SME1 was cloned due to its high copy number effect: it enabled MATa/MAT alpha diploid cells to undergo meiosis and sporulation in a vegetative medium. Disruption of SME1 resulted in a recessive Spo- phenotype. These results suggest that SME1 is a positive regulator for meiosis. DNA sequencing analysis revealed an open reading frame of 645 amino acids. An amino terminal peptide of ca 400 amino acids in the deduced protein was similar to known protein kinases. Transcription of SME1 was regulated negatively by nitrogen and glucose and positively by MATa/MAT alpha and IME1, another positive regulator gene of meiosis. By complementation analysis, SME1 was found to be identical to IME2, which had been shown to be important in meiosis. These results suggest that IME1 product stimulates meiosis by activating transcription of SME1 (IME2) and that protein phosphorylation is required for initiation of meiosis.

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To find amino acid residues which are required for glucoamylase activity, mutant glucoamylase genes were constructed by in vitro mutations of GLUlDNAencoding Saccharomycopsis fibuligera glucoamylase and introduced into Saccharomyces cerevisiae, and the resulting mutant proteins were assayed for enzymatic activities. Eighteen mutant proteins were obtained by random insertions of a BamHllinker DNA.Six out of 7 proteins with mutations in conserved regions among divergent glucoamylases showed no activities, while 8 out of ll proteins with mutations in unconserved regions had similar specific activities to a wild-type value, suggesting that the conserved regions are important to the activity. Aseries of amino-terminal deletion mutants were also constructed. A mutant protein with a deletion of only two amino acid residues from the amino terminus had a significant reduction in the activity, suggesting an essential role for the amino-terminal peptide. Ten mutant proteins with single amino acid replacements were produced by site-directed mutagenesis. Analyses for thermal stability and temperature dependency of these mutant proteins revealed that Ala81, Asp89, Trp94, Arg96, Asp97, and Trpl66 are required for wild-type levels of activities, and that at least Ala81 and Asp89 are not essential to catalytic activities, but act in thermal stability. Glucoamylase catalyzes the release of glucose from the non-reducing ends of starchy materials and other a-glucopyranosides. The enzyme is widely distributed from microorganisms to vertebrates and is important in starch fermentation industries. Recently, we cloned the glucoamylase gene GLU1from a genomic library of Saccharomycopsis fibuligera by screening Saccharomyces cerevisiae transformants capable of starch fermentation1} and determined the nucleotide