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Molecular Systems Biology 2006We have systematically made a set of precisely defined, single-gene deletions of all nonessential genes in Escherichia coli K-12. Open-reading frame coding regions were...
We have systematically made a set of precisely defined, single-gene deletions of all nonessential genes in Escherichia coli K-12. Open-reading frame coding regions were replaced with a kanamycin cassette flanked by FLP recognition target sites by using a one-step method for inactivation of chromosomal genes and primers designed to create in-frame deletions upon excision of the resistance cassette. Of 4288 genes targeted, mutants were obtained for 3985. To alleviate problems encountered in high-throughput studies, two independent mutants were saved for every deleted gene. These mutants-the 'Keio collection'-provide a new resource not only for systematic analyses of unknown gene functions and gene regulatory networks but also for genome-wide testing of mutational effects in a common strain background, E. coli K-12 BW25113. We were unable to disrupt 303 genes, including 37 of unknown function, which are candidates for essential genes. Distribution is being handled via GenoBase (http://ecoli.aist-nara.ac.jp/).
Topics: Escherichia coli; Gene Deletion; Internet; Mutation; Organisms, Genetically Modified
PubMed: 16738554
DOI: 10.1038/msb4100050 -
Cell Systems Mar 2017A systems-level understanding of Gram-positive bacteria is important from both an environmental and health perspective and is most easily obtained when high-quality,...
A systems-level understanding of Gram-positive bacteria is important from both an environmental and health perspective and is most easily obtained when high-quality, validated genomic resources are available. To this end, we constructed two ordered, barcoded, erythromycin-resistance- and kanamycin-resistance-marked single-gene deletion libraries of the Gram-positive model organism, Bacillus subtilis. The libraries comprise 3,968 and 3,970 genes, respectively, and overlap in all but four genes. Using these libraries, we update the set of essential genes known for this organism, provide a comprehensive compendium of B. subtilis auxotrophic genes, and identify genes required for utilizing specific carbon and nitrogen sources, as well as those required for growth at low temperature. We report the identification of enzymes catalyzing several missing steps in amino acid biosynthesis. Finally, we describe a suite of high-throughput phenotyping methodologies and apply them to provide a genome-wide analysis of competence and sporulation. Altogether, we provide versatile resources for studying gene function and pathway and network architecture in Gram-positive bacteria.
Topics: Amino Acids; Bacillus subtilis; Gene Deletion; Gene Library; Genomic Library; Genomics; High-Throughput Screening Assays; Sequence Deletion; Spores, Bacterial
PubMed: 28189581
DOI: 10.1016/j.cels.2016.12.013 -
Nature Communications Dec 2022Regulatory T cells (Tregs) are critically involved in neovascularization, an important compensatory mechanism in peripheral artery disease. The contribution of G protein...
Regulatory T cells (Tregs) are critically involved in neovascularization, an important compensatory mechanism in peripheral artery disease. The contribution of G protein coupled receptor 174 (GPR174), which is a regulator of Treg function and development, in neovascularization remains elusive. Here, we show that genetic deletion of GPR174 in Tregs potentiated blood flow recovery in mice after hindlimb ischemia. GPR174 deficiency upregulates amphiregulin (AREG) expression in Tregs, thereby enhancing endothelial cell functions and reducing pro-inflammatory macrophage polarization and endothelial cell apoptosis. Mechanically, GPR174 regulates AREG expression by inhibiting the nuclear accumulation of early growth response protein 1 (EGR1) via Gαs/cAMP/PKA signal pathway activation. Collectively, these findings demonstrate that GPR174 negatively regulates angiogenesis and vascular remodeling in response to ischemic injury and that GPR174 may be a potential molecular target for therapeutic interventions of ischemic vascular diseases.
Topics: Mice; Animals; Gene Deletion; Ischemia; Receptors, G-Protein-Coupled
PubMed: 36473866
DOI: 10.1038/s41467-022-35159-8 -
BMC Bioinformatics Oct 2011Paralog reduction, the loss of duplicate genes after whole genome duplication (WGD) is a pervasive process. Whether this loss proceeds gene by gene or through deletion...
BACKGROUND
Paralog reduction, the loss of duplicate genes after whole genome duplication (WGD) is a pervasive process. Whether this loss proceeds gene by gene or through deletion of multi-gene DNA segments is controversial, as is the question of fractionation bias, namely whether one homeologous chromosome is more vulnerable to gene deletion than the other.
RESULTS
As a null hypothesis, we first assume deletion events, on one homeolog only, excise a geometrically distributed number of genes with unknown mean µ, and these events combine to produce deleted runs of length l, distributed approximately as a negative binomial with unknown parameter r, itself a random variable with distribution π(·). A more realistic model requires deletion events on both homeologs distributed as a truncated geometric. We simulate the distribution of run lengths l in both models, as well as the underlying π(r), as a function of µ, and show how sampling l allows us to estimate µ. We apply this to data on a total of 15 genomes descended from 6 distinct WGD events and show how to correct the bias towards shorter runs caused by genome rearrangements. Because of the difficulty in deriving π(·) analytically, we develop a deterministic recurrence to calculate each π(r) as a function of µ and the proportion of unreduced paralog pairs.
