scholarly journals Bidirectional titration of yeast gene expression using a pooled CRISPR guide RNA approach

2020 ◽  
Vol 117 (31) ◽  
pp. 18424-18430 ◽  
Author(s):  
Emily K. Bowman ◽  
Matthew Deaner ◽  
Jan-Fang Cheng ◽  
Robert Evans ◽  
Ernst Oberortner ◽  
...  
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Essential Genes ◽  
Yeast Gene ◽  
Promoter Regions ◽  
Guide Rna ◽  
Metabolic Genes ◽  
Complementary Approach ◽  
Modulation Of Gene Expression ◽  
Generation Sequencing

Most classic genetic approaches utilize binary modifications that preclude the identification of key knockdowns for essential genes or other targets that only require moderate modulation. As a complementary approach to these classic genetic methods, we describe a plasmid-based library methodology that affords bidirectional, graded modulation of gene expression enabled by tiling the promoter regions of all 969 genes that comprise the ito977 model ofSaccharomyces cerevisiae’s metabolic network. When coupled with a CRISPR-dCas9–based modulation and next-generation sequencing, this method affords a library-based, bidirection titration of gene expression across all major metabolic genes. We utilized this approach in two case studies: growth enrichment on alternative sugars, glycerol and galactose, and chemical overproduction of betaxanthins, leading to the identification of unique gene targets. In particular, we identify essential genes and other targets that were missed by classic genetic approaches.

scholarly journals Genome-Wide Relationships between TAF1 and Histone Acetyltransferases in Saccharomyces cerevisiae

2006 ◽  
Vol 26 (7) ◽  
pp. 2791-2802 ◽  
Author(s):  
Melissa Durant ◽  
B. Franklin Pugh
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Histone Acetylation ◽  
Rna Polymerase Ii ◽  
Histone Acetyltransferases ◽  
Histone H4 ◽  
Promoter Regions ◽  
Genome Wide ◽  
Acetylated Histone H4

ABSTRACT Histone acetylation regulates gene expression, yet the functional contributions of the numerous histone acetyltransferases (HATs) to gene expression and their relationships with each other remain largely unexplored. The central role of the putative HAT-containing TAF1 subunit of TFIID in gene expression raises the fundamental question as to what extent, if any, TAF1 contributes to acetylation in vivo and to what extent it is redundant with other HATs. Our findings herein do not support the basic tenet that TAF1 is a major HAT in Saccharomyces cerevisiae, nor do we find that TAF1 is functionally redundant with other HATs, including Gcn5, Elp3, Hat1, Hpa2, Sas3, and Esa1, which is in contrast to previous conclusions regarding Gcn5. Our findings do reveal that of these HATs, only Gcn5 and Esa1 contribute substantially to gene expression genome wide. Interestingly, histone acetylation at promoter regions throughout the genome does not require TAF1 or RNA polymerase II, indicating that most acetylation is likely to precede transcription and not depend upon it. TAF1 function has been linked to Bdf1, which binds TFIID and acetylated histone H4 tails, but no linkage between TAF1 and the H4 HAT Esa1 has been established. Here, we present evidence for such a linkage through Bdf1.

Elements involved in S-adenosylmethionine-mediated regulation of the Saccharomyces cerevisiae MET25 gene

1989 ◽  
Vol 9 (8) ◽  
pp. 3292-3298
Author(s):  
D Thomas ◽  
H Cherest ◽  
Y Surdin-Kerjan
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Transcriptional Regulation ◽  
Growth Medium ◽  
Posttranscriptional Regulation ◽  
Promoter Region ◽  
Regulatory Region ◽  
Cis Elements ◽  
Promoter Regions ◽  
Activation Site

In Saccharomyces cerevisiae, the MET25 gene encodes O-acetylhomoserine sulfhydrylase. Synthesis of this enzyme is repressed by the presence of S-adenosylmethionine (AdoMet) in the growth medium. We identified cis elements required for MET25 expression by analyzing small deletions in the MET25 promoter region. The results revealed a regulatory region, acting as an upstream activation site, that activated transcription of MET25 in the absence of methionine or AdoMet. We found that, for the most part, repression of MET25 expression was due to a lack of activation at this site, reinforced by an independent repression mechanism. The activation region contained a repeated dyad sequence that is also found in the promoter regions of other unlinked but coordinately regulated genes (MET3, MET2, and SAM2). We show that the presence of the two dyads is necessary for maximal gene expression. Moreover, we demonstrate that in addition to this transcriptional regulation, a posttranscriptional regulation, probably targeted at the 5' region of mRNA, is involved in MET25 expression.

