WO1999063065A1 - A human hematopoietic cell derived rna cyclase (hrdc) gene (cbuaea12) - Google Patents

Abstract

The CBUAEA12 polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilising CBUAEA12 polypeptides and polynucleotides in therapy, and diagnostic assays for such.

Description

A Human Hematopoietic Cell Derived RNA Cyclase (HRDQ Gene (CBUAEA12)

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.

Summary of the Invention

The present invention relates to CBUAEA12, in particular CBUAEA12 polypeptides and CBUAEA12 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 of AIDS, cancer, hypertension, and hepatitis, 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 CBUAEA12 imbalance with the identified compounds. In a still further aspect, the invention relates to diagnostic assays for detecting diseases associated with inappropriate CBUAEA12 activity or levels.

Description of the Invention

In a first aspect, the present invention relates to CBUAEA12 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:l.

Polypeptides of the present invention are believed to be members of the RNA cyclase gene family of polypeptides. They are therefore of interest because this gene is homologous to the RNA 3 '- terminal phospate cyclase. In addition, RNA cyclase is very important for RNA, as it may play important roles in message transduction. This gene is highly homologous to a Drosophila yolk protein gene YP3. These properties are hereinafter referred to as "CBUAEA12 activity" or

"CBUAEA12 polypeptide activity" or "biological activity of CBUAEA12". Also included amongst these activities are antigenic and immunogenic activities of said CBUAEA12 polypeptides, in particular the antigenic and immunogenic activities of the polypeptide of SEQ ID NO:2. Preferably, a polypeptide of the present invention exhibits at least one biological activity of CBUAEA12. The polypeptides of the present mvention 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. Trie present invention also includes include variants of the aforementioned polypeptides, that is polypeptides that vary from the referents by conservative amino acid substitutions, whereby a residue is substituted by another with like characteristics. Typical such substitutions are among Ala, Val, Leu and lie; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gin; and among the basic residues Lys and Arg; or aromatic residues Phe and Tyr. Particularly preferred are variants 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 CBUAEA12 polynucleotides Such polynucleotides include isolated polynucleotides compnsing 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 compnsing 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 compnsing 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 compnsmg 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% identiy are more highly preferred, and those with at least 99% identity are most highly preferred Such polynucleotides include a polynucleotide compnsmg the polynucleotide of SEQ ID NO 1 as well as the polynucleotide of SEQ LD NO 1

The mvention also provides polynucleotides which are complementary to all the above descnbed polynucleotides The nucleotide sequence of SEQ ID NO 1 shows homology with X04754, Drosophila yolk polypeptide YP3 gene (Y L Yan, et al Nucleic Acids Res 1987, 15 67-85) The nucleotide sequence of SEQ ID NO 1 is a cDNA sequence and compnses a polypeptide encoding sequence (nucleotides 302 to 1096) encoding a polypeptide of 265 amino acids, the polypeptide of SEQ ED NO 2 The nucleotide sequence encodmg the polypeptide of SEQ ID NO 2 may be identical to the polypeptide encodmg sequence contamed 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 The polypeptide of SEQ ID NO 2 is structurally related to other proteins of the RNA cyclase gene family, having homology and/or structural similanty with P56175. a Drosophila RNA 3'-termιnal phosphate cyclase (RNA-cyclase)(P Genschick, et al EMBO J 1997, 162955-2967)

Preferred polypeptides and polynucleotides of the present mvention are expected to have, inter aha, similar biological functions/properties to their homologous polypeptides and polynucleotides Furthermore, preferred polypeptides and polynucleotides of the present mvention have at least one CBUAEA12 activity

Polynucleotides of the present mvention may be obtained, usmg standard cloning and screenmg techniques, from a cDNA hbrary denved from mRNA in cells of human umbilical cord blood, usmg the expressed sequence tag (EST) analysis (Adams, M O , 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 mvention can also be obtamed from natural sources such as genomic DNA branes or can be synthesized usmg well known and commercially available techniques

When polynucleotides of the present mvention are used for the recombmant production of polypeptides of the present mvention, the polynucleotide may mclude the coding sequence for the mature polypeptide, by itself, or the codmg sequence for the mature polypeptide in reading frame with other codmg 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 punfication of the fused polypeptide can be encoded In certain prefened embodiments of this aspect of the mvention, the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen. Inc ) and descnbed m Gentz et al , Proc Nad 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 transcnbed, non-translated sequences, splicing and polyadenylation signals, nbosome bmdmg sites and sequences that stabilize mRNA Further embodiments of the present mvention mclude polynucleotides encodmg polypeptide vanants which compnse the ammo acid sequence of SEQ ID NO 2 and m which several, for instance from 5 to 10, 1 to 5, 1 to 3, 1 to 2 or 1, ammo acid residues are substituted, deleted or added, in any combination

Polynucleotides which are identical or sufficiently identical to a nucleotide sequence contained m SEQ ID NO 1, may be used as hybndization probes for cDNA and genomic DNA or as pπmers for a nucleic acid amplification (PCR) reaction, to isolate full-length cDNAs and genomic clones encodmg polypeptides of the present mvention and to isolate cDNA and genomic clones of other genes (mcludmg genes encoding homologs and orthologs from species other than human) that have a high sequence similanty to SEQ ID NO 1 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 pnmers will generally compnse 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 encodmg a polypeptide of the present mvention, mcludmg homologs and orthologs from species other than human, may be obtamed by a process which compnses the steps of screening an appropnate hbrary under strmgent hybndization conditions with a labeled probe having the sequence of SEQ ID NO 1 or a fragment thereof, and isolating full-length cDNA and genomic clones containmg said polynucleotide sequence Such hybndization techniques are well known to the skilled artisan Preferred strmgent hybndization conditions mclude overnight incubation at 42°C m a solution compnsmg 50% formamide, 5xSSC (150mM NaCl, 15mM tπsodium 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 m 0 lx SSC at about 65°C Thus the present mvention also mcludes polynucleotides obtainable by screenmg an appropnate hbrary under stingent hybndization conditions with a labeled probe having the sequence of SEQ ID NO 1 or a fragment thereof

