WO1998010066A1 - Modulators of brca1 activity - Google Patents

Abstract

Compositions of matter consisting of a family of related nucleotide sequences that encode proteins, termed BRCA1 Modulator Proteins, that bind to the tumor suppressor gene product BRCA1, and methods of using the nucleotide sequences and the proteins encoded thereby, to diagnose and/or treat disease where the BRCA1 Modulator Proteins have an apparent molecular weight of 45-97 kdaltons and are characterized by having at least one leucine zipper domain, and optionally a zinc finger domain.

Description

MODULATORS OF BRCA1 ACTIVITY

Field of the Invention

The invention described herein relates generally to the field of human disease, and more specifically to treating and diagnosing disease based on the presence of modulators of BRCA1 activity.

Background Breast cancer is one of the leading causes of cancer deaths of women in the United States, and approximately 170,000 women are affected by the disease each year. About 5% of these reported cases are thought to result from a patient's genetic predisposition to the disease. Breast cancer is generally considered to be classifiable as early-age onset and late-age onset, the latter being defined as occurring at about age 50. Approximately 25% of patients diagnosed with breast cancer before the age of 40 are thought to be familial, and thus have an underlying genetic component. Late-age onset breast cancer is also often familial although the risks of a family member developing the disease is less compared to early-age onset if relatives have presented with the disease.

As a result of studies involving families with inherited early onset breast and ovarian cancers a gene thought to be involved in these diseases has been mapped to the long arm of chromosome 17 and termed BRCA1, or breast cancer one gene. See, Hitoyuki, T., et al., Cancer res. vol. 55: 2998-3002. Additional studies on sporadic cases of breast cancer have also established a genetic link with this disease to BRCA1 which was more precisely localized to the chromosomal region 17q21. See, Hall, }. ., et al. Science, vol. 250: 1684-1689 (1990).

Recently, the BRCA1 gene has been cloned, and shown to encode a protein having the properties of a tumor suppressor protein. See, Miki, Y., et al Science, vol. 266: 66-71; and WO96/05306. It has been known for some time that a variety of cancers are caused, at least in part, by mutations to certain normal genes, termed "proto- oncogenes." Proto-oncogenes are involved in regulating normal cell growth in ways that are only now beginning to be appreciated at the molecular level. The mutated proto-oncogenes, or cancer causing genes termed "oncogenes," disrupt normal cell growth which ultimately causes the death of the organism, if the cancer is not detected and treated in time. During normal or cancer cell growth, proto-oncogenes or oncogenes, are counterbalanced by growth-regulating proteins which regulate or try to regulate the growth of normal or cancer cells, respectively. Such proteins are termed "tumor suppressor proteins," and include BRCA1, p53, retinoblastoma protein (Rb), adenomatous polyposis coli protein (APC), Wilm's tumor 1 protein (WT1), neurofibromatosis type 1 protein (NF1), and neurofibromatosis type 2 protein (NF2). BRCA1 cDNA encodes a 1863 amino acid protein with a predicted molecular weight of approximately 207,000. See, Miki, Y., et al. (1994) Science vol. 266, pages 66- 71. The cloning and characterization of BRCA1 has facilitated establishing it as a tumor suppressor protein. For example, recent work by several investigators have shown that transfection and expression of the BRCA1 gene sequence into MCF-7 tumor cells retards tumor growth in vivo, and extends the survival time of tumor bearing animals. See, Holt, J. T., et al, (1996) Nat. Genet, vol. 12, pages 298-302. Similar results were obtained using a retroviral vector expressing wild-type BRCA1 against an established MCF-7 peritoneal tumor.

Considerable work has been done to identify those regions of BRCA1 that affects its tumor suppressor activity. It appears that different regions of the molecule may affect its tumor suppressor activity differently. For instance, near full length truncated BRCA1 proteins do not inhibit breast cancer cell growth, but do inhibit ovarian cancer cell growth. See, Holt, J. T., et al, (1996) Nat. Genet, vol. 12, pages 298-302. These observations strongly suggest that different host cell factors, presumably proteins, are interacting with different regions of BRCA1 to affect cell growth.

Over the past several years, the interactions of certain tumor suppressor proteins with host cell proteins have begun to be elucidated. See, Levin, A., Annu. Rev. Biochem. 1993, vol. 62: pages 623-651. The identification of proteins involved in these interactions will facilitate the development of novel diagnostic methods, as well as novel therapeutics for identifying and treating cancer. For example, the retinoblastoma tumor suppressor protein is phosphorylated at serine residues adjacent to a proline. The level of phosphorylation is high through S, G2, and M-phase of the cell cycle. The kinase that effects this reaction is, in turn, activated by a cyclin that regulates events in the cell cycle. Subsequently, in late mitosis, a phosphatase removes the phosphate groups from the protein, and returns the retinoblastoma tumor suppressor protein to an unphosphorylated state in Go-Gl. Clearly, the identification of drugs that can effect these interactions can be expected to play a critical role in regulating cell growth and thus be useful in the treatment of cancer.

To date, however, there have been few, if any studies on the interaction of proteins with the tumor suppressor protein, BRCA1. In order to better develop methods to diagnosis and treat both breast and ovarian cancer the identification and isolation of such proteins is critical.

Summary of the Invention A first object of the invention is to describe a family of related isolated nucleic acid sequences that encode proteins, hereinafter referred to as Modulator Proteins, that bind to the tumor suppressor protein BRCA1. A second object of the invention is to describe a family of related isolated nucleic acid sequences that encode BRCA1 Modulator Proteins having a range of molecular weights ranging from about 45-97 kdaltons, at least one leucine zipper domain, and optionally a zinc finger domain, that bind BRCA1 at a discreet sequence for Modulator Protein binding that is encompassed in the first six hundred amino acids of BRCA1. A third object of the invention is to describe a BRCA1 Modulator Protein having a calculated molecular weight of about 53 kdaltons that has one leucine zipper domain and a zinc finger domain, both domains near the amino terminal region of the molecule, that bind BRCA1 at a consenus sequence for Modulator Protein binding encompassed within the first six hundred amino acids of BRCA1. A fourth object of the invention is to describe isolated nucleic acid or protein fragments of BRCA1 Modulator Protein (s), respectively.

A fifth object of the invention is to describe host cells transformed with isolated nucleic acid sequences that encodes BRCA1 Modulator Protein(s) or fragments thereof. A sixth object of the invention is to describe vectors that contain isolated nucleic acid sequences that encode BRCA1 Modulator Protein(s) or fragments thereof.

A seventh object of the invention is to describe complexes consisting of full length or fragments of BRCA1 and BRCA1 Modulator Proteins. An eighth object of the invention is to describe methods of diagnosing disease, preferably those involving unwanted cell growth, including cancer, using isolated nucleic acid sequences, or fragments thereof, that encode a BRCA1 Modulator Protein, or fragments thereof. A ninth object of the invention is to describe an assay for identifying compounds that would have therapeutic applications for the treatment of diseases involving unwanted cell growth, including cancer.

These and other objects of the present invention will become apparent to one of ordinary skill in the art upon reading the description of the various aspects of the invention in the following specification. The foregoing and other aspects of the present invention are explained in greater detail in the drawings, detailed description, and examples set forth below.

Brief Description of the Drawings Figure 1 shows the cDNA and amino acid sequence of the BRCA1 Modulator Protein, depicted in Sequence ID No. 1, 091-21 A31.

Figure 2 shows the cDNA and amino acid sequence of the BRCA1 Modulator Protein, depicted in Sequence ID No. 3, 091-1F84.

Figure 3 shows the cDNA and amino acid sequence of the BRCA1 Modulator Protein, depicted in Sequence ID No. 5, 091-132Q20. Figure 4 shows the format of an assay to identify compounds that increase the intracellular levels of BRCA1.

Table 1 shows the regions of BRCA1 that interact with the BRCA1 Modulator Proteins 091-1F84, Sequence ID No. 3, 091-21 A31, Sequence ID No. 1 and 091-132Q20, Sequence ID No. 5. The experiment was conducted using the two-hybrid assay as described in U. S. Patent No. 5, 283, 173, or Chien et al., 1991, Proc. Natl. Acad. Sci. USA, 88:9578-9582. The cDNAs that encode 091-1F84, Sequence ID No. 3, 091-21A31, Sequence ID No. 1 and 091-132Q20, Sequence ID No. 5 were fused to the GAL 4 activation domain, and those regions of BRCA1 shown in the table were fused to the binding domain of GAL4. The "+" sign is a subjective measure of the amount of b- galactosidase activity. One "+" being the lowest, and three "+++" being the highest activity. Table 2 shows regions of the BRCA1 Modulator Protein 091-21 A31, Sequence ID No. 1 that interact with regions of BRCA1.

Detailed Description of the Invention All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Definitions At the outset it is worth noting that unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures described below are those well known and commonly employed in the art. Standard techniques are used for recombinant nucleic acid methods, polynucleotide synthesis, and microbial culture and transformation (e.g., electroporation, lipofection). Generally enzymatic reactions and purification steps are performed according to the manufacturer's specifications. The techniques and procedures are generally performed according to conventional methods in the art and various general references (see generally, Sambrook et al, Molecular Cloning: A Laboratory Manual. 2nd, edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., which is incorporated herein by reference) which are provided throughout this document. The nomenclature used herein and the laboratory procedures in analytical chemistry, organic synthetic chemistry, and pharmaceutical formulation described below are those well known and commonly employed in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical formulation and delivery, and treatment of patients.

In the formulas representing selected specific embodiments of BRCA1 or BRCA1 Modulator Proteins of the present invention, the amino- and carboxy-terminal groups, although often not specifically shown, will be understood to be in the form they would assume at physiological pH values, unless otherwise specified. Thus, the N-terminal H₂ ⁺ and C-terminal-O at physiological pH are understood to be present though not necessarily specified and shown, either in specific examples or in generic formulas. In the polypeptide notation used herein, the left-hand end of the molecule is the amino terminal end and the right-hand end is the carboxy-terminal end, in accordance with standard usage and convention. Of course, the basic and acid addition salts including those which are formed at nonphysiological pH values are also included in the compounds of the invention. The amino acid residues described herein are preferably in the "L" isomeric form. Stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids, unnatural amino acids such as a,a-distributed amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components for polypeptides of the present invention, as long as the desired functional property is retained by the polypeptide. For the peptides shown, each encoded residue where appropriate is represented by a three letter designation, corresponding to the trivial name of the conventional amino acid, in keeping with standard polypeptide nomenclature (described in T. Biol. Chem., 243:3552-59 (1969) and adopted at 37 CFR §1.822(b)(2)).

Free functional groups, including those at the carboxy- or amino-terminus, referred to as noninterfering substituents, can also be modified by amidation, acylation or other substitution, which can, for example, change the solubility of the compounds without affecting their activity. This may be particularly useful in those instances where BRCA1 Modulator Proteins are known to have certain regions that bind to BRCA1, and it is desirable to make soluble peptides from these regions. As employed throughout the disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

The term "isolated protein" referred to herein means a protein of cDNA, recombinant RNA, or synthetic origin or some combination thereof, which by virtue of its origin the "isolated protein" (1) is not substantially associated with proteins found in nature, (2) is substantially free of other proteins from the same source, e.g. free of human proteins, (3) may be expressed by a cell from a different species, or (4) does not occur in nature.

The term "naturally-occurring" as used herein as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring. The term "polynucleotide" as referred to herein means a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms of DNA. The term "oligonucleotide" referred to herein includes naturally occurring, and modified nucleotides linked together by naturally occurring, and non-naturally occurring oligonucleotide linkages. Oligonucleotides are a polynucleotide subset with 200 bases or fewer in length. Preferably oligonucleotides are 10 to 60 bases in length and most preferably 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases in length. Oligonucleotides are usually single stranded, e.g. for probes; although oligonucleotides may be double stranded, e.g. for use in the construction of a gene mutant. Oligonucleotides of the invention can be either sense or antisense oligonucleotides. The term "naturally occurring nucleotides" referred to herein includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotides" referred to herein includes nucleotides with modified or substituted sugar groups and the like. The term

"oligonucleotide linkages" referred to herein includes oligonucleotides linkages such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoroaniladate, phosphoroamidate, and the like. An oligonucleotide can include a label for detection, if desired. The term "sequence homology" referred to herein describes the proportion of base matches between two nucleic acid sequences or the proportion amino acid matches between two amino acid sequences. When sequence homology is expressed as a percentage, e.g., 50%, the percentage denotes the proportion of matches over the length of sequence from BRCA 1 that is compared to some other sequence. Gaps (in either of the two sequences) are permitted to maximize matching; gap lengths of 15 bases or less are usually used, 6 bases or less are preferred with 2 bases or less more preferred. When using oligonucleotides as probes or treatments the sequence homology between the target nucleic acid and the oligonucleotide sequence is generally not less than 17 target base matches out of 20 possible oligonucleotide base pair matches (85%); preferably not less than 9 matches out of 10 possible base pair matches (90%), and most preferably not less than 19 matches out of 20 possible base pair matches (95%). Two amino acid sequences are homologous if there is a partial or complete identity between their sequences. For example, 85% homology means that 85% of the amino acids are identical when the two sequences are aligned for maximum matching. Gaps (in either of the two sequences being matched) are allowed in maximizing matching; gap lengths of 5 or less are preferred with 2 or less being more preferred. Alternatively and preferably, two protein sequences (or polypeptide sequences derived from them of at least 30 amino acids in length) are homologous, as this term is used herein, if they have an alignment score of at more than 5 (in standard deviation units) using the program ALIGN with the mutation data matrix and a gap penalty of 6 or greater. See Dayhoff, M.O., in Atlas of Protein Sequence and Structure, 1972, volume 5, National Biomedical Research Foundation, pp. 101-110, and Supplement 2 to this volume, pp. 1-10. The two sequences or parts thereof are more preferably homologous if their amino acids are greater than or equal to 50% identical when optimally aligned using the ALIGN program. One of the properties of a BRCA1 Modulator Protein is the presence of a leucine zipper domain. The latter is defined as a stretch of amino acids rich in leucine residues, generally every seventh residue, which provide a means whereby a protein may dimerize to form either homodimers or heterodimers. Examples of proteins with leucine zippers include Jun and Fos. An optional property of a BRCA1 Modulator Protein is the presence of a zinc finger domain, preferrably of the type C,H₂C₃, C₃HC₄, or CX₂CX_{U 27}CXHX₂H or CX₂CX_{6 17}CX₂C; where C, X, and H denote cysteine, an amino acid, and histidine, respectively. The domain binds zinc ions, and is often associated with proteins that bind DNA. Such domains are readily identified using an appropriate data base known to a skilled practitioner of this art, particularly the Prosite Protein Database.

As used herein, "substantially pure" means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other macromolecular individual species in the composition), and preferably a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition will comprise more than about 80 percent of all macromolecular species present in the composition, more preferably more than about 85%, 90%, 95%, and 99%. Most preferably, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species. The phrases "Modulator Protein," "Modulator Peptide," or Modulator

Polypeptide" refer to proteins or peptides that affect the activity of the BRCA1 gene or the protein encoded by the gene. Each of these definitions is meant to encompass one or more such entities.

Chemistry terms herein are used according to conventional usage in the art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms (ed. Parker, S., 1985),

McGraw-Hill, San Francisco, incorporated herein by reference.

The production of proteins from cloned genes by genetic engineering is well known. See, e.g. U.S. Patent Number 4,761,371 to Bell et al. at column 6, line 3 to column 9, line 65. (The disclosure of all patent references cited herein is to be incorporated herein by reference.) The discussion which follows is accordingly intended as an overview of this field, and is not intended to reflect the full state of the art.

DNA regions are operably linked when they are functionally related to each other. For example: a promoter is operably linked to a coding sequence if it controls the transcription of the sequence; a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation. Generally, operably linked means contiguous and, in the case of leader sequences, contiguous and in reading frame.

Suitable host cells include prokaryotes, yeast cells, or higher eukaryotic cells. Prokaryotes include gram negative or gram positive organisms, for example Escherichia coli (E. colϊ) or Bacilli. Higher eukaryotic cells include established cell lines of mammalian origin as described below. Exemplary host cells are DH5a , £. coli W3110

(ATCC 27,325), £ coli B, £. coli X1776 (ATCC 31,537) and £. coli 294 (ATCC 31,446).

