WO1999036525A1 - Cbfblh12: a gene highly related to bovine ci-kfyi gene for ubiquinone oxireductase complex - Google Patents

Abstract

CBFBLH12 polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing CBFBLH12 polypeptides and polynucleotides in the design of protocols for the treatment of cardiovascular disease, kidney disease, metabolic-associated disorders, and autoimmune diseases, among others, and diagnostic assays for such conditions.

Description

CBFBLH12: A Gene Highly Related to Bovine CI-KFYI Gene for Ubiquinone Oxireductase

Complex

FIELD OF INVENTION

This mvention relates to newly identified polynucleotides, polypeptides encoded by them and to the use of such polynucleotides and polypeptides, and to their production More particularly, the polynucleotides and polypeptides of the present invention relate to the gene family including NADH-ubiquinone oxireductase, hereinafter referred to as CBFBLH12 The invention also relates to inhibiting or activatmg the action of such polynucleotides and polypeptides

BACKGROUND OF THE INVENTION

NADH-ubiquinone oxireductase is the first enzyme in the respiratory electron transport chain of mitochondna, and is a membrane-bound multi-subumt assembly The CI-KFYI is one of the nuclear encoded subunits, and participates in cellular respiration This indicates that the gene family including NADH-ubiquinone oxireductase has an established, proven history as therapeutic targets Clearly there is a need for identification and characterization of further members of the gene family including NADH- ubiquinone oxireductase family which can play a role n preventing, ameliorating or correcting dysfunctions or diseases, including, but not limited to. cardiovascular disease, kidney disease, metabo c- associated disorders, and autoimmune diseases

SUMMARY OF THE INVENTION

In one aspect, the mvention relates to CBFBLH12 polypeptides and recombinant matenals and methods for their production Another aspect of the invention relates to methods for using such CBFBLH12 polypeptides and polynucleotides Such uses include the treatment of cardiovascular disease, kidney disease, metabolic-associated disorders, and autoimmune diseases, among others In still another aspect, the invention relates to methods to identify agonists and antagonists usmg the matenals provided by the mvention, and treating conditions associated with CBFBLH12 imbalance with the identified compounds Yet another aspect of the invention relates to diagnostic assays for detecting diseases associated with inappropriate CBFBLH12 activity or levels

DESCRIPTION OF THE INVENTION Definitions

CONFIRMATION COflf The following definitions are provided to facilitate understanding of certain terms used frequently herein

"CBFBLH12" refers, among others, generally to a polypeptide having the ammo acid sequence set forth m SEQ ID NO 2 or an allehc variant thereof "CBFBLH12 activity or CBFBLH12 polypeptide activity" or "biological activity of the

CBFBLH12 or CBFBLH12 polypeptide" refers to the metabolic or physiologic function of said CBFBLH12 including similar activities or improved activities or these activities with decreased undesirable side-effects Also included are antigenic and lmmunogenic activities of said CBFBLH12 "CBFBLH12 gene" refers to a polynucleotide having the nucleotide sequence set forth in SEQ ID NO 1 or allehc variants thereof and/or their complements

"Antibodies" as used herein mcludes polyclonal and monoclonal antibodies, chimeπc, smgle chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other immunoglobulin expression library

"Isolated" means altered "by the hand of man" from the natural state If an "isolated" composition or substance occurs m 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 herein

"Polynucleotide" generally refers to any polyπbonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA "Polynucleotides" include, without limitation single- and double-stranded DNA, DNA that is a mixture of smgle- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of smgle- and double-stranded regions In addition, "polynucleotide" refers to triple-stranded regions compπsmg RNA or DNA or both RNA and DNA The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons "Modified" bases include, for example, tπtylated bases and unusual bases such as mosme A vaπety of modifications has been made to DNA and RNA, thus, "polynucleotide" embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells "Polynucleotide" also embraces relatively short polynucleotides, often referred to as oligonucleotides

"Polypeptide" refers to any peptide or protein compπsing two or more ammo acids joined to each other by peptide bonds or modified peptide bonds, l 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" include ammo acid sequences modified either by natural processes, such as posttranslational processing, or by chemical modification techniques which are well known the art Such modifications are well descπbed in basic texts and m more detailed monographs, as well as in a voluminous research literature Modifications can occur anywhere m a polypeptide, including the peptide backbone, the ammo acid side-chains and the ammo or carboxyl termini It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide Also, a given polypeptide may contain many types of modifications Polypeptides may be branched as a result of ubiquitmation, and they may be cyclic, with or without branching

Cyclic, branched and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods Modifications include acetylation. acylation. ADP-πbosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide deπvative, covalent attachment of a hpid or lipid deπvative, covalent attachment of phosphotidylmositol, cross-linking, cyclization, 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 ammo acids to proteins such as arg ylation, and ubiquitmation See, for instance, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed , T E Creighton, W H Freeman and Company, New York, 1993 and Wold, F , Posttranslational Protein Modifications Perspectives and Prospects, pgs 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B C Johnson, Ed , Academic Press, New York, 1983, Seifter et al , "Analysis for protein modifications and nonprotein cofactors", Meth Enzymol (1990) 182 626-646 and Rattan et al , "Protem Synthesis Posttranslational Modifications and Aging", Ann NYAcadSci (1992) 663 48-62

