Multiple GCD genes required for repression of GCN4, a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae

1986 ◽  
Vol 6 (11) ◽  
pp. 3990-3998
Author(s):  
S Harashima ◽  
A G Hinnebusch
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Lacz Gene ◽  
Gene Products ◽  
Wild Type ◽  
Double Mutation ◽  
Double Stranded Rna ◽  
Amino Acid Availability ◽  
Genes Encoding ◽  
New Alleles

GCN4 encodes a positive regulator of multiple unlinked genes encoding amino acid biosynthetic enzymes in Saccharomyces cerevisiae. Expression of GCN4 is coupled to amino acid availability by a control mechanism involving GCD1 as a negative effector and GCN1, GCN2, and GCN3 as positive effectors of GCN4 expression. We used reversion of a gcn2 gcn3 double mutation to isolate new alleles of GCD1 and mutations in four additional GCD genes which we designate GCD10, GCD11, GCD12, and GCD13. All of the mutations lead to constitutive derepression of HIS4 transcription in the absence of the GCN2+ and GCN3+ alleles. By contrast, the gcd mutations require the wild-type GCN4 allele for their derepressing effect, suggesting that each acts by influencing the level of GCN4 activity in the cell. Consistent with this interpretation, mutations in each GCD gene lead to constitutive derepression of a GCN4::lacZ gene fusion. Thus, at least five gene products are required to maintain the normal repressed level of GCN4 expression in nonstarvation conditions. Interestingly, the gcd mutations are pleiotropic and also affect growth rate in nonstarvation conditions. In addition, certain alleles lead to a loss of M double-stranded RNA required for the killer phenotype. This pleiotropy suggests that the GCD gene products contribute to an essential cellular function, in addition to, or in conjunction with, their role in GCN4 regulation.

scholarly journals Multiple GCD genes required for repression of GCN4, a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae.

1986 ◽  
Vol 6 (11) ◽  
pp. 3990-3998 ◽  
Author(s):  
S Harashima ◽  
A G Hinnebusch
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Lacz Gene ◽  
Gene Products ◽  
Wild Type ◽  
Double Mutation ◽  
Double Stranded Rna ◽  
Amino Acid Availability ◽  
Genes Encoding ◽  
New Alleles

GCN4 encodes a positive regulator of multiple unlinked genes encoding amino acid biosynthetic enzymes in Saccharomyces cerevisiae. Expression of GCN4 is coupled to amino acid availability by a control mechanism involving GCD1 as a negative effector and GCN1, GCN2, and GCN3 as positive effectors of GCN4 expression. We used reversion of a gcn2 gcn3 double mutation to isolate new alleles of GCD1 and mutations in four additional GCD genes which we designate GCD10, GCD11, GCD12, and GCD13. All of the mutations lead to constitutive derepression of HIS4 transcription in the absence of the GCN2+ and GCN3+ alleles. By contrast, the gcd mutations require the wild-type GCN4 allele for their derepressing effect, suggesting that each acts by influencing the level of GCN4 activity in the cell. Consistent with this interpretation, mutations in each GCD gene lead to constitutive derepression of a GCN4::lacZ gene fusion. Thus, at least five gene products are required to maintain the normal repressed level of GCN4 expression in nonstarvation conditions. Interestingly, the gcd mutations are pleiotropic and also affect growth rate in nonstarvation conditions. In addition, certain alleles lead to a loss of M double-stranded RNA required for the killer phenotype. This pleiotropy suggests that the GCD gene products contribute to an essential cellular function, in addition to, or in conjunction with, their role in GCN4 regulation.

scholarly journals gcd12 mutations are gcn3-dependent alleles of GCD2, a negative regulator of GCN4 in the general amino acid control of Saccharomyces cerevisiae.