Pseudomonas species, Bacillus species, and Serratia marcesans are also suitable. In an insect system, Autographa californica nuclear polyhidrosis virus ( AcNPV) may be used as a vector to express foreign genes. (E.g., see Smith et al, 1983, J. Virol.

46: 584; Smith, U.S. Patent No. 4,215,051). In a specific embodiment described below, Sf9 insect cells are infected with a baculovirus vector expressing a glu-glu epitope tagged BRCA1 Modulator construct. See, Rubinfeld, et al., J. Biol. Chem. vol. 270, no. 10, pp 5549-5555 (1995). Other epitope tags may be employed that are known in the art including a 6x histidine tag , myc, or an EE-tag (i.e. Glu-Glu-tag). "E" refers to the amino acid glutamine.

A broad variety of suitable microbial vectors are available. Generally, a microbial vector will contain an origin of replication recognized by the intended host, a promoter which will function in the host and a phenotypic selection gene such as a gene encoding proteins conferring antibiotic resistance or supplying an autotrophic requirement. Similar constructs will be manufactured for other hosts. £. coli is typically transformed using pBR322. See Bolivar et al, Gene 2, 95 (1977). pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells. Expression vectors should contain a promoter which is recognized by the host organism. This generally means a promoter obtained from the intended host. Promoters most commonly used in recombinant microbial expression vectors include the beta-lactamase (penicillinase) and lactose promoter systems (Chang et al, Nature 275, 615 (1978); and Goeddel et al, Nucleic Acids Res. 8, 4057 (1980) and EPO Application Publication Number 36,776) and the tac promoter (H. De Boer et al, Proc. Natl. Acad. Sci. USA 80, 21 (1983)). While these are commonly used, other microbial promoters are suitable. Details concerning nucleotide sequences of many promoters have been published, enabling a skilled worker to operably ligate them to DNA encoding BRCA 1 in plasmid or viral vectors (Siebenlist et al, Cell 20, 269, 1980)). The promoter and Shine-Dalgarno (SD) sequence (for prokaryotic host expression) are operably linked to the DNA encoding BRCA 1, i.e. they are positioned so as to promote transcription of the BRCA 1 messenger RNA from the DNA. The SD sequence is thought to promote binding of mRNA to the ribosome by the pairing of bases between the SD sequence and the 3' end of E. coli 16S rRNA (Steitz et al. (1979). In Biological Regulation and Development: Gene Expression (ed. R.F. Goldberger)). To express eukaryotic genes and prokaryotic genes with a weak ribosome-binding site see Sambrook et al. (1989) "Expression of cloned genes in Escherichia coli." In Molecular Cloning: A Laboratory Manual. Furthermore, a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription. A naturally occurring promoter of non-bacterial origin can also be coupled with a compatible RNA polymerase to produce high levels of expression of some genes in prokaryotes. The bacteriophage T7 RNA polymerase/promoter system is an example of a coupled promoter system (Studier et al. (1986) /. Mol. Biol. 189:113; Tabor et al. (1985) Proc. Natl. Acad. Sci. 82:1074). In addition, a hybrid promoter can also be composed of a bacteriophage promoter and an E. coli operator region (EPO Pub. No. 267,851).

BRCA1 Modulators can be expressed intracellularly. A promoter sequence can be directly linked with a BRCA1 Modulator gene or a fragment thereof, in which case the first amino acid at the N-terminus will always be a methionine, which is encoded by the ATG start codon. If desired, methionine at the N-terminus can be cleaved from the protein by in vitro incubation with cyanogen bromide or by either in vivo on in vitro incubation with a bacterial methionine N-terminal peptidase (EPO Pub. No. 219,237). Eukaryotic microbes such as yeast cultures may be transformed with suitable BRCA1 Modulator vectors. See, e.g. U.S. Patent Number 4,745,057. Saccharomyces cerevisiae is the most commonly used among lower eukaryotic host microorganisms, although a number of other strains are commonly available. Yeast vectors may contain an origin of replication from the 2 micron yeast plasmid or an autonomously replicating sequence (ARS), a promoter, DNA encoding BRCA1 Modulator, sequences for polyadenylation and transcription termination, and a selection gene.

Suitable promoting sequences in yeast vectors include the promoters for metallothionein, 3-phosphogly cerate kinase (Hitzeman et al, J. Biol. Chem. 255, 2073 (1980) or other glycolytic enzymes (Hess et al, J. Adv. Enzyme Reg. 7, 149 (1968); and Holland et al., Biochemistry 17, 4900 (1978)), such as enolase, glyceraldehyde-3- phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Suitable vectors and promotes for use in yeast expression are further described in R. Hitzman et al, EPO Publication Number 73,657. Cultures of cells derived from multicellular organisms are a desirable host for recombinant BRCA1 Modulator synthesis. In principal, any higher eukaryotic cell culture is workable, whether from vertebrate or invertebrate culture. However, mammalian cells are preferred, as illustrated in the Examples. Propagation of such cells in cell culture has become a routine procedure. See Tissue Culture, Academic Press, Kruse and Paterson, editors (1973).

The transcriptional and translational control sequences in expression vectors to be used in transforming vertebrate cells are often provided by viral sources. For example, commonly used promoters are derived from CMV, polyoma, Adenovirus 2, and Simian Virus 40 (SV40). See, e.g., U.S. Patent Number 4,599,308.

An origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from SV40 or other viral source (e.g. Polyoma, Adenovirus, VSV, or BPV), or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter may be sufficient.

Identification of BRCA1 Modulators BRC Al Modulators can be identified using several different techniques for detecting protein-protein interactions. Among the traditional methods which may be employed are co-immunoprecipitation, crosslinking and co-purification through gradients or chromatographic columns of cell lysates, or proteins obtained from cell lysates using BRCA1 to identify proteins in the lysate that interact with BRCA1. Such assays may employ full length BRCA1 or a BRCA1 peptide. Once isolated, such an intracellular protein can be identified and can, in turn, be used, in conjunction with standard techniques, to identify proteins with which it interacts. For example, at least a portion of the amino acid sequence of an intracellular protein which interacts with BRCA1, can be ascertained using techniques well known to those of skill in the art, such as the Edman degradation technique. (See, e_^g., Creighton, 1983, "Proteins: Structures and Molecular Principles", W.H. Freeman & Co., N.Y., pp.34-49). The amino acid sequence obtained may be used as a guide for the generation of oligonucleotide mixtures that can be used to screen for gene sequences encoding such intracellular proteins. Screening may be accomplished, for example, by standard hybridization or PCR techniques. Techniques for the generation of oligonucleotide mixtures and the screening are well-known. (See, e.g., Ausubel, supra., and PR Protocols: A Guide to Methods and Applications, 1990, Innis, M. et al., eds. Academic Press, Inc., New York). Additionally, methods may be employed which result in the simultaneous identification of genes which encode the intracellular proteins interacting with BRCAl. These methods include, for example, probing expression libraries, in a manner similar to the well known technique of antibody probing of λgtll libraries, using labeled BRCAl protein, or fusion protein, e.g., BRCAl fused to a marker (e.g., an enzyme, fluor, luminescent protein, or dye), or an Ig-Fc domain.

One method which detects protein interactions in vivo, the two-hybrid system is described in detail for illustration only and not by way of limitation. This system has been described ( U. S. Patent No. 5, 283, 173 Chien et al., 1991, Proc. Natl. Acad. Sci. USA, 88:9578-9582) and is commercially available from Clontech (Palo Alto, CA). Briefly, utilizing such a system, plasmids are constructed that encode two hybrid proteins: one plasmid consists of nucleotides encoding the DNA-binding domain of a transcription activator protein fused to a BRCAl nucleotide sequence encoding BRCAl, or BRCAl peptide or fusion protein, and the other plasmid consists of nucleotides encoding the transcription activator protein's activation domain fused to a cDNA encoding an unknown protein which has been recombined into this plasmid as a part of the cDNA library. The DNA-binding domain fusion plasmid and the cDNA library are transformed into a strain of the yeast Saccharomyces cerevisiae that contains a reporter gene (e.g., HIS3 or lacZ) whose regulatory region contain the transcription activator's binding site. Either hybrid protein alone cannot activate transcription of the reporter gene; the DNA-binding domain hybrid cannot because it does not provide activation function, and the activation domain hybrid cannot because it cannot localize to the activator's binding sites. Interaction of the two hybrid proteins reconstitutes the functional activator protein and results in transcriptional activation of the reporter gene, which is detected by an assay for the reporter gene product.

The two-hybrid system or related methodology may be used to screen activation domain libraries for proteins that interact with the "bait" gene product. By way of example, and not by way of limitation, preferrably BRCAl peptides, or fusion proteins are used as the bait gene product. Full length BRCAl alone can act as a transcriptional activator protein and thus cannot serve as "bait." Total genomic or cDNA sequences are fused to the DNA encoding an activation domain. This library and a plasmid encoding a hybrid of a bait BRCAl gene product fused to the DNA-binding domain are cotransformed into a yeast reporter strain, and the resulting tranformants are screened for those that have transcriptionally activated reporter gene. For example, and not by way of limitation, a bait BRCAl gene sequence, such as the open reading frame of BRCAl (or a domain of BRCAl) can be cloned into a vector such that it is translationally fused to the DNA encoding the DNA-binding domain of the GAL4 protein. These colonies are purified and the library plasmids responsible for reporter gene transcription are isolated. DNA sequencing is then used to determine the nucleotide sequence of the clones which, in turn, reveals the identity of the protein sequences encoded by the library plasmids. A cDNA library of the cell line from which proteins that interact with bait

BRCAl gene product are to be detected can be made using methods routinely practiced in the art. According to the particular system described herein, for example, the cDNA fragments can be inserted into a vector such that they are translationally fused to the transcriptional activation domain of GAL4. This library can be co-transformed along with the bait BRCAl gene-GAL4 fusion plasmid into a yeast strain which contains a lacZ gene driven by a promoter which contains GAL4 activation sequence. A cDNA encoded protein, fused to GAL4 transcriptional activation domain, that interacts with bait BRCAl gene product will reconstitute an active GAL4 protein and thereby drive expression of the HIS3 gene. Colonies which express HIS3 can be detected by their growth on petri dishes containing semi-solid agar based media lacking histidine. The cDNA can then be purified from these strains, and used to produce and isolate the bait BRCAl gene-interacting protein using techniques routinely practiced in the art.

Using the above described two-hybrid technique several BRCAl modulators were identified, and shown to share certain properties including a leucine zipper domain.

BRCAl Modulator cDNA The cDNA, and deduced amino acid sequences, of three representative BRCAl Modulator Proteins are shown in Figures 1-3. The cDNAs or the proteins that they encode are hereinafter referred to as 091-21 A31, Sequence ID No. 1, 091-1F84, Sequence ID No. 3, and 091-132Q20, Sequence ID No. 5. The cDNAs encode proteins that have calculated molecular weights in the range of about 45-97kd. Particularly noteworthy is the presence of at least one leucine zipper motif, and optionally a zinc finger domain. The BRCAl Modulator Protein nucleotide sequences of the invention include: (a)the DNA sequences shown in Figures 1-3 or contained in the cDNA clones as deposited with the American Type Culture Collection on August 14, 1996 (ATCC) under accession numbers 98141 (091-1F84, Sequence ID No. 3), 98142 (091-21 A31, Sequence ID No. 1), and 98143 (091-132Q20, Sequence ID No. 5); (b) and any nucleotide sequence that hybridizes to the complement of the DNA sequence shown in Figures 1-3 or contained in the cDNA clones as deposited with the ATCC under highly stringent conditions, e.g., hybridization to filter-bound DNA in 0.5 M NaHPO 7% sodium dodecyl sulfate (SDS), ImM EDTA at 65°C, and washing in O.lxSSC/0.1% SDS at 68°C (Ausubel F.M. et al., eds., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc., and John Wiley & sons, Inc., New York, at p. 2.10.3) and encodes a functionally equivalent gene product; and (c) any nucleotide sequence that hybridizes to the complement of the DNA sequences that encode the amino acid sequence shown in Figures 1-3 or contained in the cDNA clones as deposited with the ATCC, as described above, under less stringent conditions, such as moderately stringent conditions, g., washing in 0.2xSSC/0.1% SDS at 42°C (Ausubel et al., 1989, supra), yet which still encodes a functionally equivalent BRCAl Modulator Protein gene product. Functional equivalents include naturally occurring BRCAl Modulator Protein genes present in other species, and mutant BRCAl Modulator Protein genes whether naturally occurring or engineered which retain at least some of the functional activities of a BRCAl Modulator Protein (i.e., binding to BRCAl). The invention also includes degenerate variants of sequences (a) through (c).

The invention also includes nucleic acid molecules, preferably DNA molecules, that hybridize to, and are therefore the complements of, the nucleotide sequences (a)- through (c), in the preceding paragraph. Such hybridization conditions may be highly stringent or less highly stringent, as described above. In instances wherein the nucleic acid molecules are deoxyoligonucleotides ("oligos"), highly stringent conditions may refer, e^, to washing in 6xSSC/0.05% sodium pyrophosphate at 37°C (for 14-base oligos), 48°C (for 17-base oligos), 55°C (for 20-base oligos), and 60°C (for 23-base oligos). These nucleic acid molecules may encode or act as BRCAl Modulator gene antisense molecules, useful, for example, in gene regulation (for and/or as antisense primers in amplification reactions of BRCAl Modulator gene nucleic acid sequences). Such sequences may be used as part of ribozyme and /or triple helix sequences, also useful for BRCAl Modulator gene regulation. Still further, such molecules may be used as components of diagnostic methods whereby, for example, the presence of a particular BRCAl Modulator allele associated with uncontrolled cell growth (i.e. cancer) may be detected.

Further, it will be appreciated by one skilled in the art that a BRCAl Modulator gene homolog may be isolated from nucleic acid of an organism of interest by performing PCR using two degenerate oligonucleotide primer pools designed on the basis of amino acid sequences within the BRCAl Modulator gene product disclosed herein. The template for the reaction may be cDNA obtained by reverse transcription of mRNA prepared from, for example, human or non-human cell lines or cell types, such as breast or ovarian cells, known or suspected to express a BRCAl Modulator gene allele. The PCR product may be subcloned and sequenced to ensure that the amplified sequences represent the sequences of a BRCAl Modulator gene. The PCR fragment may then be used to isolate a full length cDNA clone by a variety of methods. For example, the amplified fragment may be labeled and used to screen a cDNA library, such as a bacteriophage cDNA library. Alternatively, the labeled fragment may be used to isolate genomic clones via the screening of a genomic library.

PCR technology may also be utilized to isolate full length cDNA sequences. For example, RNA may be isolated, following standard procedures, from an appropriate cellular source (i.e.. one known, or suspected, to express a BRCAl Modulator gene, such as, for example, from breast or ovarian cells). A reverse transcription reaction may be performed on the RNA using an oligonucleotide primer specific for the most 5' end of the amplified fragment for the priming of first strand synthesis. The resulting RNA/DNA hybrid may then be "tailed" with guanines using a standard terminal transferase reaction, the hybrid may be digested with RNAase H, and second strand synthesis may then be primed with a poly-C primer. Thus, cDNA sequences upstream of the amplified fragment may easily be isolated. For a review of cloning strategies which may be used, see e.g., Sambrook et al., 1989, supra. A cDNA of a mutant BRCAl Modulator gene may also be isolated, for example, by using PCR. In this case, the first cDNA strand may be synthesized by hybridizing an oligo-dT oligonucleotide to mRNA isolated from cells known or suspected to be expressed in an individual putatively carrying the mutant BRCAl Modulator allele, and by extending the new strand with reverse transcriptase. The second strand of the cDNA is then synthesized using an oligonucleotide that hybridizes specifically to the 5' end of the normal gene. Using these two primers, the product is then amplified via PCR, cloned into a suitable vector, and subjected to DNA sequence analysis through methods well known to those of skill in the art. By comparing the DNA sequence of the mutant BRCAl Modulator allele to that of the normal BRCAl Modulator allele, the mutation(s) responsible for the loss or alteration of function of the mutant BRCAl Modulator gene product can be ascertained.

A genomic library can be constructed using DNA obtained from an individual suspected of or known to carry the mutant BRCAl Modulator allele, or a cDNA library can be constructed using RNA from a cell type known, or suspected, to express the mutant BRCAl Modulator allele. The normal BRCAl Modulator gene or any suitable fragment thereof may then be labeled and used as a probe to identify the corresponding mutant BRCAl Modulator allele in such libraries. Clones containing the mutant BRCAl Modulator gene sequences may then be purified and subjected to sequence analysis according to methods well known to those of skill in the art.

