THE ROLE OF RECOMBINATION AND RAD52 IN MUTATION OF CHROMOSOMAL DNA TRANSFORMED INTO YEAST

While transformation is a prominent tool for genetic analysis and geno me manipulation in many organisms, transforming DNA has often been fou nd to be unstable relative to established molecules. We determined the potential for transformation-associated mutations in a 360 kb yeast c hromosome III composed primarily of unique DNA. Wild-type and rad52 Sa ccharomyces cerevisiae strains were transformed with either a homologo us chromosome III or a diverged chromosome III from S.carlsbergensis. The host strain chromosome III had a conditional centromere allowing i t to be lost on galactose medium so that recessive mutations in the tr ansformed chromosome could be identified. Following transformation of a RAD(+) strain with the homologous chromosome, there were frequent ch anges in the incoming chromosome, including large deletions and mutati ons that do not lead to detectable changes in chromosome size. Based o n results with the diverged chromosome, interchromosomal recombination al interactions were the source of many of the changes. Even though ra d52 exhibits elevated mitotic mutation rates, the percentage of transf ormed diverged chromosomes incapable of substituting for the resident chromosome was not increased in rad52 compared to the wild-type strain , indicating that the mutator phenotype does not extend to transformin g chromosomal DNA. Based on these results and our previous observation that the incidence of large mutations is reduced during the cloning o f mammalian DNA into a rad52 as compared to a RAD(+) strain, a rad52 h ost is well-suited for cloning DNA segments in which gene function mus t be maintained.