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.
Annab
L A
LA
National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA.
Kouprina
N
N
Solomon
G
G
Cable
P L
PL
Hill
D E
DE
Barrett
J C
JC
Larionov
V
V
Afshari
C A
CA
D000818:Animals; D019313:BRCA1 Protein / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D018244:Chromosomes, Artificial, Yeast; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014162:Transfection; D014170:Transformation, Genetic; D014407:Tumor Cells, Cultured
D019313:BRCA1 Protein