Additionally, an expression library can be constructed utilizing cDNA synthesized from, for example, RNA isolated from a cell type known, or suspected, to express a mutant BRCAl Modulator allele in an individual suspected of or known to carry such a mutant allele. In this manner, gene products made by the putatively mutant cell type may be expressed and screened using standard antibody screening techniques in conjunction with antibodies raised against the normal BRCAl Modulator gene product, as described, below. (For screening techniques, see, for example, Harlow, E. and Lane, eds., 1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor Press, Cold Spring Harbor.) Additionally, screening can be accomplished by screening with labeled fusion proteins. In cases where a BRCAl Modulator mutation results in an expressed gene product with altered function (e.g., as a result of a missense or a frameshift mutation), a polyclonal set of antibodies to a BRCAl Modulator are likely to cross-react with the BRCAl Modulator mutant. Such BRCAl Modulator mutants detected via their reaction with labeled antibodies can be purified and subjected to sequence analysis according to methods well known to those of skill in the art. The invention also encompasses nucleotide sequences that encode peptide fragments of a BRCAl Modulator, truncated BRCAl Modulators, and fusion proteins of a BRCAl Modulator. Nucleotides encoding fusion proteins may include but are not limited to full length BRCAl Modulators, truncated BRCAl Modulators or peptide fragments to an unrelated protein or peptide, such as for example, an epitope tag which aids in purification or detection of the resulting fusion protein; or an enzyme, fluorescent protein, luminescent protein which can be used as a marker. The preferred epitope tag is glu-glu as described by Rubinfeld, B., et al., J. Biol. Chem. vol. 270, no. 10, pp 5549-5555 (1995), and Grussenmyer, T., et al., Proc. Natl. Acad. Sci. U. S. A. vol. 82, pp. 7952-7954 (1985).

The invention also encompasses (a) DNA vectors that contain any of the foregoing BRCAl Modulator coding sequences and /or their complements (i.e., antisense); (b) DNA expression vectors that contain any of the foregoing BRCAl Modulator coding sequences operatively associated with a regulatory element that directs the expression of the coding sequences; and (c) genetically engineered host cells that contain any of the foregoing BRCAl Modulator coding sequences operatively associated with a regulatory element that directs the expression of the coding sequences in the host cell. As used herein, regulatory elements include but are not limited to inducible and non-inducible promoters, enhancers, operators and other elements known to those skilled in the art that drive and regulate expression. Such regulatory elements include but are not limited to the baculovirus promoter, cytomegalovirus hCMV immediate early gene, the early or late promoters of SV40 adenovirus, the lac system, the trp_ system, the TAC system, the TRC system, the major operator and promoter regions of phage A, the control regions of fd coat protein, the promoter for 3- phosphoglycerate kinase, the promoters of acid phosphatase, and the promoters of the yeast-mating factors. BRCAl Modulator Proteins

As mentioned above, Figures 1-3 shows the cDNA, and deduced amino acid sequences, of three representative BRCAl Modulator Proteins; 091-21A31, Sequence ID No. 1, 091-1F84, Sequence ID No. 3, and 091-132Q20, Sequence ID No. 5. 091-132Q20, Sequence ID No. 5 is not a full length sequence. The proteins have calculated molecular weights in the range of about 45-97kd. Particularly noteworthy is the presence of at least one leucine zipper motif, and optionally a zinc finger domain. For instance, 091-1F84, Sequence ID No. 3 has two leucine zippers, 091-132Q20, Sequence ID No. 5 has a single leucine zipper, while 091-21A31, Sequence ID No. 1 has a single leucine zipper and a zinc finger domain. Such domains are readily identified using the Prosite Protein Database.

The invention BRCAl Modular Proteins, peptide fragments, mutated, truncated or deleted forms of and fusion proteins of these can be prepared for a variety of uses, including but not limited to the generation of antibodies, as reagents in diagnostic assays, the identification and /or the interaction with other cellular gene products involved in cell growth, as reagents in assays for screening for compounds that can be used in the treatment of unwanted cell growth disorders, including but not limited to cancer, and as pharmaceutical reagents useful in the treatment of such diseases.

By way of example, the 091-21 A31, Sequence ID No. 1 BRCAl Modulator Protein sequence begins with a methionine in a DNA sequence context consistent with a translation initiation site. The predicted molecular mass of this BRCAl Modulator Protein is 53.3 kD. The BRCAl Modulator Protein amino acid sequences of the invention include the amino acid sequence shown in FIG. 1, or the amino acid sequence encoded by the cDNA clone, as deposited with the ATCC, as described above. Further, BRCAl Modulator Proteins of other species are encompassed by the invention. In fact, any BRCAl Modulator Protein protein encoded by the cDNAs described above, are within the scope of the invention.

The invention also encompasses proteins that are functionally equivalent to the BRCAl Modulator Protein encoded by the nucleotide sequences described above, as judged by any of a number of criteria, including but not limited to the ability to bind BRCAl, the binding affinity for BRCAl , a change in cellular metabolism or change in phenotype when the BRCAl Modulator Protein equivalent is present in an appropriate cell type (such as ovarian or breast cells). Such functionally equivalent BRCAl Modulator Protein proteins include but are not limited to additions or substitutions of amino acid residues within the amino acid sequence encoded by the BRCAl Modulator nucleotide sequences described, above, but which result in a silent change, thus producing a functionally equivalent gene product. Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and /or the amphipathic nature of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

While random mutations can be made to BRCAl Modulator DNA (using random mutagenesis techniques well known to those skilled in the art) and the resulting mutant BRCAl Modulator Proteins tested for activity, site-directed mutations of the BRCAl Modulator coding sequence can be engineered (using site-directed mutagenesis techniques well known to those skilled in the art) to generate mutant BRCAl Modulator Proteins with increased function, e.g., altered binding affinity for BRCAl.

For example, mutant BRCAl Modulator Proteins can be engineered so that regions of interspecies identity are maintained, whereas the variable residues are altered, e.g., by deletion or insertion of an amino acid residue(s) or by substitution of one or more different amino acid residues. Conservative alterations at the variable positions can be engineered in order to produce a mutant BRCAl Modulator Protein that retains function. Non-conservative changes can be engineered at these variable positions to alter function. Alternatively, where alteration of function is desired, deletion or non-conservative alterations of the conserved regions can be engineered. One of skill in the art may easily test such mutant or deleted BRCAl Modulator Proteins for these alterations in function using the teachings presented herein.

Other mutations to a BRCAl Modulator coding sequence can be made to generate BRCAl Modulator Proteins that are better suited for expression, scale up, etc. in the host cells chosen. For example, the triplet code for each amino acid can be modified to conform more closely to the preferential codon usage of the host cell's translational machinery. Peptides corresponding to one or more domains (or a portion of a domain) of a BRCAl Modulator Protein (e.g., leucine zippers, zinc fingers), truncated or deleted BRCAl Modulator Proteins (e.g., BRCAl Modulator Proteins in which portions of one or more of the above domains are deleted) as well as fusion proteins in which the full length of a BRCAl Modulator Protein, a BRCAl Modulator Protein peptide or truncated BRCAl Modulator Protein is fused to an unrelated protein are also within the scope of the invention and can be designed on the basis of a BRCAl Modulator nucleotide and BRCAl Modulator Protein amino acid sequences disclosed in this Section and above. Such fusion proteins include but are not limited to fusions to an epitope tag (such as is exemplified herein); or fusions to an enzyme, fluorescent protein, or luminescent protein which provide a marker function.

While the BRCAl Modulator Proteins and peptides can be chemically synthesized (e.g., see Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N.Y.), large polypeptides derived from the BRCAl Modulator Protein and the full length BRCAl Modulator Protein itself may advantageously be produced by recombinant DNA technology using techniques well known in the art for expressing nucleic acid containing BRCAl Modulator gene sequences and/or coding sequences. Such methods can be used to construct expression vectors containing the BRCAl Modulator nucleotide sequences described above and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. See, for example, the techniques described in Sambrook et al., 1989, supra, and Ausubel et al., 1989, supra. Alternatively, RNA capable of encoding BRCAl Modulator nucleotide sequences may be chemically synthesized using, for example, synthesizers. See, for example, the techniques described in "Oligonucleotide Synthesis", 1984, Gait, M.J. ed., IRL Press, Oxford, which is incorporated by reference herein in its entirety.

A variety of host-expression vector systems may be utilized to express the BRCAl Modulator nucleotide sequences of the invention. Where a BRCAl Modulator Protein peptide or polypeptide is a soluble secreted derivative the peptide or polypeptide can be recovered from the culture medium. If the BRCAl Modulator Protein peptide or polypeptide is not secreted, it may be isolated from the host cells. However, such engineered host cells themselves may be used in situations where it is important not only to retain the structural and functional characteristics of a BRCAl Modulator Protein, but to assess biological activity, e.g., in drug screening assays.

The expression systems that may be used for purposes of the invention include but are not limited to microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing BRCAl Modulator nucleotide sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing the BRCAl Modulator nucleotide sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the BRCAl Modulator sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing BRCAl Modulator nucleotide sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3, U937) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).

In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the BRCAl Modulator gene product being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of BRCAl Modulator Protein or for raising antibodies to the BRCAl Modulator Protein, for example, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which the BRCAl Modulator coding sequence may be ligated individually into the vector in frame with the lacZ coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). If the inserted sequence encodes a relatively small polypeptide (less than 25 kD), such fusion proteins are generally soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety. Alternatively, if the resulting fusion protein is insoluble and forms inclusion bodies in the host cell, the inclusion bodies may be purified and the recombinant protein solubilized using techniques well known to one of skill in the art.

In an insect system, Autographa californica nuclear polyhidrosis virus (AcNPV) may be used as a vector to express foreign genes. (E.g., see Smith et al., 1983, J. Virol. 46: 584; Smith, U.S. Patent No. 4,215,051). In a specific embodiment described below, Sf9 insect cells are infected with a baculovirus vectors expressing either a 6 x HIS- tagged construct, or an (EE)-tagged BRCAl Modulator construct.

In mammalian host cells, a number of viral-based expression systems may be utilized. Specific embodiments described more fully below express tagged BRCAl Modulator cDNA sequences using a CMV promoter to transiently express recombinant protein in U937 cells or in Cos-7 cells. Alternatively, retroviral vector systems well known in the art may be used to insert the recombinant expression construct into host cells. For example, retroviral vector systems for transducing hematopoietic cells are described in published PCT applications WO 96/09400 and WO 94/29438. In cases where an adenovirus is used as an expression vector, the BRCAl

Modulator nucleotide sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g.. the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing the BRCAl Modulator gene product in infected hosts. (E.g., See Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:3655-3659). Specific initiation signals may also be required for efficient translation of inserted BRCAl Modulator nucleotide sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where an entire BRCAl Modulator gene or cDNA, including its own initiation codon and adjacent sequences, is inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only a portion of the BRCAl Modulator coding sequence is inserted, exogenous translational control signals, including, perhaps, the ATG initiation codon, must be provided. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (See Bittner et al., 1987, Methods in Enzymol. 153:516-544).

In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript may be used. Such mammalian host cells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and U937 cells. For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the BRCAl Modulator sequences described above may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g.. promoter, enhancer sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form colonies which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the BRCAl Modulator gene product. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that affect the endogenous activity of the BRCAl Modulator gene product.

A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine- guanine phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can be employed in tk^", hgprt^" or aprt^" cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Natl. Acad. Sci. USA 77:3567; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1); and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147). The BRCAl Modulator gene products can also be expressed in transgenic animals. Animals of any species, including, but not limited to, mice, rats, rabbits, guinea pigs, pigs, micro-pigs, goats, and non-human primates, e.g., baboons, monkeys, and chimpanzees may be used to generate BRCAl Modulator transgenic animals.

Any technique known in the art may be used to introduce the BRCAl Modulator transgene into animals to produce the founder lines of transgenic animals. Such techniques include, but are not limited to pronuclear microinjection (Hoppe, P.C. and Wagner, T.E., 1989, U.S. Pat. No. 4,873,191); retrovirus mediated gene transfer into germ lines (Van der Putten et al, 1985, Proc. Natl. Acad. Sci., USA 82:6148-6152); gene targeting in embryonic stem cells (Thompson et al., 1989, Cell 56:313-321); electroporation of embryos (Lo, 1983, Mol Cell. Biol. 3:1803-1814); and sperm-mediated gene transfer (Lavitrano et al., 1989, Cell 57:717-723); etc. For a review of such techniques, see Gordon, 1989, Transgenic Animals, Intl. Rev. Cytol. 115:171-229, which is incorporated by reference herein in its entirety.

The present invention provides for transgenic animals that carry the BRCAl Modulator transgene in all their cells, as well as animals which carry the transgene in some, but not all their cells, Li , mosaic animals. The transgene may be integrated as a single transgene or in concatamers, e.g., head-to-head tandems or head-to-tail tandems. The transgene may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al. (Lasko, M. et al., 1992, Proc. Natl. Acad. Sci. USA 89: 6232-6236). The regulatory sequences required for such a cell- type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art. When it is desired that the BRCAl Modulator transgene be integrated into the chromosomal site of the endogenous BRCAl Modulator gene, gene targeting is preferred. Briefly, when such a technique is to be utilized, vectors containing some nucleotide sequences homologous to the endogenous BRCAl Modulator gene are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the nucleotide sequence of the endogenous BRCAl Modulator gene. In this way, the expression of the endogenous BRCAl Modulator gene may also be eliminated by inserting non-functional sequences into the endogenous gene. The transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous BRCAl Modulator gene in only that cell type, by following, for example, the teaching of Gu et al. (Gu et al., 1994, Science 265: 103-106). The regulatory sequences required for such a cell-type specific inactivation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art. Once transgenic animals have been generated, the expression of the recombinant

BRCAl Modulator gene may be assayed utilizing standard techniques. Initial screening may be accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to assay whether integration of the transgene has taken place. The level of mRNA expression of the transgene in the tissues of the transgenic animals may also be assessed using techniques which include but are not limited to Northern blot analysis of cell type samples obtained from the animal, in situ hybridization analysis, and RT-PCR. Samples of BRCAl Modulator gene-expressing tissue, may also be evaluated immunocytochemically using antibodies specific for the BRCAl Modulator transgene product, as described below. Antibodies to BRCAl Modulator Proteins

Antibodies that specifically recognize one or more epitopes of a BRCAl Modulator Protein, or epitopes of conserved variants, or peptide fragments are also encompassed by the invention. Such antibodies include but are not limited to polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab')2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope- binding fragments of any of the above.

The antibodies of the invention may be used, for example, in the detection of the BRCAl Modulator Protein in a biological sample and may, therefore, be utilized as part of a diagnostic or prognostic technique whereby patients may be tested for abnormal amounts of these proteins. Such antibodies may also be utilized in conjunction with, for example, compound screening schemes, as described herein for the evaluation of the effect of test compounds on expression and/or activity of the BRCAl Modulator Protein. Additionally, such antibodies can be used in conjunction with the gene therapy techniques described herein, to, for example, evaluate the normal and /or engineered BRCAl Modulator Protein expressing cells prior to their introduction into the patient. Such antibodies may additionally be used as a method for the inhibition of abnormal BRCAl Modulator Protein activity.

For the production of antibodies, various host animals may be immunized by injection with the BRCAl Modulator Protein, a BRCAl Modulator Protein peptide, truncated BRCAl Modulator Protein polypeptides, functional equivalents of the BRCAl Modulator Protein or mutants of the BRCAl Modulator Protein. Such host animals may include but are not limited to rabbits, mice, and rats, to name but a few. Various adjutants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjutants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of the immunized animals. Monoclonal antibodies, which are homogeneous populations of antibodies to a particular antigen, may be obtained by any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique of Kohler and Milstein, (1975, Nature 256:495-497; and U.S. Patent No. 4,376,110), the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA 80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridoma producing the mAb of this invention may be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo makes this the presently preferred method of production.

In addition, techniques developed for the production of "chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad. Sci. USA, 81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda et al., 1985, Nature, 314:452-454) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a mAb and a human immunoglobulin constant region.

