Kenneth J. Abel

The developmental pattern of Brca1 expression implies a role in differentiation of the breast and other tissues.

We have examined the developmental expression of the murine breast and ovarian cancer susceptibility gene, Brca1, to investigate its role in the control of cell growth and differentiation. Specifically, we have analysed Brca1 expression during embryonic development, in adult tissues, and during postnatal mammary gland development, particularly in response to ovarian hormones. Our results suggest that Brca1 is expressed in rapidly proliferating cell types undergoing differentiation. In the mammary gland, Brca1 expression is induced during puberty, pregnancy, and following treatment of ovariectomized animals with 17β–estradiol and progesterone. These observations imply that Brca1 is involved in the processes of proliferation and differentiation in multiple tissues, notably in the mammary gland in response to ovarian hormones.

Construction of a transcription map surrounding the BRCA1 locus of human chromosome 17

We have used a combination of methods (exon amplification, direct selection, direct screening, evolutionary conservation, island rescue-PCR, and direct sequence analysis) to survey approximately 600 kb of genomic DNA surrounding the BRCA1 gene for transcribed sequences. We have cloned a set of fragments representing at least 26 genes. The DNA sequence of these clones reveals that 5 are previously cloned genes; the precise chromosomal location of 2 was previously unknown, and 3 have been cloned and mapped by others to this interval. Three other genes, including BRCA1 itself, have recently been mapped independently to this region. Sequences from 11 genes are similar but not identical matches to known genes; 5 of these appear to be the human homologues of genes cloned from other species. Another 7 genes have no similarity with known genes. In addition, 39 putative exons and 14 expressed sequence tags have been identified and mapped to individual cosmids. This transcript map provides a detailed description of gene organization for this region of the genome.

A YAC-, P1-, and cosmid-based physical Map of the BRCA1 region on chromosome 17q21

A familial early-onset breast cancer gene (BRCA1) has been localized to chromosome 17q21. To characterize this region and to aid in the identification of the BRCA1 gene, a physical map of a region of 1.0-1.5 Mb between the EDH17B1 and the PPY loci on chromosome 17q21 was generated. The physical map is composed of a yeast artificial chromosome (YAC) and P1 phage contig with one gap. The majority of the interval has also been converted to a cosmid contig. Twenty-three PCR-based sequence-tagged sites (STSs) were mapped to these contigs, thereby confirming the order and overlap of individual clones. This complex physical map of the BRCA1 region was used to isolate genes by a number of gene identification techniques and to generate transcript maps of the region, as presented in the three accompanying manuscripts of Brody et al. (1995), Osborne-Lawrence et al. (1995), and Friedman et al. (1995).

Transcript identification in the BRCA1 candidate region

SummaryChromosome 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 BRCA1 mutation carriers. Genetic and physical maps of the BRCA1 candidate region have been largely completed and efforts are being directed at identification of candidate genes from within this region. We have begun the task of identifying transcripts from this region employing three complementary strategies. These include: 1) direct cDNA screening with cosmids derived from the BRCA1 region; 2) exon amplification; and 3) magnetic bead capture. Transcripts identified using these approaches are being characterized for: 1) tissue expression pattern; 2) the presence of genomic rearrangement in DNA derived from affected members of families believed to show linkage between breast cancer and genetic markers in the BRCA1 candidate interval; 3) altered size and/or expression pattern in RNA prepared from such individuals; and 4) homology to known genes or functional motifs. Germline mutations in affected individuals from these families will serve as presumptive evidence of BRCA1 identity.

Localization of the human homolog of the yeast cell division control 27 gene (CDC27) proximal to ITGB3 on human chromosome 17q21.3.

The human homolog of theSaccharomyces cerevisiae cell division control 27 gene (CDC27) was mapped to human chromosome 17q12-q21 using a panel of human/rodent somatic cell hybrids and localized distal to the breast cancer susceptibility gene,BRCA1, using a panel of radiation hybrids. The radiation hybrid panel indicates that the most likely position of humanCDC27 on human chromosome 17 is between the marker D17S409 and the beta 3 subunit of integrin (ITGB3). Further confirmation of this localization comes from the sequence tagged site (STS) mapping of humanCDC27 to the same yeast artificial chromosomes (YACs) positive forITGB3. The estimated distance between ITGB3 and humanCDC27 is less than 600 kb.

Isolation of Gene Sequences from the BRCA1 Region of Chromosome 17q21 by Exon Amplification

A variety of techniques now exist for the identification and isolation of gene sequences from cloned genomic DNA. We report the use of exon amplification to isolate candidate exons of genes in the chromosome 17q21 region associated with familial breast and ovarian cancer. We have used the second generation splicing vector pSPL3, which provides greater flexibility for cloning genomic fragments and which reduces the frequency of the major classes of false positive clones. In two experiments, exon amplification was performed using DNAs of approximately 170 cosmids spanning 1–2 Mb of this region. Cosmid DNAs were pooled in groups of 6–10 each. More than 2000 candidate exon clones from these experiments have been arrayed in microtiter dishes. The average size determined for nearly 400 cloned inserts was approximately 200 base pairs. Ongoing efforts to identify and eliminate clone redundancy have thus far yielded more than 100 unique exon clones. Less than 10% of the clones were found to be repetitive or to be artifacts resulting from cryptic splicing involving sequences present in the splicing vector. Thus the great majority of clones were found to be single copy and to derive from the correct chromosomal location. These exons have been used as hybridization probes to isolate cDNA clones derived from normal breast tissue. The cloned exons and corresponding cDNAs are being localized within developing cosmid contigs in order to assemble a transcription map of the region, and to position transcribed sequences with respect to critical recombinants in breast/ovarian cancer families. While database searches suggest that many of the exon sequences are unique, these searches have also identified several genes which were either mapped previously to proximal 17q or which appear to be homologs of genes in other species. Exon amplification represents a rapid and efficient means for isolating candidate gene sequences from genomic clones, facilitating efforts to identify specific genes associated with disease using positional cloning strategies. Utilization of this technique to survey large genomic regions will also assist in efforts to construct transcription maps of chromosomes.