WO2003044212A2 - Nucleic acids and polypeptides of invertebrate brca2 and methods of use - Google Patents

Abstract

BRCA2 (BRCA2) nucleic acids and proteins that have been isolated from Drosophila melanogaster and Tribolium castaneum are described. The BRCA2 nucleic acids and proteins can be used to genetically modify metazoan invertebrate organisms or cultured cells, resulting in BRCA2 expression or mis-expression. The genetically modified organisms or cells can be used in screening assays to identify candidate compounds which are potential pesticidal agents or therapeutics that interact with BRCA2 protein. They can also be used in methods for studying BRCA2 activity and identifying other genes that modulate the function of, or interact with, the BRCA2 gene.

Description

NUCLEIC ACIDS AND POLYPEPTIDES OF INVERTEBRATE BRCA2 AND METHODS OF USE

REFERENCE TO RELATED APPLICATION

This application claims priority to US provisional patent application 60/332,947, filed 11/16/2001. The contents of the prior application are hereby incorporated in its entirety.

BACKGROUND OF THE INVENTION

BRCA2 is a tumor suppressor gene that was identified (Wooster R, et al., (1995) Nature 378:789-792; Tavtigian S, et al., (1996) Nat Genet 12:333-337) in families predisposed to breast cancer. In addition to familial breast cancer, BRCA2 has also been linked to other cancers including canceros of ovary, hepatocellular, pancreas, and prostate (Gudmundsson J, et al., (1996) Am J Hum Genet 58:749-756; Thorlacius S, et al., (1996) Nat Genet 13:117-119; Katagiri T., et al., (1996) Cancer Res 56:4575-4577; Goggins M, et al., (1996) Cancer Res 56:5360-5364). Human BRCA2 gene encodes a large protein of 3418 amino acids (Tavtigian, supra), is highly expressed in rapidly proliferating cells, with the expression peaking at the Gl/S boundary of the cell cycle. BRCA2-null mouse embryos are nonviable at a very early stage of development and blastocysts derived from these embryos are very sensitive to gamma-irradiation (Sharan, S. K., et al. (1997) Nature 386: 804-810). Furthermore, BRCA2 interacts with the DNA repair protein Rad51 (Sharan, supra), specifically at the BRC repeats (Chen P-L, et al., (1998) PNAS 95:5287- 5292), and expression of BRC repeats in breast cancer cells disrupts the BRCA2-Rad51 complex and leads to radiation hypersensitivity and loss of G2/M checkpoint control (Chen C-F; et al. (1999) J Biol Chem 274: 32931-32935). Thus, BRCA2 plays a crucial role in cellular response to DNA damage. Although several mammalian and vertebrate homologs of the tumor suppressor

BRCA2 have been described, only one invertebrate homolog has been identified to date in the worm C.elegans (T07E3.5; Koonin EV, et al., (1996) Nat Gent 13:266-268). Few lines of evidence, however, have hinted at the existence of a BRCA2 homolog in any other invertebrate species, such as the fruit fly Drosophila. Indeed, a likely ortholog of BRCA2 was not found after a detailed analysis of the sequenced genome of Drosophila (Rubin GM, et al., (2000) Science 287:2204-2215). Identification of novel BRCA2 orthologues in model organisms such as Drosophila melanogaster and other insect species provides important and useful tools for genetic and molecular study and validation of these molecules as potential pharmaceutical targets. The present invention discloses novel compound targets which are members of the

BRCA2 class of proteins from the fruit fly Drosophila melanogaster and the red flour beetle tribolium castaneum.

SUMMARY OF THE INVENTION It is an object of the present invention to provide invertebrate homologs of a

BRCA2, that can be used in genetic screening methods to characterize pathways that BRCA2 may be involved in as well as other interacting genetic pathways. It is also an object of the invention to provide methods for screening compounds that interact with BRCA2 such as those that may have utility as therapeutics. These and other objects are provided by the present invention which concerns the identification and characterization of novel BRCA2 in Drosophila melanogaster and Tribolium castaneum. Isolated nucleic acid molecules are provided that comprise nucleic acid sequences encoding BRCA2 protein as well as novel fragments and derivatives thereof. Vectors and host cells comprising the BRCA2 nucleic acid molecules are also described, as well as metazoan invertebrate organisms (e.g. insects, coelomates and pseudocoelomates) that are genetically modified to express or mis-express a BRCA2 protein.

An important utility of the novel BRCA2 nucleic acids and proteins is that they can be used in screening assays to identify candidate compounds which are potential therapeutics that interact with BRCA2 proteins. Such assays typically comprise contacting a BRCA2 protein or fragment with one or more candidate molecules, and detecting any interaction between the candidate compound and the BRCA2 protein. The assays may comprise adding the candidate molecules to cultures of cells genetically engineered to express BRCA2 proteins, or alternatively, administering the candidate compound to a metazoan invertebrate organism genetically engineered to express BRCA2 protein.

The genetically engineered metazoan invertebrate animals of the invention can also be used in methods for studying BRCA2 activity. These methods typically involve detecting the phenotype caused by the expression or mis-expression of the BRCA2 protein. The methods may additionally comprise observing a second animal that has the same genetic modification as the first animal and, additionally has a mutation in a gene of interest. Any difference between the phenotypes of the two animals identifies the gene of interest as capable of modifying the function of the gene encoding the BRCA2 protein.

DETAILED DESCRIPTION OF THE INVENTION The use of invertebrate model organism genetics and related technologies can greatly facilitate the elucidation of biological pathways (Scangos, Nat. Biotechnol. (1997) 15:1220-1221; Margolis and Duyk, supra). Of particular use is the insect model organism, Drosophila melanogaster (hereinafter referred to generally as "Drosophila"). An extensive search for BRCA2 nucleic acids and their encoded proteins in Drosophila and the red flour beetle Tribolium castaneum (hereinafter referred to as Tribolium) was conducted in an attempt to identify new and useful tools for probing the function and regulation of the BRCA2 genes, and for use as targets in drug discovery. Novel BRCA2 nucleic acids and their encoded protein are identified herein. The newly identified BRCA2 nucleic acids can be used for the generation of mutant phenotypes in animal models or in living cells that can be used to study regulation of BRCA2, and the use of BRCA2 as a drug target. Due to the ability to rapidly carry out large-scale, systematic genetic screens, the use of invertebrate model organisms such as Drosophila has great utility for analyzing the expression and mis-expression of a BRCA2 protein. Thus, the invention provides a superior approach for identifying other components involved in the synthesis, activity, and regulation of BRCA2 proteins. Systematic genetic analysis of BRCA2s using invertebrate model organisms can lead to the identification and validation of compound targets directed to components of the BRCA2 pathway. Model organisms or cultured cells that have been genetically engineered to express BRCA2 can be used to screen candidate compounds for their ability to modulate BRCA2 expression or activity, and thus are useful in the identification of new drug targets, therapeutic agents, diagnostics and prognostics useful in the treatment of disorders of abnormal cell cycle checkpoint activity, such as cnacer. The details of the conditions used for the identification and or isolation of novel BRCA2 nucleic acids and proteins are described in the Examples section below. Various non-limiting embodiments of the invention, applications and uses of these novel BRCA2 genes and proteins are discussed in the following sections. The entire contents of all references, including patent applications, cited herein are incorporated by reference in their entireties for all purposes. Additionally, the citation of a reference in the preceding background section is not an admission of prior art against the claims appended hereto. BRCA2 Nucleic Acids

The invention relates generally to nucleic acid sequences of BRCA2s, and more particularly BRCA2 nucleic acid sequences of Drosophila and Tribolium, and methods of using these sequences. As described in the Examples below, nucleic acid sequences were isolated from Drosophila (SEQ ID NOs:l, 3) and Tribolium (SEQ ID NO:5) that encode BRCA2 homologs, hereinafter referred to as DBRCA2.1 for SEQ ID NO:l, DBRCA2.2 for SEQ ID NO:3, and TBRCA2 for SEQ ID NO:5. In addition to the fragments and derivatives of SEQ ID NOs: 1, 3, and 5 as described in detail below, the invention includes the reverse complements thereof. Also, the subject nucleic acid sequences, derivatives and fragments thereof may be RNA molecules comprising the nucleotide sequence of SEQ ID NOs:l, 3, and 5 (or derivative or fragment thereof) wherein the base U (uracil) is substituted for the base T (thymine). The DNA and RNA sequences of the invention can be single- or double-stranded. Thus, the term "isolated nucleic acid sequence", as used herein, includes the reverse complement, RNA equivalent, DNA or RNA single- or double-stranded sequences, and DNA/RNA hybrids of the sequence being described, unless otherwise indicated.

Fragments of the BRCA2 nucleic acid sequences can be used for a variety of purposes. Interfering RNA (RNAi) fragments, particularly double-stranded (ds) RNAi, can be used to generate loss-of-function phenotypes. BRCA2 nucleic acid fragments are also useful as nucleic acid hybridization probes and replication amplification primers. Certain "antisense" fragments, i.e. that are reverse complements of portions of the coding sequence of SEQ ID NOs: 1, 3, and 5 have utility in inhibiting the function of BRCA2 proteins. The fragments are of length sufficient to specifically hybridize with the corresponding SEQ ID NOs:l, 3, and 5. The fragments consist of or comprise at least 12, preferably at least 24, more preferably at least 36, and more preferably at least 96 contiguous nucleotides of any of SEQ ID NOs:l, 3, and 5. When the fragments are flanked by other nucleic acid sequences, the total length of the combined nucleic acid sequence is less than 15 kb, preferably less than 10 kb or less than 5kb, more preferably less than 2 kb, and in some cases, preferably less than 500 bases.

Additional preferred fragments of SEQ ID NO:l encode the BRC repeats, which are located at approximately nucleotides 1772-1877, 2075-2180, and 2300-2405, respectively. Additional preferred fragments of SEQ ID NO: 3 encode the BRC repeats, which are located at approximately nucleotides 1635-1740, 1938-2043, and 2163-2268, respectively.

Additional preferred fragments of SEQ ID NO:5 encode the BRC repeats, which are located at approximately nucleotides 369-468, 472-576, and 592-696, respectively.

The subject nucleic acid sequences may consist solely of SEQ ID NOs:l, 3, or 5 or fragments thereof. Alternatively, the subject nucleic acid sequences and fragments thereof may be joined to other components such as labels, peptides, agents that facilitate transport across cell membranes, hybridization-triggered cleavage agents or intercalating agents. The subject nucleic acid sequences and fragments thereof may also be joined to other nucleic acid sequences (i.e. they may comprise part of larger sequences) and are of synthetic/non-natural sequences and/or are isolated and/or are purified, i.e. unaccompanied by at least some of the material with which it is associated in its natural state. Preferably, the isolated nucleic acids constitute at least about 0.5%, and more preferably at least about 5% by weight of the total nucleic acid present in a given fraction, and are preferably recombinant, meaning that they comprise a non-natural sequence or a natural sequence joined to nucleotide(s) other than that which it is joined to on a natural chromosome.

