The mapping and sequencing of the human genome is a world-wide effort with many anticipated health benefits. A cornerstone of the Human Genome Project (HGP) is the use of yeast artificial chromosome (YAC) vectors for the cloning of large chromosome fragments. It is essential for the HGP that human DNA within YACs be stable within yeast. A major source of artefacts as we have shown arises from the repetitive nature of the DNA. These are potential substrates for co-cloning events and transformation- associated and mitotic recombination since recombination between diverged DNAs has been demonstrated in yeast. Some YACs exhibit considerable internal recombinational repair. We have suggested that the extent of repeats, even if diverged, may be indicated by the sensitivity of YACs to ionizing radiation induced loss and that the survival of YACs is dictated by the amount of recombinational repair between the diverged DNAs (currently under study). Small repeats that surround large inverted repeats (such as Alu's) may be sites of excision, as we have shown for Tn5 DNA in yeast. Problems in replication may also contribute significantly to YAC instability. Two potential sources of replication problems are, 1) insufficient functional origins of replication in the long tracts of mammalian DNA, and 2) possible deletions of sequences flanked by inverted repeats, which have been shown to facilitate replication bypass. We have shown that specific replication mutatants can enhance recombination between large repeats in chromosomes and can stimulate up to 100-fold excision involving small repeats in Tn5. The goals of the project are, 1) improvement of cloning methods and recipient strains, 2) improvement of YAC stability utilizing replication and recombination mutants and gene products, and 3) development of in vivo fragmentation systems for the precise physical mapping of sequences of interest within YACs. We are also merging YAC cloning systems with mammalian viral cloning systems for the easy transfer of large fragments to human cells.
