WO1999022007A1 - Cbfaie10: a human skd1 gene - Google Patents

Abstract

CBFAIE10 polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing CBFAIE10 polypeptides and polynucleotides in the design of protocols for the treatment of cancer or AIDS, among others, and diagnostic assays for such conditions.

Description

CBFAIEIO: A HUMAN SKDl GENE

FIELD OF INVENTION

This invention 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 NSF/CDC48/PASLP/TBP-1 family, hereinafter referred to as CBFAIEIO. The invention also relates to inhibiting or activating the action of such polynucleotides and polypeptides.

BACKGROUND OF THE INVENTION

The murine SKD 1 (suppressor of K+ transport growth defect) gene is a member of the ATPase family. It contains a single ATP binding site and can suppress the growth deficiency of a yeast potassium transport mutant. This indicates that the NSF/CDC48/PASLP/TBP-1 family has an established, proven history as therapeutic targets. Clearly there is a need for identification and characterization of further members of the NSF/CDC48/PASLP/TBP-1 family which can play a role in preventing, ameliorating or correcting dysfunctions or diseases, including, but not limited to, cancer or AIDS.

SUMMARY OF THE INVENTION In one aspect, the invention relates to CBFAIEIO polypeptides and recombinant materials and methods for their production. Another aspect of the invention relates to methods for using such CBFAIEIO polypeptides and polynucleotides. Such uses include the treatment of cancer or AIDS, among others. In still another aspect, the invention relates to methods to identify agonists and antagonists using the materials provided by the invention, and treating conditions associated with CBFAIEIO imbalance with the identified compounds. Yet another aspect of the invention relates to diagnostic assays for detecting diseases associated with inappropriate CBFAIEIO activity or levels.

DESCRIPTION OF THE INVENTION Definitions The following definitions are provided to facilitate understanding of certain terms used frequently herein.

"CBFAIEIO" refers, among others, generally to a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2 or an allelic variant thereof. "CBFAIEIO activity or CBFAIEIO polypeptide activity" or "biological activity of the CBFAIEIO or CBFAIEIO polypeptide" refers to the metabolic or physiologic function of said CBFAIEIO including similar activities or improved activities or these activities with decreased undesirable side-effects Also included are antigenic and lmmunogenic activities of said CBFAIEIO

"CBFAIEIO gene" refers to a polynucleotide having the nucleotide sequence set forth m SEQ ID NO 1 or alle c variants thereof and/or their complements

"Antibodies" as used herem includes polyclonal and monoclonal antibodies, chimeπc, single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other lmmunoglobuhn 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 mateπals of its natural state is "isolated", as the term is employed herein

"Polynucleotide" generally refers to any polynbonucleotide or polydeoxnbonucleot.de, which may be unmodified RNA or DNA or modified RNA or DNA "Polynucleotides" include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double- stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybπd molecules comprising 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 tπple-stranded regions compnsing RNA or DNA or both RNA and DNA The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons "Modified" bases include, for example, tπtylated bases and unusual bases such as inosine A variety of modifications has been made to DNA and RNA, thus, "polynucleotide" embraces chemically, enzymatically or metabohcally modified forms of polynucleotides as typically found m 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 compπsmg 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, ohgopeptides or o gomers, 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 in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present 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 ubiquitination, 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-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma- carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. 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, 1 83; Seifter et al, "Analysis for protein modifications and nonprotein cofactors", Meth Enzymol (1990) 182:626-646 and Rattan et al, "Protein Synthesis: Posttranslational Modifications and Aging", Ann NY AcadSci (1992) 663:48-62. "Variant" 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 variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis.

"Identity" is a measure of the identity of nucleotide sequences or amino 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, Gribskov, 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 (Carillo, H., and Lipton, D., SIAM J Applied Math (1988) 48: 1073). Methods commonly employed to determine identity or similarity 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 Carillo, H., and Lipton, D., SIAM J Applied Math (1988) 48: 1073. Methods to determine identity and similarity are codified in computer programs. Preferred computer program methods to determine identity and similarity 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 having 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 obtain 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 in 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 in one or more contiguous groups within the reference sequence.

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

Polypeptides of the Invention

In one aspect, the present invention relates to CBFAIEIO polypeptides (or CBFAIEIO proteins). The CBFAIEIO polypeptides include the polypeptide of SEQ ID NO:2; as well as polypeptides comprising the amino acid sequence of SEQ ID NO: 2; and polypeptides comprising the amino 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 included within CBFAIEIO polypeptides are polypeptides having the amino acid sequence which have at least 80% identity to the polypeptide having the amino 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 CBFAIEIO polypeptide exhibit at least one biological activity of CBFAIEIO.

The CBFAIEIO polypeptides may be in the form of the "mature" protein or may be a part of a larger protein such as a fusion protein. It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification such as multiple histidine residues, or an additional sequence for stability during recombinant production.

