scholarly journals Regulation of expression and activity of the yeast transcription factor ADR1.

1988 ◽  
Vol 8 (5) ◽  
pp. 1868-1876 ◽  
Author(s):  
H Blumberg ◽  
T A Hartshorne ◽  
E T Young
Keyword(s):  
Carbon Source ◽  
Fusion Protein ◽  
Glucose Repression ◽  
Regulatory Gene ◽  
Lacz Gene ◽  
Mrna Levels ◽  
Multicopy Plasmid ◽  
Regulation Of Expression ◽  
Yeast Saccharomyces Cerevisiae ◽  
Beta Galactosidase

Disruption of ADR1, a positive regulatory gene in the yeast Saccharomyces cerevisiae, abolished derepression of ADH2 but did not affect glucose repression of ADH2 or cell viability. The ADR1 mRNA was 5 kilobases long and had an unusually long leader containing 509 nucleotides. ADR1 mRNA levels were regulated by the carbon source in a strain-dependent fashion. beta-Galactosidase levels measured in strains carrying an ADR1-lacZ gene fusion paralleled ADR1 and ADR1-lacZ mRNA levels, indicating a lack of translational regulation of ADR1 mRNA. ADH2 was regulated by the carbon source to the same extent in all strains examined and showed complete dependence on ADR1 as well. The expression of ADR1 mRNA and an ADR1-beta-galactosidase fusion protein during glucose repression suggested that the activity of the ADR1 protein is regulated at the posttranslational level to properly regulate ADH2 expression. The ADR1-beta-galactosidase fusion protein was able to activate ADH2 expression during glucose repression but showed significantly higher levels of activation upon derepression. A similar result was obtained when ADR1 was present on a multicopy plasmid. These results suggest that low-level expression of ADR1 is required to maintain glucose repression of ADH2 and are consistent with the hypothesis that ADR1 is regulated at the posttranslational level.

Regulation of expression and activity of the yeast transcription factor ADR1

1988 ◽  
Vol 8 (5) ◽  
pp. 1868-1876
Author(s):  
H Blumberg ◽  
T A Hartshorne ◽  
E T Young
Keyword(s):  
Carbon Source ◽  
Fusion Protein ◽  
Glucose Repression ◽  
Regulatory Gene ◽  
Lacz Gene ◽  
Mrna Levels ◽  
Multicopy Plasmid ◽  
Regulation Of Expression ◽  
Yeast Saccharomyces Cerevisiae ◽  
Beta Galactosidase

Disruption of ADR1, a positive regulatory gene in the yeast Saccharomyces cerevisiae, abolished derepression of ADH2 but did not affect glucose repression of ADH2 or cell viability. The ADR1 mRNA was 5 kilobases long and had an unusually long leader containing 509 nucleotides. ADR1 mRNA levels were regulated by the carbon source in a strain-dependent fashion. beta-Galactosidase levels measured in strains carrying an ADR1-lacZ gene fusion paralleled ADR1 and ADR1-lacZ mRNA levels, indicating a lack of translational regulation of ADR1 mRNA. ADH2 was regulated by the carbon source to the same extent in all strains examined and showed complete dependence on ADR1 as well. The expression of ADR1 mRNA and an ADR1-beta-galactosidase fusion protein during glucose repression suggested that the activity of the ADR1 protein is regulated at the posttranslational level to properly regulate ADH2 expression. The ADR1-beta-galactosidase fusion protein was able to activate ADH2 expression during glucose repression but showed significantly higher levels of activation upon derepression. A similar result was obtained when ADR1 was present on a multicopy plasmid. These results suggest that low-level expression of ADR1 is required to maintain glucose repression of ADH2 and are consistent with the hypothesis that ADR1 is regulated at the posttranslational level.

