WO1998012327A2

WO1998012327A2 - Compositions and methods comprising bard1 and other brca1 binding proteins

Info

Publication number: WO1998012327A2
Application number: PCT/US1997/016842
Authority: WO
Inventors: Anne M. Bowcock; Richard Baer
Original assignee: Board Of Regents, The University Of Texas System
Priority date: 1996-09-20
Filing date: 1997-09-19
Publication date: 1998-03-26
Also published as: AU4586697A; WO1998012327A3

Abstract

Disclosed are several novel genes, identified in screening assays based upon binding to the breast cancer protein, BRCA1. The currently preferred gene and protein, termed BARD1, is a RING protein that interacts with BRCA1. The genes, proteins and other biological materials provided are envisioned for use in various cancer-related diagnostic and therapeutic methods, particularly those connected with breast, ovarian and uterine cancer.

Description

BACKGROUND OF THE INVENTION

COMPOSITIONS AND METHODS COMPRISING BARDl AND OTHER BRCAI BINDING PROTEINS

The present application claims the priority of co-pending U.S. Provisional Patent Applications Serial No. 60/025,296, filed September 20, 1996, Serial No. 60/042,61 1 , filed April 3, 1997, and Serial No. 60/042,985, filed April 4, 1997, the entire disclosures of which are incoφorated herein by reference without disclaimer.

Field of the Invention

The present invention relates generally to the field of cancer, and particularly concerns the diagnosis and treatment of breast cancer. The invention provides novel genes, proteins and related compositions that interact with the BRCAI gene product, which is known to be connected with a significant number of breast cancers. The currently preferred gene and protein of the invention is a RING protein termed BARDl. Also disclosed are various diagnostic and therapeutic methods and screening assays using the compositions of the invention.

2. Description of Related Art

Breast cancer is the most common fatal malignancy affecting women in the western world. The etiology of breast cancer is complex, and likely involves genetic, hormonal, environmental and other factors. Detailed analyses of breast cancer patients has revealed several alterations in gene expression associated with the disease. In addition to gene amplification, breast tumor development is thought to be the consequence of mutations in one or more recessive genes.

A particular breast cancer-related gene is the BRCAI gene. Germline mutations of the

BRCAI gene are found in approximately half of families that display a heritable susceptibility to breast cancer (Hall et al, 1990; Miki et al, 1994; Futreal et l, 1994; Castilla et al, 1994; Simard et al, 1994; Friedman et al, 1994). In women of these kindreds, the mutant BRCAI allele confers lifetime risks of 80-90% for breast cancer and 40-50% for ovarian cancer (Easton et al, 1993; Ford et al, 1994). The wild-type allele of BRCAI is typically lost or inactivated in the tumors that arise in these families, implying that BRCAI normally functions as a tumor- suppressor gene. Λ variety of different germline BRCAI mutations that segregate with breast cancer susceptibility have been described; these include missense mutations which produce single amino acid substitutions and, more commonly, frame-shifts or nonsense mutations which truncate the BRCAI reading frame (Miki et al, 1994; Futreal et al, 1994; Castilla et al, 1994; Simard et al, 1994; Friedman et al, 1994).

The human BRCAI gene encodes a large polypeptide of 1863 amino acids, the precise biochemical function of which is not yet known (Miki et al, 1994). A prominent feature of the protein is a RING domain that resides near its amino-terminus (residues 20-68). The RING motif, a cysteine-rich sequence found in a diverse group of regulatory proteins, adopts an interleaved structure in which two ions of zinc are coordinated by eight conserved amino acids (seven cysteines and one histidine) (Saurin et al, 1996). Thus, BRCAI can be said to have two "zinc finger domains".

It has been proposed that the zinc fingers or RING domain serves as an interface for DNA recognition or protein-protein interactions (Saurin et al, 1996), and that the BRCAI protein may be a transcription factor (Miki et al, 1994; Vogelstein and Kinzler, 1994). However, no direct evidence that BRCAI is a transcription factor has yet been presented. In fact, a detailed characterization of BRCA 1 function at the molecular level has been somewhat hindered by the lack of purified protein in amounts sufficient to conduct productive assays in vitro.

Whatever its precise function, the analysis of germline mutations in families prone to breast and ovarian cancer suggests that the RING domain may be essential for the tumor suppressor activity of BRCAI ; thus, in some kindreds the tumorigenic lesion is a single missense mutation (C61G or C64G) that specifically replaces one of the cysteine residues required for zinc coordination by the RING domain (Castilla et al, 1994; Friedman et al, 1994).

Recent studies have shown that the mouse and human homologs of BRCAI share approximately 60% amino acid identity (Bennett et al, 1995; Lane et al, 1995; Sharan et al, 1995). This degree of phylogcnetic conservation is low, especially when compared with other known tumor suppressor proteins; for example, the mouse and human counterparts of RBI, p53, APC, WTl, and NFl display amino acid identities in the range of 78-98%. Nevertheless, two regions of BRCAI are especially well conserved. The first corresponds to the amino-terminal 100 residues; this sequence encompasses the RING domain and the tumorigcnic missense mutations at C61 and C64 (Castilla et al, 1994; Friedman et al, 1994).

The second region of high conservation resides near the carboxy-terminus of BRCAI, and it also serves as a target for missense mutations associated with familial breast cancer (Sharan et al, 1995). This region includes two tandem copies of the BRCAI carboxy-tcrminal domain ("BRCT domain"), a newly-recognized amino acid motif also found in 53BP1, a mammalian polypeptide that binds the p53 tumor suppressor, and RAD9, a yeast protein that mediates cell cycle arrest in response to DNA damage (Koonin et al, 1996).

Given that the BRCAI gene and protein product are now accepted to be closely linked to familial breast cancer development, but that the function of BRCAI remains unknown, any further delineation of the properties and interactions of the BRCAI protein would be an important development. The identification of proteins that bind to BRCAI would be particularly beneficial as they themselves would likely be implicated in the breast cancer process. The cloning of genes encoding such BRCAI -binding proteins would therefore be a significant contribution towards the development of further cancer diagnostics and therapeutics.

SUMMARY OF THE INVENTION

The present invention provides several novel genes, proteins and related biological compositions developed from their ability to bind to the BRCAI protein. Methods of using the various compositions, for example, in the diagnosis, prognosis and treatment of breast, ovarian and uterine cancer are also provided.

The present invention first provides DNA segments, vectors and the like comprising at least a first isolated gene, DNA segment or coding sequence region that encodes a BARDl, B123, BE2, BE14, BE31 or BE445 protein, polypeptide, domain, peptide or any fusion protein thereof, and particularly, that encode a human BARDl, B123, BE2, BE14, BE31 or BE445 protein, domain, fragment or derivative.

As used herein in the context of the instant compositions, the term BARDl , B123, BE2,

BE14, BE31 and BE445 will be understood to include wild-type, polymorphic and mutant BARDl, B123, BE2, BE14, BE31 and BE445 sequences. Wild-type sequences are defined as the first identified sequence, polymorphic sequences are defined as naturally occurring variants of the wild-type sequence that have no effect on the expression or function of the BARDl , B123, BE2, BE14, BE31 or BE445 proteins or domains thereof, and mutant sequences are defined as changes in the wild-type sequence, either naturally occurring or introduced by the hand of man, that have an effect on either the expression and/or the function of the BARDl , B123, BE2, BE14, BE31 or BE445 proteins or domains thereof.

Thus, the invention also includes the provision of DNA segments, vectors, genes and coding sequence regions that encode BARDl, B123, BE2, BE 14, BE31 or BE445 proteins, polypeptides, domains, peptides or any fusion protein thereof, where the BARDl, B123, BE2, BE14, BE31 or BE445 protein element comprises at least one mutation in comparison to the wild-type sequence. The mutation may be deliberately introduced by the hand of man, for example, in order to test the function of the changed amino acid, e.g., in BRCAI binding, DNA binding and/or other functions. Additionally, the mutation may be a naturally occurring polymorphic change, either isolated from normal cells or introduced by the hand of man.

The BARDl, B123, BE2, BE14, BE31 or BE445 mutation may also be in a purified protein obtained directly from an aberrant cell, such as a breast, ovarian or uterine cancer cell, or may be a recombinant protein that has been changed to introduce a mutation that mirrors one identified in a patient. The mutation may result in a truncated BARDl, B123, BE2, BE14, BE31 or BE445 gene or protein, or may result in increased, decreased or undetectable levels of BARDl, B123, BE2, BE14, BE31 or BE445 gene or protein being produced. Where diagnostic or prognostic mutated BARDl, B123, BE2, BE14, BE31 or BE445 genes, proteins and antibodies are concerned the mutant gene, DNA segment, antibody or even peptide will preferably have specificity for the mutant sequence in preference to the wild-type sequence, allowing effective differentiation between the two, as may be used in diagnostic or prognostic tests for breast, ovarian or uterine cancer cells or patients, as described in more detail herein below.

The DNA segments and vectors may comprise an isolated gene or coding sequence that encodes a BARDl protein characterized as having the following properties:

being about 777, 770 or about 752 amino acids in length, preferably being 777 amino acids in length;

comprising an amino-terminal RING motif or domain, preferably characterized as comprising a cysteine-rich sequence with an interleaved structure in which two ions of zinc are coordinated by seven cysteines and one histidine, and which RING motif or domain mediates the association of BARDl with BRCAI ;

containing ankyrin repeats, which ankyrin repeats are not required for binding to BRCAI;

comprising carboxy-terminal BRCT domains that are homologous to carboxy-terminal sequences of BRCA 1 ;

being encoded by sequences on chromosome 2q;

binding to BRCA 1 , as may be assessed by one or more cellular assay systems, such as a yeast or mammalian two-hybrid system that identifies functional proteins associations in vivo; or by co-immunoprecipitation of the BRCAI and BARDl proteins from mammalian cell lysates, or by using one or more in vitro assays of protein binding;

and more preferably, characterized as binding to the amino-terminal region of BRCAI, most preferably to the BRCAI amino-terminal 101 residues that encompasses the

RING motif (residues 20-68), but as not binding to the BRCAI fragment between residues 1 and 71; and even more preferably, wherein residues 26-202 of BARDl , and most preferably, where residues 26-142 of BARDl, which include the RING motif (residues 46-90), but do not include the ankyrin repeats (residues 427-525), interact with BRCAI .

It will be understood that while the normal, native, wild-type BARDl protein is defined in terms of these properties and domains, the overall features will generally be the same for

BARDl polymoφhic and mutant proteins and domains as well. The polymorphic and mutant

BARDl genes and proteins can be understood with reference to the wild-type sequences and the exemplary mutants included herein.

The genes and DNA segments of the present invention preferably encode wild-type or polymoφhic BARDl proteins, polypeptides, domains, peptides or fusion constructs thereof where the BARDl sequence includes a contiguous amino acid sequence from SEQ ID NO:2, SEQ ID NO.21, SEQ ID NO:23, SEQ ID NO.25, SEQ ID N0.27, SEQ ID NO.29, SEQ ID NO:31 or SEQ ID NO.39, or a biologically functional equivalent thereof. The present invention also provides genes and DNA segments that encode mutant BARDl proteins, polypeptides, domains, peptides or fusion constructs thereof where the BARDl sequence includes a contiguous amino acid sequence from SEQ ID NO:33, SEQ ID NO:35 or SEQ ID NO:37, or a biologically functional equivalent thereof. As used herein, the term "contiguous amino acid sequence" will be understood to include a contiguous amino acid sequence of at least about 4, about 6, about 9, about 10, about 12, about 15 or about 20 amino acids or so.

Thus in certain aspects of the present invention, the genes and DNA segments encode wild-type BARDl proteins, polypeptides, domains, peptides or fusion constructs thereof where the wild-type BARDl sequence includes a contiguous amino acid sequence from SEQ ID NO:2 or a biologically functional equivalent thereof. Preferably, the isolated genes and coding regions will include a contiguous nucleic acid sequence from between position 75 and position 2405 of SEQ ID NO: 1 or a biologically functional equivalent thereof.

In other aspects of the present invention, the genes and DNA segments encode polymoφhic BARDl proteins, polypeptides, domains, peptides or fusion constructs thereof where the polymoφhic BARDl sequence is described as BARDl PI 43, and includes a contiguous amino acid sequence from SEQ ID NO:21 or a biologically functional equivalent thereof. Preferably, the isolated genes and coding regions will include a contiguous nucleic acid sequence from between position 75 and position 2405 of SEQ ID NO:20 or a biologically functional equivalent thereof.

In further embodiments of the present invention, the genes and DNA segments encode polymoφhic BARDl proteins, polypeptides, domains, peptides or fusion constructs thereof where the polymoφhic BARDl sequence is described as BARDl P531 , and includes a contiguous amino acid sequence from SEQ ID NO:23 or a biologically functional equivalent thereof. Preferably, the isolated genes and coding regions will include a contiguous nucleic acid sequence from between position 75 and position 2405 of SEQ ID NO:22 or a biologically functional equivalent thereof.

In yet other aspects of the present invention, the genes and DNA segments encode polymoφhic BARDl proteins, polypeptides, domains, peptides or fusion constructs thereof where the polymoφhic BARDl sequence is described as BARDl PI 121 , and includes a contiguous amino acid sequence from SEQ ID NO:25 or a biologically functional equivalent thereof. Preferably, the isolated genes and coding regions will include a contiguous nucleic acid sequence from between position 75 and position 2405 of SEQ ID NO:24 or a biologically functional equivalent thereof.

In still other embodiments of the present invention, the genes and DNA segments encode polymoφhic BARDl proteins, polypeptides, domains, peptides or fusion constructs thereof where the polymoφhic BARDl sequence is described as BARDl PΔl 140-1160, and includes a contiguous amino acid sequence from SEQ ID NO:27 or a biologically functional equivalent thereof. Preferably, the isolated genes and coding regions will include a contiguous nucleic acid sequence from between position 75 and position 2385 of SEQ ID NO:26 or a biologically functional equivalent thereof.

In alternate aspects of the present invention, the genes and DNA segments encode polymoφhic BARDl proteins, polypeptides, domains, peptides or fusion constructs thereof where the polymoφhic BARDl sequence is described as BARDl PI 592, and includes a contiguous amino acid sequence from SEQ ID NO:29 or a biologically functional equivalent thereof. Preferably, the isolated genes and coding regions will include a contiguous nucleic acid sequence from between position 75 and position 2405 of SEQ ID NO:28 or a biologically functional equivalent thereof.

In particular embodiments of the present invention, the genes and DNA segments encode polymorphic BARDl proteins, polypeptides, domains, peptides or fusion constructs thereof where the polymoφhic BARDl sequence is described as BARDl PI 765, and includes a contiguous amino acid sequence from SEQ ID NO:31 or a biologically functional equivalent thereof. Preferably, the isolated genes and coding regions will include a contiguous nucleic acid sequence from between position 75 and position 2405 of SEQ ID NO:30 or a biologically functional equivalent thereof.

In particular embodiments of the present invention, the genes and DNA segments encode polymorphic BARDl proteins, polypeptides, domains, peptides or fusion constructs thereof where the polymoφhic BARDl sequence is described as BARDl P2354, and includes a contiguous amino acid sequence from SEQ ID NO:39 or a biologically functional equivalent thereof. Preferably, the isolated genes and coding regions will include a contiguous nucleic acid sequence from between position 75 and position 2405 of SEQ ID NO:38 or a biologically functional equivalent thereof.

In certain embodiments of the present invention, the genes and DNA segments encode mutant BARDl proteins, polypeptides, domains, peptides or fusion constructs thereof where the mutant BARDl sequence is described as BARDl MQ564II, and includes a contiguous amino acid sequence from SEQ ID NO:33 or a biologically functional equivalent thereof. Preferably, the isolated genes and coding regions will include a contiguous nucleic acid sequence from between position 75 and position 2405 of SEQ ID NO:32 or a biologically functional equivalent thereof.

In other aspects of the present invention, the genes and DNA segments encode mutant BARDl proteins, polypeptides, domains, peptides or fusion constructs thereof where the mutant BARDl sequence is described as BARDl MS761N, and includes a contiguous amino acid sequence from SEQ ID NO:35 or a biologically functional equivalent thereof. Preferably, the isolated genes and coding regions will include a contiguous nucleic acid sequence from between position 75 and position 2405 of SEQ ID NO:34 or a biologically functional equivalent thereof.

In further embodiments of the present invention, the genes and DNA segments encode mutant BARDl proteins, polypeptides, domains, peptides or fusion constructs thereof where the mutant BARDl sequence is described as BARDl MR658C, and includes a contiguous amino acid sequence from SEQ ID NO:37 or a biologically functional equivalent thereof. Preferably, the isolated genes and coding regions will include a contiguous nucleic acid sequence from between position 75 and position 2405 of SEQ ID NO:36 or a biologically functional equivalent thereof.

The DNA segments and coding regions may encode wild-type, polymorphic or mutant BARDl peptides, e.g., of from about 15 to about 30 or about 50 amino acids in length or so. The BARDl peptides may be lacking in any defined BARDl activity, and may, for example, be used in generating antibodies or in other embodiments. The BARDl peptides or domains may also be deliberately engineered to include a mutation, e.g., in order to prepare antibodies that are specific for a mutated BARDl, particularly where the mutation represents one identified in a patient with breast, ovarian or endometrial cancer.

The present invention also provides DNA segments and coding regions that may encode a BARDl peptide of from about 6 to about 30 amino acids in length, the peptide having an amino acid sequence that corresponds to a wild-type BARDl sequence of a BARDl protein sequence region that is susceptible to mutations that are indicative of a malignant phenotype. Where diagnostic or prognostic BARDl genes, proteins and antibodies are concerned the gene, DNA segment, antibody or even peptide will preferably allow effective differentiation between the mutant BARDl sequence and the wild-type BARDl sequence as may be used in diagnostic or prognostic tests for breast, ovarian or uterine cancer cells or patients, as described in more detail herein below. The genes, DNA segments, vectors and coding sequence regions may also encode wild- type, polymoφhic or mutant BARDl polypeptides and peptides with certain, but necessary all, BARDl functional properties. As such genes and coding sequences encoding isolated wild- type, polymoφhic or mutant BARDl domains are provided.

The wild-type, polymoφhic or mutant BARDl domains contemplated include isolated and or purified wild-type, polymoφhic or mutant BARDl ankyrin repeat domains, including those comprising three ankyrin repeats and comprising or having the sequence of residues 427-525 from SEQ ID NO:2, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31 , SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37 or SEQ ID NO:39; isolated and/or purified BARDl BRCT-like domains, as exemplified by those comprising the BRCT domain N-terminal core motif of residues 616-653 of SEQ ID NO:2, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31 , SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37 or SEQ ID NO:39, the BRCT domain C- terminal core motif of residues 743-777 of SEQ ID NO:2, SEQ ID NO:21, SEQ ID NO.23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37 or SEQ ID NO:39, the BRCT domain of residues 616-777 of SEQ ID NO:2, SEQ ID NO:2I, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37 or SEQ ID NO:39; and isolated and/or purified BARDl RING motif domains exemplified by those comprising or having the sequence of residues 46-90 from SEQ ID NO:2, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO.27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37 or SEQ ID NO:39.

Preferred examples of domains are the BRCAI binding domains. For example, those comprising or having the sequence of residues 26-202 from SEQ ID NO:2, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37 or SEQ ID NO:39, or more preferably, those comprising or having the sequence of residues 26-142 from SEQ ID NO:2, SEQ ID NO.21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37 or SEQ ID NO:39, or any active portion of such sequences that functions to bind BRCAI. "BRCAI binding", as used herein, may be assessed by any one or more suitable in vitro, in vivo or in cellulo assays. For example, co-immunoprecipitation of the BRCAI and BARDl proteins from mammalian cell lysates, and in vitro assays of protein binding, e.g., wherein one or both of the BARDl or BRCAI components are attached to a detectable label, and/or are immobilized may be employed. Cellular assay systems, such as a yeast or mammalian two- hybrid protein association system may also be employed, as disclosed herein.

The BARDl domains may also be mutant domains, which include naturally occurring polymorphisms, mutations found in BARDl proteins in patients and, also, mutations deliberately engineered into a domain to test their function in assays. The mutant domains are also useful in antibody generation and in various in vitro and cellular assays. Engineering increased BRCAI binding is also contemplated.

The full length wild-type, polymoφhic and mutant BARDl proteins of the present invention are unusual in that they combine sequence features and motifs not previously observed in combination, e.g., RING and BRCT elements. The wild-type, polymorphic and mutant BARDl proteins of the invention may be further characterized as including domains defined as:

comprising an amino-terminal RING motif or domain that has the sequence of residues

46-90 from SEQ ID NO:2, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37 or SEQ ID NO:39;

comprising a binding domain, or "BRCAI binding domain" that has the sequence of residues 26-202 from SEQ ID NO:2, SEQ ID NO:21, SEQ ID NO:23, SEQ ID

NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37 or SEQ ID NO:39, or more preferably, that has the sequence of residues 26-142 from SEQ ID NO:2, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37 or SEQ ID NO:39, which binding domain binds to BRCAI; containing ankyrin repeats that have the sequence of residues 427-525 from SEQ ID NO:2, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37 or SEQ ID NO:39, which ankyrin repeats do not bind to BRCAI ; and

comprising carboxy-terminal BRCT domains that have a sequence between residues 605 and 777 of SEQ ID NO:2, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37 or SEQ ID NO:39, as exemplified by comprising the BRCT domain N-terminai core motif of residues 616-653 of SEQ ID NO:2, SEQ ID

NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31 , SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37 or SEQ ID NO:39 and as comprising the BRCT domain C-terminal core motif of residues 743-777 of SEQ ID NO:2, SEQ ID NO:21 , SEQ ID NO.23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ

ID NO:37 or SEQ ID NO:39.

As the full length DNA segments of the invention preferably encode wild-type, polymoφhic or mutant BARDl proteins of about 777, 770 or 752 amino acids in length, each of the sequence designations provided herein refer to the 777, 770 or 752 amino acid sequence of SEQ ID NO:2, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37 or SEQ ID NO:39. However, with proteins of shorter length, the operative domains and regions will be easily identified by virtue of the sequence and respective locations.

DNA segments, isolated genes or coding regions may also be manipulated to encode BARDl, B123, BE2, BE14, BE31 or BE445 fusion proteins or constructs in which at least one BARDl, B123, BE2, BE14, BE31 or BE445 protein sequence is operatively attached or linked to at least one distinct, selected amino acid sequence. The combination of BARDl, B123, BE2, BE14, BE31 or BE445 sequences with selected antigenic amino acid sequences; selected non- antigenic carrier amino acid sequences, for use in immunization; selected adjuvant sequences; amino acid sequences with specific binding affinity for a selected molecule; and amino acid sequences that form an active DNA binding or transactivation domain arc particularly contemplated. Certain fusion proteins may be linked together via a protease-sensitive peptide linker, allowing subsequent easy separation.

Also particularly contemplated are the combination of BARDl , B123, BE2, BE14, BE31 or BE445 sequences with a selected tumor suppressor protein or peptide. Tumor suppressor proteins contemplated for use include, but are not limited to, the retinoblastoma, p53, Wilms tumor (WT-1), DCC, neurofibromatosis type 1 (NF-1), von Hippel-Lindau (VHL) disease tumor suppressor, Maspin, Brush- 1, BRCA-1, BRCA-2 and the multiple tumor suppressor (MTS) or pi 6 proteins or peptides. Further particularly contemplated are the combination of BARDl , B123, BE2, BE14, BE31 or BE445 sequences with a selected wild-type version of a selected oncogenic protein or peptide. Wild-type oncogenic proteins contemplated for use include, but are not limited to, tyrosine kinases, both membrane-associated and cytoplasmic forms, such as members of the Src family, serine/threonine kinases, such as Mos, growth factor and receptors, such as platelet derived growth factor (PDGF), small GTPases (G proteins) including the ras family and Gs-alpha, cycl in-dependent protein kinases (cdk), members of the myc family members including c-myc, N-myc, and L-myc and bcl-2 and family members.

DNA segments and isolated genes may also be manipulated to encode BARDl, B123,

BE2, BE14, BE31 or BE445 fusion proteins or constructs in which at least one BARDl, BI23, BE2, BE 14, BE31 or BE445 protein sequence is operatively attached or linked to at least one distinct, selected BARDl, B123, BE2, BE14, BE31 or BE445 protein or peptide sequence.

The DNA segments intended for use in expression will be operatively positioned under the control of, i.e., downstream from, a promoter that directs expression of BARDl, B123, BE2, BE 14, BE31 or BE445 in a desired host cell, such as E. coli, or in certain preferred embodiments in a mammalian or human cell. The promoter may be a recombinant promoter or a promoter naturally associated with a BARDl, B123, BE2, BE 14, BE31 or BE445 gene. Recombinant vectors thus form another aspect of the present invention. The use of isolated BARDl, B123, BE2, BE14, BE31 or BE445 genes positioned, in reverse orientation, under the control of a promoter that directs the expression of an antisense product in a cell is also contemplated.

In certain aspects of the present invention, the nucleic acid segments disclosed herein further comprise a second sequence region of at least about 20 contiguous nucleotides that have the same sequence as, or are complementary to, SEQ ID NO:l , SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:l l, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26; SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129 or SEQ ID NO: 130, said sequence region and said second sequence region from spatially distant regions within SEQ ID NO: l, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:l 1, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26; SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO: I22, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129 or SEQ ID NO:130.

In the same yeast two-hybrid system used to identify BARDl, fourteen other novel genes that encode polypeptides that bind to BRCAI were identified. These are the TCL52 DNA and protein sequence (SEQ ID NO:9 and SEQ ID NO:48, respectively); TCL163 DNA and protein sequence (SEQ ID NO: 10 and SEQ ID NO:49, respectively); B223 DNA and protein sequence (SEQ ID NO:l 1 and SEQ ID NO:50, respectively); Bl 15 DNA and protein sequence (SEQ ID NO: 12 and SEQ ID NO:51, respectively); BAP28 DNA and protein sequence (SEQ ID NO: 13 and SEQ ID NO:52, respectively); B48 DNA and protein sequence (SEQ ID NO: 14 and SEQ ID NO:53, respectively); B258 DNA and protein sequence (SEQ ID NO: 15 and SEQ ID NO:54, respectively); BAP152 DNA and protein sequence (SEQ ID NO: 16 and SEQ ID NO:55, respectively); B123 DNA and protein sequence (SEQ ID NO: 17 and SEQ ID NO: 19, respectively); B268 DNA and protein sequence (SEQ ID NO: 18 and SEQ ID NO:56, respectively); BE2 DNA and protein sequence (SEQ ID NO:40 and SEQ ID NO:41, respectively); BE14 DNA and protein sequence (SEQ ID NO:42 and SEQ ID NO:43, respectively); BE31 DNA and protein sequence (SEQ ID NO:44 and SEQ ID NO:45, respectively); and BE445 DNA and protein sequence (SEQ ID NO:46 and SEQ ID NO:47, respectively).

Thus, the present invention further advantageously provides methods for identifying a human candidate tumor suppressor gene or oncogene based upon the "two hybrid screening system". One such method may be characterized as comprising the steps of:

a) obtaining a first DNA segment comprising a candidate human gene; the first DNA segment expressing a first fusion protein comprising a transcriptional transactivating domain operatively attached to the candidate protein encoded by the candidate gene;

b) obtaining a second DNA segment that expresses a second fusion protein comprising a human BRCAI or BARDl RING domain operatively attached to a DNA binding domain that binds to a defined nucleic acid sequence;

c) providing the first and second DNA segments to a eukaryotic host cell that comprises a marker gene operatively positioned downstream of the defined nucleic acid sequence; and

d) identifying a eukaryotic host cell that expresses the marker gene, thereby identifying the candidate gene as a human gene that encodes a tumor suppressor gene or oncogene.

The methods generally further comprise isolating the identified candidate human tumor suppressor gene or oncogene from the first DNA segment within the eukaryotic host cell.

The transcriptional transactivating domains used in the present invention may be the GAL4, HAPl, LEU3, PHO4, PHO2, PPRl, ARGRII, ADRl, QAIF, MAL63, LAC9, GCN4 or VP16 transcriptional transactivating domain. The fusion protein may comprise a GAL4 DNA binding domain, wherein the defined nucleic acid sequence comprises a GAL4 binding domain recognition sequence, or a lexA DNA binding domain, wherein the defined nucleic acid sequence comprises a lexO binding site sequence. In the methods, the eukaryotic host cell may be a yeast host cell (yeast two hybrid system) or a mammalian host cell.

In the two hybrid system methods of the present invention, marker genes preferred for use are chloramphenicol acetyltransferase, β-galactosidase, green fluorescent protein, β-glucuronidase or the luciferase gene, preferably the β-galactosidase gene. In other aspects, the marker genes can be genes that encode vital biological components, used in combination with strains of Saccharomyces cerevisiae that lack one or more of these genes, such that expression of one or more of the marker genes is required to produce viable colonies. Marker genes contemplated for use in these aspects of the invention are exemplified by, but not limited to, the URA3, TRP1, HIS3, LYS2,ADE1 and LEU2 genes of Saccharomyces cerevisiae.

A further explanation of the two hybrid system cloning method for identifying a human gene that encodes a candidate tumor suppressor protein or oncogene is that it generally operatively comprises the steps of:

a) obtaining a plurality of first DNA segments comprising a plurality of candidate human genes;

b) obtaining multiple copies of the second DNA segment;

c) providing the plurality of first DNA segments and multiple copies of the second

DNA segments to a population of eukaryotic host cells in an amount sufficient to provide about one first DNA segment and at least about one second DNA segment to each host cell in the population;

d) culturing the population of cells under conditions and for a period of time effective to allow marker gene expression; and e) detecting a host cell from the population that expresses the marker gene, thereby identifying the presence in the cell of a first DNA segment that comprises a candidate tumor suppressor protein or oncogene.

In a preferred method of the present invention, the plurality of candidate human genes are the plurality of genes in a B-cell, breast, ovarian or uterine DNA library. The method also generally further comprises isolating the detected cell of step (e) free from the population of cells, and isolating the candidate human gene from the first DNA segment within the cell.

The genes and DNA segments of the present invention may encode B123 proteins, polypeptides, domains, peptides or fusion constructs thereof where the B123 sequence includes a contiguous amino acid sequence from SEQ ID NO: 19, or a biologically functional equivalent thereof. Preferably, the isolated genes and coding regions will include a contiguous nucleic acid sequence from between position 46 and position 864 of SEQ ID NO: 17, or a biologically functional equivalent thereof.

The genes and DNA segments of the present invention may encode BE2 proteins, polypeptides, domains, peptides or fusion constructs thereof where the BE2 sequence includes a contiguous amino acid sequence from SEQ ID NO:4I , or a biologically functional equivalent thereof. Preferably, the isolated genes and coding regions will include a contiguous nucleic acid sequence from between position 37 and position 819 of SEQ ID NO:40, or a biologically functional equivalent thereof.

The genes and DNA segments of the present invention may encode BE 14 proteins, polypeptides, domains, peptides or fusion constructs thereof where the BE 14 sequence includes a contiguous amino acid sequence from SEQ ID NO:43, or a biologically functional equivalent thereof. Preferably, the isolated genes and coding regions will include a contiguous nucleic acid sequence from between position 1 and position 666 of SEQ ID NO:42, or a biologically functional equivalent thereof.

The genes and DNA segments of the present invention may encode BE31 proteins, polypeptides, domains, peptides or fusion constructs thereof where the BE31 sequence includes a contiguous amino acid sequence from SEQ ID NO:45, or a biologically functional equivalent thereof. Preferably, the isolated genes and coding regions will include a contiguous nucleic acid sequence from between position 1 and position 693 of SEQ ID NO:44, or a biologically functional equivalent thereof.

The genes and DNA segments of the present invention may encode BE445 proteins, polypeptides, domains, peptides or fusion constructs thereof where the BE445 sequence includes a contiguous amino acid sequence from SEQ ID NO:47, or a biologically functional equivalent thereof. Preferably, the isolated genes and coding regions will include a contiguous nucleic acid sequence from between position 1 and position 816 of SEQ ID NO:46, or a biologically functional equivalent thereof.

The genes and DNA segments of the present invention may encode TCL52 proteins, polypeptides, domains, peptides or fusion constructs thereof where the TCL52 sequence includes a contiguous amino acid sequence from SEQ ID NO:48, or a biologically functional equivalent thereof. Preferably, the isolated genes and coding regions will include a contiguous nucleic acid sequence from between position 1 and position 936 of SEQ ID NO:9, or a biologically functional equivalent thereof.

The genes and DNA segments of the present invention may encode TCL163 proteins, polypeptides, domains, peptides or fusion constructs thereof where the TCL163 sequence includes a contiguous amino acid sequence from SEQ ID NO:49, or a biologically functional equivalent thereof. Preferably, the isolated genes and coding regions will include a contiguous nucleic acid sequence from between position 7 and position 1770 of SEQ ID NO: 10, or a biologically functional equivalent thereof.

The genes and DNA segments of the present invention may encode B223 proteins, polypeptides, domains, peptides or fusion constructs thereof where the B223 sequence includes a contiguous amino acid sequence from SEQ ID NO:50, or a biologically functional equivalent thereof. Preferably, the isolated genes and coding regions will include a contiguous nucleic acid sequence from between position 1 and position 1110 of SEQ ID NO:l l, or a biologically functional equivalent thereof. The genes and DNA segments of the present invention may encode B 115 proteins, polypeptides, domains, peptides or fusion constructs thereof where the Bl 15 sequence includes a contiguous amino acid sequence from SEQ ID NO:51, or a biologically functional equivalent thereof. Preferably, the isolated genes and coding regions will include a contiguous nucleic acid sequence from between position 1 and position 1248 of SEQ ID NO: 12, or a biologically functional equivalent thereof.

The genes and DNA segments of the present invention may encode BAP28 proteins, polypeptides, domains, peptides or fusion constructs thereof where the BAP28 sequence includes a contiguous amino acid sequence from SEQ ID NO:52, or a biologically functional equivalent thereof. Preferably, the isolated genes and coding regions will include a contiguous nucleic acid sequence from between position 1 and position 1545 of SEQ ID NO:13, or a biologically functional equivalent thereof.

The genes and DNA segments of the present invention may encode B48 proteins, polypeptides, domains, peptides or fusion constructs thereof where the B48 sequence includes a contiguous amino acid sequence from SEQ ID NO:53, or a biologically functional equivalent thereof. Preferably, the isolated genes and coding regions will include a contiguous nucleic acid sequence from between position 3 and position 449 of SEQ ID NO: 14, or a biologically functional equivalent thereof.

The genes and DNA segments of the present invention may encode B258 proteins, polypeptides, domains, peptides or fusion constructs thereof where the B258 sequence includes a contiguous amino acid sequence from SEQ ID NO:54, or a biologically functional equivalent thereof. Preferably, the isolated genes and coding regions will include a contiguous nucleic acid sequence from between position 1 and position 1605 of SEQ ID NO: 15, or a biologically functional equivalent thereof.

The genes and DNA segments of the present invention may encode BAP 152 proteins, polypeptides, domains, peptides or fusion constructs thereof where the BAP 152 sequence includes a contiguous amino acid sequence from SEQ ID NO:55, or a biologically functional equivalent thereof. Preferably, the isolated genes and coding regions will include a contiguous nucleic acid sequence from between position 959 and position 2143 of SEQ ID NO: 16, or a biologically functional equivalent thereof. Alternatively, the isolated genes and coding regions will include a contiguous nucleic acid sequence from between position 2147 and position 2605 of SEQ ID NO: 16, or a biologically functional equivalent thereof.

The genes and DNA segments of the present invention may encode B268 proteins, polypeptides, domains, peptides or fusion constructs thereof where the B268 sequence includes a contiguous amino acid sequence from SEQ ID NO:56, or a biologically functional equivalent thereof. Preferably, the isolated genes and coding regions will include a contiguous nucleic acid sequence from between position 46 and position 864 of SEQ ID NO: 18, or a biologically functional equivalent thereof.

The nucleic acid segments provided by the invention are thus further characterized as including:

(a) a nucleic acid segment comprising a sequence region that consists of at least about 8, about 10, about 11, about 12, about 13, about 14, about 15, about 17 or about 20 contiguous nucleotides that have the same sequence as, or are complementary to, about 8, about 10, about 1 1, about 12, about 13, about 14, about 15, about 17 or about 20 contiguous nucleotides of SEQ ID NO: l, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:l 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID

NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129 or SEQ ID NO: 130; or

(b) a nucleic acid segment of from about 10-14, 17 or about 20 to about 20,000 nucleotides in length that specifically hybridizes to the nucleic acid segment of SEQ ID NO:l , SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:l 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO.24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO: 122, SEQ ID NO:123, SEQ ID NO: 124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO: 129 or SEQ ID NO: 130, or the complements thereof, under standard stringency, or preferably, under high stringency hybridization conditions.

Standard and high stringency hybridization conditions are well known to those of skill in the art. An exemplary, but not limiting, standard hybridization is incubated at 42°C in 50% formamide solution containing dextran sulfate for 48 hours and subjected to a final wash in 0.5X SSC, 0.1% SDS at 65°C. In addition to hybridization to Southern or northern blots, hybridization of primers for use in PCR™, as exemplified in Example XI below, is another preferred method for identification of sequences contemplated for use in the present invention.

Where the "complement" of any of the above nucleic acid segments are provided, such a complement may be functionally considered as an antisense nucleic acid, which includes nucleic acid segments positioned, in reverse orientation, under the control of a promoter that directs the expression of an antisense product. Antisense products may be used to inhibit the transcription or translation of any of the foregoing BRCAI -binding genes, in in vitro systems in order to more precisely define the cellular consequence of inhibition, or even in vivo in situations where inhibition of one or more of the foregoing BRCAI -binding genes would be believed to be result in a beneficial effect, such as an anti-cancer effect.

Mutants of each of the foregoing sequences and their encoded proteins, polypeptides, and peptides are also contemplated. The mutants may be used in the detection of physiologically relevant mutations or in further testing an functional analyses.

Segments of each of SEQ ID NO: I, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:l l, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO:128, SEQ ID NO:129 or SEQ ID NO:130, or the complements thereof, or the mutants thereof, may variously be about 10, 14, 17, 20, 25, 30, 50, 100, 200, 500, or 1000 or so nucleotides in length, up to and including the full length sequences, or even longer, as may be achieved by duplication of certain domains. Where the wild-type, polymorphic or mutant BARDl sequences of SEQ ID NO:l, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38 are concerned, sequences of at least about 1500 or about 2000 nucleotides of SEQ ID NO:l, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38, or the complement thereof are provided, up to and including the full length sequence of 2531 contiguous nucleotides of SEQ ID NO: 1 , SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38, or up to and including the full length sequence of 2510 contiguous nucleotides of SEQ ID NO:26, or the complement thereof.

Any segment may be combined into a DNA segment or vector of up to about 50,000, about 30,000, or about 20,000 basepairs in length. Segments of up to about 20,000, 15,000 or about 10,000 basepairs in length will generally be preferred, and segments of up to about 5,000 and 3,000 basepairs in length are also provided.

The nucleic acids of the present invention may also be DNA segments or RNA segments.

Nucleic acid detection kits are also provided.

The present invention further provides recombinant host cells comprising at least one

DNA segment or vector that comprises an isolated gene that encodes a BARDl, B123, BE2, BE14, BE31 or BE445 protein, polypeptide, domain, peptide or any fusion protein or mutant thereof. Prokaryotic recombinant host cells, such as E. coli, are provided, as are eukaryotic host cells, including breast, ovarian or uterine cancer cells provided with the BARDl, B123, BE2, BE14, BE31 or BE445 constructs of the invention.

The recombinant host cells may further comprise an operative BRCAI protein or active fragment or domain thereof, such as a DNA binding domain and/or a BARDl, B123, BE2, BE 14, BE31 or BE445 binding domain. Such recombinant host cells may be provided with the BRCAI in vitro, for example, to test BARDl, B123, BE2, BE 14, BE31 or BE445 and BRCAI interactions, or may naturally express BRCAI, including cells provided with BARDl, B123, BE2, BE 14, BE31 or BE445 in vivo and in vitro, either for treatment or for study.

The recombinant host cells of the present invention preferably have one or more DNA segments introduced into the cell by means of a recombinant vector, and preferably express the DNA segment to produce the encoded BARDl, B123, BE2, BE 14, BE31 or BE445 protein or peptide.

Methods of using BARDl, B123, BE2, BE14, BE31 or BE445 DNA segments are provided that comprise expressing a BARDl, B123, BE2, BE14, BE31 or BE445 DNA segment in a recombinant host cell and collecting the BARDl, B123, BE2, BE14, BE31 or BE445 protein, peptide, domain or mutant expressed by said cell. These methods may be characterized by the steps of:

(a) preparing a recombinant vector in which a BARDl, B123, BE2, BE 14, BE31 or BE445-encoding DNA segment is positioned under the control of a promoter;

(b) introducing said recombinant vector into a recombinant host cell;

(c) culturing the recombinant host cell under conditions effective to allow expression of an encoded BARDl, B123, BE2, BE14, BE31 or BE445 protein, peptide, domain or mutant; and (d) collecting said expressed BARDl, B123, BE2, BE14, BE31 or BE445 protein, peptide, domain or mutant.

Thus the present invention provides BARDl, B123, BE2, BE 14, BE31 or BE445 nucleic acid segments for use in the preparation of a recombinant BARDl, B123, BE2, BE14, BE31 or BE445 protein, polypeptide, peptide, mutant or fusion protein thereof. Thus, the use of BARDl, B123, BE2, BE14, BE31 or BE445 nucleic acid segments in the preparation of a recombinant BARDl, B123, BE2, BE14, BE31 or BE445 protein, polypeptide, peptide, mutant or fusion protein thereof is provided.

Methods for detecting BARDl, B123, BE2, BE14, BE31 or BE445 genes in cells or samples are also provided and generally comprise contacting sample nucleic acids from a sample suspected of containing BARDl, B123, BE2, BE 14, BE31 or BE445 with a nucleic acid segment that encodes a BARDl, B123, BE2, BE 14, BE31 or BE445 protein or peptide under conditions effective to allow hybridization of substantially complementary nucleic acids, and detecting the hybridized complementary nucleic acids thus formed.

The present invention also provides BARDl, B123, BE2, BE 14, BE31 or BE445 nucleic acid segments for use in the preparation of a composition for use in detecting a BARDl, B123, BE2, BE14, BE31 or BE445 nucleic acid segment. Thus, the use of BARDl, B123, BE2, BE14, BE31 or BE445 nucleic acid segments in the preparation of a composition for use in detecting a BARDl, B123, BE2, BE14, BE31 or BE445 nucleic acid segment are provided. The invention further provides BARDl nucleic acid segments for use in the preparation of a wild-type BARDl composition for use in detecting or purifying a BRCAI protein. Therefore, the use of BARDl nucleic acid segments in the preparation of a wild-type BARDl composition for use in detecting or purifying a BRCAI protein is provided.

The methods may be diagnostic of breast, ovarian or uterine cancer by detecting

BARDl, B123, BE2, BE14, BE31 or BE445 mutants as opposed to wild-type sequences. The use of both BARDl, B123, BE2, BE14, BE31 or BE445 wild-type and mutant sequences as probes or primers in such methods will naturally be included. A wild-type sequence probe or primer will be expected to bind to the native, non-mutant sequences, but not to a mutant, and vice versa. The use of a mutant-specific probe that corresponds to a mutant identified in a family member with breast cancer may be preferred in screening other family members. In any event, irrespective of the BARDl, B123, BE2, BE14, BE31 or BE445 nucleic acid segment employed, these studies will still only allow hybridization of substantially complementary nucleic acids, thus facilitating the detection only of wild-type or only mutant hybridized nucleic acid complexes.

Thus the present invention provides BARDl, B123, BE2, BE14, BE31 or BE445 compositions for use in the preparation of a diagnostic formulation for use in identifying a patient having or at risk for developing cancer. Therefore, the use of BARDl, B123, BE2,

BE14, BE31 or BE445 compositions in the preparation of a diagnostic formulation for use in identifying a patient having or at risk for developing cancer is provided.

In further embodiments, the present invention provides BARDl, B123, BE2, BE14, BE31 or BE445 proteins, polypeptides, domains, peptides, mutants and any fusion proteins thereof, including BARDl, B123, BE2, BE14, BE31 or BE445 compounds purified from natural sources, such as from mammalian and human cells, and BARDl, B123, BE2, BE 14, BE31 or BE445 prepared by recombinant means. Recombinant BARDl, B123, BE2, BE14, BE31 or BE445 proteins and peptides may be defined as being prepared by expressing a BARDl , B123, BE2, BE14, BE31 or BE445 protein or peptide in a recombinant host cell and purifying the expressed BARDl, B123, BE2, BE14, BE31 or BE445 protein or peptide away from total recombinant host cell components.

The BARDl, B123, BE2, BE14, BE31 or BE445 protein compositions, whether natural or recombinant, will generally be obtained free from total cell components, and will comprise at least one type of isolated BARDl, B123, BE2, BE 14, BE31 or BE445 protein or peptide, purified relative to the natural level in a given cell.

As stated, preferred wild-type, polymoφhic or mutant BARDl proteins may be characterized as being about 777, about 770 or about 752 amino acids in length, preferably being

777 amino acids in length; as comprising an amino-terminal RING motif or domain, preferably characterized as comprising a cysteine-rich sequence with an interleaved structure in which two ions of zinc are coordinated by seven cysteines and one histidine, and which RING motif or domain mediates the association of wild-type, polymorphic or mutant BARDl with BRCAI ; as containing ankyrin repeats, which ankyrin repeats are not required for binding to BRCAI ; as comprising carboxy-terminal BRCT domains that are homologous to carboxy-terminal sequences of BRCAI; as being encoded by sequences on chromosome 2q; and most importantly in functional terms, as binding to BRCAI .

The wild-type, polymoφhic or mutant BARDl proteins of the invention are preferably characterized as comprising an amino-terminal RING motif or domain that has the sequence of residues 46-90 from SEQ ID NO:2, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37 or SEQ ID NO:39; as comprising a BRCAI binding domain that has the sequence of residues 26- 202 from SEQ ID NO:2, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37 or SEQ ID NO:39, or more preferably, that has the sequence of residues 26-142 from SEQ ID NO:2, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37 or SEQ ID NO:39, which binding domain binds to BRCAI; as containing ankyrin repeats that have the sequence of residues 427-525 from SEQ ID NO:2, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37 or SEQ ID NO:39, which ankyrin repeats do not bind to BRCAI; and as comprising carboxy-terminal BRCT domains that have a sequence between residues 605 and 777 of SEQ ID NO:2, SEQ ID NO:21 , SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37 or SEQ ID NO:39, as exemplified by comprising the BRCT domain N-terminal core motif of residues 616-653 of SEQ ID NO:2, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37 or SEQ ID NO:39 and as comprising the BRCT domain C-terminal core motif of residues 743-777 of SEQ ID NO:2, SEQ ID NO:21 , SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37 or SEQ ID NO:39. Wild-type, polymoφhic and mutant BARDl domains and peptides are also provided by the invention, including the isolated wild-type, polymoφhic or mutant BARDl ankyrin repeat domains, isolated wild-type, polymoφhic or mutant BARDl BRCT-like domains, isolated wild- type, polymoφhic or mutant BARDl RING motif domains and the isolated wild-type, polymoφhic or mutant BARDl BRCAI -binding domains, and the non-functional antigenic peptides, as detailed hereinabove.

BARDl, B123, BE2, BE14, BE31 or BE445 fusion proteins or constructs including BARDl, B123, BE2, BE14, BE31 or BE445 sequences operatively attached to distinct, selected amino acid sequences, such as selected antigenic amino acid sequences, amino acid sequences with selected binding affinity, and DNA binding or transactivation amino acid sequences, arc also encompassed within the invention. Fusion proteins with selectably-cleavable bonds are also provided.

The present invention provides BARDl, B123, BE2, BE 14, BE31 and BE445 proteins, polypeptides, peptides, domains and fusion proteins for use in detection or purification of a BRCAI protein. Thus, the use of BARDl, B123, BE2, BE14, BE31 and BE445 proteins, polypeptides, peptides, domains and fusion proteins in detection or purification of a BRCAI protein is provided.

The BARDl, B123, BE2, BE14, BE31 or BE445 proteinaceous compositions will include the same types of mutants as described above for the nucleic acids. The use of specific mutated BARDl, B123, BE2, BE14, BE31 or BE445 peptides to prepare mutant-specific antibodies is particularly contemplated. In terms of diagnostic mutated BARDl, B123, BE2, BE14, BE31 or BE445 peptides and antibodies, these compositions will generally be more useful in regard to point mutants, whereas nucleic acid probes may be more suitable for detecting deletion, duplication, translocation and insertional mutations in addition to point mutants.

In still further embodiments, the present invention provides compositions comprising

BARDl, B123, BE2, BE14, BE31 or BE445 in combination with an operative BRCAI protein or active fragment or domain thereof. Such compositions may comprise BARDl, B123, BE2, BE 14, BE31 or BE445 in functional association with a BRCAI protein or fragment, or may even comprise one or more BARDl, B123, BE2, BE14, BE31 or BE445-BRCA1 fusion proteins.

The BARDl, B123, BE2, BE14, BE31 or BE445 proteins, polypeptides, domains, peptides and fusion proteins, as well as the BARDl , B123, BE2, BE 14, BE31 or BE445 DNA segments, vectors, isolated genes and coding sequences may also be formulated with a pharmaceutically acceptable diluent or vehicle to form a BARDl, B123, BE2, BE14, BE31 or BE445 pharmaceutical composition in accordance with this invention.

Further compositions of the present invention are antibodies, including monoclonal antibodies and antibody conjugates, that have immunospecificity for a BARDl , B123, BE2, BE 14, BE31 or BE445 protein or peptide. The antibodies may be operatively attached to a detectable label. The antibodies and antibody conjugates may be specific for mutant BARDl, B123, BE2, BE14, BE31 or BE445 proteins or peptides and allow differential binding from wild-type BARDl, B123, BE2, BE14, BE31 or BE445. Antibody detection kits are also provided.

Thus, the present invention provides BARDl, B123, BE2, BE14, BE31 and BE445 proteins, polypeptides, peptides, domains, mutants and fusion proteins thereof for use in the production of anti-BARDl , anti-B123, anti-BE2, anti-BE14, anti-BE31 and anti-BE445 antibodies. Therefore, the use of BARDl, B123, BE2, BE14, BE31 and BE445 proteins, polypeptides, peptides, domains, mutants and fusion proteins thereof in the production of anti- BARDl, anti-B123, anti-BE2, anti-BE14, anti-BE31 and anti-BE445 antibodies is provided. The anti-BARDl, anti-B123, anti-BE2, anti-BE14, anti-BE31 and anti-BE445 antibodies are also contemplated for use in the preparation of a diagnostic formulation for use in identifying a patient having or at risk for developing cancer. Thus, the use of anti-BARDl, anti-B123, anti- BE2, anti-BE14, anti-BE31 and anti-BE445 antibodies in the preparation of a diagnostic formulation for use in identifying a patient having or at risk for developing cancer is provided.

The BARDl, B123, BE2, BE14, BE31 or BE445 genes and proteins of the present invention have many utilities. For example, their BRCAI binding properties may be exploited in methods to detect BRCAI proteins. Such methods comprise contacting a sample suspected of containing a BRCAI protein with a BRCAI -binding BARDl, B123, BE2, BE 14, BE31 or BE445 protein, peptide or fusion protein, under conditions effective to allow the formation of BRCA1-BARD1, -B123, -BE2, -BE14, -BE31 or -BE445 complexes, and detecting the BRCAI -BARDl, -B123, -BE2, -BE 14, -BE31 or -BE445 complexes so formed.

Methods of purifying BRCAI proteins are also provided, which comprise contacting a composition comprising a BRCAI protein with a BRCAI -binding BARDl, B123, BE2, BE 14, BE31 or BE445 protein, peptide or fusion protein, under conditions effective to allow the formation of BRCA1-BARD1, -B123, -BE2, -BE14, -BE31 or -BE445 complexes, and obtaining the BRCAI protein from the BRCA1-BARD1, -B123, -BE2, -BE14, -BE31 or -BE445 complexes in a more purified form.

The "BRCAI -binding BARDl, B123, BE2, BE14, BE31 or BE445 protein, peptide or fusion proteins" of such methods are any BARDl, B123, BE2, BE14, BE31 or BE445 proteins or fragments sufficient to operatively bind BRCAI, using the assays and criteria disclosed herein.

Certain methods for detecting BARDl, B123, BE2, BE14, BE31 or BE445 in a sample comprise contacting a sample suspected of containing BARDl, B123, BE2, BE14, BE31 or BE445 with a first antibody that binds to a BARDl, B123, BE2, BE14, BE31 or BE445 protein or peptide, or a mutant thereof, under conditions effective to allow the formation of immune complexes, and detecting the immune complexes thus formed. In addition to their diagnostic use, these methods are also suitable for purifying BARDl, B123, BE2, BE14, BE31 or BE445, identifying BARDl, B123, BE2, BE14, BE31 or BE445 expression, in identifying engineered mutants and in titering BARDl, B123, BE2, BE14, BE31 or BE445 and/or BARDl, B123, BE2, BE14, BE31 or BE445 antibodies.

The invention further provides diagnostic methods, particularly useful in connection with breast, ovarian and uterine cancer, but also of potential usefulness in other cancers, particularly lung, colon and other cancers. Diagnostically, the present invention provides methods for identifying a patient having or at risk for developing breast, ovarian or uterine cancer, comprising determining the type or amount of BARDl, B123, BE2, BE 14, BE31 or BE445 present within a biological sample from the patient, wherein the presence of a BARDl, B123, BE2, BE 14, BE31 or BE445 mutant or an altered amount of wild-type BARDl, B123, BE2, BE14, BE31 or BE445, in comparison to a sample from a normal subject, is indicative of a patient having or at risk for developing breast, ovarian or uterine cancer.

The "type" of BARDl, B123, BE2, BE14, BE31 or BE445 may be determined, allowing mutant genes and proteins to be distinguished from wild-types. The use of mutant- and wild- type-specific nucleic acid probes is particularly contemplated. In the beginning, the use of wild- type-specific nucleic acid probes will be preferred. The identification of a particularly diagnostic mutant sequence will then lead to the increased use of that mutant sequence, cither in the population or in defined families. The use of mutant- and wild-type-specific antibodies is also contemplated, as may be prepared using mutant- and wild-type-specific BARDl, B123, BE2, BE 14, BE31 or BE445 peptides.

Where the "amount" of BARDl, B123, BE2, BE14, BE31 or BE445 is determined, a lesser amount of the natural BARDl, B123, BE2, BE 14, BE31 or BE445 protein may be indicative of the propensity to develop breast, ovarian or uterine cancer, as is typical with tumor suppressors. A greater amount of BARDl, B123, BE2, BE14, BE31 or BE445 could also be indicative of the propensity to develop breast, ovarian or uterine cancer, which situation would represent the case where the BARDl, B123, BE2, BE14, BE31 or BE445 is a dominant proto- oncogene. In any event, changes from the naturally observed range in the population will be easily detected and will have implications for disease risk and development.

The type or amount of BARDl, B123, BE2, BE14, BE31 or BE445 may be determined by means of a molecular biological assay to determine the type or amount of a nucleic acid that encodes BARDl, B123, BE2, BE14, BE31 or BE445. Such molecular biological assays will often comprise a direct or indirect step that allows a determination of the sequence of at least a portion of the BARD1-, B123-, BE2-, BE14-, BE31- or BE445 -encoding nucleic acid, which sequence can be compared to a wild-type BARDl, B123, BE2, BE 14, BE31 or BE445 sequence, such as SEQ ID NO:l, SEQ ID NO: 17, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44 or SEQ ID NO:46 or another acceptable normal allelic or polymorphic sequence, such as, in the case of BARDl , SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:2ό, SEQ ID NO:28, SEQ ID NO:30 or SEQ ID NO:38.

It is contemplated that BARDl, B123, BE2, BE14, BE31 or BE445 sequences diagnostic or prognostic for breast, ovarian, uterine or even for other forms of cancer may comprise at least one point mutation, deletion, translocation, insertion, duplication or other aberrant change.

Diagnostic RFLPs are thus also contemplated. RNase protection assays may also be employed in certain embodiments.

Diagnostic methods may be based upon the steps of:

(a) obtaining a biopsy sample from a subject or patient;

(b) contacting sample nucleic acids from the biopsy sample with an isolated BARDl, B123, BE2, BE14, BE31 or BE445 nucleic acid segment under conditions effective to allow hybridization of substantially complementary nucleic acids; and

(c) detecting, and optionally further characterizing, the hybridized complementary nucleic acids thus formed.

The methods may involve in situ detection of sample nucleic acids located within the cells of the sample. The sample nucleic acids may also be separated from the cell prior to contact. The sample nucleic acids may be DNA or RNA.

The methods may involve the use of isolated BARDl, B123, BE2, BE 14, BE31 or BE445 nucleic acid segments that comprises a radio, enzymatic or fluorescent detectable label, wherein the hybridized complementary nucleic acids are detected by detecting the label.

PCR® will often be preferred, as exemplified by the steps of: (a) contacting the sample nucleic acids with a pair of nucleic acid primers that hybridize to distant sequences from a mutant, polymoφhic or wild-type BARDl, B123, BE2, BE14, BE31 or BE445 nucleic acid sequence, the primers capable of amplifying a mutant, polymoφhic or wild-type BARDl , B123, BE2, BE 14, BE31 or BE445 nucleic acid segment when used in conjunction with a polymerase chain reaction;

(b) conducting a polymerase chain reaction to create amplification products; and

(c) detecting and characterizing the amplification products thus formed.

Diagnostic immunoassay methods are also provided, wherein the type or amount of BARDl, B123, BE2, BE14, BE31 or BE445 is determined by means of an immunoassay to determine the type or amount of a BARDl, B123, BE2, BE14, BE3 I or BE445 protein. Such methods may comprise the steps of:

(a) obtaining a biopsy sample from a subject or patient;

(b) contacting the biopsy sample with a first antibody that binds to a BARDl, B123,

BE2, BE14, BE31 or BE445 protein or peptide, or mutant, under conditions effective to allow the formation of specific immune complexes; and

(c) detecting the specific immune complexes thus formed.

The first antibody may be linked to a detectable label, wherein the immune complexes are directly detected by detecting the presence of the label. The immune complexes may also be indirectly detected by means of a second antibody linked to a detectable label, the second antibody having binding affinity for the first antibody.

Where BARDl, B123, BE2, BE14, BE31 or BE445 proves to be a tumor suppressor, the present invention also provides methods of treating cancers such as breast, ovarian or uterine cancer, comprising administering to a patient with breast, ovarian or uterine cancer a biologically effective amount of a pharmaceutically acceptable BARDl , B123, BE2, BE14, BE31 or BE445 composition

Where BARDl, B123, BE2, BE14, BE31 or BE445 proves to be an oncogene, the invention further provides methods of treating cancers such as breast, ovarian or uterine cancer, comprising administering to a patient with breast, ovarian or uterine cancer a biologically effective amount of a pharmaceutically acceptable composition that inhibits BARDl, B 123, BE2, BE14, BE31 or BE445. The composition may comprises a component that inhibits a BARDl, B123, BE2, BE14, BE31 or BE445 gene, mRNA, protein, peptide or BRCAl-BARDl , -B123, -BE2, -BE 14, -BE31 or -BE445 complex. Examples of inhibitors include antisense constructs, ribozymes, inhibitory antibodies, and recombinant vectors that express any of the foregoing BARDl, B123, BE2, BE 14, BE31 or BE445 inhibitors in mammalian cells.

The tumor suppressor-type treatment may also comprise giving BARDl, B123, BE2,

BE14, BE31 or BE445 protein or peptide compositions or BARDl, B123, BE2, BE14, BE31 or BE445 DNA segments or recombinant vectors that expresses BARDl, B123, BE2, BE 14, BE31 or BE445 proteins or peptides in the target cells. Enhancing BARDl, B 123, BE2, BE14, BE31 or BE445 transcription, translation or stability is also contemplated.

The cancer treatment methods of the present invention may be combined with any standard anti-cancer strategy, such as surgery, chemotherapy, radiotherapy and other gene therapies. The administration of a biologically effective amount of a BRCAI protein, peptide or recombinant vector composition is also contemplated.

The present invention also provides BARDl, B123, BE2, BE14, BE31 and BE445 nucleic acid segments, proteins, polypeptides, peptides, domains and fusion proteins for use in the preparation of a prophylactic formulation for administration to a patient at risk for developing cancer or a patient in the early stages of cancer. Thus, the use of BARDl, B123, BE2, BE 14, BE31 and BE445 nucleic acid segments, proteins, polypeptides, peptides, domains and fusion proteins in the preparation of a prophylactic formulation for administration to a patient at risk for developing cancer or a patient in the early stages of cancer is provided. Additionally, the present invention provides a nucleic acid segment for use in the preparation of a medicament for use in treating a patient with cancer. Therefore, the use of a nucleic acid segment in the preparation of a medicament for use in treating a patient with cancer is also provided.

In that the BARDl, B123, BE2, BE 14, BE31 or BE445 and BRCAI interaction is important for BRCAI and BARDl, B123, BE2, BE14, BE31 or BE445 function, the present invention further provides methods for identifying a BARDl, B123, BE2, BE 14, BE31, BE445 or BRCAI agonist or stimulant, or antagonist or inhibitor, comprising contacting a composition comprising BARDl, B123, BE2, BE14, BE31 or BE445 and BRCAI with a candidate substance and identifying a candidate substance that alters the binding of BARDl, B123, BE2, BE 14, BE31 or BE445 and BRCAI or that alters the activity, such as the DNA binding, transcriptional or other functional activity, of a BARD1-, B123-, BE2-, BE14-, BE31- or BE445-BRCA1 bound complex. The BARDl, B123, BE2, BE 14, BE31 or BE445 or BRCAI agonists or antagonists prepared by such as process form another aspect of the present invention, which substances may also be employed in treating breast, ovarian or uterine cancer.

Thus, the present invention also provides BARDl , B123, BE2, BE14, BE31 and BE445 proteins, polypeptides, peptides, domains and fusion proteins for use in the identification of a binding protein agonist or antagonist that alters the binding of BARDl, B123, BE2, BE14, BE31 or BE445 toBRCAl or that alters biological activity of a BRCAl-BARDl, BRCA 1-B 123, BRCA1-BE2, BRCA1-BE14, BRCA1-BE31 or BRCA1-BE445 complex. Therefore, the use of BARDl, B123, BE2, BE14, BE31 and BE445 proteins, polypeptides, peptides, domains and fusion proteins in the identification of a binding protein agonist or antagonist that alters the binding of BARDl, B123, BE2, BE14, BE31 or BE445 toBRCAl or that alters biological activity of a BRCAl-BARDl, BRCA1-B123, BRCA1-BE2, BRCA1-BE14, BRCA1-BE31 or BRCA1-BE445 complex is provided. BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 1. Mammalian two-hybrid analysis of interaction between BR304 and the candidate BRCAI -associated polypeptides. Each culture of 293 cells was transiently co- transfected with the G5LUC reporter plasmid and the two indicated expression vectors. The GAL4 expression vector encoded either the "parental" GAL4 DNA-binding domain (denoted by "+" in the GAL4 column) or the GAL4-BR304 hybrid polypeptide. The VP16 expression vector encoded either the parental VP16 transactivation domain (denoted by "+" in the VP16 column) or the indicated VP16-hybrid polypeptide. Duplicate transfections were conducted for each combination of expression plasmids, and the normalized luciferase activities obtained from each transfection are illustrated.

FIG. 2. A schematic comparison of the BRCAI and BARDl polypeptides. The map of BRCAI illustrates sequences that comprise the RING motif (20-68) and the BRCT domain (1685-1863); the N-terminal and C-terminal core motifs of the BRCT domain (residues 1699- 1736 and 1818-1855, respectively) are denoted by the solid bars marked "n" and "c", respectively. The map of the BARDl illustrates the RING motif (residues 44-90), the three ankyrin repeats (residues 427-525), and the BRCT domain (residues 605-777); the N-terminal and C-terminal core motifs of the BRCT domain (residues 616-653 and 743-777, respectively) are denoted by the solid bars marked "n" and "c", respectively. The sequences encoded by the B202 and B230 cDNA clones are indicated beneath the BARDl map. The NE (residues 26- 142) and NB (residues 26-202) segments of BARDl used in FIG. 3 are also shown.

FIG. 3. Mammalian two-hybrid analysis of the interaction between BRCAI and defined segments of the BARDl polypeptide. Each dish of 293 cells was transiently co-transfected with the G5LUC reporter plasmid, the pSV-β-galactosidase control plasmid, and the two indicated expression vectors. The GAL4 expression vector encoded either the "parental" GAL4 DNA- binding domain (denoted by "+" in the GAL4 column) or the GAL4-BR304 hybrid polypeptide. The VP16 expression vector encoded either the parental VP16 transactivation domain (denoted by "+" in the VP16 column) or the VP16-hybrid polypeptide containing segments NE (residues 26- 142) or NB (residues 26-202) of BARD 1 (see FIG. 2).

FIG. 4A and FIG. 4B. BRCAI sequences that mediate association with BARDl . FIG. 4A, mammalian two-hybrid analysis of the interaction between BARDl and defined segments of BRCAI . Each dish of 293 cells was transiently co-transfected with the G5LUC reporter plasmid, the pSV-β-galactosidase control plasmid, and the two indicated expression vectors. The VP16 expression vector encoded either the "parental" VP16 transactivation domain (denoted by "+" in the VP16 column) or VP16-NE, a hybrid polypeptide containing amino acids 26-142 of BARDl. The GAL4 expression vector encoded either the parental GAL4 DNA- binding domain (denoted by "+" in the GAL4 column) or the indicated GAL4-hybrid polypeptide; the latter contained BRCAI residues 1-147 (BR147), 1-101 (BR 101), 1-71 (BR71), or 1-45 (BR45). FIG. 4B, a reciprocal two-hybrid analysis of BARDl interaction with defined segments of BRCAI . The GAL4 expression vector encoded either the parental GAL4 DNA- binding domain (denoted by "+" in the GAL4 column) or GAL4-NE, a hybrid polypeptide containing amino acids 26-142 of BARDl . The VP16 expression vectcr encoded either the parental VP16 transactivation domain (denoted by "+" in the VP16 column) or a VP16-hybrid polypeptide containing the indicated segment of BRCAI.

FIG. 5A and FIG. 5B. Tumorigenic mutants of BRCAI fail to interact with BARDl. FIG. 5A, mammalian two-hybrid analysis of the interaction between BARDl and the mutant derivatives of BRCAI . Each dish of 293 cells was transiently co-transfected with the G5LUC reporter plasmid, the pSV-β-galactosidase control plasmid, and the two indicated expression vectors. The VP16 expression vector encoded either the parental VP16 transactivation domain (denoted by "^■+^■" in the VP16 column) or VP16-NE, a hybrid polypeptide containing amino acids 26-142 of BARDl. The GAL4 expression vector encoded either the "parental" GAL4 DNA- binding domain (denoted by "+" in the GAL4 column) or the indicated GAL4-BR304 fusion protein; the latter included wild-type BRCAI residues 1-304 (BR304; lanes 3 and 4) and variants of BR304 that bear the tumorigenic C61G or C64G mutations (lanes 5-8). FIG. 5B, co- immunoprecipitation analysis of the interaction between BARDl and the mutant derivatives of BRCAI . 293 cells were transfected with a pair of expression vectors encoding FLAG-B202 and either a wild-type or mutant derivative of FLAG-BR304. After two days the cells were lysed and the lysates were normalized for expression of FLAG-B202. Equivalent aliquots of the lysates (100 ml) were immunoprecipitated with the BRCAl-specific antiserum (lanes 2, 4, and 6) or the corresponding pre-immune serum (lanes 1, 3, and 5). The immunoprecipitates were then fractionated by SDS-PAGE, and the FLAG-B202 and FLAG-BR304 polypeptides were detected by immunoblotting with the M5 monoclonal antibody. As shown, FLAG-B202 was co-immunoprecipitated with the wild-type FLAG-BR304 (lane 2) but not with derivatives of FLAG-BR304 containing the C61G (lane 4) or C64G (lane 6) mutation. Expression of the different FLAG-BR304 derivatives was compared by immunoblotting equivalent aliquots (20 ml) of the untreated lysates with FLAG-specific M5 monoclonal antibody (Eastman Kodak) (lanes 7-9).

FIG. 6. Schematic diagram of the BARDl cDNA. The ring domain, ankyrin repeats,

BRCT domain and 5' and 3' untranslated regions are shaded as indicated. Splice sites are designated A-H. The location of the splice site according to the nucleotide sequence of the gene (GenBank Accession No. U76638) or the amino acid sequence of the protein are indicated above the diagram. Additional splice sites exist between G and H but these have not yet been determined. Mutations described in this manuscript are indicated above the cDNA diagram. Polymoφhisms are indicated below the diagram. Designations of amino acid changes are according to the nomenclature proposed by Beaudet and Tsui (1993).

SEQUENCE SUMMARY

SEQ ID NO:l BARDl DNA Sequence

SEQ ID NO:2 BARDl Amino Acid Sequence

SEQ ID NO:3 FLAG Epitope Amino Acid Sequence

SEQ ID NO:4 5' Primer for PCR Amplification of N-terminus of BRCA 1 SEQ ID NO:5 3' Primer for PCR Amplification of N-terminus of BRCA 1

SEQ ID NO:6 BARD 1 PCR Primer B202L

SEQ ID NO:7 BARD 1 PCR Primer B202R SEQ ID NO:8 HA-BR304 Amino Terminal Tag Amino Acid Sequence

SEQ ID NO:9 TCL52 DNA Sequence

SEQ ID NOilO TCL 163 DNA Sequence

SEQ ID NO: 11 B223 DNA Sequence SEQ ID NO: 12 B 115 DNA Sequence

SEQ ID NO:13 BAP28 DNA Sequence

SEQ ID NO:14 B48 DNA Sequence

SEQ ID NO:15 B258 DNA Sequence

SEQ ID NO:16 BAP 152 DNA Sequence SEQ ID NO: 17 B 123 DNA Sequence

SEQ ID NO:18 B268 DNA Sequence

SEQ ID NO: 19 B 123 Amino Acid Sequence

SEQ ID NO:20 BARDl PI 43 DNA Sequence

SEQ ID NO:21 BARDl P143 Amino Acid Sequence SEQ ID NO:22 BARDl P553 DNA Sequence

SEQ ID NO:23 BARD 1 P553 Amino Acid Sequence

SEQ ID NO:24 BARD 1 P 1121 DNA Sequence

SEQ ID NO:25 BARD 1 P 1121 Amino Acid Sequence

SEQ ID NO:26 BARDl PΔl 140-1 160 DNA Sequence SEQ ID NO:27 BARD 1 PΔ 1140- 1 160 Amino Acid Sequence

SEQ ID NO:28 BARD 1 P 1592 DNA Sequence

SEQ ID NO:29 BARDl PI 592 Amino Acid Sequence

SEQ ID NO:30 BARDl PI 765 DNA Sequence

SEQ ID NO:31 BARDl PI 765 Amino Acid Sequence SEQ ID NO:32 BARDl MQ564H DNA Sequence

SEQ ID NO:33 BARDl MQ564H Amino Acid Sequence

SEQ ID NO:34 BARD 1 MS761 N DNA Sequence

SEQ ID NO:35 BARDl MS761N Amino Acid Sequence

SEQ ID NO:36 BARDl MR658C DNA Sequence SEQ ID NO:37 BARD 1 MR658C Amino Acid Sequence

SEQ ID NO:38 BARD 1 P2354 DNA Sequence

SEQ ID NO:39 BARDl P2354 Amino Acid Sequence SEQ ID NO:40 BE2 DNA Sequence SEQ ID NO.-41 BE2 Amino Acid Sequence SEQ ID NO:42 BE 14 DNA Sequence SEQ ID NO:43 BE 14 Amino Acid Sequence SEQ ID NO:44 BE31 DNA Sequence SEQ ID NO:45 BE31 Amino Acid Sequence SEQ ID NO:46 BE445 DNA Sequence SEQ ID NO:47 BE445 Amino Acid Sequence SEQ ID NO:48 TCL52 Amino Acid Sequence SEQ ID NO:49 TCL163 Amino Acid Sequence SEQ ID NO:50 B223 Amino Acid Sequence SEQ ID NO:51 BI 15 Amino Acid Sequence SEQ ID NO:52 BAP28 Amino Acid Sequence SEQ ID NO:53 B48 Amino Acid Sequence SEQ ID NO:54 B258 Amino Acid Sequence SEQ ID NO:55 BAP 152 Amino Acid Sequence SEQ ID NO:56 B268 Amino Acid Sequence SEQ ID NO:57 BARDl PCR Primer R135S SEQ ID NO:58 BARDl PCR Primer R135AS SEQ ID NO:59 BARDl PCR Primer B202-Z IS SEQ ID NO:60 BARDl PCR Primer B202-ZAS SEQ ID NO:61 BARDl PCR Primer B202-Z1SP SEQ ID NO:62 BARDl PCR Primer B202-A SEQ ID NO:63 BARDl PCR Primer B202-N SEQ ID NO:64 BARDl PCR Primer B202-B SEQ ID NO:65 BARDl PCR Primer B202-BAS SEQ ID NO:66 BARDl PCR Primer B202-X SEQ ID NO:67 BARDl PCR Primer B202-XAS SEQ ID NO:68 BARDl PCR Primer B230-A SEQ ID NO:69 BARDl PCR Primer B230-AS SEQ ID NO:70 BARDl PCR Primer B202-Y SEQ ID NO:71 BARDl PCR Primer B202-YAS SEQ ID NO:72 BARD 1 PCR Primer B230-B

SEQ ID NO:73 BARDl PCR Primer B230-BAS

SEQ ID NO:74 BARDl PCR Primer B230-C

SEQ ID NO:75 BARDl PCR Primer B230-CAS SEQ ID NO:76 BARDl PCR Primer B230-D

SEQ ID NO:77 BARDl PCR Primer B230-DAS

SEQ ID NO:78 BARDl PCR Primer B230-PS

SEQ ID NO:79 BARDl PCR Primer B230-P

SEQ ID NO:80 BARD 1 PCR Primer B230-E SEQ ID NO:81 BARDl PCR Primer B230-EAS

SEQ ID NO:82 BARDl PCR Primer B230-F

SEQ ID NO:83 BARD 1 PCR Primer B230-FAS

SEQ ID NO:84 BARDl PCR Primer B230-FF

SEQ ID NO:85 BARD 1 PCR Primer B230-FFAS SEQ ID NO:86 BARDl PCR Primer B230-WS

SEQ ID NO:87 BARD 1 PCR Primer B230-WAS

SEQ ID NO:88 BARD 1 PCR Primer B230-G

SEQ ID NO:89 BARDl PCR Primer B230-H

SEQ ID NO:90 BARD 1 PCR Primer B230-HAS SEQ ID NO:91 BARDl PCR Primer B230-TS

SEQ ID NO:92 BARDl PCR Primer B230-TAS

SEQ ID NO:93 BARDl PCR Primer B230-US

SEQ ID NO:94 BARDl PCR Primer B230-UAS

SEQ ID NO:95 BARDl PCR Primer Rl 352S SEQ ID NO:96 BARDl PCR Primer Rl 3 AAS

SEQ ID NO:97 BARD 1 PCR Primer R 12AS

SEQ ID NO:98 BARDl PCR Primer R12BAS

SEQ ID NO:99 BARDl PCR Primer R13B5

SEQ ID NO: 100 BARDl PCR Primer R13CAS SEQ ID NO:101 BARDl PCR Primer R5C5

SEQ ID NO:102 BARDl PCR Primer B202-N1

SEQ ID NO:103 BARDl PCR Primer R5DAS SEQ ID NO: 104 BARDl PCR Primer R34D5

SEQ ID NO: 105 BARD 1 PCR Primer R34FAS

SEQ ID NO:106 BARDl PCR Primer R34F5

SEQ ID NO:107 BARDl PCR Primer R34GA5 SEQ ID NO:108 BARDl PCR Primer R36H5

SEQ ID NO: 109 BARDl PCR Primer R36EAS

SEQ ID NO:110 BARDl PCR Primer R36E5

SEQ ID NO: 111 BAP 152 Second Amino Acid Sequence

SEQ ID NO:112 BRCAI PCR Primer 4L SEQ ID NO:113 BRCAI PCR Primer 4R

SEQ ID NO:114 BRCA2 Forward PCR Primer

SEQ ID NO: 115 BRC A2 Reverse PCR Primer

SEQ ID NO:116 BARDl PCR Primer FFGS2

SEQ ID NO:117 BARDl PCR Primer B2305FGAS SEQ ID NO:118 BARDl PCR Primer 3FGR

SEQ ID NO: 119 BARD 1 PCR Primer WSGAS

SEQ ID NO:120 BARDl PCR Primer B230IXS

SEQ ID NO: 121 BARD 1 PCR Primer B230IXAS

SEQ ID NO: 122 BARDl Genomic DNA Contig 1 (Contains Exon 1) SEQ ID NO: 123 BARDl Genomic DNA Contig 2 (Contains Exon 2 and Exon3)

SEQ ID NO:124 BARDl Genomic DNA Contig 3 (Contains Exon 4)

SEQ ID NO:125 BARDl Genomic DNA Contig 4 (Contains Exon 5)

SEQ ID NO: 126 BARDl Genomic DNA Contig 5 (Contains Exon 6)

SEQ ID NO: 127 BARDl Genomic DNA Contig 6 (Contains Exon 7) SEQ ID NO:128 BARDl Genomic DNA Contig 7 (Contains Exon 8)

SEQ ID NO: 129 BARDl Genomic DNA Contig 8 (Contains Exon 9)

SEQ ID NO: 130 BARDl Genomic DNA Contig 9 (Contains Exon 10 and Exon 1 1) DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

In order to identify proteins that bind to BRCAI, the inventors first utilized the yeast two-hybrid system to identify proteins that associate with BRCAI in vivo (Fields and Song, 1989; Chien et l, 1991; Durfee et al, 1993; Haφer et al, 1993). Such analyses led to the discovery of fifteen novel genes that encode polypeptides that bind to the N-terminal 304 amino acids of BRCAI in the yeast assay.