The skilled artisan will appreciate that, m many cases, an isolated cDNA sequence will be mcomplete, m that the region codmg for the polypeptide is cut short at the 5' end of the cDNA This is a consequence of reverse transcnptase, an enzyme with inherently low 'processrvrty' (a measure of the ability of the enzyme to remain attached to the template during the polymensation reaction), failing to complete a DNA copy of the mRNA template during 1st strand cDNA synthesis

There are several methods available and well known to those skilled m 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 techmque, exemplified by the Marathon™' technology (Clontech Laboratones 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 hgated onto each end Nucleic acid amplification (PCR) is then carried out to amplify the 'missing' 5' end of the cDNA usmg a combmation of gene specific and adaptor specific oligonucleotide pnmers The PCR reaction is then repeated usmg 'nested' pnmers, that is, pnmers designed to anneal withm the amplified product (typically an adaptor specific pnmer that anneals further 3' m 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 described 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 Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). Prefened such methods include, for instance, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.

Representative examples of appropriate 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 Drosophila 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 bacteriophage, 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 retiOviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage 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 mto the lumen of the endoplasmic reticulum, the penplasmic 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 mvention is to be expressed for use m screenmg assays, it is generally preferred that the polypeptide be produced at the surface of the cell In this event, the cells may be harvested pnor to use m the screenmg assay If the polypeptide is secreted mto the medium, the medium can be recovered m order to recover and punfy the polypeptide If produced intracellularly, the cells must first be lysed before the polypeptide is recovered

Polypeptides of the present mvention can be recovered and purified from recombinant cell cultures by well-known methods mcludmg ammomum sulfate or ethanol precipitation, acid extraction, amon or cation exchange chromatography, phosphocellulose chromatography, hydrophobic mteraction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography Most preferably, high performance liquid chromatography is employed for punfication Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or punfication This mvention also relates to the use of polynucleotides of the present mvention as diagnostic reagents Detection of a mutated form of the gene charactensed by the polynucleotide of SEQ ID NO 1 which is associated with a dysfunction will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, or susceptibϋity 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 vanety of techniques

Nucleic acids for diagnosis may be obtamed from a subject's cells, such as from blood, unne, saliva, tissue biopsy or autopsy matenal The genomic DNA may be used directly for detection or may be amplified enzymatically by usmg PCR or other amplification techniques pnor to analysis RNA or cDNA may also be used in similar fashion Deletions and insertions can be detected by a change m size of the amplified product m companson to the normal genotype Pomt mutations can be identified by hybndizmg amplified DNA to labeled CBUAEA12 nucleotide sequences Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences m melting temperatures DNA sequence differences may also be detected by alterations m 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 etal , Proc Nad Acad Sci USA (1985) 85 4397-4401) In another embodiment, an anay of ohgonucleotides probes compnsmg CBUAEA12 nucleotide sequence or fragments thereof can be constructed to conduct efficient screenmg of e g , genetic mutations Array technology methods are well known and have general applicability and can be used to address a vanety of questions m molecular genetics mcludmg 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 determinmg a susceptibility to the Diseases through detection ofmutation m the CBUAEA12 gene by the methods descnbed In addition, such diseases may be diagnosed by methods compnsmg determining from a sample deπved from a subject an abnormally decreased or increased level of polypeptide or mRNA Decreased or mcreased expression can be measured at the RNA level usmg any of the methods well known m the art for the quantitation of polynucleotides, such as, for example, nucleic acid amplification, for mstance PCR, RT-PCR, RNase protection, Northern blotting and other hybndization methods Assay techniques that can be used to determine levels of a protein, such as a polypeptide of the present mvention, in a sample denved from a host are well-known to those of skill m the art Such assay methods mclude radioimmunoassays, competitive-bmdmg assays, Western Blot analysis and ELISA assays Thus m another aspect, the present mvention relates to a diagonostic kit which compnses

(a) a polynucleotide of the present mvention, preferably the nucleotide sequence of SEQ ID NO 1, or a fragment thereof ,

(b) a nucleotide sequence complementary to that of (a),

(c) a polypeptide of the present mvention, preferably the polypeptide of SEQ ID NO 2 or a fragment thereof, or

(d) an antibody to a polypeptide of the present mvention, preferably to the polypeptide of SEQ ID NO 2

It will be appreciated that m any such kit, (a), (b), (c) or (d) mav compnse a substantial component Such a kit will be of use m diagnosing a disease or suspectabihty to a disease, particularly AIDS, cancer, hypertension, and hepatitis, amongst others

The nucleotide sequences of the present mvention are also valuable for chromosome identification The sequence is specifically targeted to, and can hybndize with, a particular location on an individual human chromosome The mapping of relevant sequences to chromosomes according to the present mvention is an important first step m 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, Mende an Inhentance m 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

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 immunospecific for polypeptides of the present invention. The term "immunospecific" means that the antibodies have substantially greater affinity for the polypeptides of the invention than their affinity for other related polypeptides in the prior art. Antibodies generated against polypeptides of the present invention may be obtained by administering the polypeptides or epitope-bearing 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 hybridoma technique (Kohler, G. and Milstein, C, Nature (1975) 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al, Immunology Today (1983) 4:72) and the EBV- hybridoma 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 mvention. 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 screenmg, diagnosis and therapy A further aspect of the mvention also relates to polynucleotides encodmg such fusion protems Examples of fusion protein technology can be found in International Patent Application Nos W094/29458 and W094/22914

