Direct selection for gene replacement events in yeast

Abstract

A method that facilitates gene replacement at the HIS3 locus of Saccharomyces cerevisiae (yeast) has been developed. First, an internal region of the cloned HIS3 gene was replaced by a DNA segment containing the wild-type ribosomal protein gene, CYH2. Second, by using standard yeast transformation methods, the wild-type HIS3 locus of a cycloheximide resistant strain (cyh2r) was replaced by this his3-CYH2 substitution. The resulting strain is sensitive to cycloheximide because CYH2 is dominant to cyh2r. Third, his3 mutations cloned into integrating or replicating vectors were introduced into this strain by selecting transformants via the vector-encoded marker. Selection for cycloheximide-resistant colonies resulted in the replacement of the his3-CYH2 allele by newly introduced his3 alleles. Thus, this scheme provides for the direct selection of gene replacement events at the HIS3 locus independently of the phenotype of the cloned his3 derivatives. In principle, it can be extended to any region of the yeast genome.