WO1999046291A1 - A human glia maturation factor (gmf) beta homolog gene (cbfboe11) - Google Patents

Abstract

CBFBOE11 polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing CBFBOE11 polypeptides and polynucleotides in the design of protocols for the treatment of cancer, neurological disease, and autoimmune disease, among others, and diagnostic assays for such conditions.

Description

A Human Glia Maturation Factor (GMF) Beta Homolog gene (CBFBOEl 1)

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 Glia Maturation Factor family, hereinafter referred to as CBFBOEl 1 The invention also relates to inhibiting or activating the action of such polynucleotides and polypeptides

BACKGROUND OF THE INVENTION

Glia Maturation Factor is involved in neuron development and maturation This indicates that the Glia Maturation Factor family has an established, proven history as therapeutic targets Clearly there is a need for identification and characterization of further members of the G a Maturation Factor family which can play a role in preventing, ameliorating or correcting dysfunctions or diseases, including, but not limited to, cancer, neurological disease, and autoimmune disease

SUMMARY OF THE INVENTION

In one aspect, the invention relates to CBFBOEl 1 polypeptides and recombinant matenals and methods for their production Another aspect of the invention relates to methods for using such

CBFBOEl 1 polypeptides and polynucleotides Such uses include the treatment of cancer, neurological disease, and autoimmune disease, among others In still .mother aspect, the invention relates to methods to identify agonists and antagonists using the materials provided by the invention, and treating conditions associated with CBFBOEl 1 imbalance with the identified compounds Yet another aspect of the invention relates to diagnostic assays for detecting diseases associated with inappropnate CBFBOEl 1 activity or levels

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

"CBFBOEl 1" refers, among others, generally to a polypeptide having the ammo acid sequence set forth in SEQ ID NO 2 or an allehc variant thereof "CBFBOEl 1 activity or CBFBOEl 1 polypeptide activity" or "biological activity of the CBFBOEl 1 or CBFBOEl 1 polypeptide" refers to the metabolic or physiologic function of said CBFBOEl 1 including similar activities or improved activities or these activities with decreased undesirable side-effects. Also included are antigenic and immunogenic activities of said CBFBOEl 1. "CBFBOEl 1 gene" refers to a polynucleotide having the nucleotide sequence set forth in SEQ

ID NO: l or allelic variants thereof and/or their complements.

"Antibodies" as used herein includes polyclonal and monoclonal antibodies, chimeric, single 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 in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living animal is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein. "Polynucleotide" generally refers to any polyribonucleotide 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 single- 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 single- and double-stranded regions. In addition, "polynucleotide" refers to triple-stranded regions comprising 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, tritylated bases and unusual bases such as inosine. A variety 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 comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. "Polypeptide" refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. "Polypeptides" include amino acid sequences modified either by natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art.

2 Such modifications are well described m 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 ammo acid side-chains and the amino or carboxj 1 termini It will be appreciated that the same type of modification may be present m 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 derivative, covalent attachment of a pid or lipid deπvative, covalent attachment of phosphotidyhnositol, cross-linking, cyc zation, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxvlation, lodmation, methylation, myπstoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of ammo acids to proteins such as argmylation, 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 , "Protein Synthesis Posttranslational Modifications and Aging", Ann NYAcad Sci (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 vanant are closely similar overall and, in many regions, identical A variant and reference polypeptide may differ m amino acid sequence by one or more substitutions, additions, deletions m any combination A substituted or inserted amino acid residue may or may not be one encoded by the genetic code A vanant of a polynucleotide or polypeptide may be a naturally occurring

3 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," as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in (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 ; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S.F. et al., J. Molec. Biol. 215: 403-410 (1990). The BLAST X program is publicly available from NCBI and other sources (BIAST Manual, Altschul, S., et al. , NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et al. , J. Mol. Biol. 215: 403-410 (1990). The well known Smith Waterman algorithm may also be used to determine identity.

Preferred parameters for polypeptide sequence comparison include the following:

1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970) Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89: 10915-10919 (1992) Gap Penalty: 12 Gap Length Penalty: 4

A program useful with these parameters is publicly available as the "gap" program from Genetics Computer Group, Madison WI. The aforementioned parameters are the default parameters for polypeptide comparisons (along with no penalty for end gaps). Preferred parameters for polynucleotide comparison include the following: 1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970) Comparison matrix: matches = + 10, mismatch = 0 Gap Penalty: 50 Gap Length Penalty: 3

A program useful with these parameters is publicly available as the "gap" program from Genetics Computer Group, Madison Wl. The aforementioned parameters are the default parameters for polynucleotide comparisons.