Another aspect of the mvention relates to a method for mducmg an immunological response m a mammal which compnses moculatmg the mammal with a polypeptide of the present mvention, adequate to produce antibody and/or T cell mrmune response to protect said animal from the Diseases herembefore mentioned, amongst others Yet another aspect of the mvention relates to a method of mducmg immunological response m a mammal which compnses, delivering a polypeptide of the present mvention via a vector directing expression of the polynucleotide and coding for the polypeptide in vivo in order to mduce such an immunological response to produce antibody to protect said animal from diseases

A further aspect of the mvention relates to an lmmunological/vaccme formulation (composition) which, when mtroduced mto a mammalian host, induces an immunological response m that mammal to a polypeptide of the present mvention wherem the composition compnses a polypeptide or polynucleotide of the present mvention The vaccme formulation may further compnse a suitable earner Smce a polypeptide may be broken down m the stomach, it is preferably administered parenterally (for mstance, subcutaneous, intramuscular, mtravenous, or mtradermal injection) Formulations suitable for parenteral admimstration mclude aqueous and non-aqueous stenle injection solutions which may contain anti-oxidants, buffers, bactenostats and solutes which render the formulation mstomc with the blood of the recipient, and aqueous and non- aqueous stenle suspensions which may include suspendmg agents or thickening agents The formulations may be presented m unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dned condition requmng only the addition of the sterile liquid earner immediately pnor to use The vaccine formulation may also mclude adjuvant systems for enhancing the lmmunogenicrty of the formulation, such as oil-m 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 mvention are responsible for many biological functions, mcludmg many disease states, in particular the Diseases herembefore mentioned It is therefore desirous to devise screenmg methods to identify compounds which stimulate or which inhibit the function of the polypeptide Accordingly, in a further aspect, the present mvention provides for a method of screenmg 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 herembefore mentioned Compounds may be identified from a vanety of sources, for example, cells, cell-free preparations, chemical braπes, and natural product mixtures Such agonists, antagonists or inhibitors so-identified may be natural or modified substrates, hgands, receptors, enzymes, etc , as the case may be, of the polypeptide, or may be stmctural or functional mimetics thereof (see Coligan et al , Current Protocols in Immunology 1(2) Chapter 5 (1991)) The screenmg method may simply measure the bmdmg of a candidate compound to the polypeptide, or to cells or membranes bearing the polypeptide, or a fusion prote thereof by means of a label directly or indirectly associated with the candidate compound Alternatively, the screenmg method may mvolve competition with a labeled competitor Further, these screenmg methods may test whether the candidate compound results m a signal generated by activation or inhibition of the polypeptide, usmg detection systems appropnate to the cells bearing the polypeptide Inhibitors of activation are generally assayed m the presence of a known agomst and the effect on activation by the agomst by the presence of the candidate compound is observed Constitutively active polpypeptides may be employed m screening methods for inverse agonists or inhibitors, m the absence of an agomst or inhibitor, by testmg whether the candidate compound results in inhibition of activation of the polypeptide Further, the screenmg methods may simply compnse the steps of mixing a candidate compound with a solution containmg a polypeptide of the present mvention, to form a mixture, measunng CBUAEA12 activity m the mixture, and comparmg the CBUAEA12 activity of the mixture to a standard Fusion protems, such as those made from Fc portion and CBUAEA12 polypeptide, as hereinbefore descnbed, can also be used for high-throughput screemng 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 polynucleotides, polypeptides and antibodies to the polypeptide of the present mvention may also be used to configure screemng methods for detectmg the effect of added compounds on the production of mRNA and polypeptide m cells For example, an ELISA assay may be constructed for measunng secreted or cell associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known m the art This can be used to discover agents which may inhibit or enhance the production of polypeptide (also called antagonist or agomst, respectively) from suitably manipulated cells or tissues

The polypeptide may be used to identify membrane bound or soluble receptors, if any, through standard receptor bindmg techniques known m the art These mclude, but are not limited to, ligand bmdmg and crosslinkmg assays m which the polypeptide is labeled with a radioactive isotope (for mstance, ^1), chemically modified (for mstance, biotmylated), or fused to a peptide sequence suitable for detection or punfication, and mcubated with a source of the putative receptor (cells, cell membranes, cell supernatants, tissue extracts, bodily fluids) Other methods mclude biophysical techniques such as surface plasmon resonance and spectroscopy These screemng methods may also be used to identify agonists and antagonists of the polypeptide which compete with the bmdmg of the polypeptide to its receptors, if any Standard methods for conductmg such assays are well understood m the art

Examples of potential polypeptide antagonists mclude antibodies or, m some cases, ohgonucleotides or proteins which are closely related to the hgands, substrates, receptors, enzymes, etc , as the case may be, of the polypeptide, e g , a fragment of the hgands, substrates, receptors, enzymes, etc , or small molecules which bmd to the polypeptide of the present mvention but do not elicit a response, so that the activity of the polypeptide is prevented

Thus, m another aspect, the present mvention relates to a screemng kit for identifymg agomsts, antagonists, hgands, receptors, substrates, enzymes, etc for polypeptides of the present mvention, or compounds which decrease or enhance the production of such polypeptides, which compnses

