Science.gov

Sample records for mejorada por mutagenesis

  1. Lethal mutagenesis of viruses.

    PubMed

    Perales, Celia; Martín, Verónica; Domingo, Esteban

    2011-11-01

    Lethal mutagenesis aims at extinguishing viruses by increased mutagenesis prompted by virus-specific mutagenic agents, mainly nucleoside analogues. It is derived from the error threshold relationship of quasispecies theory, and it is slowly finding its way towards a clinical application. We summarize the current situation of research in this field of antiviral therapy. Copyright © 2011 Elsevier B.V. All rights reserved.

  2. 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.

  3. 2004 Mutagenesis Gordon Conference

    SciTech Connect

    Dr. Sue Jinks-Robertson

    2005-09-16

    Mutations are genetic alterations that drive biological evolution and cause many, if not all, human diseases. Mutation originates via two distinct mechanisms: ''vertical'' variation is de novo change of one or few bases, whereas ''horizontal'' variation occurs by genetic recombination, which creates new mosaics of pre-existing sequences. The Mutagenesis Conference has traditionally focused on the generation of mutagenic intermediates during normal DNA synthesis or in response to environmental insults, as well as the diverse repair mechanisms that prevent the fixation of such intermediates as permanent mutations. While the 2004 Conference will continue to focus on the molecular mechanisms of mutagenesis, there will be increased emphasis on the biological consequences of mutations, both in terms of evolutionary processes and in terms of human disease. The meeting will open with two historical accounts of mutation research that recapitulate the intellectual framework of this field and thereby place the current research paradigms into perspective. The two introductory keynote lectures will be followed by sessions on: (1) mutagenic systems, (2) hypermutable sequences, (3) mechanisms of mutation, (4) mutation avoidance systems, (5) mutation in human hereditary and infectious diseases, (6) mutation rates in evolution and genotype-phenotype relationships, (7) ecology, mutagenesis and the modeling of evolution and (8) genetic diversity of the human population and models for human mutagenesis. The Conference will end with a synthesis of the meeting as the keynote closing lecture.

  4. 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)

  5. 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)

  6. Mechanism of proflavin mutagenesis.

    PubMed

    Sarabhai, A; Lamfrom, H

    1969-08-01

    The mutagenic action of proflavin on bacteriophage T4 is greater in the presence of defective T4 ligase than in the presence of normal T4 ligase. This suggests that the persistence of single-strand breaks in DNA enhances proflavin mutagenesis.

  7. Monitoring the fall of large atmospheric ice conglomerations: a multianalytical approach to the study of the Mejorada del Campo megacryometeor.

    PubMed

    Orellana, Francisco Alamilla; Alegre, José Ma Ramiro; Cordero Pérez, José Carlos; Martín Redondo, Ma Paz; Delgado Huertas, Antonio; Fernández Sampedro, Ma Teresa; Menor-Salván, César; Ruiz-Bermejo, Marta; López-Vera, Fernando; Rodríguez-Losada, José A; Martinez-Frias, Jesus

    2008-04-01

    Certain local atmospheric anomalies, such as the formation of unusually large ice conglomerations (megacryometeors), have been proposed to be a potential natural hazard for people and aviation, as well as geoindicators for fingerprinting larger-scale atmospheric environmental changes. On March 13th 2007, at approximately 10:15 am, an ice chunk weighing about 10 kg fell from the clear-sky and crashed through the roof (around 15 m) of an industrial storage house in Mejorada del Campo, a town located 20 km east from Madrid. The megacryometeor monitoring follow-up and the original investigation presented here includes, for the first time, both logistic and scientific collaboration between the Laboratory of the Environment, Criminalistic Service (SECRIM, the Spanish "Guardia Civil") and academic and scientific institutions (universities and the Spanish National Research Council). We propose that the management procedure of the incident, along with the detailed scientific research and combination of analytical methodologies in different laboratories, can serve as a protocol model for other similar events.

  8. Transposon Mutagenesis in Mice

    PubMed Central

    Largaespada, David A.

    2010-01-01

    Understanding the functional landscape of the mammalian genome is the next big challenge of biomedical research. The completion of the first phases of the mouse and human genome projects, and expression analyses using microarray hybridization, generate critically important questions about the functional landscape and structure of the mammalian genome: how many genes, and of what type, are there; what kind of functional elements make up a properly functioning gene? One step in this process will be to create mutations in every identifiable mouse gene and analyze the resultant phenotypes. Transposons are being considered as tools to further initiatives to create a comprehensive resource of mutant mouse strains. Also, it may be possible to use transposons in true forward genetic screens in the mouse. The “Sleeping Beauty” (SB) transposon system is one such tool. Moreover, due to its tendency for local hopping, SB has been proposed as a method for regional saturation mutagenesis of the mouse genome. In this chapter, we review the tools and methods currently available to create mutant mice using in vivo, germline transposition in mice. PMID:19266336

  9. Theory of lethal mutagenesis for viruses.

    PubMed

    Bull, J J; Sanjuán, R; Wilke, C O

    2007-03-01

    Mutation is the basis of adaptation. Yet, most mutations are detrimental, and elevating mutation rates will impair a population's fitness in the short term. The latter realization has led to the concept of lethal mutagenesis for curing viral infections, and work with drugs such as ribavirin has supported this perspective. As yet, there is no formal theory of lethal mutagenesis, although reference is commonly made to Eigen's error catastrophe theory. Here, we propose a theory of lethal mutagenesis. With an obvious parallel to the epidemiological threshold for eradication of a disease, a sufficient condition for lethal mutagenesis is that each viral genotype produces, on average, less than one progeny virus that goes on to infect a new cell. The extinction threshold involves an evolutionary component based on the mutation rate, but it also includes an ecological component, so the threshold cannot be calculated from the mutation rate alone. The genetic evolution of a large population undergoing mutagenesis is independent of whether the population is declining or stable, so there is no runaway accumulation of mutations or genetic signature for lethal mutagenesis that distinguishes it from a level of mutagenesis under which the population is maintained. To detect lethal mutagenesis, accurate measurements of the genome-wide mutation rate and the number of progeny per infected cell that go on to infect new cells are needed. We discuss three methods for estimating the former. Estimating the latter is more challenging, but broad limits to this estimate may be feasible.

  10. In vitro models of mutagenesis.

    PubMed

    Strauss, B S; Larson, K; Sagher, D; Rabkin, S; Shenkar, R; Sahm, J

    1985-01-01

    The bypass of lesions in DNA with insertion of nucleotides opposite damaged bases has been studied as a model for mutagenesis in an in vitro system. Lesions introduced by dimethyl sulfate at adenines and by ultraviolet light at pyrimidine dimers act as termination sites on both double- and single-stranded DNA templates. Base selection opposite noninformational lesions is, in part, a property of the polymerases: different polymerases have different selectivities although all polymerases tested seem to prefer purines. The ability to insert "incorrect" bases is determined in part by the sequence 5' to the lesion on the template strand. The hypothesis that damaged purines tend to result in transversions can be applied to published data on activation of the c-ras oncogene.

  11. [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.

  12. Insertional mutagenesis and illegitimate recombination in mycobacteria.

    PubMed Central

    Kalpana, G V; Bloom, B R; Jacobs, W R

    1991-01-01

    Mycobacteria, particularly Mycobacterium tuberculosis, Mycobacterium leprae, and Mycobacterium avium, are major pathogens of man. Although insertional mutagenesis has been an invaluable genetic tool for analyzing the mechanisms of microbial pathogenesis, it has not yet been possible to apply it to the mycobacteria. To overcome intrinsic difficulties in directly manipulating the genetics of slow-growing mycobacteria, including M. tuberculosis and bacille Calmette-Guérin (BCG) vaccine strains, we developed a system for random shuttle mutagenesis. A genomic library of Mycobacterium smegmatis was subjected to transposon mutagenesis with Tn5 seq1, a derivative of Tn5, in Escherichia coli and these transposon-containing recombinant plasmids were reintroduced into mycobacterial chromosomes by homologous recombination. This system has allowed us to isolate several random auxotrophic mutants of M. smegmatis. To extend this strategy to M. tuberculosis and BCG, targeted mutagenesis was performed using a cloned BCG methionine gene that was subjected to Tn5 seq1 mutagenesis in E. coli and reintroduced into the mycobacteria. Surprisingly for prokaryotes, both BCG and M. tuberculosis were found to incorporate linear DNA fragments into illegitimate sites throughout the mycobacterial genomes at a frequency of 10(-5) to 10(-4) relative to the number of transformants obtained with autonomously replicating vectors. Thus the efficient illegitimate recombination of linear DNA fragments provides the basis for an insertional mutagenesis system for M. tuberculosis and BCG. Images PMID:2052623

  13. Lethal Mutagenesis Failure May Augment Viral Adaptation

    PubMed Central

    Paff, Matthew L.; Stolte, Steven P.; Bull, James J.

    2014-01-01

    Lethal mutagenesis, the attempt to extinguish a population by elevating its mutation rate, has been endorsed in the virology literature as a promising approach for treating viral infections. In support of the concept, in vitro studies have forced viral extinction with high doses of mutagenic drugs. However, the one known mutagenic drug used on patients commonly fails to cure infections, and in vitro studies typically find a wide range of mutagenic conditions permissive for viral growth. A key question becomes how subsequent evolution is affected if the viral population is mutated but avoids extinction—Is viral adaptation augmented rather than suppressed? Here we consider the evolution of highly mutated populations surviving mutagenesis, using the DNA phage T7. In assays using inhibitory hosts, whenever resistance mutants were observed, the mutagenized populations exhibited higher frequencies, but some inhibitors blocked plaque formation by even the mutagenized stock. Second, outgrowth of previously mutagenized populations led to rapid and potentially complete fitness recovery but polymorphism was slow to decay, and mutations exhibited inconsistent patterns of change. Third, the combination of population bottlenecks with mutagenesis did cause fitness declines, revealing a vulnerability that was not apparent from mutagenesis of large populations. The results show that a population surviving high mutagenesis may exhibit enhanced adaptation in some environments and experience little negative fitness consequences in many others. PMID:24092771

  14. 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

  15. P53 Gene Mutagenesis in Breast Cancer

    DTIC Science & Technology

    2005-03-01

    suppressor gene in sporadic breast tumours . 1991. Loss of chromosome 17 pl3 sequences and mutation of p53 Oncogene 5 :1573-1579. in human breast...COVERED March 2005 Final (I Aug 2000 - 1 Feb 2004) 4. TITLE AND SUBTITLE 5 . FUNDING NUMBERS p53 Gene Mutagenesis in Breast Cancer DAMD17-00-1-0204 6. AUTHOR...The central hypothesis of this proposal is that variability in the patterns of p 5 3 mutagenesis in breast cancer reflects differences in exposures to

  16. 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…

  17. 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…

  18. 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...

  19. 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...

  20. Genetic analysis of mutagenesis in aging Escherichia coli colonies.

    PubMed

    Taddei, F; Halliday, J A; Matic, I; Radman, M

    1997-10-01

    Bacteria live in unstructured and structured environments, experiencing feast and famine lifestyles. Bacterial colonies can be viewed as model structured environments. SOS induction and mutagenesis have been observed in aging Escherichia coli colonies, in the absence of exogenous sources of DNA damage. This cAMP-dependent mutagenesis occurring in Resting Organisms in a Structured Environment (ROSE) is unaffected by a umuC mutation and therefore differs from both targeted UV mutagenesis and recA730 (SOS constitutive) untargeted mutagenesis. As a recB mutation has only a minor effect on ROSE mutagenesis it also differs from both adaptive reversion of the lacI33 allele and from iSDR (inducible Stable DNA Replication) mutagenesis. Besides its recA and lexA dependence, ROSE mutagenesis is also uvrB and polA dependent. These genetic requirements are reminiscent of the untargeted mutagenesis in lambda phage observed when unirradiated lambda infects UV-irradiated E. coli. These mutations, which are not observed in aging liquid cultures, accumulate linearly with the age of the colonies. ROSE mutagenesis might offer a good model for bacterial mutagenesis in structured environments such as biofilms and for mutagenesis of quiescent eukaryotic cells.

  1. Final report [DNA Repair and Mutagenesis - 1999

    SciTech Connect

    Walker, Graham C.

    2001-05-30

    The meeting, titled ''DNA Repair and Mutagenesis: Mechanism, Control, and Biological Consequences'', was designed to bring together the various sub-disciplines that collectively comprise the field of DNA Repair and Mutagenesis. The keynote address was titled ''Mutability Doth Play Her Cruel Sports to Many Men's Decay: Variations on the Theme of Translesion Synthesis.'' Sessions were held on the following themes: Excision repair of DNA damage; Transcription and DNA excision repair; UmuC/DinB/Rev1/Rad30 superfamily of DNA polymerases; Cellular responses to DNA damage, checkpoints, and damage tolerance; Repair of mismatched bases, mutation; Genome-instability, and hypermutation; Repair of strand breaks; Replicational fidelity, and Late-breaking developments; Repair and mutation in challenging environments; and Defects in DNA repair: consequences for human disease and aging.

  2. 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.

  3. Fluorometric method of quantitative cell mutagenesis

    DOEpatents

    Dolbeare, F.A.

    1980-12-12

    A method for assaying a cell culture for mutagenesis is described. A cell culture is stained first with a histochemical stain, and then a fluorescent stain. Normal cells in the culture are stained by both the histochemical and fluorescent stains, while abnormal cells are stained only by the fluorescent stain. The two stains are chosen so that the histochemical stain absorbs the wavelengths that the fluorescent stain emits. After the counterstained culture is subjected to exciting light, the fluorescence from the abnormal cells is detected.

  4. Fluorometric method of quantitative cell mutagenesis

    DOEpatents

    Dolbeare, Frank A.

    1982-01-01

    A method for assaying a cell culture for mutagenesis is described. A cell culture is stained first with a histochemical stain, and then a fluorescent stain. Normal cells in the culture are stained by both the histochemical and fluorescent stains, while abnormal cells are stained only by the fluorescent stain. The two stains are chosen so that the histochemical stain absorbs the wavelengths that the fluorescent stain emits. After the counterstained culture is subjected to exciting light, the fluorescence from the abnormal cells is detected.

  5. Translesion DNA Synthesis and Mutagenesis in Eukaryotes

    PubMed Central

    Sale, Julian E.

    2013-01-01

    The structural features that enable replicative DNA polymerases to synthesize DNA rapidly and accurately also limit their ability to copy damaged DNA. Direct replication of DNA damage is termed translesion synthesis (TLS), a mechanism conserved from bacteria to mammals and executed by an array of specialized DNA polymerases. This chapter examines how these translesion polymerases replicate damaged DNA and how they are regulated to balance their ability to replicate DNA lesions with the risk of undesirable mutagenesis. It also discusses how TLS is co-opted to increase the diversity of the immunoglobulin gene hypermutation and the contribution it makes to the mutations that sculpt the genome of cancer cells. PMID:23457261

  6. AS52/GPT Mammalian Mutagenesis Assay

    DTIC Science & Technology

    1996-05-10

    dimethylnitrosamine (DMN) at 50 and 100 f.J.g/rnl was used as a 3 TLS Project Nn. A0ŗ-003: AS52/GPT Mammalian Mutagenesis Assay promutagen that requires metabolic...Chemical Source Lot No. air Air Products N/A calcium chloride Sigma 84F-0723 d imeth y !sulfoxide Fisher 933274 dimethylnitrosamine Sigma 82B0365...methanesulfonate (EMS) at 150 and 300 J.i-g/ml is used as a direct-acting mutagen for the nonactivated portion, and dimethylnitrosamine (DMN) at 150 and 300

  7. Probabilistic analysis of recessive mutagenesis screen strategies.

    PubMed

    Silver, Jeremy D; Hilton, Douglas J; Bahlo, Melanie; Kile, Benjamin T

    2007-01-01

    Random mutagenesis screens for recessive phenotypes require three generations of breeding, using either a backcross (BC) or intercross (IC) strategy. Hence, they are more costly and technically demanding than those for dominant phenotypes. Maximizing the return from these screens requires maximizing the number of mutations that are bred to homozyosity in the G(3) generation. Using a probabilistic approach, we compare different designs of screens for recessive phenotypes and the impact each one has on the number of mutations that can be effectively screened. We address the issue of BC versus IC strategies and consider genome-wide, region-specific screens and suppressor screens. We find that optimally designed BC and IC screens allow the screening of, on average, similar numbers of mutations but that interpedigree variation is more pronounced when the IC strategy is employed. By conducting a retrospective analysis of published mutagenesis screens, we show that, depending on the strategy, a threefold difference in the numbers of mutations screened per animal used could be expected. This method allows researchers to contrast, for a range of experimental designs, the cost per mutation screened and to maximize the number of mutations that one can expect to screen in a given experiment.

  8. Lethal mutagenesis in a structured environment.

    PubMed

    Steinmeyer, Shelby H; Wilke, Claus O

    2009-11-07

    We analyze how lethal mutagenesis operates in a compartmentalized host. We assume that different compartments receive different amounts of mutagen and that virions can migrate among compartments. We address two main questions: (1) To what extent can refugia, i.e., compartments that receive little mutagen, prevent extinction? (2) Does migration among compartments limit the effectiveness of refugia? We find that if there is little migration, extinction has to be achieved separately in all compartments. In this case, the total dose of mutagen administered to the host needs to be so high that the mutagen is effective even in the refugia. By contrast, if migration is extensive, then lethal mutagenesis is effective as long as the average growth in all compartments is reduced to below replacement levels. The effectiveness of migration is governed by the ratio of virion replication and death rates, R(0). The smaller R(0), the less migration is necessary to neutralize refugia and the less mutagen is necessary to achieve extinction at high migration rates.

  9. An efficient TALEN mutagenesis system in rice.

    PubMed

    Chen, Kunling; Shan, Qiwei; Gao, Caixia

    2014-08-15

    Targeted gene mutagenesis is a powerful tool for elucidating gene function and facilitating genetic improvement in rice. TALENs (transcription activator-like effector nucleases), consisting of a custom TALE DNA binding domain fused to a nonspecific FokI cleavage domain, are one of the most efficient genome engineering methods developed to date. The technology of TALENs allows DNA double-strand breaks (DSBs) to be introduced into predetermined chromosomal loci. DSBs trigger DNA repair mechanisms and can result in loss of gene function by error-prone non-homologous end joining (NHEJ), or they can be exploited to modify gene function or activity by precise homologous recombination (HR). In this paper, we describe a detailed protocol for constructing TALEN expression vectors, assessing nuclease activities in vivo using rice protoplast-based assays, generating and introducing TALEN DNAs into embryogenic calluses of rice and identifying TALEN-generated mutations at targeted genomic sites. Using these methods, T0 rice plants resulting from TALEN mutagenesis can be produced within 4-5 months.

  10. New transposon delivery plasmids for insertional mutagenesis in Bacillus anthracis

    PubMed Central

    Wilson, Adam C.; Perego, Marta; Hoch, James A.

    2007-01-01

    Two new transposon delivery vector systems utilizing Mariner and mini-Tn10 transposons have been developed for in vivo insertional mutagenesis in Bacillus anthracis and other compatible Gram-positive species. The utility of both systems was directly demonstrated through the mutagenesis of a widely used B. anthracis strain. PMID:17931726

  11. Mutagenesis assays of human amniotic fluid

    SciTech Connect

    Everson, R.B.; Milne, K.L.; Warbuton, D.; McClamrock, H.D.; Buchanan, P.D.

    1985-01-01

    Extracts of amniocentesis samples from 144 women were tested for the presence of mutagenic substances using tester strain TA1538 in the Ames Salmonella/mammalian-microsome mutagenicity test. Because the volume of amniotic fluid in these samples was limited (generally less than 10 ml), the authors investigated modifications of this mutagenesis assay that could increase its ability to detect effects from small quantities of test material. Using mutagenicity in samples of urine from smokers as a model, it appeared that improved ability to detect small amounts of mutagen could be obtained by reducing volumes of media and reagents while keeping the amount of test sample constant. Tests of amniotic fluid extracts by this modified procedure showed small increases in revertants, about 50% above dimethylsulfoxide solvent control values. The increases suggest the presence of small amounts of mutagenic material in many of the amniotic fluid samples. At the doses employed, mutagenic activity in these samples was not associated with maternal smoking.

  12. Conditional gene-trap mutagenesis in zebrafish.

    PubMed

    Maddison, Lisette A; Li, Mingyu; Chen, Wenbiao

    2014-01-01

    Zebrafish has become a widely used model for analysis of gene function. Several methods have been used to create mutations in this organism and thousands of mutant lines are available. However, all the conventional zebrafish mutations affect the gene in all cells at all time, making it difficult to determine tissue-specific functions. We have adopted a FlEx Trap approach to generate conditional mutations in zebrafish by gene-trap mutagenesis. Combined with appropriate Cre or Flp lines, the insertional mutants not only allow spatial- and temporal-specific gene inactivation but also permit spatial- and temporal-specific rescue of the disrupted gene. We provide experimental details on how to generate and use such mutations.

  13. From Chemical Mutagenesis to Post‐Expression Mutagenesis: A 50 Year Odyssey

    PubMed Central

    Wright, Tom H.; Vallée, M. Robert J.

    2016-01-01

    Abstract Site‐directed (gene) mutagenesis has been the most useful method available for the conversion of one amino acid residue of a given protein into another. Until relatively recently, this strategy was limited to the twenty standard amino acids. The ongoing maturation of stop codon suppression and related technologies for unnatural amino acid incorporation has greatly expanded access to nonstandard amino acids by expanding the scope of the translational apparatus. However, the necessity for translation of genetic changes restricts the diversity of residues that may be incorporated. Herein we highlight an alternative approach, termed post‐expression mutagenesis, which operates at the level of the very functional biomolecules themselves. Using the lens of retrosynthesis, we highlight prospects for new strategies in protein modification, alteration, and construction which will enable protein science to move beyond the constraints of the “translational filter” and lead to a true synthetic biology. PMID:27119221

  14. Germline mutagenesis mediated by Sleeping Beauty transposon system in mice

    PubMed Central

    Takeda, Junji; Keng, Vincent W; Horie, Kyoji

    2007-01-01

    Following the descovery of its transposition activity in mammalian culture systems, the Sleeping Beauty (SB) transposon has since been applied to achieve germline mutagenesis in mice. Initially, the transposition efficiency was found to be low in cultured systems, but its activity in germ cells was unexpectedly high. This difference in transposition efficiency was found to be largely dependent on chromosomal status of the host genomic DNA and transposon vector design. The SB transposon system has been found to be suitable for comprehensive mutagenesis in mice. Therefore, it is an effective tool as a forward genetics screen for tagged insertional mutagenesis in mice. PMID:18047691

  15. Heat shock and herpes virus: enhanced reactivation without untargeted mutagenesis

    SciTech Connect

    Lytle, C.D.; Carney, P.G.

    1988-01-01

    Enhanced reactivation of Ultraviolet-irradiated virus has been reported to occur in heat-shocked host cells. Since enhanced virus reactivation is often accompanied by untargeted mutagenesis, we investigated whether such mutagenesis would occur for herpes simplex virus (HSV) in CV-1 monkey kidney cells subjected to heat shock. In addition to expressing enhanced reactivation, the treated cells were transiently more susceptible to infection by unirradiated HSV. No mutagenesis of unirradiated HSV was found whether infection occurred at the time of increased susceptibility to infection or during expression of enhanced viral reactivation.

  16. History of attempts to quantify environmental mutagenesis

    SciTech Connect

    Hollaender, A.

    1981-01-01

    It became obvious in the early 1960's that the ready recognition of mutations produced by chemicals could have a profound influence on the refinement of methods to detect environmental mutagens. The experience derived over the previous 30 years in characterizing the effects of ionizing and ultraviolet radiation on the genetic mechanism came to serve us in good stead. Although the effects of chemicals are considerably more complicated and often require the analysis of individual substances, nonetheless, the area has developed rapidly in recent decades. The establishment and historical background of the International Association of Environmental Mutagen Societies (IAEMS) will be discussed. An attempt at the quantitation of chemical effects has been developed in comparison with radiation mutagenesis. As a first step, a definition of the Mutagen Burden or unavoidable exposure to chemicals will be discussed. A mathematical approach (Haynes/Eckhardt) will be considered and finally an outline for the comprehensive investigation of detailed interscience study will be made of less than six chemicals.

  17. Genomic approaches to DNA repair and mutagenesis.

    PubMed

    Wyrick, John J; Roberts, Steven A

    2015-12-01

    DNA damage is a constant threat to cells, causing cytotoxicity as well as inducing genetic alterations. The steady-state abundance of DNA lesions in a cell is minimized by a variety of DNA repair mechanisms, including DNA strand break repair, mismatch repair, nucleotide excision repair, base excision repair, and ribonucleotide excision repair. The efficiencies and mechanisms by which these pathways remove damage from chromosomes have been primarily characterized by investigating the processing of lesions at defined genomic loci, among bulk genomic DNA, on episomal DNA constructs, or using in vitro substrates. However, the structure of a chromosome is heterogeneous, consisting of heavily protein-bound heterochromatic regions, open regulatory regions, actively transcribed genes, and even areas of transient single stranded DNA. Consequently, DNA repair pathways function in a much more diverse set of chromosomal contexts than can be readily assessed using previous methods. Recent efforts to develop whole genome maps of DNA damage, repair processes, and even mutations promise to greatly expand our understanding of DNA repair and mutagenesis. Here we review the current efforts to utilize whole genome maps of DNA damage and mutation to understand how different chromosomal contexts affect DNA excision repair pathways. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. A mutagenesis study of a catalytic antibody

    SciTech Connect

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

    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 four 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.

  19. Mutagenesis during plant responses to UVB radiation.

    PubMed

    Holá, M; Vágnerová, R; Angelis, K J

    2015-08-01

    We tested an idea that induced mutagenesis due to unrepaired DNA lesions, here the UV photoproducts, underlies the impact of UVB irradiation on plant phenotype. For this purpose we used protonemal culture of the moss Physcomitrella patens with 50% of apical cells, which mimics actively growing tissue, the most vulnerable stage for the induction of mutations. We measured the UVB mutation rate of various moss lines with defects in DNA repair (pplig4, ppku70, pprad50, ppmre11), and in selected clones resistant to 2-Fluoroadenine, which were mutated in the adenosine phosphotrasferase gene (APT), we analysed induced mutations by sequencing. In parallel we followed DNA break repair and removal of cyclobutane pyrimidine dimers with a half-life τ = 4 h 14 min determined by comet assay combined with UV dimer specific T4 endonuclease V. We show that UVB induces massive, sequence specific, error-prone bypass repair that is responsible for a high mutation rate owing to relatively slow, though error-free, removal of photoproducts by nucleotide excision repair (NER). Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  20. Lethal mutagenesis in viruses and bacteria.

    PubMed

    Chen, Peiqiu; Shakhnovich, Eugene I

    2009-10-01

    In this work we study how mutations that change physical properties of cell proteins (stability) affect population survival and growth. We present a model in which the genotype is presented as a set folding free energies of cell proteins. Mutations occur upon replication, so stabilities of some proteins in daughter cells differ from those in the parent cell by amounts deduced from the distribution of mutational effects on protein stability. The genotype-phenotype relationship posits that the cell's fitness (replication rate) is proportional to the concentration of its folded proteins and that unstable essential proteins result in lethality. Simulations reveal that lethal mutagenesis occurs at a mutation rate close to seven mutations in each replication of the genome for RNA viruses and at about half that rate for DNA-based organisms, in accord with earlier predictions from analytical theory and experimental results. This number appears somewhat dependent on the number of genes in the organisms and the organism's natural death rate. Further, our model reproduces the distribution of stabilities of natural proteins, in excellent agreement with experiments. We find that species with high mutation rates tend to have less stable proteins compared to species with low mutation rates.

  1. Signature-tagged mutagenesis of Vibrio vulnificus

    PubMed Central

    YAMAMOTO, Mai; KASHIMOTO, Takashige; TONG, Ping; XIAO, Jianbo; SUGIYAMA, Michiko; INOUE, Miyuki; MATSUNAGA, Rie; HOSOHARA, Kohei; NAKATA, Kazue; YOKOTA, Kenji; OGUMA, Keiji; YAMAMOTO, Koichiro

    2015-01-01

    Vibrio vulnificus is the causative agent of primary septicemia, wound infection and gastroenteritis in immunocompromised people. In this study, signature-tagged mutagenesis (STM) was applied to identify the virulence genes of V. vulnificus. Using STM, 6,480 mutants in total were constructed and divided into 81 sets (INPUT pools); each mutant in a set was assigned a different tag. Each INPUT pool was intraperitoneally injected into iron-overloaded mice, and in vivo surviving mutants were collected from blood samples from the heart (OUTPUT pools). From the genomic DNA of mixed INPUT or OUTPUT pools, digoxigenin-labeled DNA probes against the tagged region were prepared and used for dot hybridization. Thirty tentatively attenuated mutants, which were hybridized clearly with INPUT probes but barely with OUTPUT probes, were negatively selected. Lethal doses of 11 of the 30 mutants were reduced to more than 1/100; of these, the lethal doses of 2 were reduced to as low as 1/100,000. Transposon-inserted genes in the 11 attenuated mutants were those for IMP dehydrogenase, UDP-N-acetylglucosamine-2-epimerase, aspartokinase, phosphoribosylformylglycinamidine cyclo-ligase, malate Na (+) symporter and hypothetical protein. When mice were immunized with an attenuated mutant strain into which IMP dehydrogenase had been inserted with a transposon, they were protected against V. vulnificus infection. In this study, we demonstrated that the STM method can be used to search for the virulence genes of V. vulnificus. PMID:25755021

  2. Genomic Approaches to DNA repair and Mutagenesis

    PubMed Central

    Wyrick, John J.; Roberts, Steven A.

    2015-01-01

    DNA damage is a constant threat to cells, causing cytotoxicity as well as inducing genetic alterations. The steady-state abundance of DNA lesions in a cell is minimized by a variety of DNA repair mechanisms, including DNA strand break repair, mismatch repair, nucleotide excision repair, base excision repair, and ribonucleotide excision repair. The efficiencies and mechanisms by which these pathways remove damage from chromosomes have been primarily characterized by investigating the processing of lesions at defined genomic loci, among bulk genomic DNA, on episomal DNA constructs, or using in vitro substrates. However, the structure of a chromosome is heterogeneous, consisting of heavily protein-bound heterochromatic regions, open regulatory regions, actively transcribed genes, and even areas of transient single stranded DNA. Consequently, DNA repair pathways function in a much more diverse set of chromosomal contexts than can be readily assessed using previous methods. Recent efforts to develop whole genome maps of DNA damage, repair processes, and even mutations promise to greatly expand our understanding of DNA repair and mutagenesis. Here we review the current efforts to utilize whole genome maps of DNA damage and mutation to understand how different chromosomal contexts affect DNA excision repair pathways. PMID:26411877

  3. 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.

  4. Symposium on molecular and cellular mechanisms of mutagenesis

    SciTech Connect

    Not Available

    1981-01-01

    These proceedings contain abstracts only of the 21 papers presented at the Sympsoium. The papers dealt with molecular mechanisms of mutagenesis and cellular responses to chemical and physical mutagenic agents. (ERB)

  5. 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.

  6. 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

  7. Empirical complexities in the genetic foundations of lethal mutagenesis.

    PubMed

    Bull, James J; Joyce, Paul; Gladstone, Eric; Molineux, Ian J

    2013-10-01

    From population genetics theory, elevating the mutation rate of a large population should progressively reduce average fitness. If the fitness decline is large enough, the population will go extinct in a process known as lethal mutagenesis. Lethal mutagenesis has been endorsed in the virology literature as a promising approach to viral treatment, and several in vitro studies have forced viral extinction with high doses of mutagenic drugs. Yet only one empirical study has tested the genetic models underlying lethal mutagenesis, and the theory failed on even a qualitative level. Here we provide a new level of analysis of lethal mutagenesis by developing and evaluating models specifically tailored to empirical systems that may be used to test the theory. We first quantify a bias in the estimation of a critical parameter and consider whether that bias underlies the previously observed lack of concordance between theory and experiment. We then consider a seemingly ideal protocol that avoids this bias-mutagenesis of virions-but find that it is hampered by other problems. Finally, results that reveal difficulties in the mere interpretation of mutations assayed from double-strand genomes are derived. Our analyses expose unanticipated complexities in testing the theory. Nevertheless, the previous failure of the theory to predict experimental outcomes appears to reside in evolutionary mechanisms neglected by the theory (e.g., beneficial mutations) rather than from a mismatch between the empirical setup and model assumptions. This interpretation raises the specter that naive attempts at lethal mutagenesis may augment adaptation rather than retard it.

  8. Transposon Tn916 mutagenesis in Bacillus anthracis.

    PubMed Central

    Ivins, B E; Welkos, S L; Knudson, G B; Leblanc, D J

    1988-01-01

    Mutagenesis of Bacillus anthracis by the streptococcal tetracycline resistance transposon Tn916 is described. Tn916 was transferred from Streptococcus faecalis DS16C1 to B. anthracis VNR-1 by conjugation in a standard filter mating procedure. Tetracycline-resistant (Tcr) transconjugants were obtained at a frequency of 1.6 X 10(-8) per donor CFU. When donor and recipient cells were treated with nafcillin before conjugation, the frequency was increased nearly 10-fold. Nafcillin pretreatment of donor and recipient strains was used in all subsequent conjugation experiments. S.faecalis CG110, containing multiple chromosomal insertions of Tn916, transferred the transposon to B. anthracis VNR-1 at a frequency of 9.3 x 10(-5). A Tcr B. anthracis transconjugant, strain VNR-1-tet-1, transferred Tn916 to B. anthracis UM23-1 and Bacillus subtilis BST1 at frequencies of 2.1 x 10(-4) and 5.8 X 10(-6), respectively. The transfer of Tn916 occurred only on membrane filters, since no Tcr transconjugants were obtained when strains VNR-1-tet-1 and UM23-1 were mixed and incubated in broth culture. The presence of the Tn916-associated tetM gene in Tcr B. anthracis and B. subtilis transconjugants was confirmed in hybridization experiments by using a 5-kilobase-pair DNA fragment containing the tetM gene as a probe. Of 3,000 B. anthracis UM23-1 Tcr transconjugants tested, 21 were phenylalanine auxotrophs and 2 were auxotrophic for phenylalanine, tyrosine, and tryptophan. Images PMID:2826334

  9. Endogenous mutagenesis in recombinant sulfolobus plasmids.

    PubMed

    Sakofsky, Cynthia J; Grogan, Dennis W

    2013-06-01

    Low rates of replication errors in chromosomal genes of Sulfolobus spp. demonstrate that these extreme thermoacidophiles can maintain genome integrity in environments with high temperature and low pH. In contrast to this genetic stability, we observed unusually frequent mutation of the β-D-glycosidase gene (lacS) of a shuttle plasmid (pJlacS) propagated in Sulfolobus acidocaldarius. The resulting Lac(-) mutants also grew faster than the Lac(+) parent, thereby amplifying the impact of the frequent lacS mutations on the population. We developed a mutant accumulation assay and corrections for the effects of copy number and differential growth for this system; the resulting measurements and calculations yielded a corrected rate of 5.1 × 10(-4) mutational events at the lacS gene per plasmid replication. Analysis of independent lacS mutants revealed three types of mutations: (i) G · C-to-A · T transitions, (ii) slipped-strand events, and (iii) deletions. These mutations were frequent in plasmid-borne lacS expressed at a high level but not in single-copy lacS in the chromosome or at lower levels of expression in a plasmid. Substitution mutations arose at only two of 12 potential priming sites of the DNA primase of the pRN1 replicon, but nearly all these mutations created nonsense (chain termination) codons. The spontaneous mutation rate of plasmid-borne lacS was 175-fold higher under high-expression than under low-expression conditions. The results suggest that important DNA repair or replication fidelity functions are impaired or overwhelmed in pJlacS, with results analogous to those of the "transcription-associated mutagenesis" seen in bacteria and eukaryotes.

  10. Photochemical mutagenesis: examples and toxicological relevance.

    PubMed

    Gocke, E

    2001-01-01

    Induction of DNA damage as a consequence of exposure to UV light has been established as the major cause of skin cancer. DNA molecules absorb photon energy directly for wavelengths <320 nm, and lead to well-characterized mutagenic DNA damage. Alternatively, endogenous or exogenous chemicals (sensitizers) may absorb light with the potential of subsequent energy or electron transfer, and lead indirectly to DNA damage. A few light-absorbing pharmaceuticals have long been known to cause photo(geno)toxic effects. Notably, psoralen and chlorpromazine derivatives have been established as photomutagens and the reaction mechanisms have been identified; the fluoroquinolone antibiotics have more recently been recognized as being photomutagenic. The type of DNA damage and the modulation by antioxidants indicate the involvement of reactive oxygen species (ROS), but other mechanisms are also reported for, at least, some derivatives. In routine genotoxicity studies, we observed the photomutagenic activity of a compound (Ro 19-8022) under development as an anxiolytic agent in the Ames tester strain TA102 under normal laboratory illumination conditions. Further investigations showed strong photogenotoxic activity in tests for gene mutations and chromosomal aberrations in mammalian cells. The finding led to the termination of drug development. Another example of a pharmaceutical for which photogenotoxic properties were observed during development is Ro 47-7737, a bisquinoline derivative of the antimalaria compound chloroquine. Also in this case, the photochemical reactivity contributed to the termination of the development process. The risk/benefit assessment for the described compounds has to take into account the human exposure situation, for example, the ability to avoid light exposure during treatment. Consideration of photochemical mutagenesis is specifically important for sunscreen ingredients. The active components of sunscreen lotions are efficient UV absorbers. Consequently

  11. Predicting oligonucleotide-directed mutagenesis failures in protein engineering

    PubMed Central

    Wassman, Christopher D.; Tam, Phillip Y.; Lathrop, Richard H.; Weiss, Gregory A.

    2004-01-01

    Protein engineering uses oligonucleotide-directed mutagenesis to modify DNA sequences through a two-step process of hybridization and enzymatic synthesis. Inefficient reactions confound attempts to introduce mutations, especially for the construction of vast combinatorial protein libraries. This paper applied computational approaches to the problem of inefficient mutagenesis. Several results implicated oligonucleotide annealing to non-target sites, termed ‘cross-hybridization’, as a significant contributor to mutagenesis reaction failures. Test oligonucleotides demonstrated control over reaction outcomes. A novel cross-hybridization score, quickly computable for any plasmid and oligonucleotide mixture, directly correlated with yields of deleterious mutagenesis side products. Cross-hybridization was confirmed conclusively by partial incorporation of an oligonucleotide at a predicted cross-hybridization site, and by modification of putative template secondary structure to control cross-hybridization. Even in low concentrations, cross-hybridizing species in mixtures poisoned reactions. These results provide a basis for improved mutagenesis efficiencies and increased diversities of cognate protein libraries. PMID:15585664

  12. Genetic aspects of targeted insertion mutagenesis in yeasts.

    PubMed

    Klinner, U; Schäfer, B

    2004-05-01

    Targeted insertion mutagenesis is a main molecular tool of yeast science initially applied in Saccharomyces cerevisiae. The method was extended to fission yeast Schizosaccharomyces pombe and to "non-conventional" yeast species, which show specific properties of special interest to both basic and applied research. Consequently, the behaviour of such non-Saccharomyces yeasts is reviewed against the background of the knowledge of targeted insertion mutagenesis in S. cerevisiae. Data of homologous integration efficiencies obtained with circular, ends-in or ends-out vectors in several yeasts are compared. We follow details of targeted insertion mutagenesis in order to recognize possible rate-limiting steps. The route of the vector to the target and possible mechanisms of its integration into chromosomal genes are considered. Specific features of some yeast species are discussed. In addition, similar approaches based on homologous recombination that have been established for the mitochondrial genome of S. cerevisiae are described.

  13. Fluorescent protein engineering by in vivo site-directed mutagenesis

    PubMed Central

    Ceballos, Melvys Valledor; Hu, Qinghua; Schiller, Paul; Myers, Richard S.

    2012-01-01

    Summary In vivo site-directed mutagenesis by ssDNA recombineering is a facile method to change the color of fluorescent proteins without cloning. Two different starting alleles of GFP were targeted for mutagenesis: gfpmut3* residing in the E. coli genome and egfp carried by a bacterial/mammalian dual expression lentiviral plasmid vector. Fluorescent protein spectra were shifted by subtle modification of the chromophore region and residues interacting with the chromophore of the fluorescent protein. Eight different fluorescent proteins (Violeta, Azure, Aqua, Mar, Celeste, Amarillo, Mostaza and Bronze) were isolated and shown to be useful in multicolor imaging and flow cytometry of bacteria and transgenic human stem cells. To make in vivo site-directed mutagenesis more efficient, the recombineering method was optimized using the fluorescence change as a sensitive quantitative assay for recombination. A set of rules to simplify mutant isolation by recombineering is provided. PMID:22639380

  14. Empirical Complexities in the Genetic Foundations of Lethal Mutagenesis

    PubMed Central

    Bull, James J.; Joyce, Paul; Gladstone, Eric; Molineux, Ian J.

    2013-01-01

    From population genetics theory, elevating the mutation rate of a large population should progressively reduce average fitness. If the fitness decline is large enough, the population will go extinct in a process known as lethal mutagenesis. Lethal mutagenesis has been endorsed in the virology literature as a promising approach to viral treatment, and several in vitro studies have forced viral extinction with high doses of mutagenic drugs. Yet only one empirical study has tested the genetic models underlying lethal mutagenesis, and the theory failed on even a qualitative level. Here we provide a new level of analysis of lethal mutagenesis by developing and evaluating models specifically tailored to empirical systems that may be used to test the theory. We first quantify a bias in the estimation of a critical parameter and consider whether that bias underlies the previously observed lack of concordance between theory and experiment. We then consider a seemingly ideal protocol that avoids this bias—mutagenesis of virions—but find that it is hampered by other problems. Finally, results that reveal difficulties in the mere interpretation of mutations assayed from double-strand genomes are derived. Our analyses expose unanticipated complexities in testing the theory. Nevertheless, the previous failure of the theory to predict experimental outcomes appears to reside in evolutionary mechanisms neglected by the theory (e.g., beneficial mutations) rather than from a mismatch between the empirical setup and model assumptions. This interpretation raises the specter that naive attempts at lethal mutagenesis may augment adaptation rather than retard it. PMID:23934886

  15. 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.

  16. 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.

  17. Antimutagenic effects of cinnamaldehyde on chemical mutagenesis in Escherichia coli.

    PubMed

    Ohta, T; Watanabe, K; Moriya, M; Shirasu, Y; Kada, T

    1983-02-01

    Antimutagenic effects of cinnamaldehyde on mutagenesis by chemical agents were investigated in Escherichia coli WP2 uvrA- trpE-. Cinnamaldehyde, when added to agar medium, greatly reduced the number of Trp+ revertants induced by 4-nitroquinoline 1-oxide (4-NQO) without any decrease of cell viability. This antimutagenic effect could not be explained by inactivation of 4-NQO caused by direct interaction with cinnamaldehyde. Mutagenesis by furylfuramide (AF-2) was also suppressed significantly. Mutations induced by methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) were slightly inhibited. However, cinnamaldehyde was not at all effective on the mutagenesis of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Two derivatives of cinnamaldehyde, cinnamyl alcohol and trans-cinnamic acid, did not have as strong antimutagenic effects on 4-NQO mutagenesis as cinnamaldehyde had. Because cinnamaldehyde showed marked antimutagenic effects against mutations induced by UV-mimic mutagens but not those induced by MNNG or EMS, it seems that cinnamaldehyde might act by interfering with an inducible error-prone DNA repair pathway.

  18. 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…

  19. Now and future of mouse mutagenesis for human disease models.

    PubMed

    Gondo, Yoichi

    2010-09-01

    One of the major objectives of the Human Genome Project is to understand the biological function of the gene and genome as well as to develop clinical applications for human diseases. For this purpose, the experimental validations and preclinical trails by using animal models are indispensable. The mouse (Mus musculus) is one of the best animal models because genetics is well established in the mouse and embryonic manipulation technologies are also well developed. Large-scale mouse mutagenesis projects have been conducted to develop various mouse models since 1997. Originally, the phenotype-driven mutagenesis with N-ethyl-N-nitrosourea (ENU) has been the major efforts internationally then knockout/conditional mouse projects and gene-driven mutagenesis have been following. At the beginning, simple monogenic traits in the experimental condition have been elucidated. Then, more complex traits with variety of environmental interactions and gene-to-gene interactions (epistasis) have been challenged with mutant mice. In addition, chromosomal substitution strains and collaborative cross strains are also available to elucidate the complex traits in the mouse. Altogether, mouse models with mutagenesis and various laboratory strains will accelerate the studies of functional genomics in the mouse as well as in human. Copyright © 2010 Institute of Genetics and Developmental Biology and the Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

  20. Coupled mutagenesis screens and genetic mapping in zebrafish.

    PubMed Central

    Rawls, John F; Frieda, Matthew R; McAdow, Anthony R; Gross, Jason P; Clayton, Chad M; Heyen, Candy K; Johnson, Stephen L

    2003-01-01

    Forward genetic analysis is one of the principal advantages of the zebrafish model system. However, managing zebrafish mutant lines derived from mutagenesis screens and mapping the corresponding mutations and integrating them into the larger collection of mutations remain arduous tasks. To simplify and focus these endeavors, we developed an approach that facilitates the rapid mapping of new zebrafish mutations as they are generated through mutagenesis screens. We selected a minimal panel of 149 simple sequence length polymorphism markers for a first-pass genome scan in crosses involving C32 and SJD inbred lines. We also conducted a small chemical mutagenesis screen that identified several new mutations affecting zebrafish embryonic melanocyte development. Using our first-pass marker panel in bulked-segregant analysis, we were able to identify the genetic map positions of these mutations as they were isolated in our screen. Rapid mapping of the mutations facilitated stock management, helped direct allelism tests, and should accelerate identification of the affected genes. These results demonstrate the efficacy of coupling mutagenesis screens with genetic mapping. PMID:12663538

  1. A simple mutagenesis using natural competence in Tannerella forsythia.

    PubMed

    Nishikawa, Kiyoshi; Tanaka, Yoshinobu

    2013-09-01

    We report the discovery of natural competence in Tannerella forsythia and its application to targeted chromosomal mutagenesis. Keeping T. forsythia in a biofilm throughout the procedure allowed efficient DNA uptake and allelic replacement. This simple method is cost-effective and reproducible compared with the conventional protocols using broth culture and electroporation.

  2. 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...

  3. 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…

  4. Favipiravir elicits antiviral mutagenesis during virus replication in vivo

    PubMed Central

    Arias, Armando; Thorne, Lucy; Goodfellow, Ian

    2014-01-01

    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. DOI: http://dx.doi.org/10.7554/eLife.03679.001 PMID:25333492

  5. 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...

  6. Methods for targetted mutagenesis in gram-positive bacteria

    DOEpatents

    Yang, Yunfeng

    2014-05-27

    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.

  7. Structural evidence for the rare tautomer hypothesis of spontaneous mutagenesis

    SciTech Connect

    Wang, Weina; Hellinga, Homme W.; Beese, Lorena S.

    2012-05-10

    Even though high-fidelity polymerases copy DNA with remarkable accuracy, some base-pair mismatches are incorporated at low frequency, leading to spontaneous mutagenesis. Using high-resolution X-ray crystallographic analysis of a DNA polymerase that catalyzes replication in crystals, we observe that a C {center_dot} A mismatch can mimic the shape of cognate base pairs at the site of incorporation. This shape mimicry enables the mismatch to evade the error detection mechanisms of the polymerase, which would normally either prevent mismatch incorporation or promote its nucleolytic excision. Movement of a single proton on one of the mismatched bases alters the hydrogen-bonding pattern such that a base pair forms with an overall shape that is virtually indistinguishable from a canonical, Watson-Crick base pair in double-stranded DNA. These observations provide structural evidence for the rare tautomer hypothesis of spontaneous mutagenesis, a long-standing concept that has been difficult to demonstrate directly.

  8. Insertional mutagenesis of an industrial strain of Streptococcus thermophilus.

    PubMed

    Labarre, C; Schirawski, J; van der Zwet, A; Fitzgerald, G F; van Sinderen, D

    2001-06-12

    Random mutagenesis of an industrial strain of Streptococcus thermophilus was achieved through an adapted version of a two-plasmid system. The mutagenesis strategy is based on random integration of derivatives of the non-replicative (Rep(-)) plasmid pORI19 by means of homologous recombination following a temperature shift that eliminates replication of the temperature-sensitive (Rep(ts)) helper plasmid pVE6007. In this way mutants were generated which were affected in bacteriophage sensitivity or sucrose metabolism. Homologues were identified of a protein related to folate metabolism from a bacteriophage-resistant mutant and of two subunits of an oligopeptide transport system from a mutant deficient in sucrose utilisation.

  9. Nonrandom mutagenesis. Progress report, March 1, 1981-February 28, 1982

    SciTech Connect

    Goldsby, R.A.

    1981-01-01

    The ultimate goal is the development of tools, approaches and systems which will increase our ability to detect and control mutagenesis. We have continued to develop hybrid cell lines suited to the investigation of the expression and mutagenesis of human cell surface markers. The development and characterization of the monoclonal antibody probes to identify and characterize variation in selected human cell surface antigens has continued. Human X mouse T lymphoma hybrids have proven valuable in obtaining clonal populations expressing cell surface determinants characteristic of particular differentiated cell types. We have constructed a set of human lymphocyte X mouse T lymphoma hybrids which have proven useful for the mapping of cell surface determinants to particular chromosomes.

  10. European Community research on environmental mutagenesis and carcinogenesis.

    PubMed Central

    Sors, A I

    1993-01-01

    Within the 12 Member States of the European Community (EC), environmental policy is now formulated primarily at Community level. As a result, the EC has important regulatory responsibilities for the protection of workers, consumers, and the general public from risks that may arise from environmental chemicals, foremost among them potential carcinogens and mutagens. An important part of EC environmental research and development is intended to provide a scientific basis for these regulations as well as increasing understanding of the basic mechanisms involved in environmental carcinogenesis and mutagenesis. This paper contains a brief introduction to EC environment policy and research, followed by an overview of EC chemicals control activities that are of particular relevance to the research and development program. Community-level research on environmental mutagenesis and carcinogenesis is then reviewed in some detail, including the achievements of recent projects, the scientific content of the current program, and perspectives for the future. PMID:8143645

  11. Minimizing off-Target Mutagenesis Risks Caused by Programmable Nucleases

    PubMed Central

    Ishida, Kentaro; Gee, Peter; Hotta, Akitsu

    2015-01-01

    Programmable nucleases, such as zinc finger nucleases (ZFNs), transcription activator like effector nucleases (TALENs), and clustered regularly interspersed short palindromic repeats associated protein-9 (CRISPR-Cas9), hold tremendous potential for applications in the clinical setting to treat genetic diseases or prevent infectious diseases. However, because the accuracy of DNA recognition by these nucleases is not always perfect, off-target mutagenesis may result in undesirable adverse events in treated patients such as cellular toxicity or tumorigenesis. Therefore, designing nucleases and analyzing their activity must be carefully evaluated to minimize off-target mutagenesis. Furthermore, rigorous genomic testing will be important to ensure the integrity of nuclease modified cells. In this review, we provide an overview of available nuclease designing platforms, nuclease engineering approaches to minimize off-target activity, and methods to evaluate both on- and off-target cleavage of CRISPR-Cas9. PMID:26501275

  12. 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.

  13. Specific mutagenesis of a chlorophyll-binding protein. Progress report.

    SciTech Connect

    Eaton-Rye, Dr., Julian; Shen, Gaozhong

    1990-01-01

    During the first phase of the project regarding specific mutagenesis of the chlorophyll-binding protein CP47 in photosystem II (PS II) most of the time has been devoted to (1) establishment of an optimal procedure for the reintroduction of psbB (the gene encoding CP47) carrying a site-directed mutation into the experimental organism, the cyanobacterium Synechocystis sp. PCC 6803, (2) preparations for site-directed mutagenesis, and (3) creation and analysis of chimaeric spinach/cyanobacterial CP47 mutants of Synechocystis. In the coming year, psbB constructs with site-directed mutations in potential chlorophyll-binding regions of CP47 will be introduced into the Synechocystis genome, and site-directed mutants will be characterized according to procedures described in the original project description. In addition, analysis of chimaeric CP47 mutants will be continued.

  14. Environmental mutagenesis and radiation biology: The legacy of William Morgan.

    PubMed

    Schwartz, Jeffrey L

    2017-07-25

    A symposium entitled Environmental Mutagenesis and Radiation Biology was held on September 27, 2016 to honor the memory of Dr. William F. Morgan who passed away unexpectedly on November 13, 2015. The speakers presented the latest reviews on homologous recombination repair, induced genetic instability, bystander effects, and risk estimate development. Their presentations are presented following the introduction. Copyright © 2017. Published by Elsevier B.V.

  15. Transcriptional mutagenesis: causes and involvement in tumor development

    PubMed Central

    Brégeon, Damien; Doetsch, Paul W.

