WO1999062950A1 - A human ubiquinone oxireductase subunit ci-aggg homolog gene (cbfakd10) - Google Patents

Abstract

CBFAKD10 polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing CBFAKD10 polypeptides and polynucleotides in therapy, and diagnostic assays for such.

Description

A Human Ubiquinone Oxireductase subunit CI-AGGG Homolog Gene (CBFAKDIO)

Field of the Invention

This invention relates to newly identified polypeptides and polynucleotides encodmg 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 vanous 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 charactense further genes and their related polypeptides/proteins, as targets for drug discovery

Summary of the Invention

The present invention relates to CBFAKDIO, in particular CBFAKDIO polypeptides and CBFAKD 10 polynucleotides, recombinant matenals 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, autoimmune disease, hepatitis, and diabetes, hereinafter referred to as "the Diseases", amongst others In a further aspect, the invention relates to methods for identifymg agonists and antagomsts/mhibitors using the matenals provided by the mvention, and treatmg conditions associated with CBFAKDIO imbalance with the identified compounds In a still further aspect, the mvention relates to diagnostic assays for detecting diseases associated with lnappropnate CBFAKDIO activity or levels

Description of the Invention

In a first aspect, the present invenUon relates to CBFAKDIO polypeptides Such peptides include isolated polypepϋdes 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 ammo acid of SEQ ID NO 2

Further peptides of the present invention include isolated polypeptides m which the amino acid sequence has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% identity, to the amino acid sequence of SEQ ID NO 2 over the entire length of SEQ ID NO 2 Such polypeptides include the polypeptide of SEQ ID NO 2

Further peptides of the present invention include isolated polypeptides encoded by a polynucleotide comprising the sequence contained in SEQ ID NO 1

Polypeptides of the present invention are believed to be members of the ubiquinone oxidoreductase family of polypeptides They are therefore of interest because ubiqumone oxidoreductase is a mitochondria protem complex encoded by nuclear genome, it is composed of many subumts, and it participates in metabolism These properties are hereinafter referred to as "CBFAKDIO activity" or "CBFAKDIO polypeptide activity" or "biological activity of

CBFAKDIO" Also included amongst these activities are antigenic and immunogenic activities of said CBFAKDIO 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 CBFAKD 10 The polypeptides of the present mvention may be m 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 mclude an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification such as multiple histidme residues, or an additional sequence for stability during recombinant production The present mvention also includes mclude vanants 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 charactensUcs Typical such substitutions are among Ala, Val, Leu and He, among Ser and Thr, among the acidic residues Asp and Glu, among Asn and Gin, and among the basic residues Lys and Arg, or aromatic residues Phe and Tyr Particularly preferred are vanants 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 mvention relates to CBFAKD 10 polynucleotides Such polynucleotides mclude 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 ammo 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 mclude a polynucleotide compnsing the nucleotide sequence contained in SEQ LO NO 1 encodmg the polypeptide of SEQ ID NO 2 Further polynucleotides of the present mvention include isolated polynucleotides composing 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 encodmg a polypeptide of SEQ LD 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 mvention mclude isolated polynucleotides compπsmg 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 mclude a polynucleotide compnsing the polynucleotide of SEQ ID NO 1 as well as the polynucleotide of SEQ LD NO 1

The invention also provides polynucleotides which are complementary to all the above described polynucleotides The nucleotide sequence of SEQ LD NO 1 shows homology with X63216, bovine ubiqumone oxidoreductase complex subunit CI-AGGG, (J E Walker, et al J Mol Biol 1992,226 1051-1072 ) The nucleotide sequence of SEQ ID NO 1 is a cDNA sequence and comprises a polypeptide encoding sequence (nucleotide 65 to 379) encodmg a polypeptide of 105 ammo acids, the polypeptide of SEQ ID NO 2 The nucleotide sequence encoding the polypeptide of SEQ ID NO 2 may be identical to the polypeptide encoding sequence contained m SEQ ID NO 1 or it may be a sequence other than the one contained m 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 LD NO 2 is structurally related to other proteins of the ubiqumone oxidoreductase family, having homology and/or structural similarity with Q02374, bovine ubiqumone oxidoreductase complex subunit CI-AGGG, (J E Walker, et al J Mol Bi 1992,226 1051-1072 )

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 CBFAKDIO activity

Polynucleotides of the present mvention may be obtained, using standard cloning and screening techniques, from a cDNA library denved from mRNA m cells of human umbilical cord blood, using the expressed sequence tag (EST) analysis (Adams, M D , et al Science (1991) 252 1651-1656, Adams, M D et al , Nature, (1992) 355 632-634, Adams, M D , et al , Nature (1995) 377