Alternatively, techniques described for the production of single chain antibodies (U.S. Patent 4,946,778; Bird, 1988, Science 242:423-426; Huston et al, 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-546) can be adapted to produce single chain antibodies against BRCAl Modulator Protein gene products. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Antibody fragments which recognize specific epitopes may be generated by known techniques. For example, such fragments include but are not limited to: the F(ab')2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries may be constructed (Huse et al., 1989, Science, 246:1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. Antibodies to the BRCAl Modulator Protein can, in turn, be utilized to generate anti-idiotype antibodies that "mimic" the BRCAl Modulator Protein using techniques well known to those skilled in the art. (See, e.g., Greenspan & Bona, 1993, FASEB J 7(5):437-444; and Nissinoff, 1991, J. Immunol. 147(8):2429-2438). Identification of Compounds that Increase BRCAl

Levels using BRCAl Modulators The BRCAl gene encodes a protein that has been shown to have tumor suppressor activity. See, Holt, J. T., et al, (1996) Nat. Genet, vol. 12, pages 298-302. Such studies have shown that certain cancer cells have low levels of BRCAl, and that increasing the levels causes a reversion to the normal cell phenotype. Thus, compounds that increase BRCAl levels will have significant therapeutic use for the treatment of cancer.

An aspect of the instant invention is the description of an assay using BRCAl and BRCAl Modulators that facilitates the identification of compounds that increase intracellular levels of BRCAl. One format of the assay is shown in schematic form in Figure 4. Briefly, the assay makes use of two events: firstly, BRCAl is known to be a general transcriptional activator, and secondly, BRCAl Modulators bind to BRCAl. The assay makes use of certain features of the two-hybrid assay described above. Two plasmids are constructed and transfected into a suitable cell line, preferrably a breast or ovarian cell line. A preferred breast cell line would be MCF-7. One plasmid contains the nucleotide sequence recognized by GAL4 operably linked to an activator sequence, and a reporter gene downstream of this sequence. An example of a preferred reporter gene is the gene that encodes luciferase. The second plasmid encodes and expresses the GAL4 DNA binding domain fused to a BRCAl Modulator. The preferred Modulator is 091-21 A31, Sequence ID No. 1.

The GAL4 DNA binding domain-BRCAl Modulator fusion protein binds to the GAL4 DNA binding domain on the first plasmid which, in turn, recruits any BRCAl present to form a complex consisting of GAL4 DNA binding domain-BRCAl Modulator fusion and BRCAl. As part of the complex, BRCAl is in proximity to the activator sequence which in turn initiates transcription of the reporter gene. Thus, compounds can be tested for their capacity to stimulate the production of BRCAl. Those that do will cause an increase in the reporter gene product. The above assay is schematically presented in Figure 4.

Identification of Compounds that alter BRCAl Interaction with BRCAl Modulators As mentioned above, BRCAl is a known tumor suppressor. See, Holt, J. T., et al,

(1996) Nat. Genet, vol. 12, pages 298-302. Thus compounds that affect the normal interaction of BRCAl with BRCAl Modulator Proteins may affect the tumor suppressor activity of BRCAl. The extent of the effect will, in large part, depend on the chemical properties of the compounds tested. Some may strongly disrupt the interaction of BRCAl with BRCAl Modulator Proteins, while others would have a minimal effect. The former would be reflected in a biological assay for altered tumorgenicity, while the latter would not. The converse is also true, certain compounds may strengthen the interaction of BRCAl with BRCAl Modulator Proteins, in which case the opposite biological effect would be anticipated. Thus, it is highly desirable to assay for compounds that affect BRCAl interactions with BRCAl Modulator Protein.

The basic principle of the assay systems used to identify such compounds that affect BRCAl interactions with BRCAl Modulator Proteins involves preparing a reaction mixture containing BRCAl protein, polypeptide, peptide or fusion protein as described above, and a BRCAl Modulator Protein under conditions and for a time sufficient to allow the two to interact and bind, thus forming a complex. In order to test a compound for inhibitory activity, the reaction mixture is prepared in the presence and absence of the test compound. The test compound may be initially included in the reaction mixture, or may be added at a time subsequent to the addition of the BRCAl moiety and its BRCAl Modulator Protein. Control reaction mixtures are incubated without the test compound or with a placebo. The formation of any complexes between the BRCAl moiety and the BRCAl Modulator Protein is then detected. The formation of a complex in the control reaction, but not in the reaction mixture containing the test compound, indicates that the compound interferes with the interaction of the BRCAl and the interactive BRCAl Modulator Protein. Additionally, complex formation within reaction mixtures containing the test compound and normal BRCAl protein may also be compared to complex formation within reaction mixtures containing the test compound and a mutant BRCAl. This comparison may be important in those cases wherein it is desirable to identify compounds that disrupt interactions of mutant but not normal BRCAl.

The assay for compounds that interfere with the interaction of the BRCAl and BRCAl Modulator Proteins can be conducted in a heterogeneous or homogeneous format. Heterogeneous assays involve anchoring either the BRCAl moiety or the

BRCAl Modulator Protein onto a solid phase and detecting complexes anchored on the solid phase at the end of the reaction. In homogeneous assays, the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested. For example, test compounds that interfere with the interaction by competition can be identified by conducting the reaction in the presence of the test substance; e_., by adding the test substance to the reaction mixture prior to or simultaneously with the BRCAl moiety and interactive BRCAl Modulator Protein. Alternatively, test compounds that disrupt preformed complexes, e.g. compounds with higher binding constants that displace one of the components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed. Representative formats are described briefly below.

In a heterogeneous assay system, either the BRCAl moiety or the interactive BRCAl Modulator Protein, is anchored onto a solid surface, while the non-anchored species is labeled, either directly or indirectly. In practice, microtiter plates are conveniently utilized. The anchored species may be immobilized by non-covalent or covalent attachments. Non-covalent attachment may be accomplished simply by coating the solid surface with a solution of BRCAl or BRCAl Modulator Protein and drying. Alternatively, an immobilized antibody specific for the species to be anchored may be used to anchor the species to the solid surface. The surfaces may be prepared in advance and stored.

In order to conduct the assay, the partner of the immobilized species is exposed to the coated surface with or without the test compound. After the reaction is complete, unreacted components are removed (e.g.. by washing) and any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the non-immobilized species is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the non-immobilized species is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the initially non-immobilized species (the antibody, in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody). Depending upon the order of addition of reaction components, test compounds which inhibit complex formation or which disrupt preformed complexes can be detected.

Alternatively, the reaction can be conducted in a liquid phase in the presence or absence of the test compound, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for one of the binding components to anchor any complexes formed in solution, and a labeled antibody specific for the other partner to detect anchored complexes. Again, depending upon the order of addition of reactants to the liquid phase, test compounds which inhibit complex or which disrupt preformed complexes can be identified.

In an alternate embodiment of the invention, a homogeneous assay can be used. In this approach, a preformed complex of the BRCAl moiety and the interactive BRCAl Modulator Protein is prepared in which either the BRCAl or its BRCAl Modulator Proteins is labeled, but the signal generated by the label is quenched due to formation of the complex (see, e.g., U.S. Patent No.4,109,496 by Rubenstein which utilizes this approach for immunoassays). The addition of a test substance that competes with and displaces one of the species from the preformed complex will result in the generation of a signal above background. In this way, test substances which disrupt BRCAl /intracellular BRCAl Modulator Protein interaction can be identified.

In a particular embodiment, a BRCAl fusion protein can be prepared for immobilization. For example, BRCAl or a peptide fragment can be fused to a glutathione-S-transferase (GST) gene using a fusion vector, such as pGEX-5X-l, in such a manner that its binding activity is maintained in the resulting fusion protein. The interactive BRCAl Modulator Protein can be purified and used to raise a monoclonal antibody, using methods routinely practiced in the art and described above. This antibody can be labeled with the radioactive isotope l-^I, for example, by methods routinely practiced in the art. In a heterogeneous assay, e.g., the GST-BRCA1 fusion protein can be anchored to glutathione-agarose beads. The interactive BRCAl Modulator Protein can then be added in the presence or absence of the test compound in a manner that allows interaction and binding to occur. At the end of the reaction period, unbound material can be washed away, and the labeled monoclonal antibody can be added to the system and allowed to bind to the complexed components. The interaction between the BRCAl protein and the interactive BRCAl Modulator Protein can be detected by measuring the amount of radioactivity that remains associated with the glutathione-agarose beads. A successful inhibition of the interaction by the test compound will result in a decrease in measured radioactivity.

Alternatively, the GST-BRCA1 fusion protein and the interactive BRCAl Modulator Protein can be mixed together in liquid in the absence of the solid glutathione-agarose beads. The test compound can be added either during or after the species are allowed to interact. This mixture can then be added to the glutathione- agarose beads and unbound material is washed away. Again the extent of inhibition of the BRCAl /BRCAl Modulator Protein interaction can be detected by adding the labeled antibody and measuring the radioactivity associated with the beads.

In another embodiment of the invention, these same techniques can be employed using peptide fragments that correspond to the binding domains of the BRCAl and /or the interactive or BRCAl Modulator in place of one or both of the full length proteins. Any number of methods routinely practiced in the art can be used to identify and isolate the binding domains. Such domains are discussed more fully in the examples, below. These methods include, but are not limited to, mutagenesis of the gene encoding one of the proteins and screening for disruption of binding in a co-immunoprecipitation assay. Compensating mutations in the gene encoding the second species in the complex can then be selected. Sequence analysis of the genes encoding the respective proteins will reveal the mutations that correspond to the region of the protein involved in interactive binding. The two hybrid assay may also be used, as discussed more fully in the examples below. For instance, once the gene coding for the intracellular BRCAl Modulator Protein is obtained, short gene segments can be engineered to express peptide fragments of the protein, which can then be tested for binding activity and purified or synthesized. Effective Dose

Toxicity and therapeutic efficacy of compounds identified above that affect the interaction of BRCAl with BRCAl Modulator Proteins, and thus affect cell growth can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD5_Q (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). Numerous model systems are known to the skilled practitioner of the art that can be employed to test the cell growth properties of the instant compounds including growth of cells in soft agar, and effect on tumors in vivo. Such experiments can be conducted on cells co- transfected with BRCAl and BRCAl Modulators

The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD5_Q/ED5_Q. Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC5_Q (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

The Examples which follow are illustrative of specific embodiments of the invention, and various uses thereof. They are set forth for explanatory purposes only, and are not to be taken as limiting the invention. Example 1 Identification of cDNAs that Encode BRCAl Modulator Proteins

BRCAl modulators were identified initially using the yeast two hybrid assay system described in U. S. Patent No. 5, 283, 173, or Chien et al, 1991, Proc. Natl. Acad. Sci. USA, 88:9578-9582. The assay components are also commercially available from Clontech (Palo Alto, CA).

The cDNA encoding human BRCAl (See, Miki, Y., et al Science, vol. 266: 66-71; and PCT /US95/ 10202) was digested with Mvnl-Nhel and the fragment representing BRCAl amino acids 8-1293 was fused to the GAL4 binding domain in the Smal-Nhel sites of pGBT8 plasmid, which is the pMA424 plasmid of Chien et al. as described in Proc. Natl. Acad. Sci. vol. 88: pages 9578-9582 (1991), modified by the insertion of the sequence 5'-

CCGGGGATCCCCATGGCTAGCCATATG-3' between the EcoRI and Sail unique sites. This was transformed into the yeast strain YGH1, and the YGH1 strain carrying the plasmid GAL4-BRCA1 (8-1293) was evaluated for its intrinsic ability to activate the two reporters-growth in histidine minus media and β-galactosidase activity. The YGH1 strain carrying the plasmid GAL4-BRCA1 (8-1293) was able to grow on minus histidine plates but this was controlled by the addition of 7.5mM 3-amino-l,2,4-Triazole (3AT) to the minus histidine plates and the strain had no detectable β-galactosidase activity. The YGH1 strain carrying the plasmid GAL4-BRCA1 (8-1293) was subsequently transformed with a HeLa cell cDNA library fused to the GAL4 activation domain in the pGAD plasmid (Chien et al., Proc. Natl. Acad. Sci. vol. 88: pages 9578-9582 (1991). When a cDNA encodes a protein that interacts with the BRCAl protein (amino acids 8- 1293), the YGH1 strain is expected to grow in the absence of histidine supplemented with 7.5mM 3AT and produce β-galactosidase.

Four of the 2.5 X 10⁶ transformants screened grew in the absence of histidine supplemented with 7.5mM 3 AT and had β-galactosidase activity. The plasmids recovered from these 4 yeast strains were used to re-transform the original YGH1 GAL4-BRCA1 (8-1293) strain. All the plasmids conferred the ability to grow in the absence of histidine supplemented with 7.5mM 3AT and to produce β-galactosidase. Upon subsequent screening, three of the four were found to have cDNAs that encode Modulator Proteins that clearly bound to BRCAl. One of the plasmids contained the novel cDNA encoding for the BRCAl Modulator Protein hereinafter termed, 091-21 A31, Sequence ID No. 1. The nucleotide and protein sequence are shown in Figure 1. The calculated molecular weight is about 53kd, and it has an estimated pi of 9.05. Particularly noteworthy is the presence of a zinc finger domain and a leucine zipper motif.

The nucleotide sequence of the second cDNA and amino acid sequence that it encodes, hereinafter termed, 091-1F84, Sequence ID No. 3, is shown in Figure 2. Note that this clone displays two leucine zipper domains. The protein has a calculated molecular weight of 96, 443. 3 and an estimated pi of 4.95.

The nucleotide sequence of the third cDNA and amino acid sequence that it encodes, hereinafter termed, 091-132Q20, Sequence ID No. 5, is shown in Figure 3. Note that this clone also displays a leucine zipper domain. The protein has a calculated molecular weight of 45,904. 9 and an estimated pi of 6.73. Example 2

Binding of BRCAl Domains to BRCAl Modulators Experiments were conducted to ascertain which regions of BRCAl interact with the three BRCAl Modulators described in Example 1. The experiment was conducted using the two-hybrid assay as described in U. S. Patent No. 5, 283, 173, or Chien et al., 1991, Proc. Natl. Acad. Sci. USA, 88:9578-9582. The cDNA that encodes the 091-1F84, Sequence ID No. 3, 091-21A31, Sequence ID No. 1, and 091-132Q20, Sequence ID No. 5 was fused to the GAL 4 activation domain, and those regions of BRCAl shown in Table 1 and that contain BRCAl amino acids 1-300, 1-600, or 8-1293 were fused to the binding domain of G AL4. Controls consisted of the vector, or bcl-2 fused to the GAL 4 binding domain (See, U. S. Patent 5, 539, 085).

TABLE 1 INTERACTION OF BRCAl WITH TWO HYBRID HITS (091-) The BRCAl constructs employed in the above studies were generated using restriction fragments of BRCAl, and cloning them into the plasmid pGBT8, which is a derivative of the plasmid pMA424, as described by Chien et al. in Proc. Natl. Acad. Sci. vol. 88: pages 9578-9582 (1991), modified by the insertion of the sequence 5'- CCGGGGATCCCCATGGCTAGCCATATG-3' between the EcoRI and Sail unique sites. Briefly, the construct containing the first 300 amino acids of BRCAl was generated by subcloning the Ncol-EcoRl blunted BRCAl fragment into the blunted EcoRI site of pGBT8. The BRCAl containing amino acids 8-1293 was generated as described above. Lastly, the BRCAl construct containing amino acids 1-600 was generated by subcloning the Ncol-Spel BRCAl fragment into the Ncol-Nhel site of pGBT8.

Table 1 shows those regions of BRCAl that interact with the proteins encoded by 091-1F84, Sequence ID No. 3, 091-21A31, Sequence ID No. 1, and 091-132Q20, Sequence ID No. 5. The "+" sign is a subjective measure of the amount of b-galactosidase activity. One "+" being the lowest, and three "+++" being the highest activity. It is apparent from Table 1 that the first 300 amino acids of BRCAl do not bind to any of the three BRCAl Modulators, but that all three BRCAl Modulators bind to the BRCAl construct containing the first 600 amino acids of BRCA. None of the BRCAl Modulators bind to the vector or bcl-2 controls, while all the BRCAl Modulators bound to the near full length BRCAl construct which has amino acids 8-1293. The results show that the three BRCAl Modulators preferrentially bind to the first 600 amino acids of BRCAl.