Genetics ◽  
1989 ◽  
Vol 122 (3) ◽  
pp. 543-550 ◽  
Author(s):  
C J Paddon ◽  
A G Hinnebusch
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Negative Regulator ◽  
Regulatory Function ◽  
Growth Defect ◽  
Gene Products ◽  
Wild Type ◽  
General Amino Acid Control ◽  
Translational Repressor ◽  
Acid Control

Abstract GCD12 encodes a translational repressor of the GCN4 protein, a transcriptional activator of amino acid biosynthetic genes in the yeast Saccharomyces cerevisiae. gcd12 mutations override the requirement for the GCN2 and GCN3 gene products for derepression of GCN4 expression, suggesting that GCN2 and GCN3 function indirectly as positive regulators by negative regulation of GCD12. In addition to their regulatory phenotype, gcd12 mutants are temperature-sensitive for growth (Tsm-) and, as shown here, deletion of the GCD12 gene is unconditionally lethal. Both the regulatory and the Tsm- phenotypes associated with gcd12 point mutations are completely overcome by wild-type GCN3, implying that GCN3 can promote or partially substitute for the functions of GCD12 in normal growth conditions even though it antagonizes GCD12 regulatory function in starvation conditions. The GCD12 gene has been cloned and mapped to the right arm of chromosome VII, very close to the map position reported for GCD2. We demonstrate that GCD12 and GCD2 are the same genes; however, unlike gcd12 mutations, the growth defect and constitutive derepression phenotypes associated with the gcd2-1 mutation are expressed in the presence of the wild-type GCN3 gene. These findings can be explained by either of two alternative hypotheses: (1) gcd12 mutations affect a domain of the GCD2 protein that directly interacts with GCN3, and complex formation stabilizes mutant gcd12 (but not gcd2-1) gene products; (2) gcd12 mutations selectively impair one function of GCD2 that is replaceable by GCN3, whereas gcd2-1 inactivates a different GCD2 function for which GCN3 cannot substitute. Both models imply a close interaction between these two positive and negative regulators in general amino acid control.

scholarly journals Rsp5p, a New Link between the Actin Cytoskeleton and Endocytosis in the Yeast Saccharomyces cerevisiae

2002 ◽  
Vol 22 (20) ◽  
pp. 6946-6948 ◽  
Author(s):  
Joanna Kamińska ◽  
Beata Gajewska ◽  
Anita K. Hopper ◽  
Teresa ˙Zołądek
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Actin Cytoskeleton ◽  
Cytoskeletal Proteins ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Ww Domains ◽  
Ubiquitin Protein Ligase ◽  
Growth Defects ◽  
Genes Encoding ◽  
Cytoskeleton Dynamics

ABSTRACT Rsp5p is an ubiquitin-protein ligase of Saccharomyces cerevisiae that has been implicated in numerous processes including transcription, mitochondrial inheritance, and endocytosis. Rsp5p functions at multiple steps of endocytosis, including ubiquitination of substrates and other undefined steps. We propose that one of the roles of Rsp5p in endocytosis involves maintenance and remodeling of the actin cytoskeleton. We report the following. (i) There are genetic interactions between rsp5 and several mutant genes encoding actin cytoskeletal proteins. rsp5 arp2, rsp5 end3, and rsp5 sla2 double mutants all show synthetic growth defects. Overexpressed wild-type RSP5 or mutant rsp5 genes with lesions of some WW domains suppress growth defects of arp2 and end3 cells. The defects in endocytosis, actin cytoskeleton, and morphology of arp2 are also suppressed. (ii) Rsp5p and Sla2p colocalize in abnormal F-actin-containing clumps in arp2 and pan1 mutants. Immunoprecipitation experiments confirmed that Rsp5p and Act1p colocalize in pan1 mutants. (iii) Rsp5p and Sla2p coimmunoprecipitate and partially colocalize to punctate structures in wild-type cells. These studies provide the first evidence for an interaction of an actin cytoskeleton protein with Rsp5p. (iv) rsp5-w1 mutants are resistant to latrunculin A, a drug that sequesters actin monomers and depolymerizes actin filaments, consistent with the fact that Rsp5p is involved in actin cytoskeleton dynamics.