Supp 3-174) Polynucleotides of the invention can also be obtained from natural sources such as genomic DNA libraries or can be synthesized using well known and commercially available techniques

When polynucleotides of the present invention are used for the recombinant production of polypeptides of the present invention, the polynucleotide may mclude the codmg 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 encodmg a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence, or other fusion peptide portions For example, a marker sequence which facilitates purification of the fused polypeptide can be encoded In certain preferred embodiments of this aspect of the mvention, the marker sequence is a hexa-histidine peptide, as provided m the pQE vector (Qiagen, Inc ) and descπbed m Gentz etal , Proc NatlAcadSct USA (1989) 86 821-824, or is an HA tag The polynucleotide may also contain non-coding 5' and 3' sequences, such as transcribed, non-translated sequences, splicing and polyadenylation signals, πbosome binding sites and sequences that stabilize mRNA Further embodiments of the present mvention mclude polynucleotides encodmg polypeptide vanants which compose 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, m 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 primers 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 (including genes encodmg 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 primers will generally compose at least 15 nucleotides, preferably, at least 30 nucleotides and may have at least 50 nucleotides Particularly preferred probes will have between 30 and 50 nucleotides A polynucleotide encoding a polypeptide of the present mvention, including homologs and orthologs from species other than human, may be obtamed by a process which composes the steps of screening an appropoate library under stringent hybodization conditions with a labeled probe having the sequence of SEQ LD NO 1 or a fragment thereof, and isolating full-length cDNA and genomic clones containing said polynucleotide sequence Such hybridization techniques are well known to the skilled artisan Preferred stringent hybodization conditions mclude overnight incubation at 42°C m a solution composing 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 in 0 lx SSC at about 65°C Thus the present mvention also mcludes polynucleotides obtainable by screening an appropoate library under stingent hybodization 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 incomplete, in that the region coding for the polypeptide is cut short at the 5' end of the cDNA This is a consequence of reverse transcπptase, an enzyme with inherently low 'processivity' (a measure of the ability of the enzyme to remain attached to the template during the polymerisation reaction), failing to complete a DNA copy of the mRNA template 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 Laboratories Inc ) for example, have significantly simplified the search for longer cDNAs In the Marathon™ technology, cDNAs have been prepared from mRNA extracted from a chosen tissue and an 'adaptor' sequence gated onto each end Nucleic acid amplification (PCR) is then carried out to amplify the 'missing' 5' end of the cDNA using a combmation of gene specific and adaptor specific oligonucleotide primers The PCR reaction is then repeated using 'nested' primers, that is, primers designed to anneal within the amplified product (typically an adaptor specific primer that anneals further 3' in the adaptor sequence and a gene specific primer that anneals further 5' in the known gene sequence) The products of this reaction can then be analysed by DNA sequencing and a full-length cDNA constructed either by joining the product directly to the existing cDNA to give a complete sequence, or carrying out a separate full-length PCR using the new sequence information for the design of the 5' primer

Recombinant polypeptides of the present mvention may be prepared by processes well known m the art from genetically engineered host cells composing expression systems Accordingly, m a further aspect, the present mvention relates to expression systems which compose a polynucleotide or polynucleotides of the present mvention, to host cells which are genetically engineered with such expression sytems and to the production of polypeptides of the mvention by recombinant techniques Cell-free translation systems can also be employed to produce such proteins usmg RNAs deoved from the DNA constructs of the present mvention For recombinant production, host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present mvention Introduction of polynucleotides mto host cells can be effected by methods descnbed m many standard laboratory manuals, such as Davis et al , Basic Methods m 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) Preferred such methods mclude, for instance, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic hpid-mediated transfection, electroporation, transducπon, scrape loading, ballistic introduction or infection

Representative examples of appropoate hosts mclude bacteoal cells, such as streptococci, staphylococci, E coh, 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 vaoety of expression systems can be used, for instance, chromosomal, episomal and virus-denved systems, e g , vectors deoved from bacteoal plasmids, from bacteoophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors deoved from combinations thereof, such as those deoved from plasmid and bacteoophage 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 m a host may be used The appropoate nucleotide sequence may be inserted mto an expression system by any of a vaoety of well-known and routme techniques, such as, for example, those set forth m Sambrook et al , MOLECULAR CLONING, A LABORATORY MANUAL (supra) Appropoate secretion signals may be incorporated mto the desired polypeptide to allow secretion of the translated protein mto the lumen of the endoplasmic reticulum, the peoplasmic 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 screening assays, it is generally preferred that the polypeptide be produced at the surface of the cell In this event, the cells may be harvested poor to use m the screening assay If the polypeptide is secreted into the medium, the medium can be recovered m order to recover and purify the polypeptide If produced intracellularly, the cells must first be lysed before the polypeptide is recovered

