JP2003245088A - Epo primary response gene 1, eprg1 - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide EPRG1 polypeptide and polynucleotide. <P>SOLUTION: The polypeptide comprises an amino acid sequence having at least 70% identity to the amino acid sequence derived from human. The polynucleotide comprises a nucleotide sequence encoding a polypeptide having at least 70% identity to the amino acid sequence derived from human. The EPRG1 polypeptide and polynucleotide are useful for the treatment of functional disorders or diseases including anemia, polycythemia, cancer; neutropenia, AIDS, drug-induced anemias, myelosuppression, autoimuune diseases and inflammatory diseases and diagnostic assays for such diseases. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a newly identified polypeptide, a polynucleotide encoding the polypeptide, an agonist, an antagonist which may be effective in or in the treatment of the polypeptide and the polynucleotide. And / or use in identifying compounds that are inhibitors and methods of producing the polypeptides and polynucleotides.

[0002]

BACKGROUND OF THE INVENTION There are currently radical changes in the drug discovery process. Because it is "functional genomics"
mics), that is, high-throughput genomic or gene-based biology. This approach is rapidly replacing the relatively early approaches based on "positional cloning." The phenotype, the biological function or genetic disease, will be identified and the pathogenic gene will then be located by the location of its genetic map. Functional genomics is a bioinformatic method for identifying interesting gene sequences from many molecular biology databases currently available.
It relies heavily on various tools in (s). still,
There is a need to identify and characterize as yet unknown genes and their related polypeptides / proteins as targets for drug discovery.

EPRG1 (EPO primary response gene 1)
Was isolated from a human hematopoietic cell line that requires EPO for growth. Expression of EPRG1 is induced by EPO and the presence of EPO is required to maintain its expression. EPRG1 is involved in the proliferation of EPO-dependent cells and may be important in the proliferation and development of erythroid and other hematopoietic lineages. Expression of EPRG1 is induced at high levels in human bone marrow treated with G-CSF, but significant levels of EPRG1 are expressed in fetal liver, peripheral blood leukocytes, lungs, and also in human bone marrow stimulated with EPO.
Furthermore, EPRG1 expression is induced by EPO in the presence of cycloheximide, indicating a primary gene response. As a primary response gene, EPRG1 expression is EPO
It is a direct consequence of activation of the signaling pathway following binding to the receptor. These data indicate that EPRG1 is anemia, polycythemia, cancer, neutropenia, AIDS, drug-induced anemia, myelosuppression, autoimmune disease (eg, rheumatoid arthritis, diabetes, multiple sclerosis), and It has been shown to be useful in preventing, ameliorating and treating dysfunctions or diseases including but not limited to inflammatory diseases (eg, asthma, allergies).

[0004]

The present invention provides an EPRG1,
In particular, it relates to EPRG1 polypeptides and EPRG1 polynucleotides, recombinant materials, and methods for producing the same.
In another aspect, the invention provides anemia, polycythemia, cancer, neutropenia, AIDS, drug-induced anemia, myelosuppression, autoimmune disease (eg, rheumatoid arthritis, diabetes, multiple sclerosis). ), And inflammatory diseases (eg, asthma, allergies) (hereinafter collectively referred to as “the disease”)
And methods of using the polypeptides and polynucleotides, including the treatment of In another aspect, the invention relates to methods of identifying agonists and antagonists / inhibitors using the agents provided by the invention and treating the conditions associated with EPRG1 imbalance using the identified compounds. In yet another aspect, the present invention provides for inappropriate EPRG1 activity or EPR
It relates to a diagnostic assay for detecting diseases associated with G1 levels.

In one aspect, the invention features EPRG1
Relates to polypeptides. A peptide of this kind has at least 70% identity to the amino acid sequence of SEQ ID NO: 2 over the entire length of SEQ ID NO: 2, preferably at least 80%.
An isolated polypeptide comprising an amino acid sequence having a% identity, more preferably at least 90% identity, even more preferably at least 95% identity, and most preferably at least 97-99% identity. Is included. One such polypeptide includes the amino acid sequence of SEQ ID NO: 2.

In another peptide of the present invention, the amino acid sequence thereof has at least 70% identity, preferably at least 80% identity, more preferably with the amino acid sequence of SEQ ID NO: 2 over the entire length of SEQ ID NO: 2. At least 9
Included are isolated polypeptides having 0% identity, more preferably at least 95% identity, and most preferably at least 97-99% identity. Such a polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2. Further peptides of the invention include
Included is an isolated polypeptide encoded by a polynucleotide comprising the nucleotide sequence contained in SEQ ID NO: 1.

The EPRG1 polypeptide of the present invention has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity to the amino acid sequence of SEQ ID NO: 4 over the entire length of SEQ ID NO: 4. Also included is an isolated polypeptide comprising an amino acid sequence having identity, more preferably at least 95% identity, and most preferably at least 97-99% identity. Such a polypeptide includes a polypeptide containing the amino acid sequence of SEQ ID NO: 4.

A further peptide according to the invention has an amino acid sequence of at least 70% identity, preferably at least 80% identity, more preferably at least 80% identity with the amino acid sequence of SEQ ID NO: 4 over the entire length of SEQ ID NO: 4. 90
Included are isolated polypeptides having a percent identity, more preferably at least 95% identity, and most preferably at least 97-99% identity. Such a polypeptide is the polypeptide of SEQ ID NO: 4. Further peptides of the invention include isolated polypeptides encoded by a polynucleotide comprising the nucleotide sequence contained in SEQ ID NO: 3.

The polypeptides of the present invention are considered to be members of the SOCS family. Therefore, I am interested in them. This is because genes in this family modulate activation of signal transduction pathways from cell growth and differentiation factors. These characteristics will be referred to as “E
"PRG1 activity" or "EPRG1 polypeptide activity"
Alternatively, it is referred to as “EPRG1 biological activity”. Also included among these activities are the antigenicity and immunogenicity of said EPRG1 polypeptides, in particular the antigenicity and immunogenicity of the polypeptides of SEQ ID NO: 2 or 4. The polypeptides of the present invention preferably exhibit at least one biological activity of EPRG1.

The polypeptide of the present invention may be in the form of a "mature" protein or may be part of a larger protein, such as a fusion protein. Often, it is advantageous to include additional amino acid sequences, such as secretory or leader sequences, pro-sequences, sequences useful for purification such as multiple histidine residues,
Or there are additional sequences etc. which ensure stability during recombinant production.

The present invention also includes a variant of the above-mentioned polypeptide, that is, a polypeptide which differs from the reference polypeptide by conservative amino acid substitution (one residue is replaced by another residue having similar properties). included. Typical such substitutions are between Ala, Val, Leu and Ile; Ser and Thr.
Between; acidic residues Asp and Glu; between Asn and Gln; basic residues Lys and Arg; or aromatic residues Phe and Tyr
Happens in between. In particular, several, 5-10, 1-5, 1
Variants in which ~ 3, 1-2 or 1 amino acids are substituted, deleted or added in any combination are preferred.

The polypeptide of the present invention can be produced by any appropriate method. Such polypeptides include isolated natural polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides,
Alternatively, a polypeptide produced by a combination of these methods is included. Means for producing such polypeptides are well understood in the art.

In a further aspect of the invention, the invention comprises
EPRG1 polynucleotides. Such a polynucleotide has at least 70% identity to the amino acid sequence of SEQ ID NO: 2 over the entire length of SEQ ID NO: 2,
Preferably an isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide having at least 80% identity, more preferably at least 90% identity, even more preferably at least 95% identity. . In this regard, polypeptides with at least 97% identity are more preferred, those with at least 98-99% identity are even more preferred, and polypeptides with at least 99% identity are most preferred. . As such a polynucleotide, SEQ ID NO: 1 encoding the polypeptide of SEQ ID NO: 2
A polynucleotide comprising the nucleotide sequence contained in.

A further polynucleotide of the present invention comprises at least 7 nucleotides over the entire coding region for the nucleotide sequence encoding the polypeptide of SEQ ID NO: 2.
0% identity, preferably at least 80% identity,
More preferred is an isolated polynucleotide comprising a nucleotide sequence having at least 90% identity, and even more preferably at least 95% identity. In this regard, polynucleotides having at least 97% identity are more preferred, but at least 98-
Even more preferred are those with 99% identity, and most preferred are polynucleotides with at least 99% identity.

A further polynucleotide of the invention has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity to the polynucleotide of SEQ ID NO: 1 over the entire length of SEQ ID NO: 1.
An isolated polynucleotide comprising a nucleotide sequence having at least 95% identity, more preferably at least 95% identity. In this regard, at least 9
More preferred are polynucleotides with 7% identity, even more preferred are those with at least 98-99% identity, and most preferred are polynucleotides with at least 99% identity. Such polynucleotides include a polynucleotide comprising the polynucleotide of SEQ ID NO: 1 and the polynucleotide of SEQ ID NO: 1. The invention also provides polynucleotides which are complementary to all the above polynucleotides.

The nucleotide sequence of SEQ ID NO: 1 is CIS3
Shows homology with. The nucleotide sequence of SEQ ID NO: 1 is c
DNA sequence, SEQ ID NO: 2 consisting of 225 amino acids
A polypeptide coding sequence (nucleotides 28-705) that encodes a polypeptide of Even if the nucleotide sequence encoding the polypeptide of SEQ ID NO: 2 is the same as the polypeptide coding sequence contained in SEQ ID NO: 1, due to the redundancy (degeneracy) of the genetic code, the polypeptide of SEQ ID NO: 2 is still used. It may be a sequence other than the sequence encoded and included in SEQ ID NO: 1. The polypeptide of SEQ ID NO: 2 is structurally related to other proteins of the SOCS family and has homology and / or structural similarity with CIS3.

The preferred polypeptides and polynucleotides of the invention are expected to have, inter alia, similar biological functions / properties to the homologous polypeptides and polynucleotides. Furthermore, preferred polypeptides and polynucleotides of the present invention include at least one EP
It has RG1 activity.

The invention also relates to the partial or other polynucleotides and polypeptides initially identified prior to the determination of the corresponding full-length sequences of SEQ ID NO: 1 and SEQ ID NO: 2. Therefore, in a further aspect, the invention provides (a) at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity to the nucleotide sequence of SEQ ID NO: 5 over the entire length of SEQ ID NO: 5. (B) SEQ ID NO: 5 over the entire length of SEQ ID NO: 5, comprising a polynucleotide comprising a nucleotide sequence having a% identity, more preferably at least 95% identity, and most preferably 97-99% identity. At least 70% identity to the nucleotide sequence of
Preferably a polynucleotide comprising a nucleotide sequence having at least 80% identity, more preferably at least 90% identity, even more preferably at least 95% identity, most preferably 97-99% identity. Comprises, (c) comprises the polynucleotide of SEQ ID NO: 5, (d) is the polynucleotide of SEQ ID NO: 5, or
(e) An isolated polynucleotide which is a polynucleotide complementary to the polynucleotide of (a), (b), (c) or (d) is provided.

