Reference: Symington LS, et al. (1983) Genetic recombination of homologous plasmids catalyzed by cell-free extracts of Saccharomyces cerevisiae. Cell 35(3 Pt 2):805-13

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Abstract


We have developed an in vitro system utilizing yeast cell-free extracts to catalyze recombination events between homologous plasmids containing different mutant alleles of the Tet or ARG4 genes. The reaction increased the frequency of Tcr or Arg+ transformants (recombinants) from 2 X 10(-6) to 1-3 X 10(-3). Linearizing one substrate between the two tet mutations stimulated the reaction 2 to 4 fold. The reaction required rATP, Mg++, NAD, and DTT. The rad52-1 mutation decreased the reaction between linear and circular substrates 5 to 6 fold but had little effect with circular substrates. The structures of Tcr plasmids was analyzed by restriction endonuclease mapping and was consistent with a recombination reaction involving crossing-over and gene conversion. Recombination products were also observed directly by subjecting reaction mixtures to electrophoretic analysis. These results indicate that recombination events were catalyzed by the yeast extract.

Reference Type
Journal Article | Research Support, Non-U.S. Gov't
Authors
Symington LS, Fogarty LM, Kolodner R
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