These are included herein as BARDl DNA and protein sequences (SEQ ID NO: l and SEQ ID NO:2, respectively); and also TCL52 DNA sequence (SEQ ID NO:9); TCL163 DNA sequence (SEQ ID NO: 10); B223 DNA sequence (SEQ ID NO: l 1); B l 15 DNA sequence (SEQ

ID NO: 12); BAP28 DNA sequence (SEQ ID NO: 13); B48 DNA sequence (SEQ ID NO: 14);

B258 DNA sequence (SEQ ID NO: 15); BAP 152 DNA sequence (SEQ ID NO: 16); B123 DNA and protein sequences (SEQ ID NO: 17 and SEQ ID NO: 19, respectively); B268 DNA sequence (SEQ ID NO: 18); BE2 DNA and protein sequences (SEQ ID NO:40 and SEQ ID NO:41, respectively); BE 14 DNA and protein sequences (SEQ ID NO:42 and SEQ ID NO:43, respectively); BE31 DNA and protein sequences (SEQ ID NO:44 and SEQ ID NO:45, respectively); and BE445 DNA and protein sequences (SEQ ID NO:46 and SEQ ID NO:47, respectively). Each of the genes and proteins listed above are included within all aspects of the present invention.

The yeast screening assay also led to the identification of five further gene and protein candidates for BRCAI binding. Although the sequences of these five genes have been previously reported, their potential role in BRCAI binding and/or as part of the breast cancer development pathway(s) has not previously been suggested. As such, the genes and proteins TAFII70/80 (Genbank accession nos. L25444 and U31659), filamin (X53416), STAT3/APRF (L29277), UNPH (U20657), and a human homolog of the yeast GCN5 gene product (U57317), are each included within the methodological aspects of the present invention to the extent that such methods could not previously have been contemplated. To even further increase the chances that the yeast screening assay resulted in the identification of protein interactions that are physiologically-relevant, rather than just artifactual results of over-expression of foreign proteins in yeast, the inventors used a mammalian two- hybrid assay (Dang et al., 1991). The mammalian assay appears to be especially stringent; thus, although false-negative results were observed in previous studies with this method, false- positive results have not as yet been reported (Altschul et al, 1990).

Of the fifteen analyzed candidate BRCAI -associated proteins identified by two-hybrid screening in yeast (1 1 novel and 5 known sequences), each protein tested except that encoded by a clone termed B202 failed to associate with BRCAI in the mammalian assay (the sixteenth candidate, laminin, has not yet been tested). A second independent isolate (B230) was also obtained that contained a distinct but overlapping insert of 2.5 kb. The combined B202 and B230 cDNA sequence of 2,531 bp (SEQ ID NO:l) was termed the BARDl gene, and this gene encodes the 777 and/or 752 amino acid protein of SEQ ID NO:2, also termed BARDl (named from BRCA 1 -Associated RING Domain (BARD 1 ) protein, see below).

As only BARDl registered as positive in the mammalian assay, this gene and protein are naturally the currently preferred biological compositions for use in the present invention. However, as false-negative results have been encountered previously in mammalian two-hybrid studies, the inability of the other fourteen (or fifteen) proteins to interact with BRCAI in this assay does not necessarily eliminate them as candidate BRCAI -associated factors. It is for this reason that they are still encompassed within all aspects of the present invention. Even though one or more, or even nearly all, of the additionally disclosed proteins may ultimately prove not to bind to BRCAI, this would not negate the usefulness of one or two proteins or more from the remaining candidates upon confirmation of BRCA 1 -binding properties for such proteins.

In any event, the interaction between BRCAI and BARDl was detected in both orientations of the mammalian two-hybrid system, and it was confirmed in an independent fashion by co-immunoprecipitation of these proteins from mammalian cell lysates. Furthermore, the in vivo association between these proteins was reproduced using in vitro assays of protein binding, indicating that the interaction between BRCAI and BARDl is direct. Therefore, the utility of BARDl in BRCAI binding has been rigorously shown. The BARDl protein is a novel RING protein that interacts with the amino-terminal region of BRCAI. The BRCAI -associated RING domain (BARDl) protein is encoded by sequences on chromosome 2q, and resembles BRCAI in that it possesses an amino-terminal RING motif and the carboxy-terminal BRCT domains. These features, as well as its ability to form in vivo complexes with BRCAI, indicate that BARDl gene and protein likely serves as an effector and/or a regulator of BRCAI -mediated tumor suppression.

The precise role of BARDl in tumor formation is not yet known, although this does not negate the usefulness of the BARDl compositions of the present invention, particularly and most immediately, in terms of diagnostics. On one hand, tumor suppression may be mediated by the protein complex formed by the interaction between BRCAI and BARDl . As such,

BARDl would itself function as a tumor suppressor.

The tumor suppressor model is appealing because many regulatory proteins are known to function as obligate heterodimers, including transcription factors implicated in cancer, such as the c-MYC protein (which functions as a transcription factor within the context of a c- MYC/MAX heterodimer). If BARDl is confirmed to be tumor suppressor, the provision of wild-type BARDl to a cancer cell should counteract the malignant phenotype. As such, breast cancer treatment would include administering BARDl to a patient.

On the other hand, it is well established that certain dominant proto-oncogenes promote tumorigenesis by binding and reducing the activity of tumor suppressor proteins. Prominent examples include MDM2, which binds and inhibits the tumor suppressor function of p53, and the transforming proteins encoded by certain DNA viruses (e.g., the SV40 large T antigen), that also bind and inactivate tumor suppressors such as p53 and Rb. Thus, it is formally possible that the interaction between BARDl and BRCAI would reduce the tumor suppressor function of BRCAI .

In the above scenario, the gene encoding BARDl would serve as a dominant proto- oncogene. If BARDl is confirmed to be a classical oncogene, inhibiting BARDl would be the therapeutic approach. BARDl inhibition could be achieved by providing to a cancer cell or administering to a patient any compound that inhibits the BARDl gene, mRNA or protein.

The diagnostic and therapeutic methods disclosed herein take account of both the candidate tumor suppressor and oncogenic properties of BARDl and the other BRCAI binding proteins of the present invention.

I. BARDl and Other BRCAI Binding Proteins: Genes and DNA Segments

Important aspects of the present invention concern isolated DNA segments and recombinant vectors encoding wild-type, polymoφhic or mutant BARDl, and the creation and use of recombinant host cells through the application of DNA technology, that express wild-type, polymoφhic or mutant BARDl, using sequences of SEQ ID NO: l, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:122, SEQ ID NO: 123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO: 126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO: 129 or SEQ ID NO: 130. DNA segments, recombinant vectors, recombinant host cells and expression methods involving the other BRCAI binding proteins of the present invention, using sequences of TCL52 (SEQ ID NO:9); TCL163 (SEQ ID NO: 10); B223 (SEQ ID NO:l 1); B115 (SEQ ID NO: 12); BAP28 (SEQ ID NO: 13); B48 (SEQ ID NO: 14); B258 (SEQ ID NO: 15); BAP 152 (SEQ ID NO: 16); B123 (SEQ ID NO: 17); B268 (SEQ ID NO: 18); BE2 (SEQ ID NO:40); BE14 (SEQ ID NO:42); BE31 (SEQ ID NO:44); and BE445 (SEQ ID NO:46) are also provided. Each of the foregoing genes are included within all aspects of the following description.

The present invention concerns DNA segments, isolatable from mammalian and human cells, that are free from total genomic DNA and that are capable of expressing a protein or polypeptide that has BRCAI -binding activity.

As used herein, the term "DNA segment" refers to a DNA molecule that has been isolated free of total genomic DNA of a particular species. Therefore, a DNA segment encoding BARDl refers to a DNA segment that contains wild-type, polymoφhic or mutant BARDl, TCL52, TCL163, B223, Bl 15, BAP28, B48, B258, BAP152, B123, B268, BE2, BE14, BE31 or BE445 coding sequences yet is isolated away from, or purified free from, total mammalian or human genomic DNA. Included within the term "DNA segment", are DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like.

Similarly, a DNA segment comprising an isolated or purified wild-type, polymorphic or mutant BARDl or BRCAI -binding protein gene refers to a DNA segment including wild-type, polymoφhic or mutant BARDl or BRCA 1 -binding protein coding sequences and, in certain aspects, regulatory sequences, isolated substantially away from other naturally occurring genes or protein encoding sequences. In this respect, the term "gene" is used for simplicity to refer to a functional protein, polypeptide or peptide encoding unit. As will be understood by those in the art, this functional term includes both genomic sequences, cDNA sequences and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides, domains, peptides, fusion proteins and mutants.

"Isolated substantially away from other coding sequences" means that the gene of interest, in this case the wild-type, polymoφhic or mutant BARDl gene, or other BRCAI binding protein genes, forms the significant part of the coding region of the DNA segment, and that the DNA segment does not contain large portions of naturally-occurring coding DNA, such as large chromosomal fragments or other functional genes or cDNA coding regions. Of course, this refers to the DNA segment as originally isolated, and does not exclude genes or coding regions later added to the segment by the hand of man.

In particular embodiments, the invention concerns isolated DNA segments and recombinant vectors incoφorating DNA sequences that encode a wild-type, polymoφhic or mutant BARDl protein or peptide that includes within its amino acid sequence a contiguous amino acid sequence in accordance with, or essentially as set forth in, SEQ ID NO:2, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37 or SEQ ID NO:39, corresponding to wild-type, polymoφhic or mutant human BARDl. Moreover, in other particular embodiments, the invention concerns isolated DNA segments and recombinant vectors that encode a BARDl protein or peptide that includes within its amino acid sequence the substantially full length protein sequence of SEQ ID NO:2, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37 or SEQ ID NO:39.

In other embodiments, the invention concerns isolated DNA segments and recombinant vectors incoφorating DNA sequences that encode a BRCAI binding protein or peptide that includes within its amino acid sequence a contiguous amino acid sequence in accordance with, or essentially as set forth in, any one of SEQ ID NO:48 through SEQ ID NO:56, SEQ ID NO: 19, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45 or SEQ ID NO:47, corresponding to the human BRCAI binding proteins TCL52, TCL163, B223, Bl 15, BAP28, B48, B258, BAP 152, B123, B268, BE2, BE 14, BE31 or BE445. Moreover, in other particular embodiments, the invention concerns isolated DNA segments and recombinant vectors that encode a BRCAI binding protein or peptide that includes within its amino acid sequence the substantially full length protein sequence of SEQ ID NO:48 through SEQ ID NO:56, SEQ ID NO: 19, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45 or SEQ ID NO:47.

The term "a sequence essentially as set forth in SEQ ID NO:2, SEQ ID NO:21 , SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:48 through SEQ ID NO:56, SEQ ID NO: 19, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45 or SEQ ID NO:47" means that the sequence substantially corresponds to a portion of SEQ ID NO:2, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO.29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:48 through SEQ ID NO:56, SEQ ID NO: 19, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45 or SEQ ID NO.47 and has relatively few amino acids that are not identical to, or a biologically functional equivalent of, the amino acids of SEQ ID NO:2, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:48 through SEQ ID NO:56, SEQ ID NO: 19, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO.45 or SEQ ID NO:47. The term "biologically functional equivalent" is well understood in the art and is further defined in detail herein. Accordingly, sequences that have between about 70% and about 80%; or more preferably, between about 81% and about 90%; or even more preferably, between about 91% and about 99%; of amino acids that are identical or functionally equivalent to the amino acids of SEQ ID NO:2, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:48 through SEQ ID NO:56, SEQ ID NO: 19, SEQ ID NO:41 , SEQ ID NO:43, SEQ ID NO:45 or SEQ ID NO:47 will be sequences- that are "essentially as set forth in SEQ ID NO:2, SEQ ID NO:21 , SEQ ID NO.23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO.48 through SEQ ID NO:56, SEQ ID NO: 19, SEQ ID NO:41 , SEQ ID NO:43, SEQ ID NO:45 or SEQ ID NO:47", provided the biological activity of the protein is maintained.

In certain other embodiments, the invention concerns isolated DNA segments and recombinant vectors that include within their sequence a nucleic acid sequence essentially as set forth in SEQ ID NO:l, any one of SEQ ID NO:9 through SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:122, SEQ ID NO: 123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO: 129 or SEQ ID NO:130. The term "essentially as set forth in SEQ ID NO:l, any one of SEQ ID NO:9 through SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO: 129 or SEQ ID NO: 130" is used in the same sense as described above and means that the nucleic acid sequence substantially corresponds to a portion of SEQ ID NO:l, any one of SEQ ID NO:9 through SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO: 122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO: 128, SEQ ID NO: 129 or SEQ ID NO: 130 and has relatively few codons that are not identical, or functionally equivalent, to the codons of SEQ ID NO:l, any one of SEQ ID NO:9 through SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO:124, SEQ ID NO: 125, SEQ ID NO:126, SEQ ID NO: 127, SEQ ID NO:128, SEQ ID NO:129 or SEQ ID NO:130. Again, DNA segments that encode proteins exhibiting BRCAI -binding activity will be most preferred.

The term "functionally equivalent codon" is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine or serine, and also refers to codons that encode biologically equivalent amino acids (see Table 1 , below).

Table 1 - Preferred Human DNA Codons

Amino Acids Codons

Alanine Ala A GCC GCT GCA GCG

Cysteine Cys C TGC TGT

Aspartic acid Asp D GAC GAT

Glutamic acid Glu E GAG GAA

Phenylalanine Phe F TTC TTT

Glycine Gly G GGC GGG GGA GGT

Histidine His II CAC CAT

Isoleucine He I ATC ATT ATA

Lysine Lys K AAG AAA

Leucine Leu L CTG CTC TTG CTT CTA TTA

Methionine Met M ATG

Asparagine Asn N AAC AAT

Proline Pro P CCC CCT CCA CCG

Glutamine Gin Q CAG CAA

Arginine Arg R CGC AGG CGG AGA CGA CGT

Serine Ser S AGC TCC TCT AGT TCA TCG

Threonine Thr T ACC ACA ACT ACG

Valine Val V GTG GTC GTT GTA

Tryptophan T w TGG

Tyrosine Tyr Y TAC TAT

It will also be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5' or 3' sequences, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region or may include various internal sequences, i.e., introns, which are known to occur within genes. Excepting intronic or flanking regions, and allowing for the degeneracy of the genetic code, sequences that have between about 70% and about 79%; or more preferably, between about 80%) and about 89%; or even more preferably, between about 90% and about 99%; of nucleotides that are identical to the nucleotides of SEQ ID NO:l, any one of SEQ ID NO:9 through SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO: 122, SEQ ID NO:123, SEQ ID NO:I24, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO: 128, SEQ ID NO: 129 or SEQ ID NO: 130 will be sequences that are "essentially as set forth in SEQ ID NO:l, any one of SEQ ID NO:9 through SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO.40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO.128, SEQ ID NO:129 or SEQ ID NO:130".

Sequences that are essentially the same as those set forth in SEQ ID NO:l, any one of SEQ ID NO:9 through SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129 or SEQ ID NO:130 may also be functionally defined as sequences that are capable of hybridizing to a nucleic acid segment containing the complement of SEQ ID NO:l, any one of SEQ ID NO:9 through SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129 or SEQ ID NO: 130 under relatively stringent conditions. Suitable relatively stringent hybridization conditions will be well known to those of skill in the art, as disclosed herein..

Naturally, the present invention also encompasses DNA segments that are complementary, or essentially complementary, to the sequence set forth in SEQ ID NO:l, any one of SEQ ID NO:9 through SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129 or SEQ ID NO:130. Nucleic acid sequences that are "complementary" are those that are capable of base-pairing according to the standard Watson-Crick complementarity rules. As used herein, the term "complementary sequences" means nucleic acid sequences that are substantially complementary, as may be assessed by the same nucleotide comparison set forth above, or as defined as being capable of hybridizing to the nucleic acid segment of SEQ ID NO: 1 , any one of SEQ ID NO:9 through SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO: I29 or SEQ ID NO: 130 under relatively stringent conditions such as those described herein.

The nucleic acid segments of the present invention, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.

For example, nucleic acid fragments may be prepared that include a short contiguous stretch identical to or complementary to SEQ ID NO:l, any one of SEQ ID NO:9 through SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO: 128, SEQ ID NO:129 or SEQ ID NO:130, such as about 8, about 10 to about 14, or about 15 to about 20 nucleotides, and that are up to about 20,000, or about 10,000, or about 5,000 base pairs in length, with segments of about 3,000 being preferred in certain cases. DNA segments with total lengths of about 1,000, about 500, about 200, about 100 and about 50 base pairs in length (including all intermediate lengths) are also contemplated to be useful.

It will be readily understood that "intermediate lengths", in these contexts, means any length between the quoted ranges, such as 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.; 21, 22, 23, etc.; 30, 31 , 32, etc.; 50, 51, 52, 53, etc.; 100, 101 , 102, 103, etc.; 150, 151 , 152, 153, etc.; including all integers through the 200-500; 500-1,000; 1,000-2,000; 2,000-3,000; 3,000-5,000; 5,000-10,000 ranges, up to and including sequences of about 12,001, 12,002, 13,001, 13,002, 15,000, 20,000 and the like.

The various probes and primers designed around the disclosed nucleotide sequences of the present invention may be of any length. By assigning numeric values to a sequence, for example, the first residue is 1, the second residue is 2, etc., an algorithm defining all primers can be proposed:

n to n + y

where n is an integer from 1 to the last number of the sequence and y is the length of the primer minus one, where n + y does not exceed the last number of the sequence. Thus, for a 10-mer, the probes correspond to bases 1 to 10, 2 to 11, 3 to 12 ... and so on. For a 15-mer, the probes correspond to bases 1 to 15, 2 to 16, 3 to 17 ... and so on. For a 20-mer, the probes correspond to bases 1 to 20, 2 to 21, 3 to 22 ... and so on.

It will also be understood that this invention is not limited to the particular nucleic acid and amino acid sequences of SEQ ID NO:l, any one of SEQ ID NO:9 through SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO: 129 or SEQ ID NO: 130. Recombinant vectors and isolated DNA segments may therefore variously include these coding regions themselves, coding regions bearing selected alterations or modifications in the basic coding region, or they may encode larger polypeptides that nevertheless include such coding regions or may encode biologically functional equivalent proteins or peptides that have variant amino acids sequences.

The DNA segments of the present invention encompass biologically functional equivalent BARDl and BRCAI -binding proteins and peptides. Such sequences may arise as a consequence of codon redundancy and functional equivalency that arc known to occur naturally within nucleic acid sequences and the proteins thus encoded. Alternatively, functionally equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged. Changes designed by man may be introduced through the application of site-directed mutagenesis techniques, e.g., to introduce improvements to the antigenicity of the protein or to test mutants in order to examine DNA binding activity at the molecular level.

One may also prepare fusion proteins and peptides, e.g., where the BARDl or BRCA1- binding protein coding regions are aligned within the same expression unit with other proteins or peptides having desired functions, such as for purification or immunodetection purposes (e.g., proteins that may be purified by affinity chromatography and enzyme label coding regions, respectively).

Encompassed by the invention are DNA segments encoding relatively small peptides, such as, for example, peptides of from about 15 to about 50 amino acids in length, and more preferably, of from about 15 to about 30 amino acids in length; and also larger polypeptides up to and including proteins corresponding to the full-length sequences set forth in SEQ ID NO:l, any one of SEQ ID NO:9 through SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44 or SEQ ID NO:46. B. Recombinant Vectors, Host Cells and Expression

Recombinant vectors form important further aspects of the present invention. The term "expression vector or construct" means any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed. The transcript may be translated into a protein, but it need not be. Thus, in certain embodiments, expression includes both transcription of a gene and translation of a RNA into a gene product. In other embodiments, expression only includes transcription of the nucleic acid, for example, to generate antisense constructs.

Particularly useful vectors are contemplated to be those vectors in which the coding portion of the DNA segment, whether encoding a full length protein or smaller peptide, is positioned under the transcriptional control of a promoter. A "promoter" refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. The phrases "operatively positioned", "under control" or "under transcriptional control" means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.

The promoter may be in the form of the promoter that is naturally associated with a wild-type, polymoφhic or mutant BARDl gene, or BRCAI binding protein gene, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment or exon, for example, using recombinant cloning and/or PCR technology, in connection with the compositions disclosed herein (PCR technology is disclosed in U.S. Patent 4,683,202 and U.S. Patent 4,682,195, each incorporated herein by reference).

In other embodiments, it is contemplated that certain advantages will be gained by positioning the coding DNA segment under the control of a recombinant, or heterologous, promoter. As used herein, a recombinant or heterologous promoter is intended to refer to a promoter that is not normally associated with a wild-type, polymorphic or mutant BARDl gene, or a BRCAI binding protein gene in its natural environment. Such promoters may include promoters normally associated with other genes, and/or promoters isolated from any other bacterial, viral, eukaryotic, or mammalian cell.

Naturally, it will be important to employ a promoter that effectively directs the expression of the DNA segment in the cell type, organism, or even animal, chosen for expression. The use of promoter and cell type combinations for protein expression is generally known to those of skill in the art of molecular biology, for example, see Sambrook et ul. (1989), incorporated herein by reference. The promoters employed may be constitutive, or inducible, and can be used under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins or peptides.

At least one module in a promoter functions to position the start site for RNA synthesis.

The best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.

Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription.

The particular promoter that is employed to control the expression of a nucleic acid is not believed to be critical, so long as it is capable of expressing the nucleic acid in the targeted cell. Thus, where a human cell is targeted, it is preferable to position the nucleic acid coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell. Generally speaking, such a promoter might include either a human or viral promoter. Preferred promoters include those derived from HSV, including the HNFl promoter. Another preferred embodiment is the tetracycline controlled promoter.

In various other embodiments, the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter and the Rous sarcoma virus long terminal repeat can be used to obtain high-level expression of transgenes. The use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression of a transgene is contemplated as well, provided that the levels of expression are sufficient for a given purpose. Tables 2 and 3 below list several elements/promoters which may be employed, in the context of the present invention, to regulate the expression of wild-type, polymoφhic or mutant BARDl gene or a BRCAI binding protein gene. This list is not intended to be exhaustive of all the possible elements involved in the promotion of transgene expression but, merely, to be exemplary thereof.

Enhancers were originally detected as genetic elements that increased transcription from a promoter located at a distant position on the same molecule of DNA. This ability to act over a large distance had little precedent in classic studies of prokaryotic transcriptional regulation. Subsequent work showed that regions of DNA with enhancer activity are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins.

The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization.

Additionally any promoter/enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) could also be used to drive expression of a transgene. Use of a T3, T7 or SP6 cytoplasmic expression system is another possible embodiment. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if tl e appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct.

Table 2 - Promoter and Enhancer Elements

Promoter/Enhancer References

Immunoglobulin Heavy Chain Banerji et al, 1983; Gilles et al, 1983; Grosschedl and Baltimore, 1985; Atchinson and Perry, 1986, 1987; Imler et al, 1987; Weinberger et al, 1984; Kilcdjian et al, 1988; Porton e/ α/. ; 1990

Immunoglobulin Light Chain Queen and Baltimore, 1983; Picard and Schaffner, 1984

T-Cell Receptor Luria et al, 1987; Winoto and Baltimore, 1989; Redondo et al; 1990

HLA DQ a and DQ β Sullivan and Peterlin, 1987

β-Interferon Goodboum et al, 1986; Fujita et al, 1987; Goodbourn and Maniatis, 1988

Interleukin-2 Greene et al, 1989

Interleukin-2 Receptor Greene et al, 1989; Lin et al, 1990

MHC Class II 5 Koch et al, 1989

MHC Class II HLA-DRa Sherman et l, 1989

β-Actin Kawamoto et al, 1988; Ng et al; 1989

Muscle Creatine Kinase Jaynes et al, 1988; Horlick and Benfield, 1989; Johnson et al, 1989

Prealbumin (Transthyretin) Costa et al., 1988

Elastase I Omitz et α/,, 1987

Metallothionein Karin et al, 1987; Culotta and Hamer, 1989 Table 2 - Continued

Promoter/Enhancer References

Collagenase Pinkert et al, 1987; Angel et al, 1987

Albumin Gene Pinkert et al, 1987; Tronche et al, 1989, 1990

α-Fetoprotein Godbout et al, 1988; Campere and Tilghman, 1989

t-Globin Bodine and Ley, 1987; Perez-Stable and Constantini, 1990

β-Globin Trudel and Constantini, 1987

e-fos Cohen et al, 1987

c-HA-ras Triesman, 1986; Deschamps et al, 1985

Insulin άlxxnά et al, 1985

Neural Cell Adhesion Molecule Hirsh e/ α/., 1990 (NCAM)

O-l-Anlitrypain Latimer e/ α/., 1990

H2B (TH2B) Histone Hwang et al, 1990

Mouse or Type I Collagen lϊ e et al, 1989

Glucose-Regulated Proteins Chang et al, 1989 (GRP94 and GRP78)

Rat Growth Hormone Larsen e/ o/., 1986

Human Serum Amyloid A (S AA) Edbrooke et al. , 1989

Troponin I (TN I) Yutzey et al. , 1989

Platelet-Derived Growth Factor Pech et al , 1989

Duchenne Muscular Dystrophy Kla ut et al. , 1990 Table 2 - Continued

Promoter/Enhancer References

SV40 Banerji et al. , 1981 ; Moreau et al, 1981 ; Sleigh and Lockett, 1985; Firak and Subramanian, 1986; Herr and Clarke, 1986; Imbra and Karin, 1986; Kadesch and Berg, 1986; Wang and Calamc, 1986; Ondek et al, 1987; Kuh! et al, 1987; Schaffner et al. , 1988

Polyoma Swartzendruber and Lehman, 1975; Vasseur et al, 1980; Katinka e/ α/., 1980, 1981 ; Tyndell et al, 1981 ; Dandolo et al, 1983; de Villiers et al, 1984; Hen et al, 1986; Satake et al, 1988; Campbell and Villarrcal, 1988

Retroviruses Kriegler and Botchan, 1982, 1983; Levinson et al, 1982; Kriegler e/ tf/., 1983, 1984a, b, 1988; Bosze e/ α/., 1986; Miksicek et al, 1986; Celander and Haseltine, 1987; Thiesen et al, 1988; Celander et al, 1988; Choi et al, 1988; Reisman and Rotter, 1989

Papillo a Virus Campo et al, 1983; Lusky et al, 1983; Spandidos and Wilkie, 1983; Spalholz et al, 1985; Lusky and Botchan, 1986; Cripe et al, 1987; Gloss et al, 1987; Hirochika et al, 1987; Stephens and Hcntschel, 1987; Glue et al, 1988

Hepatitis B Virus Bulla and Siddiqui, 1986; Jameel and Siddiqui, 1986; Shaul and Ben-Levy, 1987; Spandau and Lee, 1988; Vannice and Levinson, 1988

Human Immunodeficiency Virus Muesing et al, 1987; Hauber and Cullan, 1988;

Jakobovits et al, 1988; Feng and Holland, 1988; Takebe et al, 1988; Rosen et al, 1988; Berkhout et al, 1989; Laspia et al, 1989; Shaφ and Marciniak, 1989; Braddock e/ α/., 1989 Table 2 - Continued

Promoter/Enhancer References

Cytomegalovirus Weber et al, 1984; Boshart et al, 1985; Foecking and Hofstetter, 1986

Gibbon Ape Leukemia Virus Holbrook et al, 1987; Quinn et al, 1989

Table 3 - Inducible Elements

Element Inducer References

MT II Phorbol Ester (TFA) Palmiter et al, 1982; Haslinger and Karin, 1985; Searle et al, Heavy metals 1985; Stuart et al, 1985; Imagawa et al. , 1987, Karin et al. , 1987; Angel et al, 1987b; McNeall <?/ tf/., 1989

MMTV (mouse mammary Glucocorticoids Huang et α/., 1981 ; Lee et al. , tumor virus) 1981; Majors and Vannus, 1983; Chandler et al, 1983; Lee et al, 1984; Ponta et al, 1985; Sakai et al, 1988

β-Interferon poly(rI)x Tavernier et al. , 1983

poly(rc)

Adenovirus 5 E2 Ela Imperiale and Nevins, 1984

Collagenase Phorbol Ester (TPA) Angel et al, 1987a

Stromelysin Phorbol Ester (TPA) Angel et l, 1987b

SV40 Phorbol Ester (TPA) Angel et al, 1987b Table 3 - Continued

Element Inducer References

Murine MX Gene Interferon, Newcastle Disease Virus

GRP78 Gene A23187 Resendez e/ α/., 1988

ct-2-Macroglobulin IL-6 unz et al, 1989

Vimentin Serum Rittlin e/ fl/., 1989

MHC Class I Gene H-2κb Interferon Blanar e/ fl/., 1989

HSP70 Ela, SV40 Large T Antigen Taylor et al , 1989; Taylor and

Kingston, 1990a, b

Proliferin Phorbol Ester-TPA Mordacq and Linzer, 1989

Tumor Necrosis Factor FMA Hensel e/ fl/., 1989

Thyroid Stimulating Thyroid Hormone Chatterjee e /., 1989 Hormone a Gene

As indicated, it is contemplated that one may use any regulatory element to express the BARDl, B123, BE2, BE14, BE31 and BE445 genes disclosed by the present invention; however, under certain circumstances it may be desirable to use the innate promoter region associated with the gene of interest to control its expression, such as the BARDl promoter within the 5' flanking region fo the BARDl genomic clone, as disclosed in SEQ ID NO: 122. As noted above, in most cases, genes are regulated at the level of transcription by regulatory elements that are located upstream, or 5', to the genes.

In general, to identify regulatory elements for the gene of interest, one would obtain a genomic DNA segment corresponding to the region located between about 10 to 50 nucleotides up to about 2000 nucleotides or more upsteam from the transcriptional start site of the gene, i.e. the nucleotides between positions -10 and -2000. A convenient method used to obtain such a sequence is to utilize restriction enzyme(s) to excise an appropriate DNA fragment. Restriction enzyme technology is commonly used in the art and will be generally known to the skilled artisan. For example, one may use a combination of enzymes from the extensive range of known restriction enzymes to digest the genomic DNA. Analysis of the digested fragments would determine which enzyme(s) produce the desired DNA fragment. The desired region may then be excised from the genomic DNA using the enzyme(s). If desired, one may even create a particular restriction site by genetic engineering for subsequent use in ligation strategies.

Alternatively, one may choose to prepare a series of DNA fragments differentiated by size through the use of a deletion assay with linearized DNA. In such an assay, enzymes are also used to digest the genomic DNA; however, in this case, the enzymes do not recognize specific sites within the DNA but instead digest the DNA from the free end(s). In this case, a series of size differentiated DNA fragments can be achieved by stopping the enzyme reaction after specified time intervals. Of course, one may also choose to use a combination of both restriction enzyme digestion and deletion assay to obtain the desired DNA fragment(s).

Once the desired DNA fragment has been isolated, its potential to regulate a gene and determine the basic regulatory unit may be examined using any one of several conventional techniques. It is recognized that once the core regulatory region is identified, one may choose to employ a longer sequence which comprises the identified regulatory unit. This is because although the core region is all that is ultimately required, it is believed that particular advantages accrue, in terms of regulation and level of induction achieved where one employs sequences which correspond to the natural control regions over longer regions, e.g. from around 25 or so nucleotides to as many as 1000 to 1500 or so nucleotides in length. The preferred length will be in part determined by the type of expression system used and the results desired.

Numerous methods are known in the art for precisely locating regulatory units within larger DNA sequences. Most conveniently, the desired control sequence is isolated within a DNA fragments) which is subsequently modified using DNA synthesis techniques to add restriction site linkers to the fragment(s) termini. This modification readily allows the insertion of the modified DNA fragment into an expression cassette which contains a reporter gene that confers on its recombinant host cell a readily detectable phenotype that is either expressed or inhibited, as may be the case. Generally reporter genes encode a polypeptide not otherwise produced by the host cell; or a protein or factor produced by the host cell but at much lower levels; or a mutant form of a polypeptide not otherwise produced by the host cell. Preferably the reporter gene encodes an enzyme which produces a colorimetric or fluorometric change in the host cell which is detectable by in situ analysis and is a quantitative or semi-quantitative function of transcriptional activation. Exemplary reporter genes encode esterases, phosphatases, proteases and other proteins detected by activity which generates a chromophore or fluorophore as will be known to the skilled artisan. Two well-known examples of such a reporter genes are E. coli beta- galactosidase and chloramphenicol-acetyl-transferase (CAT). Alternatively, a reporter gene may render its host cell resistant to a selection agent. For example, the gene neo renders cells resistant to the antibiotic neomycin. It is contemplated that virtually any host cell system compatible with the reporter gene cassette may be used to determine the regulatory unit. Thus mammalian or other eukaryotic cells, insect, bacterial or plant cells may be used.

Once a DNA fragment containing the putative regulatory region is inserted into an expression cassette which is in turn inserted into an appropriate host cell system, using any of the techniques commonly known to those of skill in the art, the ability of the fragment to regulate the expression of the reporter gene is assessed. By using a quantitative reporter assay and analyzing a series of DNA fragments of decreasing size, for example produced by convenient restriction endonuclease sites, or through the actions of enzymes such as B AL31 , E. coli exonuclease III or mung bean nuclease, and which overlap each other a specific number of nucleotides, one may determine both the size and location of the native regulatory unit.

Of course once the core regulatory unit has been determined, one may choose to modify the regulatory unit by mutating certain nucleotides within the core unit. The effects of these modifications may be analyzed using the same reporter assay to determine whether the modifications either enhance or reduce transcription. Thus key nucleotides within the core regulatory sequence can be identified.

It is recognized that regulatory units often contain both elements that either enhance or inhibit transcription. In the case that a regulatory unit is suspected of containing both types of elements, one may use competitive DNA mobility shift assays to separately identify each element. Those of skill in the art will be familiar the use of DNA mobility shift assays.

It may also be desirable to modify the identified regulatory unit by adding additional sequences to the unit. The added sequences may include additional enhancers, promoters or even other genes. Thus one may, for example, prepare a DNA fragment that contains the native regulatory elements positioned to regulate one or more copies of the native gene and/or another gene or prepare a DNA fragment which contains not one but multiple copies of the promoter region such that transcription levels of the desired gene are relatively increased.

Turning to the expression of the wild-type, polymoφhic or mutant BARDl proteins, or the BRCAI binding proteins of the present invention , once a suitable clone or clones have been obtained, whether they be cDNA based or genomic, one may proceed to prepare an expression system. The engineering of DNA segment(s) for expression in a prokaryotic or eukaryotic system may be performed by techniques generally known to those of skill in recombinant expression. It is believed that virtually any expression system may be employed in the expression of the proteins of the present invention.

Both cDNA and genomic sequences are suitable for eukaryotic expression, as the host cell will generally process the genomic transcripts to yield functional mRNA for translation into protein. Generally speaking, it may be more convenient to employ as the recombinant gene a cDNA version of the gene. It is believed that the use of a cDNA version will provide advantages in that the size of the gene will generally be much smaller and more readily employed to transfect the targeted cell than will a genomic gene, which will typically be up to an order of magnitude larger than the cDNA gene. However, the inventor does not exclude the possibility of employing a genomic version of a particular gene where desired.

In expression, one will typically include a polyadenylation signal to effect proper polyadenylation of the transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed.

Preferred embodiments include the SV40 polyadenylation signal and the bovine growth hormone polyadenylation signal, convenient and known to function well in various target cells. Also contemplated as an element of the expression cassette is a terminator. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.

A specific initiation signal also may be required for efficient translation of coding sequences. These signals include the ATG initiation codon and adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals. It is well known that the initiation codon must be "in-frame" with the reading frame of the desired coding sequence to ensure translation of the entire insert. The exogenous translational control signals and initiation codons can be either natural or synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements.

It is proposed that wild-type, polymorphic or mutant BARDl genes, or the genes encoding BRCAI binding proteins may be co-expressed with BRCAI, wherein the proteins may be co-expressed in the same cell or wherein wild-type, polymorphic or mutant BARDl genes, or the genes encoding BRCAI binding proteins may be provided to a cell that already has BRCAI . Co-expression may be achieved by co-transfecting the cell with two distinct recombinant vectors, each bearing a copy of either the respective DNA. Alternatively, a single recombinant vector may be constructed to include the coding regions for both of the proteins, which could then be expressed in cells transfected with the single vector. In either event, the term "co- expression" herein refers to the expression of both the wild-type, polymorphic or mutant BARDl genes, or the genes encoding BRCAI binding proteins and the BRCAI proteins in the same recombinant cell.

In addition to co-expression with BRCAI , it is proposed that the wild-type, polymorphic or mutant BARDl genes, or the genes encoding BRCAI binding proteins may be co-expressed with genes encoding other selected tumor suppressor proteins or peptides. Tumor suppressor proteins contemplated for use include, but are not limited to, the retinoblastoma, p53, Wilms tumor (WT-1), DCC, neurofibromatosis type 1 (NF-1), von Hippel-Lindau (VHL) disease tumor suppressor, Maspin, Brush- 1, BRCA-2 and the multiple tumor suppressor (MTS) or pi 6 proteins or peptides. Further particularly contemplated is co-expression with a selected wild-type version of a selected oncogene. Wild-type oncogenes contemplated for use include, but are not limited to, tyrosine kinases, both membrane-associated and cytoplasmic forms, such as members of the Src family, serine/threonine kinases, such as Mos, growth factor and receptors, such as platelet derived growth factor (PDGF), small GTPases (G proteins) including the ras family and Gs- alpha, cyclin-dependent protein kinases (cdk), members of the myc family members including c- myc, N-myc, and L-myc and bcl-2 and family members.

As used herein, the terms "engineered" and "recombinant" cells are intended to refer to a cell into which an exogenous DNA segment or gene, such as a cDNA or gene encoding a BARDl or BRCAI binding protein has been introduced. Therefore, engineered cells arc distinguishable from naturally occurring cells which do not contain a recombinantly introduced exogenous DNA segment or gene. Engineered cells are thus cells having a gene or genes introduced through the hand of man. Recombinant cells include those having an introduced cDNA or genomic gene, and also include genes positioned adjacent to a promoter not naturally associated with the particular introduced gene.

To express a recombinant BARDl or BRCAI binding protein, whether mutant or wild- type, in accordance with the present invention one would prepare an expression vector that comprises a wild-type, polymoφhic or mutant BARD1-, or a BRCAI binding protein-encoding nucleic acid under the control of one or more promoters. To bring a coding sequence "under the control of a promoter, one positions the 5' end of the transcription initiation site of the transcriptional reading frame generally between about 1 and about 50 nucleotides "downstream" of (i.e., 3' of) the chosen promoter. The "upstream" promoter stimulates transcription of the DNA and promotes expression of the encoded recombinant protein. This is the meaning of "recombinant expression" in this context.

Many standard techniques are available to construct expression vectors containing the appropriate nucleic acids and transcriptional/translational control sequences in order to achieve protein or peptide expression in a variety of host-expression systems. Cell types available for expression include, but are not limited to, bacteria, such as E. coli and B. subtilis transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors. Certain examples of prokaryotic hosts are E. coli strain RR1, E. coli LE392, E. coli B, E. coli X 1776 (ATCC No. 31537) as well as E. coli W31 10 (F-, lambda-, prototrophic, ATCC No. 273325); bacilli such as Bacillus subtilis; and other enterobacteriaceas such as Salmonella typhimurium, Serratia marcescens, and various Pseudomonas species.

In general, plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts. The vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells. For example, E. coli is often transformed using derivatives of pBR322, a plasmid derived from an E. coli species. pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells. The pBR plasmid, or other microbial plasmid or phage must also contain, or be modified to contain, promoters which can be used by the microbial organism for expression of its own proteins.

In addition, phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as transforming vectors in connection with these hosts. For example, the phage lambda GEM -1 1 may be utilized in making a recombinant phage vector which can be used to transform host cells, such as E. coli LE392.

Further useful vectors include pIN vectors (Inouye et al, 1985); and pGEX vectors, for use in generating glutathione S-transferase (GST) soluble fusion proteins for later purification and separation or cleavage. Other suitable fusion proteins are those with β-galactosidase, ubiquitin, the like.

Promoters that are most commonly used in recombinant DNA construction include the β-lactamase (penicillinase), lactose and tryptophan (trp) promoter systems. While these are the most commonly used, other microbial promoters have been discovered and utilized, and details concerning their nucleotide sequences have been published, enabling those of skill in the art to ligate them functionally with plasmid vectors. The following details concerning recombinant protein production in bacterial cells, such as E. coli, are provided by way of exemplary information on recombinant protein production in general, the adaptation of which to a particular recombinant expression system will be known to those of skill in the art.

Bacterial cells, for example, E. coli, containing the expression vector are grown in any of a number of suitable media, for example, LB. The expression of the recombinant protein may be induced, e.g., by adding IPTG to the media or by switching incubation to a higher temperature. After culturing the bacteria for a further period, generally of between 2 and 24 hours, the cells are collected by centrifugation and washed to remove residual media.

The bacterial cells are then lysed, for example, by disruption in a cell homogenizer and centrifuged to separate the dense inclusion bodies and cell membranes from the soluble cell components. This centrifugation can be performed under conditions whereby the dense inclusion bodies are selectively enriched by incoφoration of sugars, such as sucrose, into the buffer and centrifugation at a selective speed.

If the recombinant protein is expressed in the inclusion bodies, as is the case in many instances, these can be washed in any of several solutions to remove some of the contaminating host proteins, then solubilized in solutions containing high concentrations of urea (e.g. 8M) or chaotropic agents such as guanidine hydrochloride in the presence of reducing agents, such as β- mercaptoethanol or DTT (dithiothreitol).

Under some circumstances, it may be advantageous to incubate the protein for several hours under conditions suitable for the protein to undergo a refolding process into a conformation which more closely resembles that of the native protein. Such conditions generally include low protein concentrations, less than 500 mg/ml, low levels of reducing agent, concentrations of urea less than 2 M and often the presence of reagents such as a mixture of reduced and oxidized glutathione which facilitate the interchange of disulfide bonds within the protein molecule. The refolding process can be monitored, for example, by SDS-PAGE, or with antibodies specific for the native molecule (which can be obtained from animals vaccinated with the native molecule or smaller quantities of recombinant protein). Following refolding, the protein can then be purified further and separated from the refolding mixture by chromatography on any of several supports including ion exchange resins, gel permeation resins or on a variety of affinity columns.

For expression in Saccharomyces, the plasmid YRp7, for example, is commonly used.

This plasmid already contains the trp\ gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-

1. The presence of the trp\ lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.

Suitable promoting sequences in yeast vectors include the promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase, glyceraldehyde-3- phosphate dehydrogenase, hexokinasc, pyruvate decarboxylase, phosphofructokinase, glucose-6- phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. In constructing suitable expression plasmids, the termination sequences associated with these genes are also ligated into the expression vector 3' of the sequence desired to be expressed to provide polyadenylation of the mRNA and termination.

Other suitable promoters, which have the additional advantage of transcription controlled by growth conditions, include the promoter region for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization.

In addition to micro-organisms, cultures of cells derived from multicellular organisms may also be used as hosts. In principle, any such cell culture is workable, whether from vertebrate or invertebrate culture. In addition to mammalian cells, these include insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus); and 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 one or more wild-type, polymorphic or mutant BARDl , or BRCAI binding protein coding sequences.

In a useful insect system, Autograph californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The wild-type, polymoφhic or mutant BARDl coding sequences or the BRCAI binding protein coding sequences are cloned into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter). Successful insertion of the coding sequences results in the inactivation of the polyhedrin gene and production of non-occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedrin gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed (e.g., U.S. Patent No. 4,215,051 , Smith, incoφorated herein by reference).

Examples of useful mammalian host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines. In addition, a host cell strain may be chosen that 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. Appropriate cells 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 such as 293 cells have already been shown to produce active BARDl .

Expression vectors for use in mammalian such cells ordinarily include an origin of replication (as necessary), a promoter located in front of the gene to be expressed, along with any necessary ribosome binding sites, RNA splice sites, polyadenylation site, and transcriptional terminator sequences. The 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 (e.g., Polyoma, Adeno, VSV, BPV) source, or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient.

The promoters may be 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). Further, it is also possible, and may be desirable, to utilize promoter or control sequences normally associated with the desired wild-iype, polymorphic or mutant BARDl or BRCAI binding protein gene sequence, provided such control sequences are compatible with the host cell systems.

A number of viral based expression systems may be utilized, for example, commonly used promoters are derived from polyoma, Adenovirus 2, and most frequently Simian Virus 40 (SV40). The early and late promoters of SV40 virus are particularly useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication. Smaller or larger SV40 fragments may also be used, provided there is included the approximately 250 bp sequence extending from the Hindlll site toward the Bgll site located in the viral origin of replication.

In cases where an adenovirus is used as an expression vector, the coding sequences 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 wild-type, polymoφhic or mutant BARDl or BRCAI binding proteins in infected hosts.

Specific initiation signals may also be required for efficient translation of wild-type, polymoφhic or mutant BARDl or BRCAI binding protein coding sequences. These signals include the ATG initiation codon and adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may additionally need to be provided. One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals. It is well known that the initiation codon must be in-frame (or 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.

In eukaryotic expression, one will also typically desire to incorporate into the transcriptional unit an appropriate polyadenylation site (e.g., 5'-AATAAA-3') if one was not contained within the original cloned segment. Typically, the poly A addition site is placed about 30 to 2000 nucleotides "downstream" of the termination site of the protein at a position prior to transcription termination.

For long-term, high-yield production of recombinant wild-type, polymorphic or mutant BARDl or BRCAI binding proteins, stable expression is preferred. For example, cell lines that stably express constructs encoding wild-type, polymoφhic or mutant BARDl or BRCAI binding proteins may be engineered. Rather than using expression vectors that contain viral origins of replication, host cells can be transformed with vectors controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of 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 foci which in turn can be cloned and expanded into cell lines.

A number of selection systems may be used, including, but not limited, to the herpes simplex virus thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase and adenine phosphoribosyltransferase genes, in tk-, hgprt- or aprt- cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for dhfr, that confers resistance to methotrexate; gpt, that confers resistance to mycophenolic acid; neo, that confers resistance to the aminoglycoside G-418; and hygro, that confers resistance to hygromycin. Animal cells can be propagated in vitro in two modes: as non-anchorage dependent cells growing in suspension throughout the bulk of the culture or as anchorage-dependent cells requiring attachment to a solid substrate for their propagation (i.e., a monolayer type of cell growth).

Non-anchorage dependent or suspension cultures from continuous established cell lines are the most widely used means of large scale production of cells and cell products. However, suspension cultured cells have limitations, such as tumorigenic potential and lower protein production than adherent cells.

Large scale suspension culture of mammalian cells in stirred tanks is a common method for production of recombinant proteins. Two suspension culture reactor designs are in wide use - the stirred reactor and the airlift reactor. The stirred design has successfully been used on an 8000 liter capacity for the production of interferon. Cells are grown in a stainless steel tank with a height-to-diameter ratio of 1 : 1 to 3: 1. The culture is usually mixed with one or more agitators, based on bladed disks or marine propeller patterns. Agitator systems offering less shear forces than blades have been described. Agitation may be driven either directly or indirectly by magnetically coupled drives. Indirect drives reduce the risk of microbial contamination through seals on stirrer shafts.

The airlift reactor, also initially described for microbial fermentation and later adapted for mammalian culture, relies on a gas stream to both mix and oxygenate the culture. The gas stream enters a riser section of the reactor and drives circulation. Gas disengages at the culture surface, causing denser liquid free of gas bubbles to travel downward in the downcomer section of the reactor. The main advantage of this design is the simplicity and lack of need for mechanical mixing. Typically, the height-to-diameter ratio is 10:1. The airlift reactor scales up relatively easily, has good mass transfer of gases and generates relatively low shear forces.

It is contemplated that the wild-type, polymorphic or mutant BARDl or BRCAI binding proteins of the invention may be "overexpressed", i.e., expressed in increased levels relative to its natural expression in cells. Such overexpression may be assessed by a variety of methods, including radio-labelling and/or protein purification. However, simple ?nd direct methods arc preferred, for example, those involving SDS/PAGE and protein staining or western blotting, followed by quantitative analyses, such as densitometric scanning of the resultant gel or blot. A specific increase in the level of the recombinant protein or peptide in comparison to the level in natural cells is indicative of overexpression, as is a relative abundance of the specific protein in relation to the other proteins produced by the host cell and, e.g., visible on a gel.

C. Nucleic Acid Detection

In addition to their use in directing the expression of the wild-type, polymorphic or mutant BARDl or BRCAI binding proteins, the nucleic acid sequences disclosed herein also have a variety of other uses. For example, they also have utility as probes or primers in nucleic acid hybridization embodiments.

Hybridization

The use of a hybridization probe of between 17 and 100 nucleotides in length allows the formation of a duplex molecule that is both stable and selective. Molecules having complementary sequences over stretches greater than 20 bases in length are generally preferred, in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of particular hybrid molecules obtained. One will generally prefer to design nucleic acid molecules having stretches of 20 to 30 nucleotides, or even longer where desired. Such fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means or by introducing selected sequences into recombinant vectors for recombinant production.

Accordingly, the nucleotide sequences of the invention may be used for their ability to selectively form duplex molecules with complementary stretches of genes or RNAs or to provide primers for amplification of DNA or RNA from tissues. Depending on the application envisioned, one will desire to employ varying conditions of hybridization to achieve varying degrees of selectivity of probe towards target sequence.

For applications requiring high selectivity, one will typically desire to employ relatively stringent conditions to form the hybrids, e.g., one will select relatively low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.10 M NaCI at temperatures of about 50°C to about 70°C. Such high stringency conditions tolerate little, if any, mismatch between the probe and the template or target strand, and would be particularly suitable for isolating specific genes or detecting specific mRNA transcripts. It is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide.

For certain applications, for example, substitution of nucleotides by site-directed mutagenesis, it is appreciated that lower stringency conditions are required. Under these conditions, hybridization may occur even though the sequences of probe and target strand are not perfectly complementary, but are mismatched at one or more positions. Conditions may be rendered less stringent by increasing salt concentration and decreasing temperature. For example, a medium stringency condition could be provided by about 0.1 to 0.25 M NaCI at temperatures of about 37°C to about 55°C, while a low stringency condition could be provided by about 0.15 M to about 0.9 M salt, at temperatures ranging from about 20°C to about 55°C. Thus, hybridization conditions can be readily manipulated depending on the desired results.

In other embodiments, hybridization may be achieved under conditions of, for example, 50 M Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂, 1.0 mM dithiothreitol, at temperatures between approximately 20°C to about 37°C. Other hybridization conditions utilized could include approximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, at temperatures ranging from approximately 40°C to about 72°C.

In certain embodiments, it will be advantageous to employ nucleic acid sequences of the present invention in combination with an appropriate means, such as a label, for determining hybridization. A wide variety of appropriate indicator means are known in the art, including fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of being detected. In preferred embodiments, one may desire to employ a fluorescent label or an enzyme tag such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmentally undesirable reagents. In the case of enzyme tags, colorimetric indicator substrates are known that can be employed to provide a detection means visible to the human eye or spectrophotometrically, to identify specific hybridization with complementary nucleic acid- containing samples. In general, it is envisioned that the hybridization probes described herein will be useful both as reagents in solution hybridization, as in PCR, for detection of expression of corresponding genes, as well as in embodiments employing a solid phase. In embodiments involving a solid phase, the test DNA (or RNA) is adsorbed or otherwise affixed to a selected matrix or surface. This fixed, single-stranded nucleic acid is then subjected to hybridization with selected probes under desired conditions. The selected conditions will depend on the particular circumstances based on the particular criteria required (depending, for example, on the G+C content, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.). Following washing of the hybridized surface to remove non-specifically bound probe molecules, hybridization is detected, or even quantified, by means of the label.

2. Amplification and PCR

Nucleic acid used as a template for amplification is isolated from cells contained in the biological sample, according to standard methodologies (Sambrook et al, 1989). The nucleic acid may be genomic DNA or fractionated or whole cell RNA. Where RNA is used, it may be desired to convert the RNA to a complementary DNA. In one embodiment, the RNA is whole cell RNA and is used directly as the template for amplification.

Pairs of primers that selectively hybridize to nucleic acids corresponding to wild-type, polymoφhic or mutant BARDl or BRCAI binding protein are contacted with the isolated nucleic acid under conditions that permit selective hybridization. The term "primer", as defined herein, is meant to encompass any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process. Typically, primers are oligonucleotides from ten to twenty base pairs in length, but longer sequences can be employed. Primers may be provided in double-stranded or single-stranded form, although the single-stranded form is preferred.

Once hybridized, the nucleic acid:primer complex is contacted with one or more enzymes that facilitate template-dependent nucleic acid synthesis. Multiple rounds of amplification, also referred to as "cycles," are conducted until a sufficient amount of amplification product is produced.

Next, the amplification product is detected. In certain applications, the detection may be performed by visual means. Alternatively, the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of incorporated radiolabel or fluorescent label or even via a system using electrical or thermal impulse signals (Affymax technology).

A number of template dependent processes are available to amplify the marker sequences present in a given template sample. One of the best known amplification methods is the polymerase chain reaction (referred to as PCR) which is described in detail in U.S. Patent Nos. 4,683,195, 4,683,202 and 4,800,159, and each incorporated herein by reference in entirety.

Briefly, in PCR, two primer sequences arc prepared that are complementary to regions on opposite complementary strands of the marker sequence. An excess of deoxynucleoside triphosphates are added to a reaction mixture along with a DNA polymerase, e.g., Taq polymerase. If the marker sequence is present in a sample, the primers will bind to the marker and the polymerase will cause the primers to be extended along the marker sequence by adding on nucleotides. By raising and lowering the temperature of the reaction mixture, the extended primers will dissociate from the marker to form reaction products, excess primers will bind to the marker and to the reaction products and the process is repeated.

A reverse transcriptase PCR amplification procedure may be performed in order to quantify the amount of mRNA amplified. Methods of reverse transcribing RNA into cDNA are well known and described in Sambrook et al, 1989. Alternative methods for reverse transcription utilize thermostable, RNA-dependent DNA polymerases. These methods are described in WO 90/07641, filed December 21, 1990, incorporated herein by reference. Polymerase chain reaction methodologies are well known in the art.

Another method for amplification is the ligase chain reaction ("LCR"), disclosed in EPA No. 320 308, incoφorated herein by reference in its entirety. In LCR, two complementary probe pairs are prepared, and in the presence of the target sequence, each pair will bind to opposite complementary strands of the target such that they abut. In the presence of a ligase, the two probe pairs will link to form a single unit. By temperature cycling, as in PCR, bound ligated units dissociate from the target and then serve as "target sequences" for ligation of excess probe pairs. U.S. Patent 4,883,750 describes a method similar to LCR for binding probe pairs to a target sequence.

Qbeta Replicase, described in PCT Application No. PCT/US87/00880, incorporated herein by reference, may also be used as still another amplification method in the present invention. In this method, a replicative sequence of RNA that has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase. The polymerase will copy the replicative sequence that can then be detected.

An isothermal amplification method, in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5'-[alpha-thio]- triphosphates in one strand of a restriction site may also be useful in the amplification of nucleic acids in the present invention.

Strand Displacement Amplification (SDA) is another method of carrying out isothermal amplification of nucleic acids which involves multiple rounds of strand displacement and synthesis, i.e., nick translation. A similar method, called Repair Chain Reaction (RCR), involves annealing several probes throughout a region targeted for amplification, followed by a repair reaction in which only two of the four bases are present. The other two bases can be added as biotinylated derivatives for easy detection. A similar approach is used in SDA. Target specific sequences can also be detected using a cyclic probe reaction (CPR). In CPR, a probe having 3' and 5' sequences of non-specific DNA and a middle sequence of specific RNA is hybridized to DNA that is present in a sample. Upon hybridization, the reaction is treated with

RNase H, and the products of the probe identified as distinctive products that are released after digestion. The original template is annealed to another cycling probe and the reaction is repeated. Still another amplification methods described in GB Application No. 2 202 328, and in PCT Application No. PCT/US89/01025, each of which is incorporated herein by reference in its entirety, may be used in accordance with the present invention. In the former application, "modified" primers are used in a PCR-like, template- and enzyme-dependent synthesis. The primers may be modified by labelling with a capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme). In the latter application, an excess of labeled probes are added to a sample. In the presence of the target sequence, the probe binds and is cleaved catalytically. After cleavage, the target sequence is released intact to be bound by excess probe. Cleavage of the labeled probe signals the presence of the target sequence.

Other nucleic acid amplification procedures include transcription-based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR Gingeras et al, PCT Application WO 88/10315, incorporated herein by reference. In NASBA, the nucleic acids can be prepared for amplification by standard phenol/chloroform extraction, heat denaturation of a clinical sample, treatment with lysis buffer and minispin columns for isolation of DNA and RNA or guanidinium chloride extraction of RNA. These amplification techniques involve annealing a primer which has target specific sequences. Following polymerization, DNA/RNA hybrids are digested with RNase H while double stranded DNA molecules are heat denatured again. In either case the single stranded DNA is made fully double stranded by addition of second target specific primer, followed by polymerization. The double- stranded DNA molecules are then multiply transcribed by an RNA polymerase such as T7 or SP6. In an isothermal cyclic reaction, the RNA's are reverse transcribed into single stranded DNA, which is then converted to double stranded DNA, and then transcribed once again with an RNA polymerase such as T7 or SP6. The resulting products, whether truncated or complete, indicate target specific sequences.

Davey et al, EPA No. 329 822 (incoφorated herein by reference in its entirety) disclose a nucleic acid amplification process involving cyclically synthesizing single-stranded RNA ("ssRNA"), ssDNA, and double-stranded DNA (dsDNA), which may be used in accordance with the present invention. The ssRNA is a template for a first primer oligonucleotide, which is elongated by reverse transcriptase (RNA-dependent DNA polymerase). The RNA is then removed from the resulting DNA:RNA duplex by the action of ribonuclease H (RNase H, an RNase specific for RNA in duplex with either DNA or RNA). The resultant ssDNA is a template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5' to its homology to the template. This primer is then extended by DNA polymerase (exemplified by the large "Klenow" fragment of E. coli DNA polymerase I), resulting in a double-stranded DNA ("dsDNA") molecule, having a sequence identical to that of the original RNA between the primers and having additionally, at one end, a promoter sequence. This promoter sequence can be used by the appropriate RNA polymerase to make many RNA copies of the DNA. These copies can then re-enter the cycle leading to very swift amplification. With proper choice of enzymes, this amplification can be done isothermally without addition of enzymes at each cycle. Because of the cyclical nature of this process, the starting sequence can be chosen to be in the form of either DNA or RNA.

Miller et al, PCT Application WO 89/06700 (incoφorated herein by reference in its entirety) disclose a nucleic acid sequence amplification scheme based on the hybridization of a promoter/primer sequence to a target single-stranded DNA ("ssDNA") followed by transcription of many RNA copies of the sequence. This scheme is not cyclic, i.e., new templates are not produced from the resultant RNA transcripts. Other amplification methods include "RACE" and "one-sided PCR" (Frohman, M.A., In: PCR PROTOCOLS: A GUIDE TO METHODS AND APPLICATIONS, Academic Press, N.Y., 1990 incoφorated by reference).

Methods based on ligation of two (or more) oligonucleotides in the presence of nucleic acid having the sequence of the resulting "di-oligonucleotide", thereby amplifying the di- oligonucleotide, may also be used in the amplification step of the present invention.

Following any amplification, it may be desirable to separate the amplification product from the template and the excess primer for the purpose of determining whether specific amplification has occurred. In one embodiment, amplification products are separated by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis using standard methods. See Sambrook e/ α/., 1989.

Alternatively, chromatographic techniques may be employed to effect separation. There are many kinds of chromatography which may be used in the present invention: adsorption, partition, ion-exchange and molecular sieve, and many specialized techniques for using them including column, paper, thin-layer and gas chromatography.

Amplification products must be visualized in order to confirm amplification of the marker sequences. One typical visualization method involves staining of a gel with ethidium bromide and visualization under UV light. Alternatively, if the amplification products are integrally labeled with radio- or fluorometrically-labeled nucleotides, the amplification products can then be exposed to x-ray film or visualized under the appropriate stimulating spectra, following separation.

In one embodiment, visualization is achieved indirectly. Following separation of amplification products, a labeled, nucleic acid probe is brought into contact with the amplified marker sequence. The probe preferably is conjugated to a chromophore but may be radiolabeled. In another embodiment, the probe is conjugated to a binding partner, such as an antibody or biotin, and the other member of the binding pair carries a detectable moiety.

In one embodiment, detection is by Southern blotting and hybridization with a labeled probe. The techniques involved in Southern blotting are well known to those of skill in the art and can be found in many standard books on molecular protocols. See Sambrook et al, 1989. Briefly, amplification products are separated by gel electrophoresis. The gel is then contacted with a membrane, such as nitrocellulose, permitting transfer of the nucleic acid and non-covalent binding. Subsequently, the membrane is incubated with a chromophore-conjugated probe that is capable of hybridizing with a target amplification product. Detection is by exposure of the membrane to x-ray film or ion-emitting detection devices.

One example of the foregoing is described in U.S. Patent No. 5,279,721, incorporated by reference herein, which discloses an apparatus and method for the automated electrophoresis and transfer of nucleic acids. The apparatus permits electrophoresis and blotting without external manipulation of the gel and is ideally suited to carrying out methods according to the present invention. All the essential materials and reagents required for detecting wild-type, polymorphic or mutant BARDl or BRCAI binding protein markers in a biological sample may be assembled together in a kit. This generally will comprise preselected primers for specific markers. Also included may be enzymes suitable for amplifying nucleic acids including various polymerases (RT, Taq, etc.), deoxynucleotides and buffers to provide the necessary reaction mixture for amplification.

Such kits generally will comprise, in suitable means, distinct containers for each individual reagent and enzyme as well as for each marker primer pair. Preferred pairs of primers for amplifying nucleic acids are selected to amplify the sequences specified in SEQ ID NO:l, SEQ ID NO:9 through SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO: 126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129 or SEQ ID NO:130.

In another embodiment, such kits will comprise hybridization probes specific for wild-type, polymoφhic or mutant BARDl or for BRCAI binding protein chosen from a group including nucleic acids corresponding to the sequences specified in SEQ ID NO: l , any one of SEQ ID NO:9 through SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129 or SEQ ID NO:130. Such kits generally will comprise, in suitable means, distinct containers for each individual reagent and enzyme as well as for each marker hybridization probe.

3. Other Assays

Other methods for genetic screening to accurately detect mutations in genomic DNA, cDNA or RNA samples may be employed, depending on the specific situation. When screening for mutations in the genomic DNA, it will be preferable to use probes or primers from intronic sequences, such as the intronic sequences disclosed herein for the BARDl gene in SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129 and SEQ ID NO: 130. In particular, mutations which are weakly expressed or are not expressed at all will still be able to be detected in the germline genomic DNA using intronic probes. Additionally, mutations which effect the splice sites of the gene will be able to be detected using intronic sequences, especially, as is the case with the BARDl gene disclosed herein, when the intron/exon borders have been defined. This is the case for each of the eleven exons of the BARDl gene, contained within the genomic contigs disclosed in SEQ ID NO: 122 (exon I, bp 2031-2188), SEQ ID NO: 123 (exon II, bp 2623-2679; exon III, bp 5421-6415), SEQ ID NO: 124 (exon IV, bp 621 -1570), SEQ ID NO: 125 (exon V, bp 451-5318), SEQ ID NO: 126 (exon VI, bp 508-680), SEQ ID NO: 127 (exon VII, bp 548-656), SEQ ID NO: 128 (exon VIII, bp 566-698), SEQ ID NO: 129 (exon IX, bp 226-318), and SEQ ID NO: 130 (exon X, bp 519-616; exon XI, bp 2019-2351).

Historically, a number of different methods have been used to detect point mutations, including denaturing gradient gel electrophoresis ("DGGE"), restriction enzyme polymoiphism analysis, chemical and enzymatic cleavage methods, and others. The more common procedures currently in use include direct sequencing of target regions amplified by PCR (see above) and single-strand conformation polymoφhism analysis ("SSCP").

Another method of screening for point mutations is based on RNase cleavage of base pair mismatches in RNA/DNA and RNA RNA heteroduplexes. As used herein, the term "mismatch" is defined as a region of one or more unpaired or mispaired nucleotides in a double- stranded RNA/RNA, RNA DNA or DNA/DNA molecule. This definition thus includes mismatches due to insertion/deletion mutations, as well as single and multiple base point mutations.

U.S. Patent No. 4,946,773 describes an RNase A mismatch cleavage assay that involves annealing single-stranded DNA or RNA test samples to an RNA probe, and subsequent treatment of the nucleic acid duplexes with RNase A. After the RNase cleavage reaction, the

RNase is inactivated by proteolytic digestion and organic extraction, and the cleavage products are denatured by heating and analyzed by electrophoresis on denaturing polyacrylamide gels. For the detection of mismatches, the single-stranded products of the RNase A treatment, electrophoretically separated according to size, are compared to similarly treated control duplexes. Samples containing smaller fragments (cleavage products) not seen in the control duplex are scored as +.

Currently available RNase mismatch cleavage assays, including those performed according to U.S. Patent No. 4,946,773, require the use of radiolabeled RNA probes. Myers and Maniatis in U.S. Patent No. 4,946,773 describe the detection of base pair mismatches using RNase A. Other investigators have described the use of E. coli enzyme, RNase I, in mismatch assays. Because it has broader cleavage specificity than RNase A, RNase I would be a desirable enzyme to employ in the detection of base pair mismatches if components can be found to decrease the extent of non-specific cleavage and increase the frequency of cleavage of mismatches. The use of RNase I for mismatch detection is described in literature from Promega Biotech. Promega markets a kit containing RNase I that is shown in their literature to cleave three out of four known mismatches, provided the enzyme level is sufficiently high.

The RNase protection assay was first used to detect and map the ends of specific mRNA targets in solution. The assay relies on being able to easily generate high specific activity radiolabeled RNA probes complementary to the mRNA of interest by in vitro transcription. Originally, the templates for in vitro transcription were recombinant plasmids containing bacteriophage promoters. The probes are mixed with total cellular RNA samples to permit hybridization to their complementary targets, then the mixture is treated with RNase to degrade excess unhybridized probe. Also, as originally intended, the RNase used is specific for single- stranded RNA, so that hybridized double-stranded probe is protected from degradation. After inactivation and removal of the RNase, the protected probe (which is proportional in amount to the amount of target mRNA that was present) is recovered and analyzed on a polyacrylamide gel.

The RNase Protection assay was adapted for detection of single base mutations. In this type of RNase A mismatch cleavage assay, radiolabeled RNA probes transcribed in vitro from wild-type sequences, are hybridized to complementary target regions derived from test samples.

The test target generally comprises DNA (either genomic DNA or DNA amplified by cloning in plasmids or by PCR ), although RNA targets (endogenous mRNA) have occasionally been used. If single nucleotide (or greater) sequence differences occur between the hybridized probe and target, the resulting disruption in Watson-Crick hydrogen bonding at that position ("mismatch") can be recognized and cleaved in some cases by single-strand specific ribonuclease. To date, RNase A has been used almost exclusively for cleavage of single-base mismatches, although RNase I has recently been shown as useful also for mismatch cleavage. There are recent descriptions of using the MutS protein and other DNA-repair enzymes for detection of single-base mismatches.

D. Mutagenesis

Site-specific mutagenesis is a technique useful in the preparation of individual peptides, or biologically functional equivalent proteins or peptides, through specific mutagenesis of the underlying DNA. The technique further provides a ready ability to prepare and test sequence variants, incoφorating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA. Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Typically, a primer of about 17 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered.

In general, the technique of site-specific mutagenesis is well known in the art. As will be appreciated, the technique typically employs a bacteriophage vector that exists in both a single stranded and double stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the Ml 3 phage. These phage vectors are commercially available and their use is generally well known to those skilled in the art. Double stranded plasmids are also routinely employed in site directed mutagenesis, which eliminates the step of transferring the gene of interest from a phage to a plasmid. In general, site-directed mutagenesis is performed by first obtaining a single-stranded vector, or melting of two strands of a double stranded vector which includes within its sequence a DNA sequence encoding the desired protein. An oligonucleotide primer bearing the desired mutated sequence is synthetically prepared. This primer is then annealed with the single- stranded DNA preparation, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected that include recombinant vectors bearing the mutated sequence arrangement.

The preparation of sequence variants of the selected gene using site-directed mutagenesis is provided as a means of producing potentially useful species and is not meant to be limiting, as there are other ways in which sequence variants of genes may be obtained. For example, recombinant vectors encoding the desired gene may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.

II. BARDl and BRCAI Binding Proteins and Peptides

In addition to its ability to bind BRCAI in vivo and in vitro, BARDl shares sequence homology with the two most conserved regions of BRCAI - the amino-terminal RING motif and the carboxy-terminal BRCT domains. Although the functional properties of the RING domain have not been clearly defined, this motif is found in a variety of proteins that regulate cell growth, including the products of tumor suppressor genes and dominant proto-oncogenes (Saurin et al, 1996).

Several different subgroups of RING proteins are now recognized. The largest of these, which includes BRCAI, features an isolated RING domain that typically resides near the amino- terminus. In other proteins, however, the RING domain forms one element of a tripartite motif that also contains a distinct zinc-binding domain (the B box) and a potential α-helical coiled- coiled sequence. The RING domain of BARDl is not found in association with a B-box or coiled-coiled sequence, and in this respect it resembles the isolated RING motif encoded by BRCAI. On the other hand, BARDl may represent a novel subgroup within the RING protein family as it is the only known member which contains ankyrin repeats.

Ankyrin repeats are found in a broad spectrum of functionally diverse proteins, and in some instances they have been implicated as sites of highly specific protein-protein interaction (Murre et al, 1989). Although the ankyrin sequences of BARDl may serve a similar function, this invention indicates that they are not required for binding to BRCAI . Instead, the sequences of BARDl and BRCAI that mediate their association appear to reside within or nearby their respective RING motifs.

The present invention shows that the ability to interact with BRCAI was retained by a segment of BARDl (residues 26-142) that includes its RING motif (residues 46-90) but lacks the ankyrin repeats (residues 427-525). Likewise, the interacting sequences of BRCAI were localized to the amino-terminal 101 residues, a segment of the protein that also encompasses the RING motif (residues 20-68).

It has been proposed that one possible function of the RING domain would be to provide a surface for protein-protein interactions (Saurin et al, 1996). ln support of this notion, BARDl does not interact with BRCAI polypeptides that have substitutions of amino acids C61 or C64 (FIG. 5A and FIG. 5B), two of the conserved cysteine residues in the RING domain that presumably participate in zinc coordination. This suggests that BARDl /BRCAI association is mediated, at least in part, by the RING domain of BRCAI . The results are also consistent with a direct heteromeric interaction between the RING domains of BRCAI and BARDl, although other examples of RING/RING dimerization have not yet been described (Saurin et al, 1996).

The minimal segment of BRCAI that successfully bound BARDl was comprised of residues 1-101. However, a smaller BRCAI segment (residues 1-71) did not interact with BARDl despite the fact that it also includes the intact RING motif (residues 20-68). Thus, BARDl binding may require multiple points of contact on BRCAI, including sequences within the BRCA 1 RING domain and sequences on its carboxy-terminal flank ( . e. , residues 72- 101 ). In any event, BRCAl/BARDl association appears to be highly specific. The yeast two- hybrid screens with the RING sequences of BRCAI and BARDl have not uncovered additional interacting RING proteins, and direct assays of binding between BRCAI or BARDl and select members of the RING family have also failed to show evidence of other RING/RING interactions.

A surprising feature of BARDl is its homology with sequences that lie near the carboxy- terminus of BRCAI . Comparisons of the mouse and human counterparts of BRCAI have established that this sequence is especially well conserved from an evolutionary standpoint, and the existence of a homologous sequence within BARDl suggests that it constitutes a discrete amino acid motif with an important but as yet unknown function.

Recently, Koonin et al. (1996) reported that this region of BRCAI is homologous to sequences that reside near the carboxy-termini of the mammalian 53BP1 and yeast RAD9 proteins. Moreover, they also showed that the conserved sequences includes two tandem copies of a novel protein motif - the BRCAI carboxy-terminal ("BRCT") domain. The function of this motif is not known. Significantly, however, the majority of tumorigenic BRCAI lesions associated with familial breast cancer result in mutation or deletion of one or both BRCT domains. Thus, these motifs are likely to play a crucial role in BRCA 1 -mediated tumor suppression. In view of the fact that BRCAI and BARDl form a stable complex in vivo, it is proposed that the tumor suppressor function of BRCAI is mediated by the combined activities of the BRCT motifs from both proteins.

The present invention therefore provides purified, and in preferred embodiments, substantially purified, BARDl and BRCAI binding proteins and peptides. The term "purified BARDl and BRCAI binding protein or peptide" as used herein, is intended to refer to a wild-type, polymoφhic or mutant BARDl, or other BRCAI binding proteinaceous composition, isolatable from mammalian cells or recombinant host cells, wherein the wild-type, polymorphic or mutant BARDl or BRCAI binding protein or peptide is purified to any degree relative to its naturally-obtainable state, i.e., relative to its purity within a cellular extract. A purified wild-type, polymoφhic or mutant BARDl or BRCAI binding protein or peptide therefore also refers to a wild-type, polymorphic or mutant BARDl or BRCAI binding protein or peptide free from the environment in which it naturally occurs.

Wild-type, polymoφhic or mutant BARDl proteins may be full length proteins, such as being 777, 770 or 752 amino acids in length. Wild-type, polymoφhic or mutant BARDl proteins, polypeptides and peptides may also be less then full length proteins, such as individual domains, regions or even epitopic peptides. Where less than full length wild-type, polymorphic or mutant BARDl proteins are concerned the most preferred will be those containing predicted immunogenic sites and those containing the functional domains identified herein.

For example, wild-type, polymoφhic or mutant BARDl protein domains consisting essentially of an amino-terminal RING motif or domain; an ankyrin repeat region or regions; or a carboxy-terminal BRCT domain or domains may be prepared. Preferred wild-type, polymoφhic or mutant BARDl protein domains or fragments will be those sufficient to bind to BRCAI, as exemplified by a BRCAI binding domain that comprises the sequence of residues 26-142 from SEQ ID NO:2, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO.31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37 or SEQ ID NO:39, and which binds to the BRCAI protein.

Generally, "purified" will refer to a wild-type, polymoφhic or mutant BARDl or

BRCAI binding protein or peptide composition that has been subjected to fractionation to remove various non-wild-type, polymoφhic or mutant BARDl or BRCAI binding protein or peptide components, and which composition substantially retains its wild-type, polymorphic or mutant BARDl or BRCAI binding activity, as may be assessed by binding to BRCAI and forming complexes with BRCAI .

Where the term "substantially purified" is used, this will refer to a composition in which the wild-type, polymorphic or mutant BARDl or BRCAI binding protein or peptide forms the major component of the composition, such as constituting about 50% of the proteins in the composition or more. In preferred embodiments, a substantially purified protein will constitute more than 60%, 70%, 80%, 90%, 95%, 99% or even more of the proteins in the composition. A polypeptide or protein that is "purified to homogeneity," as applied to the present invention, means that the polypeptide or protein has a level of purity where the polypeptide or protein is substantially free from other proteins and biological components. For example, a purified polypeptide or protein will often be sufficiently free of other protein components so that degradative sequencing may be performed successfully.

Various methods for quantifying the degree of purification of wild-type, polymorphic or mutant BARDl or BRCAI binding proteins or peptides will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific BRCAI binding activity of a fraction, or assessing the number of polypeptides within a fraction by gel electrophoresis. Assessing the number of polypeptides within a fraction by SDS/PAGE analysis will often be preferred in the context of the present invention as this is straightforward.

To purify a wild-type, polymoφhic or mutant BARDl or BRCAI binding protein or peptide a natural or recombinant composition comprising at least some wild-type, polymorphic or mutant BARDl or BRCAI binding proteins or peptides will be subjected to fractionation to remove various non-wild-type, polymoφhic or mutant BARDl or BRCAI binding components from the composition. Various techniques suitable for use in protein purification will be well known to those of skill in the art. These include, for example, precipitation with ammonium sulfate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite, lectin affinity and other affinity chromatography steps; isoelectric focusing; gel electrophoresis; and combinations of such and other techniques.

A specific example presented herein is the purification of a BARDl fusion protein using a specific binding partner. Such purification methods are routine in the art. As the present invention provides DNA sequences for BARDl proteins, any fusion protein purification method can now be practiced. This is currently exemplified by the generation of a BARDl -glutathione S-transferase fusion protein, expression in E. coli, and isolation to homogeneity using affinity chromatography on glutathione-agarose. The exemplary purification method disclosed herein represents one method to prepare a substantially purified wild-type, polymoφhic or mutant BARDl or BRCAI binding protein or peptide. This method is preferred as it results in the substantial purification of the wild-type, polymoφhic or mutant BARDl or BRCAI binding protein or peptide in yields sufficient for further characterization and use. However, given the DNA and proteins provided by the present invention, any purification method can now be employed.

Although preferred for use in certain embodiments, there is no general requirement that the wild-type, polymoφhic or mutant BARDl or BRCAI binding protein or peptide always be provided in their most purified state. Indeed, it is contemplated that less substantially purified wild-type, polymoφhic or mutant BARDl or BRCAI binding proteins or peptides, which are nonetheless enriched in wild-type, polymoφhic or mutant BARDl or BRCAI binding protein compositions, relative to the natural state, will have utility in certain embodiments. These include, for example, binding to BRCAI, as may be used to purify BRCAI ; and antibody generation where subsequent screening assays using purified wild-type, polymorphic or mutant BARDl or BRCAI binding proteins are conducted.

Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein. Inactive products also have utility in certain embodiments, such as, e.g., in antibody generation.

III. Antibodies to BARDl and Other BRCAI Binding Proteins

A. Epitopic Core Sequences

Peptides corresponding to one or more antigenic determinants, or "epitopic core regions", of wild-type, polymoφhic or mutant BARDl and the other BRCAI -binding proteins of the present invention can also be prepared. Such peptides should generally be at least five or six amino acid residues in length, will preferably be about 10, 15, 20, 25 or about 30 amino acid residues in length, and may contain up to about 35-50 residues or so. Synthetic peptides will generally be about 35 residues long, which is the approximate upper length limit of automated peptide synthesis machines, such as those available from Applied Biosystems (Foster City, CA). Longer peptides may also be prepared, e.g., by recombinant means.

