Orobanche foetida resistance in two new faba bean genotypes produced by radiation mutagenesis.

PubMed

Mejri, Sonia; Mabrouk, Yassine; Belhadj, Omrane; Saidi, Mouldi

2018-06-12

Broomrape produces serious damage to many legume crops and particularly becomes a limiting factor for faba bean (Vicia faba L.) production in the Mediterranean basin. Currently, several traditional methods of control have been developed, but none has proved to be effective for this parasite. However, breeding for resistance to this pest remains as one of the most feasible and environmentally friendly methods for managing broomrape, but the mechanisms governing the interaction between the parasite and the host are not yet well understood. Therefore, we studied behaviours and the molecular and enzymatic changes associated with resistance to Orobanche foetida in faba bean mutants that were obtained through radiation mutagenesis. Three faba bean genotypes were used in this study, the variety 'Badï 'characterized by high productivity in Orobanche-free soils and susceptibility to O. foetida and two mutant lines P2M3 and P7M3 (derived from radio mutagenesis program) selected for their higher resistance to O. foetida in field evaluation. The infection progress and the relative changes in the co culture response, the enzymatic activities changes and the efficiency of the root extract stimulants from the host plant were followed and evaluated in all genotypes. Experiments showed that low induction of seed germination is a major component of resistance in these lines against O. foetida. This is confirmed by in vitro experiments with root exudates. In parallel reduction in infection was accompanied by the continuously enhancement of the peroxidase activity, the polyphenol oxidase activity and the phenylalanine ammonia lyase activity in faba bean roots. These data suggest the contribution of these enzymes in faba bean resistance to O. foetida broomrape induced by the use of gamma rays. Management of Orobanche by way of crop selection based on these enzyme systems is a possible option.

[Radiation Anticarcinogenesis by Thiazolidine Pro-drug

NASA Technical Reports Server (NTRS)

Warters, Raymond L.; Roberts, Jeanette C.; Fain, Heidi

1999-01-01

The original goal of this work was to determine the capacity of selected aminothiols to modulate radiation induced cytotoxicity, mutagenesis and carcinogenesis in a human mammary epithelial cell line. The conclusions from this work are that WR-1065 is the "gold standard" for protection against radiation induced cytotoxicity, mutagenesis and carcinogenesis. While a potent radiation protector, WR-1065 is cytotoxic in vitro and in vivo. Our rationale for a study of the thiazolidine pro-drugs was that these compounds are neither toxic in vitro or in vivo. The results obtained during this funding period indicate that the thiazolidine pro-drugs are as potent as WR-1065 as protectors against radiation induced mutation induction, and thus presumably against radiation induced carcinogenesis. Our results indicate that the thiazolidine prodrugs are excellent candidates to test as non-toxic anticarcinogens for protecting astronauts from cancer induction during space travel.

USP7 Is a Suppressor of PCNA Ubiquitination and Oxidative-Stress-Induced Mutagenesis in Human Cells.

PubMed

Kashiwaba, Shu-ichiro; Kanao, Rie; Masuda, Yuji; Kusumoto-Matsuo, Rika; Hanaoka, Fumio; Masutani, Chikahide

2015-12-15

Mono-ubiquitinated PCNA activates error-prone DNA polymerases; therefore, strict regulation of PCNA mono-ubiquitination is crucial in avoiding undesired mutagenesis. In this study, we used an in vitro assay system to identify USP7 as a deubiquitinating enzyme of mono-ubiquitinated PCNA. Suppression of USP1, a previously identified PCNA deubiquitinase, or USP7 increased UV- and H2O2-induced PCNA mono-ubiquitination in a distinct and additive manner, suggesting that USP1 and USP7 make different contributions to PCNA deubiquitination in human cells. Cell-cycle-synchronization analyses revealed that USP7 suppression increased H2O2-induced PCNA ubiquitination throughout interphase, whereas USP1 suppression specifically increased ubiquitination in S-phase cells. UV-induced mutagenesis was elevated in USP1-suppressed cells, whereas H2O2-induced mutagenesis was elevated in USP7-suppressed cells. These results suggest that USP1 suppresses UV-induced mutations produced in a manner involving DNA replication, whereas USP7 suppresses H2O2-induced mutagenesis involving cell-cycle-independent processes such as DNA repair. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Low-dose radiation attenuates chemical mutagenesis in vivo.

PubMed

Kakinuma, Shizuko; Yamauchi, Kazumi; Amasaki, Yoshiko; Nishimura, Mayumi; Shimada, Yoshiya

2009-09-01

The biological effects of low-dose radiation are not only of social concern but also of scientific interest. The radioadaptive response, which is defined as an increased radioresistance by prior exposure to low-dose radiation, has been extensively studied both in vitro and in vivo. Here we briefly review the radioadaptive response with respect to mutagenesis, survival rate, and carcinogenesis in vivo, and introduce our recent findings of cross adaptation in mouse thymic cells, that is, the suppressive effect of repeated low-dose radiation on mutation induction by the alkylating agent N-ethyl-N-nitrosourea.

Nuclear Proximity of Mtr4 with RNA exosome restricts DNA mutational asymmetry

PubMed Central

Lim, Junghyun; Giri, Pankaj Kumar; Kazadi, David; Laffleur, Brice; Zhang, Wanwei; Grinstein, Veronika; Pefanis, Evangelos; Brown, Lewis M.; Ladewig, Erik; Martin, Ophélie; Chen, Yuling; Rabadan, Raul; Boyer, François; Rothschild, Gerson; Cogné, Michel; Pinaud, Eric; Deng, Haiteng; Basu, Uttiya

2017-01-01

SUMMARY The distribution of sense and antisense strand DNA mutations on transcribed duplex DNA contributes to the development of immune and neural systems along with the progression of cancer. Because developmentally matured B cells undergo biologically programmed strand-specific DNA mutagenesis at focal DNA/RNA hybrid structures, they make a convenient system to investigate strand-specific mutagenesis mechanisms. We demonstrate that the sense and antisense strand DNA mutagenesis at the immunoglobulin heavy chain locus and some other regions of the B cell genome depends upon localized RNA processing protein complex formation in the nucleus. Both the physical proximity and coupled activities of RNA helicase Mtr4 (and Senataxin) with the noncoding RNA processing function of RNA exosome determine the strand specific distribution of DNA mutations. Our study suggests that strand-specific DNA mutagenesis-associated mechanisms will play major roles in other undiscovered aspects of organismic development. PMID:28431250

Back to the future: revisiting HIV-1 lethal mutagenesis

PubMed Central

Dapp, Michael J.; Patterson, Steven E.; Mansky, Louis M.

2012-01-01

The concept of eliminating HIV-1 infectivity by elevating the viral mutation rate was first proposed over a decade ago, even though the general concept had been conceived earlier for RNA viruses. Lethal mutagenesis was originally viewed as a novel chemotherapeutic approach for treating HIV-1 infection in which use of a viral mutagen would over multiple rounds of replication lead to the lethal accumulation of mutations, rendering the virus population non infectious – known as the slow mutation accumulation model. There have been limitations in obtaining good efficacy data with drug leads, leaving some doubt into clinical translation. More recent studies of the APOBEC3 proteins as well as new progress in the use of nucleoside analogs for inducing lethal mutagenesis have helped to refocus attention on rapid induction of HIV-1 lethal mutagenesis in a single or limited number of replication cycles leading to a rapid mutation accumulation model. PMID:23195922

The POLD3 subunit of DNA polymerase δ can promote translesion synthesis independently of DNA polymerase ζ

PubMed Central

Hirota, Kouji; Yoshikiyo, Kazunori; Guilbaud, Guillaume; Tsurimoto, Toshiki; Murai, Junko; Tsuda, Masataka; Phillips, Lara G.; Narita, Takeo; Nishihara, Kana; Kobayashi, Kaori; Yamada, Kouich; Nakamura, Jun; Pommier, Yves; Lehmann, Alan; Sale, Julian E.; Takeda, Shunichi

2015-01-01

The replicative DNA polymerase Polδ consists of a catalytic subunit POLD1/p125 and three regulatory subunits POLD2/p50, POLD3/p66 and POLD4/p12. The ortholog of POLD3 in Saccharomyces cerevisiae, Pol32, is required for a significant proportion of spontaneous and UV-induced mutagenesis through its additional role in translesion synthesis (TLS) as a subunit of DNA polymerase ζ. Remarkably, chicken DT40 B lymphocytes deficient in POLD3 are viable and able to replicate undamaged genomic DNA with normal kinetics. Like its counterpart in yeast, POLD3 is required for fully effective TLS, its loss resulting in hypersensitivity to a variety of DNA damaging agents, a diminished ability to maintain replication fork progression after UV irradiation and a significant decrease in abasic site-induced mutagenesis in the immunoglobulin loci. However, these defects appear to be largely independent of Polζ, suggesting that POLD3 makes a significant contribution to TLS independently of Polζ in DT40 cells. Indeed, combining polη, polζ and pold3 mutations results in synthetic lethality. Additionally, we show in vitro that POLD3 promotes extension beyond an abasic by the Polδ holoenzyme suggesting that while POLD3 is not required for normal replication, it may help Polδ to complete abasic site bypass independently of canonical TLS polymerases. PMID:25628356

BATCH-GE: Batch analysis of Next-Generation Sequencing data for genome editing assessment

PubMed Central

Boel, Annekatrien; Steyaert, Woutert; De Rocker, Nina; Menten, Björn; Callewaert, Bert; De Paepe, Anne; Coucke, Paul; Willaert, Andy

2016-01-01

Targeted mutagenesis by the CRISPR/Cas9 system is currently revolutionizing genetics. The ease of this technique has enabled genome engineering in-vitro and in a range of model organisms and has pushed experimental dimensions to unprecedented proportions. Due to its tremendous progress in terms of speed, read length, throughput and cost, Next-Generation Sequencing (NGS) has been increasingly used for the analysis of CRISPR/Cas9 genome editing experiments. However, the current tools for genome editing assessment lack flexibility and fall short in the analysis of large amounts of NGS data. Therefore, we designed BATCH-GE, an easy-to-use bioinformatics tool for batch analysis of NGS-generated genome editing data, available from https://github.com/WouterSteyaert/BATCH-GE.git. BATCH-GE detects and reports indel mutations and other precise genome editing events and calculates the corresponding mutagenesis efficiencies for a large number of samples in parallel. Furthermore, this new tool provides flexibility by allowing the user to adapt a number of input variables. The performance of BATCH-GE was evaluated in two genome editing experiments, aiming to generate knock-out and knock-in zebrafish mutants. This tool will not only contribute to the evaluation of CRISPR/Cas9-based experiments, but will be of use in any genome editing experiment and has the ability to analyze data from every organism with a sequenced genome. PMID:27461955

Lon-mediated proteolysis of the Escherichia coli UmuD mutagenesis protein: in vitro degradation and identification of residues required for proteolysis

PubMed Central

Gonzalez, Martín; Frank, Ekaterina G.; Levine, Arthur S.; Woodgate, Roger

1998-01-01

Most SOS mutagenesis in Escherichia coli is dependent on the UmuD and UmuC proteins. Perhaps as a consequence, the activity of these proteins is exquisitely regulated. The intracellular level of UmuD and UmuC is normally quite low but increases dramatically in lon− strains, suggesting that both proteins are substrates of the Lon protease. We report here that the highly purified UmuD protein is specifically degraded in vitro by Lon in an ATP-dependent manner. To identify the regions of UmuD necessary for Lon-mediated proteolysis, we performed ‘alanine-stretch’ mutagenesis on umuD and followed the stability of the mutant protein in vivo. Such an approach allowed us to localize the site(s) within UmuD responsible for Lon-mediated proteolysis. The primary signal is located between residues 15 and 18 (FPLF), with an auxiliary site between residues 26 and 29 (FPSP), of the amino terminus of UmuD. Transfer of the amino terminus of UmuD (residues 1–40) to an otherwise stable protein imparts Lon-mediated proteolysis, thereby indicating that the amino terminus of UmuD is sufficient for Lon recognition and the ensuing degradation of the protein. PMID:9869642

Controlling the enantioselectivity of enzymes by directed evolution: Practical and theoretical ramifications

PubMed Central

Reetz, Manfred T.

2004-01-01

A fundamentally new approach to asymmetric catalysis in organic chemistry is described based on the in vitro evolution of enantioselective enzymes. It comprises the appropriate combination of gene mutagenesis and expression coupled with an efficient high-throughput screening system for evaluating enantioselectivity (enantiomeric excess assay). Several such cycles lead to a “Darwinistic” process, which is independent of any knowledge concerning the structure or the mechanism of the enzyme being evolved. The challenge is to choose the optimal mutagenesis methods to navigate efficiently in protein sequence space. As a first example, the combination of error-prone mutagenesis, saturation mutagenesis, and DNA-shuffling led to a dramatic enhancement of enantioselectivity of a lipase acting as a catalyst in the kinetic resolution of a chiral ester. Mutations at positions remote from the catalytically active center were identified, a surprising finding, which was explained on the basis of a novel relay mechanism. The scope and limitations of the method are discussed, including the prospect of directed evolution of stereoselective hybrid catalysts composed of robust protein hosts in which transition metal centers have been implanted. PMID:15079053

Site-specific incorporation of probes into RNA polymerase by unnatural-amino-acid mutagenesis and Staudinger-Bertozzi ligation

PubMed Central

Chakraborty, Anirban; Mazumder, Abhishek; Lin, Miaoxin; Hasemeyer, Adam; Xu, Qumiao; Wang, Dongye; Ebright, Yon W.; Ebright, Richard H.

2015-01-01

Summary A three-step procedure comprising (i) unnatural-amino-acid mutagenesis with 4-azido-phenylalanine, (ii) Staudinger-Bertozzi ligation with a probe-phosphine derivative, and (iii) in vitro reconstitution of RNA polymerase (RNAP) enables the efficient site-specific incorporation of a fluorescent probe, a spin label, a crosslinking agent, a cleaving agent, an affinity tag, or any other biochemical or biophysical probe, at any site of interest in RNAP. Straightforward extensions of the procedure enable the efficient site-specific incorporation of two or more different probes in two or more different subunits of RNAP. We present protocols for synthesis of probe-phosphine derivatives, preparation of RNAP subunits and the transcription initiation factor σ, unnatural amino acid mutagenesis of RNAP subunits and σ, Staudinger ligation with unnatural-amino-acid-containing RNAP subunits and σ, quantitation of labelling efficiency and labelling specificity, and reconstitution of RNAP. PMID:25665560

Improvement of DNA transfer frequency and transposon mutagenesis of Erwinia carotovora subsp. betavasculorum.

PubMed Central

Rella, M; Axelrood, P E; Weinhold, A R; Schroth, M N

1989-01-01

The production of antibiotics and their role in microbial competition under natural conditions can be readily studied by the use of transposon mutants. Several antibiotic-producing strains of Erwinia carotovora subsp. betavasculorum were unable to accept foreign DNA. A plasmid delivery system was developed, using ethyl methanesulfonate mutagenesis, which entailed isolating E. carotovora subsp. betavasculorum mutants able to accept foreign DNA and transfer it to other strains. This enabled transposon mutagenesis of a wild-type antibiotic-producing strain of E. carotovora subsp. betavasculorum. Twelve antibiotic-negative mutants were isolated, and one of these showed a reduction in antibiotic production in vitro. Many of these mutants also showed a reduction in their ability to macerate potato tissue. The mutants were classified into four genetic groups on the basis of their genetic and phenotypic characteristics, indicating that several genes are involved in antibiotic biosynthesis by E. carotovora subsp. betavasculorum. PMID:2543291

Characterization of the role of Fhit in maintenance of genomic integrity following low dose radiation, in vivo and in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ya Wang

2010-05-31

The major goal of this study is to determine the effects of the Fhit pathway on low dose ({le} 0.1 Gy) ionizing radiation (IR)-induced genetic instability. Reduction of Fhit protein expression is observed in most solid tumors particularly in those tumors resulting from exposure to environmental carcinogens. Therefore, characterization of the role of the Fhit-dependent pathway in preventing low dose IR-induced genetic instability will provide useful parameters for evaluating the low dose IR-induced risk of mutagenesis and carcinogenesis. We pursued 3 specific aims to study our hypothesis that the Fhit-dependent pathways maintain genomic integrity through adjusting checkpoint response and repairmore » genes expression following low dose IR. Aim 1: Determine whether Fhit interaction with RPA is necessary for Fhit to affect the cellular response to low dose IR. We combined the approaches of in vitro (GST pull-down and site-directed mutagenesis) and in vivo (observing the co-localization and immunoprecipitation of Fhit and RPA in Fhit knock out mouse cells transfected with mutant Fhit which has lost ability to interact with RPA in vitro). Aim 2: Determine the role of genes whose expression is affected by Fhit in low dose irradiated cells. We analyzed the distinct signature of gene expression in low dose irradiated Fhit-/- cells compared with Fhit+/+ cells by combining microarray, gene transfection and siRNA approaches. Aim 3: Determine the role of Fhit in genetic susceptibility to low dose IR in vivo. We compared the gene mutation frequency and the fragile site stability in the cells isolated from the Fhit+/+ and Fhit-/- mice at 1.5 years following low dose IR. These results determine the role of the Fhit-dependent pathway in maintaining genomic integrity in vitro and in vivo, which provide a basis for choosing surrogate markers in the Fhit-dependent pathway to evaluate low dose IR-induced risk of mutagenesis and carcinogenesis.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Weber, Nicholas D., E-mail: nweber@fhcrc.org; Department of Laboratory Medicine, University of Washington, Seattle, WA 98195; Aubert, Martine, E-mail: maubert@fhcrc.org

Treatment for most persistent viral infections consists of palliative drug options rather than curative approaches. This is often because long-lasting viral DNA in infected cells is not affected by current antivirals, providing a source for viral persistence and reactivation. Targeting latent viral DNA itself could therefore provide a basis for novel curative strategies. DNA cleavage enzymes can be used to induce targeted mutagenesis of specific genes, including those of exogenous viruses. Although initial in vitro and even in vivo studies have been carried out using DNA cleavage enzymes targeting various viruses, many questions still remain concerning the feasibility of thesemore » strategies as they transition into preclinical research. Here, we review the most recent findings on DNA cleavage enzymes for human viral infections, consider the most relevant animal models for several human viral infections, and address issues regarding safety and enzyme delivery. Results from well-designed in vivo studies will ideally provide answers to the most urgent remaining questions, and allow continued progress toward clinical application. - Highlights: • Recent in vitro and in vivo results for DNA cleavage enzymes targeting persistent viral infections. • Analysis of the best animal models for testing enzymes for HBV, HSV, HIV and HPV. • Challenges facing in vivo delivery of therapeutic enzymes for persistent viral infections. • Safety issues to be addressed with proper animal studies.« less

CRISPR applications in ophthalmologic genome surgery.

PubMed

Cabral, Thiago; DiCarlo, James E; Justus, Sally; Sengillo, Jesse D; Xu, Yu; Tsang, Stephen H

2017-05-01

The present review seeks to summarize and discuss the application of clustered regularly interspaced short palindromic repeats (CRISPR)-associated systems (Cas) for genome editing, also called genome surgery, in the field of ophthalmology. Precision medicine is an emerging approach for disease treatment and prevention that takes into account the variability of an individual's genetic sequence. Various groups have used CRISPR-Cas genome editing to make significant progress in mammalian preclinical models of eye disease, the basic science of eye development in zebrafish, the in vivo modification of ocular tissue, and the correction of stem cells with therapeutic applications. In addition, investigators have creatively used the targeted mutagenic potential of CRISPR-Cas systems to target pathogenic alleles in vitro. Over the past year, CRISPR-Cas genome editing has been used to correct pathogenic mutations in vivo and in transplantable stem cells. Although off-target mutagenesis remains a concern, improvement in CRISPR-Cas technology and careful screening for undesired mutations will likely lead to clinical eye therapeutics employing CRISPR-Cas systems in the near future.

Lentiviral vector-based insertional mutagenesis identifies genes associated with liver cancer

PubMed Central

Ranzani, Marco; Cesana, Daniela; Bartholomae, Cynthia C.; Sanvito, Francesca; Pala, Mauro; Benedicenti, Fabrizio; Gallina, Pierangela; Sergi, Lucia Sergi; Merella, Stefania; Bulfone, Alessandro; Doglioni, Claudio; von Kalle, Christof; Kim, Yoon Jun; Schmidt, Manfred; Tonon, Giovanni; Naldini, Luigi; Montini, Eugenio

2013-01-01

Transposons and γ-retroviruses have been efficiently used as insertional mutagens in different tissues to identify molecular culprits of cancer. However, these systems are characterized by recurring integrations that accumulate in tumor cells, hampering the identification of early cancer-driving events amongst bystander and progression-related events. We developed an insertional mutagenesis platform based on lentiviral vectors (LVV) by which we could efficiently induce hepatocellular carcinoma (HCC) in 3 different mouse models. By virtue of LVV’s replication-deficient nature and broad genome-wide integration pattern, LVV-based insertional mutagenesis allowed identification of 4 new liver cancer genes from a limited number of integrations. We validated the oncogenic potential of all the identified genes in vivo, with different levels of penetrance. Our newly identified cancer genes are likely to play a role in human disease, since they are upregulated and/or amplified/deleted in human HCCs and can predict clinical outcome of patients. PMID:23314173

Primer Extension Mutagenesis Powered by Selective Rolling Circle Amplification

PubMed Central

Huovinen, Tuomas; Brockmann, Eeva-Christine; Akter, Sultana; Perez-Gamarra, Susan; Ylä-Pelto, Jani; Liu, Yuan; Lamminmäki, Urpo

2012-01-01

Primer extension mutagenesis is a popular tool to create libraries for in vitro evolution experiments. Here we describe a further improvement of the method described by T.A. Kunkel using uracil-containing single-stranded DNA as the template for the primer extension by additional uracil-DNA glycosylase treatment and rolling circle amplification (RCA) steps. It is shown that removal of uracil bases from the template leads to selective amplification of the nascently synthesized circular DNA strand carrying the desired mutations by phi29 DNA polymerase. Selective RCA (sRCA) of the DNA heteroduplex formed in Kunkel's mutagenesis increases the mutagenesis efficiency from 50% close to 100% and the number of transformants 300-fold without notable diversity bias. We also observed that both the mutated and the wild-type DNA were present in at least one third of the cells transformed directly with Kunkel's heteroduplex. In contrast, the cells transformed with sRCA product contained only mutated DNA. In sRCA, the complex cell-based selection for the mutant strand is replaced with the more controllable enzyme-based selection and less DNA is needed for library creation. Construction of a gene library of ten billion members is demonstrated with the described method with 240 nanograms of DNA as starting material. PMID:22355397

L1 Retrotransposon Heterogeneity in Ovarian Tumor Cell Evolution.

PubMed

Nguyen, Thu H M; Carreira, Patricia E; Sanchez-Luque, Francisco J; Schauer, Stephanie N; Fagg, Allister C; Richardson, Sandra R; Davies, Claire M; Jesuadian, J Samuel; Kempen, Marie-Jeanne H C; Troskie, Robin-Lee; James, Cini; Beaven, Elizabeth A; Wallis, Tristan P; Coward, Jermaine I G; Chetty, Naven P; Crandon, Alexander J; Venter, Deon J; Armes, Jane E; Perrin, Lewis C; Hooper, John D; Ewing, Adam D; Upton, Kyle R; Faulkner, Geoffrey J

2018-06-26

LINE-1 (L1) retrotransposons are a source of insertional mutagenesis in tumor cells. However, the clinical significance of L1 mobilization during tumorigenesis remains unclear. Here, we applied retrotransposon capture sequencing (RC-seq) to multiple single-cell clones isolated from five ovarian cancer cell lines and HeLa cells and detected endogenous L1 retrotransposition in vitro. We then applied RC-seq to ovarian tumor and matched blood samples from 19 patients and identified 88 tumor-specific L1 insertions. In one tumor, an intronic de novo L1 insertion supplied a novel cis-enhancer to the putative chemoresistance gene STC1. Notably, the tumor subclone carrying the STC1 L1 mutation increased in prevalence after chemotherapy, further increasing STC1 expression. We also identified hypomethylated donor L1s responsible for new L1 insertions in tumors and cultivated cancer cells. These congruent in vitro and in vivo results highlight L1 insertional mutagenesis as a common component of ovarian tumorigenesis and cancer genome heterogeneity. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Mutation as a Stress Response and the Regulation of Evolvability

PubMed Central

Galhardo, Rodrigo S.; Hastings, P. J.; Rosenberg, Susan M.

2010-01-01

Our concept of a stable genome is evolving to one in which genomes are plastic and responsive to environmental changes. Growing evidence shows that a variety of environmental stresses induce genomic instability in bacteria, yeast, and human cancer cells, generating occasional fitter mutants and potentially accelerating adaptive evolution. The emerging molecular mechanisms of stress-induced mutagenesis vary but share telling common components that underscore two common themes. The first is the regulation of mutagenesis in time by cellular stress responses, which promote random mutations specifically when cells are poorly adapted to their environments, i.e., when they are stressed. A second theme is the possible restriction of random mutagenesis in genomic space, achieved via coupling of mutation-generating machinery to local events such as DNA-break repair or transcription. Such localization may minimize accumulation of deleterious mutations in the genomes of rare fitter mutants, and promote local concerted evolution. Although mutagenesis induced by stresses other than direct damage to DNA was previously controversial, evidence for the existence of various stress-induced mutagenesis programs is now overwhelming and widespread. Such mechanisms probably fuel evolution of microbial pathogenesis and antibiotic-resistance, and tumor progression and chemotherapy resistance, all of which occur under stress, driven by mutations. The emerging commonalities in stress-induced-mutation mechanisms provide hope for new therapeutic interventions for all of these processes. PMID:17917874

Generation of Peanut Drought Tolerant Plants by Pingyangmycin-Mediated In Vitro Mutagenesis and Hydroxyproline-Resistance Screening

PubMed Central

Qiao, Lixian; Sun, Shimeng; Hu, Xiaohui; Chen, Jing; Wang, Jingshan

2015-01-01

In order to enlarge the potential resources of drought-tolerant peanuts, we conducted in vitro mutagenesis with Pingyangmycin (PYM) as the mutagen as well as directed screening on a medium supplemented with Hydroxyproline (HYP). After being extracted from mature seeds (cv. Huayu 20), the embryonic leaflets were cultured on somatic embryogenesis-induction medium with 4 mg/L PYM and the generated embryos were successively transferred to a germination medium with 4 and then 8 mmol/L HYP to screen HYP-tolerant plantlets. After that, these plantlets were grafted and transplanted to the experimental field. In the next generation, all seeds were sown in the field, and phenotype variation and trait segregation can be observed in most of the offspring (M2 generation). The M3 generation individuals were subjected to drought stress at the seedling stages. The activities of SOD and POD were substantially increased in eight offspring of 11 HYP-tolerant, regenerated plants than in their mutagenic parents. To determine the correlation between mutant phenotypes and genomic modification, we carried out a comparison of the DNA polymorphisms between the mutagenic parents and 13 M3 generation individuals from different HYP-tolerant, regenerated plants with SSR primers. Results showed that most mutants and parent plants had signs of polymorphisms. Under drought stress, some M3 generation individuals of 10 original HYP-tolerant, regenerated plants produced more pods than the mutagenic parent; twenty individuals among them produced >60 g pods/plant. M4-generation seeds were tested for quality characteristics by Near Infrared Spectroscopy (NIS) and nine individuals with higher protein content (>30%) and 21 individuals with higher oil content (>58%) were screened. We concluded that the use of PYM-based in vitro mutagenesis in combination with directed screening with HYP is effective for the creation of potential drought-tolerant mutants of peanut. PMID:25826431

Reverse Genetics of Newcastle Disease Virus.

PubMed

Cardenas-Garcia, Stivalis; Afonso, Claudio L

2017-01-01

Reverse genetics allows for the generation of recombinant viruses or vectors used in functional studies, vaccine development, and gene therapy. This technique enables genetic manipulation and cloning of viral genomes, gene mutation through site-directed mutagenesis, along with gene insertion or deletion, among other studies. An in vitro infection-based system including the highly attenuated vaccinia virus Ankara strain expressing the T7 RNA polymerase from bacteriophage T7, with co-transfection of three helper plasmids and a full-length cDNA plasmid, was successfully developed to rescue genetically modified Newcastle disease viruses in 1999. In this chapter, the materials and the methods involved in rescuing Newcastle disease virus (NDV) from cDNA, utilizing site-directed mutagenesis and gene replacement techniques, are described in detail.

Comparative carcinogenic potencies of particulates from diesel engine exhausts, coke oven emissions, roofing tar aerosols and cigarette smoke.

PubMed Central

Albert, R E

1983-01-01

Mammalian cell mutagenesis, transformation and skin tumorigenesis assays show similar results in comparing the potencies of diesel, coke oven, roofing tar and cigarette smoke particulates. These assay results are reasonably consistent with the comparative carcinogenic potencies of coke oven and roofing tar emissions as determined by epidemiological studies. The bacterial mutagenesis assay tends to show disproportionately high potencies, particularly with diesel particulates. Results to date encourage the approach to the assessment for carcinogenic risks from diesel emissions based on the use of epidemiological data on cancer induced by coke oven emissions, roofing tar particulates and cigarette smoke with the comparative potencies of these materials determined by in vivo and in vitro bioassays. PMID:6186481

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Trosko, J.E.; Schultz, R.S.; Chang, C.C.

1980-01-01

The role on 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 to characterize the diphtheria toxin resistance marker as a useful quantitative genetic marker in human cells and tomore » isolate and characterize various DNA repair-deficient Chinese hamster cells.« less

Mutational optimization of the coelenterazine-dependent luciferase from Renilla.

PubMed

Woo, Jongchan; von Arnim, Albrecht G

2008-09-30

Renilla luciferase (RLUC) is a popular reporter enzyme for gene expression and biosensor applications, but it is an unstable enzyme whose catalytic mechanism remains to be elucidated. We titrated that one RLUC molecule can turn over about one hundred molecules of coelenterazine substrate. Mutagenesis of active site residue Pro220 extended the half-life of photon emission, yielding brighter luminescence in E. coli. Random mutagenesis uncovered two new mutations that stabilized and increased photon emission in vivo and in vitro, while ameliorating substrate inhibition. Further amended with a previously identified mutation, a new triple mutant showed a threefold improved kcat, as well as elevated luminescence in Arabidopsis. This advances the utility of RLUC as a reporter protein, biosensor, or resonance energy donor.

Mutational optimization of the coelenterazine-dependent luciferase from Renilla

PubMed Central

Woo, Jongchan; von Arnim, Albrecht G

2008-01-01

Renilla luciferase (RLUC) is a popular reporter enzyme for gene expression and biosensor applications, but it is an unstable enzyme whose catalytic mechanism remains to be elucidated. We titrated that one RLUC molecule can turn over about one hundred molecules of coelenterazine substrate. Mutagenesis of active site residue Pro220 extended the half-life of photon emission, yielding brighter luminescence in E. coli. Random mutagenesis uncovered two new mutations that stabilized and increased photon emission in vivo and in vitro, while ameliorating substrate inhibition. Further amended with a previously identified mutation, a new triple mutant showed a threefold improved kcat, as well as elevated luminescence in Arabidopsis. This advances the utility of RLUC as a reporter protein, biosensor, or resonance energy donor. PMID:18826616

Topology of evolving, mutagenized viral populations: quasispecies expansion, compression, and operation of negative selection.

PubMed

Ojosnegros, Samuel; Agudo, Rubén; Sierra, Macarena; Briones, Carlos; Sierra, Saleta; González-López, Claudia; Domingo, Esteban; Cristina, Juan

2008-07-17

The molecular events and evolutionary forces underlying lethal mutagenesis of virus (or virus extinction through an excess of mutations) are not well understood. Here we apply for the first time phylogenetic methods and Partition Analysis of Quasispecies (PAQ) to monitor genetic distances and intra-population structures of mutant spectra of foot-and-mouth disease virus (FMDV) quasispecies subjected to mutagenesis by base and nucleoside analogues. Phylogenetic and PAQ analyses have revealed a highly dynamic variation of intrapopulation diversity of FMDV quasispecies. The population diversity first suffers striking expansions in the presence of mutagens and then compressions either when the presence of the mutagenic analogue was discontinued or when a mutation that decreased sensitivity to a mutagen was selected. The pattern of mutations found in the populations was in agreement with the behavior of the corresponding nucleotide analogues with FMDV in vitro. Mutations accumulated at preferred genomic sites, and dn/ds ratios indicate the operation of negative (or purifying) selection in populations subjected to mutagenesis. No evidence of unusually elevated genetic distances has been obtained for FMDV populations approaching extinction. Phylogenetic and PAQ analysis provide adequate procedures to describe the evolution of viral sequences subjected to lethal mutagenesis. These methods define the changes of intra-population structure more precisely than mutation frequencies and Shannon entropies. PAQ is very sensitive to variations of intrapopulation genetic distances. Strong negative (or purifying) selection operates in FMDV populations subjected to enhanced mutagenesis. The quantifications provide evidence that extinction does not imply unusual increases of intrapopulation complexity, in support of the lethal defection model of virus extinction.

DEVELOPMENT OF A COMPARATIVE POTENCY METHOD FOR CANCER RISK ASSESSMENT OF COMPLEX MIXTURES USING SHORT-TERM 'IN VIVO' AND 'IN VITRO' BIOASSAYS

EPA Science Inventory

A comparative potency method for cancer risk assessment has been developed based upon a constant relative potency hypothesis. This method was developed and tested using data from a battery of short-term mutagenesis bioassays, animal tumorigenicity data and human lung cancer risk ...

Highly Efficient Targeted Mutagenesis in Mice Using TALENs

PubMed Central

Panda, Sudeepta Kumar; Wefers, Benedikt; Ortiz, Oskar; Floss, Thomas; Schmid, Bettina; Haass, Christian; Wurst, Wolfgang; Kühn, Ralf

2013-01-01

Targeted mouse mutants are instrumental for the analysis of gene function in health and disease. We recently provided proof-of-principle for the fast-track mutagenesis of the mouse genome, using transcription activator-like effector nucleases (TALENs) in one-cell embryos. Here we report a routine procedure for the efficient production of disease-related knockin and knockout mutants, using improved TALEN mRNAs that include a plasmid-coded poly(A) tail (TALEN-95A), circumventing the problematic in vitro polyadenylation step. To knock out the C9orf72 gene as a model of frontotemporal lobar degeneration, TALEN-95A mutagenesis induced sequence deletions in 41% of pups derived from microinjected embryos. Using TALENs together with mutagenic oligodeoxynucleotides, we introduced amyotrophic lateral sclerosis patient-derived missense mutations in the fused in sarcoma (Fus) gene at a rate of 6.8%. For the simple identification of TALEN-induced mutants and their progeny we validate high-resolution melt analysis (HRMA) of PCR products as a sensitive and universal genotyping tool. Furthermore, HRMA of off-target sites in mutant founder mice revealed no evidence for undesired TALEN-mediated processing of related genomic sequences. The combination of TALEN-95A mRNAs for enhanced mutagenesis and of HRMA for simplified genotyping enables the accelerated, routine production of new mouse models for the study of genetic disease mechanisms. PMID:23979585

2,6-Dithiopurine blocks toxicity and mutagenesis in human skin cells exposed to sulfur mustard analogues, 2-chloroethyl ethyl sulfide and 2-chloroethyl methyl sulfide.

PubMed

Powell, K Leslie; Boulware, Stephen; Thames, Howard; Vasquez, Karen M; MacLeod, Michael C

2010-03-15

Sulfur mustard (bis-(2-chloroethyl)sulfide) is a well-known chemical warfare agent that induces debilitating cutaneous toxicity in exposed individuals. It is also known to be carcinogenic and mutagenic because of its ability to damage DNA via electrophilic attack. We previously showed that a nucleophilic scavenger, 2,6-dithiopurine (DTP), reacts chemically with several electrophilic carcinogens, blocking DNA damage in vitro and in vivo and abolishing tumor formation in a two-stage mouse skin carcinogenesis model. To assess the potential of DTP as an antagonist of sulfur mustard, we have utilized monofunctional chemical analogues of sulfur mustard, 2-chloroethyl ethyl sulfide (CEES) and 2-chloroethyl methyl sulfide (CEMS), to induce toxicity and mutagenesis in a cell line, NCTC2544, derived from a human skin tumor. We show that DTP blocks cytotoxicity in CEMS- and CEES-treated cells when present at approximately equimolar concentration. A related thiopurine, 9-methyl-6-mercaptopurine, is similarly effective. Correlated with this, we find that DTP is transported into these cells and that adducts between DTP and CEES are found intracellularly. Using a shuttle vector-based mutagenesis system, which allows enumeration of mutations induced in the skin cells by a blue/white colony screen, we find that DTP completely abolishes the mutagenesis induced by CEMS and CEES in human cells.

Topology of evolving, mutagenized viral populations: quasispecies expansion, compression, and operation of negative selection

PubMed Central

2008-01-01

Background The molecular events and evolutionary forces underlying lethal mutagenesis of virus (or virus extinction through an excess of mutations) are not well understood. Here we apply for the first time phylogenetic methods and Partition Analysis of Quasispecies (PAQ) to monitor genetic distances and intra-population structures of mutant spectra of foot-and-mouth disease virus (FMDV) quasispecies subjected to mutagenesis by base and nucleoside analogues. Results Phylogenetic and PAQ analyses have revealed a highly dynamic variation of intrapopulation diversity of FMDV quasispecies. The population diversity first suffers striking expansions in the presence of mutagens and then compressions either when the presence of the mutagenic analogue was discontinued or when a mutation that decreased sensitivity to a mutagen was selected. The pattern of mutations found in the populations was in agreement with the behavior of the corresponding nucleotide analogues with FMDV in vitro. Mutations accumulated at preferred genomic sites, and dn/ds ratios indicate the operation of negative (or purifying) selection in populations subjected to mutagenesis. No evidence of unusually elevated genetic distances has been obtained for FMDV populations approaching extinction. Conclusion Phylogenetic and PAQ analysis provide adequate procedures to describe the evolution of viral sequences subjected to lethal mutagenesis. These methods define the changes of intra-population structure more precisely than mutation frequencies and Shannon entropies. PAQ is very sensitive to variations of intrapopulation genetic distances. Strong negative (or purifying) selection operates in FMDV populations subjected to enhanced mutagenesis. The quantifications provide evidence that extinction does not imply unusual increases of intrapopulation complexity, in support of the lethal defection model of virus extinction. PMID:18637173

Molecular dynamics simulation studies and in vitro site directed mutagenesis of avian beta-defensin Apl_AvBD2

PubMed Central

2010-01-01

Background Defensins comprise a group of antimicrobial peptides, widely recognized as important elements of the innate immune system in both animals and plants. Cationicity, rather than the secondary structure, is believed to be the major factor defining the antimicrobial activity of defensins. To test this hypothesis and to improve the activity of the newly identified avian β-defensin Apl_AvBD2 by enhancing the cationicity, we performed in silico site directed mutagenesis, keeping the predicted secondary structure intact. Molecular dynamics (MD) simulation studies were done to predict the activity. Mutant proteins were made by in vitro site directed mutagenesis and recombinant protein expression, and tested for antimicrobial activity to confirm the results obtained in MD simulation analysis. Results MD simulation revealed subtle, but critical, structural variations between the wild type Apl_AvBD2 and the more cationic in silico mutants, which were not detected in the initial structural prediction by homology modelling. The C-terminal cationic 'claw' region, important in antimicrobial activity, which was intact in the wild type, showed changes in shape and orientation in all the mutant peptides. Mutant peptides also showed increased solvent accessible surface area and more number of hydrogen bonds with the surrounding water molecules. In functional studies, the Escherichia coli expressed, purified recombinant mutant proteins showed total loss of antimicrobial activity compared to the wild type protein. Conclusion The study revealed that cationicity alone is not the determining factor in the microbicidal activity of antimicrobial peptides. Factors affecting the molecular dynamics such as hydrophobicity, electrostatic interactions and the potential for oligomerization may also play fundamental roles. It points to the usefulness of MD simulation studies in successful engineering of antimicrobial peptides for improved activity and other desirable functions. PMID:20122244

Maternal Supply of Cas9 to Zygotes Facilitates the Efficient Generation of Site-Specific Mutant Mouse Models

PubMed Central

Cebrian-Serrano, Alberto; Zha, Shijun; Hanssen, Lars; Biggs, Daniel; Preece, Christopher

2017-01-01

Genome manipulation in the mouse via microinjection of CRISPR/Cas9 site-specific nucleases has allowed the production time for genetically modified mouse models to be significantly reduced. Successful genome manipulation in the mouse has already been reported using Cas9 supplied by microinjection of a DNA construct, in vitro transcribed mRNA and recombinant protein. Recently the use of transgenic strains of mice overexpressing Cas9 has been shown to facilitate site-specific mutagenesis via maternal supply to zygotes and this route may provide an alternative to exogenous supply. We have investigated the feasibility of supplying Cas9 genetically in more detail and for this purpose we report the generation of a transgenic mice which overexpress Cas9 ubiquitously, via a CAG-Cas9 transgene targeted to the Gt(ROSA26)Sor locus. We show that zygotes prepared from female mice harbouring this transgene are sufficiently loaded with maternally contributed Cas9 for efficient production of embryos and mice harbouring indel, genomic deletion and knock-in alleles by microinjection of guide RNAs and templates alone. We compare the mutagenesis rates and efficacy of mutagenesis using this genetic supply with exogenous Cas9 supply by either mRNA or protein microinjection. In general, we report increased generation rates of knock-in alleles and show that the levels of mutagenesis at certain genome target sites are significantly higher and more consistent when Cas9 is supplied genetically relative to exogenous supply. PMID:28081254

Protein and genome evolution in Mammalian cells for biotechnology applications.

PubMed

Majors, Brian S; Chiang, Gisela G; Betenbaugh, Michael J

2009-06-01

Mutation and selection are the essential steps of evolution. Researchers have long used in vitro mutagenesis, expression, and selection techniques in laboratory bacteria and yeast cultures to evolve proteins with new properties, termed directed evolution. Unfortunately, the nature of mammalian cells makes applying these mutagenesis and whole-organism evolution techniques to mammalian protein expression systems laborious and time consuming. Mammalian evolution systems would be useful to test unique mammalian cell proteins and protein characteristics, such as complex glycosylation. Protein evolution in mammalian cells would allow for generation of novel diagnostic tools and designer polypeptides that can only be tested in a mammalian expression system. Recent advances have shown that mammalian cells of the immune system can be utilized to evolve transgenes during their natural mutagenesis processes, thus creating proteins with unique properties, such as fluorescence. On a more global level, researchers have shown that mutation systems that affect the entire genome of a mammalian cell can give rise to cells with unique phenotypes suitable for commercial processes. This review examines the advances in mammalian cell and protein evolution and the application of this work toward advances in commercial mammalian cell biotechnology.

Improving the solubility of anti-LINGO-1 monoclonal antibody Li33 by isotype switching and targeted mutagenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pepinsky, R. Blake; Silvian, Laura; Berkowitz, Steven A.

2010-11-15

Monoclonal antibodies (Mabs) are a favorite drug platform of the biopharmaceutical industry. Currently, over 20 Mabs have been approved and several hundred others are in clinical trials. The anti-LINGO-1 Mab Li33 was selected from a large panel of antibodies by Fab phage display technology based on its extraordinary biological activity in promoting oligodendrocyte differentiation and myelination in vitro and in animal models of remyelination. However, the Li33 Fab had poor solubility when converted into a full antibody in an immunoglobulin G1 framework. A detailed analysis of the biochemical and structural features of the antibody revealed several possible reasons for itsmore » propensity to aggregate. Here, we successfully applied three molecular approaches (isotype switching, targeted mutagenesis of complementarity determining region residues, and glycosylation site insertion mutagenesis) to address the solubility problem. Through these efforts we were able to improve the solubility of the Li33 Mab from 0.3 mg/mL to >50 mg/mL and reduce aggregation to an acceptable level. These strategies can be readily applied to other proteins with solubility issues.« less

Evidence that antibiotic of Pantoea agglomerans E325 is produced and active against Erwinia amylovora on stigmas of pomaceous blossoms

USDA-ARS?s Scientific Manuscript database

Pantoea agglomerans strain E325, the active ingredient in a commercial product for fire blight, previously was shown to produce a unique pH-sensitive inhibitor in vitro that is specific to E. amylovora. To evaluate antibiosis as a mode of antagonism of E325, Tn5 mutagenesis was used to generate...

Blocking phosphatidylcholine utilization in Pseudomonas aeruginosa, via mutagenesis of fatty acid, glycerol and choline degradation pathways, confirms the importance of this nutrient source in vivo.

PubMed

Sun, Zhenxin; Kang, Yun; Norris, Michael H; Troyer, Ryan M; Son, Mike S; Schweizer, Herbert P; Dow, Steven W; Hoang, Tung T

2014-01-01

Pseudomonas aeruginosa can grow to very high-cell-density (HCD) during infection of the cystic fibrosis (CF) lung. Phosphatidylcholine (PC), the major component of lung surfactant, has been hypothesized to support HCD growth of P. aeruginosa in vivo. The phosphorylcholine headgroup, a glycerol molecule, and two long-chain fatty acids (FAs) are released by enzymatic cleavage of PC by bacterial phospholipase C and lipases. Three different bacterial pathways, the choline, glycerol, and fatty acid degradation pathways, are then involved in the degradation of these PC components. Here, we identified five potential FA degradation (Fad) related fadBA-operons (fadBA1-5, each encoding 3-hydroxyacyl-CoA dehydrogenase and acyl-CoA thiolase). Through mutagenesis and growth analyses, we showed that three (fadBA145) of the five fadBA-operons are dominant in medium-chain and long-chain Fad. The triple fadBA145 mutant also showed reduced ability to degrade PC in vitro. We have previously shown that by partially blocking Fad, via mutagenesis of fadBA5 and fadDs, we could significantly reduce the ability of P. aeruginosa to replicate on FA and PC in vitro, as well as in the mouse lung. However, no studies have assessed the ability of mutants, defective in choline and/or glycerol degradation in conjunction with Fad, to grow on PC or in vivo. Hence, we constructed additional mutants (ΔfadBA145ΔglpD, ΔfadBA145ΔbetAB, and ΔfadBA145ΔbetABΔglpD) significantly defective in the ability to degrade FA, choline, and glycerol and, therefore, PC. The analysis of these mutants in the BALB/c mouse lung infection model showed significant inability to utilize PC in vitro, resulted in decreased replication fitness and competitiveness in vivo compared to the complement strain, although there was little to no variation in typical virulence factor production (e.g., hemolysin, lipase, and protease levels). This further supports the hypothesis that lung surfactant PC serves as an important nutrient for P. aeruginosa during CF lung infection.

The Effects of Di-(2-ethylhexyl)-phthalate Exposure on Fertilization and Embryonic Development In Vitro and Testicular Genomic Mutation In Vivo

PubMed Central

Gu, Yi-Hua; Liu, Miao; Xu, Yan; Yuan, Yao; Sun, Fei; Zhang, Hui-Qin; Shi, Hui-Juan

2012-01-01

The present study was undertaken to determine the reproductive hazards of Di-(2-ethylhexyl)-phthalate (DEHP) on mouse spermatozoa and embryos in vitro and genomic changes in vivo. Direct low-level DEHP exposure (1 μg/ml) on spermatozoa and embryos was investigated by in vitro fertilization (IVF) process, culture of preimplanted embryos in DEHP-supplemented medium and embryo transfer to achieve full term development. Big Blue® transgenic mouse model was employed to evaluate the mutagenesis of testicular genome with in vivo exposure concentration of DEHP (500 mg/kg/day). Generally, DEHP-treated spermatozoa (1 μg/ml, 30 min) presented reduced fertilization ability (P<0.05) and the resultant embryos had decreased developmental potential compared to DMSO controls (P<0.05). Meanwhile, the transferred 2-cell stage embryos derived from treated spermatozoa also exhibited decreased birth rate than that of control (P<0.05). When fertilized oocytes or 2-cell stage embryos were recovered by in vivo fertilization (without treatment) and then exposed to DEHP, the subsequent development proceed to blastocysts was different, fertilized oocytes were significantly affected (P<0.05) whereas developmental progression of 2-cell stage embryos was similar to controls (P>0.05). Testes of the Big Blue® transgenic mice treated with DEHP for 4 weeks indicated an approximately 3-fold increase in genomic DNA mutation frequency compared with controls (P<0.05). These findings unveiled the hazardous effects of direct low-level exposure of DEHP on spermatozoa's fertilization ability as well as embryonic development, and proved that in vivo DEHP exposure posed mutagenic risks in the reproductive organ – at least in testes, are of great concern to human male reproductive health. PMID:23226291

The effects of Di-(2-ethylhexyl)-phthalate exposure on fertilization and embryonic development in vitro and testicular genomic mutation in vivo.

PubMed

Huang, Xue-Feng; Li, Yan; Gu, Yi-Hua; Liu, Miao; Xu, Yan; Yuan, Yao; Sun, Fei; Zhang, Hui-Qin; Shi, Hui-Juan

2012-01-01

The present study was undertaken to determine the reproductive hazards of Di-(2-ethylhexyl)-phthalate (DEHP) on mouse spermatozoa and embryos in vitro and genomic changes in vivo. Direct low-level DEHP exposure (1 μg/ml) on spermatozoa and embryos was investigated by in vitro fertilization (IVF) process, culture of preimplanted embryos in DEHP-supplemented medium and embryo transfer to achieve full term development. Big Blue® transgenic mouse model was employed to evaluate the mutagenesis of testicular genome with in vivo exposure concentration of DEHP (500 mg/kg/day). Generally, DEHP-treated spermatozoa (1 μg/ml, 30 min) presented reduced fertilization ability (P<0.05) and the resultant embryos had decreased developmental potential compared to DMSO controls (P<0.05). Meanwhile, the transferred 2-cell stage embryos derived from treated spermatozoa also exhibited decreased birth rate than that of control (P<0.05). When fertilized oocytes or 2-cell stage embryos were recovered by in vivo fertilization (without treatment) and then exposed to DEHP, the subsequent development proceed to blastocysts was different, fertilized oocytes were significantly affected (P<0.05) whereas developmental progression of 2-cell stage embryos was similar to controls (P>0.05). Testes of the Big Blue® transgenic mice treated with DEHP for 4 weeks indicated an approximately 3-fold increase in genomic DNA mutation frequency compared with controls (P<0.05). These findings unveiled the hazardous effects of direct low-level exposure of DEHP on spermatozoa's fertilization ability as well as embryonic development, and proved that in vivo DEHP exposure posed mutagenic risks in the reproductive organ - at least in testes, are of great concern to human male reproductive health.

Genetically modified proteins: functional improvement and chimeragenesis

PubMed Central

Balabanova, Larissa; Golotin, Vasily; Podvolotskaya, Anna; Rasskazov, Valery

2015-01-01

This review focuses on the emerging role of site-specific mutagenesis and chimeragenesis for the functional improvement of proteins in areas where traditional protein engineering methods have been extensively used and practically exhausted. The novel path for the creation of the novel proteins has been created on the farther development of the new structure and sequence optimization algorithms for generating and designing the accurate structure models in result of x-ray crystallography studies of a lot of proteins and their mutant forms. Artificial genetic modifications aim to expand nature's repertoire of biomolecules. One of the most exciting potential results of mutagenesis or chimeragenesis finding could be design of effective diagnostics, bio-therapeutics and biocatalysts. A sampling of recent examples is listed below for the in vivo and in vitro genetically improvement of various binding protein and enzyme functions, with references for more in-depth study provided for the reader's benefit. PMID:26211369

Nuclear localization of the dystrophin-associated protein α-dystrobrevin through importin α2/β1 is critical for interaction with the nuclear lamina/maintenance of nuclear integrity.

PubMed

Aguilar, Areli; Wagstaff, Kylie M; Suárez-Sánchez, Rocío; Zinker, Samuel; Jans, David A; Cisneros, Bulmaro

2015-05-01

Although α-dystrobrevin (DB) is assembled into the dystrophin-associated protein complex, which is central to cytoskeletal organization, it has also been found in the nucleus. Here we delineate the nuclear import pathway responsible for nuclear targeting of α-DB for the first time, together with the importance of nuclear α-DB in determining nuclear morphology. We map key residues of the nuclear localization signal of α-DB within the zinc finger domain (ZZ) using various truncated versions of the protein, and site-directed mutagenesis. Pulldown, immunoprecipitation, and AlphaScreen assays showed that the importin (IMP) α2/β1 heterodimer interacts with high affinity with the ZZ domain of α-DB. In vitro nuclear import assays using antibodies to specific importins, as well as in vivo studies using siRNA or a dominant negative importin construct, confirmed the key role of IMPα2/β1 in α-DB nuclear translocation. Knockdown of α-DB expression perturbed cell cycle progression in C2C12 myoblasts, with decreased accumulation of cells in S phase and, significantly, altered localization of lamins A/C, B1, and B2 with accompanying gross nuclear morphology defects. Because α-DB interacts specifically with lamin B1 in vivo and in vitro, nuclear α-DB would appear to play a key role in nuclear shape maintenance through association with the nuclear lamina. © FASEB.

Increased efficiency of targeted mutagenesis by CRISPR/Cas9 in plants using heat stress.

PubMed

LeBlanc, Chantal; Zhang, Fei; Mendez, Josefina; Lozano, Yamile; Chatpar, Krishna; Irish, Vivian F; Jacob, Yannick

2018-01-01

The CRISPR/Cas9 system has greatly improved our ability to engineer targeted mutations in eukaryotic genomes. While CRISPR/Cas9 appears to work universally, the efficiency of targeted mutagenesis and the adverse generation of off-target mutations vary greatly between different organisms. In this study, we report that Arabidopsis plants subjected to heat stress at 37°C show much higher frequencies of CRISPR-induced mutations compared to plants grown continuously at the standard temperature (22°C). Using quantitative assays relying on green fluorescent protein (GFP) reporter genes, we found that targeted mutagenesis by CRISPR/Cas9 in Arabidopsis is increased by approximately 5-fold in somatic tissues and up to 100-fold in the germline upon heat treatment. This effect of temperature on the mutation rate is not limited to Arabidopsis, as we observed a similar increase in targeted mutations by CRISPR/Cas9 in Citrus plants exposed to heat stress at 37°C. In vitro assays demonstrate that Cas9 from Streptococcus pyogenes (SpCas9) is more active in creating double-stranded DNA breaks at 37°C than at 22°C, thus indicating a potential contributing mechanism for the in vivo effect of temperature on CRISPR/Cas9. This study reveals the importance of temperature in modulating SpCas9 activity in eukaryotes, and provides a simple method to increase on-target mutagenesis in plants using CRISPR/Cas9. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Random Mutagenesis of the Aspergillus oryzae Genome Results in Fungal Antibacterial Activity

PubMed Central

Leonard, Cory A.; Brown, Stacy D.; Hayman, J. Russell

2013-01-01

Multidrug-resistant bacteria cause severe infections in hospitals and communities. Development of new drugs to combat resistant microorganisms is needed. Natural products of microbial origin are the source of most currently available antibiotics. We hypothesized that random mutagenesis of Aspergillus oryzae would result in secretion of antibacterial compounds. To address this hypothesis, we developed a screen to identify individual A. oryzae mutants that inhibit the growth of Methicillin-resistant Staphylococcus aureus (MRSA) in vitro. To randomly generate A. oryzae mutant strains, spores were treated with ethyl methanesulfonate (EMS). Over 3000 EMS-treated A. oryzae cultures were tested in the screen, and one isolate, CAL220, exhibited altered morphology and antibacterial activity. Culture supernatant from this isolate showed antibacterial activity against Methicillin-sensitive Staphylococcus aureus, MRSA, and Pseudomonas aeruginosa, but not Klebsiella pneumonia or Proteus vulgaris. The results of this study support our hypothesis and suggest that the screen used is sufficient and appropriate to detect secreted antibacterial fungal compounds resulting from mutagenesis of A. oryzae. Because the genome of A. oryzae has been sequenced and systems are available for genetic transformation of this organism, targeted as well as random mutations may be introduced to facilitate the discovery of novel antibacterial compounds using this system. PMID:23983696

Random Mutagenesis of the Aspergillus oryzae Genome Results in Fungal Antibacterial Activity.

PubMed

Leonard, Cory A; Brown, Stacy D; Hayman, J Russell

2013-01-01

Multidrug-resistant bacteria cause severe infections in hospitals and communities. Development of new drugs to combat resistant microorganisms is needed. Natural products of microbial origin are the source of most currently available antibiotics. We hypothesized that random mutagenesis of Aspergillus oryzae would result in secretion of antibacterial compounds. To address this hypothesis, we developed a screen to identify individual A. oryzae mutants that inhibit the growth of Methicillin-resistant Staphylococcus aureus (MRSA) in vitro. To randomly generate A. oryzae mutant strains, spores were treated with ethyl methanesulfonate (EMS). Over 3000 EMS-treated A. oryzae cultures were tested in the screen, and one isolate, CAL220, exhibited altered morphology and antibacterial activity. Culture supernatant from this isolate showed antibacterial activity against Methicillin-sensitive Staphylococcus aureus, MRSA, and Pseudomonas aeruginosa, but not Klebsiella pneumonia or Proteus vulgaris. The results of this study support our hypothesis and suggest that the screen used is sufficient and appropriate to detect secreted antibacterial fungal compounds resulting from mutagenesis of A. oryzae. Because the genome of A. oryzae has been sequenced and systems are available for genetic transformation of this organism, targeted as well as random mutations may be introduced to facilitate the discovery of novel antibacterial compounds using this system.

Escherichia coli gamma-glutamyltranspeptidase mutants deficient in processing to subunits.

PubMed

Hashimoto, W; Suzuki, H; Nohara, S; Kumagai, H

1992-11-30

Arginyl residues 513 and 571 of Escherichia coli K-12 gamma-glutamyl-transpeptidase (EC 2.3.2.2) were substituted with alanyl and glycyl residues, respectively, by oligonucleotide-directed in vitro mutagenesis. Both mutants were devoid of the enzymatic activity. On Western blot analysis, we found that both mutants accumulated a gamma-glutamyltranspeptidase precursor which was not processed into large and small subunits in the periplasmic space of Escherichia coli.

Investigation of TRPV1 loss-of-function phenotypes in TRPV1 Leu206Stop mice generated by N-ethyl-N-nitrosourea mutagenesis.

PubMed

Christoph, Thomas; Kögel, Babette; Schiene, Klaus; Peters, Thomas; Schröder, Wolfgang

2018-06-02

N-ethyl-N-nitrosourea (ENU) random mutagenesis was used to generate a mouse model for the analysis of the transient receptor potential vanilloid 1 (TRPV1) cation channel. A transversion from T→A in exon 4 led to a Leu206Stop mutation generating a loss-of-function mutant. The TRPV1 agonist capsaicin was used to analyze functional and nociceptive parameters in vitro and in vivo in TRPV1 Leu206Stop mice and congenic C3HeB/FeJ controls. Capsaicin-induced [Ca 2+ ] i changes in small diameter DRG neurons were significantly diminished in TRPV1 Leu206Stop mice and administration of capsaicin induced neither hypothermia nor nocifensive behaviour in vivo. TRPV1 Leu206Stop mice were tested in the spinal nerve ligation of mononeuropathic pain and developed mechanical hypersensitivity two weeks after nerve injury. In the open field test, a significant increase in spontaneous locomotion was detected in TRPV1 Leu206Stop mice as compared to wildtype controls. TRPV1 knockout mice have been reported to carry a similar phenotype regarding capsaicin-evoked responses in vitro and in vivo. However, in contrast to TRPV1 Leu206Stop mice, TRPV1 knockout mice did not differ in spontaneous locomotion as compared to congenic C57BL/6 mice, suggesting subtle ENU-dependent or independent strain differences between TRPV1 Leu206Stop mice and their wildtype controls. In summary, these data revealed a target-related (i.e. capsaicin-evoked) phenotype of TRPV1 Leu206Stop mice closely resembling that of published TRPV1 knockout mice. However, since ENU-mutant mice are congenic with the mouse strain initially used in random mutagenesis, direct phenotypic comparison with the respective wildtype controls is possible, and the time-consuming backcrossing in lines with targeted mutations is avoided. Copyright © 2018 Elsevier Inc. All rights reserved.

Can enzyme engineering benefit from the modulation of protein motions? Lessons learned from NMR relaxation dispersion experiments.

PubMed

Doucet, Nicolas

2011-04-01

Despite impressive progress in protein engineering and design, our ability to create new and efficient enzyme activities remains a laborious and time-consuming endeavor. In the past few years, intricate combinations of rational mutagenesis, directed evolution and computational methods have paved the way to exciting engineering examples and are now offering a new perspective on the structural requirements of enzyme activity. However, these structure-function analyses are usually guided by the time-averaged static models offered by enzyme crystal structures, which often fail to describe the functionally relevant 'invisible states' adopted by proteins in space and time. To alleviate such limitations, NMR relaxation dispersion experiments coupled to mutagenesis studies have recently been applied to the study of enzyme catalysis, effectively complementing 'structure-function' analyses with 'flexibility-function' investigations. In addition to offering quantitative, site-specific information to help characterize residue motion, these NMR methods are now being applied to enzyme engineering purposes, providing a powerful tool to help characterize the effects of controlling long-range networks of flexible residues affecting enzyme function. Recent advancements in this emerging field are presented here, with particular attention to mutagenesis reports highlighting the relevance of NMR relaxation dispersion tools in enzyme engineering.

Leapfrogging: primordial germ cell transplantation permits recovery of CRISPR/Cas9-induced mutations in essential genes

PubMed Central

Fish, Margaret B.; Cho, Ken W. Y.

2016-01-01

CRISPR/Cas9 genome editing is revolutionizing genetic loss-of-function analysis but technical limitations remain that slow progress when creating mutant lines. First, in conventional genetic breeding schemes, mosaic founder animals carrying mutant alleles are outcrossed to produce F1 heterozygotes. Phenotypic analysis occurs in the F2 generation following F1 intercrosses. Thus, mutant analyses will require multi-generational studies. Second, when targeting essential genes, efficient mutagenesis of founders is often lethal, preventing the acquisition of mature animals. Reducing mutagenesis levels may improve founder survival, but results in lower, more variable rates of germline transmission. Therefore, an efficient approach to study lethal mutations would be useful. To overcome these shortfalls, we introduce ‘leapfrogging’, a method combining efficient CRISPR mutagenesis with transplantation of mutated primordial germ cells into a wild-type host. Tested using Xenopus tropicalis, we show that founders containing transplants transmit mutant alleles with high efficiency. F1 offspring from intercrosses between F0 animals that carry embryonic lethal alleles recapitulate loss-of-function phenotypes, circumventing an entire generation of breeding. We anticipate that leapfrogging will be transferable to other species. PMID:27385011

Cationic Antimicrobial Peptides Promote Microbial Mutagenesis and Pathoadaptation in Chronic Infections

PubMed Central

Limoli, Dominique H.; Rockel, Andrea B.; Host, Kurtis M.; Jha, Anuvrat; Kopp, Benjamin T.; Hollis, Thomas; Wozniak, Daniel J.

2014-01-01

Acquisition of adaptive mutations is essential for microbial persistence during chronic infections. This is particularly evident during chronic Pseudomonas aeruginosa lung infections in cystic fibrosis (CF) patients. Thus far, mutagenesis has been attributed to the generation of reactive species by polymorphonucleocytes (PMN) and antibiotic treatment. However, our current studies of mutagenesis leading to P. aeruginosa mucoid conversion have revealed a potential new mutagen. Our findings confirmed the current view that reactive oxygen species can promote mucoidy in vitro, but revealed PMNs are proficient at inducing mucoid conversion in the absence of an oxidative burst. This led to the discovery that cationic antimicrobial peptides can be mutagenic and promote mucoidy. Of specific interest was the human cathelicidin LL-37, canonically known to disrupt bacterial membranes leading to cell death. An alternative role was revealed at sub-inhibitory concentrations, where LL-37 was found to induce mutations within the mucA gene encoding a negative regulator of mucoidy and to promote rifampin resistance in both P. aeruginosa and Escherichia coli. The mechanism of mutagenesis was found to be dependent upon sub-inhibitory concentrations of LL-37 entering the bacterial cytosol and binding to DNA. LL-37/DNA interactions then promote translesion DNA synthesis by the polymerase DinB, whose error-prone replication potentiates the mutations. A model of LL-37 bound to DNA was generated, which reveals amino termini α-helices of dimerized LL-37 bind the major groove of DNA, with numerous DNA contacts made by LL-37 basic residues. This demonstrates a mutagenic role for antimicrobials previously thought to be insusceptible to resistance by mutation, highlighting a need to further investigate their role in evolution and pathoadaptation in chronic infections. PMID:24763694

Accurate RNA consensus sequencing for high-fidelity detection of transcriptional mutagenesis-induced epimutations.

PubMed

Reid-Bayliss, Kate S; Loeb, Lawrence A

2017-08-29

Transcriptional mutagenesis (TM) due to misincorporation during RNA transcription can result in mutant RNAs, or epimutations, that generate proteins with altered properties. TM has long been hypothesized to play a role in aging, cancer, and viral and bacterial evolution. However, inadequate methodologies have limited progress in elucidating a causal association. We present a high-throughput, highly accurate RNA sequencing method to measure epimutations with single-molecule sensitivity. Accurate RNA consensus sequencing (ARC-seq) uniquely combines RNA barcoding and generation of multiple cDNA copies per RNA molecule to eliminate errors introduced during cDNA synthesis, PCR, and sequencing. The stringency of ARC-seq can be scaled to accommodate the quality of input RNAs. We apply ARC-seq to directly assess transcriptome-wide epimutations resulting from RNA polymerase mutants and oxidative stress.

Epigenetic Effects of Cadmium [Abstract and Poster 2014] ...

EPA Pesticide Factsheets

We have reviewed the literature on in vitro and in vivo experiments as well as human studies on cadmium to understand the epigenetic mechanisms involved in cadmium- induced toxicity and carcinogenicity. This presentation will identify gaps in our current understanding and suggest future studies to further our knowledge. The abstract and poster are to be presented at the 45th Annual Meeting of the Environmental Mutagenesis and Genomics Society (EMGS) to be held from Septmeber 13 to 17, 2014.

DOE final report 100608.doc

DOE Office of Scientific and Technical Information (OSTI.GOV)

Golden, Susan S

2008-10-16

The aim of this project was to inactivate each locus of the genome of the cyanobacterium Synechococcus elongatus PCC 7942 and screen resulting mutants for altered circadian phenotypes. The immediate goal was to identify all open reading frames (ORFs) that contribute to circadian timing. An additional result was to create a complete archived set of mutagenesis templates, of great utility for the wider research community, that will allow inactivation of any given locus in the genome of S. elongatus. Clones that carry segments of the S. elongatus genome were saturated with transposon insertions in vitro. We completed saturation mutagenesis ofmore » the chromosome (~2800 ORFs). The positions of insertions were sequenced for 17,767 mutagenized clones. Each individual insertion into the S. elongatus DNA in a cosmid or plasmid is a substrate for mutagenesis of the genome via homologous recombination. Because the complete insertion mutation clone set is 5-7 fold redundant, we produced a streamlined set of clones that contains one insertion mutation per locus in the genome, a unigene set. All clones are archived as Escherichia coli stocks frozen in glycerol in 96-well plates at -85ºC and as replicas of these plates on Whatman CloneSaver cards. Each of the mutagenesis substrates from the unigene set has been recombined into the chromosome of wild-type S. elongatus and these cyanobacterial mutants have been archived at -85ºC as well. S. elongatus insertion mutants defective for than 1400 independent genes have screened in luciferase reporter gene backgrounds to evaluate the effect of each mutation on circadian rhythms of gene expression. For the first 700 genes tested, mutagenesis of 71 different ORFs resulted in circadian phenotypes. The mutagenesis project also created insertion mutations in the endogenous large plasmid of S. elongatus, pANL. The sequence of pANL revealed two potential addiction cassettes that appear to account for selection for plasmid persistence. Genetic experiments confirmed that these regions are present on all sub-sets of the plasmid that can replace wild-type pANL. Analysis of mutants defective in each of the remaining ~1400 genes for defects in circadian rhythms will be completed with support from another agency as part of a larger project on circadian rhythms in this cyanobacterium.« less

Pyrrole-indolinone SU11652 targets the nucleoside diphosphate kinase from Leishmania parasites.

PubMed

Vieira, Plínio Salmazo; Souza, Tatiana de Arruda Campos Brasil; Honorato, Rodrigo Vargas; Zanphorlin, Letícia Maria; Severiano, Kelven Ulisses; Rocco, Silvana Aparecida; de Oliveira, Arthur Henrique Cavalcante; Cordeiro, Artur Torres; Oliveira, Paulo Sérgio Lopes; de Giuseppe, Priscila Oliveira; Murakami, Mário Tyago

2017-07-01

Nucleoside diphosphate kinases (NDKs) are key enzymes in the purine-salvage pathway of trypanosomatids and have been associated with the maintenance of host-cell integrity for the benefit of the parasite, being potential targets for rational drug discovery and design. The NDK from Leishmania major (LmNDK) and mutants were expressed and purified to homogeneity. Thermal shift assays were employed to identify potential inhibitors for LmNDK. Calorimetric experiments, site-directed mutagenesis and molecular docking analysis were performed to validate the interaction and to evaluate the structural basis of ligand recognition. Furthermore, the anti-leishmanial activity of the newly identified and validated compound was tested in vitro against different Leishmania species. The molecule SU11652, a Sunitinib analog, was identified as a potential inhibitor for LmNDK and structural studies indicated that this molecule binds to the active site of LmNDK in a similar conformation to nucleotides, mimicking natural substrates. Isothermal titration calorimetry experiments combined with site-directed mutagenesis revealed that the residues H50 and H117, considered essential for catalysis, play an important role in ligand binding. In vitro cell studies showed that SU11652 had similar efficacy to Amphotericin b against some Leishmania species. Together, our results indicate the pyrrole-indolinone SU11652 as a promising scaffold for the rational design of new drugs targeting the enzyme NDK from Leishmania parasites. Copyright © 2017 Elsevier Inc. All rights reserved.

Comprehensive Ex Vivo Transposon Mutagenesis Identifies Genes That Promote Growth Factor Independence and Leukemogenesis.

PubMed

Guo, Yabin; Updegraff, Barrett L; Park, Sunho; Durakoglugil, Deniz; Cruz, Victoria H; Maddux, Sarah; Hwang, Tae Hyun; O'Donnell, Kathryn A

2016-02-15

Aberrant signaling through cytokine receptors and their downstream signaling pathways is a major oncogenic mechanism underlying hematopoietic malignancies. To better understand how these pathways become pathologically activated and to potentially identify new drivers of hematopoietic cancers, we developed a high-throughput functional screening approach using ex vivo mutagenesis with the Sleeping Beauty transposon. We analyzed over 1,100 transposon-mutagenized pools of Ba/F3 cells, an IL3-dependent pro-B-cell line, which acquired cytokine independence and tumor-forming ability. Recurrent transposon insertions could be mapped to genes in the JAK/STAT and MAPK pathways, confirming the ability of this strategy to identify known oncogenic components of cytokine signaling pathways. In addition, recurrent insertions were identified in a large set of genes that have been found to be mutated in leukemia or associated with survival, but were not previously linked to the JAK/STAT or MAPK pathways nor shown to functionally contribute to leukemogenesis. Forced expression of these novel genes resulted in IL3-independent growth in vitro and tumorigenesis in vivo, validating this mutagenesis-based approach for identifying new genes that promote cytokine signaling and leukemogenesis. Therefore, our findings provide a broadly applicable approach for classifying functionally relevant genes in diverse malignancies and offer new insights into the impact of cytokine signaling on leukemia development. ©2015 American Association for Cancer Research.

General method for site-directed mutagenesis in Escherichia coli O18ac:K1:H7: deletion of the inducible superoxide dismutase gene, sodA, does not diminish bacteremia in neonatal rats.

PubMed

Bloch, C A; Thorne, G M; Ausubel, F M

1989-07-01

A defined deletion in the Escherichia coli K-12 sodA gene (encoding manganese-superoxide dismutase) linked to a nontransposable selectable marker was generated by transposon Tn5 insertion in combination with in vitro mutagenesis. This mutant allele was used to replace the wild-type sodA gene in an E. coli clinical isolate of serotype O18ac:K1:H7 by bacteriophage P1 transduction. The O18ac:K1:H7 sodA mutant contained no manganese-superoxide dismutase and no hybrid manganese-iron-superoxide dismutase. The sodA mutant was more sensitive to paraquat toxicity than were the parental strain and an isogenic mutant bearing an analogously constructed sodA+ Tn5 insertion allele. In a suckling rat model for bacteremia following oral inoculation of E. coli K1, the sodA mutant was undiminished in its capabilities both to colonize the gastrointestinal tract and, surprisingly, to cause bacteremia. In conjunction with the rat model for E. coli K1 pathogenesis, the method for site-directed mutagenesis described in this paper permits determination of the role played in colonization and bacteremia by any K1 gene which either has a homolog in E. coli K-12 or can be cloned and manipulated therein.

Explosive mutation accumulation triggered by heterozygous human Pol ε proofreading-deficiency is driven by suppression of mismatch repair

PubMed Central

Campbell, Brittany B; Ungerleider, Nathan; Light, Nicholas; Wu, Tong; LeCompte, Kimberly G; Goksenin, A Yasemin; Bunnell, Bruce A; Tabori, Uri; Shlien, Adam

2018-01-01

Tumors defective for DNA polymerase (Pol) ε proofreading have the highest tumor mutation burden identified. A major unanswered question is whether loss of Pol ε proofreading by itself is sufficient to drive this mutagenesis, or whether additional factors are necessary. To address this, we used a combination of next generation sequencing and in vitro biochemistry on human cell lines engineered to have defects in Pol ε proofreading and mismatch repair. Absent mismatch repair, monoallelic Pol ε proofreading deficiency caused a rapid increase in a unique mutation signature, similar to that observed in tumors from patients with biallelic mismatch repair deficiency and heterozygous Pol ε mutations. Restoring mismatch repair was sufficient to suppress the explosive mutation accumulation. These results strongly suggest that concomitant suppression of mismatch repair, a hallmark of colorectal and other aggressive cancers, is a critical force for driving the explosive mutagenesis seen in tumors expressing exonuclease-deficient Pol ε. PMID:29488881

Explosive mutation accumulation triggered by heterozygous human Pol ε proofreading-deficiency is driven by suppression of mismatch repair.

PubMed

Hodel, Karl P; de Borja, Richard; Henninger, Erin E; Campbell, Brittany B; Ungerleider, Nathan; Light, Nicholas; Wu, Tong; LeCompte, Kimberly G; Goksenin, A Yasemin; Bunnell, Bruce A; Tabori, Uri; Shlien, Adam; Pursell, Zachary F

2018-02-28

Tumors defective for DNA polymerase (Pol) ε proofreading have the highest tumor mutation burden identified. A major unanswered question is whether loss of Pol ε proofreading by itself is sufficient to drive this mutagenesis, or whether additional factors are necessary. To address this, we used a combination of next generation sequencing and in vitro biochemistry on human cell lines engineered to have defects in Pol ε proofreading and mismatch repair. Absent mismatch repair, monoallelic Pol ε proofreading deficiency caused a rapid increase in a unique mutation signature, similar to that observed in tumors from patients with biallelic mismatch repair deficiency and heterozygous Pol ε mutations. Restoring mismatch repair was sufficient to suppress the explosive mutation accumulation. These results strongly suggest that concomitant suppression of mismatch repair, a hallmark of colorectal and other aggressive cancers, is a critical force for driving the explosive mutagenesis seen in tumors expressing exonuclease-deficient Pol ε. © 2018, Hodel et al.

Construction of a mutagenesis cartridge for poliovirus genome-linked viral protein: isolation and characterization of viable and nonviable mutants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kuhn, R.J.; Tada, H.; Ypma-Wong, M.F.

1988-01-01

By following a strategy of genetic analysis of poliovirus, the authors have constructed a synthetic mutagenesis cartridge spanning the genome-linked viral protein coding region and flanking cleavage sites in an infectious cDNA clone of the type I (Mahoney) genome. The insertion of new restriction sites within the infectious clone has allowed them to replace the wild-type sequences with short complementary pairs of synthetic oligonucleotides containing various mutations. A set of mutations have been made that create methionine codons within the genome-linked viral protein region. The resulting viruses have growth characteristics similar to wild type. Experiments that led to an alterationmore » of the tyrosine residue responsible for the linkage to RNA have resulted in nonviable virus. In one mutant, proteolytic processing assayed in vitro appeared unimpaired by the mutation. They suggest that the position of the tyrosine residue is important for genome-linked viral protein function(s).« less

Production of Trichoderma strains with pesticide-polyresistance by mutagenesis and protoplast fusion.

PubMed

Hatvani, Lóránt; Manczinger, László; Kredics, László; Szekeres, András; Antal, Zsuzsanna; Vágvölgyi, Csaba

2006-01-01

The sensitivity of two cold-tolerant Trichoderma strains belonging to the species T. harzianum and T. atroviride was determined to a series of pesticides widely used in agriculture. From the 16 pesticides tested, seven fungicides: copper sulfate, carbendazim, mancozeb, tebuconazole, imazalil, captan and thiram inhibited colony growth of the test strains significantly with minimal inhibitory concentrations of 300, 0.4, 50, 100, 100, 100 and 50 microg/ml, respectively. Mutants resistant to carbendazim and tebuconazole were produced from both wild type strains by means of UV-mutagenesis. The cross-resistance capabilities and in vitro antagonistic properties of the mutants were determined. Carbendazim-resistant mutants showed total cross-resistance to benomyl and thiabendazole at a concentration of 20 microg/ml. Intraspecific protoplast fusion was carried out between carbendazim- and tebuconazole-resistant mutants of both parental strains, and putative haploid recombinants with stable resistance to both pesticides were produced in the case of T. atroviride. These pesticide-polyresistant progenies are potential candidates for application in an integrated pest management system.