Polypeptides of the present invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, 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 purification Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification This mvention also relates to the use of polynucleotides of the present mvention as diagnostic reagents Detection of a mutated form of the gene characteπsed 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 susceptibility to a disease, which results from under-expression, over- expression or altered expression of the gene Individuals carrying mutations m the gene may be detected at the DNA level by a vaoety of techniques

Nucleic acids for diagnosis may be obtamed from a subject's cells, such as from blood, uone, 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 poor to analysis RNA or cDNA may also be used m similar fashion Deletions and insertions can be detected by a change in size of the amplified product m compaoson to the normal genotype Pomt mutations can be identified by hybodizing amplified DNA to labeled CBFAKD 10 nucleotide sequences Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures DNA sequence differences may also be detected by alterations m electrophoretic mobility of DNA fragments m gels, with or without denaturing agents, or by direct DNA sequencmg (ee, e g , Myers et al , Science ( 1985) 230 1242) Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and S 1 protection or the chemical cleavage method (see Cotton et al , Proc Nad Acad Set USA (1985) 85 4397-4401) In another embodiment, an array of ohgonucleotides probes composing CBFAKDIO nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e g , genetic mutations Array technology methods are well known and have general applicability and can be used to address a vaoety of questions m molecular genetics including gene expression, genetic linkage, and genetic vaoability (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 of mutation m the CBFAKD 10 gene by the methods descnbed In addition, such diseases may be diagnosed by methods compπsmg determinmg from a sample deoved from a subject an abnormally decreased or increased level of polypeptide or mRNA Decreased or increased expression can be measured at the RNA level usmg any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, nucleic acid amplification, for instance PCR, RT-PCR, RNase protection, Northern blottmg and other hybridization methods

Assay techniques that can be used to determine levels of a protein, such as a polypeptide of the present mvention, in a sample deoved from a host are well-known to those of skill m the art Such assay methods mclude radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays Thus in another aspect, the present invention relates to a diagonostic kit which comprises

(a) a polynucleotide of the present invention, preferably the nucleotide sequence of SEQ ID NO 1 , 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 invention, preferably to the polypeptide of SEQ ID NO 2

It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component Such a kit will be of use m diagnosmg a disease or suspectabi ty to a disease, particularly AIDS, cancer, autoimmune disease, hepatitis, and diabetes, 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 Inheotance 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 (coinheotance 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 m any normal individuals, then the mutation is likely to be the causative agent of the disease

The polypeptides of the mvention or their fragments or analogs thereof, or cells expressmg them, can also be used as immunogens to produce antibodies immunospecific for polypeptides of the present mvention The term "immunospecific" means that the antibodies have substantially greater affinity for the polypeptides of the mvention than their affinity for other related polypeptides m the poor art Antibodies generated against polypeptides of the present mvention may be obtamed by administering the polypeptides or epitope-beaong fragments, analogs or cells to an animal, preferably a non-human animal, usmg routine protocols For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used Examples mclude the hybndoma techmque (Kohler, G and Milstein, C , Nature (1975) 256 495-497), the tnoma techmque, the human B-cell hybndoma technique (Kozbor et al , Immunology Today (1983) 4 72) and the EBV- hybndoma techmque (Cole et al , MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp 77-96, Alan R Liss, Lie , 1985)

Techniques for the production of smgle chain antibodies, such as those descnbed in U S Patent No 4,946,778, can also be adapted to produce smgle 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-descnbed antibodies may be employed to isolate or to identify clones expressmg the polypeptide or to purify the polypeptides by affinity chromatography Antibodies against polypeptides of the present mvention may also be employed to treat the

Diseases, amongst others

In a further aspect, the present invention relates to genetically engineered soluble fusion proteins compnsmg 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 immunoglobulm is the constant part of the heavy cham 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 engmeenng, and to the use thereof for drug screening, diagnosis and therapy A further aspect of the invention also relates to polynucleotides encoding such fusion protems Examples of fusion protein technology can be found m International Patent Application Nos W094/29458 and W094/22914