U.S. Patent 4,554,101, (Hopp) incorporated herein by reference, teaches the identification and preparation of epitopes from primary amino acid sequences on the basis of hydrophilicity. Through the methods disclosed in Hopp, one of skill in the art would be able to identify epitopes from within an amino acid sequence such as the wild-type, polymorphic or mutant BARDl sequences disclosed herein (SEQ ID NO:2, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31 , SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37 or SEQ ID NO:39) and the other BRCAI -binding proteins encoded by the isolated nucleic acid sequences of SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: l 1, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44 or SEQ ID NO:46.

Numerous scientific publications have also been devoted to the prediction of secondary structure, and to the identification of epitopes, from analyses of amino acid sequences (Chou & Fasman, 1974a,b; 1978a,b, 1979). Any of these may be used, if desired, to supplement the teachings of Hopp in U.S. Patent 4,554,101.

Moreover, computer programs are currently available to assist with predicting antigenic portions and epitopic core regions of proteins. Examples include those programs based upon the Jameson- Wolf analysis (Jameson & Wolf, 1998; Wolf et al., 1988), the program PepPlot® (Brutlag et al, 1990; Weinberger et al, 1985), and other new programs for protein tertiary structure prediction (Fetrow & Bryant, 1993). Further commercially available software capable of carrying out such analyses is termed MacVector (IBI, New Haven, CT).

In further embodiments, major antigenic determinants of a polypeptide may be identified by an empirical approach in which portions of the gene encoding the polypeptide are expressed in a recombinant host, and the resulting proteins tested for their ability to elicit an immune response. For example, PCR can be used to prepare a range of peptides lacking successively longer fragments of the C-terminus of the protein. The immunoactivity of each of these peptides is determined to identify those fragments or domains of the polypeptide that are immunodominant. Further studies in which only a small number of amino acids are removed at each iteration then allows the location of the antigenic determinants of the polypeptide to be more precisely determined.

Another method for determining the major antigenic determinants of a polypeptide is the SPOTs™ system (Genosys Biotechnologies, Inc., The Woodlands, TX). In this method, overlapping peptides are synthesized on a cellulose membrane, which following synthesis and deprotection, is screened using a polyclonal or monoclonal antibody. The antigenic determinants of the peptides which are initially identified can be further localized by performing subsequent syntheses of smaller peptides with larger overlaps, and by eventually replacing individual amino acids at each position along the immunoreactive peptide.

Once one or more such analyses are completed, polypeptides are prepared that contain at least the essential features of one or more antigenic determinants. The peptides are then employed in the generation of antisera against the polypeptide. Minigenes or gene fusions encoding these determinants can also be constructed and inserted into expression vectors by standard methods, for example, using PCR cloning methodology.

The use of such small peptides for vaccination typically requires conjugation of the peptide to an immunogenic carrier protein, such as hepatitis B surface antigen, keyhole limpet hemocyanin or bovine serum albumin. Methods for performing this conjugation are well known in the art.

B. Antibody Generation

In certain embodiments, the present invention provides antibodies that bind with high specificity to wild-type, polymcφhic or mutant BARDl, and other BRCAI binding proteins provided herein. Thus, antibodies that bind to the protein products of the isolated nucleic acid sequences of SEQ ID NO:l, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: l l, SEQ ID NO: 12,

SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44 or SEQ ID NO:46 are provided. Antibodies specific for the wild-type and polymoφhic proteins and peptides and those specific for any one of a number of mutants are provided. As detailed above, in addition to antibodies generated against the full length proteins, antibodies may also be generated in response to smaller constructs comprising epitopic core regions, including wild-type, polymorphic and mutant epitopes.

As used herein, the term "antibody" is intended to refer broadly to any immunologic binding agent such as IgG, IgM, IgA, IgD and IgE. Generally, IgG and/or IgM are preferred because they are the most common antibodies in the physiological situation and because they arc most easily made in a laboratory setting.

Monoclonal antibodies (MAbs) are recognized to have certain advantages, e.g., reproducibility and large-scale production, and their use is generally preferred. The invention thus provides monoclonal antibodies of the human, murine, monkey, rat, hamster, rabbit and even chicken origin. Due to the ease of preparation and ready availability of reagents, murine monoclonal antibodies will often be preferred.

However, "humanized" antibodies are also contemplated, as are chimeric antibodies from mouse, rat, or other species, bearing human constant and/or variable region domains, bispecific antibodies, recombinant and engineered antibodies and fragments thereof. Methods for the development of antibodies that are "custom-tailored" to the patient's tumor are likewise known and such custom-tailored antibodies are also contemplated.

The term "antibody" is used to refer to any antibody-like molecule that has an antigen binding region, and includes antibody fragments such as Fab', Fab, F(ab')₂, single domain antibodies (DABs), Fv, scFv (single chain Fv), and the like. The techniques for preparing and using various antibody-based constructs and fragments are well known in the art. Means for preparing and characterizing antibodies are well known in the art (See, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; incorporated herein by reference).

The methods for generating monoclonal antibodies (MAbs) generally begin along the same lines as those for preparing polyclonal antibodies. Briefly, a polyclonal antibody is prepared by immunizing an animal with an immunogenic wild-type, polymoφhic or mutant BARDl or other BRCAI binding protein composition in accordance with the present invention and collecting antisera from that immunized animal.

A wide range of animal species can be used for the production of antisera. Typically the animal used for production of anti-antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or a goat. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.

As is well known in the art, a given composition may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier. Exemplary and preferred carriers arc keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers. Means for conjugating a polypeptide to a carrier protein arc well known in the art and include glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimide and bis- biazotized benzidine.

As is also well known in the art, the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Suitable adjuvants include all acceptable immunostimulatory compounds, such as cytokines, toxins or synthetic compositions.

Adjuvants that may be used include IL-1, IL-2, IL-4, IL-7, IL-12, g-interferon, GMCSP,

BCG, aluminum hydroxide, MDP compounds, such as thur-MDP and nor-MDP, CGP (MTP- PE), lipid A, and monophosphoryl lipid A (MPL). RIBI, which contains three components extracted from bacteria, MPL, trehalose dimycolate (TDM) and cell wall skeleton (CWS) in a 2% squalene/Tween 80 emulsion. MHC antigens may even be used.

Exemplary, often preferred adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant.

In addition to adjuvants, it may be desirable to coad inister biologic response modifiers (BRM), which have been shown to upregulate T cell immunity or downregulate suppressor cell activity. Such BRMs include, but are not limited to, Cimetidine (CIM; 1200 mg/d) (Smith/Kline, PA); or low-dose Cyclophosphamide (CYP; 300 mg/m ) (Johnson/ Mead, NJ) and Cytokines such as γ-interferon, IL-2, or IL-12 or genes encoding proteins involved in immune helper functions, such as B-7.

The amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization. A variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization.

A second, booster injection, may also be given. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate MAbs.

For production of rabbit polyclonal antibodies, the animal can be bled through an ear vein or alternatively by cardiac puncture. The removed blood is allowed to coagulate and then centrifuged to separate serum components from whole cells and blood clots. The serum may be used as is for various applications or else the desired antibody fraction may be purified by well- known methods, such as affinity chromatography using another antibody, a peptide bound to a solid matrix, or by using, e.g., protein A or protein G chromatography. MAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Patent 4,196,265, incorporated herein by reference. Typically, this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified wild-type, polymorphic or mutant BARDl, and other BRCAI binding protein, polypeptide, peptide or domain, be it a wild-type or mutant composition. The immunizing composition is administered in a manner effective to stimulate antibody producing cells.

The methods for generating monoclonal antibodies (MAbs) generally begin along the same lines as those for preparing polyclonal antibodies. Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep frog cells is also possible. The use of rats may provide certain advantages (Goding, 1986, pp. 60-61), but mice are preferred, with the BALB/c mouse being most preferred as this is most routinely used and generally gives a higher percentage of stable fusions.

The animals are injected with antigen, generally as described above. The antigen may be coupled to carrier molecules such as keyhole limpet hemocyanin if necessary. The antigen would typically be mixed with adjuvant, such as Freund's complete or incomplete adjuvant. Booster injections with the same antigen would occur at approximately two-week intervals.

Following immunization, somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the MAb generating protocol. These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible.

Often, a panel of animals will have been immunized and the spleen of animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe. Typically, a spleen from an immunized mouse contains approximately 5 x 10⁷ to 2 x 10⁸ lymphocytes. The antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas).

Any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, pp. 65-66, 1986; Campbell, pp. 75-83, 1984). cites). For example, where the immunized animal is a mouse, one may use P3-X63/Ag8, X63-Ag8.653, NS l/l .Ag 4 1 , Sp210-Agl4, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bui; for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with human cell fusions.

One preferred murine myeloma cell is the NS-1 myeloma cell line (also termed P3-NS-1- Ag4-1), which is readily available from the NIGMS Human Genetic Mutant Cell Repository by requesting cell line repository number GM3573. Another mouse myeloma cell line that may be used is the 8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cell line.

Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2: 1 proportion, though the proportion may vary from about 20: 1 to about 1 :1 , respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Fusion methods using Sendai virus have been described by Kohler and Milstein (1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, by Gefter et al. (1977). The use of electrically induced fusion methods is also appropriate (Goding pp. 71-74, 1986).

Fusion procedures usually produce viable hybrids at low frequencies, about 1 x 10^" to 1 x 10^" . However, this does not pose a problem, as the viable, fused hybrids are differentiated from the parental, unfused cells (particularly the unfused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium. The selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis. Where aminopterin or methotrexate is used, the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. The B cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B cells.

This culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity. The assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.

The selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide MAbs. The cell lines may be exploited for MAb production in two basic ways.

A sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion (e.g., a syngeneic mouse). Optionally, the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection. The injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can then be tapped to provide MAbs in high concentration. The individual cell lines could also be cultured in vitro, where the MAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations.

MAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography. Fragments of the monoclonal antibodies of the invention can be obtained from the monoclonal antibodies so produced by methods which include digestion with enzymes, such as pepsin or papain, and/or by cleavage of disulfide bonds by chemical reduction. Alternatively, monoclonal antibody fragments encompassed by the present invention can be synthesized using an automated peptide synthesizer.

It is also contemplated that a molecular cloning approach may be used to generate monoclonals. For this, combinatorial immunoglobulin phagemid libraries are prepared from RNA isolated from the spleen of the immunized animal, and phagemids expressing appropriate antibodies are selected by panning using cells expressing the antigen and control cells. The advantages of this approach over conventional hybridoma techniques are that approximately 10 times as many antibodies can be produced and screened in a single round, and that new specificities are generated by H and L chain combination which further increases the chance of finding appropriate antibodies.

Alternatively, monoclonal antibody fragments encompassed by the present invention can be synthesized using an automated peptide synthesizer, or by expression of full-length gene or of gene fragments in E. coli.

C. Antibody Conjugates

The present invention further provides antibodies against wild-type, polymorphic or mutant BARDl, and other BRCAI binding proteins, generally of the monoclonal type, that are linked to one or more other agents to form an antibody conjugate. Any antibody of sufficient selectivity, specificity and affinity may be employed as the basis for an antibody conjugate. Such properties may be evaluated using conventional immunological screening methodology known to those of skill in the art.

Certain examples of antibody conjugates are those conjugates in which the antibody is linked to a detectable label. "Detectable labels" are compounds or elements that can be detected due to their specific functional properties, or chemical characteristics, the use of which allows the antibody to which they are attached to be detected, and further quantified if desired. Another such example is the formation of a conjugate comprising an antibody linked to a cytotoxic or anti-cellular agent, as may be termed "immunotoxins". In the context of the present invention, immunotoxins are generally less preferred.

Antibody conjugates are thus preferred for use as diagnostic agents. Antibody diagnostics generally fall within two classes, those for use in in vitro diagnostics, such as in a variety of immunoassays, and those for use in vivo diagnostic protocols, generally known as "antibody-directed imaging". Again, antibody-directed imaging is less preferred for use with this invention.

Many appropriate imaging agents are known in the art, as are methods for their attachment to antibodies (see, e.g., U.S. patents 5,021,236 and 4,472,509, both incorporated herein by reference). Certain attachment methods involve the use of a metal chelate complex employing, for example, an organic chelating agent such a DTPA attached to the antibody (U.S. Patent 4,472,509). Monoclonal antibodies may also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with an isothiocyanate.

In the case of paramagnetic ions, one might mention by way of example ions such as chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and erbium (III), with gadolinium being particularly preferred.

Ions useful in other contexts, such as X-ray imaging, include but are not limited to lanthanum (III), gold (III), lead (II), and especially bismuth (III). In the case of radioactive isotopes for therapeutic and/or diagnostic application, one might mention astatine ", ^Hcarbon, chromium, 'chlorine, cobalt, cobalt, copper' , Eu, gallium⁶⁷, ³hydrogen, iodine¹ , iodine , iodine , indium ' , iron, phosphorus, rhenium ⁶, rhenium , selenium, sulphur, technicium and yttrium . I is often being preferred for use in certain embodiments, and technicium '" and indium are also often preferred due to their low energy and suitability for long range detection.

Radioactively labeled monoclonal antibodies of the present invention may be produced according to well-known methods in the art. For instance, monoclonal antibodies can be iodinated by contact with sodium or potassium iodide and a chemical oxidizing agent such as sodium hypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase. Monoclonal antibodies according to the invention may be labeled with technelium- m by ligand exchange process, for example, by reducing pertechnatc with stannous solution, chelating the reduced technetium onto a Sephadex column and applying the antibody to this column or by direct labeling techniques, e.g., by incubating pertechnate, a reducing agent such as SNC1₂, a buffer solution such as sodium-potassium phthalate solution, and the antibody.

Intermediary functional groups which are often used to bind radioisotopes which exist as metallic ions to antibody are diethylenetriaminepentaacetic acid (DTPA) and ethylene diaminetetracetic acid (EDTA).

Fluorescent labels include rhodamine, fluorescein isothiocyanate and renographin.

The much preferred antibody conjugates of the present invention are those intended primarily for use in vitro, where the antibody is linked to a secondary binding ligand or to an enzyme (an enzyme tag) that will generate a colored product upon contact with a chromogenic substrate. Examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase and glucose oxidase. Preferred secondary binding ligands are biotin and avidin or sfreptavidin compounds. The use of such labels is well known to those of skill in the art in light and is described, for example, in U.S. Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241; each incoφorated herein by reference. D. Immunodetection Methods

In still further embodiments, the present invention concerns immunodetection methods for binding, purifying, removing, quantifying or otherwise generally detecting biological components such as wild-type, polymoφhic or mutant BARDl , and other BRCA I binding protein components. The wild-type, polymorphic or mutant BARDl, or other BRCAI binding proteins or peptides of the present invention may be employed to detect and purify BRCAI, and antibodies prepared in accordance with the present invention, may be employed to detect wild-type, polymoφhic or mutant BARDl, or other BRCAI binding proteins or peptides. As described throughout the present application, the use of wild-type, polymorphic and mutant specific antibodies is contemplated. The steps of various useful immunodetection methods have been described in the scientific literature, such as, e.g., Nakamura et al. (1987), incorporated herein by reference.

In general, the immunobinding methods include obtaining a sample suspected of containing a wild-type, polymorphic or mutant BARDl , or other BRCAI binding protein or peptide, and contacting the sample with a first anti-wild-type, polymoφhic or mutant BARDl, or BRCAI binding protein antibody in accordance with the present invention, as the case may be, under conditions effective to allow the formation of immunocomplexes.

These methods include methods for purifying wild-type, polymorphic or mutant

BARDl, or other BRCAI binding protein, as may be employed in purifiying wild-type, polymoφhic or mutant BARDl, or other BRCAI binding protein from patients' samples or for purifying recombinantly expressed wild-type, polymorphic or mutant BARDl , or other BRCAI binding protein. In these instances, the antibody removes the antigenic wild-type, polymorphic or mutant BARDl, or other BRCAI binding protein component from a sample. The antibody

•» will preferably be linked to a solid support, such as in the form of a column matrix, and the sample suspected of containing the wild-type, polymoφhic or mutant BARDl, or other BRCAI binding protein antigenic component will be applied to the immobilized antibody. The unwanted components will be washed from the column, leaving the antigen immunocomplexed to the immobilized antibody, which wild-type, polymorphic or mutant BARDl , or other BRCAI binding protein antigen is then collected by removing the wild-type, polymorphic or mutant BARDl, or other BRCAI binding protein from the column.

The immunobinding methods also include methods for detecting or quantifying the amount of a wild-type, polymoφhic or mutant BARDl , or other BRCAI binding protein reactive component in a sample, which methods require the detection or quantification of any immune complexes formed during the binding process. Here, one would obtain a sample suspected of containing a wild-type, polymorphic or mutant BARDl, or other BRCAI binding protein or peptide, and contact the sample with an antibody against wild-type, polymorphic or mutant BARDl, or other BRCAI binding protein, and then detect or quantify the amount of immune complexes formed under the specific conditions.

In terms of antigen detection, the biological sample analyzed may be any sample that is suspected of containing a wild-type, polymoφhic or mutant BARDl, or other BRCAI binding protein-specific antigen, such as a breast, ovarian or uterine cancer tissue section or specimen, a homogenized breast, ovarian or uterine cancer tissue extract, a breast, ovarian or uterine cancer cell, separated or purified forms of any of the above wild-type, polymoφhic or mutant BARDl, or other BRCAI binding protein-containing compositions, or even any biological fluid that comes into contact with breast, ovarian or uterine cancer tissue, including blood and serum, although tissue samples and extracts are preferred.

Contacting the chosen biological sample with the antibody under conditions effective and for a period of time sufficient to allow the formation of immune complexes (primary immune complexes) is generally a matter of simply adding the antibody composition to the sample and incubating the mixture for a period of time lone enough for the antibodies to form immune complexes with, i.e., to bind to, any wild-type, polymorphic or mutant BARDl , or other BRCAI binding protein antigens present. After this time, the sample-antibody composition, such as a tissue section, ELISA plate, dot blot or western blot, will generally be washed to remove any non-specifically bound antibody species, allowing only those antibodies specifically bound within the primary immune complexes to be detected. In general, the detection of immunocomplex formation is well known in the art and may be achieved through the application of numerous approaches. These methods are generally based upon the detection of a label or marker, such as any of those radioactive, fluorescent, biological or enzymatic tags. U.S. Patents concerning the use of such labels include 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241 , each incorporated herein by reference. Of course, one may find additional advantages through the use of a secondary binding ligand such as a second antibody or a biotin/avidin ligand binding arrangement, as is known in the art.

The wild-type, polymoφhic or mutant BARDl , or other BRCAI binding protein antibody employed in the detection may itself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount of the primary immune complexes in the composition to be determined.

Alternatively, the first antibody that becomes bound within the primary immune complexes may be detected by means of a second binding ligand that has binding affinity for the antibody. In these cases, the second binding ligand may be linked to a detectable label. The second binding ligand is itself often an antibody, which may thus be termed a "secondary" antibody. The primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under conditions effective and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes are then generally washed to remove any non-specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected.

Further methods include the detection of primary immune complexes by a two step approach. A second binding ligand, such as an antibody, that has binding affinity for the antibody is used to form secondary immune complexes, as described above. After washing, the secondary immune complexes are contacted with a third binding ligand or antibody that has binding affinity for the second antibody, again under conditions effective and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes). The third ligand or antibody is linked to a detectable label, allowing detection of the tertiary immune complexes thus formed. This system may provide for signal amplification if this is desired. The immunodetection methods of the present invention have evident utility in the diagnosis or prognosis of conditions such as breast, ovarian, uterine and other forms of cancer. Here, a biological or clinical sample suspected of containing a wild-type, polymorphic or mutant BARDl, or other BRCAI binding protein, peptide or mutant is used. However, these embodiments also have applications to non-clinical samples, such as in the titering of antigen or antibody samples, in the selection of hybridomas, and the like.

In the clinical diagnosis or monitoring of patients with breast, ovarian, uterine and other forms of cancer, the detection of a BARDl or BRCAI binding protein mutant, or an alteration in the levels of BARDl or BRCAI binding protein, in comparison to the levels in a corresponding biological sample from a normal subject is indicative of a patient with breast, ovarian, uterine or another form of cancer.

However, as is known to those of skill in the art, such a clinical diagnosis would not necessarily be made on the basis of this method in isolation. Those of skill in the art are very familiar with differentiating between significant differences in types or amounts of biomarkers, which represent a positive identification, and low level or background changes of biomarkers. Indeed, background expression levels are often used to form a "cut-off above which increased detection will be scored as significant or positive.

1. ELISAs

As detailed above, immunoassays, in their most simple and direct sense, are binding assays. Certain preferred immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily appreciated that detection is not limited to such techniques, and Western blotting, dot blotting, FACS analyses, and the like may also be used.

In one exemplary ELISA, the anti-wild-type, polymoφhic or mutant BARDl, or other BRCAI binding protein antibodies of the invention are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition suspected of containing the wild-type, polymorphic or mutant BARDl, or other BRCAI binding protein antigen, such as a clinical sample, is added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound wild-type, polymoφhic or mutant BARDl, or other BRCAI binding protein antigen may be detected. Detection is generally achieved by the addition of another anti-wild-type, polymoφhic or mutant BARDl, or other BRCAI binding protein antibody that is linked to a detectable label. This type of ELISA is a simple "sandwich ELISA". Detection may also be achieved by the addition of a second anti-wild-type, polymorphic or mutant BARDl, or other BRC I binding protein antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.

In another exemplary ELISA, the samples suspected of containing the wild-type, polymoφhic or mutant BARDl , or other BRCAI binding protein antigen are immobilized onto the well surface and then contacted with the anti-wild-type, polymorphic or mutant BARDl, or other BRCAI binding protein antibodies of the invention. After binding and washing to remove non-specifically bound immune complexes, the bound anti-wild-type, polymorphic or mutant BARDl, or other BRCAI binding protein antibodies are detected. Where the initial anti- wild-type, polymoφhic or mutant BARDl , or other BRCAI binding protein antibodies are linked to a detectable label, the immune complexes may be detected directly. Again, the immune complexes may be detected using a second antibody that has binding affinity for the first anti-wild-type, polymorphic or mutant BARDl, or other BRCAI binding protein antibody, with the second antibody being linked to a detectable label.

Another ELISA in which the wild-type, polymoφhic or mutant BARDl, or other

BRCAI binding proteins or peptides are immobilized, involves the use of antibody competition in the detection. In this ELISA, labeled antibodies against wild-type, polymoφhic or mutant BARDl, or other BRCAI binding protein are added to the wells, allowed to bind, and detected by means of their label. The amount of wild-type, polymorphic or mutant BARDl, or other BRCAI binding protein antigen in an unknown sample is then determined by mixing the sample with the labeled antibodies against wild-type, polymoφhic or mutant BARDl, or other BRCAI binding protein before or during incubation with coated wells. The presence of wild-type, polymoφhic or mutant BARDl, or other BRCAI binding protein in the sample acts to reduce the amount of antibody against wild-type, polymorphic or mutant BARDl, or other BRCAI binding protein available for binding to the well and thus reduces the ultimate signal. This is also appropriate for detecting antibodies against wild-type, polymoφhic or mutant BARDl , or other BRCAI binding protein in an unknown sample, where the unlabeled antibodies bind to the antigen-coated wells and also reduces the amount of antigen available to bind the labeled antibodies.

Irrespective of the format employed, ELISAs have certain features in common, such as coating, incubating or binding, washing to remove non-specifically bound species, and detecting the bound immune complexes. These are described as follows:

In coating a plate with either antigen or antibody, one will generally incubate the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period of hours. The wells of the plate will then be washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells are then "coated" with a nonspecific protein that is antigenically neutral with regard to the test antisera. These include bovine serum albumin (BSA), casein and solutions of milk powder. The coating allows for blocking of nonspecific adsoφtion sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.

In ELISAs, it is probably more customary to use a secondary or tertiary detection means rather than a direct procedure. Thus, after binding of a protein or antibody to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the biological sample to be tested under conditions effective to allow immune complex (antigen/antibody) formation. Detection of the immune complex then requires a labeled secondary binding ligand or antibody, or a secondary binding ligand or antibody in conjunction with a labeled tertiary antibody or third binding ligand.

"Under conditions effective to allow immune complex (antigen/antibody) formation" means that the conditions preferably include diluting the antigens and antibodies with solutions such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Twecn. These added agents also tend to assist in the reduction of nonspecific background.

The "suitable" conditions also mean that the incubation is at a temperature and for a period of time sufficient to allow effective binding. Incubation steps are typically from about

1 to 2 to 4 hours, at temperatures preferably on the order of 25°C to 27°C, or may be overnight at about 4°C or so.

Following all incubation steps in an ELISA, the contacted surface is washed so as to remove non-complexed material. A preferred washing procedure includes washing with a solution such as PBS/Tween, or borate buffer. Following the formation of specific immune complexes between the test sample and the originally bound material, and subsequent washing, the occurrence of even minute amounts of immune complexes may be determined.

To provide a detecting means, the second or third antibody will have an associated label to allow detection. Preferably, this will be an enzyme that will generate color development upon incubating with an appropriate chromogenic substrate. Thus, for example, one will desire to contact and incubate the first or second immune complex with a urease, glucose oxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibody for a period of time and under conditions that favor the development of further immune complex formation (e.g., incubation for

2 hours at room temperature in a PBS-containing solution such as PBS-Tween).

After incubation with the labeled antibody, and subsequent to washing to remove unbound material, the amount of label is quantified, e.g. , by incubation with a chromogenic substrate such as urea and bromocresol purple or 2,2'-azino-di-(3-ethyl-benzthiazoIine-6- sulfonic acid [ABTS] and H₂O₂, in the case of peroxidase as the enzyme label. Quantification is then achieved by measuring the degree of color generation, e.g., using a visible spectra spectrophotometer. 2. Immunohistochemistry

The antibodies of the present invention may also be used in conjunction with both fresh- frozen and formalin-fixed, paraffin-embedded tissue blocks prepared for study by immunohistochemistry (IHC). For example, each tissue block consists of 50 mg of residual "pulverized" diabetic tissue. The method of preparing tissue blocks from these particulate specimens has been successfully used in previous IHC studies of various prognostic factors, and is well known to those of skill in the art (Brown et al, 1990; Abbondanzo et al, 1990; Allred e/ α/., 1990).

Briefly, frozen-sections may be prepared by rehydrating 50 ng of frozen "pulverized" diabetic tissue at room temperature in phosphate buffered saline (PBS) in small plastic capsules; pelleting the particles by centrifugation; resuspending them in a viscous embedding medium (OCT); inverting the capsule and pelleting again by centrifugation; snap-freezing in -70°C isopentane; cutting the plastic capsule and removing the frozen cylinder of tissue; securing the tissue cylinder on a cryostat microtome chuck; and cutting 25-50 serial sections.

Permanent-sections may be prepared by a similar method involving rehydration of the 50 mg sample in a plastic microfuge tube; pelleting; resuspending in 10% formalin for 4 hours fixation; washing/pelleting; resuspending in warm 2.5% agar; pelleting; cooling in ice water to harden the agar; removing the tissue/agar block from the tube; infiltrating and embedding the block in paraffin; and cutting up to 50 serial permanent sections.

E. Immunodetection Kits

In still further embodiments, the present invention concerns immunodetection kits for use with the immunodetection methods described above. As the wild-type, polymorphic or mutant BARDl, or other BRCAI binding protein antibodies are generally used to detect wild-type, polymoφhic or mutant BARDl, or other BRCAI binding proteins or peptides, the antibodies will preferably be included in the kit. However, kits including both such components may be provided. The immunodetection kits will thus comprise, in suitable container means, a first antibody that binds to a wild-type, polymorphic or mutant BARDl, or other BRCAI binding protein or peptide, and optionally, an immunodetection reagent and further optionally, a wild-type, polymorphic or mutant BARDl, or other BRCAI binding protein or peptide.

In preferred embodiments, monoclonal antibodies will be used. In certain embodiments, the first antibody that binds to the wild-type, polymorphic or mutant BARDl , or other BRCAI binding protein or peptide may be pre-bound to a solid support, such as a column matrix or well of a microtitre plate.

The immunodetection reagents of the kit may take any one of a variety of forms, including those detectable labels that are associated with or linked to the given antibody.

Detectable labels that are associated with or attached to a secondary binding ligand are also contemplated. Exemplary secondary ligands are those secondary antibodies that have binding affinity for the first antibody.

Further suitable immunodetection reagents for use in the present kits include the two- component reagent that comprises a secondary antibody that has binding affinity for the first antibody, along with a third antibody that has binding affinity for the second antibody, the third antibody being linked to a detectable label. As noted above, a number of exemplary labels are known in the art and all such labels may be employed in connection with the present invention.

The kits may further comprise a suitably aliquoted composition of the wild-type, polymoφhic or mutant BARDl , or other BRCAI binding protein or polypeptide, whether labeled or unlabeled, as may be used to prepare a standard curve for a detection assay.

The kits may contain antibody-label conjugates either in fully conjugated form, in the form of intermediates, or as separate moieties to be conjugated by the user of the kit. The components of the kits may be packaged either in aqueous media or in lyophilized form.

The container means of the kits will generally include at least one via!, test tube, flask, bottle, syringe or other container means, into which the antibody may be placed, and preferably, suitably aliquoted. Where wild-type, polymorphic or mutant BARDl , or other BRCAI binding protein or a second or third binding ligand or additional component is provided, the kit will also generally contain a second, third or other additional container into which this ligand or component may be placed. The kits of the present invention will also typically include a means for containing the antibody, antigen, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.

IV. Biological Functional Equivalents

As modifications and changes may be made in the structure of wild-type, polymorphic or mutant BARDl or the other BRCAl-binding genes and proteins of the present invention, and still obtain molecules having like or otherwise desirable characteristics, such biologically functional equivalents are also encompassed within the present invention.

For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies, binding sites on substrate molecules or receptors, DNA binding sites, BRCAl-binding regions, or such like. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence (or, of course, its underlying DNA coding sequence) and nevertheless obtain a protein with like (agonistic) properties. It is thus contemplated by the inventors that various changes may be made in the sequence of wild-type, polymoφhic or mutant BARDl or other BRCAl-binding proteins or peptides, or underlying DNA, without appreciable loss of their biological utility or activity.

Equally, the same considerations may be employed to create a protein or peptide with counterveiling, e.g., antagonistic properties. This is relevant to the present invention in which BARDl or other BRCAl-binding mutants or analogues may be generated. For example, a BARDl or other BRCAl-binding mutant may be generated and tested for BRCAI binding activity to identify those residues important for BRCAI and/or DNA binding. BARDl or other BRCAl-binding mutants may also be synthesized to reflect a BARDl or other BRCAl-binding mutant that occurs in the human population and that is linked to the development of breast, ovarian or uterine cancer. Such mutant proteins are particularly contemplated for use in generating mutant-specific antibodies and such mutant DNA segments may be used as mutant- specific probes and primers.

In terms of functional equivalents, it is well understood by the skilled artisan that, inherent in the definition of a "biologically functional equivalent protein or peptide or gene", is the concept that there is a limit to the number of changes that may be made within a defined portion of the molecule and still result in a molecule with an acceptable level of equivalent biological activity. Biologically functional equivalent peptides arc thus defined herein as those peptides in which certain, not most or all, of the amino acids may be substituted.

In particular, where shorter length peptides, such as RING motifs arc concerned, it is contemplated that fewer amino acids should be made within the given peptide. Longer domains may have an intermediate number of changes. The full length protein will have the most tolerance for a larger number of changes. Of course, a plurality of distinct proteins/peptides with different substitutions may easily be made and used in accordance with the invention.

It is also well understood that where certain residues are shown to be particularly important to the biological or structural properties of a protein or peptide, e.g., residues in binding regions or active sites, such residues may not generally be exchanged. This is an important consideration in the present invention, where changes in the BRCAl-binding region, the RING motif and the BRCT domains should be carefully considered and subsequently tested to ensure maintenance of biological function, where maintenance of biological function is desired. In this manner, functional equivalents are defined herein as those peptides which maintain a substantial amount of their native biological activity.

Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. An analysis of the size, shape and type of the amino acid side-chain substituents reveals that arginine, lysine and histidine are all positively charged residues; that alanine, glycine and serine are all a similar size; and that phenylalanine, tryptophan and tyrosine all have a generally similar shape. Therefore, based upon these considerations, arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine; are defined herein as biologically functional equivalents.

To effect more quantitative changes, the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are: isoleucine (+4.5); valine (+4.2); leucinc (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte & Doolittle, 1982, incorporated herein by reference). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity, particularly where the biological functional equivalent protein or peptide thereby created is intended for use in immunological embodiments, as in certain embodiments of the present invention. U.S. Patent 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e. with a biological property of the protein.

As detailed in U.S. Patent 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine

(-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). In making changes based upon similar hydrophilicity values, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

While discussion has focused on functionally equivalent polypeptides arising from amino acid changes, it will be appreciated that these changes may be effected by alteration of the encoding DNA; taking into consideration also that the genetic code is degenerate and that two or more codons may code for the same amino acid. A table of amino acids and their codons is presented herein for use in such embodiments, as well as for other uses, such as in the design of probes and primers and the like.

In addition to the wild-type, polymorphic or mutant BARDl or other BRCAI binding peptidyl compounds described herein, the inventors also contemplate that other sterically similar compounds may be formulated to mimic the key portions of the peptide structure or to interact specifically with BRCAI . Such compounds, which may be termed peptidomimetics, may be used in the same manner as the peptides of the invention and hence are also functional equivalents.

Certain mimetics that mimic elements of protein secondary structure are described in

Johnson et al. (1993). The underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orientate amino acid side chains in such a way as to facilitate molecular interactions, such as those of antibody and antigen. A peptide mimetic is thus designed to permit molecular interactions similar to the natural molecule.

Some successful applications of the peptide mimetic concept have focused on mimetics of β-turns within proteins, which are known to be highly antigenic. Likely β-turn structure within a polypeptide can be predicted by computer-based algorithms, as discussed herein. Once the component amino acids of the turn are determined, mimetics can be constructed to achieve a similar spatial orientation of the essential elements of the amino acid side chains. The generation of further structural equivalents or mimetics may be achieved by the techniques of modeling and chemical design known to those of skill in the art. The art of receptor modeling is now well known, and by such methods a chemical that binds to wild-type, polymorphic or mutant BARDl or other BRCAl-binding protein or to a BRCAI -wild-type, polymoφhic or mutant BARDl or other BRCAl-binding protein complex can be designed and then synthesized. It will be understood that all such sterically designed constructs fall within the scope of the present invention.

V. BRCAI Binding, Purification and Assays

Certain aspects of this invention concern methods for conveniently evaluating candidate substances to identify compounds capable of stimulating BRCAI binding to wild-type, polymoφhic or mutant BARDl or other BRCAI binding protein, or even transcription of wild-type, polymoφhic or mutant BARDl or other BRCAI binding protein.

Successful candidate substances may function in the absence of mutations in BARDl or another BRCAI binding protein, in which case the candidate compound may be termed a "positive stimulator" of BARDl or the other BRCAI binding protein. Alternatively, such compounds may stimulate transcription in the presence of mutated BARDl or another BRCAI binding protein, overcoming the effects of the mutation, i.e., function to oppose BARDl- or other BRCAI binding protein-mutant mediated cancer, and thus may be termed "a BARDl or other BRCAI binding protein mutant agonist". Compounds may even be discovered which combine both of these actions. Compounds of any such class will likely be useful therapeutic agents for use in treating cancer.

As BARDl and the other BRCAI binding proteins are herein shown to bind BRCAI, one method by which to identify a candidate substance capable of stimulating BARDl or other BRCAI binding protein is based upon specific protein:protein binding. Accordingly, to conduct such an assay, one may prepare a protein with a BRCAI binding domain and determine the ability of a candidate substance to increase binding to BRCAI . As BARDl and the other BRCAI binding proteins are also believed to bind DNA, most likely in the context of a complex with BRCAI, another method by which to identify a candidate substance capable of stimulating BARDl and the other BRCAI binding proteins is based upon specific protein:DNA binding. Accordingly, to conduct such an assay, one would prepare a BARDl or other BRCAI binding protein and a BRCAI protein and determine the ability of a candidate substance to increase their binding to a specific DNA segment, i.e., to increase the amount or the binding affinity of a specific protein:DNA complex.

All binding assays would be parallel assays, one of which contains the binding components alone and one of which contains the added candidate substance composition. One would perform each assay under conditions, and for a period of time, effective to allow the formation of proteimprotein complexes or protein:DNA complexes, and one would then separate the bound complexes from any unbound protein and/or DNA and measure the amount of the complexes. An increase in the amount of any bound complex formed in the presence of the candidate substance would be indicative of a candidate substance capable of promoting BARDl or other BRCAI binding protein binding to BRCAI , or BARDl or other BRCAI binding protein-BRCA 1 complex binding to DNA.

In such binding assays, the amount of the bound complex may be measured, after the removal of unbound species, by detecting a label, such as a radioactive or enzymatic label, which has been incorporated into the original wild-type, polymoφhic or mutant BARDl, other BRCAI binding protein or BRCAI protein composition or even in a DNA segment. Alternatively, one could detect the protein portion of the complex by means of an antibody directed against the protein, such as those disclosed herein.

Preferred binding assays are those in which either the BARDl or other BRCAI binding protein or the BRCAI protein is bound to a solid support and contacted with the other component to allow complex formation. Unbound protein components are then separated from the bound complexes by washing and the amount of the remaining bound complex is quantitated by detecting the label or with antibodies. Such binding assays form the basis of filter-binding and microtiter plate-type assays and can be performed in a semi-automated manner to enable analysis of a large number of candidate substances in a short period of time. Electrophoretic methods of DNA binding, such as gel-shift assays, could also be employed to separate unbound protein or DNA from bound protein:DNA complexes.

Virtually any candidate substance may be analyzed by these methods, including compounds which may interact with BRCAI or wild-type, polymorphic, mutant BARDl or other BRCAI binding protein, and also substances such as enzymes which may act by physically altering one of the structures present. Of course, any compound isolated from natural sources such as plants, animals or even marine, forest or soil samples, may be assayed, as may any synthetic chemical or recombinant protein.

Another potential method for stimulating BRCAI activity is to prepare a wild-type, polymoφhic, mutant BARDl or other BRCAI binding protein composition and to modify the protein composition in a manner effective to increase binding. The binding assays would be performed in parallel, similar to those described above, allowing the native and modified wild-type, polymorphic, mutant BARDl or other BRCAI binding protein binding to be compared. In addition to site specific mutagenesis, phosphatase and kinase enzymes may be tested, as may other agents, including proteases and chemical agents, could be employed to modify the BRCAI binding properties of wild-type, polymorphic, mutant BARDl or other BRCAI binding proteins.

Cellular assays also are available for screening candidate substances to identify those capable of stimulating wild-type, polymorphic, mutant BARDl or other BRCAI binding protein and or BRCAI -mediated transcription and gene expression. In these assays, the increased expression of any natural or heterologous gene under the control of a functional BRCA 1 and wild-type, polymoφhic, mutant BARDl or other BRCAI binding protein may be employed as a measure of stimulatory activity, although the use of reporter genes is preferred. A reporter gene is a gene that confers on its recombinant host cell a readily detectable phenotype that emerges only under specific conditions.

Reporter genes are genes which encode a polypeptide not otherwise produced by the host cell which is detectable by analysis of the cell culture, e.g., by fluoronietric, radioisotopic or spectrophotometric analysis of the cell culture. Exemplary enzymes include luciferases, transferases, esterases, phosphatases, proteases (tissue plasminogen activator or urokinase), and other enzymes capable of being detected by their physical presence or functional activity. A reporter gene often used is chloramphenicol acetyltransferase (CAT) which may be employed with a radiolabeled substrate, or luciferase, which is measured fluorometrically.

Another class of reporter genes which confer detectable characteristics on a host cell arc those which encode polypeptides, generally enzymes, which render their transformants resistant against toxins, e.g., the neo gene which protects host cells against toxic levels of the antibiotic G418, and genes encoding dihydrofolate reductasc, which confers resistance to methotrexate. Other genes of potential for use in screening assays arc those capable of transforming hosts to express unique cell surface antigens, e.g., viral env proteins such as HIV gpl20 or herpes gD, which are readily detectable by immunoassays.

The transcriptional promotion process which, in its entirety, leads to enhanced transcription is termed "activation." The mechanism by which a successful candidate substance acts is not material since the objective is to promote wild-type, polymorphic, mutant BARDl or other BRCAI binding protein and/or BRCAI -mediated gene expression, or even, to promote gene expression in the presence of mutants, by whatever means will function to do so.

To create an appropriate vector or plasmid for use in such assays one would ligate the

BRCAI and wild-type, polymorphic, mutant BARDl or other BRCAI binding protein promoter and any necessary response elements to a DNA segment encoding the reporter gene by conventional methods. The relevant promoter sequences may be obtained by in vitro synthesis or recovered from genomic DNA and should be ligated upstream of the start codon of the reporter gene. An AT-rich TATA box region should also be employed and should be located between the sequence and the reporter gene start codon. The region 3' to the coding sequence for the reporter gene will ideally contain a transcription termination and polyadenylation site. The promoter and reporter gene may be inserted into a replicable vector and transfected into a cloning host such as E. coli, the host cultured and the replicated vector recovered in order to prepare sufficient quantities of the construction for later transfection into a suitable eukaryotic host. Host cells for use in the screening assays of the present invention will generally be mammalian cells, and are preferably cell lines which may be used in connection with transient transfection studies. Cell lines should be relatively easy to grow in large scale culture. Also, they should contain as little native background as possible considering the nature of the reporter polypeptide. Examples include the Hep G2, VERO, HeLa, human embryonic kidney, 293, CHO, W138, BHK, COS-7, and MDCK cell lines, with monkey CV-1 cells being particularly preferred.

The screening assay typically is conducted by growing recombinant host cells in the presence and absence of candidate substances and determining the amount or the activity of the reporter gene. To assay for candidate substances capable of exerting their effects in the presence of mutated BARDl or other BRCAl-binding gene products, one would make serial molar proportions of such gene products that alter expression. One would ideally measure the reporter signal level after an incubation period that is sufficient to demonstrate mutant-mediated repression of signal expression in controls incubated solely with mutants. Cells containing varying proportions of candidate substances would then be evaluated for signal activation in comparison to the suppressed levels. Candidates that demonstrate dose related enhancement of reporter gene transcription or expression are then selected for further evaluation as clinical therapeutic agents.

VI. Diagnostics

As with the therapeutic methods of the present invention, the diagnostic methods are based upon the weight of evidence of the importance of BARDl and other genes identified herein, which encodes proteins that associate with BRCAI in vivo. BARDl is co-expressed with BRCAI in all breast and ovarian carcinoma lines tested. It is important to note that the BARDl /BRCAI interaction is disrupted by tumorigenic amino acid substitutions in BRCAI, indicating that the formation of a stable complex between these proteins is likely to be an essential aspect of BRCAI -mediated tumor suppression. In this light, BARDl and the other genes encoding BRCAl-binding proteins are likely to be the target of oncogenic mutations in familial or sporadic breast cancer. The diagnostic methods of the present invention generally involve determining either the type or the amount of a wild-type, polymoφhic or mutant BARDl or a BRCAI binding protein present within a biological sample from a patient suspected of having breast, ovarian or another cancer. Irrespective of the actual role of BARDl and the other BRCAI binding proteins, it will be understood that the detection of a mutant is likely to be diagnostic of cancer and that the detection of altered amounts of BARDl or one or more of the additional BRCAI binding proteins, either at the mRNA or protein level, is also likely to have diagnostic implications, particularly where there is a reasonably significant difference in amounts.

The finding of a decreased amount of wild-type, polymorphic or mutant BARDl or other

BRCAI binding protein in one, or preferably more, cancer patients, in comparison to the amount within a sample from a normal subject, will be indicative of BARDl or one or more of the other BRCAI binding proteins as a tumor suppressor. Following which, cancer in others would be similarly diagnosed by detecting a decreased amount of BARDl or other BRCAI binding protein in a sample. The finding of an increased amount of BARDl or other BRCAI binding protein in one, or preferably more, cancer patients, in comparison to the amount within a sample from a normal subject, will be indicative of BARDl or one or more of the other genes encoding a BRCAI binding proteins as an oncogene. Following which, cancer in others would be similarly diagnosed by detecting an increased amount of BARDl or other gene encoding a BRCAI binding protein in a sample.

The type or amount of a wild-type or mutant BARDl or a BRCAI binding protein present within a biological sample, such as a blood or tissue sample, may be determined by means of a molecular biological assay to determine the level of a nucleic acid that encodes such a BARDl or BRCAI binding protein, or by means of an immunoassay to determine the level of the polypeptide itself.

Any of the foregoing nucleic acid detection methods or immunodetection methods may be employed as a diagnostic methods in the context of the present invention. VII. Therapeutics

As stated above, the mechanism by which BRCAI inhibits tumor formation is not yet completely understood. Most of the BRCAI alleles that segregate with breast cancer susceptibility have frameshift or nonsense mutations that cause premature termination of protein synthesis, a relatively gross defect that provides fewer clues about the function of BRCAI polypeptides.

In some families, however, the predisposing lesion of BRCAI has been ascribed to a single amino acid substitution, such as the C61 G and C64G mutations that occur within the RING domain. It is reasonable to propose that these mutations are oncogenic, at least in part, because they prevent the in vivo association of BRCAI and BARDl or other BRCAI binding proteins. This suggests that the heteromeric BARD 1/B RCA 1 or other BRCAI binding protein/BRCAl complex has an active role in tumor suppression. This provides for two further aspects of the present invention.

First, the biochemical function of this protein complex can now be determined given that the present invention provides methods for obtaining sufficient amounts of the complex. The interaction between BARDl and BRCAI should situate their respective RING domains in close physical apposition. As such, the two domains could cooperatively perform certain functions, such as sequence-specific DNA recognition or association with other protein ligands. DNA recognition by the BARD1/BRCA1 complex is reasonable, especially since many transcription factors are known to bind DNA as obligate heterodimers (Landschulz et al, 1988; Murre et al, 1989). DNA recognition by complexes between BRCAI and other BRCAI binding proteins, even those that do not contain a RING motif, is also reasonable.

Second, upon confirmation of the active role of the heteromeric BARDl /BRCAI or other BRCAI binding protein/BRCAl complex in tumor suppression, the present invention will provide cancer therapy by provision of the appropriate wild-type gene. The therapeutic methods are based upon the weight of evidence of the importance of BARDl, which encodes a protein that associates with BRCAI in vivo, and is co-expressed with BRCAI in all breast and ovarian carcinoma lines tested. Moreover, the BARDl gene product shares homology with the two most highly conserved domains of BRCAI , both of which are common sites for germline mutations that segregate with breast cancer susceptibility. Finally, the BARD1/BRCA1 interaction is disrupted by tumorigenic amino acid substitutions in BRCAI, indicating that the formation of a stable complex between these proteins is likely to be an essential aspect of BRCAI -mediated tumor suppression.

In these aspects of the present invention, wild-type BARDl, or one of the genes encoding one of the other BRCAl-binding proteins disclosed herein, is provided to an animal with cancer, or breast, ovarian or uterine cancer, in the same manner that other tumor suppressors are provided, following identification of a cell type that lacks the tumor suppressor or that has an aberrant tumor suppressor. For example, the provision of BARDl , or one of the genes encoding one of the other BRCAl-binding proteins disclosed herein, can be considered to be analogous to the provision of p53.

Alternatively, should BARDl, or the gene encoding one of the other BRCAI binding proteins, prove to be an oncogene, as may be established by the wild-type protein binding and reducing the activity of tumor suppressor proteins, then inhibition of BARDl, or the gene encoding one of the other BRCAI binding proteins, would be adopted as a therapeutic strategy. This situation would be similar to that of MDM2, which binds and inhibits the tumor suppressor function of p53. Inhibitors would be any molecule that reduces the activity or amounts of BARDl or a gene encoding one of the other BRCAI binding proteins, including antisense, ribozymes and the like, as well as small molecule inhibitors.

Gene Therapy

The general approach to the tumor suppressor aspect of the present invention is to provide a cell with a wild-type or polymoφhic BARDl or a BRCAI binding protein, thereby peπnitting the proper regulatory activity of the proteins to take effect. While it is conceivable that tlie protein may be delivered directly, a preferred embodiment involves providing a nucleic acid encoding a BARDl or a BRCAI binding protein to the cell. Following this provision, the polypeptide is synthesized by the transcriptional and translational machinery of the cell, as well as any that may be provided by the expression construct. In providing antisense, ribozymes and other inhibitors, the preferred mode is also to provide a nucleic acid encoding the construct to the cell. All such approaches are herein encompassed within the term "gene therapy".

In various embodiments of the invention, DNA is delivered to a cell as an expression construct. Several non-viral methods for the transfer of expression constructs into cultured mammalian cells also are contemplated by the present invention. These include calcium phosphate precipitation, DEAE-dextran, electroporation, direct microinjection, DNA-loaded liposomes and lipofectamine-DNA complexes, cell sonication, gene bombardment using high velocity microprojectiles, and receptor-mediated transfection. Some of these techniques may be successfully adapted for in vivo or ex vivo use, as discussed below.

In another embodiment of the invention, the expression construct may simply consist of naked recombinant DNA or plasmids. Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane. This is particularly applicable for transfer in vitro, but it may be applied to in vivo use as well.

Another embodiment of the invention for transferring a naked DNA expression construct into cells may involve particle bombardment. This method depends on the ability to accelerate DNA coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them. Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force. The microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads.

In a further embodiment of the invention, the expression construct may be entrapped in a

Iiposome, as discussed below. Also contemplated are lipofectamine-DNA complexes. Liposome- mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful. Wong et al. (1980) demonstrated the feasibility of liposome-mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa and hepatoma cells. In certain embodiments of the invention, the Iiposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA. In other embodiments, the Iiposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-1). In yet further embodiments, the Iiposome may be complexed or employed in conjunction with both HVJ and HMG-1. In other embodiments, the delivery vehicle may comprise a ligand and a Iiposome. Where a bacterial promoter is employed in the DNA construct, it also will be desirable to include within the Iiposome an appropriate bacterial polymerase.

The ability of certain viruses to enter cells via receptor-mediated cndocytosis and to integrate into host cell genome and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign genes into mammalian cells. Preferred gene therapy vectors of the present invention will generally be viral vectors.

Retroviruses have promise as gene delivery vectors due to their ability to integrate their genes into the host genome, transferring a large amount of foreign genetic material, infecting a broad spectrum of species and cell types and of being packaged in special cell-lines (Miller, 1992).

Other viruses, such as adenovirus, herpes simplex viruses (HSV), cytomegalovirus (CMV), and adeno-associated virus (AAV), such as those described by U.S. Patent 5,139,941 , incorporated herein by reference, may also be engineered to serve as vectors for gene transfer. Although some viruses that can accept foreign genetic material are limited in the number of nucleotides they can accommodate and in the range of cells they infect, these viruses have been demonstrated to successfully effect gene expression. However, adenoviruses do not integrate their genetic material into the host genome and therefore do not require host replication for gene expression, making them ideally suited for rapid, efficient, heterologous gene expression. Techniques for preparing replication-defective infective viruses are well known in the art.

In certain further embodiments, the gene therapy vector will be HSV. A factor that makes HSV an attractive vector is the size and organization of the genome. Because HSV is large, incoφoration of multiple genes or expression cassettes is less problematic than in other smaller viral systems. In addition, the availability of different viral control sequences with varying performance (temporal, strength, etc.) makes it possible to control expression to a greater extent than in other systems. It also is an advantage that the virus has relatively few spliced messages, further easing genetic manipulations. HSV also is relatively easy to manipulate and can be grown to high titers. Thus, delivery is less of a problem, both in terms of volumes needed to attain sufficient MOI and in a lessened need for repeat dosings.

Of course, in using viral delivery systems, one will desire to purify the virion sufficiently to render it essentially free of undesirable contaminants, such as defective interfering viral particles or endotoxins and other pyrogens such that it will not cause any untoward reactions in the cell, animal or individual receiving the vector construct. A preferred means of purifying the vector involves the use of buoyant density gradients, such as cesium chloride gradient centrifugation.

Gene delivery using second generation retroviral vectors has been reported. Kasahara et l (1994) prepared an engineered variant of the Moloney murine leukemia virus, that normally infects only mouse cells, and modified an envelope protein so that the virus specifically bound to, and infected, human cells bearing the erythropoietin (EPO) receptor. This was achieved by inserting a portion of the EPO sequence into an envelope protein to create a chimeric protein with a new binding specificity.

Antisense

In an alternative embodiment, the BARDl or BRCAI binding protein nucleic acids employed may actually encode antisense constructs that hybridize, under intracellular conditions, to BARDl or BRCAI binding protein nucleic acids. The term "antisense construct" is intended to refer to nucleic acids, preferably oligonucleotides, that are complementary to the base sequences of a target DNA or RNA. Antisense oligonucleotides, when introduced into a target cell, specifically bind to their target nucleic acid and interfere with transcription, RNA processing, transport, translation and/or stability.

Antisense constructs may be designed to bind to the promoter and other control regions, exons, introns or even exon-intron boundaries of a gene. Antisense RNA constructs, or DNA encoding such antisense RNA's, may be employed to inhibit gene transcription or translation or both within a host cell, either in vitro or in vivo, such as within a host animal, including a human subject. Nucleic acid sequences which comprise "complementary nucleotides" are those which are capable of base-pairing according to the standard Watson-Crick complementarity rules. That is, that the larger purines will base pair with the smaller pyrimidines to form combinations of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T), in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA. Inclusion of less common bases such as inosinc, 5-methylcytosine, 6-methyladenine, hypoxanthine and others in hybridizing sequences does not interfere with pairing.

As used herein, the terms "complementary" means nucleic acid sequences that are substantially complementary over their entire length and have very few base mismatches. For example, nucleic acid sequences of fifteen bases in length may be termed complementary when they have a complementary nucleotide at thirteen or fourteen positions with only a single mismatch. Naturally, nucleic acid sequences which are "completely complementary" will be nucleic acid sequences which are entirely complementary throughout their entire length and have no base mismatches.

Other sequences with lower degrees of homology also arc contemplated. For example, an antisense construct which has limited regions of high homology, but also contains a non- homologous region (e.g., a ribozyme) could be designed. These molecules, though having less than 50% homology, would bind to target sequences under appropriate conditions.

While all or part of the BARDl or BRCAI binding protein gene sequence may be employed in the context of antisense construction, short oligonucleotides are easier to make and increase in vivo accessibility. However, both binding affinity and sequence specificity of an antisense oligonucleotide to its complementary target increases with increasing length. One can readily determine whether a given antisense nucleic acid is effective at targeting of the corresponding host cell gene simply by testing the constructs in vitro to determine whether the function of the endogenous gene is affected or whether the expression of related genes having complementary sequences is affected.

In certain embodiments, one may wish to employ antisense constructs which include other elements, for example, those which include C-5 propyne pyrimidines. Oligonucleotides which contain C-5 propyne analogues of uridine and cytidine have been shown to bind RNA with high affinity and to be potent antisense inhibitors of gene expression.

VIII. Pharmaceutical Compositions

A. Pharmaceutically Acceptable Carriers

Aqueous compositions of the present invention comprise an effective amount of the BARDl or other BRCAI binding agent, such as a BARDl or other BRCAI binding protein, peptide, epitopic core region, inhibitor, or such like, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Aqueous compositions of gene therapy vectors expressing any of the foregoing are also contemplated. The phrases "pharmaceutically or pharmacologically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or a human, as appropriate.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

For human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.

The biological material should be extensively dialyzed to remove undesired small molecular weight molecules and/or lyophilized for more ready formulation into a desired vehicle, where appropriate. The active compounds will then generally be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, subcutaneous, intralesional, or even intraperitoneal routes. The preparation of an aqueous composition that contains a BARDl or other BRCAI binding agent as an active component or ingredient will be known to those of skill in the art in light of the present disclosure. Typically, such compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for using to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and the preparations can also be emulsified.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

A BARDl or other BRCAI binding protein, peptide, agonist or antagonist of the present invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by die use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absoφtion, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incoφorating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and frceze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

In terms of using peptide therapeutics as active ingredients, the technology of U.S. Patents 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792; and 4,578,770, each incoφorated herein by reference, may be used.

The preparation of more, or highly, concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small tumor area.

Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.

For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCI solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035- 1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

The active BARDl- or other BRCAI binding protein-derived peptides or agents may be formulated within a therapeutic mixture to comprise about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0 or even about 10 milligrams per dose or so. Multiple doses can also be administered.

In addition to the compounds formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, e.g., tablets or other solids for oral administration; liposomal formulations; time release capsules; and any other form currently used, including cremes.

One may also use nasal solutions or sprays, aerosols or inhalants in the present invention.

Nasal solutions are usually aqueous solutions designed to be administered to the nasal passages in drops or sprays. Nasal solutions are prepared so that they are similar in many respects to nasal secretions, so that normal ciliary action is maintained. Thus, the aqueous nasal solutions usually are isotonic and slightly buffered to maintain a pH of 5.5 to 6.5.

In addition, antimicrobial preservatives, similar to those used in ophthalmic preparations, and appropriate drug stabilizers, if required, may be included in the formulation. Various commercial nasal preparations are known and include, for example, antibiotics and antihistamines and are used for asthma prophylaxis.

Additional formulations which are suitable for other modes of adminisύation include vaginal suppositories and pessaries. A rectal pessary or suppository may also be used. Suppositories are solid dosage forms of various weights and shapes, usually medicated, for insertion into the rectum, vagina or the urethra. After insertion, suppositories soften, melt or dissolve in the cavity fluids.

In general, for suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably l%-2%.

Vaginal suppositories or pessaries are usually globular or oviform and weighing about 5 g each. Vaginal medications are available in a variety of physical forms, e.g., creams, gels or liquids, which depart from the classical concept of suppositories. Vaginal tablets, however, do meet the definition, and represent convenience both of administration and manufacture.

Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate. sodium saccharine, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.

In certain defined embodiments, oral pharmaceutical compositions will comprise an inert diluent or assimilable edible carrier, or they may be enclosed in hard or soft shell gelatin capsule, or they may be compressed into tablets, or they may be incoφorated directly with the food of the diet. For oral therapeutic administration, the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 75% of the weight of the unit, or preferably between 25-60%. The amount of active compounds in such therapeutically useful compositions is such that a suitable dosage will be obtained.

The tablets, troches, pills, capsules and the like may also contain the following: a binder, as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup of elixir may contain the active compounds sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor.

It will naturally be understood that suppositories, for example, will not generally be contemplated for use in treating breast cancer. However, in the event that the proteins, peptides or other agents of the invention, or those identified by the screening methods of the present invention, are confirmed as being useful in connection with other forms of cancer, then other routes of administration and pharmaceutical compositions will be more relevant. As such, suppositories may be used in connection with colon cancer, inhalants with lung cancer and such like.

B. Liposomes and Nanocapsules

In certain embodiments, the use of liposomes and/or nanoparticles is contemplated for the introduction of wild-type, polymoφhic or mutant BARDl or other BRCAI binding protein peptides or agents, or gene therapy vectors, including both wild-type and antisense vectors, into host cells. The formation and use of liposomes is generally known to those of skill in the art, and is also described below.

Nanocapsules can generally entrap compounds in a stable and reproducible way. To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 μm) should be designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention, and such particles may be are easily made. Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles

(MLVs). MLVs generally have diameters of from 25 nm to 4 μm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 A, containing an aqueous solution in the core.

The following information may also be utilized in generating liposomal formulations. Phospholipids can form a variety of structures other than liposomes when dispersed in water, depending on the molar ratio of lipid to water. At low ratios the Iiposome is the preferred structure. The physical characteristics of liposomes depend on pll, ionic strength and the presence of divalent cations. Liposomes can show low permeability to ionic and polar substances, but at elevated temperatures undergo a phase transition which markedly alters their permeability. The phase transition involves a change from a closely packed, ordered structure, known as the gel state, to a loosely packed, less-ordered structure, known as the fluid state. This occurs at a characteristic phase-transition temperature and results in an increase in permeability to ions, sugars and drugs.

Liposomes interact with cells via four different mechanisms: Endocytosis by phagocytic cells of the reticuloendothelial system such as macrophages and neutrophils; adsoφtion to the cell surface, either by nonspecific weak hydrophobic or electrostatic forces, or by specific interactions with cell-surface components; fusion with the plasma cell membrane by insertion of the lipid bilayer of the Iiposome into the plasma membrane, with simultaneous release of liposomal contents into the cytoplasm; and by transfer of liposomal lipids to cellular or subcellular membranes, or vice versa, without any association of the Iiposome contents. Varying the Iiposome formulation can alter which mechanism is operative, although more than one may operate at the same time.

C. Kits

Therapeutic kits of the present invention are kits comprising a wild-type, polymorphic or mutant BARDl and/or other BRCAI binding protein, peptide, inhibitor, gene, vector or other BARDl or BRCAI binding protein effector. Such kits will generally contain, in suitable container means, a pharmaceutically acceptable formulation of a BARDl or BRCAI binding protein, peptide, domain, inhibitor, or a gene or vector expressing any of the foregoing in a pharmaceutically acceptable formulation, optionally comprising other anti-cancer agents. The kit may have a single container means, or it may have distinct container means for each compound.

When the components of the kit are provided in one or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred. The BARDl and BRCAI binding protein compositions may also be formulated into a syringeable composition. In which case, the container means may itself be a syringe, pipette, or other such like apparatus, from which the formulation may be applied to an infected area of the body, injected into an animal, or even applied to and mixed with the other components of the kit.

However, the components of the kit may be provided as dried powder(s). When reagents or components arc provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.

The container means will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the BARDl or BRCAI binding protein or gene or inhibitory formulation are placed, preferably, suitably allocated. Where a second anti-cancer therapeutic is provided, the kit will also generally contain a second vial or other container into which this agent may be placed. The kits may also comprise a second/third container means for containing a sterile, pharmaceutically acceptable buffer or other diluent.

The kits of the present invention will also typically include a means for containing the vials in close confinement for commercial sale, such as, e.g., injection or blow-molded plastic containers into which the desired vials are retained.

Irrespective of the number or type of containers, the kits of the invention may also comprise, or be packaged with, an instrument for assisting with the injection/administration or placement of the ultimate BARDl or BRCAI binding protein or gene composition within the body of an animal. Such an instrument may be a syringe, pipette, forceps, or any such medically approved delivery vehicle.

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLE I Methods

1. Two-hybrid screening in yeast

A cDNA fragment encoding the amino-terminal 304 residues of human BRCAI was obtained by RT-PCR™ amplification of HeLa cell RNA with flanking oligonucleotide primers: TTACCATGGATTTATCTGCTCTTCGCGTT (SEQ ID NO:4); and AAAAGTCGACTAGAATTCAGCCTTTTCTACATTCATTC (SEQ ID NO:5).

After digestion with Ncøl and Sail endonucleases, the amplified fragment was inserted into the corresponding sites of the pASl-CYH2 vector (Harper et al, 1993). The resultant plasmid (BR304/pASl-CYH2) was then used to transform yeast cells of the Y190 reporter strain (Tφ- Leu^* His^", LacZ^"). Trp prototrophs were evaluated for expression of the DBD-BR304 hybrid polypeptide (containing the GAL4 DΝA-binding domain fused to the amino-terminal 304 residues of BRCAI) by immunoblotting with 12CA5, a monoclonal antibody that recognizes the influenza hemagglutinin epitope incorporated into the expressed reading frame of pASl-CYH2 (Chien et al, 1991).

These cells were then transfected with a cDΝA library of human B cell transcripts in the pACT two-hybrid expression vector (Clontech), and approximately 11 million Trp Leu⁺ transformants were plated on a Trp/Leu His dropout medium containing 40 mM 3-aminotriazole (Durfee et al, 1993). The positive clones (His⁺LacZ⁺) were cured of the BR304/pASl-CYH2 plasmid by growth on Leu dropout plates containing 10 mg/ml cycloheximide (Harper et al, 1993).

Each of the cured clones was then subjected to a two-hybrid mating assay for protein- protein interactions with the DBD-BR304 hybrid and DBD hybrids containing sequences of two irrelevant proteins (mouse p53 and human TALI). The cDNAs that displayed a BRCAl- specific pattern of interaction in the mating assay were excised from the library plasmid (pACT), inserted into pASl-CYH2, and tested for BRCAl-specific interaction in a reciprocal two-hybrid mating assay with BR304/pACTII, an expression vector that encodes a hybrid protein (TAD- BR304) containing the transactivation domain of GAL4 fused to the amino-terminal 304 residues of BRCAI .

Three of the DBD-X hybrid proteins, including the DBD-STAT3 hybrid and two DBD- X hybrids encoded by novel cDNA sequences, could not be tested in the reciprocal yeast two- hybrid assay because they were self-activating; that is, they were able to induce expression of the LacZ reporter construct in the absence of the TAD-BR304 hybrid.

2. Two-hybrid analysis in mammalian cells Candidate cDNAs that showed a BRCAl-specific pattern of interaction in yeast were also subjected to two-hybrid analysis in mammalian cells (Dang et al, 1991 ; Hsu et al, 1994). For this puφose, each cDNA was inserted into the multiple cloning site of pVP-HA2 or pVP- FLAG, mammalian vectors designed for the expression of hybrid polypeptides that contain the transactivation domain of the heφesvirus VP16 protein. In addition, sequences encoding BRCAI residues 1-304 were inserted into pMl, a mammalian vector used for expression of hybrid proteins containing the DNA-binding domain of GAL4 (Sadowski et al, 1992). Embryonal kidney 293 cells were then co-transfected with an expression vector encoding the candidate VP16 hybrid polypeptide (3.0 mg), an expression vector encoding the GAL4-BR304 hybrid (BR304/pMl) (3.0 mg), a GAL4-responsive reporter gene (G5LUC) (1.0 mg), and the pSV-β-galactosidase control plasmid (1.5 mg). Expression vectors for mammalian two-hybrid analyses of the BARD1/BRCA1 interaction (FIG. 4A, FIG. 4B and FIG. 5A) were constructed by inserting defined cDNA segments into pVP-HA2, pVP-FLAG, or pCMV-GAL4; the latter, which is a derivative of the pCMV5 (Andersson et al, 1989) and pM2 (Andersson et al, 1989) vectors, contains a sequence encoding the FLAG epitope appended to the 3' end of the GAL4 reading frame.

3. Antibody production

The bacterial expression vector encoding GST-BRΔ304, a glutathione S-transferase fusion protein containing residues 183-304 of human BRCAI , was generated by inserting a BRCAI cDNA fragment into the Ncol/Hindlll sites of pGEX-KG. The fusion protein was then expressed in E. coli, isolated to homogeneity by affinity chromatography on glutathione- agarose, and injected into rabbits according to a standard immunization protocol. Similarly, the BARD 1 -specific antiserum was generated by immunizing rabbits with a purified GST-fusion protein containing BARDl residues 141-388. The TALl-specific antiserum (#1080) has been described (Hsu et al. , 1994).

4. Co-immunoprccipitation analysis

The TALI expression plasmid (TALl/pCMV4) has been described (Hsu et al, 1994). The expression plasmid for HA-BR304 was constructed in two steps: First, the cDNA fragment encoding residues 1-304 of human BRCAI was inserted into the NcollSall sites of pVP-HA2, a vector used for expression of VP16-fusion proteins in mammalian cell. Second, the BRCAI coding sequences were excised from pVP-HA2, along with vector sequences encoding the influenza hemagglutinin (HA) epitope, and inserted into the NoillHindlll sites of pCMV-Not, a derivative of the pCMV4 expression vector (Andersson et al, 1989).

The vectors encoding FLAG-DE12 and FLAG-B202 were also prepared in two steps: thus, the appropriate cDNA fragments were inserted into pVP-FLAG, and the cDNA fragments were then excised from pVP-FLAG, together with vector sequences encoding the FLAG epitope, and inserted into the Notl/Hindlll sites of pCMV-No/.

For co-immunoprecipitation analysis, approximately 25 x 10⁵ embryonal 293 kidney cells were seeded onto each 100 mm plate and cultured in 10 ml of growth medium (low glucose DMEM supplemented with 2 mM glutamine, 100 mg/ml penicillin G, 100 mg/ml streptomycin, and 10% fetal calf serum). After 24 hours the adherent cells were treated with the calcium phosphate transfection system according to the manufacturer's instructions (Gibco/BRL). Each 100 mm culture was transfected with 3.75 mg of the pSV-β-galactosidase control plasmid (Promega) and 7.5 mg of each expression vector; where necessary 7.5 mg of the parental pCMV4 vector was added to provide a constant DNA mass (18.75 mg) for transfection of each culture.

Two days after transfection, cell lysates were prepared in 1 ml of "low-salt NP40 buffer" (10 M HEPES pH 7.6, 250 mM NaCI, 0.1% Nonidet P-40, 5 mM EDTA) containing protease inhibitors (0.1 mg/ml aprotinin, 1 mg/ml leupeptin, 1 mg/ml pepstatin, and 1 mM PMSF), and 4 ml of immune or pre-immunc rabbit antiserum were added to each lysate. After rocking at 4°C for 1 hr, 50 ml of staphylococcal protein A-Sepharose beads (20% slurry; Pharmacia) were added to each lysate and the mixture was rocked at 4°C for an additional hour. The beads were then pelleted by brief centrifugation and washed two times in "high-salt NP40 buffer" (10 mM HEPES pH 7.6, 1.0 M NaCI, 0.1% Nonidet P-40, 5 mM EDTA) with protease inhibitors and two times in low-salt NP40 buffer with protease inhibitors.

Finally, the beads were resuspended in "loading buffer" (100 mM Tris-HCl pH 6.8, 2% SDS, 0.2%) bromophenol blue, 20% glycerol, and 5% β-mercaptoethanol), boiled for 10 minutes, and pelleted by centrifugation. The supernatant was then fractionated by electrophoresis on a SDS-15% polyacrylamide gel, and the fractionated polypeptides were electroblotted onto Hybond-ECL nitrocellulose for Western analysis by enhanced chemiluminescence (Amersham) with the FLAG-specific M5 monoclonal antibody (Eastman Kodak).

5. In vitro assays of protein-protein interaction

Expression plasmids encoding the full-length BARDl and BRCAI polypeptides were generated by inserting their respective cDNA fragments into pSP6-FLAG, a derivative of the pSPUTK vector (Stratagene) that includes coding sequences for an amino-terminal tag containing the FLAG epitope (MADYKDDDKS; SEQ ID NO:3) (Hopp et al, 1988). The

BARDl/pSP6-FLAG and BRCAl/pSP6-FLAG plasmids were then used as templates for in vitro synthesis of radiolabeled BARDl and BRCAI polypeptides, respectively, in rabbit reticulocyte lysates (Promega) containing [S ^"Jmethionine (DuPont NEN).

Expression plasmids encoding GST-fusion proteins were generated by inserting the appropriate cDNA fragments into the pGEX or pGEX-KG vectors (Smith and Johnson, 1988; Guan and Dixon, 1991). The GST fusion proteins were expressed in E. coli, purified by affinity chromatography on glutathione-agarose beads, and retained as a 50% slurry in "buffer C" (20 mM Hepes pH 7.6, 100 mM KCl, 1 mM EDTA, 1 mM dithiothreitol and 20% glycerol) with protease inhibitors (Smith and Johnson, 1988).

The loaded beads were then used directly in binding assays with radiolabeled full-length BARDl polypeptides. Thus, for each binding reaction, a 10 ml aliquot of the BARD1- programmed reticulocyte lysate was mixed with 100 ml of glutathione-agarose beads (loaded with 10 mg of the GST-fusion protein) and 890 ml of "low-salt binding buffer" (50 mM Hepes pH 7.6, 250 mM NaCI, 0.5% Nonidet P-40, 5 mM EDTA, 0.1 % bovine serum albumin, 0.5 mM dithiothreitol, 0.005% SDS, and protease inhibitors). Following a 1 hr incubation at room temperature, the beads were washed twice with low-salt binding buffer, twice with high-salt binding buffer (containing IM NaCI), and twice again with low-salt binding buffer. Finally, the beads were boiled for 10 minutes in 80 ml of loading buffer, and 40 ml of the supernatant was fractionated by electrophoresis on a SDS-10% polyacrylamide gel.

In vitro co-immunoprecipitation was performed by mixing 50 ml of rabbit reticulocyte lysate containing radiolabeled full-length FLAG-BRCA1 with 50 ml of reticulocyte lysate containing unlabeled full-length FLAG-BARD 1 or with 50 ml of an uncharged reticulocyte lysate. Each mixture was incubated at 37°C for 30 minutes in the presence of protease inhibitors. Equivalent aliquots of the mixtures (19 ml) were then diluted into 960 ml of low-salt NP40 buffer and immunoprecipitated at 4°C for 1 hour with 20 ml of staphylococcal protein A-Sepharose beads (50% slurry; Pharmacia) and 1 ml of the indicated antiserum. The beads were then pelleted by brief centrifugation and washed four times in low-salt NP40 buffer. Finally, the beads were resuspended in loading buffer, boiled for 10 minutes, and pelleted by centrifugation. The supernatant was then fractionated by electrophoresis on a SDS-6% polyacrylamide gel. 6. Expression studies

Cytoplasmic RNA was isolated from breast and ovarian cancer cell lines by a combination of NP-40 lysis and mechanical disruption before the addition of lysates to guanidinium isothiocyanatc (Sambrook et al, 1989). Total RNA was subjected to electrophoresis and blotted as described (Sambrook et al. , 1989). The probe for BARDl was purified cDNA insert from the B202 or B230 clones. The 18S probe was obtained from the

ATCC (#77242). Probes were labeled by random hexanucleotide extension with [ PjdCTP

(Amersham).

Northern blots were hybridized at 42°C in 50% formamide solution containing dextran sulfate (Oncor) for 48 hours and subjected to a final wash in 0.5X SSC, 0.1 % SDS at 65°C.

Hybridization signals were quantitated after overnight exposure to a Phosphorlmager (PI) screen using Imagequant software (Molecular Dynamics). Blots were then exposed to X-ray film; 18S was exposed for 20 minutes to the PI screen and for 2 hours to X-ray film.

7. Chromosomal localization of BARDl

The location of BARDl was determined by PCR™ amplification of a panel of monochromosomal hybrid DNAs obtained from the Coricll Institute; using the human BARDl primers:

B202L, AACAGTACAATGACTGGGCTC; SEQ ID NO:6; and B202R, TCAGCGCTTCTGCACACAGT; SEQ ID NO:7.

The location of BARDl was further refined by mapping in the Genebridge panel of DNAs from whole genome radiation hybrids.

8. Clinical Specimens

Tumor tissue, matched normal tissue and blood specimens were obtained as part of protocols approved by the University of Texas Southwestern Medical Center Human Subjects Review Board, St. Paul's Medical Center, Medical City of Dallas and The Southern division of the Cooperative Human Tissue Network. The breast cancers were primarily infiltrating ductal carcinomas. The ovarian carcinomas were of mixed histology, although the majority were papillary serous carcinomas. The following breast and ovarian cancer cell lines were obtained from the American Type Culture Collection: MCF-7, ZR75-1, BT-483, BT-20, T-47D, BT-474, 2008, OVCAR3, CAOV-3, BG-1 and 2774. The ovarian cancer line PE04 was obtained from Dr. Simon Langdon (Medical Oncology Unit, Western General Hospital, Edinburgh, Scotland). Tumors were immediately frozen in liquid nitrogen and stored at -70°C prior to RNA extraction. Buffy coat was prepared from blood. In some cases DNA was prepared from paraffin-embedded tissue. DNA, RNA and cDNA was prepared by standard procedures (Sambrook, et al, 1 89).

9. Genomic structure of BARDl A human genomic library was first screened by hybridization with fragments of BARDl cDNA (Example IV, below). Eleven hybridizing lambda clones were identified and subjected to nucleotide sequence analysis with oligonucleotide primers derived from BARDl cDNA sequence and shown in Table 4 (see Example X below).

YACs lying between D2S143 and D2S295 (The location of BARDl) were identified by accessing the Whitehead data-base. YACs containing BARDl were identified on the basis that they generated the correctly sized PCR amplification products with primers for exons for which genomic sequence was available as a result of sequencing lambda clones. These YACs were sized on pulsed-field gels and isolated as described elsewhere (Gemmill et al, 1996) and YACs 81 Od 12 and 964g6 were then subcloned into the cosmid vector sCos-1 as described (Clines et al, 1997). Hybridization of this library of approximately 5,000 cosmids with probes derived from amplification with BARDl cDNA primers described in Table 4 (B230-F/FAS, B230- FF/FFAS, B230-WS/WAS) resulted in the identification of eleven positively hybridizing cosmids. The same primers were used to sequence two of these cosmid DNAs, generating exon/intron boundary sequences for this region, for which lambda clones were not available.

10. Mutational screening for BARDl alterations cDNA was derived from tumor, matched normal tissue or cell lines. Genomic DNA was obtained from tumor tissue, matched normal tissue, cell lines, blood, and paraffin embedded tissue. SSCP was performed as described elsewhere (Orita et al, 1989; Orita et al, 1989) with oligonucleotide primers for BARDl with cDNA or genomic DNA as shown in Tables 4 and 5

(see Examples X and XI below). Briefly, PCR™ of tumor or blood DNA/cDNA was performed in 20μl volumes containing 100 ng cDNA or genomic DNA template; l χ PCR buffer (Perkin Elmer, Foster City, CA); 200 μM each dATP, dGTP, dCTP, dTTP; 10 pmoles each primer (GIBCO BRL, Grand Island, NY); 0.3μCi ³²P-dCTP (Amersham, Arlington Heights, IL); 0.5U Taq DNA polymerase (Perkin Elmer, Foster City, CA). PCR™ conditions were 30 cycles of 94°C for 30 seconds; 55°C (or as specified for annealing temperatures in Tables 4 and 5) for 30 seconds; 72°C for 30 seconds. A final extension reaction at 72°C was performed for 1 minute.

