a subject a therapeutically effective amount of a compound which activates a polypeptide of the present invention, 1 e , an agonist as described above, in combination with a pharmaceutically acceptable carrier, to thereby alleviate the abnormal condition Alternatively, gene therapy may be employed to effect the endogenous production of EPRGl by the relevant cells in the subject For example, a polynucleotide of the invention may be engineered for expression in a replication defective retroviral vector, as discussed above The retroviral expression construct may then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention such that the packaging cell now produces infectious viral particles containing the gene of interest These producer cells may be administered to a subject for engineering cells in vivo and expression of the polypeptide in vivo For an overview of gene therapy, see Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeutic Approaches, (and references cited therein) in Human Molecular Genetics, T Strachan and A P Read, BIOS Scientific Publishers Ltd ( 1996) Another approach is to administer a therapeutic amount of a polypeptide of the present invention in combination with a suitable pharmaceutical carrier In a further aspect, the present invention provides for pharmaceutical compositions comprising a therapeutically effective amount of a polypeptide, such as the soluble form of a polypeptide of the present invention, agonist/antagonist peptide or small molecule compound, in combination with a pharmaceutically acceptable carrier or excipient Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof The invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention Polypeptides and other compounds of the present invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds The composition will be adapted to the route of administration, for instance by a systemic or an oral route Preferred forms of systemic administration include injection, typically by intravenous injection Other injection routes, such as subcutaneous, intramuscular, or intrapentoneal, can be used Alternative means for systemic administration include transmucosal and transdermal administration using penetrants such as bile salts or fusidic acids or other detergents In addition, if a polypeptide or other compounds of the present invention can be formulated m an entenc or an encapsulated formulation, oral administration may also be possible Administration of these compounds may also be topical and/or localized, in the form of salves, pastes, gels, and the like The dosage range required depends on the choice of peptide or other compounds of the present invention, the route of administration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attending practitioner Suitable dosages, however, are in the range of 0 1-100 μg/kg of subject Wide variations in the needed dosage, however, are to be expected in view of the variety of compounds available and the differing efficiencies of various routes of administration For example, oral administration would be expected to require higher dosages than administration by intravenous injection Variations in these dosage levels can be adjusted using standard empirical routines for optimization, as is well understood in the art Polypeptides used in treatment can also be generated endogenously in the subject, in treatment modalities often referred to as "gene therapy" as described above Thus, for example, cells from a subject may be engineered with a polynucleotide, such as a DNA or RNA, to encode a polypeptide ex vivo, and for example, by the use of a retroviral plasmid vector The cells are then introduced into the subject Polynucleotide and polypeptide sequences form a valuable information resource with which to identify further sequences of similar homology This is most easily facilitated by storing the sequence in a computer readable medium and then using the stored data to search a sequence database using well known searching tools, such as GCG Accordingly, in a further aspect, the present invention provides for a computer readable medium having stored thereon a polynucleotide comprising the sequence of SEQ ID NO 1 , 3 or 5 and/or a polypeptide sequence encoded thereby The following definitions are provided to facilitate understanding of certain terms used frequently hereinbefore "Antibodies" as used herein includes polyclonal and monoclonal antibodies, chimeπc, single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other immunoglobulin expression library "Isolated" means altered "by the hand of man" from the natural state