Direct isolation of human BRCA2 gene by transformation-associated recombination in yeast

Abstract

Mutant forms of the BRCA2 gene contribute significantly to hereditary breast cancer. Isolation of the normal and mutant forms of the BRCA2 gene with its natural promoter would greatly facilitate analysis of the gene and its contribution to breast cancer. We have accomplished the direct isolation of the 90-kb gene from total human DNA by transformation-associated recombination in yeast using a small amount of 5' and 3' BRCA2 sequence information. Because the entire isolation procedure of a single chromosomal gene could be accomplished in approximately 2 weeks, the transformation-associated recombination cloning approach is readily applicable to studies of chromosome alterations and human genetic diseases.

Figure 1

Isolation of the human BRCA2 gene as a circular YAC using the TAR cloning method. Yeast spheroplasts were transformed with genomic human DNA along with a TAR cloning vector containing promoter (striped box) and terminal exon 27 (dotted box) sequences of BRCA2 (see Materials and Methods) at the ends of the linearized plasmid. Recombination between the sequences in the vector and BRCA2 genomic DNA led to the establishment of a circular YAC because of ARS-like sequences in the BRCA2 gene. CEN corresponds to the yeast chromosome VI centromere, and HIS3 is a selectable marker. Arrows indicate the positions of primers used for initial identification of clones containing the BRCA2 gene.

Figure 2

Characterization of circular BRCA2 YACs obtained by TAR cloning from total human DNA. Chromosomal-size DNA was isolated from transformants containing BRCA2 YACs, exposed to a low dose of γ rays (5 Krad), separated by TAFE gel electrophoresis, and blot hybridized with total human genomic DNA (6). The strong signals at the positions of the starting wells correspond to large circular molecules. The leading bands at approximately 97 kb correspond to molecules linearized by the radiation. Four subclones of isolates 27 and 32 (lanes 1–4 and 5–8, respectively) and two subclones of isolate 60 (lanes 9 and 10) were analyzed.

Figure 3

Alu-profile characterization of YACs containing BRCA2. Subclones of the three initial isolates were characterized and compared in terms of Alu profiles of restriction fragments. Total yeast DNA was isolated from each subclone and digested to completion with TaqI. Fragments were separated by gel electrophoresis, transferred to a nylon membrane, and hybridized with an Alu probe. Four subclones of isolates 27 (lanes 1–4) and 32 (lanes 5–8) and three subclones of isolate 60 (lanes 9–11) were analyzed.

Figure 4

PCR analysis of YACs containing BRCA2. The presence of BRCA2 coding region in the YAC clones was examined by PCR using 26 pairs of exon primers and 2 pairs of junction primers. Presented in this figure are PCR products obtained for exons 3, 11, and 25 and for the 5′ and 3′ TAR vector junction sequences. (We note that comparable results were obtained for the other primers, in that the PCR products with the YACs matched those of the total human DNA.) The first three lanes for each set of PCR primers correspond to products obtained from the three independent BRCA2 YAC isolates. The fourth lane corresponds to the product generated from total human DNA. (In the PCR analysis of the 3′ TAR junction some additional bands were observed with the total human DNA that were due to PCR artifacts.) The fifth lane was a control in which no DNA was added (there was no fifth lane for the 3′ TAR junction). The first and last lanes contained 123-bp ladders.