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Ultraviolet light induction of diphtheria toxin-resistant mutations in normal and DNA repair-deficient human and Chinese hamster fibroblasts.

The role of unrepaired DNA lesions in the production of mutations is suspected of contributing to the initiation phase of carcinogenesis. Since the molecular basis of mutagenesis is not understood in eukaryotic cells, development of new genetic markers for quantitative in vitro measurement of mutations for mammalian cells is needed. Furthermore, mammalian cells, genetically deficient for various DNA repair enzymes, will be needed to study the role of unrepaired DNA lesions in mutagenesis. The results in this report relate to preliminary attempts (1) to characterize the diphtheria toxin resista…

Регулирование ДИ микроцина и протеолитическое декольте являются уникальными среди известных микроцинов.

Microcin PDI подавляет разнообразие патогенных эшеричий с помощью белка-эффектора, МакпМ. В этом исследовании мы продемонстрировали, что выражение ингибиторского фенотипа вызывается при низких осмолярных условиях, а его выражение контролируется главным образом EnvZ/OmpR. two- компонентная система регулирования. Функциональные, мутагенезовые и дополняющие эксперименты были использованы для эмпирической демонстрации того, что ЭнвЗ требуется для ингибиторского фенотипа и что регулирование mcpM зависит от привязки фосфорилата OmpR к области mcpM. Фосфорилаты могут распознавать three :: различные и

Совпадение в аннотации оригинала
Microcin PDI inhibits a diversity of pathogenic Escherichia coli through the action of an effector protein, McpM. In this study we demonstrated that expression of the inhibitory phenotype is induced under low osmolarity conditions and expression is primarily controlled by the EnvZ/OmpR two-component regulatory system. Functional, mutagenesis and complementat…

Mutagenic effects of DNA-containing oncogenic viruses and malignant transformation of mammalian cells.

It was discovered in the 1970s that oncogenic viruses could induce gene mutations in mammalian cells. The phenomenon seems to be widespread: … e process of integration creates the possibility for occurrence of mutations early after infection. Mutagenesis seems to be induced by an integrated virus, though to a much smaller extent. Virus-induced mutagenesis may be connected with an activation of the cell error-prone repair systems. The sum total of the experimental data shows that virus-induced mutage …

In vitro HIV DNA integration in STAT3 drives T cell persistence-A model of HIV-associated T cell lymphoma.

Oncogenic retroviruses are known for their pathogenesis via insertional mutagenesis, in which the presence of a provirus and its transcriptional control elements alter the expression of a nearby or surrounding host gene. There are reports of proviral integration driving oncogenesis in people with HIV and the use of HIV-derived vectors for gene therapy has raised concern about oncogenic side effects. To study this issue, we used an in vitro primary human CD4 + T cell infection model developed in our laboratory to identify HIV-1 integration sites that might influence cell proliferation or surviv…

Optimizing lactose hydrolysis by computer-guided modification of the catalytic site of a wild-type enzyme.

Lactose intolerance is a serious global health problem. A lactose hydrolysis enzyme, thermostable β-galactosidase, BgaB (from Geobacillus st … for enzyme catalysis. A ligand-receptor binding model has been proposed to guide site-directed BgaB mutagenesis experiments. Based upon the model and the MD simulations, we recommend mutating Arg109, Phe341, Trp311, Asn147, Asn310, Try272, and His354 to reduce galactose product inhibition. In vitro site-directed mutagenesis experiments confirmed our predictio …

Natural Cotransformation and Multiplex Genome Editing by Natural Transformation (MuGENT) of Vibrio cholerae.

Generating mutant strains is an essential component of microbial genetics. Natural genetic transformation, a process for the uptake and inte … carless mutations in a single step. Here, we provide a detailed description of the methods used for mutagenesis of the cholera pathogen Vibrio cholerae with a particular emphasis on mutagenesis via MuGENT.

Report of a novel recurrent homozygous variant c.620A>T in three unrelated families with thiamine metabolism dysfunction syndrome 5 and review of literature.