If an "isolated" composition or substance occurs in nature, it has been changed or removed from its original environment, or both For example, a polynucleotide or a polypeptide naturally present in a living animal is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein "Polynucleotide" generally refers to any polyπbonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA "Polynucleotides" include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions In addition, "polynucleotide" refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA The term 'polynucleotide" also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons "Modified" bases include, for example tπtylated bases and unusual bases such as inosine A variety of modifications may be made to DNA and RNA thus, "polynucleotide" embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells "Polynucleotide" also embraces relatively short polynucleotides, often referred to as ohgonucleotides "Polypeptide" refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, I e , peptide isosteres "Polypeptide" refers to both short chains, commonly referred to as peptides, ohgopeptides or oligomers, and to longer chains, generally referred to as proteins Polypeptides may contain amino acids other than the 20 gene-encoded amino acids "Polypeptides" include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature Modifications may occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini It will be appreciated that the same type of modification may be present to the same or varying degrees at several sites in a given polypeptide Also, a given polypeptide may contain many types of modifications Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching Cyclic, branched and branched cyclic polypeptides may result from post-translation natural processes or may be made by synthetic methods Modifications include acetylation, acylation, ADP-πbosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidyhnositol, cross-linking, cychzation, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, lodmation, methylation, myπstoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination (see, for instance, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed , T E Creighton, W H Freeman and Company, New York, 1993, Wold, F , Post-translational Protein Modifications Perspectives and Prospects, pgs 1 - 12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B C Johnson, Ed , Academic Press, New York, 1983, Seifter et al , "Analysis for protein modifications and nonprotein cofactors", Meth Enzv ol ( 1990) 182 626-646 and Rattan et al , "Protein Synthesis Post-translational Modifications and Aging", Ann NY Acad Set ( 1992) 663 48-62) "Variant" refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains essential properties A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination A substituted or inserted amino acid residue may or may not be one encoded by the genetic code A vaπant of a polynucleotide or polypeptide may be a naturally occurring such as an allehc vaπant, or it may be a variant that is not known to occur naturally Non- naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis "Identity," as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be, as determined by compaπng the sequences In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences "Identity" can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A M , ed , Oxford University Press, New York, 1988, Biocomputing Informatics and Genome Projects, Smith, D W , ed , Academic Press, New York, 1993, Computer Analysis of Sequence Data, Part I, Griffin, A M , and Griffin, H G , eds , Humana Press, New Jersey, 1994, Sequence Analysis in Molecular Biology, von Hemje, G , Academic Press, 1987, and Sequence Analysis Primer, Gπbskov, M and Devereux, J , eds , M Stockton Press, New York, 1991 , and Caπllo, H , and Lipman, D , SIAM J Applied Math , 48 1073 ( 1988) Methods to determine identity are designed to give the largest match between the sequences tested Moreover, methods to determine identity are codified in publicly available computer programs Computer program methods to determine identity between two sequences include, but are not limited to, the GCG program package (Devereux, J , et al , Nucleic Acids Research 12(1) 387 ( 1984)), BLASTP, BLASTN, and FASTA (Atschul, S F et al , J Molec Biol 215 403-410 ( 1990) The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S , et al , NCBI NLM NIH Bethesda, MD 20894, Altschul, S , et al , J Mol Bwl 215 403-410 ( 1990) The well known Smith Waterman algorithm may also be used to determine