Derivative nucleic acid sequences of BRCA2 include sequences that hybridize to the nucleic acid sequence of SEQ ID NOs:l, 3, or 5 under stringency conditions such that the hybridizing derivative nucleic acid is related to the subject nucleic acid by a certain degree of sequence identity. A nucleic acid molecule is "hybridizable" to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule. Stringency of hybridization refers to conditions under which nucleic acids are hybridizable. The degree of stringency can be controlled by temperature, ionic strength, pH, and the presence of denaturing agents such as formamide during hybridization and washing. As used herein, the term "stringent hybridization conditions" are those normally used by one of skill in the art to establish at least a 90% sequence identity between complementary pieces of DNA or DNA and RNA. "Moderately stringent hybridization conditions" are used to find derivatives having at least 70% sequence identity. Finally, "low-stringency hybridization conditions" are used to isolate derivative nucleic acid molecules that share at least about 50% sequence identity with the subject nucleic acid sequence.

The ultimate hybridization stringency reflects both the actual hybridization conditions as well as the washing conditions following the hybridization, and it is well known in the art how to vary the conditions to obtain the desired result. Conditions routinely used are set out in readily available procedure texts (e.g., Current Protocol in Molecular Biology, Vol. 1, Chap. 2.10, John Wiley & Sons, Publishers (1994); Sambrook et al., Molecular Cloning, Cold Spring Harbor (1989)). A preferred derivative nucleic acid is capable of hybridizing to SEQ ID NOS:l, 3, or 5 under highly stringent hybridization conditions that are: prehybridization of filters containing nucleic acid for 8 hours to overnight at 65° C in a solution comprising 6X single strength citrate (SSC) (IX SSC is 0.15 M NaCl, 0.015 M Na citrate; pH 7.0), 5X Denhardt's solution, 0.05% sodium pyrophosphate and 100 μg/ml herring sperm DNA; hybridization for 18-20 hours at 65° C in a solution containing 6X SSC, IX Denhardt's solution, 100 μg/ml yeast tRNA and 0.05% sodium pyrophosphate; and washing of filters at 65° C for lh in a solution containing 0.1X SSC and 0.1% SDS (sodium dodecyl sulfate).

Derivative nucleic acid sequences that have at least about 70% sequence identity with SEQ ID NOs:l, 3, or 5are capable of hybridizing to SEQ ID NOs:l, 3, or 5under moderately stringent conditions that are: pretreatment of filters containing nucleic acid for 6 h at 40° C in a solution containing 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg/ml denatured salmon sperm DNA; hybridization for 18-20 h at 40° C in a solution containing 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon sperm DNA, and 10% (wt/vol) dextran sulfate; followed by washing twice for 1 hour at 55° C in a solution containing 2X SSC and 0.1% SDS.

Other preferred derivative nucleic acid sequences are capable of hybridizing to SEQ ID NOs:l, 3, or 5under low stringency conditions that comprise: incubation for 8 hours to overnight at 37° C in a solution comprising 20% formamide, 5 x SSC, 50 mM sodium phosphate (pH 7.6), 5X Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured sheared salmon sperm DNA; hybridization in the same buffer for 18 to 20 hours; and washing of filters in 1 x SSC at about 37° C for 1 hour.

As used herein, "percent (%) nucleic acid sequence identity" with respect to a subject sequence, or a specified portion of a subject sequence, is defined as the percentage of nucleotides in the candidate derivative nucleic acid sequence identical with the nucleotides in the subject sequence (or specified portion thereof), after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent sequence identity, as generated by the program WU-BLAST-2.0al9 (Altschul et al, J. Mol. Biol. (1997) 215:403-410; http://blast.wusd.edu/blast/README.html; hereinafter referred to generally as "BLAST") with all the search parameters set to default values. The HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched. A percent (%) nucleic acid sequence identity value is determined by the number of matching identical nucleotides divided by the sequence length for which the percent identity is being reported.

Derivative BRCA2 nucleic acid sequences usually have at least 70% sequence identity, preferably at least 80% sequence identity, more preferably at least 85% sequence identity, still more preferably at least 90% sequence identity, and most preferably at least 95% sequence identity with SEQ ID NOS:l, 3, or 5, or domain-encoding regions thereof.

In one preferred embodiment, the derivative nucleic acid encodes a polypeptide comprising a BRCA2 amino acid sequence of any of SEQ ID NOs:2, 4, or 6 or a fragment or derivative thereof as described further below under the subheading "BRCA2 proteins". A derivative BRCA2 nucleic acid sequence, or fragment thereof, may comprise 100% sequence identity with SEQ ID NOS:l, 3, or 5, but be a derivative thereof in the sense that it has one or more modifications at the base or sugar moiety, or phosphate backbone. Examples of modifications are well known in the art (Bailey, Ullmann's Encyclopedia of Industrial Chemistry (1998), 6th ed. Wiley and Sons). Such derivatives may be used to provide modified stability or any other desired property.

Another type of derivative of the subject nucleic acid sequences includes corresponding humanized sequences. A humanized nucleic acid sequence is one in which one or more codons has been substituted with a codon that is more commonly used in human genes. Preferably, a sufficient number of codons have been substituted such that a higher level expression is achieved in mammalian cells than what would otherwise be achieved without the substitutions (Wada et al, Nucleic Acids Research (1990) 18(Suppl.):2367-2411). A detailed discussion of the humanization of nucleic acid sequences is provided in U.S. Pat. No. 5,874,304 to Zolotukhin et al. Similarly, other nucleic acid derivatives can be generated with codon usage optimized for expression in other organisms, such as yeasts, bacteria, and plants, where it is desired to engineer the expression of BRCA2 proteins by using specific codons chosen according to the preferred codons used in highly expressed genes in each organism.

More specific embodiments of preferred BRCA2 protein fragments and derivatives are discussed further below in connection with specific BRCA2 proteins. Isolation, Production, And Expression of BRCA2 Nucleic Acids

Nucleic acid encoding the amino acid sequence of SEQ ID NOs: 2, 4, or 6, or fragment or derivative thereof, may be obtained from an appropriate cDNA library prepared from any eukaryotic species that encodes BRCA2 proteins such as vertebrates, preferably mammalian (e.g. primate, porcine, bovine, feline, equine, and canine species, etc.) and invertebrates, such as arthropods, particularly insects species (preferably Drosophila), acarids, Crustacea, molluscs, nematodes, and other worms. An expression library can be constructed using known methods. For example, mRNA can be isolated to make cDNA which is ligated into a suitable expression vector for expression in an isolated host cell into which it is introduced. Various screening assays can then be used to select for the gene or gene product (e.g. oligonucleotides of at least about 20 to 80 bases designed to identify the gene of interest, or labeled antibodies that specifically bind to the gene product). The gene and/or gene product can then be recovered from the host cell using known techniques.

Polymerase chain reaction (PCR) can also be used to isolate nucleic acids of the BRCA2 where oligonucleotide primers representing fragmentary sequences of interest amplify RNA or DNA sequences from a source such as a genomic or cDNA library (as described by Sambrook et al., supra). Additionally, degenerate primers for amplifying homologs from any species of interest may be used. Once a PCR product of appropriate size and sequence is obtained, it may be cloned and sequenced by standard techniques, and utilized as a probe to isolate a complete cDNA or genomic clone.

Fragmentary sequences of BRCA2 nucleic acids and derivatives may be synthesized by known methods. For example, oligonucleotides may be synthesized using an automated DNA synthesizer available from commercial suppliers (e.g. Biosearch, Novato, CA; Perkin-Elmer Applied Biosystems, Foster City, CA). Antisense RNA sequences can be produced intracellularly by transcription from an exogenous sequence, e.g. from vectors that contain antisense BRCA2 nucleic acid sequences. Newly generated sequences may be identified and isolated using standard methods. An isolated BRCA2 nucleic acid sequence can be inserted into any appropriate cloning vector, for example bacteriophages such as lambda derivatives, or plasmids such as PBR322, pUC plasmid derivatives and the Bluescript vector (Stratagene, San Diego, CA). Recombinant molecules can be introduced into host cells via transformation, transfection, infection, electroporation, etc., or into a transgenic animal such as a fly. The transformed cells can be cultured to generate large quantities of the BRCA2 nucleic acid. Suitable methods for isolating and producing the subject nucleic acid sequences are well- known in the art (Sambrook et al, supra; DNA Cloning: A Practical Approach, Vol. 1, 2, 3, 4, (1995) Glover, ed., MRL Press, Ltd., Oxford, U.K.). The nucleotide sequence encoding a BRCA2 protein or fragment or derivative thereof, can be inserted into any appropriate expression vector for the transcription and translation of the inserted protein-coding sequence. Alternatively, the necessary transcriptional and translational signals can be supplied by the native BRCA2 gene and/or its flanking regions. A variety of host-vector systems may be utilized to express the protein-coding sequence such as mammalian cell systems infected with virus (e.g. vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g. baculovirus); microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. Expression of a BRCA2 protein may be controlled by a suitable promoter/enhancer element. In addition, a host cell strain may be selected which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired.

To detect expression of the BRCA2 gene product, the expression vector can comprise a promoter operably linked to a BRCA2 gene nucleic acid, one or more origins of replication, and, one or more selectable markers (e.g. thymidine kinase activity, resistance to antibiotics, etc.). Alternatively, recombinant expression vectors can be identified by assaying for the expression of the BRCA2 gene product based on the physical or functional properties of the BRCA2 protein in in vitro assay systems (e.g. immunoassays).

The BRCA2 protein, fragment, or derivative may be optionally expressed as a fusion, or chimeric protein product (i.e. it is joined via a peptide bond to a heterologous protein sequence of a different protein). A chimeric product can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other in the proper coding frame using standard methods and expressing the chimeric product. A chimeric product may also be made by protein synthetic techniques, e.g. by use of a peptide synthesizer.

Once a recombinant that expresses the BRCA2 gene sequence is identified, the gene product can be isolated and purified using standard methods (e.g. ion exchange, affinity, and gel exclusion chromatography; centrifugation; differential solubility; electrophoresis). The amino acid sequence of the protein can be deduced from the nucleotide sequence of the chimeric gene contained in the recombinant and can thus be synthesized by standard chemical methods (Hunkapiller et al., Nature (1984) 310:105- 111). Alternatively, native BRCA2 proteins can be purified from natural sources, by standard methods (e.g. immunoaffinity purification).

