A website to identify shared genes in Saccharomyces cerevisiae homozygous deletion library screens

Transient potential receptor (TRP) channels are conserved cation channels found in most eukaryotes, known to sense a variety of chemical, thermal or mechanical stimuli. The Saccharomyces cerevisiae TRPY1 is a TRP channel with vacuolar localization involved in the cellular response to hyperosmotic shock and oxidative stress. In this study, we found that S. cerevisiae diploid cells with heterozygous deletion in TRPY1 gene are haploinsufficient when grown in synthetic media deficient in essential metal ions and that this growth defect is alleviated by non-toxic Mn2+ surplus. Using cells expressing the Ca2+-sensitive photoprotein aequorin we found that Mn2+ augmented the Ca2+ flux into the cytosol under oxidative stress, but not under hyperosmotic shock, a trait that was absent in the diploid cells with homozygous deletion of TRPY1 gene. TRPY1 activation under oxidative stress was diminished in cells devoid of Smf1 (the Mn2+-high-affinity plasma membrane transporter) but it was clearly augmented in cells lacking Pmr1 (the endoplasmic reticulum (ER)/Golgi located ATPase responsible for Mn2+ detoxification via excretory pathway). Taken together, these observations lead to the conclusion that increased levels of intracytosolic Mn2+ activate TRPY1 in the response to oxidative stress.

Download Full-text

Investigation of Bar-seq as a method to study population dynamics of Saccharomyces cerevisiae deletion library during bioreactor cultivation

Microbial Cell Factories ◽

10.1186/s12934-020-01423-z ◽

2020 ◽

Vol 19 (1) ◽

Author(s):

Maren Wehrs ◽

Mitchell G. Thompson ◽

Deepanwita Banerjee ◽

Jan-Philip Prahl ◽

Norma M. Morella ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Population Dynamics ◽

Bioreactor Cultivation ◽

Study Population ◽

Deletion Library

Download Full-text

Systematic screens of a Candida albicans homozygous deletion library decouple morphogenetic switching and pathogenicity

Nature Genetics ◽

10.1038/ng.605 ◽

2010 ◽

Vol 42 (7) ◽

pp. 590-598 ◽

Cited By ~ 419

Author(s):

Suzanne M Noble ◽

Sarah French ◽

Lisa A Kohn ◽

Victoria Chen ◽

Alexander D Johnson

Keyword(s):

Candida Albicans ◽

Homozygous Deletion ◽

Deletion Library

Download Full-text

Loss of the Homotypic Fusion and Vacuole Protein Sorting or Golgi-Associated Retrograde Protein Vesicle Tethering Complexes Results in Gentamicin Sensitivity in the Yeast Saccharomyces cerevisiae

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.50.2.587-595.2006 ◽

2006 ◽

Vol 50 (2) ◽

pp. 587-595 ◽

Cited By ~ 8

Author(s):

Mark C. Wagner ◽

Elizabeth E. Molnar ◽

Bruce A. Molitoris ◽

Mark G. Goebl

Keyword(s):

Saccharomyces Cerevisiae ◽

Intracellular Trafficking ◽

Protein Sorting ◽

Growth Conditions ◽

Yeast Saccharomyces Cerevisiae ◽

Vesicle Tethering ◽

Yeast Deletion Library ◽

Texas Red ◽

Deletion Library ◽

Logarithmic Growth

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.

Download Full-text

mSphere of Influence: Start with an Interesting Biological Phenomenon

mSphere ◽

10.1128/msphere.00299-19 ◽

2019 ◽

Vol 4 (3) ◽

Author(s):

Teresa O’Meara

Keyword(s):

Candida Albicans ◽

Single Cell Analysis ◽

Homozygous Deletion ◽

Functional Genomic ◽

Cell Analysis ◽

Biological Phenomenon ◽

Content Type ◽

Link Type ◽

New Biology ◽

Deletion Library

ABSTRACT Teresa O'Meara works in the field of functional genomics of Candida albicans, with a focus on host-pathogen interactions. In this mSphere of Influence article, she reflects on how papers entitled "Systematic Screens of a Candida albicans Homozygous Deletion Library Decouple Morphogenetic Switching and Pathogenicity" by S. M. Noble, S. French, L. A. Kohn, V. Chen, and A. D. Johnson (Nat Genet 42:590–598, 2010, https://doi.org/10.1038/ng.605) and "Exploring Quantitative Yeast Phenomics with Single-Cell Analysis of DNA Damage Foci" by E. B. Styles et al. (Cell Syst 3:264–277.e10, 2016, https://doi.org/10.1016/j.cels.2016.08.008) impacted her research and thinking through pioneering functional genomic screens. These articles show the power of combining defined mutant libraries with screens for interesting phenotypes to understand new biology.

Download Full-text

mSphere of Influence: a Systematic Approach To Dissect Virulence Traits in Candida albicans

mSphere ◽

10.1128/msphere.00258-19 ◽

2019 ◽

Vol 4 (3) ◽

Author(s):

J. Christian Pérez

Keyword(s):

Candida Albicans ◽

Systematic Approach ◽

Genetic Manipulation ◽

Homozygous Deletion ◽

Mammalian Host ◽

Genetic Screens ◽

Content Type ◽

Link Type ◽

Virulence Traits ◽

Deletion Library

ABSTRACT J. Christian Pérez studies the interplay between Candida albicans and the mammalian host. In this mSphere of Influence article, he reflects on how “Systematic screens of a Candida albicans homozygous deletion library decouple morphogenetic switching and pathogenicity” (S. M. Noble, S. French, L. A. Kohn, V. Chen, et al., Nat Genet 42:590–598, 2010, https://doi.org/10.1038/ng.605) provided tools and a blueprint for open-ended genetic screens in an organism that had been a challenge for genetic manipulation.

