scholarly journals Suboptimal Global Transcriptional Response Increases the Harmful Effects of Loss-of-Function Mutations

2020 ◽  
Author(s):  
Károly Kovács ◽  
Zoltán Farkas ◽  
Djordje Bajić ◽  
Dorottya Kalapis ◽  
Andreea Daraba ◽  
...  
Keyword(s):  
Gene Deletion ◽  
Single Gene ◽  
Transcriptional Response ◽  
Gene Inactivation ◽  
Loss Of Function ◽  
Gene Deletions ◽  
Genomic Expression ◽  
Deleterious Gene ◽  
Functionally Diverse ◽  
Harmful Effects

Abstract The fitness impact of loss-of-function mutations is generally assumed to reflect the loss of specific molecular functions associated with the perturbed gene. Here, we propose that rewiring of the transcriptome upon deleterious gene inactivation is frequently nonspecific and mimics stereotypic responses to external environmental change. Consequently, transcriptional response to gene deletion could be suboptimal and incur an extra fitness cost. Analysis of the transcriptomes of ∼1,500 single-gene deletion Saccharomyces cerevisiae strains supported this scenario. First, most transcriptomic changes are not specific to the deleted gene but are rather triggered by perturbations in functionally diverse genes. Second, gene deletions that alter the expression of dosage-sensitive genes are especially harmful. Third, by elevating the expression level of downregulated genes, we could experimentally mitigate the fitness defect of gene deletions. Our work shows that rewiring of genomic expression upon gene inactivation shapes the harmful effects of mutations.

scholarly journals Rapid and assured genetic engineering methods applied to Acinetobacter baylyi ADP1 genome streamlining

2020 ◽  
Vol 48 (8) ◽  
pp. 4585-4600
Author(s):  
Gabriel A Suárez ◽  
Kyle R Dugan ◽  
Brian A Renda ◽  
Sean P Leonard ◽  
Lakshmi Suryateja Gangavarapu ◽  
...  
Keyword(s):  
Gene Deletion ◽  
Single Gene ◽  
Acinetobacter Baylyi ◽  
Gene Deletions ◽  
Multiple Gene ◽  
Transposon Insertion ◽  
A Genome ◽  
Acinetobacter Baylyi Adp1 ◽  
Transposon Insertion Sequencing ◽  
Improved Methods

Abstract One goal of synthetic biology is to improve the efficiency and predictability of living cells by removing extraneous genes from their genomes. We demonstrate improved methods for engineering the genome of the metabolically versatile and naturally transformable bacterium Acinetobacter baylyi ADP1 and apply them to a genome streamlining project. In Golden Transformation, linear DNA fragments constructed by Golden Gate Assembly are directly added to cells to create targeted deletions, edits, or additions to the chromosome. We tested the dispensability of 55 regions of the ADP1 chromosome using Golden Transformation. The 18 successful multiple-gene deletions ranged in size from 21 to 183 kb and collectively accounted for 23.4% of its genome. The success of each multiple-gene deletion attempt could only be partially predicted on the basis of an existing collection of viable ADP1 single-gene deletion strains and a new transposon insertion sequencing (Tn-Seq) dataset that we generated. We further show that ADP1’s native CRISPR/Cas locus is active and can be retargeted using Golden Transformation. We reprogrammed it to create a CRISPR-Lock, which validates that a gene has been successfully removed from the chromosome and prevents it from being reacquired. These methods can be used together to implement combinatorial routes to further genome streamlining and for more rapid and assured metabolic engineering of this versatile chassis organism.

scholarly journals Analysis of the Contribution of cyp51 Genes to Azole Resistance in Aspergillus Section Nigri with the CRISPR-Cas9 Technique

2021 ◽  
Vol 65 (5) ◽  
Author(s):  
Alba Pérez-Cantero ◽  
Adela Martin-Vicente ◽  
Josep Guarro ◽  
Jarrod R. Fortwendel ◽  
Javier Capilla
Keyword(s):  
Single Gene ◽  
Transcriptional Response ◽  
Azole Resistance ◽  
Gene Deletions ◽  
Content Type ◽  
Ergosterol Content ◽  
Expression Studies ◽  
Gene Expression Studies ◽  
Induced Changes ◽  
The Impact