Pro-oncogene Pokemon promotes breast cancer progression by upregulating survivin expression.

PubMed

Zu, Xuyu; Ma, Jun; Liu, Hongxia; Liu, Feng; Tan, Chunyan; Yu, Lingling; Wang, Jue; Xie, Zhenhua; Cao, Deliang; Jiang, Yuyang

2011-03-10

Pokemon is an oncogenic transcription factor involved in cell growth, differentiation and oncogenesis, but little is known about its role in human breast cancer. In this study, we aimed to reveal the role of Pokemon in breast cancer progression and patient survival and to understand its underlying mechanisms. Tissue microarray analysis of breast cancer tissues from patients with complete clinicopathological data and more than 20 years of follow-up were used to evaluate Pokemon expression and its correlation with the progression and prognosis of the disease. DNA microarray analysis of MCF-7 cells that overexpress Pokemon was used to identify Pokemon target genes. Chromatin immunoprecipitation (ChIP) and site-directed mutagenesis were utilized to determine how Pokemon regulates survivin expression, a target gene. Pokemon was found to be overexpressed in 158 (86.8%) of 182 breast cancer tissues, and its expression was correlated with tumor size (P = 0.0148) and lymph node metastasis (P = 0.0014). Pokemon expression led to worse overall (n = 175, P = 0.01) and disease-related (n = 79, P = 0.0134) patient survival. DNA microarray analyses revealed that in MCF-7 breast cancer cells, Pokemon regulates the expression of at least 121 genes involved in several signaling and metabolic pathways, including anti-apoptotic survivin. In clinical specimens, Pokemon and survivin expression were highly correlated (n = 49, r = 0.6799, P < 0.0001). ChIP and site-directed mutagenesis indicated that Pokemon induces survivin expression by binding to the GT boxes in its promoter. Pokemon promotes breast cancer progression by upregulating survivin expression and thus may be a potential target for the treatment of this malignancy.

Pro-oncogene Pokemon promotes breast cancer progression by upregulating survivin expression

PubMed Central

2011-01-01

Introduction Pokemon is an oncogenic transcription factor involved in cell growth, differentiation and oncogenesis, but little is known about its role in human breast cancer. In this study, we aimed to reveal the role of Pokemon in breast cancer progression and patient survival and to understand its underlying mechanisms. Methods Tissue microarray analysis of breast cancer tissues from patients with complete clinicopathological data and more than 20 years of follow-up were used to evaluate Pokemon expression and its correlation with the progression and prognosis of the disease. DNA microarray analysis of MCF-7 cells that overexpress Pokemon was used to identify Pokemon target genes. Chromatin immunoprecipitation (ChIP) and site-directed mutagenesis were utilized to determine how Pokemon regulates survivin expression, a target gene. Results Pokemon was found to be overexpressed in 158 (86.8%) of 182 breast cancer tissues, and its expression was correlated with tumor size (P = 0.0148) and lymph node metastasis (P = 0.0014). Pokemon expression led to worse overall (n = 175, P = 0.01) and disease-related (n = 79, P = 0.0134) patient survival. DNA microarray analyses revealed that in MCF-7 breast cancer cells, Pokemon regulates the expression of at least 121 genes involved in several signaling and metabolic pathways, including anti-apoptotic survivin. In clinical specimens, Pokemon and survivin expression were highly correlated (n = 49, r = 0.6799, P < 0.0001). ChIP and site-directed mutagenesis indicated that Pokemon induces survivin expression by binding to the GT boxes in its promoter. Conclusions Pokemon promotes breast cancer progression by upregulating survivin expression and thus may be a potential target for the treatment of this malignancy. PMID:21392388

Genotoxicity assessment of an energetic propellant compound, 3-nitro-1,2,4-triazol-5-one (NTO).

PubMed

Reddy, Gunda; Song, Jian; Kirby, Paul; Lent, Emily M; Crouse, Lee C B; Johnson, Mark S

2011-02-03

3-Nitro-1,2,4-triazol-5-one (NTO) is an energetic explosive proposed for use in weapon systems, to reduce the sensitivity of warheads. In order to develop toxicity data for safety assessment, we investigated the genotoxicity of NTO, using a battery of genotoxicity tests, which included the Ames test, Chinese Hamster Ovary (CHO) cell chromosome aberration test, L5178Y TK(+/-) mouse lymphoma mutagenesis test and rat micronucleus test. NTO was not mutagenic in the Ames test or in Escherichia coli (WP2uvrA). NTO did not induce chromosomal aberrations in CHO cells, with or without metabolic activation. In the L5178Y TK(+/-) mouse lymphoma mutagenesis test, all of the NTO-treated cultures had mutant frequencies that were similar to the average frequencies of solvent control-treated cultures, indicating a negative result. Confirmatory tests for the three in vitro tests also produced negative results. The potential in vivo clastogenicity and aneugenicity of NTO was evaluated using the rat peripheral blood micronucleus test. NTO was administered by oral gavage to male and female Sprague-Dawley rats for 14 days at doses up to 2g/kg/day. Flow cytometric analysis of peripheral blood demonstrated no significant induction of micronucleated reticulocytes relative to the vehicle control (PEG-200). These studies reveal that NTO was not genotoxic in either in vitro or in vivo tests and suggest a low risk of genetic hazards associated with exposure. Copyright © 2010 Elsevier B.V. All rights reserved.

In vivo and in vitro characterization of site-specific recombination of a novel serine integrase from the temperate phage EFC-1.

PubMed

Yoon, Bohyun; Kim, Inki; Nam, Ja-Ae; Chang, Hyo-Ihl; Ha, Chang Hoon

2016-04-22

EFC-1 integrase is a site-specific recombinase that belongs to the large family of serine recombinase. In previously study, we isolated the temperate phage EFC-1, and characterized its genomic sequence. Within its genome, Orf28 was predicted encode a 464 amino acid of a putative integrase gene. In this study, EFC-1 integrase was characterized in vitro and in vivo. In vitro assay was performed using purified His-tag fusion integrase. Also, to identify which serine is involved in the catalytic domain, we used site-directed mutagenesis and analyzed by a recombination assay in vitro. In vivo assay involved PCR and confocal microscopy in HEK293 cells, and determined the minimal lengths of attP and attB sites. According to our results, the EFC-1 integrase-mediated recombination was site-specific and unidirectional system in vitro and in vivo. Serine 21 of EFC-1 integrase plays a major role in the catalytic domain, and minimal sizes of attB and attP was defined 48 and 54 bp. Our finding may help develop a useful tool for gene therapy and gene delivery system. Copyright © 2016 Elsevier Inc. All rights reserved.

Development of a database system for mapping insertional mutations onto the mouse genome with large-scale experimental data

PubMed Central

2009-01-01

Background Insertional mutagenesis is an effective method for functional genomic studies in various organisms. It can rapidly generate easily tractable mutations. A large-scale insertional mutagenesis with the piggyBac (PB) transposon is currently performed in mice at the Institute of Developmental Biology and Molecular Medicine (IDM), Fudan University in Shanghai, China. This project is carried out via collaborations among multiple groups overseeing interconnected experimental steps and generates a large volume of experimental data continuously. Therefore, the project calls for an efficient database system for recording, management, statistical analysis, and information exchange. Results This paper presents a database application called MP-PBmice (insertional mutation mapping system of PB Mutagenesis Information Center), which is developed to serve the on-going large-scale PB insertional mutagenesis project. A lightweight enterprise-level development framework Struts-Spring-Hibernate is used here to ensure constructive and flexible support to the application. The MP-PBmice database system has three major features: strict access-control, efficient workflow control, and good expandability. It supports the collaboration among different groups that enter data and exchange information on daily basis, and is capable of providing real time progress reports for the whole project. MP-PBmice can be easily adapted for other large-scale insertional mutation mapping projects and the source code of this software is freely available at http://www.idmshanghai.cn/PBmice. Conclusion MP-PBmice is a web-based application for large-scale insertional mutation mapping onto the mouse genome, implemented with the widely used framework Struts-Spring-Hibernate. This system is already in use by the on-going genome-wide PB insertional mutation mapping project at IDM, Fudan University. PMID:19958505

Compounds used to produce cloned animals are genotoxic and mutagenic in mammalian assays in vitro and in vivo

PubMed Central

Oliveira, R.J.; Mantovani, M.S.; da Silva, A.F.; Pesarini, J.R.; Mauro, M.O.; Ribeiro, L.R.

2014-01-01

The compounds 6-dimethylaminopurine and cycloheximide promote the successful production of cloned mammals and have been used in the development of embryos produced by somatic cell nuclear transfer. This study investigated the effects of 6-dimethylaminopurine and cycloheximide in vitro, using the thiazolyl blue tetrazolium bromide colorimetric assay to assess cytotoxicity, the trypan blue exclusion assay to assess cell viability, the comet assay to assess genotoxicity, and the micronucleus test with cytokinesis block to test mutagenicity. In addition, the comet assay and the micronucleus test were also performed on peripheral blood cells of 54 male Swiss mice, 35 g each, to assess the effects of the compounds in vivo. The results indicated that both 6-dimethylaminopurine and cycloheximide, at the concentrations and doses tested, were cytotoxic in vitro and genotoxic and mutagenic in vitro and in vivo, altered the nuclear division index in vitro, but did not diminish cell viability in vitro. Considering that alterations in DNA play important roles in mutagenesis, carcinogenesis, and morphofunctional teratogenesis and reduce embryonic viability, this study indicated that 6-dimethylaminopurine and cycloheximide utilized in the process of mammalian cloning may be responsible for the low embryo viability commonly seen in nuclear transfer after implantation in utero. PMID:24714812

Compounds used to produce cloned animals are genotoxic and mutagenic in mammalian assays in vitro and in vivo.

PubMed

Oliveira, R J; Mantovani, M S; Silva, A F da; Pesarini, J R; Mauro, M O; Ribeiro, L R

2014-04-01

The compounds 6-dimethylaminopurine and cycloheximide promote the successful production of cloned mammals and have been used in the development of embryos produced by somatic cell nuclear transfer. This study investigated the effects of 6-dimethylaminopurine and cycloheximide in vitro, using the thiazolyl blue tetrazolium bromide colorimetric assay to assess cytotoxicity, the trypan blue exclusion assay to assess cell viability, the comet assay to assess genotoxicity, and the micronucleus test with cytokinesis block to test mutagenicity. In addition, the comet assay and the micronucleus test were also performed on peripheral blood cells of 54 male Swiss mice, 35 g each, to assess the effects of the compounds in vivo. The results indicated that both 6-dimethylaminopurine and cycloheximide, at the concentrations and doses tested, were cytotoxic in vitro and genotoxic and mutagenic in vitro and in vivo, altered the nuclear division index in vitro, but did not diminish cell viability in vitro. Considering that alterations in DNA play important roles in mutagenesis, carcinogenesis, and morphofunctional teratogenesis and reduce embryonic viability, this study indicated that 6-dimethylaminopurine and cycloheximide utilized in the process of mammalian cloning may be responsible for the low embryo viability commonly seen in nuclear transfer after implantation in utero.

Induction of mutagenesis and transformation in BALB/c-3T3 clone A31-1 cells by diverse chemical carcinogens

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lubet, R.A.; Kouri, R.E.; Curren, R.A.

1990-01-01

BALB/c-3T3 cells were employed to examine the genotoxic potential of a variety of known chemical carcinogens. BALB/c-3T3 cells displayed a dose-dependent transformation response to a variety of carcinogens (polycyclic hydrocarbons, methylating agents, ethylating agents, aflatoxin B{sub 1} (AFT{sub 1}), and 4-nitroquinoline-N-oxide (4-NQO)). When the ability of these compounds to induce mutagenesis to resistance to the cardiac glycoside ouabain (OUA{sup R}) was examined, the authors found the short chain alkylating agents to be particularly effective mutagens, causing biologic effects at doses below those necessary to induce a transformation response. In contrast, the polycyclic hydrocarbons which were potent transforming agents were weaker,more » albeit significant, mutagens for the OUA{sup R} locus in this system, while AFB{sub 1} was quite weak. Further studies were performed with 5-azacytidine (5-AZA) and the nongenotoxic carcinogen cinnamyl anthranilate (CIN). 5-AZA was a potent transforming agent, but failed to cause mutagenesis. CIN similarly caused in vitro transformation. When a series of eight structurally diverse compounds were examined in both the BALB/c-3T3 and C3H10T1/2 mouse fibroblast transformation systems, the BALB/c-3T3 system was shown to be sensitive to a wide variety of potential carcinogens, whereas the C3H10T1/2 system proved routinely sensitive only to the polycyclic hydrocarbons.« less

The Bacteriophage P1 HumD Protein Is a Functional Homolog of the Prokaryotic UmuD′-Like Proteins and Facilitates SOS Mutagenesis in Escherichia coli

PubMed Central

McLenigan, Mary P.; Kulaeva, Olga I.; Ennis, Don G.; Levine, Arthur S.; Woodgate, Roger

1999-01-01

The Escherichia coli umuD and umuC genes comprise an operon and encode proteins that are involved in the mutagenic bypass of normally replication-inhibiting DNA lesions. UmuD is, however, unable to function in this process until it undergoes a RecA-mediated cleavage reaction to generate UmuD′. Many homologs of umuDC have now been identified. Most are located on bacterial chromosomes or on broad-host-range R plasmids. One such putative homolog, humD (homolog of umuD) is, however, found on the bacteriophage P1 genome. Interestingly, humD differs from other umuD homologs in that it encodes a protein similar in size to the posttranslationally generated UmuD′ protein and not UmuD, nor is it in an operon with a cognate umuC partner. To determine if HumD is, in fact, a bona fide homolog of the prokaryotic UmuD′-like mutagenesis proteins, we have analyzed the ability of HumD to complement UmuD′ functions in vivo as well as examined HumD’s physical properties in vitro. When expressed from a high-copy-number plasmid, HumD restored cellular mutagenesis and increased UV survival to normally nonmutable recA430 lexA(Def) and UV-sensitive ΔumuDC recA718 lexA(Def) strains, respectively. Complementing activity was reduced when HumD was expressed from a low-copy-number plasmid, but this observation is explained by immunoanalysis which indicates that HumD is normally poorly expressed in vivo. In vitro analysis revealed that like UmuD′, HumD forms a stable dimer in solution and is able to interact with E. coli UmuC and RecA nucleoprotein filaments. We conclude, therefore, that bacteriophage P1 HumD is a functional homolog of the UmuD′-like proteins, and we speculate as to the reasons why P1 might require the activity of such a protein in vivo. PMID:10559166

Molecular Targeted Therapies Using Botanicals for Prostate Cancer Chemoprevention.

PubMed

Kumar, Nagi; Chornokur, Ganna

2012-12-31

In spite of the large number of botanicals demonstrating promise as potential cancer chemopreventive agents, most have failed to prove effectiveness in clinical trials. Critical requirements for moving botanical agents to recommendation for clinical use include adopting a systematic, molecular-target based approach and utilizing the same ethical and rigorous methods that are used to evaluate other pharmacological agents. Preliminary data on a mechanistic rationale for chemoprevention activity as observed from epidemiological, in vitro and preclinical studies, phase I data of safety in suitable cohorts, duration of intervention based on time to progression of pre-neoplastic disease to cancer and using a valid panel of biomarkers representing the hypothesized carcinogenesis pathway for measuring efficacy must inform the design of clinical trials. Botanicals have been shown to influence multiple biochemical and molecular cascades that inhibit mutagenesis, proliferation, induce apoptosis, suppress the formation and growth of human cancers, thus modulating several hallmarks of carcinogenesis. These agents appear promising in their potential to make a dramatic impact in cancer prevention and treatment, with a significantly superior safety profile than most agents evaluated to date. The goal of this paper is to provide models of translational research based on the current evidence of promising botanicals with a specific focus on targeted therapies for PCa chemoprevention.

Molecular Targeted Therapies Using Botanicals for Prostate Cancer Chemoprevention

PubMed Central

Kumar, Nagi; Chornokur, Ganna

2014-01-01

In spite of the large number of botanicals demonstrating promise as potential cancer chemopreventive agents, most have failed to prove effectiveness in clinical trials. Critical requirements for moving botanical agents to recommendation for clinical use include adopting a systematic, molecular-target based approach and utilizing the same ethical and rigorous methods that are used to evaluate other pharmacological agents. Preliminary data on a mechanistic rationale for chemoprevention activity as observed from epidemiological, in vitro and preclinical studies, phase I data of safety in suitable cohorts, duration of intervention based on time to progression of pre-neoplastic disease to cancer and using a valid panel of biomarkers representing the hypothesized carcinogenesis pathway for measuring efficacy must inform the design of clinical trials. Botanicals have been shown to influence multiple biochemical and molecular cascades that inhibit mutagenesis, proliferation, induce apoptosis, suppress the formation and growth of human cancers, thus modulating several hallmarks of carcinogenesis. These agents appear promising in their potential to make a dramatic impact in cancer prevention and treatment, with a significantly superior safety profile than most agents evaluated to date. The goal of this paper is to provide models of translational research based on the current evidence of promising botanicals with a specific focus on targeted therapies for PCa chemoprevention. PMID:24527269

Silencing of diphthamide synthesis 3 (Dph3) reduces metastasis of murine melanoma.

PubMed

Wang, Lei; Shi, Yu; Ju, Peijun; Liu, Rui; Yeo, Siok Ping; Xia, Yinyan; Owlanj, Hamed; Feng, Zhiwei

2012-01-01

Melanoma is the most dangerous skin cancer due to its highly metastatic potential and resistance to chemotherapy. Currently, there is no effective treatment for melanoma once it is progressed to metastatic stage. Therefore, further study to elucidate the molecular mechanism underlying the metastasis of melanoma cells is urgently required for the improvement of melanoma treatment. In the present study, we found that diphthamide synthesis 3 (Dph3) is involved in the metastasis of B16F10 murine melanoma cells by insertional mutagenesis. We demonstrated that Dph3 disruption impairs the migration of B16F10 murine melanoma cells. The requirement of Dph3 in the migration of melanoma cells was further confirmed by gene silencing with siRNA in vitro. In corresponding to this result, overexpression of Dph3 significantly promoted the migratory ability of B16F10 and B16F0 melanoma cells. Moreover, down regulation of Dph3 expression in B16F10 melanoma cells strikingly inhibits their cellular invasion and metastasis in vivo. Finally, we found that Dph3 promotes melanoma migration and invasion through the AKT signaling pathway. To conclude, our findings suggest a novel mechanism underlying the metastasis of melanoma cells which might serve as a new intervention target for the treatment of melanoma.

Silencing of Diphthamide Synthesis 3 (Dph3) Reduces Metastasis of Murine Melanoma

PubMed Central

Wang, Lei; Shi, Yu; Ju, Peijun; Liu, Rui; Yeo, Siok Ping; Xia, Yinyan; Owlanj, Hamed; Feng, Zhiwei

2012-01-01

Melanoma is the most dangerous skin cancer due to its highly metastatic potential and resistance to chemotherapy. Currently, there is no effective treatment for melanoma once it is progressed to metastatic stage. Therefore, further study to elucidate the molecular mechanism underlying the metastasis of melanoma cells is urgently required for the improvement of melanoma treatment. In the present study, we found that diphthamide synthesis 3 (Dph3) is involved in the metastasis of B16F10 murine melanoma cells by insertional mutagenesis. We demonstrated that Dph3 disruption impairs the migration of B16F10 murine melanoma cells. The requirement of Dph3 in the migration of melanoma cells was further confirmed by gene silencing with siRNA in vitro. In corresponding to this result, overexpression of Dph3 significantly promoted the migratory ability of B16F10 and B16F0 melanoma cells. Moreover, down regulation of Dph3 expression in B16F10 melanoma cells strikingly inhibits their cellular invasion and metastasis in vivo. Finally, we found that Dph3 promotes melanoma migration and invasion through the AKT signaling pathway. To conclude, our findings suggest a novel mechanism underlying the metastasis of melanoma cells which might serve as a new intervention target for the treatment of melanoma. PMID:23185508

Intrinsically disordered chromatin protein NUPR1 binds to the C-terminal region of Polycomb RING1B

PubMed Central

Santofimia-Castaño, Patricia; Rizzuti, Bruno; Pey, Ángel L.; Soubeyran, Philippe; Vidal, Miguel; Urrutia, Raúl; Iovanna, Juan L.; Neira, José L.

2017-01-01

Intrinsically disordered proteins (IDPs) are ubiquitous in eukaryotes, and they are often associated with diseases in humans. The protein NUPR1 is a multifunctional IDP involved in chromatin remodeling and in the development and progression of pancreatic cancer; however, the details of such functions are unknown. Polycomb proteins are involved in specific transcriptional cascades and gene silencing. One of the proteins of the Polycomb complex is the Ring finger protein 1 (RING1). RING1 is related to aggressive tumor features in multiple cancer types. In this work we characterized the interaction between NUPR1 and the paralogue RING1B in vitro, in silico, and in cellulo. The interaction occurred through the C-terminal region of RING1B (C-RING1B), with an affinity in the low micromolar range (∼10 μM). The binding region of NUPR1, mapped by NMR, was a hydrophobic polypeptide patch at the 30s region of its sequence, as pinpointed by computational results and site-directed mutagenesis at Ala33. The association between C-RING1B and wild-type NUPR1 also occurred in cellulo as tested by protein ligation assays; this interaction is inhibited by trifluoperazine, a drug known to hamper binding of wild-type NUPR1 with other proteins. Furthermore, the Thr68Gln and Ala33Gln/Thr68Gln mutants had a reduction in the binding toward C-RING1B as shown by in vitro, in silico, and in cellulo studies. This is an example of a well-folded partner of NUPR1, because its other interacting proteins are also unfolded. We hypothesize that NUPR1 plays an active role in chromatin remodeling and carcinogenesis, together with Polycomb proteins. PMID:28720707

Reduced Infectivity in Cattle for an Outer Membrane Protein Mutant of Anaplasma marginale

PubMed Central

Brayton, Kelly A.; Magunda, Forgivemore; Munderloh, Ulrike G.; Kelley, Karen L.; Barbet, Anthony F.

2015-01-01

Anaplasma marginale is the causative agent of anaplasmosis in cattle. Transposon mutagenesis of this pathogen using the Himar1 system resulted in the isolation of an omp10 operon insertional mutant referred to as the omp10::himar1 mutant. The work presented here evaluated if this mutant had morphological and/or growth rate defects compared to wild-type A. marginale. Results showed that the morphology, developmental cycle, and growth in tick and mammalian cell cultures are similar for the mutant and the wild type. Tick transmission experiments established that tick infection levels with the mutant were similar to those with wild-type A. marginale and that infected ticks successfully infected cattle. However, this mutant exhibited reduced infectivity and growth in cattle. The possibility of transforming A. marginale by transposon mutagenesis coupled with in vitro and in vivo assessment of altered phenotypes can aid in the identification of genes associated with virulence. The isolation of deliberately attenuated organisms that can be evaluated in their natural biological system is an important advance for the rational design of vaccines against this species. PMID:25595772

Cytotoxicity and mutagenicity of Kevlar: an in vitro evaluation.

PubMed

Wening, J V; Marquardt, H; Katzer, A; Jungbluth, K H; Marquardt, H

1995-03-01

Toxicity and mutagenicity of Kevlar 49 (PPPT; poly-para-phenylene-terephthalamide) was tested in six strains of Salmonella typhimurium (Ames test; TA97, TA98, TA100, TA102, TA1535, TA1537) with and without an external metabolic activation system (S9), as well as in a mammalian cell mutagenesis assay using V79 Chinese hamster cells. For the Ames test, liquid preincubation, which is considered particularly sensitive, was used. The cells were incubated for 24 h at a temperature of 37 degrees C either directly with Kevlar49 or with ethanol- or chloroform-extracted Kevlar49. The experiments were performed at least twice. The Ames test with six different Salmonella typhimurium strains featuring either base pair substitution or frameshift mutations revealed no cytotoxic or mutagenic activity of Kevlar49. In the mammalian cell mutagenesis assay, using 8-azaguanine (AG) as a selective agent, Kevlar49 was also devoid of cytotoxic or mutagenic activity. Both tests have to be regarded as an initial exploratory screening due to the chosen testing conditions and should be supplemented by tests at different temperatures.

Characterizing mutagenesis in the hprt gene of rat alveolar epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Driscoll, K.E.; Deyo, L.C.; Howard, B.W.

1995-12-31

A clonal selection assay was developed for mutation in the hypoxanthine-guanine phosphoribosyl transferase (hprt) gene of rat alveolar epithelial cells. Studies were conducted to establish methods for isolation and long-term culture of rat alveolar epithelial cells. When isolated by pronase digestion purified on a Nycodenz gradient and cultured in media containing 7.5% fetal bovine serum (FBS), pituitary extract, EGF, insulin, and IGF-1, rat alveolar epithelial cells could be maintained in culture for several weeks with cell doubling times of 2-4 days. The rat alveolar epithelial cell cultures were exposed in vitro to the mutagens ethylnitrosourea (ENU) and H{sub 2}O{sub 2},more » and mutation in the hprt gene was selected for by culture in the presence of the toxic purine analog, 6-thioguanine (6TG). In vitro exposure to ENU or H{sub 2}O produced a dose-dependent increase in hprt mutation frequency in the alveolar epithelial cells. To determine if the assay system could be used to evaluate mutagenesis in alveolar type II cells after in vivo mutagen or carcinogen exposure, cells were isolated from rats treated previously with ENU or {alpha}-quartz. A significant increase in hprt mutation frequency was detected in alveolar epithelial cells obtained from rats exposed to ENU or {alpha}-quartz; the latter observation is the first demonstration that crystalline silica exposure is mutagenic in vivo. In summary, these studies show that rat alveolar epithelial cells isolated by pronase digestion and Nycodenz separation techniques and cultured in a defined media can be used in a clonal selection assay for mutation in the hprt gene. This assay demonstrates that ENU and H{sub 2}O{sub 2} in vitro and ENU and {alpha}-quartz in vivo are mutagenic for rat alveolar epithelial cells. This model should be useful for investigating the genotoxic effects of chemical and physical agents on an important lung cell target for neoplastic transformation. 41 refs., 4 figs., 3 tabs.« less

Method For Determining And Modifying Protein/Peptide Solubilty

DOEpatents

Waldo, Geoffrey S.

2005-03-15

A solubility reporter for measuring a protein's solubility in vivo or in vitro is described. The reporter, which can be used in a single living cell, gives a specific signal suitable for determining whether the cell bears a soluble version of the protein of interest. A pool of random mutants of an arbitrary protein, generated using error-prone in vitro recombination, may also be screened for more soluble versions using the reporter, and these versions may be recombined to yield variants having further-enhanced solubility. The method of the present invention includes "irrational" (random mutagenesis) methods, which do not require a priori knowledge of the three-dimensional structure of the protein of interest. Multiple sequences of mutation/genetic recombination and selection for improved solubility are demonstrated to yield versions of the protein which display enhanced solubility.

Connexins in Prostate Cancer Initiation and Progression

DTIC Science & Technology

2012-09-01

Isoleucine and A=alanine. In L212A/I213A the leucine at position 212 and isoleucine at position 213 were mutated to alanine. Similar strategy was used to...and isoleucine at the indicated amino acid residues were mutated to alanine using site-directed mutagenesis (Figure 3). Expression of Cx32 and...Its Mutants and Gap Junction Assembly Human LNCaP cells neither express Cx32 nor form functional GJs [23]. We introduced WT-Cx32 and various

Progress in Genome Editing Technology and Its Application in Plants

PubMed Central

Zhang, Kai; Raboanatahiry, Nadia; Zhu, Bin; Li, Maoteng

2017-01-01

Genome editing technology (GET) is a versatile approach that has progressed rapidly as a mechanism to alter the genotype and phenotype of organisms. However, conventional genome modification using GET cannot satisfy current demand for high-efficiency and site-directed mutagenesis, retrofitting of artificial nucleases has developed into a new avenue within this field. Based on mechanisms to recognize target genes, newly-developed GETs can generally be subdivided into three cleavage systems, protein-dependent DNA cleavage systems (i.e., zinc-finger nucleases, ZFN, and transcription activator-like effector nucleases, TALEN), RNA-dependent DNA cleavage systems (i.e., clustered regularly interspaced short palindromic repeats-CRISPR associated proteins, CRISPR-Cas9, CRISPR-Cpf1, and CRISPR-C2c1), and RNA-dependent RNA cleavage systems (i.e., RNA interference, RNAi, and CRISPR-C2c2). All these techniques can lead to double-stranded (DSB) or single-stranded breaks (SSB), and result in either random mutations via non-homologous end-joining (NHEJ) or targeted mutation via homologous recombination (HR). Thus, site-directed mutagenesis can be induced via targeted gene knock-out, knock-in, or replacement to modify specific characteristics including morphology-modification, resistance-enhancement, and physiological mechanism-improvement along with plant growth and development. In this paper, an non-comprehensive review on the development of different GETs as applied to plants is presented. PMID:28261237

Novel gene function revealed by mouse mutagenesis screens for models of age-related disease.

PubMed

Potter, Paul K; Bowl, Michael R; Jeyarajan, Prashanthini; Wisby, Laura; Blease, Andrew; Goldsworthy, Michelle E; Simon, Michelle M; Greenaway, Simon; Michel, Vincent; Barnard, Alun; Aguilar, Carlos; Agnew, Thomas; Banks, Gareth; Blake, Andrew; Chessum, Lauren; Dorning, Joanne; Falcone, Sara; Goosey, Laurence; Harris, Shelley; Haynes, Andy; Heise, Ines; Hillier, Rosie; Hough, Tertius; Hoslin, Angela; Hutchison, Marie; King, Ruairidh; Kumar, Saumya; Lad, Heena V; Law, Gemma; MacLaren, Robert E; Morse, Susan; Nicol, Thomas; Parker, Andrew; Pickford, Karen; Sethi, Siddharth; Starbuck, Becky; Stelma, Femke; Cheeseman, Michael; Cross, Sally H; Foster, Russell G; Jackson, Ian J; Peirson, Stuart N; Thakker, Rajesh V; Vincent, Tonia; Scudamore, Cheryl; Wells, Sara; El-Amraoui, Aziz; Petit, Christine; Acevedo-Arozena, Abraham; Nolan, Patrick M; Cox, Roger; Mallon, Anne-Marie; Brown, Steve D M

2016-08-18

Determining the genetic bases of age-related disease remains a major challenge requiring a spectrum of approaches from human and clinical genetics to the utilization of model organism studies. Here we report a large-scale genetic screen in mice employing a phenotype-driven discovery platform to identify mutations resulting in age-related disease, both late-onset and progressive. We have utilized N-ethyl-N-nitrosourea mutagenesis to generate pedigrees of mutagenized mice that were subject to recurrent screens for mutant phenotypes as the mice aged. In total, we identify 105 distinct mutant lines from 157 pedigrees analysed, out of which 27 are late-onset phenotypes across a range of physiological systems. Using whole-genome sequencing we uncover the underlying genes for 44 of these mutant phenotypes, including 12 late-onset phenotypes. These genes reveal a number of novel pathways involved with age-related disease. We illustrate our findings by the recovery and characterization of a novel mouse model of age-related hearing loss.

Novel gene function revealed by mouse mutagenesis screens for models of age-related disease

PubMed Central

Potter, Paul K.; Bowl, Michael R.; Jeyarajan, Prashanthini; Wisby, Laura; Blease, Andrew; Goldsworthy, Michelle E.; Simon, Michelle M.; Greenaway, Simon; Michel, Vincent; Barnard, Alun; Aguilar, Carlos; Agnew, Thomas; Banks, Gareth; Blake, Andrew; Chessum, Lauren; Dorning, Joanne; Falcone, Sara; Goosey, Laurence; Harris, Shelley; Haynes, Andy; Heise, Ines; Hillier, Rosie; Hough, Tertius; Hoslin, Angela; Hutchison, Marie; King, Ruairidh; Kumar, Saumya; Lad, Heena V.; Law, Gemma; MacLaren, Robert E.; Morse, Susan; Nicol, Thomas; Parker, Andrew; Pickford, Karen; Sethi, Siddharth; Starbuck, Becky; Stelma, Femke; Cheeseman, Michael; Cross, Sally H.; Foster, Russell G.; Jackson, Ian J.; Peirson, Stuart N.; Thakker, Rajesh V.; Vincent, Tonia; Scudamore, Cheryl; Wells, Sara; El-Amraoui, Aziz; Petit, Christine; Acevedo-Arozena, Abraham; Nolan, Patrick M.; Cox, Roger; Mallon, Anne-Marie; Brown, Steve D. M.

2016-01-01

Determining the genetic bases of age-related disease remains a major challenge requiring a spectrum of approaches from human and clinical genetics to the utilization of model organism studies. Here we report a large-scale genetic screen in mice employing a phenotype-driven discovery platform to identify mutations resulting in age-related disease, both late-onset and progressive. We have utilized N-ethyl-N-nitrosourea mutagenesis to generate pedigrees of mutagenized mice that were subject to recurrent screens for mutant phenotypes as the mice aged. In total, we identify 105 distinct mutant lines from 157 pedigrees analysed, out of which 27 are late-onset phenotypes across a range of physiological systems. Using whole-genome sequencing we uncover the underlying genes for 44 of these mutant phenotypes, including 12 late-onset phenotypes. These genes reveal a number of novel pathways involved with age-related disease. We illustrate our findings by the recovery and characterization of a novel mouse model of age-related hearing loss. PMID:27534441

GPCR-I-TASSER: A hybrid approach to G protein-coupled receptor structure modeling and the application to the human genome

PubMed Central

Zhang, Jian; Yang, Jianyi; Jang, Richard; Zhang, Yang

2015-01-01

SUMMARY Experimental structure determination remains very difficult for G protein-coupled receptors (GPCRs). We propose a new hybrid protocol to construct GPCR structure models that integrates experimental mutagenesis data with ab initio transmembrane (TM) helix assembly simulations. The method was tested on 24 known GPCRs where the ab initio TM-helix assembly procedure constructed the correct fold for 20 cases. When combined with weak-homology and sparse mutagenesis restraints, the method generated correct folds for all the tested cases with an average C-alpha RMSD 2.4 Å in the TM-regions. The new hybrid protocol was applied to model all 1026 GPCRs in the human genome, where 923 have a high confidence score that are expected to have correct folds; these contain many pharmaceutically important families with no previously solved structures, including Trace amine, Prostanoids, Releasing hormones, Melanocortins, Vasopressin and Neuropeptide Y receptors. The results demonstrate new progress on genome-wide structure modeling of transmembrane proteins. PMID:26190572

FDA toxicity databases and real-time data entry.

PubMed

Arvidson, Kirk B

2008-11-15

Structure-searchable electronic databases are valuable new tools that are assisting the FDA in its mission to promptly and efficiently review incoming submissions for regulatory approval of new food additives and food contact substances. The Center for Food Safety and Applied Nutrition's Office of Food Additive Safety (CFSAN/OFAS), in collaboration with Leadscope, Inc., is consolidating genetic toxicity data submitted in food additive petitions from the 1960s to the present day. The Center for Drug Evaluation and Research, Office of Pharmaceutical Science's Informatics and Computational Safety Analysis Staff (CDER/OPS/ICSAS) is separately gathering similar information from their submissions. Presently, these data are distributed in various locations such as paper files, microfiche, and non-standardized toxicology memoranda. The organization of the data into a consistent, searchable format will reduce paperwork, expedite the toxicology review process, and provide valuable information to industry that is currently available only to the FDA. Furthermore, by combining chemical structures with genetic toxicity information, biologically active moieties can be identified and used to develop quantitative structure-activity relationship (QSAR) modeling and testing guidelines. Additionally, chemicals devoid of toxicity data can be compared to known structures, allowing for improved safety review through the identification and analysis of structural analogs. Four database frameworks have been created: bacterial mutagenesis, in vitro chromosome aberration, in vitro mammalian mutagenesis, and in vivo micronucleus. Controlled vocabularies for these databases have been established. The four separate genetic toxicity databases are compiled into a single, structurally-searchable database for easy accessibility of the toxicity information. Beyond the genetic toxicity databases described here, additional databases for subchronic, chronic, and teratogenicity studies have been prepared.

The amino acid motif L/IIxxFE defines a novel actin-binding sequence in PDZ-RhoGEF

PubMed Central

Banerjee, Jayashree; Fischer, Christopher C.; Wedegaertner, Philip B.

2009-01-01

PDZ-RhoGEF is a member of the regulator of G protein signaling (RGS) domain-containing RhoGEFs (RGS-RhoGEFs) that link activated heterotrimeric G protein α subunits of the G12 family to activation of the small GTPase RhoA. Unique among the RGS-RhoGEFs, PDZ-RhoGEF contains a short sequence that localizes the protein to the actin cytoskeleton. In this report, we demonstrate that the actin-binding domain, located between amino acids 561–585, directly binds to F-actin in vitro. Extensive mutagenesis identifies isoleucine 568, isoleucine 569, phenylalanine 572, and glutamic acid 573 as necessary for binding to actin and for co-localization with the actin cytoskeleton in cells. These results define a novel actin-binding sequence in PDZ-RhoGEF with a critical amino acid motif of IIxxFE. Moreover, sequence analysis identifies a similar actin-binding motif in the N-terminus of the RhoGEF frabin, and, as with PDZ-RhoGEF, mutagenesis and actin interaction experiments demonstrate a motif of LIxxFE, consisting of the key amino acids leucine 23, isoleucine 24, phenylalanine 27, and glutamic acid 28. Taken together, results with PDZ-RhoGEF and frabin identify a novel actin binding sequence. Lastly, inducible dimerization of the actin-binding region of PDZ-RhoGEF revealed a dimerization-dependent actin bundling activity in vitro. PDZ-RhoGEF exists in cells as a dimer, raising the possibility that PDZ-RhoGEF could influence actin structure independent of its ability to activate RhoA. PMID:19618964

Preclinical Demonstration of Lentiviral Vector-mediated Correction of Immunological and Metabolic Abnormalities in Models of Adenosine Deaminase Deficiency

PubMed Central

Carbonaro, Denise A; Zhang, Lin; Jin, Xiangyang; Montiel-Equihua, Claudia; Geiger, Sabine; Carmo, Marlene; Cooper, Aaron; Fairbanks, Lynette; Kaufman, Michael L; Sebire, Neil J; Hollis, Roger P; Blundell, Michael P; Senadheera, Shantha; Fu, Pei-Yu; Sahaghian, Arineh; Chan, Rebecca Y; Wang, Xiaoyan; Cornetta, Kenneth; Thrasher, Adrian J; Kohn, Donald B; Gaspar, H Bobby

2014-01-01

Gene transfer into autologous hematopoietic stem cells by γ-retroviral vectors (gRV) is an effective treatment for adenosine deaminase (ADA)–deficient severe combined immunodeficiency (SCID). However, current gRV have significant potential for insertional mutagenesis as reported in clinical trials for other primary immunodeficiencies. To improve the efficacy and safety of ADA-SCID gene therapy (GT), we generated a self-inactivating lentiviral vector (LV) with a codon-optimized human cADA gene under the control of the short form elongation factor-1α promoter (LV EFS ADA). In ADA−/− mice, LV EFS ADA displayed high-efficiency gene transfer and sufficient ADA expression to rescue ADA−/− mice from their lethal phenotype with good thymic and peripheral T- and B-cell reconstitution. Human ADA-deficient CD34+ cells transduced with 1–5 × 107 TU/ml had 1–3 vector copies/cell and expressed 1–2x of normal endogenous levels of ADA, as assayed in vitro and by transplantation into immune-deficient mice. Importantly, in vitro immortalization assays demonstrated that LV EFS ADA had significantly less transformation potential compared to gRV vectors, and vector integration-site analysis by nrLAM-PCR of transduced human cells grown in immune-deficient mice showed no evidence of clonal skewing. These data demonstrated that the LV EFS ADA vector can effectively transfer the human ADA cDNA and promote immune and metabolic recovery, while reducing the potential for vector-mediated insertional mutagenesis. PMID:24256635

Long noncoding RNA GAPLINC promotes gastric cancer cell proliferation by acting as a molecular sponge of miR-378 to modulate MAPK1 expression.

PubMed

Diao, Lingyun; Wang, Shengying; Sun, Zhiguang

2018-01-01

Dysregulated long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) play key roles in the development of human cancers. LncRNA GAPLINC has been reported to be increased in gastric cancer (GC) tissues. Real-time PCR assays were used to measure expressions of GAPLINC, miR-378, and MAPK1 mRNA. Western blot assays were employed to examine MAPK1 protein expression. Cell proliferation and cell cycle were measured by CCK-8 and propidium iodide-detection assays, respectively. The interaction between GAPLINC and miR-378 was confirmed by site-directed mutagenesis and luciferase assays. Luciferase assays were also used to study whether GAPLINC was able to act as a molecular sponge of miR-378 to modulate MAPK1 expression. The lncRNA GAPLINC expression was upregulated and positively correlated with MAPK1 expression in gastric cancer tissues and cells. Additionally, lncRNA GAPLINC promoted the expression of MAPK1 and the enhancement of GC cell proliferation and cell cycle progression by LncRNA GAPLINC was dependent on MAPK1 in vitro and in vivo. Consequently, we found that miR-378 expression was inversely correlated with GAPLINC expression in GC tissues and cells. miR-378 could directly bind to GAPLINC and decreased GAPLINC expression, thus reducing MAPK1 expression. Furthermore, overexpression of miR-378 inhibited MAPK1 expression, cell proliferation, and cell cycle progression of gastric cancer cells, while these effects were abrogated by upregulating lncRNA GAPLINC expression. Taken together, lncRNA GAPLINC promotes gastric cancer cell proliferation by acting as a molecular sponge of miR-378 to modulate MAPK1 expression.

Specialization of the DNA-Cleaving Activity of a Group I Ribozyme Through In Vitro Evolution

NASA Technical Reports Server (NTRS)

Tsang, Joyce; Joyce, Gerald F.

1996-01-01

In an earlier study, an in vitro evolution procedure was applied to a large population of variants of the Tetrahymena group 1 ribozyme to obtain individuals with a 10(exp 5)-fold improved ability to cleave a target single-stranded DNA substrate under simulated physiological conditions. The evolved ribozymes also showed a twofold improvement, compared to the wild-type, in their ability to cleave a single-stranded RNA substrate. Here, we report continuation of the in vitro evolution process using a new selection strategy to achieve both enhanced DNA and diminished RNA-cleavage activity. Our strategy combines a positive selection for DNA cleavage with a negative selection against RNA binding. After 36 "generations" of in vitro evolution, the evolved population showed an approx. 100-fold increase in the ratio of DNA to RNA-cleavage activity. Site-directed mutagenesis experiment confirmed the selective advantage of two covarying mutations within the catalytic core of ribozyme that are largely responsible for this modified behavior. The population of ribozymes has now undergone a total of 63 successive generations of evolution, resulting in an average 28 mutations relative to the wild-type that are responsible for the altered phenotype.

[Corpuscular mutagenesis and its prevention].

PubMed

Daugel'-Dauge, N O; Durnev, A D; Kulakova, A V; Seredenin, S B; Velichkovskiĭ, B T

1995-01-01

The carcinogenic and mutagenic activity of dust containing chrysotile-asbestos and zeolites, as well as the role of active oxygen species in their cytotoxic and mutagenic actions are discussed. Superoxide dismutase (50 mg/ml) was demonstrated to prevent the mutagenic effects of chrysotile-asbestos and latex, catalase (20 mg/ml) to prevent the same of zeolites in experiments on cultured human whole blood. The intraperitoneal administration of dusts of chrysotile-asbestos and zeolites in a dose of 50 mg/kg to C57B1/6 mice was found to elevate the count of cells with chromosomal aberrations in the peritoneal liquid and bone marrow cells of mice, which was dependent on dust exposure time. It was revealed that ascorbic acid, rutin, chemically modified flavonoid of Scutellaria Baicalensis Georgy, drugs such as bemitil and thomersol in the broad range of concentrations (10(-7)-10(-3) M) decreased or completely reduced the clustogenic action of zeolites and chrysotile-asbestos on cultured human whole blood. The ability of bemitil (1.8-19 mg/kg) rather than the others to prevent the mutagenic effect of chrysotile-asbestos was confirmed by the method of recording chromosomal aberrations in the cells of peritoneal liquid and bone marrow in mice. The findings suggest that the mutagenic effects of the corpuscular xenobiotics under study are mediated by active oxygen species and that the use of the models in vitro and in vivo is adequate for investigations into corpuscular mutagenesis. Based on their own data and literature data, the authors have defined possible lines of further research of corpuscular mutagenesis.

Pflanzliche Zellkulturtechniken als Züchtungsschritt am Beispiel Raps

NASA Astrophysics Data System (ADS)

Hoffmann, Franz

1980-06-01

A supplementation of classical plant breeding is now necessary due to the limitations imposed by available genetic variability and the slowness of the method. Therefore, cell culture techniques could play an important role in the future. Using rape seed, in which plants derived from anther culture and in vitro mutagenesis are already field tested, it has been shown that, in this case, somatic genetics is very close to becoming a practical method. For most of the other crop plants, in particular the cereals, no such unconventional breeding techniques have yet been satisfactorily established for commercial use.

EMSA Analysis of DNA Binding By Rgg Proteins

PubMed Central

LaSarre, Breah; Federle, Michael J.

2016-01-01

In bacteria, interaction of various proteins with DNA is essential for the regulation of specific target gene expression. Electrophoretic mobility shift assay (EMSA) is an in vitro approach allowing for the visualization of these protein-DNA interactions. Rgg proteins comprise a family of transcriptional regulators widespread among the Firmicutes. Some of these proteins function independently to regulate target gene expression, while others have now been demonstrated to function as effectors of cell-to-cell communication, having regulatory activities that are modulated via direct interaction with small signaling peptides. EMSA analysis can be used to assess DNA binding of either type of Rgg protein. EMSA analysis of Rgg protein activity has facilitated in vitro confirmation of regulatory targets, identification of precise DNA binding sites via DNA probe mutagenesis, and characterization of the mechanism by which some cognate signaling peptides modulate Rgg protein function (e.g. interruption of DNA-binding in some cases). PMID:27430004

EMSA Analysis of DNA Binding By Rgg Proteins.

PubMed

LaSarre, Breah; Federle, Michael J

2013-08-20

In bacteria, interaction of various proteins with DNA is essential for the regulation of specific target gene expression. Electrophoretic mobility shift assay (EMSA) is an in vitro approach allowing for the visualization of these protein-DNA interactions. Rgg proteins comprise a family of transcriptional regulators widespread among the Firmicutes. Some of these proteins function independently to regulate target gene expression, while others have now been demonstrated to function as effectors of cell-to-cell communication, having regulatory activities that are modulated via direct interaction with small signaling peptides. EMSA analysis can be used to assess DNA binding of either type of Rgg protein. EMSA analysis of Rgg protein activity has facilitated in vitro confirmation of regulatory targets, identification of precise DNA binding sites via DNA probe mutagenesis, and characterization of the mechanism by which some cognate signaling peptides modulate Rgg protein function ( e.g. interruption of DNA-binding in some cases).

Targeted Genome Editing Using DNA-Free RNA-Guided Cas9 Ribonucleoprotein for CHO Cell Engineering.

PubMed

Shin, Jongoh; Lee, Namil; Cho, Suhyung; Cho, Byung-Kwan

2018-01-01

Recent advances in the CRISPR/Cas9 system have dramatically facilitated genome engineering in various cell systems. Among the protocols, the direct delivery of the Cas9-sgRNA ribonucleoprotein (RNP) complex into cells is an efficient approach to increase genome editing efficiency. This method uses purified Cas9 protein and in vitro transcribed sgRNA to edit the target gene without vector DNA. We have applied the RNP complex to CHO cell engineering to obtain desirable phenotypes and to reduce unintended insertional mutagenesis and off-target effects. Here, we describe our routine methods for RNP complex-mediated gene deletion including the protocols to prepare the purified Cas9 protein and the in vitro transcribed sgRNA. Subsequently, we also describe a protocol to confirm the edited genomic positions using the T7E1 enzymatic assay and next-generation sequencing.

Protein and Antibody Engineering by Phage Display

PubMed Central

Frei, J.C.; Lai, J.R.

2017-01-01

Phage display is an in vitro selection technique that allows for the rapid isolation of proteins with desired properties including increased affinity, specificity, stability, and new enzymatic activity. The power of phage display relies on the phenotype-to-genotype linkage of the protein of interest displayed on the phage surface with the encoding DNA packaged within the phage particle, which allows for selective enrichment of library pools and high-throughput screening of resulting clones. As an in vitro method, the conditions of the binding selection can be tightly controlled. Due to the high-throughput nature, rapidity, and ease of use, phage display is an excellent technological platform for engineering antibody or proteins with enhanced properties. Here, we describe methods for synthesis, selection, and screening of phage libraries with particular emphasis on designing humanizing antibody libraries and combinatorial scanning mutagenesis libraries. We conclude with a brief section on troubleshooting for all stages of the phage display process. PMID:27586328

Identification of Streptococcus sanguinis Genes Required for Biofilm Formation and Examination of Their Role in Endocarditis Virulence▿

PubMed Central

Ge, Xiuchun; Kitten, Todd; Chen, Zhenming; Lee, Sehmi P.; Munro, Cindy L.; Xu, Ping

2008-01-01

Streptococcus sanguinis is one of the pioneers in the bacterial colonization of teeth and is one of the most abundant species in the oral biofilm called dental plaque. S. sanguinis is also the most common viridans group streptococcal species implicated in infective endocarditis. To investigate the association of biofilm and endocarditis, we established a biofilm assay and examined biofilm formation with a signature-tagged mutagenesis library of S. sanguinis. Four genes that have not previously been associated with biofilm formation in any other bacterium, purB, purL, thrB, and pyrE, were putatively identified as contributing to in vitro biofilm formation in S. sanguinis. By examining 800 mutants for attenuation in the rabbit endocarditis model and for reduction in biofilm formation in vitro, we found some mutants that were both biofilm defective and attenuated for endocarditis. However, we also identified mutants with only reduced biofilm formation or with only attenuation in the endocarditis model. This result indicates that the ability to form biofilms in vitro is not associated with endocarditis virulence in vivo in S. sanguinis. PMID:18390999

Identification of Streptococcus sanguinis genes required for biofilm formation and examination of their role in endocarditis virulence.

PubMed

Ge, Xiuchun; Kitten, Todd; Chen, Zhenming; Lee, Sehmi P; Munro, Cindy L; Xu, Ping

2008-06-01

Streptococcus sanguinis is one of the pioneers in the bacterial colonization of teeth and is one of the most abundant species in the oral biofilm called dental plaque. S. sanguinis is also the most common viridans group streptococcal species implicated in infective endocarditis. To investigate the association of biofilm and endocarditis, we established a biofilm assay and examined biofilm formation with a signature-tagged mutagenesis library of S. sanguinis. Four genes that have not previously been associated with biofilm formation in any other bacterium, purB, purL, thrB, and pyrE, were putatively identified as contributing to in vitro biofilm formation in S. sanguinis. By examining 800 mutants for attenuation in the rabbit endocarditis model and for reduction in biofilm formation in vitro, we found some mutants that were both biofilm defective and attenuated for endocarditis. However, we also identified mutants with only reduced biofilm formation or with only attenuation in the endocarditis model. This result indicates that the ability to form biofilms in vitro is not associated with endocarditis virulence in vivo in S. sanguinis.

Structure and function of nucleotide sugar transporters: Current progress.

PubMed

Hadley, Barbara; Maggioni, Andrea; Ashikov, Angel; Day, Christopher J; Haselhorst, Thomas; Tiralongo, Joe

2014-06-01

The proteomes of eukaryotes, bacteria and archaea are highly diverse due, in part, to the complex post-translational modification of protein glycosylation. The diversity of glycosylation in eukaryotes is reliant on nucleotide sugar transporters to translocate specific nucleotide sugars that are synthesised in the cytosol and nucleus, into the endoplasmic reticulum and Golgi apparatus where glycosylation reactions occur. Thirty years of research utilising multidisciplinary approaches has contributed to our current understanding of NST function and structure. In this review, the structure and function, with reference to various disease states, of several NSTs including the UDP-galactose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, GDP-fucose, UDP-N-acetylglucosamine/UDP-glucose/GDP-mannose and CMP-sialic acid transporters will be described. Little is known regarding the exact structure of NSTs due to difficulties associated with crystallising membrane proteins. To date, no three-dimensional structure of any NST has been elucidated. What is known is based on computer predictions, mutagenesis experiments, epitope-tagging studies, in-vitro assays and phylogenetic analysis. In this regard the best-characterised NST to date is the CMP-sialic acid transporter (CST). Therefore in this review we will provide the current state-of-play with respect to the structure-function relationship of the (CST). In particular we have summarised work performed by a number groups detailing the affect of various mutations on CST transport activity, efficiency, and substrate specificity.

Forward and reverse mutagenesis in C. elegans

PubMed Central

Kutscher, Lena M.; Shaham, Shai

2014-01-01

Mutagenesis drives natural selection. In the lab, mutations allow gene function to be deciphered. C. elegans is highly amendable to functional genetics because of its short generation time, ease of use, and wealth of available gene-alteration techniques. Here we provide an overview of historical and contemporary methods for mutagenesis in C. elegans, and discuss principles and strategies for forward (genome-wide mutagenesis) and reverse (target-selected and gene-specific mutagenesis) genetic studies in this animal. PMID:24449699

A mutagenesis study of a catalytic antibody

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jackson, D.Y.; Prudent, J.R.; Baldwin, E.P.

1991-01-01

The authors have generated seven site-specific mutations in the genes encoding the variable region of the heavy chain domain (V{sub H}) of the phosphocholine-binding antibody S107.S107 is a member of a family of well-characterized highly homologous antibodies that bind phosphorylcholine mono- and diesters. Two of these antibodies, MOPC-167 and T15, have previously been shown to catalyze the hydrolysis of 4-nitrophenyl N-trimethylammonioethyl carbonate. Two conserved heavy-chain residues, Tyr-33 and Arg-52, were postulated to be involved in binding and hydrolysis of 4-nitrophenylcholine carbonate esters. To more precisely define the catalytic roles of these residues, three Arg-52 mutants (R52K, R52Q, R52C) and fourmore » Tyr-33 mutants (Y33H, Y33F, Y33E, Y33D) of antibody S107 were generated. The genes encoding the V{sub H} binding domain of S107 were inserted into plasmid pUC-fl, and in vitro mutagenesis was performed. These results not only demonstrate the importance of electrostatic interactions in catalysis by antibody S107 but also show that catalytic side chains can be introduced into antibodies to enhance their catalytic efficiency.« less

Mutagenesis of solvent-exposed amino acids in Photinus pyralis luciferase improves thermostability and pH-tolerance

PubMed Central

Law, G. H. Erica; Gandelman, Olga A.; Tisi, Laurence C.; Lowe, Christopher R.; Murray, James A. H.

2006-01-01

Firefly luciferase catalyses a two-step reaction, using ATP-Mg2+, firefly luciferin and molecular oxygen as substrates, leading to the efficient emission of yellow–green light. We report the identification of novel luciferase mutants which combine improved pH-tolerance and thermostability and that retain the specific activity of the wild-type enzyme. These were identified by the mutagenesis of solvent-exposed non-conserved hydrophobic amino acids to hydrophilic residues in Photinus pyralis firefly luciferase followed by in vivo activity screening. Mutants F14R, L35Q, V182K, I232K and F465R were found to be the preferred substitutions at the respective positions. The effects of these amino acid replacements are additive, since combination of the five substitutions produced an enzyme with greatly improved pH-tolerance and stability up to 45 °C. All mutants, including the mutant with all five substitutions, showed neither a decrease in specific activity relative to the recombinant wild-type enzyme, nor any substantial differences in kinetic constants. It is envisaged that the combined mutant will be superior to wild-type luciferase for many in vitro and in vivo applications. PMID:16551268

A new orange emitting luciferase from the Southern-Amazon Pyrophorus angustus (Coleoptera: Elateridae) click-beetle: structure and bioluminescence color relationship, evolutional and ecological considerations.

PubMed

Amaral, Danilo T; Oliveira, Gabriela; Silva, Jaqueline R; Viviani, Vadim R

2016-08-31

Bioluminescent click-beetles display a wide variation of bioluminescence colors ranging from green to orange, including an unusual intra-specific color variation in the Jamaican Pyrophorus plagiophthalamus. Recently, we collected individuals of the Pyrophorus angustus species from the Southern Amazon forest, in Brazil, which displays an orange light emitting abdominal lantern. This species was also previously described from Central America, but displaying a bioluminescence spectrum from 536 nm (dorsal) to 578 nm (ventral). The biogeographic variation of the bioluminescence color in this species could be an adaptation to environmental reflectance and inter/intraspecific sexual competition. Here, we cloned, sequenced, characterized and performed site-direct mutagenesis of this new orange emitting luciferase. The in vitro luciferase spectrum displayed a peak at 594 nm, KM values for ATP and d-luciferin of 160 μM and 17 μM, respectively, and an optimum pH of approximately 8.5. Comparative multialignment and site-directed mutagenesis using different color emitting click-beetle luciferases from P. angustus, Fulgeochlizus bruchi and Pyrearinus termitilluminans luciferases cloned by our group showed an integral role of residue 247 in bioluminescence color modulation.

Successful Gene Tagging in Lettuce Using the Tnt1 Retrotransposon from Tobacco

PubMed Central

Mazier, Marianne; Botton, Emmanuel; Flamain, Fabrice; Bouchet, Jean-Paul; Courtial, Béatrice; Chupeau, Marie-Christine; Chupeau, Yves; Maisonneuve, Brigitte; Lucas, Hélène

2007-01-01

The tobacco (Nicotiana tabacum) element Tnt1 is one of the few identified active retrotransposons in plants. These elements possess unique properties that make them ideal genetic tools for gene tagging. Here, we demonstrate the feasibility of gene tagging using the retrotransposon Tnt1 in lettuce (Lactuca sativa), which is the largest genome tested for retrotransposon mutagenesis so far. Of 10 different transgenic bushes carrying a complete Tnt1 containing T-DNA, eight contained multiple transposed copies of Tnt1. The number of transposed copies of the element per plant was particularly high, the smallest number being 28. Tnt1 transposition in lettuce can be induced by a very simple in vitro culture protocol. Tnt1 insertions were stable in the progeny of the primary transformants and could be segregated genetically. Characterization of the sequences flanking some insertion sites revealed that Tnt1 often inserted into genes. The progeny of some primary transformants showed phenotypic alterations due to recessive mutations. One of these mutations was due to Tnt1 insertion in the gibberellin 3β-hydroxylase gene. Taken together, these results indicate that Tnt1 is a powerful tool for insertion mutagenesis especially in plants with a large genome. PMID:17351058

DinB Upregulation Is the Sole Role of the SOS Response in Stress-Induced Mutagenesis in Escherichia coli

PubMed Central

Galhardo, Rodrigo S.; Do, Robert; Yamada, Masami; Friedberg, Errol C.; Hastings, P. J.; Nohmi, Takehiko; Rosenberg, Susan M.

2009-01-01

Stress-induced mutagenesis is a collection of mechanisms observed in bacterial, yeast, and human cells in which adverse conditions provoke mutagenesis, often under the control of stress responses. Control of mutagenesis by stress responses may accelerate evolution specifically when cells are maladapted to their environments, i.e., are stressed. It is therefore important to understand how stress responses increase mutagenesis. In the Escherichia coli Lac assay, stress-induced point mutagenesis requires induction of at least two stress responses: the RpoS-controlled general/starvation stress response and the SOS DNA-damage response, both of which upregulate DinB error-prone DNA polymerase, among other genes required for Lac mutagenesis. We show that upregulation of DinB is the only aspect of the SOS response needed for stress-induced mutagenesis. We constructed two dinB(oc) (operator-constitutive) mutants. Both produce SOS-induced levels of DinB constitutively. We find that both dinB(oc) alleles fully suppress the phenotype of constitutively SOS-“off” lexA(Ind−) mutant cells, restoring normal levels of stress-induced mutagenesis. Thus, dinB is the only SOS gene required at induced levels for stress-induced point mutagenesis. Furthermore, although spontaneous SOS induction has been observed to occur in only a small fraction of cells, upregulation of dinB by the dinB(oc) alleles in all cells does not promote a further increase in mutagenesis, implying that SOS induction of DinB, although necessary, is insufficient to differentiate cells into a hypermutable condition. PMID:19270270

Computational Redesign of Acyl-ACP Thioesterase with Improved Selectivity toward Medium-Chain-Length Fatty Acids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Grisewood, Matthew J.; Hernández-Lozada, Néstor J.; Thoden, James B.

Enzyme and metabolic engineering offer the potential to develop biocatalysts for converting natural resources to a wide range of chemicals. To broaden the scope of potential products beyond natural metabolites, methods of engineering enzymes to accept alternative substrates and/or perform novel chemistries must be developed. DNA synthesis can create large libraries of enzyme-coding sequences, but most biochemistries lack a simple assay to screen for promising enzyme variants. Our solution to this challenge is structure-guided mutagenesis, in which optimization algorithms select the best sequences from libraries based on specified criteria (i.e., binding selectivity). We demonstrate this approach by identifying medium-chain (C8–C12)more » acyl-ACP thioesterases through structure-guided mutagenesis. Medium-chain fatty acids, which are products of thioesterase-catalyzed hydrolysis, are limited in natural abundance, compared to long-chain fatty acids; the limited supply leads to high costs of C6–C10 oleochemicals such as fatty alcohols, amines, and esters. Here, we applied computational tools to tune substrate binding of the highly active ‘TesA thioesterase in Escherichia coli. We used the IPRO algorithm to design thioesterase variants with enhanced C12 or C8 specificity, while maintaining high activity. After four rounds of structure-guided mutagenesis, we identified 3 variants with enhanced production of dodecanoic acid (C12) and 27 variants with enhanced production of octanoic acid (C8). The top variants reached up to 49% C12 and 50% C8 while exceeding native levels of total free fatty acids. A comparably sized library created by random mutagenesis failed to identify promising mutants. The chain length-preference of ‘TesA and the best mutant were confirmed in vitro using acyl-CoA substrates. Molecular dynamics simulations, confirmed by resolved crystal structures, of ‘TesA variants suggest that hydrophobic forces govern ‘TesA substrate specificity. Finally, we expect the design rules that we uncovered and the thioesterase variants that we identified will be useful to metabolic engineering projects aimed at sustainable production of medium-chain-length oleochemicals.« less

Computational Redesign of Acyl-ACP Thioesterase with Improved Selectivity toward Medium-Chain-Length Fatty Acids

DOE PAGES

Grisewood, Matthew J.; Hernández-Lozada, Néstor J.; Thoden, James B.; ...

2017-04-20

Enzyme and metabolic engineering offer the potential to develop biocatalysts for converting natural resources to a wide range of chemicals. To broaden the scope of potential products beyond natural metabolites, methods of engineering enzymes to accept alternative substrates and/or perform novel chemistries must be developed. DNA synthesis can create large libraries of enzyme-coding sequences, but most biochemistries lack a simple assay to screen for promising enzyme variants. Our solution to this challenge is structure-guided mutagenesis, in which optimization algorithms select the best sequences from libraries based on specified criteria (i.e., binding selectivity). We demonstrate this approach by identifying medium-chain (C8–C12)more » acyl-ACP thioesterases through structure-guided mutagenesis. Medium-chain fatty acids, which are products of thioesterase-catalyzed hydrolysis, are limited in natural abundance, compared to long-chain fatty acids; the limited supply leads to high costs of C6–C10 oleochemicals such as fatty alcohols, amines, and esters. Here, we applied computational tools to tune substrate binding of the highly active ‘TesA thioesterase in Escherichia coli. We used the IPRO algorithm to design thioesterase variants with enhanced C12 or C8 specificity, while maintaining high activity. After four rounds of structure-guided mutagenesis, we identified 3 variants with enhanced production of dodecanoic acid (C12) and 27 variants with enhanced production of octanoic acid (C8). The top variants reached up to 49% C12 and 50% C8 while exceeding native levels of total free fatty acids. A comparably sized library created by random mutagenesis failed to identify promising mutants. The chain length-preference of ‘TesA and the best mutant were confirmed in vitro using acyl-CoA substrates. Molecular dynamics simulations, confirmed by resolved crystal structures, of ‘TesA variants suggest that hydrophobic forces govern ‘TesA substrate specificity. Finally, we expect the design rules that we uncovered and the thioesterase variants that we identified will be useful to metabolic engineering projects aimed at sustainable production of medium-chain-length oleochemicals.« less

Limited ability of DNA polymerase kappa to suppress benzo[a]pyrene-induced genotoxicity in vivo.

PubMed

Masumura, Kenichi; Toyoda-Hokaiwado, Naomi; Niimi, Naoko; Grúz, Petr; Wada, Naoko A; Takeiri, Akira; Jishage, Kou-Ichi; Mishima, Masayuki; Nohmi, Takehiko

2017-12-01

DNA polymerase kappa (Polk) is a specialized DNA polymerase involved in translesion DNA synthesis. To understand the protective roles against genotoxins in vivo, we established inactivated Polk knock-in gpt delta (inactivated Polk KI) mice that possessed reporter genes for mutations and expressed inactive Polk. In this study, we examined genotoxicity of benzo[a]pyrene (BP) to determine whether Polk actually suppressed BP-induced genotoxicity as predicted by biochemistry and in vitro cell culture studies. Seven-week-old inactivated Polk KI and wild-type (WT) mice were treated with BP at doses of 5, 15, or 50 mg/(kg·day) for three consecutive days by intragastric gavage, and mutations in the colon and micronucleus formation in the peripheral blood were examined. Surprisingly, no differences were observed in the frequencies of mutations and micronucleus formation at 5 or 50 mg/kg doses. Inactivated Polk KI mice exhibited approximately two times higher gpt mutant frequency than did WT mice only at the 15 mg/kg dose. The frequency of micronucleus formation was slightly higher in inactivated Polk KI than in WT mice at the same dose, but it was statistically insignificant. The results suggest that Polk has a limited ability to suppress BP-induced genotoxicity in the colon and bone marrow and also that the roles of specialized DNA polymerases in mutagenesis and carcinogenesis should be examined not only by in vitro assays but also by in vivo mouse studies. We also report the spontaneous mutagenesis in inactivated Polk KI mice at young and old ages. Environ. Mol. Mutagen. 58:644-653, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

FDA toxicity databases and real-time data entry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Arvidson, Kirk B.

Structure-searchable electronic databases are valuable new tools that are assisting the FDA in its mission to promptly and efficiently review incoming submissions for regulatory approval of new food additives and food contact substances. The Center for Food Safety and Applied Nutrition's Office of Food Additive Safety (CFSAN/OFAS), in collaboration with Leadscope, Inc., is consolidating genetic toxicity data submitted in food additive petitions from the 1960s to the present day. The Center for Drug Evaluation and Research, Office of Pharmaceutical Science's Informatics and Computational Safety Analysis Staff (CDER/OPS/ICSAS) is separately gathering similar information from their submissions. Presently, these data are distributedmore » in various locations such as paper files, microfiche, and non-standardized toxicology memoranda. The organization of the data into a consistent, searchable format will reduce paperwork, expedite the toxicology review process, and provide valuable information to industry that is currently available only to the FDA. Furthermore, by combining chemical structures with genetic toxicity information, biologically active moieties can be identified and used to develop quantitative structure-activity relationship (QSAR) modeling and testing guidelines. Additionally, chemicals devoid of toxicity data can be compared to known structures, allowing for improved safety review through the identification and analysis of structural analogs. Four database frameworks have been created: bacterial mutagenesis, in vitro chromosome aberration, in vitro mammalian mutagenesis, and in vivo micronucleus. Controlled vocabularies for these databases have been established. The four separate genetic toxicity databases are compiled into a single, structurally-searchable database for easy accessibility of the toxicity information. Beyond the genetic toxicity databases described here, additional databases for subchronic, chronic, and teratogenicity studies have been prepared.« less

Molecular Determinants of Mutant Phenotypes, Inferred from Saturation Mutagenesis Data.

PubMed

Tripathi, Arti; Gupta, Kritika; Khare, Shruti; Jain, Pankaj C; Patel, Siddharth; Kumar, Prasanth; Pulianmackal, Ajai J; Aghera, Nilesh; Varadarajan, Raghavan

2016-11-01

Understanding how mutations affect protein activity and organismal fitness is a major challenge. We used saturation mutagenesis combined with deep sequencing to determine mutational sensitivity scores for 1,664 single-site mutants of the 101 residue Escherichia coli cytotoxin, CcdB at seven different expression levels. Active-site residues could be distinguished from buried ones, based on their differential tolerance to aliphatic and charged amino acid substitutions. At nonactive-site positions, the average mutational tolerance correlated better with depth from the protein surface than with accessibility. Remarkably, similar results were observed for two other small proteins, PDZ domain (PSD95 pdz3 ) and IgG-binding domain of protein G (GB1). Mutational sensitivity data obtained with CcdB were used to derive a procedure for predicting functional effects of mutations. Results compared favorably with those of two widely used computational predictors. In vitro characterization of 80 single, nonactive-site mutants of CcdB showed that activity in vivo correlates moderately with thermal stability and solubility. The inability to refold reversibly, as well as a decreased folding rate in vitro, is associated with decreased activity in vivo. Upon probing the effect of modulating expression of various proteases and chaperones on mutant phenotypes, most deleterious mutants showed an increased in vivo activity and solubility only upon over-expression of either Trigger factor or SecB ATP-independent chaperones. Collectively, these data suggest that folding kinetics rather than protein stability is the primary determinant of activity in vivo This study enhances our understanding of how mutations affect phenotype, as well as the ability to predict fitness effects of point mutations. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Revised Mechanism and Improved Efficiency of the QuikChange Site-Directed Mutagenesis Method.

PubMed

Xia, Yongzhen; Xun, Luying

2017-01-01

Site-directed mutagenesis has been widely used for the substitution, addition or deletion of nucleotide residues in a defined DNA sequence. QuikChange™ site-directed mutagenesis and its related protocols have been widely used for this purpose because of convenience and efficiency. We have recently demonstrated that the mechanism of the QuikChange™ site-directed mutagenesis process is different from that being proposed. The new mechanism promotes the use of partially overlapping primers and commercial PCR enzymes for efficient PCR and mutagenesis.

Improvement of banana cv. Rasthali (Silk, AAB) against Fusarium oxysporum f.sp. cubense (VCG 0124/5) through induced mutagenesis: Determination of LD50 specific to mutagen, explants, toxins and in vitro and in vivo screening for Fusarium wilt resistance.

PubMed

Saraswathi, M S; Kannan, G; Uma, S; Thangavelu, R; Backiyarani, S

2016-05-01

Shoot tips and in vitro grown proliferating buds of banana cv. Rasthali (Silk, AAB) were treated with various concentrations and durations of chemical mutagens viz., EMS, NaN3 and DES. LD50 for shoot tips based on 50% reduction in fresh weight was determined as 2% for 3 h, 0.02% for 5 h and 0.15% for 5 h, while for proliferating buds, they were 0.6% for 30 min, 0.01% for 2 h and 0.06% for 2 h for the mutagens EMS, NaN3 and DES, respectively. Subsequently, the mutated explants were screened in vitro against fusarium wilt using selection agents like fusaric acid and culture filtrate. LD50 for in vitro selection agents calculated based on 50% survival of explants was 0.050 mM and 7% for fusaric acid and culture filtrate, respectively and beyond which a rapid decline in growth was observed. This was followed by pot screening which led to the identification of three putative resistant mutants with an internal disease score of 1 (corm completely clean, no vascular discolouration). The putative mutants identified in the present study have also been mass multiplied in vitro.

The Yeast Environmental Stress Response Regulates Mutagenesis Induced by Proteotoxic Stress

PubMed Central

Shor, Erika; Fox, Catherine A.; Broach, James R.

2013-01-01

Conditions of chronic stress are associated with genetic instability in many organisms, but the roles of stress responses in mutagenesis have so far been elucidated only in bacteria. Here, we present data demonstrating that the environmental stress response (ESR) in yeast functions in mutagenesis induced by proteotoxic stress. We show that the drug canavanine causes proteotoxic stress, activates the ESR, and induces mutagenesis at several loci in an ESR-dependent manner. Canavanine-induced mutagenesis also involves translesion DNA polymerases Rev1 and Polζ and non-homologous end joining factor Ku. Furthermore, under conditions of chronic sub-lethal canavanine stress, deletions of Rev1, Polζ, and Ku-encoding genes exhibit genetic interactions with ESR mutants indicative of ESR regulating these mutagenic DNA repair processes. Analyses of mutagenesis induced by several different stresses showed that the ESR specifically modulates mutagenesis induced by proteotoxic stress. Together, these results document the first known example of an involvement of a eukaryotic stress response pathway in mutagenesis and have important implications for mechanisms of evolution, carcinogenesis, and emergence of drug-resistant pathogens and chemotherapy-resistant tumors. PMID:23935537

A Structural Basis for the Biosynthesis of the Major Chlorogenic Acids Found in Coffee1[W][OA

PubMed Central

Lallemand, Laura A.; Zubieta, Chloe; Lee, Soon Goo; Wang, Yechun; Acajjaoui, Samira; Timmins, Joanna; McSweeney, Sean; Jez, Joseph M.; McCarthy, James G.; McCarthy, Andrew A.

2012-01-01

Chlorogenic acids (CGAs) are a group of phenolic secondary metabolites produced by certain plant species and an important component of coffee (Coffea spp.). The CGAs have been implicated in biotic and abiotic stress responses, while the related shikimate esters are key intermediates for lignin biosynthesis. Here, two hydroxycinnamoyl-coenzyme A shikimate/quinate hydroxycinnamoyl transferases (HCT/HQT) from coffee were biochemically characterized. We show, to our knowledge for the first time, that in vitro, HCT is capable of synthesizing the 3,5-O-dicaffeoylquinic acid diester, a major constituent of the immature coffee grain. In order to further understand the substrate specificity and catalytic mechanism of the HCT/HQT, we performed structural and mutagenesis studies of HCT. The three-dimensional structure of a native HCT and a proteolytically stable lysine mutant enabled the identification of important residues involved in substrate specificity and catalysis. Site-directed mutagenesis confirmed the role of residues leucine-400 and phenylalanine-402 in substrate specificity and of histidine-153 and the valine-31 to proline-37 loop in catalysis. In addition, the histidine-154-asparagine mutant was observed to produce 4-fold more dichlorogenic acids compared with the native protein. These data provide, to our knowledge, the first structural characterization of a HCT and, in conjunction with the biochemical and mutagenesis studies presented here, delineate the underlying molecular-level determinants for substrate specificity and catalysis. This work has potential applications in fine-tuning the levels of shikimate and quinate esters (CGAs including dichlorogenic acids) in different plant species in order to generate reduced or elevated levels of the desired target compounds. PMID:22822210

Quantitative evaluation of DNA damage and mutation rate by atmospheric and room-temperature plasma (ARTP) and conventional mutagenesis.

PubMed

Zhang, Xue; Zhang, Chong; Zhou, Qian-Qian; Zhang, Xiao-Fei; Wang, Li-Yan; Chang, Hai-Bo; Li, He-Ping; Oda, Yoshimitsu; Xing, Xin-Hui

2015-07-01

DNA damage is the dominant source of mutation, which is the driving force of evolution. Therefore, it is important to quantitatively analyze the DNA damage caused by different mutagenesis methods, the subsequent mutation rates, and their relationship. Atmospheric and room temperature plasma (ARTP) mutagenesis has been used for the mutation breeding of more than 40 microorganisms. However, ARTP mutagenesis has not been quantitatively compared with conventional mutation methods. In this study, the umu test using a flow-cytometric analysis was developed to quantify the DNA damage in individual viable cells using Salmonella typhimurium NM2009 as the model strain and to determine the mutation rate. The newly developed method was used to evaluate four different mutagenesis systems: a new ARTP tool, ultraviolet radiation, 4-nitroquinoline-1-oxide (4-NQO), and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) mutagenesis. The mutation rate was proportional to the corresponding SOS response induced by DNA damage. ARTP caused greater DNA damage to individual living cells than the other conventional mutagenesis methods, and the mutation rate was also higher. By quantitatively comparing the DNA damage and consequent mutation rate after different types of mutagenesis, we have shown that ARTP is a potentially powerful mutagenesis tool with which to improve the characteristics of microbial cell factories.

Electrostatically Biased Binding of Kinesin to Microtubules

PubMed Central

Zheng, Wenjun; Alonso, Maria; Huber, Gary; Dlugosz, Maciej; McCammon, J. Andrew; Cross, Robert A.

2011-01-01

The minimum motor domain of kinesin-1 is a single head. Recent evidence suggests that such minimal motor domains generate force by a biased binding mechanism, in which they preferentially select binding sites on the microtubule that lie ahead in the progress direction of the motor. A specific molecular mechanism for biased binding has, however, so far been lacking. Here we use atomistic Brownian dynamics simulations combined with experimental mutagenesis to show that incoming kinesin heads undergo electrostatically guided diffusion-to-capture by microtubules, and that this produces directionally biased binding. Kinesin-1 heads are initially rotated by the electrostatic field so that their tubulin-binding sites face inwards, and then steered towards a plus-endwards binding site. In tethered kinesin dimers, this bias is amplified. A 3-residue sequence (RAK) in kinesin helix alpha-6 is predicted to be important for electrostatic guidance. Real-world mutagenesis of this sequence powerfully influences kinesin-driven microtubule sliding, with one mutant producing a 5-fold acceleration over wild type. We conclude that electrostatic interactions play an important role in the kinesin stepping mechanism, by biasing the diffusional association of kinesin with microtubules. PMID:22140358

Stress-induced mutation via DNA breaks in Escherichia coli: A molecular mechanism with implications for evolution and medicine

PubMed Central

Rosenberg, Susan M; Shee, Chandan; Frisch, Ryan L; Hastings, P J

2012-01-01

Abstract Evolutionary theory assumed that mutations occur constantly, gradually, and randomly over time. This formulation from the “modern synthesis” of the 1930s was embraced decades before molecular understanding of genes or mutations. Since then, our labs and others have elucidated mutation mechanisms activated by stress responses. Stress-induced mutation mechanisms produce mutations, potentially accelerating evolution, specifically when cells are maladapted to their environment, that is, when they are stressed. The mechanisms of stress-induced mutation that are being revealed experimentally in laboratory settings provide compelling models for mutagenesis that propels pathogen–host adaptation, antibiotic resistance, cancer progression and resistance, and perhaps much of evolution generally. We discuss double-strand-break-dependent stress-induced mutation in Escherichia coli. Recent results illustrate how a stress response activates mutagenesis and demonstrate this mechanism's generality and importance to spontaneous mutation. New data also suggest a possible harmony between previous, apparently opposed, models for the molecular mechanism. They additionally strengthen the case for anti-evolvability therapeutics for infectious disease and cancer. PMID:22911060

Stress-induced mutation via DNA breaks in Escherichia coli: a molecular mechanism with implications for evolution and medicine.