(a) a polypeptide of the present mvention,

(b) a recombmant cell expressmg a polypeptide of the present mvention,

(c) a cell membrane expressmg a polypeptide of the present mvention, or

(d) antibody to a polypeptide of the present mvention, which polypeptide is preferably that of SEQ ID NO 2

It will be appreciated that m 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 agomst, antagonist or inhibitor of the polypeptide, by

(a) determining m the first mstance 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) synthesmg candidate compounds that are predicted to bmd 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 mvention provides methods of treating abnormal conditions such as, for mstance, AIDS, cancer, hypertension, and hepatitis, related to either an excess of, or an under- expression of, CBUAEA12 polypeptide activity

If the activity of the polypeptide is m excess, several approaches are available One approach compnses administering to a subject m need thereof an inhibitor compound (antagomst) as heremabove descnbed, optionally in combination with a pharmaceutically acceptable earner, m an amount effective to inhibit the function of the polypeptide, such as, for example, by blocking the bmdmg of hgands, 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 bmdmg the ligand, substrate, enzymes, receptors, etc m competition with endogenous polypeptide may be administered Typical examples of such competitors mclude fragments of the CBUAEA12 polypeptide

In still another approach, expression of the gene encodmg endogenous CBUAEA12 polypeptide can be inhibited usmg expression blocking techniques Known such techniques mvolve the use of antisense sequences, either internally generated or separately administered (see, for example, O'Connor, J Neurochem (1991) 56 560 m Ohgodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)) Alternatively, ohgonucleotides which form tnple 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 ohgomers can be administered per se or the relevant ohgomers can be expressed in vivo

For treating abnormal conditions related to an under-expression of CBUAEA12 and its activity, several approaches are also available One approach compnses admmistenng to a subject a therapeutically effective amount of a compound which activates a polypeptide of the present mvention, l e , an agomst as descnbed above, in combination with a pharmaceutically acceptable earner, to thereby alleviate the abnormal condition Alternatively, gene therapy may be employed to effect the endogenous production of CBUAEA12 by the relevant cells m the subject For example, a polynucleotide of the mvention may be engmeered for expression m a replication defective retroviral vector, as discussed above The retroviral expression construct may then be isolated and mtroduced mto a packaging cell transduced with a retroviral plasmid vector containmg RNA encodmg a polypeptide of the present mvention such that the packaging cell now produces infectious viral particles containmg 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) m 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 mvention in combination with a suitable pharmaceutical earner

In a further aspect, the present mvention provides for pharmaceutical compositions compnsmg a therapeutically effective amount of a polypeptide, such as the soluble form of a polypeptide of the present mvention, agonist/antagonist peptide or small molecule compound, in combination with a pharmaceutically acceptable earner or excipient Such earners mclude, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof The mvention further relates to pharmaceutical packs and kits compnsmg one or more containers filled with one or more of the ingredients of the aforementioned compositions of the mvention Polypeptides and other compounds of the present mvention may be employed alone or m conjunction with other compounds, such as therapeutic compounds

The composition will be adapted to the route of admmistration, for instance by a systemic or an oral route Preferred forms of systemic admimstration mclude injection, typically by mtravenous injection Other injection routes, such as subcutaneous, mtramuscular, or mtrapentoneal, can be used Alternative means for systemic admmistration mclude transmucosal and transdermal admimstration usmg penetrants such as bile salts or fusidic acids or other detergents In addition, if a polypeptide or other compounds of the present mvention 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 mvention, the route of admmistration, 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 vanations in the needed dosage, however, are to be expected in view of the vanety of compounds available and the differing efficiencies of vanous routes of administration For example, oral admmistration would be expected to require higher dosages than administration by mtravenous injection Vanations m these dosage levels can be adjusted usmg standard empincal routines for optimization, as is well understood m the art

Polypeptides used m treatment can also be generated endogenously m the subject, m treatment modalities often refened to as "gene therapy" as descnbed above Thus, for example, cells from a subject may be engmeered 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 mto 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 usmg the stored data to search a sequence database usmg well known searchmg tools, such as GCC Accordmgly, m a further aspect, the present mvention provides for a computer readable medium having stored thereon a polynucleotide compnsmg the sequence of SEQ ID NO 1 and/or a polypeptide sequence encoded thereby

The following definitions are provided to facilitate understanding of certain terms used frequently herembefore

"Antibodies" as used herein mcludes polyclonal and monoclonal antibodies, chimenc, smgle cham, and humamzed antibodies, as well as Fab fragments, mcludmg the products of an Fab or other immunoglobulm expression library

"Isolated" means altered "by the hand of man" from the natural state If an "isolated" composition or substance occurs m nature, it has been changed or removed from its ong al environment, or both For example, a polynucleotide or a polypeptide naturally present m a living animal is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting matenals of its natural state is "isolated", as the term is employed herem

"Polynucleotide" generally refers to any polynbonucleotide or polydeoxnbonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA "Polynucleotides" mclude, without limitation, smgle- and double-stranded DNA, DNA that is a mixture of smgle- and double- stranded regions, smgle- and double-stranded RNA, and RNA that is mixture of smgle- and double-stranded regions, hybnd molecules comprising DNA and RNA that may be smgle-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions In addition, "polynucleotide" refers to tnple-stranded regions compnsmg 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 mclude, for example, tntylated bases and unusual bases such as inosine A vaπety of modifications may be made to DNA and RNA, thus, "polynucleotide" embraces chemically, enzymatically or metabo cally modified forms of polynucleotides as typically found m nature, as well as the chemical forms of DNA and RNA charactenstic of viruses and cells "Polynucleotide" also embraces relatively short polynucleotides, often referred to as ohgonucleotides

