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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