Amplified samples were diluted 1 : 10 in formamide buffer (98% formamide, 10 mM

EDTA, pH, 8.0, 0.05% bromophenol blue, 0.05% xylene cyanol), denatured at 95°C for 5 min. then cooled rapidly to 4°C. For each sample, 4 μl was loaded onto an SSCP gel and run at 8W (constant power) for 8-16 hours in 0.6χ TBE at room temperature. Gels contained 0.5 x MDE (AT Biochem), 0.6x TBE, 240 μl 10% ammonium persulphate, 24 μl TEMED. Duplicate gels were prepared with a supplement of 10% glycerol. Gels were subjected to autoradiography with or without being dried. Film was exposed for I2-24h. with an intensifying screen.

11. DNA Sequencing of BARDl Variants Identified by SSCP

Variant bands were excised from the SSCP gel after alignment with the autoradiograph and purified with Qiaquick Gel Extraction kit (Qiagen, Santa Clarita, CA, Cat # 28706). DNA was resuspended in 20 μl H20 and 5 μl was treated with 10 units exonuclease I and 2 units shrimp alkaline phosphatase at 37°C for 15 min. Following inactivation of this reaction with heat (80°C for 15 min.), the DNA template was subjected to cycle sequencing with Thermosequenase (Amersham Life Science, Arlington Heights, IL) and α-33P-ddNTPs. Sequencing reactions were electrophoresed in 8% acrylamide/bis gels with l glycerol tolerant gel buffer at 70W constant power for 2 hours. Gels were dried and subjected to autoradiography.

12. Fish Mapping of BARDl The cytogenetic location of BARDl was obtained with fluorescence in situ hybridization

(FISH) of normal human metaphase chromosome spreads with phage DNA pooled from three of the lambda clones (R12, R5 and R35). One microgram of DNA was labeled with biotin using DOP-PCR (Telenius et al, 1992) and subjected to FISH analysis as described elsewhere (Trask, 1997; Wise et al, 1997).

13. Preparation of Normal Breast cDNA Library

Total RNA was isolated from normal breast tissue obtained during reduction mammoplasty surgery and flash frozen. Approximately 1 gram pieces of tissue (containing fat, epithelium, stroma and normal vessels, etc.) was ground in 8 ml 4 M guanidinium isothiocyanate solution by a virtishear blender. The lysate was layered over 3 ml of a 5.7 M CsCl solution and centrifuged at 32K for 18 hours at 20°C in a Beckman SW4 IT rotor.

Total RNA pellets were resuspended phenol/chloroform extracted and reprecipitated. RNA pellets were resuspended in DEPC H₂O and concentration measured by spectrophotometry at OD₂₆₀.

Aliquots of total RNA (approximately 10 μg) were electrophoresed on 1.2% agarose formaldehyde denaturing gels to assess intact status of the 28S and 18S riboso al RNAs.

Total RNA from 3 separate patients was pooled (nB 63 10.6%, nB 52 45.6%, nB 62 43.9%). The total RNA samples were not treated with DNase I before isolation of poly A⁺ RNA. Poly A⁺ RNA was isolated by two passages over oligo dT Dynabeads, with regeneration of the beads in between isolation rounds.

Approximately 5 μg of poly A RNA was used to prepare the cDNA library The library was prepared in the pACT two-hybrid expression vector (Clontech, Palo Alto, CA), and then used in the yeast two hybrid screening method as detailed in section 1 above.

EXAMPLE II Yeast two-hybrid screening with the amino-terminal sequences of BRCAI

A cDNA sequence encoding the amino-terminal 304 residues of BRCAI was amplified by RT-PCR™ and inserted into the pASl-CYH2 expression vector (Haφer et al, 1993). The resultant plasmid (BR304/pASl-CYH2) encodes a hybrid protein containing the DNA-binding domain of GAL4 fused to BRCAI residues 1-304. Yeast cells of the Y190 reporter strain (Haφer et al, 1993) were then transformed in succession with the BR304/pAS-CYH2 plasmid and with an expression library of human B cell cDNAs fused to sequences encoding the GAL4 transactivation domain (Durfee et al, 1993).

By screening approximately 1 1 million library transformants, the inventors isolated 312 clones that co-activate the GAL4-responsive HIS3 and lacZ reporter genes of Y190. Forty-six of the isolates were found to interact specifically with BRCAI in a yeast two-hybrid mating assay that employed two irrelevant proteins (mouse p53 and human TALI) as negative controls (Harper et al, 1993). Nucleotide sequence analysis revealed that the 46 isolates represent twenty-six independent cDNA clones derived from sixteen distinct mRNAs. The candidate BRCAI -associated proteins encoded by these cDNAs are comprised of eleven novel polypeptides and five known proteins; the latter include TAFII70/80 (Genbank accession nos. L25444 and U31659), filamin (X53416), STAT3/APRF (L29277), UNPH (U20657), and a human homolog of the yeast GCN5 gene product (U57317).

The eleven novel polypeptides are BARDl (SEQ ID NO:2); and the genes encoding the TCL52 (SEQ ID NO:9), TCL163 (SEQ ID NO: 10), B223 (SEQ ID NO: 1 1), Bl 15 (SEQ ID NO: 12), BAP28 (SEQ ID NO: 13), B48 (SEQ ID NO: 14), B258 (SEQ ID NO: 15), BAP 152 (SEQ ID NO: 16), B123 (SEQ ID NO: 17) and B268 (SEQ ID NO: 18) polypeptides.

Each of the candidate proteins was also tested in a reciprocal yeast two-hybrid study in which residues 1-304 of BRCAI were expressed as a fusion protein with the GAL4 transactivation domain (TAD-BR304) and the candidate cDNA sequence was expressed as a fusion with the GAL4 DNA-binding domain (DBD-X). Three of the DBD-X hybrid polypeptides were capable of activating the reporter genes in the absence of TAD-BR304, obviating their analysis in the reciprocal two-hybrid assay. However, each of the other thirteen DBD-X hybrids registered as positive in this assay; that is, reporter gene activation occurred in the presence of the TAD-BR304 hybrid but not in the presence of control hybrids, such as TAD- TALI and TAD-SV40 large T antigen. EXAMPLE III Protein-protein interactions in mammalian cells

Additional tests were conducted to determine whether any of the candidate proteins interact with BRCAI in mammalian cells. Therefore, a mammalian expression plasmid was prepared which encodes GAL4-BR304, a protein containing the DNA-binding domain of GAL4 fused to BRCAI residues 1-304. In addition, expression vectors that encode each of the candidate BRCA 1 -associated proteins as hybrids with the VP16 transactivation domain were also prepared.

The mammalian version of the two-hybrid assay was then performed by transfecting human 293 kidney cells with a GAL4-responsivc reporter gene (G5LUC) and pairwise combinations of the appropriate expression vectors (Dang et al, 1991; Hsu et al, 1994).

Transcription of the reporter gene was evaluated by measuring the luciferase activity of lysates prepared from the transfected cells.

As illustrated in FIG. 1, expression of the GAL4-BR304 hybrid did not induce significant luciferase activity in transfected 293 cells (see lane 1). Likewise, expression of VP16-B202, a VP16-hybrid that contains sequences from one of the candidate BRCA1- associated proteins, also failed to activate transcription of the G5LUC reporter gene (lane 10). However, co-expression of GAL4-BR304 and VP16-B202 generated a large increase in luciferase activity to levels more than 30-fold greater than those found with either hybrid alone (lane 9). This suggests that the BRCAI and B202 moieties of the hybrid polypeptides interact stably with one another in mammalian cells. In contrast, pairwise expression of GAL4-BR304 with each of the other six VP16-fusion proteins did not yield a measurable increase in luciferase activity (lanes 3, 5, 7, 11, 13, and 15).

To date, fifteen of the sixteen candidate BRCAI -associated proteins have been tested for interaction with BRCAI in the mammalian two-hybrid system; all of these proteins, with the exception of B202, failed to associate with BRCAI in the mammalian assay. Co-immunoprecipitation studies were carried out to confirm that the BRCAI and B202 polypeptides interact in mammalian cells. Therefore, an expression plasmid was prepared that encodes HA-BR304, a polypeptide containing the amino-terminal tag:

MAYPYDVPDYASLRS, SEQ ID NO:8, appended to residues 1-304 of BRCAI .

A plasmid was also constructed for expression of FLAG-B202, a polypeptide that includes an amino-terminal tag with the FLAG epitope, MADYKDDDDKS: SEQ ID NO:3 (Hopp et al, 1988), and 177 residues encoded by B202.

Human 293 cells were co-transfected with different combinations of these expression plasmids and, as controls, plasmids that encode two helix-loop-helix transcription factors (El 2 or TALI) that are known to form stable heterodimers in vivo (Hsu et al, 1994). Two days after transfection the cells were lysed under mild conditions. Aliquots of each lysate were immunoprecipitated with either a rabbit antiserum raised against residues 183-304 of human BRCAI , the corresponding pre-immune serum, or a TALI -specific antiserum.

To determine whether the FLAG-B202 polypeptide was co-immunoprecipitated with HA-BR304, the precipitates were fractionated by SDS-PAGE, and the presence of FLAG-B202 was determined by immunoblotting with a monoclonal antibody (M5; Eastman Kodak) that recognizes the FLAG epitope. FLAG-B202 was co-immunoprecipitated with the BRCAl- specific antiserum, but not with the corresponding pre-immune serum or with an antiserum specific for TALI. Moreover, co-immunoprecipitation of FLAG-B202 was clearly dependent on the presence of HA-BR304 since it was not observed using lysates of ceils expressing FLAG- B202 alone. Therefore, a specific in vivo association between B202 and BRCAI can be demonstrated in mammalian cells by two independent procedures, the two-hybrid assay and co- immunoprecipitation analysis.

EXAMPLE IV The BRCAI -associated RING-domain (BARDl) nrotcin

The B202 clone, which contains a cDNA insert of ~1.0 kilobasepairs, represents five of the 46 isolates obtained in the yeast two-hybrid screen. An independent isolate (B230) contained a distinct but overlapping insert of 2.5 kilobasepairs. The composite cDNA sequence of 2,531 bp (SEQ ID NO:l) derived from B202 and B230 includes a large open reading frame with at least two potential initiator codons and encodes a protein with the sequence of SEQ ID NO:2. Translation from the first two initiation methionines (residues Ml and M26) would generate polypeptides of 777 and 752 amino acids, respectively. Residue 153 of SEQ ID NO:2 is denoted with the letter "X" to reflect a difference between the sequence of B202 and B230; the corresponding triplet in these cDNAs encodes a lysine (AAA) or glutamic acid (GAA) residue, respectively. Significantly, a cysteine-rich domain (residues 46-90) that matches the consensus sequence of the RING motif of BRCAI and the PML1 and BMI-1 oncoproteins is found near the amino-termini of these polypeptides.

The BRCAI -associated RING domain protein (designated BARDl ) also contains a centrally-located sequence comprised of three tandem ankyrin repeats (residues 427-525), a 33- amino acid motif found in a variety of different regulatory proteins (Bork, 1993). In addition, when the BLAST algorithm was used to screen protein databases with the remaining BARDl sequences on the carboxy-terminal side of the ankyrin repeats (Altschul et al, 1990), a significant homology with BRCAI (and only BRCAI) was uncovered.

Moreover, the homologous region of BRCAI corresponds to the phylogenetically- conserved sequence that lies near its carboxy-terminus (Sharan et al, 1995). Recently, Koonin et al. showed that this sequence bears a weak but significant homology with the carboxy- terminal regions of the mammalian 53BP1 protein, the yeast RAD9 gene product, and two putative proteins encoded by uncharacterized cDNA clones (Koonin et al, 1996). The homologous sequences are comprised of two tandem copies of the BRCAI carboxy-terminal domain (the "BRCT domain"), a newly recognized amino acid motif of unknown function (Koonin et al, 1996).

Although homology with 53BP1 was not detected in a conventional BLAST search of existing protein databases with the BARDl sequence, the similarity of their carboxy-terminal regions becomes apparent when each is independently aligned with the BRCT domains of

BRCAI. Within each of these proteins the levels of sequence identity between the first and second copies of the BRCT domain are modest; nevertheless, the homology between the tandem copies is illustrated when the core motifs of each, which consist of a relatively well-conserved stretch of 38 amino acids, are aligned with one another (Koonin et al, 1996). Thus, BARDl and BRCAI belong to a small family of proteins that harbor BRCT domains at their carboxy- termini. Within this family BARDl and BRCAI are especially related in that they also possess an amino-terminal RING motif (FIG. 2).

EXAMPLE V In vitro analysis of the BARDl /BRCAI interaction

To examine the binding properties of BARDl and BRCAI in vitro, cDNA sequences encoding the full-length polypeptides were inserted into the pSPUTK expression vector (Stratagene) along with a short amino-terminal tag containing the FLAG epitope (MADYKDDDDKS; SEQ ID NO:3). The resultant plasmids (BARDl/pSP6-FLAG and BRCAl/pSP6-FLAG, respectively) were then used as templates for coupled in vitro transcription/translation in rabbit reticulocyte lysates.

Radiolabeled full-length BARDl polypeptides were generated by in vitro translation in a rabbit reticulocyte lysate. An aliquot (0.2 ml) of the lysate was fractionated by electrophoresis on a SDS-10% polyacrylamide gel. Additional aliquots (10 ml) were incubated with purified GST-fusion proteins loaded onto glutathione-agarose beads. The washed beads were boiled in 80 ml of loading buffer, and equivalent aliquots of the eluants (40 ml) were fractionated by electrophoresis. The binding reactions were conducted with parental GST, GST-BR304, GST- TALI , GST-E47, GST-ATF4, GST-BR184, or GST-BRD304.

Translation of BARDl/pSP6-FLAG in the presence of [ Sjmethionine generated a radiolabeled BARDl polypeptide of -97 kilodaltons. Equivalent aliquots of the radiolabeled protein were then mixed with purified glutathione S-transferase (GST) or with purified GST- fusion proteins containing various segments of BRCAI or segments of the TALI, E2A, or ATF4 transcription factors. After a short incubation, the GST proteins of each mixture were absorbed to glutathione-agarose beads.

The radiolabeled BARDl polypeptide was retained on the beads by the GST-BR304 fusion protein (which contains BRCAI residues 1-304), but not by the parental GST polypeptide or by GST fusion proteins containing irrelevant sequences from TALI, E2A, or ATF4. Moreover, in vitro binding of BARDl was observed with the GST-BR184 fusion protein (which contains BRCAI residues 1-184) but not with the GST-D304 polypeptide (which contains BRCAI residues 183-304). These results suggest that BARDl and BRCAI polypeptides interact directly to form a stable protein complex in vitro, and that the interaction is mediated by sequences within the amino-terminal 184 residues of BRCAI .

Although in most of these assays the BARD1/BRCA1 interaction was evaluated using segments of one or both polypeptides, the ability of the full-length proteins to associate with one another was also examined. For this puφose, full-length BRCAI was generated by in vitro translation in a rabbit reticulocyte lysate containing [ Sjmcthionine, while full-length BARDl was produced by in vitro translation in an unlabeled reticulocyte lysate. The radiolabeled BRCAI lysate was then incubated with the unlabeled BARDl lysate or with an uncharged reticulocyte lysate, and equivalent aliquots of the mixture were subjected to immunoprecipitation with antisera specific for BRCAI, BARDl , or TALI, or with preimmune serum as a control, and fractionated on a SDS-6% polyacrylamide gel.

As now expected, the BRCAl-specific antiserum, but not the corresponding pre-immune serum, immunoprecipitated full-length BRCAI from the mixture along with a series of smaller degradation products. Significantly, the BRCAI polypeptides were also co-immunoprecipitated from the mixture with a BARDl -specific antiserum but not with an antiserum raised against TALI. Co-immunoprecipitation of BRCAI with the BARDl -specific antiserum was clearly dependent on the presence of BARDl, since it was not observed when radiolabeled BRCAI was mixed with an unlabeled reticulocyte lysate that did not contain in v ro-translated BARDl polypeptides. These results indicate that the full-length BARDl and BRCAI polypeptides can interact to form a stable protein complex.

EXAMPLE VI

Expression and chromosomal localization of the BARDl gene

Northern hybridization revealed two major BARDl transcripts (5.9 and 4.4 kilobases) in all the breast and ovarian cancer cell lines tested (ZR-75, T-47D, BT-483, Ovcar-3, Caov3, 2774, 2008). The chromosomal location of BARDl was determined by PCR™ amplification of a panel of monochromosomal hybrid DNAs with primers specific for BARDl (B202L and B202R; SEQ ID NO:6 and SEQ ID NO:7, respectively). A single human-specific band of 230 basepairs was seen in the hybrid containing a single human chromosome 2. The location of BARDl was further refined by mapping in the Genebridge panel of DNAs from whole genome radiation hybrids. This analysis placed BARDl in the distal region of human chromosome 2q, 3.56 cR distal to D2S143 (lod >3.0) and flanked by D2S295 distally.

EXAMPLE VII

The interacting regions of BARDl

The sequences of BARDl that interact with BRCAI should be located within the shared segment encoded by both B202 and B230 (amino acid residues 8-31 1) - the two independent BARDl cDNA clones obtained in the yeast two-hybrid screen (see FIG. 2). These sequences were further localized by mammalian two-hybrid studies in which smaller segments of BARDl

(FIG. 2) were expressed as fusion proteins with the VP16 transactivation domain.

As illustrated in FIG. 3, VP16-fusion proteins containing segments NB (residues 26-202) and NE (residues 26-142), both of which encompass the RING domain of BARDl (residues 46- 90), readily activated the GAL4-responsive reporter gene when expressed in the presence of GAL4-BR304, the GAL4-fusion protein containing residues 1-304 of BRCAI (lanes 3 and 5). BRCAI association was also observed in reciprocal two-hybrid assays in which the NB and NE segments of BARDl were expressed as GAL4-fusion proteins and tested for interaction with VP16-BR304. Therefore, the interaction with BRCAI is mediated by sequences in the vicinity of the BARDl RING domain.

EXAMPLE VIII The interacting regions of BRCAI

The in vitro binding studies showed that the interacting sequences of BRCAI reside within its amino-terminal 184 residues. These sequences were further localized by mammalian two-hybrid analysis with VP16-NE, a hybrid polypeptide containing the VP16 transactivation domain fused to the NE segment of BARDl (residues 26-142). VP16-NE was tested for interaction with a panel of GAL4-hybrid proteins containing different amino-terminal segments of BRCAI .

As shown in FIG. 4A, the BR147 (residues 1-147) and BR101 (residues 1 -101) segments, both of which encompass the RING motif of BRCAI (residues 20-68), retain the ability to interact with BARDl (lanes 3 and 5). However, BARDl -association was not achieved with a smaller segment that also includes the intact RING domain (BR71 , residues 1-71) (FIG. 4A, lane 7), despite the fact that the GAL4-BR71 hybrid protein was expressed at levels comparable to those of GAL4-BR147 and GAL4-BR101, as judged by western analysis with the M5 anti-FLAG monoclonal antibody.

The same result was obtained from a reciprocal two-hybrid study in which GAL4-BR304 was tested for binding with VP16-hybrids containing different segments of BRCAI (FIG. 4B). Thus, although association between BARDl and BRCAI is mediated by sequences in the immediate vicinity of their respective RING motifs, the RING domain of BRCAI is not by itself sufficient to mediate the interaction.

EXAMPLE IX Tumorigenic missense mutations of BRCAI

The tumorigenic missense mutations of BRCAI were analyzed in regard to their effect on the BARDl/BRCAl interaction. Since the C61G and C64G mutations eliminate conserved zinc-binding cysteines from the RING motif of BRCAI, the inventors sought to determine the effect of these mutations on BARDl/BRCAl association. Therefore, C61G and C64G substitutions were incoφorated into the BR304 segment of BRCAI by site-directed mutagenesis of the corresponding cDNA fragment. Expression plasmids were then constructed to encode GAL4-BR304 hybrid polypeptides that contain either the C61G (GAL4-BR304-C61G) or C64G (GAL4-BR304-C64G) lesion.

As illustrated in FIG. 5 A, the wild-type GAL4-BR304 hybrid (lane 3), but not its mutant derivatives (lanes 5 and 7) interacted with BARDl in the mammalian two-hybrid assay, despite the fact that all three versions of the GAL4-BR304 polypeptide were expressed at comparable levels, as judged by western analysis with the M5 anti-FLAG monoclonal antibody.

The effect of the missense mutations on BARDl/BRCAl association was also evaluated by co-immunoprecipitation studies of mammalian cell lysates (FIG. 5B). Thus, 293 cells were co-transfected with expression plasmids encoding FLAG-B202 (described above) and either a wild-type or mutant derivative of FLAG-BR304, a BR304 polypeptide with an amino-terminal tag containing the FLAG epitope (MADYKDDDDKS; SEQ ID NO:3). Two days later the cells were lysed and aliquots of each lysate were immunoprecipitated with either the BRCA1- specific antiserum or the corresponding pre-immune serum.

To determine whether FLAG-B202 polypeptides were co-immunoprecipitated with FLAG-BR304, the immunoprecipitates were fractionated by SDS-PAGE, and the presence of FLAG-B202 was determined by immunoblotting with the M5 anti-FLAG monoclonal antibody. FLAG-B202 was co-immunoprecipitated with the BRCA 1 -specific antiserum when expressed in the presence of wild-type FLAG-BR304 (FIG. 5B; lane 2). In contrast, however, co- immunoprecipitation did not occur when FLAG-B202 was expressed with FLAG-BR304 derivatives containing either the C61G or C64G substitutions (lanes 4 and 6).

Together, the mammalian two-hybrid and co-immunoprecipitation studies demonstrate that the C61G and C64G mutations prevent formation of an in vivo protein complex between BRCAI and BARDl.

EXAMPLE X Genomic structure of BARDl

To obtain the genomic DNA encoding BARDl, lambda phage and cosmid libraries of human genomic or YAC DNA (YACs 81 Od 12 and 964g6) were first screened by hybridization with fragments of BARDl cDNA (Example IV, above). Eleven hybridizing lambda clones and two hybridizing BAC clones were subjected to nucleotide sequence analysis with oligonucleotide primers derived from BARDl cDNA sequence (Table 4, below). This analysis resulted in nine large contigs of genomic sequence (SEQ ID NO: 122, containing exon 1 and 5' untranslated region (UTR), which likely contains the BARDl promoter; SEQ ID NO: 123, containing exon 2 and exon 3; SEQ ID NO: 124, containing exon 4; SEQ ID NO: 125, containing exon 5; SEQ ID NO:126, containing exon 6; SEQ ID NO: 127, containing exon 7; SEQ ID NO: 128, containing exon 8; SEQ ID NO: 129, containing exon 9; and SEQ ID NO: 130, containing exon 10 and exon 11, plus 3' UTR; from the 5' end of the gene to the 3' end of the gene, respectively), which revealed that the BARDl coding sequences are derived from eleven exons distributed over at least 65 kilobases of genomic DNA.

The chromosomal origin of BARDl was then established by fluorescence in-situ hybridization (FISH) of normal human chromosomes with subclones containing BARDl genomic sequences. FISH analysis localized BARDl to bands 2q34-35, consistent with the

BARDl mapping data obtained previously with the Genbridge panel of whole genome radiation hybrid DNAs (Example VI, above).

TABLE 4 PCR™ Primers for the Amplification of BARDl Sequences from cDNA Template

Bp Forward Primer (5'>3') SEQ ID # Reverse Primer (5 >3 ) SEQ ID # PCR™ Ann. Prod. Size Temp.

44 GCGAGGAGCCTTTCATCCGA 57 CGAGCGCGGCGCGACTGT 58 154 59

149 ATGGAACCGGATGGTCGCGGT 59 TCTTCAAGTCTTGTATCCAGGC 60 205 59

145 CGCCATGGAACCAAATACA 61 TCTTCAAGTCTTGTATCCAGGC 60 209 57

340 GCCTGGATACAAGACTTGAAG 62 TTGTAGACGTCCTCCTGAACC 63 306 57

551 AAAGCTTCAGTGCAAACCCA 64 TCCAGATCTTGCAGAAGCC 65 132 53

638 CAGATGTTTCTGAGAGGGCT 66 ATTCCTCTTTGGAGTCAAATTC 67 138 55

734 GAGGCAGAAAAAGAAGATGGT 68 AGGAGCCACTTGCTAGTAAG 69 136 55

855 ATGGTGAAATAGACTTACTAGC 70 GCAGACCTTCTCAGGAGTC 71 149 55

946 AAGAGCAGGAATGAAGTAGTG 72 CTCCACTGGTGCTCAGAATG 73 163 55

1103 AGTGGAGATTTTGTTAAGCAA 74 AGGTGGTGTAGGTGGTGAA 75 159 51

1250 GGTACACCACCTTCTACATT 76 GTCTCTCCTCTATGATTTCTT 77 113 53

1311 CAATGAAGCTGTTGCCCAA 78 GTCTTTAACATTTGGATCACT 79 137 51

1427 AGTGATCCAAATGTTAAAGAC 80 CCCATTCTTGGCTGCATC 81 162 51

1550 CAAAATGACTCACCACTTCAC 82 ATCGACAGGCCGCAGACC 83 120 55

1661 CCTGTCGATTATACAGATGAT 84 AACATGAGTTACTGTACTGTC 85 234 57

1862 TATACTGAGTTTGACAGTACAG 86 CATACTTTTCTTCGTAGACATG 87 146 55

1976 B230-G:TGGGTAAAAGCATGTCTACGA 88 TCAGCGCTTCTGCACACAGT 7 126 55

2093 B230-H:GGATGCTACTTCTATTTGTG 89 GAGTCACGTCACTGTCTG 90 134 51

2179 B230-TS:CCTCAGTAGAAAGCCCAAGC 91 GCCCCTGCCGAACCCTCTC 92 154 57

2215 B230-US:GAGAGGGTTCGGCAGGGC 93 TTCAATTTCAAATGTTCATCTGGT 94 124 57

EXAMPLE XI BARDl mutation screening

The inventors used SSCP (Orita et al, 1989a; Orita et al, 1989b) to screen genomic DNA or cDNA from 48 breast tumors, 58 ovarian tumors, 60 uterine cancers (primarily endometrial), six breast cancer lines and six ovarian cancer lines and germline DNA or lymphoblastoid-derived cDNA from 67 breast/ovarian cancer patients with no observed alterations in BRCAI or BRCA2 for genetic alterations in BARDl . SSCP was performed as described elsewhere (Orita et al, 1989; Orita et al, 1989) with oligonucleotide primers for BARDl with cDNA or genomic DNA as shown in Table 4 (Example X above) and Table 5 (below).Variant bands were excised from the SSCP gel, subjected to a second round of amplification and sequenced.

TABLE 5 PCR™ Primers for the Amplification of BARDl Sequences from Genomic DNA Template

Exon Forward Primer (5'>3') SEQ ID # Reverse Primer QID# PCR™ Size Ann.Temp.

(5'>3') (bp) (°C)

I GCGAGGAGCCTTTCATCCGA 57 CGAGCGCGGCGCGACTGT 58 154 59

I ACAGTCGCGCCGCGCTCGA 95 CAGAAACTGTGCGACCCGTG 96 107 59

II AGATGTTTATCTAACAATGACTC 97 AGTTGTACTATATACATCAAACC 98 146 55

III ATTCTGCTGAATGGGTTGCTT 99 TAACTAAGAGAGATAGGGATAG 100 226 55

IVa GGAGCTCCATGTGGGAGCAA 101 AACATCTGCAGGAGGACTTGG 102 270 59

IVb CAGATGTTTCTGAGAGGGCT 66 ATTCCTCTTTGGAGTCAAATTC 67 138 55

IVc GAGGCAGAAAAAGAAGATGGT 68 AGGAGCCACTTGCTAGTAAG 69 136 55

IVd ATGGTGAAATAGACTTACTAGC 70 GCAGACCTTCTCAGGAGTC 71 149 55

IVe AAGAGCAGGAATGAAGTAGTG 72 CTCCACTGGTGCTCAGAATG 73 163 55

IVf AGTGGAGATTTTGTTAAGCAA 74 AGGTGGTGTAGGTGGTGAA 75 159 51

IVg GGTACACCACCTTCTACATT 76 TCTGAGATGGTATTTCAGAGT 103 170 53

V TGCTTTTTAATTTCCATTTTGTTC 104 AAGAACTGTAAAACACAGAAAGA 105 163 55

VI TGCTCTTTCTTATCACTTCTTTC 106 CTTGACTCAAGAATATAGGTCC 107 278 57

VII TTGAGTCGAGTCACACATTTGA 116 CTATTATGTTCCTTTCATAACCA 117 233 55

VIII TAATGTCTTTGTCTAGTCGTCTAA 1 18 GGTAGTTCTCCAAAAGGATCA 119 264 55

IX GAGTTATAAGAAGCAGGCCAA 120 ATTTCTTAATTCTCTCAAATCCAA 121 199 55

X TAGTGCTCACTTGATACTTAGT 108 CATAATAAGAACAATGAAAGTTGT 109 187 55

XIa TTGATCTGCCTTTAACAAATG 1 10 GCCCCTGCCGAACCCTCTC 92 296 57

Xlb GAGAGGGTTCGGCAGGGC 93 TTCAATTTCAAATGTTCATCTGGT 94 124 57

A. BARDl Mutations

When 58 ovarian tumors were analyzed, one (ov61) was found to harbor a missense mutation within BARDl that resulted in a glutamine to histidine (CAG to CAC; Q564H; SEQ ID NO:32 (nucleic acid) and SEQ ID NO:33 (amino acid)) change between the ankyrin repeats and the BRCT domain (FIG. 6). This patient was a woman of African-American origin who was diagnosed at age 73 with a clear cell adenocarcinoma of the ovary (stage 3A) and a synchronous infiltrating lobular carcinoma of the breast. Only the mutant allele was detected in the ovarian tumor cDNA from this individual, indicating that the wild-type transcript was either expressed at undetectable levels or was completely absent. The absence of detectable wild-type fragnments indicates that the ovarian carcinoma cells of the patient were devoid of normal BARDl polypeptides. At the time of hysterectomy six years earlier this patient had been diagnosed with an incidental stage IA endometrial clear cell tumor. It is likely that these represent two separate primary tumors of the endometrium and ovary since the initial endometrial tumor was a small focus or carcinoma confined to an endometrial polyp.

Genomic DNA extracted from paraffin-embedded tissue obtained from the three primary tumors, as well as from benign uterine tissue, were examined from this patient. SSCP analysis identified the variant allele in all samples, including normal uterine tissue, indicating that this alteration was of germ-line origin. Moreover, the wild-type allele of BARDl was absent from the genomic DNA of the ovarian tumor, explaining the loss of wild-type BARDl transcripts. Both the wild-type and mutant alleles were detected in genomic DNA of both the endometrial and breast cancers; however, histological examination indicated that a significant proportion of normal tissue had infiltrated these tumor specimens. This contaminating normal tissue could have obscured the ability to detect loss of the wild-type allele in the breast and endometrial tumors. The high degree of infiltrating normal tissue also rendered microdissection of tumor tissue from these samples impossible.

The Q564H missense alteration was not seen in over 300 individuals examined (>600 chromosomes), suggesting that this alteration is not a polymorphism. Since this patient was African American, an additional 30 African individuals (60 chromosomes) were screened for this variant. The variant was not detected, indicating that this change is unlikely to be a polymoφhism private to the African population. In light of the interaction of BARDl with BRCAI, and the observed loss of the wild-type BARDl allele in the ovarian tumor, the germline missense alteration, Q564H, may have resulted in predisposition to endometrial, breast and ovarian cancer. Additionally, since the glutamine 564 residue is conserved in the mouse sequence, it is likely to be of some importance.

A second ovarian tumor (ov208) harbored a variant within the BRCT domain (FIG. 6). This tumor was obtained from a 16 year old Caucasian female and was diagnosed as a small cell carcinoma of the ovary with neuroendocrine features. The genetic alteration in this tumor resulted in an arginine to cysteine change at amino acid 658 (R658C; SEQ ID NO:36 (nucleic acid) and SEQ ID NO:37 (amino acid)). This alteration was only seen in one other sample; an enodmelrial adenocarcinoma obtained from a 67 year old woman (utl4). This change was not seen in any other DNAs examined (>600 chromosomes). The alteration in ov208 was determined to be of germ-line origin. In this ovarian tumor sample the wild-type allele was detected, but it is not known if this was derived from contaminating normal tissue present in this tumor sample, and therefore whether the wild-type allele had been lost from the tumor itself.

As a result of the Q564H finding, the Inventors became interested in the involvement of BARDl in the development of uterine tumors and examined an additional ten for alterations. One had a serine to asparagine change at amino acid 761 in the BRCT domain (S761N; SEQ ID NO:34 (nucleic acid) and SEQ ID NO:35 (amino acid)). This alteration (S761N) occurs in the 3' end of the BRCT domain, and lies within the 30 amino acid core motif of BRCT domains adjacent to the invariant tryptophan residue. The wild-type allele was also detected in this tumor.

No mutations were seen in the germ-line DNA of the 67 breast/ovarian cancer patients.

None of these had reported BRCA1/2 mutations, although none have been screened fully for such mutations. All these patients, except one, had a family history of cancer (43 breast/ovarian, 22 breast and 2 ovarian).

Alterations of BARDl in sporadic breast and ovarian tumors appear to be a rare event.

This observation is correlated with the fact that 2q, the location of BARDl, has not been reported to undergo significant LOH in breast/ovarian cancer. However, it is possible that BARDl, like BRCAI is involved in tumorigenesis through other mechanisms such as alterations in transcript level (Thompson et al, 1995). The low frequency of genetic alterations in BARDl in breast and ovarian tumors is similar to findings for BRCAI and BRCA2. In the case of BRCAI, no genetic alterations have been detected in sporadic breast tumors. However, 10% of ovarian tumors harbor somatic mutations that result in protein truncations. In these tumors there is also loss of the wild-type allele (Hosking et al, 1995; Merajver et al, 1995).

In the case of BRCA2, four independent studies collectively identified two sporadic missense alterations and one somatic truncating mutation in 281 primary breast cancers and two somatic alterations in 185 ovarian carcinomas (Lancaster et al, 1996; Miki et al, 1996; Phelan et al, 1996; Takahashi ef o/., 1996; Teng et al, 1996; Weber et al, 1996). The alteration in one of the ovarian carcinomas was an "A" insertion in one poly(A) tract of the gene due to a mutation in the DNA mismatch repair gene hMSH2 (Takahashi et al, 1996). The second ovarian carcinoma had a missense mutation of unknown significance.

Despite the rarity of the BARDl alterations in tumors of the breast, ovary and endometrium, loss of its wild-type allele in the ovarian tumor ov61 provides evidence for a tumor-suppressor role (Haber and Harlow, 1997) for BARDl in the prevention of these cancers. The BARDl alteration in this tumor, Q564H, occurred between the BRCT domains and the ankyrin repeats. The function of the BRCT domains of BARDl is unknown, although in the case of BRCAI this region has been shown to have transactivational function (Chapman and Verma, 1996; Montiero et al, 1996).

The homology of the BRCT domain with domains in proteins such as RAD9, XRCC1 and RAD4, which are involved in cell cycle checkpoint functions in response to DNA damage (Bork et al, 1997; Callebaut and Mornon, 1997; Koonin et al, 1996), and the recent finding that BRCAI associates with another DNA repair protein, RAD51 (Scully et al, 1997), suggests that it may be important in mediating repair of DNA damage. Together with BRCAI, BARDl may be involved in cell cycle checkpoint control in response to DNA damage. The inventors have recently found further evidence for a common role for these two proteins by demonstrating thta BRCAI and BARDl co-localize in nuclear dots in the S phase of the cell cycle (Example XIV below). 90% of germ-line alterations in BRCAI and all germ-line alterations in BRCA2 that predispose to breast/ovarian cancer result in protein truncation (Shattuck-Eidens et al, 1995; Stratton, 1996). However, in the case of p53, missense mutations are the most common alteration in human breast cancer as they are in other tumors. The recently isolated PTEN/MMACl gene, which is altered in Cowden disease (Liaw et al, 1997) as well as in sporadic brain, prostate and kidney cancers (Li et al , 1997; Steck et al, 1997), has been reported to harbor both nonsense and missense mutations. These are predicted to disrupt the protein tyrosine/dual-specificity phosphatasc domain of the PTEN/MMAC gene product.

B. BARDl Polymorphisms

Seven polymoφhic sites were detected within BARDl . A description of BARDl polymoφhic sites and variants is shown in FIG. 6 and described below.

One polymorphism was detected in the first exon, 5' to the region encoding the RING domain. This mutation is a proline to serine change at amino acid 24 (P24S; SEQ ID NO:20 (nucleic acid) and SEQ ID NO:21 (amino acid)).

A second polymoφhism was detected as a result of sequencing two cDNA clones that differed at nucleotide 531. This mutation is a lysine (AAA) to glutamic acid (GAA) change at amino acid 153 (SEQ ID NO:22 (nucleic acid) and SEQ ID NO:23 (amino acid)).

Primers C/CAS amplify a region located between the RING domain and the first ankyrin repeat. Two polymoφhisms (polymoφhisms three and four) were seen within this region. The third polymoφhism is a C to G transversion at nucleotide 1 121 , generating a silent polymoφhism within a threonine codon (CCG to CGG; amino acid 351 ; SEQ ID NO:24

(nucleic acid) and SEQ ID NO:25 (amino acid)).

The fourth polymoφhism was a deletion of seven amino acids (PLPECSS) between amino acids 358 and 364 (SEQ ID NO:26 (nucleic acid) and SEQ ID NO:27 (amino acid)).

When individuals that were not selected because of a family history of breast/ovarian cancer, were examined, this deletion was seen in 2/68 individuals from the CEPH (Centre du Polymorphisme Humain) but was not detected in 40 other Caucasian individuals ascertained in the United States. This deletion appeared to be in linkage disequilibrium with the "G" allele at nucleotide 1121. This deletion was only seen in 2/216 unrelated Caucasian chromosomes where there was no significant family history of breast ovarian cancer, but was far more frequent in Africa as it was seen in 1/15 chromosomes. This accounts for its higher frequency in African-American women and in tumors from this population in general.

Interestingly, both the MCF7 cell line and the PE04 ovarian cancer cell line harbored this deletion. In both these cell lines both alleles were expressed. MCF7 was developed from a pleural effusion of a 69 year old Caucasian woman with a malignant mammary adenocarcinoma (Soule et al, 1973). PEO4 was developed from the peritoneal ascites of a Caucasian woman with an a poorly differentiated serous adenocarcinoma (Langdon et al, 1988). An African-American woman who developed ovarian endo etrioid adenocarcinoma at the age of 68 was homozygous for this deletion. However, since the frequency of this deletion is 0.067 in Africans, the frequency of homozygotes is 0.005 in African populations. The frequency of a homozygote in African-Americans would be expected to be lower than this, so that within the sample set of DNA samples from approximately 100 African- American individuals, detection of one homozygote is not an impossibility.

A fifth polymoφhism was seen in the third ankyrin repeat, and resulted in a valine to methionine change at amino acid 507 (V507M; SEQ ID N0:28 (nucleic acid) and SEQ ID NO:29 (amino acid)).

A sixth polymoφhism was located between the ankyrin repeats and the BRCT domain. This results in a cysteine to serine change at amino acid 557 as a result of a G to C transversion (C557S; SEQ ID NO:30 (nucleic acid) and SEQ ID NO:31 (amino acid)). This polymorphism was also seen in the BT474 breast cancer cell line (Lasfargues et al, 1978).

A seventh polymoφhism was located in the BRCT domain. This results in a serine to asparagine change at amino acid 761 (S761N; SEQ ID NO:38 (nucleic acid) and SEQ ID NO:39 (amino acid)). It is also possible that this alteration occurs at a much lower frequency that would be more indicative of a mutation than a polymorphism. However, gene deletions do not necessarily account for disease or cancer susceptibility. For example, a polymorphic stop codon within the 3' end of the coding sequence of BRCA2 results in loss of the 93 most terminal amino acids (Lys3326ter) with as yet no described deleterious effect (Mazoyer e/ al, 1996).

EXAMPLE XII

Other BRCAl-interacting Clones:

A. Clones Isolated From a Breast cDNA Library Four additional genes which encode proteins that interact with BRCAI were detected in the breast cDNA library using the yeast two-hybrid screening assay described in Example I above. The genes isolated were designated BE2 (SEQ ID NO:40 (nucleic acid) and SEQ ID NO:41 (amino acid)), BE 14 (SEQ ID NO:42 (nucleic acid) and SEQ ID NO:43 (amino acid)), BE31 (SEQ ID NO:44 (nucleic acid) and SEQ ID NO:45 (amino acid)) and BE445 (SEQ ID NO:46 (nucleic acid) and SEQ ID NO:47 (amino acid)).

BE2 encodes a 1.25 kb transcript in spleen, prostate, testes, small intestine, colon, and ovary. An additional transcript of approximately 1.0 kb is also seen in testes. It is also transcribed in some breast/ovarian cancer lines (Table 6, below). BE14 encodes a 4.4 kb transcript in testes.

TABLE 6 BE2 Expression in Breast and Ovarian Cancer Cell Lines

Type Cell Line Name ATCC # BE2 expression

Breast Cancer BT-474 HTB 20 -

BT-483 HTB 121 -

MDA-MB-134 VI HTB 23 -

MDAOMB-361 HTB 27 +

Ly-2 -

MCF-7 HTB 22 -

T-47D HTB 133 -

ZR-75-1 CRL 1500 -

BT-20 HTB 19 -

MDA-MB-231 HTB 26 +++

MDA-MB-436 HTB 130 +

MDA-MB-453 HTB 131 -

MDA-MB-468 HTB 132 -

MDA-MB-435S HTB 129 +++

SCC 38 +

SCC 70 +

BT-549 HTB 122 ++

SCC 202 -

SCC 712 -

SCC 1007 -

Ovarian Cancer 2008 .

2774 -

CaOv-3 HTB 75 +++

OVCAR-3 HTB 161 -

PA1 CRL 1572 +++

PE04 ++

SKOV-3 HTB 77 -H-

SW626 HTB 78 +

UCI 101 -

UCI 109 -

SCC 60 ++

SCC 1426 +

SCC 1159 ++

B. Genomic Mapping of Additional BRCAI Binding Clones

The BE2 gene was mapped with gene-specific primers and genome-wide radiation hybrids to l lp!5, the locale of a tumor suppressor gene for breast, ovarian and lung cancer (Winqvist et al, 1993). The possibility exists that this is the tumor suppressor gene that maps to this location.

The BE 14 gene was mapped with gene-specific primers and genome- wide radiation hybrids to chromosome 3q. This gene encodes a 4.4 kb transcript that we have only seen in testis. Like BRCAI, BRCA2 and BARDl , this gene is transcribed in breast cancer cells that have been starved by treatment with charcoal-stripped fetal calf serum and then supplemented with estrogen (Example XIII below). This suggests that all these genes are estrogen responsive, or are induced after the cells have been signaled to proliferate by signals created as a result of estrogen binding the estrogen receptor. This may have implications relating to the therapeutic aspects of these genes.

The B123 gene has been localized to 17pter, the locale of a tumor suppressor gene for breast cancer (Cropp et al, 1990; Lindblom et al, 1993).

EXAMPLE XIII Estrogen Responsiveness of BRCAI. BRCA2 and BARDl

A. Methods 1. Cell Culture

The previously characterized breast cancer cell lines BT-483 (Lasfargues et l, 1978) and MCF-7 were obtained from the American Type Culture Collection (ATCC No. HTB121 and HTB22). BT-483 cells were routinely cultured in RPMI 1640 media containing phenol red, 2 mM glutamine and IX antibiotic/antimycotic solution (Life Technologies, Gaithersburg, MD) supplemented with 20% fetal calf serum (FCS) (Life Technologies) and 10 μg/ml bovine insulin (Sigma, St. Louis, MO) in a humidified atmosphere containing 5% C0₂. Cells were subculturcd bi-weekly by trypsinization and the media was renewed every 2-3 days. MCF-7 cells were routinely cultured in IMEM (Improved Minimal Essential Media) containing phenol red, and 2 mM glutamine (Biofluids) supplemented with 10% FCS.

Hormone reagents 17 β-estradiol, progesterone, and trans 4'-hydroxytamoxifen were obtained from Sigma. The anti-estrogen ICI 182,780 was obtained from Alan Wakeling (ICI Pharmaceuticals). Stock solutions of each steroid were prepared in absolute ethanol and diluted directly into media.

The hormone stimulation procedure was an adaptation of the procedure described elsewhere (May and Westley, 1986). Experimental media for BT-483 cells consisted of phenol red free RPMI 1640 (Life Technologies) supplemented with 2 mM glutamine, 20% CCS, lOμg/ml bovine insulin and IX antibiotic/antimycotic solution. BT-483 cells were plated at a density of 3 x 10⁶ cells per T75 flask (Costar) in phenol red containing media. At 70-80% confluency, cells were depleted of steroids as previously described (May and Westley, 1986). Experimental media for MCF-7 cells was phenol red free IMEM (Biofluids) supplemented with 2 mM glutamine, 5% CCS and IX antibiotic/antimycotic solution. Cells were plated at 5 x 10⁶ cells per 150 mm plate (Corning) in phenol red free IMEM for 5-6 days before the refeeding with fresh media containing steroids at defined concentrations. Fetal calf serum used in hormone studies (CCS) was stripped of endogenous estrogens with dextran coated charcoal as described elsewhere (May and Westley, 1986). Dextran T-70 was obtained from Pharmacia, and acid washed, neutralized activated charcoal from Sigma.

Cycloheximide was obtained from Sigma and diluted in water to a stock concentration of 50 mM. Cycloheximide was added to culture media at a concentration of 50μM for 1 hour prior to the addition of 10 nM estradiol or 0.01% ethanol. Trypan blue was obtained from Sigma and the exclusion assay performed according to the manufacturer's protocol.

Analysis of the estrogen and progesterone receptor content of the BT-483 cell line was performed in parallel with a reference T-47D breast cancer cell line by Dr. David Zava (Aeron Biotechnology, Inc., San Leandro, California).

2. RNA Extraction and Northern Blotting

RNA was extracted from cells with guanidinium isothiocyanate as described elsewhere

(Chirgwin et al, 1979). Cytoplasmic RNA v/as isolated from BT-483 monolayers by a combination of NP-40 lysis and mechanical disruption (Sambrook et al, 1989) before the addition of lysates to guanidinium isothiocyanate. Total RNA from breast cancer cell lines was subjected to electrophoresis and blotted as described (Sambrook et al, 1989). Northern blots were hybridized separately with probes for BRCAI and BRCA2 and 18S. Since total RNA was electrophoresed and transferred for these blots, the 18S RNA levels accurately reflect the amount of total RNA loaded per lane. The probe for BRCAI was a 620 bp gel purified PCR™ product obtained with oligonucleotide primers 4L and 4R (5'-TACCCTATAAGCCAGAATCCA-3' and 5'-GGCAAACTTGTACACGAGCA-3'; SEQ ID N0:1 12 and SEQ ID N0:113, respectively) that amplified base pairs 4506-5126 of the published sequence (Miki et al, 1994). The BRCA2 probe was obtained by PCR™ amplification of genomic DNA with oligonucleotide primers 5'-GGTACTAGTGAAATCACCAGT-3' and 5'-GTGAATGCGTGCTACATTCAT (forward; SEQ ID NO:l 14 and reverse; SEQ ID NO: l 15, respectively) spanning base pairs 4880-5979 in exon 11 of the Genbank sequence (Accession # U43746, Tavtigian et al, 1996). The 18S and 36B4 probes were obtained from the American Type Culture Collection (ATCC #77242 and # 65917). Probes were labeled by random hcxanucleotide extension (Feinberg and Vogelstein, 1983) with ³²P dCTP (Amersham).

Blots were hybridized at 42°C in 50% formamide solution containing dextran sulfate (Oncor) for 48 hours and subjected to a final wash in 0.5X SSC, 0.1% SDS at 65°C. Hybridization signals were quantitated by direct exposure to a Phosphorlmager screen using Imagequant software supplied by the manufacturer (Molecular Dynamics). BRCAI and BRCA2 were exposed to the Phosphorlmager screen overnight and then exposed to x-ray film; 18S was exposed for 20 minutes to the PI screen and 2 hours to film.

B. Induction of BRCAI and BRCA2 Expression in Breast Cancer Cells Surges of the steroid hormones estrogen and progesterone occur during puberty (Drife,

1986) , the menstrual cycle (Longacre and Bartow, 1986), and pregnancy (King, 1993). These surges profoundly change the proliferation, differentiation and architecture of the breast ductal epithelium from which the most common form of breast cancer arises (Shi et al, 1994). Ductal carcinomas that are estrogen receptor positive depend on estrogen as an adjuvant to uncontrolled growth (King, 1993); however, these tumors are more differentiated, have a better prognosis (McGuire et al, 1992) and are more likely to regress with antiestrogen therapy than are estrogen receptor negative tumors. Breast tumors from women less than 40 years old have a higher rate of proliferation, are more aggressive and are more likely to be estrogen receptor negative than tumors from postmenopausal women (Marcus et al, 1994).

Estrogen modulates growth and differentiation of human breast epithelium (Drife, 1986); however, the exact pathway by which it exerts its proliferative effects has not been elucidated. Estrogen combines with the estrogen receptor to modulate the transcription of a specific subset of genes that include autocrine and paracrine polypeptide growth factors such as IGF-1 , TGF-alpha, and PDGF (Kasid and Lippman, 1987), the progesterone receptor (Horwitz and McGuire, 1978) and oncogenes such as c-myc (Dubik et al, 1987). It has been previously demonstrated that steroid hormones regulate BRCAI expression in human breast cancer cell lines (Spillman and Bowcock, 1995; Gudas et al, 1995). In vivo data for murine BTCA1 also demonstrates that the highest levels of BRCAI expression are observed in rapidly proliferating cells and in tissues that are sensitive to steroid hormones, such as the mammary gland (Marquis et al, 1995 and Lane et al, 1995).

The effect of steroid hormones on BRCAI and BRCA2 mRNA expression was examined in the estrogen receptor positive breast cancer cell lines BT-483 and MCF-7. BT-483 cells were cultured in estrogen depleted phenol-red free media for 5 days before being switched to media containing 17 β-estradiol and/or progesterone for an additional five days. The effect of estrogen or progesterone on BRCAI and BRCA2 mRNA expression in BT-483 cells were performed in triplicate and BRCAI and BRCA2 expression was quantified relative to the ethanol control.

Expression of both BRCAI and BRCA2 mRNAs was suppressed in cells cultured in steroid depleted media. A striking elevation of BRCAI and BRCA2 steady-state mRNA levels could be seen after five days of estrogen stimulation. In addition to the major BRCAI transcript of 7.8 kb, an additional minor transcript of approximately 4 kb was also induced by estrogen in a similar fashion. Estrogen upregulated BRCAI expression by approximately 17 fold and BRCA2 expression by approximately 50 fold. Similar results were seen in MCF-7 cells after severe serum deprivation. A classic effect of estrogen on breast cancer cells is its ability to increase expression of the progesterone receptor (Horwitz and McGuire, 1978). In BT-483 cells estrogen acts via an active estrogen receptor to induce both progesterone receptor mRNAs and protein; however, progesterone alone failed to induce BRCAI or BRCA2 mRNA expression in BT-483 and MCF-7 cells and the combination of estrogen and progesterone was neither synergistic nor completely antagonistic.

Both the BRCAI and BRCA2 steady-state mRNA levels are both substantially elevated after estrogen treatment in the BT-483 and MCF-7 breast cancer cell lines. The finding that BRCA2 mRNA levels were also elevated by estrogen was initially surprising. BRCA2 mutations are thought to contribute to a significant proportion of male breast cancers (Wooster et al, 1994) in addition to causing female breast cancers. Mutations in the androgen receptor have been shown to be responsible for some cases of male breast cancer (MacLean et al, 1995), and the effect of the steroid hormone testosterone on the regulation of BRCAI and BRCA2 mRNA levels is not known. However, estrogen may regulate BRCAI and BRCA2 in male breast cancers as well, because male breast cancers are more likely to be estrogen receptor positive than female breast cancers (Hecht and Winchester, 1994). In terms of histology, female and male breast carcinomas are indistinguishable (Hecht and Winchester, 1994).

The BT-483 breast cancer cell line was derived from a 23 year old woman with breast cancer (Lasfargues et al, 1978). BT-483 cells grow very slowly in culture. The doubling time of these cells is approximately 120 hours (Lasfargues et al, 1978), which is similar to the time needed for tumor doubling in vivo (Rew et al, 1992). These cells are exquisitely sensitive to estrogen, and will cease proliferation in a rich media containing steroid depleted serum (20% charcoal-stripped serum + insulin with a media change every day). In contrast, steroid deprivation of MCF-7 cells requires more drastic conditions (a very minimal media that is not changed for the first five days prior to the addition of estrogen). This treatment slows MCF-7 cell proliferation significantly and is required to demonstrate elevation of BRCA 1 and BRCA2 mRNAs by estrogen.

Failure of progesterone to affect levels of BRCAI or BRCA2 in response to estrogen in either BT-483 or MCF-7 cells is interesting because in normal breast development both estrogen and progesterone are needed to complete the proliferation and differentiation of the breast tissue. Estrogen regulates the development of the ductules and progesterone regulates the development of the lobules (King, 1993). In women with germline BRCAI mutations, although most tumors are of ductal origin, some are of lobular origin, mimicking the pattern seen in sporadic cases (Marcus et al, 1994).

Studies on proliferation of breast cancer cell lines, with combinations of estrogen and progesterone do not give such clear results (King, 1993) although progestins predominately inhibit the estrogen-induced proliferation of breast cancer cell lines (Clarke and Sutherland, 1990). In a previous study (Gudas et al, 1995), progesterone was able to induce BRCAI expression in the T-47D breast cancer cell line. However, this T-47D cell line is unusual because expression of the progesterone receptor is approximately 85 times greater in T-47D cells than in the BT-483 cells. Classic estrogen receptor positive breast cancer cell lines such as MCF-7 (Horwitz and McGuire, 1978) and BT-483 depend on estrogen induction of the progesterone receptor. Gudas et al. did not investigate the regulation of BRCAI by progesterone in the MCF-7 cell line. Herein is evidence for the primary hormone controlling the elevation of BRCAI and BRCA2 mRNAs being estrogen, not progesterone.

BRCAI and BRCA2 are both tumor suppressor genes. Inactivation of these genes in women with germline mutations is frequently by deletions revealed by a loss of heterozygosity in the tumor (Merajver et al, 1995 and Gudmundsson et al, 1995). A few families with BRCAI linked breast cancer do not have alterations in the coding sequences of BRCAI, raising the possibility of mutations in regions controlling BRCAI expression. In the absence of coding mutations in the BRCAI in sporadic breast tumors, alterations in the regulation of BRCAI expression are presumed to contribute to the cancerous phenotype (Thompson et al, 1995). Failure to induce the postulated estrogen responsive protein or alterations in the regulatory pathway involving elevation of BRCAI and BRCA2 mRNAs could result in a novel mechanism of malignant transformation through the loss of BRCAI or BRCA2 transcripts.

C. Blocking of Estrogen Induction of BRCAI and BRCA2 by An iestrogcns

Effects of estrogen mediated through the estrogen receptor can be competitively inhibited by antiestrogenic compounds. Two major classes of estrogen antagonists are nonsteroidal antiestrogens such as trans 4'-hydroxytamoxifen (4-OHT) and steroidal antiestrogens such as ICI 182,780 (Wakeling et al, 1989). While both classes of antiestrogens compete for binding to the estrogen receptor, they exert different actions on the activation of the estrogen receptor. Steroidal antiestrogens appear to prevent binding of the estrogen receptor to DNA while nonsteroidal antiestrogens fail to activate the estrogen-inducible transactivating function of the estrogen receptor protein (Green, 1990).

To confirm that the induction of BRCAI and BRCA2 mRNA expression by estrogen was mediated by the estrogen receptor, the steroidal antiestrogen ICI 182,780 and the nonsteroidal antiestrogen trans 4'-hydroxytamoxifen were used in a competitive inhibition study. The results were analyzed by northern blotting. BT-483 cells were cultured as described previously with varying amounts of estrogen and antiestrogen. The antiestrogens ICI 182,780 and 4-OHT do not induce BRCAI or BRCA2 expression. The expected estrogen mediated induction of BRCAI and BRCA2 mRNAs is seen in the absence of any antiestrogen. When the amount of estrogen was held constant and the amount of antiestrogen varied, it was found that a one hundred fold molar excess of the antiestrogen ICI 182,780 was required to inhibit the estrogen induction of BRCAI and BRCA2 mRNAs and to return their mRNA levels to a baseline level. Interestingly, a one hundred fold excess of ICI 182,780 is also the amount reported to be needed to block breast cancer cell proliferation in vivo in the presence of estradiol (Wakeling et al, 1991).

Similar results were achieved with the trans 4'-hydroxytamoxifen. A one hundred fold excess of 4-OHT shaφly reduced the amount of BRCA2 and BRCAI mRNAs. The ability of two different classes of antiestrogen to block the expression of BRCAI and BRCA2 in the presence of estradiol confirms that the expression of these genes is mediated by the estrogen receptor.

D. Time Frame of Estrogen Induction of BRCAI and BRCA2 mRNA

The time at which BRCAI and BRCA2 steady-state mRNA levels were elevated after estrogen stimulation was investigated in BT-483 cells. Cells were treated with estrogen and cytoplasmic RNA was isolated at varying times. Northern blot analysis of RNA obtained at regular time intervals for a total of ninety-six hours revealed that the initial expression levels of BRCAI were negligible and remained so during the first 18 hours following estrogen stimulation. The shaφ elevation of BRCAI mRNA between 18 and 24 hours after initial estrogen stimulation was particularly striking. This elevation persisted with continued estrogen stimulation and mRNA levels remained elevated for at least 96 hours.

The time and pattern of elevation of BRCA2 mRNA steady-state levels was remarkably similar to that demonstrated for BRCAI mRNA. Levels of BRCA2 mRNA were negligible until 24 hours, at which time a sharp increase in the amount of BRCA2 transcript was detected. The increase in BRCA2 mRNA remained constant to 96 hours and was not subject to downregulation in the continued presence of estrogen. A continuous presence of estrogen was not necessary for the induction of BRCAI and BRCA2 mRNA expression. A limited 9 hour pulse of estrogen chased with steroid depleted media was sufficient to induce BRCAI and BRCA2 mRNA expression in cells harvested 24 hours after initiation of estrogen stimulation.

The response of BRCAI and BRCA2 to estrogen occurs at the same time. In the BT-483 cell line mRNA levels of both genes are elevated 18 to 24 hours after estrogen stimulation, suggesting that they may have been coordinately regulated. This may be because they both play a role in control of the cell cycle. BRCAI has been postulated to control cell proliferation and to maintain the cell in a differentiated state (Marcus et al, 1994). Recent data (Vaughn et al., 1996 and Gudas et al, 1996) indicate that the highest levels of BRCAI mRNA and protein are seen in late GI and early S phase, suggesting a role for BRCAI in cell cycle regulation. Elevation of cyclin Dl mRNA has been observed at the same time as BRCAI and BRCA2 mRNAs are elevated, supporting this hypothesis.

E. Blocking of BRCAI and BRCA2 Estrogen Induction by Cycloheximide

The time lag between the initiation of estrogen stimulation and the increase in BRCAI and BRCA2 mRNA levels suggests that estrogen acts indirectly on these two genes. If prior synthesis of intermediate proteins is necessary, then treatment of cells with the protein inhibitor cycloheximide should block the observed increase in BRCAI and BRCA2 mRNA levels following estrogen treatment. Cells were pretreated with cycloheximide for 1 hour prior to the addition of estrogen and harvested after 24 hours of estrogen stimulation. All studies were done in triplicate. Treatment with cycloheximide before the addition of estrogen completely blocked the increase in BRCAI and BRCA2 mRNA levels. Cells treated with cycloheximide and no estrogen, as well as cells treated with no cycloheximide and no estrogen, did not result in an increase in BRCAI and BRCA2 mRNA levels. Cells treated with estrogen and no cycloheximide demonstrated the expected previously observed increase in BRCAI and BRCA2 mRNA levels. The effect of extended incubation with cycloheximide on cell viability was assayed by trypan blue exclusion and did not differ significantly between control and experimental cells, implying that the cycloheximide effect was not due to cell death. The ability of cycloheximide to block the induction of BRCAI and BRCA2 was not due to a generalized decrease in transcription, because expression of the constitutively expressed estrogen-independent 36B4 mRNA (ribosomal phosphoprotein P0; Masiakowski et al, 1982) showed no significant difference between cycloheximide treated and untreated cells.

The effect of estrogen on BRCAI and BRCA2 steady-state mRNA levels by estrogen is indirect and requires prior protein synthesis as demonstrated by the action of cycloheximide. The implication of this is that an estrogen inducible protein may coordinately elevate the levels of BRCAI and/or BRCA2 mRNAs. Alternatively, these genes may be induced by distinct estrogen induced pathways.

EXAMPLE XIV

BARDl And BRCAI In Discrete Nuclear Domains

The BRCAI tumor suppressor has been implicated in familial cases of early-onset breast and ovarian cancer (Hall et al, 1990; Miki et al, 1994). However, the biochemical functions of its protein product are not defined and the mechanism by which it counters tumor formation during normal development is not understood. The major isoform of BRCAI is a polypeptide of -220 kilodaltons that bears several recognizable amino acid motifs: these include a zinc-binding RING domain that lies near the amino terminus, two nuclear localization signals, and two tandem copies of the BRCT motif that reside at the carboxy-terminus (Miki et al, 1994; Chen et al, 1996a; Thakur et al, 1997; Koonin et al, 1996). As described herein above, BRCAI associates in vivo with BARDl . The interaction between these proteins is abolished by tumorigenic missense mutations in the RING domain of BRCAI , suggesting that tumor suppression may be mediated by a heteromeric complex of BRCAI and BARDl.

Products of the BRCAI gene are found in a broad spectrum of cell and tissue types (Miki et al, 1994; Lane et l, 1995; Marquis et al, 1995); however, its expression in most (Chen et al, 1996c; Vaughn et al, 1996a, Gudas et al, 1996; Rajan et al, 1996), but not all (Aprelikova et al, 1996), cell types is tightly regulated during cell cycle progression. In resting cells, the levels of BRCAI transcripts and polypeptides are either low or undetcctable. However, after these cells receive a mitotic stimulus the steady-state levels of BRCAI products rise in late GI, peak just prior to the onset of DNA synthesis, and persist for the duration of S phase and most of M phase. In addition, BRCAI polypeptides become hyperphosphorylated as they begin to accumulate in late GI (Chen et al, 1996c). While not conclusive, these findings suggest that BRCAI may be involved in some aspect of cell cycle regulation (Chen et al, 1996c; Vaughn et al, 1996a; Gudas et al, 1996; Rajan el al, 1996).

Recent studies indicate that BRCAI resides predominately in the nuclei of normal cells (Chen et al, 1995; Scully et al, 1996; Chen et al, 1996b; Thomas et al, 1996). During S phase, when their levels are most abundant, BRCAI polypeptides exist in distinct subnuclear bodies, termed BRCAI nuclear dots. Although the function of these dots is not known, most, but not all, co-stain with antibodies that recognize HsRad51 , a DNA-binding protein that shares extensive homology with the yeast Rad51 and E. coli RecA proteins (Scully et al, 1997). HsRad51 promotes homologous pairing and single strand exchange between DNA duplexes, and it has been implicated in a variety of nuclear processes, including DNA recombination, RNA transcription and DNA repair (Scully et al, 1997 for additional references). As such, the co- localization of BRCAI and HsRad51 to the same subnuclear structures provides important clues about BRCAI function (Scully et al, 1997).

To obtain additional insights into the function of BRCAI, the expression and subcellular distribution of BARDl was examined during cell cycle progression. In contrast to BRCAI, the steady-state levels of BARDl remain relatively constant throughout the cell cycle. Subcellular fractionation of synchronized cell populations showed that BARDl resides in the nuclei of proliferating cells, and two-color immunofluorescence with BARDl -specific antibodies revealed a punctate pattern of nuclear staining with nearly perfect co-localization of BARDl and BRCAI . However, the punctate pattern of BARDl immunostaining was observed in S-phase, but not in GI -phase, cells. Therefore, despite the presence of BARDl polypeptides in the nucleus throughout cell cycle progression, their accumulation into BRCAI nuclear dots is an S phase-specific phenomenon that may require recruitment by BRCAI . This cell cycle-dependent co-localization of BARDl and BRCAI further indicates a role for BARDl in BRCAI -mediated tumor suppression.

1. Experimental Materials

HBL-100 and T24 cell lines were obtained from the American Type Culture Collection and normal human mammary epithelial cells (HMECs) were purchased from Clonetics Corp. (San Diego, CA). Three different BARDl -specific antibody reagents were used in this study: a mouse polyclonal antiserum, a mouse monoclonal antibody, and an affinity -purified rabbit polyclonal antiserum. To prepare the latter, a cDNA fragment of human BARDl was inserted into the BamHl/Hindlll sites of the pMAL-c2 bacterial expression vector (New England Biolabs, Beverly, MA); the resultant plasmid encodes MBP-EE, a hybrid polypeptide comprised of the E. coli maltose binding protein (MBP) fused to residues 141-388 of BARDl . MBP-EE polypeptides were then purified from E. coli lysates by affinity chromatography on an amylose resin (New England Biolabs) and conjugated to CNBr-activated Sepharose 4B (Pharmacia Biotech). The rabbit polyclonal antiserum raised against GST-EE, a hybrid polypeptide containing silkworm GST fused to residues 141-388 of BARDl, was then purified by sequential affinity chromatography on HiTrap protein A-Sepharose (Pharmacia Biotech) and MBP-EE- conjugated Sepharose 4B. The BARDl -specific mouse polyclonal antiserum and monoclonal antibody were raised by immunizing mice with the GST-EE polypeptide. The monoclonal antibody was used for BARDl immunoblots (e.g., FIGs. 1 and 5). Monoclonal antibodies that recognize BRCAI (MSI 10), cyclin A (Ab-3), NuMA (Ab-1), and α-tubulin (Ab-1) were purchased from Oncogene Research Products. The CDK2 -specific antiserum (M2) was obtained from Santa Cruz Biotechnology. 2. Steady-State Levels of BARDl Remain Constant During Cell-Cycle Progression

To compare the expression of BARDl and BRCAI polypeptides with respect to the cell cycle, their steady-state levels were measured in synchronized populations of T24 bladder

2 carcinoma cells. T24 cells were arrested in GO by contact inhibition in 175 cm flasks. After at least 3 days of confluence, the cells were split 1 :10 by seeding multiple 100 mm dishes at a concentration of ~10 cells/dish. Individual cultures were harvested at various times after replating (Chen et al, 1996c). The cell cycle distribution profile of each culture was then determined by FACS analysis and protein levels were evaluated by immunoblotting.

Ten dishes were harvested at each timepoint after replating - two for FACS analysis and eight for Western analyses. To determine the cell cycle distribution at each timepoint, the contents of each dish were incubated for 10 min at room temperature in 2 ml of trypsin/EDTA solution (0.25% trypsin, 0.1% EDTA in HBSS w/o CaMg; Mediatech, Inc.). The trypsinized cells were then washed in 10 ml of growth medium (McCoy's 5A, 10% FBS) and resuspended in 1.5 ml of ice-cold PBS (w/o CaMg). After adding 3.5 ml of ice-cold 100% ethanol dropwise, the cells were fixed at 4°C for at least 16 h. The fixed cells were pelleted, resuspended in 1 ml of PI staining solution (50 μg/ml propidium iodide, 100 U/ml RNase A, 0.1 % glucose in PBS w/o CaMg), incubated for at least lh at room temperature, and analyzed on a FACScan flow cytometer (Becton Dickinson).

For Western analyses the contents of eight dishes were lysed in a total of 300 μl RIPA buffer (50 mM Tris pH 7.6, 150 mM NaCI, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) containing complete protease inhibitor cocktail (Boehringer Mannheim) and phosphatase inhibitors (5 mM β-glycerophosphate, 10 mM benzamidine, and 0.5 mM sodium orthovanadate). The lysate was vortexed for 10 min at 4°C and cleared of insoluble debris by centrifugation for 10 min at 12,000 RPM in a microfuge at 4°C. The protein concentration of each supernatant was determined using the BCA Protein Assay Reagent (Pierce). Equivalent aliquots of each lysate were subjected to Western analyses with antibodies specific for CDK2, cyclin A, BRCAI, and BARDl. Western analyses were conducted by enhanced chemiluminescence (Amersham) using 80 μg of lysate for BRCAI immunoblots and 30 μg for CDK2, cyclin A, and BARDl immunoblots. The cells display the expected expression patterns for known cell cycle regulatory molecules. For example, CDK2 is present throughout the cell cycle and its steady state levels increase modestly in S and G2/M cells. However, the levels of its regulatory subunit, cyclin A, rise dramatically after the Gl/S transition. BRCAI shows an expression profile similar to that described in a previous study of T24 cells (Chen et al, 1996c); thus, while few, if any, BRCAI products are detected in resting or GI cells, BRCAI expression increases markedly as cells enter S phase. In contrast, comparable levels of BARDl polypeptides are seen at all timepoints, indicating that BARDl expression remains relatively constant throughout the cell cycle. In addition, Western analysis of subcellular fractions from synchronized cell populations demonstrate that BARDl remains in the nuclear compartment of GI - and S-phase proliferating cells.

3. BARDl Polypeptides Reside In Discrete Subnuclear Bodies

The subcellular distribution of BARDl polypeptides was evaluated by immunofluorescent staining of unsynchronized HBL-100 cells, a human line of normal mammary epithelial cells that was presumably immortalized by transforming sequences of the SV40 papovavirus (Caron de Fromentel et al, 1985). A mouse polyclonal antiserum was prepared against residues 141-388 of BARDl, a segment that bears no homology to other known proteins. Approximately 2.5 x 10 cells were seeded onto microscope slides in a 150 mm culture dish. After 2 days, the cells were fixed with 4% paraformaldehyde for 15 min and permeabilized in 0.2% Triton X-100 for 10 min. Non-specific staining was blocked by a 60 min incubation with 2% bovine serum albumin in phosphate-buffered saline (BSA/PBS solution) and two 15 min treatments with the Avidin/Biotin Blocking Kit (Vector Laboratories, Burlington, CA). After a 60 min incubation with primary antibody, the cells were treated with 8 μg/ml biotinylated secondary antibody (Vector Laboratories) for 45 min and 20 μg/ml fluorescein avidin D (Vector Laboratories) for an additional 30 min. The cells were then treated with 100 μg/ml of RNase A in PBS for 20 min at 37°C, and with 10 μg/ml propidium iodide in PBS for an additional 20 min. The stained cells were mounted under coverslips with VECTASHIELD mounting medium (Vector Laboratories) and sealed with nail polish. Immunofluorescence was recorded using a confocal microscope equipped with a MRC-1024 Lasersharp confocal imaging system (Bio-Rad Laboratories). All the above procedures were performed at room temperature except where indicated. After staining with either the BARDl -specific antiserum or a BRCAl-specific monoclonal antibody (MSI 10; Oncogene Research Products; Scully et al, 1996), the cells were counter-stained with propidium iodide to highlight the nuclei. A characteristic pattern of BRCAI subcellular distribution was observed in which BRCAI nuclear dots appeared in some, but not all, inteφhase cells. Likewise, the BARDl -specific antiserum generated a similar pattern of punctate nuclear staining in a subset of interphase cells. The same results were also obtained using T24 colon carcinoma cells and primary human mammary epithelial cells.

4. BARDl-Containing Nuclear Foci Appear Specifically In S-Phase Cells

The nuclear dot pattern of BRCAI staining has been shown to arise specifically during S-phase of the cell cycle (Scully et al, 1997). To determine whether the subnuclear structures that stain with BARDl have a similar cell-cycle dependence, synchronized populations of T24 cells were stained with BARDl- or BRCAl-specific monoclonal antibodies. Cells harvested at 8 h (91% GI phase cells) and 20 h (56% S phase cells) after replating were analyzed. In some studies the monoclonal antibodies were pre-absorbed with an excess of the BRCAI immunogen (GST-BR304), the BARDl immunogen (GST-EE) or the parental GST polypeptide.

Cells bearing BRCAI nuclear dots were abundant in the S phase population but were rarely observed in the GI population. The specificity of BRCAI staining was confirmed in blocking studies in which the primary antibody was preabsorbed with a resin-bound protein containing silkworm glutathione S-transferase (GST) fused to the amino-terminal 304 residues of BRCAI - the same BRCAI moiety used to generate the MSI 10 monoclonal antibody (Scully et al, 1996). As expected, staining of BRCAI nuclear dots was completely abolished by preabsoφtion with the GST-BRCAl fusion protein but not with the parental GST polypeptide.

Immunofluorescence analysis of synchronized cell populations revealed that the appearance of BARDl -staining foci with respect to the cell cycle resembles that of BRCAI nuclear dots. Thus, these foci are present in most cells of the S phase population (panel f) but not the GI population. Moreover, the staining of S phase cells with BARDl -specific antibodies was ablated by preabsorption with GST-EE, a polypeptide containing GST fused to residues 141 -388 of BARDl, but not by GST itself. These data show that the BARDl -staining nuclear structure arises in an S phase-specific fashion reminiscent of the BRCAI nuclear dots.

5. BARDl And BRCAI Polypeptides Co-Localize In BRCAI Nuclear Dots If BARDl is a physiologically-relevant partner of BRCAI then the two proteins should reside in the same subcellular structures. Therefore, to determine whether the S-phase nuclear foci recognized by BARDl- and BRCAl-specific antibodies are one and the same, two-color immunofluorescence studies were conducted by staining HBL-100 cells simultaneously with an affinity-purified BARDl -specific rabbit antiserum and a mouse monoclonal antibody that recognizes either BRCAI or PML; the latter is a RING protein that resides in distinct subnuclear structures referred to as PML oncogenic domains (PODs) (Dyck, et al, 1994; Koken et al, 1994).

Cells were incubated simultaneously with the two primary antibodies for 60 min. After treatment with Texas Red-conjugated anti-rabbit goat IgG (Vector Laboratories) and biotinylated anti-mouse goat IgG (Vector Laboratories) for 45 min, the cells were incubated for an additional 30 min with fluorescein avidin D. The immunostained cells were then mounted as described above (without RNase A digestion and propidium iodide staining). A 10 μg aliquot of the BARDl- or BRCAl-specific monoclonal antibody was preabsorbed by overnight incubation at 4°C with 50 μg of either the parental GST polypeptide or the cognate immunogen (GST-EE or GST-BR304, respectively) immobilized on glutathione-agarose beads. Images of BARDl - staining (red) and BRCAI- or PML-staining (green) from the same cells were then collected both separately and conjointly.

BARDl -staining coincides almost perfectly with the BRCAI nuclear dots of HBL-100 cells. In contrast, BARDl -staining structures are distributed randomly with respect to the PML- oncogenic domains. Similar results were obtained in two-color immunofluorescence studies of normal human mammary epithelial cells. These data demonstrate that BARDl specifically co-localizes with BRCAI in the same subnuclear bodies. Co-localization of BRCAI and BARDl in nuclear dots appears to be independent of cell type and the degree of neoplastic transformation. 6. Subcellular Distribution Of BARDl Polypeptides During Cell Cycle Progression

The BARD 1 -staining nuclear foci are only apparent by immunofluorescence microscopy after the onset of S phase. Nevertheless, Western analysis of lysates from synchronized cell populations show that the steady-state levels of BARDl polypeptides remain relatively constant throughout the cell cycle. To address the question of where BARDl resides in G I -phase cells, the subcellular distribution of BARDl polypeptides was initially examined by Western analyses of nuclear, cytoplasmic, and membrane fractions prepared from asynchronous T24 cells.

To prepare whole cell lysates of unsynchronized T24 cells, the cellular contents of two η 150 mm dishes (~1.7 x 10 cells/dish) were lysed in 1 ml of RIPA buffer (containing protease and phosphatase inhibitors, as described above). Whole cell lysates of synchronized T24 cells (8h or 20 h after replating) were prepared by lysing the contents of six 150 mm dishes in 1 ml of RIPA buffer (-2.6 x 10 cells/dish). Each whole cell lysate was vortexed for 15 min at 4°C and cleared of insoluble debris by centrifugation for 10 min at 12,000 RPM in a microfuge at 4°C. To prepare membrane, cytoplasmic, and nuclear fractions from unsynchronized cells, the contents of seven 150 mm dishes (-1.7 x 10 cells/dish) were resuspended in 5 ml of hypotonic lysis buffer and processed as described (Abrams et al. , 1982).

For synchronized cells, the contents of twenty-five 150 mm dishes (-2.0 x 10 cells/dish) were resuspended in 5 ml of hypotonic lysis buffer and processed to prepare subcellular fractions (Abrams et al, 1 82). For detection of BRCAI , equivalent volumes of each fraction (corresponding to 300 μg of whole cell lysate) were immunoprecipitated with the BRCAl- specific rabbit antiserum and the immunoprecipitates were subjected to Western analysis with the BRCAl-specific MSI 10 monoclonal antibody. For detection of BARDl, NuMA, and α- tubulin, equivalent volumes of each fraction (corresponding to 10 μg of whole cell lysate) were directly evaluated by Western analysis with the appropriate monoclonal antibody.

BARDl and BRCAI were concentrated in the nuclear fraction along with the nuclear matrix protein NuMA (Lyderson et al, 1980). In contrast, α-tubulin was found exclusively in the cytoplasmic and membrane compartments, indicating that there was little, if any, cross- contamination of the nuclear compartment with cytosolic proteins. Identical results were obtained by Western analysis of subcellular fractions from synchronized populations of T24 cells harvested at 8 h (98% GI cells) and 20 h (52% S phase cells) after release from cell cycle arrest. Hence, during GI phase of the cell cycle, when BARDl is not found in BRCAI nuclear dots by immunofluorescent staining, the analysis of subcellular fractions reveals it to be predominantly a nuclear protein.