Preferred polynucleotide embodiments further include an isolated polynucleotide comprising a polynucleotide having at least a 50,60, 70, 80, 85, 90, 95, 97 or 100% identity to a polynucleotide reference sequence of SEQ ID NO: l , wherein said reference sequence may be identical to the sequence of SEQ ID NO: 1 or may include up to a certain integer number of nucleotide alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ ID NO: l by the numerical percent of the respective percent identity and subtracting that product from said total number of nucleotides in SEQ ID NO: l , or:

n_τ < x_n - (x_n • y),

wherein n_n is the number of nucleotide alterations, x_n is the total number of nucleotides in SEQ ID NO. l, and y is 0.50 for 50% , 0.60 for 60% , 0.70 for 70% , 0.80 for 80% , 0.85 for 85 % , 0.90 for 90% , 0.95 for 95% , 0.97 for 97% or 1.00 for 100% , and wherein any non-integer product of x_n and y is rounded down to the nearest integer prior to subtracting it from x_n. Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO:2 may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations. Preferred polypeptide embodiments further include an isolated polypeptide comprising a polypeptide having at least a 50,60, 70, 80, 85, 90, 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO:2, wherein said reference sequence may be identical to the sequence of SEQ ID NO: 2 or may include up to a certain integer number of amino acid alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non- conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of amino acid alterations is determined by multiplying the total number of amino acids in SEQ ID NO:2 by the numerical percent of the respective percent identity and subtracting that product from said total number of amino acids in SEQ ID NO:2, or:

n_a ≤ x_a - (x_a « y),

wherein n_a is the number of amino acid alterations, x_a is the total number of amino acids in SEQ ID NO:2, and y is 0.50 for 50% , 0.60 for 60% , 0.70 for 70% , 0.80 for 80% , 0.85 for 85 % , 0.90 for 90% , 0.95 for 95% , 0.97 for 97% or 1.00 for 100% , and wherein any non-integer product of x_a and y is rounded down to the nearest integer prior to subtracting it from x_a.

Polypeptides of the Invention

In one aspect, the present invention relates to CBFBOEl 1 polypeptides (or CBFBOEl 1 proteins). The CBFBOEl 1 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 CBFBOEl 1 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 CBFBOEl 1 polypeptide exhibit at least one biological activity of CBFBOEl 1. The CBFBOEl 1 polypeptides mav 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 CBFBOEl 1 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 CBFBOEl 1 polypeptides As with CBFBOEl 1 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 CBFBOEl 1 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 amino acid sequence of CBFBOE 1 1 polypeptides, except for deletion of a continuous senes of residues that includes the amino terminus, or a continuous senes of residues that includes the carboxyl terminus or deletion of two continuous senes of residues, one including the amino terminus and one including the carboxyl terminus Also preferred are fragments characteπzed by structural or functional attnbutes such as fragments that compnse alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming 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 preferred fragments are biologically active fragments Biologically active fragments are those that mediate CBFBOEl 1 activity, including those with a similar activity or an improved activity, or with a decreased undesirable activity Also included are those that are antigenic or lmmunogenic in an animal, especially in a human

Preferably, all of these polypeptide fragments retain the biological activity of the CBFBOEl 1, including antigenic activity Variants of the defined sequence and fragments also form part of the present invention Preferred vanants are those that vary from the referents by conservative amino acid substitutions — l e , those that substitute a residue with another of like charactenstics Typical such substitutions are among Ala, Val, Leu and He, among Ser and Thr, among the acidic residues Asp and Glu, among Asn and Gin, and among the basic residues Lys and Arg, or aromatic residues Phe and Tyr Particularly preferred are vanants in which several, 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination The CBFBOEl 1 pol peptides 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 in the art

Polynucleotides of the Invention

Another aspect of the invention relates to CBFBOEl 1 polynucleotides CBFBOEl 1 polynucleotides include isolated polynucleotides which encode the CBFBOEl 1 polypeptides and fragments, and polynucleotides closely related thereto More specifically, CBFBOEl 1 polynucleotide of the invention include a polynucleotide composing the nucleotide sequence contained in SEQ ID NO 1 encoding a

