Isolation of a functional copy of the human BRCA1 gene by transformation-associated recombination in yeast.

The BRCA1 gene, mutations of which contribute significantly to hereditary breast cancer, was not identified in the existing YAC and BAC libraries. The gene is now available only as a set of overlapping fragments that form a contig. In this work we describe direct isolation of a genomic copy of BRCA1 from human DNA by transformation-associated recombination (TAR) cloning. Despite the presence of multiple repeats, most of the primary BRCA1 YAC isolates did not contain detectable deletions and could be stably propagated in a host strain with conditional RAD52. Similar to other circular YACs, approximately 90kb BRCA1 YACs were efficiently and accurately retrofitted into bacterial artificial chromosomes (BACs) with the Neo(R) mammalian selectable marker and transferred as circular BAC/YACs in E. coli cells. The BRCA1 BAC/YAC DNAs were isolated from bacterial cells and were used to transfect mouse cells using the Neo(R) gene as selectable marker. Western blot analysis of transfectants showed that BRCA1 YACs isolated by a TAR cloning contained a functional gene. The advantage of this expression vector is that the expression of BRCA1 is generated from its own regulatory elements and does not require additional promoter elements that may result in overexpression of the protein. In contrast to the results with cDNA expression vectors, the level of BRCA1 expression from this TAR vector is stable, does not induce cell death, maintains serum regulation, and approximates the level of endogenously expressed BRCA1 in human cells. The entire isolation procedure of BRCA1 described in this paper can be accomplished in approximately 10 days and can be applied to isolation of gene from clinical material. We propose that the opportunity to directly isolate normal and mutant forms of BRCA1 will greatly facilitate analysis of the gene and its contribution to breast cancer.

Establishment and characterization of a breast cell strain containing a BRCA1 185delAG mutation.

OBJECTIVE: The aim of this study was to examine whether cells containing the heterozygous form of a BRCA1 185delAG mutation would exhibit abnormal growth or an altered response to DNA damage. METHODS: A primary culture of human mammary epithelial cells (90P) was obtained from the nontumor breast tissue of a 35-year-old patient who had undergone a mastectomy for removal of a breast tumor. These cells were immortalized (90PE6E7) following retroviral infection with HPV-16 viral E6/E7. genes. Both the 90P cell strain and the cell line were characterized for their ability to grow in culture, form colonies in soft agar, and produce tumors in athymic nude mice compared to normal breast epithelial cells containing wild-type BRCA1. 90P cells were also analyzed for cellular response to gamma radiation and H(2)O(2). RESULTS: These cells were confirmed to contain a frameshift mutation, 185delAG, of the BRCA1 gene. Despite being heterozygous for wild-type BRCA1, the 220-kDa full-size BRCA1 protein was abundantly expressed. 90P and 90PE6E7 cells grew at a similar rate and were anchorage dependent. 90PE6E7 also failed to form tumors in athymic nude mice. Finally, 90P cells exhibited a survival response similar to that of normal mammary epithelial cells to radiation damage and exposure to oxidative stress. CONCLUSION: To our knowledge the 90P cells and the 90PE6E7 cells are the first characterized, non-tumor-derived breast epithelial cells that are heterozygous for the BRCA1 germline mutation 185delAG. Our conclusion is that these BRCA1 mutant cells appear to have growth and stress response characteristics similar to those of normal human breast cells, which is consistent with the hypothesis that loss of heterozygosity must occur to impair putative BRCA1 function.

Cloning and characterization of the promoter region of human telomerase reverse transcriptase gene.

Activation of telomerase is one of the rate-limiting steps in human cell immortalization and carcinogenesis Human telomerase is composed of at least two protein subunits and an RNA component. Regulation of expression of the catalytic subunit, human telomerase reverse transcriptase (hTERT), is suggested as the major determinant of the enzymatic activity. We report here the cloning and characterization of the 5'-regulatory region of the hTERT gene. The highly GC-rich content of the 5' end of the hTERT cDNA spans to the 5'-flanking region and intron 1, making a CpG island. A 1.7-kb DNA fragment encompassing the hTERT gene promoter was placed upstream of the luciferase reporter gene and transiently transfected into human cell lines of fibroblastic and epithelial origins that differed in their expression of the endogenous hTERT gene. Endogenous hTERT-expressing cells, but not nonexpressing cells, showed high levels of luciferase activity, suggesting that the regulation of hTERT gene expression occurs mainly at the transcriptional level. Additional luciferase assays using a series of constructs containing unidirectionally deleted fragments revealed that a 59-bp region (-208 to -150) is required for the maximal promoter activity. The region contains a potential Myc oncoprotein binding site (E-box), and cotransfection of a c-myc expression plasmid markedly enhanced the promoter activity, suggesting a role of the Myc protein in telomerase activation. Identification of the regulatory regions of the hTERT promoter sequence will be essential in understanding the molecular mechanisms of positive and negative regulation of telomerase.