Global Screening of Genes Essential for Growth in High-Pressure and Cold Environments: Searching for Basic Adaptive Strategies Using a Yeast Deletion Library

ABSTRACT Gentamicin continues to be a primary antibiotic against gram-negative infections. Unfortunately, associated nephro- and ototoxicity limit its use. Our previous mammalian studies showed that gentamicin is trafficked to the endoplasmic reticulum in a retrograde manner and subsequently released into the cytosol. To better dissect the mechanism through which gentamicin induces toxicity, we have chosen to study its toxicity using the simple eukaryote Saccharomyces cerevisiae. A recent screen of the yeast deletion library identified multiple gentamicin-sensitive strains, many of which participate in intracellular trafficking. Our approach was to evaluate gentamicin sensitivity under logarithmic growth conditions. By quantifying growth inhibition in the presence of gentamicin, we determined that several of the sensitive strains were part of the Golgi-associated retrograde protein (GARP) and homotypic fusion and vacuole protein sorting (HOPS) complexes. Further evaluation of their other components showed that the deletion of any GARP member resulted in gentamicin-hypersensitive strains, while the deletion of other HOPS members resulted in less gentamicin sensitivity. Other genes whose deletion resulted in gentamicin hypersensitivity included ZUO1, SAC1, and NHX1. Finally, we utilized a Texas Red gentamicin conjugate to characterize gentamicin uptake and localization in both gentamicin-sensitive and -insensitive strains. These studies were consistent with our mammalian studies, suggesting that gentamicin toxicity in yeast results from alterations to intracellular trafficking pathways. The identification of genes whose absence results in gentamicin toxicity will help target specific pathways and mechanisms that contribute to gentamicin toxicity.

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Activator Gcn4p and Cyc8p/Tup1p Are Interdependent for Promoter Occupancy at ARG1 In Vivo

Molecular and Cellular Biology ◽

10.1128/mcb.25.24.11171-11183.2005 ◽

2005 ◽

Vol 25 (24) ◽

pp. 11171-11183 ◽

Cited By ~ 19

Author(s):

Soon-ja Kim ◽

Mark J. Swanson ◽

Hongfang Qiu ◽

Chhabi K. Govind ◽

Alan G. Hinnebusch

Keyword(s):

Amino Acid ◽

Rna Polymerase Ii ◽

Transcriptional Activation ◽

Target Genes ◽

Direct Role ◽

Valine Biosynthesis ◽

Promoter Occupancy ◽

Yeast Deletion Library ◽

Deletion Library

ABSTRACT The Cyc8p/Tup1p complex mediates repression of diverse genes in Saccharomyces cerevisiae and is recruited by DNA binding proteins specific for the different sets of repressed genes. By screening the yeast deletion library, we identified Cyc8p as a coactivator for Gcn4p, a transcriptional activator of amino acid biosynthetic genes. Deletion of CYC8 confers sensitivity to an inhibitor of isoleucine/valine biosynthesis and impairs activation of Gcn4p-dependent reporters and authentic amino acid biosynthetic target genes. Deletion of TUP1 produces similar but less severe activation defects in vivo. Although expression of Gcn4p is unaffected by deletion of CYC8, chromatin immunoprecipitation assays reveal a strong defect in binding of Gcn4p at the target genes ARG1 and ARG4 in cyc8Δ cells and to a lesser extent in tup1Δ cells. The defects in Gcn4p binding and transcriptional activation in cyc8Δ cells cannot be overcome by Gcn4p overexpression but are partially suppressed in tup1Δ cells. The impairment of Gcn4p binding in cyc8Δ and tup1Δ cells is severe enough to reduce recruitment of SAGA, Srb mediator, TATA binding protein, and RNA polymerase II to the ARG1 and ARG4 promoters, accounting for impaired transcriptional activation of these genes in both mutants. Cyc8p and Tup1p are recruited to the ARG1 and ARG4 promoters, consistent with a direct role for this complex in stimulating Gcn4p occupancy of the upstream activation sequence (UAS). Interestingly, Gcn4p also stimulates binding of Cyc8p/Tup1p at the 3′ ends of these genes, raising the possibility that Cyc8p/Tup1p influences transcription elongation. Our findings reveal a novel coactivator function for Cyc8p/Tup1p at the level of activator binding and suggest that Gcn4p may enhance its own binding to the UAS by recruiting Cyc8p/Tup1p.

