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.

Highly ordered nanoscale phosphomolybdate-grafted polyaniline/metal hybrid layered structures prepared via secondary sputtering phenomenon as high-performance pseudocapacitor electrodes

2021 ◽  
Author(s):  
Eun Seop Yoon ◽  
Bong Gill Choi ◽  
Hwan-Jin Jeon
Keyword(s):  
Electron Transfer ◽  
Aspect Ratio ◽  
Electrochemical Performance ◽  
High Aspect Ratio ◽  
High Performance ◽  
Electrode Materials ◽  
Rate Capability ◽  
High Specific Capacitance ◽  
High Rate ◽  
Large Area

Abstract The development of energy storage electrode materials is important for enhancing the electrochemical performance of supercapacitors. Despite extensive research on improving electrochemical performance with polymer-based materials, electrode materials with micro/nanostructures are needed for fast and efficient ion and electron transfer. In this work, highly ordered phosphomolybdate (PMoO)-grafted polyaniline (PMoO-PAI) deposited onto Au hole-cylinder nanopillar arrays is developed for high-performance pseudocapacitors. The three-dimensional nanostructured arrays are easily fabricated by secondary sputtering lithography, which has recently gained attention and features a high resolution of 10 nm, a high aspect ratio greater than 20, excellent uniformity/accuracy/precision, and compatibility with large area substrates. These 10nm scale Au nanostructures with a high aspect ratio of ~30 on Au substrates facilitate efficient ion and electron transfer. The resultant PMoO-PAI electrode exhibits outstanding electrochemical performance, including a high specific capacitance of 114 mF/cm2, a high-rate capability of 88%, and excellent long-term stability.

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.

Rotating wall vessel exposure alters protein secretion and global gene expression in Staphylococcus aureus

2011 ◽  
Vol 11 (2) ◽  
pp. 71-81 ◽  
Author(s):  
Helena Rosado ◽  
Alex J. O'Neill ◽  
Katy L. Blake ◽  
Meik Walther ◽  
Paul F. Long ◽  
...  
Keyword(s):  
Gene Expression ◽  
Staphylococcus Aureus ◽  
Protein Secretion ◽  
Space Station ◽  
Docking Simulation ◽  
Ligand Docking ◽  
Rotating Wall ◽  
Rotating Wall Vessel ◽  
Continuous Rotation ◽  
Low Shear

AbstractStaphylococcus aureus is routinely recovered from air and surface samples taken aboard the International Space Station (ISS) and poses a health threat to crew. As bacteria respond to the low shear forces engendered by continuous rotation conditions in a Rotating Wall Vessel (RWV) and the reduced gravitational field of near-Earth flight by altering gene expression, we examined the effect of low-shear RWV growth on protein secretion and gene expression by three S. aureus isolates. When cultured under 1 g, the total amount of protein secreted by these strains varied up to fourfold; under continuous rotation conditions, protein secretion by all three strains was significantly reduced. Concentrations of individual proteins were differentially reduced and no evidence was found for increased lysis. These data suggest that growth under continuous rotation conditions reduces synthesis or secretion of proteins. A limited number of changes in gene expression under continuous rotation conditions were noted: in all isolates vraX, a gene encoding a polypeptide associated with cell wall stress, was down-regulated. A vraX deletion mutant of S. aureus SH1000 was constructed: no differences were found between SH1000 and ΔvraX with respect to colony phenotype, viability, protein export, antibiotic susceptibility, vancomycin kill kinetics, susceptibility to cold or heat and gene modulation. An ab initio protein–ligand docking simulation suggests a major binding site for β-lactam drugs such as imipenem. If such changes to the bacterial phenotype occur during spaceflight, they will compromise the capacity of staphylococci to cause systemic infection and to circumvent antibacterial chemotherapy.

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.

High-Rate Etching of Mn-Zn Ferrite by Laser-Induced Chemical Reaction in CC12F2 Atmosphere

1990 ◽  
Vol 201 ◽  
Author(s):  
Y. F. Lu ◽  
M. Takai ◽  
S. Nagatomo ◽  
S. Namba
Keyword(s):  
Aspect Ratio ◽  
Chemical Reaction ◽  
High Aspect Ratio ◽  
Dwell Time ◽  
Gas Pressure ◽  
High Rate ◽  
Aspect Ratios ◽  
Zn Ferrite ◽  
Order Of Magnitude ◽  
Ion Laser