CONCLUSIONS
The parameter µ can be estimated based on run lengths of single-copy regions. Estimates of µ in real data do not exclude the possibility that duplicate gene deletion is largely gene by gene, although it may sometimes involve longer segments.
Topics: Chromosomes; Data Interpretation, Statistical; Evolution, Molecular; Gene Deletion; Gene Duplication; Genes, Duplicate; Genomics; Models, Genetic
PubMed: 22152148
DOI: 10.1186/1471-2105-12-S9-S5 -
Neurologic Clinics Aug 2014Facioscapulohumeral muscular dystrophy (FSHD) is a common type of adult muscular dystrophy and is divided into types 1 and 2 based on genetic mutation. Clinically, both... (Review)
Review
Facioscapulohumeral muscular dystrophy (FSHD) is a common type of adult muscular dystrophy and is divided into types 1 and 2 based on genetic mutation. Clinically, both FSHD types often show asymmetric and progressive muscle weakness affecting initially the face, shoulder, and arms followed by the distal then proximal lower extremities. Approximately 95% of patients, termed FSHD1, have a deletion of a key number of repetitive elements on chromosome 4q35. The remaining 5%, termed FSHD2, have no deletion on chromosome 4q35. Nevertheless, both types share a common downstream mechanism, making it possible for future disease-directed therapies to be effective for both FSHD types.
Topics: Gene Deletion; Humans; Muscular Dystrophy, Facioscapulohumeral
PubMed: 25037087
DOI: 10.1016/j.ncl.2014.04.003 -
Current Opinion in Genetics &... Jun 2021Copy number variants (CNVs) at specific loci have been identified as important risk factors for several neuropsychiatric disorders, such as schizophrenia, autism... (Review)
Review
Copy number variants (CNVs) at specific loci have been identified as important risk factors for several neuropsychiatric disorders, such as schizophrenia, autism spectrum disorder, intellectual disability (ID) and depression. These CNVs are individually rare (<0.5% frequency), have high effect sizes, and show pleiotropic effects for multiple neuropsychiatric disorders, which implies a shared aetiology. Neuropsychiatric CNVs are also associated with cognitive impairment and other medical morbidities, such as heart defects and obesity. As most neuropsychiatric CNVs are multigenic, it has been challenging to map their effects onto specific biological processes, although gene-set analyses have implicated genes related to the synapse and chromatin regulation. However, future whole-genome sequencing studies have potential for identifying novel single-gene CNV associations, which could provide insights into the pathophysiology underlying neuropsychiatric disorders.
Topics: DNA Copy Number Variations; Gene Deletion; Gene Duplication; Genetic Predisposition to Disease; Genome-Wide Association Study; Humans; Mental Disorders; Whole Genome Sequencing
PubMed: 33752146
DOI: 10.1016/j.gde.2021.02.014 -
Frontiers in Immunology 2022Duck plague caused by duck plague virus (DPV) is a highly contagious disease that can cause serious morbidity and death in waterfowl such as ducks and geese, and bring...
Duck plague caused by duck plague virus (DPV) is a highly contagious disease that can cause serious morbidity and death in waterfowl such as ducks and geese, and bring huge economic losses to the duck industry. In this study, on the basis of the duck plague virus gC gene deletion strain CHv-ΔgC, based on the duck plague virus bacterial artificial chromosome (BAC) platform in our laboratory, the gE gene was knocked out using the traceless deletion technology to obtain gC/gE double gene deletion candidate vaccine strain CHv-ΔgC/gE. The double gene deletion strain (CHv-ΔgC/gE) constructed in this study has greatly weakened virulence, no pathogenicity to ducks, and stable genetic characteristics and . Ducks immunized with CHv-ΔgC/gE can produce neutralizing antibodies and ELISA antibody levels comparable to those of commercial duck plague attenuated vaccine immunization, and can resist 100 LD CHv challenge of ducks, with good immune protection effect. It has the potential to be further developed into duck plague gC/gE double gene deletion, marked attenuated vaccine.
Topics: Animals; Ducks; Gene Deletion; Herpesviridae Infections; Mardivirus; Vaccines, Attenuated
PubMed: 36059553
DOI: 10.3389/fimmu.2022.963009 -
Current Genetics Apr 2021The cloning of plasmids can be time-consuming or expensive. Yet, cloning is a prerequisite for many standard experiments for the functional analysis of genes, including...