"Vaπant" as the term is used herein, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide Changes in the nucleotide sequence of the vaπant 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 m the polypeptide encoded by the reference sequence, as discussed below A typical vaπant 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, in many regions, identical A variant 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 variant of a polynucleotide or polypeptide may be a naturally occurπng such as an allehc variant, or it may be a vaπant that is not known to occur naturally Non-naturally occurπng vaπants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis

"Identity" is a measure of the identity of nucleotide sequences or ammo acid sequences In general, the sequences are aligned so that the highest order match is obtained "Identity" per se has an art-recognized meaning and can be calculated using published techniques See, e g

(COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, A M , ed , Oxford University Press, New York, 1988. BIOCOMPUTING INFORMATICS AND GENOME PROJECTS, Smith, D W , ed , Academic Press, New York, 1993, COMPUTER ANALYSIS OF SEQUENCE DATA, PART I, Griffin, A M , and Griffin, H G , eds , Humana Press, New Jersey, 1994, SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, von Heinje, G , Academic Press, 1987, and SEQUENCE ANALYSIS PRIMER, Gπbskov, M and Devereux, J , eds , M Stockton Press, New York, 1991) While there exist a number of methods to measure identity between two polynucleotide or polypeptide sequences, the term "identity" is well known to skilled artisans (Canllo, H , and Lipton, D , SIAM J Applied Math (1988) 48 1073) Methods commonly employed to determine identity or similaπty between two sequences include, but are not limited to, those disclosed in Guide to Huge Computers, Martin J

Bishop, ed , Academic Press, San Diego, 1994, and Canllo, H , and Lipton, D , SIAM J Applied Math (1988) 48 1073 Methods to determine identity and similanty are codified in computer programs Preferred computer program methods to determine identity and similanty between two sequences include, but are not limited to, GCS program package (Devereux, J , et al , Nucleic Acids Research (1984) 12(1) 387), BLASTP, BLASTN, FASTA (Atschul, S F et al , JMolec Biol (1990) 215 403) As an illustration, by a polynucleotide having a nucleotide sequence havmg at least, for example, 95% "identity" to a reference nucleotide sequence of SEQ ID NO 1 is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence of SEQ ID NO 1 In other words, to obtam a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides m the reference sequence may be inserted into the reference sequence These mutations of the reference sequence may occur at the 5 or 3 terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or m one or more contiguous groups within the reference sequence

Similarly, by a polypeptide havmg an ammo acid sequence having at least, for example, 95% "identity" to a reference ammo acid sequence of SEQ ID NO 2 is mtended that the ammo acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five ammo acid alterations per each 100 ammo acids of the reference ammo acid of SEQ ID NO 2 In other words, to obtain a polypeptide having an ammo acid sequence at least 95% identical to a reference ammo acid sequence, up to 5% of the ammo acid residues in the reference sequence may be deleted or substituted with another ammo acid, or a number of ammo acids up to 5% of the total ammo acid residues in the reference sequence may be inserted into the reference sequence These alterations of the reference sequence may occur at the ammo or carboxy terminal positions of the reference ammo acid sequence or anywhere between those terminal positions, interspersed either individually among residues m the reference sequence or one or more contiguous groups withm the reference sequence

Polypeptides of the Invention

In one aspect, the present invention relates to CBFBLH12 polypeptides (or CBFBLH12 protems) The CBFBLH12 polypeptides include the polypeptide of SEQ ID NO 2, as well as polypeptides comprising the ammo acid sequence of SEQ ID NO 2, and polypeptides compπsmg the ammo acid sequence which have at least 80% identity to that of SEQ ID NO 2 over its entire length, and still more preferably at least 90% identity, and even still more preferably at least 95% identity to SEQ ID NO 2 Furthermore, those with at least 97-99% are highly preferred Also mcluded within CBFBLH12 polypeptides are polypeptides having the ammo acid sequence which have at least 80% identity to the polypeptide having the ammo acid sequence of SEQ ID NO 2 over its entire length, and still more preferably at least 90% identity, and still more preferably at least 95% identity to SEQ ID NO 2 Furthermore, those with at least 97-99% are highly preferred Preferably CBFBLH12 polypeptide exhibit at least one biological activity of CBFBLH12

The CBFBLH12 polypeptides may be in the form of the "mature" protem or may be a part of a larger protem such as a fusion protein It is often advantageous to include an additional am o acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid m puπfication such as multiple histidine residues, or an additional sequence for stability during recombinant production Fragments of the CBFBLH12 polypeptides are also included m the invention A fragment is a polypeptide havmg an ammo acid sequence that entirely is the same as part, but not all, of the ammo acid sequence of the aforementioned CBFBLH12 polypeptides As with CBFBLH12 polypeptides, fragments may be "free-standing," or compπsed within a larger polypeptide of which they form a part or region, most preferably as a smgle continuous region Representative examples of polypeptide fragments of the mvention, mclude, for example, fragments from about ammo acid number 1-20, 21-40, 41-60, 61-80, 81-100, and 101 to the end of CBFBLH12 polypeptide In this context "about" mcludes the particularly recited ranges larger or smaller by several, 5, 4, 3, 2 or 1 ammo acid at either extreme or at both extremes