    2013-01-01

    The majority of normal cells in a human do not multiply continuously but are quiescent and devote most of their energy to gene transcription. When DNA damages in the transcribed strand of an active gene are bypassed by an RNA polymerase, they can miscode at the damaged site and produce mutant transcripts. This process known as transcriptional mutagenesis can lead to the production of mutant proteins that could be important in tumor development. PMID:21346784

  16. Transposon mutagenesis in Actinobacillus pleuropneumoniae with a Tn10 derivative.

    PubMed Central

    Tascon, R I; Rodriguez-Ferri, E F; Gutierrez-Martin, C B; Rodriguez-Barbosa, I; Berche, P; Vazquez-Boland, J A

    1993-01-01

    A transposon mutagenesis procedure functional in the gram-negative swine pathogen Actinobacillus pleuropneumoniae was developed for the first time. The technique involved the use of a suicide conjugative plasmid, pLOF/Km, carrying a mini-Tn10 with an isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible transposase located outside the mobile element (M. Herrero, V. de Lorenzo, and K. N. Timmis, J. Bacteriol. 172:6557-6567, 1990). The plasmid was mobilized from Escherichia coli to A. pleuropneumoniae through the RP4-mediated broad-host-range conjugal transfer functions provided by the chromosome of the donor strain. When IPTG was present in the mating medium, A. pleuropneumoniae CM5 transposon mutants were obtained at a frequency of 10(-5), while no mutants were detected in the absence of IPTG. Since the frequency of conjugal transfer of the RP4 plasmid from E. coli to A. pleuropneumoniae CM5 was found to be as low as 10(-4), the above result indicated that the expression level of the transposase was a critical factor for obtaining a workable efficiency of transposon mutagenesis. The transposon insertions occurred at random, as determined by Southern blotting of chromosomal DNA of randomly selected mutants and by the ability to generate mutants defective for the selected phenotypes. Almost all the mutants analyzed resulted from a single insertion of the Tn10 element. About 1.2% of the mutants resulted from the cointegration of pLOF/Km into the A. pleuropneumoniae chromosome. The applicability of this transposon mutagenesis system was verified on other A. pleuropneumoniae strains of different serotypes. The usefulness of this transposon mutagenesis system in genetic studies of A. pleuropneumoniae is discussed. Images PMID:8396122

  17. SOMA: a single oligonucleotide mutagenesis and cloning approach.

    PubMed

    Pfirrmann, Thorsten; Lokapally, Ashwin; Andréasson, Claes; Ljungdahl, Per; Hollemann, Thomas

    2013-01-01

    Modern biology research requires simple techniques for efficient and restriction site-independent modification of genetic material. Classical cloning and mutagenesis strategies are limited by their dependency on restriction sites and the use of complementary primer pairs. Here, we describe the Single Oligonucleotide Mutagenesis and Cloning Approach (SOMA) that is independent of restriction sites and only requires a single mutagenic oligonucleotide to modify a plasmid. We demonstrate the broad application spectrum of SOMA with three examples. First, we present a novel plasmid that in a standardized and rapid fashion can be used as a template for SOMA to generate GFP-reporters. We successfully use such a reporter to assess the in vivo knock-down quality of morpholinos in Xenopus laevis embryos. In a second example, we show how to use a SOMA-based protocol for restriction-site independent cloning to generate chimeric proteins by domain swapping between the two human hRMD5a and hRMD5b isoforms. Last, we show that SOMA simplifies the generation of randomized single-site mutagenized gene libraries. As an example we random-mutagenize a single codon affecting the catalytic activity of the yeast Ssy5 endoprotease and identify a spectrum of tolerated and non-tolerated substitutions. Thus, SOMA represents a highly efficient alternative to classical cloning and mutagenesis strategies.

  18. 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

  19. Maize-targeted mutagenesis: A knockout resource for maize.

    PubMed

    May, Bruce P; Liu, Hong; Vollbrecht, Erik; Senior, Lynn; Rabinowicz, Pablo D; Roh, Donna; Pan, Xiaokang; Stein, Lincoln; Freeling, Mike; Alexander, Danny; Martienssen, Rob

    2003-09-30

    We describe an efficient system for site-selected transposon mutagenesis in maize. A total of 43,776 F1 plants were generated by using Robertson's Mutator (Mu) pollen parents and self-pollinated to establish a library of transposon-mutagenized seed. The frequency of new seed mutants was between 10-4 and 10-5 per F1 plant. As a service to the maize community, maize-targeted mutagenesis selects insertions in genes of interest from this library by using the PCR. Pedigree, knockout, sequence, phenotype, and other information is stored in a powerful interactive database (maize-targeted mutagenesis database) that enables analysis of the entire population and the handling of knockout requests. By inhibiting Mu activity in most F1 plants, we sought to reduce somatic insertions that may cause false positives selected from pooled tissue. By monitoring the remaining Mu activity in the F2, however, we demonstrate that seed phenotypes depend on it, and false positives occur in lines that appear to lack it. We conclude that more than half of all mutations arising in this population are suppressed on losing Mu activity. These results have implications for epigenetic models of inbreeding and for functional genomics.

  20. GATMD: γ-Aminobutyric Acid Transporter Mutagenesis Database

    PubMed Central

    Anderson, Cynthia M.; Kidd, Patrick D.; Eskandari, Sepehr

    2010-01-01

    Since the cloning of the first γ-aminobutyric acid (GABA) transporter (GAT1; SLC6A1) from rat brain in 1990, more than 50 published studies have provided structure–function information on investigator-designed rat and mouse GAT1 mutants. To date, more than 200 of 599 GAT1 residues have been subjected to mutagenesis experiments by substitution with different amino acids, and the resulting transporter functional properties have significantly advanced our understanding of the mechanism of Na+- and Cl–-coupled GABA transport by this important member of the neurotransmitter:sodium symporter family. Moreover, many studies have addressed the functional consequences of amino acid deletion or insertion at various positions along the primary sequence. The enormity of this growing body of structure–function information has prompted us to develop GABA Transporter Mutagenesis Database (GATMD), a web-accessible, relational database of manually annotated biochemical, functional and pharmacological data reported on GAT1—the most intensely studied GABA transporter isoform. As of the last update of GATMD, 52 GAT1 mutagenesis papers have yielded 3360 experimental records, which collectively contain a total of ∼100 000 annotated parameters. Database URL: http://physiology.sci.csupomona.edu/GATMD/ PMID:21131297

  1. Use of liver cell cultures in mutagenesis studies

    SciTech Connect

    Huberman, E.; Jones, C.A.

    1980-09-30

    A sensitive cell-mediated assay has been developed for testing the mutagenesis of liver carcinogens. Mutagenesis was detected in Chinese hamster V79 cells that were cocultivated with hepatocytes isolated after collagenase/hyaluronidase digestion of rat liver slices. Mutations were characterized by resistance to ouabain and 6-thioguanine. Seven of the nitrosamines, which are potent liver carcinogens, exhibited a mutagenic response. Mutagenesis with these carcinogens could be detected at ..mu..molar doses. The polyaromatic hydrocarbon benzo(a)pyrene, which is not a liver carcinogen, but can cause fibrosarcomas, was not mutagenic in this assay, but was mutagenic in a fibroblast-mediated assay. The liver carcinogen, aflatoxin B/sub 1/, which usually does not induce fibrosarcomas, exhibited an inverse situation; it was mutagenic for V79 cells in the presence of liver cells but not in the presence of fibroblasts. We suggest that the use of various cell types, including hepatocytes prepared by the slicing method for carcinogen metabolism, and mutable V79 cells offers a sensitive assay for determining the mutagenic potential of chemical carcinogens, and may also allow a study of their organ specificity.

  2. Mutagenesis by the autoxidation of iron with isolated DNA

    SciTech Connect

    Loeb, L.A.; James, E.A.; Waltersdorph, A.M.; Klebanoff, S.J. )

    1988-06-01

    Oxygen free radicals are highly reactive species generated by many cellular oxidation-reduction processes. These radicals damage cellular constituents and have been casually implicated in the pathogenesis of many human diseases. The authors report here that oxygen free radicals generated by Fe{sup 2+} in aqueous solution are mutagenic. Aerobic incubation of {phi}X174 am3 (amber 3 mutation) DNA with Fe{sup 2+} results in decreased phage survival when the treated DNA is transfected into Escherichia coli spheroplasts. Transfection of the treated DNA into SOS-induced spheroplasts results in an increase in mutagenesis as great as 50-fold. Both killing and mutagenesis can be prevented by binding of Fe{sup 2+} with deferoxamine or by the addition of catalase or mannitol. These results suggest that DNA damage and mutagenesis brought about by Fe{sup 2+} are likely to occur by a Fenton-type mechanism. DNA sequence analysis of the Fe{sup 2+}-induced mutants indicates that reversion of the phage phenotype to wild type occurs largely by a transversion type of mutation involving substitution of deoxyadenosine for thymidine opposite a template deoxyadenosine. These findings raise the possibility that free iron localized in cellular DNA may cause mutations by the generation of oxygen free radicals.

  3. Screening for improved activity of a transglutaminase from Streptomyces mobaraensis created by a novel rational mutagenesis and random mutagenesis.

    PubMed

    Yokoyama, Keiichi; Utsumi, Hiroe; Nakamura, Takefumi; Ogaya, Daisuke; Shimba, Nobuhisa; Suzuki, Eiichiro; Taguchi, Seiichi

    2010-08-01

    Microbial transglutaminase (MTG) has been used extensively in academic research and the food industries through its cross-linking or posttranslational modification of proteins. Two enzyme engineering approaches were applied to improve MTG activity. One is a novel method of rational mutagenesis, called water-accessible surface hot-space region-oriented mutagenesis (WASH-ROM). One hundred and fifty-one point mutations were selected at 40 residues, bearing high solvent-accessibility surface area, within a 15 A space from the active site Cys64. Among them, 32 mutants showed higher specific activity than the wild type. The other is a random mutagenesis of the whole region of the MTG gene, coupled with a new plate assay screening system, using Corynebacterium Expression System CORYNEX. This in vivo system allowed us to readily distinguish the change in enzymatic activity by monitoring the intensity of enzymatic reaction-derived color zones surrounding recombinant cells. From the library of 24,000 mutants, ten were finally selected as beneficial mutants exhibiting higher specific activity than the wild type. Furthermore, we found that Ser199Ala mutant with additional N-terminal tetrapeptide showed the highest specific activity (1.7 times higher than the wild type). These various beneficial positions leading to increased specific activity of MTG were identified to achieve further enzyme improvements.

  4. EMS mutagenesis in the pea aphid Acyrthosiphon pisum.

    PubMed

    Tagu, Denis; Le Trionnaire, Gaël; Tanguy, Sylvie; Gauthier, Jean-Pierre; Huynh, Jean-René

    2014-04-16

    In aphids, clonal individuals can show distinct morphologic traits in response to environmental cues. Such phenotypic plasticity cannot be studied with classical genetic model organisms such as Caenorhabditis elegans or Drosophila melanogaster. The genetic basis of this biological process remain unknown, as mutations affecting this process are not available in aphids. Here, we describe a protocol to treat third-stage larvae with an alkylating mutagen, ethyl methanesulfonate (EMS), to generate random mutations within the Acyrthosiphon pisum genome. We found that even low concentrations of EMS were toxic for two genotypes of A. pisum. Mutagenesis efficiency was nevertheless assessed by estimating the occurrence of mutational events on the X chromosome. Indeed, any lethal mutation on the X-chromosome would kill males that are haploid on the X so that we used the proportion of males as an estimation of mutagenesis efficacy. We could assess a putative mutation rate of 0.4 per X-chromosome at 10 mM of EMS. We then applied this protocol to perform a small-scale mutagenesis on parthenogenetic individuals, which were screened for defects in their ability to produce sexual individuals in response to photoperiod shortening. We found one mutant line showing a reproducible altered photoperiodic response with a reduced production of males and the appearance of aberrant winged males (wing atrophy, alteration of legs morphology). This mutation appeared to be stable because it could be transmitted over several generations of parthenogenetic individuals. To our knowledge, this study represents the first example of an EMS-generated aphid mutant.

  5. Mitochondrial mutagenesis correlates with the local inflammatory environment in arthritis.

    PubMed

    Harty, Leonard C; Biniecka, Monika; O'Sullivan, Jacintha; Fox, Edward; Mulhall, Kevin; Veale, Douglas J; Fearon, Ursula

    2012-04-01

    To examine the association between mitochondrial mutagenesis and the proinflammatory microenvironment in patients with inflammatory arthritis. Fifty patients with inflammatory arthritis underwent arthroscopy and synovial tissue biopsies, synovial fluid and clinical assessment were obtained. Fifteen patients pre/post-TNFi therapy were also recruited. Normal synovial biopsies were obtained from 10 subjects undergoing interventional arthroscopy. Macroscopic synovitis/vascularity was measured by visual analogue scale. Cell-specific markers CD3 (T cells) and CD68 (macrophages) were quantified by immunohistology. TNFα, IL-6, IFNγ and IL-1β were measured in synovial fluids by MSD multiplex assays. Synovial tissue mitochondrial mutagenesis was quantified using a mitochondrial random mutation capture assay (RMCA). The direct effect of TNFα on oxidative stress and mitochondrial function was assessed in primary cultures of rheumatoid arthritis synovial fibroblast cells (RASFCs). Mitochondrial mutagenesis, reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and mitochondrial mass (MM) were quantified using the RMCA and specific cell fluorescent probes. A significant increase in mtDNA mutation frequency was demonstrated in inflamed synovial tissue compared with control (p<0.05), an effect that was independent of age. mtDNA mutations positively correlated with macroscopic synovitis (r=0.52, p<0.016), vascularity (r=0.54, p<0.01) and with synovial fluid cytokine levels of TNFα (r=0.74, p<0.024) and IFNγ (r=0.72, p<0.039). mtDNA mutation frequency post-TNFi therapy was significantly lower in patients with a DAS<3.2 (p<0.05) and associated with clinical and microscopic measures of disease (p<0.05). In vitro TNFα significantly induced mtDNA mutations, ROS, MM and MMP in RASFCs (all p<0.05). High mitochondrial mutations are strongly associated with synovial inflammation showing a direct link between mitochondrial mutations and key proinflammatory pathways.

  6. EMS Mutagenesis in the Pea Aphid Acyrthosiphon pisum

    PubMed Central

    Tagu, Denis; Le Trionnaire, Gaël; Tanguy, Sylvie; Gauthier, Jean-Pierre; Huynh, Jean-René

    2014-01-01

    In aphids, clonal individuals can show distinct morphologic traits in response to environmental cues. Such phenotypic plasticity cannot be studied with classical genetic model organisms such as Caenorhabditis elegans or Drosophila melanogaster. The genetic basis of this biological process remain unknown, as mutations affecting this process are not available in aphids. Here, we describe a protocol to treat third-stage larvae with an alkylating mutagen, ethyl methanesulfonate (EMS), to generate random mutations within the Acyrthosiphon pisum genome. We found that even low concentrations of EMS were toxic for two genotypes of A. pisum. Mutagenesis efficiency was nevertheless assessed by estimating the occurrence of mutational events on the X chromosome. Indeed, any lethal mutation on the X-chromosome would kill males that are haploid on the X so that we used the proportion of males as an estimation of mutagenesis efficacy. We could assess a putative mutation rate of 0.4 per X-chromosome at 10 mM of EMS. We then applied this protocol to perform a small-scale mutagenesis on parthenogenetic individuals, which were screened for defects in their ability to produce sexual individuals in response to photoperiod shortening. We found one mutant line showing a reproducible altered photoperiodic response with a reduced production of males and the appearance of aberrant winged males (wing atrophy, alteration of legs morphology). This mutation appeared to be stable because it could be transmitted over several generations of parthenogenetic individuals. To our knowledge, this study represents the first example of an EMS-generated aphid mutant. PMID:24531730

  7. [Analysis of Belamcanda chinensis with space flight mutagenesis by FTIR].

    PubMed

    Zhang, Hong-Mei; Guan, Ying; Shi, Jin-Shan

    2009-07-01

    The amorphous active components of the space mutagenesis Belamcanda chinensis and the ground group were measured, compared and analyzed. The purpose was to get a comprehensive understanding of the changes in quality of the 4th generation space mutagenesis Belamcanda chinensis, accumulate data for further studies, and try to establish the quality criterions of space mutagenesis Belamcanda chinensis. The result shows that the FTIR spectra of the space sample are almost the same as that of the ground one in terms of the main absorption peaks positions and shapes, but there are obvious differences in intensities. The intensity of the absorption peak at 1 642 and 1 318 cm(-1) of the space group was remarkably enhanced than the ground group, but at 1 541, 1 456, 1 417 and 1 051 cm(-1) it was decreased compared to the ground group. At the same time, the peak at 1 642 cm(-1) of the stretching vibration of C=O, the characteristic absorption of the keto, was remarkably enhanced. The peaks at 1 541 and 1 456 cm(-1) were assigned to C-C groups, the peak at 1 417 cm(-1) was due to the -CH2- groups, the peak at 1 318 cm(-1) was the characteristic absorption of calcium oxalate monohydrate, and the peak at 1 051 cm(-1) was assigned to C-O groups. It was shown that the relative content of flavone was increased distinctly. Space mutation breeding is conducive to breeding new varieties of highly active ingredients, it is also one of the ways to innovate germplasm resources of Chinese medicines efficiently. The effect of the space group is expected to be enhanced than the ground group, but it needs to be proved through further research.

  8. Pyrosequencing: Applicability for Studying DNA Damage-induced Mutagenesis

    PubMed Central

    Minko, Irina G.; Earley, Lauriel F.; Larlee, Kimberly E.; Lin, Ying-Chih; Lloyd, R. Stephen

    2014-01-01

    Site-specifically modified DNAs are routinely used in the study of DNA damage-induced mutagenesis. These analyses involve the creation of DNA vectors containing a lesion at a predetermined position, DNA replication, and detection of mutations at the target site. The final step has previously required the isolation of individual DNA clones, hybridization with radioactively-labeled probes, and verification of mutations by Sanger sequencing. In search for an alternative procedure that would allow direct quantification of sequence variants in a mixed population of DNA molecules, we evaluated the applicability of pyrosequencing to site-specific mutagenesis assays. The progeny DNAs were analyzed that originated from replication of N6-(deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine (MeFapy-dG)-containing vectors in primate cells, with the lesion being positioned in the 5′-GCNGG-3′ sequence context. Pyrosequencing detected ~8% G to T transversions and ~3.5% G to A transitions, a result that was in excellent agreement with frequencies previously measured by the standard procedure [Earley et al., 2013]. However, ~3.5% G to C transversions and ~2.0% deletions could not be detected by pyrosequencing. Consistent with these observations, the sensitivity of pyrosequencing for measuring the single deoxynucleotide variants differed depending on the deoxynucleotide identity, and in the given sequence contexts, was determined to be ~1-2% for A and T and ~5% for C. Pyrosequencing of other DNA isolates that were obtained following replication of MeFapy-dG-containing vectors in primate cells or Escherichia coli, identified several additional limitations. Collectively, our data demonstrated that pyrosequencing can be used for studying DNA damage-induced mutagenesis as an effective complementary experimental approach to current protocols. PMID:24962778

  9. Mutagenesis in Newts: Protocol for Iberian Ribbed Newts.

    PubMed

    Hayashi, Toshinori; Takeuchi, Takashi

    2016-01-01

    Newts have the remarkable capability of organ/tissue regeneration, and have been used as a unique experimental model for regenerative biology. The Iberian ribbed newt (Pleurodeles waltl) is suitable as a model animal. We have established methods for artificial insemination and efficient transgenesis using P. waltl newts. In addition to the transgenic technique, development of TALENs enables targeting mutagenesis in the newts. We have reported that TALENs efficiently disrupted targeted genes in newt embryos. In this chapter, we introduce a protocol for TALEN-mediated gene targeting in Iberian ribbed newts.

  10. Pollen tetrads in the detection of environmental mutagenesis

    SciTech Connect

    Mulcahy, D.L.

    1981-01-01

    Although pollen is a very sensitive indicator of environmental mutagenesis, it is also sensitive to nonmutagenic environmental stress. By analyzing pollen tetrads, rather than individual pollen grains, it is possible to distinguish between mutagenic and nonmutagenic influences. Another advantage of using pollen tetrads in mutagenicity studies is that it is possible to discriminate between pre- and post-pachytene mutations. This eliminates the mutant sector problem of a single mutational event giving rise to a large number of mutant cells. Methods of analyzing pollen tetrads are described.

  11. Chemical mutagenesis: an emerging issue for public health.

    PubMed Central

    Claxton, L D; Barry, P Z

    1977-01-01

    Chemical mutagens are recognized as prevalent in the environment and a potential threat to the health of future generations. This paper presents an overview of chemical mutagenesis as an issue for public health. Several problems in the determination of risk to human populations are discussed, including difficulties of extrapolating scientific data to humans, the latency period between exposure and recognizable genetic damage, and the large number of chemicals which must be tested. Test systems are described. Possibilities of control through federal regulation are discussed. PMID:911015

  12. 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.

  13. New approach for fish breeding by chemical mutagenesis: establishment of TILLING method in fugu (Takifugu rubripes) with ENU mutagenesis

    PubMed Central

    2013-01-01

    Background 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 INGenomes) 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. Results 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

  14. [Mechanism of arginine deiminase activity by site-directed mutagenesis].

    PubMed

    Li, Lifeng; Ni, Ye; Sun, Zhihao

    2012-04-01

    Arginine deiminase (ADI) has been studied as a potential anti-cancer agent for inhibiting arginine-auxotrophic tumors (such as melanomas and hepatocellular carcinomas) in phase III clinical trials. In this work, we studied the molecular mechanism of arginine deiminase activity by site-directed mutagenesis. Three mutation sites, A128, H404 and 1410, were introduced into wild-type ADI gene by QuikChange site-directed mutagenesis method, and four ADI mutants M1 (A128T), M2 (H404R), M3 (I410L), and M4 (A128T, H404R) were obtained. The ADI mutants were individually expressed in Escherichia coli BL21 (DE3), and the enzymatic properties of the purified mutant proteins were determined. The results show that both A128T and H404R had enhanced optimum pH, higher activity and stability of ADI under physiological condition (pH 7.4), as well as reduced K(m) value. This study provides an insight into the molecular mechanism of the ADI activity, and also the experimental evidence for the rational protein evolution in the future.

  15. Mechanisms of Base Substitution Mutagenesis in Cancer Genomes

    PubMed Central

    Bacolla, Albino; Cooper, David N.; Vasquez, Karen M.

    2014-01-01

    Cancer genome sequence data provide an invaluable resource for inferring the key mechanisms by which mutations arise in cancer cells, favoring their survival, proliferation and invasiveness. Here we examine recent advances in understanding the molecular mechanisms responsible for the predominant type of genetic alteration found in cancer cells, somatic single base substitutions (SBSs). Cytosine methylation, demethylation and deamination, charge transfer reactions in DNA, DNA replication timing, chromatin status and altered DNA proofreading activities are all now known to contribute to the mechanisms leading to base substitution mutagenesis. We review current hypotheses as to the major processes that give rise to SBSs and evaluate their relative relevance in the light of knowledge acquired from cancer genome sequencing projects and the study of base modifications, DNA repair and lesion bypass. Although gene expression data on APOBEC3B enzymes provide support for a role in cancer mutagenesis through U:G mismatch intermediates, the enzyme preference for single-stranded DNA may limit its activity genome-wide. For SBSs at both CG:CG and YC:GR sites, we outline evidence for a prominent role of damage by charge transfer reactions that follow interactions of the DNA with reactive oxygen species (ROS) and other endogenous or exogenous electron-abstracting molecules. PMID:24705290

  16. A mariner transposon vector adapted for mutagenesis in oral streptococci

    PubMed Central

    Nilsson, Martin; Christiansen, Natalia; Høiby, Niels; Twetman, Svante; Givskov, Michael; Tolker-Nielsen, Tim

    2014-01-01

    This article describes the construction and characterization of a mariner-based transposon vector designed for use in oral streptococci, but with a potential use in other Gram-positive bacteria. The new transposon vector, termed pMN100, contains the temperature-sensitive origin of replication repATs-pWV01, a selectable kanamycin resistance gene, a Himar1 transposase gene regulated by a xylose-inducible promoter, and an erythromycin resistance gene flanked by himar inverted repeats. The pMN100 plasmid was transformed into Streptococcus mutans UA159 and transposon mutagenesis was performed via a protocol established to perform high numbers of separate transpositions despite a low frequency of transposition. The distribution of transposon inserts in 30 randomly picked mutants suggested that mariner transposon mutagenesis is unbiased in S. mutans. A generated transposon mutant library containing 5000 mutants was used in a screen to identify genes involved in the production of sucrose-dependent extracellular matrix components. Mutants with transposon inserts in genes encoding glycosyltransferases and the competence-related secretory locus were predominantly found in this screen. PMID:24753509

  17. TALEN mediated somatic mutagenesis in murine models of cancer

    PubMed Central

    Zhang, Shuyuan; Li, Lin; Kendrick, Sara L.; Gerard, Robert D.; Zhu, Hao

    2014-01-01

    Cancer genome sequencing has identified numerous somatic mutations whose biological relevance is uncertain. In this study, we used genome-editing tools to create and analyze targeted somatic mutations in murine models of liver cancer. TALEN were designed against β-catenin (Ctnnb1) and Apc, two commonly mutated genes in hepatocellular carcinoma (HCC), to generate isogenic HCC cell lines. Both mutant cell lines exhibited evidence of Wnt pathway dysregulation. We asked if these TALENs could create targeted somatic mutations after hydrodynamic transfection (HDT) into mouse liver. TALENs targeting β-catenin promoted endogenous HCC carrying the intended gain-of-function mutations. However, TALENs targeting Apc were not as efficient in inducing in vivo homozygous loss-of-function mutations. We hypothesized that hepatocyte polyploidy might be protective against TALEN-induced loss of heterozygosity (LOH), and indeed Apc gene editing was less efficient in tetraploid than in diploid hepatocytes. To increase efficiency, we administered adenoviral Apc TALENs and found that we could achieve a higher mutagenesis rate in vivo. Our results demonstrate that genome-editing tools can enable the in vivo study of cancer genes and faithfully recapitulate the mosaic nature of mutagenesis in mouse cancer models. PMID:25070752

  18. Lethal Mutagenesis of Hepatitis C Virus Induced by Favipiravir

    PubMed Central

    de Ávila, Ana I.; Gallego, Isabel; Soria, Maria Eugenia; Gregori, Josep; Quer, Josep; Esteban, Juan Ignacio; Rice, Charles M.; Domingo, Esteban; Perales, Celia

    2016-01-01

    Lethal mutagenesis is an antiviral approach that consists in extinguishing a virus by an excess of mutations acquired during replication in the presence of a mutagen. Here we show that favipiravir (T-705) is a potent mutagenic agent for hepatitis C virus (HCV) during its replication in human hepatoma cells. T-705 leads to an excess of G → A and C → U transitions in the mutant spectrum of preextinction HCV populations. Infectivity decreased significantly in the presence of concentrations of T-705 which are 2- to 8-fold lower than its cytotoxic concentration 50 (CC50). Passaging the virus five times in the presence of 400 μM T-705 resulted in virus extinction. Since T-705 has undergone advanced clinical trials for approval for human use, the results open a new approach based on lethal mutagenesis to treat hepatitis C virus infections. If proven effective for HCV in vivo, this new anti-HCV agent may be useful in patient groups that fail current therapeutic regimens. PMID:27755573

  19. Lethal Mutagenesis of Hepatitis C Virus Induced by Favipiravir.

    PubMed

    de Ávila, Ana I; Gallego, Isabel; Soria, Maria Eugenia; Gregori, Josep; Quer, Josep; Esteban, Juan Ignacio; Rice, Charles M; Domingo, Esteban; Perales, Celia

    2016-01-01

    Lethal mutagenesis is an antiviral approach that consists in extinguishing a virus by an excess of mutations acquired during replication in the presence of a mutagen. Here we show that favipiravir (T-705) is a potent mutagenic agent for hepatitis C virus (HCV) during its replication in human hepatoma cells. T-705 leads to an excess of G → A and C → U transitions in the mutant spectrum of preextinction HCV populations. Infectivity decreased significantly in the presence of concentrations of T-705 which are 2- to 8-fold lower than its cytotoxic concentration 50 (CC50). Passaging the virus five times in the presence of 400 μM T-705 resulted in virus extinction. Since T-705 has undergone advanced clinical trials for approval for human use, the results open a new approach based on lethal mutagenesis to treat hepatitis C virus infections. If proven effective for HCV in vivo, this new anti-HCV agent may be useful in patient groups that fail current therapeutic regimens.

  20. Mariner-based transposon mutagenesis for Bacteroides species.

    PubMed

    Ichimura, Minoru; Uchida, Keiko; Nakayama-Imaohji, Haruyuki; Hirakawa, Hideki; Tada, Tomoyo; Morita, Hidetoshi; Yasutomo, Koji; Okazaki, Katsuichiro; Kuwahara, Tomomi

    2014-06-01

    Bacteroides is one of the most predominant groups of human gut microbiota. Recent metagenomic analyses and studies on gnotobiotic mice demonstrated the tight association of Bacteroides with epithelial function, the gut immune system and systemic metabolism in the host. The mariner family transposon shows relatively low target site specificity and has hosts ranging from prokaryotes to eukaryotes. Thereby, random mutagenesis using the mariner family transposon is expected to identify key molecules for human-Bacteroides symbiosis. In this study, we constructed the plasmid pMI07 to deliver the gene cassette (ermF/ITR), which harbors the erythromycin resistant marker (ermF) and the inverted repeat sequences (ITRs) recognized by Himar1 transposase, to Bacteroides via electrotransformation. pMI07 successfully delivered ermF/ITR to the Bacteroides genomes and generated thousands of insertion mutants/μg of pMI07 in B. thetaiotaomicron, B. fragilis, B. ovatus, and also, although to a lesser extent, B. vulgatus. Analyses of the ermF/ITR insertion sites in B. thetaiotaomicron and B. vulgatus revealed that the cassette targeted the dinucleotide TA and integrated into the genomes in an unbiased manner. The data reported here will provide useful information for transposon mutagenesis in Bacteroides species, which will enable identification of the genes responsible for their unique phenotypes. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Heritable site-specific mutagenesis using TALENs in maize.

    PubMed

    Char, Si Nian; Unger-Wallace, Erica; Frame, Bronwyn; Briggs, Sarah A; Main, Marcy; Spalding, Martin H; Vollbrecht, Erik; Wang, Kan; Yang, Bing

    2015-09-01

    Transcription activator-like effector nuclease (TALEN) technology has been utilized widely for targeted gene mutagenesis, especially for gene inactivation, in many organisms, including agriculturally important plants such as rice, wheat, tomato and barley. This report describes application of this technology to generate heritable genome modifications in maize. TALENs were employed to generate stable, heritable mutations at the maize glossy2 (gl2) locus. Transgenic lines containing mono- or di-allelic mutations were obtained from the maize genotype Hi-II at a frequency of about 10% (nine mutated events in 91 transgenic events). In addition, three of the novel alleles were tested for function in progeny seedlings, where they were able to confer the glossy phenotype. In a majority of the events, the integrated TALEN T-DNA segregated independently from the new loss of function alleles, producing mutated null-segregant progeny in T1 generation. Our results demonstrate that TALENs are an effective tool for genome mutagenesis in maize, empowering the discovery of gene function and the development of trait improvement.

  2. Mutagenesis of diploid mammalian genes by gene entrapment

    PubMed Central

    Lin, Qing; Donahue, Sarah L.; Moore-Jarrett, Tracy; Cao, Shang; Osipovich, Anna B.; Ruley, H. Earl

    2006-01-01

    The present study describes a genome-wide method for biallelic mutagenesis in mammalian cells. Novel poly(A) gene trap vectors, which contain features for direct cloning vector–cell fusion transcripts and for post-entrapment genome engineering, were used to generate a library of 979 mutant ES cells. The entrapment mutations generally disrupted gene expression and were readily transmitted through the germline, establishing the library as a resource for constructing mutant mice. Cells homozygous for most entrapment loci could be isolated by selecting for enhanced expression of an inserted neomycin-resistance gene that resulted from losses of heterozygosity (LOH). The frequencies of LOH measured at 37 sites in the genome ranged from 1.3 × 10−5 to 1.2 × 10−4 per cell and increased with increasing distance from the centromere, implicating mitotic recombination in the process. The ease and efficiency of obtaining homozygous mutations will (i) facilitate genetic studies of gene function in cultured cells, (ii) permit genome-wide studies of recombination events that result in LOH and mediate a type of chromosomal instability important in carcinogenesis, and (iii) provide new strategies for phenotype-driven mutagenesis screens in mammalian cells. PMID:17062627

  3. Mutagenesis of diploid mammalian genes by gene entrapment.

    PubMed

    Lin, Qing; Donahue, Sarah L; Moore-Jarrett, Tracy; Cao, Shang; Osipovich, Anna B; Ruley, H Earl

    2006-01-01

    The present study describes a genome-wide method for biallelic mutagenesis in mammalian cells. Novel poly(A) gene trap vectors, which contain features for direct cloning vector-cell fusion transcripts and for post-entrapment genome engineering, were used to generate a library of 979 mutant ES cells. The entrapment mutations generally disrupted gene expression and were readily transmitted through the germline, establishing the library as a resource for constructing mutant mice. Cells homozygous for most entrapment loci could be isolated by selecting for enhanced expression of an inserted neomycin-resistance gene that resulted from losses of heterozygosity (LOH). The frequencies of LOH measured at 37 sites in the genome ranged from 1.3 x 10(-5) to 1.2 x 10(-4) per cell and increased with increasing distance from the centromere, implicating mitotic recombination in the process. The ease and efficiency of obtaining homozygous mutations will (i) facilitate genetic studies of gene function in cultured cells, (ii) permit genome-wide studies of recombination events that result in LOH and mediate a type of chromosomal instability important in carcinogenesis, and (iii) provide new strategies for phenotype-driven mutagenesis screens in mammalian cells.

  4. Site-directed mutagenesis and saturation mutagenesis for the functional study of transcription factors involved in plant secondary metabolite biosynthesis.

    PubMed

    Pattanaik, Sitakanta; Werkman, Joshua R; Kong, Que; Yuan, Ling

    2010-01-01

    Regulation of gene expression is largely coordinated by a complex network of interactions between transcription factors (TFs), co-factors, and their cognate cis-regulatory elements in the genome. TFs are multidomain proteins that arise evolutionarily through protein domain shuffling. The modular nature of TFs has led to the idea that specific modules of TFs can be re-designed to regulate desired gene(s) through protein engineering. Utilization of designer TFs for the control of metabolic pathways has emerged as an effective approach for metabolic engineering. We are interested in engineering the basic helix-loop-helix (bHLH, Myc-type) transcription factors. Using site-directed and saturation mutagenesis, in combination with efficient and high-throughput screening systems, we have identified and characterized several amino acid residues critical for higher transactivation activity of a Myc-like bHLH transcription factor involved in anthocyanin biosynthetic pathway in plants. Site-directed and saturation mutagenesis should be generally applicable to engineering of all TFs.

  5. Establishment of a Counter-selectable Markerless Mutagenesis System in Veillonella atypica

    PubMed Central

    Zhou, Peng; Li, Xiaoli; Qi, Fengxia

    2015-01-01

    Using an alternative sigma factor ecf3 as target, we successfully established the first markerless mutagenesis system in the Veillonella genus. This system will be a valuable tool for mutagenesis of multiple genes for gene function analysis as well as for gene regulation studies in Veillonella. PMID:25771833

  6. Greetings from The International Association of Environmental Mutagenesis and Genomics Societies.

    PubMed

    Nohmi, Takehiko

    2015-01-01

    The International Association of Environmental Mutagenesis and Genomics Societies (IAEMGS) is an organization that promotes basic and applied research on environmental mutagenesis and genomics. In this article, as President of IAEMGS, I stress the important role of Genes and Environment to spread the voice of Asia to the international scientific community. Open access will support the journal in achieving this mission.

  7. A mouse chromosome 4 balancer ENU-mutagenesis screen isolates eleven lethal lines

    USDA-ARS?s Scientific Manuscript database

    ENU-mutagenesis is a powerful technique to identify genes regulating mammalian development. To functionally annotate the distal region of mouse chromosome 4, we performed an ENU-mutagenesis screen using a balancer chromosome targeted to this region of the genome. We isolated 11 lethal lines that map...

  8. Mutant fatty acid desaturase and methods for directed mutagenesis

    DOEpatents

    Shanklin, John [Shoreham, NY; Whittle, Edward J [Greenport, NY

    2008-01-29

    The present invention relates to methods for producing fatty acid desaturase mutants having a substantially increased activity towards substrates with fewer than 18 carbon atom chains relative to an unmutagenized precursor desaturase having an 18 carbon chain length specificity, the sequences encoding the desaturases and to the desaturases that are produced by the methods. The present invention further relates to a method for altering a function of a protein, including a fatty acid desaturase, through directed mutagenesis involving identifying candidate amino acid residues, producing a library of mutants of the protein by simultaneously randomizing all amino acid candidates, and selecting for mutants which exhibit the desired alteration of function. Candidate amino acids are identified by a combination of methods. Enzymatic, binding, structural and other functions of proteins can be altered by the method.

  9. Mutagenesis and differentiation induction in mammalian cells by environmental chemicals

    SciTech Connect

    Friedman, J.; Huberman, E.

    1980-01-01

    These studies indicate that in agreement with the somatic mutation hypothesis, chemical carcinogens: (1) are mutagenic for mammalian cells as tested in the cell-mediated assay; (2) the degree of mutagenicity is correlated with their degree of carcinogenicity; (3) that at least in cases when analyzed carefully the metabolites responsible for mutagenesis are also responsible for initiating the carcinogenic event; and (4) that a cell organ type specificity can be established using the cell-mediated assay. Studies with HL-60 cells and HO melanoma cells and those of others suggest that tumor-promoting phorbol diesters can alter cell differentiation in various cell types and that the degree of the observed alteration in the differentiation properties may be related to the potency of the phorbol esters. Thus these and similar systems may serve as models for both studies and identification of certain types of tumor promoting agents. (ERB)

  10. Environmental mutagenesis during the end-Permian ecological crisis.

    PubMed

    Visscher, Henk; Looy, Cindy V; Collinson, Margaret E; Brinkhuis, Henk; van Konijnenburg-van Cittert, Johanna H A; Kürschner, Wolfram M; Sephton, Mark A

    2004-08-31

    During the end-Permian ecological crisis, terrestrial ecosystems experienced preferential dieback of woody vegetation. Across the world, surviving herbaceous lycopsids played a pioneering role in repopulating deforested terrain. We document that the microspores of these lycopsids were regularly released in unseparated tetrads indicative of failure to complete the normal process of spore development. Although involvement of mutation has long been hinted at or proposed in theory, this finding provides concrete evidence for chronic environmental mutagenesis at the time of global ecological crisis. Prolonged exposure to enhanced UV radiation could account satisfactorily for a worldwide increase in land plant mutation. At the end of the Permian, a period of raised UV stress may have been the consequence of severe disruption of the stratospheric ozone balance by excessive emission of hydrothermal organohalogens in the vast area of Siberian Traps volcanism. Copyright 2004 The National Academy of Sciencs of the USA

  11. Environmental mutagenesis during the end-Permian ecological crisis

    PubMed Central

    Visscher, Henk; Looy, Cindy V.; Collinson, Margaret E.; Brinkhuis, Henk; van Konijnenburg-van Cittert, Johanna H. A.; Kürschner, Wolfram M.; Sephton, Mark A.

    2004-01-01

    During the end-Permian ecological crisis, terrestrial ecosystems experienced preferential dieback of woody vegetation. Across the world, surviving herbaceous lycopsids played a pioneering role in repopulating deforested terrain. We document that the microspores of these lycopsids were regularly released in unseparated tetrads indicative of failure to complete the normal process of spore development. Although involvement of mutation has long been hinted at or proposed in theory, this finding provides concrete evidence for chronic environmental mutagenesis at the time of global ecological crisis. Prolonged exposure to enhanced UV radiation could account satisfactorily for a worldwide increase in land plant mutation. At the end of the Permian, a period of raised UV stress may have been the consequence of severe disruption of the stratospheric ozone balance by excessive emission of hydrothermal organohalogens in the vast area of Siberian Traps volcanism. PMID:15282373

  12. Targeted Mutagenesis in Zebrafish Using Customized Zinc Finger Nucleases

    PubMed Central

    Foley, Jonathan E.; Maeder, Morgan L.; Pearlberg, Joseph; Joung, J. Keith; Peterson, Randall T.; Yeh, Jing-Ruey J.

    2009-01-01

    Zebrafish mutants have traditionally been obtained using random mutagenesis or retroviral insertions, methods that cannot be targeted to a specific gene and require laborious gene mapping and sequencing. Recently, we and others have shown that customized zinc finger nucleases (ZFNs) can introduce targeted frame-shift mutations with high efficiency, thereby enabling directed creation of zebrafish gene mutations. Here we describe a detailed protocol for constructing ZFN expression vectors, for generating and introducing ZFN-encoding RNAs into zebrafish embryos, and for identifying ZFN-generated mutations in targeted genomic sites. All of our vectors and methods are compatible with previously described Zinc Finger Consortium reagents for constructing engineered zinc finger arrays. Using these methods, zebrafish founders carrying targeted mutations can be identified within four months. PMID:20010934

  13. Combined Overlap Extension PCR Method for Improved Site Directed Mutagenesis

    PubMed Central

    Chong, Nikson Fatt-Ming

    2016-01-01

    The combined overlap extension PCR (COE-PCR) method developed in this work combines the strengths of the overlap extension PCR (OE-PCR) method with the speed and ease of the asymmetrical overlap extension (AOE-PCR) method. This combined method allows up to 6 base pairs to be mutated at a time and requires a total of 40–45 PCR cycles. A total of eight mutagenesis experiments were successfully carried out, with each experiment mutating between two to six base pairs. Up to four adjacent codons were changed in a single experiment. This method is especially useful for codon optimization, where doublet or triplet rare codons can be changed using a single mutagenic primer set, in a single experiment. PMID:27995143

  14. Mouse Mutagenesis Using N-Ethyl-N-Nitrosourea (ENU).

    PubMed

    Salinger, Andrew P; Justice, Monica J

    2008-04-01

    INTRODUCTIONThis protocol describes chemical mutagenesis of male mice using N-ethyl-N-nitrosourea (ENU), which is the most efficient method for obtaining mouse mutations in phenotype-driven screens. A fractionated dose of ENU, an alkylating agent, can produce a mutation rate as high as 1.5 × 10(-3) in male mouse spermatogonial stem cells. Treatment with ENU produces point mutations that provide a unique mutant resource: They reflect the consequences of single gene changes independent of position effects, provide a fine structure dissection of protein function, display a range of mutant effects from complete or partial loss of function to exaggerated function, and discover gene functions in an unbiased manner. After treatment with ENU, mice are mated in genetic screens designed to uncover mutations of interest. Screens for dominant, recessive, and modifying mutations can be performed.

  15. Statistical methods for building random transposon mutagenesis libraries.

    PubMed

    Will, Oliver

    2008-01-01

    During the construction of random transposon mutagenesis libraries, four essential statistical issues arise: (1) Computing basic probability results for number of open reading frame knockouts. (2) Estimating the number of new open reading frames that will be knockouts in the next set of clones. (3) Estimating the number of essential open reading frames. (4) Computing the probability that an open reading frame is essential given the distribution of insertions. This chapter examines these issues and evaluates potential solutions using three different approaches: Efron and Thisted's estimator, Will and Jacobs's parametric bootstrap, and Blades and Broman's Gibbs sampler. In doing so, this chapter provides guidance for using the R statistical project to solve these problems.

  16. New insights into behaviour using mouse ENU mutagenesis.

    PubMed

    Oliver, Peter L; Davies, Kay E

    2012-10-15

    Identifying genes involved in behavioural disorders in man is a challenge as the cause is often multigenic and the phenotype is modulated by environmental cues. Mouse mutants are a valuable tool for identifying novel pathways underlying specific neurological phenotypes and exploring the influence both genetic and non-genetic factors. Many human variants causing behavioural disorders are not gene deletions but changes in levels of expression or activity of a gene product; consequently, large-scale mouse ENU mutagenesis has the advantage over the study of null mutants in that it generates a range of point mutations that frequently mirror the subtlety and heterogeneity of human genetic lesions. ENU mutants have provided novel and clinically relevant functional information on genes that influence many aspects of mammalian behaviour, from neuropsychiatric endophenotypes to circadian rhythms. This review will highlight some of the most important findings that have been made using this method in several key areas of neurological disease research.

  17. Cationic Peptides Facilitate Iron-induced Mutagenesis in Bacteria.

    PubMed

    Rodríguez-Rojas, Alexandro; Makarova, Olga; Müller, Uta; Rolff, Jens

    2015-10-01

    Pseudomonas aeruginosa is the causative agent of chronic respiratory infections and is an important pathogen of cystic fibrosis patients. Adaptive mutations play an essential role for antimicrobial resistance and persistence. The factors that contribute to bacterial mutagenesis in this environment are not clear. Recently it has been proposed that cationic antimicrobial peptides such as LL-37 could act as mutagens in P. aeruginosa. Here we provide experimental evidence that mutagenesis is the product of a joint action of LL-37 and free iron. By estimating mutation rate, mutant frequencies and assessing mutational spectra in P. aeruginosa treated either with LL-37, iron or a combination of both we demonstrate that mutation rate and mutant frequency were increased only when free iron and LL-37 were present simultaneously. Colistin had the same effect. The addition of an iron chelator completely abolished this mutagenic effect, suggesting that LL-37 enables iron to enter the cells resulting in DNA damage by Fenton reactions. This was also supported by the observation that the mutational spectrum of the bacteria under LL-37-iron regime showed one of the characteristic Fenton reaction fingerprints: C to T transitions. Free iron concentration in nature and within hosts is kept at a very low level, but the situation in infected lungs of cystic fibrosis patients is different. Intermittent bleeding and damage to the epithelial cells in lungs may contribute to the release of free iron that in turn leads to generation of reactive oxygen species and deterioration of the respiratory tract, making it more susceptible to the infection.

  18. In vivo site-directed mutagenesis using oligonucleotides.

    PubMed

    Storici, F; Lewis, L K; Resnick, M A

    2001-08-01

    Functional characterization of the genes of higher eukaryotes has been aided by their expression in model organisms and by analyzing site-specific changes in homologous genes in model systems such as the yeast Saccharomyces cerevisiae. Modifying sequences in yeast or other organisms such that no heterologous material is retained requires in vitro mutagenesis together with subcloning. PCR-based procedures that do not involve cloning are inefficient or require multistep reactions that increase the risk of additional mutations. An alternative approach, demonstrated in yeast, relies on transformation with an oligonucleotide, but the method is restricted to the generation of mutants with a selectable phenotype. Oligonucleotides, when combined with gap repair, have also been used to modify plasmids in yeast; however, this approach is limited by restriction-site availability. We have developed a mutagenesis approach in yeast based on transformation by unpurified oligonucleotides that allows the rapid creation of site-specific DNA mutations in vivo. A two-step, cloning-free process, referred to as delitto perfetto, generates products having only the desired mutation, such as a single or multiple base change, an insertion, a small or a large deletion, or even random mutations. The system provides for multiple rounds of mutation in a window up to 200 base pairs. The process is RAD52 dependent, is not constrained by the distribution of naturally occurring restriction sites, and requires minimal DNA sequencing. Because yeast is commonly used for random and selective cloning of genomic DNA from higher eukaryotes such as yeast artificial chromosomes, the delitto perfetto strategy also provides an efficient way to create precise changes in mammalian or other DNA sequences.

  19. Precision Targeted Mutagenesis via Cas9 Paired Nickases in Rice

    PubMed Central

    Mikami, Masafumi; Toki, Seiichi; Endo, Masaki

    2016-01-01

    Recent reports of CRISPR- (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) mediated heritable mutagenesis in plants highlight the need for accuracy of the mutagenesis directed by this system. Off-target mutations are an important issue when considering functional gene analysis, as well as the molecular breeding of crop plants with large genome size, i.e. with many duplicated genes, and where the whole-genome sequence is still lacking. In mammals, off-target mutations can be suppressed by using Cas9 paired nickases together with paired guide RNAs (gRNAs). However, the performance of Cas9 paired nickases has not yet been fully assessed in plants. Here, we analyzed on- and off-target mutation frequency in rice calli and regenerated plants using Cas9 nuclease or Cas9 nickase with paired gRNAs. When Cas9 paired nickases were used, off-target mutations were fully suppressed in rice calli and regenerated plants. However, on-target mutation frequency also decreased compared with that induced by the Cas9 paired nucleases system. Since the gRNA sequence determines specific binding of Cas9 protein–gRNA ribonucleoproteins at the targeted sequence, the on-target mutation frequency of Cas9 paired nickases depends on the design of paired gRNAs. Our results suggest that a combination of gRNAs that can induce mutations at high efficiency with Cas9 nuclease should be used together with Cas9 nickase. Furthermore, we confirmed that a combination of gRNAs containing a one nucleotide (1 nt) mismatch toward the target sequence could not induce mutations when expressed with Cas9 nickase. Our results clearly show the effectiveness of Cas9 paired nickases in delivering on-target specific mutations. PMID:26936792

  20. CRISPR/Cas9-mediated targeted mutagenesis in Nicotiana tabacum.

    PubMed

    Gao, Junping; Wang, Genhong; Ma, Sanyuan; Xie, Xiaodong; Wu, Xiangwei; Zhang, Xingtan; Wu, Yuqian; Zhao, Ping; Xia, Qingyou

    2015-01-01

    Genome editing is one of the most powerful tools for revealing gene function and improving crop plants. Recently, RNA-guided genome editing using the type II clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system has been used as a powerful and efficient tool for genome editing in various organisms. Here, we report genome editing in tobacco (Nicotiana tabacum) mediated by the CRISPR/Cas9 system. Two genes, NtPDS and NtPDR6, were used for targeted mutagenesis. First, we examined the transient genome editing activity of this system in tobacco protoplasts, insertion and deletion (indel) mutations were observed with frequencies of 16.2-20.3% after transfecting guide RNA (gRNA) and the nuclease Cas9 in tobacco protoplasts. The two genes were also mutated using multiplexing gRNA at a time. Additionally, targeted deletions and inversions of a 1.8-kb fragment between two target sites in the NtPDS locus were demonstrated, while indel mutations were also detected at both the sites. Second, we obtained transgenic tobacco plants with NtPDS and NtPDR6 mutations induced by Cas9/gRNA. The mutation percentage was 81.8% for NtPDS gRNA4 and 87.5% for NtPDR6 gRNA2. Obvious phenotypes were observed, etiolated leaves for the psd mutant and more branches for the pdr6 mutant, indicating that highly efficient biallelic mutations occurred in both transgenic lines. No significant off-target mutations were obtained. Our results show that the CRISPR/Cas9 system is a useful tool for targeted mutagenesis of the tobacco genome.

  1. Mechanisms of uv mutagenesis in yeast and E. coli

    SciTech Connect

    Lawrence, C.; Christensen, R.; Christensen, J.R.; O'Brien, T.

    1983-01-01

    Experiments investigating ultraviolet light mutagenesis in either bakers' yeast, Saccharomyces cerevisiae, or E. coli have led to the following conclusions. First, cyclobutane pyrimidine dimers cause most mutations in both organisms; pyrimidine adducts, such as PyC, can account at best for only a small proportion. 86 percent of forward mutations induced at the E. coli lacI locus can be abolished by photoreactivation under conditions which do not alter the level of recA induction. About 75 percent of the forward mutations induced at the CAN1 locus of yeast could be removed by photoreactivation, a value that lies within the range observed previously for the reversion of CYC1 alleles (60 percent - 97 percent). Second, about 10 percent of the lacI forward mutations are untargeted, a smaller fraction than found previously for cycl-91 reversion in yeast. It is not yet clear whether the two species are really different in this respect, of whether the cycl-91 reversion site is a typical of the yeast genome at large. Third, analysis of reversion frequencies of 20 mutant alleles suggests that about 10 to 25 percent of all replication errors produced by mutagenic mechanisms in uv-irradiated yeast involve additions or deletions of base-pairs, indicating that error-prone repair does not just produce substitutions. Last, the REV1 locus in yeast is concerned with the induction of frameshift mutations at some, but not all, genetic sites, just as found previously for substitution mutations. The function of the REV3 gene is more widely, though not universally, required while the function of the RAD6 gene, like that of the recA locus in E. coli, appears to be necessary for all kinds of uv mutagenesis. E coli genes comparable to REV1 and REV3 have not yet been described; conversely, there does not yet appear to be a yeast equivalent of umuC.

  2. Structure-based design of combinatorial mutagenesis libraries.

    PubMed

    Verma, Deeptak; Grigoryan, Gevorg; Bailey-Kellogg, Chris

    2015-05-01

    The development of protein variants with improved properties (thermostability, binding affinity, catalytic activity, etc.) has greatly benefited from the application of high-throughput screens evaluating large, diverse combinatorial libraries. At the same time, since only a very limited portion of sequence space can be experimentally constructed and tested, an attractive possibility is to use computational protein design to focus libraries on a productive portion of the space. We present a general-purpose method, called "Structure-based Optimization of Combinatorial Mutagenesis" (SOCoM), which can optimize arbitrarily large combinatorial mutagenesis libraries directly based on structural energies of their constituents. SOCoM chooses both positions and substitutions, employing a combinatorial optimization framework based on library-averaged energy potentials in order to avoid explicitly modeling every variant in every possible library. In case study applications to green fluorescent protein, β-lactamase, and lipase A, SOCoM optimizes relatively small, focused libraries whose variants achieve energies comparable to or better than previous library design efforts, as well as larger libraries (previously not designable by structure-based methods) whose variants cover greater diversity while still maintaining substantially better energies than would be achieved by representative random library approaches. By allowing the creation of large-scale combinatorial libraries based on structural calculations, SOCoM promises to increase the scope of applicability of computational protein design and improve the hit rate of discovering beneficial variants. While designs presented here focus on variant stability (predicted by total energy), SOCoM can readily incorporate other structure-based assessments, such as the energy gap between alternative conformational or bound states.