Example 3 Identification of Interacting Domains of 091-21A31, Sequence ID No. 1 and BRCAl Two hybrid experiments were conducted to ascertain the regions of the BRCAl

Modulator 091-21 A31, Sequence ID No. 1 that interact with BRCAl. The assay was run essentially as described in Example 1. Transformation and growth of yeast cultures were performed essentially as described in U. S. Patent No. 5, 283, 173; Chien et al, 1991, Proc. Natl. Acad. Sci. USA, 88:9578-9582; or Spaargaren, M., et al., (1994) Biochem. J. 300, 303-307.

Briefly, the YGH1 yeast strain was co-transformed with cDNA encoding 091- 21A31, Sequence ID No. 1, or cDNA encoding 091-21A31, Sequence ID No. 1 fragments containing amino acids 78-469, 1-300, or 300-469 fused to the GAL4 activation domain. As a control, bcl-2 cDNA (See, U. S. Patent 5, 539, 085) was fused to the GAL4 activation domain. cDNAs encoding BRCAl fragments having amino acids 1-300, 1- 600, or 8-1293 were fused to the GAL4 binding domain as described in Example 2. The 091-21A31, Sequence ID No. 1 constructs were generated using the plasmids pGADGH or pGAD424; both are available from Clontech.

The 091-21A31, Sequence ID No. 1 construct containing amino acids 75-469 was generated by subcloning the EcoR -Xhol 091-21A31, Sequence ID No. 1 fragment into the EcoRl-Sall site of ρGAD424.

The 091-21A31, Sequence ID No. 1 construct containing amino acids 1-300 was generated by subcloning the BamHl-Sall 091-21A31, Sequence ID No. 1 fragment into the BamHl-Sall site of pGADGH.

The 091-21A31, Sequence ID No. 1 construct containing amino acids 300-469 was generated by subcloning the BamHl blunted-Sall 091-21 A31, Sequence ID No. 1 fragment into the Sail blunted-Xhol site of pGADGH.

Table 2 shows the results of the co-transformation studies. It is apparent that the first 300 amino acids of BRCAl do not to bind to any of three 091-21 A31, Sequence ID No. 1 fragment constructs, nor to 091-21 A31, Sequence ID No. 1. The BRCAl construct containing amino acids 1-600 does bind to 091-21 A31, Sequence ID No. 1, and to the construct containing 091-21A31, Sequence ID No. 1 amino acids 78-469, but not to the amino acid 091-21A31, Sequence ID No. 1 constructs 1-300 and 300-469. Also, the BRCAl construct having amino acids 8-1293 also binds 091-21A31, Sequence ID No. 1, the 78-469 and 1-300 amino acid constructs, but not to the 091-21 A31, Sequence ID No. 1 construct having amino acids 300-469.

TABLE 2 INTERACTION OF BRCAl WITH 091-21 Example 4 Expression and Purification of BRCAl Modulators The BRCAl Modulators were expressed in and purified from baculovirus SF9 infected cells. Methods for producing baculovirus, as well as growing SF9 cells are well known in the art, and detailed procedures can be found in M. Summers and G. Smith in "A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures," Texas Agricultural Experiment Station, Bulletin No. 1555 (May, 1987 or in EPS 127,839 to G. E. Smith and M. D. Summers.

The following constructs were generated using pAcC13 (See, Rubinfeld, B., et al. Cell 65, 1033-1042 (1991)) or pAcOG, a derivative of pAcCl3 in which the polylinker was replaced with a synthetic linker engineered to encode an initiating methionine, the Glu-Glu (See, Grussenmyer, T., et al. Proc. Natl. Acad. Sci. U.S.A. 82, 7952 (1985)) epitope tag, and a multiple cloning site containing several stop codons (See, Rubinfeld, B., et al. J. Biol. Chem, 270, 5549-5555 (1995)). The construct containing 091-21A31, Sequence ID No. 1 was generated by subcloning the Kpnl-Xbal 091-21A31, Sequence ID No. 1 fragment into pAcC13 at the Kpnl-Xbal site.

The construct containing 091-1F84, Sequence ID No. 3 was generated by subcloning the Ncol - Xbal 091-1F84, Sequence ID No. 3 fragment into pAcOGl at the Ncol-Xbal sites.

The construct containing 091-132Q20, Sequence ID No. 5 was generated by subcloning the Kpnl-Xbal fragment of 091-132Q20, Sequence ID No. 5 into pAcC13 at the Kpnl-Xbal site.

Baculovirus containing the appropriate BRCAl Modulator was produced by transfecting the above described plasmids into SF9 cells, and isolating the corresponding baculovirus using essentially the methods described in Pharmingen's cat. no. 21100D, BaculoGoldtm /Baculovirus DNA. Virus was isolated from individual plaques, and used to infect Sf9 cells. The cells were grown for 4 days, isolated by centrifugation, and cell extracts made by solubilizing the cell pellet. Briefly, recombinant Sf9 cells were pelleted, lysed in 5 volumes of [20mM Tris (pH8.0), ImM EDTA, lOμg/ml each of leupeptin, pepstatin, pefabloc, ImM aprotinin and ImM DTT] and incubated on ice for 10 minutes. NaCl was then added to a final concentration of 150mM, incubated at room temperature for 10 minutes and centrifuged. The resulting supernatant was loaded onto a 1-ml affinity column containing a mouse Glu-Glu monoclonal antibody covalently cross-linked to protein G-Sepharose. See,

Grussenmyer, T., et al., Proc. Natl. Acad. Sci. U. S. A. vol. 82, pp. 7952-7954 (1985). The column was washed with 10-15ml of lysis buffer and eluted with lOOμg of Glu-Glu peptide (EYMPME) per ml in the same buffer. Fractions were collected and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the peak fractions were pooled and based on purity subjected to further purification on HPLC columns which include Resource Q, Resource S and Resource Eth (Pharmacia). For purification of insoluble proteins, in particular 091-21 A31, Sequence ID No. 1, recombinant Sf9 cells were pelleted, lysed in 5 volumes of [20mM Tris (pH8.0), 137mM NaCl, ImM EGTA, 1.5mM MgCl,, 2%SDS, lOμg/ml each of leupeptin, pepstatin, pefabloc, ImM aprotinin and ImM DTT], incubated at room temperature for 20-30 minutes and ultra centifuged. The upper phase was removed, NaCl was adjusted to 400mM and recentrifuged. The clarified supernatent was then diluted 1:10 in 1 X TG buffer [20mM Tris (pH8.0), 137mM NaCl, ImM EGTA, 1.5mM MgCl,, 1% Triton X100, 10% glycerol, lOμg/ml each of leupeptin, pepstatin, pefabloc, ImM aprotinin and ImM DTT], filtered through a 3uM Gelman Versapore filter and loaded onto a 1-ml anti-Glu- Glu affinity column. See, Rubinfeld, B., et al., Mol. Cell. Bio. 12, 4634-4642 (1992). The column was washed with 10-15ml of 1 X TG buffer with 400mM NaCl and eluted in 1 X TG buffer with 1% SDS and lOOμg/ml Glu-Glu peptide. Fractions were analyzed by SDS-PAGE.

Example 5 Confirmation of BRCAl Modulator Protein Binding to BRCAl

To confirm the results of the two-hybrid assays described in Example 1 and further establish the binding of each of the BRCAl Modulators to BRCAl, two BRCAl constructs were generated and tested for BRCAl Modulator binding. The BRCAl constructs were Glu-Glu tagged BRCAl 5' (1-1293), and BRCAl 3' (1293-1863). The Glu- Glu epitope tag facilitated immunoaffinity purification as described in the above examples. A control construct consisted of rapGAP. This construct was made as described by Rubinfeld, B. and Polakis P., "Purification of Baculovirus-Produced Rapl GTPase-activating Proteins". In: Methods and Enzymology, W.E. Balch, Channing J. Der and Alan Hall, Eds., California: Academic Press, Inc., 255, 31-38. The BRCAl constructs were generated as follows:. pAcO BRCAl 5' (1-1293) was generated by subcloning the Ncol-Nhel BRCAl fragment into pAcO G1S Ncol-Nhel sites. pAcO BRCAl 3' (1293-1863) was generated by subcloning the Nhel blunted-Notl BRCAl fragment into pAcO G2 Stul-Notl sites. Using standard methods, the constructs were transfected into Sf9 cells. The BRCAl constructs were purified using the immunoaffinity purification methods essentially as described in the preceding Examples. For in vitro transcription /translation of the BRCAl Modulators, the following constructs were subcloned into PCANmyc, a derivative of pCDNA3 (Invitrogen) in which the polylinker was replaced with a synthetic linker engineered to encode an initiating methionine, the Myc (See, Evan, G., et al. Mol. Cell. Biol. 5, 3610 (1985)) epitope tag, and a multiple cloning site (See, Rubinfeld, B., et al. Science, 272, 1023- 1026(1996)).

The plasmid containing the BRCAl Modulator 091-1F84, Sequence ID No. 3, PCAN myc 091-1F84, Sequence ID No. 3, was generated by subcloning the Spel blunted Xhol 091-1F84, Sequence ID No. 3 fragment into PCAN myc3 EcoRV-Xhol sites. The plasmid containing the BRCAl Modulator 091-21 A31, Sequence ID No. 1,

PCAN myc 091-21A31, Sequence ID No. 1, was generated by subcloning the BamH - Xhol 091-21A31, Sequence ID No. 1 fragment into PCAN myc3 BamHl-Xhol sites. Lastly, the plasmid containing the BRCAl Modulator 091-132Q20, Sequence ID No. 5, PCAN myc 091-132Q20, Sequence ID No. 5, was generated by subcloning the EcoRl- Xhol 091-132Q20, Sequence ID No. 5 fragment into PCAN myc3 EcoRl-Xhol sites.

For in vitro binding analysis, the BRCAl Modulator cDNAs (091-1F84, Sequence ID No. 3, 091-21A31, Sequence ID No. 1, 091-132Q20, Sequence ID No. 5) were transcribed and translated in vitro in the presence of [ ⁵S]Methionine using the TNT- coupled wheat germ cell lysate system (Promega). Next, one-two μg of purified recombinant BRCAl protein, either Glu-Glu tagged BRCAl 5' (1-1293), or BRCAl 3' (1293-1863) was added to 25μl of precleared lysate along with lOμl of anti-Glu Glu coupled protein G-Sepharose beads. Following a 2 hour incubation with rocking at 4^ϋC, the beads were washed three times with 1 ml each of ice cold buffer B (20mM tris pH 7.5, 150mM NaCl, 0.5% Nonidet P-40), eluted with 20μl of SDS-PAGE sample buffer and subjected to SDS-PAGE and fluorography.

SDS-PAGE fluorography revealed that all three of the BRCAl Modulators were affinity precipitated with the construct BRCAl 5' (1-1293) but not BRCAl 3' (1293-1863). The rapGAP control also did not affinity precipitate any of the three BRCAl Modulators. Taken together these results confirm and extend the results of the two hybrid assay, and establishes that the BRCAl Modulator proteins interact with BRCAl. Example 6 Preparation of Antibody to BRCAl Modulators

For antibody production, immunoaffinity purification of BRCAl Modulators from baculovirus infected Sf9 insect cells was performed with immobilized anti-Glu- Glu antibody specific for the Glu-Glu epitope tag expressed on the recombinant soluble proteins (See, Rubinfeld, B„ et al, Mol. Cell. Bio. 12, 4634-4642 (1992)). Briefly, recombinant Sf9 cells were pelleted, lysed in 5 volumes of [20mM Tris (pH8.0), ImM EDTA, lOμg/ml each of leupeptin, pepstatin, pefabloc, ImM aprotinin and ImM DTT] and incubated on ice for 10 minutes. NaCl was then added to a final concentration of 150mM, incubated at room temperature for 10 minutes and centrifuged. Then resulting supernatant was loaded onto a 1-ml affinity column containing the Glu-Glu antibody covalently cross-linked to protein G-Sepharose. The column was washed with 10-15ml of lysis buffer and eluted with lOOμg of Glu-Glu peptide (EYMPME) per ml in the same buffer. Fractions were collected and analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE), the peak fractions were pooled and based on purity subjected to further purification on HPLC columns which include Resource Q, Resource S and Resource Eth (Pharmacia). For purification of insoluble proteins, in particular 21, recombinant Sf9 cells were pelleted, lysed in 5 volumes of [20mM Tris (pH8.0), 137mM NaCl, ImM EGTA, 1.5mM MgCl₂, 2%SDS, lOug/ml each of leupeptin, pepstatin, pefabloc, ImM aprotinin and ImM DTT], incubated at room temperature for 20-30 minutes and ultra centifuged. The upper phase was removed, NaCl was adjusted to 400mM and recentrifuged. The clarified supernatent was then diluted 1:10 in 1 X TG buffer [20mM Tris (pH8.0), 137mM NaCl, ImM EGTA, 1.5mM MgCl₂, 1% Triton X100, 10% glycerol, lOug/ml each of leupeptin, pepstatin, pefabloc, ImM aprotinin and ImM DTT], filtered through a 3um Gelman Versapore filter and loaded onto a 1-ml anti-Glu-Glu affinity column. The column was washed with 10-15ml of 1 X TG buffer with 400mM NaCl and eluted in 1 X TG buffer with 1% SDS and lOOμg/ml Glu-Glu peptide. Fractions were analyzed by SDS-PAGE, pooled and used to immunize rabbits. To produce antisera containing antibodies directed against the BRCAl

Modulators the latter are used to immunize rabbits as follows. For the BRCAl Modulator 091-21A31, Sequence ID No. 1, the immunization protocol generally consisted of two immunizations; the first was a subcutaneous injection of 0.500mg in CFA, followed by a second intramuscular injection of 0.250 mg about four weeks later in ICFA. The rabbits were bled, antisera collected and antibody purified as setforth below. BRCAl Modulator antibodies are affinity purified using BRCAl Modulator immunogens which have been coupled to a support matrix. Briefly, the BRCAl Modulator 091-21A31, Sequence ID No. 1 is coupled to CNBr activated Sepharose 6MB (Pharmacia) as follows. One ml of matrix was activated according to manufacturer's instructions (ie. resuspended in ImM H Cl, washed for 15 min. in ImM H Cl on a sintered glass filter). One mg of 091-21 A31, Sequence ID No. 1 was dialyzed against coupling buffer [0.1 M NaHC0₃ pH 8.3, 0.5M NaCl] overnight at 4°C with two changes of buffer. The dialyzed protein was then incubated with the CNBr activated Sepharose 6MB and incubated with rocking overnight at 4"C. The excess ligand was washed away with coupling buffer and any remaining active groups were blocked with IM ethanolamine at room temperature for two hours. This material was then washed with three cycles of alternating pH - each cycle consists of a wash with 0.1M acetate buffer, pH 4.0, 0.5M NaCl followed by a wash with 0.1M Tris, pH 8.0, 0.5M NaCl. The protein coupled gel matrix was then resuspended in PBS and incubated with 5ml of antibody serum with rocking overnight at 4°C. The mixture was poured into a column, allowed to drip through and washed 3 times with 15ml PBS per wash. Seven elutions with 800μl of 0.2M glycine, pH 2.5, were collected and each elution was neutralized immediately with 200μl IM K₂HP0₄. Peak fractions were combined and dialyzed into PBS Azide for storage.

