scholarly journals Quantitative Genome-Wide Analysis of Yeast Deletion Strain Sensitivities to Oxidative and Chemical Stress

2004 ◽  
Vol 5 (3) ◽  
pp. 216-224 ◽  
Author(s):  
Chandra L. Tucker ◽  
Stanley Fields
Keyword(s):  
Hydrogen Peroxide ◽  
Large Scale ◽  
Chemical Resistance ◽  
Lethal Effect ◽  
Sublethal Dose ◽  
Synthetic Lethal ◽  
Cellular Processes ◽  
Molecular Events ◽  
Genome Wide ◽  
A Cell

Understanding the actions of drugs and toxins in a cell is of critical importance to medicine, yet many of the molecular events involved in chemical resistance are relatively uncharacterized. In order to identify the cellular processes and pathways targeted by chemicals, we took advantage of the haploidSaccharomyces cerevisiaedeletion strains (Winzeleret al.,1999). Although ~4800 of the strains are viable, the loss of a gene in a pathway affected by a drug can lead to a synthetic lethal effect in which the combination of a deletion and a normally sublethal dose of a chemical results in loss of viability. WE carried out genome-wide screens to determine quantitative sensitivities of the deletion set to four chemicals: hydrogen peroxide, menadione, ibuprofen and mefloquine. Hydrogen peroxide and menadione induce oxidative stress in the cell, whereas ibuprofen and mefloquine are toxic to yeast by unknown mechanisms. Here we report the sensitivities of 659 deletion strains that are sensitive to one or more of these four compounds, including 163 multichemicalsensitive strains, 394 strains specific to hydrogen peroxide and/or menadione, 47 specific to ibuprofen and 55 specific to mefloquine.We correlate these results with data from other large-scale studies to yield novel insights into cellular function.

scholarly journals Multiplex enCas12a screens show functional buffering by paralogs is systematically absent from genome-wide CRISPR/Cas9 knockout screens

2020 ◽  
Author(s):  
Merve Dede ◽  
Megan McLaughlin ◽  
Eiru Kim ◽  
Traver Hart
Keyword(s):  
Cell Lines ◽  
Protein Complexes ◽  
Essential Genes ◽  
Systematic Analysis ◽  
Synthetic Lethal ◽  
Genome Wide ◽  
Synthetic Lethal Interactions ◽  
Crispr Screen ◽  
A Cell ◽  
Genetic Buffering

AbstractMajor efforts on pooled library CRISPR knockout screening across hundreds of cell lines have identified genes whose disruption leads to fitness defects, a critical step in identifying candidate cancer targets. However, the number of essential genes detected from these monogenic knockout screens are very low compared to the number of constitutively expressed genes in a cell, raising the question of why there are so few essential genes. Through a systematic analysis of screen data in cancer cell lines generated by the Cancer Dependency Map, we observed that half of all constitutively-expressed genes are never hits in any CRISPR screen, and that these never-essentials are highly enriched for paralogs. We investigated paralog buffering through systematic dual-gene CRISPR knockout screening by testing algorithmically defined ~400 candidate paralog pairs with the enCas12a multiplex knockout system in three cell lines. We observed 24 synthetic lethal paralog pairs which have escaped detection by monogenic knockout screens at stringent thresholds. Nineteen of 24 (79%) synthetic lethal interactions were present in at least two out of three cell lines and 14 of 24 (58%) were present in all three cell lines tested, including alternate subunits of stable protein complexes as well as functionally redundant enzymes. Together these observations strongly suggest that paralogs represent a targetable set of genetic dependencies that are systematically under-represented among cell-essential genes due to genetic buffering in monogenic CRISPR-based mammalian functional genomics approaches.

scholarly journals Non-B DNA: a major contributor to small- and large-scale variation in nucleotide substitution frequencies across the genome