Fragments of the CBFAIEIO polypeptides are also included in the invention. A fragment is a polypeptide having an amino acid sequence that entirely is the same as part, but not all, of the amino acid sequence of the aforementioned CBFAIEIO polypeptides As with CBFAIEIO polypeptides, fragments may be "free-standing," or compnsed within a larger polypeptide of which they form a part or region, most preferably as a single continuous region Representative examples of polypeptide fragments of the invention, include, for example, fragments from about amino acid number 1-20, 21-40, 41-60, 61-80, 81 - 100, and 101 to the end of CBFADΞ 10 polypeptide In this context "about" includes the particularly recited ranges larger or smaller by several, 5, 4, 3, 2 or 1 amino acid at either extreme or at both extremes

Preferred fragments include, for example, truncation polypeptides having the ammo acid sequence of CBFAIEIO polypeptides, except for deletion of a contmuous seπes of residues that mcludes the ammo terminus, or a contmuous seπes of residues that mcludes the carboxyl termmus or deletion of two contmuous seπes of residues, one including the ammo termmus and one including the carboxyl terminus Also preferred are fragments characteπzed by structural or functional attributes such as fragments that compπse alpha-he x 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 anϋgenic mdex regions Other preferred fragments are biologically active fragments Biologically active fragments are those that mediate CBFAIEIO activity, including those with a similar activity or an improved activity, or with a decreased undesirable activity Also mcluded are those that are anϋgemc or immunogenic in an animal, especially in a human Preferably, all of these polypeptide fragments retain the biological activity of the CBFAIEIO, including antigemc activity Vaπants of the defined sequence and fragments also form part of the present invention Preferred vaπants are those that vary from the referents by conservative ammo acid substitutions - 1 e , those that substitute a residue with another of like characteπstics Typical such substitutions are among Ala, Val, Leu and Ue, among Ser and Thr, among the acidic residues Asp and Glu, among Asn and Gin, and among the basic residues Lys and Arg, or aromatic residues Phe and Tyr Particularly preferred are vaπants m which several, 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination

The CBFAIE 10 polypeptides of the 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 m the art Polynucleotides of the Invention

Another aspect of the invention relates to CBFAIEIO polynucleotides. CBFAIEIO polynucleotides include isolated polynucleotides which encode the CBFAIEIO polypeptides and fragments, and polynucleotides closely related thereto. More specifically, CBFAIEIO polynucleotide of the invention include a polynucleotide comprising the nucleotide sequence contained in SEQ ID NO: 1 encoding a CBFAIEIO polypeptide of SEQ ID NO: 2, and polynucleotide having the particular sequence of SEQ ID NO: 1. CBFAIEIO polynucleotides further include a polynucleotide comprising a nucleotide sequence that has at least 80% identity over its entire length to a nucleotide sequence encoding the CBFAIEIO polypeptide of SEQ ID NO:2, and a polynucleotide comprising 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 preferred, and those with at least 95% are especially preferred. Furthermore, those with at least 97% are highly preferred and those with at least 98-99% are most highly preferred, with at least 99% being the most preferred. Also included under CBFAIEIO polynucleotides are a nucleotide sequence which has sufficient identity to a nucleotide sequence contained in SEQ ID NO:l to hybridize under conditions useable for amplification or for use as a probe or marker. The invention also provides polynucleotides which are complementary to such CBFAIEIO polynucleotides.

CBFAIEIO of the invention is structurally related to other proteins of the NSF/CDC48/PASLP/TBP-1 family, as shown by the results of sequencing the cDNA of Table 1 (SEQ ID NO: 1) encoding human CBFAIEIO. The cDNA sequence of SEQ ED NO: 1 contains an open reading frame (nucleotide number 190 to 1521) encoding a polypeptide of 444 amino acids of SEQ ED NO:2. The amino acid sequence of Table 2 (SEQ ED NO:2) has about 94% identity (using FASTA) in 444 amino acid residues with murine SKD 1 (F. Perier,et al., FEBS Letters, 351 :286-290, 1994). The nucleotide sequence of Table 1 (SEQ ED NO: 1) has about 67% identity (using FASTA) in 1978 nucleotide residues with murine SKD 1 (F. Perier,et al., FEBS Letters, 351286-290, 1994). Thus, CBFAIEIO polypeptides and polynucleotides of the present invention are expected to have, inter alia, similar biological functions/properties to their homologous polypeptides and polynucleotides, and their utility is obvious to anyone skilled in the art.

Table 1"

