Short repeated elements in the upstream regulatory region of the SUC2 gene of Saccharomyces cerevisiae.

Expression of secreted invertase from the SUC2 gene is regulated by carbon catabolite repression. Previously, an upstream regulatory region that is required for derepression of secreted invertase was identified and shown to confer glucose-repressible expression to the heterologous promoter of a LEU2-lacZ fusion. In this paper we show that tandem copies of a 32-base pair (bp) sequence from the upstream regulatory region activate expression of the same LEU2-lacZ fusion. The level of expression increased with the number of copies of the element, but was independent of their orientation; the expression from constructions containing four copies of the sequence was only twofold lower than that when the entire SUC2 upstream regulatory region was present. This activation was not significantly glucose repressible. The 32-bp sequence includes a 7-bp motif with the consensus sequence (A/C)(A/G)GAAAT that is repeated at five sites within the upstream regulatory region. Genetic evidence supporting the functional significance of this repeated motif was obtained by pseudoreversion of a SUC2 deletion mutant lacking part of the upstream region, including two copies of the 7-bp element. In three of five pseudorevertants, the mutations that restored high-level SUC2 expression altered one of the remaining copies of the 7-bp element.     

Cloning and genetic mapping of SNF1, a gene required for expression of glucose-repressible genes in Saccharomyces cerevisiae.

A functional SNF1 gene product is required to derepress expression of many glucose-repressible genes in Saccharomyces cerevisiae. Strains carrying a snf1 mutation are unable to grow on sucrose, galactose, maltose, melibiose, or nonfermentable carbon sources; utilization of these carbon sources is regulated by glucose repression. The inability of snf1 mutants to utilize sucrose results from failure to derepress expression of the structural gene for invertase at the RNA level. We isolated recombinant plasmids carrying the SNF1 gene by complementation of the snf1 defect in S. cerevisiae. A 3.5-kilobase region is common to the DNA segments cloned in five different plasmids. Transformation of S. cerevisiae with an integrating vector carrying a segment of the cloned DNA resulted in integration of the plasmid at the SNF1 locus. This result indicates that the cloned DNA is homologous to sequences at the SNF1 locus. By mapping a plasmid marker linked to SNF1 in this transformant, we showed that the SNF1 gene is located on chromosome IV. We then mapped snf1 to a position 5.6 centimorgans distal to rna3 on the right arm; snf1 is not extremely closely linked to any previously mapped mutation.     

Molecular analysis of SNF2 and SNF5, genes required for expression of glucose-repressible genes in Saccharomyces cerevisiae.

The SNF2 and SNF5 genes are required for derepression of SUC2 and other glucose-repressible genes of Saccharomyces cerevisiae in response to glucose deprivation. Previous genetic evidence suggested that SNF2 and SNF5 have functionally related roles. We cloned both genes by complementation and showed that the cloned DNA was tightly linked to the corresponding chromosomal locus. Both genes in multiple copy complemented only the cognate snf mutation. The SNF2 gene encodes a 5.7-kilobase RNA, and the SNF5 gene encodes a 3-kilobase RNA. Both RNAs contained poly(A) and were present in low abundance. Neither was regulated by glucose repression, and the level of SNF2 RNA was not dependent on SNF5 function or vice versa. Disruption of either gene at its chromosomal locus still allowed low-level derepression of secreted invertase activity, suggesting that these genes are required for high-level expression but are not directly involved in regulation. Further evidence was the finding that snf2 and snf5 mutants failed to derepress acid phosphatase, which is not regulated by glucose repression. The SNF2 and SNF5 functions were required for derepression of SUC2 mRNA.     

A glucose-repressible gene encodes acetyl-CoA hydrolase from Saccharomyces cerevisiae

Acetyl-CoA hydrolase, catalyzing the hydrolysis of acetyl-CoA, is presumably involved in regulating the intracellular acetyl-CoA pool. Recently, a yeast acetyl-CoA hydrolase was purified to homogeneity from Saccharomyces cerevisiae and partially characterized (Lee, F.-J. S., Lin, L.-W., and Smith, J. A. (1989) Eur. J. Biochem. 184, 21-28). In order to study the biological function and regulation of the acetyl-CoA hydrolase, we cloned and sequenced the full length cDNA encoding yeast acetyl-CoA hydrolase. RNA blot analysis indicates that acetyl-CoA hydrolase is encoded by a 2.5-kilobase mRNA. DNA blot analyses of genomic and chromosomal DNA reveal that the gene (so-called ACH1, acetyl-CoA hydrolase) is present as a single copy located on chromosome II. Acetyl-CoA hydrolase is established to be a mannose-containing glycoprotein, which binds concanavalin A. By measuring the levels of ACH1 mRNA and acetyl-CoA hydrolase activity in different growth phases and by examining the effects of various carbon sources, we have demonstrated that ACH1 expression is repressed by glucose.     