identity Parameters for polypeptide sequence comparison include the following 1 ) Algorithm Needleman and Wunsch, J Mol Biol 48 443-453 ( 1970) Comparison matrix BLOSSUM62 from Hentikoff and Hentikoff, Proc Natl Acad Sci USA 89 10915-10919 ( 1992) Gap Penalty 12 Gap Length Penalty 4 A program useful with these parameters is publicly available as the "gap" program from Genetics Computer Group, Madison WI The aforementioned parameters are the default parameters for peptide comparisons (along with no penalty for end gaps) Parameters for polynucleotide comparison include the following 1) Algorithm Needleman and Wunsch, J Mol Biol 48 443-453 (1970) Comparison matrix matches = +10, mismatch = 0 Gap Penalty 50 Gap Length Penalty 3 Available as The "gap" program from Genetics Computer Group, Madison WI These are the default parameters for nucleic acid comparisons A preferred meaning for "identity" for polynucleotides and polypeptides, as the case may be, are provided in ( 1) and (2) below (1 ) Polynucleotide embodiments further include an isolated polynucleotide comprising a polynucleotide sequence having at least a 50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to the reference sequence of SEQ ID NO 1 , wherein said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO 1 or may include up to a certain integer number of nucleotide alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ ID NO 1 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of nucleotides in SEQ ID NO 1 , or n n < x n - (x n • y), wherein n n is the number of nucleotide alterations, x n is the total number of nucleotides in SEQ ID NO 1 , y is 0 50 for 50%, 0 60 for 60%, 0 70 for 70%, 0 80 for 80%, 0 8 for 85%, 0 90 for 90%, 0 95 for 95%, 0 97 for 97% or 1 00 for 100%, and • is the symbol for the multiplication operator, and wherein any non-integer product of x n and y is rounded down to the nearest integer prior to subtracting it from x n Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO 2 may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations By way of example, a polynucleotide sequence of the present invention may be identical to the reference sequence of SEQ ID NO 2, that is it may be 100% identical, or it may include up to a certain integer number of amino acid alterations as compared to the reference sequence such that the percent identity is less than 100% identity Such alterations are selected from the group consisting of at least one nucleic acid deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference polynucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleic acids in the reference sequence or in one or more contiguous groups within the reference sequence The number of nucleic acid alterations for a given percent identity is determined by multiplying the total number of amino acids in SEQ ID NO 2 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of amino acids in SEQ ID NO 2, or n n < x n - (x n • y), wherein n n is the number of amino acid alterations, x n is the total number of amino acids in SEQ ID NO 2, y is, for instance 0 70 for 70%, 0 80 for 80%, 0 85 for 85% etc • is the symbol for the multiplication operator, and wherein any non-integer product of x n and y is rounded down to the nearest integer prior to subtracting it from x n (2) Polypeptide embodiments further include an isolated polypeptide comprising a polypeptide having at least a 50,60, 70, 80, 85, 90, 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO 2, wherein said polypeptide sequence may be identical to the reference sequence of SEQ ID NO 2 or may include up to a certain integer number of amino acid alterations as compared to the reference sequence wherein said alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of amino acid alterations is determined by multiplying the total number of amino acids in SEQ ID NO 2 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of ammo acids in SEQ ID NO 2, or n a ≤ x a (*a • y)- wherein n a is the number of amino acid alterations, x a is the total number of ammo acids in SEQ ID NO 2, y is 0 50 for 50%, 0 60 for 60%, 0 70 for 70%, 0 80 for 80%, 0 85 for 85%, 0 90 for 90%, 0 95 for 95%, 0 97 for 97% or 1 00 for 100%, and • is the symbol for the multiplication operator, and wherein any non-integer product of x a and y is rounded down to the nearest integer prior to subtracting it from x a By way of example, a polypeptide sequence of the present invention may be identical to the reference sequence of SEQ ID NO 2, that is it may be 100% identical, or it may include up to a certain integer number of am o acid alterations