scholarly journals Structure of the transcriptionally repressed phosphate-repressible acid phosphatase gene (PHO5) of Saccharomyces cerevisiae.

1986 ◽  
Vol 6 (1) ◽  
pp. 38-46 ◽  
Author(s):  
L W Bergman ◽  
M C Stranathan ◽  
L H Preis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Acid Phosphatase ◽  
Copy Number ◽  
Regulatory Control ◽  
Gene Copy ◽  
Growth Media ◽  
Regulatory Sequences ◽  
Mrna Levels ◽  
Acid Phosphatase Gene ◽  
Negative Supercoils

We developed a high-copy-number plasmid system containing the entire structural and regulatory sequences of the phosphate-repressible acid phosphatase (PHO5) gene and the TRP1/ARS1 replicator sequences of the yeast Saccharomyces cerevisiae to investigate the mechanism of repression-derepression of transcription. The resulting plasmid was used to transform either wild-type cells or a number of strains which contain mutations in various trans-acting regulatory loci for the production of acid phosphatase. Results of analysis of mRNA levels isolated from the transformed strains grown under repressed or derepressed conditions suggested that normal transcriptional regulation of the gene persisted, although gene copy number was significantly increased. Analysis of changes in linking number (i.e., the number of negative supercoils) of the plasmid isolated under repressed and derepressed growth conditions revealed that the transcriptionally inactive plasmid contained approximately three more negative supercoils than the transcriptionally active plasmid. This difference in topological state was similarly seen in a plasmid containing a sequence-related acid phosphatase gene (PHO11) under the same regulatory control system, but it was not seen in plasmids isolated from some strains containing mutations which caused either fully constitutive or nonderepressible production of acid phosphatase. Finally, analysis of the nucleosome positioning along the inactive gene sequence revealed that an abnormally broad internucleosomal spacer is present in a region presumed to function in the regulation of transcription by the level of Pi in the growth media.

Structure of the transcriptionally repressed phosphate-repressible acid phosphatase gene (PHO5) of Saccharomyces cerevisiae

1986 ◽  
Vol 6 (1) ◽  
pp. 38-46
Author(s):  
L W Bergman ◽  
M C Stranathan ◽  
L H Preis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Acid Phosphatase ◽  
Copy Number ◽  
Regulatory Control ◽  
Gene Copy ◽  
Growth Media ◽  
Regulatory Sequences ◽  
Mrna Levels ◽  
Acid Phosphatase Gene ◽  
Negative Supercoils

We developed a high-copy-number plasmid system containing the entire structural and regulatory sequences of the phosphate-repressible acid phosphatase (PHO5) gene and the TRP1/ARS1 replicator sequences of the yeast Saccharomyces cerevisiae to investigate the mechanism of repression-derepression of transcription. The resulting plasmid was used to transform either wild-type cells or a number of strains which contain mutations in various trans-acting regulatory loci for the production of acid phosphatase. Results of analysis of mRNA levels isolated from the transformed strains grown under repressed or derepressed conditions suggested that normal transcriptional regulation of the gene persisted, although gene copy number was significantly increased. Analysis of changes in linking number (i.e., the number of negative supercoils) of the plasmid isolated under repressed and derepressed growth conditions revealed that the transcriptionally inactive plasmid contained approximately three more negative supercoils than the transcriptionally active plasmid. This difference in topological state was similarly seen in a plasmid containing a sequence-related acid phosphatase gene (PHO11) under the same regulatory control system, but it was not seen in plasmids isolated from some strains containing mutations which caused either fully constitutive or nonderepressible production of acid phosphatase. Finally, analysis of the nucleosome positioning along the inactive gene sequence revealed that an abnormally broad internucleosomal spacer is present in a region presumed to function in the regulation of transcription by the level of Pi in the growth media.

Physiological control of repressible acid phosphatase gene transcripts in Saccharomyces cerevisiae

1983 ◽  
Vol 3 (5) ◽  
pp. 839-853
Author(s):  
K A Bostian ◽  
J M Lemire ◽  
H O Halvorson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Acid Phosphatase ◽  
Accumulation Rate ◽  
Mrna Levels ◽  
Phosphorus Metabolism ◽  
Physiological Control ◽  
Phosphatase Regulation ◽  
Gene Transcripts ◽  
Acid Phosphatase Gene ◽  
Polyphosphate Degradation

