scholarly journals Temporal analysis of general control of amino acid biosynthesis in Saccharomyces cerevisiae: role of positive regulatory genes in initiation and maintenance of mRNA derepression.

1984 ◽  
Vol 4 (3) ◽  
pp. 520-528 ◽  
Author(s):  
M D Penn ◽  
G Thireos ◽  
H Greer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Steady State ◽  
Amino Acid ◽  
Gene Product ◽  
Temporal Analysis ◽  
Regulatory Genes ◽  
Single Amino Acid ◽  
Mrna Levels ◽  
Initiation Phase ◽  
Genes Encoding

In Saccharomyces cerevisiae, starvation for a single amino acid results in the derepression of enzyme activities in multiple amino acid biosynthetic pathways. Derepression is a consequence of increased transcription of the genes encoding these enzymes. Analysis of the kinetics of mRNA elevation established that derepression occurs within 5 min of a shift of the culture from rich medium to starvation medium. Any starvation condition was sufficient to trigger an initial high mRNA elevation; however, it was the severity of starvation which determined the steady-state mRNA levels that were subsequently established. The products of the positive regulatory genes AAS101, AAS103, and AAS2 were shown to be required in the initiation phase of this response, whereas the AAS102 gene product was required to maintain the new elevated steady-state mRNA levels. The AAS101 and AAS102 genes were cloned. Consistent with their respective roles in initiation and maintenance of derepression. AAS101 mRNA was found to be expressed at high levels in both rich and starvation media, whereas AAS102 mRNA was derepressed only under starvation conditions. The derepression of AAS102 mRNA is dependent on the AAS101 gene product.

Temporal analysis of general control of amino acid biosynthesis in Saccharomyces cerevisiae: role of positive regulatory genes in initiation and maintenance of mRNA derepression

1984 ◽  
Vol 4 (3) ◽  
pp. 520-528
Author(s):  
M D Penn ◽  
G Thireos ◽  
H Greer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Steady State ◽  
Amino Acid ◽  
Gene Product ◽  
Temporal Analysis ◽  
Regulatory Genes ◽  
Single Amino Acid ◽  
Mrna Levels ◽  
Initiation Phase ◽  
Genes Encoding

In Saccharomyces cerevisiae, starvation for a single amino acid results in the derepression of enzyme activities in multiple amino acid biosynthetic pathways. Derepression is a consequence of increased transcription of the genes encoding these enzymes. Analysis of the kinetics of mRNA elevation established that derepression occurs within 5 min of a shift of the culture from rich medium to starvation medium. Any starvation condition was sufficient to trigger an initial high mRNA elevation; however, it was the severity of starvation which determined the steady-state mRNA levels that were subsequently established. The products of the positive regulatory genes AAS101, AAS103, and AAS2 were shown to be required in the initiation phase of this response, whereas the AAS102 gene product was required to maintain the new elevated steady-state mRNA levels. The AAS101 and AAS102 genes were cloned. Consistent with their respective roles in initiation and maintenance of derepression. AAS101 mRNA was found to be expressed at high levels in both rich and starvation media, whereas AAS102 mRNA was derepressed only under starvation conditions. The derepression of AAS102 mRNA is dependent on the AAS101 gene product.

New positive and negative regulators for general control of amino acid biosynthesis in Saccharomyces cerevisiae

1986 ◽  
Vol 6 (5) ◽  
pp. 1820-1829
Author(s):  
M L Greenberg ◽  
P L Myers ◽  
R C Skvirsky ◽  
H Greer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Regulatory Genes ◽  
Negative Regulator ◽  
Single Amino Acid ◽  
Mrna Levels ◽  
General Control ◽  
Positive Regulators ◽  
Direct Acting ◽  
Analog Sensitivity

