Construction and genetic characterization of temperature-sensitive mutant alleles of the yeast actin gene

Abstract

Two temperature-sensitive mutations have been constructed in the single actin gene (ACT1) of the yeast Saccharomyces cerevisiae by in vitro mutagenesis of the cloned gene followed by integrative transformation of mutagenized DNA into yeast cells. A strategy of allele replacement was used that allowed recessive mutations to be phenotypically expressed in the initial transformants, thus simplifying the screening of large numbers of independently transformed cells. After confirming that several ts mutations were located within the actin structural gene by genetic methods, these mutant alleles were cloned, and the altered amino acid residues were defined by DNA sequence analysis. The two unique mutations resulted in substitution of proline-32 with leucine and alanine-58 with threonine. In the course of isolating these mutations, the observation was made that a high proportion of yeast cells transformed with exogenous DNA by the spheroplast method are temperature sensitive for growth because of genetic changes unrelated to the transforming DNA.