In a further embodiment, the invention provides (a) at least 70% identity, preferably at least 80% identity, more preferably at least 80% identity to the nucleotide sequence of SEQ ID NO: 3 over the entire length of SEQ ID NO: 3. 90% identity, more preferably at least 95% identity,
Most preferably, it comprises a nucleotide sequence having 97-99% identity, (b) comprises the polynucleotide of SEQ ID NO: 3, (c) is at least 70% relative to the amino acid sequence of SEQ ID NO: 4 over the entire length of SEQ ID NO: 4. Identity, preferably at least 80% identity, more preferably at least 90% identity, even more preferably at least 95%
A nucleotide sequence encoding a polypeptide having an identity of, most preferably 97-99%.
(d) is the polynucleotide of SEQ ID NO: 3, or (e)
There is provided an isolated polynucleotide which is a polynucleotide complementary to the polynucleotide of (a), (b), (c) or (d).

The present invention further comprises (a) at least 7 relative to the amino acid sequence of SEQ ID NO: 4 over the entire length of SEQ ID NO: 4.
0% identity, preferably at least 80% identity,
More preferably at least 90% identity, even more preferably at least 95% identity, most preferably 97.
(B) at least 70% identity to the amino acid sequence of SEQ ID NO: 4 over the entire length of SEQ ID NO: 4, preferably at least 80% identity, more preferably, comprising an amino acid sequence having ˜99% identity. (C) comprising the amino acid sequence of SEQ ID NO: 4, having an amino acid sequence having at least 90% identity, more preferably at least 95% identity, most preferably 97-99% identity, or (d) Provided is a polypeptide which is the polypeptide of SEQ ID NO: 4 as well as a polypeptide encoded by a polynucleotide comprising the nucleotide sequence contained in SEQ ID NO: 3.

The nucleotide sequence of SEQ ID NO: 5 is an Expressed Sequence Tag:
EST) sequence. If you are a person skilled in the art, EST
It will be appreciated that there will necessarily be some nucleotide sequence read error in the sequence (Adams, MD et al., N.
ature 377 (supp) 3, 1995). Therefore, the nucleotide sequence of SEQ ID NO: 5 is subject to the same unique limits on sequence accuracy.

The polynucleotide of the present invention is a cDNA derived from mRNA in cells of human bone marrow and hematopoietic cells by standard cloning and screening.
From the library, EST analysis (Adams, MD et al., Scien
ce (1991) 252: 1651-1656; Adams, MD et al., Nature (199
2) 355: 632-634; Adams, MD et al., Nature (1995) 377 Su.
pp: 3-174). Further, the polynucleotide of the present invention can be obtained from a natural source such as a genomic DNA library, or can be synthesized by using a known technique which is commercially available.

When the polynucleotide of the present invention is used for recombinant production of the polypeptide of the present invention, the polynucleotide includes the coding sequence of the mature polypeptide alone,
Or a coding sequence for the mature polypeptide in the same reading frame as another coding sequence (eg, encoding a leader or secretory sequence, a pre-, pro- or prepro-protein sequence, or other fusion peptide moiety). Be done. For example, a marker sequence that facilitates purification of the fusion polypeptide can be encoded. In a preferred embodiment of this aspect of the invention, the marker sequence is
provided by pQE vector (Qiagen, Inc.) and Gen
tz et al., Proc. Natl. Acad. Sci. USA (1989) 86: 821-824.
A hexa-histidine peptide, or HA tag, as described in. Also, this polynucleotide is 5 '
And 3'non-coding sequences, such as transcribed but not translated sequences, splicing and polyadenylation signals, ribosome binding sites, and mRNA stabilizing sequences.

As a further specific example of the present invention, several amino acid residues, for example, 5 to 10, 1 to 5, 1 to 3, 1 to 2, or 1 amino acid residue are substituted with any combination. , A polynucleotide encoding a polypeptide variant comprising the amino acid sequence of SEQ ID NO: 2 or 4 which has been deleted or added.

A polynucleotide which is identical or substantially identical to the nucleotide sequence contained in SEQ ID NO: 1, 3 or 5 is a full-length c encoding a polypeptide of the invention.
To isolate DNA and genomic clones,
C of other genes (including genes encoding homologues and orthologs derived from species other than human) having high sequence similarity to SEQ ID NO: 1, 3 or 5.
CDNA for isolation of DNA and genomic clones
It can be used as a hybridization probe for A and genomic DNA or as a primer for nucleic acid amplification (PCR) reactions. In general, these nucleotide sequences are preferably 80%, more preferably 90%, most preferably 95% of the reference nucleotide sequence.
It is the same. Usually 15 probes or primers
It contains more than one nucleotide, preferably more than 30, and may have more than 50 nucleotides.
Particularly preferred probes are those having a range of 30-50 nucleotides.

As the polynucleotide encoding the polypeptide of the present invention (including homologues and orthologs derived from species other than human), a labeled probe containing the nucleotide sequence of SEQ ID NO: 1, 3 or 5 or a fragment thereof is used. And a suitable library is screened under stringent hybridization conditions to isolate a full-length cDNA and a genomic clone containing the polynucleotide sequence. Such hybridization techniques are known to those of skill in the art. Preferred stringent hybridization conditions are 50% formamide, 5 × SSC (150 mM NaCl,
15 mM trisodium citrate), 50 mM sodium phosphate
(pH 7.6), incubated in a solution containing 5 × Denhardt's solution, 10% dextran sulphate and 20 μg / ml denatured and sheared salmon sperm DNA at 42 ° C. overnight, then the filter in 0.1 × SSC at about 65 ° C. Including washing. Thus, the present invention uses labeled probes comprising the nucleotide sequence of SEQ ID NO: 1, 3 or 5 or fragments thereof,
It also includes a polynucleotide obtained by screening an appropriate library under stringent hybridization conditions.

As will be appreciated by those in the art, in many cases the region encoding the polypeptide will be the 5'of the cDNA.
The isolated cDNA sequence will be incomplete, as it will be truncated at the ends. It's because of reverse transcriptase, which was originally "processivity" (processi).
vity: a low measure of the enzyme's ability to remain bound to the template during polymerization) and unable to complete the DNA copy of the mRNA template during first-strand cDNA synthesis.

To obtain full-length cDNA, or short chain c
There are several methods known and available to those of skill in the art for extending DNA, such as those based on cDNA terminal rapid amplification (RACE) (eg, Frohman).
Et al., PNAS USA 85, 8998-9002, 1988). Recent improvements in the above techniques, such as those demonstrated by Marathon ^™ technology (Clontech Laboratories Inc.), have greatly facilitated the search for longer cDNAs. Marathon ^TM
In the technology, cDNA extracted from mRNA extracted from a predetermined tissue
The NA is made and an "adapter" sequence is ligated to each end. Subsequently, nucleic acid amplification (PCR) is performed using a combination of gene-specific and adapter-specific oligonucleotide primers to amplify the "deletion"5'end of the cDNA. Next, a "nested" primer, ie, a primer designed to anneal within the amplification product (typically an adapter-specific primer that anneals further 3'to the adapter sequence and an additional 5'to the known gene sequence). The PCR reaction is repeated with a gene-specific primer that anneals to the side. The product of this reaction is then D
This product was analyzed by NA sequencing and the existing cDNA
Full-length cDNA can be constructed either by direct binding to NA or by performing another full-length PCR using new sequence information for 5'primer design.

The recombinant polypeptide of the present invention can be produced from a genetically engineered host cell containing an expression system using methods known in the art. Thus, in a further aspect, the invention relates to expression systems containing one or more polynucleotides of the invention, host cells genetically engineered by the expression systems, and the production of polypeptides of the invention by recombinant techniques. Cell-free translation systems can also be used to produce proteins of this type using RNA derived from the DNA constructs of the invention.

For recombinant production, host cells are genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention. Introduction of polynucleotides into host cells is described by Davis et al., Basic Methods in Molecular Bi
ology (1986) and Sambrook et al., Molecular Cloning:
A Laboratory Manual, 2nd Ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, NY (1989)
Can be done by the methods described in many standard laboratory manuals such as Suitable such methods include, for example, calcium phosphate transfection,
DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid mediated transfection, electroporation, transduction, scrape loading, ballistic introduct
ion) or infection.

As typical examples of suitable hosts, bacterial cells (eg Streptococcus, Staphylococcus, Escherichia coli, Streptomyces, Bacillus subtilis), fungal cells (eg yeast, Aspergillus), insect cells (eg Drosophila S.
2, Spodoptera Sf9), animal cells (eg CHO, C)
OS, HeLa, C 127, 3T3, BHK, HEK 29
3, Bowes melanoma cells) and plant cells.

A wide variety of expression systems can be used. Such expression systems include, among others, chromosomal, episomal and viral derived systems such as bacterial plasmid derived, bacteriophage derived, transposon derived, yeast episome derived, insert factor derived, yeast chromosomal element derived, virus (eg baculovirus, SV40 Such as papovavirus, vaccinia virus, adenovirus, fowlpox virus, pseudorabies virus, retrovirus), and vectors derived from combinations thereof, such as plasmids such as cosmids or phagemids and genetics of bacteriophages. Some come from elements. These expression systems may include control sequences that regulate as well as direct expression. In general, any system or vector capable of maintaining, amplifying and expressing a polynucleotide for production of a polypeptide in a host may be used. Sambrook et al, Molecu
The appropriate nucleotide sequence can be inserted into the expression system by any of the well-known and routine techniques used, such as those described in lar Cloning: A Laboratory Manual (supra). In order to secrete the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space, or into the extracellular environment,
Appropriate secretory signals can be incorporated into the polypeptide of interest. These signals may be endogenous or heterologous to the polypeptide of interest.

When a polypeptide of the invention is to be expressed for use in a screening assay, it is generally preferable to produce the polypeptide on the surface of cells. In that case, cells are harvested prior to use in the screening assay. If the polypeptide is secreted into the medium, the medium is harvested to recover and purify the polypeptide. If produced intracellularly, the cells must first be lysed and then the polypeptide recovered.

