The role of recombination and RAD52 in mutation of chromosomal DNA transformed into yeast.
While transformation is a prominent tool for genetic analysis and genome manipulation in many organisms, transforming DNA has often been found to be unstable relative to established molecules. We determined the potential for transformation-associated mutations in a 360 kb yeast chromosome III composed primarily of unique DNA. Wild-type and rad52 Saccharomyces cerevisiae strains were transformed with either a homologous chromosome III or a diverged chromosome III from S. carlsbergensis. The host strain chromosome III had a conditional centromere allowing it to be lost on galactose medium so that recessive mutations in the transformed chromosome could be identified. Following transformation of a RAD+ strain with the homologous chromosome, there were frequent changes in the incoming chromosome, including large deletions and mutations that do not lead to detectable changes in chromosome size. Based on results with the diverged chromosome, interchromosomal recombinational interactions were the source of many of the changes. Even though rad52 exhibits elevated mitotic mutation rates, the percentage of transformed 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 transforming chromosomal DNA. Based on these results and our previous observation that the incidence of large mutations is reduced during the cloning of mammalian DNA into a rad52 as compared to a RAD+ strain, a rad52 host is well-suited for cloning DNA segments in which gene function must be maintained.
Larionov
V
V
Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709.
Graves
J
J
Kouprina
N
N
Resnick
M A
MA
D015825:Chromosomes, Fungal; D003470:Culture Media; D004271:DNA, Fungal / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics*; D005656:Fungal Proteins / Q000235:genetics*; D005690:Galactose; D017353:Gene Deletion; D009154:Mutation; D051721:Rad52 DNA Repair and Recombination Protein; D011995:Recombination, Genetic; D012440:Saccharomyces / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D014170:Transformation, Genetic
D003470:Culture Media; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C497197:RAD52 protein, S cerevisiae; D051721:Rad52 DNA Repair and Recombination Protein; D029701:Saccharomyces cerevisiae Proteins; D005690:Galactose