Preferred fragments mclude, for example, truncation polypeptides havmg the ammo acid sequence of CBFBLH12 polypeptides, except for deletion of a continuous senes of residues that mcludes the ammo terminus, or a continuous senes of residues that mcludes the carboxyl terminus or deletion of two continuous senes of residues, one including the ammo terminus and one including the carboxyl terminus Also prefened are fragments characterized by structural or functional attributes such as fragments that compnse alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and tum-formmg regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions Other prefened fragments are biologically active fragments Biologically active fragments are those that mediate CBFBLH12 activity, including those with a similar activity or an improved activity, or with a decreased undesirable activity Also mcluded are those that are antigenic or lmmunogenic in an animal, especially m a human

Preferably, all of these polypeptide fragments retain the biological activity of the CBFBLH12, including antigenic activity Vanants of the defined sequence and fragments also form part of the present mvention Prefened vanants are those that vary from the referents by conservative ammo acid substitutions - l e , those that substitute a residue with another of like charactenstics 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 prefened are vanants m which several, 5-10, 1-5, or 1-2 ammo acids are substituted, deleted, or added in any combination

The CBFBLH12 polypeptides of the mvention can be prepared m any suitable manner Such polypeptides mclude 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 m the art Polynucleotides of the Invention

Another aspect of the mvention relates to CBFBLH12 polynucleotides CBFBLH12 polynucleotides mclude isolated polynucleotides which encode the CBFBLH12 polypeptides and fragments, and polynucleotides closely related thereto More specifically, CBFBLH12 polynucleotide of the mvention mclude a polynucleotide compnsing the nucleotide sequence contained m SEQ ID NO 1 encodmg a

CBFBLH12 polypeptide of SEQ ID NO 2, and polynucleotide havmg the particular sequence of SEQ ID NO 1 CBFBLH12 polynucleotides further mclude a polynucleotide compnsing a nucleotide sequence that has at least 80% identity over its entire length to a nucleotide sequence encodmg the CBFBLH12 polypeptide of SEQ ID NO 2, and a polynucleotide compπsmg a nucleotide sequence that is at least 80% identical to of SEQ ID NO 1 over its entire length In this regard, polynucleotides at least 90% identical are particularly prefened, and those with at least 95% are especially prefened Furthermore, those with at least 97% are highly prefened and those with at least 98-99% are most highly prefened, with at least 99% being the most prefened Also mcluded under CBFBLH12 polynucleotides are a nucleotide sequence which has sufficient identity to a nucleotide sequence contained m SEQ ID NO 1 to hybπdize under conditions useable for amplification or for use as a probe or marker The mvention also provides polynucleotides which are complementary to such CBFBLH12 polynucleotides

CBFBLH12 of the mvention is structurally related to other proteins of the gene family including NADH-ubiquinone oxireductase, as shown by the results of sequencmg the cDNA of Table 1 (SEQ ID NO 1) encodmg human CBFBLH12 The cDNA sequence of SEQ ID NO 1 contains an open reading frame (nucleotide number 9 to 236) encodmg a polypeptide of 76 ammo acids of SEQ ID NO 2 The ammo acid sequence of Table 2 (SEQ ID NO 2) has about 78 9% identity (using FASTA) m 76 ammo acid residues with bovine CI-KFYI gene for ubiqumone oxireductase complex (J E Walker, et al , J Mol Biol 226 1051- 1072, 1992) The nucleotide sequence of Table 1 (SEQ ID NO 1) has about 80% identity (usmg FASTA) m 420 nucleotide residues with bovine CI-KFYI gene for ubiqumone oxireductase complex (J E Walker, et al , J Mol Biol 226 1051-1072, 1992) Thus, CBFBLH12 polypeptides and polynucleotides of the present mvention are expected to have, inter alia, similar biological functions/properties to their homologous polypeptides and polynucleotides, and their utility is obvious to anyone skilled m the art

Table 1'

1 GACGCAAGAT GGCGCCGTCC GCCTTGCTGC GTCCCCTTTC CCGGCTGCTG

51 GCCCCCGCCA GGCTCCCGAG CGGCCCTTCA GTGCGATCAA AGTTCTACGT

101 GCGAGAGCCG CCGAATGCCA AACCTGACTG GCTGAAAGTT GGGTTCACCT

151 TGGGCACCAC TGTCTTCTTG TGGATCTATC TCATCAAACA ACACAATGAA 201 GATATTTTAG AGTACAAAAG AAGAAATGGG CTGGAATAAA CTTTTGAAAC

251 ACTAATGTAG TATGCTCCGT ATAGTGATTG TAGCTGTTCC TCTGGATTCA

301 CCATCTGTTG AGTTGTAAAT GTGAGAGAAA AAGTTATATG TGAATATATA

351 TCAAGCCAGC ATTTGTATTT TGCATCATTA AATAAAAAGA AATAAAAAAA

401 AAAAAAAAAA AAAAAAAAAA

A nucleotide sequence of a human CBFBLH12 (SEQ ID NO 1)

Table 2^b

1 APSALLRPL SRI- APAR P SGPSVRSKFY VREPPNAKPD LKVGFTLGT

51 TVFL IYLIK QHNEDILEYK RRNGLE

An ammo acid sequence of a human CBFBLH12 (SEQ ID NO 2)