For recovering and purifying the polypeptide of the present invention from recombinant cell culture, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography. Known methods including chromatography, hydroxylapatite chromatography and lectin chromatography can be used. Most preferably,
High performance liquid chromatography is used for purification. When the polypeptide is denatured during isolation and / or purification, known techniques for refolding proteins can be used to restore the active conformation.

The present invention also relates to the use of the polynucleotide of the present invention as a diagnostic agent. Associated with dysfunction,
Detection of a variant of the gene characterized by the polynucleotide of SEQ ID NO: 1, 3 or 5 is in addition to the diagnosis of a disease caused by underexpression, overexpression or altered expression of said gene or its susceptibility to the disease. Will provide a diagnostic tool that can or can make that diagnosis. Individuals with mutations in the gene can be found at the DNA level by various techniques. The present invention also includes EPRG1 contained in a sample obtained from an individual.
Comprising comparing the polynucleotide sequence to the nucleotide sequence of SEQ ID NO: 1, 3 or 5, respectively,
A method for detecting the presence or absence of a mutation in an EPRG1 polynucleotide derived from an individual based on the nucleotide sequence of SEQ ID NO: 1, 3 or 5.

The diagnostic nucleic acid can be obtained from a biopsy or autopsy material of a subject's cells, such as blood, urine, saliva, tissue. The genomic DNA may be used directly for detection, or it may be enzymatically amplified using PCR or other amplification methods prior to analysis. RN in the same way
A or cDNA can also be used. Deletion and insertion mutations can be detected by a change in the size of the amplification product as compared to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to labeled EPRG1 nucleotide sequences. Perfectly matched sequences and mismatched duplexes can be distinguished by RNase digestion or by differences in melting temperature. Also, the difference in DNA sequence is due to DN in gels with or without denaturing agents.
A change in the electrophoretic mobility of the A fragment or direct D
It can also be detected by NA sequencing (eg Myer
S. et al., Science (1985) 230: 1242). Sequence changes at specific positions can also be confirmed by nuclease protection assays (eg RNase and S1 protection) or chemical cleavage methods (Cotton et al., Pr.
oc. Natl. Acad. Sci. USA (1985) 85: 4397-4401). In another embodiment, an array of oligonucleotide probes containing the EPRG1 nucleotide sequence or fragments thereof can be constructed, eg, for efficient screening of gene mutations. Array techniques are well known and have general applicability and have been used to solve various problems in molecular genetics, including gene expression, genetic linkage and genetic variability (eg, M Chee et al., Science, Vol.274, pp.610-613.
(1996)).

Diagnostic assays are performed by the EPR described above.
A method for diagnosing or determining the susceptibility to the disease is provided by detecting a mutation in the G1 gene. further,
A polypeptide or m from a sample obtained from the subject
Diagnosis of the disease can be made by a method of measuring an abnormal decrease or increase in the level of RNA. The decrease or increase in the expression can be achieved by a polynucleotide quantification method known in the art, for example, nucleic acid amplification (eg, PCR, RT-PCR),
RNase protection, Northern blotting,
R by any of the other hybridization methods
It can be measured at the NA level. Assay techniques for measuring the levels of proteins such as the polypeptides of the invention in a sample obtained from a host are well known to those of skill in the art. Such assay methods include radioimmunoassay, competitive binding assay, Western blot analysis, EL
ISA assay etc.

Thus, in another embodiment, the invention provides: (a) a polynucleotide of the invention (preferably a nucleotide sequence of SEQ ID NO: 1, 3 or 5) or a fragment thereof, (b) a nucleotide of (a) A nucleotide sequence complementary to the sequence, (c) the polypeptide of the present invention (preferably,
SEQ ID NO: 2 or 4) or a fragment thereof, or (d) an antibody against the polypeptide of the present invention (preferably the polypeptide of SEQ ID NO: 2 or 4).

In such a kit, (a), (b),
It will be appreciated that (c) or (d) is a substantial constituent. Such kits are susceptible to diseases or disorders, particularly anemia, polycythemia, cancer, neutropenia, AIDS, drug-induced anemia, myelosuppression, autoimmune diseases (e.g. rheumatoid arthritis, diabetes, multiple incidences). It is useful in diagnosing sclerosis) and inflammatory diseases (eg, asthma, allergy).

The nucleotide sequence of the present invention is also useful for chromosome identification. This sequence can target and hybridize to a particular location on an individual human chromosome. Generating a chromosomal map of related sequences according to the present invention is an important first step in correlating these sequences with gene-related diseases. Once a sequence is mapped to a precise chromosomal location, the physical location of that sequence on that chromosome can be correlated with genetic map data. This type of data is, for example, V. McKusick, Mendelian Inheritance in Man (J
Found online at ohns Hopkins University Welch Medical Library). Then, the relationship between the gene mapped to the same chromosomal region and the disease is confirmed by linkage analysis (co-inheritance of physically adjacent genes).

Differences in cDNA or genomic sequence between affected and unaffected individuals can also be examined. If a mutation is observed in some or all of the affected individuals, but not in any normal individual, then the mutation may be responsible for the disease.

The polypeptides of the present invention, fragments or analogs thereof, or cells expressing them can also be used as immunogens to produce antibodies immunospecific for the polypeptides of the present invention. By "immunospecific" is meant that the antibody exhibits a substantially higher affinity for a polypeptide of the invention than its affinity for other related polypeptides in the prior art.

An antibody against the polypeptide of the present invention can be obtained by administering a fragment, analog or cell containing the polypeptide or epitope to an animal (preferably an animal other than human) using a conventional protocol. For preparation of monoclonal antibodies, any technique which produces antibodies from continuous cell line cultures can be used. Examples include hybridoma technique (Kohler, G. and Milstein, C., Nature (1975) 256: 495-497), trioma technique, human B cell hybridoma technique (Kozbor
Et al., Immunology Today (1983) 4:72) and the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies an
d Cancer Therapy, pp.77-96, Alan R. Liss, Inc., 198
5) etc.

To prepare single chain antibodies to the polypeptides of the present invention, the methods for preparing single chain antibodies as described in US Pat. No. 4,946,778 can be adapted.
Also, transgenic mice, or other organisms including other mammals, may be utilized to express humanized antibodies. Using the above antibody, a clone expressing the polypeptide can be isolated and identified, or the polypeptide can be purified by affinity chromatography. Antibodies against the polypeptide of the present invention,
In particular, it may be used for the treatment of the abovementioned diseases.

In a further aspect of the invention, the invention comprises:
A genetically engineered preparation comprising the polypeptide of the present invention or a fragment thereof and various portions of constant regions of H chain or L chain of immunoglobulins of various subclasses (IgG, IgM, IgA, IgE) Related to soluble fusion proteins. As an immunoglobulin, human IgG, especially I
The constant region of the heavy chain of gG1 is preferred, in which case the fusion occurs in the hinge region. In a particular example, the Fc portion can be easily removed by incorporating a cleavage sequence that can be cleaved by blood coagulation factor Xa. Furthermore, the invention relates to methods of genetically engineering these fusion proteins and their use in drug screening, diagnosis and therapy. Also, a further aspect of the present invention relates to a polynucleotide encoding such a fusion protein. Examples of fusion protein technology are international patent applications WO94 / 29458 and WO94 / 22914
Can be found in.

A further aspect of the invention relates to a method of eliciting an immunological response in a mammal, which method is particularly sufficient to generate an antibody and / or T cell immune response to protect said animal from said disease. Comprising inoculating a mammal with a polypeptide of the invention. Yet another aspect of the invention is to elicit an immunological response such as to produce antibodies that protect the mammal from said disease.
A method of eliciting an immunological response in a mammal comprising delivering the polypeptide in vivo via a vector that directs the expression of a polynucleotide encoding the polypeptide of the invention.

A further aspect of the present invention relates to an immunological / vaccine formulation (composition) which, when introduced into a mammalian host, elicits an immunological response against the polypeptide of the present invention in the mammal. Contains a polypeptide or polynucleotide of the invention. The vaccine formulation may further include a suitable carrier. Since the polypeptide may be broken down in the stomach, it is preferably administered parenterally (eg by subcutaneous, intramuscular, intravenous or intradermal injection). Formulations suitable for parenteral administration include sterile aqueous and non-aqueous injection solutions that may contain antioxidants, buffers, bacteriostats and solutes that make this formulation isotonic with the blood of the recipient, and suspensions. There are aqueous and non-aqueous sterile suspensions which may include agents or thickeners. Such formulations may be presented in single-dose or multi-dose containers (eg, sealed ampoules and vials) and may be stored in lyophilized form with the addition of a sterile liquid carrier just prior to use. it can. The vaccine formulation may include an adjuvant system to enhance the immunogenicity of the formulation, such as an oil-in-water adjuvant system or any other adjuvant system known in the art. The dose varies depending on the specific activity of the vaccine, but can be easily determined by routine experimentation.

The polypeptides of the present invention are responsible for a variety of biological functions, including many pathological conditions, especially the diseases mentioned above. Therefore, it is desirable to develop screening methods to identify compounds that stimulate or inhibit the function of this polypeptide. Therefore, in a further aspect, the invention provides a method of screening compounds to identify compounds that stimulate or inhibit the polypeptide. Generally, agonists or antagonists are used for the therapeutic and prophylactic purposes of said diseases. Compounds can be identified from a variety of sources, such as cells, cell-free preparations, chemical libraries and mixtures of natural products. The agonist, antagonist or inhibitor thus identified may optionally be a natural or modified substrate, ligand, receptor, enzyme, etc. of the polypeptide, and also its structural or functional mimetic May be (Coli
gan et al., Current Protocols in Immunology 1 (2): Chapte
r 5 (1991)).

In the screening method, the binding of the candidate compound to the polypeptide of the present invention, the cell or membrane carrying the polypeptide, or its fusion protein is carried out using a label directly or indirectly bound to the candidate compound. And easy to measure. Alternatively, the screening method may use competition with a labeled competitor. Furthermore, in such screening methods, whether a candidate compound results in a signal generated by activation or inhibition of the polypeptide can be tested using a detection system suitable for cells carrying the polypeptide. . In general, inhibitors of activation are assayed in the presence of known agonists to determine the effect of the presence of a candidate compound on agonist activation. In a screening method for an inverse agonist or inhibitor by examining whether or not a candidate compound suppresses the activation of a polypeptide in the absence of an agonist or inhibitor,
A constitutively active polypeptide is used. In addition, these screening methods combine a candidate compound with a solution containing a polypeptide of the invention to form a mixture, measure EPRG1 activity in the mixture, and compare the EPRG1 activity of the mixture to a standard. It only needs to include the steps. Fusion proteins such as those made from the Fc portion and EPRG1 polypeptides as described above can also be used in high throughput screening assays to identify antagonists of the polypeptides of the invention (D. Bennett et al., J. Mol. . Recognition,
8: 52-58 (1995) and K. Johanson et al., J. Biol. Che.
m., 270 (16): 9459-9471 (1995)).