Heat shock-regulated production of Escherichia coli beta-galactosidase in Saccharomyces cerevisiae

1983 ◽  
Vol 3 (9) ◽  
pp. 1625-1633
Author(s):  
D B Finkelstein ◽  
S Strausberg
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Plasmid Vector ◽  
Lacz Gene ◽  
Multicopy Plasmid ◽  
Yeast Saccharomyces Cerevisiae ◽  
Reading Frame ◽  
Inducible Synthesis ◽  
Beta Galactosidase

The HSP90 gene of the yeast Saccharomyces cerevisiae encodes a heat shock-inducible protein with an Mr of 90,000 (hsp90) and unknown function. We fused DNA fragments of a known sequence (namely, either end of a 1.4-kilobase EcoRI fragment which contains the S. cerevisiae TRP1 gene) to an EcoRI site within the coding sequence of the HSP90 gene. When these fusions are introduced into S. cerevisiae they direct the synthesis of unique truncated hsp90 proteins. By determining the size and charge of these proteins we were able to deduce the translational reading frame at the (EcoRI) fusion site. This information allowed us to design and construct a well-defined in-frame fusion between the S. cerevisiae HSP90 gene and the Escherichia coli lacZ gene. When this fused gene is introduced into S. cerevisiae on a multicopy plasmid vector, it directs the heat shock-inducible synthesis of a fused protein, which is an enzymatically active beta-galactosidase. Thus, for the first time, it is possible to quantitate the heat shock response in a eucaryotic organism with a simple enzyme assay.

scholarly journals Heat shock-regulated production of Escherichia coli beta-galactosidase in Saccharomyces cerevisiae.

1983 ◽  
Vol 3 (9) ◽  
pp. 1625-1633 ◽  
Author(s):  
D B Finkelstein ◽  
S Strausberg
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Plasmid Vector ◽  
Lacz Gene ◽  
Multicopy Plasmid ◽  
Yeast Saccharomyces Cerevisiae ◽  
Reading Frame ◽  
Inducible Synthesis ◽  
Beta Galactosidase

The HSP90 gene of the yeast Saccharomyces cerevisiae encodes a heat shock-inducible protein with an Mr of 90,000 (hsp90) and unknown function. We fused DNA fragments of a known sequence (namely, either end of a 1.4-kilobase EcoRI fragment which contains the S. cerevisiae TRP1 gene) to an EcoRI site within the coding sequence of the HSP90 gene. When these fusions are introduced into S. cerevisiae they direct the synthesis of unique truncated hsp90 proteins. By determining the size and charge of these proteins we were able to deduce the translational reading frame at the (EcoRI) fusion site. This information allowed us to design and construct a well-defined in-frame fusion between the S. cerevisiae HSP90 gene and the Escherichia coli lacZ gene. When this fused gene is introduced into S. cerevisiae on a multicopy plasmid vector, it directs the heat shock-inducible synthesis of a fused protein, which is an enzymatically active beta-galactosidase. Thus, for the first time, it is possible to quantitate the heat shock response in a eucaryotic organism with a simple enzyme assay.

scholarly journals Yeast NOP2 encodes an essential nucleolar protein with homology to a human proliferation marker.

1994 ◽  
Vol 127 (6) ◽  
pp. 1799-1813 ◽  
Author(s):  
E de Beus ◽  
J S Brockenbrough ◽  
B Hong ◽  
J P Aris
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Ribosomal Protein ◽  
Nucleolar Protein ◽  
Mrna Levels ◽  
Multicopy Plasmid ◽  
Yeast Saccharomyces Cerevisiae ◽  
Protein Levels ◽  
Significant Amino Acid Sequence ◽  
Ribosomal Protein L3

We have isolated a gene (NOP2) encoding a nucleolar protein during a search for previously unidentified nuclear proteins in the yeast Saccharomyces cerevisiae. The protein encoded by NOP2 (Nop2p) has a predicted molecular mass of 70 kD, migrates at 90 kD by SDS-PAGE, and is essential for cell viability. Nop2p shows significant amino acid sequence homology to a human proliferation-associated nucleolar protein, p120. Approximately half of Nop2p exhibits 67% amino acid sequence identity to p120. Analysis of subcellular fractions indicates that Nop2p is located primarily in the nucleus, and nuclear fractionation studies suggest that Nop2p is associated with the nucleolus. Indirect immunofluorescence localization of Nop2p shows a nucleolar-staining pattern, which is heterogeneous in appearance, and a faint staining of the cytoplasm. The expression of NOP2 during the transition from stationary phase growth arrest to rapid growth was measured, and compared to the expression of TCM1, which encodes the ribosomal protein L3. Nop2p protein levels are markedly upregulated during the onset of growth, compared to the levels of ribosomal protein L3, which remain relatively constant. NOP2 mRNA levels also increase during the onset of growth, accompanied by a similar increase in the levels of TCM1 mRNA. The consequences of overexpressing NOP2 from the GAL10 promoter on a multicopy plasmid were investigated. Although NOP2 overexpression produced no discernible growth phenotype and had no effect on ribosome subunit synthesis, overexpression was found to influence the morphology of the nucleolus, as judged by electron microscopy. Overexpression caused the nucleolus to become detached from the nuclear envelope and to become more rounded and/or fragmented in appearance. These findings suggest roles for NOP2 in nucleolar function during the onset of growth, and in the maintenance of nucleolar structure.