One polynucleotide of the present mvention encodmg CBFBLH12 may be obtained using standard cloning and screening, from a cDNA library denved from mRNA m cells of human cord blood usmg 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 mvention can also be obtained from natural sources such as genomic DNA hbranes or can be synthesized using well known and commercially available techniques

The nucleotide sequence encoding CBFBLH12 polypeptide of SEQ ID NO 2 may be identical to the polypeptide encoding sequence contained in Table 1 (nucleotide number 9 to 236 of SEQ ID

NO 1), or it may be a sequence, which as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO 2

When the polynucleotides of the invention are used for the recombinant production of CBFBLH12 polypeptide, the polynucleotide may include the coding sequence for the mature polypeptide or a fragment thereof, by itself, the coding sequence for the mature polypeptide or fragment m readmg frame with other coding 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 puπficaϋon 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 m the pQE vector (Qiagen, Inc ) and descnbed in Gentz et al , Proc NatlAcadSci 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 binding sites and sequences that stabilize mRNA Further prefened embodiments are polynucleotides encodmg CBFBLH12 vanants compnse the ammo acid sequence CBFBLH12 polypeptide of Table 2 (SEQ ID NO 2) m which several, 5-10, 1-5, 1-3, 1-2 or 1 ammo acid residues are substituted, deleted or added, m any combination

The present mvention further relates to polynucleotides that hybπdize to the here above-descnbed sequences In this regard, the present mvention especially relates to polynucleotides which hybndize under stringent conditions to the herem above-descnbed polynucleotides As herem used, the term "stπngent conditions" means hybndization will occur only if there is at least 80%, and preferably at least 90%, and more preferably at least 95%, yet even more preferably 97-99% identity between the sequences

Polynucleotides of the mvention. which are identical or sufficiently identical to a nucleotide sequence contained m SEQ ID NO 1 or a fragment thereof, may be used as hybndization probes for cDNA and genomic DNA, to isolate full-length cDNAs and genomic clones encodmg CBFBLH12 polypeptide 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 the CBFBLH12 gene Such hybndization techniques are known to those of skill m the art Typically these nucleotide sequences are 80% identical, preferably 90% identical, more preferably 95% identical to that of the referent The probes generally will compnse at least 15 nucleotides Preferably, such probes will have at least 30 nucleotides and may have at least 50 nucleotides Particularly prefened probes will range between 30 and 50 nucleotides

In one embodiment, to obtam a polynucleotide encodmg CBFBLH12 polypeptide, including homologs and orthologs from species other than human, compnses the steps of screening an appropnate library under stingent hybndization conditions with a labeled probe havmg the SEQ ID NO 1 or a fragment thereof, and isolating full-length cDNA and genomic clones containmg said polynucleotide sequence Thus m another aspect, CBFBLH12 polynucleotides of the present mvention further mclude a nucleotide sequence compnsing a nucleotide sequence that hybπdize under stπngent condition to a nucleotide sequence havmg SEQ ID NO 1 or a fragment thereof Also mcluded with CBFBLH 12 polypeptides are polypeptide compnsmg ammo acid sequence encoded by nucleotide sequence obtained by the above hybndization condition Such hybndization techniques are well known to those of sbll m the art Stπngent hybndization conditions are as defined above or, alternatively, conditions under overnight mcubation 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 The polynucleotides and polypeptides of the present mvention may be employed as research reagents and matenals for discovery of treatments and diagnostics to animal and human disease

Vectors, Host Cells, Expression The present mvention also relates to vectors which compnse a polynucleotide or polynucleotides of the present mvention, and host cells which are genetically engmeered with vectors of the mvention and to the production of polypeptides of the mvention by recombmant techniques Cell-free translation systems can also be employed to produce such proteins usmg RNAs denved from the DNA constructs of the present mvention For recombinant production, host cells can be genetically engmeered to incorporate expression systems or portions thereof for polynucleotides of the present mvention Introduction of polynucleotides into host cells can be effected by methods descπbed m 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) such as calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection. catiomc hpid-mediated transfection, electroporation, transduction, scrape loadmg, ballistic introduction or infection

Representative examples of appropπate hosts mclude bactenal 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 vaπety of expression systems can be used Such systems mclude, among others, chromosomal, episomal and virus-denved systems, e g , vectors denved from bactenal plasmids, from bacteπophage, 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 denved from combinations thereof, such as those denved from plasmid and bactenophage genetic elements, such as cosmids and phagemids The expression systems may contain control regions that regulate as well as engender expression Generally, any system or vector suitable to maintain, propagate or express polynucleotides to produce a polypeptide in a host may be used The appropπate nucleotide sequence may be inserted mto an expression system by any of a vanety of well-known and routine techniques, such as, for example, those set forth m Sambrook et al , MOLECULAR CLONING, A LABORATORY MANUAL (supra)

For secretion of the translated protem mto the lumen of the endoplasmic reticulum, mto the penplasmic space or mto the extracellular environment, appropπate secretion signals may be incorporated mto the desired polypeptide These signals may be endogenous to the polypeptide or they may be heterologous signals