The biosynthesis of most amino acids in Saccharomyces cerevisiae is coregulated. Starvation for a single amino acid results in the derepression of amino acid biosynthetic enzymes in many unrelated pathways. This phenomenon, known as general control, is mediated by both positive (GCN) and negative (GCD) regulatory genes. In this paper we describe the identification and characterization of several new regulatory genes for this system, GCN6, GCN7, GCN8, GCN9, and GCD5. A mutation in the negative regulator GCD5 was isolated on the basis of its suppression of a gcn2 mutation. The effect of gcd5 is a posttranscriptional increase in histidine biosynthetic enzyme activity. Suppressors of gcd5 which are deficient in derepression were in turn isolated. Eight such mutations, defining four new positive regulatory genes (GCN6 through GCN9), were obtained. These mutations are recessive, confer sensitivity to multiple amino acid analogs, and result in decreased mRNA levels for genes under general control. The GCN6 and GCN7 gene products were shown to be positive regulators for transcription of the GCN4 gene, the most direct-acting positive regulator thus far identified. The interaction of GCN6 and GCN7 with GCN4 is fundamentally different from that of previously isolated GCN genes. It should also be noted that these gcn selections gave a completely different nonoverlapping set of mutations from earlier selections which relied on analog sensitivity. Thus, we may have identified a new class of GCN genes which are functionally distinct from GCN1 through GCN5.

scholarly journals New positive and negative regulators for general control of amino acid biosynthesis in Saccharomyces cerevisiae.

1986 ◽  
Vol 6 (5) ◽  
pp. 1820-1829 ◽  
Author(s):  
M L Greenberg ◽  
P L Myers ◽  
R C Skvirsky ◽  
H Greer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Regulatory Genes ◽  
Negative Regulator ◽  
Single Amino Acid ◽  
Mrna Levels ◽  
General Control ◽  
Positive Regulators ◽  
Direct Acting ◽  
Analog Sensitivity

The biosynthesis of most amino acids in Saccharomyces cerevisiae is coregulated. Starvation for a single amino acid results in the derepression of amino acid biosynthetic enzymes in many unrelated pathways. This phenomenon, known as general control, is mediated by both positive (GCN) and negative (GCD) regulatory genes. In this paper we describe the identification and characterization of several new regulatory genes for this system, GCN6, GCN7, GCN8, GCN9, and GCD5. A mutation in the negative regulator GCD5 was isolated on the basis of its suppression of a gcn2 mutation. The effect of gcd5 is a posttranscriptional increase in histidine biosynthetic enzyme activity. Suppressors of gcd5 which are deficient in derepression were in turn isolated. Eight such mutations, defining four new positive regulatory genes (GCN6 through GCN9), were obtained. These mutations are recessive, confer sensitivity to multiple amino acid analogs, and result in decreased mRNA levels for genes under general control. The GCN6 and GCN7 gene products were shown to be positive regulators for transcription of the GCN4 gene, the most direct-acting positive regulator thus far identified. The interaction of GCN6 and GCN7 with GCN4 is fundamentally different from that of previously isolated GCN genes. It should also be noted that these gcn selections gave a completely different nonoverlapping set of mutations from earlier selections which relied on analog sensitivity. Thus, we may have identified a new class of GCN genes which are functionally distinct from GCN1 through GCN5.

scholarly journals Negative regulatory gene for general control of amino acid biosynthesis in Saccharomyces cerevisiae.

1986 ◽  
Vol 6 (9) ◽  
pp. 3150-3155 ◽  
Author(s):  
P L Myers ◽  
R C Skvirsky ◽  
M L Greenberg ◽  
H Greer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Regulatory Gene ◽  
Negative Regulator ◽  
Single Amino Acid ◽  
Mrna Levels ◽  
General Control ◽  
Biosynthetic Genes ◽  
Isolation And Characterization ◽  
Steady State Levels

In Saccharomyces cerevisiae, many amino acid biosynthetic pathways are coregulated by a complex general control system: starvation for a single amino acid results in the derepression of amino acid biosynthetic genes in multiple pathways. Derepression of these genes is mediated by positive (GCN) and negative (GCD) regulatory genes. In this paper we describe the isolation and characterization of a previously unreported negative regulatory gene, GCD3. A gcd3 mutation is recessive to wild type, confers resistance to multiple amino acid analogs, and results in overproduction and partially constitutive elevation of mRNA levels for amino acid biosynthetic genes. Furthermore, a gcd3 mutation can overcome the derepression-deficient phenotype of mutations in the positive regulatory GCN1, GCN2, and GCN3 genes. However, the gcd3 mutation cannot overcome the derepression-deficient phenotype of a gcn4 mutation, suggesting that GCD3 acts as a negative regulator of the important GCN4 gene. Northern blot analysis confirmed this conclusion, in that the steady-state levels of GCN4 mRNA are greatly increased in a gcd3 mutant. Thus, the negative regulatory gene GCD3 plays a central role in derepression of amino acid biosynthetic genes.