2021 ◽  
Vol 49 (3) ◽  
pp. 1497-1516
Author(s):  
Wilfried M Guiblet ◽  
Marzia A Cremona ◽  
Robert S Harris ◽  
Di Chen ◽  
Kristin A Eckert ◽  
...  
Keyword(s):  
Large Scale ◽  
Nucleotide Substitution ◽  
Genetic Diseases ◽  
Nucleotide Polymorphisms ◽  
Dna Structures ◽  
Cellular Processes ◽  
Genome Wide ◽  
Dna Types ◽  
Flanking Regions

Abstract Approximately 13% of the human genome can fold into non-canonical (non-B) DNA structures (e.g. G-quadruplexes, Z-DNA, etc.), which have been implicated in vital cellular processes. Non-B DNA also hinders replication, increasing errors and facilitating mutagenesis, yet its contribution to genome-wide variation in mutation rates remains unexplored. Here, we conducted a comprehensive analysis of nucleotide substitution frequencies at non-B DNA loci within noncoding, non-repetitive genome regions, their ±2 kb flanking regions, and 1-Megabase windows, using human-orangutan divergence and human single-nucleotide polymorphisms. Functional data analysis at single-base resolution demonstrated that substitution frequencies are usually elevated at non-B DNA, with patterns specific to each non-B DNA type. Mirror, direct and inverted repeats have higher substitution frequencies in spacers than in repeat arms, whereas G-quadruplexes, particularly stable ones, have higher substitution frequencies in loops than in stems. Several non-B DNA types also affect substitution frequencies in their flanking regions. Finally, non-B DNA explains more variation than any other predictor in multiple regression models for diversity or divergence at 1-Megabase scale. Thus, non-B DNA substantially contributes to variation in substitution frequencies at small and large scales. Our results highlight the role of non-B DNA in germline mutagenesis with implications to evolution and genetic diseases.

scholarly journals Large Scale Identification of Genes Involved in Cell Surface Biosynthesis and Architecture in Saccharomyces cerevisiae

Genetics ◽  
1997 ◽  
Vol 147 (2) ◽  
pp. 435-450 ◽  
Author(s):  
Marc Lussier ◽  
Ann-Marie White ◽  
Jane Sheraton ◽  
Tiziano di Paolo ◽  
Julie Treadwell ◽  
...  
Keyword(s):  
Cell Surface ◽  
Large Scale ◽  
Cell Function ◽  
Eukaryotic Cell ◽  
Yeast Genome ◽  
Calcofluor White ◽  
Genome Database ◽  
Cellular Processes ◽  
Genome Wide ◽  
Mutant Phenotypes

The sequenced yeast genome offers a unique resource for the analysis of eukaryotic cell function and enables genome-wide screens for genes involved in cellular processes. We have identified genes involved in cell surface assembly by screening transposon-mutagenized cells for altered sensitivity to calcofluor white, followed by supplementary screens to further characterize mutant phenotypes. The mutated genes were directly retrieved from genomic DNA and then matched uniquely to a gene in the yeast genome database. Eighty-two genes with apparent perturbation of the cell surface were identified, with mutations in 65 of them displaying at least one further cell surface phenotype in addition to their modified sensitivity to calcofluor. Fifty of these genes were previously known, 17 encoded proteins whose function could be anticipated through sequence homology or previously recognized phenotypes and 15 genes had no previously known phenotype.

Seasonally hibernating phenotype assessed through transcript screening

2006 ◽  
Vol 24 (1) ◽  
pp. 13-22 ◽  
Author(s):  
Daryl R. Williams ◽  
L. Elaine Epperson ◽  
Weizhong Li ◽  
Margaret A. Hughes ◽  
Ruth Taylor ◽  
...  
Keyword(s):  
Large Scale ◽  
Cardiac Tissue ◽  
Rna Binding ◽  
Transcriptional Responses ◽  
Spermophilus Lateralis ◽  
Cellular Processes ◽  
New Genes ◽  
Molecular Events ◽  
Unbiased Gene ◽  
Transcriptional Changes