The interaction of SH2 domains with phosphotyrosine peptides is very specific for a given SH2 domain and its particular peptide substrate. The formation of this complex can be used as a basis for constructing an in vitro high throughput screen using ELISA or other means suitable for detecting the association.

Further, it is possible to construct a screening method for detecting the effect of an added compound on intracellular mRNA or polypeptide production using the polynucleotide, polypeptide or antibody against the polypeptide of the present invention. it can. For example, monoclonal or polyclonal antibodies can be used by standard methods known in the art to construct an ELISA assay for measuring the level of secretion or cell binding of a polypeptide, which may be performed on appropriately engineered cells or It can be used to search for substances that suppress or enhance the production of polypeptide from tissues (also called antagonists or agonists, respectively).

If a membrane-bound or soluble receptor is present, the polypeptide of the invention may be used to identify this type of receptor by standard receptor binding methods known in the art. Can be used. Such receptor binding methods include, but are not limited to, ligand binding and cross-linking assays in which the polypeptide is labeled with a radioactive isotope (eg, ¹²⁵ I).
Labeled, chemically modified (eg biotinylated), or fused to a peptide sequence suitable for detection and purification, and putative receptor sources (cells, cell membranes, cell supernatants, tissue extracts) , Body fluids, etc.). Other methods include biophysical methods such as surface plasmon resonance and spectroscopy. These screening methods can be performed on the polypeptide or (if present)
It can also be used to identify agonists or antagonists of the polypeptide that compete with binding to its receptor. Standard methods for conducting screening assays are well understood in the art.

Examples of potential antagonists of the polypeptides of the present invention include antibodies, in some cases oligonucleotides or proteins (eg, oligonucleotides or proteins) that are in close association with the polypeptide's ligands, substrates, receptors, enzymes, etc. , Ligands, substrates, receptors, enzymes, etc.), or small molecules that bind the polypeptide of the invention but do not induce a response (and thus interfere with the activity of the polypeptide).

Thus, in another aspect, the invention comprises:
An agonist, an antagonist of the polypeptide of the present invention,
The present invention relates to a screening kit for identifying a compound that reduces or increases the production of a ligand, a receptor, a substrate, an enzyme, etc., or a polypeptide of this type, which kit comprises (a) the polypeptide of the present invention and (b) the present invention. A recombinant cell expressing the polypeptide of the invention, (c) a cell membrane expressing the polypeptide of the invention, or (d) an antibody against the polypeptide of the invention, said polypeptide being preferred Is the polypeptide of SEQ ID NO: 2 or 4. It will be appreciated that in such a kit, (a), (b), (c) or (d) is a substantial constituent.

Those skilled in the art will appreciate that the polypeptide of the present invention may be an agonist of the polypeptide based on its structure,
It will be readily appreciated that it can also be used in methods of designing antagonists or inhibitors. This method
(a) First, analyze the three-dimensional structure of the polypeptide, (b)
Assuming a three-dimensional structure of a likely reaction site or binding site of an agonist, antagonist or inhibitor, (c) synthesizing a candidate compound expected to bind or react with the envisioned reaction site or binding site, and
(d) determining whether the candidate compound is in fact an agonist, antagonist or inhibitor. It will be further understood that this is a normal interaction process.

In a further aspect, the invention provides an EPRG
1 associated with either excess or deficiency of polypeptide activity, such as anemia, polycythemia, cancer, neutropenia,
Provides treatments for abnormal conditions such as AIDS, drug-induced anemia, myelosuppression, autoimmune diseases (eg rheumatoid arthritis, diabetes, multiple sclerosis), and inflammatory diseases (eg asthma, allergies) .

If the activity of the polypeptide is in excess, several approaches are available. One approach is to inhibit the function of the polypeptide, for example, by blocking the binding of ligands, substrates, receptors, enzymes, etc., or by reducing an abnormal condition by suppressing a second signal. An effective amount to do
It comprises administering the above inhibitor compound (antagonist) to a patient together with a pharmaceutically acceptable carrier. In another approach, soluble forms of the polypeptide that are still capable of binding the ligand, substrate, enzyme, receptor, etc. in competition with the endogenous polypeptide can be administered. A typical example of such a competitor is a fragment of EPRG1 polypeptide.

In yet another approach, expression inhibition methods can be used to suppress the expression of the gene encoding the endogenous EPRG1 polypeptide. Such known techniques require the use of antisense sequences produced in the body or administered separately (eg, Oligodeoxynucleoti).
des as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (19
88) in O'Connor, J. Neurochem (1991) 56: 560). Alternatively, oligonucleotides that form triple helices with this gene can be supplied (eg, Lee et al., Nucleic Acids Res (1979) 6: 3073;
Cooney et al., Science (1988) 241: 456; Dervan et al., Scienc.
e (1991) 251: 1360). These oligomers can be administered per se or the relevant oligomers can be expressed in vivo.

In treating abnormal conditions associated with underexpression of EPRG1 and its activity, several approaches can also be taken. One approach involves administering to a patient a therapeutically effective amount of a compound that activates a polypeptide of the invention (ie, the agonist) together with a pharmaceutically acceptable carrier to alleviate the abnormal condition. It consists of Alternatively, EPR in relevant cells of the patient
Gene therapy can be used to produce G1 endogenously. For example, the polynucleotides of the invention are genetically engineered for expression by a replication defective retroviral vector as described above. The retroviral expression construct is then isolated and the RN encoding the polypeptide of the invention.
It is introduced into packaging cells transduced with a retroviral plasmid vector containing A. as a result,
The packaging cells will produce infectious viral particles containing the gene of interest. These producer cells are administered to the patient for in vivo cell engineering and in vivo polypeptide expression. For an overview of gene therapy, see Human Molecular Genetics, T Strachan and A PRe.
Chapter in ad, BIOS Scientific Publishers Ltd (1996)
er 20, Gene Therapy and other Molecular Genetic-bas
ed Therapeutic Approaches (and references therein)
checking ... Another approach is to administer a therapeutic amount of a polypeptide of the invention together with a suitable pharmaceutical carrier.

In a further aspect, the invention provides a pharmaceutically acceptable carrier for a therapeutically effective amount of a polypeptide (eg, a soluble form of the polypeptide of the invention), an agonist or antagonist peptide, or a small molecule compound. Provided is a pharmaceutical composition containing together with an excipient. Carriers of this type include, but are not limited to, saline, 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 inventive compositions described above. The polypeptides of the invention and the other compounds may be used alone or in combination with other compounds, eg therapeutic compounds.

The pharmaceutical composition may be adapted to the route of administration, eg the systemic or oral route of administration. Suitable forms for systemic administration are infusion, typically intravenous. Other injection routes such as subcutaneous, intramuscular or intraperitoneal can also be used. Another means of systemic administration is transmucosal and transdermal administration using penetrants such as bile salts, fusidic acids and other surfactants. Furthermore, oral administration is possible provided that the polypeptide of the present invention or other compound can be formulated as an enteric solution or capsule. These compounds may be topically administered and / or localized in the form of ointments, pastes, gels and the like.

The dosage range required depends on the choice of peptide or other compound of the invention, the route of administration, the nature of the formulation, the condition of the patient and the judgment of the practitioner. However, suitable dosages are in the range of 0.1-100 μg / kg of patient weight. Wide variations in the required dosage are to be expected in view of the wide variety of compounds available and the differing efficiencies of administration routes. For example, oral administration would be expected to require higher dosages than administration by intravenous injection. Such variation in dosage levels can be adjusted using standard empirical optimization procedures, 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. For example, cells from a patient are genetically engineered ex vivo with a polynucleotide such as DNA or RNA encoding a polypeptide, eg, using a retroviral plasmid vector. Thereafter, these cells are introduced into the patient.

Polynucleotide and polypeptide sequences provide a valuable information resource with which to identify other sequences of similar homology. This is maximally facilitated by storing such sequences in a computer readable medium and then using the stored data to search a sequence database with known search tools such as GCC. Accordingly, in a further aspect, the present invention provides a computer readable medium having stored thereon a polynucleotide comprising the sequence of SEQ ID NO: 1, 3 or 5 and / or the polypeptide encoded thereby.

The following definitions are intended to facilitate understanding of terms often used in the above description. As used herein, "antibody" includes polyclonal and monoclonal antibodies, chimeric antibodies, single chain antibodies, humanized antibodies, as well as Fab fragments that include the products of Fab or other immunoglobulin expression libraries.
"Isolated" means altered "by the hand of man" from the natural state. If an "isolated" composition or substance is naturally-occurring, it has been altered or separated from its original environment, or both. For example, a polynucleotide or polypeptide naturally present in the body of a living animal is not "isolated", but a polynucleotide or polypeptide separated from its naturally occurring co-existing material is As used in the text, it is "isolated".

"Polynucleotide" generally refers to any polyribonucleotide or polydeoxyribonucleotide, which is unmodified RNA or D.
It can be NA, or modified RNA or DNA. “Polynucleotide” includes, but is not limited to, single-stranded and double-stranded DNA, DNA in which single-stranded region and double-stranded region are mixed, single-stranded and double-stranded RNA,
RNA and DNA in which single-stranded region and double-stranded region are mixed
And RNA, including hybrid molecules, which may be single-stranded or, more typically, double-stranded and may be a mixture of single-stranded and double-stranded regions. in addition,
"Polynucleotide" means RNA or DNA or RN
It means a triple-stranded region consisting of both A and DNA. The term "polynucleotide" also includes DNAs or RNAs that contain one or more modified bases, and DNAs or RNAs that have a backbone that has been modified for stability or other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. Various modifications can be made to DNA and RNA. Thus, "polynucleotide" embraces chemically, enzymatically, or metabolically modified forms of polynucleotides commonly found in nature, as well as chemical forms of DNA and RNA characteristic of viruses and cells. . "Polynucleotide" also embraces relatively short polynucleotides, often referred to as oligonucleotides.