CBFBOEl 1 polypeptide of SEQ ID NO 2, and polynucleotide having the particular sequence of SEQ ID NO 1 CBFBOEl 1 polynucleotides further include a polynucleotide composing a nucleotide sequence that has at least 80% identity over its entire length to a nucleotide sequence encoding the CBFBOEl 1 polypeptide of SEQ ID NO 2, and a polynucleotide compnsmg 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 CBFBOEl 1 polynucleotides are a nucleotide sequence which has sufficient identity to a nucleotide sequence contained in SEQ ID NO 1 to hybπdize under conditions useable for amplification or for use as a probe or marker The invention also provides polynucleotides which are complementary to such CBFBOEl 1 polynucleotides

CBFBOEl 1 of the invention is structurally related to other proteins of the Glia Maturation Factor family, as shown by the results of sequencing the cDNA of Table 1 (SEQ ID NO 1) encoding human CBFBOEl 1 The cDNA sequence of SEQ ID NO 1 contains an open reading frame (nucleotide number 5 to 430) encoding a polypeptide of 142 amino acids of SEQ ID NO 2 The amino acid sequence of Table 2 (SEQ ID NO 2) has about 82 1% identity (using FASTA) in 140 amino acid residues with human and rat GMF-beta (R Kaplan, et al , J Neurochem 57 (2), 483-490, 1991) The nucleotide sequence of Table 1 (SEQ ID NO 1) has about 70 2% identity (using FASTA) in 433 nucleotide residues with human and rat GMF-beta (R Kaplan, et al , J Neurochem 57 (2), 483-490, 1991 Thus, CBFBOEl 1 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"

1 AATCATGTCT GACTCCCTGG TGGTGTGCGA GGTAGACCCA GAGCTAACAG 51 AAAAGCTGAG GAAATTCCGC TTCCGAAAAG AGACAGACAA TGCAGCCATC

101 ATAATGAAGG TGGACAAAGA CCGGCAGATG GTGGTGCTGG AGGAAGAATT

151 TCAGAACATT TCCCCAGAGG AGCTCAAAAT GGAGTTGCCG GAGAGACAGC

201 CCAGGTTCGT GGTTTACAGC TACAAGTACG TGCATGACGA TGGCCGAGTG

251 TCCTACCCTT TGTGTTTCAT CTTCTCCAGC CCTGTGGGCT GCAAGCCGGA

301 ACAACAGATG ATGTATGCAG GGAGTAAAAA CAGGCTGGTG CAGACAGCAG

351 AGCTCACAAA GGTGTTCGAA ATCCGCACCA CTGATGACCT CACTGAGGCC

401 TGGCTCCAAG AAAAGTTGTC TTTCTTTCGT TGATCTCTGG GCTGGGGACT

451 GAATTCCTGA TGTCTGAGTC CTCAGGTGAC TGGGGACTTG GAACCCTAGG

501 ACCTGAACAA CCAAGACTTT AAATAATTTT AAATGCAAAA ACTCAGAAAA

551 AAAAAAAAAA A

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

Table 2^b

1 MSDSLWCEV DPELTEKLRK FRFRKETDNA AIIMKVDKDR QMWLEEEFQ

51 NISPEELKME LPERQPRFW YSYKYVHDDG RVSYPLCFIF SSPVGCKPEQ

101 QMMYAGSKNR LVQTAELTKV FEIRTTDDLT EAWLQEKLSF FR

An amino acid sequence of a human CBFBOEl 1 (SEQ ID NO: 2).

One polynucleotide of the present invention encoding CBFBOEl 1 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) 555:632-634; Adams, M.D., et al., Nature (1995) 377 Supp:3-174). Polynucleotides of the invention can also be obtained from natural sources such as genomic DNA libraries or can be synthesized using well known and commercially available techniques

The nucleotide sequence encoding CBFBOEl 1 polypeptide of SEQ ID NO 2 may be identical to the polypeptide encoding sequence contained in Table 1 (nucleotide number 5 to 430 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 CBFBOEl 1 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 descnbed in Gentz et al , Proc Natl Acad Set 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 preferred embodiments are polynucleotides encoding CBFBOE 11 vanants compnse the amino acid sequence CBFBOEl 1 polypeptide of Table 2 (SEQ ID 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 hybndize to the herein above-desenbed sequences In this regard, the present mvention especially relates to polynucleotides which hybndize under stringent conditions to the herein above-desenbed polynucleotides As herein used, the term "stringent 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 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 hybndization probes for cDNA and genomic DNA, to isolate full-length cDNAs and genomic clones encoding CBFBOEl 1 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 similanty to the CBFBOE 11 gene Such hybndization 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 compose 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