Hibernation is a seasonally entrained and profound phenotypic transition to conserve energy in winter. It involves significant biochemical reprogramming, although our understanding of the underpinning molecular events is fragmentary and selective. We have conducted a large-scale gene expression screen of the golden-mantled ground squirrel, Spermophilus lateralis, to identify transcriptional responses associated specifically with the summer-winter transition and the torpid-arousal transition in winter. We used 112 cDNA microarrays comprising 12,288 probes that cover at least 5,109 genes. In liver, the profiles of torpid and active states in the winter were almost identical, although we identified 102 cDNAs that were differentially expressed between winter and summer, 90% of which were downregulated in the winter states. By contrast, in cardiac tissue, 59 and 115 cDNAs were elevated in interbout arousal and torpor, respectively, relative to the summer active condition, but only 7 were common to both winter states, and during arousal none was downregulated. In brain, 78 cDNAs were found to change in winter, 44 of which were upregulated. Thus transcriptional changes associated with hibernation are qualitatively modest and, since these changes are generally less than twofold, also quantitatively modest. Unbiased Gene Ontology profiling of the transcripts suggests a winter switch to β-oxidation of lipids in liver and heart, a reduction in metabolism of toxic compounds and the urea cycle in liver, and downregulated electron transport in the brain. We identified just one strongly winter-induced transcript common to all tissues, namely an RNA-binding protein, RBM3. This analysis clearly differentiates responses of the principal tissues, identifies a large number of new genes undergoing regulation, and broadens our understanding of affected cellular processes that, in part, account for the winter-adaptive hibernating phenotype.

scholarly journals The Bright and Dark Side of DNA Methylation: A Matter of Balance

Cells ◽  
2019 ◽  
Vol 8 (10) ◽  
pp. 1243 ◽  
Author(s):  
Marta Borchiellini ◽  
Simone Ummarino ◽  
Annalisa Di Ruscio
Keyword(s):  
Dna Methylation ◽  
Developmental Stages ◽  
Germ Line ◽  
Dark Side ◽  
Epigenetic Mark ◽  
Cellular Processes ◽  
Biological Responses ◽  
Molecular Events ◽  
Genome Wide ◽  
Health Related

DNA methylation controls several cellular processes, from early development to old age, including biological responses to endogenous or exogenous stimuli contributing to disease transition. As a result, minimal DNA methylation changes during developmental stages drive severe phenotypes, as observed in germ-line imprinting disorders, while genome-wide alterations occurring in somatic cells are linked to cancer onset and progression. By summarizing the molecular events governing DNA methylation, we focus on the methods that have facilitated mapping and understanding of this epigenetic mark in healthy conditions and diseases. Overall, we review the bright (health-related) and dark (disease-related) side of DNA methylation changes, outlining how bulk and single-cell genomic analyses are moving toward the identification of new molecular targets and driving the development of more specific and less toxic demethylating agents.

scholarly journals Targeting Cancer at the Intersection of Signaling and Epigenetics

2019 ◽  
Vol 3 (1) ◽  
pp. 365-384
Author(s):  
Stephanie Guerra ◽  
Karen Cichowski
Keyword(s):  
Large Scale ◽  
Human Cancer ◽  
Biological Response ◽  
Specific Drug ◽  
Epigenetic Alterations ◽  
Synthetic Lethal ◽  
Epigenetic State ◽  
Synthetic Lethal Interactions ◽  
A Cell ◽  
Chronic Activation

While mutations resulting in the chronic activation of signaling pathways drive human cancer, the epigenetic state of a cell ultimately dictates the biological response to any given oncogenic signal. Moreover, large-scale genomic sequencing efforts have now identified a plethora of mutations in chromatin regulatory genes in human tumors, which can amplify, modify, or complement traditional oncogenic events. Nevertheless, the co-occurrence of oncogenic and epigenetic defects appears to create novel therapeutic vulnerabilities, which can be targeted by specific drug combinations. Here we discuss general mechanisms by which oncogenic and epigenetic alterations cooperate in human cancer and synthesize the field's early efforts in developing promising therapeutic combinations. Collectively, these studies reveal common themes underlying potential chemical synthetic lethal interactions and support both the expansion and refinement of this type of therapeutic approach.

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