BRCA2 Proteins

BRCA2 proteins of the invention comprise or consist of an amino acid sequence of SEQ ID NOs: 2, 4, or 6, or fragments or derivatives thereof. Compositions comprising these proteins may consist essentially of the BRCA2 protein, fragments, or derivatives, or may comprise additional components (e.g. pharmaceutically acceptable carriers or excipients, culture media, etc.).

BRCA2 protein derivatives typically share a certain degree of sequence identity or sequence similarity with SEQ ID NOs:2, 4, or 6, or a fragment thereof. As used herein, "percent (%) amino acid sequence identity" with respect to a subject sequence, or a specified portion of a subject sequence, is defined as the percentage of amino acids in the candidate derivative amino acid sequence identical with the amino acid in the subject sequence (or specified portion thereof), after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent sequence identity, as generated by BLAST (Altschul et al., supra) using the same parameters discussed above for derivative nucleic acid sequences. A % amino acid sequence identity value is determined by the number of matching identical amino acids divided by the sequence length for which the percent identity is being reported. "Percent (%) amino acid sequence similarity" is determined by doing the same calculation as for determining % amino acid sequence identity, but including conservative amino acid substitutions in addition to identical amino acids in the computation. A conservative amino acid substitution is one in which an amino acid is substituted for another amino acid having similar properties such that the folding or activity of the protein is not significantly affected. Aromatic amino acids that can be substituted for each other are phenylalanine, tryptophan, and tyrosine; interchangeable hydrophobic amino acids are leucine, isoleucine, ethionine, and valine; interchangeable polar amino acids are glutamine and asparagine; interchangeable basic amino acids arginine, lysine and histidine; interchangeable acidic amino acids aspartic acid and glutamic acid; and interchangeable small amino acids alanine, serine, cystine, threonine, and glycine. hi one preferred embodiment, a BRCA2 protein derivative shares at least 80% sequence identity or similarity, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity or similarity with a contiguous stretch of at least 25 amino acids, preferably at least 50 amino acids, more preferably at least 100 amino acids, and in some cases, the entire length of any of SEQ ID NOs:2, 4, or 6. The fragment or derivative of the BRCA2 protein is preferably "functionally active" meaning that the BRCA2 protein derivative or fragment exhibits one or more functional activities associated with a full-length, wild-type BRCA2 protein comprising the amino acid sequence of SEQ ID NOs: 2, 4, or 6. As one example, a fragment or derivative may have antigenicity such that it can be used in immunoassays, for immunization, for inhibition of BRCA2 activity, etc, as discussed further below regarding generation of antibodies to BRCA2 proteins. Preferably, a functionally active BRCA2 fragment or derivative is one that displays one or more biological activities associated with BRCA2 proteins, such as interaction with Rad51. For purposes herein, functionally active fragments also include those fragments that exhibit one or more structural features of a BRCA2, such as BRC repeats, which are located at amino acids 239-273, 340-374, and 415-449 for SEQ ID NO:2, amino acids 546-580, 647-681, and 722-756 for SEQ ID NO:4, and amino acids 124-156, 158-192, and 198-232 for SEQ ID NO:6. The functional activity of BRCA2 proteins, derivatives and fragments can be assayed by various methods known to one skilled in the art (Current Protocols in Protein Science (1998) Coligan et al, eds., John Wiley & Sons, Inc., Somerset, New Jersey). In a preferred method, which is described in detail below, a model organism, such as Drosophila, is used in genetic studies to assess the phenotypic effect of a fragment or derivative (i.e. a mutant BRCA2 protein). BRCA2 derivatives can be produced by various methods known in the art. The manipulations which result in their production can occur at the gene or protein level. For example, a cloned BRCA2 gene sequence can be cleaved at appropriate sites with restriction endonuclease(s) (Wells et al., Philos. Trans. R. Soc. London SerA (1986) 317:415), followed by further enzymatic modification if desired, isolated, and ligated in vitro, and expressed to produce the desired derivative. Alternatively, a BRCA2 gene can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or to form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in vitro modification. A variety of mutagenesis techniques are known in the art such as chemical mutagenesis, in vitro site-directed mutagenesis (Carter et al., Nucl. Acids Res. (1986) 13:4331), use of TAB^® linkers (available from Pharmacia and Upjohn, Kalamazoo, MI), etc.

At the protein level, manipulations include post translational modification, e.g. glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known technique (e.g. specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBE j, acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycin, etc.). Derivative proteins can also be chemically synthesized by use of a peptide synthesizer, for example to introduce nonclassical amino acids or chemical amino acid analogs as substitutions or additions into the BRCA2 protein sequence.

Chimeric or fusion proteins can be made comprising a BRCA2 protein or fragment thereof (preferably comprising one or more structural or functional domains of the BRCA2 protein) joined at its amino- or carboxy-terminus via a peptide bond to an amino acid sequence of a different protein. Chimeric proteins can be produced by any known method, including: recombinant expression of a nucleic acid encoding the protein (comprising a BRCA2-coding sequence joined in-frame to a coding sequence for a different protein); ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other in the proper coding frame, and expressing the chimeric product; and protein synthetic techniques, e.g. by use of a peptide synthesizer.

BRCA2 Gene Regulatory Elements

BRCA2 gene regulatory DNA elements, such as enhancers or promoters, can be used to identify tissues, cells, genes and factors that specifically control BRCA2 protein production. Analyzing components that are specific to BRCA2 protein function can lead to an understanding of how to manipulate these regulatory processes, especially therapeutic applications, as well as an understanding of how to diagnose dysfunction in these processes. Gene fusions with the BRCA2 regulatory elements can be made. For compact genes that have relatively few and small intervening sequences, such as those described herein for Drosophila, it is typically the case that the regulatory elements that control spatial and temporal expression patterns are found in the DNA immediately upstream of the coding region, extending to the nearest neighboring gene. Regulatory regions can be used to construct gene fusions where the regulatory DNAs are operably fused to a coding region for a reporter protein whose expression is easily detected, and these constructs are introduced as transgenes into the animal of choice. An entire regulatory DNA region can be used, or the regulatory region can be divided into smaller segments to identify sub- elements that might be specific for controlling expression a given cell type or stage of development. Reporter proteins that can be used for construction of these gene fusions include E. coli beta-galactosidase and green fluorescent protein (GFP). These can be detected readily in situ, and thus are useful for histological studies and can be used to sort cells that express BRCA2 proteins (O'Kane and Gehring PNAS (1987) 84(24):9123-9127; Chalfie et al, Science (1994) 263:802-805; and Cumberledge and Krasnow (1994)

Methods in Cell Biology 44: 143-159). Recombinase proteins, such as FLP or ere, can be used in controlling gene expression through site-specific recombination (Golic and Lindquist (1989) Cell 59(3):499-509; White et al, Science (1996) 271:805-807). Toxic proteins such as the reaper and hid cell death proteins, are useful to specifically ablate cells that normally express BRCA2 proteins in order to assess the physiological function of the cells (Kingston, In Current Protocols in Molecular Biology (1998) Ausubel et al, John Wiley & Sons, Inc. sections 12.0.3-12.10) or any other protein where it is desired to examine the function this particular protein specifically in cells that synthesize BRCA2 proteins. Alternatively, a binary reporter system can be used, similar to that described further below, where the BRCA2 regulatory element is operably fused to the coding region of an exogenous transcriptional activator protein, such as the GAL4 or tTA activators described below, to create a BRCA2 regulatory element "driver gene". For the other half of the binary system the exogenous activator controls a separate "target gene" containing a coding region of a reporter protein operably fused to a cognate regulatory element for the exogenous activator protein, such as UASQ or a tTA-response element, respectively. An advantage of a binary system is that a single driver gene construct can be used to activate transcription from precontracted target genes encoding different reporter proteins, each with its own uses as delineated above. BRCA2 regulatory element-reporter gene fusions are also useful for tests of genetic interactions, where the objective is to identify those genes that have a specific role in controlling the expression of BRCA2 genes, or promoting the growth and differentiation of the tissues that expresses the BRCA2 protein. BRCA2 gene regulatory DNA elements are also useful in protein-DNA binding assays to identify gene regulatory proteins that control the expression of BRCA2 genes. The gene regulatory proteins can be detected using a variety of methods that probe specific protein-DNA interactions well known to those skilled in the art (Kingston, supra) including in vivo footprinting assays based on protection of DNA sequences from chemical and enzymatic modification within living or permeabilized cells; and in vitro footprinting assays based on protection of DNA sequences from chemical or enzymatic modification using protein extracts, nitrocellulose filter-binding assays and gel electrophoresis mobility shift assays using radioactively labeled regulatory DNA elements mixed with protein extracts. Candidate BRCA2 gene regulatory proteins can be purified using a combination of conventional and DNA-affinity purification techniques. Molecular cloning strategies can also be used to identify proteins that specifically bind BRCA2 gene regulatory DNA elements. For example, a Drosophila cDNA library in an expression vector, can be screened for cDNAs that encode BRCA2 gene regulatory element DNA-binding activity. Similarly, the yeast "one-hybrid" system can be used (Li and Herskowitz, Science (1993) 262: 1870-1874; Luo et al, Biotechniques (1996) 20(4):564-568; Vidal et al, PNAS (1996) 93(19):10315-10320).

Identification of Molecules that Interact With BRCA2

A variety of methods can be used to identify or screen for molecules, such as proteins or other molecules, that interact with BRCA2 protein, or derivatives or fragments thereof. The assays may employ purified BRCA2 protein, or cell lines or model organisms such as Drosophila and C. elegans, that have been genetically engineered to express BRCA2 protein. Suitable screening methodologies are well known in the art to test for proteins and other molecules that interact with BRCA2 gene and protein (see e.g., PCT International Publication No. WO 96/34099). The newly identified interacting molecules may provide new targets for pharmaceutical agents. Any of a variety of exogenous molecules, both naturally occurring and/or synthetic (e.g., libraries of small molecules or peptides, or phage display libraries), may be screened for binding capacity. In a typical binding experiment, the BRCA2 protein or fragment is mixed with candidate molecules under conditions conducive to binding, sufficient time is allowed for any binding to occur, and assays are performed to test for bound complexes. Assays to find interacting proteins can be performed by any method known in the art, for example, immunoprecipitation with an antibody that binds to the protein in a complex followed by analysis by size fractionation of the immunoprecipitated proteins (e.g. by denaturing or nondenaturing polyacrylamide gel electrophoresis), Western analysis, non-denaturing gel electrophoresis, etc.

Another preferred method for identifying interacting proteins is a two-hybrid assay system or variation thereof (Fields and Song, Nature (1989) 340:245-246; U.S. Pat. No. 5,283,173; for review see Brent and Finley, Annu. Rev. Genet. (1997) 31 :663-704).