Effects of oxygen on invertase expression in continuous culture of recombinant Saccharomyces cerevisiae containing the SUC2 gene

The yeast SUC2 gene, cloned on a multicopy plasmid pRB58, was used to study the effect of oxygen on the invertase expression of the recombinant Saccharomyces cerevisiae. Glucose repression was not the only factor affecting the invertase expression. The results obtained from the single-stage continuous cultures under microaerobic conditions showed that invertase expression was also strongly dependent on oxygen availability, and moving from anaerobic to aerobic conditions led to a five-fold increase in specific invertase activity. However, the cell yields under anaerobic conditions were quite low compared to those under aerobic conditions. These opposite effects of oxygen on cell growth and gene expression offer a strategy for maximizing invertase productivity by a two-stage continuous culture. The first stage was operated at a low level of glucose, around 100 mg/l, under aerobic conditions in order to obtain a high yield of yeast biomass, and the second stage maintained anaerobic conditions with residual glucose levels of 50 mg/l to derepress and fully induce invertase expression. The two-stage continuous culture resulted in a 2.5-fold increase in invertase productivity over that of a single-stage continuous culture. Received: 28 July 1998 / Received revision: 22 September 1998 / Accepted: 7 November 1998     

Transposed LEU2 gene of Saccharomyces cerevisiae is regulated normally.

The repression of beta-isopropylmalate dehydrogenase, the LEU2 gene product, by leucine and leucine plus threonine was unaffected by the transposition of LEU2 from its original locus on chromosome III to a new locus within the ribosomal deoxyribonucleic acid gene cluster on chromosome XII. Since the expression of the LEU2 gene is probably controlled at a pretranslational level, we conclude that the recombinant plasmid used for transformation carries regulatory information in addition to LEU2 structural information.     

A positive regulatory gene is required for accumulation of the functional messenger RNA for the glucose-repressible alcohol dehydrogenase from Saccharomyces cerevisiae

The amount of glucose-repressible alcohol dehydrogenase is regulated by the amount of its functional messenger RNA. ADHII² protein was detected by a radioimmune assay and differentiated from ADHI, the classical ADH isozyme, by limited proteolysis with Staphylococcus aureus protease. When yeast containing the wild-type alleles for ADR2 (the ADH II structural locus) and for ADR1 (its positive regulatory gene) were pulse-labeled with [³⁵S]methionine during derepression, radioactive label accumulated in the antibody-precipitated ADHII coterminously with the appearance of ADHII activity. The kinetics of functional ADHII mRNA appearance during derepression in this strain were shown to be the same as those for ADHII protein synthesis in vivo when RNA, extracted from derepressed cells, was translated in a wheat germ cell-free translation system.The role of the positive regulatory gene, ADR1, in ADHII expression was analyzed using two strains mutated at that locus. Yeast containing the adr1-1 allele are incapable of derepressing ADHII activity. When this strain was pulselabeled with [³⁵S]methionine during derepression, approximately one-tenth to one-twentieth the level of ADHII protein synthesis was detected as in the wild-type strain. When RNA was extracted during derepression from cells containing the udr1-1 allele and translated in a wheat germ cell-free system, little functional ADHII mRNA was found to be present.The role of the ADR1 gene was further analyzed using a strain containing the ADR1-5^c allele, which allows constitutive synthesis of ADHII activity. In this strain during glucose repression. ADHII protein synthesis and amount of functional mRNA were at levels comparable to those found for the wild-type strain after complete derepression. Similar kinetics of ADHII protein synthesis and of mRNA accumulation during derepression were observed in the strain carrying the ADR1-5^c allele when compared to that carrying the ADR1 allele, but the absolute amounts were greater by three- to fourfold in cells containing the ADR1-5^c allele. These results indicate that the ADR1 gene acts to increase the level of functional ADHII mRNA during derepression.     

Control of gene expression from the SUC2 promoter of Saccharomyces cerevisiae with the aid of a glucose analyser

Summary A Saccharomyces cerevisiae strain harbouring the recombinant plasmid pSMF38TMA was cultured in a jar fermentor under the control of glucose concentration. In the recombinant plasmid, the mouse -amylase gene was fused to the S. cerevisiae SUC2 promoter. When glucose concentration in the medium was controlled at 10 g/l, the gene expression was completely repressed. On the other hand, the -amylase was produced and secreted in the medium at a very high level, around 200 mg/l as evaluated from the specific activity of commercially available human salivary amylase, when the glucose was kept at 0.15 g/l. This amount was almost 20-fold that obtained at 10 g/l glucose. The specific growth rate of the yeast in this culture was almost 60% of that attained with 10 g/l glucose. To obtain higher cell growth and productivity, the yeast was at first cultured at 2 g/l glucose and the concentration was then lowered to 0.15 g/l. By this control of the glucose concentration, on-off regulation of gene expression from the SUC promoter could be attained.     

