Role of the complex upstream region of the GDH2 gene in nitrogen regulation of the NAD-linked glutamate dehydrogenase in Saccharomyces cerevisiae

1991 ◽  
Vol 11 (12) ◽  
pp. 6229-6247
Author(s):  
S M Miller ◽  
B Magasanik
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Glutamine Synthetase ◽  
Nitrogen Source ◽  
Glutamate Dehydrogenase ◽  
Regulatory System ◽  
Sole Source ◽  
Lacz Fusion ◽  
Upstream Region ◽  
In Cells

We analyzed the upstream region of the GDH2 gene, which encodes the NAD-linked glutamate dehydrogenase in Saccharomyces cerevisiae, for elements important for the regulation of the gene by the nitrogen source. The levels of this enzyme are high in cells grown with glutamate as the sole source of nitrogen and low in cells grown with glutamine or ammonium. We found that this regulation occurs at the level of transcription and that a total of six sites are required to cause a CYC1-lacZ fusion to the GDH2 gene to be regulated in the same manner as the NAD-linked glutamate dehydrogenase. Two sites behaved as upstream activation sites (UASs). The remaining four sites were found to block the effects of the two UASs in such a way that the GDH2-CYC1-lacZ fusion was not expressed unless the cells containing it were grown under conditions favorable for the activity of both UASs. This complex regulatory system appears to account for the fact that GDH2 expression is exquisitely sensitive to glutamine, whereas the expression of GLN1, coding for glutamine synthetase, is not nearly as sensitive.

scholarly journals Role of the complex upstream region of the GDH2 gene in nitrogen regulation of the NAD-linked glutamate dehydrogenase in Saccharomyces cerevisiae.

1991 ◽  
Vol 11 (12) ◽  
pp. 6229-6247 ◽  
Author(s):  
S M Miller ◽  
B Magasanik
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Glutamine Synthetase ◽  
Nitrogen Source ◽  
Glutamate Dehydrogenase ◽  
Regulatory System ◽  
Sole Source ◽  
Lacz Fusion ◽  
Upstream Region ◽  
In Cells

We analyzed the upstream region of the GDH2 gene, which encodes the NAD-linked glutamate dehydrogenase in Saccharomyces cerevisiae, for elements important for the regulation of the gene by the nitrogen source. The levels of this enzyme are high in cells grown with glutamate as the sole source of nitrogen and low in cells grown with glutamine or ammonium. We found that this regulation occurs at the level of transcription and that a total of six sites are required to cause a CYC1-lacZ fusion to the GDH2 gene to be regulated in the same manner as the NAD-linked glutamate dehydrogenase. Two sites behaved as upstream activation sites (UASs). The remaining four sites were found to block the effects of the two UASs in such a way that the GDH2-CYC1-lacZ fusion was not expressed unless the cells containing it were grown under conditions favorable for the activity of both UASs. This complex regulatory system appears to account for the fact that GDH2 expression is exquisitely sensitive to glutamine, whereas the expression of GLN1, coding for glutamine synthetase, is not nearly as sensitive.

Regulation of glutamine-repressible gene products by the GLN3 function in Saccharomyces cerevisiae

1984 ◽  
Vol 4 (12) ◽  
pp. 2758-2766
Author(s):  
A P Mitchell ◽  
B Magasanik
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Glutamine Synthetase ◽  
Glutamate Dehydrogenase ◽  
Opposite Effect ◽  
Nuclear Gene ◽  
Yeast Saccharomyces Cerevisiae ◽  
Two Dimensional Gel Electrophoresis ◽  
Regulatory Circuit ◽  
Glutamate Dehydrogenase Activity ◽  
Glutamine Limitation