Another aspect of the mvention relates to a method for mducing an immunological response m a mammal which compnses moculating the mammal with a polypeptide of the present mvention, adequate to produce antibody and/or T cell immune response to protect said animal from the Diseases herembefore mentioned, amongst others Yet another aspect of the mvention relates to a method of inducmg immunological response in a mammal which compnses, delivering a polypeptide of the present mvention via a vector directmg expression of the polynucleotide and codmg for the polypeptide in vivo m order to induce such an immunological response to produce antibody to protect said animal from diseases

A further aspect of the mvention relates to an immunological/vaccine formulation (composition) which, when introduced mto a mammalian host, mduces an immunological response m that mammal to a polypeptide of the present invention wherem the composition comprises a polypeptide or polynucleotide of the present mvention The vaccme formulation may further compnse a suitable carrier Since a polypeptide may be broken down in the stomach, it is preferably administered parenterally (for instance, subcutaneous, intramuscular, intravenous, or lntradermal injection) Formulations suitable for parenteral administration include aqueous and non-aqueous sterile 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 sterile suspensions which may mclude suspending agents or thickening agents The formulations may be presented in umt-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dπed condition requinng only the addition of the sterile liquid carrier immediately prior to use The vaccine formulation may also include adjuvant systems for enhancing the immunogemcity of the formulation, such as oil-in water systems and other systems known in the art The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation

Polypeptides of the present mvention are responsible for many biological functions, including many disease states, m particular the Diseases herembefore mentioned It is therefore desirous to devise screening methods to identify compounds which stimulate or which inhibit the function of the polypeptide Accordingly, m a further aspect, the present mvention provides for a method of screening compounds to identify those which stimulate or which inhibit the function of the polypeptide In general, agonists or antagonists may be employed for therapeutic and prophylactic purposes for such Diseases as herembefore mentioned Compounds may be identified from a vaoety of sources, for example, cells, cell-free preparations, chemical hbranes, 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 structural or functional mimetics thereof (see Co gan et al , Current Protocols in Immunology 1(2) Chapter 5 (1991)) The screening method may simply measure the binding of a candidate compound to the polypeptide, or to cells or membranes bearing the polypeptide, or a fusion protem thereof by means of a label directly or indirectly associated with the candidate compound Alternatively, the screemng method may mvolve competition with a labeled competitor Further, these screening methods may test whether the candidate compound results m a signal generated by activation or inhibition of the polypeptide, usmg detection systems appropoate to the cells bearing the polypeptide Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed Constitutively active polpypeptides may be employed m screenmg methods for mverse agonists or inhibitors, in the absence of an agonist or inhibitor, by testmg whether the candidate compound results m inhibition of activation of the polypeptide Further, the screening methods may simply compnse the steps of mixing a candidate compound with a solution containing a polypeptide of the present invention, to form a mixture, measurmg CBFAKDIO activity m the mixture, and comparing the CBFAKDIO activity of the mixture to a standard Fusion proteins, such as those made from Fc portion and CBFAKDIO polypeptide, as herembefore described, can also be used for high-throughput screenmg assays to identify antagonists for the polypeptide of the present mvention (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 invention may also be used to configure screening methods for detecting 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 in the art This can be used to discover agents which may inhibit or enhance the production of polypeptide (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues

The polypeptide may be used to identify membrane bound or soluble receptors, if any, through standard receptor binding techniques known in the art These include, but are not limited to, hgand binding and crosshnking assays in which the polypeptide is labeled with a radioactive isotope (for instance *^■*^■■'^'), chemically modified (for instance, biotinylated), or fused to a peptide sequence suitable for detection or punfication, and incubated 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 screenmg 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 in the art

Examples of potential polypeptide antagonists mclude antibodies or, m some cases, o gonucleotides 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 invention relates to a screening kit for identifying agomsts, antagonists, hgands, receptors, substrates, enzymes, etc for polypeptides of the present invention, or compounds which decrease or enhance the production of such polypeptides, which compnses

(a) a polypeptide of the present mvention,

(b) a recombinant cell expressing a polypeptide of the present invention,

(c) a cell membrane expressing a polypeptide of the present invention, or

(d) antibody to a polypeptide of the present 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 m a method for the structure-based design of an agonist, antagonist or inhibitor of the polypeptide, by

(a) determinmg in the first instance the three-dimensional structure of the polypeptide,

(b) deducmg the three-dimensional structure for the likely reactive or bmdmg sιte(s) of an agonist, antagonist or inhibitor,