Genetic analysis of small nuclear RNAs in Saccharomyces cerevisiae: viable sextuple mutant

1988 ◽  
Vol 8 (8) ◽  
pp. 3150-3159
Author(s):  
R Parker ◽  
T Simmons ◽  
E O Shuster ◽  
P G Siliciano ◽  
C Guthrie
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Start Site ◽  
Single Copy ◽  
Gene Replacement ◽  
Null Alleles ◽  
Wild Type ◽  
Small Nuclear Rnas ◽  
Apparent Correlation ◽  
Genes Encoding ◽  
Sm Antigen

Saccharomyces cerevisiae contains at least 24 distinct small nuclear RNAs (snRNAs), several of which are known to be essential for viability and to participate in the splicing of pre-mRNAs; the RNAs in this subset contain binding sites for the Sm antigen, a hallmark of metazoan snRNAs involved in mRNA processing. In contrast, we showed previously that the single-copy genes for three other snRNAs (snR3, snR4, and snR10) are not required for viability, although cells lacking snR10 are growth impaired at low temperature. None of these RNAs associates with the Sm antigen. To assess this apparent correlation, we cloned and sequenced the genes encoding three additional non-Sm snRNAs. Comparison of these genes with nine additional yeast snRNA genes revealed a highly conserved TATA box located 92 +/- 8 nucleotides 5' of the transcriptional start site. By using the technique of gene replacement with null alleles, each of these three single copy genes was shown to be completely dispensable. We constructed multiple mutants to test the hypothesis that, individually, each of these snRNAs is nonessential because the snRNAs play functionally overlapping roles. A mutant lacking five snRNAs (snR3, snR4, snR5, snR8, snR9) was indistinguishable from the wild type, and growth of the sextuple mutant was no more impaired than that in strains lacking only snR10. This widespread dispensability of snRNAs was completely unexpected and forces us to reconsider the possible roles of these ubiquitous RNAs.

scholarly journals Acquisition of the Ability To Assimilate Mannitol by Saccharomyces cerevisiae through Dysfunction of the General Corepressor Tup1-Cyc8

2014 ◽  
Vol 81 (1) ◽  
pp. 9-16 ◽  
Author(s):  
Moeko Chujo ◽  
Shiori Yoshida ◽  
Anri Ota ◽  
Kousaku Murata ◽  
Shigeyuki Kawai
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Basis ◽  
Mutant Allele ◽  
Wild Type ◽  
Growth Data ◽  
Content Type ◽  
Corepressor Complex ◽  
Marine Biomass ◽  
Prolonged Culture ◽  
Genes Encoding

ABSTRACTSaccharomyces cerevisiaenormally cannot assimilate mannitol, a promising brown macroalgal carbon source for bioethanol production. The molecular basis of this inability remains unknown. We found that cells capable of assimilating mannitol arose spontaneously from wild-typeS. cerevisiaeduring prolonged culture in mannitol-containing medium. Based on microarray data, complementation analysis, and cell growth data, we demonstrated that acquisition of mannitol-assimilating ability was due to spontaneous mutations in the genes encoding Tup1 or Cyc8, which constitute a general corepressor complex that regulates many kinds of genes. We also showed that anS. cerevisiaestrain carrying a mutant allele ofCYC8exhibited superior salt tolerance relative to other ethanologenic microorganisms; this characteristic would be highly beneficial for the production of bioethanol from marine biomass. Thus, we succeeded in conferring the ability to assimilate mannitol onS. cerevisiaethrough dysfunction of Tup1-Cyc8, facilitating production of ethanol from mannitol.

scholarly journals Mistranslating tRNA identifies a deleterious S213P mutation in the Saccharomyces cerevisiae eco1-1 allele

2020 ◽  
Vol 98 (5) ◽  
pp. 624-630 ◽  
Author(s):  
Yanrui Zhu ◽  
Matthew D. Berg ◽  
Phoebe Yang ◽  
Raphaël Loll-Krippleber ◽  
Grant W. Brown ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Genetic Disease ◽  
Elevated Temperatures ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Standard Genetic Code ◽  
Gene Encoding ◽  
Chromatid Cohesion ◽  
Sensitive Allele