  3. The yeast environmental stress response regulates mutagenesis induced by proteotoxic stress.

    PubMed

    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.

  4. 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

  5. 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.

  6. Improved antibiotic resistance gene cassette for marker exchange mutagenesis in Ralstonia solanacearum and Burkholderia species.

    PubMed

    Um, Hae Young; Chung, Eunsook; Lee, Jai-Heon; Lee, Seon-Woo

    2011-04-01

    Marker exchange mutagenesis is a fundamental approach to understanding gene function at a molecular level in bacteria. New plasmids carrying a kanamycin resistance gene or a trimethoprim resistance gene were constructed to provide antibiotic resistance cassettes for marker exchange mutagenesis in Ralstonia solanacearum and many antibiotic-resistant Burkholderia spp. Insertion sequences present in the flanking sequences of the antibiotic resistance cassette were removed to prevent aberrant gene replacement and polar mutation during mutagenesis in wild-type bacteria. Plasmids provided in this study would be convenient for use in gene cassettes for gene replacement in other Gram-negative bacteria.

  7. Cryptococcus neoformans Virulence Gene Discovery through Insertional Mutagenesis

    PubMed Central

    Idnurm, Alexander; Reedy, Jennifer L.; Nussbaum, Jesse C.; Heitman, Joseph

    2004-01-01

    Insertional mutagenesis was applied to Cryptococcus neoformans to identify genes associated with virulence attributes. Using biolistic transformation, we generated 4,300 nourseothricin (NAT)-resistant strains, of which 590 exhibited stable resistance. We focused on mutants with defects in established virulence factors and identified two with reduced growth at 37°C, four with reduced production of the antioxidant pigment melanin, and two with an increased sensitivity to nitric oxide (NO). The NAT insertion and mutant phenotypes were genetically linked in five of eight mutants, and the DNA flanking the insertions was characterized. For the strains with altered growth at 37°C and altered melanin production, mutations were in previously uncharacterized genes, while the two NO-sensitive strains bore insertions in the flavohemoglobin gene FHB1, whose product counters NO stress. Because of the frequent instability of nourseothricin resistance associated with biolistic transformation, Agrobacterium-mediated transformation was tested. This transkingdom DNA delivery approach produced 100% stable nourseothricin-resistant transformants, and three melanin-defective strains were identified from 576 transformants, of which 2 were linked to NAT in segregation analysis. One of these mutants contained a T-DNA insertion in the promoter of the LAC1 (laccase) gene, which encodes a key enzyme required for melanin production, while the second contained an insertion in the promoter of the CLC1 gene, encoding a voltage-gated chloride channel. Clc1 and its homologs are required for ion homeostasis, and in their absence Cu+ transport into the secretory pathway is compromised, depriving laccase and other Cu+-dependent proteins of their essential cofactor. The NAT resistance cassette was optimized for cryptococcal codon usage and GC content and was then used to disrupt a mitogen-activated protein kinase gene, a predicted gene, and two putative chloride channel genes to analyze their

  8. Radiation quality and mutagenesis in human lymphoblastoid cells.

    PubMed

    Liber, Howard L; Idate, Rupa; Warner, Christy; Bailey, Susan M

    2014-10-01

    , there was no clear evidence of a dose response for bystander mutagenesis, i.e., the MF plateaued. Interestingly, the magnitudes of the bystander MFs induced by different ion/energy combinations did vary, with bystander MFs ranging from 0.8 to 2.2× higher than the background. Furthermore, the nontargeted MFs appeared to reflect a mirror image of that for direct mutagenesis.

  9. Genes Necessary for Bacterial Magnetite Biomineralization Identified by Transposon Mutagenesis

    NASA Astrophysics Data System (ADS)

    Nash, C. Z.; Komeili, A.; Newman, D. K.; Kirschvink, J. L.

    2004-12-01

    Magnetic bacteria synthesize nanoscale crystals of magnetite in intracellular, membrane-bounded organelles (magnetosomes). These crystals are preserved in the fossil record at least as far back as the late Neoproterozoic and have been tentatively identified in much older rocks (1). This fossil record may provide deep time calibration points for molecular evolution studies once the genes involved in biologically controlled magnetic mineralization (BCMM) are known. Further, a genetic and biochemical understanding of BCMM will give insight into the depositional environment and biogeochemical cycles in which magnetic bacteria play a role. The BCMM process is not well understood, though proteins have been identified from the magnetosome membrane and genetic manipulation and biochemical characterization of these proteins are underway. Most of the proteins currently thought to be involved are encoded within the mam cluster, a large cluster of genes whose products localize to the magnetosome membrane and are conserved among magnetic bacteria (2). In an effort to identify all of the genes necessary for bacterial BCMM, we undertook a transposon mutagenesis of Magnetospirillum magneticum AMB-1. Non-magnetic mutants (MNMs) were identified by growth in liquid culture followed by a magnetic assay. The insertion site of the transposon was identified two ways. First MNMs were screened with a PCR assay to determine if the transposon had inserted into the mam cluster. Second, the transposon was rescued from the mutant DNA and cloned for sequencing. The majority insertion sites are located within the mam cluster. Insertion sites also occur in operons which have not previously been suspected to be involved in magnetite biomineralization. None of the insertion sites have occurred within genes reported from previous transposon mutagenesis studies of AMB-1 (3, 4). Two of the non-mam cluster insertion sites occur in operons containing genes conserved particularly between MS-1 and MC-1. We

  10. Analysis of HIV-2 Vpx by modeling and insertional mutagenesis

    SciTech Connect

    Mahnke, Lisa A. . E-mail: lmahnke@im.wustl.edu; Belshan, Michael; Ratner, Lee . E-mail: lratner@im.wustl.edu

    2006-04-25

    Vpx facilitates HIV-2 nuclear localization by a poorly understood mechanism. We have compared Vpx to an NMR structure HIV-1 Vpr in a central helical domain and probed regions of Vpx by insertional mutagenesis. A predicted loop between helices two and three appears to be unique, overlapping with a known novel nuclear localization signal. Overall, Vpx was found to be surprisingly flexible, tolerating a series of large insertions. We found that insertion within the polyproline-containing C-terminus destabilizes nuclear localization, whereas mutating a second helix in the central domain disrupts viral packaging. Other insertional mutants in the predicted loop and in a linker region between the central domain and the C-terminus may be useful as sites of intramolecular tags as they could be packaged adequately and retained preintegration complex associated integration activity in a serum starvation assay. An unexpected result was found within a previously defined nuclear localization motif near aa 71. This mutant retained robust nuclear localization in a GFP fusion assay and was competent for preintegration complex associated nuclear import. In summary, we have modeled helical content in Vpx and assessed potential sites of intramolecular tags which may prove useful for protein-protein interactions studies.

  11. Sleeping Beauty transposon mutagenesis in rat spermatogonial stem cells.

    PubMed

    Ivics, Zoltán; Izsvák, Zsuzsanna; Medrano, Gerardo; Chapman, Karen M; Hamra, F Kent

    2011-09-08

    We describe an experimental approach for generating mutant alleles in rat spermatogonial stem cells (SSCs) using Sleeping Beauty (SB) transposon-mediated insertional mutagenesis. The protocol is based on mobilization of mutagenic gene-trap transposons from transfected plasmid vectors into the genomes of cultured stem cells. Cells with transposon insertions in expressed genes are selected on the basis of activation of an antibiotic-resistance gene encoded by the transposon. These gene-trap clones are transplanted into the testes of recipient males (either as monoclonal or polyclonal libraries); crossing of these founders with wild-type females allows the insertions to be passed to F(1) progeny. This simple, economic and user-friendly methodological pipeline enables screens for functional gene annotation in the rat, with applicability in other vertebrate models where germ line-competent stem cells have been established. The complete protocol from transfection of SSCs to the genotyping of heterozygous F(1) offspring that harbor genomic SB gene-trap insertions takes 5-6 months.

  12. Oligonucleotide-directed mutagenesis for precision gene editing.

    PubMed

    Sauer, Noel J; Mozoruk, Jerry; Miller, Ryan B; Warburg, Zachary J; Walker, Keith A; Beetham, Peter R; Schöpke, Christian R; Gocal, Greg F W

    2016-02-01

    Differences in gene sequences, many of which are single nucleotide polymorphisms, underlie some of the most important traits in plants. With humanity facing significant challenges to increase global agricultural productivity, there is an urgent need to accelerate the development of these traits in plants. oligonucleotide-directed mutagenesis (ODM), one of the many tools of Cibus' Rapid Trait Development System (RTDS(™) ) technology, offers a rapid, precise and non-transgenic breeding alternative for trait improvement in agriculture to address this urgent need. This review explores the application of ODM as a precision genome editing technology, with emphasis on using oligonucleotides to make targeted edits in plasmid, episomal and chromosomal DNA of bacterial, fungal, mammalian and plant systems. The process of employing ODM by way of RTDS technology has been improved in many ways by utilizing a fluorescence conversion system wherein a blue fluorescent protein (BFP) can be changed to a green fluorescent protein (GFP) by editing a single nucleotide of the BFP gene (CAC→TAC; H66 to Y66). For example, dependent on oligonucleotide length, applying oligonucleotide-mediated technology to target the BFP transgene in Arabidopsis thaliana protoplasts resulted in up to 0.05% precisely edited GFP loci. Here, the development of traits in commercially relevant plant varieties to improve crop performance by genome editing technologies such as ODM, and by extension RTDS, is reviewed.

  13. ENU mutagenesis in mice identifies candidate genes for hypogonadism.

    PubMed

    Weiss, Jeffrey; Hurley, Lisa A; Harris, Rebecca M; Finlayson, Courtney; Tong, Minghan; Fisher, Lisa A; Moran, Jennifer L; Beier, David R; Mason, Christopher; Jameson, J Larry

    2012-06-01

    Genome-wide mutagenesis was performed in mice to identify candidate genes for male infertility, for which the predominant causes remain idiopathic. Mice were mutagenized using N-ethyl-N-nitrosourea (ENU), bred, and screened for phenotypes associated with the male urogenital system. Fifteen heritable lines were isolated and chromosomal loci were assigned using low-density genome-wide SNP arrays. Ten of the 15 lines were pursued further using higher-resolution SNP analysis to narrow the candidate gene regions. Exon sequencing of candidate genes identified mutations in mice with cystic kidneys (Bicc1), cryptorchidism (Rxfp2), restricted germ cell deficiency (Plk4), and severe germ cell deficiency (Prdm9). In two other lines with severe hypogonadism, candidate sequencing failed to identify mutations, suggesting defects in genes with previously undocumented roles in gonadal function. These genomic intervals were sequenced in their entirety and a candidate mutation was identified in SnrpE in one of the two lines. The line harboring the SnrpE variant retains substantial spermatogenesis despite small testis size, an unusual phenotype. In addition to the reproductive defects, heritable phenotypes were observed in mice with ataxia (Myo5a), tremors (Pmp22), growth retardation (unknown gene), and hydrocephalus (unknown gene). These results demonstrate that the ENU screen is an effective tool for identifying potential causes of male infertility.

  14. A Synthetic Approach to Stop-Codon Scanning Mutagenesis

    PubMed Central

    Nie, Lihua; Lavinder, Jason J.; Sarkar, Mohosin; Stephany, Kimberly; Magliery, Thomas J.

    2011-01-01

    A general combinatorial mutagenesis strategy using common DMT-protected mononucleotide phosphoramidites and a single orthogonally-protected trinucleotide phosphoramidite (Fmoc-TAG) was developed to scan a gene with the TAG amber stop codon with complete synthetic control. In combination with stop-codon suppressors that insert natural (e.g., alanine) or unnatural (e.g., p-benzoylphenylalanine or Bpa) amino acids, a single DNA library can be used to incorporate different amino acids for diverse purposes. Here, we scanned TAG codons through part of the gene for a model four-helix bundle protein, Rop, which regulates the copy number of ColE1 plasmids. Alanine was incorporated into Rop for mapping its binding site using an in vivo activity screen, and subtle but important differences from in vitro gel-shift studies of Rop function are evident. As a test, Bpa was incorporated using a Phe14 amber mutant isolated from the scanning library. Surprisingly, Phe14Bpa Rop is weakly active, despite the critical role of Phe14 in Rop activity. Bpa is a photoaffinity label unnatural amino acid that can form covalent bonds with adjacent molecules upon UV irradiation. Irradiation of Phe14Bpa-Rop, which is a dimer in solution like wild-type Rop, results in covalent dimers, trimers and tetramers. This suggests that Phe14Bpa Rop weakly associates as a tetramer in solution and highlights the use of Bpa crosslinking as a means of trapping weak and transient interactions. PMID:21452871

  15. Combinatorial mutagenesis and selection to understand and improve yeast promoters.

    PubMed

    Berg, Laila; Strand, Trine Aakvik; Valla, Svein; Brautaset, Trygve

    2013-01-01

    Microbial promoters are important targets both for understanding the global gene expression and developing genetic tools for heterologous expression of proteins and complex biosynthetic pathways. Previously, we have developed and used combinatorial mutagenesis methods to analyse and improve bacterial expression systems. Here, we present for the first time an analogous strategy for yeast. Our model promoter is the strong and inducible P AOX1 promoter in methylotrophic Pichia pastoris. The Zeocin resistance gene was applied as a valuable reporter for mutant P AOX1 promoter activity, and we used an episomal plasmid vector to ensure a constant reporter gene dosage in the yeast host cells. This novel design enabled direct selection for colonies of recombinant cells with altered Zeocin tolerance levels originating solely from randomly introduced point mutations in the P AOX1 promoter DNA sequence. We demonstrate that this approach can be used to select for P AOX1 promoter variants with abolished glucose repression in large mutant libraries. We also selected P AOX1 promoter variants with elevated expression level under induced conditions. The properties of the selected P AOX1 promoter variants were confirmed by expressing luciferase as an alternative reporter gene. The tools developed here should be useful for effective screening, characterization, and improvement of any yeast promoters.

  16. Embryonic Lethals and T-DNA Insertional Mutagenesis in Arabidopsis.

    PubMed Central

    Errampalli, D; Patton, D; Castle, L; Mickelson, L; Hansen, K; Schnall, J; Feldmann, K; Meinke, D

    1991-01-01

    T-DNA insertional mutagenesis represents a promising approach to the molecular isolation of genes with essential functions during plant embryo development. We describe in this report the isolation and characterization of 18 mutants of Arabidopsis thaliana defective in embryo development following seed transformation with Agrobacterium tumefaciens. Random T-DNA insertion was expected to result in a high frequency of recessive embryonic lethals because many target genes are required for embryogenesis. The cointegrate Ti plasmid used in these experiments contained the nopaline synthase and neomycin phosphotransferase gene markers. Nopaline assays and resistance to kanamycin were used to estimate the number of functional inserts present in segregating families. Nine families appeared to contain a T-DNA insert either within or adjacent to the mutant gene. Eight families were clearly not tagged with a functional insert and appeared instead to contain mutations induced during the transformation process. DNA gel blot hybridization with internal and right border probes revealed a variety of rearrangements associated with T-DNA insertion. A general strategy is presented to simplify the identification of tagged embryonic mutants and facilitate the molecular isolation of genes required for plant embryogenesis. PMID:12324593

  17. Establishment of Tn5096-Based Transposon Mutagenesis in Gordonia polyisoprenivorans

    PubMed Central

    Banh, Quyen; Arenskötter, Matthias; Steinbüchel, Alexander

    2005-01-01

    The transposons Tn5, Tn10, Tn611, and Tn5096 were characterized regarding transposition in Gordonia polyisoprenivorans strain VH2. No insertional mutants were obtained employing Tn5 or Tn10. The thermosensitive plasmid pCG79 harboring Tn611 integrated into the chromosome of G. polyisoprenivorans; however, the insertional mutants were fairly unstable und reverted frequently to the wild-type phenotype. In contrast, various stable mutants were obtained employing Tn5096-mediated transposon mutagenesis. Auxotrophic mutants, mutants defective or deregulated in carotenoid biosynthesis, and mutants defective in utilization of rubber and/or highly branched isoprenoid hydrocarbons were obtained by integration of plasmid pMA5096 harboring Tn5096 as a whole into the genome. From about 25,000 isolated mutants, the insertion loci of pMA5096 were subsequently mapped in 20 independent mutants in genes which could be related to the above-mentioned metabolic pathways or to putative regulation proteins. Analyses of the genotypes of pMA5096-mediated mutants defective in biodegradation of poly(cis-1,4-isoprene) did not reveal homologues to recently identified genes coding for enzymes catalyzing the initial cleavage of poly(cis-1,4-isoprene). One rubber-negative mutant was disrupted in mcr, encoding an α-methylacyl-coenzyme A racemase. This mutant was defective in degradation of poly(cis-1,4-isoprene) and also of highly branched isoprenoid hydrocarbons. PMID:16151089

  18. Radio frequency electromagnetic fields: cancer, mutagenesis, and genotoxicity.

    PubMed

    Heynick, Louis N; Johnston, Sheila A; Mason, Patrick A

    2003-01-01

    We present critiques of epidemiologic studies and experimental investigations, published mostly in peer-reviewed journals, on cancer and related effects from exposure to nonionizing electromagnetic fields in the nominal frequency range of 3 kHz to 300 GHz of interest to Subcommittee 4 (SC4) of the International Committee on Electromagnetic Safety (ICES). The major topics discussed are presented under the headings Epidemiologic and Other Findings on Human Exposure, Mammals Exposed In Vivo, Mammalian Live Tissues and Cell Preparations Exposed In Vitro, and Mutagenesis and Genotoxicity in Microorganisms and Fruit Flies. Under each major topic, we present minireviews of papers on various specific endpoints investigated. The section on Epidemiologic and Other Findings on Human Exposure is divided into two subsections, the first on possible carcinogenic effects of exposure from emitters not in physical contact with the populations studied, for example, transmitting antennas and other devices. Discussed in the second subsection are studies of postulated carcinogenic effects from use of mobile phones, with prominence given to brain tumors from use of cellular and cordless telephones in direct physical contact with an ear of each subject. In both subsections, some investigations yielded positive findings, others had negative findings, including papers directed toward experimentally verifying positive findings, and both were reported in a few instances. Further research on various important aspects may resolve such differences. Overall, however, the preponderance of published epidemiologic and experimental findings do not support the supposition that in vivo or in vitro exposures to such fields are carcinogenic.

  19. CRISPR/Cas9-mediated targeted mutagenesis in grape

    PubMed Central

    Ban, Yusuke; Azuma, Akifumi; Onoue, Noriyuki; Moriguchi, Takaya; Yamamoto, Toshiya; Toki, Seiichi

    2017-01-01

    RNA-guided genome editing using the CRISPR/Cas9 CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) system has been applied successfully in several plant species. However, to date, there are few reports on the use of any of the current genome editing approaches in grape—an important fruit crop with a large market not only for table grapes but also for wine. Here, we report successful targeted mutagenesis in grape (Vitis vinifera L., cv. Neo Muscat) using the CRISPR/Cas9 system. When a Cas9 expression construct was transformed to embryonic calli along with a synthetic sgRNA expression construct targeting the Vitis vinifera phytoene desaturase (VvPDS) gene, regenerated plants with albino leaves were obtained. DNA sequencing confirmed that the VvPDS gene was mutated at the target site in regenerated grape plants. Interestingly, the ratio of mutated cells was higher in lower, older, leaves compared to that in newly appearing upper leaves. This result might suggest either that the proportion of targeted mutagenized cells is higher in older leaves due to the repeated induction of DNA double strand breaks (DSBs), or that the efficiency of precise DSBs repair in cells of old grape leaves is decreased. PMID:28542349

  20. CRISPR/Cas9-mediated targeted mutagenesis in grape.

    PubMed

    Nakajima, Ikuko; Ban, Yusuke; Azuma, Akifumi; Onoue, Noriyuki; Moriguchi, Takaya; Yamamoto, Toshiya; Toki, Seiichi; Endo, Masaki

    2017-01-01

    RNA-guided genome editing using the CRISPR/Cas9 CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) system has been applied successfully in several plant species. However, to date, there are few reports on the use of any of the current genome editing approaches in grape-an important fruit crop with a large market not only for table grapes but also for wine. Here, we report successful targeted mutagenesis in grape (Vitis vinifera L., cv. Neo Muscat) using the CRISPR/Cas9 system. When a Cas9 expression construct was transformed to embryonic calli along with a synthetic sgRNA expression construct targeting the Vitis vinifera phytoene desaturase (VvPDS) gene, regenerated plants with albino leaves were obtained. DNA sequencing confirmed that the VvPDS gene was mutated at the target site in regenerated grape plants. Interestingly, the ratio of mutated cells was higher in lower, older, leaves compared to that in newly appearing upper leaves. This result might suggest either that the proportion of targeted mutagenized cells is higher in older leaves due to the repeated induction of DNA double strand breaks (DSBs), or that the efficiency of precise DSBs repair in cells of old grape leaves is decreased.

  1. Site Saturation Mutagenesis Applications on Candida methylica Formate Dehydrogenase

    PubMed Central

    Özgün, Gülşah P.; Ordu, Emel B.; Tütüncü, H. Esra; Yelboğa, Emrah; Sessions, Richard B.

    2016-01-01

    In NADH regeneration, Candida methylica formate dehydrogenase (cmFDH) is a highly significant enzyme in pharmaceutical industry. In this work, site saturation mutagenesis (SSM) which is a combination of both rational design and directed evolution approaches is applied to alter the coenzyme specificity of NAD+-dependent cmFDH from NAD+ to NADP+ and increase its thermostability. For this aim, two separate libraries are constructed for screening a change in coenzyme specificity and an increase in thermostability. To alter the coenzyme specificity, in the coenzyme binding domain, positions at 195, 196, and 197 are subjected to two rounds of SSM and screening which enabled the identification of two double mutants D195S/Q197T and D195S/Y196L. These mutants increase the overall catalytic efficiency of NAD+ to 5.6 × 104-fold and 5 × 104-fold value, respectively. To increase the thermostability of cmFDH, the conserved residue at position 1 in the catalytic domain of cmFDH is subjected to SSM. The thermodynamic and kinetic results suggest that 8 mutations on the first residue can be tolerated. Among all mutants, M1L has the best residual activity after incubation at 60°C with 17%. These studies emphasize that SSM is an efficient method for creating “smarter libraries” for improving the properties of cmFDH. PMID:27847673

  2. Lethal Mutagenesis of HIV with Mutagenic Nucleoside Analogs

    NASA Astrophysics Data System (ADS)

    Loeb, Lawrence A.; Essigmann, John M.; Kazazi, Farhad; Zhang, Jue; Rose, Karl D.; Mullins, James I.

    1999-02-01

    The human immunodeficiency virus (HIV) replicates its genome and mutates at exceptionally high rates. As a result, the virus is able to evade immunological and chemical antiviral agents. We tested the hypothesis that a further increase in the mutation rate by promutagenic nucleoside analogs would abolish viral replication. We evaluated deoxynucleoside analogs for lack of toxicity to human cells, incorporation by HIV reverse transcriptase, resistance to repair when incorporated into the DNA strand of an RNA\\cdot DNA hybrid, and mispairing at high frequency. Among the candidates tested, 5-hydroxydeoxycytidine (5-OH-dC) fulfilled these criteria. In seven of nine experiments, the presence of this analog resulted in the loss of viral replicative potential after 9-24 sequential passages of HIV in human CEM cells. In contrast, loss of viral replication was not observed in 28 control cultures passaged in the absence of the nucleoside analog, nor with other analogs tested. Sequence analysis of a portion of the HIV reverse transcriptase gene demonstrated a disproportionate increase in G -> A substitutions, mutations predicted to result from misincorporation of 5-OH-dC into the cDNA during reverse transcription. Thus, "lethal mutagenesis" driven by the class of deoxynucleoside analogs represented by 5-OH-dC could provide a new approach to treating HIV infections and, potentially, other viral infections.

  3. CRISPR/Cas-mediated targeted mutagenesis in Daphnia magna.

    PubMed

    Nakanishi, Takashi; Kato, Yasuhiko; Matsuura, Tomoaki; Watanabe, Hajime

    2014-01-01

    The water flea Daphnia magna has been used as an animal model in ecology, evolution, and environmental sciences. Thanks to the recent progress in Daphnia genomics, genetic information such as the draft genome sequence and expressed sequence tags (ESTs) is now available. To investigate the relationship between phenotypes and the available genetic information about Daphnia, some gene manipulation methods have been developed. However, a technique to induce targeted mutagenesis into Daphnia genome remains elusive. To overcome this problem, we focused on an emerging genome editing technique mediated by the clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) system to introduce genomic mutations. In this study, we targeted a functionally conserved regulator of eye development, the eyeless gene in D. magna. When we injected Cas9 mRNAs and eyeless-targeting guide RNAs into eggs, 18-47% of the survived juveniles exhibited abnormal eye morphology. After maturation, up to 8.2% of the adults produced progenies with deformed eyes, which carried mutations in the eyeless loci. These results showed that CRISPR/Cas system could introduce heritable mutations into the endogenous eyeless gene in D. magna. This is the first report of a targeted gene knockout technique in Daphnia and will be useful in uncovering Daphnia gene functions.

  4. Analysis of mammalian cytochrome P450 structure and function by site-directed mutagenesis.

    PubMed

    Domanski, T L; Halpert, J R

    2001-06-01

    Over the past decade, site-directed mutagenesis has become an essential tool in the study of mammalian cytochrome P450 structure-function relationships. Residues affecting substrate specificity, cooperativity, membrane localization, and interactions with redox partners have been identified using a combination of amino-acid sequence alignments, homology modeling, chimeragenesis, and site-directed mutagenesis. As homology modeling and substrate docking technology continue to improve, the ability to predict more precise functions for specific residues will also advance, making it possible to utilize site-directed mutagenesis to test these predictions. Future studies will employ site-directed mutagenesis to learn more about cytochrome P450 substrate access channels, to define the role of residues that do not lie within substrate recognition sites, to engineer additional soluble forms of microsomal cytochromes P450 for x-ray crystallography, and to engineer more efficient enzymes for drug activation and/or bioremediation.

  5. Simple and efficient oligonucleotide-directed mutagenesis using one primer and circular plasmid DNA template.

    PubMed

    Marotti, K R; Tomich, C S

    1989-01-01

    A rapid and simple procedure for site-directed mutagenesis is described. This method uses only a single oligonucleotide primer with the double-stranded circular plasmid DNA as the template for mutagenesis. The phage T4 gene 32 product is included during primer extension in vitro to increase efficiency. Single and multiple changes as well as deletions have been obtained at an efficiency of 1-2%.

  6. Extinction of Hepatitis C Virus by Ribavirin in Hepatoma Cells Involves Lethal Mutagenesis

    PubMed Central

    Ortega-Prieto, Ana M.; Sheldon, Julie; Grande-Pérez, Ana; Tejero, Héctor; Gregori, Josep; Quer, Josep; Esteban, Juan I.; Domingo, Esteban; Perales, Celia

    2013-01-01

    Lethal mutagenesis, or virus extinction produced by enhanced mutation rates, is under investigation as an antiviral strategy that aims at counteracting the adaptive capacity of viral quasispecies, and avoiding selection of antiviral-escape mutants. To explore lethal mutagenesis of hepatitis C virus (HCV), it is important to establish whether ribavirin, the purine nucleoside analogue used in anti-HCV therapy, acts as a mutagenic agent during virus replication in cell culture. Here we report the effect of ribavirin during serial passages of HCV in human hepatoma Huh-7.5 cells, regarding viral progeny production and complexity of mutant spectra. Ribavirin produced an increase of mutant spectrum complexity and of the transition types associated with ribavirin mutagenesis, resulting in HCV extinction. Ribavirin-mediated depletion of intracellular GTP was not the major contributory factor to mutagenesis since mycophenolic acid evoked a similar decrease in GTP without an increase in mutant spectrum complexity. The intracellular concentration of the other nucleoside-triphosphates was elevated as a result of ribavirin treatment. Mycophenolic acid extinguished HCV without an intervening mutagenic activity. Ribavirin-mediated, but not mycophenolic acid-mediated, extinction of HCV occurred via a decrease of specific infectivity, a feature typical of lethal mutagenesis. We discuss some possibilities to explain disparate results on ribavirin mutagenesis of HCV. PMID:23976977

  7. Genetic Regulation of Charged Particle Mutagenesis in Human Cells

    NASA Technical Reports Server (NTRS)

    Kronenberg, Amy; Gauny, S.; Cherbonnel-Lasserre, C.; Liu, W.; Wiese, C.

    1999-01-01

    Our studies use a series of syngeneic, and where possible, isogenic human B-lymphoblastoid cell lines to assess the genetic factors that modulate susceptibility apoptosis and their impact on the mutagenic risks of low fluence exposures to 1 GeV Fe ions and 55 MeV protons. These ions are representative of the types of charged particle radiation that are of particular significance for human health in the space radiation environment. The model system employs cell lines derived from the male donor WIL-2. These cells have a single X chromosome and they are hemizygous for one mutation marker, hypoxanthine phosphoribosyltransferase (HPRT). TK6 and WTK1 cells were each derived from descendants of WIL-2 and were each selected as heterozygotes for a second mutation marker, the thymidine kinase (TK) gene located on chromosome 17q. The HPRT and TK loci can detect many different types of mutations, from single basepair substitutions up to large scale loss of heterozygosity (LOH). The single expressing copy of TK in the TK6 and WTKI cell lines is found on the same copy of chromosome 17, and this allele can be identified by a restriction fragment length polymorphism (RFLP) identified when high molecular weight DNA is digested by the SacI restriction endonuclease and hybridized against the cDNA probe for TK. A large series of polymorphic linked markers has been identified that span more than 60 cM of DNA (approx. 60 megabasepairs) and distinguish the copy of chromosome 17 bearing the initially active TK allele from the copy of chromosome 17 bearing the silent TK allele in both TK6 and WTKI cells. TK6 cells express normal p53 protein while WTKI cells express homozygous mutant p53. Expression of mutant p53 can increase susceptibility to x-ray-induced mutations. It's been suggested that the increased mutagenesis in p53 mutant cells might be due to reduced apoptosis.

  8. Tetragonal Lysozyme Interactions Studied by Site Directed Mutagenesis

    NASA Technical Reports Server (NTRS)

    Crawford, Lisa; Karr, Laurel J.; Nadarajah, Arunan; Pusey, Marc

    1999-01-01

    A number of recent experimental and theoretical studies have indicated that tetragonal lysozyme crystal growth proceeds by the addition of aggregates, formed by reversible self association of the solute molecules in the bulk solution. Periodic bond chain and atomic force microscopy studies have indicated that the probable growth unit is at minimum a 43 tetramer, and most likely an octamer composed of two complete turns about the 43 axis. If these results are correct, then there are intermolecular interactions which are only formed in the solution and others only formed at the joining of the growth unit to the crystal surface. We have set out to study these interactions, and the correctness of this hypothesis, using site directed mutagenesis of specific amino acid residues involved in the different bonds. We had initially expressed wild type lysozyme in S. cervasiae with yields of approximately 5 mg/L, which were eventually raised to approximately 40 mg/L. We are now moving the expression to the Pichia system, with anticipated yields of 300 to (3)500 mg/L, comparable to what can be obtained from egg whites. An additional advantage of using recombinant protein is the greater genetic homogeneity of the material obtained and the absence of any other contaminating egg proteins. The first mutation experiments are TYR 23 (Registered) PHE or ALA and ASN 113 (Registered) ALA or ASP. Both TYR 23 and ASN 113 form part of the postulated dimerization intermolecular binding site which lead to the formation of the 43 helix. Tyrosine also participates in an intermolecular hydrogen bond with ARG 114. The results of these and subsequent experiments will be discussed.

  9. Structure-based design of combinatorial mutagenesis libraries

    PubMed Central

    Verma, Deeptak; Grigoryan, Gevorg; Bailey-Kellogg, Chris

    2015-01-01

    The development of protein variants with improved properties (thermostability, binding affinity, catalytic activity, etc.) has greatly benefited from the application of high-throughput screens evaluating large, diverse combinatorial libraries. At the same time, since only a very limited portion of sequence space can be experimentally constructed and tested, an attractive possibility is to use computational protein design to focus libraries on a productive portion of the space. We present a general-purpose method, called “Structure-based Optimization of Combinatorial Mutagenesis” (SOCoM), which can optimize arbitrarily large combinatorial mutagenesis libraries directly based on structural energies of their constituents. SOCoM chooses both positions and substitutions, employing a combinatorial optimization framework based on library-averaged energy potentials in order to avoid explicitly modeling every variant in every possible library. In case study applications to green fluorescent protein, β-lactamase, and lipase A, SOCoM optimizes relatively small, focused libraries whose variants achieve energies comparable to or better than previous library design efforts, as well as larger libraries (previously not designable by structure-based methods) whose variants cover greater diversity while still maintaining substantially better energies than would be achieved by representative random library approaches. By allowing the creation of large-scale combinatorial libraries based on structural calculations, SOCoM promises to increase the scope of applicability of computational protein design and improve the hit rate of discovering beneficial variants. While designs presented here focus on variant stability (predicted by total energy), SOCoM can readily incorporate other structure-based assessments, such as the energy gap between alternative conformational or bound states. PMID:25611189

  10. Genetic Regulation of Charged Particle Mutagenesis in Human Cells

    NASA Technical Reports Server (NTRS)

    Kronenberg, Amy; Gauny, S.; Cherbonnel-Lasserre, C.; Liu, W.; Wiese, C.

    1999-01-01

    Our studies use a series of syngeneic, and where possible, isogenic human B-lymphoblastoid cell lines to assess the genetic factors that modulate susceptibility apoptosis and their impact on the mutagenic risks of low fluence exposures to 1 GeV Fe ions and 55 MeV protons. These ions are representative of the types of charged particle radiation that are of particular significance for human health in the space radiation environment. The model system employs cell lines derived from the male donor WIL-2. These cells have a single X chromosome and they are hemizygous for one mutation marker, hypoxanthine phosphoribosyltransferase (HPRT). TK6 and WTK1 cells were each derived from descendants of WIL-2 and were each selected as heterozygotes for a second mutation marker, the thymidine kinase (TK) gene located on chromosome 17q. The HPRT and TK loci can detect many different types of mutations, from single basepair substitutions up to large scale loss of heterozygosity (LOH). The single expressing copy of TK in the TK6 and WTKI cell lines is found on the same copy of chromosome 17, and this allele can be identified by a restriction fragment length polymorphism (RFLP) identified when high molecular weight DNA is digested by the SacI restriction endonuclease and hybridized against the cDNA probe for TK. A large series of polymorphic linked markers has been identified that span more than 60 cM of DNA (approx. 60 megabasepairs) and distinguish the copy of chromosome 17 bearing the initially active TK allele from the copy of chromosome 17 bearing the silent TK allele in both TK6 and WTKI cells. TK6 cells express normal p53 protein while WTKI cells express homozygous mutant p53. Expression of mutant p53 can increase susceptibility to x-ray-induced mutations. It's been suggested that the increased mutagenesis in p53 mutant cells might be due to reduced apoptosis.

  11. Stabilization of a prokaryotic LAT transporter by random mutagenesis.

    PubMed

    Rodríguez-Banqueri, Arturo; Errasti-Murugarren, Ekaitz; Bartoccioni, Paola; Kowalczyk, Lukasz; Perálvarez-Marín, Alex; Palacín, Manuel; Vázquez-Ibar, José Luis

    2016-04-01

    The knowledge of three-dimensional structures at atomic resolution of membrane transport proteins has improved considerably our understanding of their physiological roles and pathological implications. However, most structural biology techniques require an optimal candidate within a protein family for structural determination with (a) reasonable production in heterologous hosts and (b) good stability in detergent micelles. SteT, the Bacillus subtilis L-serine/L-threonine exchanger is the best-known prokaryotic paradigm of the mammalian L-amino acid transporter (LAT) family. Unfortunately, SteT's lousy stability after extracting from the membrane prevents its structural characterization. Here, we have used an approach based on random mutagenesis to engineer stability in SteT. Using a split GFP complementation assay as reporter of protein expression and membrane insertion, we created a library of 70 SteT mutants each containing random replacements of one or two residues situated in the transmembrane domains. Analysis of expression and monodispersity in detergent of this library permitted the identification of evolved versions of SteT with a significant increase in both expression yield and stability in detergent with respect to wild type. In addition, these experiments revealed a correlation between the yield of expression and the stability in detergent micelles. Finally, and based on protein delipidation and relipidation assays together with transport experiments, possible mechanisms of SteT stabilization are discussed. Besides optimizing a member of the LAT family for structural determination, our work proposes a new approach that can be used to optimize any membrane protein of interest. © 2016 Rodríguez-Banqueri et al.

  12. Biophysical Optimization of a Therapeutic Protein by Nonstandard Mutagenesis

    PubMed Central

    Pandyarajan, Vijay; Phillips, Nelson B.; Cox, Gabriela P.; Yang, Yanwu; Whittaker, Jonathan; Ismail-Beigi, Faramarz; Weiss, Michael A.

    2014-01-01

    Insulin provides a model for the therapeutic application of protein engineering. A paradigm in molecular pharmacology was defined by design of rapid-acting insulin analogs for the prandial control of glycemia. Such analogs, a cornerstone of current diabetes regimens, exhibit accelerated subcutaneous absorption due to more rapid disassembly of oligomeric species relative to wild-type insulin. This strategy is limited by a molecular trade-off between accelerated disassembly and enhanced susceptibility to degradation. Here, we demonstrate that this trade-off may be circumvented by nonstandard mutagenesis. Our studies employed LysB28, ProB29-insulin (“lispro”) as a model prandial analog that is less thermodynamically stable and more susceptible to fibrillation than is wild-type insulin. We have discovered that substitution of an invariant tyrosine adjoining the engineered sites in lispro (TyrB26) by 3-iodo-Tyr (i) augments its thermodynamic stability (ΔΔGu 0.5 ±0.2 kcal/mol), (ii) delays onset of fibrillation (lag time on gentle agitation at 37 °C was prolonged by 4-fold), (iii) enhances affinity for the insulin receptor (1.5 ± 0.1-fold), and (iv) preserves biological activity in a rat model of diabetes mellitus. 1H NMR studies suggest that the bulky iodo-substituent packs within a nonpolar interchain crevice. Remarkably, the 3-iodo-TyrB26 modification stabilizes an oligomeric form of insulin pertinent to pharmaceutical formulation (the R6 zinc hexamer) but preserves rapid disassembly of the oligomeric form pertinent to subcutaneous absorption (T6 hexamer). By exploiting this allosteric switch, 3-iodo-TyrB26-lispro thus illustrates how a nonstandard amino acid substitution can mitigate the unfavorable biophysical properties of an engineered protein while retaining its advantages. PMID:24993826

  13. Involvement of 5-methylcytosine in sunlight-induced mutagenesis.

    PubMed

    You, Y H; Li, C; Pfeifer, G P

    1999-10-29

    In human skin cancers, more than 30 % of all mutations in the p53 gene are transitions at dipyrimidines within the sequence context CpG, i.e. 5'-TCG and 5'-CCG, found at several mutational hotspots. Since CpGs are methylated along the p53 gene, these mutations may be derived from solar UV-induced pyrimidine dimers forming at sequences that contain 5-methylcytosine. In Xorder to define the contribution of 5-methylcytosine to sunlight-induced mutations, we have used mouse fibroblasts containing the CpG-methylated lacI transgene as a mutational target. We sequenced 182 UVC (254 nm UV)-induced mutations and 170 mutations induced by a solar UV simulator, along with 75 mutations in untreated cells. Only a few of the mutations in untreated cells were transitions at dipyrimidines, but more than 95% of the UVC and solar irradiation-induced mutations were targeted to dipyrimidine sites, the majority being transitions. After UVC irradiation, 6% of the base substitutions were at dipyrimidines containing 5-methylcytosine and only 2.2% of all mutations were transitions within this sequence context. However, 24% of the solar light-induced mutations were at dipyrimidines that contain 5-methylcytosine and most of them were transitions. Two sunlight-induced mutational hotspots at methylated CpGs correlated with sequences that form the highest levels of cyclobutane pyrimidine dimers after irradiation with sunlight but not with UVC. The data indicate that dipyrimidines that contain 5-methylcytosine are preferential targets for sunlight-induced mutagenesis in cultured mammalian cells, thus explaining the large proportion of p53 mutations at such sites in skin tumors in vivo.

  14. Trans-NIH neuroscience initiatives on mouse phenotyping and mutagenesis.

    PubMed

    Moldin, S O; Farmer, M E; Chin, H R; Battey, J F

    2001-08-01

    In the post-genomic era, the laboratory mouse will excel as a premier mammalian system to study normal and disordered biological processes, in part because of low cost, but largely because of the rich opportunities that exist for exploiting genetic tools and technologies in the mouse to systematically determine mammalian gene function. Many robust models of human disease may therefore be developed, and these in turn will provide critical clues to understanding gene function. The full potential of the mouse for understanding many of the neural and behavioral phenotypes of relevance to neuroscientists has yet to be realized. With the full anatomy of the mouse genome at hand, researchers for the first time will be able to move beyond traditional gene-by-gene approaches and take a global view of gene expression patterns crucial for neurobiological processes. In response to an action plan for mouse genomics developed on the basis of recommendations from the scientific community, seven institutes of the National Institutes of Health (NIH) initiated in 1999 a mouse genetics research program that specifically focused on neurobiology and complex behavior. The specific goals of these neuroscience initiatives are to develop high-throughput phenotyping assays and to initiate genome-wide mutagenesis projects to identify hundreds of mutant strains with heritable abnormalities of high relevance to neuroscientists. Assays and mutants generated in these efforts will be made widely available to the scientific community, and such resources will provide neuroscientists unprecedented opportunities to elucidate the molecular mechanisms of neural function and complex behavior. Such research tools ultimately will permit the manipulation and analysis of the mouse genome, as a means of gaining insight into the genetic bases of the mammalian nervous system and its complex disorders.

  15. Tetragonal Lysozyme Interactions Studied by Site Directed Mutagenesis

    NASA Technical Reports Server (NTRS)

    Crawford, Lisa; Karr, Laurel; Pusey, Marc

    1998-01-01

    A number of recent experimental and theoretical studies have indicated that tetragonal lysozyme crystal growth proceeds by the addition of aggregates, formed by reversible self association of the solute molecules in the bulk'solution. Periodic bond chain and atomic force microscopy studies have indicated that the probable growth unit is at minimum a 43 tetramer, and most likely an octamer composed of two complete turns about the 4(sub 3) axis. If these results are correct, then there are intermolecular interactions which are only formed in the solution and others only formed at the joining of the growth unit to the crystal surface. We have set out to study these interactions, and the correctness of this hypothesis, using site directed mutagenesis of specific amino acid residues involved in the different bonds. We had initially expressed wild type lysozyme in S. cervasiae with yields of approximately 5 mg/L, which were eventually raised to approximately 40 mg/L. We are now moving the expression to the Pichia system, with anticipated yields of 300 to greater than 500 mg/L, comparable to what can be obtained from egg whites. An additional advantage of using recombinant protein is the greater genetic homogeneity of the material obtained and the absence of any other contaminating egg proteins. The first mutation experiments are TYR 23 yields PHE or ALA and ASN 113 yields ALA or ASP. Both TYR 23 and ASN 113 form part of the postulated dimerization intermolecular binding site which lead to the formation of the 4(sub 3) helix. Tyrosine also participates in an intermolecular hydrogen bond with ARG 114. The results of these and subsequent experiments will be discussed.

  16. Tetragonal Lysozyme Interactions Studied by Site Directed Mutagenesis

    NASA Technical Reports Server (NTRS)

    Crawford, Lisa; Karr, Laurel J.; Nadarajah, Arunan; Pusey, Marc

    1999-01-01

    A number of recent experimental and theoretical studies have indicated that tetragonal lysozyme crystal growth proceeds by the addition of aggregates, formed by reversible self association of the solute molecules in the bulk solution. Periodic bond chain and atomic force microscopy studies have indicated that the probable growth unit is at minimum a 43 tetramer, and most likely an octamer composed of two complete turns about the 43 axis. If these results are correct, then there are intermolecular interactions which are only formed in the solution and others only formed at the joining of the growth unit to the crystal surface. We have set out to study these interactions, and the correctness of this hypothesis, using site directed mutagenesis of specific amino acid residues involved in the different bonds. We had initially expressed wild type lysozyme in S. cervasiae with yields of approximately 5 mg/L, which were eventually raised to approximately 40 mg/L. We are now moving the expression to the Pichia system, with anticipated yields of 300 to (3)500 mg/L, comparable to what can be obtained from egg whites. An additional advantage of using recombinant protein is the greater genetic homogeneity of the material obtained and the absence of any other contaminating egg proteins. The first mutation experiments are TYR 23 (Registered) PHE or ALA and ASN 113 (Registered) ALA or ASP. Both TYR 23 and ASN 113 form part of the postulated dimerization intermolecular binding site which lead to the formation of the 43 helix. Tyrosine also participates in an intermolecular hydrogen bond with ARG 114. The results of these and subsequent experiments will be discussed.

  17. Stabilization of a prokaryotic LAT transporter by random mutagenesis

    PubMed Central

    Rodríguez-Banqueri, Arturo; Errasti-Murugarren, Ekaitz; Bartoccioni, Paola; Kowalczyk, Lukasz; Perálvarez-Marín, Alex

    2016-01-01

    The knowledge of three-dimensional structures at atomic resolution of membrane transport proteins has improved considerably our understanding of their physiological roles and pathological implications. However, most structural biology techniques require an optimal candidate within a protein family for structural determination with (a) reasonable production in heterologous hosts and (b) good stability in detergent micelles. SteT, the Bacillus subtilis l-serine/l-threonine exchanger is the best-known prokaryotic paradigm of the mammalian l–amino acid transporter (LAT) family. Unfortunately, SteT’s lousy stability after extracting from the membrane prevents its structural characterization. Here, we have used an approach based on random mutagenesis to engineer stability in SteT. Using a split GFP complementation assay as reporter of protein expression and membrane insertion, we created a library of 70 SteT mutants each containing random replacements of one or two residues situated in the transmembrane domains. Analysis of expression and monodispersity in detergent of this library permitted the identification of evolved versions of SteT with a significant increase in both expression yield and stability in detergent with respect to wild type. In addition, these experiments revealed a correlation between the yield of expression and the stability in detergent micelles. Finally, and based on protein delipidation and relipidation assays together with transport experiments, possible mechanisms of SteT stabilization are discussed. Besides optimizing a member of the LAT family for structural determination, our work proposes a new approach that can be used to optimize any membrane protein of interest. PMID:26976827

  18. Generation of Enterobacter sp. YSU auxotrophs using transposon mutagenesis.

    PubMed

    Caguiat, Jonathan James

    2014-10-31

    Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can be generated using a transposome. The commercially available, Tn5-derived transposome used in this protocol consists of a linear segment of DNA containing an R6Kγ replication origin, a gene for kanamycin resistance and two mosaic sequence ends, which serve as transposase binding sites. The transposome, provided as a DNA/transposase protein complex, is introduced by electroporation into the prototrophic strain, Enterobacter sp. YSU, and randomly incorporates itself into this host's genome. Transformants are replica plated onto Luria-Bertani agar plates containing kanamycin, (LB-kan) and onto M-9 medium agar plates containing kanamycin (M-9-kan). The transformants that grow on LB-kan plates but not on M-9-kan plates are considered to be auxotrophs. Purified genomic DNA from an auxotroph is partially digested, ligated and transformed into a pir+ Escherichia coli (E. coli) strain. The R6Kγ replication origin allows the plasmid to replicate in pir+ E. coli strains, and the kanamycin resistance marker allows for plasmid selection. Each transformant possesses a new plasmid containing the transposon flanked by the interrupted chromosomal region. Sanger sequencing and the Basic Local Alignment Search Tool (BLAST) suggest a putative identity of the interrupted gene. There are three advantages to using this transposome mutagenesis strategy. First, it does not rely on the expression of a transposase gene by the host. Second, the transposome is introduced into the target host by electroporation, rather than by conjugation or by transduction and therefore is more efficient. Third, the R6Kγ replication origin makes it easy to identify the mutated gene which is partially recovered in a recombinant plasmid. This technique can be used to investigate the genes involved in other characteristics of Enterobacter sp. YSU or of a

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

    PubMed Central

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

    2016-01-01

    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 (PSD95pdz3) 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. PMID:27563054

  20. Radiation mutagenesis from molecular and genetic points of view

    SciTech Connect

    Chen, D.J.C.; Park, M.S.; Okinaka, R.T.; Jaberaboansari, A.

    1993-02-01

    An important biological effect of ionizing radiation on living organisms is mutation induction. Mutation is also a primary event in the etiology of cancer. The chain events, from induction of DNA damage by ionizing radiation to processing of these damages by the cellular repair/replication machinery, that lead to mutation are not well understood. The development of quantitative methods for measuring mutation-induction, such as the HPRT system, in cultured mammalian cells has provided an estimate of the mutagenic effects of x- and {gamma}-rays as wen as of high LET radiation in both rodent and human cells. A major conclusion from these mutagenesis data is that high LET radiation induces mutations more efficiently than g-rays. Molecular analysis of mutations induced by sparsely ionizing radiation have detected major structural alterations at the gene level. Our molecular results based on analysis of human HPRT deficient mutants induced by {gamma}-rays, {alpha}-particles and high energy charged particles indicate that higher LET radiation induce more total and large deletion mutations than {gamma}-rays. Utilizing molecular techniques including polymerase chain reaction (PCR), Single-strand conformation polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE) and Direct DNA sequencing, mutational spectra induced by ionizing radiation have been compared in different cell systems. Attempts have also been made to determine the mutagenic potential and the nature of mutation induced by low dose rate {gamma}-rays. Defective repair, in the form of either a diminished capability for repair or inaccurate repair, can lead to increased risk of heritable mutations from radiation exposure. Therefore, the effects of DNA repair deficiency on the mutation induction in mammalian cells is reviewed.

  1. Radiation mutagenesis from molecular and genetic points of view

    SciTech Connect

    Chen, D.J.C.; Park, M.S.; Okinaka, R.T.; Jaberaboansari, A.

    1993-01-01

    An important biological effect of ionizing radiation on living organisms is mutation induction. Mutation is also a primary event in the etiology of cancer. The chain events, from induction of DNA damage by ionizing radiation to processing of these damages by the cellular repair/replication machinery, that lead to mutation are not well understood. The development of quantitative methods for measuring mutation-induction, such as the HPRT system, in cultured mammalian cells has provided an estimate of the mutagenic effects of x- and [gamma]-rays as wen as of high LET radiation in both rodent and human cells. A major conclusion from these mutagenesis data is that high LET radiation induces mutations more efficiently than g-rays. Molecular analysis of mutations induced by sparsely ionizing radiation have detected major structural alterations at the gene level. Our molecular results based on analysis of human HPRT deficient mutants induced by [gamma]-rays, [alpha]-particles and high energy charged particles indicate that higher LET radiation induce more total and large deletion mutations than [gamma]-rays. Utilizing molecular techniques including polymerase chain reaction (PCR), Single-strand conformation polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE) and Direct DNA sequencing, mutational spectra induced by ionizing radiation have been compared in different cell systems. Attempts have also been made to determine the mutagenic potential and the nature of mutation induced by low dose rate [gamma]-rays. Defective repair, in the form of either a diminished capability for repair or inaccurate repair, can lead to increased risk of heritable mutations from radiation exposure. Therefore, the effects of DNA repair deficiency on the mutation induction in mammalian cells is reviewed.

  2. Exploring the potential of megaprimer PCR in conjunction with orthogonal array design for mutagenesis library construction.

    PubMed

    Tang, Lixia; Zheng, Kai; Liu, Yu; Zheng, Huayu; Wang, Hu; Song, Chunlei; Zhou, Hong

    2013-01-01

    Although megaprimer PCR mutagenesis has been used routinely in protein directed evolution, users sometimes encounter technical hurdles, particularly inefficiency during amplification when large fragments are used or the template is difficult to be amplified. Instead of methodology development, here we simply overcome the limitation by optimizing megaprimer PCR conditions via orthogonal array design of the four PCR components in three levels of each: template, primer, Mg(2+) , and dNTPs. For this, only nine PCRs need to be performed. The strategy (termed as OptiMega) was not only successfully applied for the construction of one multiple-site saturation mutagenesis library of halohydrin dehalogenase HheC, which failed to be constructed previously using the standard QuikChange™ protocol, but also expanded the construction of two high-quality random mutagenesis libraries of HheA and HheC. Most importantly, OptiMega offers a quick and simple way of constructing random mutagenesis libraries by eliminating the ligation step. Our results demonstrated that the OptiMega strategy could greatly strengthen the potential of megaprimer PCR mutagenesis for library construction.

  3. 2012 Gordon Research Conference on Mutagenesis - Formal Schedule and Speaker/Poster Program

    SciTech Connect

    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, cell 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.

  4. Signature tagged mutagenesis in the functional genetic analysis of gastrointestinal pathogens

    PubMed Central

    Cummins, Joanne; Gahan, Cormac G.M.

    2012-01-01

    Signature tagged mutagenesis is a genetic approach that was developed to identify novel bacterial virulence factors. It is a negative selection method in which unique identification tags allow analysis of pools of mutants in mixed populations. The approach is particularly well suited to functional genetic analysis of the gastrointestinal phase of infection in foodborne pathogens and has the capacity to guide the development of novel vaccines and therapeutics. In this review we outline the technical principles underpinning signature-tagged mutagenesis as well as novel sequencing-based approaches for transposon mutant identification such as TraDIS (transposon directed insertion-site sequencing). We also provide an analysis of screens that have been performed in gastrointestinal pathogens which are a global health concern (Escherichia coli, Listeria monocytogenes, Helicobacter pylori, Vibrio cholerae and Salmonella enterica). The identification of key virulence loci through the use of signature tagged mutagenesis in mice and relevant larger animal models is discussed. PMID:22555467

  5. 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.

