The protein factor which binds to the upstream activating sequence of Saccharomyces cerevisiae ENO1 gene.

Using a gel retardation assay it was shown that the 87 bp DNA fragment (UAS87) containing the upstream activating sequence (UAS) of S. cerevisiae EN01 gene and a nuclear extract gave rise to three migration-retarded species specific to UAS87. Heat- or proteinase-treatment of the nuclear extract revealed that these species were protein-DNA complexes. The precise binding region of the protein identified by DNaseI protection analysis was found to include a CCAAACA sequence which forms a dyad-symmetrical structure. The amount of one of the three migration-retarded species significantly increased when cells were grown in medium containing a gluconeogenic carbon source. The introduction of pGCR8, a multicopy plasmid containing GCR1 gene, a regulatory gene controlling the expression of several glycolytic enzymes, showed no effect on the amount of three migration-retarded species.     

A revised preparation of yeast (Saccharomyces cerevisiae) pyruvate kinase.

A revised preparation of pyruvate kinase from saccharomyces cerevisiae is reported. By purifying this cold-labile enzyme at room temperature, an improved recovery and specific activity was obtained. More than 350 mg of pure enzyme with a specific activity of 350 to 400 units/mg at 30 degrees were obtained from a pound of fresh yeast. The last step of the preparation, passage of the enzyme over Sephadex G-100, was required to remove a contaminating protease. The molecular parameters of the new preparation are: molecular weight, 209,000; four subunits of identical size; E 280 nm, 0.51; pI 6.6; and pH optimum, 6.28. Kinetic parameters are: Km for P-enolpyruvate and ADP, 0.09 and 0.18 mM in the presence of saturating Fru-1,6-P2, and 1.8 and 0.34 mM in the absence of Fru-1,6-P2; Ka for Fru-1,6-P2, 0.014 mM. No free NH2-terminal amino acid could be detected. Amino acid composition was determined and compared with other pyruvate kinase preparations.     

Identification of the upstream activating sequence of MAL and the binding sites for the MAL63 activator of Saccharomyces cerevisiae.

Maltose fermentation in Saccharomyces species requires the presence of at least one of five unlinked MAL loci: MAL1, MAL2, MAL3, MAL4, and MAL6. Each of these loci consists of a complex of genes involved in maltose metabolism; the complex includes maltase, a maltose permease, and an activator of these genes. At the MAL6 locus, the activator is encoded by the MAL63 gene. While the MAL6 locus has been the subject of numerous studies, the binding sites of the MAL63 activator have not been determined. In this study, we used Escherichia coli extracts containing the MAL63 protein to define the binding sites of the MAL63 protein in the divergently transcribed MAL61-62 promotor. When a DNA fragment containing these sites was placed upstream of a CYC1-lacZ gene, maltose induced beta-galactosidase. These sites therefore constitute an upstream activating sequence for the MAL genes.     

Bipartite structure of an early meiotic upstream activation sequence from Saccharomyces cerevisiae.

Diploid a/alpha Saccharomyces cerevisiae cells cease mitotic growth and enter meiosis in response to starvation. Expression of meiotic genes depends on the IME1 gene product, which accumulates only in meiotic cells. We report here an analysis of the regulatory region of IME2, an IME1-dependent meiotic gene. Deletion and substitution studies identified a 48-bp IME1-dependent upstream activation sequence (UAS). Activity of the UAS also requires the RIM11, RIM15, and RIM16 gene products, which are required for expression of the chromosomal IME2 promoter and for meiosis. Through a selection for suppressors that permit UAS activity in an ime1 deletion mutant, we identified recessive mutations in three genes, SIN3 (also called RPD1, UME4, and SDI1), RPD3, and UME6 (also called CAR80), that were previously known as negative regulators of other early meiotic genes. Mutational analysis of the IME2 UAS reveals two critical sequence elements: a G+C-rich sequence (called URS1), previously identified at many meiotic genes, and a newly described element, the T4C site, that we found at a subset of meiotic genes. In agreement with prior studies, URS1 mutations lead to elevated IME2 UAS activity in the absence of IME1. However, the URS1 mutations prevent any further stimulation of UAS activity by IME1. Repression through URS1 has been shown to require the UME6 gene product. We find that activation of the IME2 UAS by IME1 also requires the UME6 gene product. Thus, UME6 and the URS1 site both have dual negative and positive roles at the IME2 UAS. We propose that IME1 modifies UME6 to convert it from a negulator to a positive Regulor.     

A mitochondrial pyruvate dehydrogenase bypass in the yeast Saccharomyces cerevisiae.

Spheroplasts of the yeast Saccharomyces cerevisiae oxidize pyruvate at a high respiratory rate, whereas isolated mitochondria do not unless malate is added. We show that a cytosolic factor, pyruvate decarboxylase, is required for the non-malate-dependent oxidation of pyruvate by mitochondria. In pyruvate decarboxylase-negative mutants, the oxidation of pyruvate by permeabilized spheroplasts was abolished. In contrast, deletion of the gene (PDA1) encoding the E1alpha subunit of the pyruvate dehydrogenase did not affect the spheroplast respiratory rate on pyruvate but abolished the malate-dependent respiration of isolated mitochondria. Mutants disrupted for the mitochondrial acetaldehyde dehydrogenase gene (ALD7) did not oxidize pyruvate unless malate was added. We therefore propose the existence of a mitochondrial pyruvate dehydrogenase bypass different from the cytosolic one, where pyruvate is decarboxylated to acetaldehyde in the cytosol by pyruvate decarboxylase and then oxidized by mitochondrial acetaldehyde dehydrogenase. This pathway can compensate PDA1 gene deletion for lactate or respiratory glucose growth. However, the codisruption of PDA1 and ALD7 genes prevented the growth on lactate, indicating that each of these pathways contributes to the oxidative metabolism of pyruvate.     

Nucleotide sequence of the ADE 1 gene of the yeast Saccharomyces cerevisiae

The yeast ADE 1 gene has been cloned and sequenced. The primary structure deduced from the nucleotide sequence demonstrated that phosphoribosylaminoimidazole-succinocarboxamide synthetase is a protein with molecular weight of 34 500 D.     

Control of Herpes simplex virus thymidine kinase gene expression in Saccharomyces cerevisiae by a yeast promoter sequence

Summary This study presents the first evidence that the 5 promoter region of the Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase gene (G-3-PD) promoter will permit expression of an adjacent foreign gene. The S. cerevisiae G-3-PD promoter was linked to the herpes simplex virus — thymidine kinase (HSV-TK) gene in a shuttle plasmid capable of autonomous replication in both yeast and Escherichia coli. Since the HSV-TK gene promoter is not functional in yeast, yeast cells containing these plasmids will express the HSV-TK gene and synthesize thymidine kinase only if the yeast promoter fragment is fused to the HSV-TK gene in the proper orientation. The 5 flanking sequences necessary for the expression of heterologous eukaryotic genes in S. cerevisiae are discussed.