Anti-Cdc25 antibodies inhibit guanyl nucleotide-dependent adenylyl cyclase of Saccharomyces cerevisiae and cross-react with a 150-kilodalton mammalian protein

1992 ◽  
Vol 12 (6) ◽  
pp. 2653-2661
Author(s):  
E Gross ◽  
I Marbach ◽  
D Engelberg ◽  
M Segal ◽  
G Simchen ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fusion Protein ◽  
Adenylyl Cyclase ◽  
Gene Product ◽  
Cyclase Activity ◽  
Yeast Saccharomyces Cerevisiae ◽  
Terminal Domain ◽  
Yeast Homolog ◽  
Cdc25 Protein ◽  
Beta Galactosidase

The CDC25 gene product of the yeast Saccharomyces cerevisiae has been shown to be a positive regulator of the Ras protein. The high degree of homology between yeast RAS and the mammalian proto-oncogene ras suggests a possible resemblance between the mammalian regulator of Ras and the regulator of the yeast Ras (Cdc25). On the basis of this assumption, we have raised antibodies against the conserved C-terminal domain of the Cdc25 protein in order to identify its mammalian homologs. Anti-Cdc25 antibodies raised against a beta-galactosidase-Cdc25 fusion protein were purified by immunoaffinity chromatography and were shown by immunoblotting to specifically recognize the Cdc25 portion of the antigen and a truncated Cdc25 protein, also expressed in bacteria. These antibodies were shown both by immunoblotting and by immunoprecipitation to recognize the CDC25 gene product in wild-type strains and in strains overexpressing Cdc25. The anti-Cdc25 antibodies potently inhibited the guanyl nucleotide-dependent and, approximately 3-fold less potently, the Mn(2+)-dependent adenylyl cyclase activity in S. cerevisiae. The anti-Cdc25 antibodies do not inhibit cyclase activity in a strain harboring RAS2Val-19 and lacking the CDC25 gene product. These results support the view that Cdc25, Ras2, and Cdc35/Cyr1 proteins are associated in a complex. Using these antibodies, we were able to define the conditions to completely solubilize the Cdc25 protein. The results suggest that the Cdc25 protein is tightly associated with the membrane but is not an intrinsic membrane protein, since only EDTA at pH 12 can solubilize the protein. The anti-Cdc25 antibodies strongly cross-reacted with the C-terminal domain of the Cdc25 yeast homolog, Sdc25. Most interestingly, these antibodies also cross-reacted with mammalian proteins of approximately 150 kDa from various tissues of several species of animals. These interactions were specifically blocked by the beta-galactosidase-Cdc25 fusion protein.

Transcription of the ADH2 gene in Saccharomyces cerevisiae is limited by positive factors that bind competitively to its intact promoter region on multicopy plasmids

1987 ◽  
Vol 7 (3) ◽  
pp. 1233-1241
Author(s):  
M Irani ◽  
W E Taylor ◽  
E T Young
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Promoter Region ◽  
Glucose Repression ◽  
Regulatory Gene ◽  
Tata Box ◽  
Upstream Region ◽  
Competition Effect ◽  
Yeast Saccharomyces Cerevisiae ◽  
Upstream Activation Sequence ◽  
Activation Sequence

Transcription of the ADH2 gene in the yeast Saccharomyces cerevisiae was inhibited by excess copies of its own promoter region. This competition effect was promoter specific and required the upstream activation sequence of ADH2 as well as sequences 3' to the TATA box. Introducing excess copies of ADR1, an ADH2-specific regulatory gene, did not alleviate the competition that was observed in these circumstances during both constitutive and derepressed ADH2 expression. Excess copies of the upstream region did not release ADH2 from glucose repression, consistent with the view that ADH2 is regulated by positive trans-acting factors.

scholarly journals The HXT1 gene product of Saccharomyces cerevisiae is a new member of the family of hexose transporters.