We have examined the regulation of repressible acid phosphatase (APase; orthophosphoric-monoester phosphohydrolase [acid optimum], EC 3.1.3.2) in Saccharomyces cerevisiae at the physiological and molecular levels, through a series of repression and derepression experiments. We demonstrated that APase synthesis is tightly regulated throughout the growth phase and is influenced by exogenous and endogenous Pi pools. During growth in a nonlimiting Pi medium, APase is repressed. When external Pi becomes limiting, there is a biphasic appearance of APase mRNA and enzyme. Our data on APase mRNA half-lives and on the flux of intracellular Pi and polyphosphate during derepression are consistent with a mechanism of transcriptional autoregulation for the biphasic appearance of APase mRNA. Accordingly, preculture concentrations of Pi control the level of corepressor generated from intracellular polyphosphate degradation. When cells are fully derepressed, APase mRNA levels are constant, and the maximal linear accumulation rate of APase is observed. A scheme to integrate phosphorus metabolism and phosphatase regulation in S. cerevisiae is proposed.

scholarly journals Physiological control of repressible acid phosphatase gene transcripts in Saccharomyces cerevisiae.

1983 ◽  
Vol 3 (5) ◽  
pp. 839-853 ◽  
Author(s):  
K A Bostian ◽  
J M Lemire ◽  
H O Halvorson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Acid Phosphatase ◽  
Accumulation Rate ◽  
Mrna Levels ◽  
Phosphorus Metabolism ◽  
Physiological Control ◽  
Phosphatase Regulation ◽  
Gene Transcripts ◽  
Acid Phosphatase Gene ◽  
Polyphosphate Degradation

We have examined the regulation of repressible acid phosphatase (APase; orthophosphoric-monoester phosphohydrolase [acid optimum], EC 3.1.3.2) in Saccharomyces cerevisiae at the physiological and molecular levels, through a series of repression and derepression experiments. We demonstrated that APase synthesis is tightly regulated throughout the growth phase and is influenced by exogenous and endogenous Pi pools. During growth in a nonlimiting Pi medium, APase is repressed. When external Pi becomes limiting, there is a biphasic appearance of APase mRNA and enzyme. Our data on APase mRNA half-lives and on the flux of intracellular Pi and polyphosphate during derepression are consistent with a mechanism of transcriptional autoregulation for the biphasic appearance of APase mRNA. Accordingly, preculture concentrations of Pi control the level of corepressor generated from intracellular polyphosphate degradation. When cells are fully derepressed, APase mRNA levels are constant, and the maximal linear accumulation rate of APase is observed. A scheme to integrate phosphorus metabolism and phosphatase regulation in S. cerevisiae is proposed.

scholarly journals Signal peptide specificity in posttranslational processing of the plant protein phaseolin in Saccharomyces cerevisiae.

1987 ◽  
Vol 7 (1) ◽  
pp. 121-128 ◽  
Author(s):  
J H Cramer ◽  
K Lea ◽  
M D Schaber ◽  
R A Kramer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Acid Phosphatase ◽  
Signal Peptide ◽  
Regulatory Region ◽  
Cellular Protein ◽  
Proteolytic Processing ◽  
Coding Region ◽  
Molecular Weights ◽  
Amino Terminal ◽  
Acid Phosphatase Gene

We linked the cDNA coding region for the bean storage protein phaseolin to the promoter and regulatory region of the Saccharomyces cerevisiae repressible acid phosphatase gene (PHO5) in multicopy expression plasmids. Yeast transformants containing these plasmids expressed phaseolin at levels up to 3% of the total soluble cellular protein. Phaseolin polypeptides in S. cerevisiae were glycosylated, and their molecular weights suggested that the signal peptide had been processed. We also constructed a series of plasmids in which the phaseolin signal-peptide-coding region was either removed or replaced with increasing amounts of the amino-terminal coding region for acid phosphatase. Phaseolin polypeptides with no signal peptide were not posttranslationally modified in S. cerevisiae. Partial or complete substitution of the phaseolin signal peptide with that from acid phosphatase dramatically inhibited both signal peptide processing and glycosylation, suggesting that some specific feature of the phaseolin signal amino acid sequence was required for these modifications to occur. Larger hybrid proteins that included approximately one-half of the acid phosphatase sequence linked to the amino terminus of the mature phaseolin polypeptide did undergo proteolytic processing and glycosylation. However, these polypeptides were cleaved at several sites that are not normally used in the unaltered acid phosphatase protein.

scholarly journals Isolation and analysis of rereplicated DNA by Rerep-Seq

2020 ◽  
Vol 48 (10) ◽  
pp. e58-e58 ◽  
Author(s):  
Johannes Menzel ◽  
Philip Tatman ◽  
Joshua C Black
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genomic Instability ◽  
Copy Number ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Adaptive Potential ◽  
Developmental Abnormalities ◽  
Origin Licensing ◽  
Multiple Replication ◽  
Dna Rereplication

