scholarly journals Localization and Interaction of the Proteins Constituting the GAL Genetic Switch in Saccharomyces cerevisiae

2008 ◽  
Vol 7 (12) ◽  
pp. 2061-2068 ◽  
Author(s):  
Raymond Wightman ◽  
Rachel Bell ◽  
Richard J. Reece
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Cellular Localization ◽  
Transcriptional Control ◽  
Resonance Energy ◽  
Regulatory Proteins ◽  
Yeast Cells ◽  
Galactose Metabolism ◽  
Genetic Switch ◽  
Metabolism Regulation

ABSTRACT In Saccharomyces cerevisiae, the GAL genes encode the enzymes required for galactose metabolism. Regulation of these genes has served as the paradigm for eukaryotic transcriptional control over the last 50 years. The switch between inert and active gene expression is dependent upon three proteins—the transcriptional activator Gal4p, the inhibitor Gal80p, and the ligand sensor Gal3p. Here, we present a detailed spatial analysis of the three GAL regulatory proteins produced from their native genomic loci. Using a novel application of photobleaching, we demonstrate, for the first time, that the Gal3p ligand sensor enters the nucleus of yeast cells in the presence of galactose. Additionally, using Förster resonance energy transfer, we show that the interaction between Gal3p and Gal80p occurs throughout the yeast cell. Taken together, these data challenge existing models for the cellular localization of the regulatory proteins during the induction of GAL gene expression by galactose and suggest a mechanism for the induction of the GAL genes in which galactose-bound Gal3p moves from the cytoplasm to the nucleus to interact with the transcriptional inhibitor Gal80p.

scholarly journals Screening and Genetic Network Analysis of Genes Involved in Freezing and Thawing Resistance in DaMDHAR—Expressing Saccharomyces cerevisiae Using Gene Expression Profiling

Genes ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 219
Author(s):  
Il-Sup Kim ◽  
Woong Choi ◽  
Jonghyeon Son ◽  
Jun Hyuck Lee ◽  
Hyoungseok Lee ◽  
...  
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Transcription Factors ◽  
Stress Tolerance ◽  
Genetic Network ◽  
Yeast Cells ◽  
Enzymatic Antioxidants ◽  
Freezing And Thawing ◽  
Metal Ion Homeostasis ◽  
Transgenic Yeast

The cryoprotection of cell activity is a key determinant in frozen-dough technology. Although several factors that contribute to freezing tolerance have been reported, the mechanism underlying the manner in which yeast cells respond to freezing and thawing (FT) stress is not well established. Therefore, the present study demonstrated the relationship between DaMDHAR encoding monodehydroascorbate reductase from Antarctic hairgrass Deschampsia antarctica and stress tolerance to repeated FT cycles (FT2) in transgenic yeast Saccharomyces cerevisiae. DaMDHAR-expressing yeast (DM) cells identified by immunoblotting analysis showed high tolerance to FT stress conditions, thereby causing lower damage for yeast cells than wild-type (WT) cells with empty vector alone. To detect FT2 tolerance-associated genes, 3′-quant RNA sequencing was employed using mRNA isolated from DM and WT cells exposed to FT (FT2) conditions. Approximately 332 genes showed ≥2-fold changes in DM cells and were classified into various groups according to their gene expression. The expressions of the changed genes were further confirmed using western blot analysis and biochemical assay. The upregulated expression of 197 genes was associated with pentose phosphate pathway, NADP metabolic process, metal ion homeostasis, sulfate assimilation, β-alanine metabolism, glycerol synthesis, and integral component of mitochondrial and plasma membrane (PM) in DM cells under FT2 stress, whereas the expression of the remaining 135 genes was partially related to protein processing, selenocompound metabolism, cell cycle arrest, oxidative phosphorylation, and α-glucoside transport under the same condition. With regard to transcription factors in DM cells, MSN4 and CIN5 were activated, but MSN2 and MGA1 were not. Regarding antioxidant systems and protein kinases in DM cells under FT stress, CTT1, GTO, GEX1, and YOL024W were upregulated, whereas AIF1, COX2, and TRX3 were not. Gene activation represented by transcription factors and enzymatic antioxidants appears to be associated with FT2-stress tolerance in transgenic yeast cells. RCK1, MET14, and SIP18, but not YPK2, have been known to be involved in the protein kinase-mediated signalling pathway and glycogen synthesis. Moreover, SPI18 and HSP12 encoding hydrophilin in the PM were detected. Therefore, it was concluded that the genetic network via the change of gene expression levels of multiple genes contributing to the stabilization and functionality of the mitochondria and PM, not of a single gene, might be the crucial determinant for FT tolerance in DaMDAHR-expressing transgenic yeast. These findings provide a foundation for elucidating the DaMDHAR-dependent molecular mechanism of the complex functional resistance in the cellular response to FT stress.