American Type Culture Collection Deposits The cDNA clones that encode 091-1F84, Sequence ID No. 3, 091-21A31, Sequence

ID No. 1, and 091-132Q20, Sequence ID No. 5 were deposited with the American Type Culture Collection (ATCC) on August 14, 1996 under accession numbers 98141 (091- 1F84, Sequence ID No. 3), 98142 (091-21A31, Sequence ID No. 1), and 98143 (091- 132Q20, Sequence ID No. 5). The deposits were made under the Budapest Treaty and shall be maintained at least 30 years after the date of depost and 5 years after the date of the most recent request for the deposit. The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

SEQUENCE LISTING (1) GENERAL INFORMATION -

(1) APPLICANT. Rubinfeld, Bonnee Polakis, Paul G. Ligenfelter, Carol

Vuong, Teπlyn T. In] TITLE OF INVENTION: MODULATORS OF BRCAl ACTIVITY (ill) NUMBER OF SEQUENCES: 6 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE. ONYX Pharmaceuticals, Inc

(B) STREET- 3031 Research Drive

(C) CITY Richmond

(D) STATE: CA

(E) COUNTRY: USA (F) ZIP 94806

(v) COMPUTER READABLE FORM

(A) MEDIUM TYPE Floppy disk

(B) COMPUTER IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.30

(Vi ) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US Unknown

(B) FILING DATE-

(C) CLASSIFICATION: Utility (vm) ATTORNEY/AGENT INFORMATION:

(A) NAME: Giotta, Gregory

(B) REGISTRATION NUMBER: 32,028

(C) REFERENCE/DOCKET NUMBER- ONYX1024 GG (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE. (510) 262-8710

(B) TELEFAX- (510) 222-9758 (2) INFORMATION FOR SEQ ID NO : 1 :

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2065 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS : double

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

(ill) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 103..1512

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 : GTGGATCCCC CGGGCTGCAG GAATTCGGCA CGAGCGGCAC GAGTACGAAG CCGGACCTGT 60 AGCAGTTTCT TTGGCTGCCT GGGCCCCTTG AGTCCAGCCA TC ATG CCT ATC CGT 114

Met Pro He Arg l

GCT CTG TGC ACT ATC TGC TCC GAC TTC TTC GAT CAC TCC CGC GAC GTG 162

Ala Leu Cys Thr He Cys Ser Asp Phe Phe Asp His Ser Arg Asp Val 5 10 15 20

GCC GCC ATC CAC TGC GGC CAC ACC TTC CAC TTG CAG TGC CTA ATT CAG 210 Ala Ala He His Cys Gly His Thr Phe His Leu Gin Cys Leu He Gin

25 30 35

TGG TTT GAG ACA GCA CCA AGT CGG ACC TGC CCA CAG TGC CGA ATC CAG 258

Trp Phe Glu Thr Ala Pro Ser Arg Thr Cys Pro Gin Cys Arg He Gin 40 45 50 GTT GGC AAA AGA ACC ATT ATC AAT AAG CTC TTC TTT GAT CTT GCC CAG 306

Val Gly Lys Arg Thr He He Asn Lys Leu Phe Phe Asp Leu Ala Gin

55 60 65

GAG GAG GAG AAT GTC TTG GAT GCA GAA TTC TTA AAG AAT GAA CTG GAC 354

Glu Glu Glu Asn Val Leu Asp Ala Glu Phe Leu Lys Asn Glu Leu Asp 70 75 80

AAT GTC AGA GCC CAG CTT TCC CAG AAA GAC AAG GAG AAA CGA GAC AGC 402

Asn Val Arg Ala Gin Leu Ser Gin Lys Asp Lys Glu Lys Arg Asp Ser 85 90 95 100

CAG GTC ATC ATC GAC ACT CTG CGG GAT ACG CTG GAA GAA CGC AAT GCT 450 Gin Val He He Asp Thr Leu Arg Asp Thr Leu Glu Glu Arg Asn Ala

105 110 115

ACT GTG GTA TCT CTG CAG CAG GCC TTG GGC AAG GCC GAG ATG CTG TGC 498

Thr Val Val Ser Leu Gin Gin Ala Leu Gly Lys Ala Glu Met Leu Cys 120 125 130 TCC ACA CTG AAA AAG CAG ATG AAG TAC TTA GAG CAG CAG CAG GAT GAG 546

Ser Thr Leu Lys Lys Gin Met Lys Tyr Leu Glu Gin Gin Gin Asp Glu

135 140 145

ACC AAA CAA GCA CAA GAG GAG GCC CGC CGG CTC AGG AGC AAG ATG AAG 594

Thr Lys Gin Ala Gin Glu Glu Ala Arg Arg Leu Arg Ser Lys Met Lys 150 155 160

ACC ATG GAG CAG ATT GAG CTT CTA CTC CAG AGC CAG CGC CCT GAG GTG 642

Thr Met Glu Gin He Glu Leu Leu Leu Gin Ser Gin Arg Pro Glu Val 165 170 175 180

GAG GAG ATG ATC CGA GAC ATG GGT GTG GGA CAG TCA GCG GTG GAA CAG 690 Glu Glu Met He Arg Asp Met Gly Val Gly Gin Ser Ala Val Glu Gin

185 190 195

CTG GCT GTG TAC TGT GTG TCT CTC AAG AAA GAG TAC GAG AAT CTA AAA 738

Leu Ala Val Tyr Cys Val Ser Leu Lys Lys Glu Tyr Glu Asn Leu Lys 200 205 210 GAG GCA CGG AAG GCC TCA GGG GAG GTG GCT GAC AAG CTG AGG AAG GAT 786 Glu Ala Arg Lys Ala Ser Gly Glu Val Ala Asp Lys Leu Arg Lys Asp

215 220 225

TTG TTT TCC TCC AGA AGC AAG TTG CAG ACA GTC TAC TCT GAA TTG GAT 834 Leu Phe Ser Ser Arg Ser Lys Leu Gin Thr Val Tyr Ser Glu Leu Asp 230 235 240

CAG GCC AAG TTA GAA CTG AAG TCA GCC CAG AAG GAC TTA CAG AGT GCT 882 Gin Ala Lys Leu Glu Leu Lys Ser Ala Gin Lys Asp Leu Gin Ser Ala 245 250 255 260 GAC AAG GAA ATC ATG AGC CTG AAA AAG AAG CTA ACG ATG CTG CAG GAA 930 Asp Lys Glu He Met Ser Leu Lys Lys Lys Leu Thr Met Leu Gin Glu

265 270 275

ACC TTG AAC CTG CCA CCA GTG GCC AGT GAG ACT GTC GAC CGC CTG GTT 978 Thr Leu Asn Leu Pro Pro Val Ala Ser Glu Thr Val Asp Arg Leu Val 280 285 290

TTA GAG AGC CCA GCC CCT GTG GAG GTG AAT CTG AAG CTC CGC CGG CCA 1026 Leu Glu Ser Pro Ala Pro Val Glu Val Asn Leu Lys Leu Arg Arg Pro

295 300 305

TCC TTC CGT GAT GAT ATT GAT CTC AAT GCT ACC TTT GAT GTG GAT ACT 1074 Ser Phe Arg Asp Asp He Asp Leu Asn Ala Thr Phe Asp Val Asp Thr 310 315 320

CCC CCA GCC CGG CCC TCC AGC TCC CAG CAT GGT TAC TAC GAA AAA CTT 1122 Pro Pro Ala Arg Pro Ser Ser Ser Gin His Gly Tyr Tyr Glu Lys Leu 325 330 335 340 TGC CTA GAG AAG TCA CAC TCC CCA ATT CAG GAT GTC CCC AAG AAG ATA 1170 Cys Leu Glu Lys Ser His Ser Pro He Gin Asp Val Pro Lys Lys He

345 350 355

TGC AAA GGC CCC AGG AAG GAG TCC CAG CTC TCA CTG GGT GGC CAG AGC 1218 Cys Lys Gly Pro Arg Lys Glu Ser Gin Leu Ser Leu Gly Gly Gin Ser 360 365 370

TGT GCA GGA GAG CCA GAT GAG GAA CTG GTT GGT GCC TTC CCT ATT TTT 1266

Cys Ala Gly Glu Pro Asp Glu Glu Leu Val Gly Ala Phe Pro He Phe

375 380 385 GTC CGG AAT GCC ATC CTA GGC CAG AAA CAG CCC AAG AGG CCC AGG TCA 1314

Val Arg Asn Ala He Leu Gly Gin Lys Gin Pro Lys Arg Pro Arg Ser

390 395 400

GAG TCC TCT TGC AGC AAA GAT GTG GTA AGG ACA GGC TTC GAT GGG CTC 1362

Glu Ser Ser Cys Ser Lys Asp Val Val Arg Thr Gly Phe Asp Gly Leu 405 410 415 420

GGT GGC CGG ACA AAA TTC ATC CAG CCT ACT GAC ACA GTC ATG ATC CGC 1410

Gly Gly Arg Thr Lys Phe He Gin Pro Thr Asp Thr Val Met He Arg

425 430 435 CCA TTG CCT GTT AAG CCC AAG ACC AAG GTT AAG CAG AGG GTG AGG GTG 1458

Pro Leu Pro Val Lys Pro Lys Thr Lys Val Lys Gin Arg Val Arg Val

440 445 450

AAG ACA GTG CCT TCT CTC TTC CAG GCC AAG CTG GAC ACC TTC CTG TGG 1506 Lys Thr Val Pro Ser Leu Phe Gin Ala Lys Leu Asp Thr Phe Leu Trp 455 460 465

TCG TGA GAACAGTGAG TCTGACCAAT GGCCAGACAC ATGCCTGCAA CTTGTAGGTC 1562 Ser * 470 AAGGACTGTC CAGGCAGGGG TTTTGTGGAC AGAGCCCCAC TTTCGGGACC AGCCTGAGGT 1622

GTAAGGGCAG ACAAACAGGT GAGGGTGAGT GTGACACCCA GAGACTGCTC TTCCTGCCCT 1682

CACCCTGCCC CACTCCTACG ACTGGGAGCT GACATGACCA GCCCACTGAT CCTGTCAGCA 1742

GGTCCTGCTC CTGTTGCCAG GCTCCTGTTT A AGCCATGA TCAGATGTGG TCAGACTCTT 1802

TCTGGGCCTG GAGACCACGG TCACTTGTTG ACTGTCTCTG TGGACCAGAG TGCTTGAGGC 1862 ATCTCAGGCA GCCTCAGCCC AAGCTTCTAC CTGCCTTTGA CTTGCTTCTA GGCATAGCCT 1922

GGGCCAAGCA GGGTGGGGAA TGGAGGATAG CATGGGATGT ATGGAGAGGA TGGAAGATTT 1982

TCATGTAAAA TAAAATTAAA AAAAAAAAAA CAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 2042

AAAAAAAAAA AAAAAAACTC GAG 2065

(2) INFORMATION FOR SEQ ID NO : 2.

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH: 470 amino acids

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

(xi) SEQUENCE DESCRIPTION. SEQ ID NO 2

Met Pro He Arg Ala Leu Cys Thr He Cys Ser Asp Phe Phe Asp His 1 5 10 15 Ser Arg Asp Val Ala Ala He His Cys Gly His Thr Phe His Leu Gin 20 25 30

Cys Leu He Gin Trp Phe Glu Thr Ala Pro Ser Arg Thr Cys Pro Gin

35 40 45

Cys Arg He Gin Val Gly Lys Arg Thr He He Asn Lys Leu Phe Phe 50 55 60

Asp Leu Ala Gin Glu Glu Glu Asn Val Leu Asp Ala Glu Phe Leu Lys 65 70 75 80

Asn Glu Leu Asp Asn Val Arg Ala Gin Leu Ser Gin Lys Asp Lys Glu 85 90 95 Lys Arg Asp Ser Gin Val He He Asp Thr Leu Arg Asp Thr Leu Glu 100 105 110

Glu Arg Asn Ala Thr Val Val Ser Leu Gin Gin Ala Leu Gly Lys Ala 115 120 125 Glu Met Leu Cys Ser Thr Leu Lys Lys Gin Met Lys Tyr Leu Glu Gin

130 135 140

Gin Gin Asp Glu Thr Lys Gin Ala Gin Glu Glu Ala Arg Arg Leu Arg 145 150 155 160 Ser Lys Met Lys Thr Met Glu Gin He Glu Leu Leu Leu Gin Ser Gin

165 170 175

Arg Pro Glu Val Glu Glu Met He Arg Asp Met Gly Val Gly Gin Ser

180 185 190

Ala Val Glu Gin Leu Ala Val Tyr Cys Val Ser Leu Lys Lys Glu Tyr 195 200 205

Glu Asn Leu Lys Glu Ala Arg Lys Ala Ser Gly Glu Val Ala Asp Lys

210 215 220

Leu Arg Lys Asp Leu Phe Ser Ser Arg Ser Lys Leu Gin Thr Val Tyr 225 230 235 240 Ser Glu Leu Asp Gin Ala Lys Leu Glu Leu Lys Ser Ala Gin Lys Asp

245 250 255

Leu Gin Ser Ala Asp Lys Glu He Met Ser Leu Lys Lys Lys Leu Thr

260 265 270

Met Leu Gin Glu Thr Leu Asn Leu Pro Pro Val Ala Ser Glu Thr Val 275 280 285

Asp Arg Leu Val Leu Glu Ser Pro Ala Pro Val Glu Val Asn Leu Lys

290 295 300

Leu Arg Arg Pro Ser Phe Arg Asp Asp He Asp Leu Asn Ala Thr Phe 305 310 315 320 Asp Val Asp Thr Pro Pro Ala Arg Pro Ser Ser Ser Gin His Gly Tyr

325 330 335

Tyr Glu Lys Leu Cys Leu Glu Lys Ser His Ser Pro He Gin Asp Val

340 345 350

Pro Lys Lys He Cys Lys Gly Pro Arg Lys Glu Ser Gin Leu Ser Leu 355 360 365

Gly Gly Gin Ser Cys Ala Gly Glu Pro Asp Glu Glu Leu Val Gly Ala

370 375 380

Phe Pro He Phe Val Arg Asn Ala He Leu Gly Gin Lys Gin Pro Lys 385 390 395 400 Arg Pro Arg Ser Glu Ser Ser Cys Ser Lys Asp Val Val Arg Thr Gly

405 410 415

Phe Asp Gly Leu Gly Gly Arg Thr Lys Phe He Gin Pro Thr Asp Thr

420 425 430

Val Met He Arg Pro Leu Pro Val Lys Pro Lys Thr Lys Val Lys Gin 435 440 445

Arg Val Arg Val Lys Thr Val Pro Ser Leu Phe Gin Ala Lys Leu Asp

450 455 460

Thr Phe Leu Trp Ser * 465 470 (2) INFORMATION FOR SEQ ID NO : 3 ^■

(l) SEQUENCE CHARACTERISTICS.

(A) LENGTH. 3256 base pairs (B) TYPE- nucleic acid

(C) STRANDEDNESS . double

(D) TOPOLOGY linear (ii) MOLECULE TYPE cDNA (ix) FEATURE- (A) NAME/KEY- CDS

(B) LOCATION: 34..2541

(xi) SEQUENCE DESCRIPTION. SEQ ID NO : 3 :

GAACTAGTGG ATCCCCCGGG CTGCAGGAAT TCG GCA CGA GAA AGC TTA TCC CTT 54 Ala Arg Glu Ser Leu Ser Leu

1 5

CCC TCG ATG CTT CGG GAT GCT GCA ATT GGC ACT ACC CCT TTC TCT ACT 102 Pro Ser Met Leu Arg Asp Ala Ala He Gly Thr Thr Pro Phe Ser Thr 10 15 20 TGC TCG GTG GGG ACT TGG TTT ACT CCT TCA GCA CCA CAG GAA AAG AGT 150 Cys Ser Val Gly Thr Trp Phe Thr Pro Ser Ala Pro Gin Glu Lys Ser

25 30 35

ACA AAC ACA TCC CAG ACA GGC CTG GTT GGC ACC AAG CAC AGT ACT TCT 198 Thr Asn Thr Ser Gin Thr Gly Leu Val Gly Thr Lys His Ser Thr Ser 40 45 50 55

GAG ACA GAG CAG CTC CTG TGT GGC CGG CCT CCA GAT CTG ACT GCC TTG 246 Glu Thr Glu Gin Leu Leu Cys Gly Arg Pro Pro Asp Leu Thr Ala Leu

60 65 70

TCT CGA CAT GAC TTG GAA GAT AAC CTG CTG AGC TCT CTT GTC ATT CTG 294 Ser Arg His Asp Leu Glu Asp Asn Leu Leu Ser Ser Leu Val He Leu 75 80 85

GAG GTT CTC TCC CGC CAG CTT CGG GAC TGG AAG AGC CAG CTG GCT GTC 342 Glu Val Leu Ser Arg Gin Leu Arg Asp Trp Lys Ser Gin Leu Ala Val 90 95 100 CCT CAC CCA GAA ACC CAG GAC AGT AGC ACA CAG ACT GAC ACA TCT CAC 390 Pro His Pro Glu Thr Gin Asp Ser Ser Thr Gin Thr Asp Thr Ser His

105 110 115

AGT GGG ATA ACT AAT AAA CTT CAG CAT CTT AAG GAG AGC CAT GAG ATG 438 Ser Gly He Thr Asn Lys Leu Gin His Leu Lys Glu Ser His Glu Met 120 125 130 135

GGA CAG GCC CTA CAG CAG GCC AGA AAT GTC ATG CAA TCA TGG GTG CTT 486 Gly Gin Ala Leu Gin Gin Ala Arg Asn Val Met Gin Ser Trp Val Leu 140 145 150 ATC TCT AAA GAG CTG ATA TCC TTG CTT CAC CTA TCC CTG TTG CAT TTA 534 He Ser Lys Glu Leu He Ser Leu Leu His Leu Ser Leu Leu His Leu

