Direct selection in the BRCA1 region of human chromosome 17q21
- Univ of Texas Southwestern Medical Center, Dallas, TX (United States); and others
Direct cDNA selection was used to obtain candidate genes within the region of human chromosome 17q21 associated with early onset familial breast and ovarian cancer (BRCA1). Four sets of pooled cosmids (10 to 25 per set) derived from this region were used in the selection of cDNAs from four complex human cDNA pools: placenta, fetal head, HeLa cells, and activated T cells. Two YACs within our contig were also used in a separate selection. A reporter gene, estradiol 17 beta-hydroxysteriod dehydrogenase (EDH17B), located on one of the cosmids in the contig of the region, was monitored to observe the efficiency of the selection. A >10,000-fold enrichment of EDH17B was seen after two rounds of selection based on the number of EDH17B clones found in the resultant selected library. Selected inserts were cloned into lambda gt10, amplified with the PCR using vector primers, and dot blotted. 200 inserts have been hybridized individually to cosmids from the contig and to the cDNA dot blots. Approximately 70% of these map back to specific cosmids or YACs in the region. These PCR products were sequenced directly and analyzed for homology against each other as well as against sequences within GenBank. At least 23 new genes have been identified and isolated from this region based on sequence and hybridization overlaps. Seventeen of these cDNAs appear to be unique, two are known genes previously mapped to the region, one has homology to a known known Drosophilia gene, one is homologous to a human non-histone chromosomal protein HMG-17, and two are new members of gene families. These cDNAs are being used for mutational analyses in affected women from families with multiple cases of breast and ovarian cancer.
- OSTI ID:
- 134455
- Report Number(s):
- CONF-941009-; ISSN 0002-9297; TRN: 95:005313-1189
- Journal Information:
- American Journal of Human Genetics, Vol. 55, Issue Suppl.3; Conference: 44. annual meeting of the American Society of Human Genetics, Montreal (Canada), 18-22 Oct 1994; Other Information: PBD: Sep 1994
- Country of Publication:
- United States
- Language:
- English
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