Mutants of the yeast Saccharomyces cerevisiae have been isolated which fail to derepress glutamine synthetase upon glutamine limitation. The mutations define a single nuclear gene, GLN3, which is located on chromosome 5 near HOM3 and HIS1 and is unlinked to the structural gene for glutamine synthetase, GLN1. The three gln3 mutations are recessive, and one is amber suppressible, indicating that the GLN3 product is a positive regulator of glutamine synthetase expression. Four polypeptides, in addition to the glutamine synthetase subunit are synthesized at elevated rates when GLN3+ cultures are shifted from glutamine to glutamate media as determined by pulse-labeling and one- and two-dimensional gel electrophoresis. The response of all four proteins is blocked by gln3 mutations. In addition, the elevated NAD-dependent glutamate dehydrogenase activity normally found in glutamate-grown cells is not found in gln3 mutants. Glutamine limitation of gln1 structural mutants has the opposite effect, causing elevated levels of NAD-dependent glutamate dehydrogenase even in the presence of ammonia. We suggest that there is a regulatory circuit that responds to glutamine availability through the GLN3 product.

Multiple elements and auto-repression regulate Rox1, a repressor of hypoxic genes in Saccharomyces cerevisiae.

Genetics ◽  
1995 ◽  
Vol 139 (3) ◽  
pp. 1149-1158 ◽  
Author(s):  
J Deckert ◽  
R Perini ◽  
B Balasubramanian ◽  
R S Zitomer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Growth ◽  
Blot Analysis ◽  
Lacz Fusion ◽  
Upstream Region ◽  
Fusion Construct ◽  
Gel Retardation ◽  
Gal1 Promoter ◽  
Rna Blot Analysis

Abstract The ROX1 gene encodes a heme-induced repressor of hypoxic genes in yeast. Using RNA blot analysis and a ROX1/lacZ fusion construct that included the ROX1 upstream region and only the first codon, we discovered that Rox1 represses its own expression. Gel-retardation experiments indicated that Rox1 was capable of binding to its own upstream region. Overexpression of Rox1 from the inducible GAL1 promoter was found to be inhibitory to cell growth. Also, we found that, as reported previously, Hap1 is partially responsible for heme-induction of ROX1, but, in addition, it also may play a role in ROX1 repression in the absence of heme. There is a second repressor of anaerobic ROX1 expression that requires the general repressor Tup1/Ssn6 for its function.

The green hydra symbiosis and ammonium. I. The role of the host in ammonium assimilation and its possible regulatory significance

1986 ◽  
Vol 229 (1256) ◽  
pp. 299-314 ◽  
Keyword(s):  
Glutamine Synthetase ◽  
Glutamate Dehydrogenase ◽  
Ammonium Assimilation ◽  
Nitrogen Status ◽  
Continuous Darkness ◽  
Methionine Sulphoximine ◽  
Vacuolar Ph ◽  
Regulatory Significance

Evidence for ammonium assimilation by host and symbiont in algal─invertebrate symbioses is summarized and critically evaluated. The host from all strains of hydra studied possessed glutamine synthetase (GS) and glutamate dehydrogenase (GDH) activities. The host from associations with high maltose releasing algae (E/E, E /3N8) had high GS and low GDH activities, whereas aposymbiotic animals (EALB) and the association with a low maltose releasing alga (E/NC) had low GS and high GDH activities. The observation that symbiotic animals do not release ammonium in the light, whereas aposymbiotic animals release substantial amounts, may be explicable on the basis of variation in the ability of the host to assimilate ammonium. Thus, the photosynthetic inhibitor DCMU had no effect on ammonium release by symbiotic animals, with the possible exception of E/NC. Methionine sulphoximine (MSO) completely inhibited GS activity from EALB both in vitro and in vivo . In the presence of MSO, ammonium release was enhanced in both EALB and E/E. In continuous darkness, an increase in ammonium released by symbiotic animals (E/E) was correlated with a decrease in host GS activity. It is suggested that the evidence is consistent with host and not symbiont assimilation of ammonium. A model of symbiont regulation is proposed based on regulation of ammonium supply as a means of controlling both perialgal vacuolar pH and symbiont nitrogen status.