Abstract Changes in gene copy number contribute to genomic instability, the onset and progression of cancer, developmental abnormalities and adaptive potential. The origins of gene amplifications have remained elusive; however, DNA rereplication has been implicated as a source of gene amplifications. The inability to determine which sequences are rereplicated and under what conditions have made it difficult to determine the validity of the proposed models. Here we present Rerep-Seq, a technique that selectively enriches for rereplicated DNA in preparation for analysis by DNA sequencing that can be applied to any species. We validated Rerep-Seq by simulating DNA rereplication in yeast and human cells. Using Rerep-Seq, we demonstrate that rereplication induced in Saccharomyces cerevisiae by deregulated origin licensing is non-random and defined by broad domains that span multiple replication origins and topological boundaries.

The THI5 gene family of Saccharomyces cerevisiae: distribution of homologues among the hemiascomycetes and functional redundancy in the aerobic biosynthesis of thiamin from pyridoxine

Microbiology ◽  
2003 ◽  
Vol 149 (6) ◽  
pp. 1447-1460 ◽  
Author(s):  
Raymond Wightman ◽  
Peter A. Meacock
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Family ◽  
Biosynthetic Pathway ◽  
Growth Conditions ◽  
Anaerobic Growth ◽  
Gene Copy ◽  
Phylogenetic Study ◽  
Vitamin B ◽  
Mrna Levels ◽  
Quadruple Mutant

The THI5 gene family of Saccharomyces cerevisiae comprises four highly conserved members named THI5 (YFL058w), THI11 (YJR156c), THI12 (YNL332w) and THI13 (YDL244w). Each gene copy is located within the subtelomeric region of a different chromosome and all are homologues of the Schizosaccharomyces pombe nmt1 gene which is thought to function in the biosynthesis of hydroxymethylpyrimidine (HMP), a precursor of vitamin B1, thiamin. A comprehensive phylogenetic study has shown that the existence of THI5 as a gene family is exclusive to those yeasts of the Saccharomyces sensu stricto subgroup. To determine the function and redundancy of each of the S. cerevisiae homologues, all combinations of the single, double, triple and quadruple deletion mutants were constructed using a PCR-mediated gene-disruption strategy. Phenotypic analyses of these mutant strains have shown the four genes to be functionally redundant in terms of HMP formation for thiamin biosynthesis; each promotes synthesis of HMP from the pyridoxine (vitamin B6) biosynthetic pathway. Furthermore, growth studies with the quadruple mutant strain support a previous proposal of an alternative HMP biosynthetic pathway that operates in yeast under anaerobic growth conditions. Comparative analysis of mRNA levels has revealed subtle differences in the regulation of the four genes, suggesting that they respond differently to nutrient limitation.

Signal peptide specificity in posttranslational processing of the plant protein phaseolin in Saccharomyces cerevisiae

1987 ◽  
Vol 7 (1) ◽  
pp. 121-128
Author(s):  
J H Cramer ◽  
K Lea ◽  
M D Schaber ◽  
R A Kramer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Acid Phosphatase ◽  
Signal Peptide ◽  
Regulatory Region ◽  
Cellular Protein ◽  
Proteolytic Processing ◽  
Coding Region ◽  
Molecular Weights ◽  
Amino Terminal ◽  
Acid Phosphatase Gene

We linked the cDNA coding region for the bean storage protein phaseolin to the promoter and regulatory region of the Saccharomyces cerevisiae repressible acid phosphatase gene (PHO5) in multicopy expression plasmids. Yeast transformants containing these plasmids expressed phaseolin at levels up to 3% of the total soluble cellular protein. Phaseolin polypeptides in S. cerevisiae were glycosylated, and their molecular weights suggested that the signal peptide had been processed. We also constructed a series of plasmids in which the phaseolin signal-peptide-coding region was either removed or replaced with increasing amounts of the amino-terminal coding region for acid phosphatase. Phaseolin polypeptides with no signal peptide were not posttranslationally modified in S. cerevisiae. Partial or complete substitution of the phaseolin signal peptide with that from acid phosphatase dramatically inhibited both signal peptide processing and glycosylation, suggesting that some specific feature of the phaseolin signal amino acid sequence was required for these modifications to occur. Larger hybrid proteins that included approximately one-half of the acid phosphatase sequence linked to the amino terminus of the mature phaseolin polypeptide did undergo proteolytic processing and glycosylation. However, these polypeptides were cleaved at several sites that are not normally used in the unaltered acid phosphatase protein.

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