Upstream region of the SUC2 gene confers regulated expression to a heterologous gene in Saccharomyces cerevisiae.

Sarokin, Laura; Carlson, Marian

doi:10.1128/mcb.5.10.2521

Cited by 83 publications

(73 citation statements)

References 25 publications

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“…Previously, we showed that an ssn6 mutation causes constitutive expression of a gene fusion under the control of the SUC2 upstream region, which suggested that the upstream region mediates the repressive effect of SSN6 on transcription (36). To confirm that SSN6 exerts its regulatory effects at the RNA level, expression of SUC2 mRNA was examined in ssn6-1 and wild-type cells under glucose-repressing and derepres-sing conditions.…”

Section: Resultsmentioning

confidence: 99%

Molecular analysis of SSN6, a gene functionally related to the SNF1 protein kinase of Saccharomyces cerevisiae.

Schultz¹,

Carlson²

1987

Mol. Cell. Biol.

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214

133

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Mutations in the SSN6 gene suppress the invertase derepression defect caused by a lesion in the SNF1 protein kinase gene. We cloned the SSN6 gene of Saccharomyces cerevisiae and identified its 3.3-kilobase poly(A)-containing RNA. Disruption of the gene caused phenotypes similar to, but more severe than, those caused by missense mutations: high-level constitutivity for invertase, clumpiness, temperature-sensitive growth, aspecific mating defects, and failure of homozygous diploids to sporulate. In contrast, the presence of multiple copies of SSN6 interfered with derepression of invertase. An ssn6 mutation was also shown to cause glucose-insensitive expression of a GAL1O-lacZ fusion and maltase. The mating defects of MATa ssn6 strains were associated with production of two a-specific products, a-factor and barrier, and reduced levels of a-factor; no deficiency of MATa2 RNA was detected. We showed that ssn6 partially restored invertase expression in a cyrl-2 mutant, although ssn6 was clearly not epistatic to cyri-2. We also determined the nucleotide sequence of SSN6, which is predicted to encode a 107-kilodalton protein with stretches of polyglutamine and poly(glutamine-alanine). Possible functions of the SSN6 product are discussed.The SNFJ gene of Saccharomyces cerevisiae encodes a protein kinase that is required for derepression of many glucose-repressible genes, including the SUC2 (invertase) gene (5,8). Mutations in the SSN6 gene have been isolated as suppressors of the invertase derepression defect in a snfl mutant (6). The ssn6 mutations were found to cause highlevel constitutive (glucose-insensitive) invertase synthesis in both snfl and wild-type (SNFI) genetic backgrounds. These findings suggested that SSN6 is a negative regulator of SUC2 and may be a substrate of the SNFJ protein kinase. The pleiotropic phenotypes of ssn6 mutants indicated that SSN6 also affects expression of other genes; the mutants exhibited extreme clumpiness, a-specific mating defects, and a failure to sporulate as homozygous diploids (6). Genetic mapping has led to the finding that ssn6 is allelic to cyc8, a mutation that causes overproduction of iso-2-cytochrome c (34). Additional cyc8/ssn6 alleles have recently been isolated by Trumbly (45).We report here the cloning and molecular analysis of the SSN6 gene. We constructed null mutations to determine the phenotype of strains lacking SSN6 function, tested the effects of multiple copies of SSN6 on SUC2 expression, and determined the sequence of the gene. We showed that ssn6 affects expression of two other glucose-repressible genes and also explored the molecular basis of the mating defects of MATa ssn6 strains. MATERIALS AND METHODSStrains and genetic methods. The S. cerevisiae strains used in this study are listed in Table 1. The strains were constructed by standard genetic methods (39), and the genotypes of double mutants were confirmed by complementation analysis. Media and methods for scoring markers have been described (28). Yeast transformations were carried out as described prev...

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Section: Resultsmentioning

confidence: 99%

Molecular analysis of SSN6, a gene functionally related to the SNF1 protein kinase of Saccharomyces cerevisiae.

Schultz¹,

Carlson²

1987

Mol. Cell. Biol.