By "polypeptide" is meant a peptide or protein containing two or more amino acids linked by peptide bonds or modified peptide bonds (ie, peptide isosteres). "Polypeptide"
Refers to both short chains (commonly referred to as peptides, oligopeptides or oligomers) and long chains (commonly referred to as proteins). Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. A "polypeptide" comprises an amino acid sequence modified either by natural processes, such as post-translational processing, or by chemical modification methods known in the art. Such modifications are detailed in basic textbooks, more detailed academic and research literature. Modifications can be made anywhere in the polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. The same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. The polypeptide may be branched for ubiquitination, or cyclic with or without branching. Cyclic, branched, or branched cyclic polypeptides may result from post-translational natural processes or may be produced synthetically. Modifications include acetylation, acylation,
ADP-ribosylation, amidation, flavin covalent attachment,
Covalent bond of heme moiety, covalent bond of nucleotide or nucleotide derivative, covalent bond of lipid or lipid derivative,
Phosphatidylinositol covalent bond, crosslinking, cyclization,
Disulfide bond formation, demethylation, covalent cross-link formation, cystine formation, pyroglutamate formation, formylation, γ-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation , Oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer RNA-mediated addition of amino acids to proteins such as arginylation, ubiquitination, etc. (eg,
Proteins-Structure and Molecular Properties 2nd
Ed., TE Creighton, WH Freeman and Company, Ne
w York, 1993; Posttranslational Covalent Modificat
ion of Proteins, BC Johnson, Academic Press, N
Wold, F., Post-translational Prot in ew York, 1983
ein Modifications: Perspectives and Prospects, pg
s. 1-12; Seifter et al., “Analysis for protein modific
ations and nonprotein cofactors ", Meth Enzymol (19
90) 182: 626-646; and Rattan et al., “Protein Synthes.
is: Post-translational Modifications and Aging ", A
nn NY Acad Sci (1992) 663: 48-62).

As used herein, a “variant” is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide but retains essential properties. Typical polynucleotide variants differ in nucleotide sequence from the reference polynucleotide. Changes in the nucleotide sequence of this variant may or may not alter the amino acid sequence of the polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, deletions, additions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. Typical polypeptide variants differ in amino acid sequence from a reference polypeptide. Generally, the sequence of the reference polypeptide and the sequence of the variant are generally similar and limited to differences that are identical in many regions. Any combination of variant and reference polypeptide 1
The amino acid sequences may differ due to the above substitutions, deletions and additions. The substituted or added amino acid residue may or may not be one encoded by the genetic code. Variants of polynucleotides or polypeptides may be naturally occurring variants, such as allelic variants, or variants not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides can be made by mutagenesis or direct synthesis.

"Identity," as known in the art, is a relationship between two or more such sequences, as determined by comparing the polypeptide or polynucleotide sequences.
In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as determined by the match between such sequences. The “identity” can be calculated easily by a known method, and as such a method, for example, Computational M
olecular Biology, Lesk, AM, Oxford University
Press, New York, 1988; Biocomputing: Informatics a
nd Genome Projects, Smith, DW Edition, Academic Pres
s, New York, 1993; Computer Analysis of Sequence Da
ta, Part I, Griffin, AM and Griffin, HG ed., Hu
mana Press, New Jersey, 1994; Sequence Analysis in
Molecular Biology, von Heinje, G., Academic Pres
s, 1987; Sequence Analysis Primer, Gribskov, M. an.
d Devereux, J. Ed., M Stockton Press, New York, 199
1; and Carillo, H. and Lipman, D., SIAM J. Appli
ed Math., 48: 1073 (1988), but not limited thereto. The method of determining "identity" is
Designed to give the largest match between the sequences considered. In addition, methods to determine "identity" have been compiled into publicly available computer programs. Two
Computer program methods for determining "identity" between sequences include the GCG program package (Devereux,
J. et al., Nucleic Acids Research 12 (1): 387 (1984)), B
LASTP, BLASTN and FASTA (Atschul,
SF et al., J. Molec. Biol. 215: 403-410 (1990)), but is not limited thereto. BLAST X programs are publicly available from NCBI and other sources
(BLAST Manual, Altschul, S. et al., NCBI NLM NIH Bethesd
a, MD 20894; Altschul, S. et al., J. Mol. Biol. 215: 40.
3-410 (1990)). The well known Smith Waterman algorithm can also be used to determine identity.

Parameters for comparing polypeptide sequences include: 1) Algorithm: Needleman and Wunsch, J. Mol. Bi.
ol. 48: 443-453 (1970) Comparison matrix: Hentikoff and Hentikoff, Proc. N
B from atl. Acad. Sci. USA, 89: 10915-10919 (1992)
LOSSUM62 Gap penalty: 12 Gap length penalty: 4 A useful program with these parameters is Genet
It is publicly available as the "gap" program from the ics Computer Group (Madison WI). The above parameters are the default parameters (def
ault parameter) (there is no end gap penalty).

Suitable parameters for comparing the polynucleotide sequences include the following: 1) Algorithm: Needleman and Wunsch, J. Mol. Bi.
ol. 48: 443-453 (1970) Comparison Matrix: Match = +10, Mismatch = 0 Gap Penalty: 50 Gap Length Penalty: 3 Genetics Computer Group (Madison WI) "gap"
It is available as a program. The above parameters are the default parameters for polynucleotide comparison.

Preferred meanings for “identity” of polynucleotides and polypeptides are as follows (1) and (2)
Provided to. (1) Specific examples of the polynucleotide include at least 50, 60, 70, 80, 85, 90, and
There are isolated polynucleotides which comprise a polynucleotide sequence having 95, 97 or 100% identity.
This polynucleotide sequence may be identical to the reference sequence of SEQ ID NO: 1 or may contain up to an integer number of nucleotide mutations with respect to the reference sequence. The mutations are selected from the group consisting of deletions, substitutions (including transitions and transversions) or additions of at least one nucleotide, such mutations being 5'or 3'terminal positions of the reference nucleotide sequence, or their terminal positions. Can occur anywhere in between, and can be interspersed between the nucleotides in the reference sequence individually or as one or more contiguous groups within the reference sequence. Said number of nucleotide variations is obtained by multiplying the total number of nucleotides of SEQ ID NO: 1 by the integer defining percent identity divided by 100 and subtracting the product from the total number of nucleotides of SEQ ID NO: 1, ie following formula: _n n ≦ x n - can be obtained by (x _n · y). Where n _n is the number of nucleotide variations, x _n is the total number of nucleotides of SEQ ID NO: 1, y is 0.50 for 50% and 0 for 60%.
0.70 for 60, 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0 for 95%.
0.97 for 95, 97% or 1.00 for 100%, and · is a multiplication symbol, where the product of non-integers of x _n and y is the nearest integer before subtracting the product from x _n Round down to Alteration of the polynucleotide sequence encoding the polypeptide of SEQ ID NO: 2 may result in nonsense, missense or frameshift mutations in the coding sequence, which may result in alteration of the polypeptide encoded by the polynucleotide.

For example, the 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 may contain up to an integer number of amino acid mutations relative to the reference sequence such that the% identity is less than 100%. The mutations are selected from the group consisting of deletions, substitutions (including transitions and transversions) or additions of at least one nucleic acid, such mutations being 5'or 3'terminal positions of the reference nucleotide sequence, or their terminal positions. Can occur anywhere in between, and can be interspersed between the nucleotides in the reference sequence individually or as one or more contiguous groups within the reference sequence. To determine the number of nucleic acid variations for a given% identity, multiply the total number of amino acids in SEQ ID NO: 2 by the integer defining percent identity and divide by 100 and subtract the product from the total number of amino acids in SEQ ID NO: 2. That is, it can be obtained by the following equation: n _n ≦ x _n − (x _n · y). Where n _n is the number of amino acid mutations, x _n is the total number of amino acids of SEQ ID NO: 2, y is 0.70 for 70%, 0.80 for 85%, 85
% Is a number such as 0.85, and is a multiplication symbol, where the product of non-integers of x _n and y is rounded down to the nearest integer before subtracting the product from x _n .

(2) As a specific example of the polypeptide, at least 50 relative to the polypeptide reference sequence of SEQ ID NO: 2,
There is an isolated polypeptide that comprises a polypeptide with 60, 70, 80, 85, 90, 95, 97 or 100% identity. This polypeptide sequence may be identical to the reference sequence of SEQ ID NO: 2 or may contain up to an integer number of amino acid mutations with respect to the reference sequence. Said mutation is selected from the group consisting of a deletion, substitution (including conservative and non-conservative amino acid substitutions) or addition of at least one amino acid, such mutation being the amino or carboxy terminal position of the reference polypeptide sequence, or these termini. It may be located between any of the positions and may be interspersed among the amino acids in the reference sequence individually or as one or more contiguous groups within the reference sequence. The above-mentioned number of amino acid mutations is obtained by multiplying the total number of amino acids of SEQ ID NO: 2 by a value obtained by dividing an integer defining percent identity by 100 and multiplying the product by SEQ ID NO: 2
By subtracting from the total number of amino acids in
Following equation: n _a ≦ x _a - can be obtained by (x _a · y). In the formula, n _a is the number of amino acid mutations, x _a is the total number of amino acids in SEQ ID NO: 2, y is 0.50 for 50%, 0.60 for 70%, 70
0.70 for%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95, 97 for 95%
0.97 for% or 1.00 for 100%,
Is the multiplication symbol, where the non-integer product of x _a and y is rounded down to the nearest integer before subtracting the product from x _a .

For example, the polypeptide sequence of the present invention is a sequence
It may be identical to the reference sequence number 2. That is, it is
Even if 100% identical,% identity is less than 100%
To the reference sequence up to a certain integer amino acid mutation
May be included. The mutation is at least one amino acid.
Non-acid deletions, substitutions (including conservative and non-conservative amino acid substitutions
Mu) or addition, selected from the group consisting of
Is the amino or carboxy end of the reference polypeptide sequence
Present at the end positions or between these end positions
May be between the amino acids in the reference sequence individually, or
Scattered as one or more consecutive groups in the reference array
be able to. Of amino acid mutations for a given% identity
The number defines the percent identity in the total number of amino acids in SEQ ID NO: 2.
Multiply the integer by dividing by 100 and multiply the product by SEQ ID NO: 2
By subtracting from the total number of amino acids in
The following formula: n_a ≤ x_a -(X_a・ Y) Can be obtained by Where n_aIs an amino acid mutation
Is the number of x_aIs the total number of amino acids in SEQ ID NO: 2
Y is 0.70 for 70% and 0.80, 85 for 80%
% Is a number such as 0.85, and · is a multiplication symbol
And where x _aX is the non-integer product of x_aOr
Round down to the nearest whole number before subtracting.

A "fusion protein" is a protein encoded by two, often unrelated, fused genes or fragments thereof. As an example, EP-A
-0 464 describes fusion proteins comprising various parts of the constant region of immunoglobulin molecules and other human proteins or parts thereof. In many cases, for therapeutic and diagnostic use, it will be advantageous to use the immunoglobulin Fc region as part of a fusion protein, which may, for example, improve pharmacokinetic properties (eg EP-A-0232). See 262). On the other hand, for some uses it may be desirable to remove the Fc portion after expressing, detecting and purifying the fusion protein.