Download Full-text

Dynamic transcriptional response of Saccharomyces cerevisiae cells to copper

Scientific Reports ◽

10.1038/s41598-020-75511-w ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Sebnem Oc ◽

Serpil Eraslan ◽

Betul Kirdar

Keyword(s):

Saccharomyces Cerevisiae ◽

Cellular Response ◽

Molecular Mechanisms ◽

Clustering Algorithms ◽

Ion Homeostasis ◽

Model Organism ◽

Transcriptional Response ◽

Homozygous Deletion ◽

Suitable Model ◽

Mitochondrial Respiratory Chain Complex

Abstract Copper is a crucial trace element for all living systems and any deficiency in copper homeostasis leads to the development of severe diseases in humans. The observation of extensive evolutionary conservation in copper homeostatic systems between human and Saccharomyces cerevisiae made this organism a suitable model organism for elucidating molecular mechanisms of copper transport and homeostasis. In this study, the dynamic transcriptional response of both the reference strain and homozygous deletion mutant strain of CCC2, which encodes a Cu2+-transporting P-type ATPase, were investigated following the introduction of copper impulse to reach a copper concentration which was shown to improve the respiration capacity of CCC2 deletion mutants. The analysis of data by using different clustering algorithms revealed significantly affected processes and pathways in response to a switch from copper deficient environment to elevated copper levels. Sulfur compound, methionine and cysteine biosynthetic processes were identified as significantly affected processes for the first time in this study. Stress response, cellular response to DNA damage, iron ion homeostasis, ubiquitin dependent proteolysis, autophagy and regulation of macroautophagy, DNA repair and replication, as well as organization of mitochondrial respiratory chain complex IV, mitochondrial organization and translation were identified as significantly affected processes in only CCC2 deleted strain. The integration of the transcriptomic data with regulome revealed the differences in the extensive re-wiring of dynamic transcriptional organization and regulation in these strains.

Download Full-text

Histone H3 and H4 gene deletions in Saccharomyces cerevisiae.

The Journal of Cell Biology ◽

10.1083/jcb.106.3.557 ◽

1988 ◽

Vol 106 (3) ◽

pp. 557-566 ◽

Cited By ~ 20

Author(s):

M M Smith ◽

V B Stirling

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle Progression ◽

Histone H3 ◽

Phenotypic Expression ◽

Gene Replacement ◽

Homozygous Deletion ◽

Chromosome Loss ◽

Temperature Sensitive ◽

Wild Type ◽

Gene Deletions

The genome of haploid Saccharomyces cerevisiae contains two nonallelic sets of histone H3 and H4 genes. Strains with deletions of each of these loci were constructed by gene replacement techniques. Mutants containing deletions of either gene set were viable, however meiotic segregants lacking both histone H3 and H4 gene loci were inviable. In haploid cells no phenotypic expression of the histone gene deletions was observed; deletion mutants had wild-type growth rates, were not temperature sensitive for growth, and mated normally. However, diploids homozygous for the H3-H4 gene deletions were slightly defective in their growth and cell cycle progression. The generation times of the diploid mutants were longer than wild-type cells, the size distributions of cells from exponentially growing cultures were skewed towards larger cell volumes, and the G1 period of the mutant cells was longer than that of the wild-type diploid. The homozygous deletion of the copy-II set of H3-H4 genes in diploids also increased the frequency of mitotic chromosome loss as measured using a circular plasmid minichromosome assay.

Download Full-text

Ssd1 Is Required for Thermotolerance and Hsp104-Mediated Protein Disaggregation in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.02271-07 ◽

2008 ◽

Vol 29 (1) ◽

pp. 187-200 ◽

Cited By ~ 24

Author(s):

Snober S. Mir ◽

David Fiedler ◽

Anil G. Cashikar

Keyword(s):

Saccharomyces Cerevisiae ◽

Gene Deletion ◽

Acute Stress ◽

Protein Aggregates ◽

Yeast Gene ◽

Cell Wall Remodeling ◽

Polymorphic Gene ◽

Cellular Integrity ◽

Regulate Protein ◽

Deletion Library

ABSTRACT In the budding yeast Saccharomyces cerevisiae, the Hsp104-mediated disaggregation of protein aggregates is essential for thermotolerance and to facilitate the maintenance of prions. In humans, protein aggregation is associated with neuronal death and dysfunction in many neurodegenerative diseases. Mechanisms of aggregation surveillance that regulate protein disaggregation are likely to play a major role in cell survival after acute stress. However, such mechanisms have not been studied. In a screen using the yeast gene deletion library for mutants unable to survive an aggregation-inducing heat stress, we find that SSD1 is required for Hsp104-mediated protein disaggregation. SSD1 is a polymorphic gene that plays a role in cellular integrity, longevity, and pathogenicity in yeast. Allelic variants of SSD1 regulate the level of thermotolerance and cell wall remodeling. We have shown that Ssd1 influences the ability of Hsp104 to hexamerize, to interact with the cochaperone Sti1, and to bind protein aggregates. These results provide a paradigm for linking Ssd1-mediated cellular integrity and Hsp104-mediated disaggregation to ensure the survival of cells with fewer aggregates.

Download Full-text