Nucleic Acids Research

The Saccharomyces STA2 and SGA genes, encoding the extraceliular and intracellular sporulation-specific glucoamylase respectively, have been cloned and their transcription and regulation studied. The STA2 gene differs from the SGA gene in that it contains an extra piece of DNA, which encodes the domain for exportation of the extracellular glucoamylase. The STA2 gene produces a single 2.85 kb transcript. Transcription of the SGA gene is initiated from two different sites, yielding two transcripts of 1.95 and 2.40 kb. Transcription of both STA2 and SGA genes is repressed by the STA10 gene of Saccharomyces cerevisiae. INTRODUCTION Saccharomyces diastaticus expresses an extracellular glucoamylase which is encoded by either the STA1, STA2 or STA3 nuclear genes (1,2). Recently, the STAl and STA3 genes from S. diastaticus have been cloned and the STAl gene sequenced (3,4). It has been suggested that the extracellular glucoamylase encoded by this last gene is translated as a polypeptide of 778 aminoacids containing two domains: one comprising the NH2-terminal 388 aminoacids that would serve as a signal peptide eliminated during export, and the other one corresponding to the two subunits of the mature extracellular glucoamylase (5). In contrast to S. diastaticus, S. cerevisiae lacks these genes and, therefore, can not use starch as a carbon source (6). Nevertheless, both S. cerevisiae and S. diastaticus contain a common DNA sequence highly homologous to the STAl gene (4,7). It was shown that this homologous sequence encoded the sporulation-specific glucoamylase of S. cerevisiae (8,9,4,10). Moreover, it has been proposed that the extracellular glucoamylase gene of S. diastaticus could have evolved from the sporulation-specific glucoamylase gene (SGA) of © I RL Press Limited, Oxford, England. 470 1 Nucleic Acids Research S. cerevisiae (4) by the acquisition of extra DNA to encode the export domain of the extracellular glucoamylase. Interestingly, S. cerevisiae contains the STA10 gene which, by genetic studies, was shown to dominantly and epistatically repress the expression of the STA1, 2 and 3 genes in S. cerevisiae x S. diastaticus haploid hybrid strains (6). In this work we have studied the transcriptional products of both STA2 and SGA genes. Moreover, we have examined, both biochemically and genetically, the STA10-dependent repression of these two genes at the level of transcription in a variety of yeast strains. MATERIALS AND METHODS Yeast and bacterial strains and growth conditions The yeast strains used in this work, their origins and genotypes are listed in Table I. STA+ strains were derivatives of those described by Tamaki (1). Escherichia coli HB101 (11) was used for transformation and plasmid amplification. Yeast cells were grown on YEPD (1% yeast extract, 2% peptone, 2% glucose) or YNB (0.7% yeast nitrogen base w/o aminoacids, 2% glucose, supplemented with 40 pg/ml aminoacids when required) media. STA+ phenotype was scored on plates containing YPS medium (1% yeast extract, 2% peptone, 2% soluble starch, 2% agar). Colonies were transferred to this medium and incubated for 3-5 days before starch was precipitated by keeping the plates at 40C for 1-2 days. A clear halo appeared around the STA+ colonies. L medium (12) was used for culturing bacterial cells and, when appropiate, it was supplemented with 100 pag/ml ampicillin or 15 pg/ml tetracycline. Genetic procedures Standard yeast genetic procedures were followed for crossing, sporulation and tetrad analysis (13). DNA isolation and cloning procedures Yeast transformation was carried out as described by Hinnen et al. (14). Genomic and plasmid DNA of yeast were isolated as described by Sherman et al. (15). Bacterial transformation and