Negative regulatory gene for general control of amino acid biosynthesis in Saccharomyces cerevisiae

1986 ◽  
Vol 6 (9) ◽  
pp. 3150-3155
Author(s):  
P L Myers ◽  
R C Skvirsky ◽  
M L Greenberg ◽  
H Greer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Regulatory Gene ◽  
Negative Regulator ◽  
Single Amino Acid ◽  
Mrna Levels ◽  
General Control ◽  
Biosynthetic Genes ◽  
Isolation And Characterization ◽  
Steady State Levels

In Saccharomyces cerevisiae, many amino acid biosynthetic pathways are coregulated by a complex general control system: starvation for a single amino acid results in the derepression of amino acid biosynthetic genes in multiple pathways. Derepression of these genes is mediated by positive (GCN) and negative (GCD) regulatory genes. In this paper we describe the isolation and characterization of a previously unreported negative regulatory gene, GCD3. A gcd3 mutation is recessive to wild type, confers resistance to multiple amino acid analogs, and results in overproduction and partially constitutive elevation of mRNA levels for amino acid biosynthetic genes. Furthermore, a gcd3 mutation can overcome the derepression-deficient phenotype of mutations in the positive regulatory GCN1, GCN2, and GCN3 genes. However, the gcd3 mutation cannot overcome the derepression-deficient phenotype of a gcn4 mutation, suggesting that GCD3 acts as a negative regulator of the important GCN4 gene. Northern blot analysis confirmed this conclusion, in that the steady-state levels of GCN4 mRNA are greatly increased in a gcd3 mutant. Thus, the negative regulatory gene GCD3 plays a central role in derepression of amino acid biosynthetic genes.

scholarly journals Molecular Mechanism of Terbinafine Resistance in Saccharomyces cerevisiae

2003 ◽  
Vol 47 (12) ◽  
pp. 3890-3900 ◽  
Author(s):  
Regina Leber ◽  
Sandra Fuchsbichler ◽  
Vlasta Klobučníková ◽  
Natascha Schweighofer ◽  
Eva Pitters ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Point Mutations ◽  
Gene Replacement ◽  
Single Amino Acid ◽  
Cross Resistance ◽  
Squalene Epoxidase ◽  
Amino Acid Substitutions ◽  
Mrna Levels ◽  
In The Wild

ABSTRACT Ten mutants of the yeast Saccharomyces cerevisiae resistant to the antimycotic terbinafine were isolated after chemical or UV mutagenesis. Molecular analysis of these mutants revealed single base pair exchanges in the ERG1 gene coding for squalene epoxidase, the target of terbinafine. The mutants did not show cross-resistance to any of the substrates of various pleiotropic drug resistance efflux pumps tested. The ERG1 mRNA levels in the mutants did not differ from those in the wild-type parent strains. Terbinafine resistance was transmitted with the mutated alleles in gene replacement experiments, proving that single amino acid substitutions in the Erg1 protein were sufficient to confer the resistance phenotype. The amino acid changes caused by the point mutations were clustered in two regions of the Erg1 protein. Seven mutants carried the amino acid substitutions F402L (one mutant), F420L (one mutant), and P430S (five mutants) in the C-terminal part of the protein; and three mutants carried an L251F exchange in the central part of the protein. Interestingly, all exchanges identified involved amino acids which are conserved in the squalene epoxidases of yeasts and mammals. Two mutations that were generated by PCR mutagenesis of the ERG1 gene and that conferred terbinafine resistance mapped in the same regions of the Erg1 protein, with one resulting in an L251F exchange and the other resulting in an F433S exchange. The results strongly indicate that these regions are responsible for the interaction of yeast squalene epoxidase with terbinafine.