Codon replacement in the PGK1 gene of Saccharomyces cerevisiae: experimental approach to study the role of biased codon usage in gene expression

1987 ◽  
Vol 7 (8) ◽  
pp. 2914-2924
Author(s):  
A Hoekema ◽  
R A Kastelein ◽  
M Vasser ◽  
H A de Boer
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Steady State ◽  
Codon Usage ◽  
Experimental Approach ◽  
Mrna Translation ◽  
Yeast Cells ◽  
Expression Level ◽  
Start Codon ◽  
Mrna Levels

The coding sequences of genes in the yeast Saccharomyces cerevisiae show a preference for 25 of the 61 possible coding triplets. The degree of this biased codon usage in each gene is positively correlated to its expression level. Highly expressed genes use these 25 major codons almost exclusively. As an experimental approach to studying biased codon usage and its possible role in modulating gene expression, systematic codon replacements were carried out in the highly expressed PGK1 gene. The expression of phosphoglycerate kinase (PGK) was studied both on a high-copy-number plasmid and as a single copy gene integrated into the chromosome. Replacing an increasing number (up to 39% of all codons) of major codons with synonymous minor ones at the 5' end of the coding sequence caused a dramatic decline of the expression level. The PGK protein levels dropped 10-fold. The steady-state mRNA levels also declined, but to a lesser extent (threefold). Our data indicate that this reduction in mRNA levels was due to destabilization caused by impaired translation elongation at the minor codons. By preventing translation of the PGK mRNAs by the introduction of a stop codon 3' and adjacent to the start codon, the steady-state mRNA levels decreased dramatically. We conclude that efficient mRNA translation is required for maintaining mRNA stability in S. cerevisiae. These findings have important implications for the study of the expression of heterologous genes in yeast cells.

scholarly journals Yeast repressor alpha 2 binds to its operator cooperatively with yeast protein Mcm1.

1989 ◽  
Vol 9 (11) ◽  
pp. 5228-5230 ◽  
Author(s):  
C A Keleher ◽  
S Passmore ◽  
A D Johnson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Product ◽  
Strong Evidence ◽  
Regulatory Proteins ◽  
Yeast Cells ◽  
Specific Gene ◽  
Yeast Gene ◽  
Yeast Protein ◽  
Transcriptional Regulatory

To bring about repression of a family fo genes in Saccharomyces cerevisiae called the a-specific genes, two transcriptional regulatory proteins, alpha 2 and GRM (general regulator of matin type), bind cooperatively to an operator found upstream of each a-specific gene. To date, GRM has been defined only biochemically. In this communication we show that the product of a single yeast gene (MCM1) is sufficient to bind cooperatively with alpha 2 to the operator. We also show that antiserum raised against the MCM1 gene product recognizes GRM from yeast cells. These results, in combination with previous observations, provide strong evidence that MCM1 encodes the GRM activity.

scholarly journals Determination of the Global Pattern of Gene Expression in Yeast Cells by Intracellular Levels of Guanine Nucleotides

mBio ◽  
2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Andy Hesketh ◽  
Marta Vergnano ◽  
Stephen G. Oliver
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Carbon Source ◽  
Gene Transcription ◽  
Adenine Nucleotide ◽  
High Energy ◽  
Yeast Cells ◽  
Purine Nucleotides ◽  
Global Pattern ◽  
Content Type