155 160 165

GAA GAA GAT AAG ACT ACT GTG AGT CAG GAG TCT CGG CGT GCA GAA ACA 582 Glu Glu Asp Lys Thr Thr Val Ser Gin Glu Ser Arg Arg Ala Glu Thr 170 175 180

TTG GTC TGT TGC TGT TTT GAT TTG CTG AAG AAA TTG AGG GCA AAG CTC 630 Leu Val Cys Cys Cys Phe Asp Leu Leu Lys Lys Leu Arg Ala Lys Leu 185 190 195 CAG AGC CTC AAA GCA GAA AGG GAG GAG GCA AGG CAC AGA GAG GAA ATG 678 Gin Ser Leu Lys Ala Glu Arg Glu Glu Ala Arg His Arg Glu Glu Met 200 205 210 215

GCT CTC AGA GGC AAG GAT GCG GCA GAG ATA GTG TTG GAG GCT TTC TGT 726 Ala Leu Arg Gly Lys Asp Ala Ala Glu He Val Leu Glu Ala Phe Cys 220 225 230

GCA CAC GCC AGC CAG CGC ATC AGC CAG CTG GAA CAG GAC CTA GCA TCC 774 Ala His Ala Ser Gin Arg He Ser Gin Leu Glu Gin Asp Leu Ala Ser

235 240 245

ATG CGG GAA TTC AGA GGC CTT CTG AAG GAT GCC CAG ACC CAA CTG GTA 822 Met Arg Glu Phe Arg Gly Leu Leu Lys Asp Ala Gin Thr Gin Leu Val 250 255 260

GGG CTT CAT GCC AAG CAA GAA GAG CTG GTT CAG CAG ACA GTG AGT CTT 870 Gly Leu His Ala Lys Gin Glu Glu Leu Val Gin Gin Thr Val Ser Leu 265 270 275 ACT TCT ACC TTG CAA CAA GAC TGG AGG TCC ATG CAA CTG GAT TAT ACA 918 Thr Ser Thr Leu Gin Gin Asp Trp Arg Ser Met Gin Leu Asp Tyr Thr 280 285 290 295

ACA TGG ACA GCT TTG CTG AGT CGG TCC CGA CAA CTC ACA GAG AAA CTC 966 Thr Trp Thr Ala Leu Leu Ser Arg Ser Arg Gin Leu Thr Glu Lys Leu 300 305 310

ACA GTC AAG AGC CAG CAA GCC CTG CAG GAA CGT GAT GTG GCA ATT GAG 1014 Thr Val Lys Ser Gin Gin Ala Leu Gin Glu Arg Asp Val Ala He Glu

315 320 325

GAA AAG CAG GAG GTT TCT AGG GTG CTG GAA CAA GTC TCT GCC CAG TTA 1062 Glu Lys Gin Glu Val Ser Arg Val Leu Glu Gin Val Ser Ala Gin Leu 330 335 340

GAG GAG TGC AAA GGC CAA ACA GAA CAA CTG GAG TTG GAA AAC AGT CGT 1110 Glu Glu Cys Lys Gly Gin Thr Glu Gin Leu Glu Leu Glu Asn Ser Arg 345 350 355 CTA GCA ACA GAT CTC CGG GCT CAG TTG CAG ATT CTG GCC AAC ATG GAC 1158 Leu Ala Thr Asp Leu Arg Ala Gin Leu Gin He Leu Ala Asn Met Asp 360 365 370 375 AGC CAG CTA AAA GAG CTA CAG AGT CAG CAT ACC CAT TGT GCC CAG GAC 1206

Ser Gin Leu Lys Glu Leu Gin Ser Gin His Thr His Cys Ala Gin Asp

380 385 390

CTG GCT ATG AAG GAT GAG TTA TTC TGC CAG CTT ACC CAG AGC AAT GAG 1254 Leu Ala Met Lys Asp Glu Leu Phe Cys Gin Leu Thr Gin Ser Asn Glu 395 400 405

GAG CAG GCT GCT CAA TGG CAA AAG GAA GAG ATG GCA CTA AAA CAC ATG 1302

Glu Gin Ala Ala Gin Trp Gin Lys Glu Glu Met Ala Leu Lys His Met

410 415 420 CAG GCA GAA CTG CAG CAG CAA CAA GCT GTC CTG GCC AAA GAG GTG CGG 1350

Gin Ala Glu Leu Gin Gin Gin Gin Ala Val Leu Ala Lys Glu Val Arg

425 430 435

GAC CTG AAA GAG ACC TTG GAG TTT GCA GAC CAG GAG AAT CAG GTT GCT 1398

Asp Leu Lys Glu Thr Leu Glu Phe Ala Asp Gin Glu Asn Gin Val Ala 440 445 450 455

CAC CTG GAG CTG GGT CAG GTT GAG TGT CAA TTG AAA ACC ACA CTG GAA 1446

His Leu Glu Leu Gly Gin Val Glu Cys Gin Leu Lys Thr Thr Leu Glu

460 465 470

GTG CTC CGG GAG CGC AGC TTG CAG TGT GAG AAC CTC AAG GAC ACT GTA 1494 Val Leu Arg Glu Arg Ser Leu Gin Cys Glu Asn Leu Lys Asp Thr Val 475 480 485

GAG AAC CTA ACG GCT AAA CTG GCC AGC ACC ATA GCA GAT AAC CAG GAG 1542

Glu Asn Leu Thr Ala Lys Leu Ala Ser Thr He Ala Asp Asn Gin Glu

490 495 500 CAA GAT CTG GAG AAA ACA CGG CAG TAC TCT CAA AAG CTA AGG CTG CTG 1590

Gin Asp Leu Glu Lys Thr Arg Gin Tyr Ser Gin Lys Leu Arg Leu Leu

505 510 515

ACT GAG CAA CTA CAG AGC CTG ACT CTC TTT CTA CAG ACA AAA CTA AAG 1638

Thr Glu Gin Leu Gin Ser Leu Thr Leu Phe Leu Gin Thr Lys Leu Lys 520 525 530 535

GAG AAG ACT GAA CAA GAG ACC CTT CTG CTG AGT ACA GCC TGT CCT CCC 1686

Glu Lys Thr Glu Gin Glu Thr Leu Leu Leu Ser Thr Ala Cys Pro Pro

540 545 550

ACC CAG GAA CAC CCT CTG CCT AAT GAC AGG ACC TTC CTG GGA AGC ATC 1734 Thr Gin Glu His Pro Leu Pro Asn Asp Arg Thr Phe Leu Gly Ser He 555 560 565

TTG ACA GCA GTG GCA GAT GAA GAG CCA GAA TCA ACT CCT GTG CCC TTG 1782

Leu Thr Ala Val Ala Asp Glu Glu Pro Glu Ser Thr Pro Val Pro Leu

570 575 580 CTT GGA AGT GAC AAG AGT GCT TTC ACC CGA GTA GCA TCA ATG GTT TCC 1830

Leu Gly Ser Asp Lys Ser Ala Phe Thr Arg Val Ala Ser Met Val Ser

585 590 595 CTT CAG CCC GCA GAG ACC CCA GGC ATG GAG GAG AGC CTG GCA GAA ATG 1878 Leu Gin Pro Ala Glu Thr Pro Gly Met Glu Glu Ser Leu Ala Glu Met 600 605 610 615

AGT ATT ATG ACT ACT GAG CTT CAG AGT CTT TGT TCC CTG CTA CAA GAG 1926 Ser He Met Thr Thr Glu Leu Gin Ser Leu Cys Ser Leu Leu Gin Glu

620 625 630

TCT AAA GAA GAA GCC ATC AGG ACT CTG CAG CGA AAA ATT TGT GAG CTG 1974 Ser Lys Glu Glu Ala He Arg Thr Leu Gin Arg Lys He Cys Glu Leu 635 640 645 CAA GTT AGG CTG CAG GCC CAG GAA GAA CAG CAT CAG GAA GTC CAG AAG 2022 Gin Val Arg Leu Gin Ala Gin Glu Glu Gin His Gin Glu Val Gin Lys

650 655 660

GCA AAA GAA GCA GAC ATA GAG AAG CTG AAC CAG GCC TTG TGC TTG CGC 2070 Ala Lys Glu Ala Asp He Glu Lys Leu Asn Gin Ala Leu Cys Leu Arg 665 670 675

TAC AAG AAT GAA AAG GAG CTC CAG GAA GTG ATA CAG CAG CAG AAT GAG 2118 Tyr Lys Asn Glu Lys Glu Leu Gin Glu Val He Gin Gin Gin Asn Glu 680 685 690 695

AAG ATC CTA GAA CAG ATA GAC AAG AGT GGC GAG CTC ATA AGC CTT AGA 2166 Lys He Leu Glu Gin He Asp Lys Ser Gly Glu Leu He Ser Leu Arg

700 705 710

GAG GAG GTG ACC CAC CTT ACC CGC TCA CTT CGG CGT GCG GAG ACA GAG 2214 Glu Glu Val Thr His Leu Thr Arg Ser Leu Arg Arg Ala Glu Thr Glu 715 720 725 ACC AAA GTG CTC CAG GAG GCC CTG GCA GGC CAG CTG GAC TCC AAC TGC 2262 Thr Lys Val Leu Gin Glu Ala Leu Ala Gly Gin Leu Asp Ser Asn Cys

730 735 740

CAG CCT ATG GCC ACC AAT TGG ATC CAG GAG AAA GTG TGG CTC TCT CAG 2310 Gin Pro Met Ala Thr Asn Trp He Gin Glu Lys Val Trp Leu Ser Gin 745 750 755

GAG GTG GAC AAA CTG AGA GTG ATG TTC CTG GAG ATG AAA AAT GAG AAG 2358 Glu Val Asp Lys Leu Arg Val Met Phe Leu Glu Met Lys Asn Glu Lys 760 765 770 775

GAA AAA CTC ATG ATC AAG TTC CAG AGC CAT AGA AAT ATC CTA GAG GAG 2406 Glu Lys Leu Met He Lys Phe Gin Ser His Arg Asn He Leu Glu Glu

780 785 790

AAC CTT CGG CGC TCT GAC AAG GAG TTA GAA AAA CTA GAT GAC ATT GTT 2454 Asn Leu Arg Arg Ser Asp Lys Glu Leu Glu Lys Leu Asp Asp He Val 795 800 805 CAG CAT ATT TAT AAG ACC CTG CTC TCT ATT CCA GAG GTG GTG AGG GGA 2502 Gin His He Tyr Lys Thr Leu Leu Ser He Pro Glu Val Val Arg Gly 810 815 820 TGC AGA GAA CTA CAG GGA TTG CTG GAA TTT CTG AGC TAA GAAACTGAAA 2551 Cys Arg Glu Leu Gin Gly Leu Leu Glu Phe Leu Ser *

825 830 835

GCCAGAATCT GCTTCACCTC TTTTTACCTG CAATACCCCC TTACCCCAAT ACCAAGACCA 2611 ACTGGCATAG AGCCAACTGA GA AAATGCT ATTTAAATAA AGTGTATTTA ATGAAAACTC 2671

GTGCCGAATT CGGCACGAGC GGCACGAGCG GCACGAGCTG CAGCCATGTC TCTAGTGATC 2731

CCTGAAAAGT TCCAGCATAT TTTGCGAGTA CTCAACACCA ACATCGATGG GCGGCGGAAA 2791

ATAGCCTTTG CCATCACTGC CATTAAGGGT GTGGGCCGAA GATATGCTCA TGTGGTGTTG 2851

AGGAAAGCAG ACATTGACCT CACCAAGAGG GCGGGAGAAC TCACTGAGGA TGAGGTGGAA 2911 CGTGTGATCA CCATTATGCA GAATCCACGC CAGTACAAGA TCCCAGACTG GTTCTTGAAC 2971

AGACAGAAGG ATGTAAAGGA TGGAAAATAC AGCCAGGTCC TAGCCAATGG TCTGGACAAC 3031

AAGCTCCGTG AAGACCTGGA GCGACTGAAG AAGATTCGGG CCCATAGAGG GCTGCGTCAC 3091

TTCTGGGGCC TTCGTGTCCG AGGCCAGCAC ACCAAGACCA CTGGCCGCCG TGGCCGCACC 3151

GTGGGTGTGT CCAAGAAGAA ATAAGTCTGT AGGCCTTGTC TGTTAATAAA TAGTTTATAT 3211 ACCAAAAAAA AAAAAAAAAA ACTCGAGCAT GCATCTAGAG GGCCC 3256

(2) INFORMATION FOR SEQ ID NO 4

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH 836 ammo acids

(D) TOPOLOGY linear (n) MOLECULE TYPE, protein (xi) SEQUENCE DESCRIPTION SEQ ID NO : 4

Ala Arg Glu Ser Leu Ser Leu Pro Ser Met Leu Arg Asp Ala Ala He 1 5 10 15

Gly Thr Thr Pro Phe Ser Thr Cys Ser Val Gly Thr Trp Phe Thr Pro

20 25 30

Ser Ala Pro Gin Glu Lys Ser Thr Asn Thr Ser Gin Thr Gly Leu Val 35 40 45

Gly Thr Lys His Ser Thr Ser Glu Thr Glu Gin Leu Leu Cys Gly Arg

50 55 60

Pro Pro Asp Leu Thr Ala Leu Ser Arg His Asp Leu Glu Asp Asn Leu 65 70 75 80 Leu Ser Ser Leu Val He Leu Glu Val Leu Ser Arg Gin Leu Arg Asp

85 90 95

Trp Lys Ser Gin Leu Ala Val Pro His Pro Glu Thr Gin Asp Ser Ser

100 105 110

Thr Gin Thr Asp Thr Ser His Ser Gly He Thr Asn Lys Leu Gin His 115 120 125

Leu Lys Glu Ser His Glu Met Gly Gin Ala Leu Gin Gin Ala Arg Asn

130 135 140

Val Met Gin Ser Trp Val Leu He Ser Lys Glu Leu He Ser Leu Leu 145 150 155 160 His Leu Ser Leu Leu His Leu Glu Glu Asp Lys Thr Thr Val Ser Gin

165 170 175

Glu Ser Arg Arg Ala Glu Thr Leu Val Cys Cys Cys Phe Asp Leu Leu 180 185 190 Lys Lys Leu Arg Ala Lys Leu Gin Ser Leu Lys Ala Glu Arg Glu Glu 195 200 205

Ala Arg His Arg Glu Glu Met Ala Leu Arg Gly Lys Asp Ala Ala Glu

210 215 220

He Val Leu Glu Ala Phe Cys Ala His Ala Ser Gin Arg He Ser Gin 225 230 235 240

Leu Glu Gin Asp Leu Ala Ser Met Arg Glu Phe Arg Gly Leu Leu Lys

245 250 255

Asp Ala Gin Thr Gin Leu Val Gly Leu His Ala Lys Gin Glu Glu Leu 260 265 270 Val Gin Gin Thr Val Ser Leu Thr Ser Thr Leu Gin Gin Asp Trp Arg 275 280 285

Ser Met Gin Leu Asp Tyr Thr Thr Trp Thr Ala Leu Leu Ser Arg Ser

290 295 300

Arg Gin Leu Thr Glu Lys Leu Thr Val Lys Ser Gin Gin Ala Leu Gin 305 310 315 320

Glu Arg Asp Val Ala He Glu Glu Lys Gin Glu Val Ser Arg Val Leu

325 330 335

Glu Gin Val Ser Ala Gin Leu Glu Glu Cys Lys Gly Gin Thr Glu Gin 340 345 350 Leu Glu Leu Glu Asn Ser Arg Leu Ala Thr Asp Leu Arg Ala Gin Leu 355 360 365

Gin He Leu Ala Asn Met Asp Ser Gin Leu Lys Glu Leu Gin Ser Gin

370 375 380

His Thr His Cys Ala Gin Asp Leu Ala Met Lys Asp Glu Leu Phe Cys 385 390 395 400

Gin Leu Thr Gin Ser Asn Glu Glu Gin Ala Ala Gin Trp Gin Lys Glu

405 410 415

Glu Met Ala Leu Lys His Met Gin Ala Glu Leu Gin Gin Gin Gin Ala 420 425 430 Val Leu Ala Lys Glu Val Arg Asp Leu Lys Glu Thr Leu Glu Phe Ala 435 440 445