  6. High Frequency Targeted Mutagenesis Using Engineered Endonucleases and DNA-End Processing Enzymes

    PubMed Central

    Delacôte, Fabien; Perez, Christophe; Guyot, Valérie; Duhamel, Marianne; Rochon, Christelle; Ollivier, Nathalie; Macmaster, Rachel; Silva, George H.; Pâques, Frédéric; Daboussi, Fayza; Duchateau, Philippe

    2013-01-01

    Targeting DNA double-strand breaks is a powerful strategy for gene inactivation applications. Without the use of a repair plasmid, targeted mutagenesis can be achieved through Non-Homologous End joining (NHEJ) pathways. However, many of the DNA breaks produced by engineered nucleases may be subject to precise re-ligation without loss of genetic information and thus are likely to be unproductive. In this study, we combined engineered endonucleases and DNA-end processing enzymes to increase the efficiency of targeted mutagenesis, providing a robust and efficient method to (i) greatly improve targeted mutagenesis frequency up to 30-fold, and; (ii) control the nature of mutagenic events using meganucleases in conjunction with DNA-end processing enzymes in human primary cells. PMID:23359797

  7. An APOBEC Cytidine Deaminase Mutagenesis Pattern is Widespread in Human Cancers

    PubMed Central

    Roberts, Steven A.; Lawrence, Michael S.; Klimczak, Leszek J.; Grimm, Sara A.; Fargo, David; Stojanov, Petar; Kiezun, Adam; Kryukov, Gregory V.; Carter, Scott L.; Saksena, Gordon; Harris, Shawn; Shah, Ruchir R.; Resnick, Michael A.; Getz, Gad; Gordenin, Dmitry A.

    2013-01-01

    Recent studies indicate that a subclass of APOBEC cytidine deaminases, which convert cytosine to uracil during RNA editing and retrovirus or retrotransposon restriction, may induce mutation clusters in human tumors. We show here that throughout cancer genomes APOBEC mutagenesis is pervasive and correlates with APOBEC mRNA levels. Mutation clusters in whole-genome and exome datasets conformed to stringent criteria indicative of an APOBEC mutation pattern. Applying these criteria to 954,247 mutations in 2,680 exomes of 14 cancer types, mostly from TCGA, revealed significant presence of the APOBEC mutation pattern in bladder, cervical, breast, head and neck and lung cancers, reaching 68% of all mutations in some samples. Within breast cancer, the HER2E subtype was clearly enriched with tumors displaying the APOBEC mutation pattern, suggesting this type of mutagenesis is functionally linked with cancer development. The APOBEC mutation pattern also extended to cancer-associated genes, implying that ubiquitous APOBEC mutagenesis is carcinogenic. PMID:23852170

  8. 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.

  9. Atmospheric and room temperature plasma (ARTP) as a new powerful mutagenesis tool.

    PubMed

    Zhang, Xue; Zhang, Xiao-Fei; Li, He-Ping; Wang, Li-Yan; Zhang, Chong; Xing, Xin-Hui; Bao, Cheng-Yu

    2014-06-01

    Developing rapid and diverse microbial mutation tool is of importance to strain modification. In this review, a new mutagenesis method for microbial mutation breeding using the radio-frequency atmospheric-pressure glow discharge (RF APGD) plasma jets is summarized. Based on the experimental study, the helium RF APGD plasma jet has been found to be able to change the DNA sequences significantly, indicating that the RF APGD plasma jet would be a powerful tool for the microbial mutagenesis with its outstanding features, such as the low and controllable gas temperatures, abundant chemically reactive species, rapid mutation, high operation flexibility, etc. Then, with the RF APGD plasma generator as the core component, a mutation machine named as atmospheric and room temperature plasma (ARTP) mutation system has been developed and successfully employed for the mutation breeding of more than 40 kinds of microorganisms including bacteria, fungi, and microalgae. Finally, the prospect of the ARTP mutagenesis is discussed.

  10. 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

  11. Random UV-C mutagenesis of Scheffersomyces (formerly Pichia) stipitis NRRL Y-7124 to improve anaerobic growth on lignocellulosic sugars

    USDA-ARS?s Scientific Manuscript database

    Yeast strains for anaerobic conversion of lignocellulosic sugars to ethanol were produced from Scheffersomyces (formerly Pichia) stipitis NRRL Y-7124 using UV-C mutagenesis. Random UV-C mutagenesis potentially produces large numbers of mutations broadly and uniformly over the whole genome to genera...

  12. 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.…

  13. 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.…

  14. 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.

  15. Ribozyme Mediated gRNA Generation for In Vitro and In Vivo CRISPR/Cas9 Mutagenesis

    PubMed Central

    Ng, Ashley Shu Mei; Ingham, Philip W.

    2016-01-01

    CRISPR/Cas9 is now regularly used for targeted mutagenesis in a wide variety of systems. Here we report the use of ribozymes for the generation of gRNAs both in vitro and in zebrafish embryos. We show that incorporation of ribozymes increases the types of promoters and number of target sites available for mutagenesis without compromising mutagenesis efficiency. We have tested this by comparing the efficiency of mutagenesis of gRNA constructs with and without ribozymes and also generated a transgenic zebrafish expressing gRNA using a heat shock promoter (RNA polymerase II-dependent promoter) that was able to induce mutagenesis of its target. Our method provides a streamlined approach to test gRNA efficiency as well as increasing the versatility of conditional gene knock out in zebrafish. PMID:27832146

  16. Portable exhausters POR-004 SKID B, POR-005 SKID C, POR-006 SKID D storage plan

    SciTech Connect

    Nelson, O.D.

    1997-09-04

    This document provides a storage plan for portable exhausters POR-004 SKID B, POR-005 SKID C, AND POR-006 SKID D. The exhausters will be stored until they are needed by the TWRS (Tank Waste Remediation Systems) Saltwell Pumping Program. The storage plan provides criteria for portable exhauster storage, periodic inspections during storage, and retrieval from storage.

  17. 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

  18. Workshop on ENU Mutagenesis: Planning for Saturation, July 25-28, 2002

    SciTech Connect

    Nadeau, Joseph H

    2002-07-25

    The goal of the conference is to enhance the development of improved technologies and new approaches to the identification of genes underlying chemically-induced mutant phenotypes. The conference brings together ENU mutagenesis experts from the United States and aborad for a small, intensive workshop to consider these issues.

  19. 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...

  20. Identification of a halotolerant mutant via in vitro mutagenesis in the cyanobacterium Fremyella diplosiphon

    USDA-ARS?s Scientific Manuscript database

    Energy metabolism and photosynthetic pigment accumulation are affected by salt stress in cyanobacteria leading to cessation of growth. The effect of salinity on the fresh water cyanobacteria, Fremyella diplosiphon was investigated and mutagenesis-based efforts were undertaken to enhance salt toleran...

  1. 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...

  2. 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...

  3. In vitro mutagenesis assays as predictors of chemical carcinogenesis in mammals.

    PubMed

    Brusick, D J

    1977-01-01

    In vitro microbial mutagenesis assays coupled with mammalian activation systems offer promising technique to screen chemicals for their potential carcinogenic activity. The correlation between mutagenic and carcinogenic properties for a large array of chemicals is approximately 0.9. The best correlation exists for those carcinogens which are themselves highly electrophilic or produce electrophilic metabolites. Correlation between mutagenicity and carcinogenicity for hormonal, metallic, or physical carcinogens has been disappointing but not unexpected based on their proposed mechanisms of action. In addition to the application of in vitro mutagenesis techniques to screening chemicals for the identification of potential carcinogens, they are useful tools for investigating genetic, biochemical, and pharmacologic properties of different animal species. Studies with the chemical carcinogen dimethylnitrosamine have been conducted and show a functional relationship between mutagenesis and carcinogenesis. The assays can also be conducted using activation systems prepared from the tissues of any mammalian species. This permits a direct assessment of phylogenic extrapolation by comparing the metabolic activation capabilities of tissues from several mammalian species, including human samples. The advantages of mutagenicity testing are the short period of time required for results, the high sensitivity of the assay (microgram of nanogram quantities of chemicals can be used), and the fact that the ultimate agent can be detected biologically without first necessitating chemical identification and isolation. It appears from current studies that in vitro mutagenesis techniques may well open new avenues of investigation into some old toxicologic problems.

  4. 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

  5. Improvement of biocatalysts for industrial and environmental purposes by saturation mutagenesis.

    PubMed

    Valetti, Francesca; Gilardi, Gianfranco

    2013-10-08

    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.

  6. A quantitative model of bacterial mismatch repair as applied to studying induced mutagenesis

    NASA Astrophysics Data System (ADS)

    Belov, O. V.; Chuluunbaatar, O.; Kapralov, M. I.; Sweilam, N. H.

    2013-11-01

    The paper presents a mathematical model of the DNA mismatch repair system in Escherichia coli bacterial cells. The key pathways of this repair mechanism were simulated on the basis of modern experimental data. We have modelled in detail five main pathways of DNA misincorporation removal with different DNA exonucleases. Here we demonstrate an application of the model to problems of radiation-induced mutagenesis.

  7. Stationary-Phase Mutagenesis in Stressed Bacillus subtilis Cells Operates by Mfd-Dependent Mutagenic Pathways

    PubMed Central

    Gómez-Marroquín, Martha; Martin, Holly A.; Pepper, Amber; Girard, Mary E.; Kidman, Amanda A.; Vallin, Carmen; Yasbin, Ronald E.; Pedraza-Reyes, Mario; Robleto, Eduardo A.

    2016-01-01

    In replication-limited cells of Bacillus subtilis, Mfd is mutagenic at highly transcribed regions, even in the absence of bulky DNA lesions. However, the mechanism leading to increased mutagenesis through Mfd remains currently unknown. Here, we report that Mfd may promote mutagenesis in nutritionally stressed B. subtilis cells by coordinating error-prone repair events mediated by UvrA, MutY and PolI. Using a point-mutated gene conferring leucine auxotrophy as a genetic marker, it was found that the absence of UvrA reduced the Leu+ revertants and that a second mutation in mfd reduced mutagenesis further. Moreover, the mfd and polA mutants presented low but similar reversion frequencies compared to the parental strain. These results suggest that Mfd promotes mutagenic events that required the participation of NER pathway and PolI. Remarkably, this Mfd-dependent mutagenic pathway was found to be epistatic onto MutY; however, whereas the MutY-dependent Leu+ reversions required Mfd, a direct interaction between these proteins was not apparent. In summary, our results support the concept that Mfd promotes mutagenesis in starved B. subtilis cells by coordinating both known and previously unknown Mfd-associated repair pathways. These mutagenic processes bias the production of genetic diversity towards highly transcribed regions in the genome. PMID:27399782

  8. Highly efficient CRISPR/Cas9-mediated targeted mutagenesis of multiple genes in Populus.

    PubMed

    Tingting, Liu; Di, Fan; Lingyu, Ran; Yuanzhong, Jiang; Rui, Liu; Keming, Luo

    2015-10-01

    The typeⅡCRISPR/Cas9 system (Clustered regularly interspaced short palindromic repeats /CRISPR-associated 9) has been widely used in bacteria, yeast, animals and plants as a targeted genome editing technique. In previous work, we have successfully knocked out the endogenous phytoene dehydrogenase (PDS) gene in Populus tomentosa Carr. using this system. To study the effect of target design on the efficiency of CRISPR/Cas9-mediated gene knockout in Populus, we analyzed the efficiency of mutagenesis using different single-guide RNA (sgRNA) that target PDS DNA sequence. We found that mismatches between the sgRNA and the target DNA resulted in decreased efficiency of mutagenesis and even failed mutagenesis. Moreover, complementarity between the 3' end nucleotide of sgRNA and target DNA is especially crucial for efficient mutagenesis. Further sequencing analysis showed that two PDS homologs in Populus, PtPDS1 and PtPDS2, could be knocked out simultaneously using this system with 86.4% and 50% efficiency, respectively. These results indicated the possibility of introducing mutations in two or more endogenous genes efficiently and obtaining multi-mutant strains of Populus using this system. We have indeed generated several knockout mutants of transcription factors and structural genes in Populus, which establishes a foundation for future studies of gene function and genetic improvement of Populus.

  9. 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...

  10. 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...

  11. Using ultra-sensitive next generation sequencing to dissect DNA damage-induced mutagenesis

    PubMed Central

    Wang, Kaile; Ma, Xiaolu; Zhang, Xue; Wu, Dafei; Sun, Chenyi; Sun, Yazhou; Lu, Xuemei; Wu, Chung-I; Guo, Caixia; Ruan, Jue

    2016-01-01

    Next generation sequencing (NGS) technologies have dramatically improved studies in biology and biomedical science. However, no optimal NGS approach is available to conveniently analyze low frequency mutations caused by DNA damage treatments. Here, by developing an exquisite ultra-sensitive NGS (USNGS) platform “EasyMF” and incorporating it with a widely used supF shuttle vector-based mutagenesis system, we can conveniently dissect roles of lesion bypass polymerases in damage-induced mutagenesis. In this improved mutagenesis analysis pipeline, the initial steps are the same as in the supF mutation assay, involving damaging the pSP189 plasmid followed by its transfection into human 293T cells to allow replication to occur. Then “EasyMF” is employed to replace downstream MBM7070 bacterial transformation and other steps for analyzing damage-induced mutation frequencies and spectra. This pipeline was validated by using UV damaged plasmid after its replication in lesion bypass polymerase-deficient 293T cells. The increased throughput and reduced cost of this system will allow us to conveniently screen regulators of translesion DNA synthesis pathway and monitor environmental genotoxic substances, which can ultimately provide insight into the mechanisms of genome stability and mutagenesis. PMID:27122023

  12. 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...

  13. 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

  14. Multiple site-directed mutagenesis of more than 10 sites simultaneously and in a single round.

    PubMed

    Seyfang, Andreas; Jin, Jean Huaqian

    2004-01-15

    Site-directed mutagenesis is a powerful tool to explore the structure-function relationship of proteins, but most traditional methods rely on the mutation of only one site at a time and efficiencies drop drastically when more than three sites are targeted simultaneously. Many applications in functional proteomics and genetic engineering, including codon optimization for heterologous expression, generation of cysteine-less proteins, and alanine-scanning mutagenesis, would greatly benefit from a multiple-site mutagenesis method with high efficiency. Here we describe the development of a simple and rapid method for site-directed mutagenesis of more than 10 sites simultaneously with up to 100% efficiency. The method uses two terminal tailed primers with a unique 25-nucleotide tail each that are simultaneously annealed to template DNA together with the set of mutagenic primers in between. Following synthesis of the mutant strand by primer extension and ligation with T4 DNA polymerase and ligase, the unique mutant strand-specific tails of the terminal primers are used as anchors to specifically amplify the mutant strand by high-fidelity polymerase chain reaction. We have employed this novel method to mutate simultaneously all 9 and 11 CUG leucine codons of the Hyg and Neo resistance genes, respectively, to the Candida albicans-friendly UUG leucine codon at 100% efficiency.

  15. Random mutagenesis of the hyperthermophilic archaeon Pyrococcus furiosus using in vitro mariner transposition and natural transformation

    PubMed Central

    Guschinskaya, Natalia; Brunel, Romain; Tourte, Maxime; Lipscomb, Gina L.; Adams, Michael W. W.; Oger, Philippe; Charpentier, Xavier

    2016-01-01

    Transposition mutagenesis is a powerful tool to identify the function of genes, reveal essential genes and generally to unravel the genetic basis of living organisms. However, transposon-mediated mutagenesis has only been successfully applied to a limited number of archaeal species and has never been reported in Thermococcales. Here, we report random insertion mutagenesis in the hyperthermophilic archaeon Pyrococcus furiosus. The strategy takes advantage of the natural transformability of derivatives of the P. furiosus COM1 strain and of in vitro Mariner-based transposition. A transposon bearing a genetic marker is randomly transposed in vitro in genomic DNA that is then used for natural transformation of P. furiosus. A small-scale transposition reaction routinely generates several hundred and up to two thousands transformants. Southern analysis and sequencing showed that the obtained mutants contain a single and random genomic insertion. Polyploidy has been reported in Thermococcales and P. furiosus is suspected of being polyploid. Yet, about half of the mutants obtained on the first selection are homozygous for the transposon insertion. Two rounds of isolation on selective medium were sufficient to obtain gene conversion in initially heterozygous mutants. This transposition mutagenesis strategy will greatly facilitate functional exploration of the Thermococcales genomes. PMID:27824140

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

    PubMed Central

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

    2016-01-01

    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. PMID:27307293

  17. The utility of transposon mutagenesis for cancer studies in the era of genome editing.

    PubMed

    DeNicola, Gina M; Karreth, Florian A; Adams, David J; Wong, Chi C

    2015-10-19

    The use of transposons as insertional mutagens to identify cancer genes in mice has generated a wealth of information over the past decade. Here, we discuss recent major advances in transposon-mediated insertional mutagenesis screens and compare this technology with other screening strategies.

  18. 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.

  19. Versatile Vectors for Efficient Mutagenesis of Bradyrhizobium diazoefficiens and Other Alphaproteobacteria

    PubMed Central

    Ledermann, Raphael; Strebel, Silvan; Kampik, Clara

    2016-01-01

    ABSTRACT Analysis of bacterial gene function commonly relies on gene disruption or replacement followed by phenotypic characterization of the resulting mutant strains. Deletion or replacement of targeted regions is commonly achieved via two homologous recombination (HR) events between the bacterial genome and a nonreplicating plasmid carrying DNA fragments flanking the region to be deleted. The counterselection of clones that have integrated the entire plasmid in their genome via a single HR event is crucial in this procedure. Various genetic tools and well-established protocols are available for this type of mutagenesis in model bacteria; however, these methods are not always efficiently applicable in less established systems. Here we describe the construction and application of versatile plasmid vectors pREDSIX and pTETSIX for marker replacement and markerless mutagenesis, respectively. Apart from an array of restriction sites optimized for cloning of GC-rich DNA fragments, the vector backbone contains a constitutively expressed gene for mCherry, enabling the rapid identification of clones originating from single or double HR events by fluorescence-assisted cell sorting (FACS). In parallel, we constructed a series of plasmids from which gene cassettes providing resistance against gentamicin, kanamycin, hygromycin B, streptomycin and spectinomycin, or tetracycline were excised for use with pREDSIX-based marker replacement mutagenesis. In proof-of-concept mutagenesis experiments, we demonstrated the potential for the use of the developed tools for gene deletion mutagenesis in the nitrogen-fixing soybean symbiont Bradyrhizobium diazoefficiens (formerly Bradyrhizobium japonicum) and three additional members of the alphaproteobacteria. IMPORTANCE Mutation and phenotypic analysis are essential to the study of gene function. Efficient mutagenesis protocols and tools are available for many bacterial species, including various model organisms; however, genetic analysis of

  20. Antimutagenic effects of dehydrozingerone and its analogs on UV-induced mutagenesis in Escherichia coli.

    PubMed

    Motohashi, N; Ashihara, Y; Yamagami, C; Saito, Y

    1997-06-09

    Antimutagenic activities of dehydrozingerone, benzalacetone and its analogs substituted with the hydroxyl, methoxyl or methyl group on the benzene ring were investigated by the post-treatment for UV-induced mutagenesis in Escherichia coli WP2s (uvrA). In this assay, dehydrozingerone was a poor antimutagen. Among the test compounds except for 2-hydroxybenzalacetone, benzalacetone was the most strong antimutagen, showing that the ring substitutions decrease the antimutagenic activities. 2-Hydroxybenzalacetone was, however, more effective than benzalacetone. The antimutagenicity of 2-hydroxybenzalacetone might be dependent on the property that is known to associate inter-molecularly by hydrogen bonding between the hydroxyl group and the carbonyl group. The effects of the side chain, single, double or triple bond and its adjacent carbonyl group, were further investigated using benzylacetone with saturated carbonyl chain, benzalacetone with double-bonded carbonyl chain, 4-phenyl-3-butyn-2-one with triple-bonded carbonyl chain and trans-beta-methyl styrene with only double bond. Benzalacetone and 4-phenyl-3-butyn-2-one suppressed the UV-induced mutagenesis, but benzylacetone and trans-beta-methyl styrene scarcely inhibited the mutagenesis: an alpha, beta-double or triple-bonded carbonyl system was necessary for the antimutagenic activity. 4-Phenyl-3-butyn-2-one showed the potent antimutagenicity at one order lower concentrations than that of benzalacetone. Benzalacetone, 2-hydroxybenzalacetone and 4-phenyl-3-butyn-2-one that were effective on the UV-induced mutagenesis also decreased the gamma-induced mutagenesis in Salmonella typhimurium TA2638.

  1. 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…

  2. 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…

  3. EMS mutagenesis in mature seed-derived rice calli as a new method for rapidly obtaining TILLING mutant populations

    PubMed Central

    2014-01-01

    Background TILLING (Targeting Induced Local Lesions IN Genomes) is a reverse genetic method that combines chemical mutagenesis with high-throughput genome-wide screening for point mutation detection in genes of interest. However, this mutation discovery approach faces a particular problem which is how to obtain a mutant population with a sufficiently high mutation density. Furthermore, plant mutagenesis protocols require two successive generations (M1, M2) for mutation fixation to occur before the analysis of the genotype can begin. Results Here, we describe a new TILLING approach for rice based on ethyl methanesulfonate (EMS) mutagenesis of mature seed-derived calli and direct screening of in vitro regenerated plants. A high mutagenesis rate was obtained (i.e. one mutation in every 451 Kb) when plants were screened for two senescence-related genes. Screening was carried out in 2400 individuals from a mutant population of 6912. Seven sense change mutations out of 15 point mutations were identified. Conclusions This new strategy represents a significant advantage in terms of time-savings (i.e. more than eight months), greenhouse space and work during the generation of mutant plant populations. Furthermore, this effective chemical mutagenesis protocol ensures high mutagenesis rates thereby saving in waste removal costs and the total amount of mutagen needed thanks to the mutagenesis volume reduction. PMID:24475756

  4. The transcription elongation factor NusA is required for stress-induced mutagenesis in Escherichia coli.

    PubMed

    Cohen, Susan E; Walker, Graham C

    2010-01-12

    Stress-induced mutagenesis describes the accumulation of mutations that occur in nongrowing cells, in contrast to mutagenesis that occurs in actively dividing populations, and has been referred to as stationary-phase or adaptive mutagenesis. The most widely studied system for stress-induced mutagenesis involves monitoring the appearance of Lac(+) revertants of the strain FC40 under starvation conditions in Escherichia coli. The SOS-inducible translesion DNA polymerase DinB plays an important role in this phenomenon. Loss of DinB (DNA pol IV) function results in a severe reduction of Lac(+) revertants. We previously reported that NusA, an essential component of elongating RNA polymerases, interacts with DinB. Here we report our unexpected observation that wild-type NusA function is required for stress-induced mutagenesis. We present evidence that this effect is unlikely to be due to defects in transcription of lac genes but rather is due to an inability to adapt and mutate in response to environmental stress. Furthermore, we extended our analysis to the formation of stress-induced mutants in response to antibiotic treatment, observing the same striking abolition of mutagenesis under entirely different conditions. Our results are the first to implicate NusA as a crucial participant in the phenomenon of stress-induced mutagenesis. Copyright 2010 Elsevier Ltd. All rights reserved.

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

    PubMed

    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.

  6. 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

  7. Transversion-enriched sequence saturation mutagenesis (SeSaM-Tv+): a random mutagenesis method with consecutive nucleotide exchanges that complements the bias of error-prone PCR.

    PubMed

    Wong, Tuck Seng; Roccatano, Danilo; Loakes, David; Tee, Kang Lan; Schenk, Alexander; Hauer, Bernhard; Schwaneberg, Ulrich

    2008-01-01

    The sequence saturation mutagenesis (SeSaM) method has been advanced to a random mutagenesis method with adjustable mutational biases. SeSaM offers, for example, a bias that is complementary to error-prone (ep) PCR and is enriched in transversions (SeSaM-Tv(+)). dNTP alpha S and three degenerate bases (P, K and I) are used to control mutational bias flexibly. After quantifying incorporation rates of dPTP, dKTP and dITP by terminal transferase using a luciferase-based assay and investigating the read and/or write activities of eight DNA polymerases, a transversion-enriched protocol has been developed. In a mutant library generated using dGTP alpha S and dPTP, transversion frequencies of 16.22-22.58% (G-->T) and 6.38-9.69% (G-->C) were achieved. These mutational spectra are complementary and occur twice as frequently in comparison to standard epPCR methods employing Taq DNA polymerase. For generating more complex mutant libraries, the occurrence of consecutive nucleotide exchanges was increased by 10(5)-10(6)-fold compared to epPCR. Finally, 16.7% of all sequenced mutants contained consecutive nucleotide exchanges composed mainly of a transversion followed by a transition.

  8. Ultraviolet Mutagenesis in Bacteriophage T4 I. Irradiation of Extracellular Phage Particles

    PubMed Central

    Drake, John W.

    1966-01-01

    Drake, John W. (University of Illinois, Urbana). Ultraviolet mutagenesis in bacteriophage T4. I. Irradiation of extracellular phage particles. J. Bacteriol. 91:1775–1780. 1966.—Ultraviolet (UV) irradiation of extracellular T4 phage particles induces about 2 × 10−4r mutations per lethal hit. The mutants largely escape detection unless the irradiated phages are plated with very soft overlay agar. Multiplicity reactivation is not a prerequisite for mutagenesis. A much higher frequency of base pair substitution-type mutants is induced than is found in the spontaneous background, but sign mutants are also induced. Nearly half of the mutants map into previously identified UV hot spots. The rII mutants induced extracellularly are very similar to those induced intracellularly. The mutants also appear to result from direct radiation effects upon the bacteriophage deoxyribonucleic acid. PMID:5937237

  9. Software-supported USER cloning strategies for site-directed mutagenesis and DNA assembly.

    PubMed

    Genee, Hans Jasper; Bonde, Mads Tvillinggaard; Bagger, Frederik Otzen; Jespersen, Jakob Berg; Sommer, Morten O A; Wernersson, Rasmus; Olsen, Lars Rønn

    2015-03-20

    USER cloning is a fast and versatile method for engineering of plasmid DNA. We have developed a user friendly Web server tool that automates the design of optimal PCR primers for several distinct USER cloning-based applications. Our Web server, named AMUSER (Automated DNA Modifications with USER cloning), facilitates DNA assembly and introduction of virtually any type of site-directed mutagenesis by designing optimal PCR primers for the desired genetic changes. To demonstrate the utility, we designed primers for a simultaneous two-position site-directed mutagenesis of green fluorescent protein (GFP) to yellow fluorescent protein (YFP), which in a single step reaction resulted in a 94% cloning efficiency. AMUSER also supports degenerate nucleotide primers, single insert combinatorial assembly, and flexible parameters for PCR amplification. AMUSER is freely available online at http://www.cbs.dtu.dk/services/AMUSER/.

  10. The use of TALENs for nonhomologous end joining mutagenesis in silkworm and fruitfly.

    PubMed

    Takasu, Yoko; Tamura, Toshiki; Sajwan, Suresh; Kobayashi, Isao; Zurovec, Michal

    2014-08-15

    Transcription activator-like effector nucleases (TALENs) are custom-made enzymes designed to cut double-stranded DNA at desired locations. The DNA breaks are repaired either by error-prone non-homologous end-joining (NHEJ) pathway or via homologous recombination requiring homologous DNA as a template for the repair. TALENs are used for site-specific mutagenesis in an extended range of organisms including insects. We will describe here a simple TALEN-based mutagenesis protocol suitable for the generation of germline mutations in Bombyx mori and Drosophila melanogaster. The protocol includes assembly of specific TAL modules, in vitro synthesis of TALEN RNAs, egg microinjection and mutation detection using PCR analysis. Our procedure allows a high frequency induction of NHEJ mutations, which often allows the reception of homozygous mutants already in the G1. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. [RAD18 gene product of yeast Saccharomyces cerevisiae controls mutagenesis induced by hydrogen peroxide].

    PubMed

    Kozhina, T N; Korolev, V G

    2012-04-01

    Within eukaryotes, tolerance to DNA damage is determined primarily by the repair pathway controlled by the members of the RAD6 epistasis group. Genetic studies on a yeast Saccharomyces cerevisiae model showed that the initial stage of postreplication repair (PRR), i.e., initiation of replication through DNA damage, is controlled by Rad6-Rad18 ubiquitin-conjugating enzyme complex. Mutants of these genes are highly sensitive to various genotoxic agents and reduce the level of induced mutagenesis. In this case, the efficiency of mutagenesis suppression depends on the type of damage. In this study we showed that DNA damage induced by hydrogen peroxide at the same mutagen doses causes significantly more mutations and lethal events in the rad18 mutant cells compared to control wild-type cells.

  12. Rapid construction of mycobacterial mutagenesis vectors using ligation-independent cloning

    PubMed Central

    Balhana, Ricardo; Stoker, Neil G.; Sikder, Mahmudul Hasan; Chauviac, Francois-Xavier; Kendall, Sharon L.

    2010-01-01

    Targeted mutagenesis is one of the major tools for determining the function of a given gene and its involvement in bacterial pathogenesis. In mycobacteria, gene deletion is often accomplished by using allelic exchange techniques that commonly utilise a suicide delivery vector. We have adapted a widely-used suicide delivery vector (p1NIL) for cloning two flanking regions of a gene using ligation independent cloning (LIC). The pNILRB plasmid series produced allow a faster, more efficient and less laborious cloning procedure. In this paper we describe the making of pNILRB5, a modified version of p1NIL that contains two pairs of LIC sites flanking either a sacB or a lacZ gene. We demonstrate the success of this technique by generating 3 mycobacterial mutant strains. These vectors will contribute to more high-throughput methods of mutagenesis. PMID:20650290

  13. Transposon mutagenesis identifies genes that cooperate with mutant Pten in breast cancer progression.

    PubMed

    Rangel, Roberto; Lee, Song-Choon; Hon-Kim Ban, Kenneth; Guzman-Rojas, Liliana; Mann, Michael B; Newberg, Justin Y; Kodama, Takahiro; McNoe, Leslie A; Selvanesan, Luxmanan; Ward, Jerrold M; Rust, Alistair G; Chin, Kuan-Yew; Black, Michael A; Jenkins, Nancy A; Copeland, Neal G

    2016-11-29

    Triple-negative breast cancer (TNBC) has the worst prognosis of any breast cancer subtype. To better understand the genetic forces driving TNBC, we performed a transposon mutagenesis screen in a phosphatase and tensin homolog (Pten) mutant mice and identified 12 candidate trunk drivers and a much larger number of progression genes. Validation studies identified eight TNBC tumor suppressor genes, including the GATA-like transcriptional repressor TRPS1 Down-regulation of TRPS1 in TNBC cells promoted epithelial-to-mesenchymal transition (EMT) by deregulating multiple EMT pathway genes, in addition to increasing the expression of SERPINE1 and SERPINB2 and the subsequent migration, invasion, and metastasis of tumor cells. Transposon mutagenesis has thus provided a better understanding of the genetic forces driving TNBC and discovered genes with potential clinical importance in TNBC.

  14. Transposon mutagenesis identifies genes that cooperate with mutant Pten in breast cancer progression

    PubMed Central

    Rangel, Roberto; Lee, Song-Choon; Hon-Kim Ban, Kenneth; Guzman-Rojas, Liliana; Mann, Michael B.; Newberg, Justin Y.; McNoe, Leslie A.; Selvanesan, Luxmanan; Ward, Jerrold M.; Rust, Alistair G.; Chin, Kuan-Yew; Black, Michael A.; Jenkins, Nancy A.; Copeland, Neal G.

    2016-01-01

    Triple-negative breast cancer (TNBC) has the worst prognosis of any breast cancer subtype. To better understand the genetic forces driving TNBC, we performed a transposon mutagenesis screen in a phosphatase and tensin homolog (Pten) mutant mice and identified 12 candidate trunk drivers and a much larger number of progression genes. Validation studies identified eight TNBC tumor suppressor genes, including the GATA-like transcriptional repressor TRPS1. Down-regulation of TRPS1 in TNBC cells promoted epithelial-to-mesenchymal transition (EMT) by deregulating multiple EMT pathway genes, in addition to increasing the expression of SERPINE1 and SERPINB2 and the subsequent migration, invasion, and metastasis of tumor cells. Transposon mutagenesis has thus provided a better understanding of the genetic forces driving TNBC and discovered genes with potential clinical importance in TNBC. PMID:27849608

  15. Impact of increased mutagenesis on adaptation to high temperature in bacteriophage Qβ.

    PubMed

    Arribas, María; Cabanillas, Laura; Kubota, Kirina; Lázaro, Ester

    2016-10-01

    RNA viruses replicate with very high error rates, which makes them more sensitive to additional increases in this parameter. This fact has inspired an antiviral strategy named lethal mutagenesis, which is based on the artificial increase of the error rate above a threshold incompatible with virus infectivity. A relevant issue concerning lethal mutagenesis is whether incomplete treatments might enhance the adaptive possibilities of viruses. We have addressed this question by subjecting an RNA virus, the bacteriophage Qβ, to different transmission regimes in the presence or the absence of sublethal concentrations of the mutagenic nucleoside analogue 5-azacytidine (AZC). Populations obtained were subsequently exposed to a non-optimal temperature and analyzed to determine their consensus sequences. Our results show that previously mutagenized populations rapidly fixed a specific set of mutations upon propagation at the new temperature, suggesting that the expansion of the mutant spectrum caused by AZC has an influence on later evolutionary behavior. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Ultrafast solvation dynamics at internal sites of staphylococcal nuclease investigated by site-directed mutagenesis

    NASA Astrophysics Data System (ADS)

    Gao, Guang-Yu; Li, Yu; Wang, Wei; Wang, Shu-Feng; Dongping, Zhong; Gong, Qi-Huang

    2015-01-01

    Internal solvation of protein was studied by site-directed mutagenesis, with which an intrinsically fluorescent probe, tryptophan, is inserted into the desired position inside a protein molecule for ultrafast spectroscopic study. Here we review this unique method for protein dynamics research. We first introduce the frontiers of protein solvation, site-directed mutagenesis, protein stability and characteristics, and the spectroscopic methods. Then we present time-resolved spectroscopic dynamics of solvation dynamics inside cavities of active sites. The studies are carried out on a globular protein, staphylococcal nuclease. The solvation at sites inside the protein molecule’s cavities clearly reveals characteristics of the local environment. These solvation behaviors are directly correlated to enzyme activity. Project supported by the National Basic Research Program of China (Grant Nos. 2013CB921904, 2009CB930504, and 2013CB328700) and the National Natural Science Foundation of China (Grant Nos. 11074016, 11121091, 10934001, 61177020, 11134001, and 10828407).

  17. Targeted mutagenesis of an odorant receptor co-receptor using TALEN in Ostrinia furnacalis.

    PubMed

    Yang, Bin; Fujii, Takeshi; Ishikawa, Yukio; Matsuo, Takashi

    2016-03-01

    Genome editing using transcription activator-like effector nuclease (TALEN) has been applied for various model organisms but not yet for agricultural pest insects. In this study, TALEN-mediated mutagenesis of the gene encoding odorant receptor co-receptor (Orco) of an important agricultural pest Ostrinia furnacalis (OfurOrco) was carried out. Of the two pairs of TALEN constructs designed, one generated somatic and germline mutations at rates of 70.8% and 20.8%, respectively. Physiological and behavioral analyses using a gas chromatograph-electroantennographic detector system and a wind tunnel, respectively, revealed that antennal responses to sex pheromone components were decreased to trace levels, and behavioral responses were abolished in OfurOrco mutants. This study demonstrated that TALEN-mediated mutagenesis is applicable to pest insects, and these results will open the way for a better understanding of chemosensory systems in wild insects. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Herpesvirus mutagenesis facilitated by infectious bacterial artificial chromosomes (iBACs).

    PubMed

    Robinson, Karl E; Mahony, Timothy J

    2015-01-01

    A critical factor in the study of herpesviruses, their genes and gene functions is the capacity to derive mutants that harbor deletions, truncations, or insertions within the genetic elements of interest. Once constructed the impact of the introduced mutation on the phenotypic properties of the rescued virus can be determined in either in vitro or in vivo systems. However, the construction of such mutants by traditional virological mutagenesis techniques can be a difficult and laborious undertaking. The maintenance of a viral genome as an infectious bacterial artificial chromosome (iBAC), however, endows the capacity to manipulate the viral genome for mutagenesis studies with relative ease. Here, the construction and characterization of two gene deletion mutants of an alphaherpesvirus maintained as iBAC in combination with an inducible homologous recombination system in Escherichia coli is detailed. The methodology is generally applicable to any iBAC and is demonstrated to be a highly efficient and informative approach for mutant virus construction.

  19. Altered lipid accumulation in Nannochloropsis salina CCAP849/3 following EMS and UV induced mutagenesis

    PubMed Central

    Beacham, T.A.; Macia, V. Mora; Rooks, P.; White, D.A.; Ali, S.T.

    2015-01-01

    Microalgae have potential as a chemical feed stock in a range of industrial applications. Nannochloropsis salina was subject to EMS mutagenesis and the highest lipid containing cells selected using fluorescence-activated cell sorting. Assessment of growth, lipid content and fatty acid composition identified mutant strains displaying a range of altered traits including changes in the PUFA content and a total FAME increase of up to 156% that of the wild type strain. Combined with a reduction in growth this demonstrated a productivity increase of up to 76%. Following UV mutagenesis, lipid accumulation of the mutant cultures was elevated to more than 3 fold that of the wild type strain, however reduced growth rates resulted in a reduction in overall productivity. Changes observed are indicative of alterations to the regulation of the omega 6 Kennedy pathway. The importance of these variations in physiology for industrial applications such as biofuel production is discussed. PMID:26753128

  20. Bromination of deoxycytidine by eosinophil peroxidase: A mechanism for mutagenesis by oxidative damage of nucleotide precursors

    PubMed Central

    Henderson, Jeffrey P.; Byun, Jaeman; Williams, Michelle V.; McCormick, Michael L.; Parks, William C.; Ridnour, Lisa A.; Heinecke, Jay W.

    2001-01-01

    Oxidants generated by eosinophils during chronic inflammation may lead to mutagenesis in adjacent epithelial cells. Eosinophil peroxidase, a heme enzyme released by eosinophils, generates hypobromous acid that damages tissue in inflammatory conditions. We show that human eosinophils use eosinophil peroxidase to produce 5-bromodeoxycytidine. Flow cytometric, immunohistochemical, and mass spectrometric analyses all demonstrated that 5-bromodeoxycytidine generated by eosinophil peroxidase was taken up by cultured cells and incorporated into genomic DNA as 5-bromodeoxyuridine. Although previous studies have focused on oxidation of chromosomal DNA, our observations suggest another mechanism for oxidative damage of DNA. In this scenario, peroxidase-catalyzed halogenation of nucleotide precursors yields products that subsequently can be incorporated into DNA. Because the thymine analog 5-BrUra mispairs with guanine in DNA, generation of brominated pyrimidines by eosinophils might constitute a mechanism for cytotoxicity and mutagenesis at sites of inflammation. PMID:11172002

  1. Back to BAC: The Use of Infectious Clone Technologies for Viral Mutagenesis

    PubMed Central

    Hall, Robyn N.; Meers, Joanne; Fowler, Elizabeth; Mahony, Timothy

    2012-01-01

    Bacterial artificial chromosome (BAC) vectors were first developed to facilitate the propagation and manipulation of large DNA fragments in molecular biology studies for uses such as genome sequencing projects and genetic disease models. To facilitate these studies, methodologies have been developed to introduce specific mutations that can be directly applied to the mutagenesis of infectious clones (icBAC) using BAC technologies. This has resulted in rapid identification of gene function and expression at unprecedented rates. Here we review the major developments in BAC mutagenesis in vitro. This review summarises the technologies used to construct and introduce mutations into herpesvirus icBAC. It also explores developing technologies likely to provide the next leap in understanding these important viruses. PMID:22470833

  2. Transposon mutagenesis as an approach to improved understanding of Borrelia pathogenesis and biology

    PubMed Central

    Lin, Tao; Troy, Erin B.; Hu, Linden T.; Gao, Lihui; Norris, Steven J.

    2014-01-01

    Transposon insertion provides a method for near-random mutation of bacterial genomes, and has been utilized extensively for the study of bacterial pathogenesis and biology. This approach is particularly useful for organisms that are relatively refractory to genetic manipulation, including Lyme disease Borrelia. In this review, progress to date in the application of transposon mutagenesis to the study of Borrelia burgdorferi is reported. An effective Himar1-based transposon vector has been developed and used to acquire a sequence-defined library of nearly 4500 mutants in the infectious, moderately transformable B. burgdorferi B31 derivative 5A18NP1. Analysis of these transposon mutants using signature-tagged mutagenesis (STM) and Tn-seq approaches has begun to yield valuable information regarding the genes important in the pathogenesis and biology of this organism. PMID:24904839

  3. 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.

  4. Generation of chemically engineered ribosomes for atomic mutagenesis studies on protein biosynthesis.

    PubMed

    Erlacher, Matthias D; Chirkova, Anna; Voegele, Paul; Polacek, Norbert

    2011-05-01

    The protocol describes the site-specific chemical modification of 23S rRNA of Thermus aquaticus ribosomes. The centerpiece of this 'atomic mutagenesis' approach is the site-specific incorporation of non-natural nucleoside analogs into 23S rRNA in the context of the entire 70S ribosome. This technique exhaustively makes use of the available crystallographic structures of the ribosome for designing detailed biochemical experiments aiming at unraveling molecular insights of ribosomal functions. The generation of chemically engineered ribosomes carrying a particular non-natural 23S rRNA residue at the site of interest, a procedure that typically takes less than 2 d, allows the study of translation at the molecular level and goes far beyond the limits of standard mutagenesis approaches. This methodology, in combination with the presented tests for ribosomal functions adapted to chemically engineered ribosomes, allows unprecedented molecular insight into the mechanisms of protein biosynthesis.

  5. 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

  6. Reducing codon redundancy and screening effort of combinatorial protein libraries created by saturation mutagenesis.

    PubMed

    Kille, Sabrina; Acevedo-Rocha, Carlos G; Parra, Loreto P; Zhang, Zhi-Gang; Opperman, Diederik J; Reetz, Manfred T; Acevedo, Juan Pablo

    2013-02-15

    Saturation mutagenesis probes define sections of the vast protein sequence space. However, even if randomization is limited this way, the combinatorial numbers problem is severe. Because diversity is created at the codon level, codon redundancy is a crucial factor determining the necessary effort for library screening. Additionally, due to the probabilistic nature of the sampling process, oversampling is required to ensure library completeness as well as a high probability to encounter all unique variants. Our trick employs a special mixture of three primers, creating a degeneracy of 22 unique codons coding for the 20 canonical amino acids. Therefore, codon redundancy and subsequent screening effort is significantly reduced, and a balanced distribution of codon per amino acid is achieved, as demonstrated exemplarily for a library of cyclohexanone monooxygenase. We show that this strategy is suitable for any saturation mutagenesis methodology to generate less-redundant libraries.

  7. A Plasmid-Transposon Hybrid Mutagenesis System Effective in a Broad Range of Enterobacteria

    PubMed Central

    Monson, Rita; Smith, Debra S.; Matilla, Miguel A.; Roberts, Kevin; Richardson, Elizabeth; Drew, Alison; Williamson, Neil; Ramsay, Josh; Welch, Martin; Salmond, George P. C.

    2015-01-01

    Random transposon mutagenesis is a powerful technique used to generate libraries of genetic insertions in many different bacterial strains. Here we develop a system facilitating random transposon mutagenesis in a range of different Gram-negative bacterial strains, including Pectobacterium atrosepticum, Citrobacter rodentium, Serratia sp. ATCC39006, Serratia plymuthica, Dickeya dadantii, and many more. Transposon mutagenesis was optimized in each of these strains and three studies are presented to show the efficacy of this system. Firstly, the important agricultural pathogen D. dadantii was mutagenized. Two mutants that showed reduced protease production and one mutant producing the previously cryptic pigment, indigoidine, were identified and characterized. Secondly, the enterobacterium, Serratia sp. ATCC39006 was mutagenized and mutants incapable of producing gas vesicles, proteinaceous intracellular organelles, were identified. One of these contained a β-galactosidase transcriptional fusion within the gene gvpA1, essential for gas vesicle production. Finally, the system was used to mutate the biosynthetic gene clusters of the antifungal, anti-oomycete and anticancer polyketide, oocydin A, in the plant-associated enterobacterium, Dickeya solani MK10. The mutagenesis system was developed to allow easy identification of transposon insertion sites by sequencing, after facile generation of a replicon encompassing the transposon and adjacent DNA, post-excision. Furthermore, the system can also create transcriptional fusions with either β-galactosidase or β-glucuronidase as reporters, and exploits a variety of drug resistance markers so that multiple selectable fusions can be generated in a single strain. This system of various transposons has wide utility and can be combined in many different ways. PMID:26733980

  8. [Cloning and insertion mutagenesis of DNA fragment coding for the luminescent system of Photobacterium leiognathi].

    PubMed

    Ptitsyn, L R; Gurevich, V B; Barsanova, T G; Shenderov, A N; Khaĭkinson, M Ia

    1988-10-01

    Fragments of DNA, obtained from the luminescent bacterium Photobacterium leiognathi and inserted into the plasmid pBR322, were found to code for the luminescence expressed in E. coli cells. The genetic functions necessary for light production in E. coli are localized on a DNA fragment of about 7 kbp. The insertion mutagenesis was used to define the luminescence functions encoded by the hybrid plasmid.

  9. 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

  10. Building on the past, shaping the future: the Environmental Mutagenesis and Genomics Society.

    PubMed

    Wilson, Thomas E; DeMarini, David M; Dertinger, Stephen D; Engelward, Bevin P; Hanawalt, Philip C; MacGregor, James T; Smith-Roe, Stephanie L; Witt, Kristine L; Yauk, Carole L; Ljungman, Mats; Schwartz, Jeffrey L; Klein, Catherine B

    2013-04-01

    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 1969 and reflects the many advances in our understanding of the nature and breadth of gene-environment interactions during the intervening 43 years. Copyright © 2013 Wiley Periodicals, Inc.

  11. The mechanism of nucleotide excision repair-mediated UV-induced mutagenesis in nonproliferating cells.

    PubMed

    Kozmin, Stanislav G; Jinks-Robertson, Sue

    2013-03-01

    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.

  12. Effects of flanking base sequences on 5-bromodeoxyuridine mutagenesis in mammalian cells.

    PubMed

    Kresnak, M T; Davidson, R L

    1991-07-01

    The molecular mechanisms of incorporation-dependent, 5-bromodeoxyuridine (BrdU)-induced mutagenesis were analyzed in murine A9 cells that possess a single copy of the Escherichia coli gpt gene integrated into the chromosomal DNA as part of a shuttle vector. Four independently derived GPT- mutants with single base changes within the integrated gpt gene were utilized in BrdU-induced reversion analyses to test the relative mutability of guanine residues in four different settings: the 5' and 3' guanine residues of a GG doublet, the 3' guanine residue of a GGGG quartet, and the middle guanine residue of a GGG triplet. Two of the mutant lines possessed GG doublet sequences in which a GC----AT transition at either guanine residue of the doublet leads to restoration of GPT enzyme activity without restoring wild-type DNA sequence. Both lines were shown to be effectively reverted by BrdU incorporation-dependent mutagenesis, and sequencing of the gpt genes from numerous independently derived revertants of both lines demonstrated that greater than 90% of the revertants arose due to GC----AT transitions at the 3' guanine residue of the doublet. BrdU-induced reversion of two additional GPT- mutant lines demonstrated that the 3' guanine residue of a GGGG quartet is efficiently mutated, while the middle guanine residue of a GGG triplet sequence is at least 10-fold less mutable by BrdU incorporation-dependent mutagenesis than the 3' guanine residue of a GG doublet or GGGG quartet. All four mutant lines tested were equally revertible by treatment with the alkylating agent ethyl methane sulfonate. The results from this study define a sequence-specific mechanism for BrdU-induced, incorporation-dependent mutagenesis and demonstrate the use of reversion analysis for the determination of sequence specific effects at precise sites within a gene.

  13. Novobiocin Inhibits the Antimicrobial Resistance Acquired through DNA Damage-Induced Mutagenesis in Acinetobacter baumannii

    PubMed Central

    Jara, Luis M.; Pérez-Varela, María; Corral, Jordi; Arch, Marta; Cortés, Pilar; Bou, Germán; Barbé, Jordi

    2015-01-01

    Acinetobacter baumannii, a worldwide emerging nosocomial pathogen, acquires antimicrobial resistances in response to DNA-damaging agents, which increase the expression of multiple error-prone DNA polymerase components. Here we show that the aminocoumarin novobiocin, which inhibits the DNA damage response in Gram-positive bacteria, also inhibits the expression of error-prone DNA polymerases in this Gram-negative multidrug-resistant pathogen and, consequently, its potential acquisition of antimicrobial resistance through DNA damage-induced mutagenesis. PMID:26503651

  14. A Plasmid-Transposon Hybrid Mutagenesis System Effective in a Broad Range of Enterobacteria.

    PubMed

    Monson, Rita; Smith, Debra S; Matilla, Miguel A; Roberts, Kevin; Richardson, Elizabeth; Drew, Alison; Williamson, Neil; Ramsay, Josh; Welch, Martin; Salmond, George P C

    2015-01-01

    Random transposon mutagenesis is a powerful technique used to generate libraries of genetic insertions in many different bacterial strains. Here we develop a system facilitating random transposon mutagenesis in a range of different Gram-negative bacterial strains, including Pectobacterium atrosepticum, Citrobacter rodentium, Serratia sp. ATCC39006, Serratia plymuthica, Dickeya dadantii, and many more. Transposon mutagenesis was optimized in each of these strains and three studies are presented to show the efficacy of this system. Firstly, the important agricultural pathogen D. dadantii was mutagenized. Two mutants that showed reduced protease production and one mutant producing the previously cryptic pigment, indigoidine, were identified and characterized. Secondly, the enterobacterium, Serratia sp. ATCC39006 was mutagenized and mutants incapable of producing gas vesicles, proteinaceous intracellular organelles, were identified. One of these contained a β-galactosidase transcriptional fusion within the gene gvpA1, essential for gas vesicle production. Finally, the system was used to mutate the biosynthetic gene clusters of the antifungal, anti-oomycete and anticancer polyketide, oocydin A, in the plant-associated enterobacterium, Dickeya solani MK10. The mutagenesis system was developed to allow easy identification of transposon insertion sites by sequencing, after facile generation of a replicon encompassing the transposon and adjacent DNA, post-excision. Furthermore, the system can also create transcriptional fusions with either β-galactosidase or β-glucuronidase as reporters, and exploits a variety of drug resistance markers so that multiple selectable fusions can be generated in a single strain. This system of various transposons has wide utility and can be combined in many different ways.

  15. Construction of a guide-RNA for site-directed RNA mutagenesis utilising intracellular A-to-I RNA editing.

    PubMed

    Fukuda, Masatora; Umeno, Hiromitsu; Nose, Kanako; Nishitarumizu, Azusa; Noguchi, Ryoma; Nakagawa, Hiroyuki

    2017-02-02

    As an alternative to DNA mutagenesis, RNA mutagenesis can potentially become a powerful gene-regulation method for fundamental research and applied life sciences. Adenosine-to-inosine (A-to-I) RNA editing alters genetic information at the transcript level and is an important biological process that is commonly conserved in metazoans. Therefore, a versatile RNA-mutagenesis method can be achieved by utilising the intracellular RNA-editing mechanism. Here, we report novel guide RNAs capable of inducing A-to-I mutations by guiding the editing enzyme, human adenosine deaminase acting on RNA (ADAR). These guide RNAs successfully introduced A-to-I mutations into the target-site, which was determined by the reprogrammable antisense region. In ADAR2-over expressing cells, site-directed RNA editing could also be performed by simply introducing the guide RNA. Our guide RNA framework provides basic insights into establishing a generally applicable RNA-mutagenesis method.

  16. Construction of a guide-RNA for site-directed RNA mutagenesis utilising intracellular A-to-I RNA editing

    PubMed Central

    Fukuda, Masatora; Umeno, Hiromitsu; Nose, Kanako; Nishitarumizu, Azusa; Noguchi, Ryoma; Nakagawa, Hiroyuki

    2017-01-01

    As an alternative to DNA mutagenesis, RNA mutagenesis can potentially become a powerful gene-regulation method for fundamental research and applied life sciences. Adenosine-to-inosine (A-to-I) RNA editing alters genetic information at the transcript level and is an important biological process that is commonly conserved in metazoans. Therefore, a versatile RNA-mutagenesis method can be achieved by utilising the intracellular RNA-editing mechanism. Here, we report novel guide RNAs capable of inducing A-to-I mutations by guiding the editing enzyme, human adenosine deaminase acting on RNA (ADAR). These guide RNAs successfully introduced A-to-I mutations into the target-site, which was determined by the reprogrammable antisense region. In ADAR2-over expressing cells, site-directed RNA editing could also be performed by simply introducing the guide RNA. Our guide RNA framework provides basic insights into establishing a generally applicable RNA-mutagenesis method. PMID:28148949

  17. Dissecting partner recognition by an intrinsically disordered protein using descriptive random mutagenesis.

    PubMed

    Gruet, Antoine; Dosnon, Marion; Vassena, Andrea; Lombard, Vincent; Gerlier, Denis; Bignon, Christophe; Longhi, Sonia

    2013-09-23

    In view of getting insights into the molecular determinants of the binding efficiency of intrinsically disordered proteins (IDPs), we used random mutagenesis. As a proof of concept, we chose the interaction between the intrinsically disordered C-terminal domain of the measles virus nucleoprotein (NTAIL) and the X domain (XD) of the viral phosphoprotein and assessed how amino acid substitutions introduced at random within NTAIL affect partner recognition. In contrast with directed evolution approaches, we did not apply any selection and used the gene library approach not for production purposes but for achieving a better understanding of the NTAIL/XD interaction. For that reason, and to differentiate our approach from similar approaches that make use of systematic (i.e., targeted) mutagenesis, we propose to call it "descriptive random mutagenesis" (DRM). NTAIL variants generated by error-prone PCR were picked at random in the absence of selection pressure and were characterized in terms of sequence and binding abilities toward XD. DRM not only identified determinants of NTAIL/XD interaction that were in good agreement with previous work but also provided new insights. In particular, we discovered that the primary interaction site is poorly evolvable in terms of binding abilities toward XD. We also identified a critical NTAIL residue whose role in stabilizing the NTAIL/XD complex had previously escaped detection, and we identified NTAIL regulatory sites that dampen the interaction while being located outside the primary interaction site. Results show that DRM is a valuable approach to study binding abilities of IDPs.