Immunostaining with BARDl -specific antibodies was not observed in GI cells, despite the fact that BARDl polypeptides were readily detected by Western analysis of nuclear fractions derived from these cells. Several explanations can be invoked to account for this phenomenon. For example, the epitopes recognized by the BARDl -specific antibodies may be masked during certain stages of the cell cycle by interactions with other macromolecules. However, identical results were obtained with three different reagents raised against a substantial segment of human BARDl (residues 141-388): an affinity-purified rabbit polyclonal antiserum, a mouse polyclonal antiserum, and a mouse monoclonal antibody. Furthermore, attempts to unmask hidden epitopes with heat or high salt did not elicit BARDl -specific staining in GI cells, despite the fact that the monoclonal antibody readily detects denatured BARDl polypeptides. Thus, a more plausible explanation for this phenomenon is that BARDl polypeptides are distributed diffusely within the nuclei of GI cells at concentrations too low for immunodetection. In contrast, the S phase- dependent accumulation of BARDl into BRCAI dots presumably increases their local concentration to levels detectable by immunofluorescence microscopy.

If BARDl polypeptides are diffusely distributed in the nuclei of GI cells, then all, or at least a significant subset, of these polypeptides must be recruited into the BRCAI nuclear dots as cells progress into S phase. The re-localization of BARDl may occur independently of BRCAI, or the BARDl accumulation into the dots may require the prior formation of BRCAl/BARDl heterodimers. In this regard, determination of the nuclear distribution of BARDl in cells that lack functional BRCAI will be feasible once cell lines are established from either Brcal-null mice or breast carcinomas of BRCAI mutation carriers.

Germline mutations of either BRCAI or BRCA2 are responsible for most cases of familial breast cancer. Thus, it is intriguing to note that these genes share a number of other similarities. First, unlike most tumor suppressors, BRCAI and BRCA2 are rarely mutated in truly sporadic cases of breast cancer (Futreal et al, 1994; Lancaster et al, 1996; Teng et al, 1996; Miki et al, 1996). Second, the phylogenetic conservation of both genes is remarkably poor - for example, the mouse and human orthologs of their protein products exhibit only 58% identity at the amino acid level (Lane et al, 1995; Abel et al, 1995; Sharan et al, 1995; Bennett et al, 1995; Connor et al, 1997; Sharan et al, 1997). Third, the transcription of both genes is coordinately induced by estrogen (Example XIII, above). Fourth, the expression patterns of BRCAI and BRCA2 with respect to the cell cycle are almost indistinguishable: both are induced in late GI upon mitogenic stimulation of quiescent cells, and the levels of their gene products peak just prior to DNA synthesis (Chen et al, 1996c; Vaughn et al, 1996a; Gudas et al, 1996; Rajan et al, 1996; Vaughn et al, 1996b; Wang et al, 1997).

These intriguing parallels were underscored recently by the discovery that BRCA2 also interacts in vivo with HsRad51 (Sharan et al, 1997; Mizuta et al, 1997). Although the subcellular localization of BRCA2 has not yet been described, these findings suggest that BRCAI and BRCA2 normally serve as components of a common biochemical pathway involving the HsRad51 protein (Scully et al. , 1997; Sharan et al. , 1997; Mizuta et al. , 1997). As such, the disruption of this pathway by mutations in BRCAI or BRCA2 may be a critical step in the development of hereditary breast cancer. The specific localization of BARDl into the BRCAI nuclear dots of S phase cells suggests that it too may be an essential component of a HsRad51 -associated pathway of tumor suppression.

All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Board of Regents, University of Texas System

(B) STREET: 201 West 7th Street

(C) CITY: Austin

(D) STATE: TX

(E) COUNTRY: US

(F) POSTAL CODE (ZIP) : 78701

(G) TELEPHONE: (512) 418-3000 (H) TELEFAX: (713) 789-2679

(ii) TITLE OF INVENTION: Compositions and Methods Comprising BARDl and Other BRCAI Binding Proteins

(iii) NUMBER OF SEQUENCES: 130

(iv) 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 (EPO)

(vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 60/025,296

(B) FILING DATE: 20-SEP-1996

(vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 60/042,611

(B) FILING DATE: 03-APR-1997

(vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 60/042,985

(B) FILING DATE: 04-APR-1997

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2531 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: modified_base

(B) LOCATION: 531

(D) OTHER INFORMATION :/note= "R •^** A or G"

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 75..2405

(ix) FEATURE:

(A) NAME/KEY: modified_base

(B) LOCATION: 153

(D) OTHER INFORMATION: /note= "Xaa = Glu or Lys for both SEQ ID Nθ:l and SEQ ID NO: 2" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

GCAGCTTCCC TGTGGTTTCC CGAGGCCTCC TTGCTTCCCG CTCTCCGAGG AGCCTTTCAT 60

CCGAAGGCGG GACG ATG CCG GAT AAT CGG CAG CCG AGG AAC CGG CAG CCG 110

Met Pro Asp Asn Arg Gin Pro Arg Asn Arg Gin Pro 1 5 10

AGG ATC CGC TCC GGG AAC GAG CCT CGT TCC GCG CCC GCC ATG GAA CCG 158

Arg lie Arg Ser Gly Asn Glu Pro Arg Ser Ala Pro Ala Met Glu Pro 15 20 25

GAT GGT CGC GGT GCC TGG GCC CAC AGT CGC GCC GCG CTC GAC CGC CTG 206

Asp Gly Arg Gly Ala Trp Ala His Ser Arg Ala Ala Leu Asp Arg Leu 30 35 40

GAG AAG CTG CTG CGC TGC TCG CGT TGT ACT AAC ATT CTG AGA GAG CCT 254

Glu Lys Leu Leu Arg Cys Ser Arg Cys Thr Asn lie Leu Arg Glu Pro

45 50 55 60

GTG TGT TTA GGA GGA TGT GAG CAC ATC TTC TGT AGT AAT TGT GTA AGT 302

Val Cys Leu Gly Gly Cys Glu His lie Phe Cys Ser Asn Cys Val Ser 65 70 75

GAC TGC ATT GGA ACT GGA TGT CCA GTG TGT TAC ACC CCG GCC TGG ATA 350

Asp Cys lie Gly Thr Gly Cys Pro Val Cys Tyr Thr Pro Ala Trp lie 80 85 90

CAA GAC TTG AAG ATA AAT AGA CAA CTG GAC AGC ATG ATT CAA CTT TGT 398

Gin Asp Leu Lys lie Asn Arg Gin Leu Asp Ser Met lie Gin Leu Cys 95 100 105

AGT AAG CTT CGA AAT TTG CTA CAT GAC AAT GAG CTG TCA GAT TTG AAA 446

Ser Lys Leu Arg Asn Leu Leu His Asp Asn Glu Leu Ser Asp Leu Lys 110 115 120

GAA GAT AAA CCT AGG AAA AGT TTG TTT AAT GAT GCA GGA AAC AAG AAG 494

Glu Asp Lys Pro Arg Lys Ser Leu Phe Asn Asp Ala Gly Asn Lys Lys

125 130 135 140

AAT TCA ATT AAA ATG TGG TTT AGC CCT CGA AGT AAG RAA GTC AGA TAT 542

Asn Ser lie Lys Met Trp Phe Ser Pro Arg Ser Lys Xaa Val Arg Tyr 145 150 155

GTT GTG AGT AAA GCT TCA GTG CAA ACC CAG CCT GCA ATA AAA AAA GAT 590

Val Val Ser Lys Ala Ser Val Gin Thr Gin Pro Ala lie Lys Lys Asp 160 165 170

GCA AGT GCT CAG CAA GAC TCA TAT GAA TTT GTT TCC CCA AGT CCT CCT 638

Ala Ser Ala Gin Gin Asp Ser Tyr Glu Phe Val Ser Pro Ser Pro Pro 175 180 185

GCA GAT GTT TCT GAG AGG GCT AAA AAG GCT TCT GCA AGA TCT GGA AAA 686

Ala Asp Val Ser Glu Arg Ala Lys Lys Ala Ser Ala Arg Ser Gly Lys 190 195 200

AAG CAA AAA AAG AAA ACT TTA GCT GAA ATC AAC CAA AAA TGG AAT TTA 734

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

205 210 215 220

GAG GCA GAA AAA GAA GAT GGT GAA TTT GAC TCC AAA GAG GAA TCT AAG 782

Glu Ala Glu Lys Glu Asp Gly Glu Phe Asp Ser Lys Glu Glu Ser Lys 225 230 235 CAA AAG CTG GTA TCC TTC TGT AGC CAA CCA TCT GTT ATC TCC AGT CCT 830 Gin Lys Leu Val Ser Phe Cys Ser Gin Pro Ser Val He Ser Ser Pro 240 245 250

CAG ATA AAT GGT GAA ATA GAC TTA CTA GCA AGT GGC TCC TTG ACA GAA 878 Gin He Asn Gly Glu He Asp Leu Leu Ala Ser Gly Ser Leu Thr Glu 255 260 265

TCT GAA TGT TTT GGA AGT TTA ACT GAA GTC TCT TTA CCA TTG GCT GAG 926 Ser Glu Cys Phe Gly Ser Leu Thr Glu Val Ser Leu Pro Leu Ala Glu 270 275 280

CAA ATA GAG TCT CCA GAC ACT AAG AGC AGG AAT GAA GTA GTG ACT CCT 974 Gin He Glu Ser Pro Asp Thr Lys Ser Arg Asn Glu Val Val Thr Pro 285 290 295 300

GAG AAG GTC TGC AAA AAT TAT CTT ACA TCT AAG AAA TCT TTG CCA TTA 1022 Glu Lys Val Cys Lys Asn Tyr Leu Thr Ser Lys Lys Ser Leu Pro Leu 305 310 315

GAA AAT AAT GGA AAA CGT GGC CAT CAC AAT AGA CTT TCC AGT CCC ATT 1070 Glu Asn Asn Gly Lys Arg Gly His His Asn Arg Leu Ser Ser Pro He 320 325 330

TCT AAG AGA TGT AGA ACC AGC ATT CTG AGC ACC AGT GGA GAT TTT GTT 1118 Ser Lys Arg Cys Arg Thr Ser He Leu Ser Thr Ser Gly Asp Phe Val 335 340 345

AAG CAA ACC GTG CCC TCA GAA AAT ATA CCA TTG CCT GAA TGT TCT TCA 1166 Lys Gin Thr Val Pro Ser Glu Asn He Pro Leu Pro Glu Cys Ser Ser 350 355 360

CCA CCT TCA TGC AAA CGT AAA GTT GGT GGT ACA TCA GGG AGG AAA AAC 1214 Pro Pro Ser Cys Lys Arg Lys Val Gly Gly Thr Ser Gly Arg Lys Asn 365 370 375 380

AGT AAC ATG TCC GAT GAA TTC ATT AGT CTT TCA CCA GGT ACA CCA CCT 1262 Ser Asn Met Ser Asp Glu Phe He Ser Leu Ser Pro Gly Thr Pro Pro 385 390 395

TCT ACA TTA AGT AGT TCA AGT TAC AGG CAA GTG ATG TCT AGT CCC TCA 1310 Ser Thr Leu Ser Ser Ser Ser Tyr Arg Gin Val Met Ser Ser Pro Ser 400 405 410

GCA ATG AAG CTG TTG CCC AAT ATG GCT GTG AAA AGA AAT CAT AGA GGA 1358 Ala Met Lys Leu Leu Pro Asn Met Ala Val Lys Arg Asn His Arg Gly 415 420 425

GAG ACT TTG CTC CAT ATT GCT TCT ATT AAG GGC GAC ATA CCT TCT GTT 1406 Glu Thr Leu Leu His He Ala Ser He Lys Gly Asp He Pro Ser Val 430 435 440

GAA TAC CTT TTA CAA AAT GGA AGT GAT CCA AAT GTT AAA GAC CAT GCT 1454 Glu Tyr Leu Leu Gin Asn Gly Ser Asp Pro Asn Val Lys Asp His Ala 445 450 455 460

GGA TGG ACA CCA TTG CAT GAA GCT TGC AAT CAT GGG CAC CTG AAG GTA 1502 Gly Trp Thr Pro Leu His Glu Ala Cys Asn His Gly His Leu Lys Val 465 470 475

GTG GAA TTA TTG CTC CAG CAT AAG GCA TTG GTG AAC ACC ACC GGG TAT 1550 Val Glu Leu Leu Leu Gin His Lys Ala Leu Val Asn Thr Thr Gly Tyr 480 485 490 CAA AAT GAC TCA CCA CTT CAC GAT GCA GCC AAG AAT GGG CAC GTG GAT 1598 Gin Asn Asp Ser Pro Leu His Asp Ala Ala Lys Asn Gly His Val Asp 495 500 505

ATA GTC AAG CTG TTA CTT TCC TAT GGA GCC TCC AGA AAT GCT GTT AAT 1646 He Val Lys Leu Leu Leu Ser Tyr Gly Ala Ser Arg Asn Ala Val Asn 510 515 520

ATA TTT GGT CTG CGG CCT GTC GAT TAT ACA GAT GAT GAA AGT ATG AAA 1694 He Phe Gly Leu Arg Pro Val Asp Tyr Thr Asp Asp Glu Ser Met Lys 525 530 535 540

TCG CTA TTG CTG CTA CCA GAG AAG AAT GAA TCA TCC TCA GCT AGC CAC 1742 Ser Leu Leu Leu Leu Pro Glu Lys Asn Glu Ser Ser Ser Ala Ser His 545 550 555

TGC TCA GTA ATG AAC ACT GGG CAG CGT AGG GAT GGA CCT CTT GTA CTT 1790 Cys Ser Val Met Asn Thr Gly Gin Arg Arg Asp Gly Pro Leu Val Leu 560 565 570

ATA GGC AGT GGG CTG TCT TCA GAA CAA CAG AAA ATG CTC AGT GAG CTT 1838 He Gly Ser Gly Leu Ser Ser Glu Gin Gin Lys Met Leu Ser Glu Leu 575 580 585

GCA GTA ATT CTT AAG GCT AAA AAA TAT ACT GAG TTT GAC AGT ACA GTA 1886 Ala Val He Leu Lys Ala Lys Lys Tyr Thr Glu Phe Asp Ser Thr Val 590 595 600

ACT CAT GTT GTT GTT CCT GGT GAT GCA GTT CAA AGT ACC TTG AAG TGT 1934 Thr His Val Val Val Pro Gly Asp Ala Val Gin Ser Thr Leu Lys Cys 605 610 615 620

ATG CTT GGG ATT CTC AAT GGA TGC TGG ATT CTA AAA TTT GAA TGG GTA 1982 Met Leu Gly He Leu Asn Gly Cys Trp He Leu Lys Phe Glu Trp Val 625 630 635

AAA GCA TGT CTA CGA AGA AAA GTA TGT GAA CAG GAA GAA AAG TAT GAA 2030 Lys Ala Cys Leu Arg Arg Lys Val Cys Glu Gin Glu Glu Lys Tyr Glu 640 645 650

ATT CCT GAA GGT CCA CGC AGA AGC AGG CTC AAC AGA GAA CAG CTG TTG 2078 He Pro Glu Gly Pro Arg Arg Ser Arg Leu Asn Arg Glu Gin Leu Leu 655 660 665

CCA AAG CTG TTT GAT GGA TGC TAC TTC TAT TTG TGG GGA ACC TTC AAA 2126 Pro Lys Leu Phe Asp Gly Cys Tyr Phe Tyr Leu Trp Gly Thr Phe Lys 670 675 680

CAC CAT CCA AAG GAC AAC CTT ATT AAG CTC GTC ACT GCA GGT GGG GGC 2174 His His Pro Lys Asp Asn Leu He Lys Leu Val Thr Ala Gly Gly Gly 685 690 695 700

CAG ATC CTC AGT AGA AAG CCC AAG CCA GAC AGT GAC GTG ACT CAG ACC 2222 Gin He Leu Ser Arg Lys Pro Lys Pro Asp Ser Asp Val Thr Gin Thr 705 710 715

ATC AAT ACA GTC GCA TAC CAT GCG AGA CCC GAT TCT GAT CAG CGC TTC 2270 He Asn Thr Val Ala Tyr His Ala Arg Pro Asp Ser Asp Gin Arg Phe 720 725 730

TGC ACA CAG TAT ATC ATC TAT GAA GAT TTG TGT AAT TAT CAC CCA GAG 2318 Cys Thr Gin Tyr He He Tyr Glu Asp Leu Cys Asn Tyr H s Pro Glu 735 740 745 AGG GTT CGG CAG GGC AAA GTC TGG AAG GCT CCT TCG AGC TGG TTT ATA 2366 Arg Val Arg Gin Gly Lys Val Trp Lys Ala Pro Ser Ser Trp Phe He 750 755 760

GAC TGT GTG ATG TCC TTT GAG TTG CTT CCT CTT GAC AGC TGAATΛTTAT 2415 Asp Cys Val Met Ser Phe Glu Leu Leu Pro Leu Asp Ser 765 770 775

ACCAGATGAA CATTTCAAAT TGAATTTGCA CGGTTTGTGA GAGCCCAGTC ATTGTACTGT 2475

TTTTAATGTT CACATTTTTA CAAATAGGTA GAGTCATTCA TATTTGTCTT TGAATC 2531

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 777 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

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

Met Pro Asp Asn Arg Gin Pro Arg Asn Arg Gin Pro Arg He Arg Ser

1 5 10 15

Gly Asn Glu Pro Arg Ser Ala Pro Ala Met Glu Pro Asp Gly Arg Gly 20 25 30

Ala Trp Ala His Ser Arg Ala Ala Leu Asp Arg Leu Glu Lys Leu Leu 35 40 45

Arg Cys Ser Arg Cys Thr Asn He Leu Arg Glu Pro Val Cys Leu Gly 50 55 60

Gly Cys Glu His He Phe Cys Ser Asn Cys Val Ser Asp Cys He Gly 65 70 75 80

Thr Gly Cys Pro Val Cys Tyr Thr Pro Ala Trp He Gin Asp Leu Lys 85 90 95

He Asn Arg Gin Leu Asp Ser Met He Gin Leu Cys Ser Lys Leu Arg 100 105 110

Asn Leu Leu His Asp Asn Glu Leu Ser Asp Leu Lys Glu Asp Lys Pro 115 120 125

Arg Lys Ser Leu Phe Asn Asp Ala Gly Asn Lys Lys Asn Ser He Lys 130 135 140

Met Trp Phe Ser Pro Arg Ser Lys Xaa Val Arg Tyr Val Val Ser Lys 145 150 155 160

Ala Ser Val Gin Thr Gin Pro Ala He Lys Lys Asp Ala Ser Ala Gin 165 170 175

Gin Asp Ser Tyr Glu Phe Val Ser Pro Ser Pro Pro Ala Asp Val Ser 180 185 190

Glu Arg Ala Lys Lys Ala Ser Ala Arg Ser Gly Lys Lys Gin Lys Lys 195 200 205

Lys Thr Leu Ala Glu He Asn Gin Lys Trp Asn Leu Glu Ala Glu Lys 210 215 220 Glu Asp Gly Glu Phe Asp Ser Lys Glu Glu Ser Lys Gin Lys Leu Val 225 230 235 240

Ser Phe Cys Ser Gin Pro Ser Val He Ser Ser Pro Gin He Asn Gly 245 250 255

Glu He Asp Leu Leu Ala Ser Gly Ser Leu Thr Glu Ser Glu Cys Phe 260 265 270

Gly Ser Leu Thr Glu Val Ser Leu Pro Leu Ala Glu Gin He Glu Ser 275 280 285

Pro Asp Thr Lys Ser Arg Asn Glu Val Val Thr Pro Glu Lys Val Cys 290 295 300

Lys Asn Tyr Leu Thr Ser Lys Lys Ser Leu Pro Leu Glu Asn Asn Gly 305 310 315 320

Lys Arg Gly His His Asn Arg Leu Ser Ser Pro He Ser Lys Arg Cys 325 330 335

Arg Thr Ser He Leu Ser Thr Ser Gly Asp Phe Val Lys Gin Thr Val 340 345 350

Pro Ser Glu Asn He Pro Leu Pro Glu Cys Ser Ser Pro Pro Ser Cys 355 360 365

Lys Arg Lys Val Gly Gly Thr Ser Gly Arg Lys Asn Ser Asn Met Ser 370 375 380

Asp Glu Phe He Ser Leu Ser Pro Gly Thr Pro Pro Ser Thr Leu Ser 385 390 395 400

Ser Ser Ser Tyr Arg Gin Val Met Ser Ser Pro Ser Ala Met Lys Leu 405 410 415

Leu Pro Asn Met Ala Val Lys Arg Asn His Arg Gly Glu Thr Leu Leu 420 425 430

His He Ala Ser He Lys Gly Asp He Pro Ser Val Glu Tyr Leu Leu 435 440 445

Gin Asn Gly Ser Asp Pro Asn Val Lys Asp His Ala Gly Trp Thr Pro 450 455 460

Leu His Glu Ala Cys Asn His Gly His Leu Lys Val Val Glu Leu Leu 465 470 475 480

Leu Gin His Lys Ala Leu Val Asn Thr Thr Gly Tyr Gin Asn Asp Ser 485 490 495

Pro Leu His Asp Ala Ala Lys Asn Gly His Val Asp He Val Lys Leu 500 505 510

Leu Leu Ser Tyr Gly Ala Ser Arg Asn Ala Val Asn He Phe Gly Leu 515 520 525

Arg Pro Val Asp Tyr Thr Asp Asp Glu Ser Met Lys Ser Leu Leu Leu 530 535 540

Leu Pro Glu Lys Asn Glu Ser Ser Ser Ala Ser His Cys Ser Val Met 545 550 555 560

Asn Thr Gly Gin Arg Arg Asp Gly Pro Leu Val Leu He Gly Ser Gly 565 570 575 Leu Ser Ser Glu Gin Gin Lys Met Leu Ser Glu Leu Ala Val He Leu 580 585 590

Lys Ala Lys Lys Tyr Thr Glu Phe Asp Ser Thr Val Thr His Val Val 595 600 605

Val Pro Gly Asp Ala Val Gin Ser Thr Leu Lys Cys Met Leu Gly He 610 615 620

Leu Asn Gly Cys Trp He Leu Lys Phe Glu Trp Val Lys Ala Cys Leu 625 630 635 640

Arg Arg Lys Val Cys Glu Gin Glu Glu Lys Tyr Glu He Pro Glu Gly 645 650 655

Pro Arg Arg Ser Arg Leu Asn Arg Glu Gin Leu Leu Pro Lys Leu Phe 660 665 670

Asp Gly Cys Tyr Phe Tyr Leu Trp Gly Thr Phe Lys His His Pro Lys 675 680 685

Asp Asn Leu He Lys Leu Val Thr Ala Gly Gly Gly Gin He Leu Ser 690 695 700

Arg Lys Pro Lys Pro Asp Ser Asp Val Thr Gin Thr He Asn Thr Val 705 710 715 720

Ala Tyr His Ala Arg Pro Asp Ser Asp Gin Arg Phe Cys Thr Gin Tyr 725 730 735

He He Tyr Glu Asp Leu Cys Asn Tyr His Pro Glu Arg Val Arg Gin 740 745 750

Gly Lys Val Trp Lys Ala Pro Ser Ser Trp Phe He Asp Cys Val Met 755 760 765

Ser Phe Glu Leu Leu Pro Leu Asp Ser 770 775

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

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

Met Ala Asp Tyr Lys Asp Asp Asp Asp Lys Ser 1 5 10

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: TTACCATGGA TTTATCTGCT CTTCGCGTT 29 (2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 38 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: AAAAGTCGAC TAGAATTCAG CCTTTTCTAC ATTCATTC 38

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

( i) SEQUENCE DESCRIPTION: SEQ ID NO: 6: AACAGTACAA TGACTGGGCT C 21

(2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: TCAGCGCTTC TGCACACAGT 20

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

Met Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu Arg Ser 1 5 10 15

(2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 993 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: CCTTCGTGGC CAGAAAGCAA AGTAACAGAA TTTCTCCATC AAAGTAAATT AAAATCTTTT 60 GAAAGTGAGC GTGTTCAACT TCTGCAAGAG GAAACAGCAA GAAATCTCAC ACAGTGTCAA 120

TTGGAATGTG AAAAATATCA GAAAAAATTG GAGGTTTTAA CCAAAGAATT TTATAGTCTC 180

CAAGCCTCTT CTGAAAAACG CATTACTGAA CTTCAAGCAC AGAACTCΛGA GCATCAAGCA 240

AGGCTAGACA TTTATGAGAA ACTGGAAAAA GAGCTTGATG AAATAATAAT GCAAACTGCA 300

GAAATTGAAA ATGAAGATGA GGCTGAAAGG GTTCTTTTTT CCTACGGCTA TGGTGCTAAT 360

GTCCCCACAA CAGCCAAAAG ACGACTAAAG CAAAGTGTTC ACTTGGCAAG AAGAGTGCTT 420

CAATTAGAAA AACAAAACTC GCTGATTTTA AAAGATCTGG AACATCGAAA GGACCAAGTA 480

ACACAGCTTT CACAAGAGCT TGACAGAGCC AATTCGCTAT TAAACCAGAC TCAACAGCCT 540

TACAGGTATC TCATTGAATC AGTGCGTCAG AGAGATTCTA AGATTGATTC ACTGACGGAA 600

TCTATTGCAC AACTTGAGAA AGATGTCΛGC AACTTAAATA AAGAAAAGTC AGCTTTACTA 660

CAGACGAAGA ATCAAATGGC ATTAGATTTA GAACAACTTC TAAATCATCG TGAGGAATTG 720

GCAGCAATGA AACAGATTCT CGTTAAGATG CATAGTAAAC ATTCTGAGAA CAGCTTACTT 780

CTCACTAAAA CAGAACCAAA ACATGTGACA GAAAATCAGA AATCAAAGAC TTTGAATGTG 840

CCTAAAGAGC ATGAAGACAA TATATTTACA CCTAAACCAA CACTCTTTAC TAAAAAAGAA 900

GCACCTGAGT GGTCTAAGAA ACAAAAGATG AAGACCTAGT GTTTTGGATG GGAAGCACCT 960

GTAGACCATT ATATACTCCT GAAGTTCTTT TTC 993

(2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1770 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

CGTTCAAAGA GGGAGTTCAT TCAGGAACCT GCTAAGAATC GGCCCGGTCC CCAGACACGA 60

TCAGACCTAC TGCTGTCAGG AAGGGACTGG AATACGCTAA TTGTGGGAAA GCTTTCTCCA 120

TGGATTCGTC CAGACTCAAA AGTGGAGAAG ATTCGCAGGA ACTCCGAGGC GGCCATGTTA 180

CAGGAGCTGA ATTTTGGTGC ATATTTGGGT CTTCCAGCTT TCCTGCTGCC CCTTAATCAG 240

GAAGATAACA CCAACCTGGC CAGAGTTTTG ACCAACCACA TCCACACTGG CCAT ACTCT 300

TCCATGTTCT GGATGCGGGT ACCCTTGGTG GCACCAGAGG ACCTGAGAGA TGATATAATT 360

GAGAATGCAC CAACTACACA CACAGAGGAG TACAGTGGGG AGGAGAAAAC GTGGATGTGG 420

TGGCACAACT TCCGGACTTT GTGTGΛCTAT AGTAAGAGGA TTGCAGTGGC TCTTGAAATT 480

GGGGCTGACC TCCCATCTAA TCATGTCATT GATCGCTGGC TTGGGGAGCC CATCAAAGGA 540

GGCATTCTCC CCACTAGCAT TTCCCTGACC AATAAGAAGG GATTTCCTGT TCTTTCTAAG 600

ATGCACCAGA GGCTCATCTT CCGGCTCCTC AAGTTGGAGG TGCAGTTCAT CATCACAGGC 660

ACCAACCACC ACTCAGAGAA GGAGTTCTGC TCCTACCTCC AATACCTGGA ATACTTAAGC 720 CAGAACCGCC CTCCACCTAA TGCCTATGAA CTCTTTGACA AGGGCTATGA AGACTATCTG 780

CAGTCCCCGC TTCAGCCACT GATGGACAAT CTGGAATCTC AGACATATGA AGTGTTTGAA 840

AAGGACCCCA TCAAATACTC TCAGTACCAG CAGGCCATCT ATAAATGTCT GCTAGACCGA 900

GTACCAGAAG AGGAGAAGGA TACCAATGTC CAGGTACTGA TGGTGCTGGG AGCAGGACGG 960

GGACCCCTGG TGAACGCTTC CCTGCGGGCA GCCAAGCAGG CCGACCGGCG GATAAAGCTG 1020

TATGCTGTGG AGAAAAACCC AAATGCCGTG GTGACGCTAG AGAACTGGCA GTTTGAAGAA 1080

TGGGGAAGCC AAGTGACCGT AGTCTCATCA GACATGAGGG AATGGGTGGC TCCAGAGAAA 1140

GCAGACATCA TTGTCAGTGA GCTTCTGGGC TCATTTGCTG ACAATGAATT GTCGCCTGAG 1200

TGCCTGGATG GAGCCCAGCA CTTCCTAAAA GATGATGGTG TGAGCATCCC CGGGGAGTAC 1260

ACTTCCTTTC TGGCTCCCAT CTCTTCCTCC AAGCTGTACA ATGAGGTCCG AGCCTGTAGG 1320

GAGAAGGACC GTGACCCTGA GGCCCAGTTT GAGATGCCTT ATGTGGTACG GCTGCACAAC 1380

TTCCACCAGC TCTCTGCACC CCAGCCCTGT TTCACCTTCA GCCATCCCAA CAGAGATCCT 1440

ATGATTGACA ACAACCGCTA TTGCACCTTG GAATTTCCTG TGGAGGTGAA CACAGTACTA 1500

CATGGCTTTG CCGGCTACTT TGAGACTGTG CTTTATCAGG ACATCACTCT GAGTATCCGT 1560

CCAGAGACTC ACTCTCCTGG GATGTTCTCA TGGTTTCCCA TCCTCTTCCC TATTAAGCAG 1620

CCCATAACGG TACGTGAAGG CCAAACCATC TGTGTGCGTT TCTGGCGATG CAGCAATTCC 1680

AAGAAGGTGT GGTATGAGTG GGCTGTGACA GCACCAGTCT GTTCTGCTAT TCATAACCCC 1740

ACAGGCCGCT CATATACCAT TGGCCTCTAG 1770

(2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1345 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: modified_base

(B) LOCATION: 328

(D) OTHER INFORMATION :/note= "R = A or G"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

GAGCCCGGCC GCGGCCTGCT GGTTTCAGTG ATGGCTCATG AAGCAATGGA ATATGATGTT 60

CAGGTGCAGT TAAATCATGC CGAACAACAG CCAGCTCCTG CTGGCATGGC CAGCAGCCAA 120

GGGGGACCAG CCCTCCTCCA GCCTGTTCCT GCTGATGTGG TCAGCAGCCA GGGGGTACCA 180

TCCATCCTCC AGCCAGCTCC TGCTGAGGTG ATCAGCAGCC AAGCGACACC ACCCCTGCTC 240

CAGCCTGCTC CGCAACTGTC TGTTGACCTG ACAGAAGTGG AGGTCTTGGG AGAAGACACT 300

GTGGAGAACA TCAATCCAAG AACTTCARAA CAACATAGGC AGGGATCTGA TGGTAATCAC 360

ACCATCCCAG CATCTTCGTT GCATTCAATG ACCAACTTCA TCAGCGGACT GCAGAGACTT 420 CATGGCATGC TGGAATTCCT GAGACCTTCA TCTTCAAACC ACAGTGTAGG GCCAATGAGA 480

ACAAGAAGGA GGGTATCTGC TTCACGGAGG GCAAGAGCCG GAGGGTCTCA GAGGACAGAC 540

AGTGCCAGGT TGAGAGCACC ATTGGATGCT TACTTTCAGG TGAGCAGGAC CCAGCCTGAC 600

TTGCCAGCTA CCACTTATGA TTCAGAGACT AGGAATCCTG TATCTGAAGA GTTGCAGGTG 660

TCTAGTAGTT CTGATTCTGA CAGTGACAGC TCTGCAGAGT ATGGAGGGGT TGTTGACCAC 720

GCAGAGGAAT CTGGAGCTGT CATTTTAGAA GAGCAACTAG CAGGTGTCTC AGCAGAGCAA 780

GAAGTTACAT GTATCGATGG AGGCAAGACC CTCCCCAAAC AGCCATCTCC CCAGAAGTCT 840

GAGCCTCTGC TACCTTCTGC TTCTATGGAT GAGGAAGAAG GGGACACTTG TACAATATGT 900

CTGGAACAGT GGACCAATGC TGGGGACCAC CGGCTCTCAG CATTACGCTG TGGGCATCTC 960

TTTGGGTATA GGTGCATTTC CACGTGGCTT AAAGGACAAG TACGAAAATG TCCCCAGTGC 1020

AACAAGAAAG CCAGGCACAG TGACATTGTC GTCCTTTATG CCCGAACCCT GAGAGCTTTG 1080

GACACTAGTG AACAGGAGCG CATGAAAAGG TAGGTGGTAA GAGTATGCCT GGCTGGAATG 1140

TTCCCTTTTG GTTCATTGTA GGCACATCTG AAAAAGAAGT TATGAGTCAC TCGTAGTGAG 1200

GTTTTACTTG ACCTGTGACT TGGGATCTCT GGGGATCATT GGCAGTCTGT CTTACACTGT 1260

TATTTATAAT TCATGTCTGA TCATCTTCTT AAGGAAGTCT GCATCGTTTG CCTTATGTAG 1320

AGCATTAAAC ACAAGGATCT GGCAC 1345

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1248 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: modified_base

(B) LOCATION: 786

(D) OTHER INFORMATION: /note= "R = A or G"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

GACTACCATC AGAACTGGGG CCGTGATGGG GGTCCCCGCA GCTCCGGTGG GGGCTATGGA 60

GGGGGGCCAG CAGGGGGTCA TGGAGGTAAC CGAGGCTCCG GAGGAGGCGG CGGCGGCGGA 120

GGGGGTGGTC GAGGCGGCAG GGGCCGGCAT CCCGGGCACC TGAAAGGCCG CGAAATCGGC 180

ATGTGGTACG CGAAAAAACA GGGGCAGAAG AACAAGGAAG CGGAGAGGCA AGAGAGAGCT 240

GTAGTACACA TGGATGAACG ACGAGAAGAA CAAATTGTAC AGTTACTGAA TTCTGTTCAA 300

GCGAAGAATG ATAAAGAGTC AGAAGCACAG ATATCCTGGT TTGCTCCTGA GGATCATGGA 360

TACGGTACTG AAGTTTCTAC TAAGAACACA CCATGCTCAG AGAACAAACT TGACATCCAG 420

GAAAAGAAGT TGATAAATCA AGAAAAAAAA ATGTTTAGAA TCAGGAACAG ATCATATATT 480

GACCGAGATT CTGAGTATCT CTTGCAAGAA AATGAACCAG ATGGAACTTT AGACCAAAAA 540 TTATTGGAAG ATTTACAAAA GAAAAAAAAT GACCTTCGGT ATATTGAAAT GCAGCATTTC 600

AGAGAAAAGC TGCCTTCGTA TGGAATGCAA AAGGAATTGG TAAATTTAAT TGATAACCAT 660

CAGGTAACAG TAATAAGTGG TGAAACTGGT TGTGGCAAAA CCACTCAAGT TACTCAGTTC 720

ATTTTGGATA ACTACATTGA AAGAGGAAAA GGATCTGCTT GCAGAATAGT TTGTACTCAG 780

CCAAGRAGAA TTAGTGCCAT TTCAGTTGCG GAAAGAGTAG CTGCAGAAAG GGCAGAATCT 840

TGTGGCAGTG GTAATAGTAC TGGATATCAA ATTCGTCTCC AGAGTCGGTT GCCAAGGAAA 900

CAGGGTTCTA TCTTATACTG TACAACAGGA ATCATCCTTC AGTGGCTCCA GTCAGACCCG 960

TATTTGTCCA GTGTTAGTCA TATCGTACTT GATGAAATCC ATGAAAGAAA TCTGCAGTCA 1020

GATGTTTTAA TGACTGTTGT TAAAGACCTT CTCAATTTTC GATCTGACTT GAAAGTAATA 1080

TTGATGAGTG CAACATTGAA TGCAGAAAAG TTTTCAGAAT ATTTTGGTAA CTGTCCAATG 1140

ATACATATAC CTGGTTTTAC CTTTCCGGTT GTGGAATATC TTTTGGAAGA TGTAATTGAA 1200

AAAATAAGGT ATGTTCCAGA ACAAAAAGAA CACAGATCCC AGTTTAAG 1248

(2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1803 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: modified_base

(B) LOCATION: 1362..1771

(D) OTHER INFORMATION: /note= "N = A or C or G or T"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

AATATATCCT GGAAGAAGAC AATAGTTACC CGTTTCCTAA AACTGGTTCC AGACCTTTTG 60

GCCATTGTGC AGCGTAAGAA AAAGGAAGGG GAAGAAGAAC AAGCAATCAA CAGACAGΛCA 120

GCGTTGTATA CCTTAAAGCT TTTATGCAAG AATTTTGGTG CAGAAAATCC AGATCCTTTT 180

GTCCCAGTGC TGAGCACTGC TGTGAAACTG ATTGCTCCAG AGAGAAAGGA GGAGAAGAAT 240

GTCTTGGGAA GCGCGCTGCT GTGCATAGCA GAGGTGACCT CCACCCTGGA GGCGC GGCC 300

ATCCCCCAGC TTCCCAGCCT GATGCCATCG TTGCTGACAA CAATGAAGAA CACCAGCGAG 360

CTGGTCTCCA GCGAGGTCTA CCTGCTCAGT GCCTTGGCTG CTCTGCAGAA GGTTGTGGAG 420

ACTCTCCCGC ACTTCATCAG CCCCTATCTG GAAGGCATTC TCTCCCAGGT GATTCATCTG 480

GAGAAAATCA CTAGTGAAAT GGGTTCTGCG TCACAGGCTA ATATCCGCCT CACATCTCTT 540

AAAAAGACAC TGGCTACCAC ACTTGCACCC CGAGTCCTGT TGCCCGCCAT CAAAAAAACT 600

TACAAGCAGA TTGAGAAGAA CTGGAAGAAT CACATGGGTC CGTTTATGAG CATCTTGCAA 660

GAGCATATTG GGGCGATGAA GAAGGAAGAG CTCACCTCCC ATCAGTCTCA GCTAACCGCC 720

TTTTTCCTGG AGGCCCTGGA CTTCCGAGCC CAGCACTCTG AGAACGATCT GGAGGAAGTT 780 GGAAAAACGG AAAATTGTAT CATTGACTGT CTAGTAGCCA TGGTTGTCAA ACTTTCCGAG 840

GTCACATTCA GGCCCCTGTT CTTCAAGCTG TTTGATTGGG CTAAAACAGA AGATGCCCCA 900

AAGGACAGGT TGTTGACATT TTACAACTTG GCAGATTGCA TTGCTGAAAA GCTGAAAGGG 960

CTTTTTACTC TGTTTGCCGG CCACTTAGTG AAGCCTTTTG CTGACACCTT GGACCAGGTG 1020

AACATCTCCA AAACAGATGA AGCATTTTTT GACTCTGAAA ATGACCCTGA AAAGTGCTGC 1080

TTGCTGTTGC AGTTTATTTT GAACTGTTTA TACAAAATCT TCCTTTTTGA TACCCAGCAT 1140

TTTATAAGTA AAGAGAGAGC AGGAGCCTTG ATGATGCCTC TGGTGGATCA GCTGGAAAAC 1200

AGGCTTGGGG GAGAAGAGAA ATTCCAGGAA CGGGTGACAA AGCACCTGAT ACCATGCATC 1260

GTACAGTTTT CCGTGGCCAT GGCGGATGAC TCTCTTTGGA AACCACTGAA CTACCAGATT 1320

CTGCTAAAGA CGAGAGACTC CTCGCCTAAG GTTCGATTTG NTGCTTTGAT TACTGTGTTA 1380

GCACTGGCTG AAAAACTAAA GGAGAATTAT ATTGTCTTGC TACCAGAATC CATTCCTTTC 1440

TTAGCAGAGT TGATGGAAGA TGAATGTGAA GAAGTAGAAC ATCAGTGCCA AAAGACTATT 1500

CAGCAACTGG AAACTGTCCT GGGAGAGCCA CTCCAGAGCT ATTTCTAAGA CTTCTGTGGT 1560

GTTTCATACT CTACTCAGAG TTCACACTCA TATTTCATAT TTTTATTTTC GGGTGTTGGG 1620

TGCCATGTTA CTTTGGGTGT CTTAATACAC CTACTTGGAT TACTTACAAA TGTTTTATCA 1680

CTTCGNTACA AAATCCCCAC CTGGCTTGTG CTGNCACATA AGCCTCTCCC GCCTATCGNA 1740

TAGAGCTTGT AGAGGCCTCG CGGCCTCGAN AGATCTATTG AATCGCTAGA TACTGAAAAA 1800

ACC 1803

(2) INFORMATION FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 817 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

TGGGGGTCGT CCCTAACGGC CGCGACGCAG AGAGCGGTCA CTCCCTGGCC GAGGGGCAGG 60

CTCCTCACGG CCTCCCTGGG ACCCCAGGCG CGTCGGGAGG CGTCGTCCTC CAGCCCCGAG 120

GCCGGCGAAG GGCAGATCCG CCTCACAGAC AGTTGCGTCC AGAGGCTTTT GGAAATCACC 180

GAAGGTCAGA ATTCCTCAGG CTGCAAGTGG AGGGAGGTGG ATGCTCCGGA TTCCAATACA 240

AATTTTCACT GGATACAGTT ATCAACCCCG ACGACAGGGT ATTTGAACAG GGTGGGGCAA 300

GAGTGGTGGT TGACTCTGAT AGCTTGGCCT TCGTGAAAGG GGCCCAGGTG GACTTCAGCC 360

AAGAACTGAT CCGAAGCTCA TTTCAAGTGT TGAACAATCC TCAAGCACAG CAAGGCTGCT 420

CCTGTGGGTC ATCTTTCTCT ATCAAACTTT GATGTGATGA CTGGTGACTC TGGGATTGTC 480

ACCAGTTGTA CCAATTTGAA GAACCTGGAA TTAGTAGAAT TCTAGAAGTT TACTTCTAAT 540 CATGTCCCTC TCAATTTTAT TTCCCGCAGT CCAGGAGTGT TATGTTTTGC CACTATTATT 600

TTCAGAATGT GAAGATTTTA CTCTTGGCTT AATTTTTCCC TCCACTCAGT GCTAAGGCTG 660

AGCCTCCAGA TGCTGTTACC TCAGATTTAA TCACTGGTTG AAACTCCGTA TAATCTGTAG 720

AGCCTCCATG GCTCTAAAAT TTGGAATTAA CTTCTCTTGC CTTAAGAGCT GCTTGTACAT 780

ATGTGGATAG CTATGTATAA AAGCTTCATT TTAAAAA 817

(2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2138 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:

GGCCCGGCTG GAGGAGCCCC GACCCCAGCT CTGGTGGCGG GCAGCAGCGC CGCGGCCCCC 60

TTCCCTCACG GGGACTCGGC CCTGAACGAG CAGGAGAAGG AGTTGCAGCG GCGGCTGAAG 120

CGCCTCTACC CGGCCGTGGA CGAACAAGAG ACGCCGCTGC CTCGGTCCTG GAGCCCGAAG 180

GACAAGTTCA GCTACATCGG CCTCTCTCAG AACAACCTGC GGGTGCACTA CAAAGGTCAT 240

GGCAAAACCC CAAAAGATGC CGCGTCAGTT CGAGCCACGC ATCCAATACC AGCAGCCTGT 300

GGGATTTATT ATTTTGAAGT AAAAATTGTC AGTAAGGGAA GAGATGGTTA CATGGGAATT 360

GGTCTTTCTG CTCAAGGTGT GAACATGAAT AGACTACCAG GTTGGGATAA GCATTCATAT 420

GGTTACCATG GGGATGATGC ACATTCGTTT TGTTCTTCTG GAACTGGACA ACCTTATGGA 480

CCAACTTTCA CTACTGGTGA TGTCATTGGC TGTTGTGTTA ATCTTATCAA CAATACCTGC 540

TTTTACACCA AGAATGGACA TAGTTTAGGT ATTGCTTTCA CTGACCTACC GCCAAATTTG 600

TATCCTACTG TGGGGCTTCA AACACCAGGA GAAGTGGTCG ATGCCAATTT TGGGCAACAT 660

CCTTTCGTGT TTGATATAGA AGACTATATG CGGGAGTGGA GAACCAAAAT CCAGGCACAG 720

ATAGATCGAT TTCCTATCGG AGATCGAGAA GGAGAATGGC AGACCATGAT ACAAAAAATG 780

GTTTCATCTT ATTTAGTCCA CCATGGGTAC TGTGCCACAG CAGAGGCCTT TGCCAGATCT 840

ACAGACCAGA CCGTTCTAGA AGAATTAGCT TCCATTAAGA ATAGACAAAG AATTCAGAAA 900

TTGGTATTAG CAGGAAGAAT GGGAGAAGCC ATTGAAACAA CACAACAGTT ATACCCAAGT 960

TTACTTGAAA GAAATCCTAA TCTCCTTTTC ACATTAAAAG TGCGTCAGTT TATAGAAATG 1020

GTGAATGGTA CAGATAGTGA AGTACGATGT TTGGGAGGCC GAAGTCCAAA GTCTCAAGAC 1080

AGTTATCCTG TTAGTCCTCG ACCTTTTAGT AGTCCAAGTA TGAGCCCCAG CCATGGAATG 1140

AATATCCACA ATTTAGCATC AGGCAAAGGA AGCACCGCAC ATTTTTCAGG TTTTGAAAGT 1200

TGTAGTAATG GTGTAATATC AAATAAAGCA CATCAATCAT ATTGCCATAG TAATAAACAC 1260

CAGTCATCCA ACTTGAATGT ACCAGAACTA AACAGTATAA ATATGTCAAG ATCACAGCAA 1320

GTTAATAACT TCACCAGTAA TGATGTAGAC ATGGAAACAG ATCACTACTC CAATGGAGTT 1380 GGAGAAACTT CATCCAATGG TTTCCTAAAT GGTAGCTCTA AACATGACCA CGAAATGGAA 1440

GATTGTGACA CCGAAATGGA AGTTGATTCA AGTCAGTTGA GACGCCAGTT GTGTGGAGGA 1500

AGTCAGGCCG CCATAGAAAG AATGATCCAC TTTGGACGAG AGCTGCAAGC AATGAGTGAA 1560

CAGCTAAGGA GAGACTGTGG CAAGAACACT GCAAACAAAA AATGTTGAAG GATGCATTCA 1620

GTCTACTAGC ATATTCAGAT CCCTGGAACA GCCCAGTTGG AAATCAGCTT GACCCGATTC 1680

AGAGAGAACC TGTGTGCTCA GCTCTTAACA GTGCAA ATT AGAAACCCAC AATCTGCCAA 1740

AGCAACCTCC ACTTGCCCTA GCAATGGGAC AGGCCACACA ATGTCTAGGA CTGATGGCTC 1800

GATCAGGAAT TGGATCCTGC GCATTTGCCA CAGTGGAAGA CTACCTACAT TAGCTATGCA 1860

TTTCAAGAGC TCACACTTAT ATTGTGGCAT ATAGTCAACA TGGAAGTAGA CCAGCTCTGC 1920

TGATTTGAAA TTTAGATTTT TTAAATTATG TACTGGGGAC AGGTTTTTGT CGCTTTACAT 1980

TGCTTCCTAG TTTACAGCAT GATGCAAATG ATTTTCTAAC TTAGTGTTAG GAGAAATTAT 2040

TTTCCATCTT TAACCTCTTA GTTGTCTAAG AGTTAAATAT TACTGAATTT CAGACGTTCA 2100

AATTGATCAT CACAAATCCT TTAAAACAAT TACCTAAA 2138

(2) INFORMATION FOR SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3428 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: modified_base

(B) LOCATION: 178

(D) OTHER INFORMATION: /note= "W = A or T"

(ix) FEATURE:

(A) NAME/KEY: modified_base

(B) LOCATION: 1331..3246

(D) OTHER INFORMATION :/note= "Y = C or T"

(ix) FEATURE:

(A) NAME/KEY: modifiedjbase

(B) LOCATION: 2886..3212

(D) OTHER INFORMATION: /note= "H = A or C or T"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

GGGCCGCCCC GCGGGAAGAT GAATAAGGGC TGGCTGGAGC TGGAGAGCGA CCCAGGCCTC 60

TTCACCCTGC TCGTGGAAGA TTTCGGTGTC AAGGGGGTGC AAGTGGAGGA GATCTACGAC 120

CTTCAGAGCA AATGTCAGGG CCCTGTATAT GGATTTATCT TCCTGTTCAA ATGGATCWAA 180

GAGCGCCGGT CCCGGCGAAA GGTCTCTACC TTGGTGGATG ATACGTCCGT GATTGATGAT 240

GATATTGTGA ATAACATGTT CTTTGCCCAC CAGCTGATAC CCAACTCTTG TGCAACTCAT 300

GCCTTGCTGA GCGTGCTCCT GAACTGCAGC AGCGTGGACC TGGGACCCAC CCTGAGTCGC 360

ATGAAGGACT TCACCAAGGG TTTCAGCCCT GAGGCCCGAG CCACGCCACC TCCCTGAGAA 420 GCAGAATGGC CTTAGTGCAG TGCGGACCAT GGAGGCGTTC CACTTTGTCA GCTATGTGCC 460

TATCACAGGC CGGCTCTTTG AGCTGGATGG GCTGAAGGTC TACCCCATTG ACCATGGGCC 540

CTGGGGGGAG GACGAGGAGT GGACAGACAA GGCCCGGCGG GTCATCATGG AGCGTATCGG 600

CCTCGCCACT GCAGGGGAGC CCTACCACGA CATCCGCTTC AACCTGATGG CAGTGGTGCC 660

CGACCGCAGG ATCAAGTATG AGGCCAGGCT GCATGTGCTG AAGGTGAACC GTCAGACAGT 720

ACTAGAGGCT CTGCAGCAGC TGATAAGAGT AACACAGCCA GAGCTGATTC AGACCCACAA 780

GTCTCAAGAG TCACAGCTGC CTGAGGAGTC CAAGTCAGCC AGCAACAAGT CCCCGCTGGT 840

GCTGGAAGCA AACAGGGCCC CTGCAGCCTC TGAGGGCAAC CACACAGATG GTGCAGAGGA 900

GGCGGCTGGT TCATGCGCAC AAGCCCCATC CCACAGCCCT CCCAACAAAC CCAAGCTAGT 960

GGTGAAGCCT CCAGGCAGCA GCCTCAATGG GGTTCACCCC AACCCCACTC CCATTGTCCA 1020

GCGGCTGCCG GCCTTTCTAG ACAATCACAA TTATGCCAAG TCCCCCATGC AGGAGGAAGA 1080

AGACCTGGCG GCAGGTGTGG GCCGCAGCCG AGTTCCAGTC CGCCCACCCC AGCAGTACTC 1140

AGATGATGAG GATGACTATG AGGATGACGA GGAGGATGAC GTGCAGAACA CCAACTCTGC 1200

CCTTAGGTAT AAGGGGAAGG GAACAGGGAA GCCAGGGGCA TTGAGCGGTT CTGCTGATGG 1260

GCAACTGTCA GTGCTGCAGC CCAACACCAT CAACGTCTTG GCTGAGAAGC TCAAAGAGTC 1320

CCAGAAGGAC YTCTCAATTC CTCTGTCCAT CAAGACTAGC AGCGGGGCTG GGAGTCCGGC 1380

TGTGGCAGTG CCCACACACT CGCAGCCCTC ACCCΛCCCCC AGCAATGAGA GTACAGACAC 1440

GGCCTCTGAG ATCGGCAGTG CTTTCAACTC GCCACTGCGC TCGCCTATCC GCTCAGCCAA 1500

CCCGACGCGG CCCTCCAGCC CTGTCACCTC CCACATCTCC AAGGTGCTTT TTGGAGAGGA 1560

TGACAGCCTG CTGCGTGTTG ACTGCATACG CTACAACCGT GCTGTCCGTG ATCTGGGTCC 1620

TGTCATCAGC ACAGGCCTGC TGCACCTGGC TGAGGATGGG GTGCTGAGTC CCCTGGCGCT 1680

GACAGAGGGT GGGAAGGGTT CCTCGCCCTC CATCAGACCA ATCCAAGGCA GCCAGGGGTC 1740

CAGCAGCCCA GTGGAGAAGG AGGTCGTGGA AGCCACGGAC AGCAGAGAGA AGACGGGGAT 1800

GGTGAGGCCT GGCGAGCCCT TGAGTGGGGA GAAATACTCA CCCAAGGAGC TGCTGGCACT 1860

GCTGAAGTGT GTGGAGGCTG AGATTGCAAA CTATGAGGCG TGCCTCAAGG AGGAGGTAGA 1920

GAAGAGGAAG AAGTTCAAGA TTGATGACCA GAGAAGGACC CACAACTACG ATGAGTTCAT 1980

CTGCACCTTT ATCTCCATGC TGGCTCAGGA AGGCATGCTG GCCAACCTAG TGGAGCAGAA 2040

CATCTCCGTG CGGCGGCGCC AAGGGGTCAG CATCGGCCGG CTCCACAAGC AGCGGAAGCC 2100

TGACCGGCGG AAACGCTCTC GCCCCTACAA GGCCAAGCGC CAGTGAGGAC TGCTGGCCCT 2160

GACTCTGCAG CCCACTCTTG CCGTGTGGCC CTCACCAGGG TCCTTCCCTG CCCCACTTCC 2220

CCTTTTCCCA GTATTACTGA ATAGTCCCAG CTGGAGAGTC CAGGCCCTGG GAATGGGAGG 2280

AACCAGGCCA CATTCCTTCC ATCGTGCCCT GAGGCCTGAC ACGGCAGATC AGCCCCATAG 2340

TGCTCAGGAG GCAGCATCTG GAGTTGGGGC ACAGCGAGGT ACTGCAGCTT CCTCCACAGC 2400 CGGCTGTGGA GCAGCAGGAC CTGGCCCTTC TGCCTGGGCA GCAGAATATA TATTTTACCT 2460

ATCAGAGACA TCTATTTTTC TGGGCTCCAA CCCAACATGC CACCATGTTG ACATAAGTTC 2520

CTACCTGACT ATGCTTTCTC TCCTAAGGAG CTGTCCTGGT GGGCCCAGGT CCTTGTATCA 2580

TGCCACGGTC CCAACTACAG GGTCCTAGCT GGGGGCCTGG GTGGGCCCTG GGCTCTGGGC 2640

CCTGCTGCTC TAGCCCCAGC CΛCCAGCCTG TCCCTGTTGT AAGGAAGCCA GGTCTTCTCT 2700

CTTCATTCCT CTTAGGAGAG TGCCAAACTC AGGGACCCAG CACTGGGCTG GGTTGGGAGT 2760

AGGGTGTCCC AGTGGGGTTG GGGTGAGCAG GCTGCTGGGA TCCCATGGCC TGAGCAGAGC 2820

ATGTGGGAAC TGTTCAGTGG CCTGTGAACT GTCTTCCTTG TTCTAGCCAG GCTGTTCAAG 2880

ACTGCTHTCC ATAGCAAGGT TCTAGGGCTC TTCGCCTTCA GTGTTGTGGC CCTAGCTATG 2940

GGCCTAAATT GGGCTCTAGG TCTCTGTCCC TGGCGCTTGA GGCTCAGAAG AGCCTCTGTC 3000

CAGCCCCTCA GTATTACCAT GTCTCCCTCT CAGGGGTAGC AGAGACAGGG TTGCTTATAG 3060

GAAGCTGGCA CCACTCAGCT HTTCCTGCTA CTCCAGTTTC CTCAGCCTYT GCAAGGCACT 3120

CAGGGTGGGG GACAGCAGGA TCAAGACAAC CCGTTGGAGC CCCTGTGTTC CAGAGGACCT 3180

GATGCCAAGG GGTAATGGGC CCAGCAGTGC CTHTGGAGCC CAGGCCCCAA CACAGCCCCA 3240

TGGCCTYTGC CAGATGGCTT TGAAAAAGGT GATCCAAGCA GGCCCCTTTA TCTGTACATA 3300

GTGACTGAGT GGGGGGTGCT GGCAAGTGTG GCAGCTGCCT CTGGGCTGAG CACAGCTTGA 3360

CCCCTCTAGC CCCTGTAAAT ACTGGATCAA TGAATGAATA AAACTCTCCT AAGAATCTCC 3420

TGAAAAAA 3428

(2) INFORMATION FOR SEQ ID NO: 17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 938 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

GAGCGGGAAG CAAGGGACGA GCTACCTGGA GCGCCTCCTG TTCTTTGCAG TTCCTCCTCA 60

GATCTTAGCC TCCTGTTGGG CCCCTCTTTT CAGAGCCAGC ATTCTTTCCA GCCCCTGGAG 120

CCCAAACCAG ACCTCACTTC ATCCACAGCT GGGGCCTTCT CTGCACTTGG GGCCTTCCAT 180

CCCGATCATA GGGCAGAAAG GCCATTCCCT GAGGAAGATC CTGGACCTGA CGGGGAGGGC 240

CTCCTAAAGC AAGGGCTGCC GCCTGCTCAG CTGGAGGGCC TCAAGAATTT TTTGCACCAG 300

TTGCTGGAGA CAGTGCCCCA GAACAATGAG AACCCTTCTG TCGACCTGTT GCCCCCTAAG 360

TCTGGTCCTC TGACTGTCCC ATCTTGGGAG GAAGCCCCTC AAGTGCCACG TATTCCACCG 420

CCTGTCCACA AAACCAAAGT TCCCTTAGCC ATGGCATCCA GTCTTTTCCG GGTCCCTGAG 480

CCTCCCTCCT CCCATTCACA AGGCAGTGGT CCCAGCAGTG GTTCCCCAGA GAGAGGTGGA 540 GATGGGCTTA CATTCCCAAG GCAGCTGATG GAGGTGTCTC AACTGTTGCG ACTCTACCAG 600

GCTCGGGGCT GGGGGGCTCT GCCTGCTGAG GATCTCCTGC TCTACCTGAA GAGGCTGGAA 660

CACAGCGGGA CTGATGGCCG AGGGGATAAT GTCCCCAGAA GGAACACAGA CTCCCGCTTG 720

GGTGAGATCC CCCGGAAAGA GATTCCCTCC CAGGCTGTCC CTCGCCGCCT TGCTACAGCC 780

CCCAAGACTG AAAAACCTCC CGCACGGAAG AAAAGTGGGC ACCCTGCCCC GAGTAGCATG 840

AGGAGCCGGG GGGGAGTCTG GAGATGAGCC CCCCTACCCT CTCTCCTCTT TGTTCTCTCA 900

TTGTTGTTAT TTTAATAAAT GCTCAGTAGT CTGTAAAA 938

(2) INFORMATION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1379 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

ATGGTGAAGG TGAAGGGGCA GGTCAGCGAG ATGGCGGTGC TCCTCATCGA CCCCGAGCCT 60

CAGATTGCTG CCCTGGCCAA GAACTTCTTC AATGAGCTCT CCCACAAGGG CAACGCAATC 120

TATAATCTCC TTCCAGATAT CATCAGCCGC CTGTCAGACC CCGAGCTGGG GGTGGAGGAA 180

GAGCCTTTCC ACACCATCAT GAAACAGCTC CTCTCCTACA TCACCAAGGA CAAGCAGACA 240

GAGAGCCTGG TGGAAAAGCT GTGTCAGCGG TTCCGCACAT CCCTAACTGA GCGGCAGCAG 300

CGAGACCTGG CCTACTGTGT GTCACAGCTG CCCCTCACAG AGCGAGGCCT CCGTAAGATG 360

CTTGACAATT TTGACTGTTT TGGAGACAAA CTGTCAGATG AGTCCATCTT CAGTGCTTTT 420

TTGTCAGTTG TAGGCAAGCT GCGACGTGGG GCCAAGCCTG AGGGCAAGGC TATAATAGAT 480

GAATTTGAGC AGAAGCTTCG GGCCTGTCAT ACCAGAGGTT TGGATGGAAT CAAGGAGCTT 540

GAGATTGGCC AAGCAGGTAG CCAGAGAGCG CCATCAGCCA AGAAACCATC CACTGGTTCT 600

AGGTACCAGC CTCTGGCTTC TACAGCCTCA GACAATGACT TTGTCACACC AGAGCCCCGC 660

CGTACTACCC GTCGGCATCC AAACACCCAG CAGCGAGCTT CCAAAAAGAA ACCCAAAGTT 720

GTCTTCTCAA GTGATGAGTC CAGTGAGGAA GATCTTTCAG CAGAGATGAC AGAAGACGAG 780

ACACCCAAGA AAACAACTCC CATTCTCAGA GCATCGGCTC GCAGGCACAG ATCCTAGGAA 840

GTCTGTTCCT GTCCTCCCTG TGCAGGGTAT CCTGTAGGGT GACCTGGAAT TCGAATTCTG 900

TTTCCCTTGT AAAATATTTG TCTGTCTCTT TTTTTTAAAA AAAAAAAAGG CCGGGCACTG 960

TGGCTCACGC CTGTAATCCC AGCACTTTGC GATACCAAGG CGGGTGGATA ACCTGAGGTA 1020

GGGAGTTCGA GACCAGCCTG ACCAACATGG AGAAACCCCA TCTCTACTAA AAATΛAAAAA 1080

TTAGCCGGGC GTATTGGCGT GCGCCTGTAA TCCCAGCTAC TCAAGAGGCT GAGGCAGGAG 1140

AATCGCCTGA ACCCAGAGGC GGAGGTTGTA GTGAGCCGAA ATCACACCAT TGCACTCCAG 1200

CTTGGGCAAC AATAGCGAAC CTCCATCTCA AATTAAAAAA AAAATGCCTA CACGCTCTTT 1260 AAAATGCAAG GCTTTCTCTT AAATTAGCCT AACTGAACTG CGTTGAGCTG CTTCAACTTT 1320 GGAATATATG TTTGCCAATC TCCTTGTTTT CTAATGAATA AATGTTTTTA TATACTTTT 1379

(2) INFORMATION FOR SEQ ID NO: 19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 273 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:

Cys Ser Ser Ser Ser Asp Leu Ser Leu Leu Leu Gly Pro Ser Phe Gin 1 5 10 15

Ser Gin His Ser Phe Gin Pro Leu Glu Pro Lys Pro Asp Leu Thr Ser 20 25 30

Ser Thr Ala Gly Ala Phe Ser Ala Leu Gly Ala Phe His Pro Asp His 35 40 45

Arg Ala Glu Arg Pro Phe Pro Glu Glu Asp Pro Gly Pro Asp Gly Glu 50 55 60

Gly Leu Leu Lys Gin Gly Leu Pro Pro Ala Gin Leu Glu Gly Leu Lys 65 70 75 80

Asn Phe Leu His Gin Leu Leu Glu Thr Val Pro Gin Asn Asn Glu Asn 85 90 95

Pro Ser Val Asp Leu Leu Pro Pro Lys Ser Gly Pro Leu Thr Val Pro 100 105 110

Ser Trp Glu Glu Ala Pro Gin Val Pro Arg He Pro Pro Pro Val His 115 120 125

Lys Thr Lys Val Pro Leu Ala Met Ala Ser Ser Leu Phe Arg Val Pro 130 135 140

Glu Pro Pro Ser Ser His Ser Gin Gly Ser Gly Pro Ser Ser Gly Ser 145 150 155 160

Pro Glu Arg Gly Gly Asp Gly Leu Thr Phe Pro Arg Gin Leu Met Glu 165 170 175

Val Ser Gin Leu Leu Arg Leu Tyr Gin Ala Arg Gly Trp Gly Ala Leu 180 185 190

Pro Ala Glu Asp Leu Leu Leu Tyr Leu Lys Arg Leu Glu His Ser Gly 195 200 205

Thr Asp Gly Arg Gly Asp Asn Val Pro Arg Arg Asn Thr Asp Ser Arg 210 215 220

Leu Gly Glu He Pro Arg Lys Glu He Pro Ser Gin Ala Val Pro Arg 225 230 235 240

Arg Leu Ala Thr Ala Pro Lys Thr Glu Lys Pro Pro Ala Arg Lys Lys 245 250 255 Ser Gly His Pro Ala Pro Ser Ser Met Arg Ser Arg Gly Gly Val Trp 260 265 270

Arg

(2) INFORMATION FOR SEQ ID NO: 20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2531 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 75..2405

(ix) FEATURE:

(A) NAME/KEY: modified_base

(B) LOCATION: 531

(D) OTHER INFORMATION : /note= "R = A or G"

(ix) FEATURE:

(A) NAME/KEY: modified_base

(B) LOCATION: 153

(D) OTHER INFORMATION: /note= "Xaa = Glu or Lys for both SEQ ID NO:20 and SEQ ID NO:21"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:

GCAGCTTCCC TGTGGTTTCC CGAGGCCTCC TTGCTTCCCG CTCTCCGAGG AGCCTTTCAT 60

CCGAAGGCGG GACG ATG CCG GAT AAT CGG CAG CCG AGG AAC CGG CAG CCG 110 Met Pro Asp Asn Arg Gin Pro Arg Asn Arg Gin Pro 1 5 10

AGG ATC CGC TCC GGG AAC GAG CCT CGT TCC GCG TCC GCC ATG GAA CCG 158 Arg He Arg Ser Gly Asn Glu Pro Arg Ser Ala Ser Ala Met Glu Pro 15 20 25

GAT GGT CGC GGT GCC TGG GCC CAC AGT CGC GCC GCG CTC GAC CGC CTG 206 Asp Gly Arg Gly Ala Trp Ala His Ser Arg Ala Ala Leu Asp Arg Leu 30 35 40

GAG AAG CTG CTG CGC TGC TCG CGT TGT ACT AAC ATT CTG AGA GAG CCT 254 Glu Lys Leu Leu Arg Cys Ser Arg Cys Thr Asn He Leu Arg Glu Pro 45 50 55 60

GTG TGT TTA GGA GGA TGT GAG CAC ATC TTC TGT AGT AAT TGT GTA AGT 302 Val Cys Leu Gly Gly Cys Glu His He Phe Cys Ser Asn Cys Val Ser 65 70 75

GAC TGC ATT GGA ACT GGA TGT CCA GTG TGT TAC ACC CCG GCC TGG ATA 350 Asp Cys He Gly Thr Gly Cys Pro Val Cys Tyr Thr Pro Ala Trp He 80 85 90

CAA GAC TTG AAG ATA AAT AGA CAA CTG GAC AGC ATG ATT CAA CTT TGT 398 Gin Asp Leu Lys He Asn Arg Gin Leu Asp Ser Met He Gin Leu Cys 95 100 105

AGT AAG CTT CGA AAT TTG CTA CAT GAC AAT GAG CTG TCA GAT TTG AAA 446 Ser Lys Leu Arg Asn Leu Leu His Asp Asn Glu Leu Ser Asp Leu Lys 110 115 120 GAA GAT AAA CCT AGG AAA AGT TTG TTT AAT GAT GCA GGA AAC AAG AAG 494 Glu Asp Lys Pro Arg Lys Ser Leu Phe Asn Asp Ala Gly Asn Lys Lys 125 130 135 140

AAT TCA ATT AAA ATG TGG TTT AGC CCT CGA AGT AAG RAA GTC AGA TAT 542 Asn Ser He Lys Met Trp Phe Ser Pro Arg Ser Lys Xaa Val Arg Tyr 145 150 155

GTT GTG AGT AAA GCT TCA GTG CAA ACC CAG CCT GCA ATA AAA AAA GAT 590 Val Val Ser Lys Ala Ser Val Gin Thr Gin Pro Ala He Lys Lys Asp 160 165 170

GCA AGT GCT CAG CAA GAC TCA TAT GAA TTT GTT TCC CCA AGT CCT CCT 638 Ala Ser Ala Gin Gin Asp Ser Tyr Glu Phe Val Ser Pro Ser Pro Pro 175 180 185

GCA GAT GTT TCT GAG AGG GCT AAA AAG GCT TCT GCA AGA TCT GGA AAA 686 Ala Asp Val Ser Glu Arg Ala Lys Lys Ala Ser Ala Arg Ser Gly Lys 190 195 200

AAG CAA AAA AAG AAA ACT TTA GCT GAA ATC AAC CAA AAA TGG AAT TTA 734 Lys Gin Lys Lys Lys Thr Leu Ala Glu He Asn Gin Lys Trp Asn Leu 205 210 215 220

GAG GCA GAA AAA GAA GAT GGT GAA TTT GAC TCC AAA GAG GAA TCT AAG 782 Glu Ala Glu Lys Glu Asp Gly Glu Phe Asp Ser Lys Glu Glu Ser Lys 225 230 235

CAA AAG CTG GTA TCC TTC TGT AGC CAA CCA TCT GTT ATC TCC AGT CCT 830 Gin Lys Leu Val Ser Phe Cys Ser Gin Pro Ser Val He Ser Ser Pro 240 245 250

CCA CCT TCA TGC AAA CGT AAA GTT GGT GGT ACA TCA GGG AGG AAA AAC 1214 Pro Pro Ser Cys Lys Arg Lys Val Gly Gly Thr Ser Gly Arg Lys Asn 365 370 375 380 AGT AAC ATG TCC GAT GAA TTC ATT AGT CTT TCA CCA GGT ACA CCA CCT 1262 Ser Asn Met Ser Asp Glu Phe He Ser Leu Ser Pro Gly Thr Pro Pro 385 390 395

GTG GAA TTA TTG CTC CAG CAT AAG GCA TTG GTG AAC ACC ACC GGG TAT 1550 Val Glu Leu Leu Leu Gin His Lys Ala Leu Val Asn Thr Thr Gly Tyr 480 485 490

CAA AAT GAC TCA CCA CTT CAC GAT GCA GCC AAG AAT GGG CAC GTG GAT 1598 Gin Asn Asp Ser Pro Leu His Asp Ala Ala Lys Asn Gly His Val Asp 495 500 505

ATG CTT GGG ATT CTC AAT GGA TGC TGG ATT CTA AAA TTT GAA TGG GTA 1982 Met Leu Gly He Leu Asn Gly Cys Trp He Leu Lys Phe Glu Trp Val 625 630 635 AAA GCA TGT CTA CGA AGA AAA GTA TGT GAA CAG GAA GAA AAG TAT GAA 2030 Lys Ala Cys Leu Arg Arg Lys Val Cys Glu Gin Glu Glu Lys Tyr Glu 640 645 650

TGC ACA CAG TAT ATC ATC TAT GAA GAT TTG TGT AAT TAT CAC CCA GAG 2318 Cys Thr Gin Tyr He He Tyr Glu Asp Leu Cys Asn Tyr His Pro Glu 735 740 745

AGG GTT CGG CAG GGC AAA GTC TGG AAG GCT CCT TCG AGC TGG TTT ATA 2366 Arg Val Arg Gin Gly Lys Val Trp Lys Ala Pro Ser Ser Trp Phe He 750 755 760

GAC TGT GTG ATG TCC TTT GAG TTG CTT CCT CTT GAC AGC TGAATATTAT 2 15 Asp Cys Val Met Ser Phe Giu Leu Leu Pro Leu Asp Ser 765 770 775

ACCAGATGAA CATTTCAAAT TGAATTTGCA CGGTTTGTGA GAGCCCAGTC ATTGTACTGT 2475

TTTTAATGTT CACATTTTTA CAAATAGGTA GAGTCATTCA TATTTGTCTT TGAATC 2531

(2) INFORMATION FOR SEQ ID NO: 21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 777 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:

Met Pro Asp Asn Arg Gin Pro Arg Asn Arg Gin Pro Arg He Arg Ser 1 5 10 15

Gly Asn Glu Pro Arg Ser Ala Ser Ala Met Glu Pro Asp Gly Arg Gly 20 25 30

Ala Trp Ala His Ser Arg Ala Ala Leu Asp Arg Leu Glu Lys Leu Leu 35 40 45

Arg Cys Ser Arg Cys Thr Asn He Leu Arg Glu Pro Val Cys Leu Gly 50 55 60

Gly Cys Glu His He Phe Cys Ser Asn Cys Val Ser Asp Cys He Gly 65 70 75 80 Thr Gly Cys Pro Val Cys Tyr Thr Pro Ala Trp He Gin Asp Leu Lys 85 90 95

He Asn Arg Gin Leu Asp Ser Met He Gin Leu Cys Ser Lys Leu Arg 100 105 110

Asn Leu Leu His Asp Asn Glu Leu Ser Asp Leu Lys Glu Asp Lys Pro 115 120 125

Arg Lys Ser Leu Phe Asn Asp Ala Gly Asn Lys Lys Asn Ser He Lys 130 135 140

Met Trp Phe Ser Pro Arg Ser Lys Xaa Val Arg Tyr Val Val Ser Lys 145 150 155 160

Ala Ser Val Gin Thr Gin Pro Ala He Lys Lys Asp Ala Ser Ala Gin 165 170 175

Gin Asp Ser Tyr Glu Phe Val Ser Pro Ser Pro Pro Ala Asp Val Ser 180 185 190

Glu Arg Ala Lys Lys Ala Ser Ala Arg Ser Gly Lys Lys Gin Lys Lys 195 200 205

Lys Thr Leu Ala Glu He Asn Gin Lys Trp Asn Leu Glu Ala Glu Lys 210 215 220

Glu Asp Gly Glu Phe Asp Ser Lys Glu Glu Ser Lys Gin Lys Leu Val 225 230 235 240

Ser Phe Cys Ser Gin Pro Ser Val He Ser Ser Pro Gin He Asn Gly 245 250 255

Glu He Asp Leu Leu Ala Ser Gly Ser Leu Thr Glu Ser Glu Cys Phe 260 265 270

Gly Ser Leu Thr Glu Val Ser Leu Pro Leu Ala Glu Gin He Glu Ser 275 280 285

Pro Asp Thr Lys Ser Arg Asn Glu Val Val Thr Pro Glu Lys Val Cys 290 295 300

Lys Asn Tyr Leu Thr Ser Lys Lys Ser Leu Pro Leu Glu Asn Asn Gly 305 310 315 320

Lys Arg Gly His His Asn Arg Leu Ser Ser Pro He Ser Lys Arg Cys 325 330 335

Arg Thr Ser He Leu Ser Thr Ser Gly Asp Phe Val Lys Gin Thr Val 340 345 350

Pro Ser Glu Asn He Pro Leu Pro Glu Cys Ser Ser Pro Pro Ser Cys 355 360 365

Lys Arg Lys Val Gly Gly Thr Ser Gly Arg Lys Asn Ser Asn Met Ser 370 375 380

Asp Glu Phe He Ser Leu Ser Pro Gly Thr Pro Pro Ser Thr Leu Ser 385 390 395 400

Ser Ser Ser Tyr Arg Gin Val Met Ser Ser Pro Ser Ala Met Lys Leu 405 410 415

Leu Pro Asn Met Ala Val Lys Arg Asn His Arg Gly Glu Thr Leu Leu 420 425 430 His He Ala Ser He Lys Gly Asp He Pro Ser Val Glu Tyr Leu Leu 435 440 445

Gin Asn Gly Ser Asp Pro Asn Val Lys Asp His Ala Gly Trp Thr Pro 450 455 460

Leu His Glu Ala Cys Asn His Gly His Leu Lys Val Val Glu Leu Leu 465 470 475 480

Leu Gin His Lys Ala Leu Val Asn Thr Thr Gly Tyr Gin Asn Asp Ser 485 490 495

Pro Leu His Asp Ala Ala Lys Asn Gly His Val Asp He Val Lys Leu 500 505 510

Leu Leu Ser Tyr Gly Ala Ser Arg Asn Ala Val Asn He Phe Gly Leu 515 520 525

Arg Pro Val Asp Tyr Thr Asp Asp Glu Ser Met Lys Ser Leu Leu Leu 530 535 540

Leu Pro Glu Lys Asn Glu Ser Ser Ser Ala Ser His Cys Ser Val Met 545 550 555 560

Asn Thr Gly Gin Arg Arg Asp Gly Pro Leu Val Leu He Gly Ser Gly 565 570 575

Leu Ser Ser Glu Gin Gin Lys Met Leu Ser Glu Leu Ala Val He Leu 580 585 590

Lys Ala Lys Lys Tyr Thr Glu Phe Asp Ser Thr Val Thr His Val Val 595 600 605

Val Pro Gly Asp Ala Val Gin Ser Thr Leu Lys Cys Met Leu Gly He 610 615 620

Leu Asn Gly Cys Trp He Leu Lys Phe Glu Trp Val Lys Ala Cys Leu 625 630 635 640

Arg Arg Lys Val Cys Glu Gin Glu Glu Lys Tyr Glu He Pro Glu Gly 645 650 655

Pro Arg Arg Ser Arg Leu Asn Arg Glu Gin Leu Leu Pro Lys Leu Phe 660 665 670

Asp Gly Cys Tyr Phe Tyr Leu Trp Gly Thr Phe Lys His His Pro Lys 675 680 685

Asp Asn Leu He Lys Leu Val Thr Ala Gly Gly Gly Gin He Leu Ser 690 695 700

Arg Lys Pro Lys Pro Asp Ser Asp Val Thr Gin Thr He Asn Thr Val 705 710 715 720

Ala Tyr His Ala Arg Pro Asp Ser Asp Gin Arg Phe Cys Thr Gin Tyr 725 730 735

He He Tyr Glu Asp Leu Cys Asn Tyr His Pro Glu Arg Val Arg Gin 740 745 750

Gly Lys Val Trp Lys Ala Pro Ser Ser Trp Phe He Asp Cys Val Met 755 760 765

Ser Phe Glu Leu Leu Pro Leu Asp Ser 770 775 (2) INFORMATION FOR SEQ ID NO: 22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2531 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 75..2405

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:

GCAGCTTCCC TGTGGTTTCC CGAGGCCTCC TTGCTTCCCG CTCTCCGAGG AGCCTTTCAT 60

AGG ATC CGC TCC GGG AAC GAG CCT CGT TCC GCG CCC GCC ATG GAA CCG 158 Arg He Arg Ser Gly Asn Glu Pro Arg Ser Ala Pro Ala Met Glu Pro 15 20 25

AGT AAG CTT CGA AAT TTG CTA CAT GAC AAT GAG CTG TCA GAT TTG AAA 446 Ser Lys Leu Arg Asn Leu Leu His Asp Asn Glu Leu Ser Asp Leu Lys 110 115 120

