HIGHLY SELECTIVE ISOLATION OF HUMAN DNAS FROM RODENT-HUMAN HYBRID-CELLS AS CIRCULAR YEAST ARTIFICIAL CHROMOSOMES BY TRANSFORMATION-ASSOCIATED RECOMBINATION CLONING

Transformation-associated recombination (TAR) can be exploited in yeas t to clone human DNAs. TAR cloning was previously accomplished using o ne or two telomere-containing vectors with a common human repeat(s) th at could recombine with human DNA during transformation to generate ye ast artificial chromosomes (YACs). On basis of the proposal that broke n DNA ends are more recombinogenic than internal sequences, we have in vestigated if TAR cloning could be applied to the generation of circul ar YACs by using a single centromere vector containing various human r epeats at opposite ends. Transformation with these vectors along with human DNA led to the efficient isolation of circular YACs with a mean size of approximate to 150 kb. The circular YACs are stable and they c an be easily separated from yeast chromosomes or moved into bacterial cells if the TAR vector contains an Escherichia coli F-factor cassette . More importantly, circular TAR cloning enabled the selective isolati on of human DNAs from monochromosomal human-rodent hybrid cell lines. Although <2% of the DNA in the hybrid cells was human, as much as 80% of transformants had human DNA YACs when a TAR cloning vector containe d Alu repeats, The level of enrichment of human DNA was nearly 3000-fo ld, A comparable level of enrichment was demonstrated with DNA isolate d from a radiation hybrid cell line containing only 5 Mb of human DNA, A high selectivity of human DNA cloning was also observed for linear TAR cloning with two telomere vectors. No human-rodent chimeras were d etected among YACs generated by TAR cloning. The results with a circul ar TAR cloning vector or two vectors differed from results with a sing le-telomere vector in that the Latter often resulted in a series of te rminal deletions in linear YACs, This could provide a means for physic al mapping of cloned material.