Isolation and characterization ofSaccharomyces cerevisiae mutants with derepressed thiamine gene expression

Using a THI4-lacZ reporter gene, mutant strains have been isolated that display constitutive expression of thiamine genes in the presence of normally repressing levels of exogenous thiamine. In total, eight strains were isolated in which this derepressed expression on thiamine (Det(-)) phenotype was the result of single gene mutations. The Det- mutations of three of these strains were partially dominant in a heterozygous diploid configuration, whereas the other five were recessive. The partially dominant mutants DET1, DET12 and DET13, and the recessive mutant det2, all showed derepressed THI4-lacZ expression levels comparable to those of a fully induced normal strain. Use of other promoter-lacZ gene fusions revealed that these four mutants were pleiotropic; expression levels of all thiamine-regulated genes tested were also derepressed. Genetic analysis of the four mutants suggested that det2 and DET13 were allelic, whereas the others were at different loci; these four mutations therefore represent three different genes. None of the mutations were allelic with TH180, mutations of which have previously been shown to confer derepression on thiamine-regulated genes. Also, intracellular thiamine levels were close to normal and none of the four mutants excreted thiamine into the growth medium. All mutant strains were found to be prototrophic for thiamine and none of those tested were compromised for thiamine uptake. It is possible that some may be alleles of, or interact with, the activator gene TH13. Taken together, these results imply that DET1, det2, DET12 and DET13 represent new genes encoding negative regulators of thiamine-repressed genes. Copyright (C) 2000 John Wiley & Sons, Ltd.