GAA GAT AAA CCT AGG AAA AGT TTG TTT AAT GAT GCA GGA AAC AAG AAG 494 Glu Asp Lys Pro Arg Lys Ser Leu Phe Asn Asp Ala Gly Asn Lys Lys 125 130 135 140

AAT TCA ATT AAA ATG TGG TTT AGC CCT CGA AGT AAG GAA GTC AGA TAT 542 Asn Ser He Lys Met Trp Phe Ser Pro Arg Ser Lys Glu Val Arg Tyr 145 150 155

GCA AGT GCT CAG CAA GAC TCA TAT GAA TTT GTT TCC CCA AGT CCT CCT 638 Ala Ser Ala Gin Gin Asp Ser Tyr Glu Phe Val Ser Pro Ser Pro Pro 175 180 185 GCA GAT GTT TCT GAG AGG GCT AAA AAG GCT TCT GCA AGA TCT GGA AAA 686 Ala Asp Val Ser Glu Arg Ala Lys Lys Ala Ser Ala Arg Ser Gly Lys 190 195 200

GAG ACT TTG CTC CAT ATT GCT TCT ATT AAG GGC GAC ATA CCT TCT GTT 1406 Glu Thr Leu Leu His He Ala Ser He Lys Gly Asp He Pro Ser Val 430 435 440 GAA TAC CTT TTA CAA AAT GGA AGT GAT CCA AAT GTT AAA GAC CAT GCT 1454 Glu Tyr Leu Leu Gin Asn Gly Ser Asp Pro Asn Val Lys Asp His Ala 445 450 455 460

CAC CAT CCA AAG GAC AAC CTT ATT AAG CTC GTC ACT GCA GGT GGG GGC 2174 His His Pro Lys Asp Asn Leu He Lys Leu Val Thr Ala Gly Gly Gly 685 690 695 700 CAG ATC CTC AGT AGA AAG CCC AAG CCA GAC AGT GAC GTG ACT CAG ACC 2222 Gin He Leu Ser Arg Lys Pro Lys Pro Asp Ser Asp Val Thr Gin Thr 705 710 715

GAC TGT GTG ATG TCC TTT GAG TTG CTT CCT CTT GAC AGC TGAATATTAT 2 15 Asp Cys Val Met Ser Phe Glu Leu Leu Pro Leu Asp Ser 765 770 775

ACCAGATGAA CATTTCAAAT TGAATTTGCA CGGTTTGTGA GAGCCCAGTC ATTGTACTGT 2475

TTTTAATGTT CACATTTTTA CAAATAGGTA GAGTCATTCA TATTTGTCTT TGAATC 2531

(2) INFORMATION FOR SEQ ID NO: 23:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 777 ammo acids

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:

Met Pro Asp Asn Arg Gin Pro Arg Asn Arg Gin Pro Arg He Arg Ser 1 5 10 15

Gly Asn Glu Pro Arg Ser Ala Pro Ala Met Glu Pro Asp Gly Arg Gly 20 25 30

Ala Trp Ala His Ser Arg Ala Ala Leu Asp Arg Leu Glu Lys Leu Leu 35 40 45

Arg Cys Ser Arg Cys Thr Asn He Leu Arg Glu Pro Val Cys Leu Gly 50 55 60

Gly Cys Glu His He Phe Cys Ser Asn Cys Val Ser Asp Cys He Gly 65 70 75 80

Thr Gly Cys Pro Val Cys Tyr Thr Pro Ala Trp He Gin Asp Leu Lys 85 90 95

He Asn Arg Gin Leu Asp Ser Met He Gin Leu Cys Ser Lys Leu Arg 100 105 110

Asn Leu Leu His Asp Asn Glu Leu Ser Asp Leu Lys Glu Asp Lys Pro 115 120 125

Arg Lys Ser Leu Phe Asn Asp Ala Gly Asn Lys Lys Asn Ser He Lys 130 135 140

Met Trp Phe Ser Pro Arg Ser Lys Glu Val Arg Tyr Val Val Ser Lys 145 150 155 160 Ala Ser Val Gin Thr Gin Pro Ala He Lys Lys Asp Ala Ser Ala Gin 165 170 175

Gin Asp Ser Tyr Glu Phe Val Ser Pro Ser Pro Pro Ala Asp Val Ser 180 185 190

Glu Arg Ala Lys Lys Ala Ser Ala Arg Ser Gly Lys Lys Gin Lys Lys 195 200 205

Lys Thr Leu Ala Glu He Asn Gin Lys Trp Asn Leu Glu Ala Glu Lys 210 215 220

Glu Asp Gly Glu Phe Asp Ser Lys Glu Glu Ser Lys Gin Lys Leu Val 225 230 235 240

Ser Phe Cys Ser Gin Pro Ser Val He Ser Ser Pro Gin He Asn Gly 245 250 255

Glu He Asp Leu Leu Ala Ser Gly Ser Leu Thr Glu Ser Glu Cys Phe 260 265 270

Gly Ser Leu Thr Glu Val Ser Leu Pro Leu Ala Glu Gin He Glu Ser 275 280 285

Pro Asp Thr Lys Ser Arg Asn Glu Val Val Thr Pro Glu Lys Val Cys 290 295 300

Lys Asn Tyr Leu Thr Ser Lys Lys Ser Leu Pro Leu Glu Asn Asn Gly 305 310 315 320

Lys Arg Gly His His Asn Arg Leu Ser Ser Pro He Ser Lys Arg Cys 325 330 335

Arg Thr Ser He Leu Ser Thr Ser Gly Asp Phe Val Lys Gin Thr Val 340 345 350

Pro Ser Glu Asn He Pro Leu Pro Glu Cys Ser Ser Pro Pro Ser Cys 355 360 365

Lys Arg Lys Val Gly Gly Thr Ser Gly Arg Lys Asn Ser Asn Met Ser 370 375 380

Asp Glu Phe He Ser Leu Ser Pro Gly Thr Pro Pro Ser Thr Leu Ser 385 390 395 400

Ser Ser Ser Tyr Arg Gin Val Met Ser Ser Pro Ser Ala Met Lys Leu 405 410 415

Leu Pro Asn Met Ala Val Lys Arg Asn His Arg Gly Glu Thr Leu Leu 420 425 430

His He Ala Ser He Lys Gly Asp He Pro Ser Val Glu Tyr Leu Leu 435 440 445

Gin Asn Gly Ser Asp Pro Asn Val Lys Asp His Ala Gly Trp Thr Pro 450 455 460

Leu His Glu Ala Cys Asn His Gly His Leu Lyε Val Val Glu Leu Leu 465 470 475 480

Leu Gin His Lys Ala Leu Val Asn Thr Thr Gly Tyr Gin Asn Asp Ser 485 490 495

Pro Leu His Asp Ala Ala Lys Asn Gly His Val Asp He Val Lys Leu 500 505 510 Leu Leu Ser Tyr Gly Ala Ser Arg Asn Ala Val Asn He Phe Gly Leu 515 520 525

Arg Pro Val Asp Tyr Thr Asp Asp Glu Ser Met Lys Ser Leu Leu Leu 530 535 540

Leu Pro Glu Lys Asn Glu Ser Ser Ser Ala Ser His Cys Ser Val Met 545 550 555 560

Asn Thr Gly Gin Arg Arg Asp Gly Pro Leu Val Leu He Gly Ser Gly 565 570 575

Leu Ser Ser Glu Gin Gin Lys Met Leu Ser Glu Leu Ala Val He Leu 580 585 590

Lys Ala Lys Lys Tyr Thr Glu Phe Asp Ser Thr Val Thr His Val Val 595 600 605

Val Pro Gly Asp Ala Val Gin Ser Thr Leu Lys Cys Met Leu Gly He 610 615 620

Leu Asn Gly Cys Trp He Leu Lys Phe Glu Trp Val Lys Ala Cys Leu 625 630 635 640

Arg Arg Lys Val Cys Glu Gin Glu Glu Lys Tyr Glu He Pro Glu Gly 645 650 655

Pro Arg Arg Ser Arg Leu Asn Arg Glu Gin Leu Leu Pro Lys Leu Phe 660 665 670

Asp Gly Cys Tyr Phe Tyr Leu Trp Gly Thr Phe Lys His His Pro Lys 675 680 685

Asp Asn Leu He Lys Leu Val Thr Ala Gly Gly Gly Gin He Leu Ser 690 695 700

Arg Lys Pro Lys Pro Asp Ser Asp Val Thr Gin Thr He Asn Thr Val 705 710 715 720

Ala Tyr His Ala Arg Pro Asp Ser Asp Gin Arg Phe Cys Thr Gin Tyr 725 730 735

He He Tyr Glu Asp Leu Cys Asn Tyr His Pro Glu Arg Val Arg Gin 740 745 750

Gly Lys Val Trp Lys Ala Pro Ser Ser Trp Phe He Asp Cys Val Met 755 760 765

Ser Phe Glu Leu Leu Pro Leu Asp Ser 770 775

(2) INFORMATION FOR SEQ ID NO: 24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2531 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 75..2405 ( ix ) FEATURE :

(A) NAME/KEY: modified_base

(B) LOCATION: 531

(D) OTHER INFORMATION: /note= "R = A or G"

(ix) FEATURE:

(A) NAME/KEY: modified_base

(B) LOCATION: 153

(D) OTHER INFORMATION: /note= "Xaa = Glu or Lys for both SEQ ID NO: 24 and SEQ ID NO: 25"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:

GCAGCTTCCC TGTGGTTTCC CGAGGCCTCC TTGCTTCCCG CTCTCCGAGG AGCCTTTCAT 60

AAG CAA ACG GTG CCC TCA GAA AAT ATA CCA TTG CCT GAA TGT TCT TCA 1166 Lys Gin Thr Val Pro Ser Glu Asn He Pro Leu Pro Glu Cys Ser Ser 350 355 360

GAG ACT TTG CTC CAT ATT GCT TCT ATT AAG GGC GAC ATA CCT TCT GTT 1406 Glu Thr Leu Leu His He Ala Ser He Lys Gly Asp He Pro Ser Val 430 435 440 GAA TAC CTT TTA CAA AAT GGA AGT GAT CCA AAT GTT AAA GAC CAT GCT 1454

Glu Tyr Leu Leu Gin Asn Gly Ser Asp Pro Asn Val Lys Asp His Ala

445 450 455 460

GGA TGG ACA CCA TTG CAT GAA GCT TGC AAT CAT GGG CAC CTG AAG GTA 1502

Gly Trp Thr Pro Leu His Glu Ala Cys Asn His Gly His Leu Lys Val

465 470 475

GTG GAA TTA TTG CTC CAG CAT AAG GCA TTG GTG AAC ACC ACC GGG TAT 1550

Val Glu Leu Leu Leu Gin His Lys Ala Leu Val Asn Thr Thr Gly Tyr

480 485 490

CAA AAT GAC TCA CCA CTT CAC GAT GCA GCC AAG AAT GGG CAC GTG GAT 1598

Gin Asn Asp Ser Pro Leu His Asp Ala Ala Lys Asn Gly His Val Asp

495 500 505

ATA GTC AAG CTG TTA CTT TCC TAT GGA GCC TCC AGA AAT GCT GTT AAT 1646

He Val Lys Leu Leu Leu Ser Tyr Gly Ala Ser Arg Asn Ala Val Asn

510 515 520

ATA TTT GGT CTG CGG CCT GTC GAT TAT ACA GAT GAT GAA AGT ATG AAA 1694

He Phe Gly Leu Arg Pro Val Asp Tyr Thr Asp Asp Glu Ser Met Lys

525 530 535 540

TCG CTA TTG CTG CTA CCA GAG AAG AAT GAA TCA TCC TCA GCT AGC CAC 1742

Ser Leu Leu Leu Leu Pro Glu Lys Asn Glu Ser Ser Ser Ala Ser His

545 550 555

TGC TCA GTA ATG AAC ACT GGG CAG CGT AGG GAT GGA CCT CTT GTA CTT 1790

Cys Ser Val Met Asn Thr Gly Gin Arg Arg Asp Gly Pro Leu Val Leu

560 565 570

ATA GGC AGT GGG CTG TCT TCA GAA CAA CAG AAA ATG CTC AGT GAG CTT 1838

He Gly Ser Gly Leu Ser Ser Glu Gin Gin Lys Met Leu Ser Glu Leu

575 580 585

GCA GTA ATT CTT AAG GCT AAA AAA TAT ACT GAG TTT GAC AGT ACA GTA 1886

Ala Val He Leu Lys Ala Lys Lys Tyr Thr Glu Phe Asp Ser Thr Val

590 595 600

ACT CAT GTT GTT GTT CCT GGT GAT GCA GTT CAA AGT ACC TTG AAG TGT 1934

Thr His Val Val Val Pro Gly Asp Ala Val Gin Ser Thr Leu Lys Cys

605 610 615 620

ATG CTT GGG ATT CTC AAT GGA TGC TGG ATT CTA AAA TTT GAA TGG GTA 1982

Met Leu Gly He Leu Asn Gly Cys Trp He Leu Lys Phe Glu Trp Val

625 630 635

AAA GCA TGT CTA CGA AGA AAA GTA TGT GAA CAG GAA GAA AAG TAT GAA 2030

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

640 645 650

ATT CCT GAA GGT CCA CGC AGA AGC AGG CTC AAC AGA GAA CAG CTG TTG 2078

He Pro Glu Gly Pro Arg Arg Ser Arg Leu Asn Arg Glu Gin Leu Leu

655 660 665

CCA AAG CTG TTT GAT GGA TGC TAC TTC TAT TTG TGG GGA ACC TTC AAA 2126

Pro Lys Leu Phe Asp Gly Cys Tyr Phe Tyr Leu Trp Gly Thr Phe Lys

670 675 680

CAC CAT CCA AAG GAC AAC CTT ATT AAG CTC GTC ACT GCA GGT GGG GGC 2174

His His Pro Lys Asp Asn Leu He Lys Leu Val Thr Ala Gly Gly Gly

685 690 695 700 CAG ATC CTC AGT AGA AAG CCC AAG CCA GAC AGT GAC GTG ACT CAG ACC 2222 Gin He Leu Ser Arg Lys Pro Lys Pro Asp Ser Asp Val Thr Gin Thr 705 710 715

GAC TGT GTG ATG TCC TTT GAG TTG CTT CCT CTT GAC AGC TGAATATTAT 2415 Asp Cys Val Met Ser Phe Glu Leu Leu Pro Leu Asp Ser 765 770 775

ACCAGATGAA CATTTCAAAT TGAATTTGCA CGGTTTGTGA GAGCCCAGTC ATTGTACTGT 2475

TTTTAATGTT CACATTTTTA CAAATAGGTA GAGTCATTCA TATTTGTCTT TGAATC 2531

(2) INFORMATION FOR SEQ ID NO: 25:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 777 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:

Met Pro Asp Asn Arg Gin Pro Arg Asn Arg Gin Pro Arg He Arg Ser 1 5 10 15

Gly Asn Glu Pro Arg Ser Ala Pro Ala Met Glu Pro Asp Gly Arg Gly 20 25 30

Ala Trp Ala His Ser Arg Ala Ala Leu Asp Arg Leu Glu Lys Leu Leu 35 40 45

Arg Cys Ser Arg Cys Thr Asn He Leu Arg Glu Pro Val Cys Leu Gly 50 55 60

Gly Cys Glu His He Phe Cys Ser Asn Cys Val Ser Asp Cys He Gly 65 70 75 80

Thr Gly Cys Pro Val Cys Tyr Thr Pro Ala Trp He Gin ASD Leu Lys 85 90 95

He Asn Arg Gin Leu Asp Ser Met He Gin Leu Cys Ser Lys Leu Arg 100 105 110

Asn Leu Leu His Asp Asn Glu Leu Ser Asp Leu Lys Glu Asp Lys Pro 115 120 125

Arg Lys Ser Leu Phe Asn Asp Ala Gly Asn Lys Lys Asn Ser He Lys 130 135 140

Met Trp Phe Ser Pro Arg Ser Lys Xaa Val Arg Tyr Val Val Ser Lys 145 150 155 160 Ala Ser Val Gin Thr Gin Pro Ala He Lys Lys Asp Ala Ser Ala Gin 165 170 175

Gin Asp Ser Tyr Glu Phe Val Ser Pro Ser Pro Pro Ala Asp Val Ser 180 185 190

Glu Arg Ala Lys Lys Ala Ser Ala Arg Ser Gly Lys Lys Gin Lys Lys 195 200 205

Lys Thr Leu Ala Glu He Asn Gin Lys Trp Asn Leu Glu Ala Glu Lys 210 215 220

Glu Asp Gly Glu Phe Asp Ser Lys Glu Glu Ser Lys Gin Lys Leu Val 225 230 235 240

Ser Phe Cys Ser Gin Pro Ser Val He Ser Ser Pro Gin He Asn Gly 245 250 255

Glu He Asp Leu Leu Ala Ser Gly Ser Leu Thr Glu Ser Glu Cys Phe 260 265 270

Gly Ser Leu Thr Glu Val Ser Leu Pro Leu Ala Glu Gin He Glu Ser 275 280 285

Pro Asp Thr Lys Ser Arg Asn Glu Val Val Thr Pro Glu Lys Val Cys 290 295 300

Lys Asn Tyr Leu Thr Ser Lys Lys Ser Leu Pro Leu Glu Asn Asn Gly 305 310 315 320

Lys Arg Gly His His Asn Arg Leu Ser Ser Pro He Ser Lys Arg Cys 325 330 335

Arg Thr Ser He Leu Ser Thr Ser Gly Asp Phe Val Lys Gin Thr Val 340 345 350

Pro Ser Glu Asn He Pro Leu Pro Glu Cys Ser Ser Pro Pro Ser Cys 355 360 365

Lys Arg Lys Val Gly Gly Thr Ser Gly Arg Lys Asn Ser Asn Met Ser 370 375 380

Asp Glu Phe He Ser Leu Ser Pro Gly Thr Pro Pro Ser Thr Leu Ser 385 390 395 400

Ser Ser Ser Tyr Arg Gin Val Met Ser Ser Pro Ser Ala Met Lys Leu 405 410 415

Leu Pro Asn Met Ala Val Lys Arg Asn His Arg Gly Glu Thr Leu Leu 420 425 430

His He Ala Ser He Lys Gly Asp He Pro Ser Val Glu Tyr Leu Leu 435 440 445

Gin Asn Gly Ser Asp Pro Asn Val Lys Asp His Ala Gly Trp Thr Pro 450 455 460

Leu His Glu Ala Cys Asn His Gly His Leu Lys Val Val Glu Leu Leu 465 470 475 480

Leu Gin His Lys Ala Leu Val Asn Thr Thr Gly Tyr Gin Asn Asp Ser 485 490 495

Arg Pro Val Asp Tyr Thr Asp Asp Glu Ser Met Lys Ser Leu Leu Leu 530 535 540

Leu Pro Glu Lys Asn Glu Ser Ser Ser Ala Ser His Cys Ser Val Met 545 550 555 560

Asn Thr Gly Gin Arg Arg Asp Gly Pro Leu Val Leu He Gly Ser Gly 565 570 575

Leu Ser Ser Glu Gin Gin Lys Met Leu Ser Glu Leu Ala Val He Leu 580 585 590

Lys Ala Lys Lys Tyr Thr Glu Phe Asp Ser Thr Val Thr His al Val 595 600 605

Val Pro Gly Asp Ala Val Gin Ser Thr Leu Lys Cys Met Leu Gly He 610 615 620

Leu Asn Gly Cys Trp He Leu Lys Phe Glu Trp Val Lys Ala Cys Leu 625 630 635 640

Arg Arg Lys Val Cys Glu Gin Glu Glu Lys Tyr Glu He Pro Glu Gly 645 650 655

Pro Arg Arg Ser Arg Leu Asn Arg Glu Gin Leu Leu Pro Lys Leu Phe 660 665 670

Asp Gly Cys Tyr Phe Tyr Leu Trp Gly Thr Phe Lys His His Pro Lys 675 680 685

Asp Asn Leu He Lys Leu Val Thr Ala Gly Gly Gly Gin He Leu Ser 690 695 700

Arg Lys Pro Lys Pro Asp Ser Asp Val Thr Gin Thr He Asn Thr Val 705 710 715 720

Ala Tyr His Ala Arg Pro Asp Ser Asp Gin Arg Phe Cys Thr Gin Tyr 725 730 735

He He Tyr Glu Asp Leu Cys Asn Tyr His Pro Glu Arg Val Arg Gin 740 745 750

Gly Lys Val Trp Lys Ala Pro Ser Ser Trp Phe He Asp Cys Val Met 755 760 765

Ser Phe Glu Leu Leu Pro Leu Asp Ser 770 775

(2) INFORMATION FOR SEQ ID NO: 26:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2510 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 75..2384 ( ix ) FEATURE :

(A) NAME/KEY: modified_base

(B) LOCATION: 531

(D) OTHER INFORMATION: /note= "R = A or G"

(ix) FEATURE:

(A) NAME/KEY: modified_base

(B) LOCATION: 153

(D) OTHER INFORMATION: /note= "Xaa = Glu or Lys for both SEQ ID NO: 26 and SEQ ID NO: 27"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:

GCAGCTTCCC TGTGGTTTCC CGAGGCCTCC TTGCTTCCCG CTCTCCGAGG AGCCTTTCAT 60

CAA ATA GAG TCT CCA GAC ACT AAG AGC AGG AAT GAA GTA GTG ACT CCT^' 974 Gin He Glu Ser Pro Asp Thr Lys Ser Arg Asn Glu Val Val Thr Pro 285 290 295 300

AAG CAA ACC GTG CCC TCA GAA AAT ATA CCA CCT TCA TGC AAA CGT AAA 1166 Lys Gin Thr Val Pro Ser Glu Asn He Pro Pro Ser Cys Lys Arg Lys 350 355 360

GTT GGT GGT ACA TCA GGG AGG AAA AAC AGT AAC ATG TCC GAT GAA TTC 1214 Val Gly Gly Thr Ser Gly Arg Lys Asn Ser Asn Met Ser Asp Glu The 365 370 375 380

ATT AGT CTT TCA CCA GGT ACA CCA CCT TCT ACA TTA AGT AGT TCA AGT 1262 He Ser Leu Ser Pro Gly Thr Pro Pro Ser Thr Leu Ser Ser Ser Ser 385 390 395

TAC AGG CAA GTG ATG TCT AGT CCC TCA GCA ATG AAG CTG TTG CCC AAT 1310 Tyr Arg Gin Val Met Ser Ser Pro Ser Ala Met Lys Leu Leu Pro Asn 400 405 410

ATG GCT GTG AAA AGA AAT CAT AGA GGA GAG ACT TTG CTC CAT ATT GCT 1358 Met Ala Val Lys Arg Asn His Arg Gly Glu Thr Leu Leu His He Ala 415 420 425

TCT ATT AAG GGC GAC ATA CCT TCT GTT GAA TAC CTT TTA CAA AAT GGA 1406 Ser He Lys Gly Asp He Pro Ser Val Glu Tyr Leu Leu Gin Asn Gly 430 435 440 AGT GAT CCA AAT GTT AAA GAC CAT GCT GGA TGG ACA CCA TTG CAT GAA 1454

Ser Asp Pro Asn Val Lys Asp His Ala Gly Trp Thr Pro Leu His Glu 445 450 455 460

GCT TGC AAT CAT GGG CAC CTG AAG GTA GTG GAA TTA TTG CTC CAG CAT 1502

Ala Cys Asn His Gly His Leu Lys Val Val Glu Leu Leu Leu Gin His 465 470 475

AAG GCA TTG GTG AAC ACC ACC GGG TAT CAA AAT GAC TCA CCA CTT CAC 1550

Lys Ala Leu Val Asn Thr Thr Gly Tyr Gin Asn Asp Ser Pro Leu His

480 485 490

GAT GCA GCC AAG AAT GGG CAC GTG GAT ATA GTC AAG CTG TTA CTT TCC 1598

Asp Ala Ala Lys Asn Gly His Val Asp He Val Lys Leu Leu Leu Ser

495 500 505

TAT GGA GCC TCC AGA AAT GCT GTT AAT ATA TTT GGT CTG CGG CCT GTC 1646

Tyr Gly Ala Ser Arg Asn Ala Val Asn He Phe Gly Leu Arg Pro Val 510 515 520

GAT TAT ACA GAT GAT GAA AGT ATG AAA TCG CTA TTG CTG CTA CCA GAG 1694

Asp Tyr Thr Asp Asp Glu Ser Met Lys Ser Leu Leu Leu Leu Pro Glu 525 530 535 540

AAG AAT GAA TCA TCC TCA GCT AGC CAC TGC TCA GTA ATG AAC ACT GGG 1742

Lys Asn Glu Ser Ser Ser Ala Ser His Cys Ser Val Met Asn Thr Gly 545 550 555

CAG CGT AGG GAT GGA CCT CTT GTA CTT ATA GGC AGT GGG CTG TCT TCA 1790

Gin Arg Arg Asp Gly Pro Leu Val Leu He Gly Ser Gly Leu Ser Ser

560 565 570

GAA CAA CAG AAA ATG CTC AGT GAG CTT GCA GTA ATT CTT AAG GCT AAA 1838

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

575 580 585

AAA TAT ACT GAG TTT GAC AGT ACA GTA ACT CAT GTT GTT GTT CCT GGT 1886

Lys Tyr Thr Glu Phe Asp Ser Thr Val Thr His Val Val Val Pro Gly 590 595 600

GAT GCA GTT CAA AGT ACC TTG AAG TGT ATG CTT GGG ATT CTC AAT GGA 1934

Asp Ala Val Gin Ser Thr Leu Lys Cys Met Leu Gly He Leu Asn Gly 605 610 615 620

TGC TGG ATT CTA AAA TTT GAA TGG GTA AAA GCA TGT CTA CGA AGA AAA 1982

Cys Trp He Leu Lys Phe Glu Trp Val Lys Ala Cys Leu Arg Arg Lys 625 630 635

GTA TGT GAA CAG GAA GAA AAG TAT GAA ATT CCT GAA GGT CCA CGC AGA 2030

Val Cys Glu Gin Glu Glu Lys Tyr Glu He Pro Glu Gly Pro Arg Arg

640 645 650

AGC AGG CTC AAC AGA GAA CAG CTG TTG CCA AAG CTG TTT GAT GGA TGC 2078

Ser Arg Leu Asn Arg Glu Gin Leu Leu Pro Lys Leu Phe Asp Gly Cys

655 660 665

TAC TTC TAT TTG TGG GGA ACC TTC AAA CAC CAT CCA AAG GAC AAC CTT 2126

Tyr Phe Tyr Leu Trp Gly Thr Phe Lys His His Pro Lys Asp Asn Leu 670 675 680

ATT AAG CTC GTC ACT GCA GGT GGG GGC CAG ATC CTC AGT AGA AAG CCC 2174

He Lys Leu Val Thr Ala Gly Gly Gly Gin He Leu Ser Arg Lys Pro 685 690 695 700 AAG CCA GAC AGT GAC GTG ACT CAG ACC ATC AAT ACA GTC GCA TAC CAT 2222 Lys Pro Asp Ser Asp Val Thr Gin Thr He Asn Thr Val Ala Tyr His 705 710 715

GCG AGA CCC GAT TCT GAT CAG CGC TTC TGC ACA CAG TAT ATC ATC TAT 2270 Ala Arg Pro Asp Ser Asp Gin Arg Phe Cys Thr Gin Tyr He He Tyr 720 725 730

GAA GAT TTG TGT AAT TAT CAC CCA GAG AGG GTT CGG CAG GGC AAA GTC 2318 Glu Asp Leu Cys Asn Tyr His Pro Glu Arg Val Arg Gin Gly Lys Val 735 740 745

TGG AAG GCT CCT TCG AGC TGG TTT ATA GAC TGT GTG ATG TCC TTT GAG 2366 Trp Lys Ala Pro Ser Ser Trp Phe He Asp Cys Val Met Ser Phe Glu 750 755 760

TTG CTT CCT CTT GAC AGC TGAATATTAT ACCAGATGAA CATTTCAAAT 2414

Leu Leu Pro Leu Asp Ser 765 770

TGAATTTGCA CGGTTTGTGA GAGCCCAGTC ATTGTACTGT TTTTAATGTT CACATTTTTA 2474

CAAATAGGTA GAGTCATTCA TATTTGTCTT TGAATC 2510

(2) INFORMATION FOR SEQ ID NO: 27:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 770 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:

Met Pro Asp Asn Arg Gin Pro Arg Asn Arg Gin Pro Arg He Arg Ser 1 5 10 15

Gly Asn Glu Pro Arg Ser Ala Pro Ala Met Glu Pro Asp Gly Arg Gly 20 25 30

Ala Trp Ala His Ser Arg Ala Ala Leu Asp Arg Leu Glu Lys Leu Leu 35 40 45

Arg Cys Ser Arg Cys Thr Asn He Leu Arg Glu Pro Val Cys Leu Gly 50 55 60

Gly Cys Glu His He Phe Cys Ser Asn Cys Val Ser Asp Cys He Gly 65 70 75 80

Thr Gly Cys Pro Val Cys Tyr Thr Pro Ala Trp He Gin Asp Leu Lys 85 90 95

He Asn Arg Gin Leu Asp Ser Met He Gin Leu Cys Ser Lys Leu Arg 100 105 110

Asn Leu Leu His Asp Asn Glu Leu Ser Asp Leu Lys Glu Asp Lys Pro 115 120 125

Arg Lys Ser Leu Phe Asn Asp Ala Gly Asn Lys Lys Asn Ser He Lys 130 135 140

Gin Asp Ser Tyr Glu Phe Val Ser Pro Ser Pro Pro Ala Asp Val Ser 180 185 190

Glu Arg Ala Lys Lys Ala Ser Ala Arg Ser Gly Lys Lys Gin Lys Lys 195 200 205

Lys Thr Leu Ala Glu He Asn Gin Lys Trp Asn Leu Glu Ala Glu Lys 210 215 220

Glu Asp Gly Glu Phe Asp Ser Lys Glu Glu Ser Lys Gin Lys Leu Val 225 230 235 240

Ser Phe Cys Ser Gin Pro Ser Val He Ser Ser Pro Gin He Asn Gly 245 250 255

Glu He Asp Leu Leu Ala Ser Gly Ser Leu Thr Glu Ser Glu Cys Phe 260 265 270

Gly Ser Leu Thr Glu Val Ser Leu Pro Leu Ala Glu Gin He Glu Ser 275 280 285

Pro Asp Thr Lys Ser Arg Asn Glu Val Val Thr Pro Glu Lys Val Cys 290 295 300

Lys Asn Tyr Leu Thr Ser Lys Lys Ser Leu Pro Leu Glu Asn Asn Gly 305 310 315 320

Lys Arg Gly His His Asn Arg Leu Ser Ser Pro He Ser Lys Arg Cys 325 330 335

Arg Thr Ser He Leu Ser Thr Ser Gly Asp Phe Val Lys Gin Thr Val 340 345 350

Pro Ser Glu Asn He Pro Pro Ser Cys Lys Arg Lys Val Gly Gly Thr 355 360 365

Ser Gly Arg Lys Asn Ser Asn Met Ser Asp Glu Phe He Ser Leu Ser 370 375 380

Pro Gly Thr Pro Pro Ser Thr Leu Ser Ser Ser Ser Tyr Arg Gin Val 385 390 395 400

Met Ser Ser Pro Ser Ala Met Lys Leu Leu Pro Asn Met Ala Val Lys 405 410 415

Arg Asn His Arg Gly Glu Thr Leu Leu His He Ala Ser He Lys Gly 420 425 430

Asp He Pro Ser Val Glu Tyr Leu Leu Gin Asn Gly Ser Asp Pro Asn 435 440 445

Val Lys Asp His Ala Gly Trp Thr Pro Leu His Glu Ala Cys Asn His 450 455 460

Gly His Leu Lys Val Val Glu Leu Leu Leu Gin His Lys Ala Leu Val 465 470 475 480

Asn Thr Thr Gly Tyr Gin Asn Asp Ser Pro Leu His Asp Ala Ala Lys 485 490 495

Asn Gly His Val Asp He Val Lys Leu Leu Leu Ser Tyr Gly Ala Ser 500 505 510 Arg Asn Ala Val Asn He Phe Gly Leu Arg Pro Val Asp Tyr Thr Asp 515 520 525

Asp Glu Ser Met Lys Ser Leu Leu Leu Leu Pro Glu Lys Asn Glu Ser 530 535 540

Ser Ser Ala Ser His Cys Ser Val Met Asn Thr Gly Gin Arg Arg Asp 545 550 555 560

Gly Pro Leu Val Leu He Gly Ser Gly Leu Ser Ser Glu Gin Gin Lys 565 570 575

Met Leu Ser Glu Leu Ala Val He Leu Lys Ala Lys Lys Tyr Thr Glu 580 585 590

Phe Asp Ser Thr Val Thr His Val Val Val Pro Gly Asp Ala Val Gin 595 600 605

Ser Thr Leu Lys Cys Met Leu Gly He Leu Asn Gly Cys Trp He Leu 610 615 620

Lys Phe Glu Trp Val Lys Ala Cys Leu Arg Arg Lys Val Cys Glu Gin 625 630 635 640

Glu Glu Lys Tyr Glu He Pro Glu Gly Pro Arg Arg Ser Arg Leu Asn 645 650 655

Arg Glu Gin Leu Leu Pro Lys Leu Phe Asp Gly Cys Tyr Phe Tyr Leu 660 665 670

Trp Gly Thr Phe Lys His His Pro Lys Asp Asn Leu He Lys Leu Val 675 680 685

Thr Ala Gly Gly Gly Gin He Leu Ser Arg Lys Pro Lys Pro Asp Ser 690 695 700

Asp Val Thr Gin Thr He Asn Thr Val Ala Tyr His Ala Arg Pro Asp 705 710 715 720

Ser Asp Gin Arg Phe Cys Thr Gin Tyr He He Tyr Glu Asp Leu Cys 725 730 735

Asn Tyr His Pro Glu Arg Val Arg Gin Gly Lys Val Trp Lys Ala Pro 740 745 750

Ser Ser Trp Phe He Asp Cys Val Met Ser Phe Glu Leu Leu Pro Leu 755 760 765

Asp Ser

770

(2) INFORMATION FOR SEQ ID NO: 28:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2531 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 75..2405 ( ix ) FEATURE :

(A) NAME/KEY: modified_base

(B) LOCATION: 531

(D) OTHER INFORMATION: /note= "R = A or G"

(ix) FEATURE:

(A) NAME/KEY: modified_base

(B) LOCATION: 153

(D) OTHER INFORMATION :/note= "Xaa = Glu or Lys for both SEQ ID NO:28 and SEQ ID NO:29"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:

GCAGCTTCCC TGTGGTTTCC CGAGGCCTCC TTGCTTCCCG CTCTCCGAGG AGCCTTTCAT 60

CAA AAT GAC TCA CCA CTT CAC GAT GCA GCC AAG AAT GGG CAC ATG GAT 1598 Gin Asn Asp Ser Pro Leu His Asp Ala Ala Lys Asn Gly His Met Asp 495 500 505

ACCAGATGAA CATTTCAAAT TGAATTTGCA CGGTTTGTGA GAGCCCAGTC ATTGTACTGT 2475

TTTTAATGTT CACATTTTTA CAAATAGGTA GAGTCATTCA TATTTGTCTT TGAATC 2531

(2) INFORMATION FOR SEQ ID NO: 29:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 777 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:

Met Pro Asp Asn Arg Gin Pro Arg Asn Arg Gin Pro Arg He Arg Ser 1 5 10 15

Gly Asn Glu Pro Arg Ser Ala Pro Ala Met Glu Pro Asp Gly Arg Gly 20 25 30

Ala Trp Ala His Ser Arg Ala Ala Leu Asp Arg Leu Glu Lys Leu Leu 35 40 45

Arg Cys Ser Arg Cys Thr Asn He Leu Arg Glu Pro Val Cys Leu Gly 50 55 60

Gly Cys Glu His He Phe Cys Ser Asn Cys Val Ser Asp Cys He Gly 65 70 75 80

Thr Gly Cys Pro Val Cys Tyr Thr Pro Ala Trp He Gin Asp Leu Lys 85 90 95

He Asn Arg Gin Leu Asp Ser Met He Gin Leu Cys Ser Lys Leu Arg 100 105 110

Asn Leu Leu His Asp Asn Glu Leu Ser Asp Leu Lys Glu Asp Lys Pro 115 120 125

Arg Lys Ser Leu Phe Asn Asp Ala Gly Asn Lys Lys Asn Ser He Lys 130 135 140

Gin Asp Ser Tyr Glu Phe Val Ser Pro Ser Pro Pro Ala Asp Val Ser 180 185 190

Glu Arg Ala Lys Lys Ala Ser Ala Arg Ser Gly Lys Lys Gin Lys Lys 195 200 205

Lys Thr Leu Ala Glu He Asn Gin Lys Trp Asn Leu Glu Ala Glu Lys 210 215 220

Glu Asp Gly Glu Phe Asp Ser Lys Glu Glu Ser Lys Gin Lys Leu Val 225 230 235 240

Ser Phe Cys Ser Gin Pro Ser Val He Ser Ser Pro Gin He Asn Gly 245 250 255

Glu He Asp Leu Leu Ala Ser Gly Ser Leu Thr Glu Ser Glu Cys Phe 260 265 270

Gly Ser Leu Thr Glu Val Ser Leu Pro Leu Ala Glu Gin He Glu Ser 275 280 285

Pro Asp Thr Lys Ser Arg Asn Glu Val Val Thr Pro Glu Lys Val Cys 290 295 300

Lys Asn Tyr Leu Thr Ser Lys Lys Ser Leu Pro Leu Glu Asn Asn Gly 305 310 315 320

Lys Arg Gly His His Asn Arg Leu Ser Ser Pro He Ser Lys Arg Cys 325 330 335

Arg Thr Ser He Leu Ser Thr Ser Gly Asp Phe Val Lys Gin Thr Val 340 345 350

Pro Ser Glu Asn He Pro Leu Pro Glu Cys Ser Ser Pro Pro Ser Cys 355 360 365

Lys Arg Lys Val Gly Gly Thr Ser Gly Arg Lys Asn Ser Asn Met Ser 370 375 380

Asp Glu Phe He Ser Leu Ser Pro Gly Thr Pro Pro Ser Thr Leu Ser 385 390 395 400

Ser Ser Ser Tyr Arg Gin Val Met Ser Ser Pro Ser Ala Met Lys Leu 405 410 415

Leu Pro Asn Met Ala Val Lys Arg Asn His Arg Gly Glu Thr Leu Leu 420 425 430

His He Ala Ser He Lys Gly Asp He Pro Ser Val Glu Tyr Leu Leu 435 440 445

Gin Asn Gly Ser Asp Pro Asn Val Lys Asp His Ala Gly Trp Thr Pro 450 455 460

Leu His Glu Ala Cys Asn His Gly His Leu Lys Val Val Glu Leu Leu 465 470 475 480

Leu Gin His Lys Ala Leu Val Asn Thr Thr Gly Tyr Gin Asn Asp Ser 485 490 495

Pro Leu His Asp Ala Ala Lys Asn Gly His Met Asp He Val Lys Leu 500 505 510 Leu Leu Ser Tyr Gly Ala Ser Arg Asn Ala Val Asn He Phe Gly Leu 515 520 525

Arg Pro Val Asp Tyr Thr Asp Asp Glu Ser Met Lys Ser Leu Leu Leu 530 535 540

Leu Pro Glu Lys Asn Glu Ser Ser Ser Ala Ser His Cys Ser Val Met 545 550 555 560

Asn Thr Gly Gin Arg Arg Asp Gly Pro Leu Val Leu He Gly Ser Gly 565 570 575

Leu Ser Ser Glu Gin Gin Lys Met Leu Ser Glu Leu Ala Val He Leu 580 585 590

Lys Ala Lys Lys Tyr Thr Glu Phe Asp Ser Thr Val Thr His Val Val 595 600 605

Val Pro Gly Asp Ala Val Gin Ser Thr Leu Lys Cys Met Leu Gly He 610 615 620

Leu Asn Gly Cys Trp He Leu Lys Phe Glu Trp Val Lys Ala Cys Leu 625 630 635 640

Arg Arg Lys Val Cys Glu Gin Glu Glu Lys Tyr Glu He Pro Glu Gly 645 650 655

Pro Arg Arg Ser Arg Leu Asn Arg Glu Gin Leu Leu Pro Lys Leu Phe 660 665 670

Asp Gly Cys Tyr Phe Tyr Leu Trp Gly Thr Phe Lys His His Pro Lys 675 680 685

Asp Asn Leu He Lys Leu Val Thr Ala Gly Gly Gly Gin He Leu Ser 690 695 700

Arg Lys Pro Lys Pro Asp Ser Asp Val Thr Gin Thr He Asn Thr Val 705 710 715 720

Ala Tyr His Ala Arg Pro Asp Ser Asp Gin Arg Phe Cys Thr Gin Tyr 725 730 735

He He Tyr Glu Asp Leu Cys Asn Tyr His Pro Glu Arg Val Arg Gin 740 745 750

Gly Lys Val Trp Lys Ala Pro Ser Ser Trp Phe He Asp Cys Val Met 755 760 765

Ser Phe Glu Leu Leu Pro Leu Asp Ser 770 775

(2) INFORMATION FOR SEQ ID NO: 30:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2531 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) L0CATI0N:75..2405 ( ix ) FEATURE :

(A) NAME/KEY: modified_base

(B) LOCATION: 531

(D) OTHER INFORMATION: /note= "R = A or G"

(ix) FEATURE:

(A) NAME/KEY: modified_base

(B) LOCATION: 153

(D) OTHER INFORMATION: /note= "Xaa = Glu or Lys for both SEQ ID NO: 30 and SEQ ID NO: 31"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:

GCAGCTTCCC TGTGGTTTCC CGAGGCCTCC TTGCTTCCCG CTCTCCGAGG AGCCTTTCAT 60

CCGAAGGCGG GACG ATG CCG GAT AAT CGG CAG CCG AGG AAC CGG CAG CCG 110 Met Pro Asp Asn Arg Gin Pro Arg Asn Arg Gin Pro

1 5 10

GTG TGT TTA GGA GGA TGT GAG CAC ATC TTC TGT AGT AAT TGT GTA AGT 332 Val Cys Leu Gly Gly Cys Glu His He Phe Cys Ser Asn Cys Val Ser 65 70 75

CAA GAC TTG AAG ATA AAT AGA CAA CTG GAC AGC ATG ATT CAA CTT TGT 338 Gin Asp Leu Lys He Asn Arg Gin Leu Asp Ser Met He Gin Leu Cys 95 100 105

GAA GAT AAA CCT AGG AAA AGT TTG TTT AAT GAT GCA GGA AAC AAG AAG 434 Glu Asp Lys Pro Arg Lys Ser Leu Phe Asn Asp Ala Gly Asn Lys Lys 125 130 135 140

GTT GTG AGT AAA GCT TCA GTG CAA ACC CAG CCT GCA ATA AAA AAA GAT 530 Val Val Ser Lys Ala Ser Val Gin Thr Gin Pro Ala He Lys Lys Asp 160 165 170

CAG ATA AAT GGT GAA ATA GAC TTA CTA GCA AGT GGC TCC TTG ACA GAA 878 Gin He Asn Gly Glu He Asp Leu Leu Ala Ser Gly Ser Leu Tnr Glu 255 260 265

Glu Tyr Leu Leu Gin Asn Gly Ser Asp Pro Asn Val Lys Asp His Ala

445 450 455 460

GGA TGG ACA CCA TTG CAT GAA GCT TGC AAT CAT GGG CAC CTG AAG GTA 1502

Gly Trp Thr Pro Leu His Glu Ala Cys Asn His Gly His Leu Lys Val 465 470 475

GTG GAA TTA TTG CTC CAG CAT AAG GCA TTG GTG AAC ACC ACC GGG TAT 1550

Val Glu Leu Leu Leu Gin His Lys Ala Leu Val Asn Thr Thr Gly Tyr 480 485 490

CAA AAT GAC TCA CCA CTT CAC GAT GCA GCC AAG AAT GGG CAC GTG GAT 1598

Gin Asn Asp Ser Pro Leu His Asp Ala Ala Lys Asn Gly His Val Asp

495 500 505

ATA GTC AAG CTG TTA CTT TCC TAT GGA GCC TCC AGA AAT GCT GTT AAT 1646

He Val Lys Leu Leu Leu Ser Tyr Gly Ala Ser Arg Asn Ala Val Asn

510 515 520

ATA TTT GGT CTG CGG CCT GTC GAT TAT ACA GAT GAT GAA AGT ATG AAA 1634

He Phe Gly Leu Arg Pro Val Asp Tyr Thr Asp Asp Glu Ser Met Lys

525 530 535 540

TCG CTA TTG CTG CTA CCA GAG AAG AAT GAA TCA TCC TCA GCT AGC CAC 17.12

Ser Leu Leu Leu Leu Pro Glu Lys Asn Glu Ser Ser Ser Ala Ser His 545 550 555

TCC TCA GTA ATG AAC ACT GGG CAG CGT AGG GAT GGA CCT CTT GTA CTT 1790

Ser Ser Val Met Asn Thr Gly Gin Arg Arg Asp Gly Pro Leu Val Leu 560 565 570

ATA GGC AGT GGG CTG TCT TCA GAA CAA CAG AAA ATG CTC AGT GAG CTT 1838

He Gly Ser Gly Leu Ser Ser Glu Gin Gin Lys Met Leu Ser Glu Leu

575 580 585

GCA GTA ATT CTT AAG GCT AAA AAA TAT ACT GAG TTT GAC AGT ACA GTA 1886

Ala Val He Leu Lys Ala Lys Lys Tyr Thr Glu Phe Asp Ser Thr Val

590 595 600

ACT CAT GTT GTT GTT CCT GGT GAT GCA GTT CAA AGT ACC TTG AAG TGT 1934

Thr His Val Val Val Pro Gly Asp Ala Val Gin Ser Thr Leu Lys Cys

605 610 615 620

ATG CTT GGG ATT CTC AAT GGA TGC TGG ATT CTA AAA TTT GAA TGG GTA 1982

Met Leu Gly He Leu Asn Gly Cys Trp He Leu Lys Phe Glu Trp Val 625 630 635

AAA GCA TGT CTA CGA AGA AAA GTA TGT GAA CAG GAA GAA AAG TAT GAA 2030

Lys Ala Cys Leu Arg Arg Lys Val Cys Glu Gin Glu Glu Lys Tyr Glu 640 645 650

ATT CCT GAA GGT CCA CGC AGA AGC AGG CTC AAC AGA GAA CAG CTG TTG 20^"'8

He Pro Glu Gly Pro Arg Arg Ser Arg Leu Asn Arg Glu Gin Leu Leu

655 660 665

CCA AAG CTG TTT GAT GGA TGC TAC TTC TAT TTG TGG GGA ACC TTC AAA 2126

Pro Lys Leu Phe Asp Gly Cys Tyr Phe Tyr Leu Trp Gly Thr Phe Lys

670 675 680

CAC CAT CCA AAG GAC AAC CTT ATT AAG CTC GTC ACT GCA GGT GGG GGC 2174

His His Pro Lys Asp Asn Leu He Lys Leu Val Thr Ala Gly Gly Glv

ACCAGATGAA CATTTCAAAT TGAATTTGCA CGGTTTGTGA GAGCCCAGTC ATTGTACTGT 2475

TTTTAATGTT CACATTTTTA CAAATAGGTA GAGTCATTCA TATTTGTCTT TGAATC 2531

(2) INFORMATION FOR SEQ ID NO: 31:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 777 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:

Met Pro Asp Asn Arg Gin Pro Arg Asn Arg Gin Pro Arg He Arg Ser 1 5 10 15

Gly Asn Glu Pro Arg Ser Ala Pro Ala Met Glu Pro Asp Gly Arg Gly 20 25 30

Ala Trp Ala His Ser Arg Ala Ala Leu Asp Arg Leu Glu Lys Leu Leu 35 40 45

Arg Cys Ser Arg Cys Thr Asn He Leu Arg Glu Pro Val Cys Leu Gly 50 55 60

Gly Cys Glu His He Phe Cys Ser Asn Cys Val Ser Asp Cys He Gly 65 70 75 80

Thr Gly Cys Pro Val Cys Tyr Thr Pro Ala Trp He Gin Asp Leu Lys 85 90 95

He Asn Arg Gin Leu Asp Ser Met He Gin Leu Cys Ser Lys Leu Arg 100 105 110

Asn Leu Leu His Asp Asn Glu Leu Ser Asp Leu Lys Glu Asp Lys Pro 115 120 125

Arg Lys Ser Leu Phe Asn Asp Ala Gly Asn Lys Lys Asn Ser He Lys 130 135 140

Gin Asp Ser Tyr Glu Phe Val Ser Pro Ser Pro Pro Ala Asp Val Ser 180 185 190

Glu Arg Ala Lys Lys Ala Ser Ala Arg Ser Gly Lys Lys Gin Lys Lys 195 200 205

Lys Thr Leu Ala Glu He Asn Gin Lys Trp Asn Leu Glu Ala Glu Lys 210 215 220

Glu Asp Gly Glu Phe Asp Ser Lys Glu Glu Ser Lys Gin Lys Leu Val 225 230 235 240

Ser Phe Cys Ser Gin Pro Ser Val He Ser Ser Pro Gin He Asn Gly 245 250 255

Glu He Asp Leu Leu Ala Ser Gly Ser Leu Thr Glu Ser Glu Cys Phe 260 265 270

Gly Ser Leu Thr Glu Val Ser Leu Pro Leu Ala Glu Gin He Glu Ser 275 280 285

Pro Asp Thr Lys Ser Arg Asn Glu Val Val Thr Pro Glu Lys Val Cys 290 295 300

Lys Asn Tyr Leu Thr Ser Lys Lys Ser Leu Pro Leu Glu Asn Asn Gly 305 310 315 320

Lys Arg Gly His His Asn Arg Leu Ser Ser Pro He Ser Lys Arg Cys 325 330 335

Arg Thr Ser He Leu Ser Thr Ser Gly Asp Phe Val Lys Gin Thr Val 340 345 350

Pro Ser Glu Asn He Pro Leu Pro Glu Cys Ser Ser Pro Pro Ser Cys 355 360 365

Lys Arg Lys Val Gly Gly Thr Ser Gly Arg Lys Asn Ser Asn Met Ser 370 375 380

Asp Glu Phe He Ser Leu Ser Pro Gly Thr Pro Pro Ser Thr Leu Ser 385 390 395 400

Ser Ser Ser Tyr Arg Gin Val Met Ser Ser Pro Ser Ala Met Lys Leu 405 410 415

Leu Pro Asn Met Ala Val Lys Arg Asn His Arg Gly Glu Thr Leu Leu 420 425 430

His He Ala Ser He Lys Gly Asp He Pro Ser Val Glu Tyr Leu Leu 435 440 445

Gin Asn Gly Ser Asp Pro Asn Val Lys Asp His Ala Gly Trp Thr Pro 450 455 460

Leu His Glu Ala Cys Asn His Gly His Leu Lys Val Val Glu Leu Leu 465 470 475 480

Leu Gin His Lys Ala Leu Val Asn Thr Thr Gly Tyr Gin Asn Asp Ser 485 490 495

Arg Pro Val Asp Tyr Thr Asp Asp Glu Ser Met Lys Ser Leu Leu Leu 530 535 540

Leu Pro Glu Lys Asn Glu Ser Ser Ser Ala Ser His Ser Ser Val Met 545 550 555 560

Asn Thr Gly Gin Arg Arg Asp Gly Pro Leu Val Leu He Gly Ser Gly 565 570 575

Leu Ser Ser Glu Gin Gin Lys Met Leu Ser Glu Leu Ala Val He Leu 580 585 590

Lys Ala Lys Lys Tyr Thr Glu Phe Asp Ser Thr Val Thr His Val v_al 595 600 605

Val Pro Gly Asp Ala Val Gin Ser Thr Leu Lys Cys Met Leu Gly He 610 615 620

Leu Asn Gly Cys Trp He Leu Lys Phe Glu Trp Val Lys Ala Cys Leu 625 630 635 640

Arg Arg Lys Val Cys Glu Gin Glu Glu Lys Tyr Glu He Pro Glu Gly 645 650 655

Pro Arg Arg Ser Arg Leu Asn Arg Glu Gin Leu Leu Pro Lys Leu Phe 660 665 670

Asp Gly Cys Tyr Phe Tyr Leu Trp Gly Thr Phe Lys His His Pro Lys 675 680 685

Asp Asn Leu He Lys Leu Val Thr Ala Gly Gly Gly Gin He Leu Ser 690 695 700

Arg Lys Pro Lys Pro Asp Ser Asp Val Thr Gin Thr He Asn Thr Val 705 710 715 720

Ala Tyr His Ala Arg Pro Asp Ser Asp Gin Arg Phe Cys Thr Gin Tyr 725 730 735

He He Tyr Glu Asp Leu Cys Asn Tyr His Pro Glu Arg Val Arg Gin 740 745 750

Gly Lys Val Trp Lys Ala Pro Ser Ser Trp Phe He Asp Cys Val Met 755 760 765

Ser Phe Glu Leu Leu Pro Leu Asp Ser 770 775

(2) INFORMATION FOR SEQ ID NO: 32:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2531 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 75..2405 ( ix ) FEATURE :

(A) NAME/KEY: modified_base

(B) LOCATION: 531

(D) OTHER INFORMATIO : /note= "R = A or G"

(ix) FEATURE:

(A) NAME/KEY: modified_base

(B) LOCATION: 153

(D) OTHER INFORMATIO :/note= "Xaa = Glu or Lys for both SEQ ID NO: 32 and SEQ ID NO: 33"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:

GCAGCTTCCC TGTGGTTTCC CGAGGCCTCC TTGCTTCCCG CTCTCCGAGG AGCCTTTCAT 60

1 5 10

TGC TCA GTA ATG AAC ACT GGG CAC CGT AGG GAT GGA CCT CTT GTA CTT 1790 Cys Ser Val Met Asn Thr Gly His Arg Arg Asp Gly Pro Leu Val Leu 560 565 570

ATG CTT GGG ATT CTC AAT GGA TGC TGG ATT CTA AAA TTT GAA TGG GTA 1962 Met Leu Gly He Leu Asn Gly Cys Trp He Leu Lys Phe Glu Trp Val 625 630 635

ACCAGATGAA CATTTCAAAT TGAATTTGCA CGGTTTGTGA GAGCCCAGTC ATTGTACTGT 2475

TTTTAATGTT CACATTTTTA CAAATAGGTA GAGTCATTCA TATTTGTCTT TGAATC 2531

(2) INFORMATION FOR SEQ ID NO: 33:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 777 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:

Met Pro Asp Asn Arg Gin Pro Arg Asn Arg Gin Pro Arg He Arg Ser 1 5 10 15

Gly Asn Glu Pro Arg Ser Ala Pro Ala Met Glu Pro Asp Gly Arg Gly 20 25 30

Ala Trp Ala His Ser Arg Ala Ala Leu Asp Arg Leu Glu Lys Leu Leu 35 40 45

Arg Cys Ser Arg Cys Thr Asn He Leu Arg Glu Pro Val Cys Leu Gly 50 55 60

Gly Cys Glu His He Phe Cys Ser Asn Cys Val Ser Asp Cys He Gly 65 70 75 80

Thr Gly Cys Pro Val Cys Tyr Thr Pro Ala Trp He Gin Asp Leu Lys 85 90 95

He Asn Arg Gin Leu Asp Ser Met He Gin Leu Cys Ser Lys Leu Arg 100 105 110

Asn Leu Leu His Asp Asn Glu Leu Ser Asp Leu Lys Glu Asp Lys Pro 115 120 125

Arg Lys Ser Leu Phe Asn Asp Ala Gly Asn Lys Lys Asn Ser He Lys 130 135 140

Gin Asp Ser Tyr Glu Phe Val Ser Pro Ser Pro Pro Ala Asp Val Ser 180 185 190

Glu Arg Ala Lys Lys Ala Ser Ala Arg Ser Gly Lys Lys Gin Lys Lys 195 200 205

Lys Thr Leu Ala Glu He Asn Gin Lys Trp Asn Leu Glu Ala Glu Lys 210 215 220

Glu Asp Gly Glu Phe Asp Ser Lys Glu Glu Ser Lys Gin Lys Leu Val 225 230 235 240

Ser Phe Cys Ser Gin Pro Ser Val He Ser Ser Pro Gin He Asn Gly 245 250 255

Glu He Asp Leu Leu Ala Ser Gly Ser Leu Thr Glu Ser Glu Cys Phe 260 265 270

Gly Ser Leu Thr Glu Val Ser Leu Pro Leu Ala Glu Gin He Glu Ser 275 280 285

Pro Asp Thr Lys Ser Arg Asn Glu Val Val Thr Pro Glu Lys Val Cys 290 295 300

Lys Asn Tyr Leu Thr Ser Lys Lys Ser Leu Pro Leu Glu Asn Asn Gly 305 310 315 320

Lys Arg Gly His His Asn Arg Leu Ser Ser Pro He Ser Lys Arg Cys 325 330 335

Arg Thr Ser He Leu Ser Thr Ser Gly Asp Phe Val Lys Gin Thr Val 340 345 350

Pro Ser Glu Asn He Pro Leu Pro Glu Cys Ser Ser Pro Pro Ser Cys 355 360 365

Lys Arg Lys Val Gly Gly Thr Ser Gly Arg Lys Asn Ser Asn Met Ser 370 375 380

Asp Glu Phe He Ser Leu Ser Pro Gly Thr Pro Pro Ser Thr Leu Ser 385 390 395 400

Ser Ser Ser Tyr Arg Gin Val Met Ser Ser Pro Ser Ala Met Lys Leu 405 410 415

Leu Pro Asn Met Ala Val Lys Arg Asn His Arg Gly Glu Thr Leu Leu 420 425 430

His He Ala Ser He Lys Gly Asp He Pro Ser Val Glu Tyr Leu Leu 435 440 445

Gin Asn Gly Ser Asp Pro Asn Val Lys Asp His Ala Gly Trp Thr Pro 450 455 460

Leu His Glu Ala Cys Asn His Gly His Leu Lys Val Val Glu Leu Leu 465 470 475 480

Leu Gin His Lys Ala Leu Val Asn Thr Thr Gly Tyr Gin Asn Asp Ser 485 490 495

Arg Pro Val Asp Tyr Thr Asp Asp Glu Ser Met Lys Ser Leu Leu Leu 530 535 540

Leu Pro Glu Lys Asn Glu Ser Ser Ser Ala Ser His Cys Ser Val Met 545 550 555 560

Asn Thr Gly His Arg Arg Asp Gly Pro Leu Val Leu He Gly Ser Gly 565 570 575

Leu Ser Ser Glu Gin Gin Lys Met Leu Ser Glu Leu Ala Val He Leu 580 585 590

Lys Ala Lys Lys Tyr Thr Glu Phe Asp Ser Thr Val Thr His Val Val 595 600 605

Val Pro Gly Asp Ala Val Gin Ser Thr Leu Lys Cys Met Leu Gly He 610 615 620

Leu Asn Gly Cys Trp He Leu Lys Phe Glu Trp Val Lys Ala Cys Leu 625 630 635 640

Arg Arg Lys Val Cys Glu Gin Glu Glu Lys Tyr Glu He Pro Glu Gly 645 650 655

Pro Arg Arg Ser Arg Leu Asn Arg Glu Gin Leu Leu Pro Lys Leu Phe 660 665 670

Asp Gly Cys Tyr Phe Tyr Leu Trp Gly Thr Phe Lys His His Pro Lys 675 680 685

Asp Asn Leu He Lys Leu Val Thr Ala Gly Gly Gly Gin He Leu Ser 690 695 700

Arg Lys Pro Lys Pro Asp Ser Asp Val Thr Gin Thr He Asn Thr Val 705 710 715 720

Ala Tyr His Ala Arg Pro Asp Ser Asp Gin Arg Phe Cys Thr Gin Tyr 725 730 735

He He Tyr Glu Asp Leu Cys Asn Tyr His Pro Glu Arg Val Arg Gin 740 745 750

Gly Lys Val Trp Lys Ala Pro Ser Ser Trp Phe He Asp Cys Val Met 755 760 765

Ser Phe Glu Leu Leu Pro Leu Asp Ser 770 775

(2) INFORMATION FOR SEQ ID NO: 34:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2531 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 75..2405 ( ix ) FEATURE :

(A) NAME/KEY: modified_base

(B) LOCATION: 531

(D) OTHER INFORMATION :/note= "R = A or G"

(ix) FEATURE:

(A) NAME/KEY: modified_base

(B) LOCATION: 153

(D) OTHER INFORMATION: /note= "Xaa = Glu or Lys for both SEQ ID NO: 34 and SEQ ID NO: 35"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:

GCAGCTTCCC TGTGGTTTCC CGAGGCCTCC TTGCTTCCCG CTCTCCGAGG AGCCTTTCAT 60

1 5 10

GAG ACT TTG CTC CAT ATT GCT TCT ATT AAG GGC GAC ATA CCT TCT GTT 1406 Glu Thr Leu Leu His He Ala Ser He Lys Gly Asp He Pro Ser Val 430 435 440 GAA TAC CTT TTA CAA AAT GGA AGT GAT CCA AAT GTT AAA GAC CAT GCT 1 3

Glu Tyr Leu Leu Gin Asn Gly Ser Asp Pro Asn Val Lys Asp His Ala 445 450 455 460

GGA TGG ACA CCA TTG CAT GAA GCT TGC AAT CAT GGG CAC CTG AAG GTA 1502

Gly Trp Thr Pro Leu His Glu Ala Cys Asn His Gly His Leu Lys Val 465 470 475

GTG GAA TTA TTG CTC CAG CAT AAG GCA TTG GTG AAC ACC ACC GGG TAT 15'JO

Val Glu Leu Leu Leu Gin His Lys Ala Leu Val Asn Thr Thr Gly Tyr 480 485 490

CAA AAT GAC TCA CCA CTT CAC GAT GCA GCC AAG AAT GGG CAC GTG GAT 1598

Gin Asn Asp Ser Pro Leu His Asp Ala Ala Lys Asn Gly His Val Asp 495 500 505

ATA GTC AAG CTG TTA CTT TCC TAT GGA GCC TCC AGA AAT GCT GTT AAT 1646

He Val Lys Leu Leu Leu Ser Tyr Gly Ala Ser Arg Asn Ala Val Asn

510 515 520

ATA TTT GGT CTG CGG CCT GTC GAT TAT ACA GAT GAT GAA AGl ATG AAA 16'.4

He Phe Gly Leu Arg Pro Val Asp Tyr Thr Asp Asp Glu Ser Met Lys 525 530 535 540

TCG CTA TTG CTG CTA CCA GAG AAG AAT GAA TCA TCC TCA GCT AGC CAC 1742

Ser Leu Leu Leu Leu Pro Glu Lys Asn Glu Ser Ser Ser Ala Ser His 545 550 555

TGC TCA GTA ATG AAC ACT GGG CAG CGT AGG GAT GGA CCT CTT GTA CTT 1790

Cys Ser Val Met Asn Thr Gly Gin Arg Arg Asp Gly Pro Leu Val Leu 560 565 570

ATA GGC AGT GGG CTG TCT TCA GAA CAP. CAG AAA ATG CTC AGT GAG CTT 1828

He Gly Ser Gly Leu Ser Ser Glu Gin Gin Lys Met Leu Ser Glu Leu 575 580 585

GCA GTA ATT CTT AAG GCT AAA AAA TAT ACT GAG TTT GAC AGT ACA GTA 1886

Ala Val He Leu Lys Ala Lys Lys Tyr Thr Glu Phe Asp Ser Thr Val

590 595 600

ACT CAT GTT GTT GTT CCT GGT GAT GCA GTT CAA AGT ACC TTG AAG TGT 1924

Thr His Val Val Val Pro Gly Asp Ala Val Gin Ser Thr Leu Lys Cys 605 610 615 620

ATG CTT GGG ATT CTC AAT GGA TGC TGG ATT CTA AAA TTT GAA TGG GTA 1982

Met Leu Gly He Leu Asn Gly Cys Trp He Leu Lys Phe Glu Trp Val 625 630 635

AAA GCA TGT CTA CGA AGA AAA GTA TGT GAA CAG GAA GAA AAG TAT GAA 2030

Lys Ala Cys Leu Arg Arg Lys Val Cys Glu Gin Glu Glu Lys Tyr Glu 640 645 650

ATT CCT GAA GGT CCA CGC AGA AGC AGG CTC AAC AGA GAA CAG CTG TTG 2078

He Pro Glu Gly Pro Arg Arg Ser Arg Leu Asn Arg Glu Gin Leu Leu 655 660 665

CCA AAG CTG TTT GAT GGA TGC TAC TTC TAT TTG TGG GGA ACC TTC AAA 2126

Pro Lys Leu Phe Asp Gly Cys Tyr Phe Tyr Leu Trp Gly Thr Phe Lys

670 675 680

CAC CAT CCA AAG GAC AAC CTT ATT AAG CTC GTC ACT GCA GGT GGG GGC 2174

His His Pro Lys Asp Asn Leu He Lys Leu Val Thr Ala Gly Gly Gly 685 690 695 700 CAG ATC CTC AGT AGA AAG CCC AAG CCA GAC AGT GAC GTG ACT CAG ACC 2222 Gin He Leu Ser Arg Lys Pro Lys Pro Asp Ser Asp Val Thr Gin Thr 705 710 715

AGG GTT CGG CAG GGC AAA GTC TGG AAG GCT CCT TCG AAC TGG TTT ATA 2366 Arg Val Arg Gin Gly Lys Val Trp Lys Ala Pro Ser Asn Trp Phe He 750 755 760

ACCAGATGAA CATTTCAAAT TGAATTTGCA CGGTTTGTGA GAGCCCAGTC ATTGTACTGT 2475

TTTTAATGTT CACATTTTTA CAAATAGGTA GAGTCATTCA TATTTGTCTT TGAATC 2531

(2) INFORMATION FOR SEQ ID NO: 35:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 777 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:

Met Pro Asp Asn Arg Gin Pro Arg Asn Arg Gin Pro Arg He Arg Ser 1 5 10 15

Gly Asn Glu Pro Arg Ser Ala Pro Ala Met Glu Pro Asp Gly Arg Gly 20 25 30

Ala Trp Ala His Ser Arg Ala Ala Leu Asp Arg Leu Glu Lys Leu Leu 35 40 45

Arg Cys Ser Arg Cys Thr Asn He Leu Arg Glu Pro Val Cys Leu Gly 50 55 60

Gly Cys Glu His He Phe Cys Ser Asn Cys Val Ser Asp Cys He Gly 65 70 75 80

Thr Gly Cys Pro Val Cys Tyr Thr Pro Ala Trp He Gin Asp Leu Lys 85 90 95

He Asn Arg Gin Leu Asp Ser Met He Gin Leu Cys Ser Lys Leu Arg 100 105 110

Asn Leu Leu His Asp Asn Glu Leu Ser Asp Leu Lys Glu Asp Lys Pro 115 120 125

Arg Lys Ser Leu Phe Asn Asp Ala Gly Asn Lys Lys Asn Ser He Lys 130 135 140

Gin Asp Ser Tyr Glu Phe Val Ser Pro Ser Pro Pro Ala Asp Val Ser 180 185 190

Glu Arg Ala Lys Lys Ala Ser Ala Arg Ser Gly Lys Lys Gin Lys Lys 195 200 205

Lys Thr Leu Ala Glu He Asn Gin Lys Trp Asn Leu Glu Ala Glu Lys 210 215 220

Glu Asp Gly Glu Phe Asp Ser Lys Glu Glu Ser Lys Gin Lys Leu Val 225 230 235 240

Ser Phe Cys Ser Gin Pro Ser Val He Ser Ser Pro Gin He Asn Gly 245 250 255

Glu He Asp Leu Leu Ala Ser Gly Ser Leu Thr Glu Ser Glu Cys Phe 260 265 270

Gly Ser Leu Thr Glu Val Ser Leu Pro Leu Ala Glu Gin He Glu Ser 275 280 285

Pro Asp Thr Lys Ser Arg Asn Glu Val Val Thr Pro Glu Lys Val Cys 290 295 300

Lys Asn Tyr Leu Thr Ser Lys Lys Ser Leu Pro Leu Glu Asn Asn Gly 305 310 315 320

Lys Arg Gly His His Asn Arg Leu Ser Ser Pro He Ser Lys Arg Cys 325 330 335

Arg Thr Ser He Leu Ser Thr Ser Gly Asp Phe Val Lys Gin Thr Val 340 345 350

Pro Ser Glu Asn He Pro Leu Pro Glu Cys Ser Ser Pro Pro Ser Cys 355 360 365

Lys Arg Lys Val Gly Gly Thr Ser Gly Arg Lys Asn Ser Asn Met Ser 370 375 380

Asp Glu Phe He Ser Leu Ser Pro Gly Thr Pro Pro Ser Thr Leu Ser 385 390 395 400

Ser Ser Ser Tyr Arg Gin Val Met Ser Ser Pro Ser Ala Met Lys Leu 405 410 415

Leu Pro Asn Met Ala Val Lys Arg Asn His Arg Gly Glu Thr Leu Leu 420 425 430

His He Ala Ser He Lys Gly Asp He Pro Ser Val Glu Tyr Leu Leu 435 440 445

Gin Asn Gly Ser Asp Pro Asn Val Lys Asp His Ala Gly Trp Thr Pro 450 455 460

Leu His Glu Ala Cys Asn His Gly His Leu Lys Val Val Glu Leu Leu 465 470 475 480

Leu Gin His Lys Ala Leu Val Asn Thr Thr Gly Tyr Gin Asn Asp Ser 485 490 495

Arg Pro Val Asp Tyr Thr Asp Asp Glu Ser Met Lys Ser Leu Leu Leu 530 535 540

Leu Pro Glu Lys Asn Glu Ser Ser Ser Ala Ser His Cys Ser Val Met 545 550 555 560

Asn Thr Gly Gin Arg Arg Asp Gly Pro Leu Val Leu He Gly Ser Gly 565 570 575

Leu Ser Ser Glu Gin Gin Lys Met Leu Ser Glu Leu Ala Val He Leu 580 585 590

Lys Ala Lys Lys Tyr Thr Glu Phe Asp Ser Thr Val Thr His Val Val 595 600 605

Val Pro Gly Asp Ala Val Gin Ser Thr Leu Lys Cys Met Leu Gly He 610 615 620

Leu Asn Gly Cys Trp He Leu Lys Phe Glu Trp Val Lys Ala Cys Leu 625 630 635 640

Arg Arg Lys Val Cys Glu Gin Glu Glu Lys Tyr Glu He Pro Glu Gly 645 650 655

Pro Arg Arg Ser Arg Leu Asn Arg Glu Gin Leu Leu Pro Lys Leu Phe 660 665 670

Asp Gly Cys Tyr Phe Tyr Leu Trp Gly Thr Phe Lys His His Pro Lys 675 680 685

Asp Asn Leu He Lys Leu Val Thr Ala Gly Gly Gly Gin He Leu Ser 690 695 700

Arg Lys Pro Lys Pro Asp Ser Asp Val Thr Gin Thr He Asn Thr Val 705 710 715 720

Ala Tyr His Ala Arg Pro Asp Ser Asp Gin Arg Phe Cys Thr Gin Tyr 725 730 735

He He Tyr Glu Asp Leu Cys Asn Tyr His Pro Glu Arg Val Arg Gin 740 745 750

Gly Lys Val Trp Lys Ala Pro Ser Asn Trp Phe He Asp Cys Val Met 755 760 765

Ser Phe Glu Leu Leu Pro Leu Asp Ser 770 775

(2) INFORMATION FOR SEQ ID NO: 36:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2531 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 75..2405 ( ix ) FEATURE :

(A) NAME/KEY: modified_base

(B) LOCATION: 531

(D) OTHER INFORMATION: /note= "R = A or G"

(ix) FEATURE:

(A) NAME/KEY: modified_base

(B) LOCATION: 153

(D) OTHER INFORMATIO :/note= "Xaa = Glu or Lys for both SEQ ID NO: 36 and SEQ ID NO: 37"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:

GCAGCTTCCC TGTGGTTTCC CGAGGCCTCC TTGCTTCCCG CTCTCCGAGG AGCCTTTCAT 60

CCA CCT TCA TGC AAA CGT AAA GTT GGT GGT ACA TCA GGG AGG AAA AAC 1214 Pro Pro Ser Cys Lys Arg Lys Val Gly Gly Thr Ser Gly Arg Lvs Asn 365 370 375 ^" 380

GGA TGG ACA CCA TTG CAT GAA GCT TGC AAT CAT GGG CAC CTG AAG GTA 1Ξ02 Gly Trp Thr Pro Leu His Glu Ala Cys Asn His Gly His Leu Lys Val 465 470 475

ATT CCT GAA GGT CCA TGC AGA AGC AGG CTC AAC AGA GAA CAG CTG TTG 2078 He Pro Glu Gly Pro Cys Arg Ser Arg Leu Asn Arg Glu Gin Leu Leu 655 660 665

ACCAGATGAA CATTTCAAAT TGAATTTGCA CGGTTTGTGA GAGCCCAGTC ATTGTACTGT 2475

TTTTAATGTT CACATTTTTA CAAATAGGTA GAGTCATTCA TATTTGTCTT TGAATC 2531

(2) INFORMATION FOR SEQ ID NO: 37:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 777 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:

Met Pro Asp Asn Arg Gin Pro Arg Asn Arg Gin Pro Arg He Arg Ser 1 5 10 15

Gly Asn Glu Pro Arg Ser Ala Pro Ala Met Glu Pro Asp Gly Arg Gly 20 25 30

Ala Trp Ala His Ser Arg Ala Ala Leu Asp Arg Leu Glu Lys Leu Leu 35 40 45

Arg Cys Ser Arg Cys Thr Asn He Leu Arg Glu Pro Val Cys Leu Gly 50 55 60

Gly Cys Glu His He Phe Cys Ser Asn Cys Val Ser Asp Cys He Gly 65 70 75 80

Thr Gly Cys Pro Val Cys Tyr Thr Pro Ala Trp He Gin Asp Leu Lys 85 90 95

He Asn Arg Gin Leu Asp Ser Met He Gin Leu Cys Ser Lys Leu Arg 100 105 110

Asn Leu Leu His Asp Asn Glu Leu Ser Asp Leu Lys Glu Asp Lys Pro 115 120 125

Arg Lys Ser Leu Phe Asn Asp Ala Gly Asn Lys Lys Asn Ser He Lys 130 135 140

Gin Asp Ser Tyr Glu Phe Val Ser Pro Ser Pro Pro Ala Asp Val Ser 180 185 190

Glu Arg Ala Lys Lys Ala Ser Ala Arg Ser Gly Lys Lys Gin Lys Lys 195 200 205

Lys Thr Leu Ala Glu He Asn Gin Lys Trp Asn Leu Glu Ala Glu Lys 210 215 220

Glu Asp Gly Glu Phe Asp Ser Lys Glu Glu Ser Lys Gin Lys Leu Val 225 230 235 240

Ser Phe Cys Ser Gin Pro Ser Val He Ser Ser Pro Gin He Asn Gly 245 250 255

Glu He Asp Leu Leu Ala Ser Gly Ser Leu Thr Glu Ser Glu Cys Phe 260 265 270

Gly Ser Leu Thr Glu Val Ser Leu Pro Leu Ala Glu Gin He Glu Ser 275 280 285

Pro Asp Thr Lys Ser Arg Asn Glu Val Val Thr Pro Glu Lys Val Cys 290 295 300

Lys Asn Tyr Leu Thr Ser Lys Lys Ser Leu Pro Leu Glu Asn Asn Gly 305 310 315 320

Lys Arg Gly His His Asn Arg Leu Ser Ser Pro He Ser Lys Arg Cys 325 330 335

Arg Thr Ser He Leu Ser Thr Ser Gly Asp Phe Val Lys Gin Thr Val 340 345 350

Pro Ser Glu Asn He Pro Leu Pro Glu Cys Ser Ser Pro Pro Ser Cys 355 360 365

Lys Arg Lys Val Gly Gly Thr Ser Gly Arg Lys Asn Ser Asn Met Ser 370 375 380

Asp Glu Phe He Ser Leu Ser Pro Gly Thr Pro Pro Ser Thr Leu Ser 385 390 395 400

Ser Ser Ser Tyr Arg Gin Val Met Ser Ser Pro Ser Ala Met Lys Leu 405 410 415

Leu Pro Asn Met Ala Val Lys Arg Asn His Arg Gly Glu Thr Leu Leu 420 425 430

His He Ala Ser He Lys Gly Asp He Pro Ser Val Glu Tyr Leu Leu 435 440 445

Gin Asn Gly Ser Asp Pro Asn Val Lys Asp His Ala Gly Trp Thr Pro 450 455 460

Leu His Glu Ala Cys Asn His Gly His Leu Lys Val Val Glu Leu Leu 465 470 475 480

Leu Gin His Lys Ala Leu Val Asn Thr Thr Gly Tyr Gin Asn Asp Ser 485 490 495

Arg Pro Val Asp Tyr Thr Asp Asp Glu Ser Met Lys Ser Leu Leu Leu 530 535 540

Leu Pro Glu Lys Asn Glu Ser Ser Ser Ala Ser His Cys Ser Val Met 545 550 555 560

Asn Thr Gly Gin Arg Arg Asp Gly Pro Leu Val Leu He Gly Ser Gly 565 570 575

Leu Ser Ser Glu Gin Gin Lys Met Leu Ser Glu Leu Ala Val He Leu 580 585 590

Lys Ala Lys Lys Tyr Thr Glu Phe Asp Ser Thr Val Thr His Val Val 595 600 605

Val Pro Gly Asp Ala Val Gin Ser Thr Leu Lys Cys Met Leu Gly He 610 615 620

Leu Asn Gly Cys Trp He Leu Lys Phe Glu Trp Val Lys Ala Cys Leu 625 630 635 640

Arg Arg Lys Val Cys Glu Gin Glu Glu Lys Tyr Glu He Pro Glu Gly 645 650 655

Pro Cys Arg Ser Arg Leu Asn Arg Glu Gin Leu Leu Pro Lys Leu Phe 660 665 670

Asp Gly Cys Tyr Phe Tyr Leu Trp Gly Thr Phe Lys His His Pro Lys 675 680 685

Asp Asn Leu He Lys Leu Val Thr Ala Gly Gly Gly Gin He Leu Ser 690 695 700

Arg Lys Pro Lys Pro Asp Ser Asp Val Thr Gin Thr He Asn Thr Val 705 710 715 720

Ala Tyr His Ala Arg Pro Asp Ser Asp Gin Arg Phe Cys Thr Gin Tyr 725 730 735

He He Tyr Glu Asp Leu Cys Asn Tyr His Pro Glu Arg Val Arg Gin 740 745 750

Gly Lys Val Trp Lys Ala Pro Ser Ser Trp Phe He Asp Cys Val Met 755 760 765

Ser Phe Glu Leu Leu Pro Leu Asp Ser 770 775

(2) INFORMATION FOR SEQ ID NO: 38:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2531 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 75..2405 ( ix ) FEATURE :

