1887

Abstract

Using an optimized transformation protocol we have studied the possible interactions between transforming plasmid DNA and the genome. Plasmids consisting only of a pBR322 replicon, an antibiotic resistance marker for and the gene were shown to replicate autonomously in the yeast at an approximate copy number of 6 (copies per genome equivalent). This autonomous behaviour is probably due to an replicon-like sequence present on the gene fragment. Plasmids replicated as multimers consisting of monomers connected in a head-to-tail configuration. Two out of nine transformants analysed appeared to contain plasmid multimers in which one of the monomers contained a deletion. Plasmids containing internal or flanking regions of the genomic alcohol oxidase gene were shown to integrate by homologous single or double cross-over recombination. Both single- and multicopy (two or three) tandem integrations were observed. Targeted integration occurred in 1-22% of the cases and was only observed with plasmids linearized within the genomic sequences, indicating that homologous linear ends are recombinogenic in . In the cases in which no targeted integration occurred, double-strand breaks were efficiently repaired in a homology-independent way. Repair of double-strand breaks was precise in 50-68% of the cases. Linearization within homologous as well as nonhomologous plasmid regions stimulated transformation frequencies up to 15-fold.

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1992-11-01
2021-10-20
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