ABSTRACT Cyp51 contribution to azole resistance has been broadly studied and characterized in Aspergillus fumigatus, whereas it remains poorly investigated in other clinically relevant species of the genus, such as those of section Nigri. In this work, we aimed to analyze the impact of cyp51 genes (cyp51A and cyp51B) on the voriconazole (VRC) response and resistance of Aspergillus niger and Aspergillus tubingensis. We generated CRISPR-Cas9 cyp51A and cyp51B knockout mutants from strains with different genetic backgrounds and diverse patterns of azole susceptibility. Single-gene deletions of cyp51 genes resulted in 2- to 16-fold decreases of the VRC MIC values, which were below the VRC epidemiological cutoff value (ECV) established by the Clinical and Laboratory Standards Institute (CLSI), irrespective of their parental strains’ susceptibilities. Gene expression studies in the tested species confirmed that cyp51A participates more actively than cyp51B in the transcriptional response of azole stress. However, ergosterol quantification revealed that both enzymes comparably impact the total ergosterol content within the cell, as basal- and VRC-induced changes to ergosterol content were similar in all cases. These data contribute to our understanding of Aspergillus azole resistance, especially in non-A. fumigatus species.

scholarly journals Towards a systematic map of the functional role of protein phosphorylation

2019 ◽  
Author(s):  
Cristina Viéitez ◽  
Bede P. Busby ◽  
David Ochoa ◽  
André Mateus ◽  
Marco Galardini ◽  
...  
Keyword(s):  
Gene Deletion ◽  
Loss Of Function ◽  
Post Translational Modification ◽  
Systematic Map ◽  
Gene Deletions ◽  
Chemical Genetic ◽  
Cellular Processes ◽  
Growth Profiles ◽  
Functional Relevance ◽  
Almost All

AbstractPhosphorylation is a critical post-translational modification involved in the regulation of almost all cellular processes. However, less than 5% of thousands of recently discovered phosphorylation sites have a known function. Here, we devised a chemical genetic approach to study the functional relevance of phosphorylation in S. cerevisiae. We generated 474 phospho-deficient mutants that, along with the gene deletion library, were screened for fitness in 102 conditions. Of these, 42% exhibited growth phenotypes, suggesting these phosphosites are likely functional. We inferred their function based on the similarity of their growth profiles with that of gene deletions, and validated a subset by thermal proteome profiling and lipidomics. While some phosphomutants showed loss-of-function phenotypes, a higher fraction exhibited phenotypes not seen in the corresponding gene deletion suggestive of a gain-of-function effect. For phosphosites conserved in humans, the severity of the yeast phenotypes is indicative of their human functional relevance. This study provides a roadmap for functionally characterizing phosphorylation in a systematic manner.

scholarly journals Protein Moonlighting Revealed by Non-Catalytic Phenotypes of Yeast Enzymes

2017 ◽  
Author(s):  
Adriana Espinosa-Cantú ◽  
Diana Ascencio ◽  
Selene Herrera-Basurto ◽  
Jiewei Xu ◽  
Assen Roguev ◽  
...  
Keyword(s):  
Gene Deletion ◽  
Gene Loss ◽  
Genetic Interaction ◽  
Single Gene ◽  
Chromatin Modification ◽  
Biological Processes ◽  
Gene Deletions ◽  
Multifunctional Protein ◽  
Regulatory Processes ◽  
Catalytic Function

