scholarly journals Transcriptional Regulation of CLN3Expression by Glucose in Saccharomyces cerevisiae

1998 ◽  
Vol 180 (17) ◽  
pp. 4508-4515 ◽  
Author(s):  
Fereshteh Parviz ◽  
Duane D. Hall ◽  
David D. Markwardt ◽  
Warren Heideman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Activation ◽  
Mitotic Cycle ◽  
Specific Interaction ◽  
Point Mutations ◽  
Yeast Cells ◽  
Mrna Levels ◽  
Glucose Medium ◽  
Constant Size ◽  
Promoter Mutations

ABSTRACT In Saccharomyces cerevisiae, the transition from the G1 phase of the mitotic cycle into S phase is controlled by a set of G1 cyclins that regulate the activity of the protein kinase encoded by CDC28. Yeast cells regulate progress through the G1/S boundary in response to nutrients, moving quickly through G1 in glucose medium and more slowly in poorer medium. We have examined connections between glucose and the level of the message encoding Cln3, a G1cyclin. We found that glucose positively regulates CLN3mRNA levels through a set of repeated AAGAAAAA (A2GA5) elements within theCLN3 promoter. Mutations in these sequences reduce both transcriptional activation and specific interaction betweenCLN3 promoter elements and proteins in yeast extracts. Creation of five point mutations, replacing the G’s within these repeats with T’s, in the CLN3 promoter substantially reduces CLN3 expression in glucose medium and inhibits the ability of the cells to maintain a constant size when shifted into glucose.

Identification of an upstream activating sequence and an upstream repressible sequence of the pyruvate kinase gene of the yeast Saccharomyces cerevisiae

1989 ◽  
Vol 9 (2) ◽  
pp. 442-451
Author(s):  
M Nishizawa ◽  
R Araki ◽  
Y Teranishi
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Carbon Source ◽  
Pyruvate Kinase ◽  
Transcriptional Activation ◽  
Regulatory Elements ◽  
Noncoding Region ◽  
Yeast Cells ◽  
Kinase Gene ◽  
Yeast Saccharomyces Cerevisiae ◽  
Upstream Activating Sequence

To clarify carbon source-dependent control of the glycolytic pathway in the yeast Saccharomyces cerevisiae, we have initiated a study of transcriptional regulation of the pyruvate kinase gene (PYK). By deletion analysis of the 5'-noncoding region of the PYK gene, we have identified an upstream activating sequence (UASPYK1) located between 634 and 653 nucleotides upstream of the initiating ATG codon. The promoter activity of the PYK 5'-noncoding region was abolished when the sequence containing the UASPYK1 was deleted from the region. Synthetic UASPYK1 (26mer), in either orientation, was able to restore the transcriptional activity of UAS-depleted mutants when placed upstream of the TATA sequence located at -199 (ATG as +1). While the UASPYK1 was required for basal to intermediate levels of transcriptional activation, a sequence between -714 and -811 was found to be necessary for full activation. On the other hand, a sequence between -344 and -468 was found to be responsible for transcriptional repression of the PYK gene when yeast cells were grown on nonfermentable carbon sources. This upstream repressible sequence also repressed transcription, although to a lesser extent, when glucose was present in the medium. The possible mechanism for carbon source-dependent regulation of PYK expression through these cis-acting regulatory elements is discussed.

Suppressors of Saccharomyces cerevisiae his3 promoter mutations lacking the upstream element

1985 ◽  
Vol 5 (8) ◽  
pp. 1901-1909
Author(s):  
M A Oettinger ◽  
K Struhl
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Activation ◽  
Tata Box ◽  
Micrococcal Nuclease ◽  
Promoter Element ◽  
Wild Type ◽  
Upstream Promoter ◽  
In The Wild ◽  
Promoter Mutations ◽  
His3 Gene