The DAL81 gene product is required for induced expression of two differently regulated nitrogen catabolic genes in Saccharomyces cerevisiae.

We demonstrate that the DAL81 gene, previously thought to be specifically required for induced expression of the allantoin pathway genes in Saccharomyces cerevisiae, functions in a more global manner. The data presented show it to be required for utilization of 4-aminobutyrate as a nitrogen source and for 4-aminobutyrate-induced increases in the steady-state levels of UGA1 mRNA. The DAL81 gene encodes a 970-amino-acid protein containing sequences homologous to the Zn(II)2Cys6 motif and two stretches of polyglutamine residues. Deletion of sequences homologous to the Zn(II)2Cys6 motif did not result in a detectable loss of function. On the other hand, loss of one of the polyglutamine stretches, but not the other, resulted in a 50% loss of DAL81 function.     

Invertase SUC2 Is the Key Hydrolase for Inulin Degradation in Saccharomyces cerevisiae

Specific Saccharomyces cerevisiae strains were recently found to be capable of efficiently utilizing inulin, but genetic mechanisms of inulin hydrolysis in yeast remain unknown. Here we report functional characteristics of invertase SUC2 from strain JZ1C and demonstrate that SUC2 is the key enzyme responsible for inulin metabolism in S. cerevisiae.     

The HXT2 gene of Saccharomyces cerevisiae is required for high-affinity glucose transport.

The HXT2 gene of the yeast Saccharomyces cerevisiae was identified on the basis of its ability to complement the defect in glucose transport of a snf3 mutant when present on the multicopy plasmid pSC2. Analysis of the DNA sequence of HXT2 revealed an open reading frame of 541 codons, capable of encoding a protein of Mr 59,840. The predicted protein displayed high sequence and structural homology to a large family of procaryotic and eucaryotic sugar transporters. These proteins have 12 highly hydrophobic regions that could form transmembrane domains; the spacing of these putative transmembrane domains is also highly conserved. Several amino acid motifs characteristic of this sugar transporter family are also present in the HXT2 protein. An hxt2 null mutant strain lacked a significant component of high-affinity glucose transport when under derepressing (low-glucose) conditions. However, the hxt2 null mutation did not incur a major growth defect on glucose-containing media. Genetic and biochemical analyses suggest that wild-type levels of high-affinity glucose transport require the products of both the HXT2 and SNF3 genes; these genes are not linked. Low-stringency Southern blot analysis revealed a number of other sequences that cross-hybridize with HXT2, suggesting that S. cerevisiae possesses a large family of sugar transporter genes.     

Characterization of the heterologous invertase produced by Schizosaccharomyces pombe from the SUC2 gene of Saccharomyces cerevisiae

In order to gain information on the ability of Schizosaccharomyces pombe to process heterologous glycoproteins, the heterologous invertase, obtained from the expression in Schiz. pombe of the SUC2 gene of Saccharomyces cerevisiae , was characterized. In Schiz. pombe the heterologous invertase is secreted into the cell wall and seems to be firmly bound to this structure. After the isolation of the heterologous invertase the study of its enzymatic characteristics revealed that it is more similar to the Sacch. cerevisiae external invertase than to the Schiz. pombe invertase. However, it is glycosylated like the Schiz. pombe invertase since it reacts with the lectin from Bandeiraea simplicifolia seeds conjugated to fluorescein isothiocyanate, which indicates the presence of terminal galactose residues in the enzyme. Moreover, the presence of galactose in the heterologous invertase has been confirmed after analysis of the sugars present in its carbohydrate moiety by gas liquid chromatography.     

Organization of the SUC gene family in Saccharomyces.

The SUC gene family of yeast (Saccharomyces) includes six structural genes for invertase (SUC1 through SUC5 and SUC7) found at unlinked chromosomal loci. A given yeast strain does not usually carry SUC+ alleles at all six loci; the natural negative alleles are called suc0 alleles. Cloned SUC2 DNA probes were used to investigate the physical structure of the SUC gene family in laboratory strains, commercial wine strains, and different Saccharomyces species. The active SUC+ genes are homologous. The suc0 allele at the SUC2 locus (suc2(0) in some strains is a silent gene or pseudogene. Other SUC loci carrying suc0 alleles appear to lack SUC DNA sequences. These findings imply that SUC genes have transposed to different chromosomal locations in closely related Saccharomyces strains.