The cloning of plasmids can be time-consuming or expensive. Yet, cloning is a prerequisite for many standard experiments for the functional analysis of genes, including the generation of deletion mutants and the localization of gene products. Here, we provide Golden Gate vectors for fast and easy cloning of gene fusion as well as gene deletion vectors applicable to diverse fungi. In Golden Gate cloning, restriction and ligation occur simultaneously in a one-pot reaction. Our vector set contains recognition sites for the commonly used type IIS restriction endonuclease BsaI. We generated plasmids for C- as well as N-terminal tagging with GFP, mRFP and 3xFLAG. For gene deletion, we provide five different donor vectors for selection marker cassettes. These include standard cassettes for hygromycin B, nourseothricin and phleomycin resistance genes as well as FLP/FRT-based marker recycling cassettes for hygromycin B and nourseothricin resistance genes. To make cloning most feasible, we provide robust protocols, namely (1) an overview of cloning procedures described in this paper, (2) specific Golden Gate reaction protocols and (3) standard primers for cloning and sequencing of plasmids and generation of deletion cassettes by PCR and split-marker PCR. We show that our vector set is applicable for the biotechnologically relevant Penicillium chrysogenum and the developmental model system Sordaria macrospora. We thus expect these vectors to be beneficial for other fungi as well. Finally, the vectors can easily be adapted to organisms beyond the kingdom fungi.
Topics: Cloning, Molecular; Fungi; Gene Deletion; Gene Fusion; Genetic Engineering; Genetic Vectors; Plasmids; Sordariales
PubMed: 33367953
DOI: 10.1007/s00294-020-01143-2 -
Redox Biology Oct 2023Long-chain acyl-CoA synthetase (ACSL) 4 converts polyunsaturated fatty acids (PUFAs) into their acyl-CoAs and plays an important role in maintaining PUFA-containing...
Long-chain acyl-CoA synthetase (ACSL) 4 converts polyunsaturated fatty acids (PUFAs) into their acyl-CoAs and plays an important role in maintaining PUFA-containing membrane phospholipids. Here we demonstrated decreases in various kinds of PUFA-containing phospholipid species in ACSL4-deficient murine lung. We then examined the effects of ACSL4 gene deletion on lung injury by treating mice with two pulmonary toxic chemicals: paraquat (PQ) and methotrexate (MTX). The results showed that ACSL4 deficiency attenuated PQ-induced acute lung lesion and decreased mortality. PQ-induced lung inflammation and neutrophil migration were also suppressed in ACSL4-deficient mice. PQ administration increased the levels of phospholipid hydroperoxides in the lung, but ACSL4 gene deletion suppressed their increment. We further found that ACSL4 deficiency attenuated MTX-induced pulmonary fibrosis. These results suggested that ACSL4 gene deletion might confer protection against pulmonary toxic chemical-induced lung injury by reducing PUFA-containing membrane phospholipids, leading to the suppression of lipid peroxidation. Inhibition of ACSL4 may be promising for the prevention and treatment of chemical-induced lung injury.
Topics: Mice; Animals; Lipid Peroxidation; Lung Injury; Xenobiotics; Gene Deletion; Phospholipids; Fatty Acids, Unsaturated; Lung; Ligases
PubMed: 37586249
DOI: 10.1016/j.redox.2023.102850 -
PLoS Genetics Feb 2019Screens for epistatic interactions have long been used to characterize functional relationships corresponding to protein complexes, metabolic pathways, and other...
Screens for epistatic interactions have long been used to characterize functional relationships corresponding to protein complexes, metabolic pathways, and other functional modules. Although epistasis between adaptive mutations is also common in laboratory evolution experiments, the functional basis for these interactions is less well characterized. Here, we quantify the extent to which gene function (as determined by a genome-wide screen for epistasis among deletion mutants) influences the rate and genetic basis of compensatory adaptation in a set of 37 gene deletion mutants nested within 16 functional modules. We find that functional module has predictive power: mutants with deletions in the same module tend to adapt more similarly, on average, than those with deletions in different modules. At the same time, initial fitness also plays a role: independent of the specific functional modules involved, adaptive mutations tend to be systematically more beneficial in less-fit genetic backgrounds, consistent with a general pattern of diminishing returns epistasis. We measured epistatic interactions between initial gene deletion mutations and the mutations that accumulate during compensatory adaptation and found a general trend towards positive epistasis (i.e. mutations tend to be more beneficial in the background in which they arose). In two functional modules, epistatic interactions between the initial gene deletions and the mutations in their descendant lines caused evolutionary entrenchment, indicating an intimate functional relationship. Our results suggest that genotypes with similar epistatic interactions with gene deletion mutations will also have similar epistatic interactions with adaptive mutations, meaning that genome scale maps of epistasis between gene deletion mutations can be predictive of evolutionary dynamics.
Topics: Adaptation, Physiological; Computer Simulation; Epistasis, Genetic; Evolution, Molecular; Gene Deletion; Genes, Fungal; Genetic Fitness; Metabolic Networks and Pathways; Models, Genetic; Mutation; Saccharomyces cerevisiae
PubMed: 30768593
DOI: 10.1371/journal.pgen.1007958