10 In one embodiment, to obtain a polynucleotide encoding CBFBOEl 1 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, CBFBOEl 1 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 ID NO: 1 or a fragment thereof. Also included with CBFBOEl 1 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 described in many standard laboratory manuals, such as Davis et al. , BASIC METHODS IN MOLECULAR BIOLOGY (1986) and Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N Y. (1989) such as calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection. Representative examples of appropriate hosts include bacterial cells, such as streptococci, staphylococci, E. coli, Streptomyces and Bacillus subtilis cells; fungal cells, such as yeast cells and Aspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells; and plant cells.

1 1 A great variety of expression systems can be used. Such systems include, among others, chromosomal, episomal and virus-derived systems, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. The expression systems may contain control regions that regulate as well as engender expression. Generally, any system or vector suitable to maintain, propagate or express polynucleotides to produce a polypeptide in a host may be used. The appropriate nucleotide sequence may be inserted into an expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL (supra).

For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signals may be incorporated into the desired polypeptide. These signals may be endogenous to the polypeptide or they may be heterologous signals.

If the CBFBOEl 1 polypeptide is to be expressed for use in screening assays, generally, it is preferred that the polypeptide be produced at the surface of the cell. In this event, the cells may be harvested prior to use in the screening assay. If CBFBOEl 1 polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify the polypeptide; if produced intracellularly, the cells must first be lysed before the polypeptide is recovered.

CBFBOEl 1 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 CBFBOEl 1 polynucleotides for use as diagnostic reagents.

Detection of a mutated form of CBFBOEl 1 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 CBFBOE 11. Individuals carrying mutations in the CBFBOEl 1 gene may be detected at the DNA level by a variety of techniques.

12 Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy matenal The genomic DNA may be used directly for detection or may be amphfied enzymatically by using PCR or other amplification techniques poor 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 companson to the normal genotype Point mutations can be identified by hybndizmg amplified DNA to labeled CBFBOEl 1 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 Set USA (1985) 85 4397-4401 In another embodiment, an array of oligonucleotides probes compnsing CBFBOEl 1 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 vanety of questions in molecular genetics including gene expression, genetic linkage, and genetic vanability (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, neurological disease, and autoimmune disease, through detection of mutation in the CBFBOEl 1 gene by the methods descnbed In addition, cancer, neurological disease, and autoimmune disease, can be diagnosed by methods compnsing determining from a sample derived from a subject an abnormally decreased or increased level of CBFBOEl 1 polypeptide or CBFBOEl 1 mRNA Decreased or increased expression can be measured at the RNA level using any of the methods well known in 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

CBFBOE 11 polypeptide, in a sample denved 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 invention relates to a diagonostic kit for a disease or suspectabihty to a disease, particularly cancer, neurological disease, and autoimmune disease, which composes

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

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

13 (c) a CBFBOEl 1 polypeptide, preferably the polypeptide of SEQ ID NO 2, or a fragment thereof, or

(d) an antibody to a CBFBOEl 1 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 comprise a substantial component

Chromosome Assays

The nucleotide sequences of the present invention 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 invention is an important first step in correlating those sequences with gene associated disease Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be conelated with genetic map data Such data are found, for example, in V McKusick, Mendelian Inheotance in Man (available on line through Johns Hopkins University Welch Medical Library) The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheotance of physically adjacent genes) The differences m 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 lmmunogens to produce antibodies lmmunospecific for the CBFBOEl 1 polypeptides The term "lmmunospecific" means that the antibodies have substantiall greater affinity for the polypeptides of the invention than their affinity for other related polypeptides in the pnor art

Antibodies generated against the CBFBOEl 1 polypeptides can be obtained by administering the polypeptides or epitope-beanng 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 hybndoma technique (Kohler, G and Ivhlstein, C , Λfa/wre (1975) 256 495-497), the tnoma technique, the human B-cell hybndoma technique (Kozbor et al , Immunology Today (1983) 4 72) and the EBV-hybndoma technique (Cole et al , MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp 77-96, Alan R Liss, Inc , 1985)