ABSTRACT Correlations between gene transcription and the abundance of high-energy purine nucleotides in Saccharomyces cerevisiae have often been noted. However, there has been no systematic investigation of this phenomenon in the absence of confounding factors such as nutrient status and growth rate, and there is little hard evidence for a causal relationship. Whether transcription is fundamentally responsive to prevailing cellular energetic conditions via sensing of intracellular purine nucleotides, independently of specific nutrition, remains an important question. The controlled nutritional environment of chemostat culture revealed a strong correlation between ATP and GTP abundance and the transcription of genes required for growth. Short pathways for the inducible and futile consumption of ATP or GTP were engineered into S. cerevisiae, permitting analysis of the transcriptional effect of an increased demand for these nucleotides. During steady-state growth using the fermentable carbon source glucose, the futile consumption of ATP led to a decrease in intracellular ATP concentration but an increase in GTP and the guanylate energy charge (GEC). Expression of transcripts encoding proteins involved in ribosome biogenesis, and those controlled by promoters subject to SWI/SNF-dependent chromatin remodelling, was correlated with these nucleotide pool changes. Similar nucleotide abundance changes were observed using a nonfermentable carbon source, but an effect on the growth-associated transcriptional programme was absent. Induction of the GTP-cycling pathway had only marginal effects on nucleotide abundance and gene transcription. The transcriptional response of respiring cells to glucose was dampened in chemostats induced for ATP cycling, but not GTP cycling, and this was primarily associated with altered adenine nucleotide levels. IMPORTANCE This paper investigates whether, independently of the supply of any specific nutrient, gene transcription responds to the energy status of the cell by monitoring ATP and GTP levels. Short pathways for the inducible and futile consumption of ATP or GTP were engineered into the yeast Saccharomyces cerevisiae, and the effect of an increased demand for these purine nucleotides on gene transcription was analyzed. The resulting changes in transcription were most consistently associated with changes in GTP and GEC levels, although the reprogramming in gene expression during glucose repression is sensitive to adenine nucleotide levels. The results show that GTP levels play a central role in determining how genes act to respond to changes in energy supply and that any comprehensive understanding of the control of eukaryotic gene expression requires the elucidation of how changes in guanine nucleotide abundance are sensed and transduced to alter the global pattern of transcription.

scholarly journals Effects of Low-Shear Modeled Microgravity on Cell Function, Gene Expression, and Phenotype in Saccharomyces cerevisiae

2006 ◽  
Vol 72 (7) ◽  
pp. 4569-4575 ◽  
Author(s):  
B. Purevdorj-Gage ◽  
K. B. Sheehan ◽  
L. E. Hyman
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Cell Function ◽  
Yeast Cells ◽  
Lag Phase ◽  
Limited Information ◽  
Cellular Functions ◽  
Modeled Microgravity ◽  
Expression Of Genes ◽  
Low Shear

ABSTRACT Only limited information is available concerning the effects of low-shear modeled microgravity (LSMMG) on cell function and morphology. We examined the behavior of Saccharomyces cerevisiae grown in a high-aspect-ratio vessel, which simulates the low-shear and microgravity conditions encountered in spaceflight. With the exception of a shortened lag phase (90 min less than controls; P < 0.05), yeast cells grown under LSMMG conditions did not differ in growth rate, size, shape, or viability from the controls but did differ in the establishment of polarity as exhibited by aberrant (random) budding compared to the usual bipolar pattern of controls. The aberrant budding was accompanied by an increased tendency of cells to clump, as indicated by aggregates containing five or more cells. We also found significant changes (greater than or equal to twofold) in the expression of genes associated with the establishment of polarity (BUD5), bipolar budding (RAX1, RAX2, and BUD25), and cell separation (DSE1, DSE2, and EGT2). Thus, low-shear environments may significantly alter yeast gene expression and phenotype as well as evolutionary conserved cellular functions such as polarization. The results provide a paradigm for understanding polarity-dependent cell responses to microgravity ranging from pathogenesis in fungi to the immune response in mammals.

scholarly journals Interplay of a Ligand Sensor and an Enzyme in Controlling Expression of the Saccharomyces cerevisiae GAL Genes

2011 ◽  
Vol 11 (3) ◽  
pp. 334-342 ◽  
Author(s):  
Dariusz Abramczyk ◽  
Stacey Holden ◽  
Christopher J. Page ◽  
Richard J. Reece
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Transcriptional Control ◽  
Transcriptional Response ◽  
Content Type ◽  
Considerable Debate ◽  
Active Transcription ◽  
Gal Genes ◽  
The Subject

ABSTRACT The regulation of the Saccharomyces cerevisiae GAL genes in response to galactose as a source of carbon has served as a paradigm for eukaryotic transcriptional control over the last 50 years. Three proteins—a transcriptional activator (Gal4p), an inhibitor (Gal80p), and a ligand sensor (Gal3p)—control the switch between inert and active gene expression. The molecular mechanism by which the recognition of galactose within the cell is converted into a transcriptional response has been the subject of considerable debate. In this study, using a novel and powerful method of localizing active transcription factors within the nuclei of cells, we show that a short-lived complex between Gal4p, Gal80p, and Gal3p occurs soon after the addition of galactose to cells to activate GAL gene expression. Gal3p is subsequently replaced in this complex by Gal1p, and a Gal4p-Gal80p-Gal1p complex is responsible for the continued expression of the GAL genes. The transient role of the ligand sensor indicates that current models for the induction and continued expression of the yeast GAL genes need to be reevaluated.

