Natalya Kouprina

Specific isolation of human rDNA genes by TAR cloning

Selective cloning of human DNA in YACs from monochromosomal human/rodent hybrid cells lines and radiation hybrids can be accomplished by transformation-associated recombination (TAR) between Alu-containing vector(s) and human DNA in yeast. We have expanded this approach to the specific isolation of repetitive genes from the human genome. Highly selective isolation of human rDNA was accomplished using total human DNA and a pair of differentially marked linear TAR cloning vectors where one contained a small fragment of a human rDNA repeat and the other had an Alu repeat as targeting sequences. About half the transformants that acquired both vectors markers had YACs with human rDNA inserts. These results suggest that TAR can be applied to the general isolation of gene families and amplified region from genomic DNAs.

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.

Rapid cloning of mouse DNA as yeast artificial chromosomes by transformation-associated recombination (TAR)

Several yeast artificial chromosome (YAC) libraries have been constructed for physical mapping and characterization of the mouse genome (Larin et al. 1991; Chartier et al. 1992; Haldi et al. 1996). These complex libraries were constructed by in vitro ligation of telomere-containing vectors to mouse chromosome fragments generated by endonuclease digestion. Recently a novel approach for in vivo construction of YACs was developed. Transformation-associated recombination (TAR) in yeast has been utilized for the rapid cloning of human DNA as linear and circular YACs (Larionov et al. 1996a, 1996b). In linear TAR cloning (Larionov et al. 1996a), yeast spheroplasts are presented with gently isolated human DNA and one or two, nonreplicating vectors that contain selectable markers, a yeast telomere, and an Alu or LINE sequence(s) to serve as a ‘‘hook’’. Homologous recombination can occur between the co-transformed human DNA and repeats on the vectors to create linear YACs. Propagation of the YACs can occur because one of the vectors contains a yeast centromere and cloned human DNA typically contains sequences that can function as yeast autonomous replicating sequences (ARS). A YAC can also be established when one vector is used because a telomere can be formed at the end of cloned human DNA, possibly by (CA)n microsatellite repeats. This approach has been used to isolate human YACs ranging in size from 70 kb to >600 kb (Larionov et al. 1996a). Circular TAR cloning (Larionov et al. 1996b) differs from linear TAR cloning in that a single linear vector is used that contains a centromere and hooks at each end to generate circular YACs. Circular TAR cloning procedure has produced YACs containing human inserts up to 500 kb (Larionov et al. 1996b). Among the many utilities provided by TAR cloning is the opportunity to selectively isolate large random fragments of human DNA from rodent/human monochromosomal and radiation hybrid cell lines (Larionov et al. 1996a, 1996b). We have adapted TAR cloning for the isolation of mouse DNA as YACs. A series of linear and circular TAR vectors containing mouse repetitive elements B1 and B2 were constructed (Fig. 1). These elements were chosen because of their relative abundance in the mouse genome (80,000 and 180,000 copies respectively; Bennett et al. 1984). For linear TAR cloning the vectors contain a mouse repeat element at one end and a telomere at the other, whereas for circular TAR cloning the vectors contain mouse repeat elements at each end (Fig. 1). All vectors contain the yeast HIS3 gene as a selectable marker (except pLM1, which contains the LYS2 gene), the yeast centromere CEN6 (except pWJ522, which is acentric). Yeast spheroplasts prepared from the strain VL6-48 (the HIS3 gene is deleted) or from the strain YPH857-D1 (the entire LYS2 coding sequence and promoter region are deleted) were cotransformed with TAR vectors plus various genomic DNAs. As shown in Table 1, transformation of yeast spheroplasts by a mixture of mouse DNA plus a linear TAR cloning vector containing a B1 or B2 repeat yielded a high level of transformants, comparable to that previously reported for the cloning of human DNA with Alu-containing vectors (Larionov et al. 1996a, 1996b). Only a few transformants were obtained when mouse DNA was not included. 200 His transformants obtained with the linear vector pVCB2 and mouse genomic DNA (Table 1) were characterized by PFGE separation of chromosome size DNAs following probing with radiolabeled mouse cot-1 DNA (BRL) that was preannealed with unlabeled B2 DNA. Linear YACs (data not shown) ranging in size from 100 to >600 kb were detected in 198 of the 200 transformants examined, indicating that nearly all contained mouse DNA. Similar observations were made with transformants obtained with the linear vector pVC-B1 and mouse DNA. We also analyzed His transformants obtained with the circular TAR cloning vectors, pVC-B1-B2 and pVC-B2-B2. Large circular DNA molecules were detected in the loading wells [as expected with intact large circular molecules (Larionov et al. 1996b)] for 99 among 100 His pVC-B1-B2 and all 40 His pVC-B2-B2 transformants examined. The sizes of these circular YACs [after linearization with ionizing radiation (Larionov et al. 1996b)] ranged between 70 and 300 kb (data not shown). One hundred His transformants obtained with linear and circular TAR cloning B2 vectors were also analyzed by PCR with the primer ‘B1-21’ (Herman et al. 1991). Different fingerprints of multiple inter-B1 products were observed for all transformants (examples are shown in Fig. 2), demonstrating that different regions of the mouse genome had been cloned. The TAR cloning with B1or B2-targeting vectors was specific, since transformation was greatly reduced when chicken DNA was used instead of mouse DNA (Table 1). Transformation results also demonstrate the specificity of the B2 element for cloning of mouse DNA compared with human DNA (Table 1). In contrast, transformation of the B1-targeting vector along with human or hamster DNA occurred with the same efficiency as with mouse DNA (Table 1). In other experiments we have confirmed that mouse DNA can be selectively cloned by the circular TAR vectors pVC-B1-B2, pVC-B2-B2, and pVC-B2-B2-Neo when present in excess of heterologous DNA. On the basis of blot-hybridization analysis of His transformants obtained with pVC-B1-B2 from a mixture of mouse and chicken DNAs, 78% (60/76) contained mouse DNA. None of these transformants contained chicken DNA. From blot-hybridization analysis (with a B1 probe) of His transformants obtained from a mixture of mouse and human DNAs Correspondence to: N. Kouprina Mammalian Genome 9, 157–159 (1998).