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Towards system theory based adaptive strategies for high pressure bioprocesses

High Pressure Research ◽

10.1080/08957950601079835 ◽

2007 ◽

Vol 27 (1) ◽

pp. 7-14 ◽

Cited By ~ 10

Author(s):

A. Delgado ◽

A. Baars ◽

W. Kowalczyk ◽

R. Benning ◽

P. Kitsubun

Keyword(s):

System Theory ◽

High Pressure ◽

Adaptive Strategies

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Knockdown of vps54 aggravates tamoxifen-induced cytotoxicity in fission yeast

Genomics & Informatics ◽

10.5808/gi.21049 ◽

2021 ◽

Vol 19 (4) ◽

pp. e39

Author(s):

Sol Lee ◽

Miyoung Nam ◽

Ah-Reum Lee ◽

Seung-Tae Baek ◽

Min Jung Kim ◽

...

Keyword(s):

Fission Yeast ◽

Cancer Cell Line ◽

Drug Induced ◽

Cytotoxic Effects ◽

Action Mechanisms ◽

Er Positive ◽

Antifungal Action ◽

Microarray Screening ◽

Yeast Deletion Library ◽

Deletion Library

Tamoxifen (TAM) is an anticancer drug used to treat estrogen receptor (ER)‒positive breast cancer. However, its ER-independent cytotoxic and antifungal activities have prompted debates on its mechanism of action. To achieve a better understanding of the ER-independent antifungal action mechanisms of TAM, we systematically identified TAM-sensitive genes through microarray screening of the heterozygous gene deletion library in fission yeast (Schizosaccharomyces pombe). Secondary confirmation was followed by a spotting assay, finally yielding 13 TAM-sensitive genes under the drug-induced haploinsufficient condition. For these 13 TAM-sensitive genes, we conducted a comparative analysis of their Gene Ontology (GO) ‘biological process’ terms identified from other genome-wide screenings of the budding yeast deletion library and the MCF7breast cancer cell line. Several TAM-sensitive genes overlapped between the yeast strains and MCF7 in GO terms including ‘cell cycle’ (cdc2, rik1, pas1, and leo1), ‘signaling’ (sck2, oga1, and cki3), and ‘vesicle-mediated transport’ (SPCC126.08c, vps54, sec72, and tvp15), suggesting their roles in the ER-independent cytotoxic effects of TAM. We recently reported that the cki3 gene with the ‘signaling’ GO term was related to the ER-independent antifungal action mechanisms of TAM in yeast. In this study, we report that haploinsufficiency of the essential vps54 gene, which encodes the GARP complex subunit, significantly aggravated TAM sensitivity and led to an enlarged vesicle structure in comparison with the SP286 control strain. These results strongly suggest that the vesicle-mediated transport process might be another action mechanism of the ER-independent antifungal or cytotoxic effects of TAM.

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Population dynamics analysis of Saccharomyces cerevisiae deletion library during fed-batch cultivation using Bar-seq

10.1101/2020.04.27.064675 ◽

2020 ◽

Author(s):

Maren Wehrs ◽

Mitchell G. Thompson ◽

Deepanwita Banerjee ◽

Jan-Philip Prahl ◽

Carolina A. Barcelos ◽

...

Keyword(s):

Stress Responses ◽

Large Scale ◽

Glycogen Synthase ◽

Batch Cultivation ◽

Population Diversity ◽

Fed Batch ◽

Industrial Fermentation ◽

Biological Stress ◽

Yeast Deletion Library ◽

Deletion Library

AbstractTo understand the genetic basis of changes in strain physiology during industrial fermentation, and the corresponding roles these genes play in strain performance, we employed a barcoded yeast deletion library to assess genome-wide strain fitness across a simulated industrial fermentation regime. Our results demonstrate the utility of Bar-seq to assess fermentation associated stresses in yeast populations under industrial conditions. We find that mutant population diversity is maintained through multiple seed trains, enabling for large scale fermentation selective pressures to act upon the community. We identify specific deletion mutants that were enriched in all processes, independent of the cultivation conditions, which include MCK1, RIM11, MRK1, and YGK3 that encode homologues of mammalian glycogen synthase kinase 3 (GSK-3). Further, we show that significant changes in the population diversity during fed-batch cultivations reflect the presence of significant external stresses, such as the accumulation of the fermentative byproduct ethanol. The mutants that were lost during the time of most extreme population selection suggest that specific biological processes may be required to cope with these specific stresses. Overall our work highlights a promising avenue to identify genetic loci and biological stress responses required for fitness under industrial conditions.