Transcriptional derepression of the Saccharomyces cerevisiae HSP26 gene during heat shock

1990 ◽  
Vol 10 (12) ◽  
pp. 6362-6373
Author(s):  
R E Susek ◽  
S Lindquist
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Transcriptional Control ◽  
Critical Role ◽  
Constitutive Expression ◽  
Small Heat Shock Protein ◽  
Yeast Cells ◽  
Heat Shock Gene ◽  
General Mechanism ◽  
Regulatory Sequences

hsp26, the small heat shock protein of Saccharomyces cerevisiae, accumulates in response to heat and other types of stress. It also accumulates during the normal course of development, as cells enter stationary phase growth or begin to sporulate (S. Kurtz, J. Rossi, L. Petko, and S. Lindquist, Science 231:1154-1157, 1986). Analysis of deletion and insertion mutations demonstrated that transcriptional control plays a critical role in regulating HSP26 expression. The HSP26 promoter was found to be complex and appears to contain repressing elements as well as activating elements. Several upstream deletion mutations resulted in strong constitutive expression of HSP26. Furthermore, upstream sequences from the HSP26 gene repressed the constitutive expression of a heterologous heat shock gene. We propose that basal repression and heat-induced depression of transcription play major roles in regulating the expression of HSP26. None of the recombinant constructs that we analyzed separated cis-regulatory sequences responsible for heat shock regulation from those responsible for developmental regulation of HSP26. Depression of HSP26 transcription may be the general mechanism of HSP26 induction in yeast cells. This regulatory scheme is very different from that described for the regulation of most other heat shock genes.

Yeast repressor alpha 2 binds to its operator cooperatively with yeast protein Mcm1

1989 ◽  
Vol 9 (11) ◽  
pp. 5228-5230
Author(s):  
C A Keleher ◽  
S Passmore ◽  
A D Johnson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Product ◽  
Strong Evidence ◽  
Regulatory Proteins ◽  
Yeast Cells ◽  
Specific Gene ◽  
Yeast Gene ◽  
Yeast Protein ◽  
Transcriptional Regulatory

To bring about repression of a family fo genes in Saccharomyces cerevisiae called the a-specific genes, two transcriptional regulatory proteins, alpha 2 and GRM (general regulator of matin type), bind cooperatively to an operator found upstream of each a-specific gene. To date, GRM has been defined only biochemically. In this communication we show that the product of a single yeast gene (MCM1) is sufficient to bind cooperatively with alpha 2 to the operator. We also show that antiserum raised against the MCM1 gene product recognizes GRM from yeast cells. These results, in combination with previous observations, provide strong evidence that MCM1 encodes the GRM activity.

scholarly journals DNA-Directed Expression of an Animal Virus RNA for Replication-Dependent Colony Formation in Saccharomyces cerevisiae

2002 ◽  
Vol 76 (4) ◽  
pp. 1610-1616 ◽  
Author(s):  
B. D. Price ◽  
P. Ahlquist ◽  
L. A. Ball
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Rna Polymerase Ii ◽  
Marker Gene ◽  
Rna Replication ◽  
Yeast Cells ◽  
Northern Blot Hybridization ◽  
Marker Gene Expression ◽  
Chimeric Rna

ABSTRACT To date, the insect nodavirus flock house virus (FHV) is the only virus of a higher eukaryote that has been shown to undergo a full replicative cycle and produce infectious progeny in the yeast Saccharomyces cerevisiae. The genome of FHV is composed of two positive-sense RNA segments: RNA1, encoding the RNA replicase, and RNA2, encoding the capsid protein precursor. When yeast cells expressing FHV RNA replicase were transfected with a chimeric RNA composed of a selectable gene flanked by the termini of RNA2, the chimeric RNA was replicated and transmitted to daughter cells indefinitely. In the work reported here, we developed a system in which a selectable chimeric RNA replicon was transcribed from an inducible RNA polymerase II (polII) promoter in vivo in yeast. To render marker gene expression absolutely dependent on RNA replication, the primary polII transcript was made negative in sense and contained an intron that blocked the translation of cryptic transcripts from the opposite DNA strand. The RNA products of DNA-templated transcription, processing, and RNA replication were characterized by Northern blot hybridization and primer extension analysis. Marker gene expression and colony growth under selective conditions depended strictly on FHV RNA replication, with background colonies arising at a frequency of fewer than 1 in 108 plated cells. The utility of the system was demonstrated by introducing a second chimeric replicon and showing that at least two different selectable markers could be simultaneously expressed by means of RNA replication. This is the first example of FHV RNA1-dependent selectable marker expression initiated in vivo and will greatly facilitate the identification and characterization of the requirements and inhibitors of RNA replication.

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