GGGGGAGGGTCGGAGCTCTGGTGGAGAGAGTGTTGTCTAAAACAAGTTCCGGAAGGGAGG CTGCCCTTCGCGGTCCGAGAACCACCGGCCTCCCCAGTTTGAGGGCTGTTACCCCGTGCG CGCTTCGACGTTGCTGCTGTTGGCTCTCCTCGCCCCTCGTTCCCTTGGGAACCGCCTGGG AACTCCGCCATGTCATCCACTTCGCCCAACCTCCAGAAAGCGATAGATCTGGCTAGCAAA GCAGCGCAAGAAGACAAGGCTGGGAACTACGAAGAAGCCCTTCAGCTCTATCAGCATGCT GTGCAGTATTTTCTTCATGTCGTTAAATATGAAGCACAAGGTGATAAAGCCAAGCAAAGT ATCAGGGCAAAGTGTACAGAATATCTTGATAGAGCAGAAAAACTAAAGGAGTACCTGAAA AATAAAGAGAAAAAAGCACAGAAGCCAGTGAAAGAAGGACAGCCGAGTCCAGCAGATGAG AAGGGGAATGACAGTGATGGGGAAGGAGAATCTGATGATCCTGAAAAAAGGAAACTACAG AATCAACTTCAAGGTGCCATTGTTATAGACCGACCAAATGTGAAATGGAGTGACGTTGCT GGACTTGAAGGAGCCAAAGAAGCACTGAAAGAGGCTGTGATACTGCCTATTAAATTTCCT CATCTTTTTACAGGCAAGAGAACACCTTGGAGGGGAATCCTATTATTTGGGCCGCCTGGA ACAGGAAAGTCCTACTTAGCCAAAGCTGTAGCAACAGAAGCCAACAACTCAACATTTTTT TCAATATCTTCCTCTGATCTTGTTTCTAAGTGGCTAGGTGAAAGTGAAAAACTGGTTAAG AATTTATTCCAACTTGCCAGAGAGAACAAGCCCTCCATTATCTTCATTGATGAAATTGAT TCTCTCTGTGGTTCAAGAAGTGAAAATGAAAGTGAAGCCGCACGTAGAATTAAGACGGAG TTCCTAGTGCAAATGCAAGGGGTTGGTGTAGACAATGATGGAATTTTGGTTCTGGGAGCT ACAAATATACCCTGGGTTCTGGATTCTGCCATTAGGCGAAGATTTGAGAAACGAATTTAT ATTCCCTTGCCGGAACCCCATGCCCGAGCAGCAATGTTTAAACTGCACCTAGGGACCACT CAGAACAGTCTCACGGAAGCAGACTTTCGGGAACTTGGGAGGAAAACAGATGGTTATTCA GGGGCAGATATAGGTATCATTGTACGTGATGCCCTTATGCAGCCTGTTAGGAAAGTACAG TCAGCTACTCATTTTAAAAAGGTTCGCGGACCTTCCCGAGCTGATCCTAACCATCTTGTA GATGATCTGCTAACACCTTGCTCTCCAGGTGACCCTGGTGCCATTGAAATGACGTGGATG GATGTCCCTGGAGATAAACTTTTGGAGCCAGTTGTTTCCATGTCGGATATGTTGCGGTCA CTATCTAACACAAAACCTACAGTCAATGAACATGACTTGTTGAAATTAAAGAAGTTTACA GAAGATTTTGGTCAAGAAGGCTAAGCCAAAGACAAGGAAGATGCTTACCATATGTATTCT TTCTTTCATAGATATTTTTGTCTATTTGGATCGCATTAATTGTTTCCAGTAAAACTCTTT TACCACAGGGAAATACACATCTCACTTCAGAGTTCCATTAGGTTTTATATTGTACTTTTC CTCCATTACTTATTAAATACTCCTATTAACAAAAGGTACAAAATAACAGGTTATGAGGAA ATGAGCGATATATGAACGGCATAAAAACAGAAATTACCCAGTAAAAAGGATGTCAGAAAT TGACATACAAATATTTACAATTTTTATGAATGGTGGTCTTTGCAAAGAGCATTTATATTT TCTTTTTTTTTTACTAAAATGATATATGGGTTTATTTTATATTTTCAAAAAAATTGTTAA AC^TCATTCTTATCAATGTAAAATTTACGAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

A nucleotide sequence of a human CBFAEE 10 (SEQ ID NO: 1).

Table 2^b

MSSTSPNLQKAID--ASKAAQEDKAGNYEEA QLYQHAVQYFLHVV YΞAQGDKAKQSIRA KCTEYiDRAEK KEYLKMKEKKAQKPVKEGQPSPADEKG DSDGEGESDDPEKRKLQNQL QGAIVIDRPNV WSDVAGLEGAKEALKΞAVILPI FPHLFTG RTPWRGILLFGPPGTGK SY AKAVATEAi STFFSISSSDLVSK LGESEKLVKNLFQI-ARENKPSIIFIDEIDSLC GSRSENESEAARRIKTEFLVQMQGVGVD DGILVLGATNIPWVLDSAIRRRFEKRIYIPL PEPHARAAMFKLHLGTTQNSLTEADFRE GRKTDGYSGADIGIIVRDALMQPVR VQSAT HFKKVRGPSRADPNHLVDD LTPCSPGDPGAIEMT MDVPGDKL EPVVSMSDM RSLSN T PTVNEHDLLKLKKFTEDFGQEG

An amino acid sequence ofa human CBFAIEIO (SEQ ID NO: 2). One polynucleotide of the present invention encoding CBFAIEIO may be obtained using standard cloning and screening, from a cDNA library derived from mRNA in cells of human 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 ai, 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. The nucleotide sequence encoding CBFAIEIO polypeptide of SEQ ID NO:2 may be identical to the polypeptide encoding sequence contained in Table 1 (nucleotide number 190 to 1521 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 CBFAIE 10 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 in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence, or other fusion peptide portions. For example, a marker sequence which facilitates purification of the fused polypeptide can be encoded. In certain preferred embodiments of this aspect of the invention, the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz et al, Proc NatlAcadSci USA (1989) 86:821- 824, or is an HA tag. The polynucleotide may also contain non-coding 5' and 3' sequences, such as transcribed, non-translated sequences, splicing and polyadenylation signals, ribosome binding sites and sequences that stabilize mRNA. Further preferred embodiments are polynucleotides encoding CBFAIE 10 variants comprise the amino acid sequence CBFAEEIO polypeptide of Table 2 (SEQ ED NO:2) in which several, 5-10, 1-5, 1-3, 1-2 or 1 amino acid residues are substituted, deleted or added, in any combination.

