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The electronic form of this document may be cited in the following style:
Human Genome Program, U.S. Department of Energy, DOE Human Genome Program Contractor-Grantee Workshop IV, 1994.

Abstracts scanned from text submitted for November 1994 DOE Human Genome Program Contractor-Grantee Workshop. Inaccuracies have not been corrected.

The role of recombination and RAD52 in mutation of chromosomal size DNA transformed into yeast

Vladimir Larionov[1], Joan Graves, Natalya Kouprina, and Michael Resnick.
Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Box 12233 Research Triangle Park, NC 27709. [1]Corresponding author.

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 S. 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 small and large mutations. 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 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 [1], a rad52 host is well-suited for cloning DNA segments in which gene function must be maintained.

Support was provided in part by the Department of Energy through an interagency agreement with NIEHS and grant (1-YO2-HG-60021-01) from the NIH Human Genome Center to M.A.R.

[1]. Kouprina, N., Eldarov, M., Resnick, M., Moyzis B., Larionov, V. A model system to assess the integrity of mammalian YACs during transformation and propagation in yeast. (1994) Genomics, 21, 7-17