1991 ◽  
Vol 11 (7) ◽  
pp. 3804-3813 ◽  
Author(s):  
D A Lewis ◽  
L F Bisson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Glucose Transport ◽  
Significant Proportion ◽  
Growth Defect ◽  
Lacz Gene ◽  
Multicopy Plasmid ◽  
Hexose Transport ◽  
Yeast Saccharomyces Cerevisiae ◽  
High Affinity ◽  
Membrane Spanning

Two novel genes affecting hexose transport in the yeast Saccharomyces cerevisiae have been identified. The gene HXT1 (hexose transport), isolated from plasmid pSC7, was sequenced and found to encode a hydrophobic protein which is highly homologous to the large family of sugar transporter proteins from eucaryotes and procaryotes. Multicopy expression of the HXT1 gene restored high-affinity glucose transport to the snf3 mutant, which is deficient in a significant proportion of high-affinity glucose transport. HXT1 was unable to complement the snf3 growth defect in low copy number. The HXT1 protein was found to contain 12 putative membrane-spanning domains with a central hydrophilic domain and hydrophilic N- and C-terminal domains. The HXT1 protein is 69% identical to GAL2 and 66% identical to HXT2, and all three proteins were found to have a putative leucine zipper motif at a consensus location in membrane-spanning domain 2. Disruption of the HXT1 gene resulted in loss of a portion of high-affinity glucose and mannose transport, and wild-type levels of transport required both the HXT1 and SNF3 genes. Unexpectedly, expression of beta-galactosidase activity by using a fusion of the lacZ gene to the HXT1 promoter in a multicopy plasmid was maximal during lag and early exponential phases of growth, decreasing approximately 100-fold upon further entry into exponential growth. Deletion analysis of pSC7 revealed the presence of another gene (called ORF2) capable of suppressing the snf3 null mutant phenotype by restoring high-affinity glucose transport and increased low-affinity transport.

scholarly journals A common bacterial metabolite elicits prion-based bypass of glucose repression

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
David M Garcia ◽  
David Dietrich ◽  
Jon Clardy ◽  
Daniel F Jarosz
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Lactic Acid ◽  
Glucose Metabolism ◽  
Carbon Source ◽  
Glucose Repression ◽  
Carbon Sources ◽  
Yeast Cells ◽  
Genetic Element ◽  
Yeast Saccharomyces Cerevisiae ◽  
The Common

Robust preference for fermentative glucose metabolism has motivated domestication of the budding yeast Saccharomyces cerevisiae. This program can be circumvented by a protein-based genetic element, the [GAR+] prion, permitting simultaneous metabolism of glucose and other carbon sources. Diverse bacteria can elicit yeast cells to acquire [GAR+], although the molecular details of this interaction remain unknown. Here we identify the common bacterial metabolite lactic acid as a strong [GAR+] inducer. Transient exposure to lactic acid caused yeast cells to heritably circumvent glucose repression. This trait had the defining genetic properties of [GAR+], and did not require utilization of lactic acid as a carbon source. Lactic acid also induced [GAR+]-like epigenetic states in fungi that diverged from S. cerevisiae ~200 million years ago, and in which glucose repression evolved independently. To our knowledge, this is the first study to uncover a bacterial metabolite with the capacity to potently induce a prion.

scholarly journals Structure and regulation of KGD1, the structural gene for yeast alpha-ketoglutarate dehydrogenase.