PubMed

Rosenberg, Susan M; Shee, Chandan; Frisch, Ryan L; Hastings, P J

2012-10-01

Evolutionary theory assumed that mutations occur constantly, gradually, and randomly over time. This formulation from the "modern synthesis" of the 1930s was embraced decades before molecular understanding of genes or mutations. Since then, our labs and others have elucidated mutation mechanisms activated by stress responses. Stress-induced mutation mechanisms produce mutations, potentially accelerating evolution, specifically when cells are maladapted to their environment, that is, when they are stressed. The mechanisms of stress-induced mutation that are being revealed experimentally in laboratory settings provide compelling models for mutagenesis that propels pathogen-host adaptation, antibiotic resistance, cancer progression and resistance, and perhaps much of evolution generally. We discuss double-strand-break-dependent stress-induced mutation in Escherichia coli. Recent results illustrate how a stress response activates mutagenesis and demonstrate this mechanism's generality and importance to spontaneous mutation. New data also suggest a possible harmony between previous, apparently opposed, models for the molecular mechanism. They additionally strengthen the case for anti-evolvability therapeutics for infectious disease and cancer. Copyright © 2012 WILEY Periodicals, Inc.

Pathogenesis of leptospirosis: cellular and molecular aspects.

PubMed

Adler, Ben

2014-08-27

Leptospirosis is arguably the most widespread zoonosis; it is also a major cause of economic loss in production animals worldwide. At the level of the host animal or human, the progression of infection and the onset of disease are well documented. However, the mechanisms of pathogenesis at the cellular and molecular level remain poorly understood, mainly as a result of the lack of modern genetic tools for mutagenesis of pathogenic Leptospira spp. The recent development of transposon mutagenesis and the construction of a very small number of directed leptospiral mutants have identified a limited number of essential virulence factors. Perhaps surprisingly, many leptospiral proteins with characteristics consistent with a role in virulence have been shown to not be required for virulence in animal models, consistent with a high degree of functional redundancy in pathogenic Leptospira. A large number of putative adhesins has been reported in Leptospira, which interact with a range of host tissue components; however, almost none of these have been genetically confirmed as having an essential role in pathogenesis. Copyright © 2014 Elsevier B.V. All rights reserved.

GPCR-I-TASSER: A Hybrid Approach to G Protein-Coupled Receptor Structure Modeling and the Application to the Human Genome.

PubMed

Zhang, Jian; Yang, Jianyi; Jang, Richard; Zhang, Yang

2015-08-04

Experimental structure determination remains difficult for G protein-coupled receptors (GPCRs). We propose a new hybrid protocol to construct GPCR structure models that integrates experimental mutagenesis data with ab initio transmembrane (TM) helix assembly simulations. The method was tested on 24 known GPCRs where the ab initio TM-helix assembly procedure constructed the correct fold for 20 cases. When combined with weak homology and sparse mutagenesis restraints, the method generated correct folds for all the tested cases with an average Cα root-mean-square deviation 2.4 Å in the TM regions. The new hybrid protocol was applied to model all 1,026 GPCRs in the human genome, where 923 have a high confidence score and are expected to have correct folds; these contain many pharmaceutically important families with no previously solved structures, including Trace amine, Prostanoids, Releasing hormones, Melanocortins, Vasopressin, and Neuropeptide Y receptors. The results demonstrate new progress on genome-wide structure modeling of TM proteins. Copyright © 2015 Elsevier Ltd. All rights reserved.

In vitro replication of poliovirus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lubinski, J.M.

1986-01-01

Poliovirus is a member of the Picornaviridae whose genome is a single stranded RNA molecule of positive polarity surrounded by a proteinaceous capsid. Replication of poliovirus occurs via negative strand intermediates in infected cells using a virally encoded RNA-dependent RNA polymerase and host cell proteins. The authors have exploited the fact that complete cDNA copies of the viral genome when transfected onto susceptible cells generate virus. Utilizing the bacteriophage SP6 DNA dependent RNA polymerase system to synthesize negative strands in vitro and using these in an in vitro reaction the authors have generated full length infectious plus strands. Mutagenesis ofmore » the 5' and 3' ends of the negative and positive strands demonstrated that replication could occur either de novo or be extensions of the templates from their 3' ends or from nicks occurring during replication. The appearance of dimeric RNA molecules generated in these reactions was not dependent upon the same protein required for de novo initiation. Full length dimeric RNA molecules using a 5' /sup 32/P end-labelled oligo uridylic acid primer and positive strand template were demonstrated in vitro containing only the 35,000 Mr host protein and the viral RNA-dependent RNA polymerase. A model for generating positive strands without protein priming by cleavage of dimeric RNA molecules was proposed.« less

Limited mutagenicity of electronic cigarettes in mouse or human cells in vitro.

PubMed

Tommasi, Stella; Bates, Steven E; Behar, Rachel Z; Talbot, Prue; Besaratinia, Ahmad

2017-10-01

Electronic cigarettes (e-cig), which are promoted as safe alternatives to tobacco cigarettes or as aides to smoking cessation, are becoming increasingly popular among adult chronic smokers and adolescents experimenting with tobacco products. Despite the known presence of toxicants and carcinogens in e-cig liquid and vapor, the possible carcinogenic effects of e-cig use in humans are unknown. We have utilized two validated in vitro model systems to investigate whether e-cig vapor induces mutation in mouse or human cells. We have exposed transgenic mouse fibroblasts in vitro to e-cig vapor extracts prepared from three popular brands, and determined the induction of mutagenesis in a reporter gene, the cII transgene. Furthermore, we have treated the pSP189 plasmid with e-cig vapor extract, transfected human fibroblast cells with the e-cig-treated plasmid, and screened for the induced mutations in the supF gene. We observed no statistically significant increases in relative mutant frequency in the cII transgene or supF gene in the e-cig treated mouse or human cells, respectively. Our data indicate that e-cig vapor extracts from the selected brands and at concentrations tested in this study have limited mutagenicity in both mouse and human cells in vitro. Copyright © 2017 Elsevier B.V. All rights reserved.

A novel mouse model identifies cooperating mutations and therapeutic targets critical for chronic myeloid leukemia progression

PubMed Central

Giotopoulos, George; van der Weyden, Louise; Osaki, Hikari; Rust, Alistair G.; Gallipoli, Paolo; Meduri, Eshwar; Horton, Sarah J.; Chan, Wai-In; Foster, Donna; Prinjha, Rab K.; Pimanda, John E.; Tenen, Daniel G.; Vassiliou, George S.; Koschmieder, Steffen; Adams, David J.

2015-01-01

The introduction of highly selective ABL-tyrosine kinase inhibitors (TKIs) has revolutionized therapy for chronic myeloid leukemia (CML). However, TKIs are only efficacious in the chronic phase of the disease and effective therapies for TKI-refractory CML, or after progression to blast crisis (BC), are lacking. Whereas the chronic phase of CML is dependent on BCR-ABL, additional mutations are required for progression to BC. However, the identity of these mutations and the pathways they affect are poorly understood, hampering our ability to identify therapeutic targets and improve outcomes. Here, we describe a novel mouse model that allows identification of mechanisms of BC progression in an unbiased and tractable manner, using transposon-based insertional mutagenesis on the background of chronic phase CML. Our BC model is the first to faithfully recapitulate the phenotype, cellular and molecular biology of human CML progression. We report a heterogeneous and unique pattern of insertions identifying known and novel candidate genes and demonstrate that these pathways drive disease progression and provide potential targets for novel therapeutic strategies. Our model greatly informs the biology of CML progression and provides a potent resource for the development of candidate therapies to improve the dismal outcomes in this highly aggressive disease. PMID:26304963

A Forward Genetic Screening for Prostate Cancer Progression Genes

DTIC Science & Technology

2012-10-01

sequence  reads. For verifying  the  prevalence of insertions in tumors, PCR was  performed on  genomic  DNA corresponding to 15 insertional mutations using...and has been utilized with great effect in many organisms, from the bacterium to the fruit fly Drosophila melanogaster [1,2]. The Sleeping Beauty (SB...TX SL JC TN. References 1. Cooley L, Kelley R, Spradling A (1988) Insertional mutagenesis of the Drosophila genome with single P elements. Science

Directed molecular evolution to design advanced red fluorescent proteins.

PubMed

Subach, Fedor V; Piatkevich, Kiryl D; Verkhusha, Vladislav V

2011-11-29

Fluorescent proteins have become indispensable imaging tools for biomedical research. Continuing progress in fluorescence imaging, however, requires probes with additional colors and properties optimized for emerging techniques. Here we summarize strategies for development of red-shifted fluorescent proteins. We discuss possibilities for knowledge-based rational design based on the photochemistry of fluorescent proteins and the position of the chromophore in protein structure. We consider advances in library design by mutagenesis, protein expression systems and instrumentation for high-throughput screening that should yield improved fluorescent proteins for advanced imaging applications.

Recombination of polynucleotide sequences using random or defined primers

DOEpatents

Arnold, Frances H.; Shao, Zhixin; Affholter, Joseph A.; Zhao, Huimin H; Giver, Lorraine J.

2000-01-01

A method for in vitro mutagenesis and recombination of polynucleotide sequences based on polymerase-catalyzed extension of primer oligonucleotides is disclosed. The method involves priming template polynucleotide(s) with random-sequences or defined-sequence primers to generate a pool of short DNA fragments with a low level of point mutations. The DNA fragments are subjected to denaturization followed by annealing and further enzyme-catalyzed DNA polymerization. This procedure is repeated a sufficient number of times to produce full-length genes which comprise mutants of the original template polynucleotides. These genes can be further amplified by the polymerase chain reaction and cloned into a vector for expression of the encoded proteins.

Functional Characterization of ATM Kinase Using Acetylation-Specific Antibodies.

PubMed

Sun, Yingli; Du, Fengxia

2017-01-01

The activation of ATM is critical in the DNA double strand breaks repair pathway. Acetylation of ATM by Tip60 histone acetyltransferase (HAT) plays a key role in the activation of ATM kinase activity in response to DNA damage. ATM forms a stable complex with Tip60 through the FATC domain of ATM. Tip60 acetylates lysine3016 of ATM, and this acetylation induces the activation of ATM. Several techniques are included in the study of ATM acetylation by Tip60, such as in vitro kinase assay, systematic mutagenesis, western blots. Here, we describe how to study the acetylation of ATM using acetylation-specific antibodies.

ECB deacylase mutants

DOEpatents

Arnold, Frances H.; Shao, Zhixin; Zhao, Huimin; Giver, Lorraine J.

2002-01-01

A method for in vitro mutagenesis and recombination of polynucleotide sequences based on polymerase-catalyzed extension of primer oligonucleotides is disclosed. The method involves priming template polynucleotide(s) with random-sequences or defined-sequence primers to generate a pool of short DNA fragments with a low level of point mutations. The DNA fragments are subjected to denaturization followed by annealing and further enzyme-catalyzed DNA polymerization. This procedure is repeated a sufficient number of times to produce full-length genes which comprise mutants of the original template polynucleotides. These genes can be further amplified by the polymerase chain reaction and cloned into a vector for expression of the encoded proteins.

Recombination of polynucleotide sequences using random or defined primers

DOEpatents

Arnold, Frances H.; Shao, Zhixin; Affholter, Joseph A.; Zhao, Huimin; Giver, Lorraine J.

2001-01-01

A method for in vitro mutagenesis and recombination of polynucleotide sequences based on polymerase-catalyzed extension of primer oligonucleotides is disclosed. The method involves priming template polynucleotide(s) with random-sequences or defined-sequence primers to generate a pool of short DNA fragments with a low level of point mutations. The DNA fragments are subjected to denaturization followed by annealing and further enzyme-catalyzed DNA polymerization. This procedure is repeated a sufficient number of times to produce full-length genes which comprise mutants of the original template polynucleotides. These genes can be further amplified by the polymerase chain reaction and cloned into a vector for expression of the encoded proteins.

Unlocking the steric gate of DNA polymerase η leads to increased genomic instability in Saccharomyces cerevisiae

PubMed Central

Donigan, Katherine A.; Cerritelli, Susana M.; McDonald, John P.; Vaisman, Alexandra; Crouch, Robert J.; Woodgate, Roger

2015-01-01

DNA polymerase η (pol η) is best characterized for its ability to perform accurate and efficient translesion DNA synthesis (TLS) through cyclobutane pyrimidine dimers (CPDs). To ensure accurate bypass the polymerase is not only required to select the correct base, but also discriminate between NTPs and dNTPs. Most DNA polymerases have a conserved “steric gate” residue which functions to prevent incorporation of NMPs during DNA synthesis. Here, we demonstrate that the Phe35 residue of S. cerevisiae pol η functions as a steric gate to limit the use of ribonucleotides during polymerization both in vitro and in vivo. Unlike the related polι enzyme, wild-type pol η does not readily incorporate NMPs in vitro. In contrast, a pol η F35A mutant incorporates NMPs on both damaged and undamaged DNA in vitro with a high degree of base selectivity. An S. cerevisiae strain expressing pol η F35A (rad30-F35A) that is also deficient for nucleotide excision repair (rad1Δ) and the TLS polymerase, pol ζ (rev3Δ), is extremely sensitive to UV-light. The sensitivity is due, in part, to RNaseH2 activity, as an isogenic rnh201Δ strain is roughly 50-fold more UV-resistant than its RNH201+ counterpart. Interestingly the rad1Δ rev3Δ rad30-F35A rnh201Δ strain exhibits a significant increase in the extent of spontaneous mutagenesis with a spectrum dominated by 1 bp deletions at runs of template Ts. We hypothesize that the increased mutagenesis is due to rA incorporation at these sites and that the short poly rA tract is subsequently repaired in an error-prone manner by a novel repair pathway that is specifically targeted to polyribonucleotide tracks. These data indicate that under certain conditions, pol η can compete with the cell’s replicases and gain access to undamaged genomic DNA. Such observations are consistent with a role for pol η in replicating common fragile sites (CFS) in human cells. PMID:26340535

Unlocking the steric gate of DNA polymerase η leads to increased genomic instability in Saccharomyces cerevisiae.

PubMed

Donigan, Katherine A; Cerritelli, Susana M; McDonald, John P; Vaisman, Alexandra; Crouch, Robert J; Woodgate, Roger

2015-11-01

DNA polymerase η (pol η) is best characterized for its ability to perform accurate and efficient translesion DNA synthesis (TLS) through cyclobutane pyrimidine dimers (CPDs). To ensure accurate bypass the polymerase is not only required to select the correct base, but also discriminate between NTPs and dNTPs. Most DNA polymerases have a conserved "steric gate" residue which functions to prevent incorporation of NMPs during DNA synthesis. Here, we demonstrate that the Phe35 residue of Saccharomyces cerevisiae pol η functions as a steric gate to limit the use of ribonucleotides during polymerization both in vitro and in vivo. Unlike the related pol ι enzyme, wild-type pol η does not readily incorporate NMPs in vitro. In contrast, a pol η F35A mutant incorporates NMPs on both damaged and undamaged DNA in vitro with a high degree of base selectivity. An S.cerevisiae strain expressing pol η F35A (rad30-F35A) that is also deficient for nucleotide excision repair (rad1Δ) and the TLS polymerase, pol ζ (rev3Δ), is extremely sensitive to UV-light. The sensitivity is due, in part, to RNase H2 activity, as an isogenic rnh201Δ strain is roughly 50-fold more UV-resistant than its RNH201(+) counterpart. Interestingly the rad1Δ rev3Δ rad30-F35A rnh201Δ strain exhibits a significant increase in the extent of spontaneous mutagenesis with a spectrum dominated by 1bp deletions at runs of template Ts. We hypothesize that the increased mutagenesis is due to rA incorporation at these sites and that the short poly rA tract is subsequently repaired in an error-prone manner by a novel repair pathway that is specifically targeted to polyribonucleotide tracks. These data indicate that under certain conditions, pol η can compete with the cell's replicases and gain access to undamaged genomic DNA. Such observations are consistent with a role for pol η in replicating common fragile sites (CFS) in human cells. Published by Elsevier B.V.

Signature-tagged mutagenesis in a chicken infection model leads to the identification of a novel avian pathogenic Escherichia coli fimbrial adhesin.

PubMed

Antão, Esther-Maria; Ewers, Christa; Gürlebeck, Doreen; Preisinger, Rudolf; Homeier, Timo; Li, Ganwu; Wieler, Lothar H

2009-11-12

The extraintestinal pathogen, avian pathogenic E. coli (APEC), known to cause systemic infections in chickens, is responsible for large economic losses in the poultry industry worldwide. In order to identify genes involved in the early essential stages of pathogenesis, namely adhesion and colonization, Signature-tagged mutagenesis (STM) was applied to a previously established lung colonization model of infection by generating and screening a total of 1,800 mutants of an APEC strain IMT5155 (O2:K1:H5; Sequence type complex 95). The study led to the identification of new genes of interest, including two adhesins, one of which coded for a novel APEC fimbrial adhesin (Yqi) not described for its role in APEC pathogenesis to date. Its gene product has been temporarily designated ExPEC Adhesin I (EA/I) until the adhesin-specific receptor is identified. Deletion of the ExPEC adhesin I gene resulted in reduced colonization ability by APEC strain IMT5155 both in vitro and in vivo. Furthermore, complementation of the adhesin gene restored its ability to colonize epithelial cells in vitro. The ExPEC adhesin I protein was successfully expressed in vitro. Electron microscopy of an afimbriate strain E. coli AAEC189 over-expressed with the putative EA/I gene cluster revealed short fimbrial-like appendages protruding out of the bacterial outer membrane. We observed that this adhesin coding gene yqi is prevalent among extraintestinal pathogenic E. coli (ExPEC) isolates, including APEC (54.4%), uropathogenic E. coli (UPEC) (65.9%) and newborn meningitic E. coli (NMEC) (60.0%), and absent in all of the 153 intestinal pathogenic E. coli strains tested, thereby validating the designation of the adhesin as ExPEC Adhesin I. In addition, prevalence of EA/I was most frequently associated with the B2 group of the EcoR classification and ST95 complex of the multi locus sequence typing (MLST) scheme, with evidence of a positive selection within this highly pathogenic complex. This is the first report of the newly identified and functionally characterized ExPEC adhesin I and its significant role during APEC infection in chickens.

Identification of Potential Virulence Determinants by Himar1 Transposition of Infectious Borrelia burgdorferi B31▿

PubMed Central

Botkin, Douglas J.; Abbott, April N.; Stewart, Philip E.; Rosa, Patricia A.; Kawabata, Hiroki; Watanabe, Haruo; Norris, Steven J.

2006-01-01

Lyme disease Borrelia organisms are highly invasive spirochetes that alternate between vertebrate and arthropod hosts and that establish chronic infections and elicit inflammatory reactions in mammals. Although progress has been made in the targeted mutagenesis of individual genes in infectious Borrelia burgdorferi, the roles of the vast majority of gene products in pathogenesis remain unresolved. In this study, we examined the feasibility of using transposon mutagenesis to identify infectivity-related factors in B. burgdorferi. The transformable, infectious strain 5A18 NP1 was transformed with the spirochete-adapted Himar1 transposon delivery vector pMarGent to create a small library of 33 insertion mutants. Single mouse inoculations followed by culture of four tissue sites and serology were used to screen the mutants for infectivity phenotypes. Mutants that appeared attenuated (culture positive at some sites) or noninfectious (negative at all sites) and contained the virulence-associated plasmids lp25 and lp28-1 were examined in more extensive animal studies. Three of these mutants (including those with insertions in the putative fliG-1-encoded flagellar motor switch protein and the guaB-encoded IMP dehydrogenase) were noninfectious, whereas four clones appeared to exhibit reduced infectivity. Serological reactivity in VlsE enzyme-linked immunosorbent assays correlated with the assignment of mutants to the noninfectious or attenuated-infectivity groups. The results of this study indicate that random transposon mutagenesis of infectious B. burgdorferi is feasible and will be of value in studying the pathogenesis of Lyme disease Borrelia. PMID:17015459

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sharma, Manisha; Jamieson, Cara; Lui, Christina

β-catenin is a key mediator of Wnt signaling and its deregulated nuclear accumulation can drive cancer progression. While the central armadillo (Arm) repeats of β-catenin stimulate nuclear entry, the N- and C-terminal “tail” sequences are thought to regulate turnover and transactivation. We show here that the N- and C-tails are also potent transport sequences. The unstructured tails of β-catenin, when individually fused to a GFP-reporter, could enter and exit the nucleus rapidly in live cells. Proximity ligation assays and pull-down assays identified a weak interaction between the tail sequences and the FG-repeats of nucleoporins, consistent with a possible direct translocationmore » of β-catenin through the nuclear pore complex. Extensive alanine mutagenesis of the tail sequences revealed that nuclear translocation of β-catenin was dependent on specific uniformly distributed patches of hydrophobic residues, whereas the mutagenesis of acidic amino acids had no effect. Moreover, the mutation of hydrophobic patches within the N-tail and C-tail of full length β-catenin reduced nuclear transport rate and diminished its ability to activate transcription. We propose that the tail sequences can contribute to β-catenin transport and suggest a possible similar role for hydrophobic unstructured regions in other proteins. - Highlights: • We show that the N- and C-tails of beta-catenin possess nuclear transport activity. • Nuclear transport of the N- or C-tails requires specific hydrophobic amino acids. • Mutagenesis of the N-terminus diminished nuclear entry of full-length beta-catenin. • We propose the N-tail contributes to beta-catenin nuclear entry and transactivation.« less

Sperm DNA damage or progressive motility: which one is the better predictor of fertilization in vitro?

PubMed

Simon, Luke; Lewis, Sheena E M

2011-06-01

Sperm progressive motility has been reported to be one of the key factors influencing in vitro fertilization rates. However, recent studies have shown that sperm DNA fragmentation is a more robust predictor of assisted reproductive outcomes including reduced fertilization rates, embryo quality, and pregnancy rates. This study aimed to compare the usefulness of sperm progressive motility and DNA damage as predictive tools of in vitro fertilization rates. Here, 136 couples provided 1,767 eggs with an overall fertilization rate of 64.2%. The fertilization rate in vitro correlated with both sperm progressive motility (r² = 0.236; P = 0.002) and DNA fragmentation (r² = -0.318; P < 0.001). The relative risk of a poor fertilization rate was 9.5 times higher in sperm of men with high DNA fragmentation (>40%) compared with 2.6 times in sperm with poor motility (<40%). Further, sperm DNA fragmentation gave a higher specificity (93.3%) in predicting the fertilization rate than progressive motility (77.8%). Finally, the odds ratio to determine fertilization rate (>70%) was 4.81 (1.89-12.65) using progressive motility compared with 24.18 (5.21-154.51) using DNA fragmentation. This study shows that fertilization rates are directly dependent upon both sperm progressive motility and DNA fragmentation, but sperm DNA fragmentation is a much stronger test.

[SOS-repair--60 years].

PubMed

Zavil'gel'skiĭ, G B

2013-01-01

This review integrates 60 years of research on SOS-repair and SOS-mutagenesis in procaryotes and eucaryotes, from Jean Weigle experiment in 1953 year (mutagenesis of lambda bacteriophage in UV-irradiated bacteria) to the latest achievements in studying SOS-mutagenesis on all living organisms--Eukarya, Archaea and Bacteria. A key role in establishing of a biochemical basis for SOS-mutagenesis belonges to the finding in 1998-1999 years that specific error-prone DNA polymerases (PolV and others) catalysed translesion synthesis on damaged DNA. This review focuses on recent studies addressing the new models for SOS-induced mutagenesis in Escherichia coli and Home sapiens cells.

Phenotypic heterogeneity in a bacteriophage population only appears as stress-induced mutagenesis.

PubMed

Yosef, Ido; Edgar, Rotem; Qimron, Udi

2016-11-01

Stress-induced mutagenesis has been studied in cancer cells, yeast, bacteria, and archaea, but not in viruses. In a recent publication, we present a bacteriophage model showing an apparent stress-induced mutagenesis. We show that the stress does not drive the mutagenesis, but only selects the fittest mutants. The mechanism underlying the observed phenomenon is a phenotypic heterogeneity that resembles persistence of the viral population. The new findings, the background for the ongoing debate on stress-induced mutagenesis, and the phenotypic heterogeneity underlying a novel phage infection strategy are discussed in this short manuscript.

Use of Random and Site-Directed Mutagenesis to Probe Protein Structure-Function Relationships: Applied Techniques in the Study of Helicobacter pylori.

PubMed

Whitmire, Jeannette M; Merrell, D Scott

2017-01-01

Mutagenesis is a valuable tool to examine the structure-function relationships of bacterial proteins. As such, a wide variety of mutagenesis techniques and strategies have been developed. This chapter details a selection of random mutagenesis methods and site-directed mutagenesis procedures that can be applied to an array of bacterial species. Additionally, the direct application of the techniques to study the Helicobacter pylori Ferric Uptake Regulator (Fur) protein is described. The varied approaches illustrated herein allow the robust investigation of the structural-functional relationships within a protein of interest.

A Mutant Mouse with a Highly Specific Contextual Fear-Conditioning Deficit Found in an N-Ethyl-N-Nitrosourea (ENU) Mutagenesis Screen

ERIC Educational Resources Information Center

Pletcher, Mathew T.; Wiltshire, Tim; Tarantino, Lisa M.; Mayford, Mark; Reijmers, Leon G.; Coats, Jennifer K.

2006-01-01

Targeted mutagenesis in mice has shown that genes from a wide variety of gene families are involved in memory formation. The efficient identification of genes involved in learning and memory could be achieved by random mutagenesis combined with high-throughput phenotyping. Here, we provide the first report of a mutagenesis screen that has…

Beyond the Natural Proteome: Nondegenerate Saturation Mutagenesis-Methodologies and Advantages.

PubMed

Ferreira Amaral, M M; Frigotto, L; Hine, A V

2017-01-01

Beyond the natural proteome, high-throughput mutagenesis offers the protein engineer an opportunity to "tweak" the wild-type activity of a protein to create a recombinant protein with required attributes. Of the various approaches available, saturation mutagenesis is one of the core techniques employed by protein engineers, and in recent times, nondegenerate saturation mutagenesis is emerging as the approach of choice. This review compares the current methodologies available for conducting nondegenerate saturation mutagenesis with traditional, degenerate saturation and briefly outlines the options available for screening the resulting libraries, to discover a novel protein with the required activity and/or specificity. © 2017 Elsevier Inc. All rights reserved.

[Influence of diethyl sulfate (DES) mutagenesis on growth properties and pigment secondary metabolites of Phellinus igniarius].

PubMed

Wang, Jing; Wu, Xin-yuan; Ma, Wei; Chen, Jing; Liu, Cheng; Wu, Xiu-li

2015-06-01

The diethyl sulfate (DES) mutagenesis was chosen for the mutagenic treatment to Phellinus igniarius, and the relationship of mutagenesis time and death rate was investigated with 0.5% DES. The differences of mycelial growth speed, liquid fermentation mycelia biomass, morphology and pigment classes of secondary metabolites production speed and antioxidant activities of metabolite products were discussed. The study displayed that DES mutagenesis could change mycelial morphology without obvious effect on mycelium growth, and the DES mutagenesis improved antioxidant activities of the active ingredients of P. igniarius and had more antioxidant activity of hypoxia/sugar PC12 nerve cells than that of P. igniarius.

Multiplex Conditional Mutagenesis Using Transgenic Expression of Cas9 and sgRNAs

PubMed Central

Yin, Linlin; Maddison, Lisette A.; Li, Mingyu; Kara, Nergis; LaFave, Matthew C.; Varshney, Gaurav K.; Burgess, Shawn M.; Patton, James G.; Chen, Wenbiao

2015-01-01

Determining the mechanism of gene function is greatly enhanced using conditional mutagenesis. However, generating engineered conditional alleles is inefficient and has only been widely used in mice. Importantly, multiplex conditional mutagenesis requires extensive breeding. Here we demonstrate a system for one-generation multiplex conditional mutagenesis in zebrafish (Danio rerio) using transgenic expression of both cas9 and multiple single guide RNAs (sgRNAs). We describe five distinct zebrafish U6 promoters for sgRNA expression and demonstrate efficient multiplex biallelic inactivation of tyrosinase and insulin receptor a and b, resulting in defects in pigmentation and glucose homeostasis. Furthermore, we demonstrate temporal and tissue-specific mutagenesis using transgenic expression of Cas9. Heat-shock-inducible expression of cas9 allows temporal control of tyr mutagenesis. Liver-specific expression of cas9 disrupts insulin receptor a and b, causing fasting hypoglycemia and postprandial hyperglycemia. We also show that delivery of sgRNAs targeting ascl1a into the eye leads to impaired damage-induced photoreceptor regeneration. Our findings suggest that CRISPR/Cas9-based conditional mutagenesis in zebrafish is not only feasible but rapid and straightforward. PMID:25855067

[Stress-induced cellular adaptive mutagenesis].

PubMed

Zhu, Linjiang; Li, Qi

2014-04-01

The adaptive mutations exist widely in the evolution of cells, such as antibiotic resistance mutations of pathogenic bacteria, adaptive evolution of industrial strains, and cancerization of human somatic cells. However, how these adaptive mutations are generated is still controversial. Based on the mutational analysis models under the nonlethal selection conditions, stress-induced cellular adaptive mutagenesis is proposed as a new evolutionary viewpoint. The hypothetic pathway of stress-induced mutagenesis involves several intracellular physiological responses, including DNA damages caused by accumulation of intracellular toxic chemicals, limitation of DNA MMR (mismatch repair) activity, upregulation of general stress response and activation of SOS response. These responses directly affect the accuracy of DNA replication from a high-fidelity manner to an error-prone one. The state changes of cell physiology significantly increase intracellular mutation rate and recombination activity. In addition, gene transcription under stress condition increases the instability of genome in response to DNA damage, resulting in transcription-associated DNA mutagenesis. In this review, we summarize these two molecular mechanisms of stress-induced mutagenesis and transcription-associated DNA mutagenesis to help better understand the mechanisms of adaptive mutagenesis.

In Vitro Evolution and Affinity-Maturation with Coliphage Qβ Display

PubMed Central

Skamel, Claudia; Aller, Stephen G.; Bopda Waffo, Alain

2014-01-01

The Escherichia coli bacteriophage, Qβ (Coliphage Qβ), offers a favorable alternative to M13 for in vitro evolution of displayed peptides and proteins due to high mutagenesis rates in Qβ RNA replication that better simulate the affinity maturation processes of the immune response. We describe a benchtop in vitro evolution system using Qβ display of the VP1 G-H loop peptide of foot-and-mouth disease virus (FMDV). DNA encoding the G-H loop was fused to the A1 minor coat protein of Qβ resulting in a replication-competent hybrid phage that efficiently displayed the FMDV peptide. The surface-localized FMDV VP1 G-H loop cross-reacted with the anti-FMDV monoclonal antibody (mAb) SD6 and was found to decorate the corners of the Qβ icosahedral shell by electron microscopy. Evolution of Qβ-displayed peptides, starting from fully degenerate coding sequences corresponding to the immunodominant region of VP1, allowed rapid in vitro affinity maturation to SD6 mAb. Qβ selected under evolutionary pressure revealed a non-canonical, but essential epitope for mAb SD6 recognition consisting of an Arg-Gly tandem pair. Finally, the selected hybrid phages induced polyclonal antibodies in guinea pigs with good affinity to both FMDV and hybrid Qβ-G-H loop, validating the requirement of the tandem pair epitope. Qβ-display emerges as a novel framework for rapid in vitro evolution with affinity-maturation to molecular targets. PMID:25393763

Active site-directed double mutants of dihydrofolate reductase.

PubMed

Ercikan-Abali, E A; Mineishi, S; Tong, Y; Nakahara, S; Waltham, M C; Banerjee, D; Chen, W; Sadelain, M; Bertino, J R

1996-09-15

Variants of dihydrofolate reductase (DHFR), which confer resistance to antifolates, are used as dominant selectable markers in vitro and in vivo and may be useful in the context of gene therapy. To identify improved mutant human DHFRs with increased catalytic efficiency and decreased binding to methotrexate, we constructed by site-directed mutagenesis four variants with substitutions at both Leu22 and Phe31 (i.e., Phe22-Ser31, Tyr22-Ser31, Phe22-Gly31, and Tyr22-Gly31). Antifolate resistance has been observed previously when individual changes are made at these active-site residues. Substrate and antifolate binding properties of these "double" mutants revealed that each have greatly diminished affinity for antifolates (> 10,000-fold) yet only slightly reduced substrate affinity. Comparison of in vitro measured properties with those of single-residue variants indicates that double mutants are indeed significantly superior. This was verified for one of the double mutants that provided high-level methotrexate resistance following retrovirus-mediated gene transfer in NIH3T3 cells.

Protein and Antibody Engineering by Phage Display.

PubMed

Frei, J C; Lai, J R

2016-01-01

Phage display is an in vitro selection technique that allows for the rapid isolation of proteins with desired properties including increased affinity, specificity, stability, and new enzymatic activity. The power of phage display relies on the phenotype-to-genotype linkage of the protein of interest displayed on the phage surface with the encoding DNA packaged within the phage particle, which allows for selective enrichment of library pools and high-throughput screening of resulting clones. As an in vitro method, the conditions of the binding selection can be tightly controlled. Due to the high-throughput nature, rapidity, and ease of use, phage display is an excellent technological platform for engineering antibody or proteins with enhanced properties. Here, we describe methods for synthesis, selection, and screening of phage libraries with particular emphasis on designing humanizing antibody libraries and combinatorial scanning mutagenesis libraries. We conclude with a brief section on troubleshooting for all stages of the phage display process. © 2016 Elsevier Inc. All rights reserved.

MicroRNAs as targets for dietary and pharmacological inhibitors of mutagenesis and carcinogenesis

PubMed Central

Izzotti, Alberto; Cartiglia, Cristina; Steele, Vernon E.; De Flora, Silvio

2012-01-01

MicroRNAs (miRNAs) have been implicated in many biological processes, cancer, and other diseases. In addition, miRNAs are dysregulated following exposure to toxic and genotoxic agents. Here we review studies evaluating modulation of miRNAs by dietary and pharmacological agents, which could potentially be exploited for inhibition of mutagenesis and carcinogenesis. This review covers natural agents, including vitamins, oligoelements, polyphenols, isoflavones, indoles, isothiocyanates, phospholipids, saponins, anthraquinones and polyunsaturated fatty acids, and synthetic agents, including thiols, nuclear receptor agonists, histone deacetylase inhibitors, antiinflammatory drugs, and selective estrogen receptor modulators. As many as 145 miRNAs, involved in the control of a variety of carcinogenesis mechanisms, were modulated by these agents, either individually or in combination. Most studies used cancer cells in vitro with the goal of modifying their phenotype by changing miRNA expression profiles. In vivo studies evaluated regulation of miRNAs by chemopreventive agents in organs of mice and rats, either untreated or exposed to carcinogens, with the objective of evaluating their safety and efficacy. The tissue specificity of miRNAs could be exploited for the chemoprevention of site-specific cancers, and the study of polymorphic miRNAs is expected to predict the individual response to chemopreventive agents as a tool for developing new prevention strategies. PMID:22683846

Detection of a Putative TetR-Like Gene Related to Mycobacterium bovis BCG Growth in Cholesterol Using a gfp-Transposon Mutagenesis System

PubMed Central

Otal, Isabel; Pérez-Herrán, Esther; Garcia-Morales, Lazaro; Menéndez, María C.; Gonzalez-y-Merchand, Jorge A.; Martín, Carlos; García, María J.

2017-01-01

In vitro transposition is a powerful genetic tool for identifying mycobacterial virulence genes and studying virulence factors in relation to the host. Transposon shuttle mutagenesis is a method for constructing stable insertions in the genome of different microorganisms including mycobacteria. Using an IS1096 derivative, we have constructed the Tngfp, a transposon containing a promoterless green fluorescent protein (gfp) gene. This transposon was able to transpose randomly in Mycobacterium bovis BCG. Bacteria with a single copy of the gfp gene per chromosome from an M. bovis BCG::Tngfp library were analyzed and cells exhibiting high levels of fluorescence were detected by flow cytometry. Application of this approach allowed for the selection of a mutant, BCG_2177c::Tngfp (BCG-Tn), on the basis of high level of long-standing fluorescence at stationary phase. This BCG-Tn mutant showed some particular phenotypic features compared to the wild type strain, mainly during stationary phase, when cholesterol was used as a sole carbon source, thus supporting the relationships of the targeted gene with the regulation of cholesterol metabolism in this bacteria. This approach showed that Tngfp is a potentially useful tool for studying the involvement of the targeted loci in metabolic pathways of mycobacteria. PMID:28321208

Genome-wide identification of bacterial plant colonization genes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cole, Benjamin J.; Feltcher, Meghan E.; Waters, Robert J.

Diverse soil-resident bacteria can contribute to plant growth and health, but the molecular mechanisms enabling them to effectively colonize their plant hosts remain poorly understood. We used randomly barcoded transposon mutagenesis sequencing (RB-TnSeq) in Pseudomonas simiae, a model root-colonizing bacterium, to establish a genome-wide map of bacterial genes required for colonization of the Arabidopsis thaliana root system. We identified 115 genes (2% of all P. simiae genes) with functions that are required for maximal competitive colonization of the root system. Among the genes we identified were some with obvious colonization-related roles in motility and carbon metabolism, as well as 44more » other genes that had no or vague functional predictions. Independent validation assays of individual genes confirmed colonization functions for 20 of 22 (91%) cases tested. To further characterize genes identified by our screen, we compared the functional contributions of P. simiae genes to growth in 90 distinct in vitro conditions by RB-TnSeq, highlighting specific metabolic functions associated with root colonization genes. Here, our analysis of bacterial genes by sequence-driven saturation mutagenesis revealed a genome-wide map of the genetic determinants of plant root colonization and offers a starting point for targeted improvement of the colonization capabilities of plant-beneficial microbes.« less

Evaluation of Glucose Dehydrogenase and Pyrroloquinoline Quinine (pqq) Mutagenesis that Renders Functional Inadequacies in Host Plants.

PubMed

Naveed, Muhammad; Sohail, Younas; Khalid, Nauman; Ahmed, Iftikhar; Mumtaz, Abdul Samad

2015-08-01

The rhizospheric zone abutting plant roots usually clutches a wealth of microbes. In the recent past, enormous genetic resources have been excavated with potential applications in host plant interaction and ancillary aspects. Two Pseudomonas strains were isolated and identified through 16S rRNA and rpoD sequence analyses as P. fluorescens QAU67 and P. putida QAU90. Initial biochemical characterization and their root-colonizing traits indicated their potential role in plant growth promotion. Such aerobic systems, involved in gluconic acid production and phosphate solubilization, essentially require the pyrroloquinoline quinine (PQQ)- dependent glucose dehydrogenase (GDH) in the genome. The PCR screening and amplification of GDH and PQQ and subsequent induction of mutagenesis characterized their possible role as antioxidants as well as in growth promotion, as probed in vitro in lettuce and in vivo in rice, bean, and tomato plants. The results showed significant differences (p < or = 0.05) in parameters of plant height, fresh weight, and dry weight, etc., deciphering a clear and in fact complementary role of GDH and PQQ in plant growth promotion. Our study not only provides direct evidence of the in vivo role of GDH and PQQ in host plants but also reveals their functional inadequacy in the event of mutation at either of these loci.

Genome-wide identification of bacterial plant colonization genes

DOE PAGES

Cole, Benjamin J.; Feltcher, Meghan E.; Waters, Robert J.; ...

2017-09-22

Diverse soil-resident bacteria can contribute to plant growth and health, but the molecular mechanisms enabling them to effectively colonize their plant hosts remain poorly understood. We used randomly barcoded transposon mutagenesis sequencing (RB-TnSeq) in Pseudomonas simiae, a model root-colonizing bacterium, to establish a genome-wide map of bacterial genes required for colonization of the Arabidopsis thaliana root system. We identified 115 genes (2% of all P. simiae genes) with functions that are required for maximal competitive colonization of the root system. Among the genes we identified were some with obvious colonization-related roles in motility and carbon metabolism, as well as 44more » other genes that had no or vague functional predictions. Independent validation assays of individual genes confirmed colonization functions for 20 of 22 (91%) cases tested. To further characterize genes identified by our screen, we compared the functional contributions of P. simiae genes to growth in 90 distinct in vitro conditions by RB-TnSeq, highlighting specific metabolic functions associated with root colonization genes. Here, our analysis of bacterial genes by sequence-driven saturation mutagenesis revealed a genome-wide map of the genetic determinants of plant root colonization and offers a starting point for targeted improvement of the colonization capabilities of plant-beneficial microbes.« less

AXM mutagenesis: an efficient means for the production of libraries for directed evolution of proteins.

PubMed

Holland, Erika G; Buhr, Diane L; Acca, Felicity E; Alderman, Dawn; Bovat, Kristin; Busygina, Valeria; Kay, Brian K; Weiner, Michael P; Kiss, Margaret M

2013-08-30

Affinity maturation is an important part of the recombinant antibody development process. There are several well-established approaches for generating libraries of mutated antibody genes for affinity maturation, but these approaches are generally too laborious or expensive to allow high-throughput, parallel processing of multiple antibodies. Here, we describe a scalable approach that enables the generation of libraries with greater than 10(8) clones from a single Escherichia coli transformation. In our method, a mutated DNA fragment is produced using PCR conditions that promote nucleotide misincorporation into newly synthesized DNA. In the PCR reaction, one of the primers contains at least three phosphorothioate linkages at its 5' end, and treatment of the PCR product with a 5' to 3' exonuclease is used to preferentially remove the strand synthesized with the non-modified primer, resulting in a single-stranded DNA fragment. This fragment then serves as a megaprimer to prime DNA synthesis on a uracilated, circular, single-stranded template in a Kunkel-like mutagenesis reaction that biases nucleotide base-changes between the megaprimer and uracilated DNA sequence in favor of the in vitro synthesized megaprimer. This method eliminates the inefficient subcloning steps that are normally required for the construction of affinity maturation libraries from randomly mutagenized antibody genes. Copyright © 2013. Published by Elsevier B.V.

Tol2 transposon-mediated transgenesis in Xenopus tropicalis.

PubMed

Hamlet, Michelle R Johnson; Yergeau, Donald A; Kuliyev, Emin; Takeda, Masatoshi; Taira, Masanori; Kawakami, Koichi; Mead, Paul E

2006-09-01

The diploid frog Xenopus tropicalis is becoming a powerful developmental genetic model system. Sequencing of the X. tropicalis genome is nearing completion and several labs are embarking on mutagenesis screens. We are interested in developing insertional mutagenesis strategies in X. tropicalis. Transposon-mediated insertional mutagenesis, once used exclusively in plants and invertebrate systems, is now more widely applicable to vertebrates. The first step in developing transposons as tools for mutagenesis is to demonstrate that these mobile elements function efficiently in the target organism. Here, we show that the Medaka fish transposon, Tol2, is able to stably integrate into the X. tropicalis genome and will serve as a powerful tool for insertional mutagenesis strategies in the frog.

Fluorescence-Based Reporters for Detection of Mutagenesis in E. coli

PubMed Central

Standley, Melissa; Allen, Jennifer; Cervantes, Layla; Lilly, Joshua; Camps, Manel

2017-01-01

Mutagenesis in model organisms following exposure to chemicals is used as an indicator of genotoxicity. Mutagenesis assays are also used to study mechanisms of DNA homeostasis. The present article focuses on detection of mutagenesis in prokaryotes, which boils down to two approaches: reporter inactivation (forward mutation assay) and reversion of an inactivating mutation (reversion mutation assay). Both methods are labor-intensive, involving visual screening, quantification of colonies on solid media, or determining a Poisson distribution in liquid culture. Here we present two reversion reporters for in vivo mutagenesis that produce a quantitative output, and thus have the potential to greatly reduce the amount of test chemical and labor involved in these assays. This output is obtained by coupling a TEM β lactamase-based reversion assay with GFP fluorescence, either by placing the two genes on the same plasmid or by fusing them translationally and interrupting the N-terminus of the ORF with a stop codon. We also describe a reporter aimed at facilitating the monitoring of continuous mutagenesis in mutator strains. This reporter couples two reversion markers, allowing the temporal separation of mutation events in time, thus providing information about the dynamics of mutagenesis in mutator strains. Here, we describe these reporter systems, provide protocols for use, and demonstrate their key functional features using error-prone Pol I mutagenesis as a source of mutations. PMID:28645368

Multiple pathways for SOS-induced mutagenesis in Escherichia coli: An overexpression of dinB/dinP results in strongly enhancing mutagenesis in the absence of any exogenous treatment to damage DNA

PubMed Central

Kim, Su-Ryang; Maenhaut-Michel, Geneviéve; Yamada, Masami; Yamamoto, Yoshihiro; Matsui, Keiko; Sofuni, Toshio; Nohmi, Takehiko; Ohmori, Haruo

1997-01-01

dinP is an Escherichia coli gene recently identified at 5.5 min of the genetic map, whose product shows a similarity in amino acid sequence to the E. coli UmuC protein involved in DNA damage-induced mutagenesis. In this paper we show that the gene is identical to dinB, an SOS gene previously localized near the lac locus at 8 min, the function of which was shown to be required for mutagenesis of nonirradiated λ phage infecting UV-preirradiated bacterial cells (termed λUTM for λ untargeted mutagenesis). A newly constructed dinP null mutant exhibited the same defect for λUTM as observed previously with a dinB::Mu mutant, and the defect was complemented by plasmids carrying dinP as the only intact bacterial gene. Furthermore, merely increasing the dinP gene expression, without UV irradiation or any other DNA-damaging treatment, resulted in a strong enhancement of mutagenesis in F′lac plasmids; at most, 800-fold increase in the G6-to-G5 change. The enhanced mutagenesis did not depend on recA, uvrA, or umuDC. Thus, our results establish that E. coli has at least two distinct pathways for SOS-induced mutagenesis: one dependent on umuDC and the other on dinB/P. PMID:9391106

Fluorescence-Based Reporters for Detection of Mutagenesis in E. coli.

PubMed

Standley, Melissa; Allen, Jennifer; Cervantes, Layla; Lilly, Joshua; Camps, Manel

2017-01-01

Mutagenesis in model organisms following exposure to chemicals is used as an indicator of genotoxicity. Mutagenesis assays are also used to study mechanisms of DNA homeostasis. This chapter focuses on detection of mutagenesis in prokaryotes, which boils down to two approaches: reporter inactivation (forward mutation assay) and reversion of an inactivating mutation (reversion mutation assay). Both methods are labor intensive, involving visual screening, quantification of colonies on solid media, or determining a Poisson distribution in liquid culture. Here, we present two reversion reporters for in vivo mutagenesis that produce a quantitative output, and thus have the potential to greatly reduce the amount of test chemical and labor involved in these assays. This output is obtained by coupling a TEM β lactamase-based reversion assay with GFP fluorescence, either by placing the two genes on the same plasmid or by fusing them translationally and interrupting the N-terminus of the chimeric ORF with a stop codon. We also describe a reporter aimed at facilitating the monitoring of continuous mutagenesis in mutator strains. This reporter couples two reversion markers, allowing the temporal separation of mutation events in time, thus providing information about the dynamics of mutagenesis in mutator strains. Here, we describe these reporter systems, provide protocols for use, and demonstrate their key functional features using error-prone Pol I mutagenesis as a source of mutations. © 2017 Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yoon, Bohyun; Kim, Inki; Nam, Ja-Ae

EFC-1 integrase is a site-specific recombinase that belongs to the large family of serine recombinase. In previously study, we isolated the temperate phage EFC-1, and characterized its genomic sequence. Within its genome, Orf28 was predicted encode a 464 amino acid of a putative integrase gene. In this study, EFC-1 integrase was characterized in vitro and in vivo. In vitro assay was performed using purified His-tag fusion integrase. Also, to identify which serine is involved in the catalytic domain, we used site-directed mutagenesis and analyzed by a recombination assay in vitro. In vivo assay involved PCR and confocal microscopy in HEK293 cells, and determined the minimal lengthsmore » of attP and attB sites. According to our results, the EFC-1 integrase-mediated recombination was site-specific and unidirectional system in vitro and in vivo. Serine 21 of EFC-1 integrase plays a major role in the catalytic domain, and minimal sizes of attB and attP was defined 48 and 54 bp. Our finding may help develop a useful tool for gene therapy and gene delivery system. - Highlights: • EFC-1 integrase-mediated recombination was site-specific and unidirectional system. • Serine 21 of EFC-1 integrase plays a major role in the catalytic domain. • The functional minimal sizes of attB and attP was defined 48 and 54 bp.« less

Genome-Wide Screens Reveal New Gene Products That Influence Genetic Competence in Streptococcus mutans

PubMed Central

O'Brien, Greg; Maricic, Natalie; Kesterson, Alexandria; Grace, Megan

2017-01-01

ABSTRACT A network of genes and at least two peptide signaling molecules tightly control when Streptococcus mutans becomes competent to take up DNA from its environment. Widespread changes in the expression of genes occur when S. mutans is presented with competence signal peptides in vitro, including the increased production of the alternative sigma factor, ComX, which activates late competence genes. Still, the way that gene products that are regulated by competence peptides influence DNA uptake and cellular physiology are not well understood. Here, we developed and employed comprehensive transposon mutagenesis of the S. mutans genome, with a screen to identify mutants that aberrantly expressed comX, coupled with transposon sequencing (Tn-seq) to gain a more thorough understanding of the factors modulating comX expression and progression to the competent state. The screens effectively identified genes known to affect competence, e.g., comR, comS, comD, comE, cipB, clpX, rcrR, and ciaH, but disclosed an additional 20 genes that were not previously competence associated. The competence phenotypes of mutants were characterized, including by fluorescence microscopy to determine at which stage the mutants were impaired for comX activation. Among the novel genes studied were those implicated in cell division, the sensing of cell envelope stress, cell envelope biogenesis, and RNA stability. Our results provide a platform for determining the specific chemical and physical cues that are required for genetic competence in S. mutans, while highlighting the effectiveness of using Tn-seq in S. mutans to discover and study novel biological processes. IMPORTANCE Streptococcus mutans acquires DNA from its environment by becoming genetically competent, a physiologic state triggered by cell-cell communication using secreted peptides. Competence is important for acquiring novel genetic traits and has a strong influence on the expression of virulence-associated traits of S. mutans. Here, we used transposon mutagenesis and genomic technologies to identify novel genes involved in competence development. In addition to identifying genes previously known to be required for comX expression, 20 additional genes were identified and characterized. The findings create opportunities to diminish the pathogenic potential of S. mutans, while validating technologies that can rapidly advance our understanding of the physiology, biology, and genetics of S. mutans and related pathogens. PMID:29109185

Genome-wide screens reveal new gene products that influence genetic competence in Streptococcus mutans.

PubMed

Shields, Robert C; O'Brien, Greg; Maricic, Natalie; Kesterson, Alexandria; Grace, Megan; Hagen, Stephen J; Burne, Robert A

2017-11-06

A network of genes and at least two peptide signaling molecules tightly control when Streptococcus mutans becomes competent to take up DNA from its environment. Widespread changes in the expression of genes occur when S. mutans is presented with competence signal peptides in vitro , including increased production of the alternative sigma factor, ComX, which activates late competence genes. Still, the way that gene products that are regulated by competence peptides influence DNA uptake and cellular physiology are not well understood. Here, we developed and employed comprehensive transposon mutagenesis of the S. mutans genome with a screen to identify mutants that aberrantly expressed comX , coupled with transposon sequencing (Tn-seq) to gain a more thorough understanding of the factors modulating comX expression and progression to the competent state. The screens effectively identified genes known to affect competence, e.g. comR , comS , comD , comE , cipB , clpX , rcrR , ciaH , but disclosed an additional 20 genes that were not previously competence-associated. The competence phenotypes of mutants were characterized, including using fluorescence microscopy to determine at which stage the mutants were impaired for comX activation. Among the novel genes studied were those implicated in cell division, sensing of cell envelope stress, cell envelope biogenesis, and RNA stability. Our results provide a platform for determining the specific chemical and physical cues that are required for genetic competence in S. mutans , while highlighting the effectiveness of using Tn-seq in S. mutans to discover and study novel biological processes. IMPORTANCE Streptococcus mutans acquires DNA from its environment by becoming genetically competent, a physiologic state triggered by cell-cell communication using secreted peptides. Competence is important for acquiring novel genetic traits and has a strong influence on the expression of virulence-associated traits of S. mutans Here, we used transposon mutagenesis and genomic technologies to identify novel genes involved in competence development. In addition to identifying genes previously known to be required for comX expression, 20 additional genes were identified and characterized. The findings create opportunities to diminish the pathogenic potential of S. mutans , while validating technologies that can rapidly advance our understanding of the physiology, biology and genetics of S. mutans and related pathogens. Copyright © 2017 American Society for Microbiology.

Efficient mutagenesis by Cas9 protein-mediated oligonucleotide insertion and large-scale assessment of single-guide RNAs.

PubMed

Gagnon, James A; Valen, Eivind; Thyme, Summer B; Huang, Peng; Akhmetova, Laila; Ahkmetova, Laila; Pauli, Andrea; Montague, Tessa G; Zimmerman, Steven; Richter, Constance; Schier, Alexander F

2014-01-01

The CRISPR/Cas9 system has been implemented in a variety of model organisms to mediate site-directed mutagenesis. A wide range of mutation rates has been reported, but at a limited number of genomic target sites. To uncover the rules that govern effective Cas9-mediated mutagenesis in zebrafish, we targeted over a hundred genomic loci for mutagenesis using a streamlined and cloning-free method. We generated mutations in 85% of target genes with mutation rates varying across several orders of magnitude, and identified sequence composition rules that influence mutagenesis. We increased rates of mutagenesis by implementing several novel approaches. The activities of poor or unsuccessful single-guide RNAs (sgRNAs) initiating with a 5' adenine were improved by rescuing 5' end homogeneity of the sgRNA. In some cases, direct injection of Cas9 protein/sgRNA complex further increased mutagenic activity. We also observed that low diversity of mutant alleles led to repeated failure to obtain frame-shift mutations. This limitation was overcome by knock-in of a stop codon cassette that ensured coding frame truncation. Our improved methods and detailed protocols make Cas9-mediated mutagenesis an attractive approach for labs of all sizes.

Multiplex Conditional Mutagenesis Using Transgenic Expression of Cas9 and sgRNAs.

PubMed

Yin, Linlin; Maddison, Lisette A; Li, Mingyu; Kara, Nergis; LaFave, Matthew C; Varshney, Gaurav K; Burgess, Shawn M; Patton, James G; Chen, Wenbiao

2015-06-01

Determining the mechanism of gene function is greatly enhanced using conditional mutagenesis. However, generating engineered conditional alleles is inefficient and has only been widely used in mice. Importantly, multiplex conditional mutagenesis requires extensive breeding. Here we demonstrate a system for one-generation multiplex conditional mutagenesis in zebrafish (Danio rerio) using transgenic expression of both cas9 and multiple single guide RNAs (sgRNAs). We describe five distinct zebrafish U6 promoters for sgRNA expression and demonstrate efficient multiplex biallelic inactivation of tyrosinase and insulin receptor a and b, resulting in defects in pigmentation and glucose homeostasis. Furthermore, we demonstrate temporal and tissue-specific mutagenesis using transgenic expression of Cas9. Heat-shock-inducible expression of cas9 allows temporal control of tyr mutagenesis. Liver-specific expression of cas9 disrupts insulin receptor a and b, causing fasting hypoglycemia and postprandial hyperglycemia. We also show that delivery of sgRNAs targeting ascl1a into the eye leads to impaired damage-induced photoreceptor regeneration. Our findings suggest that CRISPR/Cas9-based conditional mutagenesis in zebrafish is not only feasible but rapid and straightforward. Copyright © 2015 by the Genetics Society of America.

Cancer CRISPR Screens In Vivo.

PubMed

Chow, Ryan D; Chen, Sidi

2018-05-01

Clustered regularly interspaced short palindromic repeats (CRISPR) screening is a powerful toolset for investigating diverse biological processes. Most CRISPR screens to date have been performed with in vitro cultures or cellular transplant models. To interrogate cancer in animal models that more closely recapitulate the human disease, autochthonous direct in vivo CRISPR screens have recently been developed that can identify causative drivers in the native tissue microenvironment. By empowering multiplexed mutagenesis in fully immunocompetent animals, direct in vivo CRISPR screens enable the rapid generation of patient-specific avatars that can guide precision medicine. This Opinion article discusses the current status of in vivo CRISPR screens in cancer and offers perspectives on future applications. Copyright © 2018 Elsevier Inc. All rights reserved.

Deciphering the Code of the Cancer Genome: Mechanisms of Chromosome Rearrangement

PubMed Central

Willis, Nicholas A.; Rass, Emilie; Scully, Ralph

2015-01-01

Chromosome rearrangement plays a causal role in tumorigenesis by contributing to the inactivation of tumor suppressor genes, the dysregulated expression or amplification of oncogenes and the generation of novel gene fusions. Chromosome breaks are important intermediates in this process. How, when and where these breaks arise and the specific mechanisms engaged in their repair strongly influence the resulting patterns of chromosome rearrangement. Here, we review recent progress in understanding how certain distinctive features of the cancer genome, including clustered mutagenesis, tandem segmental duplications, complex breakpoints, chromothripsis, chromoplexy and chromoanasynthesis may arise. PMID:26726318

Pacific Northwest Laboratory annual report for 1984 to the DOE Office of Energy Research. Part 1. Biomedical sciences

DOE Office of Scientific and Technical Information (OSTI.GOV)

Felton, D.L.

1985-02-01

Research progress is reported in the following areas: (1) evaluation of possible health effects among nuclear workers; (2) dose-effect relationship studies of carcinogenesis from both nuclear materials and complex mixtures; (3) microbial mutagenesis studies with 6-aminochrysene and benzo(a)pyrene in coal-derived complex mixtures; and (4) a variety of studies relating to noncarcinogenic and nonmutagenic endpoints, including teratology, perinatal studies and studies to determine absorption, metabolism, and doses to critical tissues and organs of coal-derived mixtures and radionuclides. Items have been individually abstracted for the data base. (ACR)

Cholinesterase structure: Identification of residues and domains affecting organophosphate inhibition and catalysis. Annual report, 6 March 1995-5 March 1996

DOE Office of Scientific and Technical Information (OSTI.GOV)

Taylor, P.W.

1996-04-01

In the initial year of the grant, we have made excellent progress in several arenas: (1) A crystal structure of a mouse acetylcholinesterase-fasciculin 2 complex has been solved. (2) Studies with enantiomeric organophosphates have yielded vital information on their binding orientation in the ground and transition states. (3) Studies in oxime reactivation of inhibited cholinesterase have uncovered the basis for enhanced reactivity of HI-6 compared to 2-PAM. (4) The interactions of fasciculin 2 with acetylcholinesterase have been studied by kinetic and site-specific mutagenesis methods.

Stress Sensors and Signal Transducers in Cyanobacteria

PubMed Central

Los, Dmitry A.; Zorina, Anna; Sinetova, Maria; Kryazhov, Sergey; Mironov, Kirill; Zinchenko, Vladislav V.

2010-01-01

In living cells, the perception of environmental stress and the subsequent transduction of stress signals are primary events in the acclimation to changes in the environment. Some molecular sensors and transducers of environmental stress cannot be identified by traditional and conventional methods. Based on genomic information, a systematic approach has been applied to the solution of this problem in cyanobacteria, involving mutagenesis of potential sensors and signal transducers in combination with DNA microarray analyses for the genome-wide expression of genes. Forty-five genes for the histidine kinases (Hiks), 12 genes for serine-threonine protein kinases (Spks), 42 genes for response regulators (Rres), seven genes for RNA polymerase sigma factors, and nearly 70 genes for transcription factors have been successfully inactivated by targeted mutagenesis in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Screening of mutant libraries by genome-wide DNA microarray analysis under various stress and non-stress conditions has allowed identification of proteins that perceive and transduce signals of environmental stress. Here we summarize recent progress in the identification of sensory and regulatory systems, including Hiks, Rres, Spks, sigma factors, transcription factors, and the role of genomic DNA supercoiling in the regulation of the responses of cyanobacterial cells to various types of stress. PMID:22294932

Mutation breeding of ornamental plants using ion beams.

PubMed

Yamaguchi, Hiroyasu

2018-01-01

Ornamental plants that have a rich variety of flower colors and shapes are highly prized in the commercial flower market, and therefore, mutant cultivars that produce different types of flowers while retaining their growth habits are in demand. Furthermore, mutation breeding is well suited for ornamental plants because many species can be easily vegetatively propagated, facilitating the production of spontaneous and induced mutants. The use of ion beams in mutation breeding has rapidly expanded since the 1990s in Japan, with the prospect that more ion beam-specific mutants will be generated. There are currently four irradiation facilities in Japan that provide ion beam irradiation for plant materials. The development of mutant cultivars using ion beams has been attempted on many ornamental plants thus far, and some species have been used to investigate the process of mutagenesis. In addition, progress is being made in clarifying the genetic mechanism for expressing important traits, which will probably result in the development of more efficient mutation breeding methods for ornamental plants. This review not only provides examples of successful mutation breeding results using ion beams, but it also describes research on mutagenesis and compares results of ion beam and gamma ray breeding using ornamental plants.

Study of the genetics and regulation of methane oxidation. Progress report, second year and a half, August 1, 1981-January 31, 1983

DOE Office of Scientific and Technical Information (OSTI.GOV)

Not Available

The purpose is to develop mutagenesis, gene transfer and cloning systems in methanotrophic bacteria, and use these techniques to study the methane oxidation genes. Although we have been successful in the first part of these objectives, the study of methane oxidation genes has proven difficult. Problems arose due to the discovery that the culture, Methylobacterium ethanolicum, is in reality a stable coculture between two methylotrophs. These partners are Methylocystis POC, an obligate methanotroph and Xanthobacter H4.14, and autotrophic methanolutilizer. The Methylocystis strain contains the three plasmids we had observed previously in methane-grown cultures, while the Xanthobacter strain contains no detectiblemore » plasmids. Therefore, our original approach to studying the methane oxidation genes, that of isolating plasmid mutants, is no longer valid. However, our discovery of the nature of this culture has led to some interesting results which show promise in elucidating the genetic structure of the methane oxidation genes in obligate methanotrophs. In addition, we have been successful in developing mutagenesis, gene transfer and cloning systems that are applicable to a wide variety of methanotrophs.« less

Mutation breeding of ornamental plants using ion beams

PubMed Central

Yamaguchi, Hiroyasu

2018-01-01

Ornamental plants that have a rich variety of flower colors and shapes are highly prized in the commercial flower market, and therefore, mutant cultivars that produce different types of flowers while retaining their growth habits are in demand. Furthermore, mutation breeding is well suited for ornamental plants because many species can be easily vegetatively propagated, facilitating the production of spontaneous and induced mutants. The use of ion beams in mutation breeding has rapidly expanded since the 1990s in Japan, with the prospect that more ion beam-specific mutants will be generated. There are currently four irradiation facilities in Japan that provide ion beam irradiation for plant materials. The development of mutant cultivars using ion beams has been attempted on many ornamental plants thus far, and some species have been used to investigate the process of mutagenesis. In addition, progress is being made in clarifying the genetic mechanism for expressing important traits, which will probably result in the development of more efficient mutation breeding methods for ornamental plants. This review not only provides examples of successful mutation breeding results using ion beams, but it also describes research on mutagenesis and compares results of ion beam and gamma ray breeding using ornamental plants. PMID:29681749

Sleeping Beauty transposon mutagenesis identifies genes that cooperate with mutant Smad4 in gastric cancer development

PubMed Central

Takeda, Haruna; Rust, Alistair G.; Ward, Jerrold M.; Yew, Christopher Chin Kuan; Jenkins, Nancy A.; Copeland, Neal G.

2016-01-01

Mutations in SMAD4 predispose to the development of gastrointestinal cancer, which is the third leading cause of cancer-related deaths. To identify genes driving gastric cancer (GC) development, we performed a Sleeping Beauty (SB) transposon mutagenesis screen in the stomach of Smad4+/− mutant mice. This screen identified 59 candidate GC trunk drivers and a much larger number of candidate GC progression genes. Strikingly, 22 SB-identified trunk drivers are known or candidate cancer genes, whereas four SB-identified trunk drivers, including PTEN, SMAD4, RNF43, and NF1, are known human GC trunk drivers. Similar to human GC, pathway analyses identified WNT, TGF-β, and PI3K-PTEN signaling, ubiquitin-mediated proteolysis, adherens junctions, and RNA degradation in addition to genes involved in chromatin modification and organization as highly deregulated pathways in GC. Comparative oncogenomic filtering of the complete list of SB-identified genes showed that they are highly enriched for genes mutated in human GC and identified many candidate human GC genes. Finally, by comparing our complete list of SB-identified genes against the list of mutated genes identified in five large-scale human GC sequencing studies, we identified LDL receptor-related protein 1B (LRP1B) as a previously unidentified human candidate GC tumor suppressor gene. In LRP1B, 129 mutations were found in 462 human GC samples sequenced, and LRP1B is one of the top 10 most deleted genes identified in a panel of 3,312 human cancers. SB mutagenesis has, thus, helped to catalog the cooperative molecular mechanisms driving SMAD4-induced GC growth and discover genes with potential clinical importance in human GC. PMID:27006499

Sleeping Beauty transposon mutagenesis identifies genes that cooperate with mutant Smad4 in gastric cancer development.

PubMed

Takeda, Haruna; Rust, Alistair G; Ward, Jerrold M; Yew, Christopher Chin Kuan; Jenkins, Nancy A; Copeland, Neal G

2016-04-05

Mutations in SMAD4 predispose to the development of gastrointestinal cancer, which is the third leading cause of cancer-related deaths. To identify genes driving gastric cancer (GC) development, we performed a Sleeping Beauty (SB) transposon mutagenesis screen in the stomach of Smad4(+/-) mutant mice. This screen identified 59 candidate GC trunk drivers and a much larger number of candidate GC progression genes. Strikingly, 22 SB-identified trunk drivers are known or candidate cancer genes, whereas four SB-identified trunk drivers, including PTEN, SMAD4, RNF43, and NF1, are known human GC trunk drivers. Similar to human GC, pathway analyses identified WNT, TGF-β, and PI3K-PTEN signaling, ubiquitin-mediated proteolysis, adherens junctions, and RNA degradation in addition to genes involved in chromatin modification and organization as highly deregulated pathways in GC. Comparative oncogenomic filtering of the complete list of SB-identified genes showed that they are highly enriched for genes mutated in human GC and identified many candidate human GC genes. Finally, by comparing our complete list of SB-identified genes against the list of mutated genes identified in five large-scale human GC sequencing studies, we identified LDL receptor-related protein 1B (LRP1B) as a previously unidentified human candidate GC tumor suppressor gene. In LRP1B, 129 mutations were found in 462 human GC samples sequenced, and LRP1B is one of the top 10 most deleted genes identified in a panel of 3,312 human cancers. SB mutagenesis has, thus, helped to catalog the cooperative molecular mechanisms driving SMAD4-induced GC growth and discover genes with potential clinical importance in human GC.

Mutagenesis: Interactions with a parallel universe.

PubMed

Miller, Jeffrey H

Unexpected observations in mutagenesis research have led to a new perspective in this personal reflection based on years of studying mutagenesis. Many mutagens have been thought to operate via a single principal mechanism, with secondary effects usually resulting in only minor changes in the observed mutation frequencies and spectra. For example, we conceive of base analogs as resulting in direct mispairing as their main mechanism of mutagenesis. Recent studies now show that in fact even these simple mutagens can cause very large and unanticipated effects both in mutation frequencies and in the mutational spectra when used in certain pair-wise combinations. Here we characterize this leap in mutation frequencies as a transport to an alternate universe of mutagenesis. Copyright © 2018 Elsevier B.V. All rights reserved.

Evolving artificial metalloenzymes via random mutagenesis

NASA Astrophysics Data System (ADS)

Yang, Hao; Swartz, Alan M.; Park, Hyun June; Srivastava, Poonam; Ellis-Guardiola, Ken; Upp, David M.; Lee, Gihoon; Belsare, Ketaki; Gu, Yifan; Zhang, Chen; Moellering, Raymond E.; Lewis, Jared C.

2018-03-01

Random mutagenesis has the potential to optimize the efficiency and selectivity of protein catalysts without requiring detailed knowledge of protein structure; however, introducing synthetic metal cofactors complicates the expression and screening of enzyme libraries, and activity arising from free cofactor must be eliminated. Here we report an efficient platform to create and screen libraries of artificial metalloenzymes (ArMs) via random mutagenesis, which we use to evolve highly selective dirhodium cyclopropanases. Error-prone PCR and combinatorial codon mutagenesis enabled multiplexed analysis of random mutations, including at sites distal to the putative ArM active site that are difficult to identify using targeted mutagenesis approaches. Variants that exhibited significantly improved selectivity for each of the cyclopropane product enantiomers were identified, and higher activity than previously reported ArM cyclopropanases obtained via targeted mutagenesis was also observed. This improved selectivity carried over to other dirhodium-catalysed transformations, including N-H, S-H and Si-H insertion, demonstrating that ArMs evolved for one reaction can serve as starting points to evolve catalysts for others.

Effect of SOS-induced levels of imuABC on spontaneous and damage-induced mutagenesis in Caulobacter crescentus.

PubMed

Alves, Ingrid R; Lima-Noronha, Marco A; Silva, Larissa G; Fernández-Silva, Frank S; Freitas, Aline Luiza D; Marques, Marilis V; Galhardo, Rodrigo S

2017-11-01

imuABC (imuAB dnaE2) genes are responsible for SOS-mutagenesis in Caulobacter crescentus and other bacterial species devoid of umuDC. In this work, we have constructed operator-constitutive mutants of the imuABC operon. We used this genetic tool to investigate the effect of SOS-induced levels of these genes upon both spontaneous and damage-induced mutagenesis. We showed that constitutive expression of imuABC does not increase spontaneous or damage-induced mutagenesis, nor increases cellular resistance to DNA-damaging agents. Nevertheless, the presence of the operator-constitutive mutation rescues mutagenesis in a recA background, indicating that imuABC are the only genes required at SOS-induced levels for translesion synthesis (TLS) in C. crescentus. Furthermore, these data also show that TLS mediated by ImuABC does not require RecA, unlike umuDC-dependent mutagenesis in E. coli. Copyright © 2017 Elsevier B.V. All rights reserved.

One-Tube-Only Standardized Site-Directed Mutagenesis: An Alternative Approach to Generate Amino Acid Substitution Collections

PubMed Central

Mingo, Janire; Erramuzpe, Asier; Luna, Sandra; Aurtenetxe, Olaia; Amo, Laura; Diez, Ibai; Schepens, Jan T. G.; Hendriks, Wiljan J. A. J.; Cortés, Jesús M.; Pulido, Rafael

2016-01-01

Site-directed mutagenesis (SDM) is a powerful tool to create defined collections of protein variants for experimental and clinical purposes, but effectiveness is compromised when a large number of mutations is required. We present here a one-tube-only standardized SDM approach that generates comprehensive collections of amino acid substitution variants, including scanning- and single site-multiple mutations. The approach combines unified mutagenic primer design with the mixing of multiple distinct primer pairs and/or plasmid templates to increase the yield of a single inverse-PCR mutagenesis reaction. Also, a user-friendly program for automatic design of standardized primers for Ala-scanning mutagenesis is made available. Experimental results were compared with a modeling approach together with stochastic simulation data. For single site-multiple mutagenesis purposes and for simultaneous mutagenesis in different plasmid backgrounds, combination of primer sets and/or plasmid templates in a single reaction tube yielded the distinct mutations in a stochastic fashion. For scanning mutagenesis, we found that a combination of overlapping primer sets in a single PCR reaction allowed the yield of different individual mutations, although this yield did not necessarily follow a stochastic trend. Double mutants were generated when the overlap of primer pairs was below 60%. Our results illustrate that one-tube-only SDM effectively reduces the number of reactions required in large-scale mutagenesis strategies, facilitating the generation of comprehensive collections of protein variants suitable for functional analysis. PMID:27548698

Random mutagenesis by error-prone pol plasmid replication in Escherichia coli.

PubMed

Alexander, David L; Lilly, Joshua; Hernandez, Jaime; Romsdahl, Jillian; Troll, Christopher J; Camps, Manel

2014-01-01

Directed evolution is an approach that mimics natural evolution in the laboratory with the goal of modifying existing enzymatic activities or of generating new ones. The identification of mutants with desired properties involves the generation of genetic diversity coupled with a functional selection or screen. Genetic diversity can be generated using PCR or using in vivo methods such as chemical mutagenesis or error-prone replication of the desired sequence in a mutator strain. In vivo mutagenesis methods facilitate iterative selection because they do not require cloning, but generally produce a low mutation density with mutations not restricted to specific genes or areas within a gene. For this reason, this approach is typically used to generate new biochemical properties when large numbers of mutants can be screened or selected. Here we describe protocols for an advanced in vivo mutagenesis method that is based on error-prone replication of a ColE1 plasmid bearing the gene of interest. Compared to other in vivo mutagenesis methods, this plasmid-targeted approach allows increased mutation loads and facilitates iterative selection approaches. We also describe the mutation spectrum for this mutagenesis methodology in detail, and, using cycle 3 GFP as a target for mutagenesis, we illustrate the phenotypic diversity that can be generated using our method. In sum, error-prone Pol I replication is a mutagenesis method that is ideally suited for the evolution of new biochemical activities when a functional selection is available.

Environmental stress induces trinucleotide repeat mutagenesis in human cells

PubMed Central

Chatterjee, Nimrat; Lin, Yunfu; Santillan, Beatriz A.; Yotnda, Patricia; Wilson, John H.

2015-01-01

The dynamic mutability of microsatellite repeats is implicated in the modification of gene function and disease phenotype. Studies of the enhanced instability of long trinucleotide repeats (TNRs)—the cause of multiple human diseases—have revealed a remarkable complexity of mutagenic mechanisms. Here, we show that cold, heat, hypoxic, and oxidative stresses induce mutagenesis of a long CAG repeat tract in human cells. We show that stress-response factors mediate the stress-induced mutagenesis (SIM) of CAG repeats. We show further that SIM of CAG repeats does not involve mismatch repair, nucleotide excision repair, or transcription, processes that are known to promote TNR mutagenesis in other pathways of instability. Instead, we find that these stresses stimulate DNA rereplication, increasing the proportion of cells with >4 C-value (C) DNA content. Knockdown of the replication origin-licensing factor CDT1 eliminates both stress-induced rereplication and CAG repeat mutagenesis. In addition, direct induction of rereplication in the absence of stress also increases the proportion of cells with >4C DNA content and promotes repeat mutagenesis. Thus, environmental stress triggers a unique pathway for TNR mutagenesis that likely is mediated by DNA rereplication. This pathway may impact normal cells as they encounter stresses in their environment or during development or abnormal cells as they evolve metastatic potential. PMID:25775519

Environmental stress induces trinucleotide repeat mutagenesis in human cells.

PubMed

Chatterjee, Nimrat; Lin, Yunfu; Santillan, Beatriz A; Yotnda, Patricia; Wilson, John H

2015-03-24

The dynamic mutability of microsatellite repeats is implicated in the modification of gene function and disease phenotype. Studies of the enhanced instability of long trinucleotide repeats (TNRs)-the cause of multiple human diseases-have revealed a remarkable complexity of mutagenic mechanisms. Here, we show that cold, heat, hypoxic, and oxidative stresses induce mutagenesis of a long CAG repeat tract in human cells. We show that stress-response factors mediate the stress-induced mutagenesis (SIM) of CAG repeats. We show further that SIM of CAG repeats does not involve mismatch repair, nucleotide excision repair, or transcription, processes that are known to promote TNR mutagenesis in other pathways of instability. Instead, we find that these stresses stimulate DNA rereplication, increasing the proportion of cells with >4 C-value (C) DNA content. Knockdown of the replication origin-licensing factor CDT1 eliminates both stress-induced rereplication and CAG repeat mutagenesis. In addition, direct induction of rereplication in the absence of stress also increases the proportion of cells with >4C DNA content and promotes repeat mutagenesis. Thus, environmental stress triggers a unique pathway for TNR mutagenesis that likely is mediated by DNA rereplication. This pathway may impact normal cells as they encounter stresses in their environment or during development or abnormal cells as they evolve metastatic potential.

The Small RNA GcvB Promotes Mutagenic Break Repair by Opposing the Membrane Stress Response

PubMed Central

Barreto, Brittany; Rogers, Elizabeth; Xia, Jun; Frisch, Ryan L.; Richters, Megan; Fitzgerald, Devon M.