The present invention further relates to polynucleotides that hybridize to the herein above- described sequences. In this regard, the present invention especially relates to polynucleotides which hybridize under stringent conditions to the herein above-described polynucleotides. As herein used, the term "stringent conditions" means hybridization 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 invention, which are identical or sufficiently identical to a nucleotide sequence contained in SEQ ID NO: 1 or a fragment thereof, may be used as hybridization probes for cDNA and genomic DNA, to isolate full-length cDNAs and genomic clones encoding CBFAIEIO polypeptide and to isolate cDNA and genomic clones of other genes (including genes encoding homologs and orthologs from species other than human) that have a high sequence similarity to the CBFAIE 10 gene. Such hybridization techniques are known to those of skill in 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 comprise at least 15 nucleotides. Preferably, such probes will have at least 30 nucleotides and may have at least 50 nucleotides. Particularly preferred probes will range between 30 and 50 nucleotides .

In one embodiment, to obtain a polynucleotide encoding CBFAIE 10 polypeptide, including homologs and orthologs from species other than human, comprises the steps of screening an appropriate library under stingent hybridization conditions with a labeled probe having the SEQ ID NO: 1 or a fragment thereof; and isolating full-length cDNA and genomic clones containing said polynucleotide sequence. Thus in another aspect, CBFAIE 10 polynucleotides of the present invention further include a nucleotide sequence comprising a nucleotide sequence that hybridize under stringent condition to a nucleotide sequence having SEQ ED NO: 1 or a fragment thereof. Also included with CBFAIE 10 polypeptides are polypeptide comprising amino acid sequence encoded by nucleotide sequence obtained by the above hybridization condition. Such hybridization techniques are well known to those of skill in the art. Stringent hybridization conditions are as defined above or, alternatively, conditions under overnight incubation at 42°C in a solution comprising: 50% formamide, 5xSSC (150mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10 % dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0. lx SSC at about 65°C. The polynucleotides and polypeptides of the present invention may be employed as research reagents and materials for discovery of treatments and diagnostics to animal and human disease.

Vectors, Host Cells, Expression

The present invention also relates to vectors which comprise a polynucleotide or polynucleotides of the present invention, and host cells which are genetically engineered with vectors of the invention and to the production of polypeptides of the invention by recombinant techniques. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. For recombinant production, host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention Introduction of polynucleotides into host cells can be effected by methods descπbed in many standard laboratory manuals, such as Davis et al, BASIC METHODS IN MOLECULAR BIOLOGY (1986) and Sambrook et al , MOLECULAR CLONING A LABORATORY MANUAL, 2nd Ed , Cold Spring Harbor Laboratory Press, Cold Spπng Harbor, N Y (1989) such as calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, rrucroinjection, cationic pid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection

Representative examples of appropπate hosts include bactenal cells, such as streptococci, staphylococci, E coh, Streptomyces and Bacillus subtihs 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-deπved systems, e g , vectors deπved 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 retroviruses, and vectors deπved from combinations thereof, such as those deπved from plasmid and bacteπophage 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 m 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 protein mto the lumen of the endoplasmic reticulum, mto the penplasmic space or mto the extracellular environment, appropnate 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 CBFAIEIO 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 pπor to use in the screenmg assay If CBFAIE 10 polypeptide is secreted mto the medium, the medium can be recovered m order to recover and punfy the polypeptide, if produced mtracellularly, the cells must first be lysed before the polypeptide is recovered CBFAIE 10 polypeptides can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification.

Diagnostic Assays This invention also relates to the use of CBFAIE 10 polynucleotides for use as diagnostic reagents. Detection of a mutated form of CBFAIE 10 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 CBFAIEIO. Individuals carrying mutations in the CBFAIEIO gene may be detected at the DNA level by a variety of techniques. Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy material. The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR or other amplification techniques prior to analysis. RNA or cDNA may also be used in similar fashion. Deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to labeled CBFAEE 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 in electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing. 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 Sci USA ( 1985) 85 : 4397-4401. In another embodiment, an array of oligonucleotides probes comprising CBFAIEIO nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e.g., genetic mutations. Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability. (See for example: M.Chee et al., Science, Vol 274, pp 610-613 (1996)).