Transcription of the Saccharomyces cerevisiae his3 gene requires an upstream promoter element and a TATA element. A strain containing his3-delta 13, an allele which deletes the upstream promoter element but contains the TATA box and intact structural gene, fails to express the gene and consequently is unable to grow in medium lacking histidine. In this paper we characterize His+ revertants of his3-delta 13 which are due to unlinked suppressor mutations. Recessive suppressors in three different ope genes allow his3-delta 13 to be expressed at wild-type levels. In all cases, the suppression is due to increased his3 transcription. However, unlike the wild-type his3 gene, whose transcripts are initiated about equally from two different sites (+1 and +12), transcription due to the ope mutations is initiated only from the +12 site, ope-mediated transcription is regulated in a novel manner; it is observed in minimal medium, but not in rich broth. Although ope mutations restore wild-type levels of transcription, his3 chromatin structure, as assayed by micrococcal nuclease sensitivity of the TATA box, resembles that found in the his3-delta 13 parent rather than in the wild-type strain. This provides further evidence that TATA box sensitivity is not correlated with transcriptional activation. ope mutations are pleiotropic in that cells have a crunchy colony morphology and lyse at 37 degrees C in conditions of normal osmolarity. ope mutations are allele specific because they fail to suppress five other his3 promoter mutations. We discuss implications concerning upstream promoter elements and propose some models for ope suppression.

The role of RAP1 in the regulation of the MAT alpha locus

1991 ◽  
Vol 11 (2) ◽  
pp. 1069-1079
Author(s):  
D Giesman ◽  
L Best ◽  
K Tatchell
Keyword(s):  
Transcriptional Activation ◽  
Consensus Sequence ◽  
Point Mutations ◽  
Yeast Cells ◽  
Effective Dosage ◽  
Upstream Region ◽  
Yeast Genome ◽  
Temperature Sensitive ◽  
Lacz Gene

The RAP1 gene of Saccharomyces cerevisiae encodes an abundant DNA-binding protein, also known as GRF1, TBA, or TUF, that binds to many sites in the yeast genome in vitro. These sites define a consensus sequence, [sequence: see text], and deletion analyses of genes that contain this sequence have implicated the involvement of RAP1 in numerous cellular processes, including gene activation and repression. The MAT alpha locus, required for determination of the alpha cell type in yeast cells, contains a RAP1 binding site; this site coincides with the MAT alpha upstream activating sequence (UAS) and is necessary for expression of the two genes encoded by the MAT alpha locus, MAT alpha 1 and MAT alpha 2. We show that the MAT alpha UAS is sufficient to activate transcription from a promoterless gene fusion of the yeast CYC1 upstream region and the lacZ gene. Constructs containing only the MAT alpha UAS generated elevated levels of beta-galactosidase activity which were indistinguishable from those of constructs containing the entire MAT alpha intergenic region. Further, the MAT alpha UAS has an intrinsic polarity of transcriptional activation; transcription of CYC1-lacZ was six- to sevenfold higher when the UAS was oriented in the direction normally associated with MAT alpha 2 transcription. Point mutations in the MAT alpha UAS that reduce MAT alpha expression three- to fivefold resulted in a bi-mating phenotype, while a mutation that reduced MAT alpha expression still further resulted in an a-mating phenotype. We isolated plasmids from a high-copy-number yeast library that suppressed the bi-mating defect of point mutations in the MAT alpha UAS, and the most effective dosage suppressor contained the gene encoding RAP1. A temperature-sensitive rap1 mutant bi-mates at the semipermissive temperature. Double mutants at rap1 and mat alpha mate exclusively as a cells, at all temperatures, and do not express detectable levels of MAT alpha RNA. These data provide evidence that the RAP1 gene product functions at the MAT alpha UAS in vivo.

Codon replacement in the PGK1 gene of Saccharomyces cerevisiae: experimental approach to study the role of biased codon usage in gene expression

1987 ◽  
Vol 7 (8) ◽  
pp. 2914-2924
Author(s):  
A Hoekema ◽  
R A Kastelein ◽  
M Vasser ◽  
H A de Boer
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Steady State ◽  
Codon Usage ◽  
Experimental Approach ◽  
Mrna Translation ◽  
Yeast Cells ◽  
Expression Level ◽  
Start Codon ◽  
Mrna Levels