All publications, including patents and patent application publications, cited herein are incorporated by reference in their entirety as if each publication was explicitly and individually indicated. Incorporated here.

In Northern blots of UT7-EPO cells, E
PRG1 is observed as a single 2.4 kb message. Low levels of EPRG1 can be detected in exponentially growing cells growing in medium supplemented with 10% FBS and rh-EPO. Overnight starvation of EPO reduces the level of messages to undetectable levels. When stimulated with EPO, EPRG1 is rapidly induced, can be easily detected in 1/2 hour, and reaches the maximum level in about 1 hour. Higher levels are obtained when cells are starved not only for EPO but also for FBS. EPRG1 expression then declines but remains at elevated levels for at least 6 hours.

EPRG1 is induced by EPO in the presence of the protein synthesis inhibitor cycloheximide, whereas EPRG1 was only added with cycloheximide.
The induction of does not occur at all. Treatment with EPO + cycloheximide results in a modest superinduction of EPRG1 message levels compared to the levels obtained when EPO is used alone. EPRG1 is also induced by TPO in the human megakaryocytic leukemia cell lines MO7e and CMK, and is hyperinduced when stimulated in the presence of cycloheximide. These observations are
It has been shown that EPRG1 is the EPO and TPO primary response gene, whose induction of expression is independent of neoprotein synthesis. Therefore, induction of EPRG1 expression must occur by direct activation of the signaling pathway by EPO and TPO.

In human bone marrow, EPRG1 is also expressed as a single 2.4 kb message on Northern blots. EPRG1 is induced in in vitro cultures by EPO treatment, but its expression is induced more strongly when stimulated with G-CSF. Elevated EPRG1 expression persisted for more than 7 days in cells in culture, indicating that EPRG1 may play an important role in the development and maturation of various hematopoietic lineages.

EPRG1 expression is RNA derived from other tissues
It was found by probing a Northern blot with. Highest levels are found in hematopoietic-related tissues including bone marrow, fetal liver, peripheral blood leukocytes and lungs. EPRG1
Is also expressed to a lesser extent in the spleen and thymus.

The 3'-UTR segment of the RNA transcript (nucleotide number 477-2115 of SEQ ID NO: 1) is useful for diagnostic purposes. The 3'-UTR is unique to a particular gene and sequence. Furthermore, 3'-
UTRs may be useful in developing screening assays to identify agents that modulate RNA stability and turnover (turnover) rates. Decreasing the steady-state level of the message will reduce the amount of translated protein in the cell and neutralize the action of that protein. Since SOCS family members are negative regulators of growth factor signaling, SOCS antagonists will function as growth factor agonists. One way to achieve this outcome is
An antisense technique to reduce or eliminate EPRG1 messages. Since RNA determinants of RNA stability are often associated with the 3'-UTR sequence, a second approach would involve modulating the cellular machinery governing EPRG1 message turnover.

Sequence Information Sequence No. 1:

SEQ ID NO: 2: Translation of SEQ ID NO: 1 in frame 1

SEQ ID NO: 3:

SEQ ID NO: 4: Translation of SEQ ID NO: 3 in frame 1

SEQ ID NO: 5:

[0088]

[Sequence list] (1) General information   (i) Applicant: Lord, Kenneth; Dillon, Susan   (ii) Title of invention: EPO primary response gene, EPRG1   (iii) Number of sequences: 5   (iv) Communication destination:      (A) Name: Ratner & Prestia      (B) Street name: P.O.Box 980      (C) City name: Valley Forge      (D) State name: PA      (E) Country name: USA      (F) Postal Code: 19482   (v) Computer-readable format:      (A) Media type: floppy disk      (B) Computer: IBM compatible      (C) Operating system: DOS      (D) Software: FastSEQ for Windows (registered trademark) Version 2.0   (vi) Data of this application:      (A) Application number:      (B) Filing date: May 1, 1998      (C) Classification:   (vii) Original application data:      (A) Application number: 60 / 045,890      (B) Filing date: May 7, 1998   (viii) Agent Information:      (A) Name: Prestia, Paul F      (B) Registration number: 23,031      (C) Reference / Document number: GP-70010   (ix) Telecommunications information:      (A) Telephone: 610-407-0700      (B) Telefax: 610-407-0701      (C) Telex:

[0089] SEQ ID NO: 1 Array length: 2342 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: cDNA Array CGCAGATCCA CGCTGGCTCC GTGCGCCATG GTCACCCACA GCAAGTTTCC CGCCGCCGGG 60 ATGAGCCGCC CCCTGGACAC CAGCCTGCGC CTCAAGACCT TCAGCTCCAA GAGCGAGTAC 120 CAGCTGGTGG TGAACGCAGT GCGCAAGCTG CAGGAGAGCG GCTTCTACTG GAGCGCAGTG 180 ACCGGCGGCG AGGCGAACCT GCTGCTCAGT GCCGAGCCCG CCGGCACCTT TCTGATCCGC 240 GACAGCTCGG ACCAGCGCCA CTTCTTCACG CTCAGCGTCA AGACCCAGTC TGGGACCAAG 300 AACCTGCGCA TCCAGTGTGA GGGGGGCAGC TTCTCTCTGC AGAGCGATCC CCGGAGCACG 360 CAGCCCGTGC CCCGCTTCGA CTGCGTGCTC AAGCTGGTGC ACCACTACAT GCCGCCCCCT 420 GGAGCCCCCT CCTTCCCCTC GCCACCTACT GAACCCTCCT CCGAGGTGCC CGAGCAGCCG 480 TCTGCCCAGC CACTCCCTGG GAGTCCCCCC AGAAGAGCCT ATTACATCTA CTCCGGGGGC 540 GAGAAGATCC CCCTGGTGTT GAGCCGGCCC CTCTCCTCCA ACGTGGCCAC TCTTCAGCAT 600 CTCTGTCGGA AGACCGTCAA CGGCCACCTG GACTCCTATG AGAAAGTCAC CCAGCTGCCG 660 GGGCCCATTC GGGAGTTCCT GGACCAGTAC GATGCCCCGC TTTAAGGGGT AAAGGGCGCA 720 AAGGGCATGG GTCGGGAGAG GGGACGCAGG CCCCTCTCCT CCGTGGCACA TGGCACAAGC 780 ACAAGAAGCC AACCAGGAGA GAGTCCTGTA GCTCTGGGGG GAACGAGGGC GGACAGGCCC 840 CTCCCTCTGC CCTCTCCCTG CAGAATGTGG CAGGCGGACC TGGAATGTGT TGGAGGGAAG 900 GGGGAGTACC ACCTGAGTCT CCAGCTTCTC CGGAGGAGCC AGCTGTCCTG GTGGGACGAT 960 AGCAACCACA AGTGGATTCT CCTTCAATTC CTCAGCTTCC CCTCTGCCTC CAAACAGGGG 1020 ACACTTCGGG AATGCTGAAC TAATGAGAAC TGCCAGGGAA TCTTCAAACT TTCCAACGGA 1080 ACTTGTTTGC TCTTTGATTT GGTTTAAACC TGAGCTGGTT GTGGAGCCTG GGAAAGGTGG 1140 AAGAGAGAGA GGTCCTGAGG GCCCCAGGGC TGCGGGCTGG CGAAGGAAAT GGTCACACCC 1200 CCCGCCCACC CCAGGCGAGG ATCCTGGTGA CATGCTCCTC TCCCTGGCTC CGGGGAGAAG 1260 GGCTTGGGGT GACCTGAAGG GAACCATCCT GGTGCCCCAC ATCCTCTCCT CCGGGACAGT 1320 CACCGAAAAC ACAGGTTCCA AAGTCTACCT GGTGCCTGAG AGCCCAGGGC CCTTCCTCCG 1380 TTTTAAGGGG GAAGCAACAT TTGGAGGGGA TGGATGGGCT GGTCAGCTGG TCTCCTTTTC 1440 CTACTCATAC TATACCTTCC TGTACCTGGG TGGATGGGGC GGGAGGATGG AGGAGACGGA 1500 CATCTTTCAC CTCAGGCTCC TGGTAGAGAA GACAGGGGAT TCTACTCTGT GCCTCCTGAC 1560 TATGTCTGGC TAAGAGATTC GCCTTAAATG CTCCCTGTCC CATGGAGAGG GACCCAGCAT 1620 AGGAAAGCCA CATACTCAGC CTGGATGGGT GGAGAGGCTG AGGGACTCAC TGGAGGGCAC 1680 CAAGCCAGCC CACAGCCAGG AAGTGGGGAG GGGGGGCGGA AACCCATGCC TCCCAGCTGA 1740 GCACTGGGAA TGTCAGCCCA GTAAGTATTG GCCAGTCAGG CGCCTCGTGG TCAGAGCAGA 1800 GCCACCAGGT CCCACTGCCC CGAGCCCTGC ACAGCCCTCC CTCCTGCCTG GGTGGGGGAG 1860 GCTGGAAGTC ATTGGAAAAG CTGGACTGCT GCCACCCCGG GTGCTCCCGC TCTGCCATAG 1920 CACTGATCAG TGACAATTTA CAGGAATGTA GCCAGCGATG GAATTACCTG GAACAGTTTT 1980 TTGTTTTTGT TTTTGTTTTT GTTTTTGTGG GGGGGGGGGCAA CTAAACAAAC ACAAAGTATT 2040 TTGTGTCAGG TATTGGGCTG GACAGGGCAC TTGTGTGTTG GGGTGGTTTT TTTCTCTATT 2100 TTTTGGTTTG TTTCTTGTTT TTTAATAATG TTTACAATCT GCCTCAATCA CTCTGTCTTT 2160 TATAAAGATT CCACCTCCAG TCCTCTCTCC TCCCCCCTAC TCAGGCCCTT GAGGCTATTA 2220 GGAGATGCTT GAAGAACTCA ACAAAATCCC AATCCAAGTC AAACTTTGCA CATATTTATA 2280 TTTATATTCA GAAAAGAAAC ATTTCAGTAA TTTATAATAA AGAGCACTAT TTTTTTAATC 2340 AN 2342