as compared to the reference sequence such that the percent identity is less than 100% identity Such alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non- conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence The number of amino acid alterations for a given % identity is determined by multiplying the total number of amino acids in SEQ ID NO 2 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of amino acids in SEQ ID NO 2, or "Λ ≤ X - ( x a * )» wherein n a is the number of amino acid alterations, x d is the total number of amino acids in SEQ ID NO 2, y is, for instance 0 70 for 70%, 0 80 for 80%, 0 85 for 85% etc , and • is the symbol for the multiplication operator, and wherein any non-integer product of x a and y is rounded down to the nearest integer prior to subtracting it from x a "Fusion protein" refers to a protein encoded by two, often unrelated, fused genes or fragments thereof In one example, EP-A-0 464 discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof In many cases, employing an immunoglobulin Fc region as a part of a fusion protein is advantageous for use in therapy and diagnosis resulting in, for example, improved pharmacokinetic properties [see, e g , EP-A 0232 262] On the other hand, for some uses it would be desirable to be able to delete the Fc part after the fusion protein has been expressed, detected and purified All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth Example 1 On northern blots of UT7-EPO cells, EPRGl is observed as a single 2 4 kb message EPRGl can be detected at low levels in log phase cells proliferating in medium supplemented with 10% FBS and rh-EPO Upon overnight starvation for EPO, message levels decay to undetectable levels EPRGl is rapidly induced upon EPO stimulation, being readily detectable at 1/2 hour, with the maximum level achieved at about 1 hour Somewhat higher levels are attained when cells have been starved for FBS as well as EPO Expression of EPRG l then decreases, but remains elevated for at least 6 hours EPRGl is induced by EPO in the presence of the protein synthesis inhibitor, cycloheximide, even though there is no induction of EPRG l when cycloheximide alone is added The treatment with EPO+cycloheximide leads to modest supeπnduction of EPRGl message levels over that obtained with EPO by itself EPRGl is also induced by TPO in human megakaryoblastic leukemia cell lines, M07e and CMK, as well as being superinduced when stimulated in the presence of cycloheximide These observations show EPRGl to be an EPO and a TPO primary response gene, whose induction of expression is not dependent on new protein synthesis Induction of EPRGl expression must therefore occur through direct activation of signalling pathway(s) by EPO and by TPO In human bone marrow, EPRGl is also expressed as a single 2 4 kb message on northern blots It is induced in in vitro culture by treatment with EPO, but expression is much more strongly induced by stimulation with G-CSF The elevated expression of EPRG l is sustained by the cells in culture for at least 7 days, showing that EPRG l likely serves an important function in the development and maturation of different hematopoietic lineages Expression of EPRGl has been found by probing northern blots with RNA from other tissues The highest levels occur in hematopoietically related ones, including bone marrow, fetal liver, peripheral blood leukocytes, and lung EPRGl is also expressed in spleen and thymus to a lesser extent The 3 -UTR segment of the RNA transcript (nucleotide #475-21 1 1 of SEQ ID NO 3) should be useful for diagnostic purposes because 3'-UTRs are unique to a gene and sequence specific In addition, the 3'-UTR may be valuable in the development of screening assays to identify agents which modulate RNA stability and turnover rate Decreasing the message's steady state level would decrease the amount of translated protein in the cell, antagonizing the protein's action Because members of the SOCS family are negative regulators of growth factor signal transduction, a SOCS antagonist would function as an agonist of the growth factor One way of achieving this result would be through antisense technology in order to decrease or eliminate the EPRGl message Because determinants of RNA stability have frequently been associated with the 3 -UTR sequence, a second means would involve modulating the cellular mechanisms governing turnover of the EPRGl message Example 2 The utility of EPRGl anti-sense ohgonucleotides is exemplified by the following example Incubation of human bone marrow cells with anti-sense orientation ohgnucleotides of