"Polypeptide" refers to any peptide or protem compnsmg two or more ammo 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 ohgomers, and to longer chains, generally referred to as protems Polypeptides may contain ammo acids other than the 20 gene-encoded ammo acids "Polypeptides" mclude ammo acid sequences modified either by natural processes, such as post-translational processmg, or by chemical modification techniques which are well known m the art Such modifications are well descnbed m basic texts and m more detailed monographs, as well as m a voluminous research literature Modifications may occur anywhere m a polypeptide, mcludmg the peptide backbone, the ammo acid side-chams and the ammo 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 m 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 mclude acetylation, acylation, ADP-nbosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide denvative, covalent attachment of a hpid or hpid denvative, covalent attachment of phosphotidylmositol, cross-linking, cychzation, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystme, formation of pyroglutamate, formylation, gamma- carboxylation, glycosylation, GPI anchor formation, hydroxylation, lodmation, methylation, mynstoylation, oxidation, proteolytic processmg, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of ammo acids to protems such as argmylation, 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 Protem 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 protem modifications and nonprotem cofactors", Meth Enzymol (1990) 182 626-646 and Rattan et al , "Protem Synthesis Post-translational Modifications and Aging", Ann NYAcad Sci (1992) 663 48-62) "Vanant" refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains essential properties A typical vanant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide Changes m the nucleotide sequence of the vanant may or may not alter the ammo acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result m ammo acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below A typical vanant of a polypeptide differs m ammo acid sequence from another, reference polypeptide Generally, differences are limited so that the sequences of the reference polypeptide and the vanant are closely similar overall and, in many regions, identical A vanant and reference polypeptide may differ m ammo acid sequence by one or more substitutions, additions, deletions m any combination A substituted or inserted ammo acid residue may or may not be one encoded by the genetic code A vanant of a polynucleotide or polypeptide may be a naturally occurring such as an allehc vanant, or it may be a vanant that is not known to occur naturally Non-naturally occurring vanants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis

""Identity," as known m the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be, as determmed by comparing the sequences In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determmed by the match between strings of such sequences

"Identity" can be readily calculated by known methods, mcludmg but not limited to those descnbed m (Computational Molecular Biology, Lesk, A M , ed , Oxford University Press, New York, 1988, Bwcomputing Informatics and Genome Projects, Smith, D W , ed , Academic Press, New York, 1993, Computer Analysis of Sequence Data, Part I, Gπffin, A M , and Gnffin, H G , eds , Humana Press, New Jersey, 1994, Sequence Analysis in Molecular Biology, von Hemje, G , Academic Press, 1987, and Sequence Analysis Pnmer, Gπbskov, M and Devereux, J , eds , M Stockton Press, New York, 1991, and Canllo, H , and Lipman, D , SLAM 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 determme identity between two sequences mclude, 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 Bwl 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 algonthm may also be used to determine identity Parameters for polypeptide sequence companson mclude the following 1) Algonthm Needleman and Wunsch, J Mol Biol 48 443-453 (1970) Companson matnx 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:l, 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_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 mvention may be identical to the reference sequence of SEQ ID NO 2, that is it may be 100% identical, or it may mclude up to a certain mteger number of ammo 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 consistmg of at least one nucleic acid deletion, substitution, mcludmg transition and transversion, or msertion, and wherem said alterations may occur at the 5 ' or 3' terminal positions of the reference polynucleotide sequence or anywhere between those terminal positions, mterspersed either individually among the nucleic acids m the reference sequence or m one or more contiguous groups withm the reference sequence The number of nucleic acid alterations for a given percent identity is determmed by multiplymg the total number of ammo acids m SEQ ID NO 2 by the mteger defining the percent identity divided by 100 and then subtractmg that product from said total number of ammo acids m SEQ ID NO 2, or

^~~n ≤ ^xn " (^xn )>

wherem n_n is the number of ammo acid alterations, x_n is the total number of ammo acids m SEQ ID NO 2, y is, for mstance 0 70 for 70%, 0 80 for 80%, 0 85 for 85% etc , • is the symbol for the multiplication operator, and wherem any non-mteger product of x_n and y is rounded down to the nearest mteger pnor to subtractmg it from x_n (2) Polypeptide embodiments further mclude an isolated polypeptide compnsmg a polypeptide havmg at least a 50,60, 70, 80, 85, 90, 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO 2, wherem said polypeptide sequence may be identical to the reference sequence of SEQ ID NO 2 or may mclude up to a certain mteger number of ammo acid alterations as compared to the reference sequence, wherem said alterations are selected from the group consisting of at least one amino acid deletion, substitution, mcludmg conservative and non- conservative substitution, or msertion, and wherem said alterations may occur at the amino- or carboxy-teiminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the ammo acids m the reference sequence or m one or more contiguous groups withm the reference sequence, and wherem said number of ammo acid alterations is determmed by multiplying the total number of ammo acids m SEQ ID NO 2 by the integer defining the percent identity divided by 100 and then subtractmg that product from said total number of ammo acids m SEQ ID NO 2, or ⁿa ≤ ^xa " (^xa * Y)>

wherem n_a is the number of ammo acid alterations, x_a is the total number of ammo acids m 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 wherem any non-mteger product of x_a and y is rounded down to the nearest mteger pnor to subtractmg it from x_a

By way of example, a polypeptide sequence of the present mvention may be identical to the reference sequence of SEQ ID NO 2, that is it may be 100% identical, or it may mclude up to a certam mteger number of ammo 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 consistmg of at least one ammo acid deletion, substitution, mcludmg conservative and non-conservative substitution, or msertion, and wherem said alterations may occur at the ammo- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, mterspersed either mdividually among the ammo acids m the reference sequence or m one or more contiguous groups withm the reference sequence The number of ammo acid alterations for a given % identity is determmed by multiplymg the total number of ammo acids m SEQ ID NO 2 by the integer defining the percent identity divided by 100 and then subtractmg that product from said total number of ammo acids in SEQ ID NO 2, or