Antibodies and immunoassays

BRCA2 proteins encoded by SEQ ID NOs:2, 4, or 6, and derivatives and fragments thereof, such as those discussed above, may be used as an immunogen to generate monoclonal or polyclonal antibodies and antibody fragments or derivatives (e.g. chimeric, single chain, Fab fragments). For example, fragments of a BRCA2 protein, preferably those identified as hydrophilic, are used as immunogens for antibody production using art-known methods such as by hybridomas; production of monoclonal antibodies in germ-free animals (PCT/US90/02545); the use of human hybridomas (Cole et al, PNAS (1983) 80:2026-2030; Cole et al, in Monoclonal Antibodies and Cancer Therapy (1985) Alan R. Liss, pp. 77-96), and production of humanized antibodies (Jones et al, Nature (1986) 321:522-525; U.S. Pat. 5,530,101). h a particular embodiment, BRCA2 polypeptide fragments provide specific antigens and/or immunogens, especially when coupled to carrier proteins. For example, peptides are covalently coupled to keyhole limpet antigen (KLH) and the conjugate is emulsified in Freund's complete adjuvant. Laboratory rabbits are immunized according to conventional protocol and bled. The presence of specific antibodies is assayed by solid phase immunosorbent assays using immobilized corresponding polypeptide. Specific activity or function of the antibodies produced may be determined by convenient in vitro, cell-based, or in vivo assays: e.g. in vitro binding assays, etc. Binding affinity may be assayed by determination of equilibrium constants of antigen-antibody association (usually at least about 10⁷ M^"1, preferably at least about 10⁸ M^"1, more preferably at least about 10⁹ M^"1).

Immunoassays can be used to identify proteins that interact with or bind to BRCA2 protein. Various assays are available for testing the ability of a protein to bind to or compete with binding to a wild-type BRCA2 protein or for binding to an anti-BRCA2 protein antibody. Suitable assays include radioimmunoassays, ELISA (enzyme linked immunosorbent assay), immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays (e.g., using colloidal gold, enzyme or radioisotope labels), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, immunoelectrophoresis assays, etc.

Identification of Potential Drug Targets Once new BRCA2 genes or BRCA2 interacting genes are identified, they can be assessed as potential drug targets.

Putative drugs and molecules can be applied onto whole insects, nematodes, and other small invertebrate metazoans, and the ability of the compounds to modulate (e.g. block or enhance) BRCA2 activity can be observed. Alternatively, the effect of various compounds on BRCA2s can be assayed using cells that have been engineered to express one or more BRCA2s and associated proteins.

Assays of Compounds on Insects

Potential compounds can be administered to insects in a variety of ways, including orally (including addition to synthetic diet, application to plants or prey to be consumed by the test organism), topically (including spraying, direct application of compound to animal, allowing animal to contact a treated surface), or by injection. Insecticides are typically very hydrophobic molecules and must commonly be dissolved in organic solvents, which are allowed to evaporate in the case of methanol or acetone, or at low concentrations can be included to facilitate uptake (ethanol, dimethyl sulfoxide).

The first step in an insect assay is usually the determination of the minimal lethal dose (MLD) on the insects after a chronic exposure to the compounds. The compounds are usually diluted in DMSO, and applied to the food surface bearing 0-48 hour old embryos and larvae. In addition to MLD, this step allows the determination of the fraction of eggs that hatch, behavior of the larvae, such as how they move /feed compared to untreated larvae, the fraction that survive to pupate, and the fraction that eclose (emergence of the adult insect from puparium). Based on these results more detailed assays with shorter exposure times may be designed, and larvae might be dissected to look for obvious morphological defects. Once the MLD is determined, more specific acute and chronic assays can be designed.

In a typical acute assay, compounds are applied to the food surface for embryos, larvae, or adults, and the animals are observed after 2 hours and after an overnight incubation. For application on embryos, defects in development and the percent that survive to adulthood are determined. For larvae, defects in behavior, locomotion, and molting may be observed. For application on adults, behavior and neurological defects are observed, and effects on fertility are noted.

For a chronic exposure assay, adults are placed on vials containing the compounds for 48 hours, then transferred to a clean container and observed for fertility, neurological defects, and death.

Assay of Compounds using Cell Cultures

Compounds that modulate (e.g. block or enhance) BRCA2 activity may also be assayed using cell culture. For example, various compounds added to cells expressing BRCA2 may be screened for their ability to modulate the activity of BRCA2 genes based upon measurements of cell survival or activity in cell cycle. Assays can be performed on cultured cells expressing endogenous normal or mutant BRCA2s. Such studies also can be performed on cells transfected with vectors capable of expressing the BRCA2s, or functional domains of one of the BRCA2s, in normal or mutant form, hi addition, to enhance the signal measured in such assays, cells may be cotransfected with genes encoding BRCA2 proteins.

For example, cell clones are generated that have tetracycline-inducible expression of BRC repeats, with GFP as markers of BRC activity (Chen C-F, et al., (1999) Biol Chem 274:32931-32935). Expression of wild type or mutant BRC is induced, in absence or presence of compounds, followed by gamma-irradiation of cells, incubation for 2 weeks. Cells are then fixed and counted.

Compounds that selectively modulate the BRCA2 are identified as potential drug candidates having BRCA2 specificity.

Identification of small molecules and compounds as potential pharmaceutical compounds from large chemical libraries requires high-throughput screening (HTS) methods (Bolger, Drug Discovery Today (1999) 4:251-253). Several of the assays mentioned herein can lend themselves to such screening methods. For example, cells or cell lines expressing wild type or mutant BRCA2 protein or its fragments, and a reporter gene can be subjected to compounds of interest, and depending on the reporter genes, interactions can be measured using a variety of methods such as color detection, fluorescence detection (e.g. GFP), autoradiography, scintillation analysis, etc.

Generation and Genetic Analysis of Animals and Cell Lines with Altered Expression of BRCA2 Gene Both genetically modified animal models (i.e. in vivo models), such as Drosophila, and in vitro models such as genetically engineered cell lines expressing or mis-expressing BRCA2 pathway genes, are useful for the functional analysis of these proteins. Model systems that display detectable phenotypes, can be used for the identification and characterization of BRCA2 pathway genes or other genes of interest and/or phenotypes associated with the mutation or mis-expression of BRCA2 pathway protein. The term "mis-expression" as used herein encompasses mis-expression due to gene mutations. Thus, a mis-expressed BRCA2 pathway protein may be one having an amino acid sequence that differs from wild-type (i.e. it is a derivative of the normal protein). A mis- expressed BRCA2 pathway protein may also be one in which one or more amino acids have been deleted, and thus is a "fragment" of the normal protein. As used herein, "mis- expression" also includes ectopic expression (e.g. by altering the normal spatial or temporal expression), over-expression (e.g. by multiple gene copies), underexpression, non-expression (e.g. by gene knockout or blocking expression that would otherwise normally occur), and further, expression in ectopic tissues. As used in the following discussion concerning in vivo and in vitro models, the term "gene of interest" refers to a BRCA2 pathway gene, or any other gene involved in regulation or modulation, or downstream effector of the BRCA2 pathway.

The in vivo and in vitro models may be genetically engineered or modified so that they 1) have deletions and/or insertions of one or more BRCA2 pathway genes, 2) harbor interfering RNA sequences derived from BRCA2 pathway genes, 3) have had one or more endogenous BRCA2 pathway genes mutated (e.g. contain deletions, insertions, rearrangements, or point mutations in BRCA2 gene or other genes in the pathway), and/or 4) contain transgenes for mis-expression of wild-type or mutant forms of such genes. Such genetically modified in vivo and in vitro models are useful for identification of genes and proteins that are involved in the synthesis, activation, control, etc. of BRCA2 pathway gene and/or gene products, and also downstream effectors of BRCA2 function, genes regulated by BRCA2, etc. The model systems can be used for testing potential pharmaceutical compounds that interact with the BRCA2 pathway, for example by administering the compound to the model system using any suitable method (e.g. direct contact, ingestion, injection, etc.) and observing any changes in phenotype, for example defective movement, lethality, etc. Various genetic engineering and expression modification methods which can be used are well-known in the art, including chemical mutagenesis, transposon mutagenesis, antisense RNAi, dsRNAi, and transgene-mediated mis-expression.

Generating Loss-of-function Mutations by Mutagenesis Loss-of-function mutations in an invertebrate metazoan BRCA2 gene can be generated by any of several mutagenesis methods known in the art (Ashburner, In Drosophila melanogaster: A Laboratory Manual (1989) , Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press: pp. 299-418; Fly pushing: The Theory and Practice of Drosophila melanogaster Genetics (1997) Cold Spring Harbor Press, Plainview, NY; The nematode C. elegans (1988) Wood, Ed., Cold Spring Harbor Laboratory Press, Cold Spring harbor, New York). Techniques for producing mutations in a gene or genome include use of radiation ( e.g., X-ray, UV, or gamma ray); chemicals (e.g., EMS, MMS, ENU, formaldehyde, etc.); and insertional mutagenesis by mobile elements including dysgenesis induced by transposon insertions, or transposon-mediated deletions, for example, male recombination, as described below. Other methods of altering expression of genes include use of transposons (e.g., P element, EP-type "overexpression trap" element, mariner element, piggyBac transposon, hermes, rninos, sleeping beauty, etc.) to misexpress genes; gene targeting by homologous recombination; antisense; double- stranded RNA interference; peptide and RNA aptamers; directed deletions; homologous recombination; dominant negative alleles; and intrabodies.

Transposon insertions lying adjacent to a gene of interest can be used to generate deletions of flanking genomic DNA, which if induced in the germline, are stably propagated in subsequent generations. The utility of this technique in generating deletions has been demonstrated and is well-known in the art. One version of the technique using collections of P element transposon induced recessive lethal mutations (P lethals) is particularly suitable for rapid identification of novel, essential genes in Drosophila (Cooley et al, Science (1988) 239:1121-1128; Spralding et al, PNAS (1995) 92:0824- 10830). Since the sequence of the P elements are known, the genomic sequence flanking each transposon insert is determined either by plasmid rescue (Hamilton et al, PNAS (1991) 88:2731-2735) or by inverse polymerase chain reaction (Rehm, website at www.fruitfly.org/methods

A more recent version of the transposon insertion technique in male Drosophila using P elements is known as P-mediated male recombination (Preston and Engels, Genetics (1996) 144:1611-1638). Gene targeting approaches using homologous recombination have proven to be successful in Drosophila (Rong and Golic, Science (2000) 288:2013-20018) and potentially provide a general method of generating directed mutations in any gene-of- interest. This method uses broken-ended extrachromosomal DNA, created in vivo, to produce homology-directed changes in a target locus. First, a "targeting construct" is designed for the gene-of-interest which allows the replacement of the normal endogenous gene with a specifically designed mutation, such as a deletion, insertion or point mutation, via homologous recombination. The targeting construct is typically carried in an appropriate transposon-mediated transgenesis vector (e.g. P element-, piggyBac-, hermes- , minos-, or mariner-based vectors) which inserts the targeting construct randomly within the genome of the organism. The targeting construct is converted to a recombinogenic extrachromosomal form by inducing the expression of separate transgenes encoding a site- specific recombinase (e.g. FLP, ere, Kw, etc.) which excises the targeting construct, and a rare-cutting site-specific endonuclease (e.g. Seel, Crel, HO, etc.) which generates recombinogenic ends that direct homologous recombination and gene replacement of the endogenous locus. Though this method has only been shown to work in Dros, it has application to worms, other animals, plants, algae etc.