14 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-desenbed antibodies may be employed to isolate or to identify clones expressing the polypeptide or to punfy the polypeptides by affinity chromatograph}

Antibodies against CBFBOEl 1 polypeptides may also be employed to treat cancer, neurological disease, and autoimmune disease, among others

Vaccines Another aspect of the invention relates to a method for inducing an lmmunological response in a mammal which comprises inoculating the mammal with CBFBOEl 1 polypeptide, or a fragment thereof, adequate to produce antibody and/or T cell immune response to protect said animal from cancer, neurological disease, and autoimmune disease, among others Yet another aspect of the invention relates to a method of inducing lmmunological response m a mammal which comprises, delivering CBFBOEl 1 polypeptide via a vector directing expression of CBFBOEl 1 polynucleotide in vivo in order to induce such an lmmunological response to produce antibody to protect said animal from diseases

Further aspect of the invention relates to an lmmunological/vaccme formulation (composition) which, when introduced into a mammalian host, induces an lmmunological response in that mammal to a CBFBOEl 1 polypeptide wherein the composition comprises a CBFBOEl 1 polypeptide or CBFBOEl 1 gene The vaccine formulation may further comprise a suitable earner Since CBFBOEl 1 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, bacteπostats and solutes which render the formulation lnstomc 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 m unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dned condition requiring only the addition of the sterile liquid earner immediately pnor to use The vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-m water systems and other systems known m the art The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation

15 Screening Assays

The CBFBOEl 1 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 CBFBOEl 1 polypeptide of the present invention Thus, polypeptides of the mvention may also be used to assess identify agomst or antagonists from, for example, cells, cell-free preparations, chemical hbranes, and natural product mixtures These agonists or antagonists may be natural or modified substrates, hgands, 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 invention See Co gan et al , Current Protocols in Immunology 1(2) Chapter 5 (1991) CBFBOE 11 polypeptides are responsible for many biological functions, including many pathologies

Accordingly, it is desirous to find compounds and drugs which stimulate CBFBOEl 1 polypeptide on the one hand and which can inhibit the function of CBFBOE 11 polypeptide on the other hand In general, agonists are employed for therapeutic and prophylactic purposes for such conditions as cancer, neurological disease, and autoimmune disease Antagonists may be employed for a vanety of therapeutic and prophylactic purposes for such conditions as cancer, neurological disease, and autoimmune disease

In general, such screening procedures may involve using appropnate cells which express the CBFBOEl 1 polypeptide or respond to CBFBOEl 1 polypeptide of the present invention Such cells include cells from mammals, yeast, Drosophila or E coll Cells which express the CBFBOEl 1 polypeptide (or cell membrane containing the expressed polypeptide) or respond to CBFBOEl 1 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 CBFBOEl 1 activity

The assays may simply test binding of a candidate compound wherein adherence to the cells beanng the CBFBOEl 1 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 CBFBOEl 1 polypeptide, using detection systems appropriate to the cells beaπng the CBFBOEl 1 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 compose the steps of mixing a candidate compound with a solution containing a CBFBOEl 1 polypeptide to form a mixture, measuring CBFBOEl 1 activity in the mixture, and comparmg the CBFBOEl 1 activity of the mixture to a standard

16 The CBFBOEl 1 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 CBFBOEl 1 mRNA and protein in cells For example, an ELISA may be constructed for measuring secreted or cell associated levels of CBFBOEl 1 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

CBFBOEl 1 (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues The CBFBOEl 1 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, gand binding and crosshnking assays in which the CBFBOEl 1 is labeled with a radioactive isotope (eg 1251), chemically modified (eg biotinylated), or fused to a peptide sequence suitable for detection or punfication, and incubated with a source of the putative receptor (cells, cell membranes, cell supernatants, tissue extracts, bodily fluids) Other methods 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 CBFBOEl 1 which compete with the bmdmg of CBFBOEl 1 to its receptors, if any Standard methods for conducting screening assays are well understood in the art

Examples of potential CBFBOEl 1 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 CBFBOEl 1 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 ehcit 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 CBFBOEl 1 polypeptides, or compounds which decrease or enhance the production of CBFBOEl 1 polypeptides, which composes (a) a CBFBOEl 1 polypeptide, preferably that of SEQ ID NO 2,