(c) synthesmg candidate compounds that are predicted to bind to or react with the deduced binding or reactive site, and

(d) testing whether the candidate compounds are indeed agonists, antagonists or inhibitors It will be further appreciated that this will normally be an interative process In a further aspect, the present mvention provides methods of treating abnormal conditions such as, for instance, AIDS, cancer, autoimmune disease, hepatitis, and diabetes, related to either an excess of, or an under-expression of, CBFAKDIO 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 (antagonist) as heremabove descnbed, optionally m combination with a pharmaceutically acceptable earner, m an amount effective to inhibit the function of the polypeptide, such as, for example, by blocking the binding 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 b dmg the ligand, substrate, enzymes, receptors, etc in competition with endogenous polypeptide may be administered Typical examples of such competitors mclude fragments of the CBFAKDIO polypeptide

In still another approach, expression of the gene encoding endogenous CBFAKDIO polypeptide can be inhibited usmg expression blocking techniques Known such techniques involve the use of antisense sequences, either internally generated or separately administered (see, for example, O'Connor, JNeurochem (1991) 56 560 in O godeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)) Alternatively, ohgonucleotides which form triple helices with the gene can be supplied (see, for example, Lee et al , Nucleic Acids Res (1979) 6 3073, Cooney et al , Science (1988) 241 456, Dervan et α/ , Science (1991) 251 1360) These ohgomers can be administered er se or the relevant o gomers can be expressed in vivo

For treating abnormal conditions related to an under-expression of CBFAKDIO and its activity, several approaches are also available One approach compnses admimstenng to a subject a therapeutically effective amount of a compound which activates a polypeptide of the present mvention, l e , an agonist 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 CBFAKDIO 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 introduced mto a packaging cell transduced with a retroviral plasmid vector containmg RNA encoding 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 m vivo and expression of the polypeptide vivo For an overview of gene therapy, see Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeutic Approaches, (and references cited therein) in Human Molecular Genetics, T Strachan and A P Read, BIOS Scientific Publishers Ltd (1996) Another approach is to administer a therapeutic amount of a polypeptide of the present mvention in combination with a suitable pharmaceutical earner

In a further aspect, the present mvention provides for pharmaceutical compositions compnsing 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 compnsing 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 in conjunction with other compounds, such as therapeutic compounds

The composition will be adapted to the route of admimstration, 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, intramuscular, or mtrapentoneal, can be used Alternative means for systemic admimstration 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 Admimstration 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 admimstration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attending practitioner Suitable dosages, however, are m the range of 0 1-100 μg/kg of subject Wide vanations in the needed dosage, however, are to be expected m view of the vanety of compounds available and the diffenng efficiencies of vanous routes of administration For example, oral admimstration would be expected to require higher dosages than admimstration by mtravenous injection Vanations m these dosage levels can be adjusted usmg standard empiπcal 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 referred 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 Accordingly, 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, chimeπc, single cham, and humamzed antibodies, as well as Fab fragments, including 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 in nature, it has been changed or removed from its original environment, or both For example, a polynucleotide or a polypeptide naturally present in a living animal is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting 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" include, without limitation, smgle- and double-stranded DNA, DNA that is a mixture of single- and double- stranded regions, smgle- and double-stranded RNA, and RNA that is mixture of smgle- and double-stranded regions, hybod molecules compnsmg DNA and RNA that may be smgle-stranded or, more typically, double-stranded or a mixture of smgle- and double-stranded regions In addition, "polynucleotide" refers to triple-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, tπtylated bases and unusual bases such as mosine A variety of modifications may be made to DNA and RNA, thus, "polynucleotide" embraces chemically, enzymatically or metabo cally modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells "Polynucleotide" also embraces relatively short polynucleotides, often referred to as ohgonucleotides