Generating Loss-of-function Phenotypes Using RNA-based Methods BRCA2 genes may be identified and/or characterized by generating loss-of- function phenotypes in animals of interest through RNA-based methods, such as antisense RNA (Schubiger and Edgar, Methods in Cell Biology (1994) 44:697-713). One form of the antisense RNA method involves the injection of embryos with an antisense RNA that is partially homologous to the gene of interest (in this case the BRCA2 gene). Another form of the antisense RNA method involves expression of an antisense RNA partially homologous to the gene of interest by operably joining a portion of the gene of interest in the antisense orientation to a powerful promoter that can drive the expression of large quantities of antisense RNA, either generally throughout the animal or in specific tissues. Antisense RNA-generated loss-of-function phenotypes have been reported previously for several Drosophila genes including cactus, pecanex, and Kriippel (LaBonne et al, Dev. Biol. (1989) 136(1):1-16; Schuh and Jackie, Genome (1989) 31(l):422-425; Geisler et al, Cell (1992) 71(4):613-621).

Loss-of-function phenotypes can also be generated by cosuppression methods (Bingham Cell (1997) 90(3):385-387; Smyth, Curr. Biol. (1997) 7(12):793-795; Que and Jorgensen, Dev. Genet. (1998) 22(1): 100-109). Cosuppression is a phenomenon of reduced gene expression produced by expression or injection of a sense strand RNA corresponding to a partial segment of the gene of interest. Cosuppression effects have been employed extensively in plants and C. elegans to generate loss-of-function phenotypes, and there is a single report of cosuppression in Drosophila, where reduced expression of the Adh gene was induced from a white- Adh transgene using cosuppression methods (Pal-Bhadra et al, Cell (1997) 90(3):479-490).

Another method for generating loss-of-function phenotypes is by double-stranded RNA interference (dsRNAi). This method is based on the interfering properties of double- stranded RNA derived from the coding regions of gene, and has proven to be of great utility in genetic studies of C. elegans (Fire et al, Nature (1998) 391:806-811), and can also be used to generate loss-of-function phenotypes in Drosophila (Kennerdell and Carthew, Cell (1998) 95:1017-1026; Misquitta and Patterson PNAS (1999) 96:1451- 1456). In one example of this method, complementary sense and antisense RNAs derived from a substantial portion of a gene of interest, such as BRCA2 gene, are synthesized in vitro. The resulting sense and antisense RNAs are annealed in an injection buffer, and the double-stranded RNA injected or otherwise introduced into animals (such as in their food or by soaking in the buffer containing the RNA). Progeny of the injected animals are then inspected for phenotypes of interest (PCT publication no. WO99/32619). In another embodiment of the method, the dsRNA can be delivered to the animal by bathing the animal in a solution containg a sufficient concentration of the dsRNA. In another embodiment of the method, dsRNA derived from BRCA2 genes can be generated in vivo by simultaneous expression of both sense and antisense RNA from appropriately positioned promoters operably fused to BRCA2 sequences in both sense and antisense orientations. In yet another embodiment of the method the dsRNA can be delivered to the animal by engineering expression of dsRNA within cells of a second organism that serves as food for the animal, for example engineering expression of dsRNA in E. coli bacteria which are fed to C. elegans, or engineering expression of dsRNA in baker's yeast which are fed to Drosophila, or engineering expression of dsRNA in transgenic plants which are fed to plant eating insects such as Leptinotarsa or Heliothis.

Recently, RNAi has been successfully used in cultured Drosophila cells to inhibit expression of targeted proteins (Dixon lab, University of Michigan, htto://dixonlab.biochem.med.unuch.edu/protocols/RNAiExperiments.html). Thus, cell lines in culture can be manipulated using RNAi both to perturb and study the function of BRCA2 pathway components and to validate the efficacy of therapeutic strategies which involve the manipulation of this pathway.

Generating Loss-of-function Phenotypes Using Peptide and RNA Aptamers Another method for generating loss-of-function phenotypes is by the use of peptide aptamers, which are peptides or small polypeptides that act as dominant inhibitors of protein function. Peptide aptamers specifically bind to target proteins, blocking their function ability (Kolonin and Finley, PNAS (1998) 95:14266-14271). Due to the highly selective nature of peptide aptamers, they may be used not only to target a specific protein, but also to target specific functions of a given protein (e.g. involvement in cellular response to DNA damage). Further, peptide aptamers may be expressed in a controlled fashion by use of promoters which regulate expression in a temporal, spatial or inducible manner. Peptide aptamers act dominantly; therefore, they can be used to analyze proteins for which loss-of-function mutants are not available. Peptide aptamers that bind with high affinity and specificity to a target protein may be isolated by a variety of techniques known in the art. In one method, they are isolated from random peptide libraries by yeast two-hybrid screens (Xu et al, PNAS (1997) 94: 12473-12478). They can also be isolated from phage libraries (Hoogenboom et al, hnmunotechnology (1998) 4:1-20) or chemically generated peptides/libraries. RNA aptamers are specific RNA ligands for proteins, that can specifically inhibit protein function of the gene (Good et al, Gene Therapy (1997) 4:45-54; Ellington, et al, Biotechnol. Annu. Rev. (1995) 1:185-214). In vitro selection methods can be used to identify RNA aptamers having a selected specificity (Bell et al, J. Biol. Chem. (1998) 273:14309-14314). It has been demonstrated that RNA aptamers can inhibit protein function in Drosophila (Shi et al, Proc. Natl. Acad. Sci USA (19999) 96:10033-10038). Accordingly, RNA aptamers can be used to decrease the expression of BRCA2 protein or derivative thereof, or a protein that interacts with the BRCA2 protein.

Transgenic animals can be generated to test peptide or RNA aptamers in vivo (Kolonin, MG, and Finley, RL, Genetics, 1998 95:4266-4271). For example, transgenic Drosophila lines expressing the desired aptamers may be generated by P element mediated transformation (discussed below). The phenotypes of the progeny expressing the aptamers can then be characterized.

Generating Loss of Function Phenotypes Using Intrabodies Intracellularly expressed antibodies, or intrabodies, are single-chain antibody molecules designed to specifically bind and inactivate target molecules inside cells. Intrabodies have been used in cell assays and in whole organisms such as Drosophila (Chen et al, Hum. Gen. Ther. (1994) 5:595-601; Hassanzadeh et al, Febs Lett. (1998) 16(1, 2):75-80 and 81-86). Inducible expression vectors can be constructed with intrabodies that react specifically with BRCA2 protein. These vectors can be introduced into model organisms and studied in the same manner as described above for aptamers.

Transgenesis Typically, transgenic animals are created that contain gene fusions of the coding regions, of the BRCA2 gene (from either genomic DNA or cDNA) or genes engineered to encode antisense RNAs, cosuppression RNAs, interfering dsRNA, RNA aptamers, peptide aptamers, or intrabodies operably joined to a specific promoter and transcriptional enhancer whose regulation has been well characterized, preferably heterologous promoters/enhancers (i.e. promoters/enhancers that are non-native to the BRCA2 pathway genes being expressed).

Methods are well known for incorporating exogenous nucleic acid sequences into the genome of animals or cultured cells to create transgenic animals or recombinant cell lines. For invertebrate animal models, the most common methods involve the use of transposable elements. There are several suitable tiansposable elements that can be used to incorporate nucleic acid sequences into the genome of model organisms. Transposable elements are particularly useful for inserting sequences into a gene of interest so that the encoded protein is not properly expressed, creating a "knock-out" animal having a loss-of- function phenotype. Techniques are well-established for the use of P element in Drosophila (Rubin and Spradling, Science (1982) 218:348-53; U.S. Pat. No. 4,670,388) and Tel in C. elegans (Zwaal et al, Proc. Natl. Acad. Sci. U.S.A. (1993) 90:7431-7435; and Caenorhabditis elegans: Modern Biological Analysis of an Organism (1995) Epstein and Shakes, Eds.). Other Tcl-like transposable elements can be used such as minos, mariner and sleeping beauty. Additionally, transposable elements that function in a variety of species, have been identified, such as PiggyBac (Thibault et al. , Insect Mol Biol (1999) 8(1):119-23), hobo, and hermes.

P elements, or marked P elements, are preferred for the isolation of loss-of- function mutations in Drosophila BRCA2 genes because of the precise molecular mapping of these genes, depending on the availability and proximity of preexisting P element insertions for use as a localized transposon source (Hamilton and Zinn, Methods in Cell Biology (1994) 44:81-94; and Wolfner and Goldberg, Methods in Cell Biology (1994) 44:33-80). Typically, modified P elements are used which contain one or more elements that allow detection of animals containing the P element. Most often, marker genes are used that affect the eye color of Drosophila, such as derivatives of the Drosophila white or rosy genes (Rubin and Spradling, Science (1982) 218(4570): 348-353; and Klemenz et al, Nucleic Acids Res. (1987) 15(10):3947-3959). However, in principle, any gene can be used as a marker that causes a reliable and easily scored phenotypic change in transgenic animals. Various other markers include bacterial plasmid sequences having selectable markers such as ampicillin resistance (Steller and Pirrotta, EMBO. J. (1985) 4:167-171); and lacZ sequences fused to a weak general promoter to detect the presence of enhancers with a developmental expression pattern of interest (Bellen et al, Genes Dev. (1989) 3(9): 1288-1300). Other examples of marked P elements useful for mutagenesis have been reported (Nucleic Acids Research (1998) 26:85-88; and http://flybase.bio.indiana.edu). A preferred method of transposon mutagenesis in Drosophila employs the "local hopping" method described by Tower et al (Genetics (1993) 133:347-359). Each new P insertion line can be tested molecularly for transposition of the P element into the gene of interest (e.g. BRCA2) by assays based on PCR. For each reaction, one PCR primer is used that is homologous to sequences contained within the P element and a second primer is homologous to the coding region or flanking regions of the gene of interest. Products of the PCR reactions are detected by agarose gel electrophoresis. The sizes of the resulting DNA fragments reveal the site of P element insertion relative to the gene of interest. Alternatively, Southern blotting and restriction mapping using DNA probes derived from genomic DNA or cDNAs of the gene of interest can be used to detect transposition events that rearrange the genomic DNA of the gene. P transposition events that map to the gene of interest can be assessed for phenotypic effects in heterozygous or homozygous mutant Drosophila.