The diagnostic assays offer a process for diagnosing or determining a susceptibility to cancer or AEDS through detection of mutation in the CBFAIE 10 gene by the methods described. In addition, cancer or AIDS, can be diagnosed by methods compπsing determining from a sample denved from a subject an abnormally decreased or increased level of CBFAIE 10 polypeptide or CBFAIE 10 mRNA Decreased or increased expression can be measured at the RNA level usmg any of the methods well known m the art for the quantitation of polynucleotides, such as, for example, PCR, RT-PCR, RNase protection, Northern blotting and other hybndization methods Assay techniques that can be used to determine levels of a protein, such as an CBFAIE 10 polypeptide, in a sample deπved from a host are well-known to those of skill in the art Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays Thus in another aspect, the present mvention relates to a diagonostic kit for a disease or suspectability to a disease, particularly cancer or A DS, which compπses

(a) a CBFAIEIO 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 CBFAIE 10 polypeptide, preferably the polypeptide of SEQ ID NO 2, or a fragment thereof, or

(d) an antibody to a CBFAIEIO polypeptide, preferably to the polypeptide of SEQ ID NO 2 It will be appreciated that in 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 hybπdize 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 conelating 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 Inheπtance 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 (coinheπtance of physically adjacent genes)

The differences in the cDNA or genomic sequence between affected and unaffected individuals can also be determined If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease.

Antibodies The polypeptides of the invention or their fragments or analogs thereof, or cells expressing them can also be used as immunogens to produce antibodies immunospecific for the CBFAIEIO polypeptides. The term "immunospecific" means that the antibodies have substantiall greater affinity for the polypeptides of the invention than their affinity for other related polypeptides in the prior art.

Antibodies generated against the CBFAIE 10 polypeptides can be obtained by administering the polypeptides or epitope-bearing fragments, analogs or cells to an animal, preferably a nonhuman, using routine protocols. For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler, G. and Mi-stein, C, Nature (1975) 256:495*497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al. , Immunology Today (1983) 4:72) and the EBV-hybridoma technique (Cole et al. , MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp. 77-96, Alan R. Liss, Inc., 1985).

Techniques for the production of single chain antibodies (U.S. Patent No. 4,946,778) can also be adapted to produce single chain antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms including other mammals, may be used to express humanized antibodies. The above-described antibodies may be employed to isolate or to identify clones expressing the polypeptide or to purify the polypeptides by affinity chromatography.

Antibodies against CBFAEEIO polypeptides may also be employed to treat cancer or AEDS, among others.

Vaccines

Another aspect of the invention relates to a method for inducing an immunological response in a mammal which comprises inoculating the mammal with CBFAEEIO polypeptide, or a fragment thereof, adequate to produce antibody and/or T cell immune response to protect said animal from cancer or AEDS, among others. Yet another aspect of the invention relates to a method of inducing immunological response in a mammal which comprises, delivering CBFAEEIO polypeptide via a vector directing expression of CBFAIEIO polynucleotide in vivo in order to induce such an immunological response to produce antibody to protect said animal from diseases.

Further aspect of the invention relates to an immunological/vaccine formulation (composition) which, when introduced into a mammalian host, induces an immunological response in that mammal to a CBFAIEIO polypeptide wherein the composition comprises a CBFAEEIO polypeptide or CBFAEEIO gene. The vaccine formulation may further comprise a suitable carrier. Since CBFAEEIO polypeptide may be broken down in the stomach, it is preferably administered parenterally (including subcutaneous, intramuscular, intravenous, intradermal etc. injection). Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation instonic with the blood of the recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use. The vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.

Screening Assays

The CBFAEEIO polypeptide of the present invention may be employed in a screening process for compounds which activate (agonists) or inhibit activation of (antagonists, or otherwise called inhibitors) the CBFAEEIO polypeptide of the present invention. Thus, polypeptides of the invention may also be used to assess identify agonist or antagonists from, for example, cells, cell-free preparations, chemical libraries, 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 invention; or may be structural or functional mimetics of the polypeptide of the present invention. See Coligan etal, Current Protocols in Immunology l(2):Chapter 5 (1991). CBFAIEIO polypeptides are responsible for many biological functions, including many pathologies.

Accordingly, it is desirous to find compounds and drugs which stimulate CBFAEEIO polypeptide on the one hand and which can inhibit the function of CBFAIE 10 polypeptide on the other hand. In general, agonists are employed for therapeutic and prophylactic purposes for such conditions as cancer or AEDS. Antagonists may be employed for a variety of therapeutic and prophylactic purposes for such conditions as cancer or AEDS.

In general, such screening procedures may involve using appropriate cells which express the CBFAIEIO polypeptide or respond to CBFAEEIO polypeptide of the present invention. Such cells include cells from mammals, yeast, Drosophila or E. coli. Cells which express the CBFAIEIO polypeptide (or cell membrane cont--ining the expressed polypeptide) or respond to CBFAEEIO 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 CBFAIEIO activity.

The assays may simply test binding of a candidate compound wherein adherence to the cells bearing the CBFAIEIO 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 in a signal generated by activation of the CBFAEEIO polypeptide, using detection systems appropriate to the cells bearing the CBFAIEIO 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.

Further, the assays may simply comprise the steps of mixing a candidate compound with a solution containing a CBFAEEIO polypeptide to form a mixture, measuring CBFAIEIO activity in the mixture, and comparing the CBFAIEIO activity of the mixture to a standard.

The CBFAEEIO 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 CBFAEEIO mRNA and protein in cells. For example, an ELISA may be constructed for measuring secreted or cell associated levels of CBFAEEIO protein using monoclonal and polyclonal antibodies by standard methods known in the art, and this can be used to discover agents which may inhibit or enhance the production of CBFAEEIO (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues.