"Polypeptide" refers to any peptide or protein comprising two or more ammo acids jomed to each other by peptide bonds or modified peptide bonds, 1 e , peptide isosteres "Polypeptide" refers to both short chains, commonly referred to as peptides, o gopeptides or ohgomers, and to longer chains, generally referred to as proteins Polypeptides may contain ammo acids other than the 20 gene-encoded ammo acids "Polypeptides" mclude amino acid sequences modified either by natural processes, such as post-translational processmg, or by chemical modification techniques which are well known in the art Such modifications are well described in basic texts and m more detailed monographs, as well as m a volummous research literature Modifications may occur anywhere m a polypeptide, including the peptide backbone, the amino acid side-chains and the am o or carboxyl termini It will be appreciated that the same type of modification may be present to the same or varying degrees at several sites in a given polypeptide Also, a given polypeptide may contain many types of modifications Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching Cyclic, branched and branched cyclic polypeptides may result from post-translation natural processes or may be made by synthetic methods Modifications mclude acetylation, acylation, ADP-πbosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a pid or pid denvative, covalent attachment of phosphotidy nositol, cross-linking, cyc zation, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystme, formation of pyroglutamate, formylation, gamma- carboxylation, glycosylation, GPI anchor formation, hydroxylation, lodination, methylation, myπstoylation, oxidation, proteolytic processmg, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to protems such as arginylation, and ubiquitination (see, for instance, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed , T E Creιghton, W H Freeman and Company, New York, 1993, Wold, F , Post-translational Protein Modifications Perspectives and Prospects, pgs 1-12 m POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B C Johnson, Ed , Academic Press, New York, 1983, Seifter et al , "Analysis for protein modifications and nonprotein cofactors", Meth Enzymol (1990) 182 626-646 and Rattan et al , "Protein Synthesis Post-translational Modifications and Aging", Ann NY AcadSci (1992) 663 48-62) "Variant" refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains essential properties A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide Changes m the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result m amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below A typical variant of a polypeptide differs m ammo acid sequence from another, reference polypeptide Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, m many regions, identical A vanant and reference polypeptide may differ m ammo acid sequence by one or more substitutions, additions, deletions m any combmation A substituted or inserted amino 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 alle c variant, or it may be a vanant that is not known to occur naturally Non-naturally occurnng variants 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 determined 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 determined by the match between strings of such sequences

"Identity" can be readily calculated by known methods, including 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, Griffin, 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 Primer, Gπbskov, M and Devereux, J , eds , M Stockton Press, New York, 1991, and Caπllo, H , and Lipman, D , SIAM J Applied Math , 48 1073 (1988) Methods to determine identity are designed to give the largest match between the sequences tested Moreover, methods to determine identity are codified in publicly available computer programs Computer program methods to determine identity between two sequences 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 NLH 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 compaπson include the following 1) Algorithm Needleman and Wunsch, J Mol Biol 48 443-453 (1970) Compaπson matrix BLOSSUM62 from Hentikoff and Hentikoff, Proc Natl Acad Sci USA 89 10915-10919 (1992) Gap Penalty 12

Gap Length Penalty 4

A program useful with these parameters is publicly available as the "gap" program from Genetics Computer Group, Madison WI The aforementioned parameters are the default parameters for peptide compaπsons (along with no penalty for end gaps) Parameters for polynucleotide comparison include the following l) Algonthm Needleman and Wunsch, J Mol Biol 48 443-453 (1970) Companson matπx 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 compansons

A preferred meaning for "identity" for polynucleotides and polypeptides, as the case may be, are provided m (1) and (2) below (1) Polynucleotide embodiments further mclude an isolated polynucleotide compnsmg 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, wherem said alterations are selected from the group consistmg 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, mterspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence, and wherem said number of nucleotide alterations is determined by multiplymg 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 l, or

ⁿn ≤ ^xn " (^xn * y),

wherem n_n is the number of nucleotide alterations, x_n is the total number of nucleotides in SEQ ID NO 1, y is 0 50 for 50%, 0 60 for 60%, 0 70 for 70%, 0 80 for 80%, 0 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 invention may be identical to the reference sequence of SEQ ID NO 2, that is it may be 100% identical, or it may include up to a certain integer number of 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 consisting of at least one nucleic acid deletion, substitution, mcludmg transition and transversion, or insertion, and wherein said alterations may occur at the 5 ' or 3' terminal positions of the reference polynucleotide sequence or anywhere between those terminal positions, mterspersed either individually among the nucleic acids m the reference sequence or in one or more contiguous groups within the reference sequence The number of nucleic acid alterations for a given percent identity is determined by multiplying the total number of ammo acids in SEQ ID NO 2 by the mteger defining the percent identity divided by 100 and then subtractmg that product from said total number of amino acids in SEQ ID NO 2, or