SEQ ID NO 6 and SEQ ID NO 7, but not with sense orientation ohgonucleotides of SEQ ID NO 8 for several hours in serum-free medium results in enhanced responsiveness of bone marrow progenitor / stem cells to optimal and suboptimal concentrations of colony stimulating factors such as G-CSF and EPO As an example, addition of G-CSF at 10 ng/ml resulted in approx 40 colonies / well (50 000 bone marrow cells/well) Marrow cells incubated with EPRGl sense ohgonucleotide of SEQ ID NO 8 resulted in an average of approx 41 colonies / well In contrast bone marrow cells incubated with EPRGl anti-sense ohgonucleotide of SEQ ID NO 7 resulted in an average of approx 61 colonies / well SEQ ID NO 6 gaCCATGGCGCACGGAGccA SEQ ID NO 7 gcTGTGGGTGACCATGGcgC SEQ ID NO 8 ccGTGCGCCATGGTCACccA Lower case nucleotide bases indicate 3 -phosphorothioate linkage

Claims: What is claimed is: An isolated polypeptide selected from the group consisting of (1) an isolated polypeptide comprising an amino acid sequence selected from the group having at least (a) 70% identity, (b) 80% identity, (c) 90% identity, or (d) 95 % identity to the amino acid sequence of SEQ ID NO 4 over the entire length of SEQ ID NO 4, (n) an isolated polypeptide comprising the amino acid sequence of SEQ ID NO 4 or (in) an isolated polypeptide which is the amino acid sequence of SEQ ID NO 4 An isolated polynucleotide selected from the group consisting of (l) an isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide that has at least (a) 70% identity, (b) 807c identity, (c) 90% identity, or (d) 957c identity, to the amino acid sequence of SEQ ID NO 4, over the entire length of SEQ ID NO 4, (n) an isolated polynucleotide comprising a nucleotide sequence that has at least (a) 707o identity (b) 807c identity, (c) 907c identity, or (d) 957c identity, over its entire length to a nucleotide sequence encoding the polypeptide of SEQ ID NO 4, (in) an isolated polynucleotide comprising a nucleotide sequence which has at least (a) 707c identity, (b) 807c identity, (c) 907c identity, or (d) 957c identity, to that of SEQ ID NO 3 over the entire length of SEQ ID NO 3, (IV) an isolated polynucleotide comprising a nucleotide sequence encoding the polypeptide of SEQ ID NO 4, (vi) an isolated polynucleotide which is the polynucleotide of SEQ ID NO 3, or (vi) an isolated polynucleotide obtainable by screening an appropπate library under stringent hybridization conditions with a labeled probe having the sequence of SEQ ID NO 3 or a fragment thereof , or a nucleotide sequence complementary to said isolated polynucleotide 3 An antibody lmmunospecific for the polypeptide of claim 1 4 A method for the treatment of a subject (I) in need of enhanced activity or expression of the polypeptide of claim 1 comprising (a) administering to the subject a therapeutically effective amount of an agonist to said polypeptide, and/or (b) providing to the subject an isolated polynucleotide comprising a nucleotide sequence encoding said polypeptide in a form so as to effect production of said polypeptide activity in vn o , or (π) having need to inhibit activity or expression of the polypeptide of claim 1 comprising (a) administering to the subject a therapeutically effective amount of an antagonist to said polypeptide and/or (b) administering to the subject a nucleic acid molecule that inhibits the expression of a nucleotide sequence encoding said polypeptide and/or (c) administering to the sub)ect a therapeutically effectiv e amount of a polypeptide that competes with said polypeptide for its hgand substrate , or receptor 5 A process for diagnosing a disease or a susceptibility to a disease in a subject related to expression or activity of the polypeptide of claim 1 in a subject comprising (a) determining the presence or absence of a mutation in the nucleotide sequence encoding said polypeptide in the genome of said subject, and/or (b) analyzing for the presence or amount of said polypeptide expression in a sample derived from said subject 6 A method for screening to identify compounds which stimulate or which inhibit the function of the polypeptide of claim 1 which compnses a method selected from the group consisting of (a) measuring the binding of a candidate compound to the polypeptide (or to the cells or membranes bearing the polypeptide) or a fusion protein thereof by means of a label directly or indirectly associated with the candidate compound, (b) measuring the binding of a candidate compound to the polypeptide (or to the cells or membranes bearing the polypeptide) or a fusion protein thereof in the presence of a labeled