2016-01-01

ABSTRACT Microbes and human cells possess mechanisms of mutagenesis activated by stress responses. Stress-inducible mutagenesis mechanisms may provide important models for mutagenesis that drives host-pathogen interactions, antibiotic resistance, and possibly much of evolution generally. In Escherichia coli, repair of DNA double-strand breaks is switched to a mutagenic mode, using error-prone DNA polymerases, via the SOS DNA damage and general (σS) stress responses. We investigated small RNA (sRNA) clients of Hfq, an RNA chaperone that promotes mutagenic break repair (MBR), and found that GcvB promotes MBR by allowing a robust σS response, achieved via opposing the membrane stress (σE) response. Cells that lack gcvB were MBR deficient and displayed reduced σS-dependent transcription but not reduced σS protein levels. The defects in MBR and σS-dependent transcription in ΔgcvB cells were alleviated by artificially increasing σS levels, implying that GcvB promotes mutagenesis by allowing a normal σS response. ΔgcvB cells were highly induced for the σE response, and blocking σE response induction restored both mutagenesis and σS-promoted transcription. We suggest that GcvB may promote the σS response and mutagenesis indirectly, by promoting membrane integrity, which keeps σE levels lower. At high levels, σE might outcompete σS for binding RNA polymerase and so reduce the σS response and mutagenesis. The data show the delicate balance of stress response modulation of mutagenesis. IMPORTANCE Mutagenesis mechanisms upregulated by stress responses promote de novo antibiotic resistance and cross-resistance in bacteria, antifungal drug resistance in yeasts, and genome instability in cancer cells under hypoxic stress. This paper describes the role of a small RNA (sRNA) in promoting a stress-inducible-mutagenesis mechanism, mutagenic DNA break repair in Escherichia coli. The roles of many sRNAs in E. coli remain unknown. This study shows that ΔgcvB cells, which lack the GcvB sRNA, display a hyperactivated membrane stress response and reduced general stress response, possibly because of sigma factor competition for RNA polymerase. This results in a mutagenic break repair defect. The data illuminate a function of GcvB sRNA in opposing the membrane stress response, and thus indirectly upregulating mutagenesis. PMID:27698081

The Small RNA GcvB Promotes Mutagenic Break Repair by Opposing the Membrane Stress Response.

PubMed

Barreto, Brittany; Rogers, Elizabeth; Xia, Jun; Frisch, Ryan L; Richters, Megan; Fitzgerald, Devon M; Rosenberg, Susan M

2016-12-15

Microbes and human cells possess mechanisms of mutagenesis activated by stress responses. Stress-inducible mutagenesis mechanisms may provide important models for mutagenesis that drives host-pathogen interactions, antibiotic resistance, and possibly much of evolution generally. In Escherichia coli, repair of DNA double-strand breaks is switched to a mutagenic mode, using error-prone DNA polymerases, via the SOS DNA damage and general (σ S ) stress responses. We investigated small RNA (sRNA) clients of Hfq, an RNA chaperone that promotes mutagenic break repair (MBR), and found that GcvB promotes MBR by allowing a robust σ S response, achieved via opposing the membrane stress (σ E ) response. Cells that lack gcvB were MBR deficient and displayed reduced σ S -dependent transcription but not reduced σ S protein levels. The defects in MBR and σ S -dependent transcription in ΔgcvB cells were alleviated by artificially increasing σ S levels, implying that GcvB promotes mutagenesis by allowing a normal σ S response. ΔgcvB cells were highly induced for the σ E response, and blocking σ E response induction restored both mutagenesis and σ S -promoted transcription. We suggest that GcvB may promote the σ S response and mutagenesis indirectly, by promoting membrane integrity, which keeps σ E levels lower. At high levels, σ E might outcompete σ S for binding RNA polymerase and so reduce the σ S response and mutagenesis. The data show the delicate balance of stress response modulation of mutagenesis. Mutagenesis mechanisms upregulated by stress responses promote de novo antibiotic resistance and cross-resistance in bacteria, antifungal drug resistance in yeasts, and genome instability in cancer cells under hypoxic stress. This paper describes the role of a small RNA (sRNA) in promoting a stress-inducible-mutagenesis mechanism, mutagenic DNA break repair in Escherichia coli The roles of many sRNAs in E. coli remain unknown. This study shows that ΔgcvB cells, which lack the GcvB sRNA, display a hyperactivated membrane stress response and reduced general stress response, possibly because of sigma factor competition for RNA polymerase. This results in a mutagenic break repair defect. The data illuminate a function of GcvB sRNA in opposing the membrane stress response, and thus indirectly upregulating mutagenesis. Copyright © 2016 Barreto et al.

[Protein interaction site of Toxoplasma gondii microneme protein 6 and aldolase determined by site-directed mutagenesis].

PubMed

Zheng, Bin; Yin, Zhi-Kui; Zhan, Xi-Mei

2014-06-01

To identify the protein interaction site of Toxoplasma gondii microneme protein 6 (MIC6) and aldolase by using site-directed mutagenesis. Based on Toxoplasma gondii MIC6 gene sequence (GenBank Accession No. AF110270), the specific primers were designed. Tryptophan (W)-348 of MIC6 C terminus (MIC6C) was mutated to valine (V) via site-directed mutagenesis. MIC6C W/V gene was obtained from cDNA library by PCR amplification and subcloned into pGEX-4T-1. The mutant protein GST-MIC6C W/V was expressed in E. coli, induced by 0.8 mmol/L IPTG, and purified by affinity chromatography. Glutathione sepharose beads were incubated with GST-MIC6C W/V and GST-MIC6C, respectively, and then incubated with T. gondii tachyzoites lysate, and bound proteins were eluted using sample buffer. Bound products were resolved by SDS-PAGE and Western blotting. Glutathione sepharose beads were incubated with GST-MIC6C W/V and GST-MIC6C, respectively, and then incubated with aldolase-His6. After incubation, the resin was washed and subjected to SDS-PAGE. The MIC6C W/N gene was obtained, and the recombinant plasmid MIC6C W/V/pGEX-4T-1 was successfully constructed. The mutant protein GST-MIC6C W/V was expressed and purified in vitro. SDS-PAGE analysis indicated that GST-MIC6C was co-precipitated with aldolase from T. gondii tachyzoites lysate or aldolase-His6, whereas GST-MIC6C W/V failed to precipitate aldolase from T. gondii tachyzoites lysate or aldolase-His6. Western blotting analysis using anti-aldolase antibody indicated that GST-MIC6C could pull-down aldolase from T. gondii tachyzoites lysate. Tryptophan (W348) was the interaction site of MIC6 and aldolase in T. gondii.

[Fermentation production of microbial catalase and its application in textile industry].

PubMed

Zhang, Dongxu; Du, Guocheng; Chen, Jian

2010-11-01

Microbial catalase is an important industrial enzyme that catalyzes the decomposition of hydrogen peroxide to water and oxygen. This enzyme has great potential of application in food, textile and pharmaceutical industries. The production of microbial catalase has been significantly improved thanks to advances in bioprocess engineering and genetic engineering. In this paper, we review the progresses in fermentation production of microbial catalase and its application in textile industry. Among these progresses, we will highlight strain isolation, substrate and environment optimization, enzyme induction, construction of engineering strains and application process optimization. Meanwhile, we also address future research trends for microbial catalase production and its application in textile industry. Molecular modification (site-directed mutagenesis and directed revolution) will endue catalase with high pH and temperature stabilities. Improvement of catalase production, based on the understanding of induction mechanism and the process control of recombinant stain fermentation, will further accelerate the application of catalase in textile industry.

Experiences with the in vivo and in vitro comet assay in regulatory testing.

PubMed

Frötschl, Roland

2015-01-01

The in vivo comet assay has recently been implemented into regulatory genotoxicity testing of pharmaceuticals with inclusion into the ICH S2R1 guidance. Regulatory genotoxicity testing aims to detect DNA alterations in form of gene mutations, larger scale chromosomal damage and recombination and aneuploidy. The ICH S2R1 guideline offers two options of standard batteries of tests for the detection of these endpoints. Both options start with an AMES assay and option 1 includes an in vitro mammalian cell assay and an in vivo micronucleus assay in rodent, whereas option 2 includes an in vivo micronucleus assay in bone marrow in rodent and a second in vivo assay in a second tissue with a second endpoint. The test recommended as second in vivo test is the comet assay in rat liver. The in vivo comet assay is considered as mature enough to ensure reliable detection of relevant in vivo genotoxicants in combination with the micronucleus test in bone marrow and the AMES assay. Although lots of research papers have been published using the in vitro comet assay, the in vitro version has not been implemented into official regulatory testing guidelines. A survey of the years 1999-2014 revealed 27 in vivo comet assays submitted to BfArM with market authorisation procedures, European and national advice procedures and clinical trial applications. In three procedures, in vitro comet assays had been submitted within the genetic toxicology packages. © The Author 2014. Published by Oxford University Press on behalf of the Mutagenesis Society. All rights reserved. For permissions, please email: journals.permissions@oup.com.

2012 Gordon Research Conference on Mutagenesis - Formal Schedule and Speaker/Poster Program

DOE Office of Scientific and Technical Information (OSTI.GOV)

Demple, Bruce

2012-08-24

The delicate balance among cellular pathways that control mutagenic changes in DNA will be the focus of the 2012 Mutagenesis Gordon Research Conference. Mutagenesis is essential for evolution, while genetic stability maintains cellular functions in all organisms from microbes to metazoans. Different systems handle DNA lesions at various times of the cell cycle and in different places within the nucleus, and inappropriate actions can lead to mutations. While mutation in humans is closely linked to disease, notably cancers, mutational systems can also be beneficial. The conference will highlight topics of beneficial mutagenesis, including full establishment of the immune system, cellmore » survival mechanisms, and evolution and adaptation in microbial systems. Equal prominence will be given to detrimental mutation processes, especially those involved in driving cancer, neurological diseases, premature aging, and other threats to human health. Provisional session titles include Branching Pathways in Mutagenesis; Oxidative Stress and Endogenous DNA Damage; DNA Maintenance Pathways; Recombination, Good and Bad; Problematic DNA Structures; Localized Mutagenesis; Hypermutation in the Microbial World; and Mutation and Disease.« less

Comparative study on the in vitro effects of Pseudomonas aeruginosa and seaweed alginates on human gut microbiota

PubMed Central

Bai, Shaofeng; Chen, Huahai; Zhu, Liying; Liu, Wei; Yu, Hongwei D.; Wang, Xin; Yin, Yeshi

2017-01-01

Alginates pertain to organic polysaccharides that have been extensively used in food- and medicine-related industries. The present study obtained alginates from an alginate overproducing Pseudomonas aeruginosa PAO1 mutant by screening transposon mutagenesis libraries. The interaction between bacterial and seaweed alginates and gut microbiota were further studied by using an in vitro batch fermentation system. Thin-layer chromatography (TLC) analysis indicated that both bacterial and seaweed alginates can be completely degraded by fecal bacteria isolated from study volunteers, indicating that a minor structural difference between bacterial and seaweed alginates (O-acetylation and lack of G-G blocks) didn’t affect the digestion of alginates by human microbiota. Although, the digestion of bacterial and seaweed alginates was attributed to different Bacteroides xylanisolvens strains, they harbored similar alginate lyase genes. Genus Bacteroides with alginate-degrading capability were enriched in growth medium containing bacterial or seaweed alginates after in vitro fermentation. Short-chain fatty acid (SCFA) production in both bacterial and seaweed alginates was also comparable, but was significantly higher than the same medium using starch. In summary, the present study has isolated an alginate-overproducing P. aeruginosa mutant strain. Both seaweed and bacterial alginates were degraded by human gut microbiota, and their regulatory function on gut microbiota was similar. PMID:28170428

Increased Antibody Affinity Confers Broad In Vitro Protection against Escape Mutants of Severe Acute Respiratory Syndrome Coronavirus

PubMed Central

Rani, Mridula; Bolles, Meagan; Donaldson, Eric F.; Van Blarcom, Thomas; Baric, Ralph; Iverson, Brent

2012-01-01

Even though the effect of antibody affinity on neutralization potency is well documented, surprisingly, its impact on neutralization breadth and escape has not been systematically determined. Here, random mutagenesis and DNA shuffling of the single-chain variable fragment of the neutralizing antibody 80R followed by bacterial display screening using anchored periplasmic expression (APEx) were used to generate a number of higher-affinity variants of the severe acute respiratory syndrome coronavirus (SARS-CoV)-neutralizing antibody 80R with equilibrium dissociation constants (KD) as low as 37 pM, a >270-fold improvement relative to that of the parental 80R single-chain variable fragment (scFv). As expected, antigen affinity was shown to correlate directly with neutralization potency toward the icUrbani strain of SARS-CoV. Additionally, the highest-affinity antibody fragment displayed 10-fold-increased broad neutralization in vitro and completely protected against several SARS-CoV strains containing substitutions associated with antibody escape. Importantly, higher affinity also led to the suppression of viral escape mutants in vitro. Escape from the highest-affinity variant required reduced selective pressure and multiple substitutions in the binding epitope. Collectively, these results support the hypothesis that engineered antibodies with picomolar dissociation constants for a neutralizing epitope can confer escape-resistant protection. PMID:22696652

Comparative study on the in vitro effects of Pseudomonas aeruginosa and seaweed alginates on human gut microbiota.

PubMed

Bai, Shaofeng; Chen, Huahai; Zhu, Liying; Liu, Wei; Yu, Hongwei D; Wang, Xin; Yin, Yeshi

2017-01-01

Alginates pertain to organic polysaccharides that have been extensively used in food- and medicine-related industries. The present study obtained alginates from an alginate overproducing Pseudomonas aeruginosa PAO1 mutant by screening transposon mutagenesis libraries. The interaction between bacterial and seaweed alginates and gut microbiota were further studied by using an in vitro batch fermentation system. Thin-layer chromatography (TLC) analysis indicated that both bacterial and seaweed alginates can be completely degraded by fecal bacteria isolated from study volunteers, indicating that a minor structural difference between bacterial and seaweed alginates (O-acetylation and lack of G-G blocks) didn't affect the digestion of alginates by human microbiota. Although, the digestion of bacterial and seaweed alginates was attributed to different Bacteroides xylanisolvens strains, they harbored similar alginate lyase genes. Genus Bacteroides with alginate-degrading capability were enriched in growth medium containing bacterial or seaweed alginates after in vitro fermentation. Short-chain fatty acid (SCFA) production in both bacterial and seaweed alginates was also comparable, but was significantly higher than the same medium using starch. In summary, the present study has isolated an alginate-overproducing P. aeruginosa mutant strain. Both seaweed and bacterial alginates were degraded by human gut microbiota, and their regulatory function on gut microbiota was similar.

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans.

PubMed

Noma, Kentaro; Jin, Yishi

2016-11-14

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

Chemical and UV Mutagenesis.

PubMed

Bose, Jeffrey L

2016-01-01

The ability to create mutations is an important step towards understanding bacterial physiology and virulence. While targeted approaches are invaluable, the ability to produce genome-wide random mutations can lead to crucial discoveries. Transposon mutagenesis is a useful approach, but many interesting mutations can be missed by these insertions that interrupt coding and noncoding sequences due to the integration of an entire transposon. Chemical mutagenesis and UV-based random mutagenesis are alternate approaches to isolate mutations of interest with the potential of only single nucleotide changes. Once a standard method, difficulty in identifying mutation sites had decreased the popularity of this technique. However, thanks to the recent emergence of economical whole-genome sequencing, this approach to making mutations can once again become a viable option. Therefore, this chapter provides an overview protocol for random mutagenesis using UV light or DNA-damaging chemicals.

DNA Polymerase ζ is essential for hexavalent chromium-induced mutagenesis

PubMed Central

O'Brien, Travis J.; Witcher, Preston; Brooks, Bradford; Patierno, Steven R.

2009-01-01

Translesion synthesis (TLS) is a unique DNA damage tolerance mechanism involved in the replicative bypass of genetic lesions in favor of uninterrupted DNA replication. TLS is critical for the generation of mutations by many different chemical and physical agents, however, there is no information available regarding the role of TLS in carcinogenic metal-induced mutagenesis. Hexavalent chromium (Cr(VI))-containing compounds are highly complex genotoxins possessing both mutagenic and clastogenic activities. The focus of this work was to determine the impact that TLS has on Cr(VI)-induced mutagenesis in S. cerevisiae. Wild-type yeast and strains deficient in TLS polymerases (i.e. Polζ (rev3), Polη (rad30)) were exposed to Cr(VI) and monitored for cell survival and forward mutagenesis at the CAN1 locus. In general, TLS deficiency had little impact on Cr(VI)-induced clonogenic lethality or cell growth. rad30 yeast exhibited higher levels of basal and induced mutagenesis compared to Wt and rev3 yeast. In contrast, rev3 yeast displayed attenuated Cr(VI)-induced mutagenesis. Moreover, deletion of REV3 in rad30 yeast (rad30 rev3) resulted in a significant decrease in basal and Cr(VI) mutagenesis relative to Wt and rad30 single mutants indicating that mutagenesis primarily depended upon Polζ. Interestingly, rev3 yeast were similar to Wt yeast in susceptibility to Cr(VI)-induced frameshift mutations. Mutational analysis of the CAN1 gene revealed that Cr(VI)-induced base substitution mutations accounted for 83.9% and 100.0% of the total mutations in Wt and rev3 yeast, respectively. Insertions and deletions comprised 16.1% of the total mutations in Cr(VI) treated Wt yeast but were not observed rev3 yeast. This work provides novel information regarding the molecular mechanisms of Cr(VI)-induced mutagenesis and is the first report demonstrating a role for TLS in the fixation of mutations induced by a carcinogenic metal. PMID:19428373

Commentary on "tissue-specific mutagenesis by N-butyl-N-(4-hydroxybutyl) nitrosamine as the basis for urothelial cell carcinogenesis." He Z, Kosinska W, Zhao ZL, Wu XR, Guttenplan JB, Department of Basic Science, New York University Dental College, NY, USA.: Mutat Res 2012;742(1-2):92-5 [Epub 2011 Dec 4].

PubMed

Scherr, Douglas S

2014-02-01

Bladder cancer is one of the few cancers that have been linked to carcinogens in the environment and tobacco smoke. Of the carcinogens tested in mouse chemical carcinogenesis models, N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) is one that reproducibly causes high-grade, invasive cancers in the urinary bladder, but not in any other tissues. However, the basis for such a high-level tissue-specificity has not been explored. Using mutagenesis in lacI (Big Blue™) mice, we show here that BBN is a potent mutagen and it causes high-level of mutagenesis specifically in the epithelial cells (urothelial) of the urinary bladder. After a 2-6-week treatment of 0.05% BBN in the drinking water, mutagenesis in urothelial cells of male and female mice was about two orders of magnitude greater than the spontaneous mutation background. In contrast, mutagenesis in smooth muscle cells of the urinary bladder was about five times lower than in urothelial tissue. No appreciable increase in mutagenesis was observed in kidney, ureter, liver or forestomach. In lacI (Big Blue™) rats, BBN mutagenesis was also elevated in urothelial cells, albeit not nearly as profoundly as in mice. This provides a potential explanation as to why rats are less prone than mice to the formation of aggressive form of bladder cancer induced by BBN. Our results suggest that the propensity to BBN-triggered mutagenesis of urothelial cells underlies its heightened susceptibility to this carcinogen and that mutagenesis induced by BBN represents a novel model for initiation of bladder carcinogenesis. Copyright © 2014 Elsevier Inc. All rights reserved.

Gene therapy for PIDs: progress, pitfalls and prospects.

PubMed

Mukherjee, Sayandip; Thrasher, Adrian J

2013-08-10

Substantial progress has been made in the past decade in treating several primary immunodeficiency disorders (PIDs) with gene therapy. Current approaches are based on ex-vivo transfer of therapeutic transgene via viral vectors to patient-derived autologous hematopoietic stem cells (HSCs) followed by transplantation back to the patient with or without conditioning. The overall outcome from all the clinical trials targeting different PIDs has been extremely encouraging but not without caveats. Malignant outcomes from insertional mutagenesis have featured prominently in the adverse events associated with these trials and have warranted intense pre-clinical investigation into defining the tendencies of different viral vectors for genomic integration. Coupled with issues pertaining to transgene expression, the therapeutic landscape has undergone a paradigm shift in determining safety, stability and efficacy of gene therapy approaches. In this review, we aim to summarize the progress made in the gene therapy trials targeting ADA-SCID, SCID-X1, CGD and WAS, review the pitfalls, and outline the recent advancements which are expected to further enhance favourable risk benefit ratios for gene therapeutic approaches in the future. Copyright © 2013 Elsevier B.V. All rights reserved.

Variable sequences outside the SAM-binding core critically influence the conformational dynamics of the SAM-III/SMK box riboswitch

PubMed Central

Lu, Changrui; Smith, Angela M; Ding, Fang; Chowdhury, Anirban; Henkin, Tina M; Ke, Ailong

2012-01-01

The SMK box (SAM-III) translational riboswitches were identified in S-adenosyl-L-methionine (SAM) synthetase metK genes in members of the Lactobacillales. This riboswitch switches between two alternative conformations in response to the intracellular SAM concentration and controls metK expression at the level of translation initiation. We previously reported the crystal structure of the SAM-bound SMK box riboswitch. In this study we combined SHAPE chemical probing with mutagenesis to probe the ligand-induced conformational switching mechanism. We revealed that while the majority of the apo SMK box RNA molecules exist in an alternatively base paired (ON) conformation, a subset of them pre-organize into a SAM-bound-like (READY) conformation, which upon SAM exposure is selectively stabilized into the SAM-bound (OFF) conformation through an induced-fit mechanism. Mutagenesis showed that the ON state is only slightly more stable than the READY state, as several single-nucleotide substitutions in a hypervariable region outside the SAM-binding core can alter the folding landscape to favor the READY state. Such SMK variants display a “constitutively-OFF” behavior both in vitro and in vivo. Time-resolved and temperature-dependent SHAPE analyses revealed adaptation of the SMK box RNA to its mesothermal working environment. The latter analysis revealed that the SAM-bound SMK box RNA follows a two-step folding/unfolding process. PMID:21549712

Histophilus somni biofilm formation in cardiopulmonary tissue of the bovine host following respiratory challenge.

PubMed

Sandal, Indra; Shao, Jian Q; Annadata, Satish; Apicella, Michael A; Boye, Mette; Jensen, Tim K; Saunders, Geoffrey K; Inzana, Thomas J

2009-02-01

Biofilms form in a variety of host sites following infection with many bacterial species. However, the study of biofilms in a host is hindered due to the lack of protocols for the proper experimental investigation of biofilms in vivo. Histophilus somni is an agent of respiratory and systemic diseases in bovines, and readily forms biofilms in vitro. In the present study the capability of H. somni to form biofilms in cardiopulmonary tissue following experimental respiratory infection in the bovine host was examined by light microscopy, transmission electron microscopy, immunoelectron microscopy of ultrathin cryosections, scanning electron microscopy of freeze-fractured samples, and fluorescent in situ hybridization. Biofilms were evident and most prominent in the myocardium, and were associated with a large amount of amorphous extracellular material. Furthermore, Pasteurella multocida was often cultured with H. somni from heart and lung samples. Transposon mutagenesis of H. somni strain 2336 resulted in the generation of mutants that expressed more or less biofilm than the parent strain. Six mutants deficient in biofilm formation had an insertion in the gene encoding for a homolog of filamentous haemagglutinin (FHA), predicted to be involved in attachment. Thus, this investigation demonstrated that H. somni is capable of forming a biofilm in its natural host, that such a biofilm may be capable of harboring other bovine respiratory disease pathogens, and that the genes responsible for biofilm formation can be identified by transposon mutagenesis.

Crizotinib-Resistant Mutants of EML4-ALK Identified Through an Accelerated Mutagenesis Screen

PubMed Central

Zhang, Sen; Wang, Frank; Keats, Jeffrey; Zhu, Xiaotian; Ning, Yaoyu; Wardwell, Scott D; Moran, Lauren; Mohemmad, Qurish K; Anjum, Rana; Wang, Yihan; Narasimhan, Narayana I; Dalgarno, David; Shakespeare, William C; Miret, Juan J; Clackson, Tim; Rivera, Victor M

2011-01-01

Activating gene rearrangements of anaplastic lymphoma kinase (ALK) have been identified as driver mutations in non-small-cell lung cancer, inflammatory myofibroblastic tumors, and other cancers. Crizotinib, a dual MET/ALK inhibitor, has demonstrated promising clinical activity in patients with non-small-cell lung cancer and inflammatory myofibroblastic tumors harboring ALK translocations. Inhibitors of driver kinases often elicit kinase domain mutations that confer resistance, and such mutations have been successfully predicted using in vitro mutagenesis screens. Here, this approach was used to discover an extensive set of ALK mutations that can confer resistance to crizotinib. Mutations at 16 residues were identified, structurally clustered into five regions around the kinase active site, which conferred varying degrees of resistance. The screen successfully predicted the L1196M, C1156Y, and F1174L mutations, recently identified in crizotinib-resistant patients. In separate studies, we demonstrated that crizotinib has relatively modest potency in ALK-positive non-small-cell lung cancer cell lines. A more potent ALK inhibitor, TAE684, maintained substantial activity against mutations that conferred resistance to crizotinib. Our study identifies multiple novel mutations in ALK that may confer clinical resistance to crizotinib, suggests that crizotinib's narrow selectivity window may underlie its susceptibility to such resistance and demonstrates that a more potent ALK inhibitor may be effective at overcoming resistance. PMID:22034911

Saturation mutagenesis reveals manifold determinants of exon definition.

PubMed

Ke, Shengdong; Anquetil, Vincent; Zamalloa, Jorge Rojas; Maity, Alisha; Yang, Anthony; Arias, Mauricio A; Kalachikov, Sergey; Russo, James J; Ju, Jingyue; Chasin, Lawrence A

2018-01-01

To illuminate the extent and roles of exonic sequences in the splicing of human RNA transcripts, we conducted saturation mutagenesis of a 51-nt internal exon in a three-exon minigene. All possible single and tandem dinucleotide substitutions were surveyed. Using high-throughput genetics, 5560 minigene molecules were assayed for splicing in human HEK293 cells. Up to 70% of mutations produced substantial (greater than twofold) phenotypes of either increased or decreased splicing. Of all predicted secondary structural elements, only a single 15-nt stem-loop showed a strong correlation with splicing, acting negatively. The in vitro formation of exon-protein complexes between the mutant molecules and proteins associated with spliceosome formation (U2AF35, U2AF65, U1A, and U1-70K) correlated with splicing efficiencies, suggesting exon definition as the step affected by most mutations. The measured relative binding affinities of dozens of human RNA binding protein domains as reported in the CISBP-RNA database were found to correlate either positively or negatively with splicing efficiency, more than could fit on the 51-nt test exon simultaneously. The large number of these functional protein binding correlations point to a dynamic and heterogeneous population of pre-mRNA molecules, each responding to a particular collection of binding proteins. © 2018 Ke et al.; Published by Cold Spring Harbor Laboratory Press.

The role of chemicals and radiation in the etiology of cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huberman, E.; Barr, S.H.

In this volume, investigators consider the mechanisms of oncogenesis, cell transformation, and carcinogen metabolism and present new findings on chemical and radiation carcinogenesis and chemically induced mutagenesis and chromosomal changes. As background to the studies of chemical and radiation carcinogenesis, the book surveys knowledge of cell transformation and carcinogen metabolism. Among the topics reviewed are the transforming genes involved in human malignancy, the genetics and epigenetics of neoplasia, and the single-hit and multi-hit concepts of hepatocarcinogenesis. Also examined are organ, species, and interindividual differences in carcinogen metabolism; chemical and biochemical dosimetry of genotoxic chemical exposure; and the role of pharmacokineticsmore » and DNA dosimetry in relating in vitro to in vivo actions of N-nitroso compounds.« less

Robust one-Tube Ω-PCR Strategy Accelerates Precise Sequence Modification of Plasmids for Functional Genomics

PubMed Central

Chen, Letian; Wang, Fengpin; Wang, Xiaoyu; Liu, Yao-Guang

2013-01-01

Functional genomics requires vector construction for protein expression and functional characterization of target genes; therefore, a simple, flexible and low-cost molecular manipulation strategy will be highly advantageous for genomics approaches. Here, we describe a Ω-PCR strategy that enables multiple types of sequence modification, including precise insertion, deletion and substitution, in any position of a circular plasmid. Ω-PCR is based on an overlap extension site-directed mutagenesis technique, and is named for its characteristic Ω-shaped secondary structure during PCR. Ω-PCR can be performed either in two steps, or in one tube in combination with exonuclease I treatment. These strategies have wide applications for protein engineering, gene function analysis and in vitro gene splicing. PMID:23335613

Expression of Herpes Simplex Virus 1 Glycoprotein B by a Recombinant Vaccinia Virus and Protection of Mice against Lethal Herpes Simplex Virus 1 Infection

NASA Astrophysics Data System (ADS)

Cantin, Edouard M.; Eberle, Richard; Baldick, Joseph L.; Moss, Bernard; Willey, Dru E.; Notkins, Abner L.; Openshaw, Harry

1987-08-01

The herpes simplex virus 1 (HSV-1) strain F gene encoding glycoprotein gB was isolated and modified at the 5' end by in vitro oligonucleotide-directed mutagenesis. The modified gB gene was inserted into the vaccinia virus genome and expressed under the control of a vaccinia virus promoter. The mature gB glycoprotein produced by the vaccinia virus recombinant was glycosylated, was expressed at the cell surface, and was indistinguishable from authentic HSV-1 gB in terms of electrophoretic mobility. Mice immunized intradermally with the recombinant vaccinia virus produced gB-specific neutralizing antibodies and were resistant to a lethal HSV-1 challenge.

Antibody VH and VL recombination using phage and ribosome display technologies reveals distinct structural routes to affinity improvements with VH-VL interface residues providing important structural diversity

PubMed Central

Groves, Maria AT; Amanuel, Lily; Campbell, Jamie I; Rees, D Gareth; Sridharan, Sudharsan; Finch, Donna K; Lowe, David C; Vaughan, Tristan J

2014-01-01

In vitro selection technologies are an important means of affinity maturing antibodies to generate the optimal therapeutic profile for a particular disease target. Here, we describe the isolation of a parent antibody, KENB061 using phage display and solution phase selections with soluble biotinylated human IL-1R1. KENB061 was affinity matured using phage display and targeted mutagenesis of VH and VL CDR3 using NNS randomization. Affinity matured VHCDR3 and VLCDR3 library blocks were recombined and selected using phage and ribosome display protocol. A direct comparison of the phage and ribosome display antibodies generated was made to determine their functional characteristics. PMID:24256948

High throughput mutagenesis for identification of residues regulating human prostacyclin (hIP) receptor expression and function.

PubMed

Bill, Anke; Rosethorne, Elizabeth M; Kent, Toby C; Fawcett, Lindsay; Burchell, Lynn; van Diepen, Michiel T; Marelli, Anthony; Batalov, Sergey; Miraglia, Loren; Orth, Anthony P; Renaud, Nicole A; Charlton, Steven J; Gosling, Martin; Gaither, L Alex; Groot-Kormelink, Paul J

2014-01-01

The human prostacyclin receptor (hIP receptor) is a seven-transmembrane G protein-coupled receptor (GPCR) that plays a critical role in vascular smooth muscle relaxation and platelet aggregation. hIP receptor dysfunction has been implicated in numerous cardiovascular abnormalities, including myocardial infarction, hypertension, thrombosis and atherosclerosis. Genomic sequencing has discovered several genetic variations in the PTGIR gene coding for hIP receptor, however, its structure-function relationship has not been sufficiently explored. Here we set out to investigate the applicability of high throughput random mutagenesis to study the structure-function relationship of hIP receptor. While chemical mutagenesis was not suitable to generate a mutagenesis library with sufficient coverage, our data demonstrate error-prone PCR (epPCR) mediated mutagenesis as a valuable method for the unbiased screening of residues regulating hIP receptor function and expression. Here we describe the generation and functional characterization of an epPCR derived mutagenesis library compromising >4000 mutants of the hIP receptor. We introduce next generation sequencing as a useful tool to validate the quality of mutagenesis libraries by providing information about the coverage, mutation rate and mutational bias. We identified 18 mutants of the hIP receptor that were expressed at the cell surface, but demonstrated impaired receptor function. A total of 38 non-synonymous mutations were identified within the coding region of the hIP receptor, mapping to 36 distinct residues, including several mutations previously reported to affect the signaling of the hIP receptor. Thus, our data demonstrates epPCR mediated random mutagenesis as a valuable and practical method to study the structure-function relationship of GPCRs.

High Throughput Mutagenesis for Identification of Residues Regulating Human Prostacyclin (hIP) Receptor Expression and Function

PubMed Central

Kent, Toby C.; Fawcett, Lindsay; Burchell, Lynn; van Diepen, Michiel T.; Marelli, Anthony; Batalov, Sergey; Miraglia, Loren; Orth, Anthony P.; Renaud, Nicole A.; Charlton, Steven J.; Gosling, Martin; Gaither, L. Alex; Groot-Kormelink, Paul J.

2014-01-01

The human prostacyclin receptor (hIP receptor) is a seven-transmembrane G protein-coupled receptor (GPCR) that plays a critical role in vascular smooth muscle relaxation and platelet aggregation. hIP receptor dysfunction has been implicated in numerous cardiovascular abnormalities, including myocardial infarction, hypertension, thrombosis and atherosclerosis. Genomic sequencing has discovered several genetic variations in the PTGIR gene coding for hIP receptor, however, its structure-function relationship has not been sufficiently explored. Here we set out to investigate the applicability of high throughput random mutagenesis to study the structure-function relationship of hIP receptor. While chemical mutagenesis was not suitable to generate a mutagenesis library with sufficient coverage, our data demonstrate error-prone PCR (epPCR) mediated mutagenesis as a valuable method for the unbiased screening of residues regulating hIP receptor function and expression. Here we describe the generation and functional characterization of an epPCR derived mutagenesis library compromising >4000 mutants of the hIP receptor. We introduce next generation sequencing as a useful tool to validate the quality of mutagenesis libraries by providing information about the coverage, mutation rate and mutational bias. We identified 18 mutants of the hIP receptor that were expressed at the cell surface, but demonstrated impaired receptor function. A total of 38 non-synonymous mutations were identified within the coding region of the hIP receptor, mapping to 36 distinct residues, including several mutations previously reported to affect the signaling of the hIP receptor. Thus, our data demonstrates epPCR mediated random mutagenesis as a valuable and practical method to study the structure-function relationship of GPCRs. PMID:24886841

Multiplex conditional mutagenesis in zebrafish using the CRISPR/Cas system.

PubMed

Yin, L; Maddison, L A; Chen, W

2016-01-01

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system is a powerful tool for genome editing in numerous organisms. However, the system is typically used for gene editing throughout the entire organism. Tissue and temporal specific mutagenesis is often desirable to determine gene function in a specific stage or tissue and to bypass undesired consequences of global mutations. We have developed the CRISPR/Cas system for conditional mutagenesis in transgenic zebrafish using tissue-specific and/or inducible expression of Cas9 and U6-driven expression of sgRNA. To allow mutagenesis of multiple targets, we have isolated four distinct U6 promoters and designed Golden Gate vectors to easily assemble transgenes with multiple sgRNAs. We provide experimental details on the reagents and applications for multiplex conditional mutagenesis in zebrafish. Copyright © 2016 Elsevier Inc. All rights reserved.

Eukaryotic DNA polymerase ζ

PubMed Central

Makarova, Alena V.; Burgers, Peter M.

2015-01-01

This review focuses on eukaryotic DNA polymerase ζ (Pol ζ), the enzyme responsible for the bulk of mutagenesis in eukaryotic cells in response to DNA damage. Pol ζ is also responsible for a large portion of mutagenesis during normal cell growth, in response to spontaneous damage or to certain DNA structures and other blocks that stall DNA replication forks. Novel insights in mutagenesis have been derived from recent advances in the elucidation of the subunit structure of Pol ζ. The lagging strand DNA polymerase δ shares the small Pol31 and Pol32 subunits with the Rev3-Rev7 core assembly giving a four subunit Pol ζ complex that is the active form in mutagenesis. Furthermore, Pol ζ forms essential interactions with the mutasome assembly factor Rev1 and with proliferating cell nuclear antigen (PCNA). These interactions are modulated by posttranslational modifications such as ubiquitination and phosphorylation that enhance translesion synthesis (TLS) and mutagenesis. PMID:25737057

Favipiravir elicits antiviral mutagenesis during virus replication in vivo.

PubMed

Arias, Armando; Thorne, Lucy; Goodfellow, Ian

2014-10-21

Lethal mutagenesis has emerged as a novel potential therapeutic approach to treat viral infections. Several studies have demonstrated that increases in the high mutation rates inherent to RNA viruses lead to viral extinction in cell culture, but evidence during infections in vivo is limited. In this study, we show that the broad-range antiviral nucleoside favipiravir reduces viral load in vivo by exerting antiviral mutagenesis in a mouse model for norovirus infection. Increased mutation frequencies were observed in samples from treated mice and were accompanied with lower or in some cases undetectable levels of infectious virus in faeces and tissues. Viral RNA isolated from treated animals showed reduced infectivity, a feature of populations approaching extinction during antiviral mutagenesis. These results suggest that favipiravir can induce norovirus mutagenesis in vivo, which in some cases leads to virus extinction, providing a proof-of-principle for the use of favipiravir derivatives or mutagenic nucleosides in the clinical treatment of noroviruses.

Optogenetic mutagenesis in Caenorhabditis elegans.

PubMed

Noma, Kentaro; Jin, Yishi

2015-12-03

Reactive oxygen species (ROS) can modify and damage DNA. Here we report an optogenetic mutagenesis approach that is free of toxic chemicals and easy to perform by taking advantage of a genetically encoded ROS generator. This method relies on the potency of ROS generation by His-mSOG, the mini singlet oxygen generator, miniSOG, fused to a histone. Caenorhabditis elegans expressing His-mSOG in the germline behave and reproduce normally, without photoinduction. Following exposure to blue light, the His-mSOG animals produce progeny with a wide range of heritable phenotypes. We show that optogenetic mutagenesis by His-mSOG induces a broad spectrum of mutations including single-nucleotide variants (SNVs), chromosomal deletions, as well as integration of extrachromosomal transgenes, which complements those derived from traditional chemical or radiation mutagenesis. The optogenetic mutagenesis expands the toolbox for forward genetic screening and also provides direct evidence that nuclear ROS can induce heritable and specific genetic mutations.

Optogenetic mutagenesis in Caenorhabditis elegans

PubMed Central

Noma, Kentaro; Jin, Yishi

2015-01-01

Reactive oxygen species (ROS) can modify and damage DNA. Here we report an optogenetic mutagenesis approach that is free of toxic chemicals and easy to perform by taking advantage of a genetically encoded ROS generator. This method relies on the potency of ROS generation by His-mSOG, the mini singlet oxygen generator, miniSOG, fused to a histone. Caenorhabditis elegans expressing His-mSOG in the germline behave and reproduce normally, without photoinduction. Following exposure to blue light, the His-mSOG animals produce progeny with a wide range of heritable phenotypes. We show that optogenetic mutagenesis by His-mSOG induces a broad spectrum of mutations including single-nucleotide variants (SNVs), chromosomal deletions, as well as integration of extrachromosomal transgenes, which complements those derived from traditional chemical or radiation mutagenesis. The optogenetic mutagenesis expands the toolbox for forward genetic screening and also provides direct evidence that nuclear ROS can induce heritable and specific genetic mutations. PMID:26632265

Toward a systems-level analysis of infection biology: a new method for conducting genetic screens in human cells.

PubMed

Stanley, Sarah A; Hung, Deborah T

2009-12-16

Loss-of-function genetic screens have facilitated great strides in our understanding of the biology of model organisms but have not been possible in diploid human cells. A recent report by Brummelkamp's group in Science describes the use of insertional mutagenesis to generate loss-of-function alleles in a largely haploid human cell line and demonstrates the versatility of this method in screens designed to investigate the host/pathogen interaction. This approach has strengths that are complementary to existing strategies and will facilitate progress toward a systems-level understanding of infectious disease and ultimately the development of new therapeutics.

Metabolic analysis of the increased adventitious rooting mutant of Artemisia annua reveals a role for the plant monoterpene borneol in adventitious root formation.

PubMed

Tian, Na; Liu, Shuoqian; Li, Juan; Xu, Wenwen; Yuan, Lin; Huang, Jianan; Liu, Zhonghua

2014-08-01

Adventitious root (AR) formation is a critical process for plant clonal propagation. The role of plant secondary metabolites in AR formation is still poorly understood. Chemical and physical mutagenesis in combination with somatic variation were performed on Artemisia annua in order to obtain a mutant with changes in adventitious rooting and composition of plant secondary metabolites. Metabolic and morphological analyses of the iar (increased adventitious rooting) mutant coupled with in vitro assays were used to elucidate the relationship between plant secondary metabolites and AR formation. The only detected differences between the iar mutant and wild-type were rooting capacity and borneol/camphor content. Consistent with this, treatment with borneol in vitro promoted adventitious rooting in wild-type. The enhanced rooting did not continue upon removal of borneol. The iar mutant displayed no significant differences in AR formation upon treatment with camphor. Together, our results suggest that borneol promotes adventitious rooting whereas camphor has no effect on AR formation. © 2013 Scandinavian Plant Physiology Society.

A potential role for RNA interference in controlling the activity of the human LINE-1 retrotransposon.

PubMed

Soifer, Harris S; Zaragoza, Adriana; Peyvan, Maany; Behlke, Mark A; Rossi, John J

2005-01-01

Long interspersed nuclear elements (LINE-1 or L1) comprise 17% of the human genome, although only 80-100 L1s are considered retrotransposition-competent (RC-L1). Despite their small number, RC-L1s are still potential hazards to genome integrity through insertional mutagenesis, unequal recombination and chromosome rearrangements. In this study, we provide several lines of evidence that the LINE-1 retrotransposon is susceptible to RNA interference (RNAi). First, double-stranded RNA (dsRNA) generated in vitro from an L1 template is converted into functional short interfering RNA (siRNA) by DICER, the RNase III enzyme that initiates RNAi in human cells. Second, pooled siRNA from in vitro cleavage of L1 dsRNA, as well as synthetic L1 siRNA, targeting the 5'-UTR leads to sequence-specific mRNA degradation of an L1 fusion transcript. Finally, both synthetic and pooled siRNA suppressed retrotransposition from a highly active RC-L1 clone in cell culture assay. Our report is the first to demonstrate that a human transposable element is subjected to RNAi.

Structure of the Cmr2 Subunit of the CRISPR-Cas RNA Silencing Complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cocozaki, Alexis I.; Ramia, Nancy F.; Shao, Yaming

Cmr2 is the largest and an essential subunit of a CRISPR RNA-Cas protein complex (the Cmr complex) that cleaves foreign RNA to protect prokaryotes from invading genetic elements. Cmr2 is thought to be the catalytic subunit of the effector complex because of its N-terminal HD nuclease domain. Here, however, we report that the HD domain of Cmr2 is not required for cleavage by the complex in vitro. The 2.3 {angstrom} crystal structure of Pyrococcus furiosus Cmr2 (lacking the HD domain) reveals two adenylyl cyclase-like and two {alpha}-helical domains. The adenylyl cyclase-like domains are arranged as in homodimeric adenylyl cyclases andmore » bind ADP and divalent metals. However, mutagenesis studies show that the metal- and ADP-coordinating residues of Cmr2 are also not critical for cleavage by the complex. Our findings suggest that another component provides the catalytic function and that the essential role by Cmr2 does not require the identified ADP- or metal-binding or HD domains in vitro.« less

Orthogonal combinatorial mutagenesis: a codon-level combinatorial mutagenesis method useful for low multiplicity and amino acid-scanning protocols

PubMed Central

Gaytán, Paul; Yáñez, Jorge; Sánchez, Filiberto; Soberón, Xavier

2001-01-01

We describe here a method to generate combinatorial libraries of oligonucleotides mutated at the codon-level, with control of the mutagenesis rate so as to create predictable binomial distributions of mutants. The method allows enrichment of the libraries with single, double or larger multiplicity of amino acid replacements by appropriate choice of the mutagenesis rate, depending on the concentration of synthetic precursors. The method makes use of two sets of deoxynucleoside-phosphoramidites bearing orthogonal protecting groups [4,4′-dimethoxytrityl (DMT) and 9-fluorenylmethoxycarbonyl (Fmoc)] in the 5′ hydroxyl. These phosphoramidites are divergently combined during automated synthesis in such a way that wild-type codons are assembled with commercial DMT-deoxynucleoside-methyl-phosphoramidites while mutant codons are assembled with Fmoc-deoxynucleoside-methyl-phosphoramidites in an NNG/C fashion in a single synthesis column. This method is easily automated and suitable for low mutagenesis rates and large windows, such as those required for directed evolution and alanine scanning. Through the assembly of three oligonucleotide libraries at different mutagenesis rates, followed by cloning at the polylinker region of plasmid pUC18 and sequencing of 129 clones, we concluded that the method performs essentially as intended. PMID:11160911

DEAD-box RNA helicase DDX23 modulates glioma malignancy via elevating miR-21 biogenesis.

PubMed

Yin, Jinlong; Park, Gunwoo; Lee, Jeong Eun; Choi, Eun Young; Park, Ju Young; Kim, Tae-Hoon; Park, Nayun; Jin, Xiong; Jung, Ji-Eun; Shin, Daye; Hong, Jun Hee; Kim, Hyunggee; Yoo, Heon; Lee, Seung-Hoon; Kim, Youn-Jae; Park, Jong Bae; Kim, Jong Heon

2015-09-01

Upregulation of microRNA-21 (miR-21) is known to be strongly associated with the proliferation, invasion, and radio-resistance of glioma cells. However, the regulatory mechanism that governs the biogenesis of miR-21 in glioma is still unclear. Here, we demonstrate that the DEAD-box RNA helicase, DDX23, promotes miR-21 biogenesis at the post-transcriptional level. The expression of DDX23 was enhanced in glioma tissues compared to normal brain, and expression level of DDX23 was highly associated with poor survival of glioma patients. Specific knockdown of DDX23 expression suppressed glioma cell proliferation and invasion in vitro and in vivo, which is similar to the function of miR-21. We found that DDX23 increased the level of miR-21 by promoting primary-to-precursor processing of miR-21 through an interaction with the Drosha microprocessor. Mutagenesis experiments critically demonstrated that the helicase activity of DDX23 was essential for the processing (cropping) of miR-21, and we further found that ivermectin, a RNA helicase inhibitor, decreased miR-21 levels by potentially inhibiting DDX23 activity and blocked invasion and cell proliferation. Moreover, treatment of ivermectin decreased glioma growth in mouse xenografts. Taken together, these results suggest that DDX23 plays an essential role in glioma progression, and might thus be a potential novel target for the therapeutic treatment of glioma. © The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Concomitant Lethal Mutagenesis of Human Immunodeficiency Virus Type 1

PubMed Central

Dapp, Michael J.; Holtz, Colleen M.; Mansky, Louis M.

2012-01-01

RNA virus population dynamics is complex, and sophisticated approaches are needed in many cases for therapeutic intervention. One such approach, termed lethal mutagenesis, is directed at targeting the virus population structure for extinction or error catastrophe. Previous studies have demonstrated the concept of this approach with human immunodeficiency virus type 1 (HIV-1) by use of chemical mutagens (i.e., 5-azacytidine) as well as by host factors with mutagenic properties (i.e., APOBEC3G). In this study, these two unrelated mutagenic agents were used concomitantly to investigate the interplay of these distinct mutagenic mechanisms. Specifically, an HIV-1 was produced from APOBEC3G (A3G)-expressing cells and used to infect permissive target cells treated with 5-azacytidine (5-AZC). Reduced viral infectivity and increased viral mutagenesis was observed with both the viral mutagen (i.e., G-to-C mutations) and the host restriction factor (i.e., G-to-A mutations); however, when combined, had complex interactions. Intriguingly, nucleotide sequence analysis revealed that concomitant HIV-1 exposure to both 5-AZC and A3G resulted in an increase of G-to-A viral mutagenesis at the expense of G-to-C mutagenesis. A3G catalytic activity was required for the diminution in G-to-C mutagenesis. Taken together, our findings provide the first demonstration for potentiation of the mutagenic effect of a cytosine analog by A3G expression, resulting in concomitant HIV-1 lethal mutagenesis. PMID:22426127

Evidence that selected amplification of a bacterial lac frameshift allele stimulates Lac(+) reversion (adaptive mutation) with or without general hypermutability.

PubMed Central

Slechta, E Susan; Liu, Jing; Andersson, Dan I; Roth, John R

2002-01-01

In the genetic system of Cairns and Foster, a nongrowing population of an E. coli lac frameshift mutant appears to specifically accumulate Lac(+) revertants when starved on medium including lactose (adaptive mutation). This behavior has been attributed to stress-induced general mutagenesis in a subpopulation of starved cells (the hypermutable state model). We have suggested that, on the contrary, stress has no direct effect on mutability but favors only growth of cells that amplify their leaky mutant lac region (the amplification mutagenesis model). Selection enhances reversion primarily by increasing the mutant lac copy number within each developing clone on the selection plate. The observed general mutagenesis is attributed to a side effect of growth with an amplification-induction of SOS by DNA fragments released from a tandem array of lac copies. Here we show that the S. enterica version of the Cairns system shows SOS-dependent general mutagenesis and behaves in every way like the original E. coli system. In both systems, lac revertants are mutagenized during selection. Eliminating the 35-fold increase in mutation rate reduces revertant number only 2- to 4-fold. This discrepancy is due to continued growth of amplification cells until some clones manage to revert without mutagenesis solely by increasing their lac copy number. Reversion in the absence of mutagenesis is still dependent on RecA function, as expected if it depends on lac amplification (a recombination-dependent process). These observations support the amplification mutagenesis model. PMID:12136002

Use of the Photoactic Ability of a Bacterium to Teach the Genetic Principles of Random Mutagenesis & Mutant Screening

ERIC Educational Resources Information Center

Din, Neena; Bird, Terry H.; Berleman, James E.

2007-01-01

In this article, the authors present a laboratory activity that relies on the use of a very versatile bacterial system to introduce the concept of how mutagenesis can be used for molecular and genetic analysis of living organisms. They have used the techniques of random mutagenesis and selection/screening to obtain strains of the organism "R.…

MMS Exposure Promotes Increased MtDNA Mutagenesis in the Presence of Replication-Defective Disease-Associated DNA Polymerase γ Variants

PubMed Central

Stumpf, Jeffrey D.; Copeland, William C.

2014-01-01

Mitochondrial DNA (mtDNA) encodes proteins essential for ATP production. Mutant variants of the mtDNA polymerase cause mutagenesis that contributes to aging, genetic diseases, and sensitivity to environmental agents. We interrogated mtDNA replication in Saccharomyces cerevisiae strains with disease-associated mutations affecting conserved regions of the mtDNA polymerase, Mip1, in the presence of the wild type Mip1. Mutant frequency arising from mtDNA base substitutions that confer erythromycin resistance and deletions between 21-nucleotide direct repeats was determined. Previously, increased mutagenesis was observed in strains encoding mutant variants that were insufficient to maintain mtDNA and that were not expected to reduce polymerase fidelity or exonuclease proofreading. Increased mutagenesis could be explained by mutant variants stalling the replication fork, thereby predisposing the template DNA to irreparable damage that is bypassed with poor fidelity. This hypothesis suggests that the exogenous base-alkylating agent, methyl methanesulfonate (MMS), would further increase mtDNA mutagenesis. Mitochondrial mutagenesis associated with MMS exposure was increased up to 30-fold in mip1 mutants containing disease-associated alterations that affect polymerase activity. Disrupting exonuclease activity of mutant variants was not associated with increased spontaneous mutagenesis compared with exonuclease-proficient alleles, suggesting that most or all of the mtDNA was replicated by wild type Mip1. A novel subset of C to G transversions was responsible for about half of the mutants arising after MMS exposure implicating error-prone bypass of methylated cytosines as the predominant mutational mechanism. Exposure to MMS does not disrupt exonuclease activity that suppresses deletions between 21-nucleotide direct repeats, suggesting the MMS-induce mutagenesis is not explained by inactivated exonuclease activity. Further, trace amounts of CdCl2 inhibit mtDNA replication but suppresses MMS-induced mutagenesis. These results suggest a novel mechanism wherein mutations that lead to hypermutation by DNA base-damaging agents and associate with mitochondrial disease may contribute to previously unexplained phenomena, such as the wide variation of age of disease onset and acquired mitochondrial toxicities. PMID:25340760

Defect of Fe-S cluster binding by DNA polymerase δ in yeast suppresses UV-induced mutagenesis, but enhances DNA polymerase ζ - dependent spontaneous mutagenesis.

PubMed

Stepchenkova, E I; Tarakhovskaya, E R; Siebler, H M; Pavlov, Y I

2017-01-01

Eukaryotic genomes are duplicated by a complex machinery, utilizing high fidelity replicative B-family DNA polymerases (pols) α, δ and ε. Specialized error-prone pol ζ, the fourth B-family member, is recruited when DNA synthesis by the accurate trio is impeded by replication stress or DNA damage. The damage tolerance mechanism dependent on pol ζ prevents DNA/genome instability and cell death at the expense of increased mutation rates. The pol switches occurring during this specialized replication are not fully understood. The loss of pol ζ results in the absence of induced mutagenesis and suppression of spontaneous mutagenesis. Disruption of the Fe-S cluster motif that abolish the interaction of the C-terminal domain (CTD) of the catalytic subunit of pol ζ with its accessory subunits, which are shared with pol δ, leads to a similar defect in induced mutagenesis. Intriguingly, the pol3-13 mutation that affects the Fe-S cluster in the CTD of the catalytic subunit of pol δ also leads to defective induced mutagenesis, suggesting the possibility that Fe-S clusters are essential for the pol switches during replication of damaged DNA. We confirmed that yeast strains with the pol3-13 mutation are UV-sensitive and defective in UV-induced mutagenesis. However, they have increased spontaneous mutation rates. We found that this increase is dependent on functional pol ζ. In the pol3-13 mutant strain with defective pol δ, there is a sharp increase in transversions and complex mutations, which require functional pol ζ, and an increase in the occurrence of large deletions, whose size is controlled by pol ζ. Therefore, the pol3-13 mutation abrogates pol ζ-dependent induced mutagenesis, but allows for pol ζ recruitment for the generation of spontaneous mutations and prevention of larger deletions. These results reveal differential control of the two major types of pol ζ-dependent mutagenesis by the Fe-S cluster present in replicative pol δ. Copyright © 2016 Elsevier B.V. All rights reserved.

MMS exposure promotes increased MtDNA mutagenesis in the presence of replication-defective disease-associated DNA polymerase γ variants.

PubMed

Stumpf, Jeffrey D; Copeland, William C

2014-10-01

Mitochondrial DNA (mtDNA) encodes proteins essential for ATP production. Mutant variants of the mtDNA polymerase cause mutagenesis that contributes to aging, genetic diseases, and sensitivity to environmental agents. We interrogated mtDNA replication in Saccharomyces cerevisiae strains with disease-associated mutations affecting conserved regions of the mtDNA polymerase, Mip1, in the presence of the wild type Mip1. Mutant frequency arising from mtDNA base substitutions that confer erythromycin resistance and deletions between 21-nucleotide direct repeats was determined. Previously, increased mutagenesis was observed in strains encoding mutant variants that were insufficient to maintain mtDNA and that were not expected to reduce polymerase fidelity or exonuclease proofreading. Increased mutagenesis could be explained by mutant variants stalling the replication fork, thereby predisposing the template DNA to irreparable damage that is bypassed with poor fidelity. This hypothesis suggests that the exogenous base-alkylating agent, methyl methanesulfonate (MMS), would further increase mtDNA mutagenesis. Mitochondrial mutagenesis associated with MMS exposure was increased up to 30-fold in mip1 mutants containing disease-associated alterations that affect polymerase activity. Disrupting exonuclease activity of mutant variants was not associated with increased spontaneous mutagenesis compared with exonuclease-proficient alleles, suggesting that most or all of the mtDNA was replicated by wild type Mip1. A novel subset of C to G transversions was responsible for about half of the mutants arising after MMS exposure implicating error-prone bypass of methylated cytosines as the predominant mutational mechanism. Exposure to MMS does not disrupt exonuclease activity that suppresses deletions between 21-nucleotide direct repeats, suggesting the MMS-induce mutagenesis is not explained by inactivated exonuclease activity. Further, trace amounts of CdCl2 inhibit mtDNA replication but suppresses MMS-induced mutagenesis. These results suggest a novel mechanism wherein mutations that lead to hypermutation by DNA base-damaging agents and associate with mitochondrial disease may contribute to previously unexplained phenomena, such as the wide variation of age of disease onset and acquired mitochondrial toxicities.

Tenofovir alafenamide demonstrates broad cross-genotype activity against wild-type HBV clinical isolates and maintains susceptibility to drug-resistant HBV isolates in vitro.

PubMed

Liu, Yang; Miller, Michael D; Kitrinos, Kathryn M

2017-03-01

Tenofovir alafenamide (TAF) is a novel prodrug of tenofovir (TFV). This study evaluated the antiviral activity of TAF against wild-type genotype A-H HBV clinical isolates as well as adefovir-resistant, lamivudine-resistant, and entecavir-resistant HBV isolates. Full length HBV genomes or the polymerase/reverse transcriptase (pol/RT) region from treatment-naïve patients infected with HBV genotypes A-H were amplified and cloned into an expression vector under the control of a CMV promoter. In addition, 11 drug resistant HBV constructs were created by site-directed mutagenesis of a full length genotype D construct. Activity of TAF was measured by transfection of each construct into HepG2 cells and assessment of HBV DNA levels following treatment across a range of TAF concentrations. TAF activity in vitro was similar against wild-type genotype A-H HBV clinical isolates. All lamivudine- and entecavir-resistant isolates and 4/5 adefovir-resistant isolates were found to be sensitive to inhibition by TAF in vitro as compared to the wild-type isolate. The adefovir-resistant isolate rtA181V + rtN236T exhibited low-level reduced susceptibility to TAF. TAF is similarly active in vitro against wild-type genotype A-H HBV clinical isolates. The TAF sensitivity results for all drug-resistant isolates are consistent with what has been observed with the parent drug TFV. The in vitro cell-based HBV phenotyping assay results support the use of TAF in treatment of HBV infected subjects with diverse HBV genotypes, in both treatment-naive and treatment-experienced HBV infected patients. Copyright © 2016 Elsevier B.V. All rights reserved.

A Simple Combinatorial Codon Mutagenesis Method for Targeted Protein Engineering.

PubMed

Belsare, Ketaki D; Andorfer, Mary C; Cardenas, Frida S; Chael, Julia R; Park, Hyun June; Lewis, Jared C

2017-03-17

Directed evolution is a powerful tool for optimizing enzymes, and mutagenesis methods that improve enzyme library quality can significantly expedite the evolution process. Here, we report a simple method for targeted combinatorial codon mutagenesis (CCM). To demonstrate the utility of this method for protein engineering, CCM libraries were constructed for cytochrome P450 BM3 , pfu prolyl oligopeptidase, and the flavin-dependent halogenase RebH; 10-26 sites were targeted for codon mutagenesis in each of these enzymes, and libraries with a tunable average of 1-7 codon mutations per gene were generated. Each of these libraries provided improved enzymes for their respective transformations, which highlights the generality, simplicity, and tunability of CCM for targeted protein engineering.

Pilot study of large-scale production of mutant pigs by ENU mutagenesis.

PubMed

Hai, Tang; Cao, Chunwei; Shang, Haitao; Guo, Weiwei; Mu, Yanshuang; Yang, Shulin; Zhang, Ying; Zheng, Qiantao; Zhang, Tao; Wang, Xianlong; Liu, Yu; Kong, Qingran; Li, Kui; Wang, Dayu; Qi, Meng; Hong, Qianlong; Zhang, Rui; Wang, Xiupeng; Jia, Qitao; Wang, Xiao; Qin, Guosong; Li, Yongshun; Luo, Ailing; Jin, Weiwu; Yao, Jing; Huang, Jiaojiao; Zhang, Hongyong; Li, Menghua; Xie, Xiangmo; Zheng, Xuejuan; Guo, Kenan; Wang, Qinghua; Zhang, Shibin; Li, Liang; Xie, Fei; Zhang, Yu; Weng, Xiaogang; Yin, Zhi; Hu, Kui; Cong, Yimei; Zheng, Peng; Zou, Hailong; Xin, Leilei; Xia, Jihan; Ruan, Jinxue; Li, Hegang; Zhao, Weiming; Yuan, Jing; Liu, Zizhan; Gu, Weiwang; Li, Ming; Wang, Yong; Wang, Hongmei; Yang, Shiming; Liu, Zhonghua; Wei, Hong; Zhao, Jianguo; Zhou, Qi; Meng, Anming

2017-06-22

N-ethyl-N-nitrosourea (ENU) mutagenesis is a powerful tool to generate mutants on a large scale efficiently, and to discover genes with novel functions at the whole-genome level in Caenorhabditis elegans, flies, zebrafish and mice, but it has never been tried in large model animals. We describe a successful systematic three-generation ENU mutagenesis screening in pigs with the establishment of the Chinese Swine Mutagenesis Consortium. A total of 6,770 G1 and 6,800 G3 pigs were screened, 36 dominant and 91 recessive novel pig families with various phenotypes were established. The causative mutations in 10 mutant families were further mapped. As examples, the mutation of SOX10 (R109W) in pig causes inner ear malfunctions and mimics human Mondini dysplasia, and upregulated expression of FBXO32 is associated with congenital splay legs. This study demonstrates the feasibility of artificial random mutagenesis in pigs and opens an avenue for generating a reservoir of mutants for agricultural production and biomedical research.

Improvement of Biocatalysts for Industrial and Environmental Purposes by Saturation Mutagenesis

PubMed Central

Valetti, Francesca; Gilardi, Gianfranco

2013-01-01

Laboratory evolution techniques are becoming increasingly widespread among protein engineers for the development of novel and designed biocatalysts. The palette of different approaches ranges from complete randomized strategies to rational and structure-guided mutagenesis, with a wide variety of costs, impacts, drawbacks and relevance to biotechnology. A technique that convincingly compromises the extremes of fully randomized vs. rational mutagenesis, with a high benefit/cost ratio, is saturation mutagenesis. Here we will present and discuss this approach in its many facets, also tackling the issue of randomization, statistical evaluation of library completeness and throughput efficiency of screening methods. Successful recent applications covering different classes of enzymes will be presented referring to the literature and to research lines pursued in our group. The focus is put on saturation mutagenesis as a tool for designing novel biocatalysts specifically relevant to production of fine chemicals for improving bulk enzymes for industry and engineering technical enzymes involved in treatment of waste, detoxification and production of clean energy from renewable sources. PMID:24970191

β-lactam antibiotics promote bacterial mutagenesis via an RpoS-mediated reduction in replication fidelity

PubMed Central

Gutierrez, A.; Laureti, L.; Crussard, S.; Abida, H.; Rodríguez-Rojas, A.; Blázquez, J.; Baharoglu, Z.; Mazel, D.; Darfeuille, F.; Vogel, J.; Matic, I.

2013-01-01

Regardless of their targets and modes of action, subinhibitory concentrations of antibiotics can have an impact on cell physiology and trigger a large variety of cellular responses in different bacterial species. Subinhibitory concentrations of β-lactam antibiotics cause reactive oxygen species production and induce PolIV-dependent mutagenesis in Escherichia coli. Here we show that subinhibitory concentrations of β-lactam antibiotics induce the RpoS regulon. RpoS-regulon induction is required for PolIV-dependent mutagenesis because it diminishes the control of DNA-replication fidelity by depleting MutS in E. coli, Vibrio cholerae and Pseudomonas aeruginosa. We also show that in E. coli, the reduction in mismatch-repair activity is mediated by SdsR, the RpoS-controlled small RNA. In summary, we show that mutagenesis induced by subinhibitory concentrations of antibiotics is a genetically controlled process. Because this mutagenesis can generate mutations conferring antibiotic resistance, it should be taken into consideration for the development of more efficient antimicrobial therapeutic strategies. PMID:23511474

High-Resolution Functional Mapping of the Venezuelan Equine Encephalitis Virus Genome by Insertional Mutagenesis and Massively Parallel Sequencing

DTIC Science & Technology

2010-10-14

High-Resolution Functional Mapping of the Venezuelan Equine Encephalitis Virus Genome by Insertional Mutagenesis and Massively Parallel Sequencing...Venezuelan equine encephalitis virus (VEEV) genome. We initially used a capillary electrophoresis method to gain insight into the role of the VEEV...Smith JM, Schmaljohn CS (2010) High-Resolution Functional Mapping of the Venezuelan Equine Encephalitis Virus Genome by Insertional Mutagenesis and

Polishing the craft of genetic diversity creation in directed evolution.

PubMed

Tee, Kang Lan; Wong, Tuck Seng

2013-12-01

Genetic diversity creation is a core technology in directed evolution where a high quality mutant library is crucial to its success. Owing to its importance, the technology in genetic diversity creation has seen rapid development over the years and its application has diversified into other fields of scientific research. The advances in molecular cloning and mutagenesis since 2008 were reviewed. Specifically, new cloning techniques were classified based on their principles of complementary overhangs, homologous sequences, overlapping PCR and megaprimers and the advantages, drawbacks and performances of these methods were highlighted. New mutagenesis methods developed for random mutagenesis, focused mutagenesis and DNA recombination were surveyed. The technical requirements of these methods and the mutational spectra were compared and discussed with references to commonly used techniques. The trends of mutant library preparation were summarised. Challenges in genetic diversity creation were discussed with emphases on creating "smart" libraries, controlling the mutagenesis spectrum and specific challenges in each group of mutagenesis methods. An outline of the wider applications of genetic diversity creation includes genome engineering, viral evolution, metagenomics and a study of protein functions. The review ends with an outlook for genetic diversity creation and the prospective developments that can have future impact in this field. © 2013. Published by Elsevier Inc. All rights reserved.

DC-Analyzer-facilitated combinatorial strategy for rapid directed evolution of functional enzymes with multiple mutagenesis sites.

PubMed

Wang, Xiong; Zheng, Kai; Zheng, Huayu; Nie, Hongli; Yang, Zujun; Tang, Lixia

2014-12-20

Iterative saturation mutagenesis (ISM) has been shown to be a powerful method for directed evolution. In this study, the approach was modified (termed M-ISM) by combining the single-site saturation mutagenesis method with a DC-Analyzer-facilitated combinatorial strategy, aiming to evolve novel biocatalysts efficiently in the case where multiple sites are targeted simultaneously. Initially, all target sites were explored individually by constructing single-site saturation mutagenesis libraries. Next, the top two to four variants in each library were selected and combined using the DC-Analyzer-facilitated combinatorial strategy. In addition to site-saturation mutagenesis, iterative saturation mutagenesis also needed to be performed. The advantages of M-ISM over ISM were that the screening effort is greatly reduced, and the entire M-ISM procedure was less time-consuming. The M-ISM strategy was successfully applied to the randomization of halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) when five interesting sites were targeted simultaneously. After screening 900 clones in total, six positive mutants were obtained. These mutants exhibited 4.0- to 9.3-fold higher k(cat) values than did the wild-type HheC toward 1,3-dichloro-2-propanol. However, with the ISM strategy, the best hit showed a 5.9-fold higher k(cat) value toward 1,3-DCP than the wild-type HheC, which was obtained after screening 4000 clones from four rounds of mutagenesis. Therefore, M-ISM could serve as a simple and efficient version of ISM for the randomization of target genes with multiple positions of interest.

Identification of Mutation Accumulation as Resistance Mechanism Emerging in First-Line Osimertinib Treatment.

PubMed

Uchibori, Ken; Inase, Naohiko; Nishio, Makoto; Fujita, Naoya; Katayama, Ryohei

2018-04-24

The survival of patients with EGFR mutation-positive lung cancer has dramatically improved since the introduction of EGFR tyrosine kinase inhibitors (EGFR-TKIs). Recently, osimertinib showed significantly prolonged progression-free survival than first-generation EGFR-TKI in first-line treatment, suggesting that a paradigm change that would move osimetinib to first-line treatment is indicated. We performed N-ethyl-N-nitrosourea (ENU) mutagenesis screening to uncover the resistant mechanism in first- and second-line osimertinib treatment. Ba/F3 cells harboring EGFR activating-mutation with or without secondary resistant mutation were exposed to ENU for 24 hours to introduce random mutations and selected with gefitinib, afatinib, or osimertinib. Mutations of emerging resistant cells were assessed. The resistance of T790M and C797S to gefitinib and osimertinib, respectively, was prevalent in the mutagenesis screening with the Ba/F3 cells harboring activating-mutation alone. From C797S/activating-mutation expressing Ba/F3, the additional T790M was a major resistant mechanism in gefitinib and afatinib selection and the additional T854A and L792H were minor resistance mechanisms only in afatinib selection. However, the additional T854A or L792H mediated resistance to all classes of EGFR-TKI. Surprisingly, no resistant clone due to secondary mutation emerged from activating-mutation alone in the gefitinib + osimertinib selection. We showed the resistance mechanism to EGFR-TKI focusing on first- and second-line osimertinib using ENU mutagenesis screening. Additional T854A and L792H on C797S/activating-mutation were found as afatinib resistance and not as gefitinib resistance. Thus, compared to afatinib, the first-generation EGFR-TKI might be preferable as second-line treatment to C797S/activating-mutation emerging after first-line osimertinib treatment. Copyright © 2018 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

Suppression of HBV replication by the expression of nickase- and nuclease dead-Cas9.

PubMed

Kurihara, Takeshi; Fukuhara, Takasuke; Ono, Chikako; Yamamoto, Satomi; Uemura, Kentaro; Okamoto, Toru; Sugiyama, Masaya; Motooka, Daisuke; Nakamura, Shota; Ikawa, Masato; Mizokami, Masashi; Maehara, Yoshihiko; Matsuura, Yoshiharu

2017-07-21

Complete removal of hepatitis B virus (HBV) DNA from nuclei is difficult by the current therapies. Recent reports have shown that a novel genome-editing tool using Cas9 with a single-guide RNA (sgRNA) system can cleave the HBV genome in vitro and in vivo. However, induction of a double-strand break (DSB) on the targeted genome by Cas9 risks undesirable off-target cleavage on the host genome. Nickase-Cas9 cleaves a single strand of DNA, and thereby two sgRNAs are required for inducing DSBs. To avoid Cas9-induced off-target mutagenesis, we examined the effects of the expressions of nickase-Cas9 and nuclease dead Cas9 (d-Cas9) with sgRNAs on HBV replication. The expression of nickase-Cas9 with a pair of sgRNAs cleaved the target HBV genome and suppressed the viral-protein expression and HBV replication in vitro. Moreover, nickase-Cas9 with the sgRNA pair cleaved the targeted HBV genome in mouse liver. Interestingly, d-Cas9 expression with the sgRNAs also suppressed HBV replication in vitro without cleaving the HBV genome. These results suggest the possible use of nickase-Cas9 and d-Cas9 with a pair of sgRNAs for eliminating HBV DNA from the livers of chronic hepatitis B patients with low risk of undesirable off-target mutation on the host genome.

Molecular control mechanisms of lysine and threonine biosynthesis in amino acid-producing corynebacteria: redirecting carbon flow.

PubMed

Malumbres, M; Martín, J F

1996-10-01

Threonine and lysine are two of the economically most important essential amino acids. They are produced industrially by species of the genera Corynebacterium and Brevibacterium. The branched biosynthetic pathway of these amino acids in corynebacteria is unusual in gene organization and in the control of key enzymatic steps with respect to other microorganisms. This article reviews the molecular control mechanisms of the biosynthetic pathways leading to threonine and lysine in corynebacteria, and their implications in the production of these amino acids. Carbon flux can be redirected at branch points by gene disruption of the competing pathways for lysine or threonine. Removal of bottlenecks has been achieved by amplification of genes which encode feedback resistant aspartokinase and homoserine dehydrogenase (obtained by in vitro directed mutagenesis).

Environmental Stress Induces Trinucleotide Repeat Mutagenesis in Human Cells by Alt-Nonhomologous End Joining Repair.

PubMed

Chatterjee, Nimrat; Lin, Yunfu; Yotnda, Patricia; Wilson, John H

2016-07-31

Multiple pathways modulate the dynamic mutability of trinucleotide repeats (TNRs), which are implicated in neurodegenerative disease and evolution. Recently, we reported that environmental stresses induce TNR mutagenesis via stress responses and rereplication, with more than 50% of mutants carrying deletions or insertions-molecular signatures of DNA double-strand break repair. We now show that knockdown of alt-nonhomologous end joining (alt-NHEJ) components-XRCC1, LIG3, and PARP1-suppresses stress-induced TNR mutagenesis, in contrast to the components of homologous recombination and NHEJ, which have no effect. Thus, alt-NHEJ, which contributes to genetic mutability in cancer cells, also plays a novel role in environmental stress-induced TNR mutagenesis. Published by Elsevier Ltd.

Evaluating the Value of Augmenting In Vitro Hazard Assessment with Exposure and Pharmacokinetics Considerations for Chemical Prioritization

EPA Science Inventory

Over time, toxicity-testing paradigms have progressed from low-throughput in vivo animal studies for limited numbers of chemicals to high-throughput (HT) in vitro screening assays for thousands of chemicals. Such HT in vitro methods, along with HT in silico predictions of popula...

Responses of antioxidant enzymes, photosynthetic pigments and carbohydrates in micropropagated Pitcairnia encholirioides L.B. Sm. (Bromeliaceae) under ex vitro water deficit and after rehydration.

PubMed

Resende, C F; Pacheco, V S; Dornellas, F F; Oliveira, A M S; Freitas, J C E; Peixoto, P H P

2018-03-22

In this study, the activities of antioxidant enzymes, photosynthetic pigments, proline and carbohydrate contents in Pitcairnia encholirioides under ex vitro conditions of water deficit were evaluated. Results show that plants under progressive water stress, previously in vitro cultured in media supplemented with 30 g L-1 sucrose and GA3, accumulated more proline and increased peroxidase (POD) activity and the contents of photosynthetic pigments and carbohydrates. For plants previously in vitro cultured with 15 g L-1 sucrose and NAA, no differences were found for proline content and there were reductions in activities of peroxidase (POD), catalase (CAT) and poliphenoloxidase (PPO), and in contents of carbohydrates, with progress of ex vitro water deficit. After rehydration, plants showed physiological recovery, with enzymatic activities and contents of metabolites similar to those found in the controls not submitted to dehydration, regardless of the previous in vitro culture conditions. These results show that micropropagated P. encholirioides has high tolerance to dehydration once in ex vitro conditions, which can ensure the survival of plants from tissue culture when transferred to its natural environment, emphasizing the importance of such biotechnology for the propagation of endangered species.

Progress and prospects: foamy virus vectors enter a new age.

PubMed

Erlwein, O; McClure, M O

2010-12-01

Foamy viruses, distantly related to the major subfamily of Retroviruses, Orthoretroviruses that include oncoviruses (for example, murine leukemia virus (MLV)) and lentiviruses (human immunodeficiency virus (HIV)), are endemic in mammalian species, but not in human populations. Humans infected by accidental or occupational exposure remain well. The virus is not transmitted to others, nor is it associated with any disease. These features added to its broad host range, efficient transduction of progenitor cells and an integration profile less likely to induce insertional mutagenesis, make these viruses attractive as vectors. Long-term reversal of disease phenotype in dogs with the genetic defect, leukocyte adhesion deficiency, by foamy virus vector therapy strengthens the case for their clinical exploitation.

The growing and glowing toolbox of fluorescent and photoactive proteins

PubMed Central

Rodriguez, Erik A.; Campbell, Robert E.; Lin, John Y.; Lin, Michael Z.; Miyawaki, Atsushi; Palmer, Amy E.; Shu, Xiaokun; Zhang, Jin

2016-01-01

Over the past 20 years, protein engineering has been extensively used to improve and modify the fundamental properties of fluorescent proteins (FPs) with the goal of adapting them for a fantastic range of applications. FPs have been modified by a combination of rational design, structure-based mutagenesis, and countless cycles of directed evolution (gene diversification followed by selection of clones with desired properties) that have collectively pushed the properties to photophysical and biochemical extremes. In this review, we attempt to provide both a summary of the progress that has been made during the past two decades, and a broad overview of the current state of FP development and applications in mammalian systems. PMID:27814948

Oxidative stress signaling to chromatin in health and disease

PubMed Central

Kreuz, Sarah; Fischle, Wolfgang

2016-01-01

Oxidative stress has a significant impact on the development and progression of common human pathologies, including cancer, diabetes, hypertension and neurodegenerative diseases. Increasing evidence suggests that oxidative stress globally influences chromatin structure, DNA methylation, enzymatic and non-enzymatic post-translational modifications of histones and DNA-binding proteins. The effects of oxidative stress on these chromatin alterations mediate a number of cellular changes, including modulation of gene expression, cell death, cell survival and mutagenesis, which are disease-driving mechanisms in human pathologies. Targeting oxidative stress-dependent pathways is thus a promising strategy for the prevention and treatment of these diseases. We summarize recent research developments connecting oxidative stress and chromatin regulation. PMID:27319358

The MurC ligase essential for peptidoglycan biosynthesis is regulated by the serine/threonine protein kinase PknA in Corynebacterium glutamicum.

PubMed

Fiuza, Maria; Canova, Marc J; Patin, Delphine; Letek, Michal; Zanella-Cléon, Isabelle; Becchi, Michel; Mateos, Luís M; Mengin-Lecreulx, Dominique; Molle, Virginie; Gil, José A

2008-12-26

The Mur ligases play an essential role in the biosynthesis of bacterial cell-wall peptidoglycan and thus represent attractive targets for the design of novel antibacterials. These enzymes catalyze the stepwise formation of the peptide moiety of the peptidoglycan disaccharide peptide monomer unit. MurC is responsible of the addition of the first residue (L-alanine) onto the nucleotide precursor UDP-MurNAc. Phosphorylation of proteins by Ser/Thr protein kinases has recently emerged as a major physiological mechanism of regulation in prokaryotes. Herein, the hypothesis of a phosphorylation-dependent mechanism of regulation of the MurC activity was investigated in Corynebacterium glutamicum. We showed that MurC was phosphorylated in vitro by the PknA protein kinase. An analysis of the phosphoamino acid content indicated that phosphorylation exclusively occurred on threonine residues. Six phosphoacceptor residues were identified by mass spectrometry analysis, and we confirmed that mutagenesis to alanine residues totally abolished PknA-dependent phosphorylation of MurC. In vitro and in vivo ligase activity assays showed that the catalytic activity of MurC was impaired following mutation of these threonine residues. Further in vitro assays revealed that the activity of the MurC-phosphorylated isoform was severely decreased compared with the non-phosphorylated protein. To our knowledge, this is the first demonstration of a MurC ligase phosphorylation in vitro. The finding that phosphorylation is correlated with a decrease in MurC enzymatic activity could have significant consequences in the regulation of peptidoglycan biosynthesis.