The coding sequences of genes in the yeast Saccharomyces cerevisiae show a preference for 25 of the 61 possible coding triplets. The degree of this biased codon usage in each gene is positively correlated to its expression level. Highly expressed genes use these 25 major codons almost exclusively. As an experimental approach to studying biased codon usage and its possible role in modulating gene expression, systematic codon replacements were carried out in the highly expressed PGK1 gene. The expression of phosphoglycerate kinase (PGK) was studied both on a high-copy-number plasmid and as a single copy gene integrated into the chromosome. Replacing an increasing number (up to 39% of all codons) of major codons with synonymous minor ones at the 5' end of the coding sequence caused a dramatic decline of the expression level. The PGK protein levels dropped 10-fold. The steady-state mRNA levels also declined, but to a lesser extent (threefold). Our data indicate that this reduction in mRNA levels was due to destabilization caused by impaired translation elongation at the minor codons. By preventing translation of the PGK mRNAs by the introduction of a stop codon 3' and adjacent to the start codon, the steady-state mRNA levels decreased dramatically. We conclude that efficient mRNA translation is required for maintaining mRNA stability in S. cerevisiae. These findings have important implications for the study of the expression of heterologous genes in yeast cells.

Molecular genetic analysis of Saccharomyces cerevisiae C1-tetrahydrofolate synthase mutants reveals a noncatalytic function of the ADE3 gene product and an additional folate-dependent enzyme

1990 ◽  
Vol 10 (11) ◽  
pp. 5679-5687
Author(s):  
C K Barlowe ◽  
D R Appling
Keyword(s):  
Saccharomyces Cerevisiae ◽  
De Novo ◽  
Point Mutations ◽  
Gene Replacement ◽  
Molecular Genetic Analysis ◽  
Yeast Cells ◽  
Yeast Saccharomyces Cerevisiae ◽  
Purine Synthesis

In eucaryotes, 10-formyltetrahydrofolate (formyl-THF) synthetase, 5,10-methenyl-THF cyclohydrolase, and NADP(+)-dependent 5,10-methylene-THF dehydrogenase activities are present on a single polypeptide termed C1-THF synthase. This trifunctional enzyme, encoded by the ADE3 gene in the yeast Saccharomyces cerevisiae, is thought to be responsible for the synthesis of the one-carbon donor 10-formyl-THF for de novo purine synthesis. Deletion of the ADE3 gene causes adenine auxotrophy, presumably as a result of the lack of cytoplasmic 10-formyl-THF. In this report, defined point mutations that affected one or more of the catalytic activities of yeast C1-THF synthase were generated in vitro and transferred to the chromosomal ADE3 locus by gene replacement. In contrast to ADE3 deletions, point mutations that inactivated all three activities of C1-THF synthase did not result in an adenine requirement. Heterologous expression of the Clostridium acidiurici gene encoding a monofunctional 10-formyl-THF synthetase in an ade3 deletion strain did not restore growth in the absence of adenine, even though the monofunctional synthetase was catalytically competent in vivo. These results indicate that adequate cytoplasmic 10-formyl-THF can be produced by an enzyme(s) other than C1-THF synthase, but efficient utilization of that 10-formyl-THF for purine synthesis requires a nonenzymatic function of C1-THF synthase. A monofunctional 5,10-methylene-THF dehydrogenase, dependent on NAD+ for catalysis, has been identified and purified from yeast cells (C. K. Barlowe and D. R. Appling, Biochemistry 29:7089-7094, 1990). We propose that the characteristics of strains expressing full-length but catalytically inactive C1-THF synthase could result from the formation of a purine-synthesizing multienzyme complex involving the structurally unchanged C1-THF synthase and that production of the necessary one-carbon units in these strains is accomplished by an NAD+ -dependent 5,10-methylene-THF dehydrogenase.

Regulation of STA1 gene expression by MAT during the life cycle of Saccharomyces cerevisiae

1989 ◽  
Vol 9 (9) ◽  
pp. 3992-3998
Author(s):  
A M Dranginis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mating Type ◽  
Yeast Cells ◽  
Specific Gene ◽  
Activity Levels ◽  
Sporulation Medium ◽  
Mrna Levels ◽  
Yeast Saccharomyces Cerevisiae ◽  
Type Locus ◽  
Diploid Cells