Asp Gin Glu Asn Gin Val Ala His Leu Glu Leu Gly Gin Val Glu Cys

450 455 460

Gin Leu Lys Thr Thr Leu Glu Val Leu Arg Glu Arg Ser Leu Gin Cys 465 470 475 480

Glu Asn Leu Lys Asp Thr Val Glu Asn Leu Thr Ala Lys Leu Ala Ser

485 490 495

Thr He Ala Asp Asn Gin Glu Gin Asp Leu Glu Lys Thr Arg Gin Tyr 500 505 510 Ser Gin Lys Leu Arg Leu Leu Thr Glu Gin Leu Gin Ser Leu Thr Leu

515 520 525

Phe Leu Gin Thr Lys Leu Lys Glu Lys Thr Glu Gin Glu Thr Leu Leu

530 535 540 Leu Ser Thr Ala Cys Pro Pro Thr Gin Glu His Pro Leu Pro Asn Asp

545 550 555 560

Arg Thr Phe Leu Gly Ser He Leu Thr Ala Val Ala Asp Glu Glu Pro

565 570 575

Glu Ser Thr Pro Val Pro Leu Leu Gly Ser Asp Lys Ser Ala Phe Thr 580 585 590

Arg Val Ala Ser Met Val Ser Leu Gin Pro Ala Glu Thr Pro Gly Met

595 600 605

Glu Glu Ser Leu Ala Glu Met Ser He Met Thr Thr Glu Leu Gin Ser

610 615 620 Leu Cys Ser Leu Leu Gin Glu Ser Lys Glu Glu Ala He Arg Thr Leu

625 630 635 640

Gin Arg Lys He Cys Glu Leu Gin Val Arg Leu Gin Ala Gin Glu Glu

645 650 655

Gin His Gin Glu Val Gin Lys Ala Lys Glu Ala Asp He Glu Lys Leu 660 665 670

Asn Gin Ala Leu Cys Leu Arg Tyr Lys Asn Glu Lys Glu Leu Gin Glu

675 680 685

Val He Gin Gin Gin Asn Glu Lys He Leu Glu Gin He Asp Lys Ser

690 695 700 Gly Glu Leu He Ser Leu Arg Glu Glu Val Thr His Leu Thr Arg Ser

705 710 715 720

Leu Arg Arg Ala Glu Thr Glu Thr Lys Val Leu Gin Glu Ala Leu Ala

725 730 735

Gly Gin Leu Asp Ser Asn Cys Gin Pro Met Ala Thr Asn Trp He Gin 740 745 750

Glu Lys Val Trp Leu Ser Gin Glu Val Asp Lys Leu Arg Val Met Phe

755 760 765

Leu Glu Met Lys Asn Glu Lys Glu Lys Leu Met He Lys Phe Gin Ser

770 775 780 His Arg Asn He Leu Glu Glu Asn Leu Arg Arg Ser Asp Lys Glu Leu

785 790 795 800

Glu Lys Leu Asp Asp He Val Gin His He Tyr Lys Thr Leu Leu Ser

805 810 815

He Pro Glu Val Val Arg Gly Cys Arg Glu Leu Gin Gly Leu Leu Glu 820 825 830

Phe Leu Ser * 835 ( 2 ) INFORMATION FOR SEQ ID NO 5

( l ) SEQUENCE CHARACTERISTICS

(A ) LENGTH 1191 base pairs

( B ) TYPE nucleic acid (C) STRANDEDNESΞ double

(D) TOPOLOGY linear (n) MOLECULE TYPE cDNA (ix) FEATURE

(A) NAME/KEY CDS (B) LOCATION 34 1191

(xi) SEQUENCE DESCRIPTION SEQ ID NO 5

GAACTAGTGG ATCCCCCGGG CTGCAGGAAT TCG GCA CGA GGC GGC GCC GAA GAG 54

Ala Arg Gly Gly Ala Glu Glu 1 5

GCG ACT GAG GCC GGA CGG GGC GGA CGG CGA CGC AGC CCG CGG CAG AAG 102 Ala Thr Glu Ala Gly Arg Gly Gly Arg Arg Arg Ser Pro Arg Gin Lys

10 15 20

TTT GAA ATT GGC ACA ATG GAA GAA GCT GGA ATT TGT GGG CTA GGG GTG 150 Phe Glu He Gly Thr Met Glu Glu Ala Gly He Cys Gly Leu Gly Val 25 30 35

AAA GCA GAT ATG TTG TGT AAC TCT CAA TCA AAT GAT ATT CTT CAA CAT 198 Lys Ala Asp Met Leu Cys Asn Ser Gin Ser Asn Asp He Leu Gin His 40 45 50 55 CAA GGC TCA AAT TGT GGT GGC ACA AGT AAC AAG CAT TCA TTG GAA GAG 246 Gin Gly Ser Asn Cys Gly Gly Thr Ser Asn Lys His Ser Leu Glu Glu

60 65 70

GAT GAA GGC AGT GAC TTT ATA ACA GAG AAC AGG AAT TTG GTG AGC CCA 294 Asp Glu Gly Ser Asp Phe He Thr Glu Asn Arg Asn Leu Val Ser Pro 75 80 85

GCA TAC TGC ACG CAA GAA TCA AGA GAG GAA ATC CCT GGG GGA GAA GCT 342 Ala Tyr Cys Thr Gin Glu Ser Arg Glu Glu He Pro Gly Gly Glu Ala

90 95 100

CGA ACA GAT CCC CCT GAT GGT CAG CAA GAT TCA GAG TGC AAC AGG AAC 390 Arg Thr Asp Pro Pro Asp Gly Gin Gin Asp Ser Glu Cys Asn Arg Asn 105 110 115

AAA GAA AAA ACT TTA GGA AAA GAA GTT TTA TTA CTG ATG CAA GCC CTA 438 Lys Glu Lys Thr Leu Gly Lys Glu Val Leu Leu Leu Met Gin Ala Leu 120 125 130 135 AAC ACC CTT TCA ACC CCA GAG GAG AAG CTG GCA GCT CTC TGT AAG AAA 486 Asn Thr Leu Ser Thr Pro Glu Glu Lys Leu Ala Ala Leu Cys Lys Lys TAT GCT GAT CTT CTG GAG GAG AGC AGG AGT GTT CAG AAG CAA ATG AAG 534 Tyr Ala Asp Leu Leu Glu Glu Ser Arg Ser Val Gin Lys Gin Met Lys

155 160 165

ATC CTG CAG AAG AAG CAA GCC CAG ATT GTG AAA GAG AAA GTT CAC TTG 582 He Leu Gin Lys Lys Gin Ala Gin He Val Lys Glu Lys Val His Leu 170 175 180

CAG AGT GAA CAT AGC AAG GCT ATC TTG GCA AGA AGC AAG CTA GAA TCT 630 Gin Ser Glu His Ser Lys Ala He Leu Ala Arg Ser Lys Leu Glu Ser 185 190 195 CTT TGC AGA GAA CTT CAG CGT CAC AAT AAG ACG TTA AAG GAG GAA AAT 678 Leu Cys Arg Glu Leu Gin Arg His Asn Lys Thr Leu Lys Glu Glu Asn 200 205 210 215

ATG CAG CAG GCA CGA GAG GAA GAA GAA CGA CGT ATA GAA GCA ACT GCA 726 Met Gin Gin Ala Arg Glu Glu Glu Glu Arg Arg He Glu Ala Thr Ala 220 225 230

CAT TTC CAG ATT ACC TTA AAT GAA ATT CAA GCC CAG CTG GAG CAG CAT 774 His Phe Gin He Thr Leu Asn Glu He Gin Ala Gin Leu Glu Gin His

235 240 245

GAC ATC CAC AAC GCC AAA CTC CGA CAG GAA AAC ATT GAG CTG GGG GAG 822 Asp He His Asn Ala Lys Leu Arg Gin Glu Asn He Glu Leu Gly Glu 250 255 260

AAG CTA AAG AAG CTC ATC GAA CAG TAC GCA CTG AGG GAA GAG CAC ATT 870 Lys Leu Lys Lys Leu He Glu Gin Tyr Ala Leu Arg Glu Glu His He 265 270 275 GAT AAG GTG TTC AAA CAT AAG GAA CTG CAA CAG CAG CTC GTG GAT GCC 918 Asp Lys Val Phe Lys His Lys Glu Leu Gin Gin Gin Leu Val Asp Ala 280 285 290 295

AAA CTG CAG CAA ACG ACA CAA CTG ATA AAA GAA GCT GAT GAA AAA CAT 966 Lys Leu Gin Gin Thr Thr Gin Leu He Lys Glu Ala Asp Glu Lys His 300 305 310

CAG AGA GAG AGA GAG TTT TTA TTA AAA GAA GCG ACA GAA TCG AGG CAC 1014 Gin Arg Glu Arg Glu Phe Leu Leu Lys Glu Ala Thr Glu Ser Arg His

315 320 325

AAA TAC GAA CAA ATG AAA CAG CAA GAA GTA CAA CTA AAA CAG CAG CTT 1062 Lys Tyr Glu Gin Met Lys Gin Gin Glu Val Gin Leu Lys Gin Gin Leu 330 335 340

TCT CTT TAT ATG GAT AAG TTT GAA GAA TTC CAG ACT ACC ATG GCA AAA 1110 Ser Leu Tyr Met Asp Lys Phe Glu Glu Phe Gin Thr Thr Met Ala Lys 345 350 355 AGC AAT GAA CTG TTT ACA ACC TTC AGA CAG GAA ATG GAA AAG ATG ACA 1158 Ser Asn Glu Leu Phe Thr Thr Phe Arg Gin Glu Met Glu Lys Met Thr 360 365 370 375 AAG AAA ATT AAA AAA AAA AAA AAA AAA CTC GAG 1191

Lys Lys He Lys Lys Lys Lys Lys Lys Leu Glu 380 385

(2) INFORMATION FOR SEQ ID NO : 6 ^•

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 386 amino acids

(B) TYPE: ammo acid (D) TOPOLOGY: linear In) MOLECULE TYPE: protein

(xi ) SEQUENCE DESCRIPTION: SEQ ID NO : 6 :

Ala Arg Gly Gly Ala Glu Glu Ala Thr Glu Ala Gly Arg Gly Gly Arg 1 5 10 15 Arg Arg Ser Pro Arg Gin Lys Phe Glu He Gly Thr Met Glu Glu Ala 20 25 30

Gly He Cys Gly Leu Gly Val Lys Ala Asp Met Leu Cys Asn Ser Gin

35 40 45

Ser Asn Asp He Leu Gin His Gin Gly Ser Asn Cys Gly Gly Thr Ser 50 55 60

Asn Lys His Ser Leu Glu Glu Asp Glu Gly Ser Asp Phe He Thr Glu

65 70 75 80

Asn Arg Asn Leu Val Ser Pro Ala Tyr Cys Thr Gin Glu Ser Arg Glu

85 90 95 Glu He Pro Gly Gly Glu Ala Arg Thr Asp Pro Pro Asp Gly Gin Gin

100 105 110

Asp Ser Glu Cys Asn Arg Asn Lys Glu Lys Thr Leu Gly Lys Glu Val

115 120 125

Leu Leu Leu Met Gin Ala Leu Asn Thr Leu Ser Thr Pro Glu Glu Lys 130 135 140

Leu Ala Ala Leu Cys Lys Lys Tyr Ala Asp Leu Leu Glu Glu Ser Arg

145 150 155 160

Ser Val Gin Lys Gin Met Lys He Leu Gin Lys Lys Gin Ala Gin He

165 170 175 Val Lys Glu Lys Val His Leu Gin Ser Glu His Ser Lys Ala He Leu

180 185 190

Ala Arg Ser Lys Leu Glu Ser Leu Cys Arg Glu Leu Gin Arg His Asn

195 200 205

Lys Thr Leu Lys Glu Glu Asn Met Gin Gin Ala Arg Glu Glu Glu Glu 210 215 220

Arg Arg He Glu Ala Thr Ala His Phe Gin He Thr Leu Asn Glu He 225 230 235 240

Gin Ala Gin Leu Glu Gin His Asp He His Asn Ala Lys Leu Arg Gin 245 250 255 Glu Asn He Glu Leu Gly Glu Lys Leu Lys Lys Leu He Glu Gin Tyr

260 265 270

Ala Leu Arg Glu Glu His He Asp Lys Val Phe Lys His Lys Glu Leu 275 280 285 Gin Gin Gin Leu Val Asp Ala Lys Leu Gin Gin Thr Thr Gin Leu He 290 295 300

Lys Glu Ala Asp Glu Lys His Gin Arg Glu Arg Glu Phe Leu Leu Lys 305 310 315 320

Glu Ala Thr Glu Ser Arg His Lys Tyr Glu Gin Met Lys Gin Gin Glu 325 330 335

Val Gin Leu Lys Gin Gin Leu Ser Leu Tyr Met Asp Lys Phe Glu Glu

340 345 350

Phe Gin Thr Thr Met Ala Lys Ser Asn Glu Leu Phe Thr Thr Phe Arg 355 360 365 Gin Glu Met Glu Lys Met Thr Lys Lys He Lys Lys Lys Lys Lys Lys 370 375 380

Leu Glu 385

Claims

We Claim:

I. A composition of matter comprising an isolated nucleic acid sequence that encodes a BRCAl Modulator Protein.

2. A composition of matter as described in claim 1 wherein said isolated nucleic acid sequence encodes a BRCAl Modulator Protein, said Modulator Protein comprising at least one leucine zipper domain and one zinc finger domain.

3. A composition of matter as described in claim 2 wherein said isolated nucleic acid sequence encodes a BRCAl Modulator Protein said BRCAl Modulator Protein comprising a molecular weight ranging from about 45-97 kd.

4. A composition of matter comprising an isolated nucleic acid sequence as described in claim 3 wherein said sequence is a cDNA sequence.

5. A composition of matter comprising an isolated nucleic acid sequence as described in claim 4 wherein cDNA sequence is selected from the group consisting of 091-21A31, Sequence ID No. 1, 091-1F84, Sequence ID No. 3, and 091-132Q20, Sequence ID No. 5.

6. A composition of matter as described in claim 1 wherein said isolated nucleic acid sequence encoding said BRCAl Modulator Protein comprises an isolated nucleic acid fragment of said nucleic acid sequence.

7. A nucleic acid sequence hybridizable to a BRCAl Modulator nucleic acid sequence of claim 1 under high stringency conditions

8. A nucleic acid sequence of claim 7, wherein said nucleic acid sequence is about 95% homologous to said BRCAl Modulator sequence.

9. A composition of matter comprising a BRCAl Modulator Protein. 10. A composition of matter as described in claim 9 wherein said BRCAl

Modulator Protein has a molecular weight ranging from about 45-97 kdaltons.

II. Isolated host cells comprising an isolated nucleic acid sequence that encodes a BRCAl Modulator Protein.

12. Vectors that comprise an isolated nucleic acid sequence that encodes a BRCAl Modulator Protein.

13. A complex comprising substantially purified BRCAl and a BRCAl Modulator Protein.

14. A host cell comprising the complex of claim 13.

15. A method of forming a complex of substantially purified BRCAl and BRCAl Modulator Protein, comprising contacting in solution substantially purified BRCAl with substantially purified BRCAl Modulator Protein, allowing sufficient time for said complex to form, and removing uncomplexed BRCAl and BRCAl Modulator Protein.

16. Isolated antibody that binds to a BRCAl Modulator.

17. A pharmaceutical composition comprising a BRCAl Modulator.

18. A pharmaceutical composition as described in claim 15 wherein said BRCAl Modulator is selected from the group consisting of 091-21 A31, Sequence ID No. 1, 091- 1F84, Sequence ID No. 3, and 091-132Q20 Sequence ID No. 5.

19. A nucleic acid sequence that encodes a protein encoded by the cDNA on deposit with the ATCC with accession no. 98141 (091-1F84, Sequence ID No. 3).

20. A nucleic acid sequence that encodes a protein encoded by the cDNA on deposit with the ATCC with accession no. 98142 (091-21A31, Sequence ID No. 1).

21. A nucleic acid sequence that encodes a protein encoded by the cDNA on deposit with the ATCC with accession no. 98143 (091-132Q20, Sequence ID No. 5).

22. A process for producing a BRCAl Modulator Protein comprising culturing a cell of claim 11 in a suitable culture medium and isolating said protein from said cell or said medium.