Saccharomyces cerevisiae gene expression changes during rotating wall vessel suspension culture

2002 ◽  
Vol 93 (6) ◽  
pp. 2171-2180 ◽  
Author(s):  
Kelly Johanson ◽  
Patricia L. Allen ◽  
Fawn Lewis ◽  
Luis A. Cubano ◽  
Linda E. Hyman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Aspect Ratio ◽  
Suspension Culture ◽  
High Aspect Ratio ◽  
High Rate ◽  
Promoter Regions ◽  
Genetic Changes ◽  
Rotating Wall ◽  
Rotating Wall Vessel

This study utilizes Saccharomyces cerevisiae to study genetic responses to suspension culture. The suspension culture system used in this study is the high-aspect-ratio vessel, one type of the rotating wall vessel, that provides a high rate of gas exchange necessary for rapidly dividing cells. Cells were grown in the high-aspect-ratio vessel, and DNA microarray and metabolic analyses were used to determine the resulting changes in yeast gene expression. A significant number of genes were found to be up- or downregulated by at least twofold as a result of rotational growth. By using Gibbs promoter alignment, clusters of genes were examined for promoter elements mediating these genetic changes. Candidate binding motifs similar to the Rap1p binding site and the stress-responsive element were identified in the promoter regions of differentially regulated genes. This study shows that, as in higher order organisms, S. cerevisiae changes gene expression in response to rotational culture and also provides clues for investigations into the signaling pathways involved in gravitational response.

Modulation of gene expression by cocktail δ-integration to improve carotenoid production in Saccharomyces cerevisiae

2018 ◽  
Vol 268 ◽  
pp. 616-621 ◽  
Author(s):  
Ryosuke Yamada ◽  
Azusa Yamauchi ◽  
Yorichika Ando ◽  
Yuki Kumata ◽  
Hiroyasu Ogino
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Carotenoid Production ◽  
Modulation Of Gene Expression

scholarly journals Dual Role of the Saccharomyces cerevisiae TEA/ATTS Family Transcription Factor Tec1p in Regulation of Gene Expression and Cellular Development

2002 ◽  
Vol 1 (5) ◽  
pp. 673-686 ◽  
Author(s):  
Tim Köhler ◽  
Stefanie Wesche ◽  
Naimeh Taheri ◽  
Gerhard H. Braus ◽  
Hans-Ulrich Mösch
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Binding Site ◽  
Target Genes ◽  
Mutational Analysis ◽  
Regulation Of Gene Expression ◽  
Control Mechanisms ◽  
Promoter Regions ◽  
Pseudohyphal Growth ◽  
Cellular Development

ABSTRACT In Saccharomyces cerevisiae, the transcription factors Tec1p and Ste12p are required for haploid invasive and diploid pseudohyphal growth. Tec1p and Ste12p have been postulated to regulate these developmental processes primarily by cooperative binding to filamentous and invasion-responsive elements (FREs), which are combined enhancer elements that consist of a Tec1p-binding site (TCS) and an Ste12p-binding site (PRE). They are present in the promoter regions of target genes, e.g., FLO11. Here, we show that Tec1p efficiently activates target gene expression and cellular development in the absence of Ste12p. We further demonstrate that TCS elements alone are sufficient to mediate Tec1p-driven gene expression by a mechanism termed TCS control that is operative even when Ste12p is absent. Mutational analysis of TEC1 revealed that TCS control, FLO11 expression, and haploid invasive growth require the C terminus of Tec1p. In contrast, the Ste12p-dependent FRE control mechanism is sufficiently executed by the N-terminal portion of Tec1p, which contains the TEA/ATTS DNA-binding domain. Our study suggests that regulation of haploid invasive and diploid pseudohyphal growth by Ste12p and Tec1p is not only executed by combinatorial control but involves additional control mechanisms in which Ste12p activates TEC1 expression via clustered PREs and where Tec1p regulates expression of target genes, e.g., FLO11, by TCS control.