STA1 encodes a secreted glucoamylase of the yeast Saccharomyces cerevisiae var. diastaticus. Glucoamylase secretion is controlled by the mating type locus MAT; a and alpha haploid yeast cells secrete high levels of the enzyme, but a/alpha diploid cells produce undetectable amounts. It has been suggested that STA1 is regulated by MATa2 (I. Yamashita, Y. Takano, and S. Fukui, J. Bacteriol. 164:769-773, 1985), which is a MAT transcript of previously unknown function. In contrast, this work shows that deletion of the entire MATa2 gene had no effect on STA1 regulation but that deletion of MATa1 sequences completely abolished mating-type control. In all cases, glucoamylase activity levels reflected STA1 mRNA levels. It appears that STA1 is a haploid-specific gene that is regulated by MATa1 and a product of the MAT alpha locus and that this regulation occurs at the level of RNA accumulation. STA1 expression was also shown to be glucose repressible. STA1 mRNA was induced in diploids during sporulation along with SGA, a closely linked gene that encodes an intracellular sporulation-specific glucoamylase of S. cerevisiae. A diploid strain with a MATa1 deletion showed normal induction of STA1 in sporulation medium, but SGA expression was abolished. Therefore, these two homologous and closely linked glucoamylase genes are induced by different mechanisms during sporulation. STA1 induction may be a response to the starvation conditions necessary for sporulation, while SGA induction is governed by the pathway by which MAT regulates sporulation. The strain containing a complete deletion of MATa2 grew, mated, and sporulated normally.

GPD1, which encodes glycerol-3-phosphate dehydrogenase, is essential for growth under osmotic stress in Saccharomyces cerevisiae, and its expression is regulated by the high-osmolarity glycerol response pathway

1994 ◽  
Vol 14 (6) ◽  
pp. 4135-4144
Author(s):  
J Albertyn ◽  
S Hohmann ◽  
J M Thevelein ◽  
B A Prior
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Osmotic Stress ◽  
Yeast Cells ◽  
Mrna Levels ◽  
Hog Pathway ◽  
Hypertonic Medium ◽  
High Osmolarity ◽  
High Osmolarity Glycerol ◽  
Enhanced Production ◽  
Glycerol 3 Phosphate Dehydrogenase

The yeast Saccharomyces cerevisiae responds to osmotic stress, i.e., an increase in osmolarity of the growth medium, by enhanced production and intracellular accumulation of glycerol as a compatible solute. We have cloned a gene encoding the key enzyme of glycerol synthesis, the NADH-dependent cytosolic glycerol-3-phosphate dehydrogenase, and we named it GPD1. gpd1 delta mutants produced very little glycerol, and they were sensitive to osmotic stress. Thus, glycerol production is indeed essential for the growth of yeast cells during reduced water availability. hog1 delta mutants lacking a protein kinase involved in osmostress-induced signal transduction (the high-osmolarity glycerol response [HOG] pathway) failed to increase glycerol-3-phosphate dehydrogenase activity and mRNA levels when osmotic stress was imposed. Thus, expression of GPD1 is regulated through the HOG pathway. However, there may be Hog1-independent mechanisms mediating osmostress-induced glycerol accumulation, since a hog1 delta strain could still enhance its glycerol content, although less than the wild type. hog1 delta mutants are more sensitive to osmotic stress than isogenic gpd1 delta strains, and gpd1 delta hog1 delta double mutants are even more sensitive than either single mutant. Thus, the HOG pathway most probably has additional targets in the mechanism of adaptation to hypertonic medium.

scholarly journals Interactions of Surfactant Proteins A and D with Saccharomyces cerevisiae and Aspergillus fumigatus

2001 ◽  
Vol 69 (4) ◽  
pp. 2037-2044 ◽  
Author(s):  
Martin J. Allen ◽  
Dennis R. Voelker ◽  
Robert J. Mason
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Aspergillus Fumigatus ◽  
Cell Walls ◽  
Specific Interaction ◽  
Surfactant Proteins ◽  
Yeast Cells ◽  
Calcium Dependent ◽  
Before And After ◽  
Carbohydrate Polymer ◽  
Defense Molecules