The CBFAEEIO protein may be used to identify membrane bound or soluble receptors, if any, through standard receptor binding techniques known in the art. These include, but are not limited to, ligand binding and crosslinking assays in which the CBFAEEIO 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 include biophysical techniques such as surface plasmon resonance and spectroscopy. In addition to being used for purification and cloning of the receptor, these binding assays can be used to identify agonists and antagonists of CBFAEEIO which compete with the binding of CBFAIEIO to its receptors, if any. Standard methods for conducting screening assays are well understood in the art. Examples of potential CBFAEEIO polypeptide antagonists include 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 CBFAIEIO polypeptide, e.g., a fragment of the ligands, substrates, receptors, enzymes, etc.; or small molecules which bind to the polypetide of the present invention but do not elicit a response, so that the activity of the polypeptide is prevented.

Thus in another aspect, the present invention relates to a screening kit for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, etc. for CBFAIEIO polypeptides; or compounds which decrease or enhance the production of CBFAEEIO polypeptides, which comprises:

(a) a CBFAEEIO polypeptide, preferably that of SEQ ID NO:2;

(b) a recombinant cell expressing a CBFAIEIO polypeptide, preferably that of SEQ ID NO:2; (c) a cell membrane expressing a CBFAIEIO polypeptide; preferably that of SEQ ID NO: 2; or

(d) antibody to a CBFAEEIO 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 invention provides methods of treating abnormal conditions such ascancer or AIDS, related to both an excess of and insufficient amounts of CBFAEEIO polypeptide activity.

If the activity of CBFAEEIO polypeptide is in excess, several approaches are available. One approach comprises administering to a subject an inhibitor compound (antagonist) as hereinabove described along with a pharmaceutically acceptable carrier in an amount effective to inhibit the function of the CBFAEEIO 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 CBFAEEIO polypeptides still capable of binding the ligand, substrate, enzymes, receptors, etc. in competition with endogenous CBFAEEIO polypeptide may be administered. Typical embodiments of such competitors comprise fragments of the CBFAIEIO polypeptide.

In still another approach, expression of the gene encoding endogenous CBFAEEIO 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 in 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 oligomers can be administered per se or the relevant oligomers can be expressed in vivo. For treating abnormal conditions related to an under-expression of CBFAIEIO and its activity, several approaches are also available One approach compπses administering to a subject a therapeutically effective amount of a compound which activates CBFAEEIO polypeptide, 1 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 CBFAIEIO by the relevant cells m the subject For example, a polynucleotide of the mvention may be engineered for expression in a replication defective retrovrral vector, as discussed above The retroviral expression construct may then be isolated and introduced mto a packaging cell transduced with a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention such that the packaging cell now produces infectious viral particles containing the gene of interest These producer cells may be administered to a subject for engineering cells in vivo and expression of the polypeptide in vivo For 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 CBFAIE 10 polypeptides in combination with a suitable pharmaceutical earner

Formulation and Administration

Peptides, such as the soluble form of CBFAEEIO polypeptides, and agonists and antagonist peptides or small molecules, may be formulated m combination with a suitable pharmaceutical earner Such formulations compπse 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 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

Prefened forms of systemic administration of the pharmaceutical compositions mclude in_jection, typically by intravenous injection Other injection routes, such as subcutaneous, intramuscular, or mtrapeπtoneal, can be used Alternative means for systemic administration mclude transmucosal and transdermal administration using penetrants such as bile salts or fusidic acids or other detergents In addition, if properly formulated m enteπc or encapsulated formulations, oral admimstration may also be possible Administration of these compounds may also be topical and/or localized, m 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 attending practitioner. Suitable dosages, however, are in the range of 0.1-100 μgkg of subject. Wide variations in the needed dosage, however, are to be expected in view of the variety of compounds available and the differing efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by intravenous injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization, as is well understood in the art.

Polypeptides used in treatment can also be generated endogenously in the subject, in treatment modalities often refened to as "gene therapy" as described above. Thus, for example, cells from a subject may be engineered with a polynucleotide, such as a DNA or RNA, to encode a polypeptide ex vivo, and for example, by the use of a retroviral plasmid vector. The cells are then introduced into the subject

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 LISTING

(1) GENERAL INFORMATION

(i) APPLICANT: MAO, MAO

(ii) TITLE OF THE INVENTION: CBFAIEIO: A HUMAN SKD1 GENE

(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-70310

(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: 1978 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

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

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

GGGGGAGGGT CGGAGCTCTG GTGGAGAGAG TGTTGTCTAA AACAAGTTCC GGAAGGGAGG 60

CTGCCCTTCG CGGTCCGAGA ACCACCGGCC TCCCCAGTTT GAGGGCTGTT ACCCCGTGCG 120

CGCTTCGACG TTGCTGCTGT TGGCTCTCCT CGCCCCTCGT TCCCTTGGGA ACCGCCTGGG 180 AACTCCGCCA TGTCATCCAC TTCGCCCAAC CTCCAGAAAG CGATAGATCT GGCTAGCAAA 240