Self Cite

214

133

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“…We also do not believe that our H2A mutations interfere with a step subsequent to transcription initiation (such as transcriptional elongation) because lacZ reporters dependent on different activators were affected differently by the H2A mutations (31). In addition, at least some of these hta1 mutations (encoding N75S K76E, L95S, and H114Q) were suppressed by ssn6 mutations (31), which are known to affect transcription initiation at SUC2 (62). We therefore favor a model in which these H2A mutations directly affect transcription initiation at SUC2.…”

Section: Discussionmentioning

confidence: 99%

A New Class of Histone H2A Mutations in Saccharomyces cerevisiae Causes Specific Transcriptional Defects In Vivo

Hirschhorn

Bortvin

Ricupero-Hovasse

et al. 1995

Molecular and Cellular Biology

107

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Nucleosomes have been shown to repress transcription both in vitro and in vivo. However, the mechanisms by which this repression is overcome are only beginning to be understood. Recent evidence suggests that in the yeast Saccharomyces cerevisiae, many transcriptional activators require the SNF/SWI complex to overcome chromatin-mediated repression. We have identified a new class of mutations in the histone H2A-encoding gene HTA1 that causes transcriptional defects at the SNF/SWI-dependent gene SUC2. Some of the mutations are semidominant, and most of the predicted amino acid changes are in or near the N-and C-terminal regions of histone H2A. A deletion that removes the N-terminal tail of histone H2A also caused a decrease in SUC2 transcription. Strains carrying these histone mutations also exhibited defects in activation by LexA-GAL4, a SNF/SWI-dependent activator. However, these H2A mutants are phenotypically distinct from snf/swi mutants. First, not all SNF/SWI-dependent genes showed transcriptional defects in these histone mutants. Second, a suppressor of snf/swi mutations, spt6, did not suppress these histone mutations. Finally, unlike in snf/swi mutants, chromatin structure at the SUC2 promoter in these H2A mutants was in an active conformation. Thus, these H2A mutations seem to interfere with a transcription activation function downstream or independent of the SNF/SWI activity. Therefore, they may identify an additional step that is required to overcome repression by chromatin.In eukaryotic cells, DNA is complexed with histones and other proteins into chromatin (see reference 75 for a review). The primary component of chromatin is the nucleosome, which consists of approximately 146 bp of DNA wrapped around an octamer of histones (two histone H2A-H2B dimers and one [H3-H4] 2 tetramer; see reference 75 for a review). A growing body of evidence from both in vivo and in vitro studies has shown that the structure of chromatin influences gene expression (see references 25 and 53 for reviews). Biochemical experiments have shown that histones can repress transcription in vitro (see reference 53 for a review) and that transcriptional activators can overcome this repression (21,41,45,79,81,82). In vivo experiments in Saccharomyces cerevisiae have shown that the loss of transcriptional activators or of activator binding sites can be suppressed by mutations in histone genes (27,30,37,57). These results suggest that one function of transcriptional activators in vivo is to antagonize chromatin-mediated repression.In vivo studies of histone mutants have also suggested that histones play a variety of roles in transcriptional regulation. Small deletions and point mutations that alter the flexible N-terminal tails of different yeast histones have been shown to cause specific changes in transcription (see reference 25 for a review). Analysis of deletions and single amino acid changes in the N-terminal region of histone H4 has demonstrated that certain changes in the H4 N terminus abolish repression of the yeast silent mating-...

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“…pression of a SUC2-LEU2-lacZ gene fusion contained in the plasmid pLS11 [20] integrated at the chromosomal URA3 locus. This gene fusion is normally regulated by glucose repression.…”

Section: Expression Of Suc2 Gene In Complete Absence Of Glucosementioning

confidence: 99%

Sudden depletion of carbon source blocks translation, but not transcription, in the yeast Saccharomyces cerevisiae

Martínez‐Pastor

Estruch

1996

FEBS Letters

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Expression of invertase in the yeast Saccharomyces cerevisiae is greatly delayed when derepression occurs in a medium that lacks a usable carbon source. The delay is not a consequence of defects in the transcription of the SUC2 gene but is due to the impossibility of translating the normal levels of mRNA generated under derepressing conditions. The inhibition of translation in the absence of glucose has to be considered when reporter genes such as E. coli lacZ are used to measure transcription in conditions of carbon source starvation.

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Upstream region of the SUC2 gene confers regulated expression to a heterologous gene in Saccharomyces cerevisiae.

Cited by 83 publications

References 25 publications

Molecular analysis of SSN6, a gene functionally related to the SNF1 protein kinase of Saccharomyces cerevisiae.

Molecular analysis of SSN6, a gene functionally related to the SNF1 protein kinase of Saccharomyces cerevisiae.

A New Class of Histone H2A Mutations in Saccharomyces cerevisiae Causes Specific Transcriptional Defects In Vivo

Sudden depletion of carbon source blocks translation, but not transcription, in the yeast Saccharomyces cerevisiae

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