INTRODUCTION: Biallelic variants in thiamine pyrophosphokinase 1 ( TPK1 ) are known to cause thiamine metabolism dysfunction syndrome 5 (THMD5). This disorder is characterized by neuroregression, ataxia and dystonia with basal ganglia abnormalities on neuroimaging. To date, 27 families have been reported with THMD5 due to variants in TPK1 . METHODS: We ascertained three individuals from three unrelated families. Singleton exome sequencing was performed on all three individuals, followed by in silico mutagenesis of the mutant TPK protein. Additionally, we reviewed the genotypic and phenotypic …

The Mouse House: a brief history of the ORNL mouse-genetics program, 1947-2009.

The large mouse genetics program at the Oak Ridge National Laboratory (ORNL) is often remembered chiefly for the germ-cell mutation-rate data it generated and their uses in estimating the risk of heritable radiation damage. In fact, it soon became a multi-faceted research effort that, over a period of almost 60 years, generated a wealth of information in the areas of mammalian mutagenesis, basic genetics (later enriched by molecular techniques), cytogenetics, reproductive biology, biochemistry of germ cells, and teratology. Research in the area of germ-cell mutagenesis explored the important p…

Biosynthesis of Pseudomonas-Derived Butenolides.

Butenolides are well-known signaling molecules in Gram-positive bacteria. Here, we describe a novel class of butenolides isolated from a Gram-negative Pseudomonas strain, the styrolides. Structure elucidation was aided by the total synthesis of styrolide A. Transposon mutagenesis enabled us to identify the styrolide biosynthetic gene cluster, and by using a homology search, we discovered the related and previously unknown acaterin biosynthetic gene cluster in another Pseudomonas species. Mutagenesis, heterologous expression, and identification of key shunt and intermediate products were crucia…

[Mechanisms of genome instability in children with periventricular leucomalacia that resulted in cerebral palsy].

The activity of free-radical oxidation and the level of genome instability in children with periventricular leucomalacia that resulted in cerebral palsy have been studied. Genome destabilization, i.e., the elevation of erythrocyte micronuclei in the peripheral blood, has been reported. There was a correlation of a number of cells with cytogenetic rearrangements with the activity of antioxidant defense enzymes and the malonaldehyde level. It has been shown that genome instability occurs during the activation of endogenous mutagenesis and reduction of antiradical and antimutagenic defense. Havin…

Patient-derived separation-of-function mutations reveal specific role for BRCA1 coiled coil domain in genome stability.

Specific inherited mutations of BRCA1 compromise homologous recombination repair and increase the risk of cancer. Here, we employ a panel of isogenic chicken DT40 cell lines for the comparative study of the main BRCA1 protein domains and of common BRCA1 founder mutations in genome stability and DNA repair. Whole genome sequencing of cell clones demonstrates that the L1407P-equivalent coiled coil domain variant is a separation-of-function mutant with an increased rate of base substitution mutagenesis but not of deletions or large-scale genomic instability, reflecting its proficiency in repairin…

DNA repair mutant pso2 of Saccharomyces cerevisiae is sensitive to intracellular acetaldehyde accumulated by disulfiram-mediated inhibition of acetaldehyde dehydrogenase.

Blocking aldehyde dehydrogenase with the drug disulfiram leads to an accumulation of intracellular acetaldehyde, which negatively affects th … s. Chronic exposure of yeast cells to disulfiram and acute exposure to acetaldehyde induced forward mutagenesis in the yeast CAN1 gene. Disulfiram-induced mutability of a pso2Delta mutant was significantly increased over that of the isogenic wild type; however, this was not found for acetaldehyde-induced mutagenesis. Spontaneous mutability at the CAN1 locus w …

Probing the active site of MIO-dependent aminomutases, key catalysts in the biosynthesis of beta-amino acids incorporated in secondary metabolites.

The tyrosine aminomutase SgTAM produces (S)-ss-tyrosine from L-tyrosine in the biosynthesis of the enediyne antitumor antibiotic C-1027. Thi … gTAM have also proved to be difficult as it is recalcitrant to perturbations to the active site via mutagenesis. An X-ray cocrystal structure of the SgTAM mutant of the catalytic base with L-tyrosine verified important substrate binding residues as well as the enzymatic base. Further mutagenesis revealed that removal of these crucial interactions renders the …

A mutation in Af4 is predicted to cause cerebellar ataxia and cataracts in the robotic mouse.