(b) a recombinant cell expressing a CBFBOEl 1 polypeptide, preferably that of SEQ ID NO 2,

(c) a cell membrane expressing a CBFBOEl 1 polypeptide, preferably that of SEQ ID NO 2, or

(d) antibody to a CBFBOEl 1 polypeptide, preferably that of SEQ ID NO 2

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

Prophylactic and Therapeutic Methods

This mvention provides methods of treating abnormal conditions such as, cancer, neurological disease, and autoimmune disease, related to both an excess of and insufficient amounts of CBFBOEl 1 polypeptide activity

17 If the activity of CBFBOEl 1 polypeptide is in excess, several approaches are available One approach compnses administering to a subject an inhibitor compound (antagonist) as hereinabove descnbed along with a pharmaceutically acceptable earner in an amount effective to inhibit the function of the CBFBOEl 1 polypeptide, such as, for example, by blocking the bmdmg of ligands, substrates, receptors, enzymes, etc , or by inhibiting a second signal, and thereby alleviating the abnormal condition In another approach, soluble forms of CBFBOEl 1 polypeptides still capable of binding the hgand, substrate, enzymes, receptors, etc in competition with endogenous CBFBOEl 1 polypeptide may be administered Typical embodiments of such competitors comprise fragments of the CBFBOEl 1 polypeptide

In still another approach, expression of the gene encoding endogenous CBFBOEl 1 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 Ohgodeoxynucleotides 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 CBFBOEl 1 and its activity, several approaches are also available One approach compnses administering to a subject a therapeutically effective amount of a compound which activates CBFBOEl 1 polypeptide, I 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 CBFBOEl 1 by the relevant cells in the subject For example, a polynucleotide of the mvention may be engineered for expression in a replication defective retroviral vector, as discussed above The retroviral expression construct may then be isolated and introduced mto a packagmg cell transduced with a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention such that the packagmg 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 CBFBOE 11 polypeptides in combination with a suitable pharmaceutical earner Formulation and Administration

Peptides, such as the soluble form of CBFBOEl 1 polypeptides, and agonists and antagonist peptides or small molecules, may be formulated in 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 include 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 invention 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 m 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, intramuscular, or lntrapentoneal, can be used Alternative means for systemic administration mclude transmucosal and transdermal administration usmg penetiants such as bile salts or fiisidic acids or other detergents In addition, if properly formulated m entenc or encapsulated formulations, oral administration may also be possible Admmistration 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 admmistration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attending practitioner Suitable dosages, however, are m the range of 0 1-100 μg/kg of subject Wide vanations m the needed dosage, however, are to be expected in view of the vanety of compounds available and the differing efficiencies of vanous routes of administration For example, oral administration would be expected to require higher dosages than administration by intravenous injection Vanations m these dosage levels can be adjusted using standard empincal routines for optimization, as is well understood m the art

Polypeptides used m treatment can also be generated endogenously in the subject, in treatment modalities often referred to as "gene therapy" as descnbed 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 to the subject

All publications, including but not limited to patents and patent applications, cited m 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

19 SEQUENCE LISTING

(1) GENERAL INFORMATION

(i) APPLICANT: FU, GANG SHEN, YU MAO, MAO WANG, YA-XIN

(ii) TITLE OF THE INVENTION: A Human Glia Maturation Factor (GMF) Beta Homolog gene (CBFBOE11)

(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

20 ( C ) REFERENCE/DOCKET NUMBER : GP-70381

( ix ) TELECOMMUNICATION INFORMATION : (A) TELEPHONE : 610- 407- 0700 ( B ) TELEFAX : 610- 407- 0701

( C ) TELEX : 846169

( 2 ) INFORMATION FOR SEQ ID NO : l :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 561 base pairs

(B) TYPE: nucleic acid

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

(ii) MOLECULE TYPE: cDNA

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

AATCATGTCT GACTCCCTGG TGGTGTGCGA GGTAGACCCA GAGCTAACAG AAAAGCTGAG 60

GAAATTCCGC TTCCGAAAAG AGACAGACAA TGCAGCCATC ATAATGAAGG TGGACAAAGA 120

CCGGCAGATG GTGGTGCTGG AGGAAGAATT TCAGAACATT TCCCCAGAGG AGCTCAAAAT 180

GGAGTTGCCG GAGAGACAGC CCAGGTTCGT GGTTTACAGC TACAAGTACG TGCATGACGA 240 TGGCCGAGTG TCCTACCCTT TGTGTTTCAT CTTCTCCAGC CCTGTGGGCT GCAAGCCGGA 300