If the CBFBLH12 polypeptide is to be expressed for use in screening assays, generally, it is prefened that the polypeptide be produced at the surface of the cell In this event, the cells may be harvested pnor to use m the screening assay If CBFBLH12 polypeptide is secreted mto the medium, the medium can be recovered m order to recover and puπfy the polypeptide, if produced intracellularly, the cells must first be lysed before the polypeptide is recovered

CBFBLH12 polypeptides can be recovered and punfied from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography Most preferably, high performance liquid chromatography is employed for puπfication Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or puπfication

Diagnostic Assays

This mvention also relates to the use of CBFBLH12 polynucleotides for use as diagnostic reagents Detection of a mutated form of CBFBLH12 gene 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 CBFBLH12 Individuals carrying mutations m the CBFBLH12 gene may be detected at the DNA level by a vaπety of techniques

Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, uπne, saliva, tissue biopsy or autopsy matenal The genomic DNA may be used directly for detection or may be amplified enzymatically by us g PCR or other amplification techniques pnor to analysis RNA or cDNA may also be used m similar fashion Deletions and insertions can be detected by a change m size of the amplified product m compaπson to the normal genotype Pomt mutations can be identified by hybπdizmg amplified DNA to labeled CBFBLH12 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 m gels, with or without denatuπng agents, or by direct DNA sequencmg See, e g , Myers et al , Science (1985) 230 1242 Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and S 1 protection or the chemical cleavage method See Cotton et al , Proc Natl Acad S i USA ( 1985) 85 4397-4401 In another embodiment, an array of oligonucleotides probes compnsmg CBFBLH12 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 apphcability and can be used to address a vanety of questions m molecular genetics including gene expression, genetic linkage, and genetic vaπabihty (See for example M Chee et al , Science, Vol 274, pp 610-613 (1996))

The diagnostic assays offer a process for diagnosing or determining a susceptibility to cardiovascular disease, kidney disease, metabolic-associated disorders, and autoimmune diseases through detection of mutation in the CBFBLH12 gene by the methods descnbed

In addition, cardiovascular disease, kidney disease, metabolic-associated disorders, and autoimmune diseases, can be diagnosed by methods compπsmg determining from a sample denved from a subject an abnormally decreased or mcreased level of CBFBLH12 polypeptide or CBFBLH12 mRNA Decreased or mcreased expression can be measured at the RNA level using any of the methods well known m the art for the quantitation of polynucleotides, such as, for example, PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods Assay techniques that can be used to determine levels of a protein, such as an CBFBLH12 polypeptide, in a sample denved 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 m another aspect, the present mvention relates to a diagonostic kit for a disease or suspectabi ty to a disease, particularly cardiovascular disease, kidney disease, metabolic-associated disorders, and autoimmune diseases, which compnses

(a) a CBFBLH12 polynucleotide, preferably the nucleotide sequence of SEQ ID NO 1, or a fragment thereof,

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

(c) a CBFBLH12 polypeptide, preferably the polypeptide of SEQ ID NO 2, or a fragment thereof, or

(d) an antibody to a CBFBLH12 polypeptide, preferably to the polypeptide of SEQ ID NO 2

It will be appreciated that m any such kit, (a), (b), (c) or (d) may compnse a substantial component

Chromosome Assays

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 in conelatmg 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 conelated with genetic map data Such data are found, for example, m V McKusick. Mende an Inhentance m Man (available on lme through Johns Hopkins University Welch Medical Library) The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheπtance of physically adjacent genes)

The differences m the cDNA or genomic sequence between affected and unaffected individuals can also be determined If a mutation is observed m 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

Antibodies

The polypeptides of the mvention or their fragments or analogs thereof, or cells expressmg them can also be used as immunogens to produce antibodies lmmunospecific for the CBFBLH12 polypeptides The term "lmmunospecific" means that the antibodies have substantiall greater affinity for the polypeptides of the mvention than their affinity for other related polypeptides m the pnor art

Antibodies generated against the CBFBLH12 polypeptides can be obtained by administering the polypeptides or epitope-beaπng fragments, analogs or cells to an animal, preferably a nonhuman, usmg routine protocols For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell lme cultures can be used Examples mclude the hybπdoma technique (Kohler, G and Milstem, C , Nature (1975) 256 495-497), the tπoma technique, the human B-cell hybπdoma technique (Kozbor et al , Immunology Toady (1983) 4 72) and the EBV-hybndoma technique (Cole et al , MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp 77-96, Alan R Liss, Inc , 1985) Techniques for the production of smgle chain antibodies (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 CBFBLH12 polypeptides may also be employed to treat cardiovascular disease, kidney disease, metabolic-associated disorders, and autoimmune diseases, among others

Vaccines

Another aspect of the invention relates to a method for mducmg an lmmunological response m a mammal which compnses inoculating the mammal with CBFBLH12 polypeptide, or a fragment thereof, adequate to produce antibody and/or T cell immune response to protect said animal from cardiovascular disease, kidney disease, metabolic-associated disorders, and autoimmune diseases, among others Yet another aspect of the invention relates to a method of inducing lmmunological response m a mammal which comprises, delivering CBFBLH12 polypeptide via a vector directing expression of CBFBLH12 polynucleotide in vivo m order to induce such an lmmunological response to produce antibody to protect said animal from diseases

