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One-step gene replacement in yeast by cotransformation.

H Rudolph·I Koenig-Rauseo·A Hinnen
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Abstract

A general method to replace chromosomal DNA sequences of Saccharomyces cerevisiae by any in vitro modified DNA sequence has been developed and was applied to the PHO5 locus on chromosome II. A recipient strain was constructed in which part of the chromosomal PHO5 sequence was substituted by the URA3 gene. Replacement of this pho5-URA3 substitution by pho5 mutant alleles was achieved in one step by cotransformation with a pho5 DNA fragment and the self-replicating plasmid YEp13, which contains the LEU2 gene as a selectable marker. Leu+ transformants were selected, and the replacement events at the PHO5 locus were detected by their Ura- phenotype (1-4% of the Leu+ were Ura-). In a similar way the PHO5 coding sequence was replaced by the sequence coding for human tissue-type plasminogen activator (t-PA).

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AllelesBase CompositionBase SequenceDNA Restriction EnzymesDNA, FungalEscherichia coliGenes, FungalPlasmidsSaccharomyces cerevisiaeTransformation, GeneticJournal Article

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H Rudolph, I Koenig-Rauseo, A Hinnen. 1985. One-step gene replacement in yeast by cotransformation.. https://doi.org/10.1016/0378-1119(85)90072-1

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