scholarly journals In silico experiments validate that twenty nucleotide spacer sequence provides precise targeting of bacterial genes in CRISPR-Cas9

2019 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
Genome Editing ◽  
In Silico ◽  
Bacterial Genome ◽  
Specific Gene ◽  
Guide Rna ◽  
Theoretical Predictions ◽  
Modulation Of Gene Expression ◽  
Target Effects

As a genome editing tool useful for modulating the expression of different genes, CRISPR-Cas9 is known for its precision in targeting specific genes. To do this, CRISPR-Cas9 utilizes a guide RNA for guiding the Cas9 endonuclease to specific stretches of the DNA for genome editing or modulation of gene expression. Guide RNA comprises a spacer sequence and a protospacer adjacent motif (PAM) sequence. Both components work together to help target Cas9 to a specific stretch of DNA within a gene. In particular, spacer sequence provides a unique address for localizing Cas9 to specific stretch of DNA. But, possibility exists that there could be off-target effects for particular spacer sequence used in guide RNA. Specifically, spacer sequence might engage in complementary base pairing with other stretches of DNA in the bacterial genome, and result in additional genome editing or modulation of gene expression at genes that are not targeted. Results from an in silico experiment conducted with the rpoH gene of Pseudomonas aeruginosa PAO1 revealed that all spacer sequences derived from different stretches of the rpoH gene did not elicit off-target effects in the genome of the bacterium. This concurs with theoretical predictions that the probability of off-target effects from a 20 nucleotide long spacer region is vanishingly small. Hence, a 20 nucleotide spacer sequence in guide RNA should provide a unique DNA address for precise targeting of specific gene in the genome of a bacterium. Collectively, off-target effects of CRISPR-Cas9 is a valid concern for both genetic engineering and genome editing applications as targeting of additional genes from the desired one would result in unpredictable physiological and biochemical impacts on the cell. Using the rpoH gene of Pseudomonas aeruginosa PAO1 as example, results from an in silico experiment examining possible off-target effects of different 20 nucleotide spacer sequence able to target the sense and antisense strand of the gene revealed no off-target effects. Specifically, each spacer sequence used could only target the intended rpoH gene, which concurs with theoretical predictions of vanishingly small possibility of off-target effects on a bacterial genome from a 20 nucleotide spacer sequence. Overall, the results highlight that use of a 20 nucleotide spacer sequence in guide RNA could offer precise targeting of specific gene in a bacterium.

scholarly journals In silico experiments validate that twenty nucleotide spacer sequence provides precise targeting of bacterial genes in CRISPR-Cas9

2019 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
Genome Editing ◽  
In Silico ◽  
Bacterial Genome ◽  
Specific Gene ◽  
Guide Rna ◽  
Theoretical Predictions ◽  
Modulation Of Gene Expression ◽  
Target Effects

As a genome editing tool useful for modulating the expression of different genes, CRISPR-Cas9 is known for its precision in targeting specific genes. To do this, CRISPR-Cas9 utilizes a guide RNA for guiding the Cas9 endonuclease to specific stretches of the DNA for genome editing or modulation of gene expression. Guide RNA comprises a spacer sequence and a protospacer adjacent motif (PAM) sequence. Both components work together to help target Cas9 to a specific stretch of DNA within a gene. In particular, spacer sequence provides a unique address for localizing Cas9 to specific stretch of DNA. But, possibility exists that there could be off-target effects for particular spacer sequence used in guide RNA. Specifically, spacer sequence might engage in complementary base pairing with other stretches of DNA in the bacterial genome, and result in additional genome editing or modulation of gene expression at genes that are not targeted. Results from an in silico experiment conducted with the rpoH gene of Pseudomonas aeruginosa PAO1 revealed that all spacer sequences derived from different stretches of the rpoH gene did not elicit off-target effects in the genome of the bacterium. This concurs with theoretical predictions that the probability of off-target effects from a 20 nucleotide long spacer region is vanishingly small. Hence, a 20 nucleotide spacer sequence in guide RNA should provide a unique DNA address for precise targeting of specific gene in the genome of a bacterium. Collectively, off-target effects of CRISPR-Cas9 is a valid concern for both genetic engineering and genome editing applications as targeting of additional genes from the desired one would result in unpredictable physiological and biochemical impacts on the cell. Using the rpoH gene of Pseudomonas aeruginosa PAO1 as example, results from an in silico experiment examining possible off-target effects of different 20 nucleotide spacer sequence able to target the sense and antisense strand of the gene revealed no off-target effects. Specifically, each spacer sequence used could only target the intended rpoH gene, which concurs with theoretical predictions of vanishingly small possibility of off-target effects on a bacterial genome from a 20 nucleotide spacer sequence. Overall, the results highlight that use of a 20 nucleotide spacer sequence in guide RNA could offer precise targeting of specific gene in a bacterium.