The MurC Ligase Essential for Peptidoglycan Biosynthesis Is Regulated by the Serine/Threonine Protein Kinase PknA in Corynebacterium glutamicum*

PubMed Central

Fiuza, Maria; Canova, Marc J.; Patin, Delphine; Letek, Michal; Zanella-Cléon, Isabelle; Becchi, Michel; Mateos, Luís M.; Mengin-Lecreulx, Dominique; Molle, Virginie; Gil, José A.

2008-01-01

The Mur ligases play an essential role in the biosynthesis of bacterial cell-wall peptidoglycan and thus represent attractive targets for the design of novel antibacterials. These enzymes catalyze the stepwise formation of the peptide moiety of the peptidoglycan disaccharide peptide monomer unit. MurC is responsible of the addition of the first residue (l-alanine) onto the nucleotide precursor UDP-MurNAc. Phosphorylation of proteins by Ser/Thr protein kinases has recently emerged as a major physiological mechanism of regulation in prokaryotes. Herein, the hypothesis of a phosphorylation-dependent mechanism of regulation of the MurC activity was investigated in Corynebacterium glutamicum. We showed that MurC was phosphorylated in vitro by the PknA protein kinase. An analysis of the phosphoamino acid content indicated that phosphorylation exclusively occurred on threonine residues. Six phosphoacceptor residues were identified by mass spectrometry analysis, and we confirmed that mutagenesis to alanine residues totally abolished PknA-dependent phosphorylation of MurC. In vitro and in vivo ligase activity assays showed that the catalytic activity of MurC was impaired following mutation of these threonine residues. Further in vitro assays revealed that the activity of the MurC-phosphorylated isoform was severely decreased compared with the non-phosphorylated protein. To our knowledge, this is the first demonstration of a MurC ligase phosphorylation in vitro. The finding that phosphorylation is correlated with a decrease in MurC enzymatic activity could have significant consequences in the regulation of peptidoglycan biosynthesis. PMID:18974047

Ribosome display: next-generation display technologies for production of antibodies in vitro.

PubMed

He, Mingyue; Khan, Farid

2005-06-01

Antibodies represent an important and growing class of biologic research reagents and biopharmaceutical products. They can be used as therapeutics in a variety of diseases. With the rapid expansion of proteomic studies and biomarker discovery, there is a need for the generation of highly specific binding reagents to study the vast number of proteins encoded by the genome. Display technologies provide powerful tools for obtaining antibodies. Aside from the preservation of natural antibody repertoires, they are capable of exploiting diversity by DNA recombination to create very large libraries for selection of novel molecules. In contrast to in vivo immunization processes, display technologies allow selection of antibodies under in vitro-defined selection condition(s), resulting in enrichment of antibodies with desired properties from large populations. In addition, in vitro selection enables the isolation of antibodies against difficult antigens including self-antigens, and this can be applied to the generation of human antibodies against human targets. Display technologies can also be combined with DNA mutagenesis for antibody evolution in vitro. Some methods are amenable to automation, permitting high-throughput generation of antibodies. Ribosome display is considered as representative of the next generation of display technologies since it overcomes the limitations of cell-based display methods by using a cell-free system, offering advantages of screening larger libraries and continuously expanding new diversity during selection. Production of display-derived antibodies can be achieved by choosing one of a variety of prokaryotic and eukaryotic cell-based expression systems. In the near future, cell-free protein synthesis may be developed as an alternative for large-scale generation of antibodies.

Immune Selection In Vitro Reveals Human Immunodeficiency Virus Type 1 Nef Sequence Motifs Important for Its Immune Evasion Function In Vivo

PubMed Central

Lee, Patricia; Ng, Hwee L.; Yang, Otto O.

2012-01-01

Human immunodeficiency virus type 1 (HIV-1) Nef downregulates major histocompatibility complex class I (MHC-I), impairing the clearance of infected cells by CD8+ cytotoxic T lymphocytes (CTLs). While sequence motifs mediating this function have been determined by in vitro mutagenesis studies of laboratory-adapted HIV-1 molecular clones, it is unclear whether the highly variable Nef sequences of primary isolates in vivo rely on the same sequence motifs. To address this issue, nef quasispecies from nine chronically HIV-1-infected persons were examined for sequence evolution and altered MHC-I downregulatory function under Gag-specific CTL immune pressure in vitro. This selection resulted in decreased nef diversity and strong purifying selection. Site-by-site analysis identified 13 codons undergoing purifying selection and 1 undergoing positive selection. Of the former, only 6 have been reported to have roles in Nef function, including 4 associated with MHC-I downregulation. Functional testing of naturally occurring in vivo polymorphisms at the 7 sites with no previously known functional role revealed 3 mutations (A84D, Y135F, and G140R) that ablated MHC-I downregulation and 3 (N52A, S169I, and V180E) that partially impaired MHC-I downregulation. Globally, the CTL pressure in vitro selected functional Nef from the in vivo quasispecies mixtures that predominately lacked MHC-I downregulatory function at the baseline. Overall, these data demonstrate that CTL pressure exerts a strong purifying selective pressure for MHC-I downregulation and identifies novel functional motifs present in Nef sequences in vivo. PMID:22553319

Progress and future prospect of in vitro spermatogenesis

PubMed Central

Ibtisham, Fahar; Wu, Jiang; Xiao, Mei; An, Lilong; Banker, Zachary; Nawab, Aamir; Zhao, Yi; Li, Guanghui

2017-01-01

Infertility has become a major health issue in the world. It affects the social life of couples and of all infertility cases; approximately 40–50% is due to “male factor” infertility. Male infertility could be due to genetic factors, environment or due to gonadotoxic treatment. Developments in reproductive biotechnology have made it possible to rescue fertility and uphold biological fatherhood. In vitro production of haploid male germ cell is a powerful tool, not only for the treatment of infertility including oligozoospermic or azoospermic patient, but also for the fertility preservation in pre-pubertal boys whose gonadal function is threatened by gonadotoxic therapies. Genomic editing of in-vitro cultured germ cells could also potentially cure flaws in spermatogenesis due to genomic mutation. Furthermore, this ex-vivo maturation technique with genomic editing may be used to prevent paternal transmission of genomic diseases. Here, we summarize the historical progress of in vitro spermatogenesis research by using organ and cell culture techniques and the future clinical application of in vitro spermatogenesis. PMID:29029549

Fungicide-induced transposon movement in Monilinia fructicola.

PubMed

Chen, Fengping; Everhart, Sydney E; Bryson, P Karen; Luo, Chaoxi; Song, Xi; Liu, Xili; Schnabel, Guido

2015-12-01

Repeated applications of fungicides with a single mode of action are believed to select for pre-existing resistant strains in a pathogen population, while the impact of sub-lethal doses of such fungicides on sensitive members of the population is unknown. In this study, in vitro evidence is presented that continuous exposure of Monilinia fructicola mycelium to some fungicides can induce genetic change in form of transposon transposition. Three fungicide-sensitive M. fructicola isolates were exposed in 12 weekly transfers of mycelia to a dose gradient of demethylation inhibitor fungicide (DMI) SYP-Z048 and quinone outside inhibitor fungicide (QoI) azoxystrobin in solo or mixture treatments. Evidence of mutagenesis was assessed by monitoring Mftc1, a multicopy transposable element of M. fructicola, by PCR and Southern blot analysis. Movement of Mftc1 was observed following azoxystrobin and azoxystrobin plus SYP-Z048 treatments in two of the three isolates, but not in the non-fungicide-treated controls. Interestingly, the upstream promoter region of MfCYP51 was a prime target for Mftc1 transposition in these isolates. Transposition of Mftc1 was verified by Southern blot in two of three isolates from another, similar experiment following prolonged, sublethal azoxystrobin exposure, although in these isolates movement of Mftc1 in the upstream MfCYP51 promoter region was not observed. More research is warranted to determine whether fungicide-induced mutagenesis may also happen under field conditions. Copyright © 2015 Elsevier Inc. All rights reserved.

Mapping the signal peptide binding and oligomer contact sites of the core subunit of the pea twin arginine protein translocase.

PubMed

Ma, Xianyue; Cline, Kenneth

2013-03-01

Twin arginine translocation (Tat) systems of thylakoid and bacterial membranes transport folded proteins using the proton gradient as the sole energy source. Tat substrates have hydrophobic signal peptides with an essential twin arginine (RR) recognition motif. The multispanning cpTatC plays a central role in Tat operation: It binds the signal peptide, directs translocase assembly, and may facilitate translocation. An in vitro assay with pea (Pisum sativum) chloroplasts was developed to conduct mutagenesis and analysis of cpTatC functions. Ala scanning mutagenesis identified mutants defective in substrate binding and receptor complex assembly. Mutations in the N terminus (S1) and first stromal loop (S2) caused specific defects in signal peptide recognition. Cys matching between substrate and imported cpTatC confirmed that S1 and S2 directly and specifically bind the RR proximal region of the signal peptide. Mutations in four lumen-proximal regions of cpTatC were defective in receptor complex assembly. Copurification and Cys matching analyses suggest that several of the lumen proximal regions may be important for cpTatC-cpTatC interactions. Surprisingly, RR binding domains of adjacent cpTatCs directed strong cpTatC-cpTatC cross-linking. This suggests clustering of binding sites on the multivalent receptor complex and explains the ability of Tat to transport cross-linked multimers. Transport of substrate proteins cross-linked to the signal peptide binding site tentatively identified mutants impaired in the translocation step.

Use of the multipurpose transposon Tn KPK2 for the mutational analysis of chromosomal regions upstream and downstream of the sipF gene in Bradyrhizobium japonicum.

PubMed

Müller, P

2004-04-01

The DNA regions upstream and downstream of the Bradyrhizobium japonicum gene sipF were cloned by in vivo techniques and subsequently sequenced. In order to study the function of the predicted genes, a new transposon for in vitro mutagenesis, Tn KPK2, was constructed. This mutagenesis system has a number of advantages over other transposons. Tn KPK2 itself has no transposase gene, making transposition events stable. Extremely short inverted repeats minimize the length of the transposable element and facilitate the determination of the nucleotide sequence of the flanking regions. Since the transposable element carries a promoterless ' phoA reporter gene, the appearance of functional PhoA fusion proteins indicates that Tn KPK2 has inserted in a gene encoding a periplasmic or secreted protein. Although such events are extremely rare, because the transposon has to insert in-frame, in the correct orientation, and at an appropriate location in the target molecule, a direct screening procedure on agar indicator plates permits the identification of candidate clones from large numbers of colonies. In this study, Tn KPK2 was used for the construction of various symbiotic mutants of B. japonicum. One of the mutant strains, A2-10, which is defective in a gene encoding a protein that comigrates with bacterioferritin ( bcpB), was found to induce the formation of small and ineffective nodules.

Molecular mechanism of the Syk activation switch.

PubMed

Tsang, Emily; Giannetti, Anthony M; Shaw, David; Dinh, Marie; Tse, Joyce K Y; Gandhi, Shaan; Ho, Hoangdung; Wang, Sandra; Papp, Eva; Bradshaw, J Michael

2008-11-21

Many immune signaling pathways require activation of the Syk tyrosine kinase to link ligation of surface receptors to changes in gene expression. Despite the central role of Syk in these pathways, the Syk activation process remains poorly understood. In this work we quantitatively characterized the molecular mechanism of Syk activation in vitro using a real time fluorescence kinase assay, mutagenesis, and other biochemical techniques. We found that dephosphorylated full-length Syk demonstrates a low initial rate of substrate phosphorylation that increases during the kinase reaction due to autophosphorylation. The initial rate of Syk activity was strongly increased by either pre-autophosphorylation or binding of phosphorylated immune tyrosine activation motif peptides, and each of these factors independently fully activated Syk. Deletion mutagenesis was used to identify regions of Syk important for regulation, and residues 340-356 of the SH2 kinase linker region were identified to be important for suppression of activity before activation. Comparison of the activation processes of Syk and Zap-70 revealed that Syk is more readily activated by autophosphorylation than Zap-70, although both kinases are rapidly activated by Src family kinases. We also studied Syk activity in B cell lysates and found endogenous Syk is also activated by phosphorylation and immune tyrosine activation motif binding. Together these experiments show that Syk functions as an "OR-gate" type of molecular switch. This mechanism of switch-like activation helps explain how Syk is both rapidly activated after receptor binding but also sustains activity over time to facilitate longer term changes in gene expression.

Control of ACAT2 liver expression by HNF4{alpha}: lesson from MODY1 patients.

PubMed

Pramfalk, C; Karlsson, E; Groop, L; Rudel, L L; Angelin, B; Eriksson, M; Parini, P

2009-08-01

ACAT2 is thought to be responsible for cholesteryl ester production in chylomicron and VLDL assembly. Recently, we identified HNF1alpha as an important regulator of the human ACAT2 promoter. Thus, we hypothesized that MODY3 (HNF1alpha gene mutations) and possibly MODY1 (HNF4alpha, upstream regulator of HNF1alpha, gene mutations) subjects may have lower VLDL esterified cholesterol. Serum analysis and lipoprotein separation using size-exclusion chromatography were performed in controls and MODY1 and MODY3 subjects. In vitro analyses included mutagenesis and cotransfections in HuH7 cells. Finally, the relevance in vivo of these findings was tested by ChIP assays in human liver. Whereas patients with MODY3 had normal lipoprotein composition, those with MODY1 had lower levels of VLDL and LDL esterified cholesterol, as well as of VLDL triglyceride. Mutagenesis revealed one important HNF4 binding site in the human ACAT2 promoter. ChIP assays and protein-to-protein interaction studies showed that HNF4alpha, directly or indirectly (via HNF1alpha), can bind to the ACAT2 promoter. We identified HNF4alpha as an important regulator of the hepatocyte-specific expression of the human ACAT2 promoter. Our results suggest that the lower levels of esterified cholesterol in VLDL- and LDL-particles in patients with MODY1 may-at least in part-be attributable to lower ACAT2 activity in these patients.

HnRNP C, YB-1 and hnRNP L coordinately enhance skipping of human MUSK exon 10 to generate a Wnt-insensitive MuSK isoform

PubMed Central

Nasrin, Farhana; Rahman, Mohammad Alinoor; Masuda, Akio; Ohe, Kenji; Takeda, Jun-ichi; Ohno, Kinji

2014-01-01

Muscle specific receptor tyrosine kinase (MuSK) is an essential postsynaptic transmembrane molecule that mediates clustering of acetylcholine receptors (AChR). MUSK exon 10 is alternatively skipped in human, but not in mouse. Skipping of this exon disrupts a cysteine-rich region (Fz-CRD), which is essential for Wnt-mediated AChR clustering. To investigate the underlying mechanisms of alternative splicing, we exploited block-scanning mutagenesis with human minigene and identified a 20-nucleotide block that contained exonic splicing silencers. Using RNA-affinity purification, mass spectrometry, and Western blotting, we identified that hnRNP C, YB-1 and hnRNP L are bound to MUSK exon 10. siRNA-mediated knockdown and cDNA overexpression confirmed the additive, as well as the independent, splicing suppressing effects of hnRNP C, YB-1 and hnRNP L. Antibody-mediated in vitro protein depletion and scanning mutagenesis additionally revealed that binding of hnRNP C to RNA subsequently promotes binding of YB-1 and hnRNP L to the immediate downstream sites and enhances exon skipping. Simultaneous tethering of two splicing trans-factors to the target confirmed the cooperative effect of YB-1 and hnRNP L on hnRNP C-mediated exon skipping. Search for a similar motif in the human genome revealed nine alternative exons that were individually or coordinately regulated by hnRNP C and YB-1. PMID:25354590

Generation and characterization of functional mutants in the translation initiation factor IF1 of Escherichia coli.

PubMed

Croitoru, Victor; Bucheli-Witschel, Margarete; Hägg, Peter; Abdulkarim, Farhad; Isaksson, Leif A

2004-02-01

Three protein factors IF1, IF2 and IF3 are involved in the initiation of translation in prokaryotes. No clear function has been assigned to the smallest of these three factors, IF1. Therefore, to investigate the role of this protein in the initiation process in Escherichia coli we have mutated the corresponding gene infA. Because IF1 is essential for cell viability and no mutant selection has so far been described, the infA gene in a plasmid was mutated by site-directed mutagenesis in a strain with a chromosomal infA+ gene, followed by deletion of this infA+ gene. Using this approach, the six arginine residues of IF1 were altered to leucine or aspartate. Another set of plasmid-encoded IF1 mutants with a cold-sensitive phenotype was collected using localized random mutagenesis. All mutants with a mutated infA gene on a plasmid and a deletion of the chromosomal infA copy were viable, except for an R65D alteration. Differences in growth phenotypes of the mutants were observed in both minimal and rich media. Some of the mutated infA genes were successfully recombined into the chromosome thereby replacing the wild-type infA+ allele. Several of these recombinants showed reduced growth rate and a partial cold-sensitive phenotype. This paper presents a collection of IF1 mutants designed for in vivo and in vitro studies on the function of IF1.

Intermolecular cleavage by UmuD-like mutagenesis proteins

PubMed Central

McDonald, John P.; Frank, Ekaterina G.; Levine, Arthur S.; Woodgate, Roger

1998-01-01

The activity of a number of proteins is regulated by self-processing reactions. Elegant examples are the cleavage of the prokaryotic LexA and λCI transcriptional repressors and the UmuD-like mutagenesis proteins. Various studies support the hypothesis that LexA and λCI cleavage reactions are predominantly intramolecular in nature. The recently described crystal structure of the Escherichia coli UmuD′ protein (the posttranslational cleavage product of the UmuD protein) suggests, however, that the region of the protein corresponding to the cleavage site is at least 50 Å away from the catalytic active site. We considered the possibility, therefore, that the UmuD-like proteins might undergo self-processing that, in contrast to LexA and λCI, occurs via an intermolecular rather than intramolecular reaction. To test this hypothesis, we introduced into E. coli compatible plasmids with mutations at either the cleavage or the catalytic site of three UmuD-like proteins. Cleavage of these proteins only occurs in the presence of both plasmids, indicating that the reaction is indeed intermolecular in nature. Furthermore, this intermolecular reaction is completely dependent upon the multifunctional RecA protein and leads to the restoration of cellular mutagenesis in nonmutable E. coli strains. Intermolecular cleavage of a biotinylated UmuD active site mutant was also observed in vitro in the presence of the wild-type UmuD′ protein, indicating that in addition to the intact UmuD protein, the normal cleavage product (UmuD′) can also act as a classical enzyme. PMID:9465040

Roles of Salmonella typhimurium umuDC and samAB in UV mutagenesis and UV sensitivity.

PubMed Central

Nohmi, T; Yamada, M; Watanabe, M; Murayama, S Y; Sofuni, T

1992-01-01

Expression of the umuDC operon is required for UV mutagenesis and most chemical mutagenesis in Escherichia coli. The closely related species Salmonella typhimurium has two sets of umuDC-like operons; the samAB operon is located in a 60-MDa cryptic plasmid, while the S. typhimurium umuDC (umuDCST) operon resides in a chromosome. The roles of these two umuDC-like operons in UV mutagenesis and UV sensitivity of S. typhimurium were investigated. A pBR322-derived plasmid carrying the samAB operon more efficiently restored UV mutability to a umuD44 strain and a umuC122::Tn5 strain of E. coli than a plasmid carrying the umuDCST operon did. When the umuDCST operon was specifically deleted from the chromosome of S. typhimurium TA2659, the resulting strain was not UV mutable and was more sensitive to the killing effect of UV irradiation than the parent strain was. Curing of the 60-MDa cryptic plasmid carrying the samAB operon did not influence the UV mutability of strain TA2659 but did increase its resistance to UV killing. A pSC101-derived plasmid carrying the samAB operon did not restore UV mutability to a umuD44 strain of E. coli, whereas pBR322- or pBluescript-derived plasmids carrying the samAB operon efficiently did restore UV mutability. We concluded that the umuDCST operon plays a major role in UV mutagenesis in S. typhimurium and that the ability of the samAB operon to promote UV mutagenesis is strongly affected by gene dosage. Possible reasons for the poor ability of samAB to promote UV mutagenesis when it is present on low-copy-number plasmids are discussed. Images PMID:1400244

Simulation and estimation of gene number in a biological pathway using almost complete saturation mutagenesis screening of haploid mouse cells.

PubMed

Tokunaga, Masahiro; Kokubu, Chikara; Maeda, Yusuke; Sese, Jun; Horie, Kyoji; Sugimoto, Nakaba; Kinoshita, Taroh; Yusa, Kosuke; Takeda, Junji

2014-11-24

Genome-wide saturation mutagenesis and subsequent phenotype-driven screening has been central to a comprehensive understanding of complex biological processes in classical model organisms such as flies, nematodes, and plants. The degree of "saturation" (i.e., the fraction of possible target genes identified) has been shown to be a critical parameter in determining all relevant genes involved in a biological function, without prior knowledge of their products. In mammalian model systems, however, the relatively large scale and labor intensity of experiments have hampered the achievement of actual saturation mutagenesis, especially for recessive traits that require biallelic mutations to manifest detectable phenotypes. By exploiting the recently established haploid mouse embryonic stem cells (ESCs), we present an implementation of almost complete saturation mutagenesis in a mammalian system. The haploid ESCs were mutagenized with the chemical mutagen N-ethyl-N-nitrosourea (ENU) and processed for the screening of mutants defective in various steps of the glycosylphosphatidylinositol-anchor biosynthetic pathway. The resulting 114 independent mutant clones were characterized by a functional complementation assay, and were shown to be defective in any of 20 genes among all 22 known genes essential for this well-characterized pathway. Ten mutants were further validated by whole-exome sequencing. The predominant generation of single-nucleotide substitutions by ENU resulted in a gene mutation rate proportional to the length of the coding sequence, which facilitated the experimental design of saturation mutagenesis screening with the aid of computational simulation. Our study enables mammalian saturation mutagenesis to become a realistic proposition. Computational simulation, combined with a pilot mutagenesis experiment, could serve as a tool for the estimation of the number of genes essential for biological processes such as drug target pathways when a positive selection of mutants is available.

A Rapid CRISPR/Cas-based Mutagenesis Assay in Zebrafish for Identification of Genes Involved in Thyroid Morphogenesis and Function.

PubMed

Trubiroha, A; Gillotay, P; Giusti, N; Gacquer, D; Libert, F; Lefort, A; Haerlingen, B; De Deken, X; Opitz, R; Costagliola, S

2018-04-04

The foregut endoderm gives rise to several organs including liver, pancreas, lung and thyroid with important roles in human physiology. Understanding which genes and signalling pathways regulate their development is crucial for understanding developmental disorders as well as diseases in adulthood. We exploited unique advantages of the zebrafish model to develop a rapid and scalable CRISPR/Cas-based mutagenesis strategy aiming at the identification of genes involved in morphogenesis and function of the thyroid. Core elements of the mutagenesis assay comprise bi-allelic gene invalidation in somatic mutants, a non-invasive monitoring of thyroid development in live transgenic fish, complementary analyses of thyroid function in fixed specimens and quantitative analyses of mutagenesis efficiency by Illumina sequencing of individual fish. We successfully validated our mutagenesis-phenotyping strategy in experiments targeting genes with known functions in early thyroid morphogenesis (pax2a, nkx2.4b) and thyroid functional differentiation (duox, duoxa, tshr). We also demonstrate that duox and duoxa crispants phenocopy thyroid phenotypes previously observed in human patients with bi-allelic DUOX2 and DUOXA2 mutations. The proposed combination of efficient mutagenesis protocols, rapid non-invasive phenotyping and sensitive genotyping holds great potential to systematically characterize the function of larger candidate gene panels during thyroid development and is applicable to other organs and tissues.

Comparison of CRISPR/Cas9 expression constructs for efficient targeted mutagenesis in rice.

PubMed

Mikami, Masafumi; Toki, Seiichi; Endo, Masaki

2015-08-01

The CRISPR/Cas9 system is an efficient tool used for genome editing in a variety of organisms. Despite several recent reports of successful targeted mutagenesis using the CRISPR/Cas9 system in plants, in each case the target gene of interest, the Cas9 expression system and guide-RNA (gRNA) used, and the tissues used for transformation and subsequent mutagenesis differed, hence the reported frequencies of targeted mutagenesis cannot be compared directly. Here, we evaluated mutation frequency in rice using different Cas9 and/or gRNA expression cassettes under standardized experimental conditions. We introduced Cas9 and gRNA expression cassettes separately or sequentially into rice calli, and assessed the frequency of mutagenesis at the same endogenous targeted sequences. Mutation frequencies differed significantly depending on the Cas9 expression cassette used. In addition, a gRNA driven by the OsU6 promoter was superior to one driven by the OsU3 promoter. Using an all-in-one expression vector harboring the best combined Cas9/gRNA expression cassette resulted in a much improved frequency of targeted mutagenesis in rice calli, and bi-allelic mutant plants were produced in the T0 generation. The approach presented here could be adapted to optimize the construction of Cas9/gRNA cassettes for genome editing in a variety of plants.

Pilot study of large-scale production of mutant pigs by ENU mutagenesis

PubMed Central

Hai, Tang; Cao, Chunwei; Shang, Haitao; Guo, Weiwei; Mu, Yanshuang; Yang, Shulin; Zhang, Ying; Zheng, Qiantao; Zhang, Tao; Wang, Xianlong; Liu, Yu; Kong, Qingran; Li, Kui; Wang, Dayu; Qi, Meng; Hong, Qianlong; Zhang, Rui; Wang, Xiupeng; Jia, Qitao; Wang, Xiao; Qin, Guosong; Li, Yongshun; Luo, Ailing; Jin, Weiwu; Yao, Jing; Huang, Jiaojiao; Zhang, Hongyong; Li, Menghua; Xie, Xiangmo; Zheng, Xuejuan; Guo, Kenan; Wang, Qinghua; Zhang, Shibin; Li, Liang; Xie, Fei; Zhang, Yu; Weng, Xiaogang; Yin, Zhi; Hu, Kui; Cong, Yimei; Zheng, Peng; Zou, Hailong; Xin, Leilei; Xia, Jihan; Ruan, Jinxue; Li, Hegang; Zhao, Weiming; Yuan, Jing; Liu, Zizhan; Gu, Weiwang; Li, Ming; Wang, Yong; Wang, Hongmei; Yang, Shiming; Liu, Zhonghua; Wei, Hong; Zhao, Jianguo; Zhou, Qi; Meng, Anming

2017-01-01

N-ethyl-N-nitrosourea (ENU) mutagenesis is a powerful tool to generate mutants on a large scale efficiently, and to discover genes with novel functions at the whole-genome level in Caenorhabditis elegans, flies, zebrafish and mice, but it has never been tried in large model animals. We describe a successful systematic three-generation ENU mutagenesis screening in pigs with the establishment of the Chinese Swine Mutagenesis Consortium. A total of 6,770 G1 and 6,800 G3 pigs were screened, 36 dominant and 91 recessive novel pig families with various phenotypes were established. The causative mutations in 10 mutant families were further mapped. As examples, the mutation of SOX10 (R109W) in pig causes inner ear malfunctions and mimics human Mondini dysplasia, and upregulated expression of FBXO32 is associated with congenital splay legs. This study demonstrates the feasibility of artificial random mutagenesis in pigs and opens an avenue for generating a reservoir of mutants for agricultural production and biomedical research. DOI: http://dx.doi.org/10.7554/eLife.26248.001 PMID:28639938

In vitro gamete derivation from pluripotent stem cells: progress and perspective.

PubMed

Nagano, Makoto C

2007-04-01

Germ cells constitute a highly specialized cell population that is indispensable for the continuation and evolution of the species. Recently, several research groups have shown that these unique cells can be produced in vitro from pluripotent stem cells. Furthermore, live births of offspring using induced germ cells have been reported in one study. These results suggest that it may be possible to investigate germ cell development ex vivo and to establish novel reproductive technologies. To this end, it is critical to assess if gamete induction processes in vitro faithfully recapitulate normal germ cell development in vivo. Here, this issue is discussed with a focus on the germ line specification and the sex-specific development of pre- and postnatal germ cells. The aim of this paper is to concisely summarize the past progress and to present some future issues for the investigation into in vitro gamete production from pluripotent stem cells.

A conformational switch high-throughput screening assay and allosteric inhibition of the flavivirus NS2B-NS3 protease

PubMed Central

Liu, Binbin; Zhang, Jing; Koetzner, Cheri A.; Jones, Susan A.; Lin, Qishan

2017-01-01

The flavivirus genome encodes a single polyprotein precursor requiring multiple cleavages by host and viral proteases in order to produce the individual proteins that constitute an infectious virion. Previous studies have revealed that the NS2B cofactor of the viral NS2B-NS3 heterocomplex protease displays a conformational dynamic between active and inactive states. Here, we developed a conformational switch assay based on split luciferase complementation (SLC) to monitor the conformational change of NS2B and to characterize candidate allosteric inhibitors. Binding of an active-site inhibitor to the protease resulted in a conformational change of NS2B and led to significant SLC enhancement. Mutagenesis of key residues at an allosteric site abolished this induced conformational change and SLC enhancement. We also performed a virtual screen of NCI library compounds to identify allosteric inhibitors, followed by in vitro biochemical screening of the resultant candidates. Only three of these compounds, NSC135618, 260594, and 146771, significantly inhibited the protease of Dengue virus 2 (DENV2) in vitro, with IC50 values of 1.8 μM, 11.4 μM, and 4.8 μM, respectively. Among the three compounds, only NSC135618 significantly suppressed the SLC enhancement triggered by binding of active-site inhibitor in a dose-dependent manner, indicating that it inhibits the conformational change of NS2B. Results from virus titer reduction assays revealed that NSC135618 is a broad spectrum flavivirus protease inhibitor, and can significantly reduce titers of DENV2, Zika virus (ZIKV), West Nile virus (WNV), and Yellow fever virus (YFV) on A549 cells in vivo, with EC50 values in low micromolar range. In contrast, the cytotoxicity of NSC135618 is only moderate with CC50 of 48.8 μM on A549 cells. Moreover, NSC135618 inhibited ZIKV in human placental and neural progenitor cells relevant to ZIKV pathogenesis. Results from binding, kinetics, Western blot, mass spectrometry and mutagenesis experiments unambiguously demonstrated an allosteric mechanism for inhibition of the viral protease by NSC135618. PMID:28542603

ENU mutagenesis to generate genetically modified rat models.

PubMed

van Boxtel, Ruben; Gould, Michael N; Cuppen, Edwin; Smits, Bart M G

2010-01-01

The rat is one of the most preferred model organisms in biomedical research and has been extremely useful for linking physiology and pathology to the genome. However, approaches to genetically modify specific genes in the rat germ line remain relatively scarce. To date, the most efficient approach for generating genetically modified rats has been the target-selected N-ethyl-N-nitrosourea (ENU) mutagenesis-based technology. Here, we describe the detailed protocols for ENU mutagenesis and mutant retrieval in the rat model organism.

Facile Site-Directed Mutagenesis of Large Constructs Using Gibson Isothermal DNA Assembly.

PubMed

Yonemoto, Isaac T; Weyman, Philip D

2017-01-01

Site-directed mutagenesis is a commonly used molecular biology technique to manipulate biological sequences, and is especially useful for studying sequence determinants of enzyme function or designing proteins with improved activity. We describe a strategy using Gibson Isothermal DNA Assembly to perform site-directed mutagenesis on large (>~20 kbp) constructs that are outside the effective range of standard techniques such as QuikChange II (Agilent Technologies), but more reliable than traditional cloning using restriction enzymes and ligation.

Identification of pH-sensing Sites in the Light Harvesting Complex Stress-related 3 Protein Essential for Triggering Non-photochemical Quenching in Chlamydomonas reinhardtii

DOE PAGES

Ballottari, Matteo; Truong, Thuy B.; De Re, Eleonora; ...

2016-01-27

Light harvesting complex stress-related 3 (LHCSR3) is the protein essential for photoprotective excess energy dissipation (non-photochemical quenching, NPQ) in the model green alga Chlamydomonas reinhardtii. Activation of NPQ requires low pH in the thylakoid lumen, which is induced in excess light conditions and sensed by lumen-exposed acidic residues. In this work we have used site-specific mutagenesis in vivo and in vitro for identification of the residues in LHCSR3 that are responsible for sensing lumen pH. Lumen-exposed protonatable residues, aspartate and glutamate, were mutated to asparagine and glutamine, respectively. By expression in a mutant lacking all LHCSR isoforms, residues Asp 117,more » Glu 221, and Glu 224 were shown to be essential for LHCSR3-dependent NPQ induction in C. reinhardtii. Analysis of recombinant proteins carrying the same mutations refolded in vitro with pigments showed that the capacity of responding to low pH by decreasing the fluorescence lifetime, present in the wild-type protein, was lost. Consistent with a role in pH sensing, the mutations led to a substantial reduction in binding the NPQ inhibitor dicyclohexylcarbodiimide.« less

Efficient transposition of the Tnt1 tobacco retrotransposon in the model legume Medicago truncatula.

PubMed

d'Erfurth, Isabelle; Cosson, Viviane; Eschstruth, Alexis; Lucas, Helene; Kondorosi, Adam; Ratet, P

2003-04-01

The tobacco element, Tnt1, is one of the few active retrotransposons in plants. Its transposition is activated during protoplast culture in tobacco and tissue culture in the heterologous host Arabidopsis thaliana. Here, we report its transposition in the R108 line of Medicago truncatula during the early steps of the in vitro transformation-regeneration process. Two hundred and twenty-five primary transformants containing Tnt1 were obtained. Among them, 11.2% contained only transposed copies of the element, indicating that Tnt1 transposed very early and efficiently during the in vitro transformation process, possibly even before the T-DNA integration. The average number of insertions per transgenic line was estimated to be about 15. These insertions were stable in the progeny and could be separated by segregation. Inspection of the sequences flanking the insertion sites revealed that Tnt1 had no insertion site specificity and often inserted in genes (one out of three insertions). Thus, our work demonstrates the functioning of an efficient transposable element in leguminous plants. These results indicate that Tnt1 can be used as a powerful tool for insertion mutagenesis in M. truncatula.

Identification of pH-sensing Sites in the Light Harvesting Complex Stress-related 3 Protein Essential for Triggering Non-photochemical Quenching in Chlamydomonas reinhardtii

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ballottari, Matteo; Truong, Thuy B.; De Re, Eleonora

Light harvesting complex stress-related 3 (LHCSR3) is the protein essential for photoprotective excess energy dissipation (non-photochemical quenching, NPQ) in the model green alga Chlamydomonas reinhardtii. Activation of NPQ requires low pH in the thylakoid lumen, which is induced in excess light conditions and sensed by lumen-exposed acidic residues. In this work we have used site-specific mutagenesis in vivo and in vitro for identification of the residues in LHCSR3 that are responsible for sensing lumen pH. Lumen-exposed protonatable residues, aspartate and glutamate, were mutated to asparagine and glutamine, respectively. By expression in a mutant lacking all LHCSR isoforms, residues Asp 117,more » Glu 221, and Glu 224 were shown to be essential for LHCSR3-dependent NPQ induction in C. reinhardtii. Analysis of recombinant proteins carrying the same mutations refolded in vitro with pigments showed that the capacity of responding to low pH by decreasing the fluorescence lifetime, present in the wild-type protein, was lost. Consistent with a role in pH sensing, the mutations led to a substantial reduction in binding the NPQ inhibitor dicyclohexylcarbodiimide.« less

In vivo evidence for translesion synthesis by the replicative DNA polymerase δ

PubMed Central

Hirota, Kouji; Tsuda, Masataka; Mohiuddin; Tsurimoto, Toshiki; Cohen, Isadora S.; Livneh, Zvi; Kobayashi, Kaori; Narita, Takeo; Nishihara, Kana; Murai, Junko; Iwai, Shigenori; Guilbaud, Guillaume; Sale, Julian E.; Takeda, Shunichi

2016-01-01

The intolerance of DNA polymerase δ (Polδ) to incorrect base pairing contributes to its extremely high accuracy during replication, but is believed to inhibit translesion synthesis (TLS). However, chicken DT40 cells lacking the POLD3 subunit of Polδ are deficient in TLS. Previous genetic and biochemical analysis showed that POLD3 may promote lesion bypass by Polδ itself independently of the translesion polymerase Polζ of which POLD3 is also a subunit. To test this hypothesis, we have inactivated Polδ proofreading in pold3 cells. This significantly restored TLS in pold3 mutants, enhancing dA incorporation opposite abasic sites. Purified proofreading-deficient human Polδ holoenzyme performs TLS of abasic sites in vitro much more efficiently than the wild type enzyme, with over 90% of TLS events resulting in dA incorporation. Furthermore, proofreading deficiency enhances the capability of Polδ to continue DNA synthesis over UV lesions both in vivo and in vitro. These data support Polδ contributing to TLS in vivo and suggest that the mutagenesis resulting from loss of Polδ proofreading activity may in part be explained by enhanced lesion bypass. PMID:27185888

Sequential folding of UmuC by the Hsp70 and Hsp60 chaperone complexes of Escherichia coli.

PubMed

Petit, M A; Bedale, W; Osipiuk, J; Lu, C; Rajagopalan, M; McInerney, P; Goodman, M F; Echols, H

1994-09-23

Replication-blocking lesions generate a signal in Escherichia coli that leads to the induction of the multigene SOS response. Among the SOS-induced genes are umuD and umuC, whose products are necessary for the increased mutation rate in induced bacteria. The mutations are likely to result from replication across the DNA lesion, and such a bypass event has been reconstituted in vitro (Rajagopalan, M., L, C., Woodgate, R., O'Donnel, M., Goodman, M. F., Echols, H. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 10777-10781). In this work, we show that the chaperone proteins promote the proper folding of UmuC protein in vitro. We treated purified and inactive UmuC with Hsp70 and Hsp60. After Hsp70 treatment, the DNA binding activity of UmuC was recovered, but the ability to promote replication across DNA lesions was not. However, lesion bypass activity was recovered upon further treatment with Hsp60. The biological significance of such a folding pathway for UmuC protein is strengthened by in vivo evidence for a role of DnaK in UV-induced mutagenesis.

Demonstration of GTG as an alternative initiation codon for the serpin endopin 2B-2.

PubMed

Hwang, Shin-Rong; Garza, Christina Z; Wegrzyn, Jill L; Hook, Vivian Y H

2005-02-18

This study demonstrates GTG as a novel, alternative initiation codon for translation of bovine endopin 2B-2, a serpin protease inhibitor. Molecular cDNA cloning revealed the endopin 2B-1 and endopin 2B-2 isoforms that are predicted to inhibit papain and elastase. Notably, GTG was demonstrated as the initiation codon for endopin 2B-2, whereas endopin 2B-1 possesses ATG as its initiation codon. GTG mediated in vitro translation of 46kDa endopin 2B-2. GTG also mediated translation of EGFP by in vitro translation and by expression in mammalian cells. Notably, mutagenesis of GTG to GTC resulted in the absence of EGFP expression in cells. GTG produced a lower level of protein expression compared to ATG. The use of GTG as an initiation codon to direct translation of endopin 2B, as well as the heterologous protein EGFP, demonstrates the role of GTG in the regulation of mRNA translation in mammalian cells. Significantly, further analyses of mammalian genomes based on GTG as an alternative initiation codon may predict new candidate gene products expressed by mammalian and human genomes.

Production of medakafish chimeras from a stable embryonic stem cell line.

PubMed

Hong, Y; Winkler, C; Schartl, M

1998-03-31

Embryonic stem (ES) cell lines provide a unique tool for introducing targeted or random genetic alterations through gene replacement, insertional mutagenesis, and gene addition because they offer the possibility for in vitro selection for the desired, but extremely rare, recombinant genotypes. So far only mouse blastocyst embryos are known to have the competence to give rise to such ES cell lines. We recently have established a stable cell line (Mes1) from blastulae of the medakafish (Oryzias latipes) that shows all characteristics of mouse ES cells in vitro. Here, we demonstrate that Mes1 cells also have the competence for chimera formation; 90% of host blastulae transplanted with Mes1 cells developed into chimeric fry. This high frequency was not compromised by cryostorage or DNA transfection of the donor cells. The Mes1 cells contributed to numerous organs derived from all three germ layers and differentiated into various types of functional cells, most readily observable in pigmented chimeras. These features suggest the possibility that Mes1 cells may be a fish equivalent of mouse ES cells and that medaka can be used as another system for the application of the ES cell technology.

Putting on the brakes: Bacterial impediment of wound healing

PubMed Central

Brothers, Kimberly M.; Stella, Nicholas A.; Hunt, Kristin M.; Romanowski, Eric G.; Liu, Xinyu; Klarlund, Jes K.; Shanks, Robert M. Q.

2015-01-01

The epithelium provides a crucial barrier to infection, and its integrity requires efficient wound healing. Bacterial cells and secretomes from a subset of tested species of bacteria inhibited human and porcine corneal epithelial cell migration in vitro and ex vivo. Secretomes from 95% of Serratia marcescens, 71% of Pseudomonas aeruginosa, 29% of Staphylococcus aureus strains, and other bacterial species inhibited epithelial cell migration. Migration of human foreskin fibroblasts was also inhibited by S. marcescens secretomes indicating that the effect is not cornea specific. Transposon mutagenesis implicated lipopolysaccharide (LPS) core biosynthetic genes as being required to inhibit corneal epithelial cell migration. LPS depletion of S. marcescens secretomes with polymyxin B agarose rendered secretomes unable to inhibit epithelial cell migration. Purified LPS from S. marcescens, but not from Escherichia coli or S. marcescens strains with mutations in the waaG and waaC genes, inhibited epithelial cell migration in vitro and wound healing ex vivo. Together these data suggest that S. marcescens LPS is sufficient for inhibition of epithelial wound healing. This study presents a novel host-pathogen interaction with implications for infections where bacteria impact wound healing and provides evidence that secreted LPS is a key factor in the inhibitory mechanism. PMID:26365869

Genomic insights into the broad antifungal activity, plant-probiotic properties, and their regulation, in Pseudomonas donghuensis strain SVBP6

PubMed Central

Iriarte, Andrés; Valverde, Claudio Fabián

2018-01-01

Plant-growth promotion has been linked to the Pseudomonas genus since the beginning of this research field. In this work, we mined the genome of an Argentinean isolate of the recently described species P. donghuensis. Strain SVBP6, isolated from bulk soil of an agricultural plot, showed a broad antifungal activity and several other plant-probiotic activities. As this species has been recently described, and it seems like some plant-growth promoting (PGP) traits do not belong to the classical pseudomonads toolbox, we decide to explore the SVBP6 genome via an bioinformatic approach. Genome inspection confirmed our previous in vitro results about genes involved in several probiotic activities. Other genetic traits possibly involved in survival of SVBP6 in highly competitive environments, such as rhizospheres, were found. Tn5 mutagenesis revealed that the antifungal activity against the soil pathogen Macrophomina phaseolina was dependent on a functional gacS gene, from the regulatory cascade Gac-Rsm, but it was not due to volatile compounds. Altogether, our genomic analyses and in vitro tests allowed the phylogenetic assignment and provided the first insights into probiotic properties of the first P. donghuensis isolate from the Americas. PMID:29538430

Production of medakafish chimeras from a stable embryonic stem cell line

PubMed Central

Hong, Yunhan; Winkler, Christoph; Schartl, Manfred

1998-01-01

Embryonic stem (ES) cell lines provide a unique tool for introducing targeted or random genetic alterations through gene replacement, insertional mutagenesis, and gene addition because they offer the possibility for in vitro selection for the desired, but extremely rare, recombinant genotypes. So far only mouse blastocyst embryos are known to have the competence to give rise to such ES cell lines. We recently have established a stable cell line (Mes1) from blastulae of the medakafish (Oryzias latipes) that shows all characteristics of mouse ES cells in vitro. Here, we demonstrate that Mes1 cells also have the competence for chimera formation; 90% of host blastulae transplanted with Mes1 cells developed into chimeric fry. This high frequency was not compromised by cryostorage or DNA transfection of the donor cells. The Mes1 cells contributed to numerous organs derived from all three germ layers and differentiated into various types of functional cells, most readily observable in pigmented chimeras. These features suggest the possibility that Mes1 cells may be a fish equivalent of mouse ES cells and that medaka can be used as another system for the application of the ES cell technology. PMID:9520425

The 2-Cys Peroxiredoxin Alkyl Hydroperoxide Reductase C Binds Heme and Participates in Its Intracellular Availability in Streptococcus agalactiae*

PubMed Central

Lechardeur, Delphine; Fernandez, Annabelle; Robert, Bruno; Gaudu, Philippe; Trieu-Cuot, Patrick; Lamberet, Gilles; Gruss, Alexandra

2010-01-01

Heme is a redox-reactive molecule with vital and complex roles in bacterial metabolism, survival, and virulence. However, few intracellular heme partners were identified to date and are not well conserved in bacteria. The opportunistic pathogen Streptococcus agalactiae (group B Streptococcus) is a heme auxotroph, which acquires exogenous heme to activate an aerobic respiratory chain. We identified the alkyl hydroperoxide reductase AhpC, a member of the highly conserved thiol-dependent 2-Cys peroxiredoxins, as a heme-binding protein. AhpC binds hemin with a Kd of 0.5 μm and a 1:1 stoichiometry. Mutagenesis of cysteines revealed that hemin binding is dissociable from catalytic activity and multimerization. AhpC reductase activity was unchanged upon interaction with heme in vitro and in vivo. A group B Streptococcus ahpC mutant displayed attenuation of two heme-dependent functions, respiration and activity of a heterologous catalase, suggesting a role for AhpC in heme intracellular fate. In support of this hypothesis, AhpC-bound hemin was protected from chemical degradation in vitro. Our results reveal for the first time a role for AhpC as a heme-binding protein. PMID:20332091

Economical analysis of saturation mutagenesis experiments

PubMed Central

Acevedo-Rocha, Carlos G.; Reetz, Manfred T.; Nov, Yuval

2015-01-01

Saturation mutagenesis is a powerful technique for engineering proteins, metabolic pathways and genomes. In spite of its numerous applications, creating high-quality saturation mutagenesis libraries remains a challenge, as various experimental parameters influence in a complex manner the resulting diversity. We explore from the economical perspective various aspects of saturation mutagenesis library preparation: We introduce a cheaper and faster control for assessing library quality based on liquid media; analyze the role of primer purity and supplier in libraries with and without redundancy; compare library quality, yield, randomization efficiency, and annealing bias using traditional and emergent randomization schemes based on mixtures of mutagenic primers; and establish a methodology for choosing the most cost-effective randomization scheme given the screening costs and other experimental parameters. We show that by carefully considering these parameters, laboratory expenses can be significantly reduced. PMID:26190439

Genome-wide comparison of ultraviolet and ethyl methanesulphonate mutagenesis methods for the brown alga Ectocarpus.

PubMed

Godfroy, Olivier; Peters, Akira F; Coelho, Susana M; Cock, J Mark

2015-12-01

Ectocarpus has emerged as a model organism for the brown algae and a broad range of genetic and genomic resources are being generated for this species. The aim of the work presented here was to evaluate two mutagenesis protocols based on ultraviolet irradiation and ethyl methanesulphonate treatment using genome resequencing to measure the number, type and distribution of mutations generated by the two methods. Ultraviolet irradiation generated a greater number of genetic lesions than ethyl methanesulphonate treatment, with more than 400 mutations being detected in the genome of the mutagenised individual. This study therefore confirms that the ultraviolet mutagenesis protocol is suitable for approaches that require a high density of mutations, such as saturation mutagenesis or Targeting Induced Local Lesions in Genomes (TILLING). Copyright © 2015 Elsevier B.V. All rights reserved.

Targeted Mutagenesis in Rice Using TALENs and the CRISPR/Cas9 System.

PubMed

Endo, Masaki; Nishizawa-Yokoi, Ayako; Toki, Seiichi

2016-01-01

Sequence-specific nucleases (SSNs), such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system, are powerful tools for understanding gene function and for developing novel traits in plants. In plant species for which transformation and regeneration systems using protoplasts are not yet established, direct delivery to nuclei of SSNs either in the form of RNA or protein is difficult. Thus, Agrobacterium-mediated transformation of SSN expression constructs in cultured cells is a practical means of delivering targeted mutagenesis in some plant species including rice. Because targeted mutagenesis occurs stochastically in transgenic cells and SSN-mediated targeted mutagenesis often leads to no selectable phenotype, identification of highly mutated cell lines is a critical step in obtaining regenerated plants with desired mutations.

CRISPR/Cas9 mediates efficient conditional mutagenesis in Drosophila.

PubMed

Xue, Zhaoyu; Wu, Menghua; Wen, Kejia; Ren, Menda; Long, Li; Zhang, Xuedi; Gao, Guanjun

2014-09-05

Existing transgenic RNA interference (RNAi) methods greatly facilitate functional genome studies via controlled silencing of targeted mRNA in Drosophila. Although the RNAi approach is extremely powerful, concerns still linger about its low efficiency. Here, we developed a CRISPR/Cas9-mediated conditional mutagenesis system by combining tissue-specific expression of Cas9 driven by the Gal4/upstream activating site system with various ubiquitously expressed guide RNA transgenes to effectively inactivate gene expression in a temporally and spatially controlled manner. Furthermore, by including multiple guide RNAs in a transgenic vector to target a single gene, we achieved a high degree of gene mutagenesis in specific tissues. The CRISPR/Cas9-mediated conditional mutagenesis system provides a simple and effective tool for gene function analysis, and complements the existing RNAi approach. Copyright © 2014 Xue et al.

Structural and biochemical studies on Vibrio cholerae Hsp31 reveals a novel dimeric form and Glutathione-independent Glyoxalase activity

PubMed Central

Dey, Sanjay

2017-01-01

Vibrio cholerae experiences a highly hostile environment at human intestine which triggers the induction of various heat shock genes. The hchA gene product of V. cholerae O395, referred to a hypothetical intracellular protease/amidase VcHsp31, is one such stress-inducible homodimeric protein. Our current study demonstrates that VcHsp31 is endowed with molecular chaperone, amidopeptidase and robust methylglyoxalase activities. Through site directed mutagenesis coupled with biochemical assays on VcHsp31, we have confirmed the role of residues in the vicinity of the active site towards amidopeptidase and methylglyoxalase activities. VcHsp31 suppresses the aggregation of insulin in vitro in a dose dependent manner. Through crystal structures of VcHsp31 and its mutants, grown at various temperatures, we demonstrate that VcHsp31 acquires two (Type-I and Type-II) dimeric forms. Type-I dimer is similar to EcHsp31 where two VcHsp31 monomers associate in eclipsed manner through several intersubunit hydrogen bonds involving their P-domains. Type-II dimer is a novel dimeric organization, where some of the intersubunit hydrogen bonds are abrogated and each monomer swings out in the opposite directions centering at their P-domains, like twisting of wet cloth. Normal mode analysis (NMA) of Type-I dimer shows similar movement of the individual monomers. Upon swinging, a dimeric surface of ~400Å2, mostly hydrophobic in nature, is uncovered which might bind partially unfolded protein substrates. We propose that, in solution, VcHsp31 remains as an equilibrium mixture of both the dimers. With increase in temperature, transformation to Type-II form having more exposed hydrophobic surface, occurs progressively accounting for the temperature dependent increase of chaperone activity of VcHsp31. PMID:28235098

Rhodococcus equi.

PubMed

Meijer, Wim G; Prescott, John F

2004-01-01

Rhodococcus equi is an important cause of subacute or chronic abscessating bronchopneumonia of foals up to 3-5 months of age. It shares the lipid-rich cell wall envelope characteristic of the mycolata, including Mycobacterium tuberculosis, as well as the ability of pathogenic members of this group to survive within macrophages. The possession of a large virulence plasmid in isolates recovered from pneumonic foals is crucial for virulence. The plasmid contains an 27 kb pathogenicity island (PI) that encodes seven related virulence-associated proteins (Vaps), including the immunodominant surface-expressed protein, VapA. Only PI genes are differentially expressed when the organism is grown in macrophages in vitro. Ten of the PI genes, including six Vap genes, have signal sequences, suggesting that they are exported from the cell to interact with the macrophage. Different PI genes are regulated by temperature, pH, iron, oxidative stress and probably also by magnesium, all environmental changes encountered after environmental R. equi are inhaled in dust and are ingested into macrophages in the lung. The basis of pathogenicity of R. equi is its ability to multiply in and eventually to destroy alveolar macrophages. Infectivity is largely or exclusively limited to cells of the monocyte-macrophage lineage. Current evidence suggests that infection of foals with virulent R. equi results in some foals in subversion of cell-mediated immunity and development of an ineffective and sometimes lethal Th2-based immune response. Significant progress has been made recently in the development of R. equi-E. coli shuttle vectors, transformation and random and site specific mutagenesis procedures, all of which will be important in molecular dissection of the mechanisms by which R. equi subverts normal macrophage killing mechanisms and cell-mediated immunity.

Ionization in liquids. Progress report, September 1, 1977-April 30, 1981

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bakale, G.

1980-12-19

Quasifree electrons simulate the behavior of unsolvated or dry electrons in aqueous media including the special case of biological systems. A model of direct radiosensitization was developed based on dry charge-carriers having an extended lifetime in the sheath of structured water that surrounds polar biomolecules. In this model, the pre-solvation lifetimes of dry electrons increased with an increase in the rotational times of solvent molecules. During the development of this model, an increasing number of radiosensitizers were found to be carcinogenic. Measurement of the k/sub e/'s of known carcinogens and noncarcinogens revealed that carcinogens attached quasifree electrons at diffusion-controlled rates,more » whereas the k/sub e/'s of noncarcinogens were significantly less. To explore the k/sub e/-carcinogenicity correlation further, a study of quasifree electron attachment to the water pools of reversed micelles was conducted. The degree of structuredness of the water pools which determines the k/sub e/ of the reversed micellar systems was also controlled. Another approach to controlling the microenvironment of quasifree electrons in biological systems was done in studies of radiation-induced damage to DNA in concentrated DNA solutions. The high concentration of DNA introduces more structure into the solutions than that occurring in typical in vitro experiments. The structural enhancement by DNA extends the lifetime of unsolvated charge-carriers. The DNA-damaging effects of radiolyticaly produced charge-carriers were also determined in studies of synergistic mutagenesis in bacteria simultaneously exposed to ionizing radiation and electrophilic chemical carcinogens. The attachment-detachment equilibrium of nicotine in hexane solutions was also studied. Both the kinetics and the thermodynamics of electron reactions were studied. (ERB)« less

Effective lethal mutagenesis of influenza virus by three nucleoside analogs.

PubMed

Pauly, Matthew D; Lauring, Adam S

2015-04-01

Lethal mutagenesis is a broad-spectrum antiviral strategy that exploits the high mutation rate and low mutational tolerance of many RNA viruses. This approach uses mutagenic drugs to increase viral mutation rates and burden viral populations with mutations that reduce the number of infectious progeny. We investigated the effectiveness of lethal mutagenesis as a strategy against influenza virus using three nucleoside analogs, ribavirin, 5-azacytidine, and 5-fluorouracil. All three drugs were active against a panel of seasonal H3N2 and laboratory-adapted H1N1 strains. We found that each drug increased the frequency of mutations in influenza virus populations and decreased the virus' specific infectivity, indicating a mutagenic mode of action. We were able to drive viral populations to extinction by passaging influenza virus in the presence of each drug, indicating that complete lethal mutagenesis of influenza virus populations can be achieved when a sufficient mutational burden is applied. Population-wide resistance to these mutagenic agents did not arise after serial passage of influenza virus populations in sublethal concentrations of drug. Sequencing of these drug-passaged viral populations revealed genome-wide accumulation of mutations at low frequency. The replicative capacity of drug-passaged populations was reduced at higher multiplicities of infection, suggesting the presence of defective interfering particles and a possible barrier to the evolution of resistance. Together, our data suggest that lethal mutagenesis may be a particularly effective therapeutic approach with a high genetic barrier to resistance for influenza virus. Influenza virus is an RNA virus that causes significant morbidity and mortality during annual epidemics. Novel therapies for RNA viruses are needed due to the ease with which these viruses evolve resistance to existing therapeutics. Lethal mutagenesis is a broad-spectrum strategy that exploits the high mutation rate and the low mutational tolerance of most RNA viruses. It is thought to possess a higher barrier to resistance than conventional antiviral strategies. We investigated the effectiveness of lethal mutagenesis against influenza virus using three different drugs. We showed that influenza virus was sensitive to lethal mutagenesis by demonstrating that all three drugs induced mutations and led to an increase in the generation of defective viral particles. We also found that it may be difficult for resistance to these drugs to arise at a population-wide level. Our data suggest that lethal mutagenesis may be an attractive anti-influenza strategy that warrants further investigation. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Random oligonucleotide mutagenesis: application to a large protein coding sequence of a major histocompatibility complex class I gene, H-2DP.

PubMed Central

Murray, R; Pederson, K; Prosser, H; Muller, D; Hutchison, C A; Frelinger, J A

1988-01-01

We have used random oligonucleotide mutagenesis (or saturation mutagenesis) to create a library of point mutations in the alpha 1 protein domain of a Major Histocompatibility Complex (MHC) molecule. This protein domain is critical for T cell and B cell recognition. We altered the MHC class I H-2DP gene sequence such that synthetic mutant alpha 1 exons (270 bp of coding sequence), which contain mutations identified by sequence analysis, can replace the wild type alpha 1 exon. The synthetic exons were constructed from twelve overlapping oligonucleotides which contained an average of 1.3 random point mutations per intact exon. DNA sequence analysis of mutant alpha 1 exons has shown a point mutant distribution that fits a Poisson distribution, and thus emphasizes the utility of this mutagenesis technique to "scan" a large protein sequence for important mutations. We report our use of saturation mutagenesis to scan an entire exon of the H-2DP gene, a cassette strategy to replace the wild type alpha 1 exon with individual mutant alpha 1 exons, and analysis of mutant molecules expressed on the surface of transfected mouse L cells. Images PMID:2903482

Gene discovery by chemical mutagenesis and whole-genome sequencing in Dictyostelium.

PubMed

Li, Cheng-Lin Frank; Santhanam, Balaji; Webb, Amanda Nicole; Zupan, Blaž; Shaulsky, Gad

2016-09-01

Whole-genome sequencing is a useful approach for identification of chemical-induced lesions, but previous applications involved tedious genetic mapping to pinpoint the causative mutations. We propose that saturation mutagenesis under low mutagenic loads, followed by whole-genome sequencing, should allow direct implication of genes by identifying multiple independent alleles of each relevant gene. We tested the hypothesis by performing three genetic screens with chemical mutagenesis in the social soil amoeba Dictyostelium discoideum Through genome sequencing, we successfully identified mutant genes with multiple alleles in near-saturation screens, including resistance to intense illumination and strong suppressors of defects in an allorecognition pathway. We tested the causality of the mutations by comparison to published data and by direct complementation tests, finding both dominant and recessive causative mutations. Therefore, our strategy provides a cost- and time-efficient approach to gene discovery by integrating chemical mutagenesis and whole-genome sequencing. The method should be applicable to many microbial systems, and it is expected to revolutionize the field of functional genomics in Dictyostelium by greatly expanding the mutation spectrum relative to other common mutagenesis methods. © 2016 Li et al.; Published by Cold Spring Harbor Laboratory Press.

Natural selection underlies apparent stress-induced mutagenesis in a bacteriophage infection model.

PubMed

Yosef, Ido; Edgar, Rotem; Levy, Asaf; Amitai, Gil; Sorek, Rotem; Munitz, Ariel; Qimron, Udi

2016-04-18

The emergence of mutations following growth-limiting conditions underlies bacterial drug resistance, viral escape from the immune system and fundamental evolution-driven events. Intriguingly, whether mutations are induced by growth limitation conditions or are randomly generated during growth and then selected by growth limitation conditions remains an open question(1). Here, we show that bacteriophage T7 undergoes apparent stress-induced mutagenesis when selected for improved recognition of its host's receptor. In our unique experimental set-up, the growth limitation condition is physically and temporally separated from mutagenesis: growth limitation occurs while phage DNA is outside the host, and spontaneous mutations occur during phage DNA replication inside the host. We show that the selected beneficial mutations are not pre-existing and that the initial slow phage growth is enabled by the phage particle's low-efficiency DNA injection into the host. Thus, the phage particle allows phage populations to initially extend their host range without mutagenesis by virtue of residual recognition of the host receptor. Mutations appear during non-selective intracellular replication, and the frequency of mutant phages increases by natural selection acting on free phages, which are not capable of mutagenesis.

The substitution of cysteine 17 of recombinant human G-CSF with alanine greatly enhanced its stability.

PubMed

Ishikawa, M; Iijima, H; Satake-Ishikawa, R; Tsumura, H; Iwamatsu, A; Kadoya, T; Shimada, Y; Fukamachi, H; Kobayashi, K; Matsuki, S

1992-02-01

Human recombinant granulocyte-colony stimulating factor (rhG-CSF) has one free cysteine at position 17 and has two disulfide bridges (Cys36-Cys42 and Cys64-Cys74). The Cys17 of rhG-CSF was substituted with Gly, Ala, Ser, Ile, Tyr, Arg, and Pro, or deleted using site-directed mutagenesis in order to improve its thermostability. With the exception of Pro17-rhG-CSF, all mutant proteins retained biological activity which promotes the growth of mouse bone marrow cells in vitro. Among these mutant proteins, Ala17-rhG-CSF had more than 5 times higher stability than rhG-CSF. But Ser17-rhG-CSF had almost same stability as rhG-CSF and other mutant proteins had only lower stability.

Theories of Lethal Mutagenesis: From Error Catastrophe to Lethal Defection.

PubMed

Tejero, Héctor; Montero, Francisco; Nuño, Juan Carlos

2016-01-01

RNA viruses get extinct in a process called lethal mutagenesis when subjected to an increase in their mutation rate, for instance, by the action of mutagenic drugs. Several approaches have been proposed to understand this phenomenon. The extinction of RNA viruses by increased mutational pressure was inspired by the concept of the error threshold. The now classic quasispecies model predicts the existence of a limit to the mutation rate beyond which the genetic information of the wild type could not be efficiently transmitted to the next generation. This limit was called the error threshold, and for mutation rates larger than this threshold, the quasispecies was said to enter into error catastrophe. This transition has been assumed to foster the extinction of the whole population. Alternative explanations of lethal mutagenesis have been proposed recently. In the first place, a distinction is made between the error threshold and the extinction threshold, the mutation rate beyond which a population gets extinct. Extinction is explained from the effect the mutation rate has, throughout the mutational load, on the reproductive ability of the whole population. Secondly, lethal defection takes also into account the effect of interactions within mutant spectra, which have been shown to be determinant for the understanding the extinction of RNA virus due to an augmented mutational pressure. Nonetheless, some relevant issues concerning lethal mutagenesis are not completely understood yet, as so survival of the flattest, i.e. the development of resistance to lethal mutagenesis by evolving towards mutationally more robust regions of sequence space, or sublethal mutagenesis, i.e., the increase of the mutation rate below the extinction threshold which may boost the adaptability of RNA virus, increasing their ability to develop resistance to drugs (including mutagens). A better design of antiviral therapies will still require an improvement of our knowledge about lethal mutagenesis.

Mutagenesis and phenotyping resources in zebrafish for studying development and human disease

PubMed Central

Varshney, Gaurav Kumar

2014-01-01

The zebrafish (Danio rerio) is an important model organism for studying development and human disease. The zebrafish has an excellent reference genome and the functions of hundreds of genes have been tested using both forward and reverse genetic approaches. Recent years have seen an increasing number of large-scale mutagenesis projects and the number of mutants or gene knockouts in zebrafish has increased rapidly, including for the first time conditional knockout technologies. In addition, targeted mutagenesis techniques such as zinc finger nucleases, transcription activator-like effector nucleases and clustered regularly interspaced short sequences (CRISPR) or CRISPR-associated (Cas), have all been shown to effectively target zebrafish genes as well as the first reported germline homologous recombination, further expanding the utility and power of zebrafish genetics. Given this explosion of mutagenesis resources, it is now possible to perform systematic, high-throughput phenotype analysis of all zebrafish gene knockouts. PMID:24162064

Modification of Antibody Function by Mutagenesis.

PubMed

Dasch, James R; Dasch, Amy L

2017-09-01

The ability to "fine-tune" recombinant antibodies by mutagenesis separates recombinant antibodies from hybridoma-derived antibodies because the latter are locked with respect to their properties. Recombinant antibodies can be modified to suit the application: Changes in isotype, format (e.g., scFv, Fab, bispecific antibodies), and specificity can be made once the heavy- and light-chain sequences are available. After immunoglobulin heavy and light chains for a particular antibody have been cloned, the binding site-namely, the complementarity determining regions (CDR)-can be manipulated by mutagenesis to obtain antibody variants with improved properties. The method described here is relatively simple, uses commercially available reagents, and is effective. Using the pComb3H vector, a commercial mutagenesis kit, PfuTurbo polymerase (Agilent), and two mutagenic primers, a library of phage with mutagenized heavy and light CDR3 can be obtained. © 2017 Cold Spring Harbor Laboratory Press.

Genetic and biochemical analysis of the interaction of Bacillus subtilis CodY with branched-chain amino acids.

PubMed

Villapakkam, Anuradha C; Handke, Luke D; Belitsky, Boris R; Levdikov, Vladimir M; Wilkinson, Anthony J; Sonenshein, Abraham L

2009-11-01

Bacillus subtilis CodY protein is a DNA-binding global transcriptional regulator that responds to branched-chain amino acids (isoleucine, leucine, and valine) and GTP. Crystal structure studies have shown that the N-terminal region of the protein includes a GAF domain that contains a hydrophobic pocket within which isoleucine and valine bind. This region is well conserved in CodY homologs. Site-directed mutagenesis was employed to understand the roles of some of the residues in the GAF domain and hydrophobic pocket in interaction with isoleucine and GTP. The F40A, F71E, and F98A forms of CodY were inactive in vivo. They were activatable by GTP but to a much lesser extent by branched-chain amino acids in vitro. The CodY mutant R61A retained partial repression of target promoters in vivo and was able to respond to GTP in vitro but also responded poorly to branched-chain amino acids in vitro unless GTP was simultaneously present. Thus, the GAF domain includes residues essential for full activation of CodY by branched-chain amino acids, but these residues are not critical for activation by GTP. Binding studies with branched-chain amino acids and their analogs revealed that an amino group at position 2 and a methyl group at position 3 of valine are critical components of the recognition of the amino acids by CodY.

Computer Simulation of Developmental Processes and Toxicities (SOT)

EPA Science Inventory

Rationale: Recent progress in systems toxicology and synthetic biology have paved the way to new thinking about in vitro/in silico modeling of developmental processes and toxicities, both for embryological and reproductive impacts. Novel in vitro platforms such as 3D organotypic ...

Extinction of Zika virus and Usutu virus by lethal mutagenesis reveals different patterns of sensitivity to three mutagenic drugs.

PubMed

Bassi, Maria Rosaria; Sempere, Raquel Navarro; Meyn, Prashansa; Polacek, Charlotta; Arias, Armando

2018-06-18

Flaviviruses constitute an increasing source of public health concern with growing numbers of pathogens causing disease, and a geographic spread to temperate climates. Despite a large body of evidence supporting mutagenesis as a conceivable antiviral strategy, there is currently no data on the sensitivity to increased mutagenesis for Zika virus (ZIKV) and Usutu virus (USUV), two emerging flaviviral threats. In this study, we demonstrate that both viruses are sensitive to three ribonucleosides that have shown mutagenic activity against other RNA viruses - favipiravir, ribavirin and 5-fluorouracil - while they remain unaffected by a mutagenic deoxyribonucleoside. Serial cell culture passages of ZIKV in the presence of these compounds resulted in the rapid extinction of infectivity, suggesting elevated sensitivity to mutagenesis. USUV extinction was achieved when a 10-fold dilution was applied between every passage, but not in experiments involving undiluted virus, indicating an overall lower susceptibility than ZIKV. Although both viruses are inhibited by the same three drugs, ZIKV is relatively more susceptive to serial passage in the presence of purine analogues (favipiravir and ribavirin) while USUV replication is suppressed more efficiently by 5-fluorouracil. These differences in sensitivity typically correlate with the increases in the mutation frequencies observed in each nucleoside treatment. These results are relevant to the development of efficient therapies based on lethal mutagenesis, and support the rational selection of different mutagenic nucleosides for each pathogen. We will discuss the implications of these results to the fidelity of flavivirus replication, and the design of antiviral therapies based on lethal mutagenesis. Copyright © 2018 Bassi et al.

A high-throughput shotgun mutagenesis approach to mapping B-cell antibody epitopes.

PubMed

Davidson, Edgar; Doranz, Benjamin J

2014-09-01

Characterizing the binding sites of monoclonal antibodies (mAbs) on protein targets, their 'epitopes', can aid in the discovery and development of new therapeutics, diagnostics and vaccines. However, the speed of epitope mapping techniques has not kept pace with the increasingly large numbers of mAbs being isolated. Obtaining detailed epitope maps for functionally relevant antibodies can be challenging, particularly for conformational epitopes on structurally complex proteins. To enable rapid epitope mapping, we developed a high-throughput strategy, shotgun mutagenesis, that enables the identification of both linear and conformational epitopes in a fraction of the time required by conventional approaches. Shotgun mutagenesis epitope mapping is based on large-scale mutagenesis and rapid cellular testing of natively folded proteins. Hundreds of mutant plasmids are individually cloned, arrayed in 384-well microplates, expressed within human cells, and tested for mAb reactivity. Residues are identified as a component of a mAb epitope if their mutation (e.g. to alanine) does not support candidate mAb binding but does support that of other conformational mAbs or allows full protein function. Shotgun mutagenesis is particularly suited for studying structurally complex proteins because targets are expressed in their native form directly within human cells. Shotgun mutagenesis has been used to delineate hundreds of epitopes on a variety of proteins, including G protein-coupled receptor and viral envelope proteins. The epitopes mapped on dengue virus prM/E represent one of the largest collections of epitope information for any viral protein, and results are being used to design better vaccines and drugs. © 2014 John Wiley & Sons Ltd.

GERM-LINE SPECIFIC FACTORS IN CHEMICAL MUTAGENESIS

EPA Science Inventory

Chemical mutagenesis test results ave not revealed evidence of germ-line specific mutagens. owever, conventional assays have indicated that there are male-female differences in mutagenic response, as well as quantitative/qualitative differences in induced mutations which depend u...

Computer Simulation of Mutagenesis.

ERIC Educational Resources Information Center

North, J. C.; Dent, M. T.

1978-01-01

A FORTRAN program is described which simulates point-substitution mutations in the DNA strands of typical organisms. Its objective is to help students to understand the significance and structure of the genetic code, and the mechanisms and effect of mutagenesis. (Author/BB)

Dissecting protein function: an efficient protocol for identifying separation-of-function mutations that encode structurally stable proteins.

PubMed

Lubin, Johnathan W; Rao, Timsi; Mandell, Edward K; Wuttke, Deborah S; Lundblad, Victoria

2013-03-01

Mutations that confer the loss of a single biochemical property (separation-of-function mutations) can often uncover a previously unknown role for a protein in a particular biological process. However, most mutations are identified based on loss-of-function phenotypes, which cannot differentiate between separation-of-function alleles vs. mutations that encode unstable/unfolded proteins. An alternative approach is to use overexpression dominant-negative (ODN) phenotypes to identify mutant proteins that disrupt function in an otherwise wild-type strain when overexpressed. This is based on the assumption that such mutant proteins retain an overall structure that is comparable to that of the wild-type protein and are able to compete with the endogenous protein (Herskowitz 1987). To test this, the in vivo phenotypes of mutations in the Est3 telomerase subunit from Saccharomyces cerevisiae were compared with the in vitro secondary structure of these mutant proteins as analyzed by circular-dichroism spectroscopy, which demonstrates that ODN is a more sensitive assessment of protein stability than the commonly used method of monitoring protein levels from extracts. Reverse mutagenesis of EST3, which targeted different categories of amino acids, also showed that mutating highly conserved charged residues to the oppositely charged amino acid had an increased likelihood of generating a severely defective est3(-) mutation, which nevertheless encoded a structurally stable protein. These results suggest that charge-swap mutagenesis directed at a limited subset of highly conserved charged residues, combined with ODN screening to eliminate partially unfolded proteins, may provide a widely applicable and efficient strategy for generating separation-of-function mutations.

Mutagenesis of the Phosphatidylinositol 4,5-Bisphosphate (Pip2) Binding Site in the Nh2-Terminal Domain of Ezrin Correlates with Its Altered Cellular Distribution

PubMed Central

Barret, Cécile; Roy, Christian; Montcourrier, Philippe; Mangeat, Paul; Niggli, Verena

2000-01-01

The cytoskeleton-membrane linker protein ezrin has been shown to associate with phosphatidyl-inositol 4,5-bisphosphate (PIP2)-containing liposomes via its NH2-terminal domain. Using internal deletions and COOH-terminal truncations, determinants of PIP2 binding were located to amino acids 12–115 and 233–310. Both regions contain a KK(X)nK/RK motif conserved in the ezrin/radixin/moesin family. K/N mutations of residues 253 and 254 or 262 and 263 did not affect cosedimentation of ezrin 1-333 with PIP2-containing liposomes, but their combination almost completely abolished the capacity for interaction. Similarly, double mutation of Lys 63, 64 to Asn only partially reduced lipid interaction, but combined with the double mutation K253N, K254N, the interaction of PIP2 with ezrin 1-333 was strongly inhibited. Similar data were obtained with full-length ezrin. When residues 253, 254, 262, and 263 were mutated in full-length ezrin, the in vitro interaction with the cytoplasmic tail of CD44 was not impaired but was no longer PIP2 dependent. This construct was also expressed in COS1 and A431 cells. Unlike wild-type ezrin, it was not any more localized to dorsal actin-rich structures, but redistributed to the cytoplasm without strongly affecting the actin-rich structures. We have thus identified determinants of the PIP2 binding site in ezrin whose mutagenesis correlates with an altered cellular localization. PMID:11086008

Tautomerism provides a molecular explanation for the mutagenic properties of the anti-HIV nucleoside 5-aza-5,6-dihydro-2'-deoxycytidine.

PubMed

Li, Deyu; Fedeles, Bogdan I; Singh, Vipender; Peng, Chunte Sam; Silvestre, Katherine J; Simi, Allison K; Simpson, Jeffrey H; Tokmakoff, Andrei; Essigmann, John M

2014-08-12

Viral lethal mutagenesis is a strategy whereby the innate immune system or mutagenic pool nucleotides increase the error rate of viral replication above the error catastrophe limit. Lethal mutagenesis has been proposed as a mechanism for several antiviral compounds, including the drug candidate 5-aza-5,6-dihydro-2'-deoxycytidine (KP1212), which causes A-to-G and G-to-A mutations in the HIV genome, both in tissue culture and in HIV positive patients undergoing KP1212 monotherapy. This work explored the molecular mechanism(s) underlying the mutagenicity of KP1212, and specifically whether tautomerism, a previously proposed hypothesis, could explain the biological consequences of this nucleoside analog. Establishing tautomerism of nucleic acid bases under physiological conditions has been challenging because of the lack of sensitive methods. This study investigated tautomerism using an array of spectroscopic, theoretical, and chemical biology approaches. Variable temperature NMR and 2D infrared spectroscopic methods demonstrated that KP1212 existed as a broad ensemble of interconverting tautomers, among which enolic forms dominated. The mutagenic properties of KP1212 were determined empirically by in vitro and in vivo replication of a single-stranded vector containing a single KP1212. It was found that KP1212 paired with both A (10%) and G (90%), which is in accord with clinical observations. Moreover, this mutation frequency is sufficient for pushing a viral population over its error catastrophe limit, as observed before in cell culture studies. Finally, a model is proposed that correlates the mutagenicity of KP1212 with its tautomeric distribution in solution.

Comparison of Polyphenol Profile and Antimutagenic and Antioxidant Activities in Two Species Used as Source of Solidaginis herba - Goldenrod.

PubMed

Woźniak, Dorota; Ślusarczyk, Sylwester; Domaradzki, Krzysztof; Dryś, Andrzej; Matkowski, Adam

2018-04-01

European Pharmacopoeia accepts two equivalent species Solidago canadensis L. and S. gigantea Aiton as goldenrod (Solidaginis herba). We compared phytochemical profile of both species from invasive populations in Poland. Further, we compared in vitro antimutagenic and antioxidant activities of solvent extracts from aerial (AP) and underground parts (UP). In S. gigantea, flavonoid profile was dominated by quercetin glycosides, with quercitrin as the major compound. In S. canadensis, quercetin and kaempferol rutinosides were two major constituents. Caffeoylquinic acids (CQAs) were less diverse with 5-CQA as a main compound. In UP, over 20 putative diterpenoids were detected, mostly unidentified. Several CQAs were present in higher amounts than in AP. Antioxidant and antimutagenic activities were different between species and organs, with the strongest inhibition of lipid peroxidation by Et 2 O and AcOEt fractions from AP of both species (IC 50 13.33 - 16.89 μg/mL) and BuOH fraction from S. gigantea UP (IC 50  = 13.32 μg/mL). Chemical mutagenesis was completely inhibited by non-polar fractions, but oxidative mutagenesis was inhibited up to 35% only by S. canadensis. No clear relationship was found between chemical profiles and antimutagenic activity. In conclusion, both species have diverse activity and their phytochemical profiles should be considered in quality evaluation. UP of these weeds can also provide potential chemopreventive substances for further studies. © 2018 Wiley-VHCA AG, Zurich, Switzerland.