ⁿa ^{≤ x}a (^χ _a • y)»

wherem n_a is the number of ammo acid alterations, x_a is the total number of amino acids in SEQ ID NO 2, y is, for mstance 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-mteger product of x_a and y is rounded down to the nearest mteger pnor to subtractmg it from x_a

"Fusion protem" refers to a protein encoded by two, often unrelated, fused genes or fragments thereof In one example, EP-A-0 464 discloses fusion protems compnsmg various portions of constant region of immunoglobulin molecules together with another human protein or part thereof In many cases, employmg an immunoglobulm Fc region as a part of a fusion protem is advantageous for use m therapy and diagnosis resulting m, 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.

SEQUENCE INFORMATION SEQ ID NO:l

1 CTCAGCTACG CAGGTGCAAC TTCTTGCGCC AACGTCTGGT CCTGTCTACC

51 CTGAGCGGGC GCCCCGTCAA AATCCGAAAG ATTCGGGCCA GAGACGACAA

101 CCCGGGCCTC CGAGATTTTG AAGCCAGCTT CATAAGGCTA TTGGACAAAT

151 AACGAATGGT TCTCGAATTG AAATAAACCA AACAGGAACA ACCTTATATT

201 ATCAGCCTGG CCTCCTGTAT GGTGGATCTG TGGAACATGA CTGTAGCGTC

251 CTTCGTGGCA TTGGGTATTA CCTGGAGAGT CTTCTTTGCT TGGCTCCATT

301 TATGAAGCAC CCGTTAAAAA TAGTTCTACG AGGAGTGACC AATGATCAGG

351 TTGACCCTTC AGTTGATGTT CTTAAGGCAA CAGCACTCCC TTTGTTGAAA

401 CAATTTGGGA TTGATGGTGA ATCATTTGAA CTGAAGATTG TGCGACGGGG

451 AATGCCTCCC GGAGGAGGAG GCGAAGTGGT TTTCTCATGT CCTGTGAGGA

501 AGGTCTTGAA GCCCATTCAA CTCACAGATC CAGGAAAAAT CAAACGTATT

551 AGAGGAATGG CGTACTCTGT ACGTGTGTCA CCTCAGATGG CGAACCGGAT

601 TGTGGATTCT GCAAGGAGCA TCCTCAACAA GTTCATACCT GATATCTATA

651 TTTACACAGA TCACATGAAA GGAGTCAACT CTGGGAAGTC TCCGGGCTTT

701 GGGTTGTCAC TGGTTGCTGA GACCACCAGT GGCACCTTCC TCAGTGCTGA

751 ACTGGCCTCC AACCCCCAGG GCCAGGGAGC AGCAGTACTT CCAGAGGACC

801 TTGGCAGGAA CTGTGCCCGG CTGCTGCTGG AGGAAATCTA CAGGGGTGGA

851 TGCGTAGACT CGACCAACCA AAGCCTGGCG CTACTACTCA TGACCCTTGG

901 ACAGCGGGAT GTTTCCAAAG TCCTGCTAGG CCCTCTCTCT CCCTACACGA 951 TAGAATTTTT GCGGCATTTG AAGAGCTTTT TCCAGATTAT GTTTAAAATT

1001 GAAACCAAGC CATGTGGTGA AGAACTCAAG GGTGGGGATA AAGTGCTGAT

1051 GACCTGTGTT GGCATTGGTT TCTCCAACCT TAGCAAGACC CTCAAGTGAT

1101 AACCATCACA AGATAAGGCC CCAATGCCTA CAGACAAAGC AGAAGCTGCC

1151 ACGGACACCA ATGGGACCAA GTCCAAATGG ATTAATCCAG GACAGAATAG

1201 CCACTTGCTT AATTTTCTGT GAAGAAATAT CAATATACAA ATAAAAGACA

1251 TCCCTGTAGC ATATGGTTTC CAGCTGTTTC TCCAGTGGCA TTGCCATTGC

1301 CCAGGAGGGG CCCAGTCACC ATGAGAGCTC CCTTGCCTTA CCTGGAGGAA

1351 GGATGTGCCT TCAGGCCACA GTCGTGCTGC TAGAACAGTC TCATAGCTGC

1401 AGTTCAGCTG TGCTTCCTCA GCCTACTATC ATAGGCTTCC TCAGCCCTCT

1451 GTCATATGCT GTTTTCCAAA CCTGTGGAGT CTGTTACTGT TCTTTCTGCA

1501 AGGACTCACC TCCTTGAGCC TTGGTTTTTG TTGTAGGGAT TAAATGAGAT

1551 AATATGAGTG GCAGCTCTTC ATGAGTCCTG CAGTGCTAAG CAAATGTCAG

1601 AAATTGGTGT ATTAGACTAT TTATCTTTGA TCTTCTGAAT GGATTGCTGT

1651 CATGGACACG GACACGGATC TTCATCTGGT TCATTGTATT TATATGTGAG

1701 GGATGGATGG CTGCGGGGCT CCAAGTAAGT TATTGGGATG TTTTTTATAT

1751 TCCAGGTGTG CTGTACATTC TTATTTTATT TTCACAATAG CTCTGTGATG

1801 TAAATGCTAT CTCCATGAGA AAATTCATAA AGGGTGTTTT GTTCCTTTGA

1851 AATGTATAAT GTAAAGACAT TAAATCTCCT CATTTAGTGA AAAAAAAAAA