In another embodiment, Drosophila lines carrying P insertions in the gene of interest, can be used to generate localized deletions using known methods (Kaiser, Bioassays (1990) 12(6):297-301; Harnessing the power of Drosophila genetics, In

Drosophila melanogaster: Practical Uses in Cell and Molecular Biology, Goldstein and Fyrberg, Eds., Academic Press, Inc. San Diego, California). This is particularly useful if no P element transpositions are found that disrupt the gene of interest. Briefly, flies containing P elements inserted near the gene of interest are exposed to a further round of transposase to induce excision of the element. Progeny in which the transposon has excised are typically identified by loss of the eye color marker associated with the transposable element. The resulting progeny will include flies with either precise or imprecise excision of the P element, where the imprecise excision events often result in deletion of genomic DNA neighboring the site of P insertion. Such progeny are screened by molecular techniques to identify deletion events that remove genomic sequence from the gene of interest, and assessed for phenotypic effects in heterozygous and homozygous mutant Drosophila.

Recently a transgenesis system has been described that may have universal applicability in all eye-bearing animals and which has been proven effective in delivering transgenes to diverse insect species (Berghammer et ah, Nature (1999) 402:370-371). This system includes: an artificial promoter active in eye tissue of all animal species, preferably containing three Pax6 binding sites positioned upstream of a TATA box (3xP3; Sheng et al, Genes Devel. (1997) 11:1122-1131); a strong and visually detectable marker gene, such as GFP or other autofluorescent protein genes (Pasher et ah, Gene (1992) 111:229-233; U.S. Pat. No 5,491,084); and promiscuous vectors capable of delivering transgenes to a broad range of animal species. Examples of promiscuous vectors include transposon-based vectors derived from Hermes, PiggyBac, or mariner, and vectors based on pantropic VSVc-pseudotyped retroviruses (Bums et ah, hi Vitro Cell Dev Biol Anim (1996) 32:78-84; Jordan et ah, Insect Mol Biol (1998) 7: 215-222; U.S. Pat. No. 5,670,345). Thus, since the same transgenesis system can be used in a variety of phylogenetically diverse animals, comparative functional studies are greatly facilitated, which is especially helpful in evaluating new applications to pest management.

In addition to creating loss-of-function phenotypes, transposable elements can be used to incorporate the gene of interest, or mutant or derivative thereof, as an additional gene into any region of an animal's genome resulting in mis-expression (including over- expression) of the gene. A preferred vector designed specifically for misexpression of genes in transgenic Drosophila, is derived from pGMR (Hay et al, Development (1994) 120:2121-2129), is 9Kb long, and contains: an origin of replication for E. coli; an ampicillin resistance gene; P element transposon 3' and 5' ends to mobilize the inserted sequences; a White marker gene; an expression unit comprising the TATA region of hsp70 enhancer and the 3'untranslated region of α-tubulin gene. The expression unit contains a first multiple cloning site (MCS) designed for insertion of an enhancer and a second MCS located 500 bases downstream, designed for the insertion of a gene of interest. As an alternative to transposable elements, homologous recombination or gene targeting techniques can be used to substitute a gene of interest for one or both copies of the animal's homologous gene. The transgene can be under the regulation of either an exogenous or an endogenous promoter element, and be inserted as either a minigene or a large genomic fragment. In one application, gene function can be analyzed by ectopic expression, using, for example, Drosophila (Brand et al, Methods in Cell Biology (1994) 44:635- 654) or C. elegans (Mello and Fire, Methods in Cell Biology (1995) 48:451-482). Examples of well-characterized heterologous promoters that may be used to create the transgenic animals include heat shock promoters/enhancers, which are useful for temperature induced mis-expression, hi Drosophila, these include the hsp 70 and hsp83 genes, and in C. elegans, include hsp 16-2 and hsp 16-41. Tissue specific promoters/enhancers are also useful, and in Drosophila, include eyeless (Mozer and Benzer, Development (1994) 120:1049-1058), sevenless (Bowtell et al, PNAS (1991) 88(15):6853-6857), and gZαss-responsive promoters/enhancers (Quiring et al., Science (1994) 265:785-789) which are useful for expression in the eye; and enhancers/promoters derived from the dpp or vestigal genes which are useful for expression in the wing (Staehling-Hampton et al, Cell Growth Differ. (1994) 5(6):585-593; Kim et al, Nature (1996) 382:133-138). Finally, where it is necessary to restrict the activity of dominant active or dominant negative transgenes to regions where the pathway is normally active, it may be useful to use endogenous promoters of genes in the pathway, such as the BRCA2 pathway genes.

In Drosophila, binary control systems that employ exogenous DNA are useful when testing the mis-expression of genes in a wide variety of developmental stage-specific and tissue-specific patterns. Two examples of binary exogenous regulatory systems include the UAS/GAL4 system from yeast (Hay et al, PNAS (1997) 94(10):5195-5200; Ellis et al, Development (1993) 119(3):855-865), and the "Tet system" derived from E. coli (Bello et al., Development (1998) 125:2193-2202). The UAS/GAL4 system is a well- established and powerful method of mis-expression in Drosophila which employs the UAS_G upstream regulatory sequence for control of promoters by the yeast GAL4 transcriptional activator protein (Brand and Perrimon, Development (1993) 118(2):401- 15). In this approach, transgenic Drosophila, termed "target" lines, are generated where the gene of interest to be mis-expressed is operably fused to an appropriate promoter controlled by UAS_G- Other transgenic Drosophila strains, termed "driver" lines, are generated where the GAL4 coding region is operably fused to promoters/enhancers that direct the expression of the GAL4 activator protein in specific tissues, such as the eye, wing, nervous system, gut, or musculature. The gene of interest is not expressed in the target lines for lack of a transcriptional activator to drive transcription from the promoter joined to the gene of interest. However, when the UAS-target line is crossed with a GAL4 driver line, mis-expression of the gene of interest is induced in resulting progeny in a specific pattern that is characteristic for that GAL4 line. The technical simplicity of this approach makes it possible to sample the effects of directed mis-expression of the gene of interest in a wide variety of tissues by generating one transgenic target line with the gene of interest, and crossing that target line with a panel of pre-existing driver lines. In the "Tet" binary control system, transgenic Drosophila driver lines are generated where the coding region for a tetracycline-controlled transcriptional activator (tTA) is operably fused to promoters/enhancers that direct the expression of tTA in a tissue-specific and/or developmental stage-specific manner. The driver lines are crossed with transgenic Drosophila target lines where the coding region for the gene of interest to be mis-expressed is operably fused to a promoter that possesses a tTA-responsive regulatory element. When the resulting progeny are supplied with food supplemented with a sufficient amount of tetracycline, expression of the gene of interest is blocked. Expression of the gene of interest can be induced at will simply by removal of tetracycline from the food. Also, the level of expression of the gene of interest can be adjusted by varying the level of tetracycline in the food. Thus, the use of the Tet system as a binary control mechanism for mis-expression has the advantage of providing a means to control the amplitude and timing of mis-expression of the gene of interest, in addition to spatial control. Consequently, if a gene of interest (e.g. a BRCA2 gene) has lethal or deleterious effects when mis-expressed at an early stage in development, such as the embryonic or larval stages, the function of the gene of interest in the adult can still be assessed by adding tetracycline to the food during early stages of development and removing tetracycline later so as to induce mis-expression only at the adult stage.

Dominant negative mutations, by which the mutation causes a protein to interfere with the normal function of a wild-type copy of the protein, and which can result in loss- of-function or reduced-function phenotypes in the presence of a normal copy of the gene, can be made using known methods (Hershkowitz, Nature (1987) 329:219-222). In the case of active monomeric proteins, overexpression of an inactive form, achieved, for example, by linking the mutant gene to a highly active promoter, can cause competition for natural substrates or ligands sufficient to significantly reduce net activity of the normal protein. Alternatively, changes to active site residues can be made to create a virtually irreversible association with a target.

Assays for Change in Gene Expression Various expression analysis techniques may be used to identify genes which are differentially expressed between a cell line or an animal expressing a wild type BRCA2 gene compared to another cell line or animal expressing a mutant BRCA2 gene. Such expression profiling techniques include differential display, serial analysis of gene expression (SAGE), transcript profiling coupled to a gene database query, nucleic acid array technology, subtractive hybridization, and proteome analysis (e.g. mass- spectrometry and two-dimensional protein gels). Nucleic acid array technology may be used to determine a global (i.e., genome- wide) gene expression pattern in a normal animal for comparison with an animal having a mutation in BRCA2 gene. Gene expression profiling can also be used to identify other genes (or proteins) that may have a functional relation to BRCA2 (e.g. may participate in a signaling pathway with the BRCA2 gene). The genes are identified by detecting changes in their expression levels following mutation, i.e., insertion, deletion or substitution in, or over-expression, under-expression, mis-expression or knock-out, of the BRCA2 gene.

Phenotypes Associated With BRCA2 Pathway Gene Mutations

After isolation of model animals carrying mutated or mis-expressed BRCA2 pathway genes or inhibitory RNAs, animals are carefully examined for phenotypes of interest. For analysis of BRCA2 pathway genes that have been mutated (i.e. deletions, insertions, and/or point mutations) animal models that are both homozygous and heterozygous for the altered BRCA2 pathway gene are analyzed. Examples of specific phenotypes that may be investigated include lethality; sterility; feeding behavior, perturbations in neuromuscular function including alterations in motility, and alterations in sensitivity to pharmaceuticals. Some phenotypes more specific to flies include alterations in: adult behavior such as, flight ability, walking, grooming, phototaxis, mating or egg- laying; alterations in the responses of sensory organs, changes in the morphology, size or number of adult tissues such as, eyes, wings, legs, bristles, antennae, gut, fat body, gonads, and musculature; larval tissues such as mouth parts, cuticles, internal tissues or imaginal discs; or larval behavior such as feeding, molting, crawling, or puparian formation; or developmental defects in any germline or embryonic tissues. Some phenotypes more specific to nematodes include: locomotory, egg laying, chemosensation, male mating, and intestinal expulsion defects. In various cases, single phenotypes or a combination of specific phenotypes in model organisms might point to specific genes or a specific pathway of genes, which facilitate the cloning process. Genomic sequences containing a BRCA2 pathway gene can be used to confirm whether an existing mutant insect or worm line corresponds to a mutation in one or more BRCA2 pathway genes, by rescuing the mutant phenotype. Briefly, a genomic fragment containing the BRCA2 pathway gene of interest and potential flanking regulatory regions can be subcloned into any appropriate insect (such as Drosophila) transformation vector, and injected into the animals. For Drosophila, an appropriate helper plasmid is used in the injections to supply transposase for transposon-based vectors. Resulting germline transformants are crossed for complementation testing to an existing or newly created panel of Drosophila lines whose mutations have been mapped to the vicinity of the gene of interest (Fly Pushing: The Theory and Practice of Drosophila Genetics, supra). If a mutant line is discovered to be rescued by this genomic fragment, as judged by complementation of the mutant phenotype, then the mutant line likely harbors a mutation in the BRCA2 pathway gene. This prediction can be further confirmed by sequencing the BRCA2 pathway gene from the mutant line to identify the lesion in the BRCA2 pathway gene.