Functional and structural characterization of the pentapeptide insertion of Theileria annulata lactate dehydrogenase by site-directed mutagenesis, comparative modeling and molecular dynamics simulations.

PubMed

Erdemir, Aysegul; Mutlu, Ozal

2017-06-01

Lactate dehydrogenase (LDH) is an important metabolic enzyme in glycolysis and it has been considered as the main energy source in many organisms including apicomplexan parasites. Differences at the active site loop of the host and parasite LDH's makes this enzyme an attractive target for drug inhibitors. In this study, five amino acid insertions in the active site pocket of Theileria annulata LDH (TaLDH) were deleted by PCR-based site-directed mutagenesis, expression and activity analysis of mutant and wild type TaLDH enzymes were performed. Removal of the insertion at the active site loop caused production of an inactive enzyme. Furthermore, structures of wild and mutant enzymes were predicted by comparative modeling and the importance of the insertions at the active site loop were also assigned by molecular docking and dynamics simulations in order to evaluate essential role of this loop for the enzymatic activity. Pentapeptide insertion removal resulted in loss of LDH activity due to deletion of Trp96 and conformational change of Arg98 because of loop instability. Analysis of wild type and mutant enzymes with comparative molecular dynamics simulations showed that the fluctuations of the loop residues increase in mutant enzyme. Together with in silico studies, in vitro results revealed that active site loop has a vital role in the enzyme activity and our findings promise hope for the further drug design studies against theileriosis and other apicomplexan parasite diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

Improved antimicrobial activity of Pediococcus acidilactici against Salmonella Gallinarum by UV mutagenesis and genome shuffling.

PubMed

Han, Geon Goo; Song, Ahn Ah; Kim, Eun Bae; Yoon, Seong-Hyun; Bok, Jin-Duck; Cho, Chong-Su; Kil, Dong Yong; Kang, Sang-Kee; Choi, Yun-Jaie

2017-07-01

Pediococcus acidilactici is a widely used probiotic, and Salmonella enterica serovar Gallinarum (SG) is a significant pathogen in the poultry industry. In this study, we improved the antimicrobial activity of P. acidilactici against SG using UV mutation and genome shuffling (GS). To improve antimicrobial activity against SG, UV mutagenesis was performed against wild-type P. acidilactici (WT), and five mutants showed improved antimicrobial activity. To further improve antimicrobial activity, GS was performed on five UV mutants. Following GS, four mutants showed improved antimicrobial activity compared with the UV mutants and WT. The antimicrobial activity of GS1 was highest among the mutants; however, the activity was reduced when the culture supernatant was treated with proteinase K, suggesting that the improved antimicrobial activity is due to a proteinous substance such as bacteriocin. To validate the activity of GS1 in vivo, we designed multi-species probiotics and performed broiler feeding experiments. Groups consisted of no treatment (NC), avilamycin-treated (PC), probiotic group 1 containing WT (T1), and probiotic group 2 containing GS1 (T2). In broiler feeding experiments, coliform bacteria were significantly reduced in T2 compared with NC, PC, and T1. The cecal microbiota was modulated and pathogenic bacteria were reduced by GS1 oral administration. In this study, GS1 showed improved antimicrobial activity against SG in vitro and reduced pathogenic bacteria in a broiler feeding experiment. These results suggest that GS1 can serve as an efficient probiotic, as an alternative to antibiotics in the poultry industry.

Progress in bio-manufacture of platelets for transfusion.

PubMed

Heazlewood, Shen Y; Nilsson, Susan K; Cartledge, Kellie; Be, Cheang Ly; Vinson, Andrew; Gel, Murat; Haylock, David N

2017-11-01

Blood transfusion services face an ever-increasing demand for donor platelets to meet clinical needs. Whilst strategies for increasing platelet storage life and improving the efficiency of donor platelet collection are important, in the longer term, platelets generated by bio-manufacturing processes will be required to meet demands. Production of sufficient numbers of in vitro-derived platelets for transfusion represents a significant bioengineering challenge. In this review, we highlight recent progress in this area of research and outline the main technical and biological obstacles that need to be met before this becomes feasible and economic. A critical consideration is assurance of the functional properties of these cells as compared to their fresh, donor collected, counterparts. We contend that platelet-like particles and in vitro-derived platelets that phenotypically resemble fresh platelets must deliver the same functions as these cells upon transfusion. We also note recent progress with immortalized megakaryocyte progenitor cell lines, molecular strategies for reducing expression of HLA Class I to generate universal donor platelets and the move to early clinical studies with in vitro-derived platelets.

What Mutagenesis Can and Cannot Reveal About Allostery.

PubMed

Carlson, Gerald M; Fenton, Aron W

2016-05-10

Allosteric regulation of protein function is recognized to be widespread throughout biology; however, knowledge of allosteric mechanisms, the molecular changes within a protein that couple one binding site to another, is limited. Although mutagenesis is often used to probe allosteric mechanisms, we consider herein what the outcome of a mutagenesis study truly reveals about an allosteric mechanism. Arguably, the best way to evaluate the effects of a mutation on allostery is to monitor the allosteric coupling constant (Qax), a ratio of the substrate binding constants in the absence versus presence of an allosteric effector. A range of substitutions at a given residue position in a protein can reveal when a particular substitution causes gain-of-function, which addresses a key challenge in interpreting mutation-dependent changes in the magnitude of Qax. Thus, whole-protein mutagenesis studies offer an acceptable means of identifying residues that contribute to an allosteric mechanism. With this focus on monitoring Qax, and keeping in mind the equilibrium nature of allostery, we consider alternative possibilities for what an allosteric mechanism might be. We conclude that different possible mechanisms (rotation-of-solid-domains, movement of secondary structure, side-chain repacking, changes in dynamics, etc.) will result in different findings in whole-protein mutagenesis studies. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Mismatch repair deficiency does not enhance ENU mutagenesis in the zebrafish germ line.

PubMed

Feitsma, Harma; de Bruijn, Ewart; van de Belt, Jose; Nijman, Isaac J; Cuppen, Edwin

2008-07-01

S(N)1-type alkylating agents such as N-ethyl-N-nitrosourea (ENU) are very potent mutagens. They act by transferring their alkyl group to DNA bases, which, upon mispairing during replication, can cause single base pair mutations in the next replication cycle. As DNA mismatch repair (MMR) proteins are involved in the recognition of alkylation damage, we hypothesized that ENU-induced mutation rates could be increased in a MMR-deficient background, which would be beneficial for mutagenesis approaches. We applied a standard ENU mutagenesis protocol to adult zebrafish deficient in the MMR gene msh6 and heterozygous controls to study the effect of MMR on ENU-induced DNA damage. Dose-dependent lethality was found to be similar for homozygous and heterozygous mutants, indicating that there is no difference in ENU resistance. Mutation discovery by high-throughput dideoxy resequencing of genomic targets in outcrossed progeny of the mutagenized fish did also not reveal any differences in germ line mutation frequency. These results may indicate that the maximum mutation load for zebrafish has been reached with the currently used, highly optimized ENU mutagenesis protocol. Alternatively, the MMR system in the zebrafish germ line may be saturated very rapidly, thereby having a limited effect on high-dose ENU mutagenesis.

Approaches towards the enhanced production of Rapamycin by Streptomyces hygroscopicus MTCC 4003 through mutagenesis and optimization of process parameters by Taguchi orthogonal array methodology.

PubMed

Dutta, Subhasish; Basak, Bikram; Bhunia, Biswanath; Sinha, Ankan; Dey, Apurba

2017-05-01

The present research was conducted to define the approaches for enhanced production of rapamycin (Rap) by Streptomyces hygroscopicus microbial type culture collection (MTCC) 4003. Both physical mutagenesis by ultraviolet ray (UV) and chemical mutagenesis by N-methyl-N-nitro-N-nitrosoguanidine (NTG) have been applied successfully for the improvement of Rap production. Enhancing Rap yield by novel sequential UV mutagenesis technique followed by fermentation gives a significant difference in getting economically scalable amount of this industrially important macrolide compound. Mutant obtained through NTG mutagenesis (NTG-30-27) was found to be superior to others as it initially produced 67% higher Rap than wild type. Statistical optimization of nutritional and physiochemical parameters was carried out to find out most influential factors responsible for enhanced Rap yield by NTG-30-27 which was performed using Taguchi orthogonal array approach. Around 72% enhanced production was achieved with nutritional factors at their assigned level at 23 °C, 120 rpm and pH 7.6. Results were analysed in triplicate basis where validation and purification was carried out using high performance liquid chromatography. Stability study and potency of extracted Rap was supported by turbidimetric assay taking Candida albicans MTCC 227 as test organism.

Novel Escherichia coli umuD′ Mutants: Structure-Function Insights into SOS Mutagenesis

PubMed Central

McLenigan, Mary; Peat, Thomas S.; Frank, Ekaterina G.; McDonald, John P.; Gonzalez, Martín; Levine, Arthur S.; Hendrickson, Wayne A.; Woodgate, Roger

1998-01-01

Although it has been 10 years since the discovery that the Escherichia coli UmuD protein undergoes a RecA-mediated cleavage reaction to generate mutagenically active UmuD′, the function of UmuD′ has yet to be determined. In an attempt to elucidate the role of UmuD′ in SOS mutagenesis, we have utilized a colorimetric papillation assay to screen for mutants of a hydroxylamine-treated, low-copy-number umuD′ plasmid that are unable to promote SOS-dependent spontaneous mutagenesis. Using such an approach, we have identified 14 independent umuD′ mutants. Analysis of these mutants revealed that two resulted from promoter changes which reduced the expression of wild-type UmuD′, three were nonsense mutations that resulted in a truncated UmuD′ protein, and the remaining nine were missense alterations. In addition to the hydroxylamine-generated mutants, we have subcloned the mutations found in three chromosomal umuD1, umuD44, and umuD77 alleles into umuD′. All 17 umuD′ mutants resulted in lower levels of SOS-dependent spontaneous mutagenesis but varied in the extent to which they promoted methyl methanesulfonate-induced mutagenesis. We have attempted to correlate these phenotypes with the potential effect of each mutation on the recently described structure of UmuD′. PMID:9721309

In Vitro Tumor Models: Advantages, Disadvantages, Variables, and Selecting the Right Platform.

PubMed

Katt, Moriah E; Placone, Amanda L; Wong, Andrew D; Xu, Zinnia S; Searson, Peter C

2016-01-01

In vitro tumor models have provided important tools for cancer research and serve as low-cost screening platforms for drug therapies; however, cancer recurrence remains largely unchecked due to metastasis, which is the cause of the majority of cancer-related deaths. The need for an improved understanding of the progression and treatment of cancer has pushed for increased accuracy and physiological relevance of in vitro tumor models. As a result, in vitro tumor models have concurrently increased in complexity and their output parameters further diversified, since these models have progressed beyond simple proliferation, invasion, and cytotoxicity screens and have begun recapitulating critical steps in the metastatic cascade, such as intravasation, extravasation, angiogenesis, matrix remodeling, and tumor cell dormancy. Advances in tumor cell biology, 3D cell culture, tissue engineering, biomaterials, microfabrication, and microfluidics have enabled rapid development of new in vitro tumor models that often incorporate multiple cell types, extracellular matrix materials, and spatial and temporal introduction of soluble factors. Other innovations include the incorporation of perfusable microvessels to simulate the tumor vasculature and model intravasation and extravasation. The drive toward precision medicine has increased interest in adapting in vitro tumor models for patient-specific therapies, clinical management, and assessment of metastatic potential. Here, we review the wide range of current in vitro tumor models and summarize their advantages, disadvantages, and suitability in modeling specific aspects of the metastatic cascade and drug treatment.

In Vitro Tumor Models: Advantages, Disadvantages, Variables, and Selecting the Right Platform

PubMed Central

Katt, Moriah E.; Placone, Amanda L.; Wong, Andrew D.; Xu, Zinnia S.; Searson, Peter C.

2016-01-01

In vitro tumor models have provided important tools for cancer research and serve as low-cost screening platforms for drug therapies; however, cancer recurrence remains largely unchecked due to metastasis, which is the cause of the majority of cancer-related deaths. The need for an improved understanding of the progression and treatment of cancer has pushed for increased accuracy and physiological relevance of in vitro tumor models. As a result, in vitro tumor models have concurrently increased in complexity and their output parameters further diversified, since these models have progressed beyond simple proliferation, invasion, and cytotoxicity screens and have begun recapitulating critical steps in the metastatic cascade, such as intravasation, extravasation, angiogenesis, matrix remodeling, and tumor cell dormancy. Advances in tumor cell biology, 3D cell culture, tissue engineering, biomaterials, microfabrication, and microfluidics have enabled rapid development of new in vitro tumor models that often incorporate multiple cell types, extracellular matrix materials, and spatial and temporal introduction of soluble factors. Other innovations include the incorporation of perfusable microvessels to simulate the tumor vasculature and model intravasation and extravasation. The drive toward precision medicine has increased interest in adapting in vitro tumor models for patient-specific therapies, clinical management, and assessment of metastatic potential. Here, we review the wide range of current in vitro tumor models and summarize their advantages, disadvantages, and suitability in modeling specific aspects of the metastatic cascade and drug treatment. PMID:26904541

29 CFR 1990.103 - Definitions.

Code of Federal Regulations, 2012 CFR

2012-07-01

... Health and Human Services, or designee. Director of NCI means the Director of the National Cancer... means the induction of heritable changes in the genetic material of either somatic or germinal cells..., Neurospora or Drosophila melanogaster; (3) Mutagenesis in mammalian somatic cells; (4) Mutagenesis in...

29 CFR 1990.103 - Definitions.

Code of Federal Regulations, 2013 CFR

2013-07-01

... Health and Human Services, or designee. Director of NCI means the Director of the National Cancer... means the induction of heritable changes in the genetic material of either somatic or germinal cells..., Neurospora or Drosophila melanogaster; (3) Mutagenesis in mammalian somatic cells; (4) Mutagenesis in...

29 CFR 1990.103 - Definitions.

Code of Federal Regulations, 2011 CFR

2011-07-01

... Health and Human Services, or designee. Director of NCI means the Director of the National Cancer... means the induction of heritable changes in the genetic material of either somatic or germinal cells..., Neurospora or Drosophila melanogaster; (3) Mutagenesis in mammalian somatic cells; (4) Mutagenesis in...

29 CFR 1990.103 - Definitions.

Code of Federal Regulations, 2014 CFR

2014-07-01

... Health and Human Services, or designee. Director of NCI means the Director of the National Cancer... means the induction of heritable changes in the genetic material of either somatic or germinal cells..., Neurospora or Drosophila melanogaster; (3) Mutagenesis in mammalian somatic cells; (4) Mutagenesis in...

CYTOTOXICITY AND MUTAGENESIS METHODS FOR EVALUATING TOXICITY REMOVAL FROM WASTEWATERS

EPA Science Inventory

This project was a feasibility study of the effectiveness of a mammalian cell cytotoxicity assay and a mammalian cell mutagenesis assay for monitoring the toxicity and mutagenicity of influent and effluent wastewater at treatment plants. In the cytotoxicity assay, ambient samples...

Simultaneous site-directed mutagenesis of duplicated loci in soybean using a single guide RNA.

PubMed

Kanazashi, Yuhei; Hirose, Aya; Takahashi, Ippei; Mikami, Masafumi; Endo, Masaki; Hirose, Sakiko; Toki, Seiichi; Kaga, Akito; Naito, Ken; Ishimoto, Masao; Abe, Jun; Yamada, Tetsuya

2018-03-01

Using a gRNA and Agrobacterium-mediated transformation, we performed simultaneous site-directed mutagenesis of two GmPPD loci in soybean. Mutations in GmPPD loci were confirmed in at least 33% of T 2 seeds. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system is a powerful tool for site-directed mutagenesis in crops. Using a single guide RNA (gRNA) and Agrobacterium-mediated transformation, we performed simultaneous site-directed mutagenesis of two homoeologous loci in soybean (Glycine max), GmPPD1 and GmPPD2, which encode the orthologs of Arabidopsis thaliana PEAPOD (PPD). Most of the T 1 plants had heterozygous and/or chimeric mutations for the targeted loci. The sequencing analysis of T 1 and T 2 generations indicates that putative mutation induced in the T 0 plant is transmitted to the T 1 generation. The inheritable mutation induced in the T 1 plant was also detected. This result indicates that continuous induction of mutations during T 1 plant development increases the occurrence of mutations in germ cells, which ensures the transmission of mutations to the next generation. Simultaneous site-directed mutagenesis in both GmPPD loci was confirmed in at least 33% of T 2 seeds examined. Approximately 19% of double mutants did not contain the Cas9/gRNA expression construct. Double mutants with frameshift mutations in both GmPPD1 and GmPPD2 had dome-shaped trifoliate leaves, extremely twisted pods, and produced few seeds. Taken together, our data indicate that continuous induction of mutations in the whole plant and advancing generations of transgenic plants enable efficient simultaneous site-directed mutagenesis in duplicated loci in soybean.

Roles for the yeast RAD18 and RAD52 DNA repair genes in UV mutagenesis.

PubMed

Armstrong, J D; Chadee, D N; Kunz, B A

1994-11-01

Experimental evidence indicates that although the Saccharomyces cerevisiae RAD18 and RAD52 genes are not required for nucleotide excision repair, they function in the processing of UV-induced DNA damage in yeast. Conflicting statements regarding the UV mutability of strains deleted for RAD18 prompted us to re-examine the influence of RAD18, and RAD52, on UV mutagenesis. To do so, we characterized mutations induced by UV in SUP4-o, a yeast suppressor tRNA gene. SUP4-o was maintained on a plasmid in isogenic strains that either carried one of two different rad18 deletions (rad18 delta) or had RAD52 disrupted. Both rad18 deletions decreased the frequency of UV-induced SUP4-o mutations to levels close to those for spontaneous mutagenesis in the rad18 delta backgrounds, and prevented a net increase in mutant yield. A detailed analysis of mutations isolated after UV irradiation of one of the rad18 delta strains uncovered little evidence of the specificity features typical for UV mutagenesis in the isogenic repair-proficient (RAD) parent (e.g., predominance of G.C-->A.T transitions). Evidently, UV induction of SUP4-o mutations is highly dependent on the RAD18 gene. Compared to the RAD strain, disruption of RAD52 reduced the frequency and yield of UV mutagenesis by about two-thirds. Closer inspection revealed that 80% of this reduction was due to a decrease in the frequency of G.C-->A.T transitions. In addition, there were differences in the distributions and site specificities of single base-pair substitutions. Thus, RAD52 also participates in UV mutagenesis of a plasmid-borne gene in yeast, but to a lesser extent than RAD18.

Atypical Role for PhoU in Mutagenic Break Repair under Stress in Escherichia coli

PubMed Central

Aponyi, Ildiko; Vera Cruz, Diana; Ray, Mellanie P.; Rosenberg, Susan M.

2015-01-01

Mechanisms of mutagenesis activated by stress responses drive pathogen/host adaptation, antibiotic and anti-fungal-drug resistance, and perhaps much of evolution generally. In Escherichia coli, repair of double-strand breaks (DSBs) by homologous recombination is high fidelity in unstressed cells, but switches to a mutagenic mode using error-prone DNA polymerases when the both the SOS and general (σS) stress responses are activated. Additionally, the σE response promotes spontaneous DNA breakage that leads to mutagenic break repair (MBR). We identified the regulatory protein PhoU in a genetic screen for functions required for MBR. PhoU negatively regulates the phosphate-transport and utilization (Pho) regulon when phosphate is in excess, including the PstB and PstC subunits of the phosphate-specific ABC transporter PstSCAB. Here, we characterize the PhoU mutation-promoting role. First, some mutations that affect phosphate transport and Pho transcriptional regulation decrease mutagenesis. Second, the mutagenesis and regulon-expression phenotypes do not correspond, revealing an apparent new function(s) for PhoU. Third, the PhoU mutagenic role is not via activation of the σS, SOS or σE responses, because mutations (or DSBs) that restore mutagenesis to cells defective in these stress responses do not restore mutagenesis to phoU cells. Fourth, the mutagenesis defect in phoU-mutant cells is partially restored by deletion of arcA, a gene normally repressed by PhoU, implying that a gene(s) repressed by ArcA promotes mutagenic break repair. The data show a new role for PhoU in regulation, and a new regulatory branch of the stress-response signaling web that activates mutagenic break repair in E. coli. PMID:25961709

The p21 and PCNA partnership: a new twist for an old plot.

PubMed

Prives, Carol; Gottifredi, Vanesa

2008-12-15

The contribution of error-prone DNA polymerases to the DNA damage response has been a subject of great interest in the last decade. Error-prone polymerases are required for translesion DNA synthesis (TLS), a process that involves synthesis past a DNA lesion. Under certain circumstances, TLS polymerases can achieve bypass with good efficiency and fidelity. However, they can also in some cases be mutagenic, and so negative regulators of TLS polymerases would have the important function of inhibiting their recruitment to undamaged DNA templates. Recent work from Livneh's and our groups have provided evidence regarding the role of the cyclin kinase inhibitor p21 as a negative regulator of TLS. Interestingly, both the cyclin dependent kinase (CDK) and proliferating cell nuclear antigen (PCNA) binding domains of p21 are involved in different aspects of the modulation of TLS, affecting both the interaction between PCNA and the TLS-specific pol eta as well as PCNA ubiquitination status. In line with this, p21 was shown to reduce the efficiency but increase the accuracy of TLS. Hence, in absence of DNA damage p21 may work to impede accidental loading of pol eta to undamaged DNA and avoid consequential mutagenesis. After UV irradiation, when TLS plays a decisive role, p21 is progressively degraded. This might allow gradual release of replication fork blockage by TLS polymerases. For these reasons, in higher eukaryotes p21 might represent a key regulator of the equilibrium between mutagenesis and cell survival.

Progress of targeted genome modification approaches in higher plants.

PubMed

Cardi, Teodoro; Neal Stewart, C

2016-07-01

Transgene integration in plants is based on illegitimate recombination between non-homologous sequences. The low control of integration site and number of (trans/cis)gene copies might have negative consequences on the expression of transferred genes and their insertion within endogenous coding sequences. The first experiments conducted to use precise homologous recombination for gene integration commenced soon after the first demonstration that transgenic plants could be produced. Modern transgene targeting categories used in plant biology are: (a) homologous recombination-dependent gene targeting; (b) recombinase-mediated site-specific gene integration; (c) oligonucleotide-directed mutagenesis; (d) nuclease-mediated site-specific genome modifications. New tools enable precise gene replacement or stacking with exogenous sequences and targeted mutagenesis of endogeneous sequences. The possibility to engineer chimeric designer nucleases, which are able to target virtually any genomic site, and use them for inducing double-strand breaks in host DNA create new opportunities for both applied plant breeding and functional genomics. CRISPR is the most recent technology available for precise genome editing. Its rapid adoption in biological research is based on its inherent simplicity and efficacy. Its utilization, however, depends on available sequence information, especially for genome-wide analysis. We will review the approaches used for genome modification, specifically those for affecting gene integration and modification in higher plants. For each approach, the advantages and limitations will be noted. We also will speculate on how their actual commercial development and implementation in plant breeding will be affected by governmental regulations.

Imaging C. elegans embryos using an epifluorescent microscope and open source software.

PubMed

Verbrugghe, Koen J C; Chan, Raymond C

2011-03-24

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples(1,2). Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage(3), thus providing an ideal experiment model for studying questions in cell biology(4,5)and development(6-9). C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis(10,11)) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis(12-15)). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters(16,17). These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo(18-21). In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

The Ebola virus matrix protein penetrates into the plasma membrane: a key step in viral protein 40 (VP40) oligomerization and viral egress.

PubMed

Adu-Gyamfi, Emmanuel; Soni, Smita P; Xue, Yi; Digman, Michelle A; Gratton, Enrico; Stahelin, Robert V

2013-02-22

Ebola, a fatal virus in humans and non-human primates, has no Food and Drug Administration-approved vaccines or therapeutics. The virus from the Filoviridae family causes hemorrhagic fever, which rapidly progresses and in some cases has a fatality rate near 90%. The Ebola genome encodes seven genes, the most abundantly expressed of which is viral protein 40 (VP40), the major Ebola matrix protein that regulates assembly and egress of the virus. It is well established that VP40 assembles on the inner leaflet of the plasma membrane; however, the mechanistic details of plasma membrane association by VP40 are not well understood. In this study, we used an array of biophysical experiments and cellular assays along with mutagenesis of VP40 to investigate the role of membrane penetration in VP40 assembly and egress. Here we demonstrate that VP40 is able to penetrate specifically into the plasma membrane through an interface enriched in hydrophobic residues in its C-terminal domain. Mutagenesis of this hydrophobic region consisting of Leu(213), Ile(293), Leu(295), and Val(298) demonstrated that membrane penetration is critical to plasma membrane localization, VP40 oligomerization, and viral particle egress. Taken together, VP40 membrane penetration is an important step in the plasma membrane localization of the matrix protein where oligomerization and budding are defective in the absence of key hydrophobic interactions with the membrane.

CHEMICAL MUTAGENESIS AND CARCINOGENESIS: INCORPORATION OF MECHANISTIC DATA INTO RISK ASSESSMENT

EPA Science Inventory

CHEMICAL MUTAGENESIS AND CARCINOGENESIS: INCORPORATION OF MECHANISTIC DATA INTO RISK ASSESSMENT

The current understanding of cancer as a genetic disease, requiring a specific set of genomic alterations for a normal cell to form a metastatic tumor, has provided the oppor...

CHALLENGES FOR THE FUTURE IN ENVIRONMENTAL MUTAGENESIS

EPA Science Inventory

CHALLENGES FOR THE FUTURE IN ENVIRONMENTAL MUTAGENESIS
Michael D. Waters
US Environmental Protection Agency, MD-51A, Research Triangle Park, NC 27711 USA

Our rapidly growing understanding of the structure of the human genome is forming the basis for numerous new...

Faux Mutagenesis: Teaching Troubleshooting through Controlled Failure

ERIC Educational Resources Information Center

Hartberg, Yasha

2006-01-01

By shifting pedagogical goals from obtaining successful mutations to teaching students critical troubleshooting skills, it has been possible to introduce site-directed mutagenesis into an undergraduate teaching laboratory. Described in this study is an inexpensive laboratory exercise in which students follow a slightly modified version of…

In vitro manipulation of mammalian gametes and embryos: what are we learning from animal settings

USDA-ARS?s Scientific Manuscript database

Since the birth of the first baby conceived by in vitro fertilization (Louise Brown) in 1978, significant progress has been achieved in the application of assisted reproductive technology (ART) in clinical settings. A significant accumulation of knowledge obtained through various research endeavors ...

Study of radiation effects on mammalian cells in vitro

NASA Technical Reports Server (NTRS)

Sinclair, W. K.

1968-01-01

Radiation effect on single cells and cell populations of Chinese hamster lung tissue is studied in vitro. The rate and position as the cell progresses through the generation cycle shows division delay, changes in some biochemical processes in the cell, chromosomal changes, colony size changes, and loss of reproductive capacity.

Combined mutagenesis of Rhodosporidium toruloides for improved production of carotenoids and lipids.

PubMed

Zhang, Chaolei; Shen, Hongwei; Zhang, Xibin; Yu, Xue; Wang, Han; Xiao, Shan; Wang, Jihui; Zhao, Zongbao K

2016-10-01

To improve production of lipids and carotenoids by the oleaginous yeast Rhodosporidium toruloides by screening mutant strains. Upon physical mutagenesis of the haploid strain R. toruloides np11 with an atmospheric and room temperature plasma method followed by chemical mutagenesis with nitrosoguanidine, a mutant strain, R. toruloides XR-2, formed dark-red colonies on a screening plate. When cultivated in nitrogen-limited media, XR-2 cells grew slower but accumulated 0.23 g lipids/g cell dry wt and 0.75 mg carotenoids/g CDW. To improve its production capacity, different amino acids and vitamins were supplemented. p-Aminobenzoic acid and tryptophan had beneficial effects on cell growth. When cultivated in nitrogen-limited media in the presence of selected vitamins, XR-2 accumulated 0.41 g lipids/g CDW and 0.69 mg carotenoids/g CDW. A mutant R. toruloides strain with improved production profiles for lipids and carotenoids was obtained, indicating its potential to use combined mutagenesis for a more productive phenotype.

Regulation of error-prone translesion synthesis by Spartan/C1orf124

PubMed Central

Kim, Myoung Shin; Machida, Yuka; Vashisht, Ajay A.; Wohlschlegel, James A.; Pang, Yuan-Ping; Machida, Yuichi J.

2013-01-01

Translesion synthesis (TLS) employs low fidelity polymerases to replicate past damaged DNA in a potentially error-prone process. Regulatory mechanisms that prevent TLS-associated mutagenesis are unknown; however, our recent studies suggest that the PCNA-binding protein Spartan plays a role in suppression of damage-induced mutagenesis. Here, we show that Spartan negatively regulates error-prone TLS that is dependent on POLD3, the accessory subunit of the replicative DNA polymerase Pol δ. We demonstrate that the putative zinc metalloprotease domain SprT in Spartan directly interacts with POLD3 and contributes to suppression of damage-induced mutagenesis. Depletion of Spartan induces complex formation of POLD3 with Rev1 and the error-prone TLS polymerase Pol ζ, and elevates mutagenesis that relies on POLD3, Rev1 and Pol ζ. These results suggest that Spartan negatively regulates POLD3 function in Rev1/Pol ζ-dependent TLS, revealing a previously unrecognized regulatory step in error-prone TLS. PMID:23254330

Identification of α-amylase by random and specific mutagenesis of Texcoconibacillus texcoconensis 13CCT strain isolated from extreme alkaline-saline soil of the former Lake Texcoco (Mexico).

PubMed

Bello-López, Juan Manuel; Navarro-Noya, Yendi E; Gómez-Acata, Selene; Hernández-Montañez, Zahuiti; Dendooven, Luc

2014-05-01

The alkaline α-amylase produced by Texcoconibacillus texcoconensis 13CC(T) strain was identified by random mutagenesis and confirmed by directed mutagenesis. A transposon mutagenesis approach was taken to identify the gene responsible for the degradation of starch in T. texcoconensis 13CC(T) strain. The deduced amino acids of the amy gene had a 99% similarity with those of Bacillus selenitireducens MLS10 and 97% with those of Paenibacillus curdlanolyticus YK9. The enzyme showed a maximum activity of 131.1 U/mL at 37 °C and pH 9.5 to 10.5. In situ activity of the enzyme determined by polyacrylamide gel electrophoresis showed only one band with amylolytic activity. This is the first report of a bacterium isolated from the extreme alkaline-saline soil of the former Lake Texcoco (Mexico) with amylolytic activity in alkaline conditions while its potential as a source of amylases for the industry is discussed.

Role of drosophila in chemical mutagenesis testing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nix, C.E.; Brewen, B.

1978-01-01

An important question facing our society is the impact of numerous chemical insults on the health of man and his environment. Faced with a staggering array of chemicals and enormous testing costs, only a few chemicals can be tested for possible carcinogenic effects. Recent results with the Salmonella/mammalian microsome mutagenesis bioassay system demonstrate a striking correlation between carcinogenicity and mutagenicity of many chemical compounds and offer the possibility that mutagenesis assay systems can provide a quick identification of potential carcinogens. Results from microbial assays can serve as a guideline for further mutagenesis testing as well as identify those compounds requiringmore » more extensive analysis in mammalian systems. Reliance on the results from a single mutagenic assay system is rather risky. It would be preferable to use a battery of tests (the tier approach) which would include the rapid microbial assays as well as mammalian systems. Also the use of Drosophila as a bridge between the microbial and mammalian assays has many desirable features which are discussed.« less

Induction of mutagenesis and alterations in gene expression by tumorigenic chemicals

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huberman, E.

1979-01-01

To determine the relationship between mutagenesis and carcinogenesis, a series of eleven polycyclic hydrocarbons with different degrees of carcinogenicity were tested in the cell-mediated mutagenesis assay for the induction of ouabain-resistant mutants. Four carcinogenic hydrocarbons induced ouabain-resistant mutants; five noncarcinogenic hydrocarbons were not mutagenic. Results indicated that there was a relationship between mutagenesis and the degree of carcinogenicity of polycyclic hydrocarbons after enhancement of their metabolism by aminophylline. To study liver carcinogens a system was developed for cocultivating primary liver cells and V79 hamster cells. In this system the nitrosamines and aflatoxins were metabolized by liver cells to intermediates thatmore » were mutagenic to the V79 cells. In experiments using human cells, tumor-promoting phorbol esters induced terminal differentiation while in other studies, in which avian and murine cells were employed, they inhibited differentiation. The results imply that human cells may respond differently from mouse and chicken cells to the biological effects of phorbol diesters. (HLW)« less

A method for multi-codon scanning mutagenesis of proteins based on asymmetric transposons.

PubMed

Liu, Jia; Cropp, T Ashton

2012-02-01

Random mutagenesis followed by selection or screening is a commonly used strategy to improve protein function. Despite many available methods for random mutagenesis, nearly all generate mutations at the nucleotide level. An ideal mutagenesis method would allow for the generation of 'codon mutations' to change protein sequence with defined or mixed amino acids of choice. Herein we report a method that allows for mutations of one, two or three consecutive codons. Key to this method is the development of a Mu transposon variant with asymmetric terminal sequences. As a demonstration of the method, we performed multi-codon scanning on the gene encoding superfolder GFP (sfGFP). Characterization of 50 randomly chosen clones from each library showed that more than 40% of the mutants in these three libraries contained seamless, in-frame mutations with low site preference. By screening only 500 colonies from each library, we successfully identified several spectra-shift mutations, including a S205D variant that was found to bear a single excitation peak in the UV region.

mtDNA Mutagenesis Disrupts Pluripotent Stem Cell Function by Altering Redox Signaling

PubMed Central

Hämäläinen, Riikka H.; Ahlqvist, Kati J.; Ellonen, Pekka; Lepistö, Maija; Logan, Angela; Otonkoski, Timo; Murphy, Michael P.; Suomalainen, Anu

2015-01-01

Summary mtDNA mutagenesis in somatic stem cells leads to their dysfunction and to progeria in mouse. The mechanism was proposed to involve modification of reactive oxygen species (ROS)/redox signaling. We studied the effect of mtDNA mutagenesis on reprogramming and stemness of pluripotent stem cells (PSCs) and show that PSCs select against specific mtDNA mutations, mimicking germline and promoting mtDNA integrity despite their glycolytic metabolism. Furthermore, mtDNA mutagenesis is associated with an increase in mitochondrial H2O2, reduced PSC reprogramming efficiency, and self-renewal. Mitochondria-targeted ubiquinone, MitoQ, and N-acetyl-L-cysteine efficiently rescued these defects, indicating that both reprogramming efficiency and stemness are modified by mitochondrial ROS. The redox sensitivity, however, rendered PSCs and especially neural stem cells sensitive to MitoQ toxicity. Our results imply that stem cell compartment warrants special attention when the safety of new antioxidants is assessed and point to an essential role for mitochondrial redox signaling in maintaining normal stem cell function. PMID:26027936

Variant-aware saturating mutagenesis using multiple Cas9 nucleases identifies regulatory elements at trait-associated loci.

PubMed

Canver, Matthew C; Lessard, Samuel; Pinello, Luca; Wu, Yuxuan; Ilboudo, Yann; Stern, Emily N; Needleman, Austen J; Galactéros, Frédéric; Brugnara, Carlo; Kutlar, Abdullah; McKenzie, Colin; Reid, Marvin; Chen, Diane D; Das, Partha Pratim; A Cole, Mitchel; Zeng, Jing; Kurita, Ryo; Nakamura, Yukio; Yuan, Guo-Cheng; Lettre, Guillaume; Bauer, Daniel E; Orkin, Stuart H

2017-04-01

Cas9-mediated, high-throughput, saturating in situ mutagenesis permits fine-mapping of function across genomic segments. Disease- and trait-associated variants identified in genome-wide association studies largely cluster at regulatory loci. Here we demonstrate the use of multiple designer nucleases and variant-aware library design to interrogate trait-associated regulatory DNA at high resolution. We developed a computational tool for the creation of saturating-mutagenesis libraries with single or multiple nucleases with incorporation of variants. We applied this methodology to the HBS1L-MYB intergenic region, which is associated with red-blood-cell traits, including fetal hemoglobin levels. This approach identified putative regulatory elements that control MYB expression. Analysis of genomic copy number highlighted potential false-positive regions, thus emphasizing the importance of off-target analysis in the design of saturating-mutagenesis experiments. Together, these data establish a widely applicable high-throughput and high-resolution methodology to identify minimal functional sequences within large disease- and trait-associated regions.

Single-step generation of rabbits carrying a targeted allele of the tyrosinase gene using CRISPR/Cas9.

PubMed

Honda, Arata; Hirose, Michiko; Sankai, Tadashi; Yasmin, Lubna; Yuzawa, Kazuaki; Honsho, Kimiko; Izu, Haruna; Iguchi, Atsushi; Ikawa, Masahito; Ogura, Atsuo

2015-01-01

Targeted genome editing of nonrodent mammalian species has provided the potential for highly accurate interventions into gene function in humans and the generation of useful animal models of human diseases. Here we show successful clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (Cas)-mediated gene targeting via circular plasmid injection in rabbits. The rabbit tyrosinase gene (TYR) was effectively disrupted, and we confirmed germline transmission by pronuclear injection of a circular plasmid expressing humanized Cas9 (hCas9) and single-guide RNA. Direct injection into pronuclear stage zygotes was possible following an in vitro validation assay. Neither off-target mutagenesis nor hCas9 transgenesis was detected in any of the genetically targeted pups and embryos examined. Gene targeting with this rapid and simplified strategy will help accelerate the development of translational research using other nonrodent mammalian species.

Thymidine Phosphorylase is Angiogenic and Promotes Tumor Growth

NASA Astrophysics Data System (ADS)

Moghaddam, Amir; Zhang, Hua-Tang; Fan, Tai-Ping D.; Hu, De-En; Lees, Vivien C.; Turley, Helen; Fox, Stephen B.; Gatter, Kevin C.; Harris, Adrian L.; Bicknell, Roy

1995-02-01

Platelet-derived endothelial cell growth factor was previously identified as the sole angiogenic activity present in platelets; it is now known to be thymidine phosphorylase (TP). The effect of TP on [methyl-^3H]thymidine uptake does not arise from de novo DNA synthesis and the molecule is not a growth factor. Despite this, TP is strongly angiogenic in a rat sponge and freeze-injured skin graft model. Neutralizing antibodies and site-directed mutagenesis confirmed that the enzyme activity of TP is a condition for its angiogenic activity. The level of TP was found to be elevated in human breast tumors compared to normal breast tissue (P < 0.001). Overexpression of TP in MCF-7 breast carcinoma cells had no effect on growth in vitro but markedly enhanced tumor growth in vivo. These data and the correlation of expression in tumors with malignancy identify TP as a target for antitumor strategies.

The Bridge Helix of RNA polymerase acts as a central nanomechanical switchboard for coordinating catalysis and substrate movement.

PubMed

Weinzierl, Robert O J

2011-01-01

The availability of in vitro assembly systems to produce recombinant archaeal RNA polymerases (RNAPs) offers one of the most powerful experimental tools for investigating the still relatively poorly understood molecular mechanisms underlying RNAP function. Over the last few years, we pioneered new robot-based high-throughput mutagenesis approaches to study structure/function relationships within various domains surrounding the catalytic center. The Bridge Helix domain, which appears in numerous X-ray structures as a 35-amino-acid-long alpha helix, coordinates the concerted movement of several other domains during catalysis through kinking of two discrete molecular hinges. Mutations affecting these kinking mechanisms have a direct effect on the specific catalytic activity of RNAP and can in some instances more than double it. Molecular dynamics simulations have established themselves as exceptionally useful for providing additional insights and detailed models to explain the underlying structural motions.

Assessment of antimutagenic and genotoxic potential of senna (Cassia angustifolia Vahl.) aqueous extract using in vitro assays.

PubMed

Silva, C R; Monteiro, M R; Rocha, H M; Ribeiro, A F; Caldeira-de-Araujo, A; Leitão, A C; Bezerra, R J A C; Pádula, M

2008-02-01

Senna (Cassia angustifolia Vahl.) is widely used as a laxative, although potential side effects, such as toxicity and genotoxicity, have been reported. This study evaluated genotoxic and mutagenic effects of senna aqueous extract (SAE) by means of four experimental assays: inactivation of Escherichia coli cultures; bacterial growth inhibition; reverse mutation test (Mutoxitest) and DNA strand break analysis in plasmid DNA. Our results demonstrated that SAE produces single and double strand breaks in plasmid DNA in a cell free system. On the other hand, SAE was not cytotoxic or mutagenic to Escherichia coli strains tested. In effect, SAE was able to avoid H(2)O(2)-induced mutagenesis and toxicity in Escherichia coli IC203 (uvrA oxyR) and IC205 (uvrA mutM) strains, pointing to a new antioxidant/antimutagenic action of SAE.

Analysis of the Mutations in the Active Site of the RNA-Dependent RNA Polymerase of Human Parainfluenza Virus Type 3 (HPIV3)

PubMed Central

Malur, Achut G.; Gupta, Neera K.; De, Bishnu P.; Banerjee, Amiya K.

2002-01-01

The large protein (L) of the human parainfluenza virus type 3 (HPIV3) is the functional RNA-dependent RNA polymerase, which possesses highly conserved residues QGDNQ located within motif C of domain III comprising the putative polymerase active site. We have characterized the role of the QGDNQ residues as well as the residues flanking this region in the polymerase activity of the L protein by site-directed mutagenesis and examining the polymerase activity of the wild-type and mutant L proteins by an in vivo minigenome replication assay and an in vitro mRNA transcription assay. All mutations in the QGDNQ residues abolished transcription while mutations in the flanking residues gave rise to variable polymerase activities. These observations support the contention that the QGDNQ sequence is absolutely required for the polymerase activity of the HPIV3 RNA-dependent RNA polymerase. PMID:12064576

Amyloid-β Peptide Induces Prion Protein Amyloid Formation: Evidence for Its Widespread Amyloidogenic Effect.

PubMed

Honda, Ryo

2018-04-12

Transmissible spongiform encephalopathy is associated with misfolding of prion protein (PrP) into an amyloid β-rich aggregate. Previous studies have indicated that PrP interacts with Alzheimer's disease amyloid-β peptide (Aβ), but it remains elusive how this interaction impacts on the misfolding of PrP. This study presents the first in vitro evidence that Aβ induces PrP-amyloid formation at submicromolar concentrations. Interestingly, systematic mutagenesis of PrP revealed that Aβ requires no specific amino acid sequences in PrP, and induces the misfolding of other unrelated proteins (insulin and lysozyme) into amyloid fibrils in a manner analogous to PrP. This unanticipated nonspecific amyloidogenic effect of Aβ indicates that this peptide might be involved in widespread protein aggregation, regardless of the amino acid sequences of target proteins, and exacerbate the pathology of many neurodegenerative diseases. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Single-step generation of rabbits carrying a targeted allele of the tyrosinase gene using CRISPR/Cas9

PubMed Central

Honda, Arata; Hirose, Michiko; Sankai, Tadashi; Yasmin, Lubna; Yuzawa, Kazuaki; Honsho, Kimiko; Izu, Haruna; Iguchi, Atsushi; Ikawa, Masahito; Ogura, Atsuo

2014-01-01

Targeted genome editing of nonrodent mammalian species has provided the potential for highly accurate interventions into gene function in humans and the generation of useful animal models of human diseases. Here we show successful clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (Cas)-mediated gene targeting via circular plasmid injection in rabbits. The rabbit tyrosinase gene (TYR) was effectively disrupted, and we confirmed germline transmission by pronuclear injection of a circular plasmid expressing humanized Cas9 (hCas9) and single-guide RNA. Direct injection into pronuclear stage zygotes was possible following an in vitro validation assay. Neither off-target mutagenesis nor hCas9 transgenesis was detected in any of the genetically targeted pups and embryos examined. Gene targeting with this rapid and simplified strategy will help accelerate the development of translational research using other nonrodent mammalian species. PMID:25195632

Characterization of choline trimethylamine-lyase expands the chemistry of glycyl radical enzymes.

PubMed

Craciun, Smaranda; Marks, Jonathan A; Balskus, Emily P

2014-07-18

The recently identified glycyl radical enzyme (GRE) homologue choline trimethylamine-lyase (CutC) participates in the anaerobic conversion of choline to trimethylamine (TMA), a widely distributed microbial metabolic transformation that occurs in the human gut and is linked to disease. The proposed biochemical function of CutC, C-N bond cleavage, represents new reactivity for the GRE family. Here we describe the in vitro characterization of CutC and its activating protein CutD. We have observed CutD-mediated formation of a glycyl radical on CutC using EPR spectroscopy and have demonstrated that activated CutC processes choline to trimethylamine and acetaldehyde. Surveys of potential alternate CutC substrates uncovered a strict specificity for choline. Homology modeling and mutagenesis experiments revealed essential CutC active site residues. Overall, this work establishes that CutC is a GRE of unique function and a molecular marker for anaerobic choline metabolism.

Carcinogens induce reversion of the mouse pink-eyed unstable mutation

PubMed Central

Schiestl, Robert H.; Aubrecht, Jiri; Khogali, Fathia; Carls, Nicholas

1997-01-01

Deletions and other genome rearrangements are associated with carcinogenesis and inheritable diseases. The pink-eyed unstable (pun) mutation in the mouse is caused by duplication of a 70-kb internal fragment of the p gene. Spontaneous reversion events in homozygous pun/pun mice occur through deletion of a duplicated sequence. Reversion events in premelanocytes in the mouse embryo detected as black spots on the gray fur of the offspring were inducible by the carcinogen x-rays, ethyl methanesulfonate, methyl methanesulfonate, ethyl nitrosourea, benzo[a]pyrene, trichloroethylene, benzene, and sodium arsenate. The latter three carcinogens are not detectable with several in vitro or in vivo mutagenesis assays. We studied the molecular mechanism of the carcinogen-induced reversion events by cDNA analysis using reverse transcriptase–PCR method and identified the induced reversion events as deletions. DNA deletion assays may be sensitive indicators for carcinogen exposure. PMID:9114032

In Silico Identification of Novel APRIL Peptide Antagonists and Binding Insights by Molecular Modeling and Immunosorbent Assays.

PubMed

Silva, Joao H M da; Calmon-Hamaty, Flavia; Savino, Wilson; Hahne, Michael; Caffarena, Ernesto R

2015-01-01

The "A proliferation inducing ligand" protein (APRIL) is a cytokine over-expressed in many transformed and tumoral cells acting onto two distinct receptors of the Tumoral Necrosis Factor B cell maturation antigen (BCMA) and the transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI). We herein describe, through a detailed computational approach, the molecular interactions between TACI and its ligands APRIL and another structurally similar protein called B-cell activating factor (BAFF) by means of molecular dynamics. Dynamical analysis suggests R84 and D85 residues from TACI as possible mutation candidates, yielding increased affinity between TACI and APRIL. The association of computational simulations, site directed mutagenesis and peptide design could be a powerful tool, driving to better in vitro experiments. Our results contribute to the elucidation of APRIL signaling and help clarify the effects of blocking interaction between APRIL and its receptors through the use of particular peptides.

Insights into an evolutionary strategy leading to antibiotic resistance

NASA Astrophysics Data System (ADS)

Hou, Chun-Feng D.; Liu, Jian-Wei; Collyer, Charles; Mitić, Nataša; Pedroso, Marcelo Monteiro; Schenk, Gerhard; Ollis, David L.

2017-01-01

Metallo-β-lactamases (MBLs) with activity towards a broad-spectrum of β-lactam antibiotics have become a major threat to public health, not least due to their ability to rapidly adapt their substrate preference. In this study, the capability of the MBL AIM-1 to evade antibiotic pressure by introducing specific mutations was probed by two alternative methods, i.e. site-saturation mutagenesis (SSM) of active site residues and in vitro evolution. Both approaches demonstrated that a single mutation in AIM-1 can greatly enhance a pathogen’s resistance towards broad spectrum antibiotics without significantly compromising the catalytic efficiency of the enzyme. Importantly, the evolution experiments demonstrated that relevant amino acids are not necessarily in close proximity to the catalytic centre of the enzyme. This observation is a powerful demonstration that MBLs have a diverse array of possibilities to adapt to new selection pressures, avenues that cannot easily be predicted from a crystal structure alone.

Structural and enzymatic characterization of a host-specificity determinant from Salmonella

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kohler, Amanda C.; Spanò, Stefania; Galán, Jorge E.

The Salmonella effector protein GtgE functions as a cysteine protease to cleave a subset of the Rab-family GTPases and to prevent delivery of antimicrobial agents to the Salmonella-containing vacuole. GtgE is an effector protein from Salmonella Typhimurium that modulates trafficking of the Salmonella-containing vacuole. It exerts its function by cleaving the Rab-family GTPases Rab29, Rab32 and Rab38, thereby preventing the delivery of antimicrobial factors to the bacteria-containing vacuole. Here, the crystal structure of GtgE at 1.65 Å resolution is presented, and structure-based mutagenesis and in vivo infection assays are used to identify its catalytic triad. A panel of cysteine proteasemore » inhibitors were examined and it was determined that N-ethylmaleimide, antipain and chymostatin inhibit GtgE activity in vitro. These findings provide the basis for the development of novel therapeutic strategies to combat Salmonella infections.« less

EMMPRIN/CD147 is a novel coreceptor of VEGFR-2 mediating its activation by VEGF.

PubMed

Khayati, Farah; Pérez-Cano, Laura; Maouche, Kamel; Sadoux, Aurélie; Boutalbi, Zineb; Podgorniak, Marie-Pierre; Maskos, Uwe; Setterblad, Niclas; Janin, Anne; Calvo, Fabien; Lebbé, Céleste; Menashi, Suzanne; Fernandez-Recio, Juan; Mourah, Samia

2015-01-01

EMMPRIN/CD147 is mainly known for its protease inducing function but a role in promoting tumor angiogenesis has also been demonstrated. This study provides evidence that EMMPRIN is a new coreceptor for the VEGFR-2 tyrosine kinase receptor in both endothelial and tumor cells, as it directly interacts with it and regulates its activation by its VEGF ligand, signalling and functional consequences both in vitro and in vivo. Computational docking analyses and mutagenesis studies identified a molecular binding site in the extracellular domain of EMMPRIN located close to the cell membrane and containing the amino acids 195/199. EMMPRIN is overexpressed in cancer and hence is able to further potentiate VEGFR-2 activation, suggesting that a combinatory therapy of an antiangiogenic drug together with an inhibitor of EMMPRIN/VEGFR-2 interaction may have a greater impact on inhibiting angiogenesis and malignancy.

EMMPRIN/CD147 is a novel coreceptor of VEGFR-2 mediating its activation by VEGF

PubMed Central

Khayati, Farah; Pérez-Cano, Laura; Maouche, Kamel; Sadoux, Aurélie; Boutalbi, Zineb; Podgorniak, Marie-Pierre; Maskos, Uwe; Setterblad, Niclas; Janin, Anne; Calvo, Fabien; Lebbé, Céleste; Menashi, Suzanne; Fernandez-Recio, Juan; Mourah, Samia

2015-01-01

EMMPRIN/CD147 is mainly known for its protease inducing function but a role in promoting tumor angiogenesis has also been demonstrated. This study provides evidence that EMMPRIN is a new coreceptor for the VEGFR-2 tyrosine kinase receptor in both endothelial and tumor cells, as it directly interacts with it and regulates its activation by its VEGF ligand, signalling and functional consequences both in vitro and in vivo. Computational docking analyses and mutagenesis studies identified a molecular binding site in the extracellular domain of EMMPRIN located close to the cell membrane and containing the amino acids 195/199. EMMPRIN is overexpressed in cancer and hence is able to further potentiate VEGFR-2 activation, suggesting that a combinatory therapy of an antiangiogenic drug together with an inhibitor of EMMPRIN/VEGFR-2 interaction may have a greater impact on inhibiting angiogenesis and malignancy. PMID:25825981

Mutagenic and genotoxic potential of direct electric current in Escherichia coli and Salmonella thyphimurium strains.

PubMed

Gomes, Marina das Neves; Cardoso, Janine Simas; Leitão, Alvaro Costa; Quaresma, Carla Holandino

2016-05-01

Direct electric current has several therapeutic uses such as antibacterial and antiprotozoal action, tissues scarring and regeneration, as well as tumor treatment. This method has shown promising results in vivo and in vitro, with significant efficacy and almost no side effects. Considering lack of studies regarding direct electric current mutagenic and/or genotoxic effects, the present work evaluated both aspects by using five different bacterial experimental assays: survival of repair-deficient mutants, Salmonella-histidine reversion mutagenesis (Ames test), forward mutations to rifampicin resistance, phage reactivation, and lysogenic induction. In these experimental conditions, cells were submitted to an approach that allows evaluation of anodic, cathodic, and electro-ionic effects generated by 2 mA of direct electric current, with doses ranging from 0.36 to 3.60 Coulombs. Our results showed these doses did not induce mutagenic or genotoxic effects. © 2016 Wiley Periodicals, Inc.

Mutagenesis of Trichoderma Viride by Ultraviolet and Plasma

NASA Astrophysics Data System (ADS)

Yao, Risheng; Li, Manman; Deng, Shengsong; Hu, Huajia; Wang, Huai; Li, Fenghe

2012-04-01

Considering the importance of a microbial strain capable of increased cellulase production, a mutant strain UP4 of Trichoderma viride was developed by ultraviolet (UV) and plasma mutation. The mutant produced a 21.0 IU/mL FPase which was 98.1% higher than that of the parent strain Trichoderma viride ZY-1. In addition, the effect of ultraviolet and plasma mutagenesis was not merely simple superimposition of single ultraviolet mutation and single plasma mutation. Meanwhile, there appeared a capsule around some of the spores after the ultraviolet and plasma treatment, namely, the spore surface of the strain became fuzzy after ultraviolet or ultraviolet and plasma mutagenesis.

Sites of instability in the human TCF3 (E2A) gene adopt G-quadruplex DNA structures in vitro

PubMed Central

Williams, Jonathan D.; Fleetwood, Sara; Berroyer, Alexandra; Kim, Nayun; Larson, Erik D.

2015-01-01

The formation of highly stable four-stranded DNA, called G-quadruplex (G4), promotes site-specific genome instability. G4 DNA structures fold from repetitive guanine sequences, and increasing experimental evidence connects G4 sequence motifs with specific gene rearrangements. The human transcription factor 3 (TCF3) gene (also termed E2A) is subject to genetic instability associated with severe disease, most notably a common translocation event t(1;19) associated with acute lymphoblastic leukemia. The sites of instability in TCF3 are not randomly distributed, but focused to certain sequences. We asked if G4 DNA formation could explain why TCF3 is prone to recombination and mutagenesis. Here we demonstrate that sequences surrounding the major t(1;19) break site and a region associated with copy number variations both contain G4 sequence motifs. The motifs identified readily adopt G4 DNA structures that are stable enough to interfere with DNA synthesis in physiological salt conditions in vitro. When introduced into the yeast genome, TCF3 G4 motifs promoted gross chromosomal rearrangements in a transcription-dependent manner. Our results provide a molecular rationale for the site-specific instability of human TCF3, suggesting that G4 DNA structures contribute to oncogenic DNA breaks and recombination. PMID:26029241

Hexokinase 2 from Saccharomyces cerevisiae: regulation of oligomeric structure by in vivo phosphorylation at serine-14.

PubMed

Behlke, J; Heidrich, K; Naumann, M; Müller, E C; Otto, A; Reuter, R; Kriegel, T

1998-08-25

Homodimeric hexokinase 2 from Saccharomyces cerevisiae is known to have two sites of phosphorylation: for serine-14 the modification in vivo increases with glucose exhaustion [Kriegel et al. (1994) Biochemistry 33, 148-152], while for serine-157 it occurs in vitro with ATP in the presence of nonphosphorylateable five-carbon analogues of glucose [Heidrich et al. (1997) Biochemistry 36, 1960-1964]. We show now by site-directed mutagenesis and sedimentation analysis that serine-14 phosphorylation affects the oligomeric state of hexokinase, its substitution by glutamate causing complete dissociation; glutamate exchange for serine-157 does not. Phosphorylation of wild-type hexokinase at serine-14 likewise causes dissociation in vitro. In view of the higher glucose affinity of monomeric hexokinase and the high hexokinase concentration in yeast [Womack, F., and Colowick, S. P. (1978) Arch. Biochem. Biophys. 191, 742-747; Mayes, E. L., Hoggett, J. G., and Kellett, G. L. (1983) Eur. J. Biochem. 133, 127-134], we speculate that the in vivo phosphorylation at serine-14 as transiently occurring in glucose derepression might provide a mechanism to improve glucose utilization from low level and/or that nuclear localization of the monomer might be involved in the signal transduction whereby glucose causes catabolite repression.

Methylation mediated by an anthocyanin, O-methyltransferase, is involved in purple flower coloration in Paeonia

PubMed Central

Du, Hui; Wu, Jie; Ji, Kui-Xian; Zeng, Qing-Yin; Bhuiya, Mohammad-Wadud; Su, Shang; Shu, Qing-Yan; Ren, Hong-Xu; Liu, Zheng-An; Wang, Liang-Sheng

2015-01-01

Anthocyanins are major pigments in plants. Methylation plays a role in the diversity and stability of anthocyanins. However, the contribution of anthocyanin methylation to flower coloration is still unclear. We identified two homologous anthocyanin O-methyltransferase (AOMT) genes from purple-flowered (PsAOMT) and red-flowered (PtAOMT) Paeonia plants, and we performed functional analyses of the two genes in vitro and in vivo. The critical amino acids for AOMT catalytic activity were studied by site-directed mutagenesis. We showed that the recombinant proteins, PsAOMT and PtAOMT, had identical substrate preferences towards anthocyanins. The methylation activity of PsAOMT was 60 times higher than that of PtAOMT in vitro. Interestingly, this vast difference in catalytic activity appeared to result from a single amino acid residue substitution at position 87 (arginine to leucine). There were significant differences between the 35S::PsAOMT transgenic tobacco and control flowers in relation to their chromatic parameters, which further confirmed the function of PsAOMT in vivo. The expression levels of the two homologous AOMT genes were consistent with anthocyanin accumulation in petals. We conclude that AOMTs are responsible for the methylation of cyanidin glycosides in Paeonia plants and play an important role in purple coloration in Paeonia spp. PMID:26208646

In silico-designed novel non-peptidic ABAD LD hot spot mimetics reverse Aβ-induced mitochondrial impairments in vitro.

PubMed

Viswanath, Ambily Nath Indu; Kim, TaeHun; Jung, Seo Yun; Lim, Sang Min; Pae, Ae Nim

2017-12-01

Present work aimed to introduce non-peptidic ABAD loop D (L D ) hot spot mimetics as ABAD-Aβ inhibitors. A full-length atomistic model of ABAD-Aβ complex was built as a scaffold to launch the lead design and its topology later verified by cross-checking the computational mutagenesis results with that of in vitro data. Thereafter, the interactions of prime Aβ-binding L D residues-Tyr101, Thr108, and Thr110-were translated into specific pharmacophore features and this hypothesis subsequently used as a virtual screen query. ELISA-based screening of 20 hits identified two promising lead candidates, VC15 and VC19 with an IC 50 of 4.4 ± 0.3 and 9.6 ± 0.1 μm, respectively. They productively reversed Aβ-induced mitochondrial dysfunctions such as mitochondrial membrane potential loss (JC-1 assay), toxicity (MTT assay), and ATP reduction (ATP assay) in addition to increased cell viabilities. This is the first reporting of L D hot spot-centric in silico scheme to discover novel compounds with promising ABAD-Aβ inhibitory potential. These chemotypes are proposed for further structural optimization to derive novel Alzheimer's disease (AD) therapeutics. © 2017 John Wiley & Sons A/S.

Insertional mutagenesis using Tnt1 retrotransposon in potato

USDA-ARS?s Scientific Manuscript database

Potato is the third most important food crop in the world. However, genetics and genomics research of potato has lagged behind many major crop species due to its autotetraploidy and a highly heterogeneous genome. Insertional mutagenesis using T-DNA or transposable elements, which is available in sev...

Efficient genome-wide detection and cataloging of EMS-induced mutations using exome capture and next-generation sequencing

USDA-ARS?s Scientific Manuscript database

Chemical mutagenesis efficiently generates phenotypic variation in otherwise homogeneous genetic backgrounds, enabling functional analysis of genes. Advances in mutation detection have brought the utility of induced mutant populations on par with those produced by insertional mutagenesis, but system...

Methods for targetted mutagenesis in gram-positive bacteria

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Yunfeng

The present invention provides a method of targeted mutagenesis in Gram-positive bacteria. In particular, the present invention provides a method that effectively integrates a suicide integrative vector into a target gene in the chromosome of a Gram-positive bacterium, resulting in inactivation of the target gene.

Synthetic polyester-hydrolyzing enzymes from thermophilic actinomycetes.

PubMed

Wei, Ren; Oeser, Thorsten; Zimmermann, Wolfgang

2014-01-01

Thermophilic actinomycetes produce enzymes capable of hydrolyzing synthetic polyesters such as polyethylene terephthalate (PET). In addition to carboxylesterases, which have hydrolytic activity predominantly against PET oligomers, esterases related to cutinases also hydrolyze synthetic polymers. The production of these enzymes by actinomycetes as well as their recombinant expression in heterologous hosts is described and their catalytic activity against polyester substrates is compared. Assays to analyze the enzymatic hydrolysis of synthetic polyesters are evaluated, and a kinetic model describing the enzymatic heterogeneous hydrolysis process is discussed. Structure-function and structure-stability relationships of actinomycete polyester hydrolases are compared based on molecular dynamics simulations and recently solved protein structures. In addition, recent progress in enhancing their activity and thermal stability by random or site-directed mutagenesis is presented. © 2014 Elsevier Inc. All rights reserved.

Enhanced mutagenesis parallels enhanced reactivation of herpes virus in a human cell line.

PubMed Central

Lytle, C D; Knott, D C

1982-01-01

U.v. irradiation of human NB-E cells results in enhanced mutagenesis and enhanced reactivation of u.v.-irradiated H-1 virus grown in those cells ( Cornelis et al., 1982). This paper reports a similar study using herpes simplex virus (HSV) in NB-E cells. The mutation frequency of HSV (resistance of virus plaque formation to 40 micrograms/ml iododeoxycytidine ) increased approximately linearly with exposure of the virus to u.v. radiation. HSV grown in unirradiated cells gave a slope of 1.8 X 10(-5)m2/J, with 3.2 X 10(-5)m2/J for HSV grown in cells irradiated (3 J/m2) 24 h before infection. There was no evidence for mutagenesis of unirradiated virus by irradiated cells, as seen with H-1 virus. Enhanced reactivation of irradiated HSV in parallel cultures increased virus survival, manifested as a change in slope of the final component of the two-component survival curve from a D0 of 27 J/m2 in unirradiated cells to 45 J/m2 in irradiated cells. Thus, enhanced mutagenesis and enhanced reactivation occurred for irradiated HSV in NB-E cells. The difference in the enhanced mutagenesis of HSV (dependent on damaged DNA sites) and of H-1 virus (primarily independent of damaged DNA sites) is discussed in terms of differences in DNA polymerases. PMID:6329698

A site-directed mutagenesis method particularly useful for creating otherwise difficult-to-make mutants and alanine scanning.

PubMed

Wan, Haisu; Li, Yongwen; Fan, Yu; Meng, Fanrong; Chen, Chen; Zhou, Qinghua

2012-01-15

Site-directed mutagenesis has become routine in molecular biology. However, many mutants can still be very difficult to create. Complicated chimerical mutations, tandem repeats, inverted sequences, GC-rich regions, and/or heavy secondary structures can cause inefficient or incorrect binding of the mutagenic primer to the target sequence and affect the subsequent amplification. In theory, these problems can be avoided by introducing the mutations into the target sequence using mutagenic fragments and so removing the need for primer-template annealing. The cassette mutagenesis uses the mutagenic fragment in its protocol; however, in most cases it needs to perform two rounds of mutagenic primer-based mutagenesis to introduce suitable restriction enzyme sites into templates and is not suitable for routine mutagenesis. Here we describe a highly efficient method in which the template except the region to be mutated is amplified by polymerase chain reaction (PCR) and the type IIs restriction enzyme-digested PCR product is directly ligated with the mutagenic fragment. Our method requires no assistance of mutagenic primers. We have used this method to create various types of difficult-to-make mutants with mutagenic frequencies of nearly 100%. Our protocol has many advantages over the prevalent QuikChange method and is a valuable tool for studies on gene structure and function. Copyright © 2011 Elsevier Inc. All rights reserved.

Comparing Different Strategies in Directed Evolution of Enzyme Stereoselectivity: Single- versus Double-Code Saturation Mutagenesis.

PubMed

Sun, Zhoutong; Lonsdale, Richard; Li, Guangyue; Reetz, Manfred T

2016-10-04

Saturation mutagenesis at sites lining the binding pockets of enzymes constitutes a viable protein engineering technique for enhancing or inverting stereoselectivity. Statistical analysis shows that oversampling in the screening step (the bottleneck) increases astronomically as the number of residues in the randomization site increases, which is the reason why reduced amino acid alphabets have been employed, in addition to splitting large sites into smaller ones. Limonene epoxide hydrolase (LEH) has previously served as the experimental platform in these methodological efforts, enabling comparisons between single-code saturation mutagenesis (SCSM) and triple-code saturation mutagenesis (TCSM); these employ either only one or three amino acids, respectively, as building blocks. In this study the comparative platform is extended by exploring the efficacy of double-code saturation mutagenesis (DCSM), in which the reduced amino acid alphabet consists of two members, chosen according to the principles of rational design on the basis of structural information. The hydrolytic desymmetrization of cyclohexene oxide is used as the model reaction, with formation of either (R,R)- or (S,S)-cyclohexane-1,2-diol. DCSM proves to be clearly superior to the likewise tested SCSM, affording both R,R- and S,S-selective mutants. These variants are also good catalysts in reactions of further substrates. Docking computations reveal the basis of enantioselectivity. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Targeting fibroblast growth factor receptor signaling inhibits prostate cancer progression.

PubMed

Feng, Shu; Shao, Longjiang; Yu, Wendong; Gavine, Paul; Ittmann, Michael

2012-07-15

Extensive correlative studies in human prostate cancer as well as studies in vitro and in mouse models indicate that fibroblast growth factor receptor (FGFR) signaling plays an important role in prostate cancer progression. In this study, we used a probe compound for an FGFR inhibitor, which potently inhibits FGFR-1-3 and significantly inhibits FGFR-4. The purpose of this study is to determine whether targeting FGFR signaling from all four FGFRs will have in vitro activities consistent with inhibition of tumor progression and will inhibit tumor progression in vivo. Effects of AZ8010 on FGFR signaling and invasion were analyzed using immortalized normal prostate epithelial (PNT1a) cells and PNT1a overexpressing FGFR-1 or FGFR-4. The effect of AZ8010 on invasion and proliferation in vitro was also evaluated in prostate cancer cell lines. Finally, the impact of AZ8010 on tumor progression in vivo was evaluated using a VCaP xenograft model. AZ8010 completely inhibits FGFR-1 and significantly inhibits FGFR-4 signaling at 100 nmol/L, which is an achievable in vivo concentration. This results in marked inhibition of extracellular signal-regulated kinase (ERK) phosphorylation and invasion in PNT1a cells expressing FGFR-1 and FGFR-4 and all prostate cancer cell lines tested. Treatment in vivo completely inhibited VCaP tumor growth and significantly inhibited angiogenesis and proliferation and increased cell death in treated tumors. This was associated with marked inhibition of ERK phosphorylation in treated tumors. Targeting FGFR signaling is a promising new approach to treating aggressive prostate cancer.

Random T-DNA mutagenesis identifies a Cu-Zn-superoxide dismutase gene as a virulence factor of Sclerotinia sclerotiorum

USDA-ARS?s Scientific Manuscript database

The Ascomycetous fungus Sclerotinia sclerotiorum is a devastating pathogen capable of infecting more than 400 plant species including many economically important crops. In order to gain a better mechanistic understanding of its non-specific host-pathogen interactions, random mutagenesis through Agro...

Cellobiohydrolase I gene and improved variants

DOEpatents

Adney, William S [Golden, CO; Decker, Stephen R [Berthoud, CO; Mc Carter, Suzanne [San Carlos, CA; Baker, John O [Golden, CO; Nieves, Raphael [Lakewood, CO; Himmel, Michael E [Littleton, CO; Vinzant, Todd B [Golden, CO

2008-05-20

The disclosure provides a method for preparing an active exoglucanase in a heterologous host of eukaryotic origin. The method includes mutagenesis to reduce glycosylation of the exoglucanase when expressed in a heterologous host. It is further disclosed a method to produce variant cellobiohydrolase that is stable at high temperature through mutagenesis.

Targeted mutagenesis using CRISPR/Cas in inbred potatoes

USDA-ARS?s Scientific Manuscript database

Targeted mutagenesis using sequence-specific nucleases (SSNs) has been well established in several important crop species, but is in need of improvement in potato (Solanum tuberosum L.). For over a century, potatoes have been bred as autotetraploids (2n = 4x = 48), relying on F1 selections and clona...

What Can a Micronucleus Teach? Learning about Environmental Mutagenesis

ERIC Educational Resources Information Center

Linde, Ana R.; Garcia-Vazquez, Eva

2009-01-01

The micronucleus test is widely employed in environmental health research. It can also be an excellent tool for learning important concepts in environmental health. In this article we present an inquiry-based laboratory exercise where students explore several theoretical and practical aspects of environmental mutagenesis employing the micronucleus…

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells (HSPCs) in Developing Zebrafish Embryos.

PubMed

Berrun, A C; Stachura, D L

2017-11-30

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell lineages that comprise mature blood. Isolation and identification of these HSPCs is difficult because they are defined ex post facto; they can only be defined after their differentiation into specific cell lineages. Over the past few decades, the zebrafish (Danio rerio) has become a model organism to study hematopoiesis. Zebrafish embryos develop ex utero, and by 48 h post-fertilization (hpf) have generated definitive HSPCs. Assays to assess HSPC differentiation and proliferation capabilities have been developed, utilizing transplantation and subsequent reconstitution of the hematopoietic system in addition to visualizing specialized transgenic lines with confocal microscopy. However, these assays are cost prohibitive, technically difficult, and time consuming for many laboratories. Development of an in vitro model to assess HSPCs would be cost effective, quicker, and present fewer difficulties compared to previously described methods, allowing laboratories to quickly assess mutagenesis and drug screens that affect HSPC biology. This novel in vitro assay to assess HSPCs is performed by plating dissociated whole zebrafish embryos and adding exogenous factors that promote only HSPC differentiation and proliferation. Embryos are dissociated into single cells and plated with HSPC-supportive colony stimulating factors that cause them to generate colony forming units (CFUs) that arise from a single progenitor cell. These assays should allow more careful examination of the molecular pathways responsible for HSPC proliferation, differentiation, and regulation, which will allow researchers to understand the underpinnings of vertebrate hematopoiesis and its dysregulation during disease.

An in vitro recombination-based reverse genetic system for rapid mutagenesis of structural genes of the Japanese encephalitis virus.

PubMed

Du, Ruikun; Wang, Manli; Hu, Zhihong; Wang, Hualin; Deng, Fei

2015-10-01

Japanese encephalitis virus (JEV) is one of the most common pathogens of severe viral encephalitis, which is a severe threat to human health. Despite instability of the JEV genome in bacteria, many strategies have been developed to establish molecular clone systems of JEV, providing convenient tools for studying the virus life cycle and virus-host interactions. In this study, we adapted an In-Fusion enzyme-based in vitro recombination method to construct a reverse genetic system of JEV, thereby providing a rapid approach to introduce mutations into the structural genes. A truncated genome without the structural genes was constructed as the backbone, and the complementary segment containing the structural genes was recombined in vitro, which was then transfected directly into virus-permissive cells. The progeny of the infectious virus was successfully detected in the supernatant of the transfected cells, and showed an identical phenotype to its parental virus. To provide a proof-of-principle, the 12 conserved cysteine residues in the envelope (E) protein of JEV were respectively mutated using this approach, and all mutations resulted in a complete failure to generate infectious virus. However, a leucine-tophenylanine mutation at amino acid 107 of the E protein did not interfere with the production of the infectious virus. These results suggested that all 12 cysteines in the E protein are essential for the JEV life cycle. In summary, a novel reverse genetic system of JEV was established for rapidly introducing mutations into structural genes, which will serve as a useful tool for functional studies.

A single heterologously expressed plant cellulose synthase isoform is sufficient for cellulose microfibril formation in vitro

PubMed Central

Purushotham, Pallinti; Cho, Sung Hyun; Díaz-Moreno, Sara M.; Kumar, Manish; Nixon, B. Tracy; Bulone, Vincent; Zimmer, Jochen

2016-01-01

Plant cell walls are a composite material of polysaccharides, proteins, and other noncarbohydrate polymers. In the majority of plant tissues, the most abundant polysaccharide is cellulose, a linear polymer of glucose molecules. As the load-bearing component of the cell wall, individual cellulose chains are frequently bundled into micro and macrofibrils and are wrapped around the cell. Cellulose is synthesized by membrane-integrated and processive glycosyltransferases that polymerize UDP-activated glucose and secrete the nascent polymer through a channel formed by their own transmembrane regions. Plants express several different cellulose synthase isoforms during primary and secondary cell wall formation; however, so far, none has been functionally reconstituted in vitro for detailed biochemical analyses. Here we report the heterologous expression, purification, and functional reconstitution of Populus tremula x tremuloides CesA8 (PttCesA8), implicated in secondary cell wall formation. The recombinant enzyme polymerizes UDP-activated glucose to cellulose, as determined by enzyme degradation, permethylation glycosyl linkage analysis, electron microscopy, and mutagenesis studies. Catalytic activity is dependent on the presence of a lipid bilayer environment and divalent manganese cations. Further, electron microscopy analyses reveal that PttCesA8 produces cellulose fibers several micrometers long that occasionally are capped by globular particles, likely representing PttCesA8 complexes. Deletion of the enzyme’s N-terminal RING-finger domain almost completely abolishes fiber formation but not cellulose biosynthetic activity. Our results demonstrate that reconstituted PttCesA8 is not only sufficient for cellulose biosynthesis in vitro but also suffices to bundle individual glucan chains into cellulose microfibrils. PMID:27647898

A single heterologously expressed plant cellulose synthase isoform is sufficient for cellulose microfibril formation in vitro.

PubMed

Purushotham, Pallinti; Cho, Sung Hyun; Díaz-Moreno, Sara M; Kumar, Manish; Nixon, B Tracy; Bulone, Vincent; Zimmer, Jochen

2016-10-04

Plant cell walls are a composite material of polysaccharides, proteins, and other noncarbohydrate polymers. In the majority of plant tissues, the most abundant polysaccharide is cellulose, a linear polymer of glucose molecules. As the load-bearing component of the cell wall, individual cellulose chains are frequently bundled into micro and macrofibrils and are wrapped around the cell. Cellulose is synthesized by membrane-integrated and processive glycosyltransferases that polymerize UDP-activated glucose and secrete the nascent polymer through a channel formed by their own transmembrane regions. Plants express several different cellulose synthase isoforms during primary and secondary cell wall formation; however, so far, none has been functionally reconstituted in vitro for detailed biochemical analyses. Here we report the heterologous expression, purification, and functional reconstitution of Populus tremula x tremuloides CesA8 (PttCesA8), implicated in secondary cell wall formation. The recombinant enzyme polymerizes UDP-activated glucose to cellulose, as determined by enzyme degradation, permethylation glycosyl linkage analysis, electron microscopy, and mutagenesis studies. Catalytic activity is dependent on the presence of a lipid bilayer environment and divalent manganese cations. Further, electron microscopy analyses reveal that PttCesA8 produces cellulose fibers several micrometers long that occasionally are capped by globular particles, likely representing PttCesA8 complexes. Deletion of the enzyme's N-terminal RING-finger domain almost completely abolishes fiber formation but not cellulose biosynthetic activity. Our results demonstrate that reconstituted PttCesA8 is not only sufficient for cellulose biosynthesis in vitro but also suffices to bundle individual glucan chains into cellulose microfibrils.

Lentiviral and targeted cellular barcoding reveals ongoing clonal dynamics of cell lines in vitro and in vivo

PubMed Central

2014-01-01

Background Cell lines are often regarded as clonal, even though this simplifies what is known about mutagenesis, transformation and other processes that destabilize them over time. Monitoring these clonal dynamics is important for multiple areas of biomedical research, including stem cell and cancer biology. Tracking the contributions of individual cells to large populations, however, has been constrained by limitations in sensitivity and complexity. Results We utilize cellular barcoding methods to simultaneously track the clonal contributions of tens of thousands of cells. We demonstrate that even with optimal culturing conditions, common cell lines including HeLa, K562 and HEK-293 T exhibit ongoing clonal dynamics. Starting a population with a single clone diminishes but does not eradicate this phenomenon. Next, we compare lentiviral and zinc-finger nuclease barcode insertion approaches, finding that the zinc-finger nuclease protocol surprisingly results in reduced clonal diversity. We also document the expected reduction in clonal complexity when cells are challenged with genotoxic stress. Finally, we demonstrate that xenografts maintain clonal diversity to a greater extent than in vitro culturing of the human non-small-cell lung cancer cell line HCC827. Conclusions We demonstrate the feasibility of tracking and quantifying the clonal dynamics of entire cell populations within multiple cultured cell lines. Our results suggest that cell heterogeneity should be considered in the design and interpretation of in vitro culture experiments. Aside from clonal cell lines, we propose that cellular barcoding could prove valuable in modeling the clonal behavior of heterogeneous cell populations over time, including tumor populations treated with chemotherapeutic agents. PMID:24886633

Current concepts on Escherichia coli K1 translocation of the blood-brain barrier.

