New SNF genes, GAL11 and GRR1 affect SUC2 expression in Saccharomyces cerevisiae.

Abstract The first step in the biosynthesis of leucine is catalyzed by alpha-isopropylmalate (alpha-IPM) synthase. In the yeast Saccharomyces cerevisiae, LEU4 encodes the isozyme responsible for the majority of alpha-IPM synthase activity. Yeast strains that bear disruption alleles of LEU4, however, are Leu+ and exhibit a level of synthase activity that is 20% of the wild type. To identify the gene or genes that encode this remaining activity, a leu4 disruption strain was mutagenized. The mutations identified define three new complementation groups, designated leu6, leu7 and leu8. Each of these new mutations effect leucine auxotrophy only if a leu4 mutation is present and each results in loss of alpha-IPM synthase activity. Further analysis suggests that LEU7 and LEU8 are candidates for the gene or genes that encode an alpha-IPM synthase activity. The results demonstrate that multiple components determine the residual alpha-IPM synthase activity in leu4 gene disruption strains of S. cerevisiae.

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Yeast mutants sensitive to antimicrotubule drugs define three genes that affect microtubule function.

Genetics ◽

10.1093/genetics/124.2.251 ◽

1990 ◽

Vol 124 (2) ◽

pp. 251-262 ◽

Cited By ~ 8

Author(s):

T Stearns ◽

M A Hoyt ◽

D Botstein

Keyword(s):

Chromosome Loss ◽

Gene Products ◽

Wild Type ◽

Tubulin Genes ◽

New Genes ◽

Complementation Groups ◽

Yeast Mutants ◽

Antimicrotubule Drugs ◽

Tubulin Mutations ◽

Microtubule Function

Abstract Three new genes affecting microtubule function in Saccharomyces cerevisiae were isolated by screening for mutants displaying supersensitivity to the antimicrotubule drug benomyl. Such mutants fall into six complementation groups: TUB1, TUB2 and TUB3, the three tubulin genes of yeast, and three new genes, which we have named CIN1, CIN2 and CIN4. Mutations in each of the CIN genes were also independently isolated by screening for mutants with increased rates of chromosome loss. Strains bearing mutations in the CIN genes are approximately tenfold more sensitive than wild type to both benomyl and to the related antimicrotubule drug, nocodazole. This phenotype is recessive for all alleles isolated. The CIN1, CIN2 and CIN4 genes were cloned by complementation of the benomyl-supersensitive phenotype. Null mutants of each of the genes are viable, and have phenotypes similar to those of the point mutants. Genetic evidence for the involvement of the CIN gene products in microtubule function comes from the observation that some tubulin mutations are suppressed by cin mutations, while other tubulin mutations are lethal in combination with cin mutations. Additional genetic experiments with cin mutants suggest that the three genes act together in the same pathway or structure to affect microtubule function.

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TWO CHROMOSOMAL GENES REQUIRED FOR KILLING EXPRESSION IN KILLER STRAINS OF SACCHAROMYCES CEREVISIAE

Genetics ◽

10.1093/genetics/82.3.429 ◽

1976 ◽

Vol 82 (3) ◽

pp. 429-442

Author(s):

Reed B Wickner ◽

Michael J Leibowitz

Keyword(s):

Saccharomyces Cerevisiae ◽

Molecular Weight ◽

Mutant Phenotype ◽

Wild Type ◽

Double Stranded Rna ◽

Killer Strain ◽

Chromosomal Genes ◽

Complementation Groups ◽

Killer Plasmid ◽

Type Strains

ABSTRACT The killer character of yeast is determined by a 1.4 × 106 molecular weight double-stranded RNA plasmid and at least 12 chromosomal genes. Wild-type strains of yeast that carry this plasmid (killers) secrete a toxin which is lethal only to strains not carrying this plasmid (sensitives). —— We have isolated 28 independent recessive chromosomal mutants of a killer strain that have lost the ability to secrete an active toxin but remain resistant to the effects of the toxin and continue to carry the complete cytoplasmic killer genome. These mutants define two complementation groups, kex1 and kex2. Kex1 is located on chromosome VII between ade5 and lys5. Kex2 is located on chromosome XIV, but it does not show meiotic linkage to any gene previously located on this chromosome. —— When the killer plasmid of kex1 or kex2 strains is eliminated by curing with heat or cycloheximide, the strains become sensitive to killing. The mutant phenotype reappears among the meiotic segregants in a cross with a normal killer. Thus, the kex phenotype does not require an alteration of the killer plasmid. —— Kex1 and kex2 strains each contain near-normal levels of the 1.4 × 106 molecular weight double-stranded RNA, whose presence is correlated with the presence of the killer genome.