Identification of Genes That Modify BRCA2 Genes

The characterization of new phenotypes created by mutations or misexpression in BRCA2 genes enables one to test for genetic interactions between BRCA2 genes and other genes that may participate in the same, related, or interacting genetic or biochemical pathway(s). Individual genes can be used as starting points in large-scale genetic modifier screens as described in more detail below. Alternatively, RNAi methods can be used to simulate loss-of-function mutations in the genes being analyzed. It is of particular interest to investigate whether there are any interactions of BRCA2 genes with other well- characterized genes, particularly genes involved in cellular response to DNA damage.

Genetic Modifier Screens

A genetic modifier screen using invertebrate model organisms is a particularly preferred method for identifying genes that interact with BRCA2 genes, because large numbers of animals can be systematically screened making it more possible that interacting genes will be identified. In Drosophila, a screen of up to about 10,000 animals is considered to be a pilot-scale screen. Moderate-scale screens usually employ about 10,000 to about 50,000 flies, and large-scale screens employ greater than about 50,000 flies. In a genetic modifier screen, animals having a mutant phenotype due to a mutation in or misexpression of one or more BRCA2 genes are further mutagenized, for example by chemical mutagenesis or transposon mutagenesis.

The procedures involved in typical Drosophila genetic modifier screens are well- known in the art (Wolfner and Goldberg, Methods in Cell Biology (1994) 44:33-80; and Karim et al, Genetics (1996) 143:315-329). The procedures used differ depending upon the precise nature of the mutant allele being modified. If the mutant allele is genetically recessive, as is commonly the situation for a loss-of-function allele, then most typically males, or in some cases females, which carry one copy of the mutant allele are exposed to an effective mutagen, such as EMS, MMS, ENU, triethylamine, diepoxyalkanes, ICR-170, formaldehyde, X-rays, gamma rays, or ultraviolet radiation. The mutagenized animals are crossed to animals of the opposite sex that also carry the mutant allele to be modified. In the case where the mutant allele being modified is genetically dominant, as is commonly the situation for ectopically expressed genes, wild type males are mutagenized and crossed to females carrying the mutant allele to be modified.

The progeny of the mutagenized and crossed flies that exhibit either enhancement or suppression of the original phenotype are presumed to have mutations in other genes, called "modifier genes", that participate in the same phenotype-generating pathway. These progeny are immediately crossed to adults containing balancer chromosomes and used as founders of a stable genetic line, hi addition, progeny of the founder adult are retested under the original screening conditions to ensure stability and reproducibility of the phenotype. Additional secondary screens may be employed, as appropriate, to confirm the suitability of each new modifier mutant line for further analysis.

Standard techniques used for the mapping of modifiers that come from a genetic screen in Drosophila include meiotic mapping with visible or molecular genetic markers; male-specific recombination mapping relative to P-element insertions; complementation analysis with deficiencies, duplications, and lethal P-element insertions; and cytological analysis of chromosomal aberrations (Fly Pushing: Theory and Practice of Drosophila Genetics, supra; Drosophila: A Laboratory Handbook, supra). Genes corresponding to modifier mutations that fail to complement a lethal P-element may be cloned by plasmid rescue of the genomic sequence surrounding that P-element. Alternatively, modifier genes may be mapped by phenotype rescue and positional cloning (Sambrook et ah, supra). Newly identified modifier mutations can be tested directiy for interaction with other genes of interest known to be involved or implicated with BRCA2 genes using methods described above.

The modifier mutations may also be used to identify "complementation groups". Two modifier mutations are considered to fall within the same complementation group if animals carrying both mutations in trans exhibit essentially the same phenotype as animals that are homozygous for each mutation individually and, generally are lethal when in trans to each other (Fly Pushing: The Theory and Practice of Drosophila Genetics, supra). Generally, individual complementation groups defined in this way correspond to individual genes.

When BRCA2 modifier genes are identified, homologous genes in other species can be isolated using procedures based on cross-hybridization with modifier gene DNA probes, PCR-based strategies with primer sequences derived from the modifier genes, and/or computer searches of sequence databases. For therapeutic applications related to the function of BRCA2 genes, human and rodent homologs of the modifier genes are of particular interest.

Although the above-described Drosophila genetic modifier screens are quite powerful and sensitive, some genes that interact with BRCA2 genes may be missed in this approach, particularly if there is functional redundancy of those genes. This is because the vast majority of the mutations generated in the standard mutagenesis methods will be loss- of-function mutations, whereas gain-of-function mutations that could reveal genes with functional redundancy will be relatively rare. Another method of genetic screening in Drosophila has been developed that focuses specifically on systematic gain-of-function genetic screens (Rorth et ah, Development (1998) 125:1049-1057). This method is based on a modular mis-expression system utilizing components of the GAL4/UAS system (described above) where a modified P element, termed an "enhanced P" (EP) element, is genetically engineered to contain a GAL4-responsive UAS element and promoter. Any other transposons can also be used for this system. The resulting transposon is used to randomly tag genes by insertional mutagenesis (similar to the method of P element mutagenesis described above). Thousands of transgenic Drosophila strains, termed EP lines, can be generated, each containing a specific UAS-tagged gene. This approach takes advantage of the preference of P elements to insert at the 5'-ends of genes. Consequently, many of the genes that are tagged by insertion of EP elements become operably fused to a GAL4-regulated promoter, and increased expression or mis-expression of the randomly tagged gene can be induced by crossing in a GAL4 driver gene.

Systematic gain-of-function genetic screens for modifiers of phenotypes induced by mutation or mis-expression of a BRCA2 gene can be performed by crossing several thousand Drosophila EP lines individually into a genetic background containing a mutant or mis-expressed BRCA2 gene, and further containing an appropriate GAL4 driver transgene. It is also possible to remobilize the EP elements to obtain novel insertions. The progeny of these crosses are then analyzed for enhancement or suppression of the original mutant phenotype as described above. Those identified as having mutations that interact with the BRCA2 gene can be tested further to verify the reproducibility and specificity of this genetic interaction. EP insertions that demonstrate a specific genetic interaction with a mutant or mis-expressed BRCA2 gene, have a physically tagged BRCA2 which can be identified and sequenced using PCR or hybridization screening methods, allowing the isolation of the genomic DNA adjacent to the position of the EP element insertion.

EXAMPLES

The following examples describe the isolation and cloning of the nucleic acid sequence of SEQ ID NOS : 1 , 3, or 5, and how these sequences, and derivatives and fragments thereof, as well as other BRCA2 pathway nucleic acids and gene products can be used for genetic studies to elucidate mechanisms of the BRCA2 pathway as well as the discovery of potential pharmaceutical agents that interact with the pathway.

These Examples are provided merely as illustrative of various aspects of the invention and should not be construed to limit the invention in any way.

Example 1: Preparation of Drosophila cDNA Library

A Drosophila expressed sequence tag (EST) cDNA library was prepared as follows. Tissue from mixed stage embryos (0-20 hour), imaginal disks and adult fly heads were collected and total RNA was prepared. Mitochondrial rRNA was removed from the total RNA by hybridization with biotinylated rRNA specific oligonucleotides and the resulting RNA was selected for polyadenylated mRNA. The resulting material was then used to construct a random primed library. First strand cDNA synthesis was primed using a six nucleotide random primer. The first strand cDNA was then tailed with terminal transferase to add approximately 15 dGTP molecules. The second strand was primed using a primer which contained a Notl site followed by a 13 nucleotide C-tail to hybridize to the G-tailed first strand cDNA. The double stranded cDNA was ligated with BstXl adaptors and digested with Notl. The cDNA was then fractionated by size by electrophoresis on an agarose gel and the cDNA greater than 700 bp was purified. The cDNA was ligated with Notl, BstXl digested pCDNA-sk+ vector (a derivative of pBluescript, Stratagene) and used to transform E. coli (XLlblue). The final complexity of the library was 6 X 10⁶ independent clones.

The cDNA library was normalized using a modification of the method described by Bonaldo et al. (Genome Research (1996) 6:791-806). Biotinylated driver was prepared from the cDNA by PCR amplification of the inserts and allowed to hybridize with single stranded plasmids of the same library. The resulting double-stranded forms were removed using strepavidin magnetic beads, the remaining single stranded plasmids were converted to double stranded molecules using Sequenase (Amersham, Arlington Hills, IL), and the plasmid DNA stored at -20°C prior to transformation. Aliquots of the normalized plasmid library were used to transform E. coli (XLlblue or DH10B), plated at moderate density, and the colonies picked into a 384- well master plate containing bacterial growth media using a Qbot robot (Genetix, Christchurch, UK). The clones were allowed to grow for 24 hours at 37° C then the master plates were frozen at -80° C for storage. The total number of colonies picked for sequencing from the normalized library was 240,000. The master plates were used to inoculate media for growth and preparation of DNA for use as template in sequencing reactions. The reactions were primarily carried out with primer that initiated at the 5' end of the cDNA inserts. However, a minor percentage of the clones were also sequenced from the 3' end. Clones were selected for 3' end sequencing based on either further biological interest or the selection of clones that could extend assemblies of contiguous sequences ("contigs") as discussed below. DNA sequencing was carried out using ABI377 automated sequencers and used either ABI FS, dirhodamine or BigDye chemistries (Applied Biosystems, Inc., Foster City, CA).

Analysis of sequences were done as follows: the traces generated by the automated sequencers were base-called using the program "Phred" (Gordon, Genome Res. (1998) 8: 195-202), which also assigned quality values to each base. The resulting sequences were trimmed for quality in view of the assigned scores. Vector sequences were also removed. Each sequence was compared to all other fly EST sequences using the BLAST program and a filter to identify regions of near 100% identity. Sequences with potential overlap were then assembled into contigs using the programs "Phrap", "Phred" and "Consed" (Phil Green, University of Washington, Seattle, Washington; htto://bozeman.mbt.washington.edu/phrap.docs/phrap.html). The resulting assemblies were then compared to existing public databases and homology to known proteins was then used to direct translation of the consensus sequence. Where no BLAST homology was available, the statistically most likely translation based on codon and hexanucleotide preference was used. The Pfam (Bateman et al, Nucleic Acids Res. (1999) 27:260-262) and Prosite (Hofmann et ah, Nucleic Acids Res. (1999) 27(1):215-219) collections of protein domains were used to identify motifs in the resulting translations. The contig sequences were archived in an Oracle-based relational database (FlyTag™, Exelixis Pharmaceuticals, Inc., South San Francisco, CA).