The robotic mouse is an autosomal dominant mutant that arose from a large-scale chemical mutagenesis program. It has a jerky, ataxic gait and develops adult-onset Purkinje cell loss in the cerebellum in a striking region-specific pattern, as well as cataracts. Genetic and physical mapping of the disease locus led to the identification of a missense mutation in a highly conserved region of Af4, a putative transcription factor that has been previously implicated in leukemogenesis. We demonstrate that Af4 is specifically expressed in Purkinje cells, and we hypothesize that the expression of mutan…

Stimulation of Replication Template-Switching by DNA-Protein Crosslinks.

Covalent DNA protein crosslinks (DPCs) are common lesions that block replication. We examine here the consequence of DPCs on mutagenesis involving replicational template-switch reactions in Escherichia coli. 5-Azacytidine (5-azaC) is a potent mutagen for template-switching. This effect is dependent on DNA cytosine methylase (Dcm), implicating the Dcm-DNA covalent complex trapped by 5-azaC as the initiator for mutagenesis. The leading strand of replication is more mutable than the lagging strand, which can be explained by blocks to the replicative helicase and/or fork regression. We find that t…

Construction of targeted insertion mutations in Francisella tularensis subsp. novicida.

Francisella tularensis is one of the most deadly bacterial agents, yet most of the genetic determinants of pathogenesis are still unknown. We have developed an efficient targeted mutagenesis strategy in the model organism F. tularensis subsp. novicida by utilizing universal priming of optimized antibiotic resistance cassettes and splicing by overlap extension (SOE). This process enables fast and efficient construction of targeted insertion mutations in F. tularensis subsp. novicida that have characteristics of nonpolar mutations; optimized targeted mutagenesis strategies will promote the study…

Zebrafish myelopoiesis and blood cell development.

The zebrafish (Danio rerio) animal model offers a unique opportunity to discover novel genes required for the control of normal vertebrate myeloid cell development. It is well suited for both developmental and genetic analyses: eg, genome-wide chemical mutagenesis screens have led to the identification of specific new genes affecting vertebrate erythropoiesis. Mutants defective in one or more hematopoietic functions will be useful as models of human disease and will assist in the elucidation of lineage-specific developmental programs. By using a combination of forward genetic mutagenesis scree…

Transposon Sequencing in Leptospira interrogans.

The development of random transposon mutagenesis in L. interrogans has enabled the creation of large libraries of mutants, facilitating the identification of genes involved in key phenotypes such as virulence. However, phenotypic screening of individual mutants from these libraries remains time-consuming and labor-intensive. Here, we describe a transposon-sequencing protocol (Tn-Seq) that combines random transposon mutagenesis with high-throughput sequencing to simultaneously assess fitness changes across a large collection of L. interrogans mutants under defined conditions. Inoculating indivi…

Structural basis of the interaction between the meningitis pathogen Streptococcus suis adhesin Fhb and its human receptor.

The recently identified Streptococcus suis adhesin factor H-binding protein (Fhb) targets the host cellular receptor glycolipid GbO3 through its N terminus. However, it is unclear how Fhb interacts with its receptor. Here, we determined the complex structure of factor H-binding protein receptor-binding domain (Fhb RBD) with Gb2, an analog of its receptor, revealing that Gb2 binds in a pocket of the β sandwich core domain. We identified the key residues for Fhb RBD receptor binding using mutagenesis and isothermal titration calorimetry. Mutagenesis analyses indicated that Fhb binds to Gb2 mainl…

rumAB genes from SXT/R391 ICEs confer UV-induced mutability to Proteus mirabilis hosts and improve conjugation after UV irradiation.

Proteus mirabilis is one of the Enterobacteriales species that has been deemed as non-mutable by DNA damaging agents. A genomic analysis of P. mirabilis genomes shows that this species often does not carry pol V-encoding genes in its chromosome, which are responsible for SOS mutagenesis in other bacteria. On the other hand, the highly active umuDC homologs rumAB are carried in the mobile integrative conjugative elements (ICEs) from the SXT/R391 family that are frequently found in this species. Here we show that isolates devoid of SXT/R391 are indeed weakly or non-mutable by UV-light exposure, …