Further aspect of the mvention relates to an lmmunological/vaccme formulation (composition) which, when mtroduced into a mammalian host, induces an lmmunological response in that mammal to a CBFBLH12 polypeptide wherem the composition comprises a CBFBLH12 polypeptide or CBFBLH12 gene The vaccine formulation may further compnse a suitable earner Smce CBFBLH12 polypeptide may be broken down m the stomach, it is preferably administered parenterally (including subcutaneous, intramuscular, mtravenous, intradermal etc injection) Formulations suitable for parenteral administration mclude aqueous and non-aqueous stenle injection solutions which may contain anti- oxidants, buffers, bacteπostats and solutes which render the formulation mstonic with the blood of the recipient, and aqueous and non-aqueous stenle suspensions which may mclude suspending 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 m a freeze-dπed condition requmng only the addition of the stenle liquid earner immediately pnor to use The vaccme formulation may also mclude adjuvant systems for enhancmg 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 vaccme and can be readily determined by routine experimentation

Screening Assays The CBFBLH12 polypeptide of the present mvention may be employed m a screening process for compounds which activate (agonists) or inhibit activation of (antagonists, or otherwise called inhibitors) the CBFBLH12 polypeptide of the present mvention Thus, polypeptides of the mvention may also be used to assess identify agonist or antagonists from, for example, cells, cell-free preparations, chemical hbraπes, and natural product mixtures These agonists or antagonists may be natural or modified substrates, ligands, receptors, enzymes, etc , as the case may be, of the polypeptide of the present mvention, or may be structural or functional mimetics of the polypeptide of the present mvention See Coligan et al , Current Protocols in Immunology 1(2) Chapter 5 (1991)

CBFBLH12 polypeptides are responsible for many biological functions, including many pathologies Accordingly, it is desirous to find compounds and drugs which stimulate CBFBLH12 polypeptide on the one hand and which can inhibit the function of CBFBLH 12 polypeptide on the other hand In general, agonists are employed for therapeutic and prophylactic purposes for such conditions as cardiovascular disease, kidney disease, metabolic-associated disorders, and autoimmune diseases Antagonists may be employed for a vaπety of therapeutic and prophylactic purposes for such conditions as cardiovascular disease, kidney disease, metabolic-associated disorders, and autoimmune diseases In general, such screening procedures may involve usmg appropnate cells which express the CBFBLH12 polypeptide or respond to CBFBLH12 polypeptide of the present mvention Such cells mclude cells from mammals, yeast, Drosophila or E cob Cells which express the CBFBLH12 polypeptide (or cell membrane containing the expressed polypeptide) or respond to CBFBLH12 polypeptide are then contacted with a test compound to observe binding, or stimulation or inhibition of a functional response The ability of the cells which were contacted with the candidate compounds is compared with the same cells which were not contacted for CBFBLH12 activity

The assays may simply test binding of a candidate compound wherem adherence to the cells beaπng the CBFBLH12 polypeptide is detected by means of a label directly or indirectly associated with the candidate compound or in an assay involving competition with a labeled competitor Further, these assays may test whether the candidate compound results m a signal generated by activation of the CBFBLH12 polypeptide, using detection systems appropπate to the cells beaπng the CBFBLH12 polypeptide Inhibitors of activation are generally assayed m the presence of a known agonist and the effect on activation by the agomst by the presence of the candidate compound is observed Further, the assays may simply compnse the steps of mixing a candidate compound with a solution containing a CBFBLH12 polypeptide to form a mixture, measuring CBFBLH12 activity m the mixture, and comparing the CBFBLH12 activity of the mixture to a standard

The CBFBLH12 cDNA, protein and antibodies to the protein may also be used to configure assays for detecting the effect of added compounds on the production of CBFBLH12 mRNA and protem in cells For example, an ELISA may be constructed for measunng secreted or cell associated levels of CBFBLH12 protein using monoclonal and polyclonal antibodies by standard methods known m the art, and this can be used to discover agents which may inhibit or enhance the production of CBFBLH12 (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues

The CBFBLH12 protem may be used to identify membrane bound or soluble receptors, if any, through standard receptor binding techniques known m the art These mclude, but are not limited to, gand binding and crosslinkmg assays in which the CBFBLH12 is labeled with a radioactive isotope (eg 1251), chemically modified (eg biotinylated), or fused to a peptide sequence suitable for detection or purification, and incubated with a source of the putative receptor (cells, cell membranes, cell supernatants, tissue extracts, bodily fluids) Other methods mclude biophysical techniques such as surface plasmon resonance and spectroscopy In addition to being used for purification and cloning of the receptor, these bindmg assays can be used to identify agonists and antagonists of CBFBLH12 which compete with the bindmg of CBFBLH12 to its receptors, if any Standard methods for conducting screening assays are well understood in the art Examples of potential CBFBLH12 polypeptide antagonists mclude antibodies or, in some cases, oligonucleotides or proteins which are closely related to the ligands, substrates, receptors, enzymes, etc , as the case may be, of the CBFBLH12 polypeptide, e g , a fragment of the ligands, substrates, receptors, enzymes, etc , or small molecules which bmd to the polypetide 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 screenmg kit for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, etc for CBFBLH12 polypeptides, or compounds which decrease or enhance the production of CBFBLH12 polypeptides, which comprises (a) a CBFBLH12 polypeptide, preferably that of SEQ ID NO 2, (b) a recombmant cell expressmg a CBFBLH12 polypeptide, preferably that of SEQ ID NO 2,