GCAGCGCAAG AAGACAAGGC TGGGAACTAC GAAGAAGCCC TTCAGCTCTA TCAGCATGCT 300

GTGCAGTATT TTCTTCATGT CGTTAAATAT GAAGCACAAG GTGATAAAGC CAAGCAAAGT 360

ATCAGGGCAA AGTGTACAGA ATATCTTGAT AGAGCAGAAA AACTAAAGGA GTACCTGAAA 420

AATAAAGAGA AAAAAGCACA GAAGCCAGTG AAAGAAGGAC AGCCGAGTCC AGCAGATGAG 480 AAGGGGAATG ACAGTGATGG GGAAGGAGAA TCTGATGATC CTGAAAAAAG GAAACTACAG 540

AATCAACTTC AAGGTGCCAT TGTTATAGAC CGACCAAATG TGAAATGGAG TGACGTTGCT 600

GGACTTGAAG GAGCCAAAGA AGCACTGAAA GAGGCTGTGA TACTGCCTAT TAAATTTCCT 660

CATCTTTTTA CAGGCAAGAG AACACCTTGG AGGGGAATCC TATTATTTGG GCCGCCTGGA 720

ACAGGAAAGT CCTACTTAGC CAAAGCTGTA GCAACAGAAG CCAACAACTC AACATTTTTT 780 TCAATATCTT CCTCTGATCT TGTTTCTAAG TGGCTAGGTG AAAGTGAAAA ACTGGTTAAG 840

AATTTATTCC AACTTGCCAG AGAGAACAAG CCCTCCATTA TCTTCATTGA TGAAATTGAT 900

TCTCTCTGTG GTTCAAGAAG TGAAAATGAA AGTGAAGCCG CACGTAGAAT TAAGACGGAG 960

TTCCTAGTGC AAATGCAAGG GGTTGGTGTA GACAATGATG GAATTTTGGT TCTGGGAGCT 1020

ACAAATATAC CCTGGGTTCT GGATTCTGCC ATTAGGCGAA GATTTGAGAA ACGAATTTAT 1080 ATTCCCTTGC CGGAACCCCA TGCCCGAGCA GCAATGTTTA AACTGCACCT AGGGACCACT 1140

CAGAACAGTC TCACGGAAGC AGACTTTCGG GAACTTGGGA GGAAAACAGA TGGTTATTCA 1200

GGGGCAGATA TAGGTATCAT TGTACGTGAT GCCCTTATGC AGCCTGTTAG GAAAGTACAG 1260

TCAGCTACTC ATTTTAAAAA GGTTCGCGGA CCTTCCCGAG CTGATCCTAA CCATCTTGTA 1320

GATGATCTGC TAACACCTTG CTCTCCAGGT GACCCTGGTG CCATTGAAAT GACGTGGATG 1380 GATGTCCCTG GAGATAAACT TTTGGAGCCA GTTGTTTCCA TGTCGGATAT GTTGCGGTCA 1440

CTATCTAACA CAAAACCTAC AGTCAATGAA CATGACTTGT TGAAATTAAA GAAGTTTACA 1500

GAAGATTTTG GTCAAGAAGG CTAAGCCAAA GACAAGGAAG ATGCTTACCA TATGTATTCT 1560

TTCTTTCATA GATATTTTTG TCTATTTGGA TCGCATTAAT TGTTTCCAGT AAAACTCTTT 1620 TACCACAGGG AAATACACAT CTCACTTCAG AGTTCCATTA GGTTTTATAT TGTACTTTTC 1680

CTCCATTACT TATTAAATAC TCCTATTAAC AAAAGGTACA AAATAACAGG TTATGAGGAA 1740

ATGAGCGATA TATGAACGGC ATAAAAACAG AAATTACCCA GTAAAAAGGA TGTCAGAAAT 1800

TGACATACAA ATATTTACAA TTTTTATGAA TGGTGGTCTT TGCAAAGAGC ATTTATATTT 1860 TCTTTTTTTT TTACTAAAAT GATATATGGG TTTATTTTAT ATTTTCAAAA AAATTGTTAA 1920

ACATCATTCT TATCAATGTA AAATTTACGA AAAAAAAAAA AAAAAAAAAA AAAAAAAA 1978

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 444 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 Ser Thr Ser Pro Asn Leu Gin Lys Ala lie Asp Leu Ala Ser 1 5 10 15

Lys Ala Ala Gin Glu Asp Lys Ala Gly Asn Tyr Glu Glu Ala Leu Gin

20 25 30

Leu Tyr Gin His Ala Val Gin Tyr Phe Leu His Val Val Lys Tyr Glu 35 40 45 Ala Gin Gly Asp Lys Ala Lys Gin Ser lie Arg Ala Lys Cys Thr Glu 50 55 60

Tyr Leu Asp Arg Ala Glu Lys Leu Lys Glu Tyr Leu Lys Asn Lys Glu 65 70 75 80

Lys Lys Ala Gin Lys Pro Val Lys Glu Gly Gin Pro Ser Pro Ala Asp 85 90 95

Glu Lys Gly Asn Asp Ser Asp Gly Glu Gly Glu Ser Asp Asp Pro Glu

100 105 110

Lys Arg Lys Leu Gin Asn Gin Leu Gin Gly Ala lie Val lie Asp Arg 115 120 125 Pro Asn Val Lys Trp Ser Asp Val Ala Gly Leu Glu Gly Ala Lys Glu 130 135 140