ABSTRACTA single gene can partake in several biological processes, and therefore gene deletions can lead to different—sometimes unexpected—phenotypes. However, it is not always clear whether such pleiotropy reflects the loss of a unique molecular activity involved in different processes or the loss of a multifunctional protein. Here, usingSaccharomyces cerevisiaemetabolism as a model, we systematically test the null hypothesis that enzyme phenotypes depend on a single annotated molecular function, namely their catalysis. We screened a set of carefully selected genes by quantifying the contribution of catalysis to gene-deletion phenotypes under different environmental conditions. While most phenotypes were explained by loss of catalysis, 30% could be readily complemented by a catalytically-inactive enzyme. Such non-catalytic phenotypes were frequent in the Alt1 and Bat2 transaminases and in the isoleucine/valine-biosynthetic enzymes Ilv1 and Ilv2, suggesting novel "moonlighting" activities in these proteins. Furthermore, differential genetic-interaction profiles of gene-deletion and catalytic mutants indicated thatILV1is functionally associated to regulatory processes, specifically to chromatin modification. Our systematic study shows that gene-loss phenotypes and their genetic interactions are frequently not driven by the loss of an annotated catalytic function, underscoring the moonlighting nature of cellular metabolism.

scholarly journals Mutability of demographic noise in microbial range expansions

2021 ◽  
Author(s):  
QinQin Yu ◽  
Matti Gralka ◽  
Marie-Cécilia Duvernoy ◽  
Megan Sousa ◽  
Arbel Harpak ◽  
...  
Keyword(s):  
Gene Deletion ◽  
Genetic Manipulation ◽  
Single Gene ◽  
Population Level ◽  
Growth Conditions ◽  
Establishment Probability ◽  
In The Wild ◽  
Order Of Magnitude ◽  
Demographic Noise ◽  
Single Gene Deletion

AbstractDemographic noise, the change in the composition of a population due to random birth and death events, is an important driving force in evolution because it reduces the efficacy of natural selection. Demographic noise is typically thought to be set by the population size and the environment, but recent experiments with microbial range expansions have revealed substantial strain-level differences in demographic noise under the same growth conditions. Many genetic and phenotypic differences exist between strains; to what extent do single mutations change the strength of demographic noise? To investigate this question, we developed a high-throughput method for measuring demographic noise in colonies without the need for genetic manipulation. By applying this method to 191 randomly-selected single gene deletion strains from the E. coli Keio collection, we find that a typical single gene deletion mutation decreases demographic noise by 8% (maximal decrease: 81%). We find that the strength of demographic noise is an emergent trait at the population level that can be predicted by colony-level traits but not cell-level traits. The observed differences in demographic noise from single gene deletions can increase the establishment probability of beneficial mutations by almost an order of magnitude (compared to in the wild type). Our results show that single mutations can substantially alter adaptation through their effects on demographic noise and suggest that demographic noise can be an evolvable trait of a population.

scholarly journals CHOP and c-JUN up-regulate the mutant Z α1-antitrypsin, exacerbating its aggregation and liver proteotoxicity

2020 ◽  
Vol 295 (38) ◽  
pp. 13213-13223
Author(s):  
Sergio Attanasio ◽  
Rosa Ferriero ◽  
Gwladys Gernoux ◽  
Rossella De Cegli ◽  
Annamaria Carissimo ◽  
...  
Keyword(s):  
Liver Disease ◽  
Er Stress ◽  
Proteinase Inhibitors ◽  
Pediatric Population ◽  
Transcriptional Response ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Primary Role ◽  
Loss Of Function ◽  
Human Livers