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An efficient positive selection procedure for the isolation of peroxisomal import and peroxisome assembly mutants of Saccharomyces cerevisiae.

Genetics ◽

10.1093/genetics/135.3.731 ◽

1993 ◽

Vol 135 (3) ◽

pp. 731-740 ◽

Cited By ~ 8

Author(s):

Y Elgersma ◽

M van den Berg ◽

H F Tabak ◽

B Distel

Keyword(s):

Saccharomyces Cerevisiae ◽

Oleic Acid ◽

Protein Import ◽

Chimeric Protein ◽

Selection Procedure ◽

Peroxisome Biogenesis ◽

Wild Type ◽

Peroxisomal Protein ◽

Complementation Groups ◽

Resistance Protein

Abstract To study peroxisome biogenesis, we developed a procedure to select for Saccharomyces cerevisiae mutants defective in peroxisomal protein import or peroxisome assembly. For this purpose, a chimeric gene was constructed encoding the bleomycin resistance protein linked to the peroxisomal protein luciferase. In wild-type cells this chimeric protein is imported into the peroxisome, which prevents the neutralizing interaction of the chimeric protein with its toxic phleomycin ligand. Peroxisomal import and peroxisome assembly mutants are unable to import this chimeric protein into their peroxisomes. This enables the bleomycin moiety of the chimeric protein to bind phleomycin, thereby preventing its toxicity. The selection is very efficient: upon mutagenesis, 84 (10%) of 800 phleomycin resistant colonies tested were unable to grow on oleic acid. This rate could be increased to 25% using more stringent selection conditions. The selection procedure is very specific; all oleic acid non utilizing (onu) mutants tested were disturbed in peroxisomal import and/or peroxisome assembly. The pas (peroxisome assembly) mutants that have been used for complementation analysis represent 12 complementation groups including three novel ones, designated pas20, pas21 and pas22.

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Disruption of regulatory gene GAL80 in Saccharomyces cerevisiae: effects on carbon-controlled regulation of the galactose/melibiose pathway genes

Molecular and Cellular Biology ◽

10.1128/mcb.4.8.1521-1527.1984 ◽

1984 ◽

Vol 4 (8) ◽

pp. 1521-1527

Author(s):

T E Torchia ◽

R W Hamilton ◽

C L Cano ◽

J E Hopper

Keyword(s):

Saccharomyces Cerevisiae ◽

Catabolite Repression ◽

Carbon Catabolite Repression ◽

Regulatory Gene ◽

Negative Regulator ◽

Null Mutation ◽

Null Mutant ◽

Wild Type

In Saccharomyces cerevisiae, the transcriptional expression of the galactose-melibiose catabolic pathway genes is under the control of at least three regulatory genes, GAL4, GAL80, and GAL3. We have isolated the GAL80 gene and have studied the effect of a null mutation on the carbon-controlled regulation of the MEL1 and GAL cluster genes. The null mutation was achieved in vivo by replacing the chromosomal wild-type GAL80 allele with an in vitro-created GAL80 deletion-disruption mutation. Enzyme activities and RNA levels for the GAL cluster and MEL1 genes were constitutively expressed in the null mutant strain grown on glycerol-lactate and were higher than in the isogenic wild-type yeast strain when compared after growth on galactose. Carbon catabolite repression of the GAL cluster and MEL1 genes, which occurs at the level of transcription, is retained in the null mutant. Deletion of the GAL80 gene in a gal4 cell does not restore GAL cluster and MEL1 gene expression. The data demonstrate that (i) the GAL80 protein is a purely negative regulator, (ii) the GAL80 protein does not mediate carbon catabolite repression, and (iii) the GAL4 protein is not simply an antagonizer of GAL80-mediated repression.

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A mutation allowing an mRNA secondary structure diminishes translation of Saccharomyces cerevisiae iso-1-cytochrome c.