Mistranslation occurs when an amino acid not specified by the standard genetic code is incorporated during translation. Since the ribosome does not read the amino acid, tRNA variants aminoacylated with a non-cognate amino acid or containing a non-cognate anticodon dramatically increase the frequency of mistranslation. In a systematic genetic analysis, we identified a suppression interaction between tRNASerUGG, G26A, which mistranslates proline codons by inserting serine, and eco1-1, a temperature sensitive allele of the gene encoding an acetyltransferase required for sister chromatid cohesion. The suppression was partial, with a tRNA that inserts alanine at proline codons and not apparent for a tRNA that inserts serine at arginine codons. Sequencing of the eco1-1 allele revealed a mutation that would convert the highly conserved serine 213 within β7 of the GCN5-related N-acetyltransferase core to proline. Mutation of P213 in eco1-1 back to the wild-type serine restored the function of the enzyme at elevated temperatures. Our results indicate the utility of mistranslating tRNA variants to identify functionally relevant mutations and identify eco1 as a reporter for mistranslation. We propose that mistranslation could be used as a tool to treat genetic disease.

scholarly journals Amino Acid Transport and Metabolism in Mycobacteria: Cloning, Interruption, and Characterization of anl-Arginine/γ-Aminobutyric Acid Permease inMycobacterium bovis BCG

2000 ◽  
Vol 182 (4) ◽  
pp. 919-927 ◽  
Author(s):  
Anjali Seth ◽  
Nancy D. Connell
Keyword(s):  
Amino Acid ◽  
Mutant Strain ◽  
Nitrogen Sources ◽  
Single Copy ◽  
Aminobutyric Acid ◽  
Cosmid Library ◽  
Wild Type ◽  
Carbon And Nitrogen ◽  
Genes Encoding ◽  
Type Gene

ABSTRACT Genes encoding l-arginine biosynthetic and transport proteins have been shown in a number of pathogenic organisms to be important for metabolism within the host. In this study we describe the cloning of a gene (Rv0522) encoding an amino acid transporter fromMycobacterium bovis BCG and the effects of its deletion onl-arginine transport and metabolism. The Rv0522 gene of BCG was cloned from a cosmid library by using primers homologous to therocE gene of Bacillus subtilis, a putative arginine transporter. A deletion mutant strain was constructed by homologous recombination with the Rv0522 gene interrupted by a selectable marker. The mutant strain was complemented with the wild-type gene in single copy. Transport analysis of these strains was conducted using 14C-labeled substrates. Greatly reduced uptake of l-arginine and γ-aminobutyric acid (GABA) but not of lysine, ornithine, proline, or alanine was observed in the mutant strain compared to the wild type, grown in Middlebrook 7H9 medium. However, when the strains were starved for 24 h or incubated in a minimal salts medium containing 20 mM arginine (in which even the parent strain does not grow),l-[14C]arginine uptake by the mutant but not the wild-type strain increased strongly. Exogenousl-arginine but not GABA, lysine, ornithine, or alanine was shown to be toxic at concentrations of 20 mM and above to wild-type cells growing in optimal carbon and nitrogen sources such as glycerol and ammonium. l-Arginine supplied in the form of dipeptides showed no toxicity at concentrations as high as 30 mM. Finally, the permease mutant strain showed no defect in survival in unactivated cultured murine macrophages compared with wild-type BCG.

Eight UCA codons differentially affect the expression of the lacZ gene in the divE42 mutant of Escherichia coli

2000 ◽  
Vol 46 (6) ◽  
pp. 577-583 ◽  
Author(s):  
Takashi Kubo ◽  
Toshiko Aiso ◽  
Reiko Ohki
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Temperature Sensitive ◽  
Lacz Gene ◽  
Permissive Temperature ◽  
Wild Type ◽  
Double Mutation ◽  
Mutant Genes ◽  
Beta Galactosidase ◽  
Serine Codons