  18. UV-Induced Mutagenesis in Escherichia coli SOS Response: A Quantitative Model

    PubMed Central

    Krishna, Sandeep; Maslov, Sergei; Sneppen, Kim

    2007-01-01

    Escherichia coli bacteria respond to DNA damage by a highly orchestrated series of events known as the SOS response, regulated by transcription factors, protein–protein binding, and active protein degradation. We present a dynamical model of the UV-induced SOS response, incorporating mutagenesis by the error-prone polymerase, Pol V. In our model, mutagenesis depends on a combination of two key processes: damage counting by the replication forks and a long-term memory associated with the accumulation of UmuD′. Together, these provide a tight regulation of mutagenesis, resulting, we show, in a “digital” turn-on and turn-off of Pol V. Our model provides a compact view of the topology and design of the SOS network, pinpointing the specific functional role of each of the regulatory processes. In particular, we suggest that the recently observed second peak in the activity of promoters in the SOS regulon (Friedman et al., 2005, PLoS Biology 3(7): e238) is the result of positive feedback from Pol V to RecA filaments. PMID:17367202

  19. Analysis of the antimutagenic effect of cinnamaldehyde on chemically induced mutagenesis in Escherichia coli.

    PubMed

    Ohta, T; Watanabe, K; Moriya, M; Shirasu, Y; Kada, T

    1983-01-01

    The antimutagenic effect of cinnamaldehyde on mutagenesis was investigated using ten kinds of chemical mutagen in Escherichia coli WP2s (uvr A-). In addition, the frequency of mutation induction by each mutagen in an SOS repair deficient (umuC-) strain was compared with that in a wild-type (umuC+) strain. Cinnamaldehyde greatly suppressed the umuC-dependent mutagenesis induced by 4-nitroquinoline 1-oxide (4-NQO), furylfuramide or captan. However, cinnamaldehyde was less effective against the umuC-independent mutagenesis by alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine and ethylmethanesulfonate. On the other hand, no inhibitory effect of cinnamaldehyde was observed on prophage induction or tif-mediated filamentous growth. These results suggest that a cinnamaldehyde does not prevent the induction of the SOS functions. Despite the decrease in the number of revertants, a remarkable increase was observed in the survival of 4-NQO-treated WP2s cells after exposure to cinnamaldehyde. The reactivation of survival suggests the promotion of some DNA repair system by cinnamaldehyde. This enhancement of survival was also observed in uvr B, polA, recF or umuC mutants and less in lexA or recB, C mutants. However, it was not observed in recA mutants. Therefore, we assume that cinnamaldehyde may enhance an error-free recombinational repair system by acting on recA-enzyme activity.

  20. Targeted mutagenesis in cotton (Gossypium hirsutum L.) using the CRISPR/Cas9 system

    PubMed Central

    Chen, Xiugui; Lu, Xuke; Shu, Na; Wang, Shuai; Wang, Junjuan; Wang, Delong; Guo, Lixue; Ye, Wuwei

    2017-01-01

    The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 system has been widely used for genome editing in various plants because of its simplicity, high efficiency and design flexibility. However, to our knowledge, there is no report on the application of CRISPR/Cas9-mediated targeted mutagenesis in cotton. Here, we report the genome editing and targeted mutagenesis in upland cotton (Gossypium hirsutum L., hereafter cotton) using the CRISPR/Cas9 system. We designed two guide RNAs to target distinct sites of the cotton Cloroplastos alterados 1 (GhCLA1) and vacuolar H+-pyrophosphatase (GhVP) genes. Mutations in these two genes were detected in cotton protoplasts. Most of the mutations were nucleotide substitutions, with one nucleotide insertion and one substitution found in GhCLA1 and one deletion found in GhVP in cotton protoplasts. Subsequently, the two vectors were transformed into cotton shoot apexes through Agrobacterium-mediated transformation, resulting in efficient target gene editing. Most of the mutations were nucleotide deletions, and the mutation efficiencies were 47.6–81.8% in transgenic cotton plants. Evaluation using restriction-enzyme-PCR assay and sequence analysis detected no off-target mutations. Our results indicated that the CRISPR/Cas9 system was an efficient and specific tool for targeted mutagenesis of the cotton genome. PMID:28287154

  1. Lack of Mutational Hot Spots during Decitabine-Mediated HIV-1 Mutagenesis

    PubMed Central

    Rawson, Jonathan M. O.; Landman, Sean R.; Reilly, Cavan S.; Bonnac, Laurent; Patterson, Steven E.

    2015-01-01

    Decitabine has previously been shown to induce lethal mutagenesis of human immunodeficiency virus type 1 (HIV-1). However, the factors that determine the susceptibilities of individual sequence positions in HIV-1 to decitabine have not yet been defined. To investigate this, we performed Illumina high-throughput sequencing of multiple amplicons prepared from proviral DNA that was recovered from decitabine-treated cells infected with HIV-1. We found that decitabine induced an ≈4.1-fold increase in the total mutation frequency of HIV-1, primarily due to a striking ≈155-fold increase in the G-to-C transversion frequency. Intriguingly, decitabine also led to an ≈29-fold increase in the C-to-G transversion frequency. G-to-C frequencies varied substantially (up to ≈80-fold) depending upon sequence position, but surprisingly, mutational hot spots (defined as upper outliers within the mutation frequency distribution) were not observed. We further found that every single guanine position examined was significantly susceptible to the mutagenic effects of decitabine. Taken together, these observations demonstrate for the first time that decitabine-mediated HIV-1 mutagenesis is promiscuous and occurs in the absence of a clear bias for mutational hot spots. These data imply that decitabine-mediated G-to-C mutagenesis is a highly effective antiviral mechanism for extinguishing HIV-1 infectivity. PMID:26282416

  2. Insertional mutagenesis by a hybrid piggyBac and sleeping beauty transposon in the rat.

    PubMed

    Furushima, Kenryo; Jang, Chuan-Wei; Chen, Diane W; Xiao, Ningna; Overbeek, Paul A; Behringer, Richard R

    2012-12-01

    A hybrid piggyBac/Sleeping Beauty transposon-based insertional mutagenesis system that can be mobilized by simple breeding was established in the rat. These transposons were engineered to include gene trap sequences and a tyrosinase (Tyr) pigmentation reporter to rescue the albinism of the genetic background used in the mutagenesis strategy. Single-copy transposon insertions were transposed into the rat genome by co-injection of plasmids carrying the transposon and RNA encoding piggyBac transposase into zygotes. The levels of transgenic Tyr expression were influenced by chromosomal context, leading to transgenic rats with different pigmentation that enabled visual genotyping. Transgenic rats designed to ubiquitously express either piggyBac or Sleeping Beauty transposase were generated by standard zygote injection also on an albino background. Bigenic rats carrying single-copy transposons at known loci and transposase transgenes exhibited coat color mosaicism, indicating somatic transposition. PiggyBac or Sleeping Beauty transposase bigenic rats bred with wild-type albino rats yielded offspring with pigmentation distinct from the initial transposon insertions as a consequence of germline transposition to new loci. The germline transposition frequency for Sleeping Beauty and piggyBac was ∼10% or about one new insertion per litter. Approximately 50% of the insertions occurred in introns. Chimeric transcripts containing endogenous and gene trap sequences were identified in Gabrb1 mutant rats. This mutagenesis system based on simple crosses and visual genotyping can be used to generate a collection of single-gene mutations in the rat.

  3. Insertional Mutagenesis by a Hybrid PiggyBac and Sleeping Beauty Transposon in the Rat

    PubMed Central

    Furushima, Kenryo; Jang, Chuan-Wei; Chen, Diane W.; Xiao, Ningna; Overbeek, Paul A.; Behringer, Richard R.

    2012-01-01

    A hybrid piggyBac/Sleeping Beauty transposon-based insertional mutagenesis system that can be mobilized by simple breeding was established in the rat. These transposons were engineered to include gene trap sequences and a tyrosinase (Tyr) pigmentation reporter to rescue the albinism of the genetic background used in the mutagenesis strategy. Single-copy transposon insertions were transposed into the rat genome by co-injection of plasmids carrying the transposon and RNA encoding piggyBac transposase into zygotes. The levels of transgenic Tyr expression were influenced by chromosomal context, leading to transgenic rats with different pigmentation that enabled visual genotyping. Transgenic rats designed to ubiquitously express either piggyBac or Sleeping Beauty transposase were generated by standard zygote injection also on an albino background. Bigenic rats carrying single-copy transposons at known loci and transposase transgenes exhibited coat color mosaicism, indicating somatic transposition. PiggyBac or Sleeping Beauty transposase bigenic rats bred with wild-type albino rats yielded offspring with pigmentation distinct from the initial transposon insertions as a consequence of germline transposition to new loci. The germline transposition frequency for Sleeping Beauty and piggyBac was ∼10% or about one new insertion per litter. Approximately 50% of the insertions occurred in introns. Chimeric transcripts containing endogenous and gene trap sequences were identified in Gabrb1 mutant rats. This mutagenesis system based on simple crosses and visual genotyping can be used to generate a collection of single-gene mutations in the rat. PMID:23023007

  4. Sleeping Beauty mutagenesis in a mouse medulloblastoma model defines networks that discriminate between human molecular subgroups.

    PubMed

    Genovesi, Laura A; Ng, Ching Ging; Davis, Melissa J; Remke, Marc; Taylor, Michael D; Adams, David J; Rust, Alistair G; Ward, Jerrold M; Ban, Kenneth H; Jenkins, Nancy A; Copeland, Neal G; Wainwright, Brandon J

    2013-11-12

    The Sleeping Beauty (SB) transposon mutagenesis screen is a powerful tool to facilitate the discovery of cancer genes that drive tumorigenesis in mouse models. In this study, we sought to identify genes that functionally cooperate with sonic hedgehog signaling to initiate medulloblastoma (MB), a tumor of the cerebellum. By combining SB mutagenesis with Patched1 heterozygous mice (Ptch1(lacZ/+)), we observed an increased frequency of MB and decreased tumor-free survival compared with Ptch1(lacZ/+) controls. From an analysis of 85 tumors, we identified 77 common insertion sites that map to 56 genes potentially driving increased tumorigenesis. The common insertion site genes identified in the mutagenesis screen were mapped to human orthologs, which were used to select probes and corresponding expression data from an independent set of previously described human MB samples, and surprisingly were capable of accurately clustering known molecular subgroups of MB, thereby defining common regulatory networks underlying all forms of MB irrespective of subgroup. We performed a network analysis to discover the likely mechanisms of action of subnetworks and used an in vivo model to confirm a role for a highly ranked candidate gene, Nfia, in promoting MB formation. Our analysis implicates candidate cancer genes in the deregulation of apoptosis and translational elongation, and reveals a strong signature of transcriptional regulation that will have broad impact on expression programs in MB. These networks provide functional insights into the complex biology of human MB and identify potential avenues for intervention common to all clinical subgroups.

  5. 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.

  6. CRISPR/Cas9-mediated mutagenesis of the RIN locus that regulates tomato fruit ripening.

    PubMed

    Ito, Yasuhiro; Nishizawa-Yokoi, Ayako; Endo, Masaki; Mikami, Masafumi; Toki, Seiichi

    2015-11-06

    Site-directed mutagenesis using genetic approaches can provide a wealth of resources for crop breeding as well as for biological research. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated 9 endonuclease (CRISPR/Cas9) system is a novel strategy used to induce mutations in a specific genome region; the system functions in a variety of organisms, including plants. Here, we report application of the CRISPR/Cas9 system to efficient mutagenesis of the tomato genome. In this study, we targeted the tomato RIN gene, which encodes a MADS-box transcription factor regulating fruit ripening. Three regions within the gene were targeted and mutations consisting either of a single base insertion or deletion of more than three bases were found at the Cas9 cleavage sites in T0 regenerated plants. The RIN-protein-defective mutants produced incomplete-ripening fruits in which red color pigmentation was significantly lower than that of wild type, while heterologous mutants expressing the remaining wild-type gene reached full-ripening red color, confirming the important role of RIN in ripening. Several mutations that were generated at three independent target sites were inherited in the T1 progeny, confirming the applicability of this mutagenesis system in tomato. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. DNA repair and sequence context affect 1O2-induced mutagenesis in bacteria

    PubMed Central

    Agnez-Lima, L. F.; Napolitano, R. L.; Fuchs, R. P. P.; Mascio, P. Di; Muotri, A. R.; Menck, C. F. M.

    2001-01-01

    Electronic excited molecular oxygen (singlet oxygen, 1O2) is known to damage DNA, yielding mutations. In this work, the mutagenicity induced by 1O2 in a defined sequence of DNA was investigated after replication in Escherichia coli mutants deficient for nucleotide and base excision DNA repair pathways. For this purpose a plasmid containing a 1O2-damaged 14 base oligonucleotide was introduced into E.coli by transfection and mutations were screened by hybridization with an oligonucleotide with the original sequence. Mutagenesis was observed in all strains tested, but it was especially high in the BH20 (fpg), AYM57 (fpg mutY) and AYM84 (fpg mutY uvrC) strains. The frequency of mutants in the fpg mutY strain was higher than in the triple mutant fpg mutY uvrC, suggesting that activity of the UvrABC excinuclease can favor the mutagenesis of these lesions. Additionally, most of the mutations were G→T and G→C transversions, but this was dependent on the position of the guanine in the sequence and on repair deficiency in the host bacteria. Thus, the kind of repair and the mutagenesis associated with 1O2-induced DNA damage are linked to the context of the damaged sequence. PMID:11433036

  8. In vitro Inactivation of Latent HSV by Targeted Mutagenesis Using an HSV-specific Homing Endonuclease

    PubMed Central

    Aubert, Martine; Boyle, Nicole M; Stone, Daniel; Stensland, Laurence; Huang, Meei-Li; Magaret, Amalia S; Galetto, Roman; Rawlings, David J; Scharenberg, Andrew M; Jerome, Keith R

    2014-01-01

    Following acute infection, herpes simplex virus (HSV) establishes latency in sensory neurons, from which it can reactivate and cause recurrent disease. Available antiviral therapies do not affect latent viral genomes; therefore, they do not prevent reactivation following therapy cessation. One possible curative approach involves the introduction of DNA double strand breaks in latent HSV genomes by rare-cutting endonucleases, leading to mutagenesis of essential viral genes. We tested this approach in an in vitro HSV latency model using the engineered homing endonuclease (HE) HSV1m5, which recognizes a sequence in the HSV-1 gene UL19, encoding the virion protein VP5. Coexpression of the 3′-exonuclease Trex2 with HEs increased HE-mediated mutagenesis frequencies up to sixfold. Following HSV1m5/Trex2 delivery with adeno-associated viral (AAV) vectors, the target site was mutated in latent HSV genomes with no detectable cell toxicity. Importantly, HSV production by latently infected cells after reactivation was decreased after HSV1m5/Trex2 exposure. Exposure to histone deacetylase inhibitors prior to HSV1m5/Trex2 treatment increased mutagenesis frequencies of latent HSV genomes another two- to fivefold, suggesting that chromatin modification may be a useful adjunct to gene-targeting approaches. These results support the continuing development of HEs and other nucleases (ZFNs, TALENs, CRISPRs) for cure of chronic viral infections. PMID:24496438

  9. A mouse chromosome 4 balancer ENU-mutagenesis screen isolates eleven lethal lines

    PubMed Central

    Boles, Melissa K; Wilkinson, Bonney M; Maxwell, Andrea; Lai, Lihua; Mills, Alea A; Nishijima, Ichiko; Salinger, Andrew P; Moskowitz, Ivan; Hirschi, Karen K; Liu, Bin; Bradley, Allan; Justice, Monica J

    2009-01-01

    Background ENU-mutagenesis is a powerful technique to identify genes regulating mammalian development. To functionally annotate the distal region of mouse chromosome 4, we performed an ENU-mutagenesis screen using a balancer chromosome targeted to this region of the genome. Results We isolated 11 lethal lines that map to the region of chromosome 4 between D4Mit117 and D4Mit281. These lines form 10 complementation groups. The majority of lines die during embryonic development between E5.5 and E12.5 and display defects in gastrulation, cardiac development, and craniofacial development. One line displayed postnatal lethality and neurological defects, including ataxia and seizures. Conclusion These eleven mutants allow us to query gene function within the distal region of mouse chromosome 4 and demonstrate that new mouse models of mammalian developmental defects can easily and quickly be generated and mapped with the use of ENU-mutagenesis in combination with balancer chromosomes. The low number of mutations isolated in this screen compared with other balancer chromosome screens indicates that the functions of genes in different regions of the genome vary widely. PMID:19267930

  10. Ascorbate enhances u. v. -mutagenesis in E. coli but inhibits it in Chinese hamster cells

    SciTech Connect

    Rossman, T.G.; Klein, C.B.; Naslund, M.

    1986-05-01

    Ascorbic acid (vitamin C) causes an increase in the mutation frequency of u.v.-irradiated Escherichia coli WP2. The enhancement occurs at all u.v. fluences, and is dependent upon the ascorbate concentration in the medium. A maximum effect (approximately 8- to 13-fold) is seen at 100-150 micrograms/ml, although some enhancement can be seen even at 10 micrograms/ml. The comutagenic effect of ascorbate with u.v. in E. coli is dependent upon peptone, a constituent of nutrient broth. The enhancement of u.v.-mutagenesis by ascorbate is absent in strains WP2s (uvrA) and WP6 (polA), suggesting that ascorbate affects the repair of pyrimidine dimers. The opposite results are observed for u.v.-mutagenesis in Chinese hamster V79 cells. The presence of ascorbate (50 micrograms/ml) during u.v. irradiation does not enhance the u.v. effect, but rather decreases it approximately 30%. These results are discussed with regard to differences in the mechanism of u.v.-mutagenesis and DNA repair in bacterial and mammalian cells.

  11. Pseudorabies virus glycoprotein C attachment-proficient revertants isolated through a simple, targeted mutagenesis scheme.

    PubMed

    Rue, Cary A; Ryan, Patrick

    2008-07-01

    Pseudorabies virus (PRV) glycoprotein C (gC) initiates virus attachment to cells by binding to heparan sulfate (HS) proteoglycans. The gC:HS interaction is not essential since gC null mutants still infect; however, they are more easily removed from cells during the initial stages of infection. The expendability of gC has facilitated a genetic mapping of the HS-binding domain, which is composed of three independent heparin-binding domains (HBDs) of six to eight amino acids each. Previous results suggested that at least one of the HBDs (HBD 1) functioned in a context-dependent manner. To define the context better, a reversion analysis was performed in which a defective gC containing a nonfunctional but intact HBD 1 regained HS-binding ability. To increase the reversion frequency, an efficient method for targeted, yet random mutagenesis of the gC gene was developed. The method involves random mutagenesis of a plasmid-borne copy of gC, and highly efficient recombination of the plasmid-borne genes into the virus genome at the site of a double-strand break in the viral gC locus. Revertants were recovered readily, and their gC alleles suggested that HS-binding could be restored by several different amino acid substitutions. This approach should be applicable to targeted mutagenesis of other herpesvirus genes.

  12. Are transversion mutations better? A Mutagenesis Assistant Program analysis on P450 BM-3 heme domain.

    PubMed

    Wong, Tuck Seng; Roccatano, Danilo; Schwaneberg, Ulrich

    2007-01-01

    Directed evolution represents a versatile tool to tailor enzyme properties to needs in industrial applications and to understand structure-function relationships. Genetic diversity is commonly generated using error-prone PCR. Exploration of sequence space by random mutagenesis strongly favors transitions when enzyme-based mutagenesis methods are employed (Wong, T. S., Zhurina, D., Schwaneberg, U., Comb. Chem. High Throughput Screen. 2006, 9, 271-288). The genetic code has been organized in a manner that limits chemical diversity when a single transition mutation occurs in a codon (Wong, T. S., Roccatano, D., Schwaneberg, U., Biocatal. Biotransformation 2006, in press). Are transitions more beneficial than transversions for adapting biocatalysts to non-natural process conditions? In a statistical analysis performed with the Mutagenesis Assistant Program (MAP), we compared the consequences of transition and transversion bias on amino acid substitution patterns of the P450 BM-3 heme domain. For the analysis, we used a recently introduced benchmarking system consisting of a protein structure indicator, an amino acid diversity indicator with a codon diversity coefficient, and a chemical diversity indicator. A detailed analysis for the P450 BM-3 heme domain showed that an ideal transversion bias generates more diverse amino acid substitution patterns with a significantly different chemical composition than an ideal transition bias. Emphasis is given on the theoretical analysis with a brief discussion on potential implication of transition and transversion bias in directed evolution experiments.

  13. Targeted mutagenesis in cotton (Gossypium hirsutum L.) using the CRISPR/Cas9 system.

    PubMed

    Chen, Xiugui; Lu, Xuke; Shu, Na; Wang, Shuai; Wang, Junjuan; Wang, Delong; Guo, Lixue; Ye, Wuwei

    2017-03-13

    The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 system has been widely used for genome editing in various plants because of its simplicity, high efficiency and design flexibility. However, to our knowledge, there is no report on the application of CRISPR/Cas9-mediated targeted mutagenesis in cotton. Here, we report the genome editing and targeted mutagenesis in upland cotton (Gossypium hirsutum L., hereafter cotton) using the CRISPR/Cas9 system. We designed two guide RNAs to target distinct sites of the cotton Cloroplastos alterados 1 (GhCLA1) and vacuolar H(+)-pyrophosphatase (GhVP) genes. Mutations in these two genes were detected in cotton protoplasts. Most of the mutations were nucleotide substitutions, with one nucleotide insertion and one substitution found in GhCLA1 and one deletion found in GhVP in cotton protoplasts. Subsequently, the two vectors were transformed into cotton shoot apexes through Agrobacterium-mediated transformation, resulting in efficient target gene editing. Most of the mutations were nucleotide deletions, and the mutation efficiencies were 47.6-81.8% in transgenic cotton plants. Evaluation using restriction-enzyme-PCR assay and sequence analysis detected no off-target mutations. Our results indicated that the CRISPR/Cas9 system was an efficient and specific tool for targeted mutagenesis of the cotton genome.

  14. 5-fluorouracil in lethal mutagenesis of foot-and-mouth disease virus.

    PubMed

    Agudo, Rubén; Arias, Armando; Domingo, Esteban

    2009-06-01

    5-fluorouracil (FU) is a pyrimidine analogue extensively used in cancer chemotherapy. FU can be metabolized into 5-fluorouridine-triphosphate, which can be used as substrate for viral RNA-dependent RNA polymerases. This results in the incorporation of mutations into viral RNA. Accumulation of mutations may lead to loss of virus infectivity, in a process known as lethal mutagenesis. RNA virus pathogens are particularly difficult to control because they are highly mutable, and mutants resistant to antiviral agents are readily selected. Here, we review the basic principles of lethal mutagenesis as an antiviral approach, and the participation of FU in its development. Recent studies with foot-and-mouth disease virus indicate that FU can act both as an inhibitor and as a mutagen during foot-and-mouth disease virus replication. This dual activity renders FU an adequate drug for lethal mutagenesis. We suggest that structural and biochemical studies can contribute to the lead to new design of base or nucleoside analogues targeted specifically to viral polymerases.

  15. Mouse ENU Mutagenesis to Understand Immunity to Infection: Methods, Selected Examples, and Perspectives

    PubMed Central

    Caignard, Grégory; Eva, Megan M.; van Bruggen, Rebekah; Eveleigh, Robert; Bourque, Guillaume; Malo, Danielle; Gros, Philippe; Vidal, Silvia M.

    2014-01-01

    Infectious diseases are responsible for over 25% of deaths globally, but many more individuals are exposed to deadly pathogens. The outcome of infection results from a set of diverse factors including pathogen virulence factors, the environment, and the genetic make-up of the host. The completion of the human reference genome sequence in 2004 along with technological advances have tremendously accelerated and renovated the tools to study the genetic etiology of infectious diseases in humans and its best characterized mammalian model, the mouse. Advancements in mouse genomic resources have accelerated genome-wide functional approaches, such as gene-driven and phenotype-driven mutagenesis, bringing to the fore the use of mouse models that reproduce accurately many aspects of the pathogenesis of human infectious diseases. Treatment with the mutagen N-ethyl-N-nitrosourea (ENU) has become the most popular phenotype-driven approach. Our team and others have employed mouse ENU mutagenesis to identify host genes that directly impact susceptibility to pathogens of global significance. In this review, we first describe the strategies and tools used in mouse genetics to understand immunity to infection with special emphasis on chemical mutagenesis of the mouse germ-line together with current strategies to efficiently identify functional mutations using next generation sequencing. Then, we highlight illustrative examples of genes, proteins, and cellular signatures that have been revealed by ENU screens and have been shown to be involved in susceptibility or resistance to infectious diseases caused by parasites, bacteria, and viruses. PMID:25268389

  16. [IXR1 and HMO1 genes jointly control the level of spontaneous mutagenesis in yeast Saccharomyces cerevisiae].

    PubMed

    Fedorov, D V; Koval'tsova, S V; Peshekhonov, V T; Korolev, V G

    2010-06-01

    The yeast genes IXR1 and HMO1 encode proteins belonging to the family of chromatin nonhistone proteins, which are able to recognize and bind to irregular DNA structures. The full deletion of gene IXR1 leads to an increase in cell resistance to the lethal action of UV light, gamma-rays, and MMS, increases spontaneous mutagenesis and significantlly decreases the level of UV-induced mutations. It was earlier demonstrated in our works that the hmo 1 mutation renders cells sensitive to the lethal action of cisplatin and virtually does not affect the sensitivity to UV light. Characteristically, the rates of spontaneous and UV-induced mutagenesis in the mutant are increased. Epistatic analysis of the double mutation hmo 1 ixr1 demonstrated that the interaction of these genes in relation to the lethal effect of cisplatin and UV light, as well as UV-induced mutagenesis, is additive. This suggests that the products of genes HMO1 and IXR1 participate in different repair pathways. The ixr1 mutation significantly increases the rate of spontaneous mutagenesis mediated by replication errors, whereas mutation hmo 1 increases the rate of repair mutagenesis. In wild-type cells, the level of spontaneous mutagenesis was nearly one order of magnitude lower than that obtained in cells of the double mutant. Consequently, the combined activity of the Hmo 1 and the Ixr1 proteins provides efficient correction of both repair and replication errors.

  17. Dietary effects of soy isoflavones daidzein and genistein on 7,12-dimethylbenz[a]anthracene-induced mammary mutagenesis and carcinogenesis in ovariectomized Big Blue transgenic rats.

    PubMed

    Manjanatha, Mugimane G; Shelton, Sharon; Bishop, Michelle E; Lyn-Cook, Lascelles E; Aidoo, Anane

    2006-12-01

    The major constituents of isoflavones, daidzein (DZ) and genistein (GE) are known to interact with the alpha and beta estrogen receptors (ERalpha/beta) in several tissues including mammary. In this study, we used ovariectomy (OVX) to model menopause and determined the effects of DZ, GE or 17beta-estradiol (E2) exposures on chemically induced mutagenesis and carcinogenesis in the mammary glands of female Big Blue (BB) transgenic rats. The rats were fed control diet containing the isoflavones and E2 and treated with a single oral dose of 7,12-dimethylbenz[a]anthracene (DMBA) at PND 50. Animals were sacrificed at 16 or 20 weeks post-carcinogen treatment to assess mutant frequencies (MFs) and histopathological parameters, respectively. The isoflavones or E2 supplementation alone resulted in modest increases in the lacI MF that were not significantly different from the MFs measured in rats fed the control diet alone. DMBA exposure, however, induced significant increases in the lacI MFs in the mammary of both OVX and ovary intact (INT) rats and Hprt MFs in spleen lymphocytes (P=0.01). In general, feeding the isoflavones or E2 separately did not cause any significant changes in DMBA-induced mutagenicity in the mammary. However, feeding the isoflavone mixture (DZG) resulted in a significant reduction in the DMBA-induced lacI MFs (P=0.05). Cell proliferation as measured by PCNA immunohistochemistry was increased in both OVX and INT rats exposed to DMBA as compared with rats fed control diet (P=0.05). Mammary histology indicated that hyperplasia was induced in most of the treatment groups including control. Although DMBA treatment did not induce mammary tumors in the OVX rats, adenoma and adenocarcinoma were detected in the mammary glands of INT rats.

  18. Mutation Induced Extinction in Finite Populations: Lethal Mutagenesis and Lethal Isolation

    PubMed Central

    Wylie, C. Scott; Shakhnovich, Eugene I.

    2012-01-01

    Reproduction is inherently risky, in part because genomic replication can introduce new mutations that are usually deleterious toward fitness. This risk is especially severe for organisms whose genomes replicate “semi-conservatively,” e.g. viruses and bacteria, where no master copy of the genome is preserved. Lethal mutagenesis refers to extinction of populations due to an unbearably high mutation rate (U), and is important both theoretically and clinically, where drugs can extinguish pathogens by increasing their mutation rate. Previous theoretical models of lethal mutagenesis assume infinite population size (N). However, in addition to high U, small N can accelerate extinction by strengthening genetic drift and relaxing selection. Here, we examine how the time until extinction depends jointly on N and U. We first analytically compute the mean time until extinction (τ) in a simplistic model where all mutations are either lethal or neutral. The solution motivates the definition of two distinct regimes: a survival phase and an extinction phase, which differ dramatically in both how τ scales with N and in the coefficient of variation in time until extinction. Next, we perform stochastic population-genetics simulations on a realistic fitness landscape that both (i) features an epistatic distribution of fitness effects that agrees with experimental data on viruses and (ii) is based on the biophysics of protein folding. More specifically, we assume that mutations inflict fitness penalties proportional to the extent that they unfold proteins. We find that decreasing N can cause phase transition-like behavior from survival to extinction, which motivates the concept of “lethal isolation.” Furthermore, we find that lethal mutagenesis and lethal isolation interact synergistically, which may have clinical implications for treating infections. Broadly, we conclude that stably folded proteins are only possible in ecological settings that support sufficiently large

  19. Illegitimate recombination: an efficient method for random mutagenesis in Mycobacterium avium subsp. hominissuis.

    PubMed

    Khattak, Faisal Asghar; Kumar, Ashutosh; Kamal, Elisabeth; Kunisch, Ralph; Lewin, Astrid

    2012-09-11

    The genus Mycobacterium (M.) comprises highly pathogenic bacteria such as M. tuberculosis as well as environmental opportunistic bacteria called non-tuberculous mycobacteria (NTM). While the incidence of tuberculosis is declining in the developed world, infection rates by NTM are increasing. NTM are ubiquitous and have been isolated from soil, natural water sources, tap water, biofilms, aerosols, dust and sawdust. Lung infections as well as lymphadenitis are most often caused by M. avium subsp. hominissuis (MAH), which is considered to be among the clinically most important NTM. Only few virulence genes from M. avium have been defined among other things due to difficulties in generating M. avium mutants. More efforts in developing new methods for mutagenesis of M. avium and identification of virulence-associated genes are therefore needed. We developed a random mutagenesis method based on illegitimate recombination and integration of a Hygromycin-resistance marker. Screening for mutations possibly affecting virulence was performed by monitoring of pH resistance, colony morphology, cytokine induction in infected macrophages and intracellular persistence. Out of 50 randomly chosen Hygromycin-resistant colonies, four revealed to be affected in virulence-related traits. The mutated genes were MAV_4334 (nitroreductase family protein), MAV_5106 (phosphoenolpyruvate carboxykinase), MAV_1778 (GTP-binding protein LepA) and MAV_3128 (lysyl-tRNA synthetase LysS). We established a random mutagenesis method for MAH that can be easily carried out and combined it with a set of phenotypic screening methods for the identification of virulence-associated mutants. By this method, four new MAH genes were identified that may be involved in virulence.

  20. Engineered Zymomonas mobilis for salt tolerance using EZ-Tn5-based transposon insertion mutagenesis system.

    PubMed

    Wang, Jing-Li; Wu, Bo; Qin, Han; You, Yang; Liu, Song; Shui, Zong-Xia; Tan, Fu-Rong; Wang, Yan-Wei; Zhu, Qi-Li; Li, Yan-Bin; Ruan, Zhi-Yong; Ma, Ke-Dong; Dai, Li-Chun; Hu, Guo-Quan; He, Ming-Xiong

    2016-06-10

    The cell growth and ethanol yield of Zymomonas mobilis may be detrimentally affected by salt stress frequently present in some biomass-based fermentation systems, leading to a decrease in the rate of sugar conversion to ethanol or other bioproducts. To address this problem, improving the salt tolerance of Z. mobilis is a desirable way. However, limited progress has been made in development of Z. mobilis with higher salt tolerance for some technical challenges in the past decades. Recently, transposon insertion mutant system has been widely used as a novel genetic tool in many organisms to develop mutant strains. In this study, Tn5-based transposon insertion mutagenesis system firstly used for construction of higher salt tolerance strain in Z. mobilis. Approximately 200 Z. mobilis ZM4 mutants were generated by using Tn5-based transposon mutagenesis system. The mutant strain ZMT2 with improved salt tolerance phenotype was obtained by screening on RM agar plates with additional 1 % NaCl. Strain ZMT2 was confirmed to exhibit better fermentation performance under NaCl stress than wild type of strain ZM4. The transposon insertion was located in ZMO1122 (himA) by genome walking. Discruption of himA gene showed that himA may play an important role in response to salt tolerance in Z. mobils. The mutant strain ZMT2 with a transposon insertion in himA gene of the genome showed obviously higher sugar conversion rate to ethonal under up to 2 % NaCl stress than did the wild ZM4 strain. Besides, ZMT2 exhibited shared fermentative capabilities with wild ZM4 strain under no or low NaCl stress. This report firstly showed that himA played a role in responding to NaCl stress. Furthermore, the result indicated that Tn5-based transposon mutagenesis system was a feasible tool not only for genetic engineering in Z. mobilis strain improvement, but also in tapping resistent genes.

  1. Inactivation and mutagenesis of herpes virus by photodynamic treatment with therapeutic dyes.

    PubMed

    Lytle, C D; Carney, P G; Felten, R P; Bushar, H F; Straight, R C

    1989-09-01

    Dyes which photosensitize membranes may be clinically useful for photodynamic treatment (PDT) of Herpes simplex virus (HSV) infections. It is important to determine whether the enveloped HSV can be inactivated via membrane damage without affecting the genetic material. Selection of appropriate PDT conditions, including the choice of dye, could minimize viral mutagenesis. We determined the mutagenesis caused by PDT employing three membrane-photosensitizing dyes of potential use in cancer photochemotherapy (Photofrin II, polyhematoporphyrin esters, zinc phthalocyanine tetrasulfonates) and a DNA-photosensitizing dye (proflavine sulfate). The effects were compared to those caused by exposure of HSV to ultraviolet radiation (UV). The procedure consisted of incubating HSV with microgram/ml (microM) concentrations of the dye, irradiating the samples with broad spectrum visible/near-UV radiation (Daylight fluorescent lamps) and assaying the survival of the treated HSV. Zinc phthalocyanine was the most potent dye per absorbed photon for inactivating HSV. In parallel with determination of survival, progeny of the surviving virus were grown for determination of mutagenesis. The progeny virus was harvested and subsequently assayed in the presence and absence of 40 micrograms/ml iododeoxycytidine (ICrd) to determine the frequency of mutation to ICrd resistance. Mutation frequencies were determined for progeny from the 1-4% survival level. For PDT with each membrane-photosensitizing dye, only zinc phthalocyanine increased the mutation frequency over the untreated control. This increase was less than 2-fold. Proflavine increased the mutation frequency 2-3 fold over the untreated control. Ultraviolet produced a 15-20 fold increase over the untreated control.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. CRISPR/Cas9-mediated targeted mutagenesis in the liverwort Marchantia polymorpha L.

    PubMed

    Sugano, Shigeo S; Shirakawa, Makoto; Takagi, Junpei; Matsuda, Yoriko; Shimada, Tomoo; Hara-Nishimura, Ikuko; Kohchi, Takayuki

    2014-03-01

    Targeted genome modification technologies are key tools for functional genomics. The clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease Cas9 system (CRISPR/Cas9) is an emerging technology for targeted genome modification. The CRISPR/Cas9 system consists of a short guide RNA (gRNA), which specifies the target genome sequence, and the Cas9 protein, which has endonuclease activity. The CRISPR/Cas9 system has been applied to model animals and flowering plants, including rice, sorghum, wheat, tobacco and Arabidopsis. Here, we report the application of CRISPR/Cas9 to targeted mutagenesis in the liverwort Marchantia polymorpha L., which has emerged as a model species for studying land plant evolution. The U6 promoter of M. polymorpha was identified and cloned to express the gRNA. The target sequence of the gRNA was designed to disrupt the gene encoding auxin response factor 1 (ARF1) in M. polymorpha. Using Agrobacterium-mediated transformation, we isolated stable mutants in the gametophyte generation of M. polymorpha. CRISPR/Cas9-based site-directed mutagenesis in vivo was achieved using either the Cauliflower mosaic virus 35S or M. polymorpha EF1α promoter to express Cas9. Isolated mutant individuals showing an auxin-resistant phenotype were not chimeric. Moreover, stable mutants were produced by asexual reproduction of T1 plants. Multiple arf1 alleles were easily established using CRIPSR/Cas9-based targeted mutagenesis. Our results provide a rapid and simple approach for molecular genetics in M. polymorpha, and raise the possibility that CRISPR/Cas9 may be applied to a wide variety of plant species.

  3. Transposon mutagenesis identifies genes driving hepatocellular carcinoma in a chronic hepatitis B mouse model

    PubMed Central

    Bard-Chapeau, Emilie A.; Nguyen, Anh-Tuan; Rust, Alistair G.; Sayadi, Ahmed; Lee, Philip; Chua, Belinda Q; New, Lee-Sun; de Jong, Johann; Ward, Jerrold M.; Chin, Christopher KY.; Chew, Valerie; Toh, Han Chong; Abastado, Jean-Pierre; Benoukraf, Touati; Soong, Richie; Bard, Frederic A.; Dupuy, Adam J.; Johnson, Randy L.; Radda, George K.; Chan, Eric CY.; Wessels, Lodewyk FA.; Adams, David J.

    2014-01-01

    The most common risk factor for developing hepatocellular carcinoma (HCC) is chronic infection with hepatitis B virus (HBV). To better understand the evolutionary forces driving HCC we performed a near saturating transposon mutagenesis screen in a mouse HBV model of HCC. This screen identified 21 candidate early stage drivers, and a bewildering number (2860) of candidate later stage drivers, that were enriched for genes mutated, deregulated, or that function in signaling pathways important for human HCC, with a striking 1199 genes linked to cellular metabolic processes. Our study provides a comprehensive overview of the genetic landscape of HCC. PMID:24316982

  4. Genetically engineered insertional mutagenesis in mice to model cancer: Sleeping Beauty.

    PubMed

    Howell, Viive M; Colvin, Emily K

    2014-01-01

    The ability to accurately model human cancer in mice enables in vivo examination of the biological mechanisms related to cancer initiation and progression as well as preclinical testing of new anticancer treatments and potential targets. The emergence of the genetically engineered Sleeping Beauty system of insertional mutagenesis has led to the development of a new generation of genetic mouse models of cancer and identification of novel cancer-causing genes. This chapter reviews the published cancer models of Sleeping Beauty and strategies using available strains to generate several models of cancer.

  5. Construction, characterization, and mutagenesis of an anti-fluorescein single chain antibody idiotype family.

    PubMed

    Denzin, L K; Voss, E W

    1992-05-05

    In addition to crystallographic studies that determined antigen contact residues for monoclonal anti-fluorescein (Fl) antibody 4-4-20 (Ka = 2.5 x 10(10) M-1), primary structure comparisons revealed idiotypically cross-reactive monoclonal antibodies (mAbs) 9-40 (Ka = 4.4 x 10(7) M-1), 12-40 (Ka = 4.0 x 10(8) M-1), and 5-14 (Ka = 2.4 x 10(8) M-1) possessed identical Fl contact residues, with the exception of L34His for L34Arg. Site-specific mutagenesis of single chain antibody (SCA) 4-4-20 in which L34Arg was changed to L34His resulted in approximately 1000- and 3-fold decreases in binding affinity and Qmax (maximum quenching of bound Fl), respectively, which suggested that L34Arg was directly involved in increased binding affinity and fluorescence quenching. Therefore, substitution of Arg for His at residue L34 in mAbs 9-40, 12-40, and 5-14 should result in increased binding affinity and Qmax. To facilitate site-specific mutagenesis studies, single chain derivatives of mAbs 9-40, 12-40, and 5-14 were constructed. Following expression in Escherichia coli, characterization of the SCAs demonstrated that when compared with the respective parental mAb, the SCAs possessed identical binding affinities and similar Qmax and lambda max (absorption profiles of bound Fl) values. These results validated SCA 9-40, 12-40, and 5-14 for use in site-directed mutagenesis studies. Results of mutagenesis studies indicated that substitution of L34Arg into the active sites of 9-40, 12-40, and 5-14 was not enough to produce 4-4-20-like binding characteristics. Therefore, the following single chain mutants were constructed: 9-40L34Arg/L46Val, 12-40L34Arg/L46Val and 5-14L34Arg/L46Val, 9-40L34Arg/L46Val/H101Asp and 4-4-20H101Ala. Results demonstrated that these mutations were not able to render the mutant SCAs with increased binding affinity and fluorescence quenching values. Collectively, these results suggest that the combining sites of mAb 9-40, 12-40, and 5-14 may possess different active

  6. Structure of the sodium channel pore revealed by serial cysteine mutagenesis.

    PubMed Central

    Pérez-García, M T; Chiamvimonvat, N; Marban, E; Tomaselli, G F

    1996-01-01

    The pores of voltage-gated cation channels are formed by four intramembrane segments that impart selectivity and conductance. Remarkably little is known about the higher order structure of these critical pore-lining or P segments. Serial cysteine mutagenesis reveals a pattern of side-chain accessibility that contradicts currently favored structural models based on alpha-helices or beta-strands. Like the active sites of many enzymes of known structure, the sodium channel pore consists of irregular loop regions. Images Fig. 1 Fig. 4 PMID:8552626

  7. Transversion-specific purine analogue mutagens and the mechanism of hydroxylaminopurine mutagenesis.

    PubMed

    Murray, V

    1987-04-01

    The tryptophan synthetase gene A series of mutants in E. coli has been used to examine the mutational specificity of over 80 purine base analogues. 4 purine analogues have been discovered that solely cause transversions. Evidence is presented that hydroxylaminopurine mutagenesis is caused by a covalent reaction of these compounds with DNA. The transversion-causing purine analogues are derivatives of 2-aminopurine (2AP) and 2,6-diaminopurine (2,6DAP). They stimulate the full reversion frequency of those trp A which can revert through an AT----CG transversion. 8 purine base analogues have been found that induce the AT----CG transversion at the trp A88 site; and 2-amino-6-methylaminopurine (2A6MAP) stimulates by 124-fold, 2-amino-6-ethylaminopurine by 20-fold, 2-methylaminopurine (2MAP) by 9.4-fold, 2,6-bismethylaminopurine by 25-fold, 2AP by 230-fold, 2,6DAP by 15-fold, 2.6-diaminopurine riboside by 5-fold, and 2-hydroxylaminopurine by 11-fold. The last 4 analogues also cause transitions. 2A6MAP, 2-amino-6-ethylaminopurine and 2,6-bismethylaminopurine stimulate only the AT----CG transversion while 2MAP additionally gives rise to AT----TA transversions. By testing other negative 2AP derivatives, the structural requirements necessary for AT----CG transversion mutagenesis have been determined. All 12 hydroxylaminopurine base analogues tested, 2,6-dimethoxyaminopurine and 2-hydrazinopurine were found to cause transition mutations. All of the compounds stimulated the AT----GC transition (by up to 1000-fold) and 11 of the 14 base analogues raised the GT----AT transition (by up to 450-fold). On increasing the hydroxylaminopurine concentration, the mutation frequency also increased concomitantly. Since 6-hydroxylamino-9-methylpurine and 6-methylhydroxylaminopurine cause transitions, the mechanism of hydroxylaminopurine mutagenesis cannot be entirely due to an alteration in tautomeric equilibria or "wobble" type base mispairing. It is proposed that a major mechanism for

  8. Isolation of Magnetospirillum magneticum AMB-1 mutants defective in bacterial magnetic particle synthesis by transposon mutagenesis.

    PubMed

    Wahyudi, A T; Takeyama, H; Matsunaga, T

    2001-01-01

    Nonmagnetic mutants of Magnetospirillum magneticum AMB-1 were recovered following mini-Tn5 transposon mutagenesis. Transconjugants with kanamycin resistance were obtained at a frequency of 2.7 x 10(-7) per recipient. Of 3327 transconjugants, 62 were defective for bacterial magnetic particle (BMP) synthesis. The frequency of independent transposition events for nonmagnetic mutants was about 1.4% in transconjugants. Further analysis of DNA sequences flanking transposon by inverted polymerase chain reaction allowed isolation of at least 10 genes or DNA sequences involved in BMP synthesis in M. magneticum AMB-1.

  9. Enhancing effect of heterocyclic amines and beta-carbolines on UV or chemically induced mutagenesis in E. coli.

    PubMed

    Shimoi, K; Kawabata, H; Tomita, I

    1992-08-01

    Most heterocyclic amines and beta-carbolines--harman, norharman, harmine, harmaline--enhanced UVC (254 nm) induced mutagenesis without microsomal activation in E. coli B/r WP2. 3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) was most effective and increased UVAB (295-400 nm) induced mutations as well as UVC induced ones. Trp-P-1 enhanced the frequencies of mutations induced by not only UV but also 4-nitroquinoline-1-oxide (4NQO) or 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF2), while it showed little effect on N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or gamma-ray induced mutagenesis. Trp-P-1 decreased the survival of UVC irradiated cells of CM571recA. However, these effects of Trp-P-1 on UVC induced mutagenesis and lethality were not observed in WP2suvrA which is excision repair deficient. The alkaline sucrose gradient sedimentation analysis demonstrated that Trp-P-1 blocked the incision step in DNA excision repair. Further, pretreatment with Trp-P-1 before UVC irradiation showed no effect on UVC induced mutagenesis. Similar effects were also seen in the case of harman or norharman. These results suggest that heterocyclic amines and beta-carbolines inhibit DNA excision repair directly or indirectly, thus enhancing UV or chemically induced mutagenesis.

  10. [Rational screen of high kelimycin-producing strain by combined conventional mutagenesis and high-throughput screen method].

    PubMed

    Liu, Xinxing; Li, Ping; Zhao, Xiaofeng; Wang, Yonghong; Zhang, Siliang

    2013-07-04

    Kelimycin, is a new macrolide antibiotic drug obtained through genetic engineering approaches. With 4"-O-isovalerylspiramycins as the major components, was produced by genetically engineered Streptomyces spiramyceticus transformed with 4"-O-acyltransferase gene from S. mycarofaciens. Improve the efficiency of strain fermentation, to meet the needs of industrial production. The enhanced kelimycin-producing strain was obtained by applying various conventional mutagenesis approaches, and high-throughput screen methods, including protoplast mutagenesis by ultraviolet, mutagenesis by diethyl sulfate and UV-reactivation, valine content resistance screen and enrichment of improved strains. A strategy for positive mutant enrichment was developed after mutagenesis and before high-throughput screen. Finally, the high-producing strain WSJ-1-7-49-133-82-18-43 was obtained and its potency in shake flask increased by 56% compared to the original strain. The potency in 500 L pilot fermenter increased by 61%. This study shows that the screening industrial production strains can be enhanced effectively by combining multiple conventional mutagenesis and high-throughput screen methods.

  11. Reciprocal relationship between mouse germ-cell mutagenesis and basic genetics: from early beginnings to future opportunities.

    PubMed

    Russell, L B

    1989-01-01

    The scientific foundations for several mammalian germ-line mutagenesis tests in common use today were laid in the 1930s, 1940s, and early 1950s. Subsequent developments in the field have had multiple objectives: detection of mutagenicity of environmental agents (which has led to the development of numerous methodologies), identification of biological and physical factors that affect mutation yield, analysis of the structural nature of the genetic alterations, and assessment of the organismic effects of various types of mutations. Mutagenesis studies have made numerous contributions to basic genetics by generating mutant types that led to elucidation of sex-determining mechanisms in mammals; formulation of the single-active-, or inactive-, X-chromosome hypothesis; correlation of genetic and cytological maps; discovery of genetic "imprinting" phenomena; study of developmental pathways and cell lineages, etc. Particularly useful are sets of complexly overlapping deletions that have been recovered in radiation mutagenesis studies, propagated in breeding stocks, and genetically analyzed; these have constituted prerequisites for molecular genetic studies aimed at development of the DNA structure-function relationships for important genomic regions. Mutagenesis experiments have also served to identify mutagens that are particularly effective in inducing specific types of genetic lesions desired for basic studies. Reciprocally, basic genetics has contributed to the development of mutagenesis tests and has enhanced the value of the specific-locus test by adding to its quantitative capabilities the capability for qualitatively characterizing the actions of mutagens.

  12. Control of mammalian cell mutagenesis and differentiation by chemicals which initiate or promote tumor formation

    SciTech Connect

    Jones, C. A.; Huberman, E.

    1980-01-01

    A cell-mediated mutagenesis assay was developed to predict the potential carcinogenic hazard of some environmental chemicals. In this assay, Chinese hamster V79 cells, which are susceptible to mutagenesis, are co-cultivated with cells capable of metabolizing chemical carcinogens. Use of this assay made it possible to demonstrate a relationship between the degree of carcinogenicity and mutagenicity of a series of polycyclic hydrocarbons and nitrosamines and to study the organ specificity exhibited by some chemical carcinogens. However, most short-term in vitro assays are designed to detect mutagenic activity and therefore do not detect tumor promoting agents which are devoid of this activity. By analyzing various markers of terminal differentiation in cultured human melanoma and myeloid leukemia cells, we have established a relationship between the activity of a series of tumor promoting phorbol diesters in the mouse skin and their ability to induce terminal differentiation. We suggest that measuring alterations in the differentiation characteristics of some cultured cells may represent an approach by which environmental tumor promoting agents can be studied and detected.

  13. An ENU mutagenesis screen identifies novel and known genes involved in epigenetic processes in the mouse

    PubMed Central

    2013-01-01

    Background We have used a sensitized ENU mutagenesis screen to produce mouse lines that carry mutations in genes required for epigenetic regulation. We call these lines Modifiers of murine metastable epialleles (Mommes). Results We report a basic molecular and phenotypic characterization for twenty of the Momme mouse lines, and in each case we also identify the causative mutation. Three of the lines carry a mutation in a novel epigenetic modifier, Rearranged L-myc fusion (Rlf), and one gene, Rap-interacting factor 1 (Rif1), has not previously been reported to be involved in transcriptional regulation in mammals. Many of the other lines are novel alleles of known epigenetic regulators. For two genes, Rlf and Widely-interspaced zinc finger (Wiz), we describe the first mouse mutants. All of the Momme mutants show some degree of homozygous embryonic lethality, emphasizing the importance of epigenetic processes. The penetrance of lethality is incomplete in a number of cases. Similarly, abnormalities in phenotype seen in the heterozygous individuals of some lines occur with incomplete penetrance. Conclusions Recent advances in sequencing enhance the power of sensitized mutagenesis screens to identify the function of previously uncharacterized factors and to discover additional functions for previously characterized proteins. The observation of incomplete penetrance of phenotypes in these inbred mutant mice, at various stages of development, is of interest. Overall, the Momme collection of mouse mutants provides a valuable resource for researchers across many disciplines. PMID:24025402

  14. An inducible tool for random mutagenesis in Aspergillus niger based on the transposon Vader.

    PubMed

    Paun, Linda; Nitsche, Benjamin; Homan, Tim; Ram, Arthur F; Kempken, Frank

    2016-07-01

    The ascomycete Aspergillus niger is widely used in the biotechnology, for instance in producing most of the world's citric acid. It is also known as a major food and feed contaminant. While generation of gene knockouts for functional genomics has become feasible in ku70 mutants, analyzing gene functions or metabolic pathways remains a laborious task. An unbiased transposon-based mutagenesis approach may aid this process of analyzing gene functions by providing mutant libraries in a short time. The Vader transposon is a non-autonomous DNA-transposon, which is activated by the homologous tan1-transposase. However, in the most commonly used lab strain of A. niger (N400 strain and derivatives), we found that the transposase, encoded by the tan1 gene, is mutated and inactive. To establish a Vader transposon-based mutagenesis system in the N400 background, we expressed the functional transposase of A. niger strain CBS 513.88 under the control of an inducible promoter based on the Tet-on system, which is activated in the presence of the antibiotic doxycycline (DOX). Increasing amounts of doxycycline lead to higher Vader excision frequencies, whereas little to none activity of Vader was observed without addition of doxycycline. Hence, this system appears to be suitable for producing stable mutants in the A. niger N400 background.