Identification of a Functional Domain Within the Essential Core of Histone H3 That Is Required for Telomeric and HM Silencing in Saccharomyces cerevisiae

Genetics ◽  
2003 ◽  
Vol 163 (1) ◽  
pp. 447-452 ◽  
Author(s):  
Jeffrey S Thompson ◽  
Marilyn L Snow ◽  
Summer Giles ◽  
Leslie E McPherson ◽  
Michael Grunstein
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Amino Acid Substitution ◽  
Histone H3 ◽  
Single Amino Acid ◽  
Functional Domain ◽  
Partial Loss ◽  
Histone Fold ◽  
Gal1 Promoter ◽  
Substitution Mutations

Abstract Fourteen novel single-amino-acid substitution mutations in histone H3 that disrupt telomeric silencing in Saccharomyces cerevisiae were identified, 10 of which are clustered within the α1 helix and L1 loop of the essential histone fold. Several of these mutations cause derepression of silent mating locus HML, and an additional subset cause partial loss of basal repression at the GAL1 promoter. Our results identify a new domain within the essential core of histone H3 that is required for heterochromatin-mediated silencing.

scholarly journals Underproduction of the Largest Subunit of RNA Polymerase II Causes Temperature Sensitivity, Slow Growth, and Inositol Auxotrophy in Saccharomyces cerevisiae

Genetics ◽  
1996 ◽  
Vol 142 (3) ◽  
pp. 737-747 ◽  
Author(s):  
Jacques Archambault ◽  
David B Jansma ◽  
James D Friesen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Steady State ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Reporter Gene ◽  
Growth Medium ◽  
Temperature Sensitivity ◽  
Slow Growth ◽  
Yeast Saccharomyces Cerevisiae ◽  
Genes Encoding

Abstract In the yeast Saccharomyces cerevisiae, mutations in genes encoding subunits of RNA polymerase II (RNAPII) often give rise to a set of pleiotropic phenotypes that includes temperature sensitivity, slow growth and inositol auxotrophy. In this study, we show that these phenotypes can be brought about by a reduction in the intracellular concentration of RNAPII. Underproduction of RNAPII was achieved by expressing the gene (RPO21), encoding the largest subunit of the enzyme, from the LEU2 promoter or a weaker derivative of it, two promoters that can be repressed by the addition of leucine to the growth medium. We found that cells that underproduced RPO21 were unable to derepress fully the expression of a reporter gene under the control of the INO1 UAS. Our results indicate that temperature sensitivity, slow growth and inositol auxotrophy is a set of phenotypes that can be caused by lowering the steady-state amount of RNAPII; these results also lead to the prediction that some of the previously identified RNAPII mutations that confer this same set of phenotypes affect the assembly/stability of the enzyme. We propose a model to explain the hypersensitivity of INO1 transcription to mutations that affect components of the RNAPII transcriptional machinery.

scholarly journals SEN1, a positive effector of tRNA-splicing endonuclease in Saccharomyces cerevisiae.

1992 ◽  
Vol 12 (5) ◽  
pp. 2154-2164 ◽  
Author(s):  
D J DeMarini ◽  
M Winey ◽  
D Ursic ◽  
F Webb ◽  
M R Culbertson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Gene Product ◽  
Signal Sequence ◽  
Null Alleles ◽  
Nucleoside Triphosphate ◽  
Temperature Sensitive ◽  
Yeast Saccharomyces Cerevisiae ◽  
Amino Terminal

The SEN1 gene, which is essential for growth in the yeast Saccharomyces cerevisiae, is required for endonucleolytic cleavage of introns from all 10 families of precursor tRNAs. A mutation in SEN1 conferring temperature-sensitive lethality also causes in vivo accumulation of pre-tRNAs and a deficiency of in vitro endonuclease activity. Biochemical evidence suggests that the gene product may be one of several components of a nuclear-localized splicing complex. We have cloned the SEN1 gene and characterized the SEN1 mRNA, the SEN1 gene product, the temperature-sensitive sen1-1 mutation, and three SEN1 null alleles. The SEN1 gene corresponds to a 6,336-bp open reading frame coding for a 2,112-amino-acid protein (molecular mass, 239 kDa). Using antisera directed against the C-terminal end of SEN1, we detect a protein corresponding to the predicted molecular weight of SEN1. The SEN1 protein contains a leucine zipper motif, consensus elements for nucleoside triphosphate binding, and a potential nuclear localization signal sequence. The carboxy-terminal 1,214 amino acids of the SEN1 protein are essential for growth, whereas the amino-terminal 898 amino acids are dispensable. A sequence of approximately 500 amino acids located in the essential region of SEN1 has significant similarity to the yeast UPF1 gene product, which is involved in mRNA turnover, and the mouse Mov-10 gene product, whose function is unknown. The mutation that creates the temperature-sensitive sen1-1 allele is located within this 500-amino-acid region, and it causes a substitution for an amino acid that is conserved in all three proteins.

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.

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