Analysis of High Dimensionality Yeast Gene Expression Data Using Data Mining

2012 ◽  
Vol 197 ◽  
pp. 515-522
Author(s):  
Mazin Aouf ◽  
Liwan Liyanage
Keyword(s):  
Gene Expression ◽  
Data Mining ◽  
Hydrogen Peroxide ◽  
Transcription Factors ◽  
Gene Expression Data ◽  
Process Model ◽  
Expression Data ◽  
Yeast Gene ◽  
Promoter Regions ◽  
Biological Studies

Data Mining is the process of discovering interesting knowledge from large amounts of data stored either in databases, data warehouses, or other information repositories. From biological studies, the Yeast Proteome Database (YPD) is a model for the organization and presentation of genome-wide functional data. Accordingly, a yeast gene expression which is a unicellular DNA is selected which contains 6103 genes and the database combined with a number of related dataset to create a general dataset. DNA-binding transcriptional regulators interpret the genome’s regulatory code by binding to specific sequences to induce or repress gene expression. The gene products including RNA and protein are responsible for the development and functioning of all living membranes by 2 steps process, transcription and translation. Various transcription factors control gene transcription by binding to the promoter regions. Translation is the production of proteins from mRNA produced in transcription. In this study, out of the 169 transcription factors known to access yeast, we are considering those thought to be involved in the response of Hydrogen Peroxide (H2O2). They are 22 transcription factors. Each one is partitioned to 3 parts: TF with No H2O2, TF with Low H2O2 and TF with High H2O2. The aim of this paper was to enhance the effectiveness of the integration of hydrogen peroxide response data related to yeast gene expression data to obtain a protein response process model and to label a set of important genes related to this approach.

scholarly journals Elements involved in S-adenosylmethionine-mediated regulation of the Saccharomyces cerevisiae MET25 gene.

1989 ◽  
Vol 9 (8) ◽  
pp. 3292-3298 ◽  
Author(s):  
D Thomas ◽  
H Cherest ◽  
Y Surdin-Kerjan
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Transcriptional Regulation ◽  
Growth Medium ◽  
Posttranscriptional Regulation ◽  
Promoter Region ◽  
Regulatory Region ◽  
Cis Elements ◽  
Promoter Regions ◽  
Activation Site

In Saccharomyces cerevisiae, the MET25 gene encodes O-acetylhomoserine sulfhydrylase. Synthesis of this enzyme is repressed by the presence of S-adenosylmethionine (AdoMet) in the growth medium. We identified cis elements required for MET25 expression by analyzing small deletions in the MET25 promoter region. The results revealed a regulatory region, acting as an upstream activation site, that activated transcription of MET25 in the absence of methionine or AdoMet. We found that, for the most part, repression of MET25 expression was due to a lack of activation at this site, reinforced by an independent repression mechanism. The activation region contained a repeated dyad sequence that is also found in the promoter regions of other unlinked but coordinately regulated genes (MET3, MET2, and SAM2). We show that the presence of the two dyads is necessary for maximal gene expression. Moreover, we demonstrate that in addition to this transcriptional regulation, a posttranscriptional regulation, probably targeted at the 5' region of mRNA, is involved in MET25 expression.

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