(A) NAME/KEY: modified_base

(B) LOCATION: 531

(D) OTHER INFORMATION :/note= "R = A or G"

(ix) FEATURE:

(A) NAME/KEY: modified_base

(B) LOCATION: 153

(D) OTHER INFORMATIO :/note= "Xaa = Glu or Lys for both SEQ ID NO: 38 and SEQ ID NO: 39"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:

GCAGCTTCCC TGTGGTTTCC CGAGGCCTCC TTGCTTCCCG CTCTCCGAGG AGCCTTTCAT 60

GAG ACT TTG CTC CAT ATT GCT TCT ATT AAG GGC GAC ATA CCT TCT GTT 1406 Glu Thr Leu Leu His He Ala Ser He Lys Gly Asp He Pro Ser Val 430 435 440 GAA TAC CTT TTA CAA AAT GGA AGT GAT CCA AAT GTT AAA GAC CAT GCT 1<54

Glu Tyr Leu Leu Gin Asn Gly Ser Asp Pro Asn Val Lys Asp His Ala 445 450 455 460

GGA TGG ACA CCA TTG CAT GAA GCT TGC AAT CAT GGG CAC CTG AAG GTA 1502

Gly Trp Thr Pro Leu His Glu Ala Cys Asn His Gly His Leu Lys VaJ 465 470 475

GTG GAA TTA TTG CTC CAG CAT AAG GCA TTG GTG AAC ACC ACC GGG TAT 1550

Val Glu Leu Leu Leu Gin His Lys Ala Leu Val Asn Thr Thr Gly Tyr 480 485 490

CAA AAT GAC TCA CCA CTT CAC GAT GCA GCC AAG AAT GGG CAC GTG GAT 1598

Gin Asn Asp Ser Pro Leu His Asp Ala Ala Lys Asn Gly His Val Asp 495 500 505

ATA GTC AAG CTG TTA CTT TCC TAT GGA GCC TCC AGA AAT GCT GTT AAT l€ 46

He Val Lys Leu Leu Leu Ser Tyr Gly Ala Ser Arg Asn Ala Val Asn

510 515 520

ATA TTT GGT CTG CGG CCT GTC GAT TAT ACA GAT GAT GAA AGT ATG AAA 1694

He Phe Gly Leu Arg Pro Val Asp Tyr Thr Asp Asp Glu Ser Met Lys 525 530 535 540

TCG CTA TTG CTG CTA CCA GAG AAG AAT GAA TCA TCC TCA GCT AGC CAC 1742

Ser Leu Leu Leu Leu Pro Glu Lys Asn Glu Ser Ser Ser Ala Ser His 545 550 555

TGC TCA GTA ATG AAC ACT GGG CAG CGT AGG GAT GGA CCT CTT GTA CTT 17S0

Cys Ser Val Met Asn Thr Gly Gin Arg Arg Asp Gly Pro Leu Val Leu 560 565 570

ATA GGC AGT GGG CTG TCT TCA GAA CAA CAG AAA ATG CTC AGT GAG CTT 1838

He Gly Ser Gly Leu Ser Ser Glu Gin Gin Lys Met Leu Ser Glu Leu 575 580 585

GCA GTA ATT CTT AAG GCT AAA AAA TAT ACT GAG TTT GAC AGT ACA GTA 1886

Ala Val He Leu Lys Ala Lys Lys Tyr Thr Glu Phe Asp Ser Thr Val

590 595 600

ACT CAT GTT GTT GTT CCT GGT GAT GCA GTT CAA AGT ACC TTG AAG TGT 1934

Thr His Val Val Val Pro Gly Asp Ala Val Gin Ser Thr Leu Lys Cys 605 610 615 620

ATG CTT GGG ATT CTC AAT GGA TGC TGG ATT CTA AAA TTT GAA TGG GTA 1982

Met Leu Gly He Leu Asn Gly Cys Trp He Leu Lys Phe Glu Trp Val 625 630 635

AAA GCA TGT CTA CGA AGA AAA GTA TGT GAA CAG GAA GAA AAG TAT GAA 2030

Lys Ala Cys Leu Arg Arg Lys Val Cys Glu Gin Glu Glu Lys Tyr Glu 640 645 650

ATT CCT GAA GGT CCA CGC AGA AGC AGG CTC AAC AGA GAA CAG CTG TTG 2078

He Pro Glu Gly Pro Arg Arg Ser Arg Leu Asn Arg Glu Gin Leu Leu 655 660 665

CCA AAG CTG TTT GAT GGA TGC TAC TTC TAT TTG TGG GGA ACC TTC AAA 2126

Pro Lys Leu Phe Asp Gly Cys Tyr Phe Tyr Leu Trp Gly Thr Phe Lys

670 675 680

CAC CAT CCA AAG GAC AAC CTT ATT AAG CTC GTC ACT GCA GGT GGG GGC 2174

ACCAGATGAA CATTTCAAAT TGAATTTGCA CGGTTTGTGA GAGCCCAGTC ATTGTACTGT 2475

TTTTAATGTT CACATTTTTA CAAATAGGTA GAGTCATTCA TATTTGTCTT TGAATC 2531

(2) INFORMATION FOR SEQ ID NO: 39:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 777 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:

Met Pro Asp Asn Arg Gin Pro Arg Asn Arg Gin Pro Arg He Arg Ser 1 5 10 15

Gly Asn Glu Pro Arg Ser Ala Pro Ala Met Glu Pro Asp Gly Arg Gly 20 25 30

Ala Trp Ala His Ser Arg Ala Ala Leu Asp Arg Leu Glu Lys Leu Leu 35 40 45

Arg Cys Ser Arg Cys Thr Asn He Leu Arg Glu Pro Val Cys Leu Gly 50 55 60

Gly Cys Glu His He Phe Cys Ser Asn Cys Val Ser Asp Cys He Gly 65 70 75 80

Thr Gly Cys Pro Val Cys Tyr Thr Pro Ala Trp He Gin Asp Leu Lys 85 90 95

He Asn Arg Gin Leu Asp Ser Met He Gin Leu Cys Ser Lys Leu Arg 100 105 110

Asn Leu Leu His Asp Asn Glu Leu Ser Asp Leu Lys Glu Asp Lys Pro 115 120 125

Arg Lys Ser Leu Phe Asn Asp Ala Gly Asn Lys Lys Asn Ser He Lys 130 135 140

Gin Asp Ser Tyr Glu Phe Val Ser Pro Ser Pro Pro Ala Asp Val Ser 180 185 190

Glu Arg Ala Lys Lys Ala Ser Ala Arg Ser Gly Lys Lys Gin Lys Lys 195 200 205

Lys Thr Leu Ala Glu He Asn Gin Lys Trp Asn Leu Glu Ala Glu Lys 210 215 220

Glu Asp Gly Glu Phe Asp Ser Lys Glu Glu Ser Lys Gin Lys Leu Val 225 230 235 240

Ser Phe Cys Ser Gin Pro Ser Val He Ser Ser Pro Gin He Asn Gly 245 250 255

Glu He Asp Leu Leu Ala Ser Gly Ser Leu Thr Glu Ser Glu Cys Phe 260 265 270

Gly Ser Leu Thr Glu Val Ser Leu Pro Leu Ala Glu Gin He Glu Ser 275 280 285

Pro Asp Thr Lys Ser Arg Asn Glu Val Val Thr Pro Glu Lys Val Cys 290 295 300

Lys Asn Tyr Leu Thr Ser Lys Lys Ser Leu Pro Leu Glu Asn Asn Gly 305 310 315 320

Lys Arg Gly His His Asn Arg Leu Ser Ser Pro He Ser Lys Arg Cys 325 330 335

Arg Thr Ser He Leu Ser Thr Ser Gly Asp Phe Val Lys Gin Thr Val 340 345 350

Pro Ser Glu Asn He Pro Leu Pro Glu Cys Ser Ser Pro Pro Ser Cys 355 360 365

Lys Arg Lys Val Gly Gly Thr Ser Gly Arg Lys Asn Ser Asn Met Ser 370 375 380

Asp Glu Phe He Ser Leu Ser Pro Gly Thr Pro Pro Ser Thr Leu Ser 385 390 395 400

Ser Ser Ser Tyr Arg Gin Val Met Ser Ser Pro Ser Ala Met Lys Leu 405 410 415

Leu Pro Asn Met Ala Val Lys Arg Asn His Arg Gly Glu Thr Leu Leu 420 425 430

His He Ala Ser He Lys Gly Asp He Pro Ser Val Glu Tyr Leu Leu 435 440 445

Gin Asn Gly Ser Asp Pro Asn Val Lys Asp His Ala Gly Trp Thr Pro 450 455 460

Leu H s Glu Ala Cys Asn His Gly His Leu Lys Val Val Glu Leu Leu 465 470 475 480

Leu Gin His Lys Ala Leu Val Asn Thr Thr Gly Tyr Gin Asn Asp Ser 485 490 495

Arg Pro Val Asp Tyr Thr Asp Asp Glu Ser Met Lys Ser Leu Leu Leu 530 535 540

Leu Pro Glu Lys Asn Glu Ser Ser Ser Ala Ser His Cys Ser Val Met 545 550 555 560

Asn Thr Gly Gin Arg Arg Asp Gly Pro Leu Val Leu He Gly Ser Gly 565 570 575

Leu Ser Ser Glu Gin Gin Lys Met Leu Ser Glu Leu Ala Val He Leu 580 585 590

Lys Ala Lys Lys Tyr Thr Glu Phe Asp Ser Thr Val Thr His Val Val 595 600 605

Val Pro Gly Asp Ala Val Gin Ser Thr Leu Lys Cys Met Leu Gly He 610 615 620

Leu Asn Gly Cys Trp He Leu Lys Phe Glu Trp Val Lys Ala Cys Leu 625 630 635 640

Arg Arg Lys Val Cys Glu Gin Glu Glu Lys Tyr Glu He Pro Glu Gly 645 650 655

Pro Arg Arg Ser Arg Leu Asn Arg Glu Gin Leu Leu Pro Lys Leu Phe 660 665 670

Asp Gly Cys Tyr Phe Tyr Leu Trp Gly Thr Phe Lys His His Pro Lys 675 680 685

Asp Asn Leu He Lys Leu Val Thr Ala Gly Gly Gly Gin He Leu Ser 690 695 700

Arg Lys Pro Lys Pro Asp Ser Asp Val Thr Gin Thr He Asn Thr Val 705 - 710 715 720

Ala Tyr His Ala Arg Pro Asp Ser Asp Gin Arg Phe Cys Thr Gin Tyr 725 730 735

He He Tyr Glu Asp Leu Cys Asn Tyr His Pro Glu Arg Val Arg Gin 740 745 750

Gly Lys Val Trp Lys Ala Pro Ser Asn Trp Phe He Asp Cys Val Met 755 760 765

Ser Phe Glu Leu Leu Pro Leu Asp Ser 770 775

(2) INFORMATION FOR SEQ ID NO: 40:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1083 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 37..819 ( ix ) FEATURE :

(A) NAME/KEY: modifιed_base

(B) LOCATION: 346..378

(D) OTHER INFORMATION: /note= "R = A or G"

(ix) FEATURE:

(A) NAME/KEY: modifιed_base

(B) LOCATION: 690

(D) OTHER INFORMATION: /note= "W = A or T"

(ix) FEATURE:

(A) NAME/KEY: modifιed_base

(B) LOCATION: 104

(D) OTHER INFORMATION :/note= "Xaa =- Ala or Ser or Pro or Thr for both SEQ ID NO: 40 and 41"

(ix) FEATURE:

(A) NAME/KEY: modifιed_base

(B) LOCATION: 114

(D) OTHER INFORMATION: /note= "Xaa = Gly for both SEQ ID NO: 40 and SEQ ID NO: 1"

(ix) FEATURE:

(A) NAME/KEY: modifιed_base

(B) LOCATION: 218

(D) OTHER INFORMATION: /note= "Xaa = Ala for both SEQ ID NO: 40 and SEQ ID NO: 41"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:

CAGTTGCAGG CAGACGGAGC AGAGCGGTCA GGGATC ATG AGG GAG AGT GCG TTG 54

Met Arg Glu Ser Ala Leu

1 5

GAG CCG GGG CCT GTG CCC GAG GCG CCG GCG GGG GGT CCC GTG CAC GCC 102 Glu Pro Gly Pro Val Pro Glu Ala Pro Ala Gly Gly Pro Val His Ala 10 15 20

GTG ACG GTG GTG ACC CTG CTG GAG AAG CTG GCC TCC ATG CTG GAG ACT 150 Val Thr Val Val Thr Leu Leu Glu Lys Leu Ala Ser Met Leu Glu Thr 25 30 35

CTG CGG GAG CGG CAG GGA GGC CTG GCT CGA AGG CAG GGA GGC CTG GCA 198 Leu Arg Glu Arg Gin Gly Gly Leu Ala Arg Arg Gin Gly Gly Leu Ala 40 45 50

GGG TCC GTG CGC CGC ATC CAG AGC GGC CTG GGC GCT CTG AGT CGC AGC 246 Gly Ser Val Arg Arg He Gin Ser Gly Leu Gly Ala Leu Ser Arg Ser 55 60 65 70

CAC GAC ACC ACC AGC AAC ACC TTG GCG CAG CTG CTG GCC AAG GCG GAG 2 -.4 His Asp Thr Thr Ser Asn Thr Leu Ala Gin Leu Leu Ala Lys Ala Glu 75 80 85

CGC GTG AGC TCG CAC GCC AAC GCC GCC CAA GAG CGC GCG GTG CGC CGC 342 Arg Val Ser Ser His Ala Asn Ala Ala Gin Glu Arg Ala Val Arg Arg 90 95 100

GCA RCC CAG GTG CAG CGG CTG GAG GCC AAC CAC GGR CTG CTG GTG GCG 3S0 Ala Xaa Gin Val Gin Arg Leu Glu Ala Asn H s Xaa Leu Leu Val Ala 105 110 115

CGC GGG AAG CTC CAC GTT CTG CTC TTC AAG GAG GAG GGT GAA GTC CCA 438 Arg Gly Lys Leu His Val Leu Leu Phe Lys Glu Glu Gly Glu Val Pro 120 125 130 GCC AGC GCT TTC CAG AAG GCA CCA GAG CCC TTG GGC CCG GCG GAC CAG 486 Ala Ser Ala Phe Gin Lys Ala Pro Glu Pro Leu Gly Pro Ala Asp Gin 135 140 145 150

TCC GAG CTG GGC CCA GAG CAG CTG GAG GCC GAA GTT GGA GAG AGC TCG 534 Ser Glu Leu Gly Pro Glu Gin Leu Glu Ala Glu Val Gly Glu Ser Ser 155 160 165

GAC GAG GAG CCG GTG GAG TCC AGG GCC CAG CGG CTG CGG CGC ACC GGA 582 Asp Glu Glu Pro Val Glu Ser Arg Ala Gin Arg Leu Arg Arg Thr Gly 170 175 180

TTG CAG AAG GTA CAG AGC CTC CGA AGG GCC CTT TCG GGC CGG AAA GGC 630 Leu Gin Lys Val Gin Ser Leu Arg Arg Ala Leu Ser Gly Arg Lys Gly 185 190 195

CCT GCA GCG CCA CCG CCC ACC CCG GTC AAG CCG CCT CGC CTT GGG CCT 678 Pro Ala Ala Pro Pro Pro Thr Pro Val Lys Pro Pro Arg Leu Gly Pro 200 205 ^' 210

GGC CGG AGC GCW GAA GCC CAG CCG GAA GCC CAG CCT GCG CTG GAG CCC 726

Gly Arg Ser Xaa Glu Ala Gin Pro Glu Ala Gin Pro Ala Leu Glu Pro 215 220 225 230

ACG CTG GAG CCA GAG CCT CCG CAG GAC ACC GAG GAA GAT CCC GGG AGA 774 Thr Leu Glu Pro Glu Pro Pro Gin Asp Thr Glu Glu Asp Pro Gly Arg 235 240 245

CCT GGG GCT GCC GAA GAA GCT CTG CTC CAA ATG GAG AGT GTA GCC 819

Pro Gly Ala Ala Glu Glu Ala Leu Leu Gin Met Glu Ser Val Ala 250 255 260

TGAGGGCTGG TGTTGCCTGC CTCCCCTGTG CTTGTGCCTT GTCCCAAAAT AAATCCTTTC 879

AGAATGTAGC ACTCACGCCC TAATAAGGAG CGAATCCTAC ATCCACCAAG GCGGGCGCTC 939

TGGCCCTCCC TTCCTTAAGC CCAGTCCTGT GTCCTCTGAA AGAGGTGCAG TCACTCACAC 999

CTGCTTGCGC TCACCATCAA TAAAAGTAAT TTCACCCGAA AAAAAAAAAA AAAAAAAAAA 1059

AAAAAAAAAA AAAAAAAAAA AAAA 1083

(2) INFORMATION FOR SEQ ID NO: 41:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 261 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:

Met Arg Glu Ser Ala Leu Glu Pro Gly Pro Val Pro Glu Ala Pro Ala 1 5 10 15

Gly Gly Pro Val His Ala Val Thr Val Val Thr Leu Leu Glu Lys Leu 20 25 30

Ala Ser Met Leu Glu Thr Leu Arg Glu Arg Gin Gly Gly Leu Ala Arg 35 40 45

Arg Gin Gly Gly Leu Ala Gly Ser Val Arg Arg He Gin Ser Gly Leu 50 55 60 Gly Ala Leu Ser Arg Ser His Asp Thr Thr Ser Asn Thr Leu Ala Gin 65 70 75 80

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

Glu Arg Ala Val Arg Arg Ala Xaa Gin Val Gin Arg Leu Glu Ala Asn 100 105 110

His Xaa Leu Leu Val Ala Arg Gly Lys Leu His Val Leu Leu Phe Lys 115 120 125

Glu Glu Gly Glu Val Pro Ala Ser Ala Phe Gin Lys Ala Pro Glu Pro 130 135 140

Leu Gly Pro Ala Asp Gin Ser Glu Leu Gly Pro Glu Gin Leu Glu Ala 145 150 155 160

Glu Val Gly Glu Ser Ser Asp Glu Glu Pro Val Glu Ser Arg Ala Gin 165 170 175

Arg Leu Arg Arg Thr Gly Leu Gin Lys Val Gin Ser Leu Arg Arg Ala 180 185 190

Leu Ser Gly Arg Lys Gly Pro Ala Ala Pro Pro Pro Thr Pro Val Lys 195 200 205

Pro Pro Arg Leu Gly Pro Gly Arg Ser Xaa Glu Ala Gin Pro Glu Ala 210 215 220

Gin Pro Ala Leu Glu Pro Thr Leu Glu Pro Glu Pro Pro Gin Asp Thr 225 230 235 240

Glu Glu Asp Pro Gly Arg Pro Gly Ala Ala Glu Glu Ala Leu Leu Gin 245 250 255

Met Glu Ser Val Ala 260

(2) INFORMATION FOR SEQ ID NO: 42:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1326 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..666

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:

GGA ATT CCT GCT GTA CCA TGC CAT GCT CCC TCT CAT TCT GAA TCT CAG 48 Gly He Pro Ala Val Pro Cys His Ala Pro Ser His Ser Glu Ser Gin 1 5 10 15

GCA ACT CCT CAT TCT AGT TAT GGC TTA TGT ACC TCC ACC CCA GTC TGG 96 Ala Thr Pro His Ser Ser Tyr Gly Leu Cys Thr Ser Thr Pro Val Trp 20 25 30

TCA CTT CAG CGG CCA CCC TGC CCT CCA AAG GTT CAT TCT GAA GTT CAA 144 Ser Leu Gin Arg Pro Pro Cys Pro Pro Lys Val His Ser Glu Val Gin 35 40 45 ACT GAT GGC AAC AGT CAG TTT GCA TCA CAA GGT AAA ACA GTT TCT GCA 192 Thr Asp Gly Asn Ser Gin Phe Ala Ser Gin Gly Lys Thr Val Ser Ala 50 55 60

ACC TGT ACT GAT GTT CTA CGG AAT TCA TTT AAT ACC AGT CCT GGA GTT 240 Thr Cys Thr Asp Val Leu Arg Asn Ser Phe Asn Thr Ser Pro Gly Val 65 70 75 80

CCA TGT AGC CTG CCC AAA ACT GAC ATA TCA GCT ATT CCA ACA TTG CAG 288 Pro Cys Ser Leu Pro Lys Thr Asp He Ser Ala He Pro Thr Leu Gin 85 90 95

CAA CTG GGC CTT GTT AAT GGA ATT CTG CCA CAA CAA GGA ATT CAT AAG 336 Gin Leu Gly Leu Val Asn Gly He Leu Pro Gin Gin Gly He His Lys 100 105 110

GAA ACA GAC CTA CTA AAA TGT ATT CAA ACA TAT TTG TCT CTT TTT CGA 384 Glu Thr Asp Leu Leu Lys Cys He Gin Thr Tyr Leu Ser Leu Phe Arg 115 120 125

TCT CAT GGA AAA GAA ACG CAT CTG GAC AGT CAG ACA CAC CGA AGC CCT 432 Ser His Gly Lys Glu Thr His Leu Asp Ser Gin Thr His Arg Ser Pro 130 135 140

ACT CAG TCA CAA CCA GCT TTC TTG GCC ACT AAT GAA GAA AAA TGT GCC 480 Thr Gin Ser Gin Pro Ala Phe Leu Ala Thr Asn Glu Glu Lys Cys Ala 145 150 155 160

AGA GAG CAA ATT AGA GAG GCC ACA AGT GAA AGA AAG GAT TTA AAC ATA 528 Arg Glu Gin He Arg Glu Ala Thr Ser Glu Arg Lys Asp Leu Asn He 165 170 175

CAT GTG CGA GAT ACA AAA ACA GTG AAG GAT GTA CAG AAG GCA AAA AAT 576 His Val Arg Asp Thr Lys Thr Val Lys Asp Val Gin Lys Ala Lys Asn 180 185 190

GTG AAC AAG ACA GCT GAA AAA GTT AGA ATT ATA AAA TAT TTG TTG GGA 624 Val Asn Lys Thr Ala Glu Lys Val Arg He He Lys Tyr Leu Leu Gly 195 200 205

GAG CTC AAG GCC CTG GTA GCA GAA CAA GGT AGA TGG GAC TTA 666

Glu Leu Lys Ala Leu Val Ala Glu Gin Gly Arg Trp Asp Leu 210 215 220

TAACTTTCTG TAGTATGGTG TTATACTAAA TAGCAATGTC ATGTTATTTA GCTATCATTT 726

AAATGGAGTT TGTGGTATTT TCCATAGAAC TGTGTTTTGA GCTAATAAGA AAATGAGTTC 786

TACTTATTGT ATTATTTTTT AAGTTTTGAT CCCTTCTTTC CTGTGGATTT AAAATGCGTT 846

TGAGAATATC AAACATTCAG TCTTTTGCTT GCAAGTGTGT ATTTATTCTG CTTGATAATA 906

GACCTTGAAA AGAGTCAACC AAAGAGAATT TGGACAGATA AAAATTTTAA TTAGAGAATG 966

CCTATAAATG ATTAACTCCC TGAGTAGACT GATTATTCTT CCTGTTTTAA AAAGATGCAG 1026

AGAATTCTTT CCTGTCACTT CTTTAATAGC CAACTGTTAG ATTGTTTAAC AAATCTCACT 1086

TTGAGAAGTA ACGCATACCT TCTTATGCCC TTTTCAGTGT ATTTTTAGGA CTTTTTTTCT 1146

TAAATCAAGG TGTTTCTGAG CCAGATTCTA TTCATTTGTT TCCATTCTGT ATATGTATTC 1206

TATAGTAATG GCTTTTGCTT GAAATGAGTT ACAGTTTTGT CATCTTGGAA ACACAGTAAT 1266

TGATTTTGGA AGCATTGATT GAATACCTAA CGTTTGCAGA CCAAAAAAAA AAAAAAAAAA 1326 (2) INFORMATION FOR SEQ ID NO: 43:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 222 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:

Gly He Pro Ala Val Pro Cys His Ala Pro Ser His Ser Glu Ser Gin 1 5 10 15

Ala Thr Pro His Ser Ser Tyr Gly Leu Cys Thr Ser Thr Pro Val Trp 20 25 30

Ser Leu Gin Arg Pro Pro Cys Pro Pro Lys Val His Ser Glu Val Gin 35 40 45

Thr Asp Gly Asn Ser Gin Phe Ala Ser Gin Gly Lys Thr Val Ser Ala 50 55 60

Thr Cys Thr Asp Val Leu Arg Asn Ser Phe Asn Thr Ser Pro Gly Val 65 70 75 80

Pro Cys Ser Leu Pro Lys Thr Asp He Ser Ala He Pro Thr Leu Gin 85 90 95

Gin Leu Gly Leu Val Asn Gly He Leu Pro Gin Gin Gly He His Lys 100 105 110

Glu Thr Asp Leu Leu Lys Cys He Gin Thr Tyr Leu Ser Leu Phe Arg 115 120 125

Ser His Gly Lys Glu Thr His Leu Asp Ser Gin Thr His Arg Ser Pro 130 135 140

Thr Gin Ser Gin Pro Ala Phe Leu Ala Thr Asn Glu Glu Lys Cys Ala 145 150 155 160

Arg Glu Gin He Arg Glu Ala Thr Ser Glu Arg Lys Asp Leu Asn He 165 170 175

His Val Arg Asp Thr Lys Thr Val Lys Asp Val Gin Lys Ala Lvs Asn 180 185 190

Val Asn Lys Thr Ala Glu Lys Val Arg He He Lys Tyr Leu Leu Gly 195 200 205

Glu Leu Lys Ala Leu Val Ala Glu Gin Gly Arg Trp Asp Leu 210 215 220

(2) INFORMATION FOR SEQ ID NO: 44:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 834 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

( ix ) FEATURE :

(A) NAME/KEY : CDS

(B) LOCATION : ! . . 693 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:

GAA AAT GAA AAA ATA GTG GAA ACA TAC AGG GGA AAG GAA ACA GAA TAT 48 Glu Asn Glu Lys He Val Glu Thr Tyr Arg Gly Lys Glu Thr Glu Tyr 1 5 10 15

CAA GCG TTA CAA GAG ACT AAC ATG AAG TTT TCT ATG ATG CTG CGA GAA 96 Gin Ala Leu Gin Glu Thr Asn Met Lys Phe Ser Met Met Leu Arg Glu 20 25 30

AAA GAG TTT GAG TGC CAC TCA ATG AAG GAG AAG GCT CTT GCT TTT GAA 144 Lys Glu Phe Glu Cys His Ser Met Lys Glu Lys Ala Leu Ala Phe Glu 35 40 45

CAG CTA TTG AAA GAG AAA GAA CAG GGC AAG ACT GGA GAG TTA AAT CAG 192 Gin Leu Leu Lys Glu Lys Glu Gin Gly Lys Thr Gly Glu Leu Asn Gin 50 55 60

CTT TTA AAT GCA GTT AAA TCA ATG CAG GAG AAG ACA GTT GTG TTT CAA 240 Leu Leu Asn Ala Val Lys Ser Met Gin Glu Lys Thr Val Val Phe Gin 65 70 75 80

CAG GAG AGA GAC CAA GTC ATG TTG GCC CTG AAA CAA AAA CAA ATG GAA 288 Gin Glu Arg Asp Gin Val Met Leu Ala Leu Lys Gin Lys Gin Met Glu 85 90 95

AAT ACT GCC CTA CAG AAT GAG GTT CAA CGT TTA CGT GAC AAA GAA TTT 336 Asn Thr Ala Leu Gin Asn Glu Val Gin Arg Leu Arg Asp Lys Glu Phe 100 105 110

CGT TCA AAC CAA GAG CTA GAG AGA TTG CGT AAT CAT CTT TTA GAA TCA 384 Arg Ser Asn Gin Glu Leu Glu Arg Leu Arg Asn His Leu Leu Glu Ser 115 120 125

GAA GAT TCT TAT ACC CGT GAA GCT TTG GCT GCA GAA GAT AGA GAG GCT 432 Glu Asp Ser Tyr Thr Arg Glu Ala Leu Ala Ala Glu Asp Arg Glu Ala 130 135 140

AAA CTA AGA AAG AAA GTC ACA GTA TTG GAG GAA AAG CTA GTT TCA TCC 480 Lys Leu Arg Lys Lys Val Thr Val Leu Glu Glu Lys Leu Val Ser Ser 145 150 155 160

TCT AAT GCA ATG GAA AAT GCA AGC CAT CAA GCC AGT GTG CAG GTA GAG 528 Ser Asn Ala Met Glu Asn Ala Ser His Gin Ala Ser Val Gin Val Glu 165 170 175

TCA TTG CAA GAA CAG TTG AAT GTA GTT TCC AAG CAA AGG GAT GAA ACT 576 Ser Leu Gin Glu Gin Leu Asn Val Val Ser Lys Gin Arg Asp Glu Thr 180 185 190

GCG CTG CAG CTT TCT GTC TCT CAG GAA CAA GTA AAG CAG TAT GCT CTG 624 Ala Leu Gin Leu Ser Val Ser Gin Glu Gin Val Lys Gin Tyr Ala Leu 195 200 205

TCA CTG GCC AAC CTG CAG ATG GTA CTA GAG CAT TTC CAA CAA GAG GAA 672 Ser Leu Ala Asn Leu Gin Met Val Leu Glu His Phe Gin Gin Glu Glu 210 215 220

AAA GCT ATG TAT TCT GCT GAA CTCGAAAAGC AAAAAAAAAA AAAAAAAACT 723

Lys Ala Met Tyr Ser Ala Glu 225 230

CGAGAGATCT ATGAATCGTA GATACTGAAA AACCCCGCAA GTTCACTTCA ACTGTGCATC 783

GTGCACCATC TCAATTTCTT TCATTTATAC ATCGTTTTGC CTTCTTTTAT G 834 (2) INFORMATION FOR SEQ ID NO: 45:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 231 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:

Glu Asn Glu Lys He Val Glu Thr Tyr Arg Gly Lys Glu Thr Glu Tyr 1 5 10 15

Gin Ala Leu Gin Glu Thr Asn Met Lys Phe Ser Met Met Leu Arg Glu 20 25 30

Lys Glu Phe Glu Cys His Ser Met Lys Glu Lys Ala Leu Ala Phe Glu 35 40 45

Gin Leu Leu Lys Glu Lys Glu Gin Gly Lys Thr Gly Glu Leu Asn Gin 50 55 60

Leu Leu Asn Ala Val Lys Ser Met Gin Glu Lys Thr Val Val Phe Gin 65 70 75 80

Gin Glu Arg Asp Gin Val Met Leu Ala Leu Lys Gin Lys Gin Met Glu 85 90 95

Asn Thr Ala Leu Gin Asn Glu Val Gin Arg Leu Arg Asp Lys Glu Phe 100 105 110

Arg Ser Asn Gin Glu Leu Glu Arg Leu Arg Asn His Leu Leu Glu Ser 115 120 125

Glu Asp Ser Tyr Thr Arg Glu Ala Leu Ala Ala Glu Asp Arg Glu Ala 130 135 140

Lys Leu Arg Lys Lys Val Thr Val Leu Glu Glu Lys Leu Val Ser Ser 145 150 155 160

Ser Asn Ala Met Glu Asn Ala Ser His Gin Ala Ser Val Gin Val Glu 165 170 175

Ser Leu Gin Glu Gin Leu Asn Val Val Ser Lys Gin Arg Asp Glu Thr 180 185 190

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

Ser Leu Ala Asn Leu Gin Met Val Leu Glu His Phe Gin Gin Glu Glu 210 215 220

Lys Ala Met Tyr Ser Ala Glu 225 230

(2) INFORMATION FOR SEQ ID NO: 46:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 898 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ix ) FEATURE :

(A) NAME/KEY: CDS

(B) LOCATION:!..816

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:

CTC GTG CCG CGA GAC CCT GAG CCA GAG CAG GCT GGG CCC AGC TCT GGA 48 Leu Val Pro Arg Asp Pro Glu Pro Glu Gin Ala Gly Pro Ser Ser Gly 1 5 10 15

GTC ACG AAC AGG TGC CCG TTC CTC CTG GAC AAT TGC CTT GGC ACA TCT 96 Val Thr Asn Arg Cys Pro Phe Leu Leu Asp Asn Cys Leu Gly Thr Ser 20 25 30

CAG TGG CCC CCA AGG CGA CGA CGC AAG CAG CTG TTC ACC CTG CAG ACG 144 Gin Trp Pro Pro Arg Arg Arg Arg Lys Gin Leu Phe Thr Leu Gin Thr 35 40 45

GTG AAC TCC AAT GGG ACC AGC GAC CGC ACA ACC TCC CCT GAA GAA GTC 192 Val Asn Ser Asn Gly Thr Ser Asp Arg Thr Thr Ser Pro Glu Glu Val 50 55 60

CAT GCC CAG CCG TAC ATT GCT ATC GAC TGG GAG CCA GAG ATG AAG AAG 240 His Ala Gin Pro Tyr He Ala He Asp Trp Glu Pro Glu Met Lys Lys 65 70 75 80

CGT TAC TAT GAC GAG GTA GAG GCT GAG GGC TAC GTG AAG CAT GAC TGC 288 Arg Tyr Tyr Asp Glu Val Glu Ala Glu Gly Tyr Val Lys His Asp Cys 85 90 95

GTC GGG TAC GTG ATG AAG AAG GCT CCC GTG CGG CTG CAG GAG TGC ATT 336 Val Gly Tyr Val Met Lys Lys Ala Pro Val Arg Leu Gin Glu Cys He 100 105 110

GAG CTC TTC ACC ACT GTG GAG ACC CTG GAG AAG GAA AAC CCC TGG TAC 384 Glu Leu Phe Thr Thr Val Glu Thr Leu Glu Lys Glu Asn Pro Trp Tyr 115 120 125

TGC CCT TCC TGC AAG CAG CAC CAG CTG GCA ACC AAG AAG CTG GAC CTG 432 Cys Pro Ser Cys Lys Gin His Gin Leu Ala Thr Lys Lys Leu Asp Leu 130 135 140

TGG ATG CTG CCG GAG ATT CTC ATC ATC CAC CTG AAA CGC TTT TCC TAC 480 Trp Met Leu Pro Glu He Leu He He His Leu Lys Arg Phe Ser Tyr 145 150 155 160

ACC AAG TTC TCC CGA GAG AAG CTG GAC ACC CTC GTG GAG TTT CCT ATC 528 Thr Lys Phe Ser Arg Glu Lys Leu Asp Thr Leu Val Glu Phe Pro He 165 170 175

CGG GAC CTG GAC TTC TCT GAG TTT GTC ATC CAG CCA CAG AAT GAG TCG 576 Arg Asp Leu Asp Phe Ser Glu Phe Val He Gin Pro Gin Asn Glu Ser 180 185 190

AAT CCG GAG CTG TAC AAA TAT GAC CTC ATC GCG GTT TCC AAC CAT TAT 624 Asn Pro Glu Leu Tyr Lys Tyr Asp Leu He Ala Val Ser Asn His Tyr 195 200 205

GGG GGC ATG CGT GAT GGA CAC TAC ACA ACA TTT GCC TGC AAC AAG GAC 672 Gly Gly Met Arg Asp Gly His Tyr Thr Thr Phe Ala Cys Asn Lys Asp 210 215 220

AGC GGC CAG TGG CAC TAC TTT GAT GAC AAC AGC GTC TCC CCT GTC AAT 720 Ser Gly Gin Trp His Tyr Phe Asp Asp Asn Ser Val Ser Pro Val Asn 225 230 235 240 GAG AAT CAG ATC GAG TCC AAG GCA GCC TAT GTC CTC TTC TAC CAA CGC ^"'68 Glu Asn Gin He Glu Ser Lys Ala Ala Tyr Val Leu Phe Tyr Gin Arg 245 250 255

CAG GAC GTG GCG CGA CGC CTG CTG TCC CCG GCC GGC TCA TCT GGC GCC 816 Gin Asp Val Ala Arg Arg Leu Leu Ser Pro Ala Gly Ser Ser Gly Ala 260 ^" 265 270

CCAGCCTCCC CTGCCTGCAG CTCCCCACCC AGCTCTGAGT TCATGGATGT TAATTGAG G £76

CCCTGGGTCC TGCCACAGAA AA 698

(2) INFORMATION FOR SEQ ID NO: 47:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 272 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:

Leu Val Pro Arg Asp Pro Glu Pro Glu Gin Ala Gly Pro Ser Ser Gly 1 5 10 15

Val Thr Asn Arg Cys Pro Phe Leu Leu Asp Asn Cys Leu Gly Thr Ser 20 25 30

Gin Trp Pro Pro Arg Arg Arg Arg Lys Gin Leu Phe Thr Leu Gin Thr 35 40 45

Val Asn Ser Asn Gly Thr Ser Asp Arg Thr Thr Ser Pro Glu Glu Val 50 55 60

His Ala Gin Pro Tyr He Ala He Asp Trp Glu Pro Glu Met Lys Lys 65 70 75 80

Arg Tyr Tyr Asp Glu Val Glu Ala Glu Gly Tyr Val Lys His Asp Cys 85 90 95

Val Gly Tyr Val Met Lys Lys Ala Pro Val Arg Leu Gin Glu Cys He 100 105 110

Glu Leu Phe Thr Thr Val Glu Thr Leu Glu Lys Glu Asn Pro Trp Tyr 115 120 125

Cys Pro Ser Cys Lys Gin His Gin Leu Ala Thr Lys Lys Leu Asp Leu 130 135 140

Trp Met Leu Pro Glu He Leu He He His Leu Lys Arg Phe Ser Tyr 145 150 155 160

Thr Lys Phe Ser Arg Glu Lys Leu Asp Thr Leu Val Glu Phe Pro He 165 170 175

Arg Asp Leu Asp Phe Ser Glu Phe Val He Gin Pro Gin Asn Glu Ser 180 185 190

Asn Pro Glu Leu Tyr Lys Tyr Asp Leu He Ala Val Ser Asn His Tyr 195 200 205

Gly Gly Met Arg Asp Gly His Tyr Thr Thr Phe Ala Cys Asn Lys Asp 210 215 220 Ser Gly Gin Trp His Tyr Phe Asp Asp Asn Ser Val Ser Pro Val Asn 225 230 235 240

Glu Asn Gin He Glu Ser Lys Ala Ala Tyr Val Leu Phe Tyr Gin Arg 245 250 255

Gin Asp Val Ala Arg Arg Leu Leu Ser Pro Ala Gly Ser Ser Gly Ala 260 265 270

(2) INFORMATION FOR SEQ ID NO: 48:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 312 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:

Pro Ser Trp Pro Glu Ser Lys Val Thr Glu Phe Leu His Gin Ser Lys 1 5 10 15

Leu Lys Ser Phe Glu Ser Glu Arg Val Gin Leu Leu Gin Glu Glu Thr 20 25 30

Ala Arg Asn Leu Thr Gin Cys Gin Leu Glu Cys Glu Lys Tyr Gin Lys 35 40 45

Lys Leu Glu Val Leu Thr Lys Glu Phe Tyr Ser Leu Gin Ala Ser Ser 50 55 60

Glu Lys Arg He Thr Glu Leu Gin Ala Gin Asn Ser Glu His Gin Ala 65 70 75 80

Arg Leu Asp He Tyr Glu Lys Leu Glu Lys Glu Leu Asp Glu He He 85 90 95

Met Gin Thr Ala Glu He Glu Asn Glu Asp Glu Ala Glu Arg Val Leu 100 105 110

Phe Ser Tyr Gly Tyr Gly Ala Asn Val Pro Thr Thr Ala Lys Arg Arg 115 120 125

Leu Lys Gin Ser Val His Leu Ala Arg Arg Val Leu Gin Leu Glu Lys 130 135 140

Gin Asn Ser Leu He Leu Lys Asp Leu Glu His Arg Lys Asp Gin Val 145 150 155 160

Thr Gin Leu Ser Gin Glu Leu Asp Arg Ala Asn Ser Leu Leu Asn Gin 165 170 175

Thr Gin Gin Pro Tyr Arg Tyr Leu He Glu Ser Val Arg Gin Arg Asp 180 185 190

Ser Lys He Asp Ser Leu Thr Glu Ser He Ala Gin Leu Glu Lys Asp 195 200 205

Val Ser Asn Leu Asn Lys Glu Lys Ser Ala Leu Leu Gin Thr Lys Asn 210 215 220

Gin Met Ala Leu Asp Leu Glu Gin Leu Leu Asn His Arg Glu Glu Leu 225 230 235 240 Ala Ala Met Lys Gin He Leu Val Lys Met His Ser Lys His Ser Glu 245 250 255

Asn Ser Leu Leu Leu Thr Lys Thr Glu Pro Lys His Val Thr Glu Asn 260 265 270

Gin Lys Ser Lys Thr Leu Asn Val Pro Lys Glu His Glu Asp Asn He 275 280 285

Phe Thr Pro Lys Pro Thr Leu Phe Thr Lys Lys Glu Ala Pro Glu Trp 290 295 300

Ser Lys Lys Gin Lys Met Lys Thr 305 310

(2) INFORMATION FOR SEQ ID NO: 49:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 587 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49:

Lys Arg Glu Phe He Gin Glu Pro Ala Lys Asn Aig Pro Gly Pro Gin 1 5 10 15

Thr Arg Ser Asp Leu Leu Leu Ser Gly Arg Asp Trp Asn Thr Leu He 20 25 30

Val Gly Lys Leu Ser Pro Trp He Arg Pro Asp Ser Lys Val Glu Lys 35 40 45

He Arg Arg Asn Ser Glu Ala Ala Met Leu Gin Glu Leu As i Phe Gly 50 55 60

Ala Tyr Leu Gly Leu Pro Ala Phe Leu Leu Pro Leu Asn Gin Glu Asp 65 70 75 80

Asn Thr Asn Leu Ala Arg Val Leu Thr Asn His He His Thr Gly His 85 90 95

His Ser Ser Met Phe Trp Met Arg Val Pro Leu Val Ala Pro Glu Asp 100 105 110

Leu Arg Asp Asp He He Glu Asn Ala Pro Thr Thr His Thr Glu Glu 115 120 125

Tyr Ser Gly Glu Glu Lys Thr Trp Met Trp Trp His Asn Phe Arg Thr 130 135 140

Leu Cys Asp Tyr Ser Lys Arg He Ala Val Ala Leu Glu He Gly Ala 145 150 155 160

Asp Leu Pro Ser Asn His Val He Asp Arg Trp Leu Gly Glu Pro He 165 170 175

Lys Ala Ala He Leu Pro Thr Ser He Phe Leu Thr Asn Lys Lys Gly 180 185 190

Phe Pro Val Leu Ser Lys Met His Gin Arg Leu He Phe Arg Leu Leu 195 200 205 Lys Leu Glu Val Gin Phe He He Thr Gly Thr Asn His His Ser Glu 210 215 220

Lys Glu Phe Cys Ser Tyr Leu Gin Tyr Leu Glu Tyr Leu Ser Gin Asn 225 230 235 240

Arg Pro Pro Pro Asn Ala Tyi Glu Leu Phe Ala Lys Gly Tyr Glu Asp 245 250 255

Tyr Leu Gin Ser Pro Leu Gin Pro Leu Met Asp Asn Leu Glu Ser Gin 260 265 270

Thr Tyr Glu Val Phe Glu Lys Asp Pro He Lys Tyr Ser Gin Tyr Gin 275 280 285

Gin Ala He Tyr Lys Cys Leu Leu Asp Arg Val Pro Glu Glu Glu Lys 290 295 300

Asp Thr Asn Val Gin Val Leu Met Val Leu Gly Ala Gly Arg Gly Pro 305 310 315 320

Leu Val Asn Ala Ser Leu Arg Ala Ala Lys Gin Ala Asp Arg Arg He 325 330 335

Lys Leu Tyr Ala Val Glu Lys Asn Pro Asn Ala Val Val Thr Leu Glu 340 345 350

Asn Trp Gin Phe Glu Glu Trp Gly Ser Gin Val Thr Val Val Ser Ser 355 360 365

Asp Met Arg Glu Trp Val Ala Pro Glu Lys Ala Asp He He Val Ser 370 375 380

Glu Leu Leu Gly Ser Phe Ala Asp Asn Glu Leu Ser Pro Glu Cys Leu 385 390 395 400

Asp Gly Ala Gin His Phe Leu Lys Asp Asp Gly Val Ser He Pro Gly 405 410 415

Glu Tyr Thr Ser Phe Leu Ala Pro He Ser Ser Ser Lys Leu Tyr Asn 420 425 430

Glu Val Arg Ala Cys Arg Glu Lys Asp Arg Asp Pro Glu Ala Gin Phe 435 440 445

Glu Met Pro Tyr Val Val Arg Leu His Asn Phe His Gin Leu Ser Ala 450 455 460

Pro Gin Pro Cys Phe Thr Phe Ser His Pro Asn Arg Asp Pro Met He 465 470 475 ^" 480

Asp Asn Asn Arg Tyr Cys Thr Leu Glu Phe Pro Val Glu Val Asn Thr 485 490 495

Val Leu His Gly Phe Ala Gly Tyr Phe Glu Thr Val Leu Tyr Gin Asp 500 505 510

He Thr Leu Ser He Arg Pro Glu Thr His Ser Pro Gly Met Phe Ser 515 520 525

Trp Phe Pro He Leu Phe Pro He Lys Gin Pro He Thr Val Arg Glu 530 535 540

Gly Gin Thr He Cys Val Arg Phe Trp Arg Cys Ser Asn Ser Lys Lys 545 550 555 560 Val Trp Tyr Glu Trp Ala Val Thr Ala Pro Val Cys Ser Ala He His 565 570 575

Asn Pro Thr Gly Arg Ser Tyr Thr He Gly Leu 580 585

(2) INFORMATION FOR SEQ ID NO: 50:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 370 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 110..111

(D) OTHER INFORMATION: /note= "Xaa =- Glu or Lys"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50:

Glu Pro Gly Arg Gly Leu Leu Val Ser Val Met Ala His Glu Ala Met 1 5 10 15

Glu Tyr Asp Val Gin Val Gin Leu Asn His Ala Glu Gin Gin Pro Ala 20 25 30

Pro Ala Gly Met Ala Ser Ser Gin Gly Gly Pro Ala Leu Leu Gin Pro 35 40 45

Val Pro Ala Asp Val Val Ser Ser Gin Gly Val Pro Ser He Leu Gin 50 55 60

Pro Ala Pro Ala Glu Val He Ser Ser Gin Ala Thr Pro Pro Leu Leu 65 70 75 80

Gin Pro Ala Pro Gin Leu Ser Val Asp Leu Thr Glu Val Glu Val Leu 85 90 95

Gly Glu Asp Thr Val Glu Asn He Asn Pro Arg Thr Ser Xaa Gin His 100 105 110

Arg Gin Gly Ser Asp Gly Asn His Thr He Pro Ala Ser Ser Leu His 115 120 125

Ser Met Thr Asn Phe He Ser Gly Leu Gin Arg Leu His Gly Met Leu 130 135 140

Glu Phe Leu Arg Pro Ser Ser Ser Asn His Ser Val Gly Pro Met Arg 145 150 155 160

Thr Arg Arg Arg Val Ser Ala Ser Arg Arg Ala Arg Ala Gly Gly Ser 165 170 175

Gin Arg Thr Asp Ser Ala Arg Leu Arg Ala Pro Leu Asp Ala Tyr Phe 180 185 190

Gin Val Ser Arg Thr Gin Pro Asp Leu Pro Ala Thr Thr Tyr Asp Ser 195 200 205

Glu Thr Arg Asn Pro Val Ser Glu Glu Leu Gin Val Ser Ser Ser Ser 210 215 220 Asp Ser Asp Ser Asp Ser Ser Ala Glu Tyr Gly Gly Val Val Asp Gin 225 230 235 240

Ala Glu Glu Ser Gly Ala Val He Leu Glu Glu Gin Leu Ala Gly Val 245 250 255

Ser Ala Glu Gin Glu Val Thr Cys He Asp Gly Gly Lys Thr Leu Pro 260 265 270

Lys Gin Pro Ser Pro Gin Lys Ser Glu Pro Leu Leu Pro Ser Ala Ser 275 280 285

Met Asp Glu Glu Glu Gly Asp Thr Cys Thr He Cys Leu Glu Gin Trp 290 295 300

Thr Asn Ala Gly Asp His Arg Leu Ser Ala Leu Arg Cys Gly His Leu 305 310 315 320

Phe Gly Tyr Arg Cys He Ser Thr Trp Leu Lys Gly Gin Val Arg Lys 325 330 335

Cys Pro Gin Cys Asn Lys Lys Ala Arg His Ser Asp He Val Val Leu 340 345 350

Tyr Ala Arg Thr Leu Arg Ala Leu Asp Thr Ser Glu Gin Glu Arg Met 355 360 365

Lys Arg 370

(2) INFORMATION FOR SEQ ID NO: 51:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 416 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51:

Asp Tyr His Gin Asn Trp Gly Arg Asp Gly Gly Pro Arg Ser Ser Gly 1 5 10 15

Gly Gly Tyr Gly Gly Gly Pro Ala Gly Gly His Gly Gly Asn Arg Gly 20 25 30

Ser Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Arg Gly Gly Arg Gly 35 40 45

Arg His Pro Gly His Leu Lys Gly Arg Glu He Gly Met Trp Tyr Ala 50 55 60

Lys Lys Gin Gly Gin Lys Asn Lys Glu Ala Glu Arg Gin Glu Arg Ala 65 70 75 80

Val Val His Met Asp Glu Arg Arg Glu Glu Gin He Val Gin Leu Leu 85 90 95

Asn Ser Val Gin Ala Lys Asn Asp Lys Glu Ser Glu Ala Gin He Ser 100 105 110

Trp Phe Ala Pro Glu Asp His Gly Tyr Gly Thr Glu Val Ser Thr Lys 115 120 125 Asn Thr Pro Cys Ser Glu Asn Lys Leu Asp He Gin Glu Lys Lys Leu 130 135 140

He Asn Gin Glu Lys Lys Mot Phe Arg He Arg Asn Arg Ser Tyr He 145 150 155 160

Asp Arg Asp Ser Glu Tyr Leu Leu Gin Glu Asn Glu Pro Asp Gly Thr 165 170 175

Leu Asp Gin Lys Leu Leu Glu Asp Leu Gin Lys Lys Lys Asn Asp Leu 180 185 190

Arg Tyr He Glu Met Gin His Phe Arg Glu Lys Leu Pro Ser Tyr Gly 195 200 205

Met Gin Lys Glu Leu Val Asn Leu He Asp Asn His Gin Val Thr Val 210 215 220

He Ser Gly Glu Thr Gly Cys Gly Lys Thr Thr Gin Val Thr Gin Phe 225 230 235 240

He Leu Asp Asn Tyr He Glu Arg Gly Lys Gly Ser Ala Cys Arg He 245 250 255

Val Cys Thr Gin Pro Arg Arg He Ser Ala He Ser Val Ala Glu Arg 260 265 270

Val Ala Ala Glu Arg Ala Glu Ser Cys Gly Ser Gly Asn Ser Thr Gly 275 280 285

Tyr Gin He Arg Leu Gin Ser Arg Leu Pro Arg Lys Gin Gly Ser He 290 295 300

Leu Tyr Cys Thr Thr Gly He He Leu Gin Trp Leu Gin Ser Asp Pro 305 310 315 320

Tyr Leu Ser Ser Val Ser His He Val Leu Asp Glu He His Glu Arg 325 330 335

Asn Leu Gin Ser Asp Val Leu Met Thr Val Val Lys Asp Leu Leu Asn 340 345 350

Phe Arg Ser Asp Leu Lys Val He Leu Met Ser Ala Thr Leu Asn Ala 355 360 365

Glu Lys Phe Ser Glu Tyr Phe Gly Asn Cys Pro Met He His He Pro 370 375 380

Gly Phe Thr Phe Pro Val Val Glu Tyr Leu Leu Glu Asp Val He Glu 385 390 395 400

Lys He Arg Tyr Val Pro Glu Gin Lys Glu His Arg Ser Gin Phe Lys 405 410 415

(2) INFORMATION FOR SEQ ID NO: 52:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 515 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52:

Asn He Ser Trp Lys Lys Thr He Val Thr Arg Phe Leu Lys Leu Val 1 5 10 15

Pro Asp Leu Leu Ala He Val Gin Arg Lys Lys Lys Glu Gly Glu Glu 20 25 30

Glu Gin Ala He Asn Arg Gin Thr Ala Leu Tyr Thr Leu Lys Leu Leu 35 40 45

Cys Lys Asn Phe Gly Ala Glu Asn Pro Asp Pro Phe Val Pro Val Leu 50 55 60

Ser Thr Ala Val Lys Leu He Ala Pro Glu Arg Lys Glu Glu Lys Asn 65 70 75 80

Val Leu Gly Ser Ala Leu Leu Cys Met Ala Glu Val Thr Ser Thr Leu 85 90 95

Glu Ala Leu Ala He Pro Gin Leu Pro Ser Leu Met Pro Ser Leu Leu 100 105 110

Thr Thr Met Lys Asn Thr Ser Glu Leu Val Ser Ser Glu Val Tyr Leu 115 120 125

Leu Ser Ala Leu Ala Ala Leu Gin Lys Val Val Glu Thr Leu Pro His 130 135 140

Phe He Ser Pro Tyr Leu Glu Gly He Leu Ser Gin Val He His Leu 145 150 155 160

Glu Lys He Thr Ser Glu Met Gly Ser Ala Ser Gin Ala Asn He Arg 165 170 175

Leu Thr Ser Leu Lys Lys Thr Leu Ala Thr Thr Leu Ala Pro Arg Val 180 185 190

Leu Leu Pro Ala He Lys Lys Thr Tyr Lys Gin He Glu Lys Asn Trp 195 200 205

Lys Asn His Met Gly Pro Phe Met Ser He Leu Gin Glu His He Gly 210 215 220

Ala Met Lys Lys Glu Glu Leu Thr Ser His Gin Ser Gin Leu Thr Ala 225 230 235 240

Phe Phe Leu Glu Ala Leu Asp Phe Arg Ala Gin His Ser Glu Asn Asp 245 250 255

Leu Glu Glu Val Gly Lys Thr Glu Asn Cys He He Asp Cys Leu Val 260 265 270

Ala Met Val Val Lys Leu Ser Glu Val Thr Phe Arg Pro Leu Phe Phe 275 280 285

Lys Leu Phe Asp Trp Ala Lys Thr Glu Asp Ala Pro Lys Asp Arg Leu 290 295 300

Leu Thr Phe Tyr Asn Leu Ala Asp Cys He Ala Glu Lys Leu Lys Gly 305 310 315 320

Leu Phe Thr Leu Phe Ala Gly His Leu Val Lys Pro Phe Ala Asp Thr 325 330 335 Leu Asp Gin Val Asn He Ser Lys Thr Asp Glu Ala Phe Phe Asp Ser 340 345 350

Glu Asn Asp Pro Glu Lys Cys Cys Leu Leu Leu Gin Phe He Leu Asn 355 360 365

Cys Leu Tyr Lys He Phe Leu Phe Asp Thr Gin His Phe He Ser Lys 370 375 380

Glu Arg Ala Gly Ala Leu Met Met Pro Leu Val Asp Gin Leu Glu Asn 385 390 395 400

Arg Leu Gly Gly Glu Glu Lys Phe Gin Glu Arg Val Thr Lys His Leu 405 410 415

He Pro Cys He Ala Gin Phe Ser Val Ala Met Ala Asp Asp Ser Leu 420 425 430

Trp Lys Pro Leu Asn Tyr Gin He Leu Leu Lys Thr Arg Asp Ser Ser 435 440 445

Pro Lys Val Arg Phe Ala Ala Leu He Thr Val Leu Ala Leu Ala Glu 450 455 460

Lys Leu Lys Glu Asn Tyr He Val Leu Leu Pro Glu Ser He Pro Phe 465 470 475 480

Leu Ala Glu Leu Met Glu Asp Glu Cys Glu Glu Val Glu His Gin Cys 485 490 495

Gin Lys Thr He Gin Gin Leu Glu Thr Val Leu Gly Glu Pro Leu Gin 500 505 510

Ser Tyr Phe 515

(2) INFORMATION FOR SEQ ID NO: 53:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 149 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53:

Gly Val Val Pro Asn Gly Arg Asp Ala Glu Ser Gly His Ser Leu Ala 1 5 10 15

Glu Gly Gin Ala Pro His Gly Leu Pro Gly Thr Pro Gly Ala Ser Gly 20 25 30

Gly Val Val Leu Gin Pro Arg Gly Arg Arg Arg Ala Asp Pro Pro His 35 40 45

Arg Gin Leu Arg Pro Glu Ala Phe Gly Asn His Arg Arg Ser Glu Phe 50 55 60

Leu Arg Leu Gin Val Glu Gly Gly Gly Cys Ser Gly Phe Gin Tyr Lys 65 70 75 80

Phe Ser Leu Asp Thr Val He Asn Pro Asp Asp Arg Val Phe Giu Gin 85 90 95 Gly Gly Ala Arg Val Val Val Asp Ser Asp Ser Leu Ala Phe Val Lys 100 105 110

Gly Ala Gin Val Asp Phe Ser Gin Glu Leu He Arg Ser Ser Phe Gin 115 120 125

Val Leu Asn Asn Pro Gin Ala Gin Gin Gly Cys Ser Cys Gly Ser Ser 130 135 140

Phe Ser He Lys Leu 145

(2) INFORMATION FOR SEQ ID NO: 54:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 535 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54:

Gly Pro Ala Gly Gly Ala Pro Thr Pro Ala Leu Val Ala Gly Ser Ser 1 5 10 15

Ala Ala Ala Pro Phe Pro His Gly Asp Ser Ala Leu Asn Glu Gin Glu 20 25 30

Lys Glu Leu Gin Arg Arg Leu Lys Arg Leu Tyr Pro Ala Val Asp Glu 35 40 45

Gin Glu Thr Pro Leu Pro Arg Ser Trp Ser Pro Lys Asp Lys Phe Ser 50 55 60

Tyr He Gly Leu Ser Gin Asn Asn Leu Arg Val His Tyr Lys Gly His 65 70 75 80

Gly Lys Thr Pro Lys Asp Ala Ala Ser Val Arg Ala Thr His Pro He 85 90 95

Pro Ala Ala Cys Gly He Tyr Tyr Phe Glu Val Lys He Val Ser Lys 100 105 110

Gly Arg Asp Gly Tyr Met Gly He Gly Leu Ser Ala Gin Gly Val Asn 115 120 125

Met Asn Arg Leu Pro Gly Trp Asp Lys His Ser Tyr Gly Tyr His Gly 130 135 140

Asp Asp Gly His Ser Phe Cys Ser Ser Gly Thr Gly Gin Pro Tyr Gly 145 150 155 160

Pro Thr Phe Thr Thr Gly Asp Val He Gly Cys Cys Val Asn Leu He 165 170 175

Asn Asn Thr Cys Phe Tyr Thr Lys Asn Gly His Ser Leu Gly He Ala 180 185 190

Phe Thr Asp Leu Pro Pro Asn Leu Tyr Pro Thr Val Gly Leu Gin Thr 195 200 205

Pro Gly Glu Val Val Asp Ala Asn Phe Gly Gin His Pro Phe Val Phe 210 215 220 Asp He Glu Asp Tyr Met Arg Glu Trp Arg Thr Lys He Gin Ala Gin 225 230 235 240

He Asp Arg Phe Pro He Gly Asp Arg Glu Gly Glu Trp Gin Thr Met 245 250 255

He Gin Lys Met Val Ser Ser Tyr Leu Val His His Gly Tyr Cys Ala 260 265 270

Thr Ala Glu Ala Phe Ala Arg Ser Thr Asp Gin Thr Val Leu Glu Glu 275 280 285

Leu Ala Ser He Lys Asn Arg Gin Arg He Gin Lys Leu Val Leu Ala 290 295 300

Gly Arg Met Gly Glu Ala He Glu Thr Thr Gin Gin Leu Tyr Pro Ser 305 310 315 320

Leu Leu Glu Arg Asn Pro Asn Leu Leu Phe Thr Leu Lys Val Arg Gin 325 330 335

Phe He Glu Met Val Asn Gly Thr Asp Ser Glu Val Arg Cys Leu Gly 340 345 350

Gly Arg Ser Pro Lys Ser Gin Asp Ser Tyr Pro Val Ser Pro Arg Pro 355 360 365

Phe Ser Ser Pro Ser Met Ser Pro Ser His Gly Met Asn He His Asn 370 375 380

Leu Ala Ser Gly Lys Gly Ser Thr Ala His Phe Ser Gly Phe Glu Ser 385 390 395 400

Cys Ser Asn Gly Val He Ser Asn Lys Ala His Gin Ser Tyr Cys His 405 410 415

Ser Asn Lys His Gin Ser Ser Asn Leu Asn Val Pro Glu Leu Asn Ser 420 425 430

He Asn Met Ser Arg Ser Gin Gin Val Asn Asn Phe Thr Ser Asn Asp 435 440 445

Val Asp Met Glu Thr Asp His Tyr Ser Asn Gly Val Gly Glu Thr Ser 450 455 460

Ser Asn Gly Phe Leu Asn Gly Ser Ser Lys His Asp His Glu Met Glu 465 470 475 480

Asp Cys Asp Thr Glu Met Glu Val Asp Ser Ser Gin Leu Arg Arg Gin 485 490 495

Leu Cys Gly Gly Ser Gin Ala Ala He Glu Arg Met He His Phe Gly 500 505 510

Arg Glu Leu Gin Ala Met Ser Glu Gin Leu Arg Arg Asp Cys Gly Lys 515 520 525

Asn Thr Ala Asn Lys Lys Cys 530 535 (2) INFORMATION FOR SEQ ID NO: 55:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 395 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:

Val Val Lys Pro Pro Gly Ser Ser Leu Asn Gly Val His Pro Asn Pro 1 5 10 15

Thr Pro He Val Gin Arg Leu Pro Ala Phe Leu Asp Asn His Asn Tyr 20 25 30

Ala Lys Ser Pro Met Gin Glu Glu Glu Asp Leu Ala Ala Gly Val Gly 35 40 45

Arg Ser Arg Val Pro Val Arg Pro Pro Gin Gin Tyr Ser Asp Asp Glu 50 55 60

Asp Asp Tyr Glu Asp Asp Glu Glu Asp Asp Val Gin Asn Thr Asn Ser 65 70 75 80

Ala Leu Arg Tyr Lys Gly Lys Gly Thr Gly Lys Pro Gly Ala Leu Ser 85 90 95

Gly Ser Ala Asp Gly Gin Leu Ser Val Leu Gin Pro Asn Thr He Asn 100 105 110

Val Leu Ala Glu Lys Leu Lys Glu Ser Gin Lys Asp Leu Ser He Pro 115 120 125

Leu Ser He Lys Thr Ser Ser Gly Ala Gly Ser Pro Ala Val Ala Val 130 135 140

Pro Thr His Ser Gin Pro Ser Pro Thr Pro Ser Asn Glu Ser Thr Asp 145 150 155 160

Thr Ala Ser Glu He Gly Ser Ala Phe Asn Ser Pro Leu Arg Ser Pro 165 170 175

He Arg Ser Ala Asn Pro Thr Arg Pro Ser Ser Pro Val Th_- Ser His 180 185 190

He Ser Lys Val Leu Phe Gly Glu Asp Asp Ser Leu Leu Arg Val Asp 195 200 205

Cys He Arg Tyr Asn Arg Ala Val Arg Asp Leu Gly Pro Val He Ser 210 215 220

Thr Gly Leu Leu His Leu Ala Glu Asp Glv Val Leu Ser Pro Leu Ala 225 230 ^" 235 240

Leu Thr Glu Gly Gly Lys Gly Ser Ser Pro Ser He Arg Pro He Gin 245 250 255

Gly Ser Gin Gly Ser Ser Ser Pro Val Glu Lys Glu Val Val Glu Ala 260 265 270

Thr Asp Ser Arg Glu Lys Thr Gly Met Val Arg Pro Gly Glu Pro Leu 275 280 285 Ser Gly Glu Lys Tyr Ser Pro Lys Glu Leu Leu Ala Leu Leu Lys Cys 290 295 300

Val Glu Ala Glu He Ala Asn Tyr Glu Ala Cys Leu Lys Glu Glu Val 305 310 315 320

Glu Lys Arg Lys Lys Phe Lys He Asp Asp Gin Arg Arg Thr His Asn 325 330 335

Tyr Asp Glu Phe He Cys Thr Phe He Ser Met Leu Ala Gin Glu Gly 340 345 350

Met Leu Ala Asn Leu Val Glu Gin Asn He Ser Val Arg Arg Arg Gin 355 360 365

Gly Ala Ser He Gly Arg Leu His Lys Gin Arg Lys Pro Asp Arg Arg 370 375 380

Lys Arg Ser Arg Pro Tyr Lys Ala Lys Arg Gin 385 390 395

(2) INFORMATION FOR SEQ ID MO: 56:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 278 ammo acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56:

Met Val Lys Val Lys Gly Gin Val Ser Glu Met Ala Val Leu Leu He 1 5 10 15

Asp Pro Glu Pro Gin He Ala Ala Leu Ala Lys Asn Phe Phe Asn Glu 20 25 30

Leu Ser His Lys Gly Asn Ala He Tyr Asn Leu Leu Pro Asp He He 35 40 45

Ser Arg Leu Ser Asp Pro Glu Leu Gly Val Glu Glu Glu Pro Phe His 50 55 60

Thr He Met Lys Gin Leu Leu Ser Tyr He Thr Lys Asp Lys Gin Thr 65 70 75 80

Glu Ser Leu Val Glu Lys Leu Cys Gin Arg Phe Arg Thr Ser Leu Thr 85 90 95

Glu Arg Gin Gin Arg Asp Leu Ala Tyr Cys Val Ser Gin Leu Pro Leu 100 105 110

Thr Glu Arg Gly Leu Arg Lys Met Leu Asp Asn Phe Asp Cys Phe Gly

115 120 ^" 125

Asp Lys Leu Ser Asp Glu Ser He Phe Ser Ala Phe Leu Ser Val Val 130 135 140

Gly Lys Leu Arg Arg Gly Ala Lys Pro Glu Gly Lys Ala He He Asp 145 150 155 160

Glu Phe Glu Gin Lys Leu Arg Ala Cys His Thr Arg Gly Leu Asp Gly 165 170 175 He Lys Glu Leu Glu He Gly Gin Ala Gly Ser Gin Arg Ala Pro Ser 180 185 190

Ala Lys Lys Pro Ser Thr Gly Ser Arg Tyr Gin Pro Leu Ala Ser Thr 195 200 205

Ala Ser Asp Asn Asp Phe Val Thr Pro Glu Pro Arg Arg Thr Thr Arg 210 215 220

Arg His Pro Asn Thr Gin Gin Arg Ala Ser Lys Lys Lys Pro Lys Val 225 230 235 240

Val Phe Ser Ser Asp Glu Ser Ser Glu Glu Asp Leu Ser Ala Glu Met 245 250 255

Thr Glu Asp Glu Thr Pro Lys Lys Thr Thr Pro He Leu Arg Ala Ser 260 265 270

Ala Arg Arg His Arg Ser 275

(2) INFORMATION FOR SEQ ID NO: 57:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57: GCGAGGAGCC TTTCATCCGA 20

(2) INFORMATION FOR SEQ ID NO: 58:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58: CGAGCGCGGC GCGACTGT 18

(2) INFORMATION FOR SEQ ID NO: 59:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59: ATGGAACCGG ATGGTCGCGG T 21 (2) INFORMATION FOR SEQ ID NO: 60:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60: TCTTCAAGTC TTGTATCCAG GC 22

(2) INFORMATION FOR SEQ ID NO: 61:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61: CGCCATGGAA CCAAATACA 19

(2) INFORMATION FOR SEQ ID NO: 62:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62: GCCTGGATAC AAGACTTGAA G 21

(2) INFORMATION FOR SEQ ID NO: 63:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63: TTGTAGACGT CCTCCTGAAC C 21

(2) INFORMATION FOR SEQ ID NO: 64:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64: AAAGCTTCAG TGCAAACCCA 20 (2) INFORMATION FOR SEQ ID NO: 65:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65: TCCAGATCTT GCAGAAGCC 19

(2) INFORMATION FOR SEQ ID NO: 66:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 66. CAGATGTTTC TGAGAGGGCT 20

(2) INFORMATION FOR SEQ ID NO: 67:

( ) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic ac d

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(X ) SEQUENCE DESCRIPTION: SEQ ID NO: 67: ATTCCTCTTT GGAGTCAAAT TC 22

(2) INFORMATION FOR SEQ ID NO: 68:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 68: GAGGCAGAAA AAGAAGATGG T 21

(2) INFORMATION FOR SEQ ID NO: 69:

(ι) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 69: AGGAGCCACT TGCTAGTAAG 20 (2) INFORMATION FOR SEQ ID NO: 70:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 70: ATGGTGAAAT AGACTTACTA GC 22

(2) INFORMATION FOR SEQ ID NO: 71:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 71: GCAGACCTTC TCAGGAGTC 19

(2) INFORMATION FOR SEQ ID NO: 72:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 72: AAGAGCAGGA ATGAAGTAGT G 21

(2) INFORMATION FOR SEQ ID NO: 73:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 73: CTCCACTGGT GCTCAGAATG 20

(2) INFORMATION FOR SEQ ID NO: 74:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 74: AGTGGAGATT TTGTTAAGCA A 21 (2) INFORMATION FOR SEQ ID NO: 75.