Ala Leu Lys Glu Ala Val lie Leu Pro lie Lys Phe Pro His Leu Phe 145 150 155 160

Thr Gly Lys Arg Thr Pro Trp Arg Gly lie Leu Leu Phe Gly Pro Pro 165 170 175

Gly Thr Gly Lys Ser Tyr Leu Ala Lys Ala Val Ala Thr Glu Ala Asn

180 185 190

Asn Ser Thr Phe Phe Ser lie Ser Ser Ser Asp Leu Val Ser Lys Trp 195 200 205 Leu Gly Glu Ser Glu Lys Leu Val Lys Asn Leu Phe Gin Leu Ala Arg

210 215 220 Glu Asn Lys Pro Ser lie lie Phe lie Asp Glu He Asp Ser Leu Cys 225 230 235 240

Gly Ser Arg Ser Glu Asn Glu Ser Glu Ala Ala Arg Arg He Lys Thr

245 250 255

Glu Phe Leu Val Gin Met Gin Gly Val Gly Val Asp Asn Asp Gly He 260 265 270 Leu Val Leu Gly Ala Thr Asn He Pro Trp Val Leu Asp Ser Ala He

275 280 285

Arg Arg Arg Phe Glu Lys Arg He Tyr He Pro Leu Pro Glu Pro His

290 295 300 Ala Arg Ala Ala Met Phe Lys Leu His Leu Gly Thr Thr Gin Asn Ser 305 310 315 320

Leu Thr Glu Ala Asp Phe Arg Glu Leu Gly Arg Lys Thr Asp Gly Tyr

325 330 335

Ser Gly Ala Asp He Gly He He Val Arg Asp Ala Leu Met Gin Pro 340 345 350 Val Arg Lys Val Gin Ser Ala Thr His Phe Lys Lys Val Arg Gly Pro

355 360 365

Ser Arg Ala Asp Pro Asn His Leu Val Asp Asp Leu Leu Thr Pro Cys

370 375 380 Ser Pro Gly Asp Pro Gly Ala He Glu Met Thr Trp Met Asp Val Pro 385 390 395 400

Gly Asp Lys Leu Leu Glu Pro Val Val Ser Met Ser Asp Met Leu Arg

405 410 415

Ser Leu Ser Asn Thr Lys Pro Thr Val Asn Glu His Asp Leu Leu Lys 420 425 430 Leu Lys Lys Phe Thr Glu Asp Phe Gly Gin Glu Gly 435 440

Claims

What is claimed is:

1. An isolated polynucleotide comprising a nucleotide sequence that has at least 80% identity over its entire length to a nucleotide sequence encoding the CBFAIE 10 polypeptide of SEQ ED 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 in SEQ ID NO: 1 encoding the CBFAEEIO polypeptide of SEQ ED N02.

3. The polynucleotide of claim 1 wherein said polynucleotide comprises a nucleotide sequence that is at least 80% 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 comprising an expression system, wherein said expression system is capable of producing a CBFAEEIO polypeptide comprising an amino acid sequence, which has at least 95% identity with the polypeptide of SEQ ID NO:2 when said expression system is present in a compatible host cell.

7. A host cell comprising the expression system of claim 6.

8. A process for producing a CBFAEEIO polypeptide comprising culturing a host of claim 7 under conditions sufficient for the production of said polypeptide and recovering the polypeptide from the culture.

9. A process for producing a cell which produces a CBFAEEIO polypeptide thereof comprising transforming or transfecting a host cell with the expression system of claim 6 such that the host cell, under appropriate culture conditions, produces a CBFAIEIO polypeptide.

10 A CBFAIEIO polypeptide comprising an ammo acid sequence which is at least 95% identical to the amino acid sequence of SEQ ID NO 2 over its entire length

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

12 An antibody immunospecific for the CBFAEEIO polypeptide of claim 10

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

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

(b) providing to the subject an isolated polynucleotide compπsmg a nucleotide sequence that has at least 80% identity to a nucleotide sequence encodmg the CBFAIEIO polypeptide of SEQ ED NO 2 over its entire length, or a nucleotide sequence complementary to said nucleotide sequence in a form so as to effect production of said polypeptide activity m vivo

14 A method for the treatment of a subject having need to inhibit activity or expression of CBFAEEIO polypeptide of claim 10 compnsmg (a) administering to the subject a therapeutically effective amount of an antagonist to said polypeptide, and/or

(b) admimsteπng to the subject a nucleic acid molecule that inhibits the expression of the 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 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 CBFAEEIO polypeptide of claim 10 in a subject compπsmg

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

(b) analyzing for the presence or amount of the CBFAIEIO polypeptide expression m a sample denved from said subject

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

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

(b) observing the binding, or stimulation or inhibition of a functional response; or comparing the abihty of the cells (or cell membrane) which were contacted with the candidate compounds with the same cells which were not contacted for CBFAEEIO 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 CBFAIE 10 polypeptide .