α1-Antitrypsin (AAT) encoded by the SERPINA1 gene is an acute-phase protein synthesized in the liver and secreted into the circulation. Its primary role is to protect lung tissue by inhibiting neutrophil elastase. The Z allele of SERPINA1 encodes a mutant AAT, named ATZ, that changes the protein structure and leads to its misfolding and polymerization, which cause endoplasmic reticulum (ER) stress and liver disease through a gain-of-function toxic mechanism. Hepatic retention of ATZ results in deficiency of one of the most important circulating proteinase inhibitors and predisposes to early-onset emphysema through a loss-of-function mechanism. The pathogenetic mechanisms underlying the liver disease are not completely understood. C/EBP-homologous protein (CHOP), a transcription factor induced by ER stress, was found among the most up-regulated genes in livers of PiZ mice that express ATZ and in human livers of patients homozygous for the Z allele. Compared with controls, juvenile PiZ/Chop−/− mice showed reduced hepatic ATZ and a transcriptional response indicative of decreased ER stress by RNA-Seq analysis. Livers of PiZ/Chop−/− mice also showed reduced SERPINA1 mRNA levels. By chromatin immunoprecipitations and luciferase reporter–based transfection assays, CHOP was found to up-regulate SERPINA1 cooperating with c-JUN, which was previously shown to up-regulate SERPINA1, thus aggravating hepatic accumulation of ATZ. Increased CHOP levels were detected in diseased livers of children homozygous for the Z allele. In summary, CHOP and c-JUN up-regulate SERPINA1 transcription and play an important role in hepatic disease by increasing the burden of proteotoxic ATZ, particularly in the pediatric population.

scholarly journals Single Gene Deletions of Orexin, Leptin, Neuropeptide Y, and Ghrelin Do Not Appreciably Alter Food Anticipatory Activity in Mice

PLoS ONE ◽  
2011 ◽  
Vol 6 (3) ◽  
pp. e18377 ◽  
Author(s):  
Keith M. Gunapala ◽  
Christian M. Gallardo ◽  
Cynthia T. Hsu ◽  
Andrew D. Steele
Keyword(s):  
Neuropeptide Y ◽  
Single Gene ◽  
Gene Deletions ◽  
Food Anticipatory Activity ◽  
Anticipatory Activity

scholarly journals Rapid and assured genetic engineering methods applied to Acinetobacter baylyi ADP1 genome streamlining

2019 ◽  
Author(s):  
Gabriel A. Suárez ◽  
Kyle R. Dugan ◽  
Brian A. Renda ◽  
Sean P. Leonard ◽  
Lakshmi S. Gangavarapu ◽  
...  
Keyword(s):  
Gene Knockout ◽  
Single Gene ◽  
Golden Gate ◽  
Acinetobacter Baylyi ◽  
Gene Deletions ◽  
Multiple Gene ◽  
A Genome ◽  
Strain Collection ◽  
Acinetobacter Baylyi Adp1 ◽  
Improved Methods

ABSTRACTOne goal of synthetic biology is to improve the efficiency and predictability of living cells by removing extraneous genes from their genomes. We demonstrate improved methods for engineering the genome of the metabolically versatile and naturally transformable bacterium Acinetobacter baylyi ADP1 and apply them to a genome streamlining project. In Golden Transformation, linear DNA fragments constructed by Golden Gate Assembly are directly added to cells to create targeted deletions, edits, or additions to the chromosome. We tested the dispensability of 55 regions of the ADP1 chromosome using Golden Transformation. The 19 successful multiple-gene deletions ranged in size from 21 to 183 kilobases and collectively accounted for 24.6% of its genome. Deletion success could only be partially predicted on the basis of a single-gene knockout strain collection and a new Tn-Seq experiment. We further show that ADP1’s native CRISPR/Cas locus is active and can be retargeted using Golden Transformation. We reprogrammed it to create a CRISPR-Lock, which validates that a gene has been successfully removed from the chromosome and prevents it from being reacquired. These methods can be used together to implement combinatorial routes to further genome streamlining and for more rapid and assured metabolic engineering of this versatile chassis organism.

scholarly journals Zebrafish Cre/lox regulated UFlip alleles generated by CRISPR/Cas targeted integration provide cell-type specific conditional gene inactivation

2021 ◽  
Author(s):  
Maira P. Almeida ◽  
Sekhar Kambakam ◽  
Fang Liu ◽  
Zhitao Ming ◽  
Jordan M. Welker ◽  
...  
Keyword(s):  
Gene Function ◽  
Cre Recombinase ◽  
Gene Trap ◽  
Gene Inactivation ◽  
Loss Of Function ◽  
Cell Type ◽  
Active Gene ◽  
Indel Mutation ◽  
Targeted Integration ◽  
Cell Type Specific

