Isolation of the catalase T structural gene of Saccharomyces cerevisiae by functional complementation.
The catalase T structural gene of Saccharomyces cerevisiae was cloned by functional complementation of a mutation causing specific lack of the enzyme (cttl). Catalase T-deficient mutants were obtained by UV mutagenesis of an S. cerevisiae strain bearing the cas1 mutation, which causes insensitivity of catalase T to glucose repression. Since the second catalase protein of S. cerevisiae, catalase A, is completely repressed on 10% glucose, catalase T-deficient mutant colonies could be detected under such conditions. A cttl mutant was transformed with an S. cerevisiae gene library in plasmid YEp13. Among the catalase T-positive clones, four contained overlapping DNA fragments according to restriction analysis. Hybridization selection of yeast mRNA binding specifically to one of the cloned DNAs, translation of this mRNA in cell-free protein synthesis systems, and demonstration of catalase T protein formation by specific immunoadsorption showed that the catalase T structural gene had been cloned. By subcloning, the gene was located within a 3.5-kilobase S. cerevisiae DNA fragment. As in wild-type cells, catalase T synthesis in cttl mutant cells transformed with plasmids containing this fragment is sensitive to glucose repression. By DNA-RNA hybridization, catalase T transcripts were shown to be present in oxygen-adapting cells but absent from heme-deficient cells.
D002374:Catalase / Q000235:genetics*; D003001:Cloning, Molecular; D005796:Genes; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D009154:Mutation; D009693:Nucleic Acid Hybridization; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D014170:Transformation, Genetic
D002374:Catalase