[0090] SEQ ID NO: 2 Array length: 225 Sequence type: Amino acid Number of chains: Single chain Topology: linear Sequence type: Protein Array  Met Val Thr His Ser Lys Phe Pro Ala Ala Gly Met Ser Arg Pro Leu   1 5 10 15  Asp Thr Ser Leu Arg Leu Lys Thr Phe Ser Ser Lys Ser Glu Tyr Gln              20 25 30  Leu Val Val Asn Ala Val Arg Lys Leu Gln Glu Ser Gly Phe Tyr Trp          35 40 45  Ser Ala Val Thr Gly Gly Glu Ala Asn Leu Leu Leu Ser Ala Glu Pro      50 55 60  Ala Gly Thr Phe Leu Ile Arg Asp Ser Ser Asp Gln Arg His Phe Phe  65 70 75 80  Thr Leu Ser Val Lys Thr Gln Ser Gly Thr Lys Asn Leu Arg Ile Gln                  85 90 95  Cys Glu Gly Gly Ser Phe Ser Leu Gln Ser Asp Pro Arg Ser Thr Gln              100 105 110  Pro Val Pro Arg Phe Asp Cys Val Leu Lys Leu Val His His Tyr Met          115 120 125  Pro Pro Pro Gly Ala Pro Ser Phe Pro Ser Pro Pro Thr Glu Pro Ser      130 135 140  Ser Glu Val Pro Glu Gln Pro Ser Ala Gln Pro Leu Pro Gly Ser Pro  145 150 155 160  Pro Arg Arg Ala Tyr Tyr Ile Tyr Ser Gly Gly Glu Lys Ile Pro Leu                  165 170 175  Val Leu Ser Arg Pro Leu Ser Ser Asn Val Ala Thr Leu Gln His Leu              180 185 190  Cys Arg Lys Thr Val Asn Gly His Leu Asp Ser Tyr Glu Lys Val Thr          195 200 205  Gln Leu Pro Gly Pro Ile Arg Glu Phe Leu Asp Gln Tyr Asp Ala Pro      210 215 220  Leu  225

[0091] SEQ ID NO: 3 Array length: 2111 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: cDNA Array GAATTCGCGA ACAGCTCGGA CCAGCGGCAC TTCTTTCAGC TCAGCGTCAA GACCCAGTCT 60 GGGACCAAGA ACCTGCGCAT CCAATGTGAG GGGGGCAGCT TCTGTCTGCA GAGCGATCCC 120 CGGAAGACGC AGCCCGTGCC CCGCGTCGAC TGCGTGCTGA AGCTGGTGCA CCACTACATG 180 CCGCCCCCTG GAGCCCCCTC CTTCCCCTCG CCACCTACTG AACCCTCCTC CGAGGTGCCC 240 GAGCAGCCGT CTGCCCAGCC ACTCCCTGGG AGTCCCCCCA GAAGAGCCTA TTACATCTAC 300 TCCGGGGGCG AGAAGATCCC CCTGGTGTTG AGCCGGCCCC TCTCCTCCAA CGTGGCCACT 360 CTTCAGCATC TCTGTCGGAA GACCGTCAAC GGCCACCTGG ACTCCTATGA GAAAGTCACC 420 CAGCTGCCGG GGCCCATTCG GGAGTTCCTG GACCAGTACG ATGCCCCGCT TTAAGGGGTA 480 AAGGGCGCAA AGGGCATGGG TCGGGAGAGG GGACGCAGGC CCCTCTCCTC CGTGGCACAT 540 GGCACAAGCA CAAGAAGCCA ACCAGGAGAG AGTCCTGTAG CTCTGGGGGG AACGAGGGCG 600 GACAGGCCCC TCCCTCTGCC CTCTCCCTGC AGAATGTGGC AGGCGGACCT GGAATGTGTT 660 GGAGGGAAGG GGGAGTACCA CCTGAGTCTC CAGCTTCTCC GGAGGAGCCA GCTGTCCTGG 720 TGGGACGATA GCAACCACAA GTGGATTCTC CTTCAATTCC TCAGCTTCCC CTCTGCCTCC 780 AAACAGGGGA CACTTCGGGA ATGCTGAACT AATGAGAACT GCCAGGGAAT CTTCAAACTT 840 TCCAACGGAA CTTGTTTGCT CTTTGATTTG GTTTAAACCT GAGCTGGTTG TGGAGCCTGG 900 GAAAGGTGGA AGAGAGAGAG GTCCTGAGGG CCCCAGGGCT GCGGGCTGGC GAAGGAAATG 960 GTCACACCCC CCGCCCACCC CAGGCGAGGA TCCTGGTGAC ATGCTCCTCT CCCTGGCTCC 1020 GGGGAGAAGG GCTTGGGGTG ACCTGAAGGG AACCATCCTG GTGCCCCACA TCCTCTCCTC 1080 CGGGACAGTC ACCGAAAACA CAGGTTCCAA AGTCTACCTG GTGCCTGAGA GCCCAGGGCC 1140 CTTCCTCCGT TTTAAGGGGG AAGCAACATT TGGAGGGGAC GGATGGGCTG GTCAGCTGGT 1200 CTCCTTTTCC TACTCATACT ATACCTTCCT GTACCTGGGT GGATGGGGCG GGAGGATGGA 1260 GGAGACGGGA CATCTTTCAC CTCAGGCTCC TGGTAGAGAA GACAGGGGAT TCTACTCTGT 1320 GCCTCCTGAC TATGTCTGGC TAAGAGATTC GCCTTAAATG CTCCCTGTCC CATGGAGAGG 1380 GACCCAGCAT AGGAAAGCCA CATACTCAGC CTGGATGGGT GGAGAGGCTG AGGGACTCAC 1440 TGGAGGGCAC CAAGCCAGCC CACAGCCAGG GAAGTGGGGA GGGGGGGCGG AAACCCATGC 1500 CTCCCAGCTG AGCACTGGGA ATGTCAGCCC AGTAAGTATT GGCCAGTCAG GCGCCTCGTG 1560 GTCAGAGCAG AGCCACCAGG TCCCACTGCC CCGAGCCCTG CACAGCCCTC CCTCCTGCCT 1620 GGGTGGGGGA RGCTGGAAGT CATTGGAAAA GCTGGACTGC TGCCACCCCG GGTGCTCCCG 1680 CTCTGCCATA GCACTGATCA GTGACAATTT ACAGGAATGT AGCCAGCGAT GGAATTACCT 1740 GGAACAGTTT TTTGTTTTTG TTTTTGTTTT TGTTTTTGTG GGGGGGGGCA ACTAAACAAA 1800 CACAAAGTAT TTTGTGTCAG GTATTGGGCT GGACAGGGCA CTTGTGTGTT GGGGTGGTTT 1860 TTTTCTCTAT TTTTTGGTTT GTTTCTTGTT TTTTAATAAT GTTTACAATC TGCCTCAATC 1920 ACTCTGTCTT TTATAAAGAT TCCACCTCCA GTCCTCTCTC CTCCCCCCTA CTCAGGCCCT 1980 TGAGGTTATT AGGAGATGCT TGAAGAACTC AACAAAATCC CAATCCAAGT CAAACTTTGC 2040 ACATATTTAT ATTTATATTC AGAAAAGAAA CATTTCAGTA ATTTATAATA AAGAGCACTA 2100 TTTTTTTAAC G 2111

[0092] SEQ ID NO: 4 Sequence length: 157 Sequence type: Amino acid Number of chains: Single chain Topology: linear Sequence type: Protein Array  Glu Phe Ala Asn Ser Ser Asp Gln Arg His Phe Phe Gln Leu Ser Val   1 5 10 15  Lys Thr Gln Ser Gly Thr Lys Asn Leu Arg Ile Gln Cys Glu Gly Gly              20 25 30  Ser Phe Cys Leu Gln Ser Asp Pro Arg Lys Thr Gln Pro Val Pro Arg          35 40 45  Val Asp Cys Val Leu Lys Leu Val His Arg Ser Gly Ala Pro Ala Gly      50 55 60  Ala Pro Ser Phe Pro Ser Pro Pro Thr Glu Pro Ser Ser Glu Val Pro  65 70 75 80  Glu Gln Pro Ser Ala Gln Pro Leu Pro Gly Ser Pro Pro Arg Arg Ala                  85 90 95  Tyr Tyr Ile Tyr Ser Gly Gly Glu Lys Ile Pro Leu Val Leu Ser Arg              100 105 110  Pro Leu Ser Ser Asn Val Ala Thr Leu Gln His Leu Cys Arg Lys Thr          115 120 125  Val Asn Gly His Leu Asp Ser Tyr Glu Lys Val Thr Gln Leu Pro Gly      130 135 140  Pro Ile Arg Glu Phe Leu Asp Gln Tyr Asp Ala Pro Leu  145 150 155