1989 ◽  
Vol 9 (6) ◽  
pp. 2695-2705 ◽  
Author(s):  
B Repetto ◽  
A Tzagoloff
Keyword(s):  
Nuclear Dna ◽  
Lacz Gene ◽  
Multicopy Plasmid ◽  
Wild Type ◽  
Core Element ◽  
Plasmid Library ◽  
Alpha Ketoglutarate Dehydrogenase ◽  
Wild Type Yeast ◽  
Beta Galactosidase ◽  
Ketoglutarate Dehydrogenase

Nuclear respiratory-defective mutants of Saccharomyces cerevisiae have been screened for lesions in the mitochondrial alpha-ketoglutarate dehydrogenase complex. Strains assigned to complementation group G70 were ascertained to be deficient in enzyme activity due to mutations in the KGD1 gene coding for the alpha-ketoglutarate dehydrogenase component of the complex. The KGD1 gene has been cloned by transformation of a representative kgd1 mutant, C225/U1, with a recombinant plasmid library of wild-type yeast nuclear DNA. Transformants containing the gene on a multicopy plasmid had three- to four-times-higher alpha-ketoglutarate dehydrogenase activity than did wild-type S. cerevisiae. Substitution of the chromosomal copy of KGD1 with a disrupted allele (kgd1::URA3) induced a deficiency in alpha-ketoglutarate dehydrogenase. The sequence of the cloned region of DNA which complements kgd1 mutants was found to have an open reading frame of 3,042 nucleotides capable of coding for a protein of Mw 114,470. The encoded protein had 38% identical residues with the reported sequence of alpha-ketoglutarate dehydrogenase from Escherichia coli. Two lines of evidence indicated that transcription of KGD1 is catabolite repressed. Higher steady-state levels of KGD1 mRNA were detected in wild-type yeast grown on the nonrepressible sugar galactose than in yeast grown on high glucose. Regulation of KGD1 was also studied by fusing different 5'-flanking regions of KGD1 to the lacZ gene of E. coli and measuring the expression of beta-galactosidase in yeast. Transformants harboring a fusion of 693 nucleotides of the 5'-flanking sequence expressed 10 times more beta-galactosidase activity when grown under derepressed conditions. The response to the carbon source was reduced dramatically when the same lacZ fusion was present in a hap2 or hap3 mutant. The promoter element(s) responsible for the regulated expression of KGD1 has been mapped to the -354 to -143 region. This region contained several putative activation sites with sequences matching the core element proposed to be essential for binding of the HAP2 and HAP3 regulatory proteins.

scholarly journals The signal for glucose repression of the lactose-galactose regulon is amplified through subtle modulation of transcription of the Kluyveromyces lactis Kl-GAL4 activator gene.

1992 ◽  
Vol 12 (5) ◽  
pp. 1924-1931 ◽  
Author(s):  
N Kuzhandaivelu ◽  
W K Jones ◽  
A K Martin ◽  
R C Dickson
Keyword(s):  
Glucose Repression ◽  
Rotational Symmetry ◽  
Kluyveromyces Lactis ◽  
Regulatory Gene ◽  
Regulatory Protein ◽  
Genetic Regulation ◽  
Structural Genes ◽  
Important Principle ◽  
Biological Phenomena ◽  
Beta Galactosidase

Induction of the lactose-galactose regulon is strongly repressed by glucose in some but not all strains of Kluyveromyces lactis. We show here that in strongly repressed strains, two to three times less Kl-GAL4 mRNA is synthesized and that expression of structural genes in the regulon such as LAC4, the structural gene for beta-galactosidase, is down regulated 40-fold or more. Comparative analysis of strains having a strong or weak repression phenotype revealed a two-base difference in the promoter of the Kl-GAL4 (also called LAC9) positive regulatory gene. This two-base difference is responsible for the strong versus the weak repression phenotype. The two base changes are symmetrically located in a DNA sequence having partial twofold rotational symmetry (14 of 21 bases). We hypothesize that this region functions as a sensitive regulatory switch, an upstream repressor sequence (URS). According to our model, the presence of glucose in the culture medium signals, by an unidentified pathway, a repressor protein to bind the URS. Binding reduces transcription of the Kl-GAL4 gene so that the concentration of the Kl-GAL4 protein falls below the level needed for induction of LAC4 and other genes in the regulon. For strains showing weak glucose repression, we hypothesize that the two base changes in the URS reduce repressor binding so that the regulon is not repressed. Our results illustrate an important principle of genetic regulation: a small (2- to 3-fold) change in the concentration of a regulatory protein can produce a large (40-fold or greater) change in expression of structural genes. This mechanism of signal amplification could play a role in many biological phenomena that require regulated transcription.

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