Isolation of the catalase T structural gene of Saccharomyces cerevisiae by functional complementation.

Abstract

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.

A cis-acting element present in multiple genes serves as a repressor protein binding site for the yeast CAR1 gene.

“…DNA fragments containing these sequences were demonstrated to possess two characteristics expected of a repressor binding site as described above. In (2,11). These results are consistent with our interpretation.…”

The Saccharomyces cerevisiae ADR1 gene is a positive regulator of transcription of genes encoding peroxisomal proteins.

“…Essentially, strain WS13-9A, which carries a defect in the gene encoding the cytosolic catalase T, was transformed with a genomic yeast DNA library (28) in vector YEp13, and transformant colonies were screened for catalase activity on plates containing 10% glucose. One of the catalase-positive clones isolated (YEpl3-78) was demonstrated to produce catalase A on 10% glucose (Table 2), whereas the others contained the CTTI gene encoding catalase T (33). After the CTAI gene had been cloned (13), the nature of the DNA fragment cloned in plasmid YEp13-78 which interferes with glucose repression of CTAI transcription was further investigated.…”

“…The isolation of clone YEp13-78 as a multicopy suppressor of CTAI glucose repression has been described previously (33). Essentially, strain WS13-9A, which carries a defect in the gene encoding the cytosolic catalase T, was transformed with a genomic yeast DNA library (28) in vector YEp13, and transformant colonies were screened for catalase activity on plates containing 10% glucose.…”

Control of Saccharomyces cerevisiae catalase T gene (CTT1) expression by nutrient supply via the RAS-cyclic AMP pathway.

“…The yeast Saccharomyces cerevisiae contains two different hemoprotein catalases, the peroxisomal catalase A (32), which is encoded by the CTAI gene (11), and the cytoplasmic catalase T (33), encoded by CTTJ (35). Like the CYC1 (iso-1-cytochrome c) gene, the two catalase genes have been reported to be controlled positively by oxygen and hence and negatively by glucose repression (18).…”