(c) a cell membrane expressmg a CBFBLH12 polypeptide, preferably that of SEQ ID NO 2, or

(d) antibody to a CBFBLH12 polypeptide, preferably that of SEQ ID NO 2

It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component

Prophylactic and Therapeutic Methods

This mvention provides methods of treating abnormal conditions such as, cardiovascular disease, kidney disease, metabolic-associated disorders, and autoimmune diseases, related to both an excess of and insufficient amounts of CBFBLH12 polypeptide activity

If the activity of CBFBLH12 polypeptide is m excess, several approaches are available One approach compnses administering to a subject an inhibitor compound (antagonist) as heremabove descnbed along with a pharmaceutically acceptable earner m an amount effective to inhibit the function of the CBFBLH12 polypeptide, such as. for example, by blocking the binding of ligands, substrates, receptors, enzymes, etc , or by inhibiting a second signal, and thereby alleviating the abnormal condition In another approach, soluble forms of CBFBLH12 polypeptides still capable of binding the hgand, substrate, enzymes, receptors, etc in competition with endogenous CBFBLH12 polypeptide may be admimstered Typical embodiments of such competitors compnse fragments of the CBFBLH12 polypeptide

In still another approach, expression of the gene encoding endogenous CBFBLH12 polypeptide can be inhibited using expression blocking techniques Known such techniques involve the use of antisense sequences, either internally generated or separately administered See, for example, O'Connor, J Neurochem (1991) 56 560 m Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression. CRC Press, Boca Raton, FL (1988) Alternatively, oligonucleotides which form triple helices with the gene can be supplied See, for example, Lee et al , Nucleic Acids Res (1979) 6 3073, Cooney et al , Science (1988) 241 456, Dervan et al , Science (1991) 251 1360 These 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 CBFBLH12 and its activity, several approaches are also available One approach compnses administering to a subject a therapeutically effective amount of a compound which activates CBFBLH 12 polypeptide, 1 e , an agonist as descnbed above, m combination with a pharmaceutically acceptable earner, to thereby alleviate the abnormal condition Alternatively, gene therapy may be employed to effect the endogenous production of CBFBLH 12 by the relevant cells in the subject For example, a polynucleotide of the mvention may be engmeered for expression in 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 plasrmd vector containmg RNA encodmg a polypeptide of the present mvention such that the packaging cell now produces infectious viral particles containmg the gene of mterest These producer cells may be administered to a subject for engineering cells n vivo and expression of the polypeptide in vivo For 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 CBFBLH 12 polypeptides m combination with a suitable pharmaceutical earner

Formulation and Administration

Peptides, such as the soluble form of CBFBLH12 polypeptides, and agonists and antagonist peptides or small molecules, may be formulated m combination with a suitable pharmaceutical earner Such formulations compnse a therapeutically effective amount of the polypeptide or compound, and a pharmaceutically acceptable earner or excipient Such earners mclude but are not limited to. saline, buffered saline, dextrose, water, glycerol, ethanol. and combinations thereof Formulation should suit the mode of administration, and is well within the skill of the art The mvention further relates to pharmaceutical packs and kits compπsmg 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

Prefened forms of systemic administration of the pharmaceutical compositions mclude injection, typically by intravenous injection Other injection routes, such as subcutaneous, mtramuscular, or intrapentoneal, can be used Alternative means for systemic administration mclude transmucosal and transdermal administration usmg penetrants such as bile salts or fusidic acids or other detergents In addition, if properly formulated m enteπc or encapsulated formulations, 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, the route of administration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attendmg practitioner. Suitable dosages, however, are in the range of 0.1-100 μg/kg of subject. Wide vanations m the needed dosage, however, are to be expected m view of the vaπety of compounds available and the differing efficiencies of vaπous routes of administration. For example, oral administration would be expected to require higher dosages than administration by intravenous injection Variations m these dosage levels can be adjusted usmg standard empirical routines for optimization, as is well understood in the art

Polypeptides used m treatment can also be generated endogenously in the subject, in treatment modal-ties 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 plas id vector. The cells are then introduced mto the subject.

All publications, including but not limited to patents and patent applications, cited m this specification are herem 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 LISTING

(1) GENERAL INFORMATION

(i) APPLICANT: SHANGHAI SECOND MEDICAL UNIVERSITY/RUI-JIN HOSPITAL

(ii) TITLE OF THE INVENTION: CBFBLH12 : A Gene Highly Related to Bovine CI-KFYI Gene for Ubiguinone Oxireductase Complex

(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-70352

(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: 420 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