PubMed

Xie, Yi; Kim, Kee Jun; Kim, Kwang Sik

2004-11-01

The mortality and morbidity associated with neonatal gram-negative meningitis have remained significant despite advances in antimicrobial chemotherapy. Escherichia coli K1 is the most common gram-negative organism causing neonatal meningitis. Our incomplete knowledge of the pathogenesis of this disease is one of the main reasons for this high mortality and morbidity. We have previously established both in vitro and in vivo models of the blood-brain barrier (BBB) using human brain microvascular endothelial cells (HBMEC) and hematogenous meningitis in neonatal rats, respectively. With these in vitro and in vivo models, we have shown that successful crossing of the BBB by circulating E. coli requires a high-degree of bacteremia, E. coli binding to and invasion of HBMEC, and E. coli traversal of the BBB as live bacteria. Our previous studies using TnphoA, signature-tagged mutagenesis and differential fluorescence induction identified several E. coli K1 determinants such as OmpA, Ibe proteins, AslA, TraJ and CNF1 contributing to invasion of HBMEC in vitro and traversal of the blood-brain barrier in vivo. We have shown that some of these determinants interact with specific receptors on HBMEC, suggesting E. coli translocation of the BBB is the result of specific pathogen-host cell interactions. Recent studies using functional genomics techniques have identified additional E. coli K1 factors that contribute to the high degree of bacteremia and HBMEC binding/invasion/transcytosis. In this review, we summarize the current knowledge on the mechanisms underlying the successful E. coli translocation of the BBB.

3D bioprinting: improving in vitro models of metastasis with heterogeneous tumor microenvironments

PubMed Central

Albritton, Jacob L.

2017-01-01

ABSTRACT Even with many advances in treatment over the past decades, cancer still remains a leading cause of death worldwide. Despite the recognized relationship between metastasis and increased mortality rate, surprisingly little is known about the exact mechanism of metastatic progression. Currently available in vitro models cannot replicate the three-dimensionality and heterogeneity of the tumor microenvironment sufficiently to recapitulate many of the known characteristics of tumors in vivo. Our understanding of metastatic progression would thus be boosted by the development of in vitro models that could more completely capture the salient features of cancer biology. Bioengineering groups have been working for over two decades to create in vitro microenvironments for application in regenerative medicine and tissue engineering. Over this time, advances in 3D printing technology and biomaterials research have jointly led to the creation of 3D bioprinting, which has improved our ability to develop in vitro models with complexity approaching that of the in vivo tumor microenvironment. In this Review, we give an overview of 3D bioprinting methods developed for tissue engineering, which can be directly applied to constructing in vitro models of heterogeneous tumor microenvironments. We discuss considerations and limitations associated with 3D printing and highlight how these advances could be harnessed to better model metastasis and potentially guide the development of anti-cancer strategies. PMID:28067628

3D bioprinting: improving in vitro models of metastasis with heterogeneous tumor microenvironments.

PubMed

Albritton, Jacob L; Miller, Jordan S

2017-01-01

Even with many advances in treatment over the past decades, cancer still remains a leading cause of death worldwide. Despite the recognized relationship between metastasis and increased mortality rate, surprisingly little is known about the exact mechanism of metastatic progression. Currently available in vitro models cannot replicate the three-dimensionality and heterogeneity of the tumor microenvironment sufficiently to recapitulate many of the known characteristics of tumors in vivo Our understanding of metastatic progression would thus be boosted by the development of in vitro models that could more completely capture the salient features of cancer biology. Bioengineering groups have been working for over two decades to create in vitro microenvironments for application in regenerative medicine and tissue engineering. Over this time, advances in 3D printing technology and biomaterials research have jointly led to the creation of 3D bioprinting, which has improved our ability to develop in vitro models with complexity approaching that of the in vivo tumor microenvironment. In this Review, we give an overview of 3D bioprinting methods developed for tissue engineering, which can be directly applied to constructing in vitro models of heterogeneous tumor microenvironments. We discuss considerations and limitations associated with 3D printing and highlight how these advances could be harnessed to better model metastasis and potentially guide the development of anti-cancer strategies. © 2017. Published by The Company of Biologists Ltd.

Boron-doped diamond nano/microelectrodes for biosensing and in vitro measurements.

PubMed

Dong, Hua; Wang, Shihua; Galligan, James J; Swain, Greg M

2011-01-01

Since the fabrication of the first diamond electrode in the mid 1980s, repid progress has been made on the development and application of this new type of electrode material. Boron-doped diamond (BDD) electrodes exhibit outstanding properties compared to oxygen-containing sp2 carbon electrodes. These properties make BDD electrodes an ideal choice for use in complex samples. In recent years, BDD microelectrodes have been applied to in vitro measurements of biological molecules in tissues and cells. This review will summarize recent progress in the development and applications of BDD electrodes in bio-sensing and in vitro measurements of biomolecules. In the first section, the methods for BDD diamond film deposition and BDD microelectrodes preparation are described. This is followed by a description and discussion of several approaches for characterization of the BDD electrode surface structure, morphology, and electrochemical activity. Further, application of BDD microelectrodes for use in the in vitro analysis of norepinephrine (NE), serotonin (5-HT), nitric oxide (NO), histamine, and adenosine from tissues are summarized and finally some of the remaining challenges are discussed.

Boron-doped diamond nano/microelectrodes for bio-sensing and in vitro measurements

PubMed Central

Dong, Hua; Wang, Shihua; Galligan, James J.; Swain, Greg M.

2015-01-01

Since the fabrication of the first diamond electrode in the mid 1980s, repid progress has been made on the development and application of this new type of electrode material. Boron-doped diamond (BDD) electrodes exhibit outstanding properties compared to oxygen-containing sp2 carbon electrodes. These properties make BDD electrodes an ideal choice for use in complex samples. In recent years, BDD microelectrodes have been applied to in vitro and in vivo measurements of biological molecules in animals, tissues and cells. This review will summarize recent progress in the development and applications of BDD electrodes in bio-sensing and in vitro measurements of biomolecules. In the first section, the methods for BDD nanocrystalline diamond film deposition and BDD microelectrodes preparation are described. This is followed by a description and discussion of several approaches for characterization of the BDD electrode surface structure, morphology, and electrochemical activity. Further, application of BDD microelectrodes for use in the in vitro analysis of norepinephrine (NE), serotonin (5-HT), nitric oxide (NO), histamine, and adenosine from tissues are summarized and finally some of the remaining challenges are discussed. PMID:21196394

Soluble expression, purification and characterization of the full length IS2 Transposase.

PubMed

Lewis, Leslie A; Astatke, Mekbib; Umekubo, Peter T; Alvi, Shaheen; Saby, Robert; Afrose, Jehan

2011-10-27

The two-step transposition pathway of insertion sequences of the IS3 family, and several other families, involves first the formation of a branched figure-of-eight (F-8) structure by an asymmetric single strand cleavage at one optional donor end and joining to the flanking host DNA near the target end. Its conversion to a double stranded minicircle precedes the second insertional step, where both ends function as donors. In IS2, the left end which lacks donor function in Step I acquires it in Step II. The assembly of two intrinsically different protein-DNA complexes in these F-8 generating elements has been intuitively proposed, but a barrier to testing this hypothesis has been the difficulty of isolating a full length, soluble and active transposase that creates fully formed synaptic complexes in vitro with protein bound to both binding and catalytic domains of the ends. We address here a solution to expressing, purifying and structurally analyzing such a protein. A soluble and active IS2 transposase derivative with GFP fused to its C-terminus functions as efficiently as the native protein in in vivo transposition assays. In vitro electrophoretic mobility shift assay data show that the partially purified protein prepared under native conditions binds very efficiently to cognate DNA, utilizing both N- and C-terminal residues. As a precursor to biophysical analyses of these complexes, a fluorescence-based random mutagenesis protocol was developed that enabled a structure-function analysis of the protein with good resolution at the secondary structure level. The results extend previous structure-function work on IS3 family transposases, identifying the binding domain as a three helix H + HTH bundle and explaining the function of an atypical leucine zipper-like motif in IS2. In addition gain- and loss-of-function mutations in the catalytic active site define its role in regional and global binding and identify functional signatures that are common to the three dimensional catalytic core motif of the retroviral integrase superfamily. Intractably insoluble transposases, such as the IS2 transposase, prepared by solubilization protocols are often refractory to whole protein structure-function studies. The results described here have validated the use of GFP-tagging and fluorescence-based random mutagenesis in overcoming this limitation at the secondary structure level.

Soluble expression, purification and characterization of the full length IS2 Transposase

PubMed Central

2011-01-01

Background The two-step transposition pathway of insertion sequences of the IS3 family, and several other families, involves first the formation of a branched figure-of-eight (F-8) structure by an asymmetric single strand cleavage at one optional donor end and joining to the flanking host DNA near the target end. Its conversion to a double stranded minicircle precedes the second insertional step, where both ends function as donors. In IS2, the left end which lacks donor function in Step I acquires it in Step II. The assembly of two intrinsically different protein-DNA complexes in these F-8 generating elements has been intuitively proposed, but a barrier to testing this hypothesis has been the difficulty of isolating a full length, soluble and active transposase that creates fully formed synaptic complexes in vitro with protein bound to both binding and catalytic domains of the ends. We address here a solution to expressing, purifying and structurally analyzing such a protein. Results A soluble and active IS2 transposase derivative with GFP fused to its C-terminus functions as efficiently as the native protein in in vivo transposition assays. In vitro electrophoretic mobility shift assay data show that the partially purified protein prepared under native conditions binds very efficiently to cognate DNA, utilizing both N- and C-terminal residues. As a precursor to biophysical analyses of these complexes, a fluorescence-based random mutagenesis protocol was developed that enabled a structure-function analysis of the protein with good resolution at the secondary structure level. The results extend previous structure-function work on IS3 family transposases, identifying the binding domain as a three helix H + HTH bundle and explaining the function of an atypical leucine zipper-like motif in IS2. In addition gain- and loss-of-function mutations in the catalytic active site define its role in regional and global binding and identify functional signatures that are common to the three dimensional catalytic core motif of the retroviral integrase superfamily. Conclusions Intractably insoluble transposases, such as the IS2 transposase, prepared by solubilization protocols are often refractory to whole protein structure-function studies. The results described here have validated the use of GFP-tagging and fluorescence-based random mutagenesis in overcoming this limitation at the secondary structure level. PMID:22032517

HTP-OligoDesigner: An Online Primer Design Tool for High-Throughput Gene Cloning and Site-Directed Mutagenesis.

PubMed

Camilo, Cesar M; Lima, Gustavo M A; Maluf, Fernando V; Guido, Rafael V C; Polikarpov, Igor

2016-01-01

Following burgeoning genomic and transcriptomic sequencing data, biochemical and molecular biology groups worldwide are implementing high-throughput cloning and mutagenesis facilities in order to obtain a large number of soluble proteins for structural and functional characterization. Since manual primer design can be a time-consuming and error-generating step, particularly when working with hundreds of targets, the automation of primer design process becomes highly desirable. HTP-OligoDesigner was created to provide the scientific community with a simple and intuitive online primer design tool for both laboratory-scale and high-throughput projects of sequence-independent gene cloning and site-directed mutagenesis and a Tm calculator for quick queries.

The mechanism of folding robustness revealed by the crystal structure of extra-superfolder GFP.

PubMed

Choi, Jae Young; Jang, Tae-Ho; Park, Hyun Ho

2017-01-01

Stability of green fluorescent protein (GFP) is sometimes important for a proper practical application of this protein. Random mutagenesis and targeted mutagenesis have been used to create better-folded variants of GFP, including recently reported extra-superfolder GFP. Our aim was to determine the crystal structure of extra-superfolder GFP, which is more robustly folded and stable than GFP and superfolder GFP. The structural and structure-based mutagenesis analyses revealed that some of the mutations that created extra-superfolder GFP (F46L, E126K, N149K, and S208L) contribute to folding robustness by stabilizing extra-superfolder GFP with various noncovalent bonds. © 2016 Federation of European Biochemical Societies.

Site-directed mutagenesis of serine 158 demonstrates its role in spinach leaf sucrose-phosphate synthase modulation

NASA Technical Reports Server (NTRS)

Toroser, D.; McMichael, R. Jr; Krause, K. P.; Kurreck, J.; Sonnewald, U.; Stitt, M.; Huber, S. C.; Davies, E. (Principal Investigator)

1999-01-01

Site-directed mutagenesis of spinach sucrose-phosphate synthase (SPS) was performed to investigate the role of Ser158 in the modulation of spinach leaf SPS. Tobacco plants expressing the spinach wild-type (WT), S158A, S158T and S157F/S158E SPS transgenes were produced. Expression of transgenes appeared not to reduce expression of the tobacco host SPS. SPS activity in the WT and the S158T SPS transgenics showed light/dark modulation, whereas the S158A and S157F/S158E mutants were not similarly light/dark modulated: the S158A mutant enzyme was not inactivated in the dark, and the S157F/S158E was not activated in the light. The inability to modulate the activity of the S158A mutant enzyme by protein phosphorylation was demonstrated in vitro. The WT spinach enzyme immunopurified from dark transgenic tobacco leaves had a low initial activation state, and could be activated by PP2A and subsequently inactivated by SPS-kinase plus ATP. Rapid purification of the S158A mutant enzyme from dark leaves of transgenic plants using spinach-specific monoclonal antibodies yielded enzyme that had a high initial activation state, and pre-incubation with leaf PP2A or ATP plus SPS-kinase (the PKIII enzyme) caused little modulation of activity. The results demonstrate the regulatory significance of Ser158 as the major site responsible for dark inactivation of spinach SPS in vivo, and indicate that the significance of phosphorylation is the introduction of a negative charge at the Ser158 position.

Structure of the Bifunctional Acyltransferase/Decarboxylase LnmK from the Leinamycin Biosynthetic Pathway Revealing Novel Activity for a Double-Hot-Dog Fold

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lohman, Jeremy R.; Bingman, Craig A.; George N. Phillips Jr.

The β-branched C3 unit in leinamycin biosynthesis is installed by a set of four proteins, LnmFKLM. In vitro biochemical investigation confirmed that LnmK is a bifunctional acyltransferase/decarboxylase (AT/DC) that catalyzes first self-acylation using methylmalonyl-CoA as a substrate and subsequently transacylation of the methylmalonyl group to the phosphopantetheinyl group of the LnmL acyl carrier protein [Liu, T., Huang, Y., and Shen, B. (2009) J. Am. Chem. Soc. 131, 6900–6901]. LnmK shows no sequence homology to proteins of known function, representing a new family of AT/DC enzymes. Here we report the X-ray structure of LnmK. LnmK is homodimer with each of themore » monomers adopting a double-hot-dog fold. Cocrystallization of LnmK with methylmalonyl-CoA revealed an active site tunnel terminated by residues from the dimer interface. But, to canonical AT and ketosynthase enzymes that employ Ser or Cys as an active site residue, none of these residues are found in the vicinity of the LnmK active site. Instead, three tyrosines were identified, one of which, Tyr62, was established, by site-directed mutagenesis, to be the most likely active site residue for the AT activity of LnmK. Moreover, LnmK represents the first AT enzyme that employs a Tyr as an active site residue and the first member of the family of double-hot-dog fold enzymes that displays an AT activity known to date. The LnmK structure sets the stage for probing of the DC activity of LnmK through site-directed mutagenesis. These findings highlight natural product biosynthetic machinery as a rich source of novel enzyme activities, mechanisms, and structures.« less

Structural Basis for Certain Naturally Occurring Bioflavonoids to Function as Reducing Co-Substrates of Cyclooxygenase I and II

PubMed Central

Zhu, Bao Ting

2010-01-01

Background Recent studies showed that some of the dietary bioflavonoids can strongly stimulate the catalytic activity of cyclooxygenase (COX) I and II in vitro and in vivo, presumably by facilitating enzyme re-activation. In this study, we sought to understand the structural basis of COX activation by these dietary compounds. Methodology/Principal Findings A combination of molecular modeling studies, biochemical analysis and site-directed mutagenesis assay was used as research tools. Three-dimensional quantitative structure-activity relationship analysis (QSAR/CoMFA) predicted that the ability of bioflavonoids to activate COX I and II depends heavily on their B-ring structure, a moiety known to be associated with strong antioxidant ability. Using the homology modeling and docking approaches, we identified the peroxidase active site of COX I and II as the binding site for bioflavonoids. Upon binding to this site, bioflavonoid can directly interact with hematin of the COX enzyme and facilitate the electron transfer from bioflavonoid to hematin. The docking results were verified by biochemical analysis, which reveals that when the cyclooxygenase activity of COXs is inhibited by covalent modification, myricetin can still stimulate the conversion of PGG2 to PGE2, a reaction selectively catalyzed by the peroxidase activity. Using the site-directed mutagenesis analysis, we confirmed that Q189 at the peroxidase site of COX II is essential for bioflavonoids to bind and re-activate its catalytic activity. Conclusions/Significance These findings provide the structural basis for bioflavonoids to function as high-affinity reducing co-substrates of COXs through binding to the peroxidase active site, facilitating electron transfer and enzyme re-activation. PMID:20808785

RIP1-HAT1-SIRT Complex Identification and Targeting in Treatment and Prevention of Cancer.

PubMed

Carafa, Vincenzo; Nebbioso, Angela; Cuomo, Francesca; Rotili, Dante; Cobellis, Gilda; Bontempo, Paola; Baldi, Alfonso; Spugnini, Enrico P; Citro, Gennaro; Chambery, Angela; Russo, Rosita; Ruvo, Menotti; Ciana, Paolo; Maravigna, Luca; Shaik, Jani; Radaelli, Enrico; De Antonellis, Pasquale; Tarantino, Domenico; Pirolli, Adele; Ragno, Rino; Zollo, Massimo; Stunnenberg, Hendrik G; Mai, Antonello; Altucci, Lucia

2018-03-13

Purpose: Alteration in cell death is a hallmark of cancer. A functional role regulating survival, apoptosis, and necroptosis has been attributed to RIP1/3 complexes. Experimental Design: We have investigated the role of RIP1 and the effects of MC2494 in cell death induction, using different methods as flow cytometry, transcriptome analysis, immunoprecipitation, enzymatic assays, transfections, mutagenesis, and in vivo studies with different mice models. Results: Here, we show that RIP1 is highly expressed in cancer, and we define a novel RIP1/3-SIRT1/2-HAT1/4 complex. Mass spectrometry identified five acetylations in the kinase and death domain of RIP1. The novel characterized pan-SIRT inhibitor, MC2494, increases RIP1 acetylation at two additional sites in the death domain. Mutagenesis of the acetylated lysine decreases RIP1-dependent cell death, suggesting a role for acetylation of the RIP1 complex in cell death modulation. Accordingly, MC2494 displays tumor-selective potential in vitro , in leukemic blasts ex vivo, and in vivo in both xenograft and allograft cancer models. Mechanistically, MC2494 induces bona fide tumor-restricted acetylated RIP1/caspase-8-mediated apoptosis. Excitingly, MC2494 displays tumor-preventive activity by blocking 7,12-dimethylbenz(α)anthracene-induced mammary gland hyperproliferation in vivo Conclusions: These preventive features might prove useful in patients who may benefit from a recurrence-preventive approach with low toxicity during follow-up phases and in cases of established cancer predisposition. Thus, targeting the newly identified RIP1 complex may represent an attractive novel paradigm in cancer treatment and prevention. Clin Cancer Res; 1-15. ©2018 AACR. ©2018 American Association for Cancer Research.

Tautomerism provides a molecular explanation for the mutagenic properties of the anti-HIV nucleoside 5-aza-5,6-dihydro-2′-deoxycytidine

PubMed Central

Li, Deyu; Fedeles, Bogdan I.; Singh, Vipender; Peng, Chunte Sam; Silvestre, Katherine J.; Simi, Allison K.; Simpson, Jeffrey H.; Tokmakoff, Andrei; Essigmann, John M.

2014-01-01

Viral lethal mutagenesis is a strategy whereby the innate immune system or mutagenic pool nucleotides increase the error rate of viral replication above the error catastrophe limit. Lethal mutagenesis has been proposed as a mechanism for several antiviral compounds, including the drug candidate 5-aza-5,6-dihydro-2′-deoxycytidine (KP1212), which causes A-to-G and G-to-A mutations in the HIV genome, both in tissue culture and in HIV positive patients undergoing KP1212 monotherapy. This work explored the molecular mechanism(s) underlying the mutagenicity of KP1212, and specifically whether tautomerism, a previously proposed hypothesis, could explain the biological consequences of this nucleoside analog. Establishing tautomerism of nucleic acid bases under physiological conditions has been challenging because of the lack of sensitive methods. This study investigated tautomerism using an array of spectroscopic, theoretical, and chemical biology approaches. Variable temperature NMR and 2D infrared spectroscopic methods demonstrated that KP1212 existed as a broad ensemble of interconverting tautomers, among which enolic forms dominated. The mutagenic properties of KP1212 were determined empirically by in vitro and in vivo replication of a single-stranded vector containing a single KP1212. It was found that KP1212 paired with both A (10%) and G (90%), which is in accord with clinical observations. Moreover, this mutation frequency is sufficient for pushing a viral population over its error catastrophe limit, as observed before in cell culture studies. Finally, a model is proposed that correlates the mutagenicity of KP1212 with its tautomeric distribution in solution. PMID:25071207

Random Mutagenesis of the Multidrug Transporter AcrB from Escherichia coli for Identification of Putative Target Residues of Efflux Pump Inhibitors

PubMed Central

Kohler, Samay; Buck, Annika; Dambacher, Christine; König, Armin; Bohnert, Jürgen A.; Kern, Winfried V.

2014-01-01

Efflux is an important mechanism of bacterial multidrug resistance (MDR), and the inhibition of MDR pumps by efflux pump inhibitors (EPIs) could be a promising strategy to overcome MDR. 1-(1-Naphthylmethyl)-piperazine (NMP) and phenylalanine-arginine-β-naphthylamide (PAβN) are model EPIs with activity in various Gram-negative bacteria expressing AcrB, the major efflux pump of Escherichia coli, or similar homologous pumps of the resistance-nodulation-cell division class. The aim of the present study was to generate E. coli AcrB mutants resistant to the inhibitory action of the two model EPIs and to identify putative EPI target residues in order to better understand mechanisms of pump inhibition. Using an in vitro random mutagenesis approach focusing on the periplasmic domain of AcrB, we identified the double mutation G141D N282Y, which substantially compromised the synergistic activity of NMP with linezolid, was associated with similar intracellular linezolid concentrations in the presence and absence of NMP, and did not impair the intrinsic MICs of various pump substrates and dye accumulation. We propose that these mutations near the outer face of the distal substrate binding pocket reduce NMP trapping. Other residues found to be relevant for efflux inhibition by NMP were G288 and A279, but mutations at these sites also changed the susceptibility to several pump substrates. Unlike with NMP, we were unable to generate AcrB periplasmic domain mutants with resistance or partial resistance to the EPI activity of PAβN, which is consistent with the modes of action of PAβN differing from those of NMP. PMID:25182653

Determination of the binding mode for the cyclopentapeptide CXCR4 antagonist FC131 using a dual approach of ligand modifications and receptor mutagenesis

PubMed Central

Thiele, S; Mungalpara, J; Steen, A; Rosenkilde, M M; Våbenø, J

2014-01-01

Background and Purpose The cyclopentapeptide FC131 (cyclo(-L-Arg1-L-Arg2-L-2-Nal3-Gly4-D-Tyr5-)) is an antagonist at the CXC chemokine receptor CXCR4, which plays a role in human immunodeficiency virus infection, cancer and stem cell recruitment. Binding modes for FC131 in CXCR4 have previously been suggested based on molecular docking guided by structure–activity relationship (SAR) data; however, none of these have been verified by in vitro experiments. Experimental Approach Heterologous 125I-12G5-competition binding and functional assays (inhibition of CXCL12-mediated activation) of FC131 and three analogues were performed on wild-type CXCR4 and 25 receptor mutants. Computational modelling was used to rationalize the experimental data. Key Results The Arg2 and 2-Nal3 side chains of FC131 interact with residues in TM-3 (His113, Asp171) and TM-5 (hydrophobic pocket) respectively. Arg1 forms charge-charge interactions with Asp187 in ECL-2, while D-Tyr5 points to the extracellular side of CXCR4. Furthermore, the backbone of FC131 interacts with the chemokine receptor-conserved Glu288 via two water molecules. Intriguingly, Tyr116 and Glu288 form a H-bond in CXCR4 crystal structures and mutation of either residue to Ala abolishes CXCR4 activity. Conclusions and Implications Ligand modification, receptor mutagenesis and computational modelling approaches were used to identify the binding mode of FC131 in CXCR4, which was in agreement with binding modes suggested from previous SAR studies. Furthermore, insights into the mechanism for CXCR4 activation by CXCL12 were gained. The combined findings will facilitate future design of novel CXCR4 antagonists. PMID:25039237

Analysis of the function of E. coli 23S rRNA helix-loop 69 by mutagenesis

PubMed Central

Liiv, Aivar; Karitkina, Diana; Maiväli, Ülo; Remme, Jaanus

2005-01-01

Background The ribosome is a two-subunit enzyme known to exhibit structural dynamism during protein synthesis. The intersubunit bridges have been proposed to play important roles in decoding, translocation, and the peptidyl transferase reaction; yet the physical nature of their contributions is ill understood. An intriguing intersubunit bridge, B2a, which contains 23S rRNA helix 69 as a major component, has been implicated by proximity in a number of catalytically important regions. In addition to contacting the small ribosomal subunit, helix 69 contacts both the A and P site tRNAs and several translation factors. Results We scanned the loop of helix 69 by mutagenesis and analyzed the mutant ribosomes using a plasmid-borne IPTG-inducible expression system. We assayed the effects of 23S rRNA mutations on cell growth, contribution of mutant ribosomes to cellular polysome pools and the ability of mutant ribosomes to function in cell-free translation. Mutations A1912G, and A1919G have very strong growth phenotypes, are inactive during in vitro protein synthesis, and under-represented in the polysomes. Mutation Ψ1917C has a very strong growth phenotype and leads to a general depletion of the cellular polysome pool. Mutation A1916G, having a modest growth phenotype, is apparently defective in the assembly of the 70S ribosome. Conclusion Mutations A1912G, A1919G, and Ψ1917C of 23S rRNA strongly inhibit translation. Mutation A1916G causes a defect in the 50S subunit or 70S formation. Mutations Ψ1911C, A1913G, C1914A, Ψ1915C, and A1918G lack clear phenotypes. PMID:16053518

Enzymatic recognition of DNA damage induced by UVB-photosensitized titanium dioxide and biological consequences in Saccharomyces cerevisiae: evidence for oxidatively DNA damage generation.

PubMed

Pinto, A Viviana; Deodato, Elder L; Cardoso, Janine S; Oliveira, Eliza F; Machado, Sérgio L; Toma, Helena K; Leitão, Alvaro C; de Pádula, Marcelo

2010-06-01

Although titanium dioxide (TiO(2)) has been considered to be biologically inert, finding use in cosmetics, paints and food colorants, recent reports have demonstrated that when TiO(2) is attained by UVA radiation oxidative genotoxic and cytotoxic effects are observed in living cells. However, data concerning TiO(2)-UVB association is poor, even if UVB radiation represents a major environmental carcinogen. Herein, we investigated DNA damage, repair and mutagenesis induced by TiO(2) associated with UVB irradiation in vitro and in vivo using Saccharomyces cerevisiae model. It was found that TiO(2) plus UVB treatment in plasmid pUC18 generated, in addition to cyclobutane pyrimidine dimers (CPDs), specific damage to guanine residues, such as 8-oxo-7,8-dihydroguanine (8-oxoG) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG), which are characteristic oxidatively generated lesions. In vivo experiments showed that, although the presence of TiO(2) protects yeast cells from UVB cytotoxicity, high mutation frequencies are observed in the wild-type (WT) and in an ogg1 strain (deficient in 8-oxoG and FapyG repair). Indeed, after TiO(2) plus UVB treatment, induced mutagenesis was drastically enhanced in ogg1 cells, indicating that mutagenic DNA lesions are repaired by the Ogg1 protein. This effect could be attenuated by the presence of metallic ion chelators: neocuproine or dipyridyl, which partially block oxidatively generated damage occurring via Fenton reactions. Altogether, the results indicate that TiO(2) plus UVB potentates UVB oxidatively generated damage to DNA, possibly via Fenton reactions involving the production of DNA base damage, such as 8-oxo-7,8-dihydroguanine. Copyright 2010 Elsevier B.V. All rights reserved.

Efficient CRISPR/Cas9-based genome editing in carrot cells.

PubMed

Klimek-Chodacka, Magdalena; Oleszkiewicz, Tomasz; Lowder, Levi G; Qi, Yiping; Baranski, Rafal

2018-04-01

The first report presenting successful and efficient carrot genome editing using CRISPR/Cas9 system. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas9) is a powerful genome editing tool that has been widely adopted in model organisms recently, but has not been used in carrot-a model species for in vitro culture studies and an important health-promoting crop grown worldwide. In this study, for the first time, we report application of the CRISPR/Cas9 system for efficient targeted mutagenesis of the carrot genome. Multiplexing CRISPR/Cas9 vectors expressing two single-guide RNA (gRNAs) targeting the carrot flavanone-3-hydroxylase (F3H) gene were tested for blockage of the anthocyanin biosynthesis in a model purple-colored callus using Agrobacterium-mediated genetic transformation. This approach allowed fast and visual comparison of three codon-optimized Cas9 genes and revealed that the most efficient one in generating F3H mutants was the Arabidopsis codon-optimized AteCas9 gene with up to 90% efficiency. Knockout of F3H gene resulted in the discoloration of calli, validating the functional role of this gene in the anthocyanin biosynthesis in carrot as well as providing a visual marker for screening successfully edited events. Most resulting mutations were small Indels, but long chromosome fragment deletions of 116-119 nt were also generated with simultaneous cleavage mediated by two gRNAs. The results demonstrate successful site-directed mutagenesis in carrot with CRISPR/Cas9 and the usefulness of a model callus culture to validate genome editing systems. Given that the carrot genome has been sequenced recently, our timely study sheds light on the promising application of genome editing tools for boosting basic and translational research in this important vegetable crop.

A Conserved UDP-Glucose Dehydrogenase Encoded outside the hasABC Operon Contributes to Capsule Biogenesis in Group A Streptococcus

PubMed Central

Cole, Jason N.; Aziz, Ramy K.; Kuipers, Kirsten; Timmer, Anjuli M.; Nizet, Victor

2012-01-01

Group A Streptococcus (GAS) is a human-specific bacterial pathogen responsible for serious morbidity and mortality worldwide. The hyaluronic acid (HA) capsule of GAS is a major virulence factor, contributing to bloodstream survival through resistance to neutrophil and antimicrobial peptide killing and to in vivo pathogenicity. Capsule biosynthesis has been exclusively attributed to the ubiquitous hasABC hyaluronan synthase operon, which is highly conserved across GAS serotypes. Previous reports indicate that hasA, encoding hyaluronan synthase, and hasB, encoding UDP-glucose 6-dehydrogenase, are essential for capsule production in GAS. Here, we report that precise allelic exchange mutagenesis of hasB in GAS strain 5448, a representative of the globally disseminated M1T1 serotype, did not abolish HA capsule synthesis. In silico whole-genome screening identified a putative HasB paralog, designated HasB2, with 45% amino acid identity to HasB at a distant location in the GAS chromosome. In vitro enzymatic assays demonstrated that recombinant HasB2 is a functional UDP-glucose 6-dehydrogenase enzyme. Mutagenesis of hasB2 alone slightly decreased capsule abundance; however, a ΔhasB ΔhasB2 double mutant became completely acapsular. We conclude that HasB is not essential for M1T1 GAS capsule biogenesis due to the presence of a newly identified HasB paralog, HasB2, which most likely resulted from gene duplication. The identification of redundant UDP-glucose 6-dehydrogenases underscores the importance of HA capsule expression for M1T1 GAS pathogenicity and survival in the human host. PMID:22961854

Building on the Past, Shaping the Future: The Environmental Mutagenesis and Genomics Society

EPA Science Inventory

In late 2012 the members of the Environmental Mutagen Society voted to change its name to the Environmental Mutagenesis and Genomics Society. Here we describe the thought process that led to adoption of the new name, which both respects the rich history of a Society founded in 19...

Rational development of an attenuated recombinant cyprinid herpesvirus 3 vaccine using prokaryotic mutagenesis and in vivo bioluminescent imaging

USDA-ARS?s Scientific Manuscript database

Cyprinid herpesvirus 3 (CyHV-3) is causing severe economic losses worldwide in the carp industry, and a safe and efficacious attenuated vaccine compatible with mass vaccination is needed. We produced single deleted recombinants using prokaryotic mutagenesis. When producing a recombinant lacking open...

HTLV-1 Tax Effects on Cellular Mitotic Regulation

DTIC Science & Technology

2007-04-12

39 2 MutagenesisTax mutagenesis and selection for tax mutants...43 Isolation of tax mutants that do not cause growth arrest in S. cerevisiae. .......... 43 Tax mutants selected in W303a are functional in...114). One currently-approved therapeutic agent , Indinavir, is an effective inhibitor of both proteases. New protease inhibitors are currently being

Alleles conferring improved fiber quality from EMS mutagenesis of elite cotton genotypes

USDA-ARS?s Scientific Manuscript database

The elite gene pool of cotton (Gossypium spp.) has less diversity than those of most other major crops, making identification of novel alleles important to ongoing crop improvement. A total of 3,164 M5 lines resulting from ethyl methanesulfonate mutagenesis of two G. hirsutum breeding lines, TAM 94L...

Deletion mutagenesis identifies a haploinsufficient role for gamma-zein in opaque-2 endosperm modification

USDA-ARS?s Scientific Manuscript database

Quality Protein Maize (QPM) is a hard kernel variant of the high-lysine mutant, opaque-2. Using gamma irradiation, we created opaque QPM variants to identify opaque-2 modifier genes and to investigate deletion mutagenesis combined with Illumina sequencing as a maize functional genomics tool. A K0326...

Insertional engineering of chromosomes with Sleeping Beauty transposition: an overview.

PubMed

Grabundzija, Ivana; Izsvák, Zsuzsanna; Ivics, Zoltán

2011-01-01

Novel genetic tools and mutagenesis strategies based on the Sleeping Beauty (SB) transposable element are currently under development with a vision to link primary DNA sequence information to gene functions in vertebrate models. By virtue of its inherent capacity to insert into DNA, the SB transposon can be developed into powerful tools for chromosomal manipulations. Mutagenesis screens based on SB have numerous advantages including high throughput and easy identification of mutated alleles. Forward genetic approaches based on insertional mutagenesis by engineered SB transposons have the advantage of providing insight into genetic networks and pathways based on phenotype. Indeed, the SB transposon has become a highly instrumental tool to induce tumors in experimental animals in a tissue-specific -manner with the aim of uncovering the genetic basis of diverse cancers. Here, we describe a battery of mutagenic cassettes that can be applied in conjunction with SB transposon vectors to mutagenize genes, and highlight versatile experimental strategies for the generation of engineered chromosomes for loss-of-function as well as gain-of-function mutagenesis for functional gene annotation in vertebrate models.

Quantitative Missense Variant Effect Prediction Using Large-Scale Mutagenesis Data.

PubMed

Gray, Vanessa E; Hause, Ronald J; Luebeck, Jens; Shendure, Jay; Fowler, Douglas M

2018-01-24

Large datasets describing the quantitative effects of mutations on protein function are becoming increasingly available. Here, we leverage these datasets to develop Envision, which predicts the magnitude of a missense variant's molecular effect. Envision combines 21,026 variant effect measurements from nine large-scale experimental mutagenesis datasets, a hitherto untapped training resource, with a supervised, stochastic gradient boosting learning algorithm. Envision outperforms other missense variant effect predictors both on large-scale mutagenesis data and on an independent test dataset comprising 2,312 TP53 variants whose effects were measured using a low-throughput approach. This dataset was never used for hyperparameter tuning or model training and thus serves as an independent validation set. Envision prediction accuracy is also more consistent across amino acids than other predictors. Finally, we demonstrate that Envision's performance improves as more large-scale mutagenesis data are incorporated. We precompute Envision predictions for every possible single amino acid variant in human, mouse, frog, zebrafish, fruit fly, worm, and yeast proteomes (https://envision.gs.washington.edu/). Copyright © 2017 Elsevier Inc. All rights reserved.

Lethal mutagenesis: targeting the mutator phenotype in cancer.

PubMed

Fox, Edward J; Loeb, Lawrence A

2010-10-01

The evolution of cancer and RNA viruses share many similarities. Both exploit high levels of genotypic diversity to enable extensive phenotypic plasticity and thereby facilitate rapid adaptation. In order to accumulate large numbers of mutations, we have proposed that cancers express a mutator phenotype. Similar to cancer cells, many viral populations, by replicating their genomes with low fidelity, carry a substantial mutational load. As high levels of mutation are potentially deleterious, the viral mutation frequency is thresholded at a level below which viral populations equilibrate in a traditional mutation-selection balance, and above which the population is no longer viable, i.e., the population undergoes an error catastrophe. Because their mutation frequencies are fine-tuned just below this error threshold, viral populations are susceptible to further increases in mutational load and, recently this phenomenon has been exploited therapeutically by a concept that has been termed lethal mutagenesis. Here we review the application of lethal mutagenesis to the treatment of HIV and discuss how lethal mutagenesis may represent a novel therapeutic approach for the treatment of solid cancers. Copyright © 2010 Elsevier Ltd. All rights reserved.

Induction of Pectinase Hyper Production by Multistep Mutagenesis Using a Fungal Isolate--Aspergillus flavipes.

PubMed

Akbar, Sabika; Prasuna, R Gyana; Khanam, Rasheeda

2014-04-01

Aspergillus flavipes, a slow growing pectinase producing ascomycete, was isolated from soil identified and characterised in the previously done preliminary studies. Optimisation studies revealed that Citrus peel--groundnut oil cake [CG] production media is the best media for production of high levels of pectinase up to 39 U/ml using wild strain of A. flavipes. Strain improvement of this isolated strain for enhancement of pectinase production using multistep mutagenesis procedure is the endeavour of this project. For this, the wild strain of A. flavipes was treated with both physical (UV irradiation) and chemical [Colchicine, Ethidium bromide, H2O2] mutagens to obtain Ist generation mutants. The obtained mutants were assayed and differentiated basing on pectinase productivity. The better pectinase producing strains were further subjected to multistep mutagenesis to attain stability in mutants. The goal of this project was achieved by obtaining the best pectinase secreting mutant, UV80 of 45 U/ml compared to wild strain and sister mutants. This fact was confirmed by quantitatively analysing 3rd generation mutants obtained after multistep mutagenesis.

The Novel Mouse Mutation Oblivion Inactivates the PMCA2 Pump and Causes Progressive Hearing Loss

PubMed Central

de Angelis, Martin Hrabé; Fuchs, Helmut; Lim, Dmitry; Ortolano, Saida; Ingham, Neil J.; Brini, Marisa; Carafoli, Ernesto; Mammano, Fabio; Steel, Karen P.

2008-01-01

Progressive hearing loss is common in the human population, but we have few clues to the molecular basis. Mouse mutants with progressive hearing loss offer valuable insights, and ENU (N-ethyl-N-nitrosourea) mutagenesis is a useful way of generating models. We have characterised a new ENU-induced mouse mutant, Oblivion (allele symbol Obl), showing semi-dominant inheritance of hearing impairment. Obl/+ mutants showed increasing hearing impairment from post-natal day (P)20 to P90, and loss of auditory function was followed by a corresponding base to apex progression of hair cell degeneration. Obl/Obl mutants were small, showed severe vestibular dysfunction by 2 weeks of age, and were completely deaf from birth; sensory hair cells were completely degenerate in the basal turn of the cochlea, although hair cells appeared normal in the apex. We mapped the mutation to Chromosome 6. Mutation analysis of Atp2b2 showed a missense mutation (2630C→T) in exon 15, causing a serine to phenylalanine substitution (S877F) in transmembrane domain 6 of the PMCA2 pump, the resident Ca2+ pump of hair cell stereocilia. Transmembrane domain mutations in these pumps generally are believed to be incompatible with normal targeting of the protein to the plasma membrane. However, analyses of hair cells in cultured utricular maculae of Obl/Obl mice and of the mutant Obl pump in model cells showed that the protein was correctly targeted to the plasma membrane. Biochemical and biophysical characterisation showed that the pump had lost a significant portion of its non-stimulated Ca2+ exporting ability. These findings can explain the progressive loss of auditory function, and indicate the limits in our ability to predict mechanism from sequence alone. PMID:18974863

Cloning-Independent and Counterselectable Markerless Mutagenesis System in Streptococcus mutans▿

PubMed Central

Xie, Zhoujie; Okinaga, Toshinori; Qi, Fengxia; Zhang, Zhijun; Merritt, Justin

2011-01-01

Insertion duplication mutagenesis and allelic replacement mutagenesis are among the most commonly utilized approaches for targeted mutagenesis in bacteria. However, both techniques are limited by a variety of factors that can complicate mutant phenotypic studies. To circumvent these limitations, multiple markerless mutagenesis techniques have been developed that utilize either temperature-sensitive plasmids or counterselectable suicide vectors containing both positive- and negative-selection markers. For many species, these techniques are not especially useful due to difficulties of cloning with Escherichia coli and/or a lack of functional negative-selection markers. In this study, we describe the development of a novel approach for the creation of markerless mutations. This system employs a cloning-independent methodology and should be easily adaptable to a wide array of Gram-positive and Gram-negative bacterial species. The entire process of creating both the counterselection cassette and mutation constructs can be completed using overlapping PCR protocols, which allows extremely quick assembly and eliminates the requirement for either temperature-sensitive replicons or suicide vectors. As a proof of principle, we used Streptococcus mutans reference strain UA159 to create markerless in-frame deletions of 3 separate bacteriocin genes as well as triple mutants containing all 3 deletions. Using a panel of 5 separate wild-type S. mutans strains, we further demonstrated that the procedure is nearly 100% efficient at generating clones with the desired markerless mutation, which is a considerable improvement in yield compared to existing approaches. PMID:21948849

Incorporation of a lambda phage recombination system and EGFP detection to simplify mutagenesis of Herpes simplex virus bacterial artificial chromosomes

PubMed Central

Schmeisser, Falko; Weir, Jerry P

2007-01-01

Background Targeted mutagenesis of the herpesvirus genomes has been facilitated by the use of bacterial artificial chromosome (BAC) technology. Such modified genomes have potential uses in understanding viral pathogenesis, gene identification and characterization, and the development of new viral vectors and vaccines. We have previously described the construction of a herpes simplex virus 2 (HSV-2) BAC and the use of an allele replacement strategy to construct HSV-2 recombinants. While the BAC mutagenesis procedure is a powerful method to generate HSV-2 recombinants, particularly in the absence of selective marker in eukaryotic culture, the mutagenesis procedure is still difficult and cumbersome. Results Here we describe the incorporation of a phage lambda recombination system into an allele replacement vector. This strategy enables any DNA fragment containing the phage attL recombination sites to be efficiently inserted into the attR sites of the allele replacement vector using phage lambda clonase. We also describe how the incorporation of EGFP into the allele replacement vector can facilitate the selection of the desired cross-over recombinant BACs when the allele replacement reaction is a viral gene deletion. Finally, we incorporate the lambda phage recombination sites directly into an HSV-2 BAC vector for direct recombination of gene cassettes using the phage lambda clonase-driven recombination reaction. Conclusion Together, these improvements to the techniques of HSV BAC mutagenesis will facilitate the construction of recombinant herpes simplex viruses and viral vectors. PMID:17501993

In vitro Studies of Sandfly Fever Viruses and Their Potential Significance for Vaccine Development.

DTIC Science & Technology

1981-02-01

34 - "’: / AD In vitro Studies of Sandfly Fever Viruses and Their Potential Significance for Vaccine Development Annual Progress Report by CO Jonathan F. Smith...Significance for Vaccine 1 gR1 Development 6 WPW"a EOTHmaei AUTHOV&PSI CONTRACt OR GRANT NUMSERWa Jonahan . Smth, h.D.DAMD-17-78-C-8056 P6EftJrPORim...antibodies, post-translational processing, Immunoprecipitation, antigen purification, In vitro translation, passive transfer, vaccines _26. AEISTRACTMO

Use of Synthetic Peptides and Anti-Idiotypes for Controlling Human Immunodeficiency Virus Infections

DTIC Science & Technology

1993-08-27

example, studies by others have suggested that Iy-I. isolates which readily form syncytia in vitro may be associated with a more rapid rate of disease ...isolates which readily form syncytia in vitro may be associated with a more rapid rate of disease progression in vivo. Using fluorescent intracellular...will be useful reagents in testing sara for neutralizing activity in vitro. 0. Longitudinal analysis of the humoral inmune response to RIV-I gPi60

Site-directed mutagenesis in Petunia × hybrida protoplast system using direct delivery of purified recombinant Cas9 ribonucleoproteins.

PubMed

Subburaj, Saminathan; Chung, Sung Jin; Lee, Choongil; Ryu, Seuk-Min; Kim, Duk Hyoung; Kim, Jin-Soo; Bae, Sangsu; Lee, Geung-Joo

2016-07-01

Site-directed mutagenesis of nitrate reductase genes using direct delivery of purified Cas9 protein preassembled with guide RNA produces mutations efficiently in Petunia × hybrida protoplast system. The clustered, regularly interspaced, short palindromic repeat (CRISPR)-CRISPR associated endonuclease 9 (CRISPR/Cas9) system has been recently announced as a powerful molecular breeding tool for site-directed mutagenesis in higher plants. Here, we report a site-directed mutagenesis method targeting Petunia nitrate reductase (NR) gene locus. This method could create mutations efficiently using direct delivery of purified Cas9 protein and single guide RNA (sgRNA) into protoplast cells. After transient introduction of RNA-guided endonuclease (RGEN) ribonucleoproteins (RNPs) with different sgRNAs targeting NR genes, mutagenesis at the targeted loci was detected by T7E1 assay and confirmed by targeted deep sequencing. T7E1 assay showed that RGEN RNPs induced site-specific mutations at frequencies ranging from 2.4 to 21 % at four different sites (NR1, 2, 4 and 6) in the PhNR gene locus with average mutation efficiency of 14.9 ± 2.2 %. Targeted deep DNA sequencing revealed mutation rates of 5.3-17.8 % with average mutation rate of 11.5 ± 2 % at the same NR gene target sites in DNA fragments of analyzed protoplast transfectants. Further analysis from targeted deep sequencing showed that the average ratio of deletion to insertion produced collectively by the four NR-RGEN target sites (NR1, 2, 4, and 6) was about 63:37. Our results demonstrated that direct delivery of RGEN RNPs into protoplast cells of Petunia can be exploited as an efficient tool for site-directed mutagenesis of genes or genome editing in plant systems.

A Technique for the Growth of Epidermal Sheets Obtained from Patients Undergoing Reduction Mammoplasty.

DTIC Science & Technology

1991-03-06

flask with Dispase, a neutral protease and attached to petrolatum gauze. At this point the sheets could be used as skin grafts . Progress: 1...a composite in vitro skin graft . We believe that development of an artificial dermis as part of a composite in vitro graft is where our future

Gastric cancer-derived MSC-secreted PDGF-DD promotes gastric cancer progression.

PubMed

Huang, Feng; Wang, Mei; Yang, Tingting; Cai, Jie; Zhang, Qiang; Sun, Zixuan; Wu, Xiaodan; Zhang, Xu; Zhu, Wei; Qian, Hui; Xu, Wenrong

2014-11-01

This study was designed to investigate the role of PDGF-DD secreted by gastric cancer-derived mesenchymal stem cells (GC-MSCs) in human gastric cancer progression. Gastric cancer cells were indirectly co-cultured with GC-MSCs in a transwell system. The growth and migration of gastric cancer cells were evaluated by cell colony formation assay and transwell migration assay, respectively. The production of PDGF-DD in GC-MSCs was determined by using Luminex and ELISA. Neutralization of PDGFR-β by su16f and siRNA interference of PDGF-DD in GC-MSCs was used to demonstrate the role of PDGF-DD produced by GC-MSCs in gastric cancer progression. GC-MSC conditioned medium promoted gastric cancer cell proliferation and migration in vitro and in vivo. Co-culture with GC-MSCs increased the phosphorylation of PDGFR-β in SGC-7901 cells. Neutralization of PDGFR-β by su16f blocked the promoting role of GC-MSC conditioned medium in gastric cancer cell proliferation and migration. Recombinant PDGF-DD duplicated the effects of GC-MSC conditioned medium on gastric cancer cells. Knockdown of PDGF-DD in GC-MSCs abolished its effects on gastric cancer cells in vitro and in vivo. PDGF-DD secreted by GC-MSCs is capable of promoting gastric cancer cell progression in vitro and in vivo. Targeting the PDGF-DD/PDGFR-β interaction between MSCs and gastric cancer cells may represent a novel strategy for gastric cancer therapy.

From Green to Blue: Site-Directed Mutagenesis of the Green Fluorescent Protein to Teach Protein Structure-Function Relationships

ERIC Educational Resources Information Center

Giron, Maria D.; Salto, Rafael

2011-01-01

Structure-function relationship studies in proteins are essential in modern Cell Biology. Laboratory exercises that allow students to familiarize themselves with basic mutagenesis techniques are essential in all Genetic Engineering courses to teach the relevance of protein structure. We have implemented a laboratory course based on the…

Enhanced toxicity of Bacillus thuringiensis Cry3A 8-endotoxin in coleopterans by mutagenesis in recetor binding loop

Treesearch

Sheng-Jiun Wu; Noah C. Koller; Deborah L. Miller; Leah S. Bauer; Donald H. Dean

2000-01-01

We used site-directed mutagenesis to modify the Bacillus cry3A gene in amino acid residues 350-354. Two mutant toxins, A1 (R345A, Y350F, Y351F) and A2 (R345A,DeltaY350, DeltaY351, showed significantly improved...

UV-C mutagenesis of Kluyveromyces marxianus NRRL Y-1109 strain for improved anaerobic growth at elevated temperature on pentose and hexose sugars

USDA-ARS?s Scientific Manuscript database

More robust industrial yeast strains from Kluyveromyces marxianus NRRL Y-1109 and have been produced using UV-C irradiation specifically for anaerobic conversion of lignocellulosic sugar streams to fuel ethanol at elevated temperature (45°C). This type of random mutagenesis offers the possibility o...

Pla2g12b and Hpn Are Genes Identified by Mouse ENU Mutagenesis That Affect HDL Cholesterol

PubMed Central

Aljakna, Aleksandra; Choi, Seungbum; Savage, Holly; Hageman Blair, Rachael; Gu, Tongjun; Svenson, Karen L.; Churchill, Gary A.; Hibbs, Matt; Korstanje, Ron

2012-01-01

Despite considerable progress understanding genes that affect the HDL particle, its function, and cholesterol content, genes identified to date explain only a small percentage of the genetic variation. We used N-ethyl-N-nitrosourea mutagenesis in mice to discover novel genes that affect HDL cholesterol levels. Two mutant lines (Hlb218 and Hlb320) with low HDL cholesterol levels were established. Causal mutations in these lines were mapped using linkage analysis: for line Hlb218 within a 12 Mbp region on Chr 10; and for line Hlb320 within a 21 Mbp region on Chr 7. High-throughput sequencing of Hlb218 liver RNA identified a mutation in Pla2g12b. The transition of G to A leads to a cysteine to tyrosine change and most likely causes a loss of a disulfide bridge. Microarray analysis of Hlb320 liver RNA showed a 7-fold downregulation of Hpn; sequencing identified a mutation in the 3′ splice site of exon 8. Northern blot confirmed lower mRNA expression level in Hlb320 and did not show a difference in splicing, suggesting that the mutation only affects the splicing rate. In addition to affecting HDL cholesterol, the mutated genes also lead to reduction in serum non-HDL cholesterol and triglyceride levels. Despite low HDL cholesterol levels, the mice from both mutant lines show similar atherosclerotic lesion sizes compared to control mice. These new mutant mouse models are valuable tools to further study the role of these genes, their affect on HDL cholesterol levels, and metabolism. PMID:22912808

Concise review: managing genotoxicity in the therapeutic modification of stem cells.

PubMed

Baum, Christopher; Modlich, Ute; Göhring, Gudrun; Schlegelberger, Brigitte

2011-10-01

The therapeutic use of procedures for genetic stem cell modification is limited by potential adverse events related to uncontrolled mutagenesis. Prominent findings have been made in hematopoietic gene therapy, demonstrating the risk of clonal, potentially malignant outgrowth on the basis of mutations acquired during or after therapeutic genome modification. The incidence and the growth rate of insertional mutants have been linked to the "stemness" of the target cells and vector-related features such as the integration pattern, the architecture, and the exact content of transgene cassettes. Milieu factors supporting the survival and expansion of mutants may eventually allow oncogenic progression. Similar concerns apply for medicinal products based on pluripotent stem cells. Focusing on the genetic stress induced by insertional mutagenesis and culture adaptation, we propose four conclusions. (a) Mutations occurring in the production of stem cell-based medicines may be unavoidable and need to be classified according to their risk to trigger the formation of clones that are sufficiently long-lived and mitotically active to acquire secondary transforming mutations. (b) The development of rational prevention strategies depends upon the identification of the specific mutations forming such "dominant clones" (which can also be addressed as cancer stem cell precursors) and a better knowledge of the mechanisms underlying their creation, expansion, and homeostatic control. (c) Quantitative assay systems are required to assess the practical value of preventive actions. (d) Improved approaches for the genetic modification of stem cells can address all critical steps in the origin and growth control of mutants. Copyright © 2011 AlphaMed Press.

Keratinolytic protease: a green biocatalyst for leather industry.

PubMed

Fang, Zhen; Yong, Yang-Chun; Zhang, Juan; Du, Guocheng; Chen, Jian

2017-11-01

Depilation/unhairing is the crucial but heavy pollution process in leather industry. Traditional inorganic sulfide treatment was the most widely used depilation technique in the past decades, which was usually detrimental to leather quality and resulted in serious environmental pollution. Using biocatalysts to substitute inorganic sulfide showed great advantages in environment protection and unhairing efficiency. Keratinolytic protease is one of the excellent biocatalysts to hydrolyze disulfide bond-rich proteins of hair and has little damage to leather. Biological treatment with keratinolytic proteases could largely reduce the quantity and toxicity of wastewater effluent from the leather industry. But low thermostability and substrate specificity or specific activity of these enzymes limited their practical application. Therefore, recent progresses on protein engineering strategies (site-directed mutagenesis, protein fusion, N/C-terminus truncation, and domain swapping) used to enhance the keratinolytic enzyme performance were presented.

Clinical and Molecular Features of POLG-Related Mitochondrial Disease

PubMed Central

Stumpf, Jeffrey D.; Saneto, Russell P.; Copeland, William C.

2013-01-01

The inability to replicate mitochondrial genomes (mtDNA) by the mitochondrial DNA polymerase (pol γ) leads to a subset of mitochondrial diseases. Many mutations in POLG, the gene that encodes pol γ, have been associated with mitochondrial diseases such as myocerebrohepatopathy spectrum (MCHS) disorders, Alpers-Huttenlocher syndrome, myoclonic epilepsy myopathy sensory ataxia (MEMSA), ataxia neuropathy spectrum (ANS), and progressive external ophthalmoplegia (PEO). This chapter explores five important topics in POLG-related disease: (1) clinical symptoms that identify and distinguish POLG-related diseases, (2) molecular characterization of defects in polymerase activity by POLG disease variants, (3) the importance of holoenzyme formation in disease presentation, (4) the role of pol γ exonuclease activity and mutagenesis in disease and aging, and (5) novel approaches to therapy and avoidance of toxicity based on primary research in pol γ replication. PMID:23545419

Characterization in vitro and in vivo of progressively adriamycin-resistant B16-BL6 mouse melanoma cells.

PubMed

Ganapathi, R; Grabowski, D; Schmidt, H; Bell, D; Melia, M

1987-07-01

Adriamycin (ADR)-resistant sublines of B16-BL6 mouse melanoma selected by exposure to increasing concentrations of ADR were characterized in vitro for growth properties and in vivo for tumorigenicity and pulmonary metastases. The progressively resistant sublines adapted to grow in the presence of 0.025, 0.05, 0.1, and 0.25 microgram/ml ADR in monolayer culture were found to be 5-, 10-, 20-, and 40-fold ADR-resistant, respectively, compared to the parental sensitive cells, using a soft-agar colony assay and continuous ADR treatment for 7 days. The doubling time in monolayer culture of the parent sensitive and progressively ADR-resistant sublines of B16-BL6 melanoma cells was approximately 16-18 h. Although the colony-forming efficiency in soft agar of parental sensitive cells was only 0.5-4%, the 5-, 10-, 20-, and 40-fold ADR-resistant sublines had colony-forming efficiencies of 15, 20, 30, and 77%, respectively. Tumorigenicity in C57BL/6 mice of progressively ADR-resistant sublines was similar to parental sensitive cells following s.c. and i.p. implantation of 10(5)-10(6) tumor cells. Experimental pulmonary metastases were significantly lower in ADR-resistant sublines with progressive resistance. Additionally, unlike the parental sensitive and 5-fold ADR-resistant B16-BL6 cells, the 10-, 20-, and 40-fold ADR-resistant sublines were spontaneously nonmetastatic. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunochemical detection of P-glycoprotein revealed the presence of a Mr 170,000 plasma membrane glycoprotein in the 40-fold ADR-resistant subline and its counterpart maintained for 1 year in ADR-free medium. Results from this study suggest that progressively ADR-resistant B16-BL6 mouse melanoma cells selected in vitro demonstrate a marked increase in colony formation in soft agar and a decrease in the ability to produce pulmonary metastases, without alterations in tumorigenicity.

Human Papilloma Virus in Melanoma Biopsy Specimens and Its Relation to Melanoma Progression

PubMed Central

Dréau, Didier; Culberson, Cathy; Wyatt, Sharon; Holder, Walter D.

2000-01-01

Objectives To evaluate melanoma biopsy specimens for human papilloma virus (HPV) and determine the relation between the presence of HPV, in vitro growth, and clinical progression of melanoma in the patients from whom the biopsy specimens were derived. Summary Background Data Ultraviolet radiation from sun exposure appears to be the primary causal agent in the development of cutaneous melanoma. However, other agents, including HPV, as observed in different epithelial carcinomas, may also play a role in melanoma development and progression. Methods Twelve melanoma biopsy specimens obtained from 12 patients with AJCC stage III and IV melanoma were stained with antibodies against gp-100 (HMB-45) and S-100 protein to confirm melanoma diagnosis and with a polyclonal HPV antibody. After mechanical dissociation, the melanoma specimen cells’ ability to grow in vitro was assessed. Patients were evaluated for melanoma progression with physical examination, complete blood count, and liver function tests every 3 months and a chest radiograph every 6 months. Results All biopsy specimens were positive for S-100, and nine (75%) were positive for gp-100. Seven of 12 (58%) were positive for HPV by immunohistochemistry. In vitro, none of the HPV-negative tumor cells grew from the tumor biopsies, whereas five of seven (71%) of the HPV-positive melanoma tumor cells grew very well. All patients with HPV-positive tumor cells had recurrences and died of melanoma progression, whereas four of five (80%) patients with HPV-negative tumor cells remained alive and without melanoma recurrence. Conclusions The presence of HPV was found in 58% of the biopsy specimens obtained from patients with stage III and IV melanoma and correlated with rapid melanoma progression. HPV may serve as a cofactor in the development of melanoma and may modulate a more aggressive phenotype in HPV-containing melanoma cells. PMID:10767787

Structural insights into Cydia pomonella pheromone binding protein 2 mediated prediction of potentially active semiochemicals

NASA Astrophysics Data System (ADS)

Tian, Zhen; Liu, Jiyuan; Zhang, Yalin

2016-03-01

Given the advantages of behavioral disruption application in pest control and the damage of Cydia pomonella, due progresses have not been made in searching active semiochemicals for codling moth. In this research, 31 candidate semiochemicals were ranked for their binding potential to Cydia pomonella pheromone binding protein 2 (CpomPBP2) by simulated docking, and this sorted result was confirmed by competitive binding assay. This high predicting accuracy of virtual screening led to the construction of a rapid and viable method for semiochemicals searching. By reference to binding mode analyses, hydrogen bond and hydrophobic interaction were suggested to be two key factors in determining ligand affinity, so is the length of molecule chain. So it is concluded that semiochemicals of appropriate chain length with hydroxyl group or carbonyl group at one head tended to be favored by CpomPBP2. Residues involved in binding with each ligand were pointed out as well, which were verified by computational alanine scanning mutagenesis. Progress made in the present study helps establish an efficient method for predicting potentially active compounds and prepares for the application of high-throughput virtual screening in searching semiochemicals by taking insights into binding mode analyses.

Structural insights into Cydia pomonella pheromone binding protein 2 mediated prediction of potentially active semiochemicals

PubMed Central

Tian, Zhen; Liu, Jiyuan; Zhang, Yalin

2016-01-01

Given the advantages of behavioral disruption application in pest control and the damage of Cydia pomonella, due progresses have not been made in searching active semiochemicals for codling moth. In this research, 31 candidate semiochemicals were ranked for their binding potential to Cydia pomonella pheromone binding protein 2 (CpomPBP2) by simulated docking, and this sorted result was confirmed by competitive binding assay. This high predicting accuracy of virtual screening led to the construction of a rapid and viable method for semiochemicals searching. By reference to binding mode analyses, hydrogen bond and hydrophobic interaction were suggested to be two key factors in determining ligand affinity, so is the length of molecule chain. So it is concluded that semiochemicals of appropriate chain length with hydroxyl group or carbonyl group at one head tended to be favored by CpomPBP2. Residues involved in binding with each ligand were pointed out as well, which were verified by computational alanine scanning mutagenesis. Progress made in the present study helps establish an efficient method for predicting potentially active compounds and prepares for the application of high-throughput virtual screening in searching semiochemicals by taking insights into binding mode analyses. PMID:26928635

The Saccharomyces cerevisiae RAD9, RAD17, RAD24 and MEC3 genes are required for tolerating irreparable, ultraviolet-induced DNA damage.

PubMed Central

Paulovich, A G; Armour, C D; Hartwell, L H

1998-01-01

In wild-type Saccharomyces cerevisiae, a checkpoint slows the rate of progression of an ongoing S phase in response to exposure to a DNA-alkylating agent. Mutations that eliminate S phase regulation also confer sensitivity to alkylating agents, leading us to suggest that, by regulating the S phase rate, cells are either better able to repair or better able to replicate damaged DNA. In this study, we determine the effects of mutations that impair S phase regulation on the ability of excision repair-defective cells to replicate irreparably UV-damaged DNA. We assay survival after UV irradiation, as well as the genetic consequences of replicating a damaged template, namely mutation and sister chromatid exchange induction. We find that RAD9, RAD17, RAD24, and MEC3 are required for UV-induced (although not spontaneous) mutagenesis, and that RAD9 and RAD17 (but not REV3, RAD24, and MEC3) are required for maximal induction of replication-dependent sister chromatid exchange. Therefore, checkpoint genes not only control cell cycle progression in response to damage, but also play a role in accommodating DNA damage during replication. PMID:9725831

The Saccharomyces cerevisiae RAD9, RAD17, RAD24 and MEC3 genes are required for tolerating irreparable, ultraviolet-induced DNA damage.

PubMed

Paulovich, A G; Armour, C D; Hartwell, L H

1998-09-01

In wild-type Saccharomyces cerevisiae, a checkpoint slows the rate of progression of an ongoing S phase in response to exposure to a DNA-alkylating agent. Mutations that eliminate S phase regulation also confer sensitivity to alkylating agents, leading us to suggest that, by regulating the S phase rate, cells are either better able to repair or better able to replicate damaged DNA. In this study, we determine the effects of mutations that impair S phase regulation on the ability of excision repair-defective cells to replicate irreparably UV-damaged DNA. We assay survival after UV irradiation, as well as the genetic consequences of replicating a damaged template, namely mutation and sister chromatid exchange induction. We find that RAD9, RAD17, RAD24, and MEC3 are required for UV-induced (although not spontaneous) mutagenesis, and that RAD9 and RAD17 (but not REV3, RAD24, and MEC3) are required for maximal induction of replication-dependent sister chromatid exchange. Therefore, checkpoint genes not only control cell cycle progression in response to damage, but also play a role in accommodating DNA damage during replication.

Replication protein A (RPA) hampers the processive action of APOBEC3G cytosine deaminase on single-stranded DNA.

PubMed

Lada, Artem G; Waisertreiger, Irina S-R; Grabow, Corinn E; Prakash, Aishwarya; Borgstahl, Gloria E O; Rogozin, Igor B; Pavlov, Youri I

2011-01-01

Editing deaminases have a pivotal role in cellular physiology. A notable member of this superfamily, APOBEC3G (A3G), restricts retroviruses, and Activation Induced Deaminase (AID) generates antibody diversity by localized deamination of cytosines in DNA. Unconstrained deaminase activity can cause genome-wide mutagenesis and cancer. The mechanisms that protect the genomic DNA from the undesired action of deaminases are unknown. Using the in vitro deamination assays and expression of A3G in yeast, we show that replication protein A (RPA), the eukaryotic single-stranded DNA (ssDNA) binding protein, severely inhibits the deamination activity and processivity of A3G. We found that mutations induced by A3G in the yeast genomic reporter are changes of a single nucleotide. This is unexpected because of the known property of A3G to catalyze multiple deaminations upon one substrate encounter event in vitro. The addition of recombinant RPA to the oligonucleotide deamination assay severely inhibited A3G activity. Additionally, we reveal the inverse correlation between RPA concentration and the number of deaminations induced by A3G in vitro on long ssDNA regions. This resembles the "hit and run" single base substitution events observed in yeast. Our data suggest that RPA is a plausible antimutator factor limiting the activity and processivity of editing deaminases in the model yeast system. Because of the similar antagonism of yeast RPA and human RPA with A3G in vitro, we propose that RPA plays a role in the protection of the human genome cell from A3G and other deaminases when they are inadvertently diverged from their natural targets. We propose a model where RPA serves as one of the guardians of the genome that protects ssDNA from the destructive processive activity of deaminases by non-specific steric hindrance.

Spermatogonial stem cells from domestic animals: progress and prospects.

PubMed

Zheng, Yi; Zhang, Yaqing; Qu, Rongfeng; He, Ying; Tian, Xiue; Zeng, Wenxian

2014-03-01

Spermatogenesis, an elaborate and male-specific process in adult testes by which a number of spermatozoa are produced constantly for male fertility, relies on spermatogonial stem cells (SSCs). As a sub-population of undifferentiated spermatogonia, SSCs are capable of both self-renewal (to maintain sufficient quantities) and differentiation into mature spermatozoa. SSCs are able to convert to pluripotent stem cells during in vitro culture, thus they could function as substitutes for human embryonic stem cells without ethical issues. In addition, this process does not require exogenous transcription factors necessary to produce induced-pluripotent stem cells from somatic cells. Moreover, combining genetic engineering with germ cell transplantation would greatly facilitate the generation of transgenic animals. Since germ cell transplantation into infertile recipient testes was first established in 1994, in vivo and in vitro study and manipulation of SSCs in rodent testes have been progressing at a staggering rate. By contrast, their counterparts in domestic animals, despite the failure to reach a comparable level, still burgeoned and showed striking advances. This review outlines the recent progressions of characterization, isolation, in vitro propagation, and transplantation of spermatogonia/SSCs from domestic animals, thereby shedding light on future exploration of these cells with high value, as well as contributing to the development of reproductive technology for large animals.

Progress in Assessing Air Pollutant Risks from In Vitro Exposures: Matching Ozone Dose and Effect in Human Air Way Cells

EPA Science Inventory

In vitro exposures to air pollutants could, in theory, facilitate a rapid and detailed assessment of molecular mechanisms of toxicity. However, it is difficult to ensure that the dose of a gaseous pollutant to cells in tissue culture is similar to that of the same cells during in...

The Mechanism of Nucleotide Excision Repair-Mediated UV-Induced Mutagenesis in Nonproliferating Cells

PubMed Central

Kozmin, Stanislav G.; Jinks-Robertson, Sue

2013-01-01

Following the irradiation of nondividing yeast cells with ultraviolet (UV) light, most induced mutations are inherited by both daughter cells, indicating that complementary changes are introduced into both strands of duplex DNA prior to replication. Early analyses demonstrated that such two-strand mutations depend on functional nucleotide excision repair (NER), but the molecular mechanism of this unique type of mutagenesis has not been further explored. In the experiments reported here, an ade2 adeX colony-color system was used to examine the genetic control of UV-induced mutagenesis in nondividing cultures of Saccharomyces cerevisiae. We confirmed a strong suppression of two-strand mutagenesis in NER-deficient backgrounds and demonstrated that neither mismatch repair nor interstrand crosslink repair affects the production of these mutations. By contrast, proteins involved in the error-prone bypass of DNA damage (Rev3, Rev1, PCNA, Rad18, Pol32, and Rad5) and in the early steps of the DNA-damage checkpoint response (Rad17, Mec3, Ddc1, Mec1, and Rad9) were required for the production of two-strand mutations. There was no involvement, however, for the Pol η translesion synthesis DNA polymerase, the Mms2-Ubc13 postreplication repair complex, downstream DNA-damage checkpoint factors (Rad53, Chk1, and Dun1), or the Exo1 exonuclease. Our data support models in which UV-induced mutagenesis in nondividing cells occurs during the Pol ζ-dependent filling of lesion-containing, NER-generated gaps. The requirement for specific DNA-damage checkpoint proteins suggests roles in recruiting and/or activating factors required to fill such gaps. PMID:23307894

Modification of a deoxynivalenol-antigen-mimicking nanobody to improve immunoassay sensitivity by site-saturation mutagenesis.

PubMed

Qiu, Yu-Lou; He, Qing-Hua; Xu, Yang; Wang, Wei; Liu, Yuan-Yuan

2016-01-01

A nanobody (N-28) which can act as a deoxynivalenol (DON) antigen has been generated, and its residues Thr102-Ser106 were identified to bind with anti-DON monoclonal antibody by alanine-scanning mutagenesis. Site-saturation mutagenesis was used to analyze the plasticity of five residues and to improve the sensitivity of the N-28-based immunoassay. After mutagenesis, three mutants were selected by phage immunoassay and were sequenced. The half-maximal inhibitory concentrations of the immunoassay based on mutants N-28-T102Y, N-28-V103L, and N-28-Y105F were 24.49 ± 1.0, 51.83 ± 2.5, and 35.65 ± 1.6 ng/mL, respectively, showing the assay was, respectively, 3.2, 1.5, and 2.2 times more sensitive than the wild-type-based assay. The best mutant, N-28-T102Y, was used to develop a competitive phage ELISA to detect DON in cereals with high specificity and accuracy. In addition, the structural properties of N-28-T102Y and N-28 were investigated, revealing that the affinity of N-28-T102Y decreased because of increased steric hindrance with the large side chain. The lower-binding-affinity antigen mimetic may contribute to the improvement of the sensitivity of competitive immunoassays. These results demonstrate that nanobodies would be a favorable tool for engineering. Moreover, our results have laid a solid foundation for site-saturation mutagenesis of antigen-mimicking nanobodies to improve immunoassay sensitivity for small molecules.

Targeted Mutagenesis of Duplicated Genes in Soybean with Zinc-Finger Nucleases1[W][OA

PubMed Central

Curtin, Shaun J.; Zhang, Feng; Sander, Jeffry D.; Haun, William J.; Starker, Colby; Baltes, Nicholas J.; Reyon, Deepak; Dahlborg, Elizabeth J.; Goodwin, Mathew J.; Coffman, Andrew P.; Dobbs, Drena; Joung, J. Keith; Voytas, Daniel F.; Stupar, Robert M.

2011-01-01

We performed targeted mutagenesis of a transgene and nine endogenous soybean (Glycine max) genes using zinc-finger nucleases (ZFNs). A suite of ZFNs were engineered by the recently described context-dependent assembly platform—a rapid, open-source method for generating zinc-finger arrays. Specific ZFNs targeting DICER-LIKE (DCL) genes and other genes involved in RNA silencing were cloned into a vector under an estrogen-inducible promoter. A hairy-root transformation system was employed to investigate the efficiency of ZFN mutagenesis at each target locus. Transgenic roots exhibited somatic mutations localized at the ZFN target sites for seven out of nine targeted genes. We next introduced a ZFN into soybean via whole-plant transformation and generated independent mutations in the paralogous genes DCL4a and DCL4b. The dcl4b mutation showed efficient heritable transmission of the ZFN-induced mutation in the subsequent generation. These findings indicate that ZFN-based mutagenesis provides an efficient method for making mutations in duplicate genes that are otherwise difficult to study due to redundancy. We also developed a publicly accessible Web-based tool to identify sites suitable for engineering context-dependent assembly ZFNs in the soybean genome. PMID:21464476

Transcription coupled repair deficiency protects against human mutagenesis and carcinogenesis: Personal Reflections on the 50th anniversary of the discovery of xeroderma pigmentosum.

PubMed

Cleaver, James E

2017-10-01

Xeroderma pigmentosum (XP) patients who lack the main damage recognition protein for global genome repair (GGR), XPC, have greatly increased skin cancer rates and elevated mutation frequencies originating from unrepaired ultraviolet photoproducts in the nontranscribed regions of the genome and in nontranscribed strands of expressed genes. But they show no increased mutations in transcribed strands. In contrast, cancer is absent from Cockayne syndrome (CS) patients that have defective transcription coupled repair (TCR) despite severe photosensitivity, CS patients remarkably show no elevation of UV induced mutagenesis implying that defective TCR may be protective against mutagenesis and carcinogenesis. Mutation avoidance in CS is postulated to occur through arrested transcription that generates a tripled stranded R loop consisting of DNA double strands and a nascent mRNA strand. R loops result in S phase apoptosis or activation of ATM kinase that causes a delay in DNA replication until TCR, or transcript cleavage by TFIIS or RNAaseH, relieves the transcription block. Resumption of replication then occurs on repaired DNA without concomitant mutagenesis. Copyright © 2017 Elsevier B.V. All rights reserved.

Live Births from Domestic Dog (Canis familiaris) Embryos Produced by In Vitro Fertilization

PubMed Central

Nagashima, Jennifer B.; Sylvester, Skylar R.; Nelson, Jacquelyn L.; Cheong, Soon Hon; Mukai, Chinatsu; Lambo, Colleen; Flanders, James A.; Meyers-Wallen, Vicki N.; Songsasen, Nucharin; Travis, Alexander J.

2015-01-01

Development of assisted reproductive technologies (ART) in the dog has resisted progress for decades, due to their unique reproductive physiology. This lack of progress is remarkable given the critical role ART could play in conserving endangered canid species or eradicating heritable disease through gene-editing technologies—an approach that would also advance the dog as a biomedical model. Over 350 heritable disorders/traits in dogs are homologous with human conditions, almost twice the number of any other species. Here we report the first live births from in vitro fertilized embryos in the dog. Adding to the practical significance, these embryos had also been cryopreserved. Changes in handling of both gametes enabled this progress. The medium previously used to capacitate sperm excluded magnesium because it delayed spontaneous acrosome exocytosis. We found that magnesium significantly enhanced sperm hyperactivation and ability to undergo physiologically-induced acrosome exocytosis, two functions essential to fertilize an egg. Unlike other mammals, dogs ovulate a primary oocyte, which reaches metaphase II on Days 4–5 after the luteinizing hormone (LH) surge. We found that only on Day 6 are oocytes consistently able to be fertilized. In vitro fertilization of Day 6 oocytes with sperm capacitated in medium supplemented with magnesium resulted in high rates of embryo development (78.8%, n = 146). Intra-oviductal transfer of nineteen cryopreserved, in vitro fertilization (IVF)-derived embryos resulted in seven live, healthy puppies. Development of IVF enables modern genetic approaches to be applied more efficiently in dogs, and for gamete rescue to conserve endangered canid species. PMID:26650234

In Vitro Modeling of Mechanics in Cancer Metastasis

PubMed Central

2017-01-01

In addition to a multitude of genetic and biochemical alterations, abnormal morphological, structural, and mechanical changes in cells and their extracellular environment are key features of tumor invasion and metastasis. Furthermore, it is now evident that mechanical cues alongside biochemical signals contribute to critical steps of cancer initiation, progression, and spread. Despite its importance, it is very challenging to study mechanics of different steps of metastasis in the clinic or even in animal models. While considerable progress has been made in developing advanced in vitro models for studying genetic and biological aspects of cancer, less attention has been paid to models that can capture both biological and mechanical factors realistically. This is mainly due to lack of appropriate models and measurement tools. After introducing the central role of mechanics in cancer metastasis, we provide an outlook on the emergence of novel in vitro assays and their combination with advanced measurement technologies to probe and recapitulate mechanics in conditions more relevant to the metastatic disease. PMID:29457129

Understanding the Impact of 2D and 3D Fibroblast Cultures on In Vitro Breast Cancer Models

PubMed Central

Sung, Kyung Eun; Su, Xiaojing; Berthier, Erwin; Pehlke, Carolyn; Friedl, Andreas; Beebe, David J.

2013-01-01

The utilization of 3D, physiologically relevant in vitro cancer models to investigate complex interactions between tumor and stroma has been increasing. Prior work has generally focused on the cancer cells and, the role of fibroblast culture conditions on tumor-stromal cell interactions is still largely unknown. Here, we focus on the stroma by comparing functional behaviors of human mammary fibroblasts (HMFs) cultured in 2D and 3D and their effects on the invasive progression of breast cancer cells (MCF10DCIS.com). We identified increased levels of several paracrine factors from HMFs cultured in 3D conditions that drive the invasive transition. Using a microscale co-culture model with improved compartmentalization and sensitivity, we demonstrated that HMFs cultured in 3D intensify the promotion of the invasive progression through the HGF/c-Met interaction. This study highlights the importance of the 3D stromal microenvironment in the development of multiple cell type in vitro cancer models. PMID:24124550

Local energetic frustration affects the dependence of green fluorescent protein folding on the chaperonin GroEL.