  15. A shuttle mutagenesis system for tagging genes in the yeast Yarrowia lipolytica.

    PubMed

    Neuvéglise, C; Nicauda, J M; Ross-Macdonald, P; Gaillardin, C

    1998-06-15

    A shuttle mutagenesis system was developed for the dimorphic yeast Yarrowia lipolytica. This system combines transposon insertions generated in Escherichia coli with the transformation of yeast with the Tn-mutagenized DNA. The mini-transposon mTn-3xHA/GFP, used in Saccharomyces cerevisiae for producing stable insertions, was adapted for use in the yeast Y. lipolytica. The mTnYl1 transposon (for mini-Tn of Y. lipolytica) confers resistance to tetracycline in E. coli. It also contains the Y. lipolytica URA3 gene for selection of yeast transformants, and the coding sequence for the S65T mutant form of GFP. The rare cutter endonuclease, I-SceI, restriction site, which enables identification of the chromosomal localization of mutagenized genes, was also incorporated. mTnYl1 was first tested on the ACO1 gene, which encodes an Acyl CoA oxidase isozyme. The mutagenesis system was further validated on a Y. lipolytica genomic DNA library constructed in a pHSS6 derivative vector. Mutants with a particular morphology or defective for alkane, fatty acids and oil degradation were obtained.

  16. Mechanisms of mutagenesis: DNA replication in the presence of DNA damage

    PubMed Central

    Liu, Binyan; Xue, Qizhen; Tang, Yong; Cao, Jia; Guengerich, F. Peter; Zhang, Huidong

    2017-01-01

    Environmental mutagens cause DNA damage that disturbs replication and produces mutations, leading to cancer and other diseases. We discuss mechanisms of mutagenesis resulting from DNA damage, from the level of DNA replication by a single polymerase to the complex DNA replisome of some typical model organisms (including bacteriophage T7, T4, Sulfolobus solfataricus, E. coli, yeast and human). For a single DNA polymerase, DNA damage can affect replication in three major ways: reducing replication fidelity, causing frameshift mutations, and blocking replication. For the DNA replisome, protein interactions and the functions of accessory proteins can yield rather different results even with a single DNA polymerase. The mechanism of mutation during replication performed by the DNA replisome is a long-standing question. Using new methods and techniques, the replisomes of certain organisms and human cell extracts can now be investigated with regard to the bypass of DNA damage. In this review, we consider the molecular mechanism of mutagenesis resulting from DNA damage in replication at the levels of single DNA polymerases and complex DNA replisomes, including translesion DNA synthesis. PMID:27234563

  17. The roles of transcription and genotoxins underlying p53 mutagenesis in vivo.

    PubMed

    Wright, Barbara E; Schmidt, Karen H; Hunt, Aaron T; Lodmell, J Stephen; Minnick, Michael F; Reschke, Dennis K

    2011-10-01

    Transcription drives supercoiling which forms and stabilizes single-stranded (ss) DNA secondary structures with loops exposing G and C bases that are intrinsically mutable and vulnerable to non-enzymatic hydrolytic reactions. Since many studies in prokaryotes have shown direct correlations between the frequencies of transcription and mutation, we conducted in silico analyses using the computer program, mfg, which simulates transcription and predicts the location of known mutable bases in loops of high-stability secondary structures. Mfg analyses of the p53 tumor suppressor gene predicted the location of mutable bases and mutation frequencies correlated with the extent to which these mutable bases were exposed in secondary structures. In vitro analyses have now confirmed that the 12 most mutable bases in p53 are in fact located in predicted ssDNA loops of these structures. Data show that genotoxins have two independent effects on mutagenesis and the incidence of cancer: Firstly, they activate p53 transcription, which increases the number of exposed mutable bases and also increases mutation frequency. Secondly, genotoxins increase the frequency of G-to-T transversions resulting in a decrease in G-to-A and C mutations. This precise compensatory shift in the 'fate' of G mutations has no impact on mutation frequency. Moreover, it is consistent with our proposed mechanism of mutagenesis in which the frequency of G exposure in ssDNA via transcription is rate limiting for mutation frequency in vivo.

  18. Ionizing radiation-induced mutagenesis: radiation studies in Neurospora predictive for results in mammalian cells

    NASA Technical Reports Server (NTRS)

    Evans, H. H.; DeMarini, D. M.

    1999-01-01

    Ionizing radiation was the first mutagen discovered and was used to develop the first mutagenicity assay. In the ensuing 70+ years, ionizing radiation became a fundamental tool in understanding mutagenesis and is still a subject of intensive research. Frederick de Serres et al. developed and used the Neurospora crassa ad-3 system initially to explore the mutagenic effects of ionizing radiation. Using this system, de Serres et al. demonstrated the dependence of the frequency and spectra of mutations induced by ionizing radiation on the dose, dose rate, radiation quality, repair capabilities of the cells, and the target gene employed. This work in Neurospora predicted the subsequent observations of the mutagenic effects of ionizing radiation in mammalian cells. Modeled originally on the mouse specific-locus system developed by William L. Russell, the N. crassa ad-3 system developed by de Serres has itself served as a model for interpreting the results in subsequent systems in mammalian cells. This review describes the primary findings on the nature of ionizing radiation-induced mutagenesis in the N. crassa ad-3 system and the parallel observations made years later in mammalian cells.

  19. MouseNet database: digital management of a large-scale mutagenesis project.

    PubMed

    Pargent, W; Heffner, S; Schäble, K F; Soewarto, D; Fuchs, H; Hrabé de Angelis, M

    2000-07-01

    The Munich ENU Mouse Mutagenesis Screen is a large-scale mutant production, phenotyping, and mapping project. It encompasses two animal breeding facilities and a number of screening groups located in the general area of Munich. A central database is required to manage and process the immense amount of data generated by the mutagenesis project. This database, which we named MouseNet(c), runs on a Sybase platform and will finally store and process all data from the entire project. In addition, the system comprises a portfolio of functions needed to support the workflow management of the core facility and the screening groups. MouseNet(c) will make all of the data available to the participating screening groups, and later to the international scientific community. MouseNet(c) will consist of three major software components:* Animal Management System (AMS)* Sample Tracking System (STS)* Result Documentation System (RDS)MouseNet(c) provides the following major advantages:* being accessible from different client platforms via the Internet* being a full-featured multi-user system (including access restriction and data locking mechanisms)* relying on a professional RDBMS (relational database management system) which runs on a UNIX server platform* supplying workflow functions and a variety of plausibility checks.

  20. Transposon and Deletion Mutagenesis of Genes Involved in Perchlorate Reduction in Azospira suillum PS

    PubMed Central

    Melnyk, Ryan A.; Clark, Iain C.; Liao, Annette; Coates, John D.

    2013-01-01

    ABSTRACT Although much work on the biochemistry of the key enzymes of bacterial perchlorate reduction, chlorite dismutase, and perchlorate reductase has been published, understanding of the molecular mechanisms of this metabolism has been somewhat hampered by the lack of a clear model system amenable to genetic manipulation. Using transposon mutagenesis and clean deletions, genes important for perchlorate reduction in Azospira suillum PS have been identified both inside and outside the previously described perchlorate reduction genomic island (PRI). Transposon mutagenesis identified 18 insertions in 11 genes that completely abrogate growth via reduction of perchlorate but have no phenotype during denitrification. Of the mutants deficient in perchlorate reduction, 14 had insertions that were mapped to eight different genes within the PRI, highlighting its importance in this metabolism. To further explore the role of these genes, we also developed systems for constructing unmarked deletions and for complementing these deletions. Using these tools, every core gene in the PRI was systematically deleted; 8 of the 17 genes conserved in the PRI are essential for perchlorate respiration, including 3 genes that comprise a unique histidine kinase system. Interestingly, the other 9 genes in the PRI are not essential for perchlorate reduction and may thus have unknown functions during this metabolism. We present a model detailing our current understanding of perchlorate reduction that incorporates new concepts about this metabolism. PMID:24381299

  1. Transposon mutagenesis identifies genetic drivers of BrafV600E melanoma

    PubMed Central

    Mann, Michael B; Black, Michael A; Jones, Devin J; Ward, Jerrold M; Yew, Christopher Chin Kuan; Newberg, Justin Y; Dupuy, Adam J; Rust, Alistair G; Bosenberg, Marcus W; McMahon, Martin; Print, Cristin G; Copeland, Neal G; Jenkins, Nancy A

    2016-01-01

    Although nearly half of human melanomas harbor oncogenic BRAFV600E mutations, the genetic events that cooperate with these mutations to drive melanogenesis are still largely unknown. Here we show that Sleeping Beauty (SB) transposon-mediated mutagenesis drives melanoma progression in BrafV600E mutant mice and identify 1,232 recurrently mutated candidate cancer genes (CCGs) from 70 SB-driven melanomas. CCGs are enriched in Wnt, PI3K, MAPK and netrin signaling pathway components and are more highly connected to one another than predicted by chance, indicating that SB targets cooperative genetic networks in melanoma. Human orthologs of >500 CCGs are enriched for mutations in human melanoma or showed statistically significant clinical associations between RNA abundance and survival of patients with metastatic melanoma. We also functionally validate CEP350 as a new tumor-suppressor gene in human melanoma. SB mutagenesis has thus helped to catalog the cooperative molecular mechanisms driving BRAFV600E melanoma and discover new genes with potential clinical importance in human melanoma. PMID:25848750

  2. UV mutagenesis of Cupriavidus necator for extracellular production of (R)-3-hydroxybutyric acid.

    PubMed

    Ugwu, C U; Tokiwa, Y; Aoyagi, H; Uchiyama, H; Tanaka, H

    2008-07-01

    Ultraviolet (UV) mutagenesis was carried out to obtain mutant strains of Cupriavidus necator that could produce (R)-3-hydroxybutyric acid [(R)-3-HB] in the culture supernatant. C. necator (formerly known as Ralstonia eutropha) was subjected to UV radiation to generate mutants that are capable of producing (R)-3-HB in the culture supernatant. Results indicated that UV mutagen disrupted the phbB (phbB knock-out) and thus, promoted production of (R)-3-HB in mutant strains. Inclusion of acetoacetate esters (carbonyl compounds) in the culture broth led to increased production of (R)-3-HB. Thus, acetoacetyl-CoA (an intermediate of the PHB synthetic pathway) might have been converted to acetoacetate, which in the presence of (R)-3-HB dehydrogenase and NADPH/NADP(+), resulted in extracellular production of (R)-3-HB. UV mutagenesis proved to be a satisfactory method in generating interesting mutants for extracellular production of (R)-3-HB. Extracellular production of (R)-3-HB upon addition of acetoacetate esters would suggest a likely (R)-3-HB biosynthetic pathway in C. necator. Mutants obtained in this study are very useful for production of (R)-3-HB. For the first time, the production of (R)-3-HB by C. necator via acetoacetate is reported.

  3. Molecular dynamics simulation and site-directed mutagenesis of alcohol acyltransferase: a proposed mechanism of catalysis.

    PubMed

    Morales-Quintana, Luis; Nuñez-Tobar, María Ximena; Moya-León, María Alejandra; Herrera, Raúl

    2013-10-28

    Aroma in Vasconcellea pubescens fruit is determined by esters, which are the products of catalysis by alcohol acyltransferase (VpAAT1). VpAAT1 protein structure displayed the conserved HxxxD motif facing the solvent channel in the center of the structure. To gain insight into the role of these catalytic residues, kinetic and site-directed mutagenesis studies were carried out in VpAAT1 protein. Based on dead-end inhibition studies, the kinetic could be described in terms of a ternary complex mechanism with the H166 residue as the catalytic base. Kinetic results showed the lowest Km value for hexanoyl-CoA. Additionally, the most favorable predicted substrate orientation was observed for hexanoyl-CoA, showing a coincidence between kinetic studies and molecular docking analysis. Substitutions H166A, D170A, D170N, and D170E were evaluated in silico. The solvent channel in all mutant structures was lost, showing large differences with the native structure. Molecular docking and molecular dynamics simulations were able to describe unfavored energies for the interaction of the mutant proteins with different alcohols and acyl-CoAs. Additionally, in vitro site-directed mutagenesis of H166 and D170 in VpAAT1 induced a loss of activity, confirming the functional role of both residues for the activity, H166 being directly involved in catalysis.

  4. Improving the activity of the subtilisin nattokinase by site-directed mutagenesis and molecular dynamics simulation.

    PubMed

    Weng, Meizhi; Deng, Xiongwei; Bao, Wei; Zhu, Li; Wu, Jieyuan; Cai, Yongjun; Jia, Yan; Zheng, Zhongliang; Zou, Guolin

    2015-09-25

    Nattokinase (NK), a bacterial serine protease from Bacillus subtilis var. natto, is a potential cardiovascular drug exhibiting strong fibrinolytic activity. To broaden its commercial and medical applications, we constructed a single-mutant (I31L) and two double-mutants (M222A/I31L and T220S/I31L) by site-directed mutagenesis. Active enzymes were expressed in Escherichia coli with periplasmic secretion and were purified to homogeneity. The kinetic parameters of enzymes were examined by spectroscopy assay and isothermal titration calorimetry (ITC), and their fibrinolytic activities were determined by fibrin plate method. The substitution of Leu(31) for Ile(31) resulted in about 2-fold enhancement of catalytic efficiency (Kcat/KM) compared with wild-type NK. The specific activities of both double-mutants (M222A/I31L and T220S/I31L) were significantly increased when compared with the single-mutants (M222A and T220S) and the oxidative stability of M222A/I31L mutant was enhanced with respect to wild-type NK. This study demonstrates the feasibility of improving activity of NK by site-directed mutagenesis and shows successful protein engineering cases to improve the activity of NK as a potent therapeutic agent.

  5. Mechanisms of mutagenesis: Analysis through the use of alcohol dehydrogenase in Drosophila: Final report

    SciTech Connect

    Sofer, W.H.

    1986-12-01

    Our original objective was to understand the mechanism of mutagenesis of several important mutagens in higher organisms. Our approach was to try to deduce this mechanism by working backwards from its final effects. The strategy that we used in an effort to carry out our studies was to make mutations in the alcohol dehydrogenase gene of Drosophila melanogaster and sequence the modified genes. Most of our work was focused on an array of mutants that we had induced with formaldehyde, a potent mutagen in Drosophila, and with ethyl methane sulfonate. Over the course of the project period we cloned and sequenced the ADH gene from four formalde-induced mutants and from one EMS mutant. We showed that the four formaldehyde-induced mutants contained small deletions within the protein-coding region of their ADH genes ranging in size from between 6 and 34 bp. The one EMS-induced mutant was shown by DNA sequencing to bear an AT to GC sequence change at a tryptophan codon near the c-terminal coding portion of the gene. These results have significantly increased our understanding of the mechanism(s) of mutagenesis in higher organisms. 20 refs., 1 fig.

  6. Ionizing radiation-induced mutagenesis: radiation studies in Neurospora predictive for results in mammalian cells

    NASA Technical Reports Server (NTRS)

    Evans, H. H.; DeMarini, D. M.

    1999-01-01

    Ionizing radiation was the first mutagen discovered and was used to develop the first mutagenicity assay. In the ensuing 70+ years, ionizing radiation became a fundamental tool in understanding mutagenesis and is still a subject of intensive research. Frederick de Serres et al. developed and used the Neurospora crassa ad-3 system initially to explore the mutagenic effects of ionizing radiation. Using this system, de Serres et al. demonstrated the dependence of the frequency and spectra of mutations induced by ionizing radiation on the dose, dose rate, radiation quality, repair capabilities of the cells, and the target gene employed. This work in Neurospora predicted the subsequent observations of the mutagenic effects of ionizing radiation in mammalian cells. Modeled originally on the mouse specific-locus system developed by William L. Russell, the N. crassa ad-3 system developed by de Serres has itself served as a model for interpreting the results in subsequent systems in mammalian cells. This review describes the primary findings on the nature of ionizing radiation-induced mutagenesis in the N. crassa ad-3 system and the parallel observations made years later in mammalian cells.

  7. Generation of a glucose de-repressed mutant of Trichoderma reesei using disparity mutagenesis.

    PubMed

    Iwakuma, Hidekazu; Koyama, Yoshiyuki; Miyachi, Ayako; Nasukawa, Masashi; Matsumoto, Hitoshi; Yano, Shuntaro; Ogihara, Jun; Kasumi, Takafumi

    2016-01-01

    We obtained a novel glucose de-repressed mutant of Trichoderma reesei using disparity mutagenesis. A plasmid containing DNA polymerase δ lacking proofreading activity, and AMAI, an autonomously replicating sequence was introduced into T. reesei ATCC66589. The rate of mutation evaluated with 5-fluoroorotic acid resistance was approximately 30-fold higher than that obtained by UV irradiation. The transformants harboring incompetent DNA polymerase δ were then selected on 2-deoxyglucose agar plates with hygromycin B. The pNP-lactoside hydrolyzing activities of mutants were 2 to 5-fold higher than the parent in liquid medium containing glucose. Notably, the amino acid sequence of cre1, a key gene involved in glucose repression, was identical in the mutant and parent strains, and further, the cre1 expression levels was not abolished in the mutant. Taken together, these results demonstrate that the strains of T. reesei generated by disparity mutagenesis are glucose de-repressed variants that contain mutations in yet-unidentified factors other than cre1.

  8. Characterization of a metagenome-derived protease from contaminated agricultural soil microorganisms and its random mutagenesis.

    PubMed

    Jiang, Chengjian; Zhang, Liang; Li, Fajia; Meng, Can; Zeng, Rong; Deng, Jie; Shen, Peihong; Ou, Qian; Wu, Bo

    2017-04-05

    Proteases are typical key enzymes that hydrolyze proteins into amino acids and peptides. Numerous proteases have been studied, but the discovery of metagenome-derived proteases is still significant for both commercial applications and basic research. An unexplored protease gene sep1A was identified by function-based screening from a plasmid metagenomic library derived from uncultured contaminated agricultural soil microorganisms. The putative protease gene was subcloned into pET-32a (+) vector and overexpressed in E. coli BL21(DE3) pLysS, then the recombinant protein was purified to homogeneity. The detailed biochemical characterization of the Sep1A protein was performed, including its molecular characterization, specific activity, pH-activity profile, metal ion-activity profile, and enzyme kinetic assays. Furthermore, the protein engineering approach of random mutagenesis via error-prone PCR was applied on the original Sep1A protein. Biochemical characterization demonstrated that the purified recombinant Ep48 protein could hydrolyze casein. Compared with the original Sep1A protein, the best variant of Ep48 in the random mutagenesis library, with the Gln307Leu and Asp391Gly changes, exhibited 2.62-fold activity at the optimal reaction conditions of 50 °C and pH 9.0. These results are the first step toward a better understanding of the properties of Sep1A protein. Protein engineering with error-prone PCR paves the way toward the metagenome-derived genes for biotechnological applications.

  9. Transcription-dependent cytosine deamination: a novel mechanism in ultraviolet light-induced mutagenesis

    PubMed Central

    Hendriks, Giel; Calléja, Fabienne; Besaratinia, Ahmad; Vrieling, Harry; Pfeifer, Gerd P.; Mullenders, Leon H.F.; Jansen, Jacob G.; de Wind, Niels

    2009-01-01

    Summary Skin cancer is the most ubiquitous cancer type in the Caucasian population and its incidence is increasing rapidly [1]. Transcribed, proliferation-related, genes in dermal stem cells are targets for the induction of ultraviolet light (UV)-induced mutations that drive carcinogenesis. We have recently found that gene transcription increases UV-induced mutagenesis in mammalian stem cells, suggesting a role of transcription in skin carcinogenesis [2]. Here we show that transcription-associated, UV-induced, nucleotide substitutions are caused by increased deamination of cytosines to uracil within photolesions at the transcribed strand, presumably at sites of stalled transcription complexes. Additionally, via an independent mechanism, transcription of UV-damaged DNA induces the generation of intragenic deletions. We provide evidence that transcription-coupled nucleotide excision repair (TC-NER) provides protection against both classes of transcription-associated mutagenesis. Combined, these results unveil the existence of two mutagenic pathways, operating specifically at the transcribed DNA strand of active genes. Moreover, these results uncover a novel role for TC-NER in the suppression of UV light-induced genome aberrations and provide a rationale for the efficient induction of apoptosis by stalled transcription complexes. PMID:20045328

  10. Sleeping Beauty transposon insertional mutagenesis based mouse models for cancer gene discovery

    PubMed Central

    Moriarity, Branden S; Largaespada, David A

    2016-01-01

    Large-scale genomic efforts to study human cancer, such as the cancer gene atlas (TCGA), have identified numerous cancer drivers in a wide variety of tumor types. However, there are limitations to this approach, the mutations and expression or copy number changes that are identified are not always clearly functionally relevant, and only annotated genes and genetic elements are thoroughly queried. The use of complimentary, nonbiased, functional approaches to identify drivers of cancer development and progression is ideal to maximize the rate at which cancer discoveries are achieved. One such approach that has been successful is the use of the Sleeping Beauty (SB) transposon-based mutagenesis system in mice. This system uses a conditionally expressed transposase and mutagenic transposon allele to target mutagenesis to somatic cells of a given tissue in mice to cause random mutations leading to tumor development. Analysis of tumors for transposon common insertion sites (CIS) identifies candidate cancer genes specific to that tumor type. While similar screens have been performed in mice with the PiggyBac (PB) transposon and viral approaches, we limit extensive discussion to SB. Here we discuss the basic structure of these screens, screens that have been performed, methods used to identify CIS. PMID:26051241

  11. Creatine supplementation normalizes mutagenesis of mitochondrial DNA as well as functional consequences.

    PubMed

    Berneburg, Mark; Gremmel, Tobias; Kürten, Viola; Schroeder, Peter; Hertel, Ines; von Mikecz, Anna; Wild, Susanne; Chen, Min; Declercq, Lieve; Matsui, Mary; Ruzicka, Thomas; Krutmann, Jean

    2005-08-01

    Mutations of mitochondrial (mt) DNA play a role in neurodegeneration, normal aging, premature aging of the skin (photoaging), and tumors. We and others could demonstrate that mtDNA mutations can be induced in skin cells in vitro and in normal human skin in vivo by repetitive, sublethal ultraviolet (UV)-A-irradiation. These mutations are mediated by singlet oxygen and persist in human skin as long-term biomarkers of UV exposure. Although mtDNA exclusively encodes for the respiratory chain, involvement of the energy metabolism in mtDNA mutagenesis and a protective role of the energy precursor creatine have thus far not been shown. We assessed the amount of a marker mutation of mtDNA, the so-called common deletion, by real-time PCR. Induction of the common deletion was paralleled by a measurable decrease of oxygen consumption, mitochondrial membrane potential, and ATP content, as well as an increase of matrix metalloproteinase-1. Mitochondrial mutagenesis as well as functional consequences could be normalized by increasing intracellular creatine levels. These data indicate that increase of the energy precursor creatine protects from functionally relevant, aging-associated mutations of mitochondrial DNA.

  12. Simple-MSSM: a simple and efficient method for simultaneous multi-site saturation mutagenesis.

    PubMed

    Cheng, Feng; Xu, Jian-Miao; Xiang, Chao; Liu, Zhi-Qiang; Zhao, Li-Qing; Zheng, Yu-Guo

    2017-04-01

    To develop a practically simple and robust multi-site saturation mutagenesis (MSSM) method that enables simultaneously recombination of amino acid positions for focused mutant library generation. A general restriction enzyme-free and ligase-free MSSM method (Simple-MSSM) based on prolonged overlap extension PCR (POE-PCR) and Simple Cloning techniques. As a proof of principle of Simple-MSSM, the gene of eGFP (enhanced green fluorescent protein) was used as a template gene for simultaneous mutagenesis of five codons. Forty-eight randomly selected clones were sequenced. Sequencing revealed that all the 48 clones showed at least one mutant codon (mutation efficiency = 100%), and 46 out of the 48 clones had mutations at all the five codons. The obtained diversities at these five codons are 27, 24, 26, 26 and 22, respectively, which correspond to 84, 75, 81, 81, 69% of the theoretical diversity offered by NNK-degeneration (32 codons; NNK, K = T or G). The enzyme-free Simple-MSSM method can simultaneously and efficiently saturate five codons within one day, and therefore avoid missing interactions between residues in interacting amino acid networks.

  13. A mutagenesis and screening strategy to generate optimally thermostabilized membrane proteins for structural studies.

    PubMed

    Magnani, Francesca; Serrano-Vega, Maria J; Shibata, Yoko; Abdul-Hussein, Saba; Lebon, Guillaume; Miller-Gallacher, Jennifer; Singhal, Ankita; Strege, Annette; Thomas, Jennifer A; Tate, Christopher G

    2016-08-01

    The thermostability of an integral membrane protein (MP) in detergent solution is a key parameter that dictates the likelihood of obtaining well-diffracting crystals that are suitable for structure determination. However, many mammalian MPs are too unstable for crystallization. We developed a thermostabilization strategy based on systematic mutagenesis coupled to a radioligand-binding thermostability assay that can be applied to receptors, ion channels and transporters. It takes ∼6-12 months to thermostabilize a G-protein-coupled receptor (GPCR) containing 300 amino acid (aa) residues. The resulting thermostabilized MPs are more easily crystallized and result in high-quality structures. This methodology has facilitated structure-based drug design applied to GPCRs because it is possible to determine multiple structures of the thermostabilized receptors bound to low-affinity ligands. Protocols and advice are given on how to develop thermostability assays for MPs and how to combine mutations to make an optimally stable mutant suitable for structural studies. The steps in the procedure include the generation of ∼300 site-directed mutants by Ala/Leu scanning mutagenesis, the expression of each mutant in mammalian cells by transient transfection and the identification of thermostable mutants using a thermostability assay that is based on binding of an (125)I-labeled radioligand to the unpurified, detergent-solubilized MP. Individual thermostabilizing point mutations are then combined to make an optimally stable MP that is suitable for structural biology and other biophysical studies.

  14. Engineering stress tolerance of Escherichia coli by stress-induced mutagenesis (SIM)-based adaptive evolution.

    PubMed

    Zhu, Linjiang; Cai, Zhen; Zhang, Yanping; Li, Yin

    2014-01-01

    Microbial tolerance to toxic products and biomass hydrolysates is a challenge for the production of fuels and chemicals from renewable resources. To improve cellular tolerance to these environmental stresses, a novel adaptive evolutionary strategy based on stress-induced mutagenesis (SIM) was developed using non-dividing cells. The concept of this method was proved using Escherichia coli FC40 as a model strain, which was used to quantitatively evaluate the rate of SIM. By deleting either the mutL or mutS gene to disturb the mismatch repair activity of the host cells, the SIM rate under stressful conditions increased by 92- and 57-fold, respectively. A periodic SIMbased adaptive evolution procedure, which synchronized the mutagenesis and the selection process in a single plate-incubation step, was then developed using the mutL-deleted mutant. E. coli mutants tolerant to high concentrations of butanol (13 g/L), NaCl (95 g/L), and high temperature (50°C) were obtained. These results indicate that stress-induced adaptive evolution in non-dividing cells is an effective approach that can improve microbial tolerance against various stresses and generate robust microbial strains suitable for production of fuels and chemicals.

  15. Development of a Dunaliella tertiolecta Strain with Increased Zeaxanthin Content Using Random Mutagenesis

    PubMed Central

    Kim, Minjae; Ahn, Junhak; Jeon, Hancheol; Jin, EonSeon

    2017-01-01

    Zeaxanthin is a xanthophyll pigment that is regarded as one of the best carotenoids for the prevention and treatment of degenerative diseases. In the worldwide natural products market, consumers prefer pigments that have been produced from biological sources. In this study, a Dunaliella tertiolecta strain that has 10–15% higher cellular zeaxanthin content than the parent strain (zea1), was obtained by random mutagenesis using ethyl methanesulfonate (EMS) as a mutagen. This mutant, mp3, was grown under various salinities and light intensities to optimize culture conditions for zeaxanthin production. The highest cellular zeaxanthin content was observed at 1.5 M NaCl and 65–85 μmol photons·m−2·s−1, and the highest daily zeaxanthin productivity was observed at 0.6 M NaCl and 140–160 μmol photons·m−2·s−1. The maximal yield of zeaxanthin from mp3 in fed-batch culture was 8 mg·L−1, which was obtained at 0.6 M NaCl and 140–160 μmol photons·m−2·s−1. These results suggest that random mutagenesis with EMS is useful for generating D. tertiolecta strains with increased zeaxanthin content, and also suggest optimal culture conditions for the enhancement of biomass and zeaxanthin production by the zeaxanthin accumulating mutant strains. PMID:28635662

  16. The effect of chronic alcohol consumption on mitochondrial DNA mutagenesis in human blood.

    PubMed

    von Wurmb-Schwark, N; Ringleb, A; Schwark, T; Broese, T; Weirich, S; Schlaefke, D; Wegener, R; Oehmichen, M

    2008-01-01

    The 4977bp deletion of mitochondrial DNA (mtDNA) is known to accumulate with increasing age in post mitotic tissues. Recently, studies came out detecting this specific alteration also in fast replicating cells, e.g. in blood or skin tissue, often in correlation to specific diseases or -- specifically in skin -- external stressors such as UV radiation. In this study, we investigated mitochondrial mutagenesis in 69 patients with a chronic alcoholic disease and 46 age matched controls with a moderate drinking behavior. Two different fragments, specific for total and for deleted mtDNA (dmtDNA) were amplified in a duplex-PCR. A subsequent fragment analysis was performed and for relative quantification, the quotient of the peak areas of amplification products specific for deleted and total mtDNA was determined. Additionally, a real time PCR was performed to quantify mtDNA copy number. The relative amount of 4977bp deleted mtDNA in alcoholics was significantly increased compared to controls. On the other hand, no difference regarding the mtDNA/nuclear DNA ratio in both investigated groups was detected. Additionally, no age dependence could be found nor in alcoholics, neither in the control group. These findings indicate that mtDNA mutagenesis in blood can be influenced by stressors such as alcohol. Ethanol seems to be a significant factor to alter mitochondrial DNA in blood and might be an additional contributor for the cellular aging process.

  17. The disparity mutagenesis model predicts rescue of living things from catastrophic errors

    PubMed Central

    Furusawa, Mitsuru

    2014-01-01

    In animals including humans, mutation rates per generation exceed a perceived threshold, and excess mutations increase genetic load. Despite this, animals have survived without extinction. This is a perplexing problem for animal and human genetics, arising at the end of the last century, and to date still does not have a fully satisfactory explanation. Shortly after we proposed the disparity theory of evolution in 1992, the disparity mutagenesis model was proposed, which forms the basis for an explanation for an acceleration of evolution and species survival. This model predicts a significant increase of the mutation threshold values if the fidelity difference in replication between the lagging and leading strands is high enough. When applied to biological evolution, the model predicts that living things, including humans, might overcome the lethal effect of accumulated deleterious mutations and be able to survive. Artificially derived mutator strains of microorganisms, in which an enhanced lagging-strand-biased mutagenesis was introduced, showed unexpectedly high adaptability to severe environments. The implications of the striking behaviors shown by these disparity mutators will be discussed in relation to how living things with high mutation rates can avoid the self-defeating risk of excess mutations. PMID:25538731

  18. Restriction enzyme-free construction of random gene mutagenesis libraries in Escherichia coli.

    PubMed

    Pai, Jen C; Entzminger, Kevin C; Maynard, Jennifer A

    2012-02-15

    Directed evolution relies on both random and site-directed mutagenesis of individual genes and regulatory elements to create variants with altered activity profiles for engineering applications. Central to these experiments is the construction of large libraries of related variants. However, a number of technical hurdles continue to limit routine construction of random mutagenesis libraries in Escherichia coli, in particular, inefficiencies during digestion and ligation steps. Here, we report a restriction enzyme-free approach to library generation using megaprimers termed MegAnneal. Target DNA is first exponentially amplified using error-prone polymerase chain reaction (PCR) and then linearly amplified with a single 3' primer to generate long, randomly mutated, single-stranded megaprimers. These are annealed to single-stranded dUTP-containing template plasmid and extended with T7 polymerase to create a complementary strand, and the resulting termini are ligated with T4 DNA ligase. Using this approach, we are able to reliably generate libraries of approximately 10⁷ colony-forming units (cfu)/μg DNA/transformation in a single day. We have created MegAnneal libraries based on three different single-chain antibodies and identified variants with enhanced expression and ligand-binding affinity. The key advantages of this approach include facile amplification, restriction enzyme-free library generation, and a significantly reduced risk of mutations outside the targeted region and wild-type contamination as compared with current methods.

  19. Transposon mutagenesis identifies genetic drivers of Braf(V600E) melanoma.

    PubMed

    Mann, Michael B; Black, Michael A; Jones, Devin J; Ward, Jerrold M; Yew, Christopher Chin Kuan; Newberg, Justin Y; Dupuy, Adam J; Rust, Alistair G; Bosenberg, Marcus W; McMahon, Martin; Print, Cristin G; Copeland, Neal G; Jenkins, Nancy A

    2015-05-01

    Although nearly half of human melanomas harbor oncogenic BRAF(V600E) mutations, the genetic events that cooperate with these mutations to drive melanogenesis are still largely unknown. Here we show that Sleeping Beauty (SB) transposon-mediated mutagenesis drives melanoma progression in Braf(V600E) mutant mice and identify 1,232 recurrently mutated candidate cancer genes (CCGs) from 70 SB-driven melanomas. CCGs are enriched in Wnt, PI3K, MAPK and netrin signaling pathway components and are more highly connected to one another than predicted by chance, indicating that SB targets cooperative genetic networks in melanoma. Human orthologs of >500 CCGs are enriched for mutations in human melanoma or showed statistically significant clinical associations between RNA abundance and survival of patients with metastatic melanoma. We also functionally validate CEP350 as a new tumor-suppressor gene in human melanoma. SB mutagenesis has thus helped to catalog the cooperative molecular mechanisms driving BRAF(V600E) melanoma and discover new genes with potential clinical importance in human melanoma.

  20. Mutagenesis Is Elevated in Male Germ Cells Obtained from DNA Polymerase-beta Heterozygous Mice1

    PubMed Central

    Allen, Diwi; Herbert, Damon C.; McMahan, C. Alex; Rotrekl, Vladimir; Sobol, Robert W.; Wilson, Samuel H.; Walter, Christi A.

    2008-01-01

    Gametes carry the DNA that will direct the development of the next generation. By compromising genetic integrity, DNA damage and mutagenesis threaten the ability of gametes to fulfill their biological function. DNA repair pathways function in germ cells and serve to ameliorate much DNA damage and prevent mutagenesis. High base excision repair (BER) activity is documented for spermatogenic cells. DNA polymerase-beta (POLB) is required for the short-patch BER pathway. Because mice homozygous null for the Polb gene die soon after birth, mice heterozygous for Polb were used to examine the extent to which POLB contributes to maintaining spermatogenic genomic integrity in vivo. POLB protein levels were reduced only in mixed spermatogenic cells. In vitro short-patch BER activity assays revealed that spermatogenic cell nuclear extracts obtained from Polb heterozygous mice had one third the BER activity of age-matched control mice. Polb heterozygosity had no effect on the BER activities of somatic tissues tested. The Polb heterozygous mouse line was crossed with the lacI transgenic Big Blue mouse line to assess mutant frequency. The spontaneous mutant frequency for mixed spermatogenic cells prepared from Polb heterozygous mice was 2-fold greater than that of wild-type controls, but no significant effect was found among the somatic tissues tested. These results demonstrate that normal POLB abundance is necessary for normal BER activity, which is critical in maintaining a low germline mutant frequency. Notably, spermatogenic cells respond differently than somatic cells to Polb haploinsufficiency.. PMID:18650495

  1. Efficient targeted mutagenesis of rice and tobacco genomes using Cpf1 from Francisella novicida

    PubMed Central

    Endo, Akira; Masafumi, Mikami; Kaya, Hidetaka; Toki, Seiichi

    2016-01-01

    CRISPR/Cas9 systems are nowadays applied extensively to effect genome editing in various organisms including plants. CRISPR from Prevotella and Francisella 1 (Cpf1) is a newly characterized RNA-guided endonuclease that has two distinct features as compared to Cas9. First, Cpf1 utilizes a thymidine-rich protospacer adjacent motif (PAM) while Cas9 prefers a guanidine-rich PAM. Cpf1 could be used as a sequence-specific nuclease to target AT-rich regions of a genome that Cas9 had difficulty accessing. Second, Cpf1 generates DNA ends with a 5′ overhang, whereas Cas9 creates blunt DNA ends after cleavage. “Sticky” DNA ends should increase the efficiency of insertion of a desired DNA fragment into the Cpf1-cleaved site using complementary DNA ends. Therefore, Cpf1 could be a potent tool for precise genome engineering. To evaluate whether Cpf1 can be applied to plant genome editing, we selected Cpf1 from Francisella novicida (FnCpf1), which recognizes a shorter PAM (TTN) within known Cpf1 proteins, and applied it to targeted mutagenesis in tobacco and rice. Our results show that targeted mutagenesis had occurred in transgenic plants expressing FnCpf1 with crRNA. Deletions of the targeted region were the most frequently observed mutations. Our results demonstrate that FnCpf1 can be applied successfully to genome engineering in plants. PMID:27905529

  2. In vitro mutagenesis and identification of mutants via ISSR in lily (Lilium longiflorum).

    PubMed

    Xi, Mengli; Sun, Lina; Qiu, Shuai; Liu, Juanjuan; Xu, Jin; Shi, Jisen

    2012-06-01

    An efficient in vitro mutagenesis protocol for Lilium longiflorum Thunb. cv. White fox has been established. The effect of 6-BA and NAA on adventitious bud formation from the bulblet-scale thin cell layers was tested. Results showed that the optimal medium for adventitious bud induction is MS basal medium supplemented with 2.0 mg/l 6-BA and 0.1 mg/l NAA. The differentiation frequency and the average number of adventitious buds reached 95.55% and 3.00, respectively. Various doses (0.0, 0.5, 1.0, 1.5, 2.0, and 2.5 Gy) of gamma rays were applied to investigate the effect of radiation on adventitious bud formation from bulblet-scale thin cell layers. The forming capacity of the adventitious buds significantly decreased with the increase of radiation dose. The results suggested that the optimal irradiation dose is 1.0 Gy. Dose of 1.0 Gy treatment resulted in 55.33% survival of irradiated bulblet-scale thin cell layers and 39.27% mutagenesis rate. The genetic variations among the morphological mutants were evaluated by DNA fingerprinting using ISSR molecular marker. The genetic variation frequency reached 36.06% using seven ISSR primers. Out of the 50 mutant lines transferred to the greenhouse, 9 were observed to have significantly different morphological characters than those of the controls.

  3. Insight into Binding of Phosphodiesterase-9A Selective Inhibitors by Crystal Structures and Mutagenesis

    SciTech Connect

    Wang, H.; Luo, X; Ye, M; Hou, J; Robinson, H; Ke, H

    2010-01-01

    PDE9 inhibitors have been studied as therapeutics for treatment of cardiovascular diseases, diabetes, and neurodegenerative disorders. To illustrate the inhibitor selectivity, the crystal structures of the PDE9A catalytic domain in complex with the enantiomers of PDE9 inhibitor 1-(2-chlorophenyl)-6-(3,3,3-trifluoro-2-methylpropyl)-1H-pyrazolo[3,4-d]pyrimidine-4(5H)-one ((R)-BAY73-6691 or (S)-BAY73-6691, 1r or 1s) were determined and mutagenesis was performed. The structures showed that the fluoromethyl groups of 1r and 1s had different orientations while the other parts of the inhibitors commonly interacted with PDE9A. These differences may explain the slightly different affinity of 1r (IC{sub 50} = 22 nM) and 1s (IC{sub 50} = 88 nM). The mutagenesis experiments revealed that contribution of the binding residues to the inhibitor sensitivity varies dramatically, from few-fold to 3 orders of magnitude. On the basis of the crystal structures, a hypothesized compound that simulates the recently published PDE9 inhibitors was modeled to provide insight into the inhibitor selectivity.

  4. Phenotypic mutants of the intracellular actinomycete Rhodococcus equi created by in vivo Himar1 transposon mutagenesis.

    PubMed

    Ashour, Joseph; Hondalus, Mary K

    2003-04-01

    Rhodococcus equi is a facultative intracellular opportunistic pathogen of immunocompromised people and a major cause of pneumonia in young horses. An effective live attenuated vaccine would be extremely useful in the prevention of R. equi disease in horses. Toward that end, we have developed an efficient transposon mutagenesis system that makes use of a Himar1 minitransposon delivered by a conditionally replicating plasmid for construction of R. equi mutants. We show that Himar1 transposition in R. equi is random and needs no apparent consensus sequence beyond the required TA dinucleotide. The diversity of the transposon library was demonstrated by the ease with which we were able to screen for auxotrophs and mutants with pigmentation and capsular phenotypes. One of the pigmentation mutants contained an insertion in a gene encoding phytoene desaturase, an enzyme of carotenoid biosynthesis, the pathway necessary for production of the characteristic salmon color of R. equi. We identified an auxotrophic mutant with a transposon insertion in the gene encoding a putative dual-functioning GTP cyclohydrolase II-3,4-dihydroxy-2-butanone-4-phosphate synthase, an enzyme essential for riboflavin biosynthesis. This mutant cannot grow in minimal medium in the absence of riboflavin supplementation. Experimental murine infection studies showed that, in contrast to wild-type R. equi, the riboflavin-requiring mutant is attenuated because it is unable to replicate in vivo. The mutagenesis methodology we have developed will allow the characterization of R. equi virulence mechanisms and the creation of other attenuated strains with vaccine potential.

  5. Mechanisms of mutagenesis: DNA replication in the presence of DNA damage.

    PubMed

    Liu, Binyan; Xue, Qizhen; Tang, Yong; Cao, Jia; Guengerich, F Peter; Zhang, Huidong

    2016-01-01

    Environmental mutagens cause DNA damage that disturbs replication and produces mutations, leading to cancer and other diseases. We discuss mechanisms of mutagenesis resulting from DNA damage, from the level of DNA replication by a single polymerase to the complex DNA replisome of some typical model organisms (including bacteriophage T7, T4, Sulfolobus solfataricus, Escherichia coli, yeast and human). For a single DNA polymerase, DNA damage can affect replication in three major ways: reducing replication fidelity, causing frameshift mutations, and blocking replication. For the DNA replisome, protein interactions and the functions of accessory proteins can yield rather different results even with a single DNA polymerase. The mechanism of mutation during replication performed by the DNA replisome is a long-standing question. Using new methods and techniques, the replisomes of certain organisms and human cell extracts can now be investigated with regard to the bypass of DNA damage. In this review, we consider the molecular mechanism of mutagenesis resulting from DNA damage in replication at the levels of single DNA polymerases and complex DNA replisomes, including translesion DNA synthesis. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Caffeine enhanced measurement of mutagenesis by low levels of [gamma]-irradiation in human lymphocytes

    SciTech Connect

    Puck, T.P.; Johnson, R.; Waldren, C.A. ); Morse, H. )

    1993-09-01

    The well-known action of caffeine in synergizing mutagenesis (including chromosome aberrations) of agents like ionizing radiation by inhibition of cellular repair processes has been incorporated into a rapid procedure for detection of mutagenicity with high sensitivity. Effects of 5-10 rads of [gamma]-irradiation, which approximate the human lifetime dose accumulation from background radiation, can be detected in a two-day procedure using an immortalized human WBC culture. Chromosomally visible lesions are scored on cells incubated for 2 h after irradiation in the presence and absence of 1.0 mg/ml of caffeine. An eightfold amplification of scorable lesions is achieved over the action of radiation alone. This approach provides a closer approximation to absolute mutagenicity unmitigated by repair processes, which can vary in different situations. It is proposed that mutagenesis testing of this kind, using caffiene or other repair-inhibitory agents, be employed to identify mutagens in their effective concentrations to which human populations may be exposed; to detect agents such as caffeine that may synergize mutagenic actions and pose epidemiologic threats; and to discover effective anti-mutagens. Information derived from the use of such procedures may help prevent cancer and newly acquired genetic disease.

  7. Mutagenesis and functional selection protocols for directed evolution of proteins in E. coli.

    PubMed

    Troll, Chris; Alexander, David; Allen, Jennifer; Marquette, Jacob; Camps, Manel

    2011-03-16

    The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular signatures, or to evolve proteins in the laboratory. Here, we present a protocol that allows the generation of large (>10(11)) mutant libraries for a given target sequence. This method is based on replication of a ColE1 plasmid encoding the desired sequence by a low-fidelity variant of DNA polymerase I (LF-Pol I). The target plasmid is transformed into a mutator strain of E. coli and plated on solid media, yielding between 0.2 and 1 mutations/kb, depending on the location of the target gene. Higher mutation frequencies are achieved by iterating this process of mutagenesis. Compared to alternative methods of mutagenesis, our protocol stands out for its simplicity, as no cloning or PCR are involved. Thus, our method is ideal for mutational labeling of plasmids or other Pol I templates or to explore large sections of sequence space for the evolution of activities not present in the original target. The tight spatial control that PCR or randomized oligonucleotide-based methods offer can also be achieved through subsequent cloning of specific sections of the library. Here we provide protocols showing how to create a random mutant library and how to establish drug-based selections in E. coli to identify mutants exhibiting new biochemical activities.

  8. Mutagenesis by cytostatic alkylating agents in yeast strains of differing repair capacities.

    PubMed

    Ruhland, A; Brendel, M

    1979-05-01

    Reversion of two nulcear ochre nonsense alleles and cell inactivation induced by mono-, bi-, and tri-functional alkylating agents and by UV has been investigated in stationary-phase haploid cells of yeast strains with differing capacities for DNA repair. The ability to survive alkylation damage is correlated with UV repair capacity, a UV-resistant and UV-mutable strain (RAD REV) being least and a UV-sensitive and UV-nonmutable strain (radi rev3) most sensitive. Mutagenicity of alkylating agents is highest in the former and is abolished in the latter strain. Deficiency in excision repair (rad1 rad2) or in the RAD18 function does not lead to enhanced mutability. Mutagenesis by the various agents is characterized by a common pattern of induction of locus-specific revertants and suppressor mutants. Induction kinetics are mostly linear, but UV-induced reversion in the RAD REV strain follows higher-than-linear (probably "quadratic") kinetics. The alkylating agent cyclophosphamide, usually considered inactive without metabolic conversion, reduces colony-forming ability and induces revertants in a manner similar but not identical to the other chemicals tested. These findings are taken to support the concept of mutagenesis by misrepair after alkylation, which albeit sharing common features with the mechanism of UV-induced reversion, can be distinguished therefrom.

  9. PAX5 is a tumor suppressor in mouse mutagenesis models of acute lymphoblastic leukemia

    PubMed Central

    Dang, Jinjun; Wei, Lei; de Ridder, Jeroen; Su, Xiaoping; Rust, Alistair G.; Roberts, Kathryn G.; Payne-Turner, Debbie; Cheng, Jinjun; Ma, Jing; Qu, Chunxu; Wu, Gang; Song, Guangchun; Huether, Robert G.; Schulman, Brenda; Janke, Laura; Zhang, Jinghui; Downing, James R.; van der Weyden, Louise; Adams, David J.

    2015-01-01

    Alterations of genes encoding transcriptional regulators of lymphoid development are a hallmark of B-progenitor acute lymphoblastic leukemia (B-ALL) and most commonly involve PAX5, encoding the DNA-binding transcription factor paired-box 5. The majority of PAX5 alterations in ALL are heterozygous, and key PAX5 target genes are expressed in leukemic cells, suggesting that PAX5 may be a haploinsufficient tumor suppressor. To examine the role of PAX5 alterations in leukemogenesis, we performed mutagenesis screens of mice heterozygous for a loss-of-function Pax5 allele. Both chemical and retroviral mutagenesis resulted in a significantly increased penetrance and reduced latency of leukemia, with a shift to B-lymphoid lineage. Genomic profiling identified a high frequency of secondary genomic mutations, deletions, and retroviral insertions targeting B-lymphoid development, including Pax5, and additional genes and pathways mutated in ALL, including tumor suppressors, Ras, and Janus kinase-signal transducer and activator of transcription signaling. These results show that in contrast to simple Pax5 haploinsufficiency, multiple sequential alterations targeting lymphoid development are central to leukemogenesis and contribute to the arrest in lymphoid maturation characteristic of ALL. This cross-species analysis also validates the importance of concomitant alterations of multiple cellular growth, signaling, and tumor suppression pathways in the pathogenesis of B-ALL. PMID:25855603

  10. The disparity mutagenesis model predicts rescue of living things from catastrophic errors.

    PubMed

    Furusawa, Mitsuru

    2014-01-01

    In animals including humans, mutation rates per generation exceed a perceived threshold, and excess mutations increase genetic load. Despite this, animals have survived without extinction. This is a perplexing problem for animal and human genetics, arising at the end of the last century, and to date still does not have a fully satisfactory explanation. Shortly after we proposed the disparity theory of evolution in 1992, the disparity mutagenesis model was proposed, which forms the basis for an explanation for an acceleration of evolution and species survival. This model predicts a significant increase of the mutation threshold values if the fidelity difference in replication between the lagging and leading strands is high enough. When applied to biological evolution, the model predicts that living things, including humans, might overcome the lethal effect of accumulated deleterious mutations and be able to survive. Artificially derived mutator strains of microorganisms, in which an enhanced lagging-strand-biased mutagenesis was introduced, showed unexpectedly high adaptability to severe environments. The implications of the striking behaviors shown by these disparity mutators will be discussed in relation to how living things with high mutation rates can avoid the self-defeating risk of excess mutations.

  11. Site directed mutagenesis of StSUT1 reveals target amino acids of regulation and stability.

    PubMed

    Krügel, Undine; Wiederhold, Elena; Pustogowa, Jelena; Hackel, Aleksandra; Grimm, Bernhard; Kühn, Christina

    2013-11-01

    Plant sucrose transporters (SUTs) are functional as sucrose-proton-cotransporters with an optimal transport activity in the acidic pH range. Recently, the pH optimum of the Solanum tuberosum sucrose transporter StSUT1 was experimentally determined to range at an unexpectedly low pH of 3 or even below. Various research groups have confirmed these surprising findings independently and in different organisms. Here we provide further experimental evidence for a pH optimum at physiological extrema. Site directed mutagenesis provides information about functional amino acids, which are highly conserved and responsible for this extraordinary increase in transport capacity under extreme pH conditions. Redox-dependent dimerization of the StSUT1 protein was described earlier. Here the ability of StSUT1 to form homodimers was demonstrated by heterologous expression in Lactococcus lactis and Xenopus leavis using Western blots, and in plants by bimolecular fluorescence complementation. Mutagenesis of highly conserved cysteine residues revealed their importance in protein stability. The accessibility of regulatory amino acid residues in the light of StSUT1's compartmentalization in membrane microdomains is discussed.

  12. Identification of 17 hearing impaired mouse strains in the TMGC ENU-mutagenesis screen

    SciTech Connect

    Kermany, Mohammad; Parker, Lisan; Guo, Yun-Kai; Miller, Darla R; Swanson, Douglas J; Yoo, Tai-June; Goldowitz, Daniel; Zuo, Jian

    2006-01-01

    The Tennessee Mouse Genome Consortium (TMGC) employed an N-ethyl-N-nitrosourea (ENU)-mutagenesis scheme to identify mouse recessive mutants with hearing phenotypes. We employed auditory brainstem responses (ABR) to click and 8, 16, and 32 kHz stimuli and screened 285 pedigrees (1819 mice of 8-11 weeks old in various mixed genetic backgrounds) each bred to carry a homozygous ENU-induced mutation. To define mutant pedigrees, we measured P12 mice per pedigree in P2 generations and used a criterion where the mean ABR threshold per pedigree was two standard deviations above the mean of all offspring from the same parental strain. We thus identified 17 mutant pedigrees (6%), all exhibiting hearing loss at high frequencies (P16 kHz) with an average threshold elevation of 30-35 dB SPL. Interestingly, four mutants showed sex-biased hearing loss and six mutants displayed wide range frequency hearing loss. Temporal bone histology revealed that six of the first nine mutants displayed cochlear morphological defects: degeneration of spiral ganglia, spiral ligament fibrocytes or inner hair cells (but not outer hair cells) mostly in basal turns. In contrast to other ENU-mutagenesis auditory screens, our screen identified high-frequency, mild and sex-biased hearing defects. Further characterization of these 17 mouse models will advance our understanding of presbycusis and noise-induced hearing loss in humans.