Molecular and Cellular Biology ◽

10.1128/mcb.5.8.1839 ◽

1985 ◽

Vol 5 (8) ◽

pp. 1839-1846 ◽

Cited By ~ 79

Author(s):

S B Baim ◽

D F Pietras ◽

D C Eustice ◽

F Sherman

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acid ◽

Secondary Structure ◽

Cytochrome C ◽

Mrna Secondary Structure ◽

Wild Type ◽

Stable Secondary Structure ◽

Yeast Saccharomyces Cerevisiae ◽

Wild Type Level ◽

Altered Protein

The CYC1-239-O mutation in the yeast Saccharomyces cerevisiae produces a -His-Leu- replacement of the normal -Ala-Gly- sequence at amino acid positions 5 and 6, which lie within a dispensable region of iso-1-cytochrome c; this mutation can accommodate the formation of a hairpin structure at the corresponding site in the mRNA. The amount of the altered protein was diminished to 20% of the wild-type level, whereas the amount of the mRNA remained normal. However, in contrast to the normal CYC1+ mRNA that is associated mainly with four to seven ribosomes, the bulk of the CYC1-239-O mRNA is associated with one to four ribosomes. These results suggest that the stable secondary structure within the translated region of the CYC1 mRNA diminishes translation by inhibiting elongation.

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Disruption of regulatory gene GAL80 in Saccharomyces cerevisiae: effects on carbon-controlled regulation of the galactose/melibiose pathway genes.

Molecular and Cellular Biology ◽

10.1128/mcb.4.8.1521 ◽

1984 ◽

Vol 4 (8) ◽

pp. 1521-1527 ◽

Cited By ~ 65

Author(s):

T E Torchia ◽

R W Hamilton ◽

C L Cano ◽

J E Hopper

Keyword(s):

Saccharomyces Cerevisiae ◽

Catabolite Repression ◽

Carbon Catabolite Repression ◽

Regulatory Gene ◽

Negative Regulator ◽

Null Mutation ◽

Null Mutant ◽

Wild Type

In Saccharomyces cerevisiae, the transcriptional expression of the galactose-melibiose catabolic pathway genes is under the control of at least three regulatory genes, GAL4, GAL80, and GAL3. We have isolated the GAL80 gene and have studied the effect of a null mutation on the carbon-controlled regulation of the MEL1 and GAL cluster genes. The null mutation was achieved in vivo by replacing the chromosomal wild-type GAL80 allele with an in vitro-created GAL80 deletion-disruption mutation. Enzyme activities and RNA levels for the GAL cluster and MEL1 genes were constitutively expressed in the null mutant strain grown on glycerol-lactate and were higher than in the isogenic wild-type yeast strain when compared after growth on galactose. Carbon catabolite repression of the GAL cluster and MEL1 genes, which occurs at the level of transcription, is retained in the null mutant. Deletion of the GAL80 gene in a gal4 cell does not restore GAL cluster and MEL1 gene expression. The data demonstrate that (i) the GAL80 protein is a purely negative regulator, (ii) the GAL80 protein does not mediate carbon catabolite repression, and (iii) the GAL4 protein is not simply an antagonizer of GAL80-mediated repression.

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Springing into Action: Reg2 Negatively Regulates Snf1 Protein Kinase and Facilitates Recovery from Prolonged Glucose Starvation in Saccharomyces cerevisiae

Applied and Environmental Microbiology ◽

10.1128/aem.00154-16 ◽

2016 ◽

Vol 82 (13) ◽

pp. 3875-3885 ◽

Cited By ~ 6

Author(s):