AbstractMaskless dry etching of Mn-Zn ferrite in dich1orodif1uoromethane (CC12F2) by Ar+-ion laser (514.5 nm-line) irradiation has been investigated to obtain high etching rates and aspect-ratios of etched grooves. The etching reaction was found to be thermochemical and caused by Cl radicals thermally decomposed from CCl2F2 gas. High etching rates of up to 360 μm/s, which is about one order of magnitude higher than that in a CCl4 gas and even higher than that in a H3PO4 solution, have been achieved. A high aspect-ratio of up to 12 was obtained. Definite gas pressure and dwell time are necessary to fabricate a smooth groove.

scholarly journals Optimized suspension culture: the rotating-wall vessel

2001 ◽  
Vol 281 (1) ◽  
pp. F12-F25 ◽  
Author(s):  
T. G. Hammond ◽  
J. M. Hammond
Keyword(s):  
Suspension Culture ◽  
Solid Body ◽  
Cultured Cells ◽  
Terminal Velocity ◽  
Culture Conditions ◽  
Culture Vessel ◽  
Body Rotation ◽  
Solid Body Rotation ◽  
Rotating Wall ◽  
Rotating Wall Vessel

Suspension culture remains a popular modality, which manipulates mechanical culture conditions to maintain the specialized features of cultured cells. The rotating-wall vessel is a suspension culture vessel optimized to produce laminar flow and minimize the mechanical stresses on cell aggregates in culture. This review summarizes the engineering principles, which allow optimal suspension culture conditions to be established, and the boundary conditions, which limit this process. We suggest that to minimize mechanical damage and optimize differentiation of cultured cells, suspension culture should be performed in a solid-body rotation Couette-flow, zero-headspace culture vessel such as the rotating-wall vessel. This provides fluid dynamic operating principles characterized by 1) solid body rotation about a horizontal axis, characterized by colocalization of cells and aggregates of different sedimentation rates, optimally reduced fluid shear and turbulence, and three-dimensional spatial freedom; and 2) oxygenation by diffusion. Optimization of suspension culture is achieved by applying three tradeoffs. First, terminal velocity should be minimized by choosing microcarrier beads and culture media as close in density as possible. Next, rotation in the rotating-wall vessel induces both Coriolis and centrifugal forces, directly dependent on terminal velocity and minimized as terminal velocity is minimized. Last, mass transport of nutrients to a cell in suspension culture depends on both terminal velocity and diffusion of nutrients. In the transduction of mechanical culture conditions into cellular effects, several lines of evidence support a role for multiple molecular mechanisms. These include effects of shear stress, changes in cell cycle and cell death pathways, and upstream regulation of secondary messengers such as protein kinase C. The discipline of suspension culture needs a systematic analysis of the relationship between mechanical culture conditions and biological effects, emphasizing cellular processes important for the industrial production of biological pharmaceuticals and devices.

Expression of renal cell protein markers is dependent on initial mechanical culture conditions

2002 ◽  
Vol 92 (2) ◽  
pp. 691-700 ◽  
Author(s):  
Nancy L. Cowger ◽  
Edmund Benes ◽  
Patricia L. Allen ◽  
Timothy G. Hammond
Keyword(s):  
Vitamin D ◽  
Suspension Culture ◽  
Vitamin D Receptor ◽  
Membrane Trafficking ◽  
Membrane Damage ◽  
Culture Conditions ◽  
Cell Protein ◽  
Protein Markers ◽  
Rotating Wall ◽  
Rotating Wall Vessel

The rotating wall vessel is optimized for suspension culture, with laminar flow and adequate nutrient delivery, but minimal shear. However, higher shears may occur in vivo. During rotating wall vessel cultivation of human renal cells, size and density of glass-coated microcarrier beads were changed to modulate initial shear. Renal-specific proteins were assayed after 2 days. Flow cytometry antibody binding analysis of vitamin D receptor demonstrated peak expression at intermediate shears, with 30% reduction outside this range. Activity of cathepsin C showed the inverse pattern, lowest at midshear, with twofold increases at either extreme. Dipeptidyl-peptidase IV had no shear dependence, suggesting that the other results are specific, not universal, changes in membrane trafficking or protein synthesis. On addition of dextran, which changes medium density and viscosity but not shear, vitamin D receptor assay showed no differences from controls. Neither cell cycle, apoptosis/necrosis indexes, nor lactate dehydrogenase release varied between experiments, confirming that the changes are primary, not secondary to cell cycling or membrane damage. This study provides direct evidence that mechanical culture conditions modulate protein expression in suspension culture.

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.

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