engleski

Linearized integrative single-stranded plasmids in yeast transformation

We used the single-stranded nonreplicative plasmid pOM that was linearized with restriction endonuclease EcoRV to transform yeast protoplasts. Linearization was achieved after hybridization of the plasmidic DNA with appropriate 17-mer and was confirmed by both agarose gel electrophoresis and transformation of the bacterium Escherichia coli. Circular and linearized plasmids were shown to transform yeast with comparable efficiencies, but the spectrum of transformants obtained in these two cases was remarkably different. Namely, only 3/30 transformants analyzed by Southern blot hybridization were due to the plasmid integration by homologous recombination, while more than 30% of transformants were produced by conversion of the Ty element (6, 1 kb) that was present in the ura3 gene. Additional experiments revealed that the large majority of transformation events resulted from non-specific (or less-specific) plasmid integrations. A possible role of single-stranded tails in homologous recombination will be discussed.

yeast; transformation; single-stranded DNA

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