Example 2: Cloning of BRCA2 Nucleic Acid Sequences

Unless otherwise noted, the PCR conditions used for cloning the BRCA2 nucleic acid sequence was as follows: A denaturation step of 94° C, 5 min; followed by 35 cycles of: 94° C 1 min, 55° C 1 min 72° C 1 min; then, a final extension at 72° C 10 min.

All DNA sequencing reactions were performed using standard protocols for the BigDye sequencing reagents (Applied Biosystems, Inc.) and products were analyzed using ABI 377 DNA sequencers. Trace data obtained from the ABI 377 DNA sequencers was analyzed and assembled into contigs using the Phred-Phrap programs.

For Tribolium, Well-separated, single colonies were streaked on a plate and end- sequenced to verify the clones. Single colonies were picked and the enclosed plasmid DNA was purified using Qiagen REAL Preps (Qiagen, Inc., Valencia, CA). Samples were then digested with appropriate enzymes to excise insert from vector and determine size. Clones were then sequenced using a combination of primer walking and in vitro transposon tagging strategies.

For primer walking, primers were designed to the known DNA sequences in the clones, using the Primer-3 software (Steve Rozen, Helen J. Skaletsky (1998) Primer3. Code available online at website www- genome.wi.mit.edu/genome_software/other/primer3.html.). These primers were then used in sequencing reactions to extend the sequence until the full sequence of the insert was determined.

The GPS-1 Genome Priming System in vitro transposon kit (New England Biolabs, ie, Beverly, MA) was used for transposon-based sequencing, following manufacturer's protocols. Briefly, multiple DNA templates with randomly interspersed primer-binding sites were generated. These clones were prepared by picking 24 colonies/clone into a Qiagen REAL Prep to purify DNA and sequenced by using supplied primers to perform bidirectional sequencing from both ends of transposon insertion. Sequences were then assembled using Phred/Phrap and analyzed using Consed.

Ambiguities in the sequence were resolved by resequencing several clones. This effort resulted in a contiguous nucleotide sequence of 2.3 kilobases in length, encompassing an open reading frame (ORF) of 2271 nucleotides encoding a predicted protein of 756 amino acids. The ORF extends from base 1-2271 of SEQ ID NO:5. For Drosophila, genomic sequencing of BAC clones led to a gene prediction for

DBRCA2.2 (SEQ ID NO:3 for DNA and SEQ ID NO:4 for protein). DBRCA2.2 is an open reading frame of 2844 nucleotides, encoding a predicted protein of 947 amino acids. Sequences from the predicted gene DBRCA2.2 were used to probe and isolate the clone GM03728 from Berkeley Drosophila EST library (website at www.fruitfly.org). Sequencing of this clone led to a contiguous nucleotide sequence of 3.2 kilobases in length, encompassing an open reading frame (ORF) of 1920 nucleotides encoding a predicted protein of 640 amino acids. The ORF extends from base 1058-2977 of SEQ ID NO:l.

Example 3: Analysis of BRCA2 Nucleic Acid Sequences

Upon completion of cloning, the sequences were analyzed using the Pfam and Prosite programs. BRCA2 repeat domains were identified for each sequence. For DBRCA2.1 (SEQ ID NO:2), these repeats are at amino acids 239-273, 340-374, and 415- 449. For DBRCA2.2 (SEQ ID NO: 4), these repeats are at amino acids 546-580, 647-681, and 722-756. For TBRCA2 (SEQ ID NO:6), the repeats are at amino acids 124-156, 158- 192, and 198-232. Nucleotide and amino acid sequences for each of the BRCA2 nucleic acid sequences and their encoded proteins were searched against all available nucleotide and amino acid sequences in the public databases, using BLAST (Altschul et ah, supra). Table 1 below summarizes the results. The 5 most similar sequences, where available, are listed for each BRCA2 gene.

TABLE 1 I DNA BLAST, DBRCA2.1, SEQ ID NO: 1 |

The closest homolog predicted by BLAST analysis for each sequence is a mammalian BRCA2. Example 4: Cell survival and cell cycle checkpoint analysis

Cell clones are generated that express a GFP-BRC4 fusion protein, employeing the tetracycline-inducible expression system controlled by a tet-responsive promoter (Gossen M and Bujard H (1992) PNAS 89:5547-5551). For cell survival assays, cells are seeded in identical plates at 5000 cells/plate in medium with tetracycline (1 μg/ml). Expression of the wild-type or mutant GFP-BRC4 repeat is induced by removing tetracycline 24 h after seeding. Twenty four hours after induction of GFP-BRC4 expression, cells are gamma-irradiated with 3 Gy. After incubation for 14 days, cells are fixed and stained with 2% methylene blue in 50% of ethanol for colony counting. Averages and standard deviations are determined from eight plates.

For cell cycle checkpoint analysis, cells in logarithmic growth are mock-exposed or -irradiated (12 Gy). After 24 h, cells are labeled with 10 μM BrdUrd for 4 h and fixed for BrdUrd staining using a Cell Proliferation Kit (Amersham Pharmacia Biotech). BrdUrd-positive cells are quantified, and expressed as a fraction of the total cells. For the G2/M checkpoint, cells are irradiated to 3 Gy, fixed with 4% paraformaldehyde at indicated time and stained with DAPI for counting mitotic cells in prophase, metaphase, anaphase, and telophase. Alternately, cells are irradiated with 4-16 Gy and processed for analysis of mitotic cells after 1 hr.

Example 5: BRCA2 expression patterns in Drosophila

BRCA2 expression levels in cDNA libraries and cell lines derived from Drosophila were assessed by TaqMan (Perkin Elmer Corp./Applied Biosystems, Foster City, Calif) Quantitative Reverse-transcriptase PCR (RT-PCR) (Livak KJ, et al., PCR Methods Appl. 1995 Jun;4(6):357-62). Briefly, reverse transcribed cDNA from total RNA was produced from each S2 and Dmel2 Drosophila cell lines (S2 is available from American Type Culture Collection (ATCC), Cat#CRL-1963; Dmel 2 is available from Life Technologies, Cat#10831). Embryonic, adult, and larval cDNA Drosophila libraries were obtained from Berkeley Drosophila Genome Project (BDGP, website at www.fruitfly.org). Primers were made using SEQ ID NO: 1. Procedures for primer design and PCR conditions were according to manufacturer's protocols.

The results of these experiments indicate that BRCA2 has high expression levels in embryonic and adult cDNA libraries, medium expression levels in S2 and Dmel2 cells, and low expression levels in larval cDNA libraries. Example 6: BRCA2 RNAi in DmeI2 cells

RNAi was used to knock down levels of BRCA2 in Dmel2 Drosophila cells. Briefly, PCR primers based on BRCA2 sequences of SEQ JD NO: 1 were employed to produce PCR products of about 1 kb, using an embryonic Drosophila cDNA library (Example 5) as template, hi vitro transcription (Ambion, TX) was then used to produce single stranded (ss) RNA, which was then annealed to produce double stranded (ds) RNA. Cultured Drosophila embryonic serum-free medium adapted Dmel2 cell line was treated with BRCA2 dsRNA and allowed to incubate for 2 days. RNA was then extracted from the untreated (control) and treated cells (Qiagen, Valencia, CA), and reverse transcribed to produce cDNA (ABI kit, Applied Biosystems, Foster City, CA). The reverse transcribed cDNA was used as a template for RT-PCR using Taqman experiments. Taqman primers and procedures were produced and performed according to manufacturer's suggestions. The results show that BRCA2 RNAi in Dmel2 cells reduces BRCA2 mRNA levels by 80%.

Claims

WHAT IS CLAIMED IS:

1. An isolated nucleic acid molecule of less than about 15 kb in size comprising a nucleic acid sequence selected from the group consisting of: (a) a nucleic acid sequence that encodes a polypeptide comprising amino acids of

SEQ ID NOS:2, 4, or 6; and

(b) the complement of the nucleic acid sequence of (a).

2. The isolated nucleic acid molecule of Claim 1 which is capable of hybridizing to a nucleic acid sequence selected from the group consisting of SEQ ID NOs:l, 3, or 5 using highly stringent hybridization conditions.

3. The isolated nucleic acid molecule of Claim 1 wherein said nucleic acid sequence encodes the entire sequence of SEQ ID NOs:2, 4, or 6.

4. The isolated nucleic acid molecule of Claim 1 wherein said nucleic acid sequence encodes at least one BRC repeat of the BRCA2.

5. A vector comprising the nucleic acid molecule of Claim 1.

6. An isolated host cell comprising the vector of Claim 7.

7. A process for producing a BRCA2 protein comprising culturing the host cell of Claim 8 under conditions suitable for expression of said BRCA2 protein and recovering said protein.

8. A method for detecting a candidate compound that interacts with a BRCA2 protein or fragment thereof, said method comprising contacting said BRCA2 protein or fragment with one or more candidate molecules, and detecting any interaction between said candidate compound and said BRCA2 protein or fragment; wherein the amino acid sequence of said BRCA2 protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:2, 4, or 6.

9. The method of Claim 8 wherein said candidate compound is a putative pharmaceutical agent.

10. The method of Claim 8 wherein said contacting comprises administering said candidate compound to cultured host cells that have been genetically engineered to express said BRCA2 protein.

11. The method of Claim 8 wherein said contacting comprises administering said candidate compound to a metazoan invertebrate organism that has been genetically engineered to express a BRCA2 protein.

12. A first animal that is an insect that has been genetically modified to express or mis- express a BRCA2 protein, or the progeny of said animal that has inherited said BRCA2 protein expression or mis-expression, wherein said BRCA2 protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:2, 4, or 6.

13. A method for studying BRCA2 activity comprising detecting the phenotype caused by the expression or mis-expression of said BRCA2 protein in the first animal of Claim 12.

14. The method of Claim 13 additionally comprising observing a second animal having the same genetic modification as said first animal which causes said expression or mis-expression of said BRCA2 protein, and wherein said second animal additionally comprises a mutation in a gene of interest, wherein differences, if any, between the phenotype of said fhst animal and the phenotype of said second animal identifies the gene of interest as capable of modifying the function of the gene encoding said BRCA2 protein.

15. The method of Claim 13 additionally comprising administering one or more candidate compounds to said animal or its progeny and observing any changes in BRCA2 activity of said animal or its progeny.