Chromosome 17q12-21 is known to contain a gene (or genes) which confers susceptibility to early-onset breast cancer and ovarian cancer (BRCA1). Identification and isolation of BRCA1 will likely provide the basis for increased understanding of the pathogenesis of breast and ovarian cancer, the development of targeted diagnostic and therapeutic approaches and a means of screening women at risk of being gene carriers. The thyroid hormone receptor-alpha (THRA1) locus and the anonymous DNA segment D17S579 flank this region, thought to be approximately 2 centimorgans (cM) in length. We have cloned approximately 80% of this region in yeast artificial chromosomes (YACs) and identified cosmids spanning the majority of these YACs using PCR amplification of inter-Alu segments. We are now prepared to begin the task of identifying transcripts from this region in order to isolate BRCA1. We propose to employ a variety of transcript-identification strategies in order to achieve this goal. These include: 1) Direct cDNA screening with YACs and cosmids from within the BRCA1 region. cDNA libraries will be derived from normal breast and ovarian tissue for BRCA1 candidate transcripts. Candidate transcripts will be analyzed by expected tissue distribution and the presence of germline mutations in affected individuals from families thought to be linked to BRCA1. 2) Exon trapping strategies. Cosmids from the BRCA1 region will be subcloned into plasmid vectors designed to assist in the identification of exon/intron splice junctions within cloned DNA segments. Plasmid subclones containing exons will be used to screen appropriate cDNA libraries for BRCA1 candidate transcripts. 3) Solution hybridization of cosmid DNA PCR-amplified using biotinylated primers with cDNA from breast or ovarian tissue. These double-stranded DNA complexes will be conjugated to magnetic beads for rapid isolation and analyzed as described above. 4) Direct screening of breast and ovarian cDNA libraries with microdissected material from the BRCA1 region. 5) Subtractive hybridization. We plan to combine a PCR-based method for fingerprinting mRNA from specific tissues with the technique utilizing magnetic beads for isolation of cDNAs from individual cosmids. This approach should allow selective identification of unique mRNA fragments from the BRCA1 region which are present in normal breast and ovary but absent in tumors arising from these tissues. Current evidence suggests that BRCA1 is a tumor suppressor gene, thus transcripts encoded by genes in the BRCA1 region which demonstrate this pattern of expression are excellent candidates for BRCA1.
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