The molecular and genetic characterization of the human genome has required the isolation of large human chromosome fragments. With the yeast S. cerevisiae it is routinely possible to randomly isolate molecules that are several hundred kb as artificial chromosomes (YACs). While YACs have proven useful for developing large contig libraries several types of errors are produced in the present commonly used YAC cloning systems. They include chimeras (co-cloned DNA sequences from different regions of the genome), rearrangements during transformation and instabilities during growth. Over the last year we have identified many of the sources of artifacts and have provided strains and approaches that help to reduce the problems. Specifically, we have shown that i) the frequency of co-penetration of chromosomal size DNAs during transformation is high and co-transformed YACs readily recombine giving rise to chimeras ii) Alu sequences are a source of transformation- associated recombination between YAC DNAs, iii) the recombination between two incoming YAC DNA molecules is decreased in a rad52 host strain, iv) the integrity of YACs during mitotic propagation can be greatly improved using recombination-deficient host strains, particularly a rad52 mutant. Thus, we demonstrated through these systematic studies that a rad52 mutant, as compared to a wild-type strain, can greatly improve the fidelity during cloning and propagation of human DNA as YACs. In addition we have developed a novel system for the generation of YACs. The system which is based on transformation-associated recombination (TAR) greatly simplifies the cloning of large segments of chromosomal DNA from a variety of organisms. We have demonstrated that this system can be used to specifically isolate human DNA from a mouse/human monochromosomal hybrid cell line.