ABSTRACT Surfactant proteins A (SP-A) and D (SP-D) are members of the collectin family of calcium-dependent lectins and are important pulmonary host defense molecules. Human SP-A and SP-D and rat SP-D bind to Aspergillus fumigatus conidia, but the ligand remains unidentified. To identify a fungal ligand for SP-A and/or SP-D, we examined the interactions of the proteins with Saccharomyces cerevisiae. SP-D but not SP-A bound yeast cells, and EDTA inhibited the binding. SP-D also aggregated yeast cells and isolated yeast cell walls. Treating yeast cells to remove cell wall mannoprotein did not reduce SP-D binding, and SP-D failed to aggregate chitin. However, SP-D aggregated yeast glucan before and after treatment with a β(1→3)-glucanase, suggesting a specific interaction between the collectin and β(1→6)-glucan. In support of this idea, SP-D-induced yeast aggregation was strongly inhibited by pustulan [a β(1→6)-linked glucose homopolymer] but was not inhibited by laminarin [a β(1→3)-linked glucose homopolymer]. Additionally, pustulan but not laminarin strongly inhibited SP-D binding to A. fumigatus. The pustulan concentration for 50% inhibition of SP-D binding to A. fumigatus is 1.0 ± 0.3 μM glucose equivalents. Finally, SP-D showed reduced binding to the β(1→6)-glucan-deficient kre6 yeast mutant. Taken together, these observations demonstrate that β(1→6)-glucan is an important fungal ligand for SP-D and that glycosidic bond patterns alone can determine if an extended carbohydrate polymer is recognized by SP-D.

scholarly journals Suppressors of Saccharomyces cerevisiae his3 promoter mutations lacking the upstream element.

1985 ◽  
Vol 5 (8) ◽  
pp. 1901-1909 ◽  
Author(s):  
M A Oettinger ◽  
K Struhl
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Activation ◽  
Tata Box ◽  
Micrococcal Nuclease ◽  
Promoter Element ◽  
Wild Type ◽  
Upstream Promoter ◽  
In The Wild ◽  
Promoter Mutations ◽  
His3 Gene

Transcription of the Saccharomyces cerevisiae his3 gene requires an upstream promoter element and a TATA element. A strain containing his3-delta 13, an allele which deletes the upstream promoter element but contains the TATA box and intact structural gene, fails to express the gene and consequently is unable to grow in medium lacking histidine. In this paper we characterize His+ revertants of his3-delta 13 which are due to unlinked suppressor mutations. Recessive suppressors in three different ope genes allow his3-delta 13 to be expressed at wild-type levels. In all cases, the suppression is due to increased his3 transcription. However, unlike the wild-type his3 gene, whose transcripts are initiated about equally from two different sites (+1 and +12), transcription due to the ope mutations is initiated only from the +12 site, ope-mediated transcription is regulated in a novel manner; it is observed in minimal medium, but not in rich broth. Although ope mutations restore wild-type levels of transcription, his3 chromatin structure, as assayed by micrococcal nuclease sensitivity of the TATA box, resembles that found in the his3-delta 13 parent rather than in the wild-type strain. This provides further evidence that TATA box sensitivity is not correlated with transcriptional activation. ope mutations are pleiotropic in that cells have a crunchy colony morphology and lyse at 37 degrees C in conditions of normal osmolarity. ope mutations are allele specific because they fail to suppress five other his3 promoter mutations. We discuss implications concerning upstream promoter elements and propose some models for ope suppression.

scholarly journals Mitochondrial instability in a strain ofSaccharomyces cerevisiae

1978 ◽  
Vol 32 (2) ◽  
pp. 171-182 ◽  
Author(s):  
I. H. Evans ◽  
D. Wilkie
Keyword(s):  
Saccharomyces Cerevisiae ◽  
High Frequency ◽  
Glucose Repression ◽  
Point Mutations ◽  
Nuclear Gene ◽  
Fermentable Sugars ◽  
Glucose Medium ◽  
Haploid Strain ◽  
Respiratory Impairment ◽  
Mutator Effect

SUMMARYA haploid strain ofSaccharomyces cerevisiaehas been described which, on glucose medium, segregates vegetatively a high frequency of mutants with different degrees of respiratory impairment. The range of mutants seemingly encompasses both non-revertible ρ- petites and revertible point mutations resembling leakymit- mutations. The segregants have aberrant cytochrome contents and reduced growth capabilities on fermentable sugars other than glucose; these defects apparently correlate with the degree of respiratory impairment. Genetic analysis of this mutator phenomenon has implicated a nuclear gene which appears to show specificity of interaction with the mitochondrial genome as well as a requirement for glucose repression. The mutator effect seems to extend also to the loci in mitochondrial DNA for resistance to the antibiotics erythromycin and oligomycin.

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