1901 AAAAAAAAAA AAAAAAAAA SEQ ID NO:2

1 MKHPLKIVLR GVTNDQVDPS VDVLKATALP LLKQFGIDGE SFE KIVRRG

51 MPPGGGGEW FSCPVRKVLK PIQLTDPGKI KRIRGMAYSV RVSPQMANRI

101 VDSARSILNK FIPDIYIYTD HMKGVNSGKS PGFGLSLVAE TTSGTFLSAE

151 LASNPQGQGA AVLPEDLGRN CARLLLEEIY RGGCVDSTNQ SLALLLMTLG

201 QRDVSKVLLG PLSPYTIEFL RHLKSFFQIM FKIETKPCGE ELKGGDKVLM

251 TCVGIGFSNL SKTLK

SEQUENCE LISTING

(1) GENERAL INFORMATION

(i) APPLICANT: SHANGHAI SECOND MEDICAL UNIVERSITY

(ii) TITLE OF THE INVENTION: A HUMAN HEMATOPOIETIC CELL DERIVED RNA CYCLASE (HRDC) GENE (CBUAEA12)

(iii) NUMBER OF SEQUENCES: 2

(iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: RATNER & PRESTIA

(B) STREET: P.O. BOX 980

(C) CITY: VALLEY FORGE

(D) STATE: PA

(E) COUNTRY: USA (F) ZIP: 19482

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Diskette

(B) COMPUTER: IBM Compatible (C) OPERATING SYSTEM: DOS

(D) SOFTWARE: FastSEQ for Windows Version 2.0

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: TO BE ASSIGNED (B) FILING DATE:

(C) CLASSIFICATION: UNKNOWN

(vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE:

(viii) ATTORNEY/AGENT INFORMATION: (A) NAME: PRESTIA, PAUL F

(B) REGISTRATION NUMBER: 23,031

(C) REFERENCE/DOCKET NUMBER: GP-70444 (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 610-407-0700

(B) TELEFAX: 610-407-0701

(C) TELEX: 846169

(2) INFORMATION FOR SEQ ID NO : 1 :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1919 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 :

CTCAGCTACG CAGGTGCAAC TTCTTGCGCC AACGTCTGGT CCTGTCTACC CTGAGCGGGC 60

GCCCCGTCAA AATCCGAAAG ATTCGGGCCA GAGACGACAA CCCGGGCCTC CGAGATTTTG 120 AAGCCAGCTT CATAAGGCTA TTGGACAAAT AACGAATGGT TCTCGAATTG AAATAAACCA 180

AACAGGAACA ACCTTATATT ATCAGCCTGG CCTCCTGTAT GGTGGATCTG TGGAACATGA 240

CTGTAGCGTC CTTCGTGGCA TTGGGTATTA CCTGGAGAGT CTTCTTTGCT TGGCTCCATT 300

TATGAAGCAC CCGTTAAAAA TAGTTCTACG AGGAGTGACC AATGATCAGG TTGACCCTTC 360

AGTTGATGTT CTTAAGGCAA CAGCACTCCC TTTGTTGAAA CAATTTGGGA TTGATGGTGA 420 ATCATTTGAA CTGAAGATTG TGCGACGGGG AATGCCTCCC GGAGGAGGAG GCGAAGTGGT 480

TTTCTCATGT CCTGTGAGGA AGGTCTTGAA GCCCATTCAA CTCACAGATC CAGGAAAAAT 540

CAAACGTATT AGAGGAATGG CGTACTCTGT ACGTGTGTCA CCTCAGATGG CGAACCGGAT 600

TGTGGATTCT GCAAGGAGCA TCCTCAACAA GTTCATACCT GATATCTATA TTTACACAGA 660

TCACATGAAA GGAGTCAACT CTGGGAAGTC TCCGGGCTTT GGGTTGTCAC TGGTTGCTGA 720 GACCACCAGT GGCACCTTCC TCAGTGCTGA ACTGGCCTCC AACCCCCAGG GCCAGGGAGC 780

AGCAGTACTT CCAGAGGACC TTGGCAGGAA CTGTGCCCGG CTGCTGCTGG AGGAAATCTA 840

CAGGGGTGGA TGCGTAGACT CGACCAACCA AAGCCTGGCG CTACTACTCA TGACCCTTGG 900

ACAGCGGGAT GTTTCCAAAG TCCTGCTAGG CCCTCTCTCT CCCTACACGA TAGAATTTTT 960

GCGGCATTTG AAGAGCTTTT TCCAGATTAT GTTTAAAATT GAAACCAAGC CATGTGGTGA 1020 AGAACTCAAG GGTGGGGATA AAGTGCTGAT GACCTGTGTT GGCATTGGTT TCTCCAACCT 1080

TAGCAAGACC CTCAAGTGAT AACCATCACA AGATAAGGCC CCAATGCCTA CAGACAAAGC 1140

AGAAGCTGCC ACGGACACCA ATGGGACCAA GTCCAAATGG ATTAATCCAG GACAGAATAG 1200

CCACTTGCTT AATTTTCTGT GAAGAAATAT CAATATACAA ATAAAAGACA TCCCTGTAGC 1260

ATATGGTTTC CAGCTGTTTC TCCAGTGGCA TTGCCATTGC CCAGGAGGGG CCCAGTCACC 1320 ATGAGAGCTC CCTTGCCTTA CCTGGAGGAA GGATGTGCCT TCAGGCCACA GTCGTGCTGC 1380