ACAACAGATG ATGTATGCAG GGAGTAAAAA CAGGCTGGTG CAGACAGCAG AGCTCACAAA 360

GGTGTTCGAA ATCCGCACCA CTGATGACCT CACTGAGGCC TGGCTCCAAG AAAAGTTGTC 420

TTTCTTTCGT TGATCTCTGG GCTGGGGACT GAATTCCTGA TGTCTGAGTC CTCAGGTGAC 480

TGGGGACTTG GAACCCTAGG ACCTGAACAA CCAAGACTTT AAATAATTTT AAATGCAAAA 540 ACTCAGAAAA AAAAAAAAAA A 561

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 142 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:2:

21 Met Ser Asp Ser Leu Val Val Cys Glu Val Asp Pro Glu Leu Thr Glu

1 5 10 15

Lys Leu Arg Lys Phe Arg Phe Arg Lys Glu Thr Asp Asn Ala Ala lie 20 25 30 lie Met Lys Val Asp Lys Asp Arg Gin Met Val Val Leu Glu Glu Glu 35 40 45

Phe Gin Asn lie Ser Pro Glu Glu Leu Lys Met Glu Leu Pro Glu Arg

50 55 60

Gin Pro Arg Phe Val Val Tyr Ser Tyr Lys Tyr Val His Asp Asp Gly 65 70 75 80

Arg Val Ser Tyr Pro Leu Cys Phe lie Phe Ser Ser Pro Val Gly Cys

85 90 95

Lys Pro Glu Gin Gin Met Met Tyr Ala Gly Ser Lys Asn Arg Leu Val 100 105 110 Gin Thr Ala Glu Leu Thr Lys Val Phe Glu lie Arg Thr Thr Asp Asp 115 120 125

Leu Thr Glu Ala Trp Leu Gin Glu Lys Leu Ser Phe Phe Arg 130 135 140

22

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 CBFBOEl 1 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 in SEQ ID NO: 1 encoding the CBFBOEl 1 polypeptide of SEQ ID 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 CBFBOEl 1 polypeptide comprising an amino acid sequence, which has at least 83% 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 CBFBOEl 1 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 CBFBOEl 1 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 CBFBOEl 1 polypeptide.

23

10. A CBFBOEl 1 polypeptide comprising an amino acid sequence which is at least 83% identical to the amino acid sequence of SEQ ID NO:2 over its entire length.

1 1. The polypeptide of claim 10 which comprises the amino acid sequence of SEQ ID NO:2.

12. An antibody immunospecific for the CBFBOEl 1 polypeptide of claim 10.

13. A method for the treatment of a subject in need of enhanced activity or expression of CBFBOEl 1 polypeptide of claim 10 comprising:

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

(b) providing to the subject an isolated polynucleotide comprising a nucleotide sequence that has at least 80% identity to a nucleotide sequence encoding the CBFBOEl 1 polypeptide of SEQ ID 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 in vivo.

14. A method for the treatment of a subject having need to inhibit activity or expression of CBFBOEl 1 polypeptide of claim 10 comprising: (a) administering to the subject a therapeutically effective amount of an antagonist to said polypeptide; and/or

(b) administering to the subject a nucleic acid molecule that inhibits the expression of 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 ligand, substrate , or receptor.

15. A process for diagnosing a disease or a susceptibility to a disease in a subject related to expression or activity of CBFBOEl 1 polypeptide of claim 10 in a subject comprising:

(a) determining the presence or absence of a mutation in the nucleotide sequence encoding said CBFBOEl 1 polypeptide in the genome of said subject; and/or

(b) analyzing for the presence or amount of the CBFBOEl 1 polypeptide expression in a sample derived from said subject.

24

16. A method for identifying compounds which inhibit (antagonize) or agonize the CBFBOEl 1 polypeptide of claim 10 which comprises:

(a) contacting a candidate compound with cells which express the CBFBOEl 1 polypeptide (or cell membrane expressing CBFBOEl 1 polypeptide) or respond to CBFBOEl 1 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 CBFBOEl 1 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 CBFBOEl 1 polypeptide.

25