  13. An ENU mutagenesis screen identifies novel and known genes involved in epigenetic processes in the mouse.

    PubMed

    Daxinger, Lucia; Harten, Sarah K; Oey, Harald; Epp, Trevor; Isbel, Luke; Huang, Edward; Whitelaw, Nadia; Apedaile, Anwyn; Sorolla, Anabel; Yong, Joan; Bharti, Vandhana; Sutton, Joanne; Ashe, Alyson; Pang, Zhenyi; Wallace, Nathan; Gerhardt, Daniel J; Blewitt, Marnie E; Jeddeloh, Jeffrey A; Whitelaw, Emma

    2013-01-01

    We have used a sensitized ENU mutagenesis screen to produce mouse lines that carry mutations in genes required for epigenetic regulation. We call these lines Modifiers of murine metastable epialleles (Mommes). We report a basic molecular and phenotypic characterization for twenty of the Momme mouse lines, and in each case we also identify the causative mutation. Three of the lines carry a mutation in a novel epigenetic modifier, Rearranged L-myc fusion (Rlf), and one gene, Rap-interacting factor 1 (Rif1), has not previously been reported to be involved in transcriptional regulation in mammals. Many of the other lines are novel alleles of known epigenetic regulators. For two genes, Rlf and Widely-interspaced zinc finger (Wiz), we describe the first mouse mutants. All of the Momme mutants show some degree of homozygous embryonic lethality, emphasizing the importance of epigenetic processes. The penetrance of lethality is incomplete in a number of cases. Similarly ,abnormalities in phenotype seen in the heterozygous individuals of some lines occur with incomplete penetrance. Recent advances in sequencing enhance the power of sensitized mutagenesis screens to identify the function of previously uncharacterized factors and to discover additional functions for previously characterized proteins. The observation of incomplete penetrance of phenotypes in these inbred mutant mice, at various stages of development, is of interest. Overall, the Momme collection of mouse mutants provides a valuable resource for researchers across many disciplines.

  14. Sleeping Beauty transposon insertional mutagenesis based mouse models for cancer gene discovery.

    PubMed

    Moriarity, Branden S; Largaespada, David A

    2015-02-01

    Large-scale genomic efforts to study human cancer, such as the cancer gene atlas (TCGA), have identified numerous cancer drivers in a wide variety of tumor types. However, there are limitations to this approach, the mutations and expression or copy number changes that are identified are not always clearly functionally relevant, and only annotated genes and genetic elements are thoroughly queried. The use of complimentary, nonbiased, functional approaches to identify drivers of cancer development and progression is ideal to maximize the rate at which cancer discoveries are achieved. One such approach that has been successful is the use of the Sleeping Beauty (SB) transposon-based mutagenesis system in mice. This system uses a conditionally expressed transposase and mutagenic transposon allele to target mutagenesis to somatic cells of a given tissue in mice to cause random mutations leading to tumor development. Analysis of tumors for transposon common insertion sites (CIS) identifies candidate cancer genes specific to that tumor type. While similar screens have been performed in mice with the PiggyBac (PB) transposon and viral approaches, we limit extensive discussion to SB. Here we discuss the basic structure of these screens, screens that have been performed, methods used to identify CIS.

  15. Reconstitutional Mutagenesis of the Maize P Gene by Short-Range Ac Transpositions

    PubMed Central

    Moreno, M. A.; Chen, J.; Greenblatt, I.; Dellaporta, S. L.

    1992-01-01

    The tendency for Ac to transpose over short intervals has been utilized to develop insertional mutagenesis and fine structure genetic mapping strategies in maize. We recovered excisions of Ac from the P gene and insertions into nearby chromosomal sites. These closely linked Ac elements reinserted into the P gene, reconstituting over 250 unstable variegated alleles. Reconstituted alleles condition a variety of variegation patterns that reflect the position and orientation of Ac within the P gene. Molecular mapping and DNA sequence analyses have shown that reinsertion sites are dispersed throughout a 12.3-kb chromosomal region in the promoter, exons and introns of the P gene, but in some regions insertions sites were clustered in a nonrandom fashion. Transposition profiles and target site sequence data obtained from these studies have revealed several features of Ac transposition including its preference for certain target sites. These results clearly demonstrate the tendency of Ac to transpose to nearby sites in both proximal and distal directions from the donor site. With minor modifications, reconstitutional mutagenesis should be applicable to many Ac-induced mutations in maize and in other plant species and can possibly be extended to other eukaryotic transposon systems as well. PMID:1325389

  16. Construction of a horseradish peroxidase resistant toward hydrogen peroxide by saturation mutagenesis.

    PubMed

    Asad, Sedigheh; Dastgheib, Seyed Mohammad Mehdi; Khajeh, Khosro

    2016-11-01

    Horseradish peroxidase (HRP) with a variety of potential biotechnological applications is still isolated from the horseradish root as a mixture of different isoenzymes with different biochemical properties. There is an increasing demand for preparations of high amounts of pure enzyme but its recombinant production is limited because of the lack of glycosylation in Escherichia coli and different glycosylation patterns in yeasts which affects its stability parameters. The goal of this study was to increase the stability of non-glycosylated enzyme, which is produced in E. coli, toward hydrogen peroxide via mutagenesis. Asparagine 268, one of the N-glycosylation sites of the enzyme, has been mutated via saturation mutagenesis using the megaprimer method. Modification and miniaturization of previously described protocols enabled screening of a library propagated in E. coli XJb (DE3). The library of mutants was screened for stability toward hydrogen peroxide with azinobis (ethylbenzthiazoline sulfonate) as a reducing substrate. Asn268Gly mutant, the top variant from the screening, exhibited 18-fold increased stability toward hydrogen peroxide and twice improved thermal stability compared with the recombinant HRP. Moreover, the substitution led to 2.5-fold improvement in the catalytic efficiency with phenol/4-aminoantipyrine. Constructed mutant represents a stable biocatalyst, which may find use in medical diagnostics, biosensing, and bioprocesses. © 2015 International Union of Biochemistry and Molecular Biology, Inc.

  17. Mutagenesis by host antimicrobial peptides: insights into microbial evolution during chronic infections

    PubMed Central

    Limoli, Dominique H.; Wozniak, Daniel J.

    2014-01-01

    Antimicrobial peptides (AMPs) are produced by the mammalian immune system to fight invading pathogens. The best understood function of AMPs is to integrate into the membranes of microbes, thereby disrupting and killing cells. However, a recent study [PLoS Pathogens (2014) 10, e1004083] provides evidence that at subinhibitory levels, AMPs promote mutations in bacterial DNA, which enhance bacterial survival. In particular, in the bacterium Pseudomonas aeruginosa, one AMP called LL-37 can promote mutations, which enable the bacteria to overproduce a protective sugar coating, a process called mucoid conversion. P. aeruginosa mucoid conversion is a major risk factor for those suffering from cystic fibrosis (CF), one of the most common lethal, heritable diseases in the US. LL-37 was found to produce mutations by penetrating the bacterial cell and binding to bacterial DNA. It was proposed that LL-37 binding DNA disrupts normal DNA replication and potentiates mutations. Importantly, LL-37 induced mutagenesis was also found to promote resistance to rifampicin in both P. aeruginosa and E. coli. This suggests that AMP-induced mutagenesis may be important for a broad range of chronic diseases and pathogens. PMID:28357249

  18. Comparative Analysis of Context-Dependent Mutagenesis Using Human and Mouse Models

    PubMed Central

    Medvedeva, Sofya A.; Panchin, Alexander Y.; Alexeevski, Andrey V.; Spirin, Sergey A.; Panchin, Yuri V.

    2013-01-01

    Substitution rates strongly depend on their nucleotide context. One of the most studied examples is the excess of C > T mutations in the CG context in various groups of organisms, including vertebrates. Studies on the molecular mechanisms underlying this mutation regularity have provided insights into evolution, mutagenesis, and cancer development. Recently several other hypermutable motifs were identified in the human genome. There is an increased frequency of T > C mutations in the second position of the words ATTG and ATAG and an increased frequency of A > C mutations in the first position of the word ACAA. For a better understanding of evolution, it is of interest whether these mutation regularities are human specific or present in other vertebrates, as their presence might affect the validity of currently used substitution models and molecular clocks. A comprehensive analysis of mutagenesis in 4 bp mutation contexts requires a vast amount of mutation data. Such data may be derived from the comparisons of individual genomes or from single nucleotide polymorphism (SNP) databases. Using this approach, we performed a systematical comparison of mutation regularities within 2–4 bp contexts in Mus musculus and Homo sapiens and uncovered that even closely related organisms may have notable differences in context-dependent mutation regularities. PMID:24058920

  19. Efficient and Heritable Targeted Mutagenesis in Mosses Using the CRISPR/Cas9 System.

    PubMed

    Nomura, Toshihisa; Sakurai, Tetsuya; Osakabe, Yuriko; Osakabe, Keishi; Sakakibara, Hitoshi

    2016-12-01

    Targeted genome modification by RNA-guided nucleases derived from the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9) system has seen rapid development in many organisms, including several plant species. In the present study, we succeeded in introducing the CRISPR/Cas9 system into the non-model organism Scopelophila cataractae, a moss that exhibits heavy metal tolerance, and the model organism Physcomitrella patens Utilizing the process by which moss plants regenerate from protoplasts, we conducted targeted mutagenesis by expression of single-chain guide RNA (sgRNA) and Cas9 in protoplasts. Using this method, the acquisition rate of strains exhibiting phenotypic changes associated with the target genes was approximately 45-69%, and strains with phenotypic changes exhibited various insertion and deletion mutations. In addition, we report that our method is capable of multiplex targeted mutagenesis (two independent genes) and also permits the efficient introduction of large deletions (∼3 kbp). These results demonstrate that the CRISPR/Cas9 system can be used to accelerate investigations of bryology and land plant evolution.

  20. [Analysis of the 4th generation Scutellaria baicalensis Georgi with space mutagenesis via FTIR spectroscopy].

    PubMed

    Ding, Xi-feng; Gao, Hua-na; Guo, Xi-hua; Wang, Zhi-zhou; Yang, La-huz; Guan, Ying; Shi, Jin-shan

    2009-05-01

    Determination and analysis for the Scutellaria baicalensis Georgi with space mutagenesis and the ground group were carried out with FTIR for the first time in order to fully understand the quality changes of the 4th generation of space mutagenesis of Scutellaria baicalensis Georgi and do further research. The result shows that for the FTIR spectra of the two samples the position and shape of main absorption peaks are approximately the same, indicating that the major components and the structures of space group remain intact, but the intensities of the absorption peaks are obviously different, with the intensities of the space group significantly enhanced compared to the ground group, especially for the flavonoid compounds (baicalin, baicalein, wogonoside, wogonin and wogonoside etc), which are the main active components of the Scutellaria baicalensis Georgi, the absorption peak intensities at 3391, 1655 and 1069 cm(-1) are obviously stronger than those of the ground group, so the contents of flavonoid compounds and other components are higher, and the amorphous active components are optimized. Space mutation breeding is conducive to breeding new varieties of highly active components, and it is also one of the ways to innovate Chinese medicines germplasm resources efficiently.

  1. Mutagenesis breeding of high echinocandin B producing strain and further titer improvement with culture medium optimization.

    PubMed

    Zou, Shu-Ping; Zhong, Wei; Xia, Chao-Jie; Gu, Ya-Nan; Niu, Kun; Zheng, Yu-Guo; Shen, Yin-Chu

    2015-10-01

    A combination of microbial strain improvement and statistical optimization is investigated to maximize echinocandin B (ECB) production from Aspergillus nidulans ZJB-0817. A classical sequential mutagenesis was studied first by using physical (ultraviolet irradiation at 254 nm) and chemical mutagens (lithium chloride and sodium nitrite). Mutant strain ULN-59 exhibited 2.1-fold increase in ECB production to 1583.1 ± 40.9 mg/L when compared with the parent strain (750.8 ± 32.0 mg/L). This is the first report where mutagenesis is applied in Aspergillus to improve ECB production. Further, fractional factorial design and central composite design were adopted to optimize the culture medium for increasing ECB production by the mutant ULN-59. Results indicated that four culture media including peptone, K2HPO4, mannitol and L-ornithine had significant effects on ECB production. The optimized medium provided another 1.4-fold increase in final ECB concentration to 2285.6 ± 35.6 mg/L compared to the original medium. The results of this study indicated the combined application of a classical mutation and medium optimization can improve effectively ECB production from A. nidulans and could be a promising tool to improve other secondary metabolites production by fungal strains.

  2. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants.

    PubMed

    Hehle, Verena K; Paul, Matthew J; Roberts, Victoria A; van Dolleweerd, Craig J; Ma, Julian K-C

    2016-04-01

    This study examined the degradation pattern of a murine IgG1κ monoclonal antibody expressed in and extracted from transformedNicotiana tabacum Gel electrophoresis of leaf extracts revealed a consistent pattern of recombinant immunoglobulin bands, including intact and full-length antibody, as well as smaller antibody fragments. N-terminal sequencing revealed these smaller fragments to be proteolytic cleavage products and identified a limited number of protease-sensitive sites in the antibody light and heavy chain sequences. No strictly conserved target sequence was evident, although the peptide bonds that were susceptible to proteolysis were predominantly and consistently located within or near to the interdomain or solvent-exposed regions in the antibody structure. Amino acids surrounding identified cleavage sites were mutated in an attempt to increase resistance. Different Guy's 13 antibody heavy and light chain mutant combinations were expressed transiently inN. tabacumand demonstrated intensity shifts in the fragmentation pattern, resulting in alterations to the full-length antibody-to-fragment ratio. The work strengthens the understanding of proteolytic cleavage of antibodies expressed in plants and presents a novel approach to stabilize full-length antibody by site-directed mutagenesis.-Hehle, V. K., Paul, M. J., Roberts, V. A., van Dolleweerd, C. J., Ma, J. K.-C. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants. © The Author(s).

  3. Involvement of a joker mutation in a polymerase-independent lethal mutagenesis escape mechanism.

    PubMed

    Agudo, Rubén; de la Higuera, Ignacio; Arias, Armando; Grande-Pérez, Ana; Domingo, Esteban

    2016-07-01

    We previously characterized a foot-and-mouth disease virus (FMDV) with three amino acid replacements in its polymerase (3D) that conferred resistance to the mutagenic nucleoside analogue ribavirin. Here we show that passage of this mutant in the presence of high ribavirin concentrations resulted in selection of viruses with the additional replacement I248T in 2C. This 2C substitution alone (even in the absence of replacements in 3D) increased FMDV fitness mainly in the presence of ribavirin, prevented an incorporation bias in favor of A and U associated with ribavirin mutagenesis, and conferred the ATPase activity of 2C decreased sensitivity to ribavirin-triphosphate. Since in previous studies we described that 2C with I248T was selected under different selective pressures, this replacement qualifies as a joker substitution in FMDV evolution. The results have identified a role of 2C in nucleotide incorporation, and have unveiled a new polymerase-independent mechanism of virus escape to lethal mutagenesis.

  4. Picomolar affinity fibronectin domains engineered utilizing loop length diversity, recursive mutagenesis, and loop shuffling.

    PubMed

    Hackel, Benjamin J; Kapila, Atul; Wittrup, K Dane

    2008-09-19

    The 10th type III domain of human fibronectin (Fn3) has been validated as an effective scaffold for molecular recognition. In the current work, it was desired to improve the robustness of selection of stable, high-affinity Fn3 domains. A yeast surface display library of Fn3 was created in which three solvent-exposed loops were diversified in terms of amino acid composition and loop length. The library was screened by fluorescence-activated cell sorting to isolate binders to lysozyme. An affinity maturation scheme was developed to rapidly and broadly diversify populations of clones by random mutagenesis as well as homologous recombination-driven shuffling of mutagenized loops. The novel library and affinity maturation scheme combined to yield stable, monomeric Fn3 domains with 3 pM affinity for lysozyme. A secondary affinity maturation identified a stable 1.1 pM binder, the highest affinity yet reported for an Fn3 domain. In addition to extension of the affinity limit for this scaffold, the results demonstrate the ability to achieve high-affinity binding while preserving stability and the monomeric state. This library design and affinity maturation scheme is highly efficient, utilizing an initial diversity of 2x10(7) clones and screening only 1x10(8) mutants (totaled over all affinity maturation libraries). Analysis of intermediate populations revealed that loop length diversity, loop shuffling, and recursive mutagenesis of diverse populations are all critical components.

  5. Rational and random mutagenesis of firefly luciferase to identify an efficient emitter of red bioluminescence

    NASA Astrophysics Data System (ADS)

    Branchini, Bruce R.; Southworth, Tara L.; Khattak, Neelum F.; Murtiashaw, Martha H.; Fleet, Sarah E.

    2004-06-01

    Firefly luciferase, which emits yellow-green (557 nm) light, and the corresponding cDNA have been used successfully as a bioluminescence reporter of gene expression. One particularly exciting application is in the area of in vivo bioluminescence imaging. Our interest is in developing improved reagents by identifying Photinus pyralis luciferase mutants that efficiently emit red bioluminescence. In this way, the proven advantages of the P. pyralis protein can be combined with the potential advantages of a red-shifted emitter. Using site-directed mutagenesis techniques, we have identified many mutants emitting red bioluminescence. Unfortunately, these enzymes generally have significantly decreased bioluminescence activity. Interestingly, we discovered a mutation, Ile351Ala, that produced a moderate 16 nm red-shift, while maintaining excellent bioluminescence activity. We then undertook a random mutagenesis approach to identify luciferase mutants that emit further red-shifted bioluminescence with minimal loss of activity. Libraries of mutants were created using an error-prone PCR method and the Ile351Ala luciferase mutant as the template DNA. The libraries were screened by in vivo bacterial assays and the promising mutants were purified to enable accurate determination of bioluminescence emission spectra and total bioluminescence activity. We will report the characterization results, including the identification of the randomly altered amino acids, of several mutants that catalyze bioluminescence with emission maxima of approximately 600 nm.

  6. Phenotypic Mutants of the Intracellular Actinomycete Rhodococcus equi Created by In Vivo Himar1 Transposon Mutagenesis

    PubMed Central

    Ashour, Joseph; Hondalus, Mary K.

    2003-01-01

    Rhodococcus equi is a facultative intracellular opportunistic pathogen of immunocompromised people and a major cause of pneumonia in young horses. An effective live attenuated vaccine would be extremely useful in the prevention of R. equi disease in horses. Toward that end, we have developed an efficient transposon mutagenesis system that makes use of a Himar1 minitransposon delivered by a conditionally replicating plasmid for construction of R. equi mutants. We show that Himar1 transposition in R. equi is random and needs no apparent consensus sequence beyond the required TA dinucleotide. The diversity of the transposon library was demonstrated by the ease with which we were able to screen for auxotrophs and mutants with pigmentation and capsular phenotypes. One of the pigmentation mutants contained an insertion in a gene encoding phytoene desaturase, an enzyme of carotenoid biosynthesis, the pathway necessary for production of the characteristic salmon color of R. equi. We identified an auxotrophic mutant with a transposon insertion in the gene encoding a putative dual-functioning GTP cyclohydrolase II-3,4-dihydroxy-2-butanone-4-phosphate synthase, an enzyme essential for riboflavin biosynthesis. This mutant cannot grow in minimal medium in the absence of riboflavin supplementation. Experimental murine infection studies showed that, in contrast to wild-type R. equi, the riboflavin-requiring mutant is attenuated because it is unable to replicate in vivo. The mutagenesis methodology we have developed will allow the characterization of R. equi virulence mechanisms and the creation of other attenuated strains with vaccine potential. PMID:12670990

  7. Structure-antimutagenic activity relationships of benzalacetone derivatives against UV-induced mutagenesis in E. coli WP2uvrA and gamma-induced mutagenesis in Salmonella typhimurium TA2638.

    PubMed

    Motohashi, N; Ashihara, Y; Yamagami, C; Saito, Y

    2001-03-01

    The antimutagenic activities of benzalacetone (4-phenyl-3-buten-2-one) and its structurally-related compounds were evaluated through their use as post-treatments for the UV-induced mutagenesis in Escherichia coli WP2s (uvrA) and the gamma-induced mutagenesis in Salmonella typhimurium TA2638, the latter of which is sensitive to oxidants. Structure-activity relationships were studied between IC(50) activity values, i.e. the dose (micromol/ml) at which the mutation frequency is reduced to 50% of the control, and electronic and hydrophobicity properties of the studied molecules. Benzalacetone and benzalacetone analogs, cinnamaldehyde and trans-1,1,1-trifluoro-4-phenyl-3-buten-2-one (TF), inhibited both forms of mutagenesis, but methyl cinnamate, cinnamic acid and cinnamamide did not. The IC(50) values of TF, for UV-induced mutagenesis and gamma-induced mutagenesis, were 0.028 and 0.045 micromol/ml, respectively, and one order of magnitude lower than those of cinnamaldehyde and benzalacetone. The three antimutagenic analogs listed in order of decreasing activity are: TF>cinnamaldehyde>benzalacetone. This order is proportional to the electron-withdrawing property of the terminal group attached to an alpha,beta-unsaturated carbonyl moiety in the side chain that is known to play an important role in the antimutagenicities of benzalacetone and related compounds. In UV-induced mutagenesis in E. coli WP2s, mono-substituted benzalacetones - the ring-substituents of which have electron-withdrawing properties - showed antimutagenic activity that correlated with their electronic property. In gamma-induced mutagenesis in S. typhimurium TA2638, the antimutagenic activities of mono-substituted benzalacetones were proportional to the substituent hydrophobicities (pi). The different effects on both the mutation-induced systems is suggested to be related to the relative permeability of the cell membranes and the different sensitivities to mutagens between E. coli WP2s and S

  8. Transposon-directed base-exchange mutagenesis (TDEM): a novel method for multiple-nucleotide substitutions within a target gene.

    PubMed

    Kim, Yun Cheol; Lee, Hui Sun; Yoon, Sukjoon; Morrison, Sherie L

    2009-06-01

    In this report we describe transposon-directed base-exchange mutagenesis (TDEM), an efficient and controllable method for introducing a mutation into a gene. Each round of TDEM can remove up to 11 base pairs from a randomly selected site within the target gene and replace them with any length of DNA of predetermined sequence. Therefore, the number of bases to be deleted and inserted can be independently regulated providing greater versatility than existing methods of transposon-based mutagenesis. Subsequently, multiple rounds of mutagenesis will provide a diverse mutant library that contains multiple mutations throughout the gene. Additionally, we developed a simple frame-checking procedure that eliminates nonfunctional mutants containing frameshifts or stop codons. As a proof of principle, we used TDEM to generate mutant lacZalpha lacking alpha-complementation activity and recovered active revertants using a second round of TDEM. Furthermore, a single round of TDEM yielded unique, inactive mutants of ccdB.

  9. 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

  10. Use of a simian virus 40-based shuttle vector to analyze enhanced mutagenesis in mitomycin C-treated monkey cells.

    PubMed Central

    Roilides, E; Munson, P J; Levine, A S; Dixon, K

    1988-01-01

    When monkey cells were treated with mitomycin C 24 h before transfection with UV-irradiated pZ189 (a simian virus 40-based shuttle vector), there was a twofold increase in the frequency of mutations in the supF gene of the vector. These results suggest the existence of an enhancible mutagenesis pathway in mammalian cells. However, DNA sequence analysis of the SupF- mutants suggested no dramatic changes in the mechanisms of mutagenesis due to mitomycin C treatment of the cells. PMID:2851732

  11. Use of a simian virus 40-based shuttle vector to analyze enhanced mutagenesis in mitomycin C-treated monkey cells

    SciTech Connect

    Roilides, E.; Munson, P.J.; Levine, A.S.; Dixon, K.

    1988-09-01

    When monkey cells were treated with mitomycin C 24 h before transfection with UV-irradiated pZ189 (a simian virus 40-based shuttle vector), there was a twofold increase in the frequency of mutations in the supF gene of the vector. These results suggest the existence of an enhancible mutagenesis pathway in mammalian cells. However, DNA sequence analysis of the SupF- mutants suggested no dramatic changes in the mechanisms of mutagenesis due to mitomycin C treatment of the cells.

  12. Characterization of highly efficient heavy-ion mutagenesis in Arabidopsis thaliana

    PubMed Central

    2011-01-01

    Background Heavy-ion mutagenesis is recognised as a powerful technology to generate new mutants, especially in higher plants. Heavy-ion beams show high linear energy transfer (LET) and thus more effectively induce DNA double-strand breaks than other mutagenic techniques. Previously, we determined the most effective heavy-ion LET (LETmax: 30.0 keV μm-1) for Arabidopsis mutagenesis by analysing the effect of LET on mutation induction. However, the molecular structure of mutated DNA induced by heavy ions with LETmax remains unclear. Knowledge of the structure of mutated DNA will contribute to the effective exploitation of heavy-ion beam mutagenesis. Results Dry Arabidopsis thaliana seeds were irradiated with carbon (C) ions with LETmax at a dose of 400 Gy and with LET of 22.5 keV μm-1 at doses of 250 Gy or 450 Gy. The effects on mutation frequency and alteration of DNA structure were compared. To characterise the structure of mutated DNA, we screened the well-characterised mutants elongated hypocotyls (hy) and glabrous (gl) and identified mutated DNA among the resulting mutants by high-resolution melting curve, PCR and sequencing analyses. The mutation frequency induced by C ions with LETmax was two-fold higher than that with 22.5 keV μm-1 and similar to the mutation frequency previously induced by ethyl methane sulfonate. We identified the structure of 22 mutated DNAs. Over 80% of the mutations caused by C ions with both LETs were base substitutions or deletions/insertions of less than 100 bp. The other mutations involved large rearrangements. Conclusions The C ions with LETmax showed high mutation efficiency and predominantly induced base substitutions or small deletions/insertions, most of which were null mutations. These small alterations can be determined by single-nucleotide polymorphism (SNP) detection systems. Therefore, C ions with LETmax might be useful as a highly efficient reverse genetic system in conjunction with SNP detection systems, and will be

  13. Chromium (VI) potentiates mutagenesis by sodium azide but not ethyl methanesulfonate

    SciTech Connect

    LaVelle, J.M.; Witmer, C.M.

    1984-01-01

    A fluctuation test using Salmonella typhimurium strain 1535 has been used in an experimental protocol to assess biological effects of interactions between chromium (VI), such as K/sub 2/CrO/sub 4/, and two DNA-damaging agents, ethyl methanesulfonate (EMS), and sodium azide. For the combination of K/sub 2/CrO/sub 4/ and NaN/sub 3/, the response was significantly greater than expected suggesting a possible potentiation of mutagenesis. The opposite (a less-than-additive response) was found for the K/sub 2/CrO/sub 4//EMS combination. Both effects were found to be dose related to the concentration of potassium chromate used. Toxicity of the compounds or their combinations to the bacterial could not explain the results.

  14. Detection of occupational and environmental exposures by bacterial mutagenesis assays of human body fluids.

    PubMed

    Everson, R B

    1986-08-01

    Assays of human body fluids provide a means to document human exposure to mutagens in the environment. In contrast to measurements of ambient levels, these assays demonstrate absorption of mutagens and provide estimates of minimal systemic doses. For most studies reviewed here, specimens of urine were concentrated by adsorption to columns of XAD-2 resin or by liquid partition extraction prior to the mutagenesis assays. The resulting extracts most commonly were analyzed for mutagenicity using the Salmonella/mammalian microsomal plate assay. Less frequently used assays included bacterial fluctuation tests instead of the plate assay and assays for the induction of sister chromatid exchanges in cultured mammalian cells. In addition to reviewing literature reports where body fluids were tested, the advantages, disadvantages, and potential role of this approach will be briefly discussed and compared with other approaches to the identification of mutagenic hazards in the workplace.

  15. Detection of occupational and environmental exposures by bacterial mutagenesis assays of human body fluids

    SciTech Connect

    Everson, R.B.

    1986-08-01

    Assays of human body fluids provide a means to document human exposure to mutagens in the environment. In contrast to measurements of ambient levels, these assays demonstrate absorption of mutagens and provide estimates of minimal systemic doses. For most studies reviewed here, specimens of urine were concentrated by adsorption to columns of XAD-2 resin or by liquid partition extraction prior to the mutagenesis assays. The resulting extracts most commonly were analyzed for mutagenicity using the Salmonella/mammalian microsomal plate assay. Less frequently used assays included bacterial fluctuation tests instead of the plate assay and assays for the induction of sister chromatid exchanges in cultured mammalian cells. In addition to reviewing literature reports where body fluids were tested, the advantages, disadvantages, and potential role of this approach will be briefly discussed and compared with other approaches to the identification of mutagenic hazards in the workplace.

  16. Characterisation of the Rab binding properties of Rab coupling protein (RCP) by site-directed mutagenesis.

    PubMed

    Lindsay, Andrew J; McCaffrey, Mary W

    2004-07-30

    Rab coupling protein (RCP) is a member of the Rab11-family of interacting proteins (Rab11-FIPs). Family members are characterised by their ability to interact with Rab11. This property is mediated by a conserved Rab binding domain (RBD) located at their carboxy-termini. Several Rab11-FIPs can also interact with other small GTPases. RCP interacts with Rab4 in addition to Rab11. To dissect out the individual properties of the Rab4 and Rab11 interactions with RCP, conserved amino acids within the RBD of RCP were mutated by site-directed mutagenesis. The effect of these mutations on Rab4 and Rab11 binding, and the intracellular localisation of RCP, was examined. Our results indicate that Rab11, rather than Rab4, mediates the intracellular localisation of RCP, and that the class I Rab11-FIPs compete for binding to Rab11.

  17. Spectrum of Bmp5 Mutations from Germline Mutagenesis Experiments in Mice

    PubMed Central

    Marker, P. C.; Seung, K.; Bland, A. E.; Russell, L. B.; Kingsley, D. M.

    1997-01-01

    Over 40 years of mutagenesis experiments using the mouse specific-locus test have produced a large number of induced germline mutations at seven loci, among them the short ear locus. We have previously shown that the short ear locus encodes bone morphogenetic protein 5 (BMP5), a member of a large family of secreted signaling molecules that play key roles in axis formation, tissue differentiation, mesenchymal-epithelial interactions, and skeletal development. Here we examine 24 chemical- and radiation-induced mutations at the short ear locus. Sequence changes in the Bmp5 open reading frame confirm the importance of cysteine residues in the function of TGFβ superfamily members. The spectrum of N-ethyl-N-nitrosourea-induced mutations also provides new information about the basepair, sequence context, and strand specificity of germline mutations in mammals. PMID:9071596

  18. 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.

  19. Evaluating Risks of Insertional Mutagenesis by DNA Transposons in Gene Therapy

    PubMed Central

    Hackett, Perry B.; Largaespada, David A.; Switzer, Kirsten C.; Cooper, Laurence J.N.

    2013-01-01

    Investigational therapy can be successfully undertaken using viral- and non-viral-mediated ex vivo gene transfer. Indeed, recent clinical trials have established the potential for genetically modified T cells to improve and restore health. Recently the Sleeping Beauty (SB) transposon/transposase system has been applied in clinical trials to stably insert a chimeric antigen receptor (CAR) to redirect T-cell specificity. We discuss the context in which the SB system can be harnessed for gene therapy and describe the human application of SB-modified CAR+ T cells. We have focused on theoretical issues relating to insertional mutagenesis in the context of human genomes that are naturally subjected to remobilization of transposons and the experimental evidence over the last decade of employing SB transposons for defining genes that induce cancer. These findings are put into the context of the use of SB transposons in the treatment of human disease. PMID:23313630

  20. Arenaviruses and Lethal Mutagenesis. Prospects for New Ribavirin-based Interventions

    PubMed Central

    Moreno, Héctor; Grande-Pérez, Ana; Domingo, Esteban; Martín, Verónica

    2012-01-01

    Lymphocytic choriomeningitis virus (LCMV) has contributed to unveil some of the molecular mechanisms of lethal mutagenesis, or loss of virus infectivity due to increased mutation rates. Here we review these developments, and provide additional evidence that ribavirin displays a dual mutagenic and inhibitory activity on LCMV that can be relevant to treatment designs. Using 5-fluorouracil as mutagenic agent and ribavirin either as inhibitor or mutagen, we document an advantage of a sequential inhibitor-mutagen administration over the corresponding combination treatment to achieve a low LCMV load in cell culture. This advantage is accentuated in the concentration range in which ribavirin acts mainly as an inhibitor, rather than as mutagen. This observation reinforces previous theoretical and experimental studies in supporting a sequential inhibitor-mutagen administration as a possible antiviral design. Given recent progress in the development of new inhibitors of arenavirus replication, our results suggest new options of ribavirin-based anti-arenavirus treatments. PMID:23202505

  1. Analysis of acidic surface of Haloferax mediterranei glucose dehydrogenase by site-directed mutagenesis.

    PubMed

    Esclapez, J; Pire, C; Bautista, V; Martínez-Espinosa, R M; Ferrer, J; Bonete, M J

    2007-03-06

    Generally, halophilic enzymes present a characteristic amino acid composition, showing an increase in the content of acidic residues and a decrease in the content of basic residues, particularly lysines. The latter decrease appears to be responsible for a reduction in the proportion of solvent-exposed hydrophobic surface. This role was investigated by site-directed mutagenesis of glucose dehydrogenase from Haloferax mediterranei, in which surface aspartic residues were changed to lysine residues. From the biochemical analysis of the mutant proteins, it is concluded that the replacement of the aspartic residues by lysines results in slightly less halotolerant proteins, although they retain the same enzymatic activities and kinetic parameters compared to the wild type enzyme.

  2. In vivo mutagenesis reveals that OriL is essential for mitochondrial DNA replication

    PubMed Central

    Wanrooij, Sjoerd; Miralles Fusté, Javier; Stewart, James B; Wanrooij, Paulina H; Samuelsson, Tore; Larsson, Nils-Göran; Gustafsson, Claes M; Falkenberg, Maria

    2012-01-01

    The mechanisms of mitochondrial DNA replication have been hotly debated for a decade. The strand-displacement model states that lagging-strand DNA synthesis is initiated from the origin of light-strand DNA replication (OriL), whereas the strand-coupled model implies that OriL is dispensable. Mammalian mitochondria cannot be transfected and the requirements of OriL in vivo have therefore not been addressed. We here use in vivo saturation mutagenesis to demonstrate that OriL is essential for mtDNA maintenance in the mouse. Biochemical and bioinformatic analyses show that OriL is functionally conserved in vertebrates. Our findings strongly support the strand-displacement model for mtDNA replication. PMID:23090476

  3. Cloning of human epidermal growth factor as a bacterial secretory protein, its properties and mutagenesis

    SciTech Connect

    Engler, D.A.; Matsunami, R.K.; Campion, S.R.; Foote, R.S.; Mural, R.J.; Larimer, F.W.; Stevens, A.; Niyogi, S.K.

    1987-05-01

    A chimeric gene, containing the DNA coding for the human epidermal growth factor (EGF) and that for the signal peptide of E. coli alkaline phosphatase, was constructed by the annealing and subsequent ligation of appropriate DNA oligonucleotides synthesized in an automated DNA synthesizer. The gene was then cloned into a bacterial plasmid under the transcriptional control of the E. coli trp-lac (tac) promoter, and then transformed into E. coli. Following induction with isopropylthiogalactoside, the secretion of EGF into the E. coli periplasmic space and some into the growth medium was confirmed by its specific binding to the EGF receptor and stimulation of the EGF receptor tyrosine kinase activity. The size and physicochemical properties of the purified protein mimicked those of authentic human EGF. Studies of structure/function relationships by specific alterations of targeted amino acid residues in the EGF molecule have been initiated by utilizing site-directed mutagenesis.

  4. Site-directed mutagenesis in bacteriorhodopsin mutants and their characterization for bioelectrical and biotechnological equipment.

    PubMed

    Saeedi, Pardis; Moosaabadi, Jafar Mohammadian; Sebtahmadi, Seyed Sina; Behmanesh, Mehrdad; Mehrabadi, Jalil Fallah

    2012-03-01

    Bacteriorhodopsin (BR) mutagenesis plays an important role in the development of BR-based materials and tools with enhanced optical and electrical properties. Previously reported protocols for generating BR mutations are inefficient for the preparation and purification of mutant proteins. Therefore, a series of BR mutations were generated by using improved methods, which are described in further detail. The functional activity of the recombinant proteins was confirmed by spectroscopic and electrochemical assays. Modified proteins with different wavelengths and activities form a foundation for color-sensitive sensors and can be utilized to produce unique bioelectrical and biotechnological tools and materials. The proton-pumping activity of the generated mutant D85E was normal, indicating that the mutant could be used in light batteries. However, mutants D85Q and D85N were almost inactive; and D85N had a prolonged M state, suggesting that it could be utilized in light memories.

  5. Site-directed mutagenesis of possible catalytic residues of cellobiose 2-epimerase from Ruminococcus albus.

    PubMed

    Ito, Shigeaki; Hamada, Shigeki; Ito, Hiroyuki; Matsui, Hirokazu; Ozawa, Tadahiro; Taguchi, Hidenori; Ito, Susumu

    2009-07-01

    The cellobiose 2-epimerase from Ruminococcus albus (RaCE) catalyzes the epimerization of cellobiose and lactose to 4-O-beta-D-glucopyranosyl-D-mannose and 4-O-beta-D-galactopyranosyl-D-mannose (epilactose). Based on the sequence alignment with N-acetyl-D-glucosamine 2-epimerases of known structure and on a homology-modeled structure of RaCE, we performed site-directed mutagenesis of possible catalytic residues in the enzyme, and the mutants were expressed in Escherichia coli cells. We found that R52, H243, E246, W249, W304, E308, and H374 were absolutely required for the activity of RaCE. F114 and W303 also contributed to catalysis. These residues protruded into the active-site cleft in the model (alpha/alpha)(6) core barrel structure.

  6. Development of an inducible transposon system for efficient random mutagenesis in Clostridium acetobutylicum

    PubMed Central

    Zhang, Ying; Xu, Shu; Chai, Changsheng; Yang, Sheng; Jiang, Weihong; Minton, Nigel P.; Gu, Yang

    2016-01-01

    Clostridium acetobutylicum is an industrially important Gram-positive organism, which is capable of producing economically important chemicals in the ABE (Acetone, Butanol and Ethanol) fermentation process. Renewed interests in the ABE process necessitate the availability of additional genetics tools to facilitate the derivation of a greater understanding of the underlying metabolic and regulatory control processes in operation through forward genetic strategies. In this study, a xylose inducible, mariner-based, transposon system was developed and shown to allow high-efficient random mutagenesis in the model strain ATCC 824. Of the thiamphenicol resistant colonies obtained, 91.9% were shown to be due to successful transposition of the catP-based mini-transposon element. Phenotypic screening of 200 transposon clones revealed a sporulation-defective clone with an insertion in spo0A, thereby demonstrating that this inducible transposon system can be used for forward genetic studies in C. acetobutylicum. PMID:27001972

  7. Systematic analysis of the kalimantacin assembly line NRPS module using an adapted targeted mutagenesis approach.

    PubMed

    Uytterhoeven, Birgit; Appermans, Kenny; Song, Lijiang; Masschelein, Joleen; Lathouwers, Thomas; Michiels, Chris W; Lavigne, Rob

    2016-04-01

    Kalimantacin is an antimicrobial compound with strong antistaphylococcal activity that is produced by a hybrid trans-acyltransferase polyketide synthase/nonribosomal peptide synthetase system in Pseudomonas fluorescens BCCM_ID9359. We here present a systematic analysis of the substrate specificity of the glycine-incorporating adenylation domain from the kalimantacin biosynthetic assembly line by a targeted mutagenesis approach. The specificity-conferring code was adapted for use in Pseudomonas and mutated adenylation domain active site sequences were introduced in the kalimantacin gene cluster, using a newly adapted ligation independent cloning method. Antimicrobial activity screens and LC-MS analyses revealed that the production of the kalimantacin analogues in the mutated strains was abolished. These results support the idea that further insight in the specificity of downstream domains in nonribosomal peptide synthetases and polyketide synthases is required to efficiently engineer these strains in vivo.

  8. Inducible pathway is required for mutagenesis in Salmonella typhimurium LT2

    SciTech Connect

    Orrego, C.; Eisenstadt, E.

    1987-06-01

    UV mutability of Salmonella typhimurium LT2 was eliminated in the presence of a multicopy plasmid carrying the Escherichia coli lexA/sup +/ gene. This result suggests that inducible, SOS-like functions are required for UV mutagenesis in S. typhimurium. S. typhimurium strains carrying either point or deletion mutations in topA had previously been shown to lose their mutability by UV or methyl methanesulfonate. Mitomycin C induction of the Phi(mucB'-lacZ') fusion (a DNA damage-inducible locus carried on plasmid pSE205) in S. typhimurium topA was normal, suggesting that RecA is activated in topA mutants. These observations lead the authors deduce that S. typhimurium has at least one DNA damage-inducible locus in addition to recA that is required for UV mutability.

  9. Enhancement of astaxanthin production from Haematococcus pluvialis mutants by three-stage mutagenesis breeding.

    PubMed

    Wang, Ni; Guan, Bin; Kong, Qing; Sun, Han; Geng, Zhaoyan; Duan, Liangfei

    2016-10-20

    Haematococcus pluvialis was modified for higher astaxanthin production compatible with the superiorities of high biomass and high activity by three-stage mutagenesis breeding. UV irradiation mutants named UV11-4 made an increase on cell dry weight, but showed a longer growth circle than the wild type. On the basis of UV mutants, ethyl methane sulphonate (EMS) mutants E2-5 cut down the latent phase, brought forward and extended the logarithmic phase. The inhibitor diphenylamine (DPA) was employed to screen high-yield astaxanthin producer by the color change of colonies from green to red on solid medium. Via the contravariant cultivation, proliferation and transformation, the mutant DPA12-2 possessed an 1.7-fold astaxanthin production compared to the wild type, reaching 47.21±3.30mg/g dry cells. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Intrinsic and environmental mutagenesis drive diversification and persistence of Pseudomonas aeruginosa in chronic lung infections.

    PubMed

    Rodríguez-Rojas, Alexandro; Oliver, Antonio; Blázquez, Jesús

    2012-01-01

    Pseudomonas aeruginosa is a versatile opportunistic pathogen causing a wide variety of hospital-acquired acute infections in immunocompromised patients as well as chronic respiratory infections in patients suffering from cystic fibrosis or other chronic respiratory diseases. Several traits contribute to its ability to colonize and persist in the lungs of chronically infected patients, including development of high resistance to antimicrobials and hypermutability, biofilm growth, and alginate hyperproduction, or a customized pathogenicity, which may include the loss of classical virulence factors and metabolic changes. Here we argue that a combination of both intrinsic and environmental mutagenesis leads to a high number of mutant variants in the population. The conducive environment then triggers a positive feedback loop leading to adaptation and persistence of P. aeruginosa, rendering these chronic infections almost impossible to eradicate.

  11. The role of flexibility and molecular shape in the crystallization of proteins by surface mutagenesis.

    PubMed

    Devedjiev, Yancho D

    2015-02-01

    Proteins are dynamic systems and interact with their environment. The analysis of crystal contacts in the most accurately determined protein structures (d < 1.5 Å) reveals that in contrast to current views, static disorder and high side-chain entropy are common in the crystal contact area. These observations challenge the validity of the theory that presumes that the occurrence of well ordered patches of side chains at the surface is an essential prerequisite for a successful crystallization event. The present paper provides evidence in support of the approach for understanding protein crystallization as a process dependent on multiple factors, each with its relative contribution, rather than a phenomenon driven by a few dominant physicochemical characteristics. The role of the molecular shape as a factor in the crystallization of proteins by surface mutagenesis is discussed.

  12. The role of the bacterial mismatch repair system in SOS-induced mutagenesis: a theoretical background.

    PubMed

    Belov, Oleg V; Chuluunbaatar, Ochbadrakh; Kapralov, Mikhail I; Sweilam, Nasser H

    2013-09-07

    A theoretical study is performed of the possible role of the methyl-directed mismatch repair system in the ultraviolet-induced mutagenesis of Escherichia coli bacterial cells. For this purpose, mathematical models of the SOS network, translesion synthesis and mismatch repair are developed. Within the proposed models, the key pathways of these repair systems were simulated on the basis of modern experimental data related to their mechanisms. Our model approach shows a possible mechanistic explanation of the hypothesis that the bacterial mismatch repair system is responsible for attenuation of mutation frequency during ultraviolet-induced SOS response via removal of the nucleotides misincorporated by DNA polymerase V (the UmuD'2C complex). Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. Genetic recombination as a major cause of mutagenesis in the human globin gene clusters.

    PubMed

    Borg, Joseph; Georgitsi, Marianthi; Aleporou-Marinou, Vassiliki; Kollia, Panagoula; Patrinos, George P

    2009-12-01

    Homologous recombination is a frequent phenomenon in multigene families and as such it occurs several times in both the alpha- and beta-like globin gene families. In numerous occasions, genetic recombination has been previously implicated as a major mechanism that drives mutagenesis in the human globin gene clusters, either in the form of unequal crossover or gene conversion. Unequal crossover results in the increase or decrease of the human globin gene copies, accompanied in the majority of cases with minor phenotypic consequences, while gene conversion contributes either to maintaining sequence homogeneity or generating sequence diversity. The role of genetic recombination, particularly gene conversion in the evolution of the human globin gene families has been discussed elsewhere. Here, we summarize our current knowledge and review existing experimental evidence outlining the role of genetic recombination in the mutagenic process in the human globin gene families.

  14. Genetic modification through oligonucleotide-mediated mutagenesis. A GMO regulatory challenge?

    PubMed

    Breyer, Didier; Herman, Philippe; Brandenburger, Annick; Gheysen, Godelieve; Remaut, Erik; Soumillion, Patrice; Van Doorsselaere, Jan; Custers, René; Pauwels, Katia; Sneyers, Myriam; Reheul, Dirk

    2009-01-01

    In the European Union, the definition of a GMO is technology-based. This means that a novel organism will be regulated under the GMO regulatory framework only if it has been developed with the use of defined techniques. This approach is now challenged with the emergence of new techniques. In this paper, we describe regulatory and safety issues associated with the use of oligonucleotide-mediated mutagenesis to develop novel organisms. We present scientific arguments for not having organisms developed through this technique fall within the scope of the EU regulation on GMOs. We conclude that any political decision on this issue should be taken on the basis of a broad reflection at EU level, while avoiding discrepancies at international level.

  15. Identification of catalytically essential residues in Escherichia coli esterase by site-directed mutagenesis.

    PubMed

    Haruki, M; Oohashi, Y; Mizuguchi, S; Matsuo, Y; Morikawa, M; Kanaya, S

    1999-07-09

    Escherichia coli esterase (EcE) is a member of the hormone-sensitive lipase family. We have analyzed the roles of the conserved residues in this enzyme (His103, Glu128, Gly163, Asp164, Ser165, Gly167, Asp262, Asp266 and His292) by site-directed mutagenesis. Among them, Gly163, Asp164, Ser165, and Gly167 are the components of a G-D/E-S-A-G motif. We showed that Ser165, Asp262, and His292 are the active-site residues of the enzyme. We also showed that none of the other residues, except for Asp164, is critical for the enzymatic activity. The mutation of Asp164 to Ala dramatically reduced the catalytic efficiency of the enzyme by the factor of 10(4) without seriously affecting the substrate binding. This residue is probably structurally important to make the conformation of the active-site functional.

  16. [Mutagenesis of the human histamine H1 receptor and design of new antihistamine agents].

    PubMed

    Differding, E; Gillard, M; Moguilevsky, N; Varsalona, F; Noyer, M; Daliers, J; Goldstein, S; Neuwels, M; Lassoie, M A; Guillaume, J P; Bascour, M; Bollen, A; Hénichart, J P

    1996-01-01

    The binding cavity of histamine and histamine antagonists is explored using site directed mutagenesis of the human histamine H1 receptor and the amino acids involved in ligand binding are identified. Whereas Asp107 and Phe199 are important for both agonists and antagonists, two additional amino acids (Asn198 and Trp103) are required for efficient histamine binding. The binding site of antagonists is best defined as resulting from a strong ionic bond to Asp107, an orthogonal interaction between one of the aromatic rings with Phe199, and probably a hydrophobic interaction between the second aromatic ring and the lipophilic amino acids of the upper part of TMIV and TMV. This is consistent with structure-activity data of most described antagonists.

  17. Spectrum of Bmp5 mutations from germline mutagenesis experiments in mice

    SciTech Connect

    Marker, P.C.; Kwonjune Seung; Bland, A.E.

    1997-02-01

    Over 40 years of mutagenesis experiments using the mouse specific-locus test have produced a large number of induced germline mutations at seven loci, among them the short ear locus. We have previously shown that the short ear locus encodes bone morphogenetic protein 5 (BMP5), a member of a large family of secreted signaling molecules that play key roles in axis formation, tissue differentiation, mesenchymal-epithelial interactions, and skeletal development. Here we examine 24 chemical- and radiation-induced mutations at the short ear locus. Sequence changes in the Bmp5 open reading frame confirm the importance of cysteine residues in the function of TGF{beta} superfamily members. The spectrum of N-ethyl-N-nitrosourea-induced mutations also provides new information about the basepair, sequence context, and strand specificity of germline mutations in mammals. 52 refs., 3 figs., 2 tabs.

  18. Site-specific mutagenesis of the calcium-binding photoprotein aequorin

    PubMed Central

    Tsuji, Frederick I.; Inouye, Satoshi; Goto, Toshio; Sakaki, Yoshiyuki

    1986-01-01

    The luminescent protein aequorin from the jellyfish Aequoria victoria emits light by an intramolecular reaction in the presence of a trace amount of Ca2+. In order to understand the mechanism of the reaction, a study of structure-function relationships was undertaken with respect to modifying certain of its amino acid residues. This was done by carrying out oligonucleotide-directed site-specific mutagenesis of apoaequorin cDNA and expressing the mutagenized cDNA in Escherichia coli. Amino acid substitutions were made at the three Ca2+-binding sites, the three cysteines, and a histidine in one of the hydrophobic regions. Subsequent assay of the modified aequorin showed that the Ca2+-binding sites, the cysteines, and probably the histidine all play a role in the bioluminescence reaction of aequorin. PMID:16593774

  19. Mutagenesis as a Tool in Plant Genetics, Functional Genomics, and Breeding

    PubMed Central

    Sikora, Per; Chawade, Aakash; Larsson, Mikael; Olsson, Johanna; Olsson, Olof

    2011-01-01

    Plant mutagenesis is rapidly coming of age in the aftermath of recent developments in high-resolution molecular and biochemical techniques. By combining the high variation of mutagenised populations with novel screening methods, traits that are almost impossible to identify by conventional breeding are now being developed and characterised at the molecular level. This paper provides a comprehensive overview of the various techniques and workflows available to researchers today in the field of molecular breeding, and how these tools complement the ones already used in traditional breeding. Both genetic (Targeting Induced Local Lesions in Genomes; TILLING) and phenotypic screens are evaluated. Finally, different ways of bridging the gap between genotype and phenotype are discussed. PMID:22315587

  20. Arenaviruses and lethal mutagenesis. Prospects for new ribavirin-based interventions.

    PubMed

    Moreno, Héctor; Grande-Pérez, Ana; Domingo, Esteban; Martín, Verónica

    2012-11-06

    Lymphocytic choriomeningitis virus (LCMV) has contributed to unveil some of the molecular mechanisms of lethal mutagenesis, or loss of virus infectivity due to increased mutation rates. Here we review these developments, and provide additional evidence that ribavirin displays a dual mutagenic and inhibitory activity on LCMV that can be relevant to treatment designs. Using 5-fluorouracil as mutagenic agent and ribavirin either as inhibitor or mutagen, we document an advantage of a sequential inhibitor-mutagen administration over the corresponding combination treatment to achieve a low LCMV load in cell culture. This advantage is accentuated in the concentration range in which ribavirin acts mainly as an inhibitor, rather than as mutagen. This observation reinforces previous theoretical and experimental studies in supporting a sequential inhibitor-mutagen administration as a possible antiviral design. Given recent progress in the development of new inhibitors of arenavirus replication, our results suggest new options of ribavirin-based anti-arenavirus treatments.

  1. Molecular basis of transcriptional fidelity and DNA lesion-induced transcriptional mutagenesis

    PubMed Central

    Xu, Liang; Da, Lintai; Plouffe, Steven W.; Chong, Jenny; Kool, Eric; Wang, Dong

    2014-01-01

    Maintaining high transcriptional fidelity is essential for life. Some DNA lesions lead to significant changes in transcriptional fidelity. In this review, we will summarize recent progress towards understanding the molecular basis of RNA polymerase II (Pol II) transcriptional fidelity and DNA lesion-induced transcriptional mutagenesis. In particular, we will focus on the three key checkpoint steps of controlling Pol II transcriptional fidelity: insertion (specific nucleotide selection and incorporation), extension (differentiation of RNA transcript extension of a matched over mismatched 3'-RNA terminus), and proofreading (preferential removal of misincorporated nucleotides from the 3'-RNA end). We will also discuss some novel insights into the molecular basis and chemical perspectives of controlling Pol II transcriptional fidelity through structural, computational, and chemical biology approaches. PMID:24767259

  2. Molecular basis of transcriptional fidelity and DNA lesion-induced transcriptional mutagenesis.

    PubMed

    Xu, Liang; Da, Linati; Plouffe, Steven W; Chong, Jenny; Kool, Eric; Wang, Dong

    2014-07-01

    Maintaining high transcriptional fidelity is essential for life. Some DNA lesions lead to significant changes in transcriptional fidelity. In this review, we will summarize recent progress towards understanding the molecular basis of RNA polymerase II (Pol II) transcriptional fidelity and DNA lesion-induced transcriptional mutagenesis. In particular, we will focus on the three key checkpoint steps of controlling Pol II transcriptional fidelity: insertion (specific nucleotide selection and incorporation), extension (differentiation of RNA transcript extension of a matched over mismatched 3'-RNA terminus), and proofreading (preferential removal of misincorporated nucleotides from the 3'-RNA end). We will also discuss some novel insights into the molecular basis and chemical perspectives of controlling Pol II transcriptional fidelity through structural, computational, and chemical biology approaches. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Agrobacterium-mediated transformation (AMT) of Trichoderma reesei as an efficient tool for random insertional mutagenesis.