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 75: AGGTGGTGTA GGTGGTGAA 19

(2) INFORMATION FOR SEQ ID NO: 76:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION. SEQ ID NO: 76: GGTACACCAC CTTCTACATT 20

(2) INFORMATION FOR SEQ ID NO: 77:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 77: GTCTCTCCTC TATGATTTCT T 21

(2) INFORMATION FOR SEQ ID NO: 78:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 78: CAATGAAGCT GTTGCCCAA 19

(2) INFORMATION FOR SEQ ID NO: 79:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 79: GTCTTTAACA TTTGGATCAC T 21 (2) INFORMATION FOR SEQ ID NO: 80:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic ac d

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(Xl ) SEQUENCE DESCRIPTION: SEQ ID NO: 80* AGTGATCCAA ATGTTAAAGA C 21

(2) INFORMATION FOR SEQ ID NO: 81:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 81: AGTGATCCAA ATGTTAAAGA C 21

(2) INFORMATION FOR SEQ ID NO: 82:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 82: CAAAATGACT CACCACTTCA C 21

(2) INFORMATION FOR SEQ ID NO: 83:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 83: ATCGACAGGC CGCAGACC 18

(2) INFORMATION FOR SEQ ID NO: 84:

( ) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 84: CCTGTCGATT ATACAGATGA T 21 (2) INFORMATION FOR SEQ ID NO: 85:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 85: AACATGAGTT ACTGTACTGT C 21

(2) INFORMATION FOR SEQ ID NO: 86:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 86: TATACTGAGT TTGACAGTAC AG 22

(2) INFORMATION FOR SEQ ID NO: 87:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 87: CATACTTTTC TTCGTAGACA TG 22

(2) INFORMATION FOR SEQ ID NO: 88:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 88: TGGGTAAAAG CATGTCTACG A 21

(2) INFORMATION FOR SEQ ID NO: 89:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 89: GGATGCTACT TCTATTTGTG 20 (2) INFORMATION FOR SEQ ID NO: 90:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 90: GAGTCACGTC ACTGTCTG 18

(2) INFORMATION FOR SEQ ID NO: 91:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 91: CCTCAGTAGA AAGCCCAAGC 20

(2) INFORMATION FOR SEQ ID NO: 92:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 92: GCCCCTGCCG AACCCTCTC 19

(2) INFORMATION FOR SEQ ID NO: 93:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 93: GAGAGGGTTC GGCAGGGC 18

(2) INFORMATION FOR SEQ ID NO: 94:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 94: TTCAATTTCA AATGTTCATC TGGT 24 (2) INFORMATION FOR SEQ ID NO: 95:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 95: ACAGTCGCGC CGCGCTCGA 19

(2) INFORMATION FOR SEQ ID NO: 96:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 96: CAGAAACTGT GCGACCCGTG 20

(2) INFORMATION FOR SEQ ID NO: 97:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 97: AGATGTTTAT CTAACAATGA CTC 23

(2) INFORMATION FOR SEQ ID NO: 98:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 98: AGTTGTACTA TA ACATCAA ACC 23

(2) INFORMATION FOR SEQ ID NO: 99:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 99: ATTCTGCTGA ATGGGTTGCT T 21 (2) INFORMATION FOR SEQ ID NO: 100:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 100: TAACTAAGAG AGATAGGGAT AG 22

(2) INFORMATION FOR SEQ ID NO: 101:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 101 GGAGCTCCAT GTGGGAGCAA 20

(2) INFORMATION FOR SEQ ID NO: 102:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 102: AACATCTGCA GGAGGACTTG G 21

(2) INFORMATION FOR SEQ ID NO: 103:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 103: TCTGAGATGG TATTTCAGAG T 21

(2) INFORMATION FOR SEQ ID NO: 104:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION. SEQ ID NO: 104: TGCTTTTTAA TTTCCATTTT GTTC 24 (2) INFORMATION FOR SEQ ID NO: 105:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 105: AAGAACTGTA AAACACAGAA AGA 23

(2) INFORMATION FOR SEQ ID NO: 106:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 106: TGCTCTTTCT TATCACTTCT TTC 23

(2) INFORMATION FOR SEQ ID NO: 107:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 107: CTTGACTCAA GAATATAGGT CC 22

(2) INFORMATION FOR SEQ ID NO: 108:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 108: TAGTGCTCAC TTGATACTTA GT 22

(2) INFORMATION FOR SEQ ID NO: 109:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 109: CATAATAAGA ACAATGAAAG TTGT 24 (2) INFORMATION FOR SEQ ID NO: 110:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 110: TTGATCTGCC TTTAACAAAT G 21

(2) INFORMATION FOR SEQ ID NO: 111:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 153 ammo acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 111:

Gly Leu Leu Ala Leu Thr Leu Gin Pro Thr Leu Ala Val Trp Pro Ser 1 5 10 15

Pro Gly Ser Phe Pro Ala Pro Leu Pro Leu Phe Pro Val Leu Leu Asn 20 25 30

Ser Pro Ser Trp Arg Val Gin Ala Leu Gly Met Gly Gly Thr Arg Pro 35 40 45

His Ser Phe His Arg Ala Leu Arg Pro Asp Thr Ala Asp Gin Pro His 50 55 60

Ser Ala Gin Glu Ala Ala Ser Gly Val Gly Ala Gin Arg Gly Thr Ala 65 70 75 80

Ala Ser Ser Thr Ala Gly Cys Gly Ala Ala Gly Pro Gly Pro Ser Ala 85 90 95

Trp Ala Ala Glu Tyr He Phe Tyr Leu Ser Glu Thr Ser He Phe Leu 100 105 110

Gly Ser Asn Pro Thr Cys His His Val Asp He Ser Ser Tyr Leu Thr 115 120 125

Met Leu Ser Leu Leu Arg Ser Cys Pro Gly Gly Pro Arg Ser Leu Tyr 130 135 140

His Ala Thr Val Pro Thr Thr Gly Ser 145 150

(2) INFORMATION FOR SEQ ID NO: 112:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 112: TACCCTATAA GCCAGAATCC A 21 (2) INFORMATION FOR SEQ ID NO: 113:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 113: GGCAAACTTG TACACGAGCA 20

(2) INFORMATION FOR SEQ ID NO: 114:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 114: GGTACTAGTG AAATCACCAG T 21

(2) INFORMATION FOR SEQ ID NO: 115:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 115: GTGAATGCGT GCTACATTCA T 21

(2) INFORMATION FOR SEQ ID NO: 116:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 116: TTGAGTCGAG TCACACATTT GA 22

(2) INFORMATION FOR SEQ ID NO: 117:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 117: CTATTATGTT CCTTTCATAA CCA 23 (2) INFORMATION FOR SEQ ID NO: 118:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 118: TAATGTCTTT GTCTAGTCGT CTAA 24

(2) INFORMATION FOR SEQ ID NO: 119:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 119: GGTAGTTCTC CAAAAGGATC A 21

(2) INFORMATION FOR SEQ ID NO: 120:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 120: GAGTTATAAG AAGCAGGCCA A 21

(2) INFORMATION FOR SEQ ID NO: 121:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 121: ATTTCTTAAT TCTCTCAAAT CCAA 24

(2) INFORMATION FOR SEQ ID NO: 122:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2856 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: modified_base

(B) LOCATION: 233

(D) OTHER INFORMATION :/note= "H = A, C or T" ( ix ) FEATURE :

(A) NAME/KEY: modified_base

(B) LOCATION: 359

(D) OTHER INFORMATIO :/note= "Y = C or T"

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 2031..2188

(D) OTHER INFORMATIO :/note= "Exon I"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 122:

TGCCCTCATA ACCCCATCTA AATTTAACTA CCACCCAAAG GTCCCCTCCA AATACTATCA 60

CGTTGGGGTT ACAACTTTTA ATATATGAAT TTGGGGGTGA CACTACAGAT ACTAATGCCC 120

ATTTCATAGG GTCCCTATAA GGCTTAAGGC AGGTATTAAC ATAGGAAAGC ACTTAAAGCT 180

GGGTCTGGCT TGGGTAGGTA GTTCAATGAT TCAACAAACA CTGAGCACCT ACHTGGAGCC 240

AAGCACTGCA TGTGCCACAT GAAGCGATAT TGGGAAATGA GTCACATGCA GCCAATCTCT 300

GGCCTTTTGG AGGTTTTGAA CTAGAAGGGG ACACGCACAT AATCGTATGT GTGTGTATYT 360

ACATACACAG GTGATATGAT GCACCTGAGA GAATCCAGTC TAGGAACTAG GAAAACCTTT 420

AAGGAGTGAT ACTTCAGCTG TATTCTGAAG GATGAGGAAT GGAGAGGAGG CCAATTCCAG 480

GTTCCAAAGT GAATCTTTGC GCAAAAGCCA TGAGGCAGCA AGGTGCAGGG GCTTTTAA^G 540

ACCTAAGGGA ACGCACTGTG GGGTTGGGAC CATGATGGCC AGAGGAGGTT TTGACAAGAG 600

GCTAGTCAAA GAGCAGAGAA AAACTTTAAG GAGTTTTAAG AAAGGGAAGT GCCACGATGA 660

GTTTTGTGTT TGGAAATGTT TTAGGCGGCC ACACCGCAGT CTCGGGACTG GCTGGCACCT 720

GGATAGACAC TTGGATATCA GCTAGAAAGC TACGACAGGA AACCAGGCGA GGACGAGGCA 780

ACTGGGGATG TTGTGGAGCA GAGACGGAGA GAAAATGGGT GCATTCGAGA CAAGTTAGGG 840

GGAAAAAATG CAAGGACTTG GAAATGAACT TGGGGCGCGG CAGGAAGGCA TGACGGGTTG 900

CTCTGTAGGT CTTATCTGTA AATTACGGCG ATCAGTGAAA GATCTGGAGG AGGAAGGTGG 960

ACACACTCTC TAACAAAAAA AACCCTTTTT GAAATTTTAT ACCAATATTT TAAAAGTAAA 1020

CCAGATCTTT TCAGACATGC CTTTGAGCTG ATATTTGTTA ACTAGTTAGA ATTAGAAACT 1080

TTCCTTATTT TTACTCAGTT ACAATATACG CCACAGCTGA GGTGAGAGGA AAGAAAAGGT 1140

TGCTTTCTTA GGAACAAAGA GTGGTACCTT CAGTATCGTG GGCAAAGCTT TTCCAAGTCC 1200

AACAGTAGTC AAAACAGCGC TTTTTATAAA TAACACTCAG CTAAAAGTTT CTGGGTTTGT 1260

GATTGTTCCA ACGGTTAAGC TCGGATGAGG GTCCCTGGAG TCGTAGCTCC CGGGAAACGT 1320

CGACTGGCTT TCCACCTGGA CTTCATCCGT CCAGGCAGCC CAGAGGGGCT TCAGGCCCCG 1380

CCCGCTCTCC TGCCAACTAC AGCCTCGCGA CTGCGCTCAG CCTTCAGGCC CCGCCCCTTC 1440

GGTCAAGCGG CGTGCTCTCA CTGCACGGCG CCTGGGCCCC GCGCGCCGGG ACCTCGGTTT 1500

CAGCCGTCCT GTCCTGCCCC GAGGCCCCTA GGCCCCGCCC CTGGGCCCCG CGCGCCAGGA 1560

CTTCGGTTTC GACCGTCCTG TCCCGCCCCG AGGCTCCTAG GCCCCGCCCC CTCTGTCCCC 1620 GGCGTGTTCT CGCGGCTCCG CCCCTAGGAC CCGCGCGCCG GGACTTTGGC AAGTTTCAGC 1680

CGTCCGGCCC CGCCCCCTCG GTCCCACGGC TCTCGCGGCC CCTCCCCTAA GTCCCACACG 1740

CCGGGACTTT GGCAAGTTTC AGCCTCCAGC CCCACCCCTA GGTCCCGCCC ACTCGGCCAG 1800

CGGCTGGCTC TCGCGGCCCC GCCCCTGTGC CCTGCGAGTC CCTATTTTGG GΛGCATTGCG 1860

GCCGCCGTGC CCCGCCCCTC CCCGCGCACC CCGCCCCTCT GGCGGCCCGC CGTCCCAGAC 1920

GCGGGAAGAG CTTGGCCGGT TTCGAGTCGC TGGCCTGCAG CTTCCCTGTG GTTTCCCGAG 1980

GCCTCCTTGC TTCCCGCTCT GCGAGGAGCC TTTCATCCGA AGGCGGGACG ATGCCGGATA 2040

ATCGGCAGCC GAGGAACCGG CAGCCGAGGA TCCGCTCCGG GAACGAGCCT CGTTCCGCGC 2100

CCGCCATGGA ACCGGATGGT CGCGGTGCCT GGGCCCACAG TCGCGCCGCG CTCGACCGCC 2160

TGGAGAAGCT GCTGCGCTGC TCGCGTTGGT AAAGACGGAG CTTCTTGGGG GTGGCTGCGA 2220

GGGCACGGGT CGCACAGTTT CTGGGGGCGG CAGAATCTTT TCAAATCTTC CGTTTCCTCC 2280

TTCCGTTCCC GCGCTGCAGT CGGGTCGGCG TGCGGTTAGC ACCTGCCGGG GGATATAGTA 2340

TTAACAACTT CTGCTTCTCA TTCACTTTAT TTTTGGGCGA CTTACCGGCC TCCCCTTGCC 2400

CTGAATCCAA CTGAAACGGT AGTTTTTGAA CTTCAGCGGG CTGAAGAACC GTCTGGAGGT 2460

GTGGCTAAAA AAATGTTCAT CCCGGTCGCG CCTCCAGAGT TTGAATCGGG CTGGGGTGGG 2520

GCTGAGGCTT CTGCATTTTT TACCCGGCCC TGGATTACCC CGCTGCTTTC CGGGAGCTGT 2580

GGCGAATTGG GCTGGCGGGC CGCCCCGGAG ACCCTCTAAA TTAGAAGCAG CTGCCACTCT 2640

AAGTTAAACT GGCCTTTTTG ACATTTTCTC CGTGCCAGCT TTTTCGAGTG AGATGGGATG 2700

GAGCATCGGA TATCTACCAT AGTTGTAGAT TGAAGATGGC ACGGAATTTC TCATTTTCTT 2760

AGTTTGCTCA AAAGACTGTA TGTCTGGTGT CCCCGCTCTT AGTGATGCTG TTTATTGTTT 2820

TCCTTCATGC TGTCACATTA TGGGAGTCCT CTCAGG 2856

(2) INFORMATION FOR SEQ ID NO: 123:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8804 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY : misc_feature

(B ) LOCATION : 2623. . 2679

(D) OTHER INFORMATION: /note= "Exon II"

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 5421..6415

(D) OTHER INFORMATION: /note= "Exon III^*'

(ix) FEATURE:

(A) NAME/KEY: modified_base

(B) LOCATION :one-of (387, 699)

(D) OTHER INFORMATIO :/note= "R = A or G" ( ix ) FEATURE :

(A) NAME/KEY: modified_base

(B) LOCATION :one-of (2743, 5777, 5783)

(D) OTHER INFORMATION: /note= "W = A or T"

( ix ) FEATURE :

(A) NAME/KEY : modi f i ed_base

( B ) LOCATION : 4763

(D) OTHER INFORMATION: /note= "Y = C or T"

( ix ) FEATURE :

(A ) NAME/KEY : modi f ied_base

( B ) LOCATION : 6867

(D) OTHER INFORMATION: /note= "H = A, C or T"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 123:

GAGCTCGAGG ATCAAAACTG TGTTTTTTCT GTGTCAAAGG AGAGATTACC TGTTCATGTG 60

TAGATGCAAA TGAGAGAAGA AAGGCAAGTA TTATGAATAT CTAGTGCTTT GGGAGGCAGA 120

AAAGATATGG GAACTATAGG TAGAGTAGCA CATTTTAGGA AGAGTGACTA AAGGAGATCG 180

AATTTTCCTC TTAGACTCTA GGAAGGGTAA GATGACTGGT GAAGAGATAA AAATGTATTT 240

AGGTGTTAAA AAACTTACAC CATTAAGTTC CAGTAAAGTT AATGAGATGA GGAAGCATAG 300

AGATTGTTTT GAATAGCCAT CTATTCATTT GTTTGTTTAT TTATAGAATA TTGAATACAT 360

TACTTTGGTA GAGATACAAA CATGAARAGG CTACCAAATA AATTTTATGT CTTTATTTTA 420

TTTTATTTTA ATATTTTTGA GACAGAGTCT GGCTCTGTCA CTCAGGCTGG AGTGCAGGGG 480

TGTAATCTCG GCTCACCTCA ACCTGTGCCT CCTGGGTTCA AGTGATTCTT GTGCCTCAGC 540

CTCCCCAGTT CCTGGGATTA CAGGCGTGCA CTACCATGCC TGGCTAATTT TTATATTTTT 600

AAAATTTTAT TTATTTATTT TTGATACAGG GTCTCGCTCT GTCACCCAGT CTGGAGTGCA 660

GTGGCTCAAT CTTGGCTCAG TGCAACCTCC ACCTTCCARG TTCAAGTGAT TCTTGTGCCT 720

CAGCCTCCCA AGTTGTTGGC ATTACAGACA TGCACCACCA CACCTGGCTA ATTTTTGTAT 780

TTTTAGTAGA GACAGGGTTT CACCATGTTG CCCAGGCCAG TCAAGCTCCT GGCCTCAAGT 840

GATCCTCTCA CCTCGGCCTC CCAAAGTGCT AGGATTACAG GCATGAGCCT CAGTACCCGG 900

TTGCTACCAA ATGAATTTAT AGAGAGAAGT TTATTCATCT TTCTTTCCTT TTTTTTTTTT 960

TTTTTCTTTT TTTAATAGTG GTTATTCGCA GCCTGAGTGG GCAGGGAAGA AGTAGACTCT 1020

GGGGCCTATC TAGCATTATA GATTGGCATC CATGAGTGTC TAAGAGGTCA GCACAATTAG 1080

GTAGTGGTGA AGGTGGCTTG GAAATAGTTA ACTCTGGCCT GGGCTGGATA GGGAATCCAA 1140

GGTGCTAGGA AACTGAATGG ACTTCTTTCA AGGTAGAGAG CAACCTGAAG GTGGAGGTTT 1200

GGCCAGGGCG ATGTAATAAA AGAGGTAAAG GAAGGATATT ATGATAGGAG GAGCTTGCCA 1260

CGAAATAGAA TTCAGTACAC TTGATGGGGA AAAGGAGGTT AGGTTTGCTA GGTCAAATAA 1320

CAAAAATTAT AGACACAGTA TGAATGTTAA AAGAGATCAT GTTTATTGGC AGAGTCACAA 1380

GTCATATTTT TATCTGTACG TTTTACATAC TGTGATACAA AAATCAGACA TGGTGAGTTT 1440

TTAAAAAGTA ATAGGTTTTG TAACGTCAGT GAGCACCATT TTCAGTATCT TGAGAATAAC 1500 ATTGTGGGTG TGTACTGTTT TTCGCAATCT TTCTGAAAAT CTCAATTTCA CTTGAATGTT 1560

CATATCTATT AAGATGTGCA GTGTGATACT TTCAATCTTT CAAGAAATGT GAAGAATTGT 1620

TTTTATTGAT ACGTGGTATG TGTACAGAGG TAATTTAATA ATAATGGTGT TAAATATCTA 1680

AAGGTTTTAG AGTTAGTATA ATAAACCAAG CCAGAAAAAG TGCTCATCAT TTAAAAGGCT 1740

TTACTTCTCT GGGTACATTA CATCCATTGA GTATAATGTC TTGGTGTGTA TTTATTAGTA 1800

TCATTACTTT GTTAGGATTA ACAAATGTAG CAGGTATTTT GATGGACAGT ATTAGAAATA 1860

TTTTGTTTCT GTGTCTCTTT GCTCACTCAC TGCAGGGAAC TTCATTCTAT CTGCAGACAC 1920

ACTGCTAATC TGATGATTTC TGTTTACATT TGCTTAAAGA ACAATTAACT TTCTGTGAAC 1980

TGAAAGAATG GCTGCATTTT TCACAATATA TCTGTGAAAA ATTGATGGCT ACTTTACAGT 2040

AGTAAGAAAC ACATGTTTTT TTTAATTGGG AAACCTTGGA TTCTTACGGT CAAAATGACT 2100

AGATATCCTG GTTTGTCTGG GGCAGTCCTG GTTTATTCCT GTTGTCCCAG CATCCTATTC 2160

AATTATCACT CCTCTTCACT CTCAGAAGTT TTCTCATTTG GATGATACGT TATATGGTCA 2220

TTCTACGTGT GGGACACATT TTGAGGTATA TGGTCTACAC TTTGAATCAT AAGGGGAAGA 2280

TACACCTTAG TAGTTGAAAG AAGCAGCCAG TAGTTGGTTT TGCCTTAAGA GGTTTAGAGA 2340

TACTTGAACA AAATTGTTCC TTTCTCCAAC TTTCTAGAAA ATACATTTTA AAATGTATTA 2400

CAACTTGTAC CCAGTTTGGT GGTTACTATT TAAAATCATC AGGTATGTTA TGGTACAATA 2460

TTTAACCAGG GAGTAACAGC CTTTCAAATG AATGCATCCT TAATACCTTC TGCTTTGAGA 2520

AAGTGAGAAA TATGGTAGTG TTGGGCCTTG GATGAAATAT TGGGTGTGAG ATGTTTATCT 2580

AACAATGACT CAAATCTTGA TTGTTTTAAT TTCTTCAAAC AGTACTAACA TTCTGAGAGA 2640

GCCTGTGTGT TTAGGAGGAT GTGAGCACAT CTTCTGTAGG TAAGTAATTA CGGTTTGATG 2700

TATATAGTAC AACTGTATTT TTTACTAGΛT AACAATCATG TA TCTTGTT GATAATAATG 2760

GTACTTGATG TTTGTGTAAC TTTCAAAGTC TGCAAAGTAA CCTATTGTAC ATTATCTTAG 2820

TTTGATCTTT AAAACTGTGT CTTAATTTAC AAATAAGAAA ACCAAAGCTC AAAGAGGATG 2880

ACTTGTCCAA GTTACAAAGC TTAGTAGACA GCTTTGCCAA ATTGGAGGAA AAAAATTAAT 2940

GCCTTTTATA TAATTCAAAT AGATGTTTTT AAATTTTCCA GTTAAATTTT G.AATCTAGAC 3000

TCAAAATTGT GAAAGTATAG GTCTTACAGT TATTATATTA GTTTCCTAAG GCTGCCATAG 3060

CAAAGAACCA AAAACTGGGT GGCTTAGAAC AACAGAAATG TATTGTCTCT CTAGTTCTGT 3120

AGGCTAGGAG TCTAAGATCA AGATGATGAC AGGGTTGGTT CCTTCTGTGG GCTGTGAGGG 3180

AGAATCTGCT CCTTGCCTTT CCCCTAGCAA TCTTTGCTGT TCCTTGACTT GGAGATGTAT 3240

CACTCTGGTC TTTGCTTTCA TGTACACCTG GCATTCTTCC TTGAGTGCTT GCTTCTCTCT 3300

GTGTCAAATT TCCTCTTTTC ATACGAATAC CACTTATATT AAGGGCTCAC CCTAATGTTC 3360

TCATCTTAAC TTGATCGTCT AGAGCCTCTT TTTCCAAATT AGGTCACATT CACAGTAACT 3420

GAGGGGTGAA GACTCAAACA TCTTTTTGGG GGACACAATT CAGTGCATAA CAGTTATTGT 3480 GAAATTATAT CCATGTGATG GCCCTGGCTT ACAGGTCAGA AGATTAGATT TTTATCTCTT 3540

ATTTCTTGTT CAGGAGAAAT CAGTTGAGAG ATTAAGTGTC TTAGTTTAAT TGCCTATGAG 3600

ACAGGGAAAA TATAGTCTCC TTTGAATTAA TCTTTTAATT ATTTCCAGTT ATGAGTATGT 3660

TACTGTCTGA CATGAACAAA TACATTCGGA AATTTGAGCA TATATGAAAA TAACCTTGTG 3720

TATTACTTCA AGGGCTAAGG TTGTCTGGGA GGCAGCTATG TTTTGGGTCC CAATTTTGTT 3780

CCTGGAAAAA CAGATCTGTT ATACGAGGGA TATCCAGCAG GAGGATGGGG GGTAGGAGAT 3840

GGAACCTGTT GCCTACCAGA TACGTCTGTT TGGATGATGA AAGAAAAGGC ATTTTAAAGT 3900

TGGTGTGTTC AGAGCTGACC TCTTGATCAT CCTCCTCTTC AGCCTTTTCC TGCCTGGGTA 3960

ATCTTCATCA CAGTAAATGG TACCAGTTAC TGCTAGGTTG CTTATCCCAC ATCGCTAAGT 4020

TCTGTCAGTT CTGCCTCAAA ATGTGTCCTG AATCTTGAAT TTTTCCACCT TTTTTCTGAA 4080

TTTTCTACAG TTTATAGTTT GTTCCAAGCC ATCATTGTAT TATCTCTGCT CATACTATTG 4140

TGATAGGTTC ATAGCTGGTC TTCCCTTTTT GGCATCATCA CCCCTTCCTC ATTTTCTACA 4200

TTCTTGACAG TGATCTTTGA AAAATCACGA TAATATCCAT ATCATTACCC TGCTTAAACT 4260

CTTTAGTGGG TTCCTATTGC AGTTAAAATA AAATCCAAGC TCTGCCCTCT GGTCTGCAAA 4320

ACCCTGTATG GGACCTAGAA CCTGTCTTCC TCTTGGACGT CATTTACCTC TGGCTCACAC 4380

TGTTCCAGCA CTTCTCCTTT TAGCTCATTC AATACACCAA ATTCACTCCC GCCCCAGGAC 4440

TTTGGCACTT GCTATTTCTT CCACGTGAAA TGTACTTATG CCAGATTTCT GTGAGGCTTA 4500

CTTTTTACCA GTTATATATC AGCTGAAATG TCACTGCCTC CAAAACGCCT CCCATGGATC 4560

TGCTTACGAA ATGATGCCCA CTCCCTGTCC CCACTCTGTA GCAAGCAGAT GTATTTTGTT 4620

TCAGCTGACA GATGATGGGT TTGCTCACTG TATACAATAG GTCAGTCACA AAGCTGCTAG 4680

AGCATAATGA ACTCCATGAC ATTTTATGCA AAGTTGGTTT GTGTGTGTGG TGGGGTGGGG 4740

GATCCATACG TTTAATCAGA TTYTAAAGGA CTCTGTGACT TAAAACAACC AATCAAAGGT 4800

TTGAGACACG AGAACATCTT AAAAGAGAAA ATGATTATGT GTATATGACA TGAACGTAGG 4860

AAAGAATTCA TTCAGTGCAG TTTTGATTTG TTTTGATTAT ATGATGCCAA AAATTATGGT 4920

AGCTGTTTTT ATTCATGCAG ATTTTGAGTT AAAACTGCTC AGCAATGAGG TTTTAAAATG 4980

ATTTGACATA GCTCAGTTCA ACTGATAAAG GTAATTCATC TACTCTCTAA GATACAATTT 5040

AAGACATGTG GCAGGGGTGC TCAAACATTT TGAGCATTCT TCAGAATTGA GAACCATAAA 5100

GAGCTTTTGT GTGTTCATGC ATTTACCGTA CAAAACTGAG ACTTTTTAAA AAAAGAATTA 5160

ATTTAAAAAT AATAAACCCA TTAATACAAA CACAAATAAC ACTTTGTAAG GAAAAATAAC 5220

AATTTTAACA AATCAAAGAA AAAAATAGAG TGGCACTGTT GTACATTTTT GCAAATCTCT 5280

GTCTTGCTGA ATGGAAAACA GCTACATTCT CAACTTATAT ATTCAGTCTA TTGTGATACT 5340

TTTTTGATTG AAGAAAATCT GGCTTTATAC ATAATTGTAG TTGGAAGAAG TATTGAAGTA 5400

TTTTCAGTAG CTTTTTTAGA TAATTGTGGC TATTCTTCTT TAATACTACA CCAAAACTTG 5460 ACAAGTGCTT TTTTATTTTT ATTTTTTTGT GAAGACAAGA GTCTCACTCT GTCACCCAGG 5520

CTGGAGTGCA GTGGCGCGAT CATGGCTCAC TACAGCCTCA ACTTCTTGGC TCGGGTGATC 5580

ATCCAATCTC ACTTTCTGAG TAGCTGAGAC TACAGGTGCG CCCCACCACG CCTGGCTAAT 5640

TTTTGTAGTT TTTTGTAGAG ACAAGGTTTT GCCATGTTGC CCATGATGGT CTTGAACTCC 5700

TGGGCTCAAG CGATTCTCTT GTCTCGGCCT CCCAAAGTGC TGAGATTACA GGAATGAATC 5760

ACCATGCCTG GCCAACWAGT GCWGTTTTCT TTAGGTTCAT CACAGTGTGG AATCTGAAAC 5820

TATATCAATA AATTTTTATA CTCTGTTACA TGAAATTTCA TTGGTTTATC TAGTACTTTG 5880

AATTTATCTG TTACTCAGCA TGATTTTATG ACATCACACA CGGGTCATTT GAGAAATACT 5940

CACTGAGCTA TACAGGTCCA CCGAAAAATG ACATTTTTTT CAGAATTCCA AATTAATGTA 6000

TATAAAATAA TTTGGGAATA GTTTATAAAA TGTTATAGAA ATATAAAATA CTAGTCTGAT 6060

CTGAGTACTA GTGCAGTGCT GGACAAGTAA CTTAAATAAC TGGATCTCAT TTTCTTCATC 6120

CAAAAATGAG GTGGTAGTTC TTGATGAGTT GATTTCAGAT TGTATTTTAC TGATAATTAA 6180

GTATTGCACA AATGATTTAA ATTGCATGAG TAATCAGTTT TACATATTTT TTTGTGTTGG 6240

GGTTCCAAGT TAGAGTTCTT AACTACTAGC AAACAAATGT ACAGAAGATC CTTTGCTAAA 6300

GAAAGTTGAC ATTTATTGAC CCCAGTGACA TTTTTTGAAT TAGATGGTCC CAAAAGTCTC 6360

TTCCAGCTCT GGTATGGGTT ACAGATTCAT TTTACAATTT TTTTTGAGTT ACATTCTGTC 6420

AGAAATCATG CTAGAAGCCA AGGATACAAG AATTAGAAAT GGCATAGGTT TTGTTTGAGG 6480

ATAGTTCATA TTGAGTAAGA ATCCTCTCTG CCTACAGAGG ATTGGGTCCT GTGACAAGGA 6540

ATGTCCTGTG GTGCTTGGGG AAGATGTGGC TTTTCAACTG TTACATTACT TACTCAGTCC 6600

TACTGGAACC CATCTGGTGA GGCCAATGAA GGAGGAGATT TATAGAATTC TTATTCTGGA 6660

ATTCACAGAT GGGCTTGTGG GGGGGATCGT GAATCCCTCT TTTTACATGΛ GTATGTATAG 6720

ATTTTCATCA GATTTGCAAA AGGTCTATAA ACCCCAAAAT TAGAAAAACT CTTCTAACTC 6780

TGAAACTTTA GTCCTTAGAT AACCCAGGTA ATTGAGTCTA CAGGTTTTAA AATTGTCTGA 6840

AAAAGTCAAA GATCTTTTCC CAAAAGHTAC TTTAAAGCCT TGAGATACTA GTCCCAAAAC 6900

AAAACAAAAG GTCTTTCCCA GTGTCTGCAG TAGTTTTGGG TATTTTCATG CAATGTTAAG 6960

ATAGAAAAAG TTAGGATGCA CGACTACTAT GCATTAGGCA TTCTATCTTT CTGTTACATC 7020

TCTGTAACCT TTAGAAGCTA CAGATTTATT TGTAGGGAAA ATTATGCAGA CTAATAATCA 7080

GGCTAAGTAA AGTCTCTTCA TAGCAAATTA CATGAGCAAC CTTAGATTTG ATGTATGTAT 7140

TTTACTCTTT AAAACAGTAT TCAACAAGGA TATTACAATT GACCATTGTA TGTTAGAATA 7200

ACCTCTGCTC CATTTATTTC TGTTCAAACT GTTTAGTTTT TGGAATTAAA TTCTGCTGAA 7260

TGGGTTGCTT fTTTTTTTTT TTTTAATTAT TTTAAAGTAA TTGTGTAAGT GACTGCATTG 7320

GAACTGGATG TCCAGTGTGT TACACCCCGG CCTGGATACA AGACTTGAAG ATAAATAGAC 7380

AACTGGACAG CATGATTCAA CTTTGTAGTA AGCTTCGAAA TTTGCTACAT GACAATGAGC 7440 TGTCAGGTAA GAACTATCCC TATCTCTCTT AGTTAAATTC ATCAGTTAAA AACTGATGAA 7500

TTCATATTCA TAAAGTATAT AAAACATCTA TCTGGAGTTC TGGAATACGT ATTTCAGATT 7560

TTAAAATCTG TAGGTTTTTT _TTTTχττTTT TTAAATAGCC ATTGAGTCTC TCTATGTTGT 7620

CCAAGCCGGA CTTGAACTCC TGCACTCAAG GGATTCCCCC CACCTCAGCC TCCCTGGTAC 7680

ATGCATGTGA CACACCCGGT GGTTTTTTGG AAGATCATTT TGAGAAATGA CGGGAACCAT 7740

AGAGATAATT AAAGTCTAAC CCCCTTATTT TATAGTTGAG AAATCAAGGT CCAGAGAGGT 7800

GAAATAACCT TGCCTACAGC CGCATGTGTA GTTAGTACTA GAAGCTGGAG AAATAAAGCC 7860

CATTTCTGAC TGTAAATCCA GGAACTTTTT TGCTGCTTTA CATTGCCTGC TTTTTACATT 7920

TATGATAACT TCTGCAGAAT ATATATGGAA TAGTGATTTT GGCCTTAATA GCACTTAACT 7980

CACTTAAATA CACTTTCCCA AGATAGAATA CGACATTTTT CCATGGATCA CTTTTCTAGA 8040

CTGAATTAAT TAGATGAGCA CATTTTGAAA GAGCAGAAAT CTAATATTCA TTTTCTTTTC 8100

TATTTAAGTG GGGGTTAACG TTTTTTAAAT TGTCCTTAGA CTCTGTGTAT TAACTGTGTA 8160

TCTTTCCATT GTGCTTTCTG CTGAGAACTA ATTAACTGTG GAAAGAGAGT AAAATGTTTT 8220

GTATCTTCAT AATTGGATAT TAGAGTTGTC TTTTTATTGA CCAGATCATG CTATTTTAGT 8280

GTGTGTTTGT AGAAGAACCT GTTCTTGACT GGCAGGATGC CATGGATGAT TGATAATGCT 8340

CATATAAAAT TGTTAGATTT CTATTTTTAA ATCTTTTGTT CTTAGAATCC TGCCATGATG 8400

TCTTATTGTC AGCTAAAGAT GAAGTTGATT ATACAAAATA AATAAATGTG GCAAAAACTT 8460

TGAGTCTTAC CACAGGTCCA TATTTTTAAG AATTAGAACT TAAATCACTT TATACCTATT 8520

AAAATTTTCC TTCAGCTAGT TATCTGCGTC ATTGTATTCA ACTCCATTCT TTTATGATAA 8580

AATGTGCTGT AGTGCAGAAG TTTTTCTTAC TTTCAGATGT AACTATACAC ACACATTTTT 8640

AAAAGTTGCC GTTTTTTAAA AATGATATTG TAGTTGTAAA CCTTTTTAAG AACACACTGA 8700

AGAAAAATTC TGTCATTAGT TCATCTGAAC TCTTCTTATT TAAAGACAGT ACTGACAATG 8760

TTTATTTTCT AGTTAAAAGA GATTGGTGTG TGATCCTCGA GCTC 8804

(2) INFORMATION FOR SEQ ID NO: 124:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2111 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: modified_base

(B) LOCATION: 1899

(D) OTHER INFORMATION :/note= "D = A, G or T"

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 621..1570

(D) OTHER INFORMATIO :/note= "Exon IV" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 124:

CCCTTTATTC ATTTTGTCAG AGATACGTTT TGAAGTCATA TAGTATGAGT CCTCCAACTT 60

GGTTCTTCAG TATTATGTTG GTTGTTCTAA GTCTTTTGCC TTTCAAATTG TACCTTTCTT 120

TTTCCGTGTA AATTTTATAA TTGCTTGTCG AGTTTTACAA ACAAGCCTAC TGGAAATTAC 180

ATTGGGATTT GTTATATCTG TAAATCTGTT TGGAACAATT GGCATCTTAA CAATATTGTT 240

ATAGTTCATG AACATGGTAT ATCTCTCCAT TTGTGTAGAA TTCTTTAATA AGTATTTTGT 300

AATTCTTAGC AAACAGAACT TATACATTCT GTTAGATTTG TATTTTATGG GTTTTTTTGG 360

GTACTATATT TGGAATATAA GTTTCATGTT TTGTTCTCTG CTATATCTTC AGTCTCTAGG 420

ATATGGCACA AAGTCGGATT CAGTAAGTAT TGAATGAGTG AATATCTGCT ATCAAAGAGT 480

TCACACTCTA GGAGCTGAGA AAGAAGTACA TAATTAAAAG ATGATACACT TTAGGGGAAC 540

TGTAAACAAA ATTCTTCGGG AGCTCCATGT GGGAGCAATA AATTTCATGT AACAGATTTC 600

TTTTTCTTTT TTTCTGTCAG ATTTGAAAGA AGATAAACCT AGGAAAAGTT TGTTTAATGA 660

TGCAGGAAAC AAGAAGAATT CAATTAAAAT GTGGTTTAGC CCTCGAAGTA AGAAAGTCAG 720

ATATGTTGTG AGTAAAGCTT CAGTGCAAAC CCAGCCTGCA ATAAAAAAAG ATGCAAGTGC 780

TCAGCAAGAC TCATATGAAT TTGTTTCCCC AAGTCCTCCT GCAGATGTTT CTGAGAGGGC 840

TAAAAAGGCT TCTGCAAGAT CTGGAAAAAA GCAAAAAAAG AAAACTTTΛG CTGAAATCAA 900

CCAAAAATGG AATTTAGAGG CAGAAAAAGA AGATGGTGAA TTTGACTCCA AAGAGGAATC 960

TAAGCAAAAG CTGGTATCCT TCTGTAGCCA ACCATCTGTT ATCTCCAGTC CTCAGATAAA 1020

TGGTGAAATA GACTTACTAG CAAGTGGCTC CTTGACAGAA TCTGAATGTT TTGGAAGTTT 1080

AACTGAAGTC TCTTTACCAT TGGCTGAGCA AATAGAGTCT CCAGACACTA AGAGCAGGAA 1140

TGAAGTAGTG ACTCCTGAGA AGGTCTGCAA AAATTATCTT ACATCTAAGA AATCTTTGCC 1200

ATTAGAAAAT AATGGAAAAC GTGGCCATCA CAATAGACTT TCCAGTCCCA TTTCTAAGAG 1260

ATGTAGAACC AGCATTCTGA GCACCAGTGG AGATTTTGTT AAGCAAACGG TGCCCTCAGA 1320

AAATATACCA TTGCCTGAAT GTTCTTCACC ACCTTCATGC AAACGTAAAG TTGGTGGTAC 1380

ATCAGGGAGC AAAAACAGTA ACATGTCCGA TGAATTCATT AGTCTTTCAC CAGGTACACC 1440

ACCTTCTACA TTAAGTAGTT CAAGTTACAG GCGAGTGATG TCTAGTCCCT CAGCAATGAA 1500

GCTGTTGCCC AATATGGCTG TGAAAAGAAA TCATAGAGGA GAGACTTTGC TCCATATTGC 1560

TTCTATTAAG GTAGGATGCT TACTCTGAAA TACCATCTCA GAATGAGGCC AACTATAAAG 1620

CAATTTCTTT GCAGTTTTTG AAAAATGGCA TAGGATTACT AGGATAATTA ACCTTTCACA 1680

GACATGATAC TTCCTCTGAA CCAGAGAAGC CAGATTCACA GGGAGAGCAT CTCTACTTCA 1740

GTTGGAGCAG TGGCCCCTGA GTCTGGGCGC ATGATCTTGT AGGAGAAAAC CAATATTTGA 1800

ATATTTCAGC TTTTATTTTG CCAAGTGCTT TTGCTTTTGT CTATTTTACC TTCAGTTTTT 1860

ATCATTTTGT TTACCTGTCT TCATGCTTTA TGAATGTADA CAATTGCTAA GTTATTACAG 1920 GCAACAATGT TTACTTAGTA AAAAAGCCCA TATTTACCAT CCAAATTCAA CCAAAATTT& 1980

GAAGGTTGAA AGATGTGGTC TGTACATTTC TCCAATGACC GGGACATTTG ACTATCAGAA 2040

ATGGCTCCTC CAGTTCACCA CAAAGGAGCT GCTTTTTACC CTACAATCAG CTGTTCCTTT 2100

TACTGACCTG T 2111

(2) INFORMATION FOR SEQ ID NO: 125:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1098 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: mιsc_feature

(B) LOCATION: 451..531

(D) OTHER INFORMATION: /note= "Exon V"

(xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 125:

TTCTTTTTTG TTTTGTTTTT TTGAGACGGA GTCTTGCTCT GTGGCCCAGG CTGGAGTGCA 60

GTGACATGAT CTTGGTTCAC TGCAACCTCT GCCTCCTGGG CTCAAGTGAT TCTCCTGCCT 120

CAGCCTCCCA AGTAGCTGGG ATTACAGGCT GGCACCACCA TGCCCGACTA GTTTTGTATT 180

TTTAGTAGAG ACGGGGTTTC ACCATGTTGA CCAGGCTGGT CTCAAACTCC TGACCTCAGG 240

TGATCCACCC GCCTCGGCCT CTCAAAGTGT TGGGATTACA GGCGTGAGCC ACCACACCCG 300

GCCTAATAAT TTATTAACTC ATGAACAGTA GCCTTAAGAG AAAACGATTT AAGTTTTACT 360

TTATATTGAA GAAGGCAGCA TTTAAAAAAG CTCAATATTT TCCTTTCTTT CCTTAATGCT 420

TTTTAATTTC CATTTTGTTC ATTTTTCTAG GGCGACATAC CTTCTGTTGA ATACCTTTTA 480

CAAAATGGAA GTGATCCAAA TGTTAAAGAC CATGCTGGAT GGACACCATT GGTAGTTGTC 540

TGGTTTTTAT TCTCATTCTT TCTGTGTTTT ACAGTTCTTA TAGTTTATAG TTATGTAGTT 600

GTCTATATAT CATCCTCTGC CACATATACT CTTTTTAGTC TGAAGAACTT ATGTTTTCAT 660

CAAGTATGAG AACATGATTA CTTTCCTTCT AGCTTTTCAT TTGTGACAGG CAAGAAATTG 720

GTTACCTTTT GACAGACTAC CTTTAGATTT AGGAATCCAT TTGTACTGTA CTGCAGAATT 780

TAGCTAATGT CTAGAGGTAA CAGCTACAGC TGACATCAGG CTCCATTCTG TAGCACTGCA 840

TGTCACTGGA ACCAAATTTC TTGGAACAAA AAGAGGTCGG AGGAACTGAG TATAGGAAAG 900

TGATCACAAG GAAGTAATTC TCACTGAGGG TCTATCTTAG CCTCACTTAT ACCCTATCCA 960

ATTGTAGATA TATAACGCAG TAGAAATCTT TGCTTACATT GAACATTTTT AAAGGTCTTT 1020

GCTCATTATT ACTAAAAAAG TGTGAAGCAT AATCTGGAAA CAGAATGACA CAAATGCTTG 1080

GAAACAATTG GTATGTAG 1098 (2) INFORMATION FOR SEQ ID NO: 126:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1756 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 508..680

(D) OTHER INFORMATIO : /note= "Exon VI"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 126:

TTGTATTCCT ACTCGTGTTA TTTCTCTTAT CAATAACAGC AGCCATACAG CAATTTGTAG 60

GTTCAGCAAA TAGCTTGTCT TAAAAGCATT CCTTCACGGA TACTTACTTT GTTGCATGAT 120

ATGTGTATGT ACTGGTACAG ATTTTATGTA TCATCTTGTT ATTAAATATG TAGACTTTTT 180

TTCTTAATGT GTTACATTTA TTGTAGAACA TTTAAGGAGC TACCGTAGGT TTAAAACTAC 240

ATTTTCTTCT AAAAAAAAGA AAAGTGCTTG ACCCAAGGCT CAAATGAGAA TAGCCTTTCT 300

TTTTTTATGA GTTACACAGA TCTTGATTGA AAGATTATTA ATAGTAACTT TCACTCTGTC 360

AGCAACTTAT AGTGTTTTTG AGTATTTAGG TAACAATAAA TTTACTGCCT GACGTTTACA 420

TTTATTTTTC TAAAGTGTGA TATTATAATA TCATCCATTG CTCTTTCTTA TCACTTCTTT 480

CACTTCTTTT TCAAAAAATT TAATTAGCAT GAAGCTTGCA ATCATGGGCA CCTGAAGGTA 540

GTGGAATTAT TGCTCCAGCA TAAGGCATΪG GTGAACACCA CCGGGTATCA AAATGACTCA 600

CCACTTCACG ATGCAGCCAA GAATGGGCAC GTGGATATAG TCAAGCTGTT ACTTTCCTAT 660

GGAGCCTCCA GAAATGCTGT GTAAGTAGTT CAATGTAAAA ATTATTTTTA AAATGGACCT 720

ATATTCTTGA GTCAAGGTGT GTGATAAAGC AGACTTTAAT AGTCAAGTTG ATGGCTTTCT 780

TCACTTTCAC AACTAAAATT AGATGTGATC ATCACATTCT GCACTCATAA TCAGCATTCA 840

TGCCCTTTCT CTTTATGATA CAGTTGGTCC TTCATATTCT TGGGTTCTAC ACTTGAGGAT 900

CCAGCCAACT GCAGATCAAA AATAATTGGG AAATATCAAT GACAGATCGG ATAAAGAAAA 960

TGTGTTACAT ATATACCATG GAATACTATG CAACTACAAA AAAGAATGAG ATCATGTTTT 1020

TTTGTGGGCA CATGATGGAG CTGGAGGCCA TTATCCTTAG TAAACTAACG CATGAACAGA 1080

AAACCAAATA CCGCATGTTC TCACTTATAA GTGAGAGCTA AATGATGAGA ATTCATGAAC 1140

ACAAAGAAGG GAACAACAGA CACCAGAGTC TACTTGAGTG TGGAGGGTGG GAGGAGGGAG 1200

AGGAGCAGAA AAAGTAACTA TTAGGTACTA GGCTCTATAC CTGCGTGGTG AAATAATCTG 1260

TACAACACAC CCCCGTTACA CAAGTTTACC TATATAACAA ACCTTCACAA CTTAAATAAA 1320

AACCTAGAAT AAAAGTTTAA AAAGGGAAAA AAAAATAACA CTACGATAAT AAGTAATATA 1380

GGTAAAACAA TATAGTATAA ATATTTATAC AGCATTTCAT TCTATTAGGT ATTACAAGTA 1440

ATCTGGAGAT GATTTAAAGT ATACGGGAGG ATGTATGTAG TTTACAAGTA AATACTATGC 1500

CATCTTTTAT AAGAAACTTG AGCAGTGGCA CATTTTGACA TCACAGGGGT TGAGGAACCA 1560 ATTCCCCATG GATAGCAATG GGGATAATTG TGCTGACATA TTTGGGGGAG ATTTACTTTC 1620

TTAATTCAGA AACAGTTGTC AATTTTGGAA GCTTTCATTT AATGGAAAAA TTTACTTAGT 1680

GTTTATATTC TGTAGATTGA TTTACACTTT AATAAGCAGT TATTGTAGAA ATAATTATTT 1740

TGTATGCTTC CTAATA 1756

(2) INFORMATION FOR SEQ ID NO: 127:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1190 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 5 8..656

(D) OTHER INFORMATIO :/note= "Exon VII"

( ix ) FEATURE :

(A) NAME/KEY : modif ied_base

( B ) LOCATION : 1023

(D) OTHER INFORMATION: /note= "W = A or T"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 127:

TGAGCCATAA TACTTTGTTC TGCGATGGTT GTGATTATTA TAGGTTATTG TATGCACACA 60

TGTTTAAATT AATTTTTAAA GTACCCTGTT AACTATATTA TTAAACTGTT TGTTATGTGG 120

CATAATTTTC CTTCTAGTAG AACAAAATCC CTGTCCTGTG AATTTATCTA ATTTTTTATT 180

GGTTTATAAA GACTATATGG CCTATAATAG CTATAGTAAA TGATTTTTAT TGGCATTTGA 240

AAGTCTGTCA CTTATAGTGA TTGGTGATTA TGAAGCCATA TTTTAATATG AATAAGAATG 300

CAGAATACAG TTGTGAAAAA TTCATAATAC TATATTCAGT AAAAACAATC CCTATAATCT 360

GATGTCAAAC TGAAATTTTA CATCATTTCT CCTTTGAGTT CAGCAGCTTT TGATTCTAGA 420

TTCTTCTGCC TAATATGAGT TCTGAGTAAT TTATTTTAGT TAAAATTGTA TATTATTAAG 480

GATGTTGAAA AATTGAGTCG AGTCACACAT TTGACTTACT TAAACACATC TGCACTTATT 540

TTACCAGTAA TATATTTGGT CTGCGGCCTG TCGATTATAC AGATGATGAA AGTATGAAAT 600

CGCTATTGCT GCTACCAGAG AAGAATGAAT CATCCTCAGC TAGCCACTGC TCAGTAGTAA 660

GTATGGATTT AGCTTTGGGA CATTTATATA TTTTATTAAA ATTGGTTATG AAAGGAACAT 720

AATAGAAAAA TTTCCATTTG ACCAATTGCT TACATTCACC AAACAATTAT TGAGCACTTC 780

CTGAGTATTA GCTACTGTGG ATTCAAAGAC ATAATCACAG TACGACCATC TAGAAATACT 840

TATTGAGCCC ACTCTGTATT TTAGGCAGCA TTCATAAAAC AATGAATATG ACTGGTAGAA 900

CTCTTATTCT CAGGGAGGAG CTTACCATCT GAGAGGTAGG AAAGAGACAA ACTGTAAATA 960

TTGAACTAAT ATAAATAAAA TAATTTCAGA CACTTAGACA TGAGTGTTTC GAAGATGTTG 1020

TAWAGTGTCG TTGGGTGGAG GTAGTGGGCT TCTGCAAGGC CATTGCTTTA GCTAGGGTCA 1080 CAGAGTGGAG CCTTAAAGCA CTGATTTGAA TTGAAACCTG AATGTTGAAG TGAGGAGGCT 1140 GCCAGGTGAC TATCTGGAGG ACACAGTGTA TAGGCCCTTC ACTGAATGAG 1190

(2) INFORMATION FOR SEQ ID NO: 128:

( ) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 915 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: mιsc_feature

(B) LOCATION: 566..698

(D) OTHER INFORMATION: /note= "Exon VIII"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 128:

CTTACTATTT ATGGATCTGA TCTCCTAAGT TTTGAATTAA CTTGTCTGTT TTTATCTTTT 60

CCTAGTTTTG AGGGGTTACT ATTTTGATGC TAATTTGTTT TCTATCTTTG AGGTCAGCAC 120

TGTTCTAGAA GCCTTGGCAT TCTTTGATTT TTCAGATAAT CTCAGTTTAA ACTAAACAAG 180

TTTGATTTTA ACTCTATTGG GACAAGTTAG TGGAGGTGGA ATAGGGAATT GCTGATTTTA 240

AGTGGATATT TTAAGTTACT TGGGAAAAGA AAAAGACTTA CTGGTGACTG AATGAAGTAA 300

AACCCTAGAG AGACCCAATT TAAAATTGAA GAAATGAGAT GCCCCTGGGT ATAGAGAGCT 360

ATCACAATTG ACATTTTCTT GAGGGAAAAA TAAAGAGAAA AAAATTATTT AAAAGGTTCT 420

GGGTGTAGAT TCAATGGAAA TAATTGAAAA TTATTAGAGT AAACTAAGTA ATGAAATTCA 480

AGCTTATATC AAGTAACAGT CTGTTTAATG TCTTTGTCTA GTCGTCTAAT GTTTTTAACA 540

CTGGTATCTC CTTTTATATT AACAGATGAA CACTGGGCAG CGTAGGGATG GACCTCTTGT 600

ACTTATAGGC AGTGGGCTGT CTTCAGAACA ACAGAAAATG CTCAGTGAGC TTGCAGTAAT 660

TCTTAAGGCT AAAAAATATA CTGAGTTTGA CAGTACAGGT GAGGATTTTG AATTTTGGGA 720

GGTGGGGTAG AAAAAATGTT AAATAGATGA TCCTTTTGGA GAACTACCTT TGATAATTTA 780

CATATGTTTT AACCATTGGG AGATGGCTGT ATACTTTGCA TCTTGTAATA AATCTAAATT 840

TTTTTTCAGT AATAAACTAC TTATAGACAA CAACGTAGTT AGGAAATGTA AAGTTTAAAG 900

GTTTGCATAT ATTTT 915

(2) INFORMATION FOR SEQ ID NO: 129:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 464 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 226..318

(D) OTHER INFORMATION :/note= "Exon IX" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 129:

CAATATGGCT TTAAGATATA TGGTTTATGA TCTGATTTTT TATATTGATG GCCAGGTTAG 60

AGAACTAGAT ACTAAATAGA AGTAGTCTTA CACTTAAGTG TAAAAATTGT TGCCTTTGAA 120

GATTCAGATA TAAGCTTACA AAATATAGAT GAGTTATAAG AAGCAGGCCA AAGAAATACT 180

TTGGCTTGTA TCTTTCTTTC TCTTACTGCT TTTTTTGTAT TTTAGTAACT CATGTTGTTG 240

TTCCTGGTGA TGCAGTTCAA AGTACCTTGA AGTGTATGCT TGGGATTCTC AATGGATGCT 300

GGATTCTAAA ATTTGAATGT AAGTGTTGGA TTTGAGAGAA TTAAGAAATG AATTAGACTA 360

GTTTTGTTTT TCATGGTTAT TAATGCCTGT GATTAAGGAA CTTGATGTTA ATTTTCTTAC 420

CTCTGGTTAG TCACTGCATT TTGGAAAAGC TTCTGGCTGG GCGC 464

(2) INFORMATION FOR SEQ ID NO: 130:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4334 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

{ ix ) FEATURE :

(A) NAME/KEY: misc_feature

(B) LOCATION: 519..616

(D) OTHER INFORMATION: /note= "Exon X"

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 2019..2351

(D) OTHER INFORMATIO :/note= "Exon XI"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 130:

CCCTGTTGTG TGGCTAGCTG AGCTTGGTGC TGTAGACTAA AGCACATTCC TTCATGTCAA 60

ATCACTTACA GTTTAACAGA CGATTAGACA TATAACTGTC AAAATAAGCA GTATAGATGG 120

TAAGTGCTCA GTTTAGGTTA TTGTGTCATG GACTTTTTAT TCACCTTAAT TTTGGGTAAT 180

TGCTATGAGT GGAAATGTAG ACTTTTATTT TTGTCTTTGA AATAGTATCC TGGCTTAGAT 240

TTTTCAGAAA GGAGATTAAA ATTACAGTTA GTGTTCAGTA CTAACTTATG GCTTAATCCT 300

CCAAATAAAG AGTTTTTTAA AATATTTTCT TTATATGGGA AAACCAGTTG TATTACATTT 360

TGTTTTGGCA TAAGTAAGAT TTCTGTTTGC ATTTTAGAAT AATACTTAAA AACTGCCATG 420

AAGAAGAAAA ACCACTTAGG TAAATTGCTT GATTTTAATG AGAGAGATAT AGTGCTCACT 480

TGATACTTAG TTTGCTTTAA TTCTTGTGTT TTTGTCAGGG GTAAAAGCAT GTCTACGAAG 540

AAAAGTATGT GAACAGGAAG AAAAGTATGA AATTCCTGAA GGTCCACGCA GAAGCAGGCT 600

CAACAGAGAA CAGCTGGTAT TTTTCTTTTA ATACAACTTT CATTGTTCTT ATTATGACAT 660

ACTATTATTA TCACCATCAG GAAGAACTTC TGCCCTTTCA ACAGCTACAG GTGACTGATT 720

AAAATTTTAA TTGTGCTTAT TTCAAGCACT TGATTCTGAA AGATGATCAC GATGAGCAGT 780

AAAATCCAGA AGGTAATAAT TTCATACTGT TAATGGATTT TTGGCATCTT GAACATTGCC 840 ATAAACCTTT CAGAATCTGA GGTAAATCTC AGATACAGGA AGTAGCTTGA AAGAAGACTT 900

ACAGCTGCTG CTTGGATTTA GTTACCATAT GTCTCTATGG CCACATATTG TAGCTTTAAT 960

GGATAATATC GCATTATCCT GTTGATATTA TATAAGTATA TTAGAAGTCA CAAAGAAAAT 1020

TTCCATAGAA GGGAATTATG AAACTTTTTT TATTTCCAAC GAGCATACGG AAGTATGTTT 1080

CATAGCTAAT TGGATCCCTA GCCTCAGCAC AAAAATCTTT TGTGCCCCGT GAATACATTT 1140

CTGGAACCCT GGAGGGCACA CCCCCATGGT GGCTGCCCTG GAGACCTTAG GTTGGTAATA 1200

TGTAAGGACC TGAATGTGGA TGGGCAGAAT TGGATAAAAG TCCACGGAAA AGATGTTACT 1260

CTTGTAATTT AATAATGTTT AGCCTGGTGT CTCTGAAGCC TATTTCAAAT AAGCTAGGAG 1320

TTGTGGAGGC TTTAAGTCCC ACCAAATAAG CATAAACATC CTGATGAAAA AAGTTTGATG 1380

AATAGTTTGT TTTTTTCTTT ATACCAAGCA TATCTAAAAT TTTAGAAGAG TGAAAAGGAA 1440

CCGAGATGGT GACTGAATCT TAGGGAAAAA ATTGTAAATA GGAAGCCCCT ATTTGCCTAA 1500

GTATTTTTCT TGATCCAGTT AGTATGCTTG AAATATAACT TGTCCCAGCA CCTCATTAAG 1560

TAGCTTCTTA GCTGCTCATA ATTGTTACAG ATGGAGCATT CCTAATCCAA CATCTAAAAT 1620

GCTCCAAAAT CCAAAACTTT TTGAGCTTTG ACATGATGCC ACAAGTGGAA AATTCCACAC 1680

CTGACCTCAT GTGACAGGTC ACGGTCAGAA CACAGTCAAA ATTTTGTTTC ATGCACAAAA 1740

TTACTGAAGA TATTGTATAA AATTACTTCA GGCTATGTGC ATAAGGTGTA CAAGAAACAA 1800

ACGAATTTTG TGTTTAGGCT TGAGCCTCAT CCTTAAGATA CCTCATGTAT ATGCAAATTT 1860

TCCAAAACCC AAAAAATTTC TGAATCTGAA ATGCTTCTCG TCCAAATGTT TCAGGTAAGG 1920

GATATTCAAC TTGTATTTTT ATTTTCCTCA TTCATATACA GTGTTTTTGA ATACAGTATT 1980

TTGATCTGCC TTTAACAAAT GTTTTCTCAT TATTTCAGTT GCCAAAGCTG TTTGATGGAT 2040

GCTACTTCTA TTTGTGGGGA ACCTTCAAAC ACCATCCAAA GGACAACCTT ATTAAGCTCG 2100

TCACTGCAGG TGGGGGCCAG ATCCTCAGTA GAAAGCCCAA GCCAGACAGT GACGTGACTC 2160

AGACCATCAA TACAGTCGCA TACCATGCGA GACCCGATTC TGATCAGCGC TTCTGCACAC 2220

AGTATATCAT CTATGAAGAT TTGTGTAATT ATCACCCAGA GAGGGTTCGG CAGGGCAAAG 2230

TCTGGAAGGC TCCTTCGAGC TGGTTTATAG ACTGTGTGAT GTCCTTTGAG TTGCTTCCTC 2340

TTGACAGCTG AATATTATAC CAGATGAACA TTTCAAATTG AATTTGCACG GTTTGTGAGA 2400

GCCCAGTCAT TGTACTGTTT TTAATGTTCA CATTTTTACA AATAGGTAGA GTCATTCATA 2460

TTTGTCTTTG AATCAAAAAA AAAAAAAAAA AGTCTAATGC CAGATTAGGA ATTCATGTTG 2520

TGTTTACCAT TTAGAAGCTG GGATTGCTTT TAAAGGTTTT TCTTTTTAAA ATTGGCATGT 2580

TTTTGATTTA TCATGTCTTT CTATTCAGAT TATTGGGTAT CAAAGATTAA TGAGGACACC 2640

AGAATCTTGG TTAAATAGAC AAGTGGTATC ATTACTGTTT GAGTCTTTTA ATATTCTCCA 2700

TACCTGCCAC CAGTGAAAAA ACTTGCCTTT TTTTTTTTTT TTTTTTTAGT AAACAGAATA 2760

TTATCAAACA ATTTATTTTG GCTTTATTGA AAAAAGAGTA TTTGGTCTAA ATGTGCCACC 2820 ATAGGTGTTA AATTCTCCTA TCTGCATTTG TCTTTATCCT ATATTGTGTT CATTTCTTTT 2880

CTTAATAATT TACTTTGTTG TGTCTTTCTA CACTTTCATC CCTGTTTTTT ATCTTGTATA 2940

TCATCAGGAA ATTGTGATTT AATCATTAAC ATTGGTTTTT TTGTGTGTGT GGTAAAAATC 3000

AACACTAGGC TCATGGTACA TATTTTTATT CTGTACATTT GCTTGTAACT ATCAATTTGT 3060

AACTCTGTTT ATCTACTACA TGTGTATATA TACTTAGAGC ATTTTCTCTA ACACATTTTA 3120

ATGTTAGTAT TTTTTAAAAG GTCTGACCAG TCTAGCAAAT TGTCAGTCCA ACGTCATTAC 3180

TTTAAATTAA GAAGCAGTCT TCTTCTGGTA AACCTTGTTG GTATTTGTAA AATAATTTTG 3240

AAGGTCTTAA TTTCTTCCTT TGTAAAAGGA AAAGGTTTTT TTTAAAGTTT TTAGGTTGGC 3300

ATGGAGGCAG AAGTTGGTGA TTACTTGATT TACAACAGAT TTTTTCCAGA TCATACAAAA 3360

GGCCATACAG TAAGTATAGA AGTAGGTATG GGGAGGGCTT ACTAATATCA AATAGGCAAG 3420

GCCTTAGTGA GTGGGCAGGA TACCACTTGA GAGTGGCCAG ATCTGGGGAG GTTACTCTGC 3480

TCTGGGTGCT CTCATTCATG AATCGACAAG GATACATTAG ATTATTTTGA AACATTTTTT 3540

TAAGAAGCAG AATTCTTTAA TAATTCCTTC CTAGACATTG AATATACTTA TAAAATTAAA 3600

GACTTGGGGA AGGAGACACT GAGAGACTTG CCAGTTTGGT TCCTCATGAA CAAAAGAGGA 3660

CAGTTTGATA ACTACCAGAA TAGAATATCC CTAGTTTTAA AATAGTGAGA ATCTCTGAAG 3720

TTCATCAACA TCTTAAGATG CACTTACTTG AAAGTTTGAG ATTCTGTTTA TCATTTGAAA 3780

ACACATTTTG CTTTAATTCT TTCTTTGACA TGTTGTTTTT TCATATCAAG AAATATATGA 3840

ACAAAATAAT AACCTTTTGA CCCTGACCTT GCTGGGTGAA TTAGCTCTGA AACACTCTCT 3900

ACAACCAGTA ATGCATTTGT CCCACATTTC ATTCTGATAG AAAATGAACA CCATAGCACC 3960

AAACAAAAAT CCGAGGCGTT AGATAATGTC TGGATTAAAT AATTTAAGAC TCTCTAGGAT 4020

TTTGGTTGTC ATTTTTTATT TATAACAGAC TTTAAGTCAC TTTCTGTTGC CTCATAGGTC 4080

ACATTTTAGA CAGGTTTGTG TCTGTTCCTT GCATCTGAAT TCCTGATTGT AAAGACACCT 4140

ATGAGGTCTC TTAGTTTTTG TCATTCATTT TCTTGGTTTA TCACCCCTCC CTTCTTTTTG 4200

TTGTTTTTCC CTGACTGTTA AGCAGTTTCA TCTTTGCTTT TGTTAAATAT TTGACAGCAG 4260

TTAGTTTGTG TTAAGCTCTT GAAACTTGTG ATTGTACTTT CTGTGTAGAT ATACATGTAA 4320

TTATTTTTTA TTTT 4334

Claims

CLAIMS:

1. A nucleic acid segment comprising an isolated DNA sequence that encodes a BARDl,

B123, BE2, BE 14, BE31 or BE445 protein, polypeptide or peptide.

2. The nucleic acid segment of claim 1, comprising an isolated DNA sequence that encodes a BARDl protein, polypeptide, peptide or mutant thereof.

3. The nucleic acid segment of claim 2, comprising an isolated DNA sequence that encodes a BARDl protein characterized as:

(a) being between about 752 and about 777 amino acids in length;

(b) comprising an amino-terminal RING motif or domain that mediates the association of BARDl with the protein BRCAI ;

(c) containing ankyrin repeats that are not required for binding to the protein BRCAI ;

(d) comprising carboxy-terminal BRCT domains that are homologous to the carboxy-terminal sequences of the protein BRCAI ;

(e) being encoded by sequences on human chromosome 2q; and

(f) binding to the amino-terminal region of the protein BRCA 1.

4. The nucleic acid segment of claim 2 or 3, comprising an isolated DNA sequence that encodes an isolated BARDl domain.

5. The nucleic acid segment of claim 4, comprising an isolated DNA sequence that encodes an isolated BARDl ankyrin repeat, BARDl BRCT-like, BARDl RING motif or BARDl BRCAl-binding domain.

6. The nucleic acid segment of any one of claims 2 to 5, comprising an isolated DNA sequence that encodes a wild type BARDl protein or peptide that includes a contiguous amino acid sequence of at least about six amino acids from the sequence of SEQ ID NO:2, SEQ ID NO.21, SEQ ID NO.23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31 or SEQ ID NO:39.

7. The nucleic acid segment of any one of claims 2 to 6, comprising an isolated DNA sequence that encodes a BARDl protein or peptide that includes a contiguous amino acid sequence of at least about six amino acids from SEQ ID NO:2.

8. The nucleic acid segment of any one of claims 2 to 6, comprising an isolated DNA sequence that encodes a BARDl protein or peptide that includes a contiguous amino acid sequence of at least about six amino acids from the sequence of SEQ ID NO:21 , SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31 or SEQ ID NO:39.

9. The nucleic acid segment of any one of claims 2 to 6, comprising an isolated DNA sequence that includes a contiguous nucleic acid sequence from between position 75 and position 2406 of SEQ ID NO:l, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO.30 or SEQ ID NO:38, or from between position 75 and position 2385 of SEQ ID NO:26.

10. The nucleic acid segment of any one of claims 2 to 6, comprising an isolated DNA sequence that encodes a full length wild type BARDl protein having the contiguous amino acid sequence of SEQ ID NO:2, SEQ ID NO:21 , SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31 or SEQ ID NO:39.

11. The nucleic acid segment of claim 10, having the DNA sequence of SEQ ID NO:l, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30 or SEQ ID NO:38.

12. The nucleic acid segment of any one of claims 2 to 5, comprising an isolated DNA sequence that encodes a mutant BARDl protein or peptide that includes a contiguous amino acid sequence of at least about six amino acids from SEQ ID NO:33, SEQ ID NO:35 or SEQ ID NO:37.

13. The nucleic acid segment of claim 12, comprising an isolated DNA sequence that includes a contiguous nucleic acid sequence from between position 75 and position 2406 of SEQ ID NO:32, SEQ ID NO:34 or SEQ ID NO:36.

14. The nucleic acid segment of claim 12, comprising an isolated DNA sequence that encodes a full length mutant BARDl protein having the contiguous amino acid sequence of SEQ ID NO:33, SEQ ID NO:35 or SEQ ID NO:37.

15. The nucleic acid segment of claim 14, having the DNA sequence of SEQ ID NO:32, SEQ ID NO:34 or SEQ ID NO:36.

16. The nucleic acid segment of claim 12, comprising an isolated DNA sequence that encodes a mutant BARDl peptide of from about six to about thirty amino acids in length, the peptide including at least one amino acid that is different to the amino acid in the corresponding position within the wild type BARDl protein sequence, the difference being a mutation that is indicative of a malignant phenotype.

17. The nucleic acid segment of claim 1, comprising an isolated DNA sequence characterized as:

(a) a B123 DNA sequence encoding a B123 protein or peptide that includes a contiguous amino acid sequence of at least about six amino acids from SEQ ID NO:19;

(b) a BE2 DNA sequence encoding a BE2 protein or peptide that includes a contiguous amino acid sequence of at least about six amino acids from SEQ ID NO.41 ;

(c) a BE14 DNA sequence encoding a BE14 protein or peptide that includes a contiguous amino acid sequence of at least about six amino acids from SEQ ID

NO:43;

(d) a BE31 DNA sequence encoding a BE31 protein or peptide that includes a contiguous amino acid sequence of at least about six amino acids from SEQ ID NO:45; or

(e) a BE445 DNA sequence encoding a BE445 protein or peptide that includes a contiguous amino acid sequence of at least about six amino acids from SEQ ID NO:47.

18. The nucleic acid segment of claim 17, wherein said isolated DNA sequence is characterized as:

(a) a B123 DNA sequence that includes a contiguous nucleic acid sequence from between position 46 and position 864 of SEQ ID NO: 17;

(b) a BE2 DNA sequence that includes a contiguous nucleic acid sequence from between position 37 and position 819 of SEQ ID NO:40;

(c) a BE 14 DNA sequence that includes a contiguous nucleic acid sequence from between position 1 and position 666 of SEQ ID NO:42;

(d) a BE31 DNA sequence that includes a contiguous nucleic acid sequence from between position 1 and position 693 of SEQ ID NO:44; or

(e) a BE445 DNA sequence that includes a contiguous nucleic acid sequence from between position 1 and position 816 of SEQ ID NO:46.

19. The nucleic acid segment of claim 18, characterized as:

(a) a B 123 DNA sequence having the contiguous DNA sequence of SEQ ID NO: 17;

(b) a BE2 DNA sequence having the contiguous DNA sequence of SEQ ID NO:40;

(c) a BE14 DNA sequence having the contiguous DNA sequence of SEQ ID NO:42;

(d) a BE31 DNA sequence having the contiguous DNA sequence of SEQ ID NO:44; or

(e) a BE445 DNA sequence having the contiguous DNA sequence of SEQ ID NO:46.

20. The nucleic acid segment of any preceding claim, wherein said nucleic acid segment comprises a first DNA coding region that encodes a first protein or peptide selected from BARDl, B123, BE2, BE14, BE31 or BE445 and a second DNA coding region that encodes a second, distinct selected protein or peptide.

21. The nucleic acid segment of claim 20, wherein said second DNA coding region encodes a selected tumor suppressor protein or peptide.

22. The nucleic acid segment of claim 20 or 21 , wherein said first DNA coding region is operatively linked in frame to said second DNA coding region, said first and second DNA coding regions encoding a fusion protein.

23. A nucleic acid segment characterized as:

(a) a nucleic acid segment comprising a sequence region that consists of at least about 20 contiguous nucleotides that have the same sequence as, or are complementary to, about 20 contiguous nucleotides of SEQ ID NO: l, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:l 1, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26;

SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO: 129 or SEQ ID NO: 130; or (b) a nucleic acid segment of from about 20 to about 20,000 nucleotides in length that hybridizes to the nucleic acid segment of SEQ ID NO:l, SEQ ID NO:9,

SEQ ID NO:10, SEQ ID NO: l l , SEQ ID NO: 12, SEQ ID NO:13, SEQ ID

NO:14, SEQ ID NO:15, SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26; SEQ ID

NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO.46, SEQ ID NO:122, SEQ ID NO: 123, SEQ ID NO:I24, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO: 127, SEQ ID NO:i28, SEQ ID NO: 129 or SEQ ID NO: 130; or the complements thereof, under standard hybridization conditions.

24. The nucleic acid segment of claim 23, wherein the segment comprises a sequence region of at least about 20 contiguous nucleotides from SEQ ID NO:l, SEQ ID NO:9, SEQ ID NO: 10,

SEQ ID NO: 1 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26; SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID

NO: 127, SEQ ID NO: 128, SEQ ID NO: 129 or SEQ ID NO: 130; or the complements thereof.

25. The nucleic acid segment of claim 23, wherein the segment hybridizes to the nucleic acid segment of SEQ ID NO:l, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: l 1, SEQ ID NO: 12,

SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26; SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34: SEQ ID NO:36; SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO.44, SEQ ID NO:46, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID

NO: 129 or SEQ ID NO: 130, or the complements thereof, under standard hybridization conditions.

26. The nucleic acid segment of claim 23, wherein the segment comprises a sequence region of at least about 20 contiguous nucleotides from SEQ ID NO:l , or the complement thereof; or wherein the segment hybridizes to the nucleic acid segment of SEQ ID NO:l , or the complement thereof, under standard hybridization conditions.

27. The nucleic acid segment of claim 23, wherein the segment comprises a sequence region of at least about 20 contiguous nucleotides from SEQ ID NO:20, SEQ ID NO:22, SEQ ID

NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30 or SEQ ID NO:38, or the complements thereof; or wherein the segment hybridizes to the nucleic acid segment of SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30 or SEQ ID NO:38, or the complements thereof, under standard hybridization conditions.

28. The nucleic acid segment of claim 23, wherein the segment comprises a sequence region of at least about 20 contiguous nucleotides from SEQ ID NO:32, SEQ ID NO:34 or SEQ ID NO:36, or the complements thereof; or wherein the segment hybridizes to the nucleic acid segment of SEQ ID NO:32, SEQ ID NO:34 or SEQ ID NO:36, or the complements thereof, under standard hybridization conditions.

29. The nucleic acid segment of claim 23, wherein the segment comprises a sequence region of at least about 20 contiguous nucleotides from SEQ ID NO: 17, SEQ ID NO:40, SEQ ID NO.42, SEQ ID NO:44 or SEQ ID NO:46, or the complements thereof; or wherein the segment hybridizes to the nucleic acid segment of SEQ ID NO: 17, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44 or SEQ ID NO:46, or the complements thereof, under standard hybridization conditions.

30. The nucleic acid segment of claim 23, wherein the segment comprises a sequence region of at least about 20 contiguous nucleotides from SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:l 1 , SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO: 16 or SEQ ID NO: 18, or the complements thereof; or wherein the segment hybridizes to the nucleic acid segment of SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: l 1 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 18, or the complements thereof, under standard hybridization conditions.

31. The nucleic acid segment of claim 24, wherein the segment comprises a sequence region of at least about 25, about 30, about 50, about 100 or about 500 contiguous nucleotides from SEQ ID NO:l, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:l 1, SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26; SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42,

SEQ ID NO:44 or SEQ ID NO:46; or the complements thereof.

32. The nucleic acid segment of claim 25, wherein the hybridizing segment is about 30, about 50, about 100, about 500, about 1,000, about 3,000, about 5,000, about 10,000 or about

15,000 nucleotides in length.

33. The nucleic acid segment of claim 31, wherein the segment comprises a sequence region that consists of about 2531 contiguous nucleotides of SEQ ID NO: 1 , SEQ ID NO:20, SEQ ID

NO:22, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:30 or SEQ ID NO:38, or of about 2510 contiguous nucleotides of SEQ ID NO:26, or the complements thereof.

34. The nucleic acid segment of claim 31, wherein the segment comprises a sequence region that consists of about 2531 contiguous nucleotides of SEQ ID NO:32, SEQ ID NO:34 or SEQ ID NO: 36, or the complements thereof.

35. The nucleic acid segment of claim 31, wherein the segment comprises a sequence region that consists of about 938 contiguous nucleotides of SEQ ID NO: 17, about 1083 contiguous nucleotides of SEQ ID NO:40, about 1326 contiguous nucleotides of SEQ ID NO:42, about 834 contiguous nucleotides of SEQ ID NO:44 or about 898 contiguous nucleotides of SEQ ID NO:46, or the complements thereof.

36. The nucleic acid segment of any one of claims 23 to 35, further defined as a DNA segment.

37. The nucleic acid segment of any one of claims 23 to 35, further defined as a RNA segment.

38. The nucleic acid segment of any preceding claim, operatively positioned under the control of a promoter.

39. The nucleic acid segment of any preceding claim, comprised within a recombinant vector.

40. The nucleic acid segment of any one of claims 23-37, further comprising a second sequence region of at least about 20 contiguous nucleotides that have the same sequence as, or are complementary to, SEQ ID NO:l, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:l 1, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26; SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO:128, SEQ ID NO: 129 or SEQ ID NO: 130, said sequence region and said second sequence region from spatially distant regions within SEQ ID NO:l, SEQ ID NO:9, SEQ ID NO: 10. SEQ ID NO:l 1 , SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26; SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO: 122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO: 126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129 or SEQ ID NO:130..

41. A nucleic acid segment in accordance with any one of claims 1 to 22 for use in the preparation of a recombinant BARDl, B123, BE2, BE14, BE31 or BE445 protein, polypeptide. peptide, mutant or fusion protein thereof.

42. Use of a nucleic acid segment in accordance with any one of claims 1 to 22 in the preparation of a recombinant BARDl, B123, BE2, BE 14, BE31 or BE445 protein, polypeptide, peptide, mutant or fusion protein thereof.

43. A composition comprising at least a first nucleic acid segment in accordance with any one of claims 1 to 40 for use in the preparation of a composition for use in detecting a BARDl, B123, BE2, BE14, BE31 or BE445 nucleic acid sequence.

44. Use of a composition comprising at least a first nucleic acid segment in accordance with any one of claims 1 to 40 in the preparation of a composition for use in detecting a BARDl, B123, BE2, BE14, BE31 or BE445 nucleic acid sequence.

45. A nucleic acid segment in accordance with any one of claims 2 to 1 1 for use in the preparation of a wild type BARDl composition for use in detecting or purifying a BRCAI protein.

46. Use of a nucleic acid segment in accordance with any one of claims 2 to 1 1 in the preparation of a wild type BARDl composition for use in detecting or purifying a BRCAI protein.

47. A composition comprising at least a first nucleic acid segment in accordance with any one of claims 1 to 40 for use in the preparation of a diagnostic formulation for use in identifying a patient having or at risk for developing cancer.

48. Use of a composition comprising at least a first nucleic acid segment in accordance with any one of claims 1 to 40 in the preparation of a diagnostic formulation for use in identifying a patient having or at risk for developing cancer.

49. A method of using a nucleic acid segment that comprises an isolated BARDl, B123, BE2, BE14, BE31 or BE445 DNA sequence, the method comprising expressing said nucleic acid segment in a recombinant host cell to prepare a BARDl, B123, BE2, BE 14, BE31 or BE445 protein or peptide expression product in said cell.

50. A method for detecting BARDl, B123, BE2, BE14, BE31 or BE445 in a sample, comprising contacting sample nucleic acids from a sample suspected of containing BARDl, B123, BE2, BE14, BE31 or BE445 with a nucleic acid segment that encodes a BARDl, B123, BE2, BE 14, BE31 or BE445 protein or peptide, respectively, under conditions effective to allow hybridization of substantially complementary nucleic acids, and detecting the hybridized complementary nucleic acids thus formed.

51. A method of detecting a BRCAI protein, comprising contacting a sample suspected of containing a BRCAI protein with a BRCAl-binding protein selected from a BARDl, B123, BE2, BE14, BE31 or BE445 protein, peptide or fusion protein, under conditions effective to allow the formation of BRCAI -BRCAI -binding protein complexes, and detecting the BRCA1- BRCA1 -binding protein complexes so formed.

52. A method of purifying a BRCAI protein, comprising contacting a composition comprising a BRCAI protein with a BRCAl-binding protein selected from a BARDl, B123, BE2, BE14, BE31 or BE445 protein, peptide or fusion protein, under conditions effective to allow the formation of BRCAI -BRCAI -binding protein complexes, and obtaining the BRCAI protein from said BRCAI -BRCAI -binding protein complexes.

53. A method for identifying a patient having or at risk for developing cancer, comprising determining the type or amount of BARDl, B123, BE2, BE14, BE3 ! or BE445 present within a biological sample from said patient, wherein the presence of a BARDl, B123, BE2, BE14, BE31 or BE445 mutant or an altered amount of wild type BARDl, B123, BE2, BE14, BE31 or

BE445 in comparison to a sample from a normal subject, is indicative of a patient having or at risk for developing cancer.

54. A recombinant host cell comprising a nucleic acid segment in accordance with any one of claims 1 to 40.

55. A composition comprising an isolated BARDl , B123, BE2, BE!4, BE31 or BE445 protein, polypeptide, peptide, domain, mutant or fusion protein thereof.

56. The composition of claim 55, comprising an isolated BARDl protein, polypeptide. peptide, domain or fusion protein thereof that includes a contiguous amino acid sequence of at least about six amino acids from SEQ ID NO:2, SEQ ID NO:21 , SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31 , SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37 or SEQ ID NO:39.

57. A BARDl protein, polypeptide, peptide, domain, mutant or fusion protein thereof for use in the preparation of an anti-BARDl antibody.

58. Use of a BARDl protein, polypeptide, peptide, domain, mutant or fusion protein thereof in the preparation of an anti-BARDl antibody.

59. A BARDl protein, polypeptide, peptide, domain or fusion protein thereof for use in the detection or purification of a BRCAI protein.

60. Use of a BARDl protein, polypeptide, peptide, domain or fusion protein thereof in the detection or purification of a BRCAI protein.

61. A BARDl protein, polypeptide, peptide, domain or fusion protein thereof for use in the identification of a binding protein agonist or antagonist that alters the binding of BARDl to

BRCAI or that alters a biological activity of a BRCAl-BARDl complex.

62. Use of a BARDl protein, polypeptide, peptide, domain or fusion protein thereof in the identification of a binding protein agonist or antagonist that alters the binding of BARDl to BRCAI or that alters a biological activity of a BRCAl-BARDl complex.

63. A method for identifying a binding protein agonist or antagonist, comprising contacting a composition comprising BRCAI and either BARDl , B123, BE2, BE14, BE31 or BE445, with a candidate substance and identifying a candidate substance that alters the binding of BARDl, B123, BE2, BE14, BE31 or BE445 to BRCAI or that alters a biological activity of a complex comprising BRCAI and either BARDl , B 123, BE2, BE 14, BE31 or BE445.

64. An antibody having immunospecificity for a BARDl, B123, BE2, BE 14, BE31 or BE445 protein or peptide.

65. An anti-BARDl antibody for use in the preparation of a diagnostic formulation for use in identifying a patient having or at risk for developing cancer.

66. Use of an anti-BARDl antibody in the preparation of a diagnostic formulation for use in identifying a patient having or at risk for developing cancer.

67. A method for detecting BARDl, B123, BE2, BE 14, BE3 ! or BE445 in a sample, comprising contacting a sample suspected of containing BARDl, B123, BE2, BE14, BE31 or BE445 with a first antibody that binds to a BARDl , B323, BE2, BE14, BE31 or BE445 protein or peptide, respectively, under conditions effective to allow the formation of immune complexes, and detecting the immune complexes thus formed.

68. A method of identifying a candidate tumor suppressor gene or oncogene, comprising the steps of:

(a) obtaining a first DNA segment comprising a candidate gene; said first DNA segment expressing a first fusion protein comprising a transcriptional transactivating domain operatively attached to the candidate protein encoded by said candidate gene;

(b) obtaining a second DNA segment that expresses a second fusion protein comprising a BRCAI or BARDl RING domain operatively attached to a

DNA binding domain that binds to a defined nucleic acid sequence;

(c) providing said first and second DNA segments to a eukaryotic host cell that comprises a marker gene operatively positioned downstream of said defined nucleic acid sequence; and

(d) identifying a eukaryotic host cell that expresses said marker gene, thereby identifying said candidate gene as a candidate tumor suppressor gene or oncogene.

69. The method of claim 68, wherein said second DNA segment in step (b) expresses a second fusion protein comprising a BRCAI RING domain.

70. The method of claim 68, wherein said second DNA segment in step (b) expresses a second fusion protein comprising a BARDl RING domain.

71. The method of claim 68, wherein said method further comprises isolating the candidate tumor suppressor gene or oncogene identified in step (d) from said first DNA segment.

72. The method of claim 68, wherein said first fusion protein comprises a GAL4 or a VP16 transcriptional transactivating domain.

73. The method of claim 72, wherein said second fusion protein comprises a GAL4 DNA binding domain and wherein said defined nucleic acid sequence comprises a GAL4 binding domain recognition sequence.

74. The method of claim 68, wherein said eukaryotic host cell is a yeast host cell.

75. The method of claim 68, wherein said eukaryotic host cell is a mammalian host cell.

76. The method of claim 68, wherein said method comprises the steps of:

(a) obtaining a plurality of first DNA segments comprising a plurality of candidate tumor suppressor genes or oncogenes;

(b) obtaining multiple copies of said second DNA segment;

(c) providing said plurality of first DNA segments and multiple copies of said second DNA segments to a population of said eukaryotic host cells in an amount sufficient to provide about one first DNA segment and at least about one second DNA segment to each host cell in said population;

(d) culturing said population of cells under conditions and for a period of time effective to allow marker gene expression; and

(e) detecting a host cell from said population that expresses said marker gene, thereby identifying the presence in said cell of a first DNA segment that comprises a candidate tumor suppressor gene or oncogene.