The ability to regulate gene activity spatially and temporally is essential to investigate cell type specific gene function during development and in postembryonic processes and disease models. The Cre/lox system has been widely used for performing cell and tissue-specific conditional analysis of gene function in zebrafish, but simple and efficient methods for isolation of stable, Cre/lox regulated alleles are lacking. Here we applied our GeneWeld CRISPR/Cas9 short homology-directed targeted integration strategy to generate floxed conditional alleles that provide robust gene knockdown and strong loss of function phenotypes. A universal targeting vector, UFlip, with sites for cloning short 24-48 bp homology arms flanking a floxed mRFP gene trap plus secondary reporter cassette, was integrated into an intron in hdac1, rbbp4, and rb1. Active, gene off orientation hdac1-UFlip-Off and rb1-UFlip-Off integration alleles result in >99% reduction of gene expression in homozygotes and recapitulate known indel loss of function phenotypes. Passive, gene on orientation rbbp4-UFlip-On and rb1-UFlip-On integration alleles do not cause phenotypes in trans-heterozygous combination with an indel mutation. Cre recombinase injection leads to recombination at alternating pairs of loxP and lox2272 sites, inverting and locking the cassette into the active, gene off orientation, and the expected mutant phenotypes. In combination with our endogenous neural progenitor Cre drivers we demonstrate rbbp4-UFlip-On and rb1-UFlip-On gene inactivation phenotypes can be restricted to specific neural cell populations. Replacement of the UFlip mRFP primary reporter gene trap with a 2A-RFP in rbbp4-UFlip-Off, or 2A-KalTA4 in rb1-UFlip-Off, shows strong RFP expression in wild type or UAS:RFP injected embryos, respectively. Together these results validate a simplified approach for efficient isolation of highly mutagenic Cre/lox responsive conditional gene alleles to advance zebrafish Cre recombinase genetics.

scholarly journals Damage Signaling by Extracellular Nucleotides: A Role for Cyclic Nucleotides in Elevating Cytosolic Free Calcium?

2021 ◽  
Vol 12 ◽  
Author(s):  
Jian Sun ◽  
Youzheng Ning ◽  
Limin Wang ◽  
Katie A. Wilkins ◽  
Julia M. Davies
Keyword(s):  
Cyclic Nucleotides ◽  
Transcriptional Response ◽  
Extracellular Nucleotides ◽  
Free Calcium ◽  
Loss Of Function ◽  
Cyclic Nucleotide Gated Channels ◽  
Molecular Pattern ◽  
Defense Response Genes ◽  
Pathogen Attack ◽  
Camp And Cgmp Production

Extracellular ATP (eATP) is now held to be a constitutive damage-associated molecular pattern (DAMP) that is released by wounding, herbivory or pathogen attack. The concentration of eATP must be tightly regulated as either depletion or overload leads to cell death. In Arabidopsis thaliana, sensing of eATP is by two plasma membrane legume-like lectin serine–threonine receptor kinases (P2K1 and P2K2), although other receptors are postulated. The transcriptional response to eATP is dominated by wound- and defense-response genes. Wounding and pathogen attack can involve the cyclic nucleotides cyclic AMP (cAMP) and cyclic GMP (cGMP) which, in common with eATP, can increase cytosolic-free Ca2+ as a second messenger. This perspective on DAMP signaling by eATP considers the possibility that the eATP pathway involves production of cyclic nucleotides to promote opening of cyclic nucleotide-gated channels and so elevates cytosolic-free Ca2+. In silico analysis of P2K1 and P2K2 reveals putative adenylyl and guanylyl kinase sequences that are the hallmarks of “moonlighting” receptors capable of cAMP and cGMP production. Further, an Arabidopsis loss of function cngc mutant was found to have an impaired increase in cytosolic-free Ca2+ in response to eATP. A link between eATP, cyclic nucleotides, and Ca2+ signaling therefore appears credible.

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