Marcin Maziarz ◽

Aishwarya Shevade ◽

LaKisha Barrett ◽

Sergei Kuchin

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Kinase ◽

Carbon Source ◽

Glucose Deprivation ◽

Regulatory Subunit ◽

Glucose Starvation ◽

Wild Type ◽

Glucose Limitation ◽

Content Type ◽

Glucose Depletion

ABSTRACTGlucose is the preferred carbon source for the yeastSaccharomyces cerevisiae. Glucose limitation activates Snf1 protein kinase, a key regulator of energy homeostasis that promotes utilization of alternative carbon sources and enforces energy conservation. Snf1 activation requires phosphorylation of its T-loop threonine (Thr210) by upstream kinases. When glucose is abundant, Snf1 is inhibited by Thr210 dephosphorylation. This involves the function of the type 1 protein phosphatase Glc7, which is targeted to Snf1 by a regulatory subunit, Reg1. Thereg1mutation causes increased Snf1 activity and mimics various aspects of glucose limitation, including slower growth. Reg2 is another Glc7 regulatory subunit encoded by a paralogous gene,REG2. Previous evidence indicated that thereg2mutation exacerbates the Snf1-dependent slow-growth phenotype caused byreg1, suggesting a link between Reg2 and Snf1. Here, we explore this link in more detail and present evidence that Reg2 contributes to Snf1 Thr210 dephosphorylation. Consistent with this role, Reg2 interacts with wild-type Snf1 but not with nonphosphorylatable Snf1-T210A. Reg2 accumulation increases in a Snf1-dependent manner during prolonged glucose deprivation, and glucose-starved cells lacking Reg2 exhibit delayed Snf1 Thr210 dephosphorylation and slower growth recovery upon glucose replenishment. Accordingly, cells lacking Reg2 are outcompeted by wild-type cells in the course of several glucose starvation/replenishment cycles. Collectively, our results support a model in which Reg2-Glc7 contributes to the negative control of Snf1 in response to glucose refeeding after prolonged starvation. The competitive growth advantage provided by Reg2 underscores the evolutionary significance of this paralog forS. cerevisiae.IMPORTANCEThe ability of microorganisms to respond to stress is essential for their survival. However, rapid recovery from stress could be equally crucial in competitive environments. Therefore, a wise stress response program should prepare cells for quick recovery upon reexposure to favorable conditions. Glucose is the preferred carbon source for the yeastS. cerevisiae. Glucose depletion activates the stress response protein kinase Snf1, which functions to limit energy-consuming processes, such as growth. We show that prolonged glucose deprivation also leads to Snf1-dependent accumulation of Reg2 and that this protein helps to inhibit Snf1 and to accelerate growth recovery upon glucose replenishment. Cells lacking Reg2 are readily outcompeted by wild-type cells during glucose depletion/replenishment cycles. Thus, while prolonged glucose deprivation might seem to put yeast cells “on their knees,” concomitant accumulation of Reg2 helps configure the cells into a “sprinter's crouch start position” to spring into action once glucose becomes available.

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Bent DNA functions as a replication enhancer in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.8.7.2763 ◽

1988 ◽

Vol 8 (7) ◽

pp. 2763-2769 ◽

Cited By ~ 44

Author(s):

J S Williams ◽

T T Eckdahl ◽

J N Anderson

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Bending ◽

Direct Test ◽

Wild Type ◽

Bent Dna ◽

Unusual Structure ◽

Wild Type Level ◽

Common Features ◽

Sequence Patterns ◽

Per Se

Previous studies have demonstrated that bent DNA is a conserved property of Saccharomyces cerevisiae autonomously replicating sequences (ARSs). Here we showed that bending elements are contained within ARS subdomains identified by others as replication enhancers. To provide a direct test for the function of this unusual structure, we analyzed the ARS activity of plasmids that contained synthetic bent DNA substituted for the natural bending element in yeast ARS1. The results demonstrated that deletion of the natural bending locus impaired ARS activity which was restored to a near wild-type level with synthetic bent DNA. Since the only obvious common features of the natural and synthetic bending elements are the sequence patterns that give rise to DNA bending, the results suggest that the bent structure per se is crucial for ARS function.

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A General Method for Identifying Recessive Diploid-Specific Mutations in Saccharomyces cerevisiae, Its Application to the Isolation of Mutants Blocked at Intermediate Stages of Meiotic Prophase and Characterization of a New Gene SAE2

Genetics ◽

10.1093/genetics/146.3.797 ◽

1997 ◽

Vol 146 (3) ◽

pp. 797-816 ◽

Cited By ~ 8

Author(s):

Andrew H Z McKee ◽

Nancy Kleckner

Keyword(s):

Saccharomyces Cerevisiae ◽

Meiotic Prophase ◽

Temperature Sensitive ◽

Yeast Saccharomyces Cerevisiae ◽

New Approach ◽

Strand Breaks ◽

Recessive Mutations ◽

New Genes ◽

New Gene

We describe a general new approach for identifying recessive mutations that affect diploid strains of yeast Saccharomyces cerevisiae and the application of this method to the identification of mutations that confer an intermediate block in meiotic prophase chromosome metabolism. The method uses a temperature-sensitive conjugation mutation ste7-1 in combination with homothallism. The mutations of interest confer a defect in spore formation that is dependent upon a gene required for initiation of meiotic recombination and development of meiosis-specific chromosome structure (SPO11). Identified in this screen were null mutations of the DMC1 gene, nonnull mutations of RAD50 (rad50S, and mutations in three new genes designated SAE1, SAE2 and SAE3 (Sporulation in the Absence of Spo Eleven). Molecular characterization of the SAE2 gene and characterization of meiotic and mitotic phenotypes of sae2 mutants are also presented. The phenotypes conferred by a sae2 null mutation are virtually indistinguishable from those conferred by the previously identified nonnull mutations of RAD50 (rad50S). Most notably, both mutations confer only weak sensitivity to the radiomimetic agent methyl methane sulfonate (MMS) but completely block resection and turnover of meiosis-specific double-strand breaks. These observations provide further evidence that this constellation of phenotypes identifies a specific molecular function.

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