Isolation and functional analysis of sporulation-induced transcribed sequences from Saccharomyces cerevisiae

Abstract

Strains of the yeast Saccharomyces cerevisiae that are heterozygous for the mating-type locus (MATa/MAT alpha) undergo meiosis and spore formation when they are starved for nitrogen and are provided with a nonfermentable carbon source such as potassium acetate. Haploids and diploids homozygous for the mating-type locus (MAT alpha/MAT alpha or MATa/MATa) are asporogenous and undergo neither meiosis nor spore formation when incubated under the same conditions. A small number of genes produce transcripts that appear to be induced specifically in sporulating cells. These transcripts either are not found or are present at much lower levels both in vegetatively growing cells and in cells from asporogenous strains that have been incubated in sporulation medium. Several genes complementary to these MATa/MAT alpha-dependent sporulation-induced transcripts were isolated from a gene-size insert yeast-lambda recombinant DNA library, by differential-plaque filter hybridization. An attempt was made to determine the function of three of these genes by mutating them in the yeast genome with in vitro mutagenesis and one-step gene replacement techniques. One gene was extensively disrupted by both a 0.3-kilobase deletion and the insertion of two large DNA sequences at different sites within the gene. Surprisingly, this compound mutation did not appear to affect meiosis or the production of viable ascospores, indicating that this gene was dispensable for differentiation. The other two genes were disrupted by simple insertion mutations at a site where it was possible that they might still possess some gene activity. These mutations also did not appear to affect sporulation. These results suggest that not all sporulation-induced genes are essential for meiosis and the production of viable ascospores under the conditions examined.