PubMed

Bandyopadhyay, Boudhayan; Goldenzweig, Adi; Unger, Tamar; Adato, Orit; Fleishman, Sarel J; Unger, Ron; Horovitz, Amnon

2017-12-15

The GroE chaperonin system in Escherichia coli comprises GroEL and GroES and facilitates ATP-dependent protein folding in vivo and in vitro Proteins with very similar sequences and structures can differ in their dependence on GroEL for efficient folding. One potential but unverified source for GroEL dependence is frustration, wherein not all interactions in the native state are optimized energetically, thereby potentiating slow folding and misfolding. Here, we chose enhanced green fluorescent protein as a model system and subjected it to random mutagenesis, followed by screening for variants whose in vivo folding displays increased or decreased GroEL dependence. We confirmed the altered GroEL dependence of these variants with in vitro folding assays. Strikingly, mutations at positions predicted to be highly frustrated were found to correlate with decreased GroEL dependence. Conversely, mutations at positions with low frustration were found to correlate with increased GroEL dependence. Further support for this finding was obtained by showing that folding of an enhanced green fluorescent protein variant designed computationally to have reduced frustration is indeed less GroEL-dependent. Our results indicate that changes in local frustration also affect partitioning in vivo between spontaneous and chaperonin-mediated folding. Hence, the design of minimally frustrated sequences can reduce chaperonin dependence and improve protein expression levels. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Characterization of Site-Specific Mutations in a Short-Chain-Length/Medium-Chain-Length Polyhydroxyalkanoate Synthase: In Vivo and In Vitro Studies of Enzymatic Activity and Substrate Specificity

PubMed Central

Chuah, Jo-Ann; Tomizawa, Satoshi; Yamada, Miwa; Tsuge, Takeharu; Doi, Yoshiharu

2013-01-01

Saturation point mutagenesis was carried out at position 479 in the polyhydroxyalkanoate (PHA) synthase from Chromobacterium sp. strain USM2 (PhaCCs) with specificities for short-chain-length (SCL) [(R)-3-hydroxybutyrate (3HB) and (R)-3-hydroxyvalerate (3HV)] and medium-chain-length (MCL) [(R)-3-hydroxyhexanoate (3HHx)] monomers in an effort to enhance the specificity of the enzyme for 3HHx. A maximum 4-fold increase in 3HHx incorporation and a 1.6-fold increase in PHA biosynthesis, more than the wild-type synthase, was achieved using selected mutant synthases. These increases were subsequently correlated with improved synthase activity and increased preference of PhaCCs for 3HHx monomers. We found that substitutions with uncharged residues were beneficial, as they resulted in enhanced PHA production and/or 3HHx incorporation. Further analysis led to postulations that the size and geometry of the substrate-binding pocket are determinants of PHA accumulation, 3HHx fraction, and chain length specificity. In vitro activities for polymerization of 3HV and 3HHx monomers were consistent with in vivo substrate specificities. Ultimately, the preference shown by wild-type and mutant synthases for either SCL (C4 and C5) or MCL (C6) substrates substantiates the fundamental classification of PHA synthases. PMID:23584780

Conserved Residues Lys57 and Lys401 of Protein Disulfide Isomerase Maintain an Active Site Conformation for Optimal Activity: Implications for Post-Translational Regulation

PubMed Central

Caba, Cody; Ali Khan, Hyder; Auld, Janeen; Ushioda, Ryo; Araki, Kazutaka; Nagata, Kazuhiro; Mutus, Bulent

2018-01-01

Despite its study since the 1960's, very little is known about the post-translational regulation of the multiple catalytic activities performed by protein disulfide isomerase (PDI), the primary protein folding catalyst of the cell. This work identifies a functional role for the highly conserved CxxC-flanking residues Lys57 and Lys401 of human PDI in vitro. Mutagenesis studies have revealed these residues as modulating the oxidoreductase activity of PDI in a pH-dependent manner. Non-conservative amino acid substitutions resulted in enzyme variants upwards of 7-fold less efficient. This attenuated activity was found to translate into a 2-fold reduction of the rate of electron shuttling between PDI and the intraluminal endoplasmic reticulum oxidase, ERO1α, suggesting a functional significance to oxidative protein folding. In light of this, the possibility of lysine acetylation at residues Lys57 and Lys401 was assessed by in vitro treatment using acetylsalicylic acid (aspirin). A total of 28 acetyllysine residues were identified, including acLys57 and acLys401. The kinetic behavior of the acetylated protein form nearly mimicked that obtained with a K57/401Q double substitution variant providing an indication that acetylation of the active site-flanking lysine residues can act to reversibly modulate PDI activity. PMID:29541639

Combining a CD20 Chimeric Antigen Receptor and an Inducible Caspase 9 Suicide Switch to Improve the Efficacy and Safety of T Cell Adoptive Immunotherapy for Lymphoma

PubMed Central

Budde, Lihua E.; Berger, Carolina; Lin, Yukang; Wang, Jinjuan; Lin, Xubin; Frayo, Shani E.; Brouns, Shaunda A.; Spencer, David M.; Till, Brian G.; Jensen, Michael C.; Riddell, Stanley R.; Press, Oliver W.

2013-01-01

Modification of T cells with chimeric antigen receptors (CAR) has emerged as a promising treatment modality for human malignancies. Integration of co-stimulatory domains into CARs can augment the activation and function of genetically targeted T cells against tumors. However, the potential for insertional mutagenesis and toxicities due to the infused cells have made development of safe methods for removing transferred cells an important consideration. We have genetically modified human T cells with a lentiviral vector to express a CD20-CAR containing both CD28 and CD137 co-stimulatory domains, a “suicide gene” relying on inducible activation of caspase 9 (iC9), and a truncated CD19 selectable marker. Rapid expansion (2000 fold) of the transduced T cells was achieved in 28 days after stimulation with artificial antigen presenting cells. Transduced T cells exhibited effective CD20-specific cytotoxic activity in vitro and in a mouse xenograft tumor model. Activation of the iC9 suicide switch resulted in efficient removal of transduced T cells both in vitro and in vivo. Our work demonstrates the feasibility and promise of this approach for treating CD20+ malignancies in a safe and more efficient manner. A phase I clinical trial using this approach in patients with relapsed indolent B-NHL is planned. PMID:24358223

The plant G box promoter sequence activates transcription in Saccharomyces cerevisiae and is bound in vitro by a yeast activity similar to GBF, the plant G box binding factor.

PubMed Central

Donald, R G; Schindler, U; Batschauer, A; Cashmore, A R

1990-01-01

G box and I box sequences of the Arabidopsis thaliana ribulose-bisphosphate-1,5-carboxylase small subunit (RBCS) promoter are required for expression mediated by the Arabidopsis rbcS-1A promoter in transgenic tobacco plants and are bound in vitro by factors from plant nuclear extracts termed GBF and GA-1, respectively. We show here that a -390 to -60 rbcS-1A promoter fragment containing the G box and two I boxes activates transcription from a truncated iso-1-cytochrome c (CYC1) gene promoter in Saccharomyces cerevisiae. Mutagenesis of either the rbcS-1A G box or both I box sequences eliminated the expression mediated by this fragment. When polymerized, I box oligonucleotides were also capable of enhancing expression from the truncated CYC1 promoter. Single-copy G box sequences from the Arabidopsis rbcS-1A, Arabidopsis Adh and tomato rbcS-3A promoters were more potent activators and were used in mobility shift assays to identify a DNA binding activity in yeast functionally similar to GBF. In methylation interference experiments, the binding specificity of the yeast protein was indistinguishable from that obtained with plant nuclear extracts. Images Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:2161333

Opposite Stereoselectivities of Dirigent Proteins in Arabidopsis and Schizandra Species

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Kye-Won; Moinuddin, Syed G. A.; Atwell, Kathleen M.

2012-08-01

How stereoselective monolignol-derived phenoxy radical-radical coupling reactions are differentially biochemically orchestrated in planta, whereby for example they afford (+)- and (-)-pinoresinols, respectively, is both a fascinating mechanistic and evolutionary question. In earlier work, biochemical control of (+)-pinoresinol formation had been established to be engendered by a (+)-pinoresinol-forming dirigent protein in Forsythia intermedia, whereas the presence of a (-)-pinoresinol-forming dirigent protein was indirectly deduced based on the enantiospecificity of downstream pinoresinol reductases (AtPrRs) in Arabidopsis thaliana root tissue. In this study of 16 putative dirigent protein homologs in Arabidopsis, AtDIR6, AtDIR10, and AtDIR13 were established to be root-specific using a β-glucuronidasemore » reporter gene strategy. Of these three, in vitro analyses established that only recombinant AtDIR6 was a (-)-pinoresinol-forming dirigent protein, whose physiological role was further confirmed using overexpression and RNAi strategies in vivo. Interestingly, its closest homolog, AtDIR5, was also established to be a (-)-pinoresinol-forming dirigent protein based on in vitro biochemical analyses. Both of these were compared in terms of properties with a (+)-pinoresinol-forming dirigent protein from Schizandra chinensis. In this context, sequence analyses, site-directed mutagenesis, and region swapping resulted in identification of putative substrate binding sites/regions and candidate residues controlling distinct stereoselectivities of coupling modes.« less

Altered Gag Polyprotein Cleavage Specificity of Feline Immunodeficiency Virus/Human Immunodeficiency Virus Mutant Proteases as Demonstrated in a Cell-Based Expression System

PubMed Central

Lin, Ying-Chuan; Brik, Ashraf; de Parseval, Aymeric; Tam, Karen; Torbett, Bruce E.; Wong, Chi-Huey; Elder, John H.

2006-01-01

We have used feline immunodeficiency virus (FIV) protease (PR) as a mutational system to study the molecular basis of substrate-inhibitor specificity for lentivirus PRs, with a focus on human immunodeficiency virus type 1 (HIV-1) PR. Our previous mutagenesis studies demonstrated that discrete substitutions in the active site of FIV PR with structurally equivalent residues of HIV-1 PR dramatically altered the specificity of the mutant PRs in in vitro analyses. Here, we have expanded these studies to analyze the specificity changes in each mutant FIV PR expressed in the context of the natural Gag-Pol polyprotein ex vivo. Expression mutants were prepared in which 4 to 12 HIV-1-equivalent substitutions were made in FIV PR, and cleavage of each Gag-Pol polyprotein was then assessed in pseudovirions from transduced cells. The findings demonstrated that, as with in vitro analyses, inhibitor specificities of the mutants showed increased HIV-1 PR character when analyzed against the natural substrate. In addition, all of the mutant PRs still processed the FIV polyprotein but the apparent order of processing was altered relative to that observed with wild-type FIV PR. Given the importance of the order in which Gag-Pol is processed, these findings likely explain the failure to produce infectious FIVs bearing these mutations. PMID:16873240

Furaldehyde substrate specificity and kinetics of Saccharomyces cerevisiae alcohol dehydrogenase 1 variants.

PubMed

Laadan, Boaz; Wallace-Salinas, Valeria; Carlsson, Åsa Janfalk; Almeida, João Rm; Rådström, Peter; Gorwa-Grauslund, Marie F

2014-08-09

A previously discovered mutant of Saccharomyces cerevisiae alcohol dehydrogenase 1 (Adh1p) was shown to enable a unique NADH-dependent reduction of 5-hydroxymethylfurfural (HMF), a well-known inhibitor of yeast fermentation. In the present study, site-directed mutagenesis of both native and mutated ADH1 genes was performed in order to identify the key amino acids involved in this substrate shift, resulting in Adh1p-variants with different substrate specificities. In vitro activities of the Adh1p-variants using two furaldehydes, HMF and furfural, revealed that HMF reduction ability could be acquired after a single amino acid substitution (Y295C). The highest activity, however, was reached with the double mutation S110P Y295C. Kinetic characterization with both aldehydes and the in vivo primary substrate acetaldehyde also enabled to correlate the alterations in substrate affinity with the different amino acid substitutions. We demonstrated the key role of Y295C mutation in HMF reduction by Adh1p. We generated and kinetically characterized a group of protein variants using two furaldehyde compounds of industrial relevance. Also, we showed that there is a threshold after which higher in vitro HMF reduction activities do not correlate any more with faster in vivo rates of HMF conversion, indicating other cell limitations in the conversion of HMF.

Bioassaying Putative RNA-Binding Motifs in a Protein Encoded by a Gene That Influences Courtship and Visually Mediated Behavior in Drosophila: In Vitro Mutagenesis of Nona

PubMed Central

Stanewsky, R.; Fry, T. A.; Reim, I.; Saumweber, H.; Hall, J. C.

1996-01-01

The no-on-transient-A (nonA) gene of Drosophila melanogaster influences vision, courtship song, and viability. The nonA-encoded polypeptide is inferred to bind single-stranded nucleic acids. Although sequence-analysis of NONA implies that it belongs to a special interspecific family of this protein type, it does contain two classical RNA recognition motifs (RRM). Their behavioral significance was assayed by generating transgenic strains that were singly or multiply mutated within the relatively N-terminal motif (RRM1) or within RRM2. Neither class of mutation affected NONA binding to polytene chromosomes. The former mutations led to extremely low viability, accompanied by diminished adult longevities that were much worse than for a nonA-null mutant, implying that faulty interpolypeptide interactions might accompany the effects of the amino-acid substitutions within RRM1. All in vitro-mutated types caused optomotor blindness and an absence of transient spikes in the electroretinogram. Courtship analysis discriminated between the effects of the mutations: the RRM2-mutated type generated song pulses and trains that tended to be mildly mutant. These phenotypic abnormalities reinforce the notion that nonA's ubiquitous expression has its most important consequences in the optic lobes, the thoracic ganglia, or both, depending in part on the nonA allele. PMID:8722780

Combining a CD20 chimeric antigen receptor and an inducible caspase 9 suicide switch to improve the efficacy and safety of T cell adoptive immunotherapy for lymphoma.

PubMed

Budde, Lihua E; Berger, Carolina; Lin, Yukang; Wang, Jinjuan; Lin, Xubin; Frayo, Shani E; Brouns, Shaunda A; Spencer, David M; Till, Brian G; Jensen, Michael C; Riddell, Stanley R; Press, Oliver W

2013-01-01

Modification of T cells with chimeric antigen receptors (CAR) has emerged as a promising treatment modality for human malignancies. Integration of co-stimulatory domains into CARs can augment the activation and function of genetically targeted T cells against tumors. However, the potential for insertional mutagenesis and toxicities due to the infused cells have made development of safe methods for removing transferred cells an important consideration. We have genetically modified human T cells with a lentiviral vector to express a CD20-CAR containing both CD28 and CD137 co-stimulatory domains, a "suicide gene" relying on inducible activation of caspase 9 (iC9), and a truncated CD19 selectable marker. Rapid expansion (2000 fold) of the transduced T cells was achieved in 28 days after stimulation with artificial antigen presenting cells. Transduced T cells exhibited effective CD20-specific cytotoxic activity in vitro and in a mouse xenograft tumor model. Activation of the iC9 suicide switch resulted in efficient removal of transduced T cells both in vitro and in vivo. Our work demonstrates the feasibility and promise of this approach for treating CD20(+) malignancies in a safe and more efficient manner. A phase I clinical trial using this approach in patients with relapsed indolent B-NHL is planned.

Potential late health effects of depleted uranium and tungsten used in armor-piercing munitions: comparison of neoplastic transformation and genotoxicity with the known carcinogen nickel.

PubMed

Miller, Alexandra C; Xu, Jiaquan; Stewart, Michael; Prasanna, Pataje G S; Page, Natalie

2002-02-01

Limited data exist to permit an accurate assessment of risks for carcinogenesis and mutagenesis from embedded fragments or inhaled particulates of depleted uranium (DU). Ongoing studies have been designed to provide information about the carcinogenic potential of DU using in vitro and in vivo assessments of morphological transformation as well as cytogenetic, mutagenic, and oncogenic effects. For comparison, we also examined tungsten alloys used in military projectiles and the known carcinogen nickel. Quantitative and qualitative in vitro transformation studies were done to assess the carcinogenic potential of radiation and chemical hazards. Using a human osteosarcoma cell model, we demonstrated that soluble and insoluble DU compounds can transform cells to the tumorigenic phenotype, as characterized by morphological, biochemical, and oncogenic changes consistent with tumor cell behavior. Tungsten alloys and nickel were also shown to be neoplastic transforming agents, although at a frequency less than that of DU. Sister chromatid exchange, micronuclei, and alkaline filter elution assays showed DU and tungsten alloys were genotoxic. Exposure to a nontoxic, nontransforming dose of DU induced a small but statistically significant increase in the number of dicentrics formed in cells. These results suggest that long-term exposure to DU or tungsten alloys could be critical to the development of neoplastic disease in humans and that additional studies are needed.

Random transposon mutagenesis of the Saccharopolyspora erythraea genome reveals additional genes influencing erythromycin biosynthesis

PubMed Central

Fedashchin, Andrij; Cernota, William H.; Gonzalez, Melissa C.; Leach, Benjamin I.; Kwan, Noelle; Wesley, Roy K.; Weber, J. Mark

2015-01-01

A single cycle of strain improvement was performed in Saccharopolyspora erythraea mutB and 15 genotypes influencing erythromycin production were found. Genotypes generated by transposon mutagenesis appeared in the screen at a frequency of ∼3%. Mutations affecting central metabolism and regulatory genes were found, as well as hydrolases, peptidases, glycosyl transferases and unknown genes. Only one mutant retained high erythromycin production when scaled-up from micro-agar plug fermentations to shake flasks. This mutant had a knockout of the cwh1 gene (SACE_1598), encoding a cell-wall-associated hydrolase. The cwh1 knockout produced visible growth and morphological defects on solid medium. This study demonstrated that random transposon mutagenesis uncovers strain improvement-related genes potentially useful for strain engineering. PMID:26468041

Targeted mutagenesis in sea urchin embryos using TALENs.

PubMed

Hosoi, Sayaka; Sakuma, Tetsushi; Sakamoto, Naoaki; Yamamoto, Takashi

2014-01-01

Genome editing with engineered nucleases such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) has been reported in various animals. We previously described ZFN-mediated targeted mutagenesis and insertion of reporter genes in sea urchin embryos. In this study, we demonstrate that TALENs can induce mutagenesis at specific genomic loci of sea urchin embryos. Injection of TALEN mRNAs targeting the HpEts transcription factor into fertilized eggs resulted in the impairment of skeletogenesis. Sequence analyses of the mutations showed that deletions and/or insertions occurred at the HpEts target site in the TALEN mRNAs-injected embryos. The results suggest that targeted gene disruption using TALENs is feasible in sea urchin embryos. © 2013 The Authors Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists.

Rapid and Programmable Protein Mutagenesis Using Plasmid Recombineering.

PubMed

Higgins, Sean A; Ouonkap, Sorel V Y; Savage, David F

2017-10-20

Comprehensive and programmable protein mutagenesis is critical for understanding structure-function relationships and improving protein function. There is thus a need for robust and unbiased molecular biological approaches for the construction of the requisite comprehensive protein libraries. Here we demonstrate that plasmid recombineering is a simple and robust in vivo method for the generation of protein mutants for both comprehensive library generation as well as programmable targeting of sequence space. Using the fluorescent protein iLOV as a model target, we build a complete mutagenesis library and find it to be specific and comprehensive, detecting 99.8% of our intended mutations. We then develop a thermostability screen and utilize our comprehensive mutation data to rapidly construct a targeted and multiplexed library that identifies significantly improved variants, thus demonstrating rapid protein engineering in a simple protocol.

A highly efficient transposon mutagenesis system for the tomato pathogen Clavibacter michiganensis subsp. michiganensis.

PubMed

Kirchner, O; Gartemann, K H; Zellermann, E M; Eichenlaub, R; Burger, A

2001-11-01

A transposon mutagenesis system for Clavibacter michiganensis subsp. michiganensis was developed based on antibiotic resistance transposons that were derived from the insertion element IS1409 from Arthrobacter sp. strain TM1 NCIB12013. As a prerequisite, the electroporation efficiency was optimized by using unmethylated DNA and treatment of the cells with glycine such that about 5 x 10(6) transformants per microg of DNA were generally obtained. Electroporation of C. michiganensis subsp. michiganensis with a suicide vector carrying transposon Tn1409C resulted in approximately 1 x 10(3) transposon mutants per pg of DNA and thus is suitable for saturation mutagenesis. Analysis of Tn1409C insertion sites suggests a random mode of transposition. Transposition of Tn1409C was also demonstrated for other subspecies of C. michiganensis.

Novel Random Mutagenesis Method for Directed Evolution.

PubMed

Feng, Hong; Wang, Hai-Yan; Zhao, Hong-Yan

2017-01-01

Directed evolution is a powerful strategy for gene mutagenesis, and has been used for protein engineering both in scientific research and in the biotechnology industry. The routine method for directed evolution was developed by Stemmer in 1994 (Stemmer, Proc Natl Acad Sci USA 91, 10747-10751, 1994; Stemmer, Nature 370, 389-391, 1994). Since then, various methods have been introduced, each of which has advantages and limitations depending upon the targeted genes and procedure. In this chapter, a novel alternative directed evolution method which combines mutagenesis PCR with dITP and fragmentation by endonuclease V is described. The kanamycin resistance gene is used as a reporter gene to verify the novel method for directed evolution. This method for directed evolution has been demonstrated to be efficient, reproducible, and easy to manipulate in practice.

Pseudomonas aeruginosa overexpression system of nitric oxide reductase for in vivo and in vitro mutational analyses.

PubMed

Yamagiwa, Raika; Kurahashi, Takuya; Takeda, Mariko; Adachi, Mayuho; Nakamura, Hiro; Arai, Hiroyuki; Shiro, Yoshitsugu; Sawai, Hitomi; Tosha, Takehiko

2018-05-01

Membrane-integrated nitric oxide reductase (NOR) reduces nitric oxide (NO) to nitrous oxide (N 2 O) with protons and electrons. This process is essential for the elimination of the cytotoxic NO that is produced from nitrite (NO 2 - ) during microbial denitrification. A structure-guided mutagenesis of NOR is required to elucidate the mechanism for NOR-catalyzed NO reduction. We have already solved the crystal structure of cytochrome c-dependent NOR (cNOR) from Pseudomonas aeruginosa. In this study, we then constructed its expression system using cNOR-gene deficient and wild-type strains for further functional study. Characterizing the variants of the five conserved Glu residues located around the heme/non-heme iron active center allowed us to establish how the anaerobic growth rate of cNOR-deficient strains expressing cNOR variants correlates with the in vitro enzymatic activity of the variants. Since bacterial strains require active cNOR to eliminate cytotoxic NO and to survive under denitrification conditions, the anaerobic growth rate of a strain with a cNOR variant is a good indicator of NO decomposition capability of the variants and a marker for the screening of functionally important residues without protein purification. Using this in vivo screening system, we examined the residues lining the putative proton transfer pathways for NO reduction in cNOR, and found that the catalytic protons are likely transferred through the Glu57 located at the periplasmic protein surface. The homologous cNOR expression system developed here is an invaluable tool for facile identification of crucial residues in vivo, and for further in vitro functional and structural studies. Copyright © 2018 Elsevier B.V. All rights reserved.

The carboxyl-terminal region of staphylococcal enterotoxin type A is required for a fully active molecule.

PubMed Central

Hufnagle, W O; Tremaine, M T; Betley, M J

1991-01-01

Staphylococcal enterotoxin type A (SEA) gene (sea+) mutations were constructed by exonuclease III digestion or cassette mutagenesis. Five different sea mutations that had 1, 3, 7, 39, and 65 codons deleted from the 3' end of sea+ were identified and confirmed by restriction enzyme and nucleotide sequence analyses. Each of these sea mutations was constructed in Escherichia coli and transferred to Staphylococcus aureus by using the plasmid vector pC194. Culture supernatants from the parent S. aureus strain that lacked an enterotoxin gene (negative controls) and from derivatives that contained either sea+ (positive control) or a sea mutation were examined for in vitro sensitivity to degradation by monkey stomach lavage fluid, the ability to cause emesis when administered by an intragastric route to rhesus monkeys, and the ability to induce T-cell proliferation and by Western immunoblot analysis and a gel double-diffusion assay with polyclonal antibodies prepared against SEA. Altered SEAs corresponding to the predicted sizes were visualized by Western blot analysis of culture supernatants for each of the staphylococcal derivatives that contained a sea mutation. The altered SEA that lacked the C-terminal amino acid residue behaved like SEA in all of the assays performed. The altered SEA that lacked the three C-terminal residues of SEA caused T-cell proliferation but was not emetic; this altered SEA was degraded in vitro by monkey stomach lavage fluid and did not reach in the gel double diffusion assay. Altered SEAs that lacked 7, 39, or 65 carboxyl-terminal residues were degraded by stomach lavage fluid in vitro, did not produce an emetic response, and did not induce T-cell proliferation or form a visible reaction in the gel double-diffusion assay. Images PMID:1903773

The pathogen-related yeast protein Pry1, a member of the CAP protein superfamily, is a fatty acid-binding protein

PubMed Central

Darwiche, Rabih; Mène-Saffrané, Laurent; Gfeller, David; Asojo, Oluwatoyin A.; Schneiter, Roger

2017-01-01

Members of the CAP superfamily (cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins), also known as SCP superfamily (sperm-coating proteins), have been implicated in many physiological processes, including immune defenses, venom toxicity, and sperm maturation. Their mode of action, however, remains poorly understood. Three proteins of the CAP superfamily, Pry1, -2, and -3 (pathogen related in yeast), are encoded in the Saccharomyces cerevisiae genome. We have shown previously that Pry1 binds cholesterol in vitro and that Pry function is required for sterol secretion in yeast cells, indicating that members of this superfamily may generally bind sterols or related small hydrophobic compounds. On the other hand, tablysin-15, a CAP protein from the horsefly Tabanus yao, has been shown to bind leukotrienes and free fatty acids in vitro. Therefore, here we assessed whether the yeast Pry1 protein binds fatty acids. Computational modeling and site-directed mutagenesis indicated that the mode of fatty acid binding is conserved between tablysin-15 and Pry1. Pry1 bound fatty acids with micromolar affinity in vitro, and its function was essential for fatty acid export in cells lacking the acyl-CoA synthetases Faa1 and Faa4. Fatty acid binding of Pry1 is independent of its capacity to bind sterols, and the two sterol- and fatty acid-binding sites are nonoverlapping. These results indicate that some CAP family members, such as Pry1, can bind different lipids, particularly sterols and fatty acids, at distinct binding sites, suggesting that the CAP domain may serve as a stable, secreted protein domain that can accommodate multiple ligand-binding sites. PMID:28365570

Variations in seed protein content of cotton (Gossypium hirsutum L.) mutant lines by in vivo and in vitro mutagenesis.

PubMed

Muthusamy, Annamalai; Jayabalan, Narayanasamy

2013-01-01

The present work describes the influence of gamma irradiation (GR), ethyl methane sulphonate (EMS) and sodium azide (SA) treatment on yield and protein content of selected mutant lines of cotton. Seeds of MCU 5 and MCU 11 were exposed to gamma rays (GR), ethyl methane sulphonate (EMS) and sodium azide (SA). Lower dose of gamma irradiation (100-500 Gy), 10-50 mM EMS and SA at lower concentration effectively influences in improving the yield and protein content. Significant increase in yield (258.9 g plant(-1)) and protein content (18.63 mg g(-1) d. wt.) as compared to parental lines was noted in M2 generations. During the subsequent field trials, number of mutant lines varied morphologically in terms of yield as well as biochemical characters such as protein. The selected mutant lines were bred true to their characters in M3 and M4 generations. The significant increase in protein content and profiles of the mutant lines with range of 10.21-18.63 mg g(-1). The SDS-PAGE analysis of mutant lines revealed 9 distinct bands of different intensities with range of 26-81 kDa. The difference in intensity of bands was more (41, 50 and 58 kDa) in the mutant lines obtained from in vitro mutation than in vivo mutation. Significance of such stimulation in protein content correlated with yielding ability of the mutant lines of cotton in terms of seed weight per plant. The results confirm that in cotton it is possible to enhance the both yield and biochemical characters by in vivo and in vitro mutagenic treatments.

Trends in the development of microfluidic cell biochips for in vitro hepatotoxicity.

PubMed

Baudoin, Régis; Corlu, Anne; Griscom, Laurent; Legallais, Cécile; Leclerc, Eric

2007-06-01

Current developments in the technological fields of liver tissue engineering, bioengineering, biomechanics, microfabrication and microfluidics have lead to highly complex and pertinent new tools called "cell biochips" for in vitro toxicology. The purpose of "cell biochips" is to mimic organ tissues in vitro in order to partially reduce the amount of in vivo testing. These "cell biochips" consist of microchambers containing engineered tissue and living cell cultures interconnected by a microfluidic network, which allows the control of microfluidic flows for dynamic cultures, by continuous feeding of nutrients to cultured cells and waste removal. Cell biochips also allow the control of physiological contact times of diluted molecules with the tissues and cells, for rapid testing of sample preparations or specific addressing. Cell biochips can be situated between in vitro and in vivo testing. These types of systems can enhance functionality of cells by mimicking the tissue architecture complexities when compared to in vitro analysis but at the same time present a more rapid and simple process when compared to in vivo testing procedures. In this paper, we first introduce the concepts of microfluidic and biochip systems based on recent progress in microfabrication techniques used to mimic liver tissue in vitro. This includes progress and understanding in biomaterials science (cell culture substrate), biomechanics (dynamic cultures conditions) and biology (tissue engineering). The development of new "cell biochips" for chronic toxicology analysis of engineered tissues can be achieved through the combination of these research domains. Combining these advanced research domains, we then present "cell biochips" that allow liver chronic toxicity analysis in vitro on engineered tissues. An extension of the "cell biochip" idea has also allowed "organ interactions on chip", which can be considered as a first step towards the replacement of animal testing using a combined liver/lung organ model.

Variants in EXOSC9 Disrupt the RNA Exosome and Result in Cerebellar Atrophy with Spinal Motor Neuronopathy.

PubMed

Burns, David T; Donkervoort, Sandra; Müller, Juliane S; Knierim, Ellen; Bharucha-Goebel, Diana; Faqeih, Eissa Ali; Bell, Stephanie K; AlFaifi, Abdullah Y; Monies, Dorota; Millan, Francisca; Retterer, Kyle; Dyack, Sarah; MacKay, Sara; Morales-Gonzalez, Susanne; Giunta, Michele; Munro, Benjamin; Hudson, Gavin; Scavina, Mena; Baker, Laura; Massini, Tara C; Lek, Monkol; Hu, Ying; Ezzo, Daniel; AlKuraya, Fowzan S; Kang, Peter B; Griffin, Helen; Foley, A Reghan; Schuelke, Markus; Horvath, Rita; Bönnemann, Carsten G

2018-05-03

The exosome is a conserved multi-protein complex that is essential for correct RNA processing. Recessive variants in exosome components EXOSC3, EXOSC8, and RBM7 cause various constellations of pontocerebellar hypoplasia (PCH), spinal muscular atrophy (SMA), and central nervous system demyelination. Here, we report on four unrelated affected individuals with recessive variants in EXOSC9 and the effect of the variants on the function of the RNA exosome in vitro in affected individuals' fibroblasts and skeletal muscle and in vivo in zebrafish. The clinical presentation was severe, early-onset, progressive SMA-like motor neuronopathy, cerebellar atrophy, and in one affected individual, congenital fractures of the long bones. Three affected individuals of different ethnicity carried the homozygous c.41T>C (p.Leu14Pro) variant, whereas one affected individual was compound heterozygous for c.41T>C (p.Leu14Pro) and c.481C>T (p.Arg161 ∗ ). We detected reduced EXOSC9 in fibroblasts and skeletal muscle and observed a reduction of the whole multi-subunit exosome complex on blue-native polyacrylamide gel electrophoresis. RNA sequencing of fibroblasts and skeletal muscle detected significant >2-fold changes in genes involved in neuronal development and cerebellar and motor neuron degeneration, demonstrating the widespread effect of the variants. Morpholino oligonucleotide knockdown and CRISPR/Cas9-mediated mutagenesis of exosc9 in zebrafish recapitulated aspects of the human phenotype, as they have in other zebrafish models of exosomal disease. Specifically, portions of the cerebellum and hindbrain were absent, and motor neurons failed to develop and migrate properly. In summary, we show that variants in EXOSC9 result in a neurological syndrome combining cerebellar atrophy and spinal motoneuronopathy, thus expanding the list of human exosomopathies. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

IκB kinaseα/β control biliary homeostasis and hepatocarcinogenesis in mice by phosphorylating the cell-death mediator receptor-interacting protein kinase 1.

PubMed

Koppe, Christiane; Verheugd, Patricia; Gautheron, Jérémie; Reisinger, Florian; Kreggenwinkel, Karina; Roderburg, Christoph; Quagliata, Luca; Terracciano, Luigi; Gassler, Nikolaus; Tolba, René H; Boege, Yannick; Weber, Achim; Karin, Michael; Luedde, Mark; Neumann, Ulf P; Weiskirchen, Ralf; Tacke, Frank; Vucur, Mihael; Trautwein, Christian; Lüscher, Bernhard; Preisinger, Christian; Heikenwalder, Mathias; Luedde, Tom

2016-10-01

The IκB-Kinase (IKK) complex-consisting of the catalytic subunits, IKKα and IKKβ, as well as the regulatory subunit, NEMO-mediates activation of the nuclear factor κB (NF-κB) pathway, but previous studies suggested the existence of NF-κB-independent functions of IKK subunits with potential impact on liver physiology and disease. Programmed cell death is a crucial factor in the progression of liver diseases, and receptor-interacting kinases (RIPKs) exerts strategic control over multiple pathways involved in regulating novel programmed cell-death pathways and inflammation. We hypothesized that RIPKs might be unrecognized targets of the catalytic IKK-complex subunits, thereby regulating hepatocarcinogenesis and cholestasis. In this present study, mice with specific genetic inhibition of catalytic IKK activity in liver parenchymal cells (LPCs; IKKα/β(LPC-KO) ) were intercrossed with RIPK1(LPC-KO) or RIPK3(-/-) mice to examine whether RIPK1 or RIPK3 might be downstream targets of IKKs. Moreover, we performed in vivo phospho-proteome analyses and in vitro kinase assays, mass spectrometry, and mutagenesis experiments. These analyses revealed that IKKα and IKKβ-in addition to their known function in NF-κB activation-directly phosphorylate RIPK1 at distinct regions of the protein, thereby regulating cell viability. Loss of this IKKα/β-dependent RIPK1 phosphorylation in LPCs inhibits compensatory proliferation of hepatocytes and intrahepatic biliary cells, thus impeding HCC development, but promoting biliary cell paucity and lethal cholestasis. IKK-complex subunits transmit a previously unrecognized signal through RIPK1, which is fundamental for the long-term consequences of chronic hepatic inflammation and might have potential implications for future pharmacological strategies against cholestatic liver disease and cancer. (Hepatology 2016;64:1217-1231). © 2016 by the American Association for the Study of Liver Diseases.

Metastatic melanoma moves on: translational science in the era of personalized medicine.

PubMed

Levesque, Mitchell P; Cheng, Phil F; Raaijmakers, Marieke I G; Saltari, Annalisa; Dummer, Reinhard

2017-03-01

Progress in understanding and treating metastatic melanoma is the result of decades of basic and translational research as well as the development of better in vitro tools for modeling the disease. Here, we review the latest therapeutic options for metastatic melanoma and the known genetic and non-genetic mechanisms of resistance to these therapies, as well as the in vitro toolbox that has provided the greatest insights into melanoma progression. These include next-generation sequencing technologies and more complex 2D and 3D cell culture models to functionally test the data generated by genomics approaches. The combination of hypothesis generating and hypothesis testing paradigms reviewed here will be the foundation for the next phase of metastatic melanoma therapies in the coming years.

Interleukin-30: A novel microenvironmental hallmark of prostate cancer progression.

PubMed

Di Carlo, Emma

2014-01-01

Metastatic prostate cancer is a leading cause of cancer-related death in men worldwide. We have recently discovered that IL-30 shapes the microenvironment of prostate cancer and tumor-draining lymph nodes to favor tumor progression. IL-30 supports tumor growth in vitro, and IL-30 expression in prostate cancer patients is associated with high tumor grade and metastatic stage of disease. Thus, IL-30 may constitute a valuable target for modern therapeutic approaches to hamper prostate cancer progression.

The Role of ERK1/2 in the Progression of Anti-Androgen Resistance of MtDNA Deficient Prostate Cancer

DTIC Science & Technology

2012-03-01

proto-oncogenic pathway, it is plausible that the mitoGPS is a ubiquitous (patho) physiological response to the etiology and/or progression of a broad...the mitochondrion as a direct physiological source of hypoxia in an in vitro system. Our results demonstrate that the reduction of the mitochondrial...ubiquitous (patho) physiological response to the etiology and/or progression of a broad spectrum of human diseases that are attributed to respiratory

Chemical mutagenesis in laboratory mammals. A bibliography on the effects of chemicals on germ cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Von Halle, E.S.

1973-09-01

A list of references is presented on chemical mutagenesis in laboratory mammals. The references relate primarily to chemical effects on germ cells. Only references to the use of chemicals or chemicals and radiation are included. The publication includes a citation index, agent index, chemical abstracts registry number index, organism index, KWIC index, author index, and first author index. (ERB)

PCR-mediated site-directed mutagenesis.

PubMed

Carey, Michael F; Peterson, Craig L; Smale, Stephen T

2013-08-01

Unlike traditional site-directed mutagenesis, this protocol requires only a single PCR step using full plasmid amplification to generate point mutants. The method can introduce small mutations into promoter sites and is even better suited for introducing single or double mutations into proteins. It is elegant in its simplicity and can be applied quite easily in any laboratory using standard protein expression vectors and commercially available reagents.

Targeted Mutagenesis of Guinea Pig Cytomegalovirus Using CRISPR/Cas9-Mediated Gene Editing.

PubMed

Bierle, Craig J; Anderholm, Kaitlyn M; Wang, Jian Ben; McVoy, Michael A; Schleiss, Mark R

2016-08-01

The cytomegaloviruses (CMVs) are among the most genetically complex mammalian viruses, with viral genomes that often exceed 230 kbp. Manipulation of cytomegalovirus genomes is largely performed using infectious bacterial artificial chromosomes (BACs), which necessitates the maintenance of the viral genome in Escherichia coli and successful reconstitution of virus from permissive cells after transfection of the BAC. Here we describe an alternative strategy for the mutagenesis of guinea pig cytomegalovirus that utilizes clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome editing to introduce targeted mutations to the viral genome. Transient transfection and drug selection were used to restrict lytic replication of guinea pig cytomegalovirus to cells that express Cas9 and virus-specific guide RNA. The result was highly efficient editing of the viral genome that introduced targeted insertion or deletion mutations to nonessential viral genes. Cotransfection of multiple virus-specific guide RNAs or a homology repair template was used for targeted, markerless deletions of viral sequence or to introduce exogenous sequence by homology-driven repair. As CRISPR/Cas9 mutagenesis occurs directly in infected cells, this methodology avoids selective pressures that may occur during propagation of the viral genome in bacteria and may facilitate genetic manipulation of low-passage or clinical CMV isolates. The cytomegalovirus genome is complex, and viral adaptations to cell culture have complicated the study of infection in vivo Recombineering of viral bacterial artificial chromosomes enabled the study of recombinant cytomegaloviruses. Here we report the development of an alternative approach using CRISPR/Cas9-based mutagenesis in guinea pig cytomegalovirus, a small-animal model of congenital cytomegalovirus disease. CRISPR/Cas9 mutagenesis can introduce the same types of mutations to the viral genome as bacterial artificial chromosome recombineering but does so directly in virus-infected cells. CRISPR/Cas9 mutagenesis is not dependent on a bacterial intermediate, and defined viral mutants can be recovered after a limited number of viral genome replications, minimizing the risk of spontaneous mutation. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Structure-Based and Random Mutagenesis Approaches Increase the Organophosphate-Degrading Activity of a Phosphotriesterase Homologue from Deinococcus radiodurans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hawwa, Renda; Larsen, Sonia D.; Ratia, Kiira

2010-11-09

An enzyme from the amidohydrolase family from Deinococcus radiodurans (Dr-OPH) with homology to phosphotriesterase has been shown to exhibit activity against both organophosphate (OP) and lactone compounds. We have characterized the physical properties of Dr-OPH and have found it to be a highly thermostable enzyme, remaining active after 3 h of incubation at 60 C and withstanding incubation at temperatures up to 70 C. In addition, it can withstand concentrations of at least 200 mg/mL. These properties make Dr-OPH a promising candidate for development in commercial applications. However, compared to the most widely studied OP-degrading enzyme, that from Pseudomonas diminuta,more » Dr-OPH has low hydrolytic activity against certain OP substrates. Therefore, we sought to improve the OP-degrading activity of Dr-OPH, specifically toward the pesticides ethyl and methyl paraoxon, using structure-based and random approaches. Site-directed mutagenesis, random mutagenesis, and site-saturation mutagenesis were utilized to increase the OP-degrading activity of Dr-OPH. Out of a screen of more than 30,000 potential mutants, a total of 26 mutant enzymes were purified and characterized kinetically. Crystal structures of w.t. Dr-OPH, of Dr-OPH in complex with a product analog, and of 7 mutant enzymes were determined to resolutions between 1.7 and 2.4 {angstrom}. Information from these structures directed the design and production of 4 additional mutants for analysis. In total, our mutagenesis efforts improved the catalytic activity of Dr-OPH toward ethyl and methyl paraoxon by 126- and 322-fold and raised the specificity for these two substrates by 557- and 183-fold, respectively. Our work highlights the importance of an iterative approach to mutagenesis, proving that large rate enhancements are achieved when mutations are made in already active mutants. In addition, the relationship between the kinetic parameters and the introduced mutations has allowed us to hypothesize on those factors most important for maintaining the structure and function of the enzyme.« less

New approach for fish breeding by chemical mutagenesis: establishment of TILLING method in fugu (Takifugu rubripes) with ENU mutagenesis.

PubMed

Kuroyanagi, Miwa; Katayama, Takashi; Imai, Tadashi; Yamamoto, Yoshihisa; Chisada, Shin-ichi; Yoshiura, Yasutoshi; Ushijima, Tomokazu; Matsushita, Tomonao; Fujita, Masashi; Nozawa, Aoi; Suzuki, Yuzuru; Kikuchi, Kiyoshi; Okamoto, Hiroyuki

2013-11-13

In fish breeding, it is essential to discover and generate fish exhibiting an effective phenotype for the aquaculture industry, but screening for natural mutants by only depending on natural spontaneous mutations is limited. Presently, reverse genetics has become an important tool to generate mutants, which exhibit the phenotype caused by inactivation of a gene. TILLING (Targeting Induced Local Lesions IN Genomes) is a reverse genetics strategy that combines random chemical mutagenesis with high-throughput discovery technologies for screening the induced mutations in target genes. Although the chemical mutagenesis has been used widely in a variety of model species and also genetic breeding of microorganisms and crops, the application of the mutagenesis in fish breeding has been only rarely reported. In this study, we developed the TILLING method in fugu with ENU mutagenesis and high-resolution melting (HRM) analysis to detect base pair changes in target sequences. Fugu males were treated 3 times at weekly intervals with various ENU concentrations, and then the collected sperm after the treatment was used to fertilize normal female for generating the mutagenized population (F1). The fertilization and the hatching ratios were similar to those of the control and did not reveal a dose dependency of ENU. Genomic DNA from the harvested F1 offspring was used for the HRM analysis. To obtain a fish exhibiting a useful phenotype (e.g. high meat production and rapid growth), fugu myostatin (Mstn) gene was examined as a target gene, because it has been clarified that the mstn deficient medaka exhibited double-muscle phenotype in common with MSTN knockout mice and bovine MSTN mutant. As a result, ten types of ENU-induced mutations were identified including a nonsense mutation in the investigated region with HRM analysis. In addition, the average mutation frequency in fugu Mstn gene was 1 mutant per 297 kb, which is similar to values calculated for zebrafish and medaka TILLING libraries. These results demonstrate that the TILLING method in fugu was established. We anticipate that this TILLING approach can be used to generate a wide range of mutant alleles, and be applicable to many farmed fish that can be chemically mutagenized.

In vivo testing of chemicals that undergo substantial biotransformation: An opportunity to advance in vitro-in vivo metabolism extrapolation procedures for fish

EPA Science Inventory

This is one of several keynote presentations given at the beginning of a one-day workshop on in vivo testing with fish. The purpose of this talk is to describe recent progress on using in vitro data to predict metabolism impacts on chemical accumulation by fish. The focus of th...

The Cbf5-Nop10 Complex is a Molecular Bracket that Organizes Box H/ACA RNPs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hamma, Tomoko; Reichow, Steve L.; Varani, Gabriele

2005-12-01

Box H/ACA ribonucleoprotein particles (RNPs) catalyze RNA pseudouridylation and direct processing of ribosomal RNA, and are essential architectural components of vertebrate telomerases. H/ACA RNPs comprise four proteins and a multihelical RNA. Two proteins, Cbf5 and Nop10, suffice for basal enzymatic activity in an archaeal in vitro system. We now report their cocrystal structure at 1.95-A resolution. We find that archaeal Cbf5 can assemble with yeast Nop10 and with human telomerase RNA, consistent with the high sequence identity of the RNP componenets between archaea and eukarya. Thus, the Cbf5-Nop10 architecture is phylogenetically conserved. The structure shows how Nop10 buttresses the activemore » site of Cbf5, and it reveals two basic troughs that bidirectionally extend the active site cleft. Mutagenesis results implicate an adjacent basic patch in RNA binding. This tripartite RNA-binding surface may function as a molecular bracket that organizes the multihelical H/ACA and telomerase RNAs.« less

Development of a Humanized Monoclonal Antibody with Therapeutic Potential against West Nile Virus

PubMed Central

Oliphant, Theodore; Engle, Michael; Nybakken, Grant E.; Doane, Chris; Johnson, Syd; Huang, Ling; Gorlatov, Sergey; Mehlhop, Erin; Marri, Anantha; Chung, Kyung Min; Ebel, Gregory D.; Kramer, Laura D.; Fremont, Daved H.; Diamond, Michael S.

2006-01-01

Neutralization of West Nile virus (WNV) in vivo correlates with the development of an antibody response against the viral envelope (E) protein. Using random mutagenesis and yeast surface display, we defined individual contact residues of 14 newly generated mAbs against domain III of the WNV E protein. MAbs that strongly neutralized WNV localized to a surface patch on the lateral face of domain III. Convalescent antibodies from human patients who had recovered from WNV infection also detected this epitope. One mAb, E16, neutralized 10 different strains in vitro, and demonstrated therapeutic efficacy in mice, even when administered as a single dose 5 d after infection. A humanized version of E16 was generated that retained antigen specificity, avidity, and neutralizing activity. In post-exposure therapeutic trials in mice, a single dose of humanized E16 protected mice against WNV-induced mortality, and thus, may be a viable treatment option against WNV infection in humans. PMID:15852016

Development of an Efficient Genome Editing Method by CRISPR/Cas9 in a Fish Cell Line.

PubMed

Dehler, Carola E; Boudinot, Pierre; Martin, Samuel A M; Collet, Bertrand

2016-08-01

CRISPR/Cas9 system has been used widely in animals and plants to direct mutagenesis. To date, no such method exists for fish somatic cell lines. We describe an efficient procedure for genome editing in the Chinook salmon Oncorhynchus tshawytscha CHSE. This cell line was genetically modified to firstly overexpress a monomeric form of EGFP (cell line CHSE-E Geneticin resistant) and additionally to overexpress nCas9n, a nuclear version of Cas9 (cell line CHSE-EC, Hygromycin and Geneticin resistant). A pre-validated sgRNA was produced in vitro and used to transfect CHSE-EC cells. The EGFP gene was disrupted in 34.6 % of cells, as estimated by FACS and microscopy. The targeted locus was characterised by PCR amplification, cloning and sequencing of PCR products; inactivation of the EGFP gene by deletions in the expected site was validated in 25 % of clones. This method opens perspectives for functional genomic studies compatible with high-throughput screening.

Msp1 Is a Membrane Protein Dislocase for Tail-Anchored Proteins.

PubMed

Wohlever, Matthew L; Mateja, Agnieszka; McGilvray, Philip T; Day, Kasey J; Keenan, Robert J

2017-07-20

Mislocalized tail-anchored (TA) proteins of the outer mitochondrial membrane are cleared by a newly identified quality control pathway involving the conserved eukaryotic protein Msp1 (ATAD1 in humans). Msp1 is a transmembrane AAA-ATPase, but its role in TA protein clearance is not known. Here, using purified components reconstituted into proteoliposomes, we show that Msp1 is both necessary and sufficient to drive the ATP-dependent extraction of TA proteins from the membrane. A crystal structure of the Msp1 cytosolic region modeled into a ring hexamer suggests that active Msp1 contains a conserved membrane-facing surface adjacent to a central pore. Structure-guided mutagenesis of the pore residues shows that they are critical for TA protein extraction in vitro and for functional complementation of an msp1 deletion in yeast. Together, these data provide a molecular framework for Msp1-dependent extraction of mislocalized TA proteins from the outer mitochondrial membrane. Copyright © 2017 Elsevier Inc. All rights reserved.

cDNA cloning, expression, and mutagenesis of a PR-10 protein SPE-16 from the seeds of Pachyrrhizus erosus.

PubMed

Wu, Fang; Yan, Ming; Li, Yikun; Chang, Shaojie; Song, Xiaomin; Zhou, Zhaocai; Gong, Weimin

2003-12-19

SPE-16 is a new 16kDa protein that has been purified from the seeds of Pachyrrhizus erosus. It's N-terminal amino acid sequence shows significant sequence homology to pathogenesis-related class 10 proteins. cDNA encoding 150 amino acids was cloned by RT-PCR and the gene sequence proved SPE-16 to be a new member of PR-10 family. The cDNA was cloned into pET15b plasmid and expressed in Escherichia coli. The bacterially expressed SPE-16 also demonstrated ribonuclease-like activity in vitro. Site-directed mutation of three conserved amino acids E95A, E147A, Y150A, and a P-loop truncated form were constructed and their different effects on ribonuclease activities were observed. SPE-16 is also able to bind the fluorescent probe 8-anilino-1-naphthalenesulfonate (ANS) in the native state. The ANS anion is a much-utilized "hydrophobic probe" for proteins. This binding activity indicated another biological function of SPE-16.

Dual Functions of Lip6 and Its Regulation of Lipid Metabolism in the Oleaginous Fungus Mucor circinelloides.

PubMed

Zan, Xinyi; Tang, Xin; Chu, Linfang; Song, Yuanda

2018-03-21

Although multiple roles of lipases have been reported in yeasts and microalgae, the functions of lipases have not been studied in oleaginous filamentous fungi. Lipase Lip6 has been reported in the oleaginous filamentous fungus Mucor circinelloides with the consensus lipase motif GXSXG and the typical acyltransferase motif of H-(X) 4 -D. To demonstrate that Lip6 might play dual roles as a lipase and an acyltransferase, we performed site-directed mutagenesis in the lipase motif and the acyltransferase motif of Lip6. Mutation in the lipase motif increased cell biomass by 12%-18% and promoted lipid accumulation by 9%-24%, while mutation in the acyltransferase motif induced lipid degradation. In vitro, purified Lip6 had a slight lipase activity but had a stronger phospholipid:DAG acyltransferase activity. Enzyme activity assays in vivo and phospholipid synthesis pathway analysis suggested that phosphatidyl serine and phosphatidyl ethanolamine can be the supplier of a fatty acyl moiety to form TAG in M. circinelloides.

Deciphering the structural framework of glycine receptor anchoring by gephyrin

PubMed Central

Kim, Eun Young; Schrader, Nils; Smolinsky, Birthe; Bedet, Cécile; Vannier, Christian; Schwarz, Günter; Schindelin, Hermann

2006-01-01

Glycine is the major inhibitory neurotransmitter in the spinal cord and brain stem. Gephyrin is required to achieve a high concentration of glycine receptors (GlyRs) in the postsynaptic membrane, which is crucial for efficient glycinergic signal transduction. The interaction between gephyrin and the GlyR involves the E-domain of gephyrin and a cytoplasmic loop located between transmembrane segments three and four of the GlyR β subunit. Here, we present crystal structures of the gephyrin E-domain with and without the GlyR β-loop at 2.4 and 2.7 Å resolutions, respectively. The GlyR β-loop is bound in a symmetric ‘key and lock' fashion to each E-domain monomer in a pocket adjacent to the dimer interface. Structure-guided mutagenesis followed by in vitro binding and in vivo colocalization assays demonstrate that a hydrophobic interaction formed by Phe 330 of gephyrin and Phe 398 and Ile 400 of the GlyR β-loop is crucial for binding. PMID:16511563

Lysine acetylation sites in bovine foamy virus transactivator BTas are important for its DNA binding activity.

PubMed

Chang, Rui; Tan, Juan; Xu, Fengwen; Han, Hongqi; Geng, Yunqi; Li, Yue; Qiao, Wentao

2011-09-15

Cellular acetylation signaling is important for viral gene regulation, particularly during the transactivation of retroviruses. The regulatory protein of bovine foamy virus (BFV), BTas, is a transactivator that augments viral gene transcription from both the long terminal repeat (LTR) promoter and the internal promoter (IP). In this study, we report that the histone acetyltransferase (HAT), p300, specifically acetylates BTas both in vivo and in vitro. Further studies demonstrated that BTas acetylation markedly enhances its transactivation activity. Mutagenesis analysis identified three lysines at positions 66, 109 and 110 in BTas that are acetylated by p300. The K110R mutant lost its binding to BFV promoter as well as its ability to activate BFV promoter. The acetylation of K66 and K109 may contribute to increased BTas binding ability. These results suggest that the p300-acetylated lysines of BTas are important for transactivation of BFV promoters and therefore have an important role in BFV replication. Copyright © 2011 Elsevier Inc. All rights reserved.

Molecular Imaging of Human Embryonic Stem Cells Stably Expressing Human PET Reporter Genes After Zinc Finger Nuclease-Mediated Genome Editing.

PubMed

Wolfs, Esther; Holvoet, Bryan; Ordovas, Laura; Breuls, Natacha; Helsen, Nicky; Schönberger, Matthias; Raitano, Susanna; Struys, Tom; Vanbilloen, Bert; Casteels, Cindy; Sampaolesi, Maurilio; Van Laere, Koen; Lambrichts, Ivo; Verfaillie, Catherine M; Deroose, Christophe M

2017-10-01

Molecular imaging is indispensable for determining the fate and persistence of engrafted stem cells. Standard strategies for transgene induction involve the use of viral vectors prone to silencing and insertional mutagenesis or the use of nonhuman genes. Methods: We used zinc finger nucleases to induce stable expression of human imaging reporter genes into the safe-harbor locus adeno-associated virus integration site 1 in human embryonic stem cells. Plasmids were generated carrying reporter genes for fluorescence, bioluminescence imaging, and human PET reporter genes. Results: In vitro assays confirmed their functionality, and embryonic stem cells retained differentiation capacity. Teratoma formation assays were performed, and tumors were imaged over time with PET and bioluminescence imaging. Conclusion: This study demonstrates the application of genome editing for targeted integration of human imaging reporter genes in human embryonic stem cells for long-term molecular imaging. © 2017 by the Society of Nuclear Medicine and Molecular Imaging.

Identification of striated muscle activator of Rho signaling (STARS) as a novel calmodulin target by a newly developed genome-wide screen.

PubMed

Furuya, Yusui; Denda, Miwako; Sakane, Kyohei; Ogusu, Tomoko; Takahashi, Sumio; Magari, Masaki; Kanayama, Naoki; Morishita, Ryo; Tokumitsu, Hiroshi

2016-07-01

To search for novel target(s) of the Ca(2+)-signaling transducer, calmodulin (CaM), we performed a newly developed genome-wide CaM interaction screening of 19,676 GST-fused proteins expressed in human. We identified striated muscle activator of Rho signaling (STARS) as a novel CaM target and characterized its CaM binding ability and found that the Ca(2+)/CaM complex interacted stoichiometrically with the N-terminal region (Ala13-Gln35) of STARS in vitro as well as in living cells. Mutagenesis studies identified Ile20 and Trp33 as the essential hydrophobic residues in CaM anchoring. Furthermore, the CaM binding deficient mutant (Ile20Ala, Trp33Ala) of STARS further enhanced its stimulatory effect on SRF-dependent transcriptional activation. These results suggest a connection between Ca(2+)-signaling via excitation-contraction coupling and the regulation of STARS-mediated gene expression in muscles. Copyright © 2016 Elsevier Ltd. All rights reserved.

Genome shuffling of Lactobacillus plantarum C88 improves adhesion.

PubMed

Zhao, Yujuan; Duan, Cuicui; Gao, Lei; Yu, Xue; Niu, Chunhua; Li, Shengyu

2017-01-01

Genome shuffling is an important method for rapid improvement in microbial strains for desired phenotypes. In this study, ultraviolet irradiation and nitrosoguanidine were used as mutagens to enhance the adhesion of the wild-type Lactobacillus plantarum C88. Four strains with better property were screened after mutagenesis to develop a library of parent strains for three rounds of genome shuffling. Fusants F3-1, F3-2, F3-3, and F3-4 were screened as the improved strains. The in vivo and in vitro tests results indicated that the population after three rounds of genome shuffling exhibited improved adhesive property. Random Amplified Polymorphic DNA results showed significant differences between the parent strain and recombinant strains at DNA level. These results suggest that the adhesive property of L. plantarum C88 can be significantly improved by genome shuffling. Improvement in the adhesive property of bacterial cells by genome shuffling enhances the colonization of probiotic strains which further benefits to exist probiotic function.

Efficient, footprint-free human iPSC genome editing by consolidation of Cas9/CRISPR and piggyBac technologies.

PubMed

Wang, Gang; Yang, Luhan; Grishin, Dennis; Rios, Xavier; Ye, Lillian Y; Hu, Yong; Li, Kai; Zhang, Donghui; Church, George M; Pu, William T

2017-01-01

Genome editing of human induced pluripotent stem cells (hiPSCs) offers unprecedented opportunities for in vitro disease modeling and personalized cell replacement therapy. The introduction of Cas9-directed genome editing has expanded adoption of this approach. However, marker-free genome editing using standard protocols remains inefficient, yielding desired targeted alleles at a rate of ∼1-5%. We developed a protocol based on a doxycycline-inducible Cas9 transgene carried on a piggyBac transposon to enable robust and highly efficient Cas9-directed genome editing, so that a parental line can be expeditiously engineered to harbor many separate mutations. Treatment with doxycycline and transfection with guide RNA (gRNA), donor DNA and piggyBac transposase resulted in efficient, targeted genome editing and concurrent scarless transgene excision. Using this approach, in 7 weeks it is possible to efficiently obtain genome-edited clones with minimal off-target mutagenesis and with indel mutation frequencies of 40-50% and homology-directed repair (HDR) frequencies of 10-20%.

The Prp19 WD40 Domain Contains a Conserved Protein Interaction Region Essential for its Function

PubMed Central

Vander Kooi, Craig W.; Ren, Liping; Xu, Ping; Ohi, Melanie D.; Gould, Kathleen L.; Chazin, Walter J.

2010-01-01

Summary Prp19 is a member of the WD40-repeat family of E3 ubiquitin ligases and a conserved eukaryotic RNA splicing factor essential for activation and stabilization of the spliceosome. To understand the role of the WD40 repeat domain of Prp19 we have determined its structure using X-ray crystallography. The domain has a distorted seven bladed WD40 architecture with significant asymmetry due to irregular packing of blades one and seven into the core of the WD40 domain. Structure-based mutagenesis identified a highly conserved surface centered around blade five that is required for the physical interaction between Prp19 and Cwc2, another essential splicing factor. This region is found to be required for Prp19 function and yeast viability. Experiments in vitro and in vivo demonstrate that two molecules of Cwc2 bind to the Prp19 tetramer. These coupled structural and functional studies provide a model for the functional architecture of Prp19. PMID:20462492

Nutraceutical potential of hemp (Cannabis sativa L.) seeds and sprouts.

PubMed

Frassinetti, Stefania; Moccia, Eleonora; Caltavuturo, Leonardo; Gabriele, Morena; Longo, Vincenzo; Bellani, Lorenza; Giorgi, Gianluca; Giorgetti, Lucia

2018-10-01

In this study the antioxidant effect of Cannabis sativa L. seeds and sprouts (3 and 5 days of germination) was evaluated. Total polyphenols, flavonoids and flavonols content, when expressed on dry weight basis, were highest in sprouts; ORAC and DPPH (in vitro assays), CAA-RBC (cellular antioxidant activity in red blood cells) and hemolysis test (ex vivo assays) evidenced a good antioxidant activity higher in sprouts than in seeds. Untargeted analysis by high resolution mass spectrometry in negative ion mode allowed the identification of main polyphenols (caffeoyltyramine, cannabisin A, B, C) in seeds and of ω-6 (linoleic acid) in sprouts. Antimutagenic effect of seeds and sprouts extracts evidenced a significant decrease of mutagenesis induced by hydrogen peroxide in Saccharomyces cerevisiae D7 strain. In conclusion our results show that C. sativa seeds and sprouts exert beneficial effects on yeast and human cells and should be further investigated as a potential functional food. Copyright © 2018. Published by Elsevier Ltd.

Photoswitchable red fluorescent protein with a large Stokes shift

PubMed Central

Piatkevich, Kiryl D.; English, Brian P.; Malashkevich, Vladimir N.; Xiao, Hui; Almo, Steven C.; Singer, Robert H.; Verkhusha, Vladislav V.

2014-01-01

SUMMARY Subclass of fluorescent proteins, large Stokes shift fluorescent proteins, is characterized by their increased spread between the excitation and emission maxima. Here we report a photoswitchable variant of a red fluorescent protein with a large Stokes shift, PSLSSmKate, which initially exhibits excitation/emission at 445/622 nm, but irradiation with violet light photoswitches PSLSSmKate into a common red form with excitation/emission at 573/621 nm. We characterize spectral, photophysical and biochemical properties of PSLSSmKate in vitro and in mammalian cells, and determine its crystal structure in the large Stokes shift form. Mass-spectrometry, mutagenesis and spectroscopic analysis of PSLSSmKate allow us to propose molecular mechanisms for the large Stokes shift, pH dependence and light-induced chromophore transformation. We demonstrate applicability of PSLSSmKate to superresolution PALM microscopy and protein dynamics in live cells. Given its promising properties, we expect that PSLSSmKate-like phenotype will be further used for photoactivatable imaging and tracking multiple populations of intracellular objects. PMID:25242289

Photoswitchable red fluorescent protein with a large Stokes shift.

PubMed

Piatkevich, Kiryl D; English, Brian P; Malashkevich, Vladimir N; Xiao, Hui; Almo, Steven C; Singer, Robert H; Verkhusha, Vladislav V

2014-10-23

A subclass of fluorescent proteins (FPs), large Stokes shift (LSS) FP, are characterized by increased spread between excitation and emission maxima. We report a photoswitchable variant of a red FP with an LSS, PSLSSmKate, which initially exhibits excitation and emission at 445 and 622 nm, but violet irradiation photoswitches PSLSSmKate into a common red form with excitation and emission at 573 and 621 nm. We characterize spectral, photophysical, and biochemical properties of PSLSSmKate in vitro and in mammalian cells and determine its crystal structure in the LSS form. Mass spectrometry, mutagenesis, and spectroscopy of PSLSSmKate allow us to propose molecular mechanisms for the LSS, pH dependence, and light-induced chromophore transformation. We demonstrate the applicability of PSLSSmKate to superresolution photoactivated localization microscopy and protein dynamics in live cells. Given its promising properties, we expect that PSLSSmKate-like phenotype will be further used for photoactivatable imaging and tracking multiple populations of intracellular objects.

Structure and Function of the Glycopeptide N-methyltransferase MtfA, a Tool for the Biosynthesis of Modified Glycopeptide Antibiotics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Rong; Lamb, Sherry S.; Zakeri, Bijan

2009-06-01

There is a considerable interest in the modification of existing antibiotics to generate new antimicrobials. Glycopeptide antibiotics (GPAs) are effective against serious Gram-positive bacterial pathogens including methicillin-resistant Staphylococcus aureus. However, resistance to these antibiotics is becoming a serious problem requiring new strategies. We show that the Amycolatopsis orientalis (S)-adenosyl-L-methionine-dependent methyltransferase MtfA, from the vancomycin-class GPA chloroeremomycin biosynthetic pathway, catalyzes in vivo and in vitro methyl transfer to generate methylated GPA derivatives of the teicoplanin class. The crystal structure of MtfA complexed with (S)-adenosyl-L-methionine, (S)-adenosylhomocysteine, or sinefungin inhibitor, coupled with mutagenesis, identified His228 as a likely general base required for methylmore » transfer to the N terminus of the glycopeptide. Computational docking and molecular dynamics simulations were used to model binding of demethyl-vancomycin aglycone to MtfA. These results demonstrate its utility as a tool for engineering methylated analogs of GPAs.« less

Enzymatic reduction of disulfide bonds in lysosomes: Characterization of a Gamma-interferon-inducible lysosomal thiol reductase (GILT)

NASA Astrophysics Data System (ADS)

Arunachalam, Balasubramanian; Phan, Uyen T.; Geuze, Hans J.; Cresswell, Peter

2000-01-01

Proteins internalized into the endocytic pathway are usually degraded. Efficient proteolysis requires denaturation, induced by acidic conditions within lysosomes, and reduction of inter- and intrachain disulfide bonds. Cytosolic reduction is mediated enzymatically by thioredoxin, but the mechanism of lysosomal reduction is unknown. We describe here a lysosomal thiol reductase optimally active at low pH and capable of catalyzing disulfide bond reduction both in vivo and in vitro. The active site, determined by mutagenesis, consists of a pair of cysteine residues separated by two amino acids, similar to other enzymes of the thioredoxin family. The enzyme is a soluble glycoprotein that is synthesized as a precursor. After delivery into the endosomal/lysosomal system by the mannose 6-phosphate receptor, N- and C-terminal prosequences are removed. The enzyme is expressed constitutively in antigen-presenting cells and induced by IFN-γ in other cell types, suggesting a potentially important role in antigen processing.

Even an old technique is suitable in the molecular world of science: the everted sac preparation turns 60 years old.

PubMed

Hamilton, Kirk L

2014-04-15

An old proverb states "necessity is the mother of invention." This certainly holds true in science as one pursues research questions. Experimental techniques have evolved as scientists have asked more specific research questions. Indeed, techniques such as the Ussing chamber, the perfused renal tubule preparation, patch-clamp, polymerase chain reaction, and site-directed mutagenesis have been developed over the past 60 years. However, sometimes, simple techniques may be useful and still very informative, and this certainly applies to intestinal physiology. Indeed, Gerald Wiseman and Thomas Hastings Wilson described the intestinal everted sac preparation some 60 years ago. Since then, this technique has been used for numerous research purposes including determining ion, amino acid, water and solute transport across the intestinal epithelium; and drug metabolism, absorption, and interactions in pharmaceutical/pharmacological research and even in education. This article provides a historical review of the development of the in vitro intestinal preparation that led to the everted sac preparation and its use in science.

Discovery and antiplatelet activity of a selective PI3Kβ inhibitor (MIPS-9922).

PubMed

Zheng, Zhaohua; Pinson, Jo-Anne; Mountford, Simon J; Orive, Stephanie; Schoenwaelder, Simone M; Shackleford, David; Powell, Andrew; Nelson, Erin M; Hamilton, Justin R; Jackson, Shaun P; Jennings, Ian G; Thompson, Philip E

2016-10-21

A series of amino-substituted triazines were developed and examined for PI3Kβ inhibition and anti-platelet function. Structural adaptations of a morpholine ring of the prototype pan-PI3K inhibitor ZSTK474 yielded PI3Kβ selective compounds, where the selectivity largely derives from an interaction with the non-conserved Asp862 residue, as shown by site directed mutagenesis. The most PI3Kβ selective inhibitor from the series was studied in detail through a series of in vitro and in vivo functional studies. MIPS-9922, 10 potently inhibited ADP-induced washed platelet aggregation. It also inhibited integrin αIIbβ3 activation and αIIbβ3 dependent platelet adhesion to immobilized vWF under high shear. It prevented arterial thrombus formation in the in vivo electrolytic mouse model of thrombosis without inducing prolonged bleeding or excess blood loss. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Carney, H.J.; Hass, B.S.

In order to test the thousands of man-made chemicals in the environment for carcinogenic and genetic hazards, a multitude of short-term screening tests has been developed to complement long-term mammalian bioassays and epidemiological studies. These tests cover a broad spectrum of organisms, and include the use of naked and viral nucleic acids, bacteria, fungi, higher plants, insects in vitro mammalian cell cultures (cell transformation, cell-mediated mutagenesis, DNA repair, and chromosome aberration tests) and live mammals. Assay end points include effects on nucleic acids, DNA repair synthesis, point or gene mutation, structural and numerical chromosome aberrations, cytological alterations, and in vitromore » cell transformation. The present review describes and compares these assays. In addition, it discusses their historical development, the problems and limitations associated with their use, and their implementation in comprehensive testing programs. It is intended to provide overview and specific information to the laboratory that is in the process of establishing genetic toxicological systems. (The literature is reviewed to January 1978.)« less

Characterization of the Annonaceous acetogenin, annonacinone, a natural product inhibitor of plasminogen activator inhibitor-1

NASA Astrophysics Data System (ADS)

Pautus, Stéphane; Alami, Mouad; Adam, Fréderic; Bernadat, Guillaume; Lawrence, Daniel A.; de Carvalho, Allan; Ferry, Gilles; Rupin, Alain; Hamze, Abdallah; Champy, Pierre; Bonneau, Natacha; Gloanec, Philippe; Peglion, Jean-Louis; Brion, Jean-Daniel; Bianchini, Elsa P.; Borgel, Delphine

2016-11-01

Plasminogen activator inhibitor-1 (PAI-1) is the main inhibitor of the tissue type and urokinase type plasminogen activators. High levels of PAI-1 are correlated with an increased risk of thrombotic events and several other pathologies. Despite several compounds with in vitro activity being developed, none of them are currently in clinical use. In this study, we evaluated a novel PAI-1 inhibitor, annonacinone, a natural product from the Annonaceous acetogenins group. Annonacinone was identified in a chromogenic screening assay and was more potent than tiplaxtinin. Annonacinone showed high potency ex vivo on thromboelastography and was able to potentiate the thrombolytic effect of tPA in vivo in a murine model. SDS-PAGE showed that annonacinone inhibited formation of PAI-1/tPA complex via enhancement of the substrate pathway. Mutagenesis and molecular dynamics allowed us to identify annonacinone binding site close to helix D and E and β-sheets 2A.

3D Printing of Tissue Engineered Constructs for In Vitro Modeling of Disease Progression and Drug Screening.

PubMed

Vanderburgh, Joseph; Sterling, Julie A; Guelcher, Scott A

2017-01-01

2D cell culture and preclinical animal models have traditionally been implemented for investigating the underlying cellular mechanisms of human disease progression. However, the increasing significance of 3D vs. 2D cell culture has initiated a new era in cell culture research in which 3D in vitro models are emerging as a bridge between traditional 2D cell culture and in vivo animal models. Additive manufacturing (AM, also known as 3D printing), defined as the layer-by-layer fabrication of parts directed by digital information from a 3D computer-aided design file, offers the advantages of simultaneous rapid prototyping and biofunctionalization as well as the precise placement of cells and extracellular matrix with high resolution. In this review, we highlight recent advances in 3D printing of tissue engineered constructs that recapitulate the physical and cellular properties of the tissue microenvironment for investigating mechanisms of disease progression and for screening drugs.

3D Printing of Tissue Engineered Constructs for in vitro Modeling of Disease Progression and Drug Screening

PubMed Central

Vanderburgh, Joseph; Sterling, Julie A.

2016-01-01

2D cell culture and preclinical animal models have traditionally been implemented for investigating the underlying cellular mechanisms of human disease progression. However, the increasing significance of 3D versus 2D cell culture has initiated a new era in cell culture research in which 3D in vitro models are emerging as a bridge between traditional 2D cell culture and in vivo animal models. Additive manufacturing (AM, also known as 3D printing), defined as the layer-by-layer fabrication of parts directed by digital information from a 3D computer-aided design (CAD) file, offers the advantages of simultaneous rapid prototyping and biofunctionalization as well as the precise placement of cells and extracellular matrix with high resolution. In this review, we highlight recent advances in 3D printing of tissue engineered constructs (TECs) that recapitulate the physical and cellular properties of the tissue microenvironment for investigating mechanisms of disease progression and for screening drugs. PMID:27169894

Differences in temporal aspects of mutagenesis and cytotoxicity in Chinese hamster cells treated with methylating agents and thymidine.

PubMed Central

Peterson, A R; Peterson, H

1982-01-01

Equitoxic concentrations of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and methyl methanesulfonate (MeMes) produced different frequencies of 8-azaguanine-resistant mutants and different amounts of N7-methylguanine, O6-methylguanine (m6G), and N3-methyladenine in the DNA of V79 Chinese hamster cells. Thus, neither the cytotoxicities nor the mutagenicities of these methylating agents could be attributed solely to nitrogen or to oxygen methylations in the DNA. However, MNNG produced 12-fold more m6G and 5-fold more mutants than did MeMes, indicating that a substantial part of the MNNG-induced mutations resulted from m6G--thymine mispairing during DNA replication. The expression as mutants of mutagenic oxygen methylations in the DNA of cells treated with MNNG was enhanced by thymidine (dThd) and deoxycytidine (dCyd), but these nucleosides did not significantly enhance MeMes-induced mutagenesis. The cytotoxicities of MNNG and MeMes were also increased by 10 microM dThd in proportion to the amount of m6G in the DNA. These increases in cytotoxicity were abolished by dCyd, which did not greatly reduce the dThd-induced enhancements of mutagenesis. Moreover, when dThd was present only during the 2-hr treatment with MNNG, maximal cytotoxicity occurred, but MNNG-induced mutagenesis was not increased. Maximal mutagenesis occurred when the dThd was present throughout the first doubling time of the MNNG-treated cells. Thus, the expression of the cytotoxicity and the mutagenicity associated with m6G in the DNA of V79 cells occurred by quite different mechanisms. PMID:6951203

Differences in temporal aspects of mutagenesis and cytotoxicity in Chinese hamster cells treated with methylating agents and thymidine.

PubMed

Peterson, A R; Peterson, H

1982-03-01

Equitoxic concentrations of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and methyl methanesulfonate (MeMes) produced different frequencies of 8-azaguanine-resistant mutants and different amounts of N7-methylguanine, O6-methylguanine (m6G), and N3-methyladenine in the DNA of V79 Chinese hamster cells. Thus, neither the cytotoxicities nor the mutagenicities of these methylating agents could be attributed solely to nitrogen or to oxygen methylations in the DNA. However, MNNG produced 12-fold more m6G and 5-fold more mutants than did MeMes, indicating that a substantial part of the MNNG-induced mutations resulted from m6G--thymine mispairing during DNA replication. The expression as mutants of mutagenic oxygen methylations in the DNA of cells treated with MNNG was enhanced by thymidine (dThd) and deoxycytidine (dCyd), but these nucleosides did not significantly enhance MeMes-induced mutagenesis. The cytotoxicities of MNNG and MeMes were also increased by 10 microM dThd in proportion to the amount of m6G in the DNA. These increases in cytotoxicity were abolished by dCyd, which did not greatly reduce the dThd-induced enhancements of mutagenesis. Moreover, when dThd was present only during the 2-hr treatment with MNNG, maximal cytotoxicity occurred, but MNNG-induced mutagenesis was not increased. Maximal mutagenesis occurred when the dThd was present throughout the first doubling time of the MNNG-treated cells. Thus, the expression of the cytotoxicity and the mutagenicity associated with m6G in the DNA of V79 cells occurred by quite different mechanisms.

Amino acid substitutions in random mutagenesis libraries: lessons from analyzing 3000 mutations.

PubMed

Zhao, Jing; Frauenkron-Machedjou, Victorine Josiane; Kardashliev, Tsvetan; Ruff, Anna Joëlle; Zhu, Leilei; Bocola, Marco; Schwaneberg, Ulrich

2017-04-01

The quality of amino acid substitution patterns in random mutagenesis libraries is decisive for the success in directed evolution campaigns. In this manuscript, we provide a detailed analysis of the amino acid substitutions by analyzing 3000 mutations of three random mutagenesis libraries (1000 mutations each; epPCR with a low-mutation and a high-mutation frequency and SeSaM-Tv P/P) employing lipase A from Bacillus subtilis (bsla). A comparison of the obtained numbers of beneficial variants in the mentioned three random mutagenesis libraries with a site saturation mutagenesis (SSM) (covering the natural diversity at each amino acid position of BSLA) concludes the diversity analysis. Seventy-six percent of the SeSaM-Tv P/P-generated substitutions yield chemically different amino acid substitutions compared to 64% (epPCR-low) and 69% (epPCR-high). Unique substitutions from one amino acid to others are termed distinct amino acid substitutions. In the SeSaM-Tv P/P library, 35% of all theoretical distinct amino acid substitutions were found in the 1000 mutation library compared to 25% (epPCR-low) and 26% (epPCR-high). Thirty-six percent of distinct amino acid substitutions found in SeSaM-Tv P/P were unobtainable by epPCR-low. Comparison with the SSM library showed that epPCR-low covers 15%, epPCR-high 18%, and SeSaM-Tv P/P 21% of obtainable beneficial amino acid positions. In essence, this study provides first insights on the quality of epPCR and SeSaM-Tv P/P libraries in terms of amino acid substitutions, their chemical differences, and the number of obtainable beneficial amino acid positions.