Physiological and genetic analysis of the carbon regulation of the NAD-dependent glutamate dehydrogenase of Saccharomyces cerevisiae

1991 ◽  
Vol 11 (9) ◽  
pp. 4455-4465
Author(s):  
P W Coschigano ◽  
S M Miller ◽  
B Magasanik
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nitrogen Source ◽  
Glutamate Dehydrogenase ◽  
Carbon Sources ◽  
Growth Conditions ◽  
Neighboring Element ◽  
Carbon Regulation ◽  
Activation Of Transcription ◽  
Regulated Expression ◽  
Nonfermentable Carbon Source

We found that cells of Saccharomyces cerevisiae have an elevated level of the NAD-dependent glutamate dehydrogenase (NAD-GDH; encoded by the GDH2 gene) when grown with a nonfermentable carbon source or with limiting amounts of glucose, even in the presence of the repressing nitrogen source glutamine. This regulation was found to be transcriptional, and an upstream activation site (GDH2 UASc) sufficient for activation of transcription during respiratory growth conditions was identified. This UAS was found to be separable from a neighboring element which is necessary for the nitrogen source regulation of the gene, and strains deficient for the GLN3 gene product, required for expression of NAD-GDH during growth with the activating nitrogen source glutamate, were unaffected for the expression of NAD-GDH during growth with activating carbon sources. Two classes of mutations which prevented the normal activation of NAD-GDH in response to growth with nonfermentable carbon sources, but which did not affect the nitrogen-regulated expression of NAD-GDH, were found and characterized. Carbon regulation of GDH2 was found to be normal in hxk2, hap3, and hap4 strains and to be only slightly altered in a ssn6 strain; thus, in comparison with the regulation of previously identified glucose-repressed genes, a new pathway appears to be involved in the regulation of GDH2.

The URE2 gene product of Saccharomyces cerevisiae plays an important role in the cellular response to the nitrogen source and has homology to glutathione s-transferases

1991 ◽  
Vol 11 (2) ◽  
pp. 822-832 ◽  
Author(s):  
P W Coschigano ◽  
B Magasanik
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Molecular Weight ◽  
Glutamine Synthetase ◽  
Nitrogen Source ◽  
Gene Product ◽  
Cellular Response ◽  
Transcriptional Level ◽  
Structural Genes ◽  
Glutathione S Transferases

The URE2 gene of Saccharomyces cerevisiae has been cloned and sequenced. It encodes a predicted polypeptide of 354 amino acids with a molecular weight of 40,226. Deletion of the first 63 amino acids does not have any effect on the function of the protein. Studies with disruption alleles of the URE2 and GLN3 genes showed that both genes regulate GLN1 and GDH2, the structural genes for glutamine synthetase and NAD-linked glutamate dehydrogenase, respectively, at the transcriptional level, but expression of the regulatory genes does not appear to be regulated. Active URE2 gene product was required for the inactivation of glutamine synthetase upon addition of glutamine to cells growing with glutamate as the source of nitrogen. The predicted URE2 gene product has homology to glutathione S-transferases. The gene has been mapped to chromosome XIV, 5.9 map units from petX and 3.4 map units from kex2.

Conservative replication of double-stranded RNA in Saccharomyces cerevisiae by displacement of progeny single strands

1984 ◽  
Vol 4 (8) ◽  
pp. 1618-1626
Author(s):  
R A Sclafani ◽  
W L Fangman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
G1 Phase ◽  
Double Stranded Rna ◽  
Virus Like Particles ◽  
Protein Encoding ◽  
In Cells

Saccharomyces cerevisiae contains two double-stranded RNA (dsRNA) molecules, L and M, encapsulated in virus-like particles. After cells are transferred from dense (13C 15N) to light (12C 14N) medium, only two density classes of dsRNA are found, fully light (LL) and fully dense (HH). Cells contain single-stranded copies of both dsRNAs and, at least for L dsRNA, greater than 99% of these single strands are the positive protein-encoding strand. Single-stranded copies of L and M dsRNA accumulate rapidly in cells arrested in the G1 phase. These results parallel previous observations on L dsRNA synthesis and are consistent with a role of the positive single strands as intermediates in dsRNA replication. We propose that new positive strands are displaced from parental molecules and subsequently copied to produce the completely new duplexes.

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