[0093] SEQ ID NO: 5 Array length: 2342 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: cDNA Array CGCAGATCCA CGCTGGCTCC GTGCGCCATG GTCACCCACA GCAAGTTTCC CGCCGCCGGG 60 ATGAGCCGCC CCCTGGACAC CAGCCTGCGC CTCAAGACCT TCAGCTCCAA GAGCGAGTAC 120 CAGCTGGTGG TGAACGCAGT GCGCAAGCTG CAGGAGAGCG GCTTCTACTG GAGCGCAGTG 180 ACCGGCGGCG AGGCGAACCT GCTGCTCAGT GCCGAGCCCG CCGGCACCTT TCTGATCCGC 240 GACAGCTCGG ACCAGCGCCA CTTCTTCACG CTCAGCGTCA AGACCCAGTC TGGGACCAAG 300 AACCTGCGCA TCCAGTGTGA GGGGGGCAGC TTCTCTCTGC AGAGCGATCC CCGGAGCACG 360 CAGCCCGTGC CCCGCTTCGA CTGCGTGCTC AAGCTGGTGC ACCACTACAT GCCGCCCCCT 420 GGAGCCCCCT CCTTCCCCTC GCCACCTACT GAACCCTCCT CCGAGGTGCC CGAGCAGCCG 480 TCTGCCCAGC CACTCCCTGG GAGTCCCCCC AGAAGAGCCT ATTACATCTA CTCCGGGGGC 540 GAGAAGATCC CCCTGGTGTT GAGCCGGCCC CTCTCCTCCA ACGTGGCCAC TCTTCAGCAT 600 CTCTGTCGGA AGACCGTCAA CGGCCACCTG GACTCCTATG AGAAAGTCAC CCAGCTGCCG 660 GGGCCCATTC GGGAGTTCCT GGACCAGTAC GATGCCCCGC TTTAAGGGGT AAAGGGCGCA 720 AAGGGCATGG GTCGGGAGAG GGGACGCAGG CCCCTCTCCT CCGTGGCACA TGGCACAAGC 780 ACAAGAAGCC AACCAGGAGA GAGTCCTGTA GCTCTGGGGG GAACGAGGGC GGACAGGCCC 840 CTCCCTCTGC CCTCTCCCTG CAGAATGTGG CAGGCGGACC TGGAATGTGT TGGAGGGAAG 900 GGGGAGTACC ACCTGAGTCT CCAGCTTCTC CGGAGGAGCC AGCTGTCCTG GTGGGACGAT 960 AGCAACCACA AGTGGATTCT CCTTCAATTC CTCAGCTTCC CCTCTGCCTC CAAACAGGGG 1020 ACACTTCGGG AATGCTGAAC TAATGAGAAC TGCCAGGGAA TCTTCAAACT TTCCAACGGA 1080 ACTTGTTTGC TCTTTGATTT GGTTTAAACC TGAGCTGGTT GTGGAGCCTG GGAAAGGTGG 1140 AAGAGAGAGA GGTCCTGAGG GCCCCAGGGC TGCGGGCTGG CGAAGGAAAT GGTCACACCC 1200 CCCGCCCACC CCAGGCGAGG ATCCTGGTGA CATGCTCCTC TCCCTGGCTC CGGGGAGAAG 1260 GGCTTGGGGT GACCTGAAGG GAACCATCCT GGTGCCCCAC ATCCTCTCCT CCGGGACAGT 1320 CACCGAAAAC ACAGGTTCCA AAGTCTACCT GGTGCCTGAG AGCCCAGGGC CCTTCCTCCG 1380 TTTTAAGGGG GAAGCAACAT TTGGAGGGGA TGGATGGGCT GGTCAGCTGG TCTCCTTTTC 1440 CTACTCATAC TATACCTTCC TGTACCTGGG TGGATGGGGC GGGAGGATGG AGGAGACGGA 1500 CATCTTTCAC CTCAGGCTCC TGGTAGAGAA GACAGGGGAT TCTACTCTGT GCCTCCTGAC 1560 TATGTCTGGC TAAGAGATTC GCCTTAAATG CTCCCTGTCC CATGGAGAGG GACCCAGCAT 1620 AGGAAAGCCA CATACTCAGC CTGGATGGGT GGAGAGGCTG AGGGACTCAC TGGAGGGCAC 1680 CAAGCCAGCC CACAGCCAGG AAGTGGGGAG GGGGGGCGGA AACCCATGCC TCCCAGCTGA 1740 GCACTGGGAA TGTCAGCCCA GTAAGTATTG GCCAGTCAGG CGCCTCGTGG TCAGAGCAGA 1800 GCCACCAGGT CCCACTGCCC CGAGCCCTGC ACAGCCCTCC CTCCTGCCTG GGTGGGGGAG 1860 GCTGGAAGTC ATTGGAAAAG CTGGACTGCT GCCACCCCGG GTGCTCCCGC TCTGCCATAG 1920 CACTGATCAG TGACAATTTA CAGGAATGTA GCCAGCGATG GAATTACCTG GAACAGTTTT 1980 TTGTTTTTGT TTTTGTTTTT GTTTTTGTGG GGGGGGGGGCAA CTAAACAAAC ACAAAGTATT 2040 TTGTGTCAGG TATTGGGCTG GACAGGGCAC TTGTGTGTTG GGGTGGTTTT TTTCTCTATT 2100 TTTTGGTTTG TTTCTTGTTT TTTAATAATG TTTACAATCT GCCTCAATCA CTCTGTCTTT 2160 TATAAAGATT CCACCTCCAG TCCTCTCTCC TCCCCCCTAC TCAGGCCCTT GAGGCTATTA 2220 GGAGATGCTT GAAGAACTCA ACAAAATCCC AATCCAAGTC AAACTTTGCA CATATTTATA 2280 TTTATATTCA GAAAAGAAAC ATTTCAGTAA TTTATAATAA AGAGCACTAT TTTTTTAATC 2340 AN 2342

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 7/00 A61P 11/06 4C084 7/06 19/02 4H045 11/06 25/28 19/02 29 / 00 101 25/28 31/18 29/00 101 35/00 31/18 37/00 35/00 37/08 37/00 43/00 111 37/08 C07K 14/47 43/00 111 16/18 C07K 14 / 47 C12N 1/15 16/18 1/19 C12N 1/15 1/21 1/19 C12P 21/02 C 1/21 C12Q 1/68 A 5/10 G01N 33/15 Z C12P 21/02 33/50 Z C12Q 1/68 33/53 D G01N 33/15 M 33/50 C12N 15/00 ZNAA 33/53 5/00 A A61K 37/02 (72) Inventor Susan Bee. Dillon United States 19425 Chester Springs, Pennsylvania, Turamoa Circle 1209 F-term (reference) 2G045 AA34 AA35 DA13 DA14 DA36 FB02 4B024 AA01 BA44 CA04 GA11 HA12 4B063 QA12 QA17 QA18 QA19 QQ42 QQ52 QR19 CC25BQ4BQ0BQQBQQBQQQA QRQR QRQR QR QR QR QR AA93Y AB01 AC14 BA02 CA24 CA44 CA46 4C084 AA01 AA02 AA06 AA07 AA13 AA17 BA01 BA02 BA08 BA22 CA18 DC50 MA01 NA14 ZA021 ZA511 ZA551 ZA591 ZB071 ZB091 ZB111 ZB131 ZB151 ZB261 ZC351 ZC412 ZC422 ZC551 4H045 AA10 AA11 AA20 AA30 BA10 CA40 DA76 DA86 EA22 EA24 EA28 EA50 FA74

Claims (16)

[Claims] 1. An isolated polypeptide selected from (i) to (iii) below: (i) has at least 70% identity to the amino acid sequence of SEQ ID NO: 4 over the entire length of SEQ ID NO: 4. A polypeptide comprising the amino acid sequence having, (ii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 4, and (iii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 4. 2. An isolated polynucleotide selected from the following (i) to (vi), or a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence thereof: (i) a sequence spanning the entire length of SEQ ID NO: 4 A polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 4, (ii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 4 A polynucleotide comprising a nucleotide sequence having at least 70% identity over its entire length to the encoding nucleotide sequence, (iii) at least 7 relative to the nucleotide sequence of SEQ ID NO: 3 over the entire length of SEQ ID NO: 3
A polynucleotide comprising a nucleotide sequence having 0% identity, (iv) a polynucleotide comprising a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 4, (v) a nucleotide sequence of SEQ ID NO: 3 And a (vi) a polynucleotide obtained by screening a suitable library with a labeled probe having the nucleotide sequence of SEQ ID NO: 3 or a fragment thereof under stringent hybridization conditions. 3. An antibody immunospecific for the polypeptide of claim 1. 4. A pharmaceutical composition according to (i) or (ii) below: (i) A pharmaceutical composition for treating a subject in need of enhancing the activity or expression of the polypeptide according to claim 1. Which comprises (a) a therapeutically effective amount of an agonist for said polypeptide and / or (b) a nucleotide sequence encoding said polypeptide in a form which results in the in vivo production of said polypeptide activity. A pharmaceutical composition containing a polynucleotide; or (ii) a pharmaceutical composition for treating a subject in need of suppressing the activity or expression of the polypeptide according to claim 1, comprising (a) treatment An effective amount of an antagonist to the polypeptide, and / or (b) a nucleic acid molecule that suppresses the expression of a nucleotide sequence encoding the polypeptide, and / or (c) a therapeutically effective amount of the polypeptide. Peptide ligands, substrates or polypeptide that competes with said polypeptide for receptor, which contains the pharmaceutical composition. 5. A method for examining a disease associated with expression or activity of the polypeptide of claim 1 in a subject or susceptibility to said disease, comprising: (a) Determining whether there is a mutation in the nucleotide sequence encoding the peptide, and / or (b) analyzing the presence or amount of expression of said polypeptide in a sample obtained from said subject. Method. 6. A screening method for identifying a compound that stimulates or suppresses the function of the polypeptide according to claim 1, which comprises (a) a candidate compound and the polypeptide (or a protein carrying the polypeptide). Cell or its membrane) or its fusion protein is measured by a label directly or indirectly bound to the candidate compound, (b) the candidate compound and the polypeptide (or the polypeptide) Binding to cells or their membranes) or their fusion proteins, in the presence of a labeled competitor, (c) the candidate compound results in a signal generated by activation or inhibition of the polypeptide. Whether or not the candidate compound and the polypeptide are included in the detection system suitable for cells or cell membranes carrying the polypeptide. And a solution is prepared to prepare a mixture, the activity of the polypeptide in the mixture is measured, and the activity of the mixture is compared to a standard, and (e) the candidate compound is the polypeptide in the cell. A screening method comprising a method selected from the group consisting of detecting the effect on the production of the mRNA encoding the polypeptide and the polypeptide. 7. An antagonist of the polypeptide of claim 1. 8. An expression system containing a polynucleotide capable of producing the polypeptide of claim 1 when the expression system described below is present in a compatible host cell. 9. A host cell which, under suitable 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 its entire length. A method for producing a recombinant host cell, comprising transforming or transfecting a cell with the expression system according to claim 8. 10. A recombinant host cell obtained by the method of claim 9. 11. SEQ ID NO: 4 spanning the entire length of SEQ ID NO: 4
The membrane of a recombinant host cell according to claim 10, which expresses a polypeptide comprising an amino acid sequence having at least 70% identity to the amino acid sequence of. 12. A method according to claim 10, comprising culturing the host cell of claim 10 under conditions sufficient to produce the polypeptide and recovering the polypeptide from its culture medium. Production method. 13. An isolated polynucleotide selected from (a) to (d) below: (a) has at least 70% identity to the nucleotide sequence of SEQ ID NO: 5 over the entire length of SEQ ID NO: 5. A polynucleotide comprising the nucleotide sequence having, (b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 5, (c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 5, and (d) (a), (b ) Or a polynucleotide complementary to the polynucleotide of (c). 14. An isolated polynucleotide selected from (a) to (d) below: (a) has at least 70% identity to the polynucleotide of SEQ ID NO: 1 over the entire length of SEQ ID NO: 1. A polynucleotide comprising the nucleotide sequence having, (b) a polynucleotide comprising the polynucleotide of SEQ ID NO: 1, (c) a polynucleotide of SEQ ID NO: 1, and (d)
A polynucleotide complementary to the polynucleotide of (a), (b) or (c). 15. An isolated polypeptide of (a) or (b) below: (a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 4,
Or (b) a polypeptide comprising an amino acid sequence of SEQ ID NO: 4 in which at least one amino acid has been deleted, substituted or added, and having EPRG1 activity. 16. A polynucleotide encoding the polypeptide according to claim 15.