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.
A fusion gene which encoded a polypeptide comprised of 1116 amino acids was constructed using the alpha-amylase and glucoamylase cDNAs of Aspergillus shirousamii. When the fusion gene was expressed in Saccharomyces cerevisiae using a yeast expression plasmid under the control of the yeast ADH1 promoter, a bifunctional fusion protein (145 kDa) having both alpha-amylase and glucoamylase activities was secreted into the culture medium. The fusion protein had higher raw-starch-digesting activity than those of the original alpha-amylase and glucoamylase, and adsorbed onto raw starch like the glucoamylase. It was suggested that the characteristics are a result of the raw-starch-affinity site in the glucoamylase domain of the fusion protein.
Mutants of Saccharomyces diastaticus with defects in starch fermentation were isolated. In addition to the mutations in the STA1structural gene for glucoamylase (stal) and in the transcription factor (garni), we recovered two new complementation groups, designated gam2 and gam3. These starch-nonfermenting mutants secreted little or no glucoamylase. The garni and gam2mutants were pleiotropically defective in use of nonfermentable carbon sources, and consequently did not undergo meiosis and sporulation. No pleiotrophy was detected in the garni mutant. RNAblot analysis revealed that GAM2 and GAMS are also required for transcription of STAL
Deletion analysis was used to identify sequences upstream of the STA1 gene of Saccharomyces diastaticus that are required for its expression. Our analysis revealed that STA1has at least three upstream activation sequences (UAS): UAS1 and UAS2-2 were required for full transcriptional activity, while deletions removing UAS2-1 were still transcribed but the transcripts failed to be translated. These sequences contained both A + T-rich and inverted repeat sequences that might serve as componentsof the upstream activation sites. Wealso showedthat either deletion of a TATA homologue or insertion of palindromic DNAwithin the region between the TATAbox and the mRNA start sites had no effect on the level of STA1transcription but the transcripts failed to initiate properly.Amonga numberof Saccharomycesspecies, S. diastaticus is notable for its ability to secrete glucoamylase and to ferment starch.1} The enzyme is encoded by STA1,2'3) and is a major protein in the culture medium.4'5) This organism is closely related to S. cerevisiae, a starch-nonfermenting species, since haploid cells of these species are able to mate, and they are genetically similar. Both tetrad analysis6'7) of the cross between these species and mutational analysis8) revealed that expression of STA1 is regulated positively by GAM1 (formerly AMY2) or negatively by INHI and MAT.Recently, we have shown that these regulatory genes exert their functions at either the transcriptional or post-transcriptional level (accompanying paper). It is not known how these regulatory proteins act at the upstream promoter region of STAL Previous studies on the eukaryotic promoters have revealed a number of control elements which are required for faithful transcription.9* One element, the TATAbox, found in the 5'-flanking regions of virtually all eukaryotic genes, has been shown to be required for setting the site of transcription initation10'n) and, in some cases,12) maintain-749 ing levels of transcription. A second element, the upstream activation sequence, has been found in the 5' noncoding regions far upstream from the TATAbox. Deletion of these sequences greatly reduces levels of transcription.9) To date, however, the precise role of these elements and molecular mechanisms for transcriptional activation are obscure.For this paper, we undertook a detailed internal deletion analysis of the promoter region of STA1to identify the control elements involved in efficient expression of the gene. Our results demonstrate that the STA1 promoter contains at least three UASs: two are required for levels of transcription, while the remaining one does not affect the level of transcription but is required for generating translatable mRNA.We also showed that the TATA box and its flanking sequences are important for accurate transcription initiation.
Materials and MethodsConstruction of mutant plasmids. Xhol linker DNA(5'-CCTCGAGG-3 ) was randomly inserted by the method of Heffron et al.l3) into the plasmid pSTAl-7-183) which carries STA1, LEIQ, URA3, and a 2-fim replicon.
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