In the divE mutant, which has a temperature-sensitive mutation in the tRNA1Ser gene, the synthesis of beta-galactosidase is dramatically decreased at the non-permissive temperature. In Escherichia coli, the UCA codon is only recognized by tRNA1Ser. Several genes containing UCA codons are normally expressed at 42°C in the divE mutant. Therefore, it is unlikely that the defect is due to the general translational deficiency of the mutant tRNA1Ser. In this study, we constructed mutant lacZ genes, in which one or several UCA codons at eight positions were replaced with other serine codons such as UCU or UCC, and we examined the expression of these mutant genes in the divE mutant. We found that a single UCA codon at position 6 or 462 was sufficient to cause the same level of reduced beta-galactosidase synthesis as that of the wild-type lacZ gene, and that the defect in beta-galactosidase synthesis was accompanied by a low level of lacZ mRNA. It was also found that introduction of an rne-1 pnp-7 double mutation restored the expression of mutant lacZ genes with only UCA codons at position 6 or 462. A polarity suppressor mutation in the rho gene had no effect on the defect in lacZ gene expression in the divE mutant. We propose a model to explain these results.Key words: divE gene, tRNA1Ser, lacZ gene expression, UCA codon.

scholarly journals Biological role of the general control of amino acid biosynthesis in Saccharomyces cerevisiae.

1981 ◽  
Vol 1 (7) ◽  
pp. 584-593 ◽  
Author(s):  
P Niederberger ◽  
G Miozzari ◽  
R Hütter
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Amino Acid Biosynthesis ◽  
Wild Type ◽  
General Control ◽  
Acid Biosynthesis ◽  
Mutant Strains ◽  
In The Wild ◽  
Amino Acid Imbalance

The biological role of the "general control of amino acid biosynthesis" has been investigated by analyzing growth and enzyme levels in wild-type, bradytrophic, and nonderepressing mutant strains of Saccharomyces cerevisiae. Amino acid limitation was achieved by using either bradytrophic mutations or external amino acid imbalance. In the wild-type strain noncoordinate derepression of enzymes subject to the general control has been found. Derepressing factors were in the order of 2 to 4 in bradytrophic mutant strains grown under limiting conditions and only in the order of 1.5 to 2 under the influence of external amino acid imbalance. Nonderepressing mutations led to slower growth rates under conditions of amino acid limitation, and no derepression of enzymes under the general control was observed. The amino acid pools were found to be very similar in the wild type and in nonderepressing mutant strains under all conditions tested. Our results indicate that the general control affects all branched amino acid biosynthetic pathways, namely, those of the aromatic amino acids and the aspartate family, the pathways for the basic amino acids lysine, histidine, and arginine, and also the pathways of serine and valine biosyntheses.

scholarly journals A suppressor of an RNA polymerase II mutation of Saccharomyces cerevisiae encodes a subunit common to RNA polymerases I, II, and III.

1990 ◽  
Vol 10 (12) ◽  
pp. 6123-6131 ◽  
Author(s):  
J Archambault ◽  
K T Schappert ◽  
J D Friesen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Essential Gene ◽  
Single Amino Acid ◽  
Temperature Sensitive ◽  
Gene Products ◽  
Gene Encoding ◽  
Temperature Sensitive Mutation

RNA polymerase II (RNAPII) is a complex multisubunit enzyme responsible for the synthesis of pre-mRNA in eucaryotes. The enzyme is made of two large subunits associated with at least eight smaller polypeptides, some of which are common to all three RNA polymerase species. We have initiated a genetic analysis of RNAPII by introducing mutations in RPO21, the gene encoding the largest subunit of RNAPII in Saccharomyces cerevisiae. We have used a yeast genomic library to isolate plasmids that can suppress a temperature-sensitive mutation in RPO21 (rpo21-4), with the goal of identifying gene products that interact with the largest subunit of RNAPII. We found that increased expression of wild-type RPO26, a single-copy, essential gene encoding a 155-amino-acid subunit common to RNAPI, RNAPII, and RNAPIII, suppressed the rpo21-4 temperature-sensitive mutation. Mutations were constructed in vitro that resulted in single amino acid changes in the carboxy-terminal portion of the RPO26 gene product. One temperature-sensitive mutation, as well as some mutations that did not by themselves generate a phenotype, were lethal in combination with rpo21-4. These results support the idea that the RPO26 and RPO21 gene products interact.

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