competitor, (c) testing whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide, using detection systems appropriate to the cells or cell membranes bearing the polypeptide, (d) mixing a candidate compound with a solution containing a polypeptide of claim 1 , to form a mixture, measuring activity of the polypeptide in the mixture, and comparing the activity of the mixture to a standard, or (e) detecting the effect of a candidate compound on the production of mRNA encoding said polypeptide and said polypeptide in cells, using for instance, an ELISA assay 7 An agonist or an antagonist of the polypeptide of claim 1 8 An expression system comprising a polynucleotide capable of producing a polypeptide of claim 1 when said expression system is present in a compatible host cell 9 A process for producing a recombinant host cell comprising transforming or transfecting a cell with the expression system of claim 8 such that the host cell, under appropπate culture conditions, produces a polypeptide comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO 4 over the entire length of SEQ ID NO 4 10 A recombinant host cell produced by the process of claim 9 1 1 A membrane of a recombinant host cell of claim 10 expressing a polypeptide comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO 4 over the entire length of SEQ ID NO 4 12 A process for producing a polypeptide comprising cultuπng a host cell of claim 10 under conditions sufficient for the production of said polypeptide and recovering the polypeptide from the culture 13 An isolated polynucleotide selected form the group consisting of (a) an isolated polynucleotide comprising a nucleotide sequence which has at least 70%, 80%, 907c, 957c, 977c identity to SEQ ID NO 5 over the entire length of SEQ ID NO 5, (b) an isolated polynucleotide comprising the polynucleotide of SEQ ID NO 5, (c) the polynucleotide of SEQ ID NO 5, or (d) a polynucleotide which is complementary to a polynucleotide of (a), (b) or (c) 14 An isolated polynucleotide selected form the group consisting of (a) an isolated polynucleotide comprising a nucleotide sequence which has at least 707c, 807c, 907c, 957c, 977c identity to SEQ ID NO 1 over the entire length of SEQ ID NO 1 , (b) an isolated polynucleotide comprising the polynucleotide of SEQ ID NO 1 , (c) the polynucleotide of SEQ ID NO 1 , or (d) a polynucleotide which is complementary to a polvnucleotide of (a), (b), or (c) 16 A method for treating cytopenia comprising adminstering to a patient in need thereof an effective amount of EPRGl inhibitor 17 The method of claim 16 further comprising coadministeπng with cyotokιne(s) 18 The method of claim 17 in which the cytokine is G-CSF, EPO, TPO, IL-l 1 , IL-3, PEG- rHuMGDF, FLT-3, NESP, or NEUPOGEN SD 19 An EPRGl inhibitor of claim 16 which is ASO comprising the sequence of SEQ ID NO 6 or 7 20. A method for increasing the effectiveness and engraftment of hematopoietic stem cell transplantation by treating human hematopoietic progenitor / stem cells with an inhibitor of EPRGl , comprising: (a) mixing a EPRGl inhibitor with a solution containing hematopoietic progenitor and stem cells, ex vivo, followed by transplantation of the hematopoietic cells into a patient in need thereof; or (b) mixing a EPRGl inhibitor with a solution containing hematopoietic cells in an ex vivo stem cell expansion culture system followed by transplantation of the hematopoietic cells into the patient; or (c) administering EPRGl inhibitor in vivo as an adjunct to hematopoietic stem cell transplantation or during the treatment of cytopenia. 21. The method of claim 20 in which EPRGl inhibitor is ASO comprising the sequence of SEQ ID NO: 6 or 7. 22. A method for increasing the efficiency of gene therapy transfer into hematopoietic stem cells comprising, (a) isolating hematopoietic stem cells or modified stem cell lines; and (b) incubating with EPRGl inhibitor before transfection with plasmid vectors containing any corrective gene of interest useful in treating a hereditary genetic diseases. 23. The method of claim 22 in which EPRGl inhibitor is ASO comprising the sequence of SEQ ID NO: 6 or 7. 24. The ASO comprising the sequence of SEQ ID NO: 6 or 7.

Inventors: DILLON SUSAN B, KING ANDREW G, LORD KENNETH A, DILLON, SUSAN, B., KING, ANDREW, G., LORD, KENNETH, A.
Applicants: SMITHKLINE BEECHAM CORP, SMITHKLINE BEECHAM CORPORATION
Application year: 2000
Main classification: C12N
IPC codes: A61K31, A61K38, A61K39, A61K45, A61K48, A61P3, A61P5, A61P7, A61P11, A61P25, A61P29, A61P31, A61P35, A61P37, A61P43, C07K14, C07K16, C12N1, C12N5, C12N15, C12P21, C12Q1, G01N33