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High-pressure freezing of cells in suspension for low-voltage scanning electron microscopy (LVSEM)

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100084624 ◽

1991 ◽

Vol 49 ◽

pp. 64-65

Author(s):

Marek Malecki ◽

James Pawley ◽

Hans Ris

Keyword(s):

Electron Microscopy ◽

High Pressure ◽

Low Voltage ◽

Culture Media ◽

Cell Structure ◽

Freeze Substitution ◽

Ice Crystal ◽

High Pressure Freezing ◽

Cell Culture Media ◽

Voltage Scanning

The ultrastructure of cells suspended in physiological fluids or cell culture media can only be studied if the living processes are stopped while the cells remain in suspension. Attachment of living cells to carrier surfaces to facilitate further processing for electron microscopy produces a rapid reorganization of cell structure eradicating most traces of the structures present when the cells were in suspension. The structure of cells in suspension can be immobilized by either chemical fixation or, much faster, by rapid freezing (cryo-immobilization). The fixation speed is particularly important in studies of cell surface reorganization over time. High pressure freezing provides conditions where specimens up to 500μm thick can be frozen in milliseconds without ice crystal damage. This volume is sufficient for cells to remain in suspension until frozen. However, special procedures are needed to assure that the unattached cells are not lost during subsequent processing for LVSEM or HVEM using freeze-substitution or freeze drying. We recently developed such a procedure.

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Prolonged Fixation Studies For Spaceflight

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100072101 ◽

1973 ◽

Vol 31 ◽

pp. 332-333

Author(s):

Robert Corbett ◽

Delbert E. Philpott ◽

Sam Black

Keyword(s):

High Pressure ◽

Living Systems ◽

Prolonged Storage ◽

Time Intervals ◽

Early Signs ◽

Large Numbers

Observation of subtle or early signs of change in spaceflight induced alterations on living systems require precise methods of sampling. In-flight analysis would be preferable but constraints of time, equipment, personnel and cost dictate the necessity for prolonged storage before retrieval. Because of this, various tissues have been stored in fixatives and combinations of fixatives and observed at various time intervals. High pressure and the effect of buffer alone have also been tried.Of the various tissues embedded, muscle, cartilage and liver, liver has been the most extensively studied because it contains large numbers of organelles common to all tissues (Fig. 1).

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Cryosectioning plant roots prepared by high-pressure freezing

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100127748 ◽

1987 ◽

Vol 45 ◽

pp. 662-663

Author(s):

R.E. Crang ◽

M. Mueller ◽

K. Zierold

Keyword(s):

High Pressure ◽

Cell Walls ◽

Plant Tissues ◽

Ice Crystal ◽

High Pressure Freezing ◽

Vitreous State ◽

Radial Depth ◽

Irregular Topography ◽

Considerable Depth ◽

Conventional Freezing

Obtaining frozen-hydrated sections of plant tissues for electron microscopy and microanalysis has been considered difficult, if not impossible, due primarily to the considerable depth of effective freezing in the tissues which would be required. The greatest depth of vitreous freezing is generally considered to be only 15-20 μm in animal specimens. Plant cells are often much larger in diameter and, if several cells are required to be intact, ice crystal damage can be expected to be so severe as to prevent successful cryoultramicrotomy. The very nature of cell walls, intercellular air spaces, irregular topography, and large vacuoles often make it impractical to use immersion, metal-mirror, or jet freezing techniques for botanical material.However, it has been proposed that high-pressure freezing (HPF) may offer an alternative to the more conventional freezing techniques, inasmuch as non-cryoprotected specimens may be frozen in a vitreous, or near-vitreous state, to a radial depth of at least 0.5 mm.

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High-pressure freezing and freeze substitution of plant tissue

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100124148 ◽

1992 ◽

Vol 50 (1) ◽

pp. 748-749

Author(s):

William P. Sharp ◽

Robert W. Roberson

Keyword(s):

High Pressure ◽

Cell Structure ◽

Leaf Tissue ◽

Freeze Substitution ◽

Crystal Formation ◽

Ice Crystal ◽

High Pressure Freezing ◽

Cell Components ◽

Cold Metal ◽

Brome Grass

The aim of ultrastructural investigation is to analyze cell architecture and relate a functional role(s) to cell components. It is known that aqueous chemical fixation requires seconds to minutes to penetrate and stabilize cell structure which may result in structural artifacts. The use of ultralow temperatures to fix and prepare specimens, however, leads to a much improved preservation of the cell’s living state. A critical limitation of conventional cryofixation methods (i.e., propane-jet freezing, cold-metal slamming, plunge-freezing) is that only a 10 to 40 μm thick surface layer of cells can be frozen without distorting ice crystal formation. This problem can be allayed by freezing samples under about 2100 bar of hydrostatic pressure which suppresses the formation of ice nuclei and their rate of growth. Thus, 0.6 mm thick samples with a total volume of 1 mm3 can be frozen without ice crystal damage. The purpose of this study is to describe the cellular details and identify potential artifacts in root tissue of barley (Hordeum vulgari L.) and leaf tissue of brome grass (Bromus mollis L.) fixed and prepared by high-pressure freezing (HPF) and freeze substitution (FS) techniques.

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