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.

Catalase T deficient mutants of Saccharomyces cerevisiae.

A method for the isolation of catalase T deficient mutants of Saccharomyces cerevisiae is described. Ten mutants lacking catalase T and belonging to 5 complementation groups were isolated. CTT1 locus was identified as the structural gene for catalase T. It is under the control of CTT2, CTT3 and CTT4 loci.

Isolation of the catalase A gene of Saccharomyces cerevisiae by complementation of the cta1 mutation.

As a first step in an analysis of the DNA regions involved in the control of the catalase A gene of Saccharomyces cerevisiae by glucose, heme, and oxygen this gene has been cloned. Catalase A-deficient mutants were obtained by UV mutagenesis of a ctt1 mutant strain specifically lacking catalase T. All the catalase A-deficient mutants obtained fall into one complementation group. The single recessive mutation causing specific lack of catalase A was designated cta1. Several overlapping DNA fragments complementing the cta1 mutation were obtained by transforming ctt1 cta1 double mutants with a yeast gene library in vector YEp13. Hybrid selection of RNA with the help of one of the cloned DNAs followed by in vitro translation of this RNA and identification of the protein synthesized with catalase A-specific antibodies showed that the catalase A structural gene has been cloned. A single copy of this gene is present in the yeast genome. Transcription of the catalase A gene cloned into vector YEp13 is repressed by glucose. The DNA isolated hybridizes to a 1.6 kb polyA+-RNA virtually absent from heme-deficient cells, presumably catalase A mRNA.