ⁿn ≤ *n " (^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 instance 0 70 for 70%, 0 80 for 80%, 0 85 for 85% etc , • is the symbol for the multiplication operator, and wherein any non-integer product of x_n and y is rounded down to the nearest mteger prior to subtractmg it from x_n (2) Polypeptide embodiments further include an isolated polypeptide comprising a polypeptide having at least a 50,60, 70, 80, 85, 90, 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO 2, 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 ammo acid deletion, substitution, including conservative and non- conservative substitution, or insertion, and wherein said alterations may occur at the ammo- or carboxy-termmal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the ammo acids in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of amino acid alterations is determmed by multiplying the total number of ammo acids in SEQ ID NO 2 by the mteger defining the percent identity divided by 100 and then subtracting that product from said total number of amino acids in SEQ ID NO 2, or ⁿa ^{≤ x}a " (^xa * y)>

wherem n_a is the number of ammo acid alterations, x_a is the total number of am o 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 prior 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 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 consisting of at least one ammo acid deletion, substitution, mcludmg conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the ammo- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, mterspersed either individually among the ammo acids m the reference sequence or m one or more contiguous groups withm the reference sequence The number of am o acid alterations for a given % identity is determined by multiplymg the total number of amino acids in SEQ ID NO 2 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of ammo acids in SEQ ID NO 2, or

n_a ≤ x_a ( _a • y)>

wherein n_a is the number of ammo acid alterations, x_a is the total number of amino acids m SEQ ID NO 2, y is, for instance 0 70 for 70%, 0 80 for 80%, 0 85 for 85% etc , and • is the symbol for the multiplication operator, and wherein any non-mteger product of x_a and y is rounded down to the nearest integer prior to subtracting it from x_a

"Fusion protein" refers to a protein encoded by two, often unrelated, fused genes or fragments thereof In one example, EP-A-0 464 discloses fusion proteins comprising various portions of constant region of lmmunoglobuhn molecules together with another human protein or part thereof In many cases, employing an immunoglobulm Fc region as a part of a fusion protein is advantageous for use in 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 Nθ : l

1 CGCCGGGGAA GCGAAGTAGG CAGGGGCGAG GCGGCTGGGG ACCGCGGGGC

51 GGACGGGAGC GAGTATGTCC GCTCTGACTC GGCTGGCGTC TTTCGCTCGC

101 GTTGGAGGCC GCCTTTTCAG AAGCGGcTGC GCACGGACTG CTGGAGATGG

151 TGGAGTCCGT CATGCCGGTG GTGGTGTGCA CATTGAGCCC CGGTATAGAC

201 AGTTCCCCCA GCTGACCAGA TCCCAGGTGT TCCAGAGCGA GTTCTTCAGC

251 GGACTCATGT GGTTCTGGAT TCTCTGGCGC TTTTGGCATG ACTCAGAAGA

301 GGTGCTGGGT CACTTTCCGT ATCCTGATCC TTCCCAGTGG ACAGATGAAG

351 AATTAGGTAT CCCTCCTGAT GATGAAGACT GAAGGTGTAG ACTCAGCCTC

401 ACTCTGTACA AGAGCCAGGT GAGAATTTCA AGGATTATCG ACTTCATATT

451 GCACATTAAA GTTACAAATT AAAGTGGCTT GGTCAAGAAT GAgAAAAAAA

501 AAAAAAAAA

SEQ ID NO : 2 1 MSALTRLASF ARVGGRLFRS GCARTAGDGG VRHAGGGVHI EPRYRQFPQL

51 TRSQVFQSEF FSGLMWFWI RFWHDSEEV LGHFPYPDPS QWTDEELGIP

101 PDDED SEQUENCE LISTING

(1) GENERAL INFORMATION

(i) APPLICANT: SHANGHAI SECOND MEDICAL UNIVERSITY

(ii) TITLE OF THE INVENTION: A Human Ubiquinone Oxireductase subunit CI-AGGG Homolog Gene (CBFAKDIO)

(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) Z I P : 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-70440 (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 610-407-0700

(B) TELEFAX: 610-407-0701

(C) TELEX: 846169

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 509 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

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

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

CGCCGGGGAA GCGAAGTAGG CAGGGGCGAG GCGGCTGGGG ACCGCGGGGC GGACGGGAGC 60 GAGTATGTCC GCTCTGACTC GGCTGGCGTC TTTCGCTCGC GTTGGAGGCC GCCTTTTCAG 120

AAGCGGCTGC GCACGGACTG CTGGAGATGG TGGAGTCCGT CATGCCGGTG GTGGTGTGCA 180

CATTGAGCCC CGGTATAGAC AGTTCCCCCA GCTGACCAGA TCCCAGGTGT TCCAGAGCGA 240

GTTCTTCAGC GGACTCATGT GGTTCTGGAT TCTCTGGCGC TTTTGGCATG ACTCAGAAGA 300

GGTGCTGGGT CACTTTCCGT ATCCTGATCC TTCCCAGTGG ACAGATGAAG AATTAGGTAT 360 CCCTCCTGAT GATGAAGACT GAAGGTGTAG ACTCAGCCTC ACTCTGTACA AGAGCCAGGT 420