TAGAACAGTC TCATAGCTGC AGTTCAGCTG TGCTTCCTCA GCCTACTATC ATAGGCTTCC 1440

TCAGCCCTCT GTCATATGCT GTTTTCCAAA CCTGTGGAGT CTGTTACTGT TCTTTCTGCA 1500

AGGACTCACC TCCTTGAGCC TTGGTTTTTG TTGTAGGGAT TAAATGAGAT AATATGAGTG 1560 GCAGCTCTTC ATGAGTCCTG CAGTGCTAAG CAAATGTCAG AAATTGGTGT ATTAGACTAT 1620

TTATCTTTGA TCTTCTGAAT GGATTGCTGT CATGGACACG GACACGGATC TTCATCTGGT 1680

TCATTGTATT TATATGTGAG GGATGGATGG CTGCGGGGCT CCAAGTAAGT TATTGGGATG 1740

TTTTTTATAT TCCAGGTGTG CTGTACATTC TTATTTTATT TTCACAATAG CTCTGTGATG 1800 TAAATGCTAT CTCCATGAGA AAATTCATAA AGGGTGTTTT GTTCCTTTGA AATGTATAAT 1860

GTAAAGACAT TAAATCTCCT CATTTAGTGA AAAAAAAAAA AAAAAAAAAA AAAAAAAAA 1919

(2) INFORMATION FOR SEQ ID NO : 2 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 265 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2 :

Met Lys His Pro Leu Lys lie Val Leu Arg Gly Val Thr Asn Asp Gin 1 5 10 15

Val Asp Pro Ser Val Asp Val Leu Lys Ala Thr Ala Leu Pro Leu Leu

20 25 30

Lys Gin Phe Gly lie Asp Gly Glu Ser Phe Glu Leu Lys He Val Arg 35 40 45 Arg Gly Met Pro Pro Gly Gly Gly Gly Glu Val Val Phe Ser Cys Pro 50 55 60

Val Arg Lys Val Leu Lys Pro He Gin Leu Thr Asp Pro Gly Lys He 65 70 75 80

Lys Arg He Arg Gly Met Ala Tyr Ser Val Arg Val Ser Pro Gin Met 85 90 95

Ala Asn Arg He Val Asp Ser Ala Arg Ser He Leu Asn Lys Phe He

100 105 110

Pro Asp He Tyr He Tyr Thr Asp His Met Lys Gly Val Asn Ser Gly 115 120 125 Lys Ser Pro Gly Phe Gly Leu Ser Leu Val Ala Glu Thr Thr Ser Gly 130 135 140

Thr Phe Leu Ser Ala Glu Leu Ala Ser Asn Pro Gin Gly Gin Gly Ala 145 150 155 160

Ala Val Leu Pro Glu Asp Leu Gly Arg Asn Cys Ala Arg Leu Leu Leu 165 170 175

Glu Glu He Tyr Arg Gly Gly Cys Val Asp Ser Thr Asn Gin Ser Leu

180 185 190

Ala Leu Leu Leu Met Thr Leu Gly Gin Arg Asp Val Ser Lys Val Leu 195 200 205

Leu Gly Pro Leu Ser Pro Tyr Thr He Glu Phe Leu Arg His Leu Lys

210 215 220

Ser Phe Phe Gin He Met Phe Lys He Glu Thr Lys Pro Cys Gly Glu 225 230 235 240

Glu Leu Lys Gly Gly Asp Lys Val Leu Met Thr Cys Val Gly He Gly

245 250 255

Phe Ser Asn Leu Ser Lys Thr Leu Lys 260 265

Claims

What is claimed is:

1. An isolated polypeptide selected from the group consisting of:

(i) 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:2 over the entire length of SEQ ID

NO:2; (ii) an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:2 or (iii) an isolated polypeptide which is the amino acid sequence of SEQ ID NO:2.

2. An isolated polynucleotide selected from the group consisting of:

(i) an isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide that has at least

(a) 70% identity;

(b) 80% identity; (c) 90% identity; or

(d) 95% identity; to the amino acid sequence of SEQ ID NO:2, over the entire length of SEQ ID NO:2; (ii) an isolated polynucleotide comprising a nucleotide sequence that has at least:

(a) 70% identity (b) 80% identity;

(c) 90% identity; or

(d) 95% identity; over its entire length to a nucleotide sequence encoding the polypeptide of SEQ ID NO:2; (iii) an isolated polynucleotide comprising a nucleotide sequence which has at least:

(a) 70% identity;

(b) 80% identity;

(c) 90% identity; or

(d) 95% identity; to that of SEQ ID NO: 1 over the entire length of SEQ ID NO: 1; (iv) an isolated polynucleotide comprising a nucleotide sequence encoding the polypeptide of SEQ ID NO:2;

(vi) an isolated polynucleotide which is the polynucleotide of SEQ ID NO: 1 ; or (vi) an isolated polynucleotide obtainable by screening an appropriate hbrary under stringent hybridization conditions with a labelled probe having the sequence of SEQ ID NO: 1 or a fragment thereof. ; or a nucleotide sequence complementary to said isolated polynucleotide.

3. An antibody immunospecific 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 vivo.; or

(ii) 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 subject a therapeutically effective amount of a polypeptide that competes with said polypeptide for its ligand, 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 comprises 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 comparmg 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 producmg 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 appropriate culture conditions, produces a polypeptide comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO:2 over the entire length of SEQ ID NO:2.

10. A recombinant host cell produced by the process of claim 9.

11. 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: 2 over the entire length of SEQ ID NO:2.

12. A process for producing a polypeptide comprising culturing a host cell of claim 10 under conditions sufficient for the production of said polypeptide and recovering the polypeptide from the culture.