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

GACGCAAGAT GGCGCCGTCC GCCTTGCTGC GTCCCCTTTC CCGGCTGCTG GCCCCCGCCA 60

GGCTCCCGAG CGGCCCTTCA GTGCGATCAA AGTTCTACGT GCGAGAGCCG CCGAATGCCA 120

AACCTGACTG GCTGAAAGTT GGGTTCACCT TGGGCACCAC TGTCTTCTTG TGGATCTATC 180

TCATCAAACA ACACAATGAA GATATTTTAG AGTACAAAAG AAGAAATGGG CTGGAATAAA 240

CTTTTGAAAC ACTAATGTAG TATGCTCCGT ATAGTGATTG TAGCTGTTCC TCTGGATTCA 300

CCATCTGTTG AGTTGTAAAT GTGAGAGAAA AAGTTATATG TGAATATATA TCAAGCCAGC 360

ATTTGTATTT TGCATCATTA AATAAAAAGA AATAAAAAAA AAAAAAAAAA AAAAAAAAAA 420

(2) INFORMATION FOR SEQ ID NO: 2

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 76 ammo acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

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

Met Ala Pro Ser Ala Leu Leu Arg Pro Leu Ser Arg Leu Leu Ala Pro 1 5 10 15 Ala Arg Leu Pro Ser Gly Pro Ser Val Arg Ser Lys Phe Tyr Val Arg 20 25 30

Glu Pro Pro Asn Ala Lys Pro Asp Trp Leu Lys Val Gly Phe Thr Leu 35 40 45 Gly Thr Thr Val Phe Leu Trp lie Tyr Leu lie Lys Gin His Asn Glu

50 55 60

Asp lie Leu Glu Tyr Lys Arg Arg Asn Gly Leu Glu

65 70 75

Claims

What is claimed is:

1 An isolated polynucleotide compπsmg a nucleotide sequence that has at least 81% identity over its entire length to a nucleotide sequence encodmg the CBFBLH12 polypeptide of SEQ ID NO 2, or a nucleotide sequence complementary to said isolated polynucleotide

2 The polynucleotide of claim 1 wherein said polynucleotide comprises the nucleotide sequence contained m SEQ ID NO 1 encodmg the CBFBLH12 polypeptide of SEQ ID N02

3 The polynucleotide of claim 1 wherem said polynucleotide comprises a nucleotide sequence that is at least 81% identical to that of SEQ ID NO 1 over its entire length

4 The polynucleotide of claim 3 which is polynucleotide of SEQ ID NO 1

5 The polynucleotide of claim 1 which is DNA or RNA

6 A DNA or RNA molecule compnsmg an expression system, wherein said expression system is capable of producmg a CBFBLH12 polypeptide compnsmg an ammo acid sequence, which has at least 80% identity with the polypeptide of SEQ ID NO 2 when said expression system is present in a compatible host cell

7 A host cell compnsmg the expression system of claim 6

8 A process for producing a CBFBLH12 polypeptide compnsing cultunng a host of claim 7 under conditions sufficient for the production of said polypeptide and recoveπng the polypeptide from the culture

9 A process for producmg a cell which produces a CBFBLH12 polypeptide thereof comprising transformmg or transfecting a host cell with the expression system of claim 6 such that the host cell, under appropriate culture conditions, produces a CBFBLH 12 polypeptide

10 A CBFBLH12 polypeptide compnsmg an ammo acid sequence which is at least 80% identical to the ammo acid sequence of SEQ ID NO 2 over its entire length

11 The polypeptide of claim 10 which compnses the ammo acid sequence of SEQ ID NO 2

12 An antibody lmmunospecific for the CBFBLH 12 polypeptide of claim 10

13 A method for the treatment of a subject in need of enhanced activity or expression of CBFBLH12 polypeptide of claim 10 compπsmg

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

(b) providing to the subject an isolated polynucleotide compπsmg a nucleotide sequence that has at least 81% identity to a nucleotide sequence encodmg the CBFBLH12 polypeptide of SEQ ID NO 2 over its entire length, or a nucleotide sequence complementary to said nucleotide sequence m a form so as to effect production of said polypeptide activity in vivo

14 A method for the treatment of a subject havmg need to inhibit activity or expression of CBFBLH12 polypeptide of claim 10 compπsmg. (a) administenng to the subject a therapeutically effective amount of an antagonist to said polypeptide, and/or

(b) administenng to the subject a nucleic acid molecule that inhibits the expression of the nucleotide sequence encodmg said polypeptide, and/or

(c) administering to the subject a therapeutically effective amount of a polypeptide that competes with said polypeptide for its hgand, substrate , or receptor

15 A process for diagnosing a disease or a susceptibility to a disease m a subject related to expression or activity of CBFBLH12 polypeptide of claim 10 in a subject compnsing

(a) determining the presence or absence of a mutation m the nucleotide sequence encodmg said CBFBLH12 polypeptide m the genome of said subject, and or

(b) analyzing for the presence or amount of the CBFBLH 12 polypeptide expression m a sample derived from said subject

16. A method for identifying compounds which inhibit (antagonize) or agonize the CBFBLH12 polypeptide of claim 10 which comprises:

(a) contacting a candidate compound with cells which express the CBFBLH12 polypeptide (or cell membrane expressing CBFBLH12 polypeptide) or respond to CBFBLH12 polypeptide; and

(b) observing the binding, or stimulation or inhibition of a functional response; or comparing the ability of the cells (or cell membrane) which were contacted with the candidate compounds with the same cells which were not contacted for CBFBLH12 polypeptide activity.

17. An agonist identified by the method of claim 16.

18. An antagonist identified by the method of claim 16.

19. A recombinant host cell produced by a method of Claim 9 or a membrane thereof expressing a CBFBLH12 polypeptide.