GAGAATTTCA AGGATTATCG ACTTCATATT GCACATTAAA GTTACAAATT AAAGTGGCTT 480

GGTCAAGAAT GAGAAAAAAA AAAAAAAAA 509

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 105 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

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

Met Ser Ala Leu Thr Arg Leu Ala Ser Phe Ala Arg Val Gly Gly Arg 1 5 10 15

Leu Phe Arg Ser Gly Cys Ala Arg Thr Ala Gly Asp Gly Gly Val Arg 20 25 30 His Ala Gly Gly Gly Val His lie Glu Pro Arg Tyr Arg Gin Phe Pro

35 40 45

Gin Leu Thr Arg Ser Gin Val Phe Gin Ser Glu Phe Phe Ser Gly Leu

50 55 60 Met Trp Phe Trp lie Leu Trp Arg Phe Trp His Asp Ser Glu Glu Val

65 70 75 80

Leu Gly His Phe Pro Tyr Pro Asp Pro Ser Gin Trp Thr Asp Glu Glu

85 90 95

Leu Gly lie Pro Pro Asp Asp Glu Asp 100 105

Claims

What is claimed is:

1 An isolated polypeptide selected from the group consisting of

(1) an isolated polypeptide compnsmg an ammo acid sequence selected from the group havmg at least

(a) 70% identity,

(b) 80% identity,

(c) 90% identity, or

(d) 95% identity to the ammo acid sequence of SEQ ID NO 2 over the entire length of SEQ ID

NO 2, (n) an isolated polypeptide comprising the ammo acid sequence of SEQ ID NO 2 or (in) an isolated polypeptide which is the amino acid sequence of SEQ ID NO 2

2 An isolated polynucleotide selected from the group consisting of

(l) an isolated polynucleotide compnsmg a nucleotide sequence encodmg a polypeptide that has at least

(a) 70% identity,

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

(d) 95% identity, to the ammo acid sequence of SEQ ID NO 2, over the entire length of SEQ LD NO 2, (n) an isolated polynucleotide compnsmg a nucleotide sequence that has at least (a) 70%o identity (b) 80% identity,

(c) 90% identity, or

(d) 95% identity, over its entire length to a nucleotide sequence encoding the polypeptide of SEQ LD NO 2, (in) an isolated polynucleotide compnsmg 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 compnsing a nucleotide sequence encodmg the polypeptide of SEQ LD NO 2,

(vi) an isolated polynucleotide which is the polynucleotide of SEQ ID NO 1 , or (vi) an isolated polynucleotide obtainable by screenmg an appropnate hbrary under strmgent hybπdization conditions with a labeled probe having the sequence of SEQ LD NO 1 or a fragment thereof , or a nucleotide sequence complementary to said isolated polynucleotide

An antibody immunospecific for the polypeptide of claim 1

A method for the treatment of a subject

(l) m need of enhanced activity or expression of the polypeptide of claim 1 comprising

(a) administering to the subject a therapeutically effective amount of an agomst to said polypeptide, and/or

(b) providing to the subject an isolated polynucleotide compnsmg a nucleotide sequence encoding said polypeptide in a form so as to effect production of said polypeptide activity n vivo , or

(n) 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 compnsing

(a) determining the presence or absence of a mutation m 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 m a sample derived from said subject 6 A method for screenmg to identify compounds which stimulate or which inhibit the function of the polypeptide of claim 1 which compnses a method selected from the group consisting of

(a) measunng 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) measurmg 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) testmg whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide, using detection systems appropnate 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, measunng activity of the polypeptide in the mixture, and comparing the activity of the mixture to a standard, or

(e) detecting the effect of a candidate compound on the production of mRNA encodmg said polypeptide and said polypeptide m cells, using for instance, an ELISA assay

7 An agonist or an antagonist of the polypeptide of claim 1

8 An expression system compnsmg a polynucleotide capable of producing a polypeptide of claim 1 when said expression system is present in a compatible host cell

9 A process for producmg a recombmant host cell comprising transforming or transfectmg a cell with the expression system of claim 8 such that the host cell, under appropnate culture conditions, produces a polypeptide compnsmg an ammo acid sequence having at least 70% identity to the ammo 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 ammo acid sequence having at least 70% identity to the ammo 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.