    PubMed

    Zhong, Yao Hua; Wang, Xiao Li; Wang, Tian Hong; Jiang, Qiao

    2007-01-01

    Filamentous fungus Trichoderma reesei QM9414 was successfully transformed with Agrobacterium tumefaciens AGL-1 for random integration of transforming DNA (T-DNA). Co-cultivation of T. reesei conidia or protoplasts with A. tumefaciens in the presence of acetosyringone resulted in the formation of hygromycin B-resistant fungal colonies with high transformation frequency. Nine randomly selected resistant clones were proved to be stable through mitotic cell division. The integration of the hph gene into T. reesei genome was determined by PCR and dot blot analysis. Transgenic T. reesei strains were analyzed using TAIL-PCR for their T-DNA contents. The results showed that T-DNA inserts occurred evidently by fusing DNA at T-DNA borders via random recombination, which suggests that Agrobacterium-mediated transformation is a potentially powerful tool towards tagged mutagenesis and gene transfer technology for T. reesei.

  4. Evaluating risks of insertional mutagenesis by DNA transposons in gene therapy.

    PubMed

    Hackett, Perry B; Largaespada, David A; Switzer, Kirsten C; Cooper, Laurence J N

    2013-04-01

    Investigational therapy can be successfully undertaken using viral- and nonviral-mediated ex vivo gene transfer. Indeed, recent clinical trials have established the potential for genetically modified T cells to improve and restore health. Recently, the Sleeping Beauty (SB) transposon/transposase system has been applied in clinical trials to stably insert a chimeric antigen receptor (CAR) to redirect T-cell specificity. We discuss the context in which the SB system can be harnessed for gene therapy and describe the human application of SB-modified CAR(+) T cells. We have focused on theoretical issues relating to insertional mutagenesis in the context of human genomes that are naturally subjected to remobilization of transposons and the experimental evidence over the last decade of employing SB transposons for defining genes that induce cancer. These findings are put into the context of the use of SB transposons in the treatment of human disease.

  5. Excavating the Genome: Large Scale Mutagenesis Screening for the Discovery of New Mouse Models

    PubMed Central

    Sundberg, John P.; Dadras, Soheil S.; Silva, Kathleen A.; Kennedy, Victoria E.; Murray, Stephen A.; Denegre, James; Schofield, Paul N.; King, Lloyd E.; Wiles, Michael; Pratt, C. Herbert

    2016-01-01

    Technology now exists for rapid screening of mutated laboratory mice to identify phenotypes associated with specific genetic mutations. Large repositories exist for spontaneous mutants and those induced by chemical mutagenesis, many of which have never been studied or comprehensively evaluated. To supplement these resources, a variety of techniques have been consolidated in an international effort to create mutations in all known protein coding genes in the mouse. With targeted embryonic stem cell lines now available for almost all protein coding genes and more recently CRISPR/Cas9 technology, large-scale efforts are underway to create novel mutant mouse strains and to characterize their phenotypes. However, accurate diagnosis of skin, hair, and nail diseases still relies on careful gross and histological analysis. While not automated to the level of the physiological phenotyping, histopathology provides the most direct and accurate diagnosis and correlation with human diseases. As a result of these efforts, many new mouse dermatological disease models are being developed. PMID:26551941

  6. Oligonucleotide-directed mutagenesis as a general and powerful method for studies of protein function.

    PubMed Central

    Dalbadie-McFarland, G; Cohen, L W; Riggs, A D; Morin, C; Itakura, K; Richards, J H

    1982-01-01

    We have used oligonucleotide-directed mutagenesis to make a specific change in the beta-lactamase (EC 3.5.2.6) (ampicillin resistance) gene of the plasmid pBR322. Evidence suggests that the active site for this enzyme may include a serine-threonine dyad (residues 70 and 71). By priming in vitro DNA synthesis with a chemically synthesized 16-base oligodeoxyribonucleotide, we have inverted the Ser-Thr dyad to Thr-Ser and thereby generated a mutant with an ampicillin-sensitive phenotype. This "double-mismatch" method is relatively simple and also very general because detection of mutants is at the level of DNA and involves only colony hybridization. Accordingly, the procedure can be applied to any DNA sequence and does not depend on the phenotype of the mutant. Images PMID:6983070

  7. The hidden side of unstable DNA repeats: mutagenesis at a distance

    PubMed Central

    Shah, Kartik A.; Mirkin, Sergei M.

    2015-01-01

    Structure-prone DNA repeats are common components of genomic DNA in all kingdoms of life. In humans, these repeats are linked to genomic instabilities that result in various hereditary disorders, including many cancers. It has long been known that DNA repeats are not only highly polymorphic in length but can also cause chromosomal fragility and stimulate gross chromosomal rearrangements, i.e. deletions, duplications, inversions, translocations and more complex shuffles. More recently, it has become clear that inherently unstable DNA repeats dramatically elevate mutation rates in surrounding DNA segments and that these mutations can occur upto ten kilobases away from the repetitive tract, a phenomenon we call repeat-induced mutagenesis (RIM). This review describes experimental data that led to the discovery and characterization of RIM and discusses the molecular mechanisms that could account for this phenomenon. PMID:25956860

  8. Germ cell comparative Drosophila mutagenesis: sensitivity and mutation pattern in chemically treated stem cells

    SciTech Connect

    Abrahamson, S.; Houtchens, K.; Li Jia, X.; Foureman, P.

    1983-01-01

    Mutagenesis studies on Drosophila oogonial cells with methylnitrosourea, dimethylnitrosamine, and diethylnitrosamine revealed unexpectedly high rates of sex-linked recessive lethals relative to other male and female germ cell stages. Indeed, the oogonial mutation rates with chemicals are higher than with massive x-ray or neutron exposures of oogonia. Analysis of the distribution of lethals per treated female suggests most of the mutations recovered are of independent origin, with very small levels of clustering of identical mutations. In the male stem cell population (spermatogonia) on the other hand, the distribution of lethals is primarily nonrandom and highly clustered. The nature of the mutational endpoint and the different pattern of germ cell development in the two sexes are the probable causes of this difference. The oogonial sensitivity to chemical mutagens may have important bearing on strategies for assessing human hazard.

  9. The role of flexibility and molecular shape in the crystallization of proteins by surface mutagenesis

    PubMed Central

    Devedjiev, Yancho D.

    2015-01-01

    Proteins are dynamic systems and interact with their environment. The analysis of crystal contacts in the most accurately determined protein structures (d < 1.5 Å) reveals that in contrast to current views, static disorder and high side-chain entropy are common in the crystal contact area. These observations challenge the validity of the theory that presumes that the occurrence of well ordered patches of side chains at the surface is an essential prerequisite for a successful crystallization event. The present paper provides evidence in support of the approach for understanding protein crystallization as a process dependent on multiple factors, each with its relative contribution, rather than a phenomenon driven by a few dominant physicochemical characteristics. The role of the molecular shape as a factor in the crystallization of proteins by surface mutagenesis is discussed. PMID:25664789

  10. [Preparation of catalitaxol-deficient defective strains by using ultraviolet mutagenesis].

    PubMed

    Hu, Dan; Tang, Kexuan; Miao, Zhiqi

    2017-05-25

    Secondary metabolites of endophytic fungi FSN002 from Juglans mandshurica Maxim have excellent liver cancer resistance. Preparation of mutant strains is an important means to study the biosynthesis mechanism of catalitaxol. Fungal spores germinating young hyphae with 4 to 6 cells after culturing for 13 hours were used as starting materials of ultraviolet (UV) mutagenesis. UV light intensity and irradiation time have a linear relationship with fungal mortality. The two factors had no obvious interactions. When UV light was 90 000 μJ/cm2 and irradiation time for 6 s, the mortality of fungi was around 95%. Under the optimization mutation condition, two mutant strains were obtained, of which one lost the synthesis ability of catalitaxol completely, and the another synthetized only 16% catalitaxol of the wild strain. Our findings may serve basis for further study on the biosynthesis mechanism and efficient production of catalitaxol.

  11. Comparative mutagenesis of human cells in vitro and in vivo. Progress report and financial statement

    SciTech Connect

    Thilly, W.G.

    1983-01-01

    Our laboratory has undertaken the study of mutagenesis from two perspectives. First, we are devising methods to measure mutation in human beings and to recognize specific patterns of mutation indicative of exposures to particular environmental agents. Second, we are studying the basic question of how chemicals induce mutation and also their potential effects on gene regulation and expression. Specifically we report results which show the applicability of measuring mutation frequencies at small loci in a human lymphoblast cell line in the generation of mutational spectra. Our experiments in the area of gene regulation have revealed that 5-azacytidine can reverse the previously discovered effect of BUdR in inducing pseudomutation at the TK locus. In addition, we have developed an improvement in the performance of human lymphoblast mutation assays by devising means to measure plating efficiency at high cell densities.

  12. In vivo Elimination of Parental Clones in General and Site-directed Mutagenesis

    PubMed Central

    Holland, Erika G.; Acca, Felicity E.; Belanger, Kristina M.; Bylo, Mary E.; Kay, Brian K.; Weiner, Michael P.; Kiss, Margaret M.

    2015-01-01

    The Eco29k I restriction endonuclease is a Sac II isoschizomer that recognizes the sequence 5’-CCGCGG-3’ and is encoded, along with the Eco29k I methylase, in the Escherichia coli strain 29k. We have expressed the Eco29k I restriction-methylation system (RM2) in E. coli strain TG1 to produce the strain AXE688. We have developed a directed molecular evolution (DME) mutagenesis method that uses Eco29k I to restrict incoming parental DNA in transformed cells. Using our DME method, we have demonstrated that our AXE688 strain results in mutated directed molecular evolution libraries with diversity greater than 107 from a single transformation and with greater than 90% recombinant clones. PMID:25523926

  13. In vivo elimination of parental clones in general and site-directed mutagenesis.

    PubMed

    Holland, Erika G; Acca, Felicity E; Belanger, Kristina M; Bylo, Mary E; Kay, Brian K; Weiner, Michael P; Kiss, Margaret M

    2015-02-01

    The Eco29k I restriction endonuclease is a Sac II isoschizomer that recognizes the sequence 5'-CCGCGG-3' and is encoded, along with the Eco29k I methylase, in the Escherichia coli strain 29k. We have expressed the Eco29k I restriction-methylation system (RM2) in E. coli strain TG1 to produce the strain AXE688. We have developed a directed molecular evolution (DME) mutagenesis method that uses Eco29k I to restrict incoming parental DNA in transformed cells. Using our DME method, we have demonstrated that our AXE688 strain results in mutated directed molecular evolution libraries with diversity greater than 10(7) from a single transformation and with greater than 90% recombinant clones. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Chemical mutagenesis--a promising technique to increase metal concentration and extraction in sunflowers.

    PubMed

    Nehnevajova, Erika; Herzig, Rolf; Federer, Guido; Erismann, Karl-Hans; Schwitzguébel, Jean-Paul

    2007-01-01

    Since most of the metal-hyperaccumulating wild plants only produce very low biomass and many high-yielding crops accumulate only moderate amounts of metals, the current research is mainly focused on overcoming these limitations and the optimization of metal phytoextraction. The main goal of the present study was the improvement of metal concentration and extraction properties of Helianthus annuus L by chemical mutagenesis (the non-GMO approach). Sunflowers--hybrid cultivar Salut and inbred lines-were treated with the chemical mutagen ethyl methanesulfonate (EMS). The effect of chemical mutagenesis on metal concentration in and extraction by new sunflower M1 and M2 mutants was directly assessed on a metal-contaminated field in Raft, Switzerland. Mutants of the M2 generation showed a 2-3 times higher metal shoot concentration than the control plants. The best M2 sunflower "giant mutant" 14/185/04 showed a significantly enhanced metal extraction ability: 7.5 times for Cd, 9.2 times for Zn, and 8.2 times for Pb in aboveground parts, as compared to the control plants. Theoretical calculations for the phytoextraction potential of new sunflower variants note that the best sunflower mutant can produce up to 26 t dry matter per hectare and remove 13.3 kg Zn per hectare and year at the sewage sludge contaminated site of Raft; that is a gain factor of 9 compared to Zn extraction by sunflower controls. Furthermore, the use of sunflower oil and biomass for technical purposes (lubricants, biodiesel, biogas) should produce an additional value and improve the economical balance of phytoextraction.

  15. Improving the chitinolytic activity of Bacillus pumilus SG2 by random mutagenesis.

    PubMed

    Vahed, Majid; Motalebi, Ebrahim; Rigi, Garshasb; Akbari Noghabi, Kambiz; Soudi, Mohammad Reza; Sadeghi, Mehdi; Ahmadian, Gholamreza

    2013-11-28

    Bacillus pumilus SG2, a halotolerant strain, expresses two major chitinases designated ChiS and ChiL that were induced by chitin and secreted into the supernatant. The present work aimed to obtain a mutant with higher chitinolytic activity through mutagenesis of Bacillus pumilus SG2 using a combination of UV irradiation and nitrous acid treatment. Following mutagenesis and screening on chitin agar and subsequent formation of halos, the mutated strains were examined for degradation of chitin under different conditions. A mutant designated AV2-9 was selected owing to its higher chitinase activity. To search for possible mutations in the whole operon including ChiS and ChiL, the entire chitinase operon, including the intergenic region, promoter, and two areas corresponding to the ChiS and ChiL ORF, was suquenced. Nucleotide sequence analysis of the complete chitinase operon from the SG2 and AV2-9 strains showed the presence of a mutation in the catalytic domain (GH18) of chitinase (ChiL). The results demonstrated that a single base change had occurred in the ChiL sequence in AV2- 9. The wild-type chitinase, ChiL, and the mutant (designated ChiLm) were cloned, expressed, and purified in E. coli. Both enzymes showed similar profiles of activity at different ranges of pH, NaCl concentration, and temperature, but the mutant enzyme showed approximately 30% higher catalytic activity under all the conditions tested. The results obtained in this study showed that the thermal stability of chitinase increased in the mutant strain. Bioinformatics analysis was performed to predict changes in the stability of proteins caused by mutation.

  16. Therapeutic genome mutagenesis using synthetic donor DNA and triplex-forming molecules.

    PubMed

    Reza, Faisal; Glazer, Peter M

    2015-01-01

    Genome mutagenesis can be achieved in a variety of ways, though a select few are suitable for therapeutic settings. Among them, the harnessing of intracellular homologous recombination affords the safety and efficacy profile suitable for such settings. Recombinagenic donor DNA and mutagenic triplex-forming molecules co-opt this natural recombination phenomenon to enable the specific, heritable editing and targeting of the genome. Editing the genome is achieved by designing the sequence-specific recombinagenic donor DNA to have base mismatches, insertions, and deletions that will be incorporated into the genome when it is used as a template for recombination. Targeting the genome is similarly achieved by designing the sequence-specific mutagenic triplex-forming molecules to further recruit the recombination machinery thereby upregulating its activity with the recombinagenic donor DNA. This combination of extracellularly introduced, designed synthetic molecules and intercellularly ubiquitous, evolved natural machinery enables the mutagenesis of chromosomes and engineering of whole genomes with great fidelity while limiting nonspecific interactions. Herein, we demonstrate the harnessing of recombinagenic donor DNA and mutagenic triplex-forming molecular technology for potential therapeutic applications. These demonstrations involve, among others, utilizing this technology to correct genes so that they become physiologically functional, to induce dormant yet functional genes in place of non-functional counterparts, to place induced genes under regulatory elements, and to disrupt genes to abrogate a cellular vulnerability. Ancillary demonstrations of the design and synthesis of this recombinagenic and mutagenic molecular technology as well as their delivery and assayed interaction with duplex DNA reveal a potent technological platform for engineering specific changes into the living genome.

  17. Transposon Mutagenesis of Mycobacterium marinum Identifies a Locus Linking Pigmentation and Intracellular Survival

    PubMed Central

    Gao, Lian-Yong; Groger, Richard; Cox, Jeffery S.; Beverley, Stephen M.; Lawson, Elise H.; Brown, Eric J.

    2003-01-01

    Pathogenic mycobacteria survive and replicate within host macrophages, but the molecular mechanisms involved in this necessary step in the pathogenesis of infection are not completely understood. Mycobacterium marinum has recently been used as a model for aspects of the pathogenesis of tuberculosis because of its close genetic relationship to Mycobacterium tuberculosis and because of similarities in the pathology and course of infection caused by this organism in its natural hosts, fish and frogs, with tuberculosis in humans. In order to advance the utility of the M. marinum model, we have developed efficient transposon mutagenesis of the organism by using a Drosophila melanogaster mariner-based transposon. To determine the efficiency of transposition, we have analyzed pigmentation mutants from the transposon mutant library. In addition to insertions in four known genes in the pathway of pigment biosynthesis, two insertions in novel genes were identified in our mutant library. One of these is in a putative inhibitor of the carotenoid biosynthesis pathway. The second unexpected insertion is in an intergenic region between two genes homologous to Rv2603c and Rv2604c of M. tuberculosis. In addition to a pigmentation defect, this mutant showed increased susceptibility to singlet oxygen and grew poorly in murine macrophages. Complementation with M. tuberculosis genomic DNA encompassing Rv2603c to Rv2606c corrected the pigmentation and growth defects of the mutant. These data demonstrate the utility of mariner-based transposon mutagenesis of M. marinum and that M. marinum can be used to study the function of M. tuberculosis genes involved in intracellular survival and replication. PMID:12540574

  18. Development of Therapeutic Chimeric Uricase by Exon Replacement/Restoration and Site-Directed Mutagenesis

    PubMed Central

    Xie, Guangrong; Yang, Weizhen; Chen, Jing; Li, Miaomiao; Jiang, Nan; Zhao, Baixue; Chen, Si; Wang, Min; Chen, Jianhua

    2016-01-01

    The activity of urate oxidase was lost during hominoid evolution, resulting in high susceptibility to hyperuricemia and gout in humans. In order to develop a more “human-like” uricase for therapeutic use, exon replacement/restoration and site-directed mutagenesis were performed to obtain porcine–human uricase with higher homology to deduced human uricase (dHU) and increased uricolytic activity. In an exon replacement study, substitution of exon 6 in wild porcine uricase (wPU) gene with corresponding exon in dhu totally abolished its activity. Substitutions of exon 5, 3, and 1–2 led to 85%, 60%, and 45% loss of activity, respectively. However, replacement of exon 4 and 7–8 did not significantly change the enzyme activity. When exon 5, 6, and 3 in dhu were replaced by their counterparts in wpu, the resulting chimera H1-2P3H4P5-6H7-8 was active, but only about 28% of wPU. Multiple sequence alignment and homology modeling predicted that mutations of E24D and E83G in H1-2P3H4P5-6H7-8 were favorable for further increase of its activity. After site-directed mutagenesis, H1-2P3H4P5-6H7-8 (E24D & E83G) with increased homology (91.45%) with dHU and higher activity and catalytic efficiency than the FDA-approved porcine–baboon chimera (PBC) was obtained. It showed optimum activity at pH 8.5 and 35 °C and was stable in a pH range of 6.5–11.0 and temperature range of 20–40 °C. PMID:27213357

  19. Evaluation of conformational epitopes on thyroid peroxidase by antipeptide antibody binding and mutagenesis

    PubMed Central

    GORA, M; GARDAS, A; WIKTOROWICZ, W; HOBBY, P; WATSON, P F; WEETMAN, A P; SUTTON, B J; BANGA, J P

    2004-01-01

    Autoantibodies to thyroid peroxidase (TPO) recognize predominantly conformational epitopes, which are restricted to two distinct determinants, termed immunodominant domain region (IDR) A and B. These dominant determinants reside in the region with structural homology to myeloperoxidase (MPO)-like domain and may extend into the adjacent complement control protein (CCP) domain. We have explored the location of these determinants on the MPO-like domain of the structural model of TPO, by identifying exposed hydrophilic loops that are potential candidates for the autoantigenic sites, generating rabbit antipeptide antisera, and competing with well characterized murine monoclonal antibodies (mabs) specific for these two IDRs. We recently defined the location of IDR-B, and here report our findings on the location of IDR-A and its relationship to IDR-B, defined with a new panel of 15 antipeptide antisera. Moreover, in combination with single amino acid replacements by in vitro mutagenesis, we have defined the limits of the IDR-B region on the TPO model. The combination of antisera to peptides P12 (aa 549–563), P14 (aa 599–617) and P18 (aa 210–225) inhibited the binding of the mab specific for IDR-A (mab 2) by 75. The same combination inhibited the binding of autoantibodies to native TPO from 67 to 94% (mean 81·5%) at autoantibody levels of 5 IU. Fabs prepared from the antipeptide IgG and pooled in this combination were also effective in competition assays, thus defining the epitopes more precisely. IDR-A was found to lie immediately adjacent to IDR-B and thus the two immunodominant epitopes form an extended patch on the surface of TPO. Finally, by single amino acid mutagenesis, we show that IDR-B extends to residue N642, thus further localizing the boundary of this autoantigenic region on the structural model. PMID:15030525

  20. Trapping Cardiac Recessive Mutants via Expression-based Insertional Mutagenesis Screening

    PubMed Central

    Ding, Yonghe; Liu, Weibin; Deng, Yun; Jomok, Beninio; Yang, Jingchun; Huang, Wei; Clark, Karl J.; Zhong, Tao P.; Lin, Xueying; Ekker, Stephen C.; Xu, Xiaolei

    2013-01-01

    Rationale Mutagenesis screening is a powerful genetic tool for probing biological mechanisms underlying vertebrate development and human diseases. However, the increased colony management efforts in vertebrates impose a significant challenge for identifying genes affecting a particular organ such as the heart, especially those exhibiting adult phenotypes upon depletion. Objective We aim to develop a facile approach that streamlines colony management efforts via enriching cardiac mutants, which enables us to screen for adult phenotypes. Methods and Results The transparency of the zebrafish embryos enabled us to score 67 stable transgenic lines generated from an insertional mutagenesis screen using a transposon-based protein trapping vector. Fifteen lines with cardiac monomeric red fluorescent protein (mRFP) reporter expression were identified. We defined the molecular nature for 10 lines and bred them to homozygosity, which led to the identification of one embryonic lethal, one larval lethal, and one adult recessive mutant exhibiting cardiac hypertrophy at one year of age. Further characterization of these mutants uncovered an essential function of methionine adenosyltransferase II, alpha a (mat2aa) in cardiogenesis, an essential function of mitochondrial ribosomal protein S18B (mrps18b) in cardiac mitochondrial homeostasis, as well as a function of DnaJ (Hsp40) homolog, subfamily B, member 6b (dnajb6b) in adult cardiac hypertrophy. Conclusions We demonstrate that transposon-based gene trapping is an efficient approach for identifying both embryonic and adult recessive mutants with cardiac expression. The generation of a Zebrafish Insertional Cardiac (ZIC) mutant collection shall facilitate the annotation of a vertebrate cardiac genome, as well as enable heart-based adult screens. PMID:23283723

  1. DNA-Reactive Protein Monoepoxides Induce Cell Death and Mutagenesis in Mammalian Cells

    PubMed Central

    Tretyakova, Natalia Y.; Michaelson-Richie, Erin D.; Gherezghiher, Teshome B.; Kurtz, Jamie; Ming, Xun; Wickramaratne, Susith; Campion, Melissa; Kanugula, Sreenivas; Pegg, Anthony E.; Campbell, Colin

    2013-01-01

    Although cytotoxic alkylating agents possessing two electrophilic reactive groups are thought to act by cross-linking cellular biomolecules, their exact mechanisms of action have not been established. In cells, these compounds form a mixture of DNA lesions including nucleobase monoadducts, interstrand and intrastrand cross-links, and DNA-protein cross-links (DPCs). Interstrand DNA-DNA cross-links block replication and transcription by preventing DNA strand separation, contributing to toxicity and mutagenesis. In contrast, potential contributions of drug-induced DPCs are poorly understood. To gain insight into the biological consequences of DPC formation, we generated DNA-reactive protein reagents and examined their toxicity and mutagenesis in mammalian cells. Recombinant human O6-alkylguanine DNA alkyltransferase (AGT) protein or its variants (C145A and K125L) were treated with 1,2,3,4-diepoxybutane to yield proteins containing 2-hydroxy-3,4-epoxybutyl groups on cysteine residues. Gel shift and mass spectrometry experiments confirmed that epoxide-functionalized AGT proteins formed covalent DPC but no other types of nucleobase damage when incubated with duplex DNA. Introduction of purified AGT monoepoxides into mammalian cells via electroporation generated AGT-DNA cross-links and induced cell death and mutations at the hypoxanthine-guanine phosphoribosyltransferase gene. Lower numbers of DPC lesions and reduced levels of cell death were observed when using protein monoepoxides generated from an AGT variant that fails to accumulate in the cell nucleus (K125L), suggesting that nuclear DNA damage is required for toxicity. Taken together, these results indicate that AGT protein monoepoxides produce cytotoxic and mutagenic DPC lesions within chromosomal DNA. More generally, these data suggest that covalent DPC lesions contribute to the cytotoxic and mutagenic effects of bis-electrophiles. PMID:23566219

  2. Mutagenesis of bacteriophage IKe major coat protein transmembrane domain: role of an interfacial proline residue.

    PubMed

    Williams, K A; Deber, C M

    1993-10-15

    The transmembrane (TM) domain of the 53-residue major coat protein of the M13-related bacteriophage IKe (residues 24-42: LISQTWPVVTTVVVAGVLI) has been subjected to randomized mutagenesis to probe the conformation and stability of the TM domain, as well as the effect of structurally-important residues such as proline. TM mutants were obtained by the Eckstein method of site-directed mutagenesis using the IKe genome as template so as to eliminate the need for subcloning. Over 40 single- and double-site viable mutants of bacteriophage IKe were isolated. Every residue in the TM segment, except the highly conserved Trp29, could be mutated to at least one other residue; polar and charged mutations occurred in the TM segment adjacent to the N-terminal domain (residues 24-28), while non-polar substitutions predominated in the C-terminal portion (residues 30-42). The Pro30 locus tolerated four mutations-Ala, Gly, Cys, and Ser- which represent the four side chains of least volume. Mutant coat proteins obtained directly from the phage in milligram quantities were studied by circular dichroism spectroscopy and SDS-PAGE gels. Wild type IKe coat protein solubilized in sodium deoxycholate micelles was found to occur as an alpha-helical, monomeric species which is stable at 95 degrees C, whereas the mutant Pro30-->Gly undergoes an irreversible conformational transition at ca. 90 degrees C to an aggregated beta-sheet structure. The result that Pro30 stabilizes the TM helix in the micellar membrane suggests a sterically-restricted location for the wild type Pro pyrrolidine side chain in the bulky Trp-Pro-Val triad, where it may be positioned to direct the initiation of the subsequent TM core domain helix.

  3. High Throughput Random Mutagenesis and Single Molecule Real Time Sequencing of the Muscle Nicotinic Acetylcholine Receptor

    PubMed Central

    Groot-Kormelink, Paul J.; Ferrand, Sandrine; Kelley, Nicholas; Bill, Anke; Freuler, Felix; Imbert, Pierre-Eloi; Marelli, Anthony; Gerwin, Nicole; Sivilotti, Lucia G.; Miraglia, Loren; Orth, Anthony P.; Oakeley, Edward J.; Schopfer, Ulrich; Siehler, Sandra

    2016-01-01

    High throughput random mutagenesis is a powerful tool to identify which residues are important for the function of a protein, and gain insight into its structure-function relation. The human muscle nicotinic acetylcholine receptor was used to test whether this technique previously used for monomeric receptors can be applied to a pentameric ligand-gated ion channel. A mutant library for the α1 subunit of the channel was generated by error-prone PCR, and full length sequences of all 2816 mutants were retrieved using single molecule real time sequencing. Each α1 mutant was co-transfected with wildtype β1, δ, and ε subunits, and the channel function characterized by an ion flux assay. To test whether the strategy could map the structure-function relation of this receptor, we attempted to identify mutations that conferred resistance to competitive antagonists. Mutant hits were defined as receptors that responded to the nicotinic agonist epibatidine, but were not inhibited by either α-bungarotoxin or tubocurarine. Eight α1 subunit mutant hits were identified, six of which contained mutations at position Y233 or V275 in the transmembrane domain. Three single point mutations (Y233N, Y233H, and V275M) were studied further, and found to enhance the potencies of five channel agonists tested. This suggests that the mutations made the channel resistant to the antagonists, not by impairing antagonist binding, but rather by producing a gain-of-function phenotype, e.g. increased agonist sensitivity. Our data show that random high throughput mutagenesis is applicable to multimeric proteins to discover novel functional mutants, and outlines the benefits of using single molecule real time sequencing with regards to quality control of the mutant library as well as downstream mutant data interpretation. PMID:27649498

  4. Gene transfer and mutagenesis mediated by Sleeping Beauty transposon in Nile tilapia (Oreochromis niloticus).

    PubMed

    He, Xiaozhen; Li, Jie; Long, Yong; Song, Guili; Zhou, Peiyong; Liu, Qiuxiang; Zhu, Zuoyan; Cui, Zongbin

    2013-10-01

    The success of gene transfer has been demonstrated in many of vertebrate species, whereas the efficiency of producing transgenic animals remains pretty low due to the random integration of foreign genes into a recipient genome. The Sleeping Beauty (SB) transposon is able to improve the efficiency of gene transfer in zebrafish and mouse, but its activity in tilapia (Oreochromis niloticus) has yet to be characterized. Herein, we demonstrate the potential of using the SB transposon system as an effective tool for gene transfer and insertional mutagenesis in tilapia. A transgenic construct pT2/tiHsp70-SB11 was generated by subcloning the promoter of tilapia heat shock protein 70 (tiHsp70) gene, the SB11 transposase gene and the carp β-actin gene polyadenylation signal into the second generation of SB transposon. Transgenic tilapia was produced by microinjection of this construct with in vitro synthesized capped SB11 mRNA. SB11 transposon was detected in 28.89 % of founders, 12.9 % of F1 and 43.75 % of F2. Analysis of genomic sequences flanking integrated transposons indicates that this transgenic tilapia line carries two copies of SB transposon, which landed into two different endogenous genes. Induced expression of SB11 gene after heat shock was detected using reverse transcription PCR in F2 transgenic individuals. In addition, the Cre/loxP system was introduced to delete the SB11 cassette for stabilization of gene interruption and bio-safety. These findings suggest that the SB transposon system is active and can be used for efficient gene transfer and insertional mutagenesis in tilapia.

  5. 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.

  6. Iterative saturation mutagenesis: a powerful approach to engineer proteins by systematically simulating Darwinian evolution.

    PubMed

    Acevedo-Rocha, Carlos G; Hoebenreich, Sabrina; Reetz, Manfred T

    2014-01-01

    Iterative saturation mutagenesis (ISM) is a widely applicable and powerful strategy for the efficient directed evolution of enzymes. First, one or more amino acid positions from the chosen enzyme are assigned to multi-residue sites (i.e., groups of amino acids or "multisites"). Then, the residues in each multisite are mutated with a user-defined randomization scheme to all canonical amino acids or a reduced amino acid alphabet. Subsequently, the genes of chosen variants (usually the best but not necessarily) are used as templates for saturation mutagenesis at other multisites, and the process is repeated until the desired degree of biocatalyst improvement has been achieved. Addressing multisites iteratively results in a so-called ISM scheme or tree with various upward branches or pathways. The systematic character of ISM simulates in vitro the natural process of Darwinian evolution: variation (library creation), selection (library screening), and amplification (template chosen for the next round of randomization). However, the main feature of ISM that distinguishes it from other directed evolution methods is the systematic probing of a defined segment of the protein sequence space, as it has been shown that ISM is much more efficient in terms of biocatalyst optimization than random methods such as error-prone PCR. In addition, ISM trees have also shed light on the emergence of epistasis, thereby rationally improving the strategies for evolving better enzymes. ISM was developed to improve catalytic properties such as rate, substrate scope, stereo- and regioselectivity using the Combinatorial Active-site Saturation Test (CAST), as well as chemical and thermal stability employing the B-Factor Iterative Test (B-FIT). However, ISM can also be invoked to manipulate such protein properties as binding affinity among other possibilities, including protein-protein interactions. Herein, we provide general guidelines for ISM, using CAST as the case study in the quest to

  7. Site-Directed Mutagenesis to Improve Sensitivity of a Synthetic Two-Component Signaling System

    PubMed Central

    Kuldell, Natalie

    2016-01-01

    Two-component signaling (2CS) systems enable bacterial cells to respond to changes in their local environment, often using a membrane-bound sensor protein and a cytoplasmic responder protein to regulate gene expression. Previous work has shown that Escherichia coli’s natural EnvZ/OmpR 2CS could be modified to construct a light-sensing bacterial photography system. The resulting bacterial photographs, or “coliroids,” rely on a phosphotransfer reaction between Cph8, a synthetic version of EnvZ that senses red light, and OmpR. Gene expression changes can be visualized through upregulation of a LacZ reporter gene by phosphorylated OmpR. Unfortunately, basal LacZ expression leads to a detectable reporter signal even when cells are grown in the light, diminishing the contrast of the coliroids. We performed site-directed mutagenesis near the phosphotransfer site of Cph8 to isolate mutants with potentially improved image contrast. Five mutants were examined, but only one of the mutants, T541S, increased the ratio of dark/light gene expression, as measured by β-galactosidase activity. The ratio changed from 2.57 fold in the starting strain to 5.59 in the T541S mutant. The ratio decreased in the four other mutant strains we examined. The phenotype observed in the T541S mutant strain may arise because the serine sidechain is chemically similar but physically smaller than the threonine sidechain. This may minimally change the protein’s local structure, but may be less sterically constrained when compared to threonine, resulting in a higher probability of a phosphotransfer event. Our initial success pairing synthetic biology and site-directed mutagenesis to optimize the bacterial photography system’s performance encourages us to imagine further improvements to the performance of this and other synthetic systems, especially those based on 2CS signaling. PMID:26799494

  8. Genome-wide kinetics of DNA excision repair in relation to chromatin state and mutagenesis.

    PubMed

    Adar, Sheera; Hu, Jinchuan; Lieb, Jason D; Sancar, Aziz

    2016-04-12

    We recently developed a high-resolution genome-wide assay for mapping DNA excision repair named eXcision Repair-sequencing (XR-seq) and have now used XR-seq to determine which regions of the genome are subject to repair very soon after UV exposure and which regions are repaired later. Over a time course, we measured repair of the UV-induced damage of cyclobutane pyrimidine dimers (CPDs) (at 1, 4, 8, 16, 24, and 48 h) and (6-4)pyrimidine-pyrimidone photoproducts [(6-4)PPs] (at 5 and 20 min and 1, 2, and 4 h) in normal human skin fibroblasts. Each type of damage has distinct repair kinetics. The (6-4)PPs are detected as early as 5 min after UV treatment, with the bulk of repair completed by 4 h. Repair of CPDs, which we previously showed is intimately coupled to transcription, is slower and in certain regions persists even 2 d after UV irradiation. We compared our results to the Encyclopedia of DNA Elements data regarding histone modifications, chromatin state, and transcription. For both damage types, and for both transcription-coupled and general excision repair, the earliest repair occurred preferentially in active and open chromatin states. Conversely, repair in regions classified as "heterochromatic" and "repressed" was relatively low at early time points, with repair persisting into the late time points. Damage that remains during DNA replication increases the risk for mutagenesis. Indeed, late-repaired regions are associated with a higher level of cancer-linked mutations. In summary, we show that XR-seq is a powerful approach for studying relationships among chromatin state, DNA repair, genome stability, mutagenesis, and carcinogenesis.

  9. Engineering of LadA for enhanced hexadecane oxidation using random- and site-directed mutagenesis.

    PubMed

    Dong, Yanpeng; Yan, Jiang; Du, Huiqian; Chen, Miao; Ma, Ting; Feng, Lu

    2012-05-01

    LadA, a monooxygenase catalyzing the oxidation of n-alkanes to 1-alkanols, is the key enzyme for the degradation of long-chain alkanes (C(15)-C(36)) in Geobacillus thermodenitrificans NG80-2. In this study, random- and site-directed mutagenesis were performed to enhance the activity of the enzyme. By screening 7,500 clones from random-mutant libraries for enhanced hexadecane hydroxylation activity, three mutants were obtained: A102D, L320V, and F146C/N376I. By performing saturation site-directed mutagenesis at the 102, 320, 146, and 376 sites, six more mutants (A102E, L320A, F146Q/N376I, F146E/N376I, F146R/N376I, and F146N/N376I) were generated. Kinetic studies showed that the hydroxylation activity of purified LadA mutants on hexadecane was 2-3.4-fold higher than that of the wild-type enzyme, with the activity of F146N/N376I being the highest. Effects of the mutations on optimum temperature, pH, and heat stability of LadA were also investigated. A complementary study showed that Pseudomonas fluorescens KOB2Δ1 strains expressing the LadA mutants grew more rapidly with hexadecane than the strain expressing wild-type LadA, confirming the enhanced activity of LadA mutants in vivo. Structural changes resulting from the mutations were analyzed and the correlation between structural changes and enzyme activity was discussed. The mutants generated in this study are potentially useful for the treatment of environmental oil pollution and in other bioconversion processes.

  10. Transposon insertional mutagenesis in mice identifies human breast cancer susceptibility genes and signatures for stratification.

    PubMed

    Chen, Liming; Jenjaroenpun, Piroon; Pillai, Andrea Mun Ching; Ivshina, Anna V; Ow, Ghim Siong; Efthimios, Motakis; Zhiqun, Tang; Tan, Tuan Zea; Lee, Song-Choon; Rogers, Keith; Ward, Jerrold M; Mori, Seiichi; Adams, David J; Jenkins, Nancy A; Copeland, Neal G; Ban, Kenneth Hon-Kim; Kuznetsov, Vladimir A; Thiery, Jean Paul

    2017-03-14

    Robust prognostic gene signatures and therapeutic targets are difficult to derive from expression profiling because of the significant heterogeneity within breast cancer (BC) subtypes. Here, we performed forward genetic screening in mice using Sleeping Beauty transposon mutagenesis to identify candidate BC driver genes in an unbiased manner, using a stabilized N-terminal truncated β-catenin gene as a sensitizer. We identified 134 mouse susceptibility genes from 129 common insertion sites within 34 mammary tumors. Of these, 126 genes were orthologous to protein-coding genes in the human genome (hereafter, human BC susceptibility genes, hBCSGs), 70% of which are previously reported cancer-associated genes, and ∼16% are known BC suppressor genes. Network analysis revealed a gene hub consisting of E1A binding protein P300 (EP300), CD44 molecule (CD44), neurofibromin (NF1) and phosphatase and tensin homolog (PTEN), which are linked to a significant number of mutated hBCSGs. From our survival prediction analysis of the expression of human BC genes in 2,333 BC cases, we isolated a six-gene-pair classifier that stratifies BC patients with high confidence into prognostically distinct low-, moderate-, and high-risk subgroups. Furthermore, we proposed prognostic classifiers identifying three basal and three claudin-low tumor subgroups. Intriguingly, our hBCSGs are mostly unrelated to cell cycle/mitosis genes and are distinct from the prognostic signatures currently used for stratifying BC patients. Our findings illustrate the strength and validity of integrating functional mutagenesis screens in mice with human cancer transcriptomic data to identify highly prognostic BC subtyping biomarkers.

  11. Thymidine-induced mutations in mammalian cells: sequence specificity and implications for mutagenesis in vivo.

    PubMed Central

    Kresnak, M T; Davidson, R L

    1992-01-01

    Imbalances in the intracellular nucleotide precursor pools in mammalian cells can result in the induction of mutations during the DNA replication process. By using a shuttle vector system developed in our laboratory, we have analyzed the sequence specificity of mutations induced in mouse A9 cells by exposure of the cells to a high concentration of thymidine. The target for mutagenesis in these studies was the bacterial gpt gene stably integrated into the chromosomal DNA of the mouse cells. Previous studies in this laboratory had generated a large panel of xanthine guanine phosphoribosyl-transferase (EC 2.4.2.22)-negative mutant lines that possess single-base mutations within the gpt coding sequence. This study utilized four xanthine guanine phosphoribosyltransferase-negative mutant lines to assess the frequency of mutation induced by thymidine at guanine residues in four sequence contexts: the 5' and 3' guanine residues of a GG doublet, the middle guanine residue of a GGG triplet, and the 3' guanine residue of a GGGG quartet. The results of this study demonstrate that treatment of cultured cells with a high concentration of thymidine can result in G.C----A.T transition mutations that occur preferentially at the 3' guanine residue of a run of two or more adjacent guanines. Guanine residues flanked on their 3' side by other guanine residues are severalfold less mutable by thymidine than are guanine residues flanked on their 3' side by a different base. This study demonstrates a sequence-specific mode for thymidine-induced mutations and suggests implications for mutagenesis in vivo. PMID:1557389

  12. Efficient targeted mutagenesis of the chordate Ciona intestinalis genome with zinc-finger nucleases.

    PubMed

    Kawai, Narudo; Ochiai, Hiroshi; Sakuma, Tetsushi; Yamada, Lixy; Sawada, Hitoshi; Yamamoto, Takashi; Sasakura, Yasunori

    2012-06-01

    Zinc-finger nucleases (ZFNs) are engineered nucleases that induce DNA double-strand breaks (DSBs) at target sequences. They have been used as tools for generating targeted mutations in the genomes of multiple organisms in both animals and plants. The DSB induced by ZFNs is repaired by non-homologous end joining (NHEJ) or by homologous recombination (HR) mechanisms. Non-homologous end joining induces some errors because it is independent of a reference DNA sequence. Through the NHEJ mechanism, ZFNs generate insertional or deletional mutations at the target sequence. We examined the usability, specificity and toxicity of ZFNs in the basal chordate Ciona intestinalis. As the target of ZFNs, we chose an enhanced green fluorescent protein (EGFP) gene artificially inserted in the C. intestinalis genome because this locus is neutral for the development and growth of C. intestinalis, and the efficiency of mutagenesis with ZFNs can thus be determined without any bias. We introduced EGFP -ZFN mRNAs into the embryos of an EGFP -transgenic line and observed the mutation frequency in the target site of EGFP . We also examined the effects of the EGFP -ZFNs at off-target sites resembling the EGFP target sequence in the C. intestinalis genome in order to examine the specificity of ZFNs. We further investigated the influence of ZFNs on embryogenesis, and showed that adequate amounts of ZFNs, which do not disrupt embryogenesis, can efficiently induce mutations on the on-target site with less effect on the off-target sites. This suggests that target mutagenesis with ZFNs will be a powerful technique in C. intestinalis.

  13. Development of Therapeutic Chimeric Uricase by Exon Replacement/Restoration and Site-Directed Mutagenesis.

    PubMed

    Xie, Guangrong; Yang, Weizhen; Chen, Jing; Li, Miaomiao; Jiang, Nan; Zhao, Baixue; Chen, Si; Wang, Min; Chen, Jianhua

    2016-05-20

    The activity of urate oxidase was lost during hominoid evolution, resulting in high susceptibility to hyperuricemia and gout in humans. In order to develop a more "human-like" uricase for therapeutic use, exon replacement/restoration and site-directed mutagenesis were performed to obtain porcine-human uricase with higher homology to deduced human uricase (dHU) and increased uricolytic activity. In an exon replacement study, substitution of exon 6 in wild porcine uricase (wPU) gene with corresponding exon in dhu totally abolished its activity. Substitutions of exon 5, 3, and 1-2 led to 85%, 60%, and 45% loss of activity, respectively. However, replacement of exon 4 and 7-8 did not significantly change the enzyme activity. When exon 5, 6, and 3 in dhu were replaced by their counterparts in wpu, the resulting chimera H1-2P₃H₄P5-6H7-8 was active, but only about 28% of wPU. Multiple sequence alignment and homology modeling predicted that mutations of E24D and E83G in H1-2P₃H₄P5-6H7-8 were favorable for further increase of its activity. After site-directed mutagenesis, H1-2P₃H₄P5-6H7-8 (E24D & E83G) with increased homology (91.45%) with dHU and higher activity and catalytic efficiency than the FDA-approved porcine-baboon chimera (PBC) was obtained. It showed optimum activity at pH 8.5 and 35 °C and was stable in a pH range of 6.5-11.0 and temperature range of 20-40 °C.

  14. 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

  15. Efficient targeted mutagenesis in medaka using custom-designed transcription activator-like effector nucleases.

    PubMed

    Ansai, Satoshi; Sakuma, Tetsushi; Yamamoto, Takashi; Ariga, Hiroyoshi; Uemura, Norihito; Takahashi, Ryosuke; Kinoshita, Masato

    2013-03-01

    Transcription activator-like effector nucleases (TALENs) have become powerful tools for targeted genome editing. Here we demonstrate efficient targeted mutagenesis in medaka (Oryzias latipes), which serves as an excellent vertebrate model for genetics and genomics. We designed and constructed a pair of TALENs targeting the medaka DJ-1 gene, a homolog of human DJ-1 (PARK7). These TALENs induced a number of insertions and deletions in the injected embryos with extremely high efficiency. This induction of mutations occurred in a dose-dependent manner. All screened G0 fish injected with the TALENs transmitted the TALEN-induced mutations to the next generation with high efficiency (44-100%). We also confirmed that these TALENs induced site-specific mutations because none of the mutations were found at potential off-target sites. In addition, the DJ-1 protein was lost in DJ-1(Δ7/Δ7) fish that carried a TALEN-induced frameshift mutation in both alleles. We also investigated the effect of the N- and C-terminal regions of the transcription activator-like (TAL) effector domain on the gene-disrupting activity of DJ1-TALENs and found that 287 amino acids at the N terminus and 63 amino acids at the C terminus of the TAL domain exhibited the highest disrupting activity in the injected embryos. Our results suggest that TALENs enable us to rapidly and efficiently establish knockout medaka strains. This is the first report of targeted mutagenesis in medaka using TALENs. The TALEN technology will expand the potential of medaka as a model system for genetics and genomics.

  16. Protective effect of basil (Ocimum basilicum L.) against oxidative DNA damage and mutagenesis.

    PubMed

    Berić, Tanja; Nikolić, Biljana; Stanojević, Jasna; Vuković-Gacić, Branka; Knezević-Vukcević, Jelena

    2008-02-01

    Mutagenic and antimutagenic properties of essential oil (EO) of basil and its major constituent Linalool, reported to possess antioxidative properties, were examined in microbial tests. In Salmonella/microsome and Escherichia. coli WP2 reversion assays both derivatives (0.25-2.0 microl/plate) showed no mutagenic effect. Salmonella. typhimurium TA98, TA100 and TA102 strains displayed similar sensitivity to both basil derivatives as non-permeable E. coli WP2 strains IC185 and IC202 oxyR. Moreover, the toxicity of basil derivatives to WP2 strains did not depend on OxyR function. The reduction of t-BOOH-induced mutagenesis by EO and Linalool (30-60%) was obtained in repair proficient strains of the E. coli K12 assay (Nikolić, B., Stanojević, J., Mitić, D., Vuković-Gacić, B., Knezević-Vukcević, J., Simić, D., 2004. Comparative study of the antimutagenic potential of vitamin E in different E. coli strains. Mutat. Res. 564, 31-38), as well as in E. coli WP2 IC202 strain. EO and Linalool reduced spontaneous mutagenesis in mismatch repair deficient E. coli K12 strains (27-44%). In all tests, antimutagenic effect of basil derivatives was comparable with that obtained with model antioxidant vitamin E. Linalool and vitamin E induced DNA strand breaks in Comet assay on S. cerevisiae 3A cells, but at non-genotoxic concentrations (0.075 and 0.025 microg/ml, respectively) they reduced the number of H(2)O(2)-induced comets (45-70% Linalool and 80-93% vitamin E). Obtained results indicate that antigenotoxic potential of basil derivatives could be attributed to their antioxidative properties.

  17. Predicting Resistance by Mutagenesis: Lessons from 45 Years of MBC Resistance

    PubMed Central

    Hawkins, Nichola J.; Fraaije, Bart A.

    2016-01-01

    When a new fungicide class is introduced, it is useful to anticipate the resistance risk in advance, attempting to predict both risk level and potential mechanisms. One tool for the prediction of resistance risk is laboratory selection for resistance, with the mutational supply increased through UV or chemical mutagenesis. This enables resistance to emerge more rapidly than in the field, but may produce mutations that would not emerge under field conditions. The methyl benzimidazole carbamates (MBCs) were the first systemic single-site agricultural fungicides, and the first fungicides affected by rapid evolution of target-site resistance. MBC resistance has now been reported in over 90 plant pathogens in the field, and laboratory mutants have been studied in nearly 30 species. The most common field mutations, including β-tubulin E198A/K/G, F200Y and L240F, have all been identified in laboratory mutants. However, of 28 mutations identified in laboratory mutants, only nine have been reported in the field. Therefore, the predictive value of mutagenesis studies would be increased by understanding which mutations are likely to emerge in the field. Our review of the literature indicates that mutations with high resistance factors, and those found in multiple species, are more likely to be reported in the field. However, there are many exceptions, possibly due to fitness penalties. Whether a mutation occurred in the same species appears less relevant, perhaps because β-tubulin is highly conserved so functional constraints are similar across all species. Predictability of mutations in other target sites will depend on the level and conservation of constraints. PMID:27895632

  18. Saturation mutagenesis of putative catalytic residues of benzoylformate decarboxylase provides a challenge to the accepted mechanism

    PubMed Central

    Yep, Alejandra; Kenyon, George L.; McLeish, Michael J.

    2008-01-01

    Benzoylformate decarboxylase from Pseudomonas putida (PpBFDC) is a thiamin diphosphate-dependent enzyme that carries out the nonoxidative decarboxylation of aromatic 2-keto acids. The x-ray structure of PpBFDC suggested that Ser-26, His-70, and His-281 would play important roles in its catalytic mechanism, and the S26A, H70A, and H281A variants all exhibited greatly impaired catalytic activity. Based on stopped-flow studies with the alanine mutants, it was proposed that the histidine residues acted as acid-base catalysts, whereas Ser-26 was involved in substrate binding and played a significant, albeit less well defined, role in catalysis. While developing a saturation mutagenesis protocol to examine residues involved in PpBFDC substrate specificity, we tested the procedure on His-281. To our surprise, we found that His-281, which is thought to be necessary for protonation of the carbanion/enamine intermediate, could be replaced by phenyl alanine with only a 5-fold decrease in kcat. Even more surprising were our subsequent observations (i) that His-70 could be replaced by threonine or leucine with approximately a 30-fold decrease in kcat/Km compared with a 4,000-fold decrease for the H70A variant and (ii) that Ser-26, which forms hydrogen bonds with the substrate carboxylate, could be replaced by threonine, leucine, or methionine without significant loss of activity. These results call into question the assigned roles for Ser-26, His-70, and His-281. Further, they demonstrate the danger in assigning catalytic function based solely on results with alanine mutants and show that saturation mutagenesis is a valuable tool in assessing the role and relative importance of putative catalytic residues. PMID:18398009

  19. Efficient Targeted Mutagenesis in Medaka Using Custom-Designed Transcription Activator-Like Effector Nucleases

    PubMed Central

    Ansai, Satoshi; Sakuma, Tetsushi; Yamamoto, Takashi; Ariga, Hiroyoshi; Uemura, Norihito; Takahashi, Ryosuke; Kinoshita, Masato

    2013-01-01

    Transcription activator-like effector nucleases (TALENs) have become powerful tools for targeted genome editing. Here we demonstrate efficient targeted mutagenesis in medaka (Oryzias latipes), which serves as an excellent vertebrate model for genetics and genomics. We designed and constructed a pair of TALENs targeting the medaka DJ-1 gene, a homolog of human DJ-1 (PARK7). These TALENs induced a number of insertions and deletions in the injected embryos with extremely high efficiency. This induction of mutations occurred in a dose-dependent manner. All screened G0 fish injected with the TALENs transmitted the TALEN-induced mutations to the next generation with high efficiency (44–100%). We also confirmed that these TALENs induced site-specific mutations because none of the mutations were found at potential off-target sites. In addition, the DJ-1 protein was lost in DJ-1Δ7/Δ7 fish that carried a TALEN-induced frameshift mutation in both alleles. We also investigated the effect of the N- and C-terminal regions of the transcription activator-like (TAL) effector domain on the gene-disrupting activity of DJ1-TALENs and found that 287 amino acids at the N terminus and 63 amino acids at the C terminus of the TAL domain exhibited the highest disrupting activity in the injected embryos. Our results suggest that TALENs enable us to rapidly and efficiently establish knockout medaka strains. This is the first report of targeted mutagenesis in medaka using TALENs. The TALEN technology will expand the potential of medaka as a model system for genetics and genomics. PMID:23288935

  20. The Origin of Mutants Under Selection: How Natural Selection Mimics Mutagenesis (Adaptive Mutation)

    PubMed Central

    Maisnier-Patin, Sophie; Roth, John R.

    2015-01-01

    Selection detects mutants but does not cause mutations. Contrary to this dictum, Cairns and Foster plated a leaky lac mutant of Escherichia coli on lactose medium and saw revertant (Lac+) colonies accumulate with time above a nongrowing lawn. This result suggested that bacteria might mutagenize their own genome when growth is blocked. However, this conclusion is suspect in the light of recent evidence that revertant colonies are initiated by preexisting cells with multiple copies the conjugative F′lac plasmid, which carries the lac mutation. Some plated cells have multiple copies of the simple F′lac plasmid. This provides sufficient LacZ activity to support plasmid replication but not cell division. In nongrowing cells, repeated plasmid replication increases the likelihood of a reversion event. Reversion to lac+ triggers exponential cell growth leading to a stable Lac+ revertant colony. In 10% of these plated cells, the high-copy plasmid includes an internal tandem lac duplication, which provides even more LacZ activity—sufficient to support slow growth and formation of an unstable Lac+ colony. Cells with multiple copies of the F′lac plasmid have an increased mutation rate, because the plasmid encodes the error-prone (mutagenic) DNA polymerase, DinB. Without DinB, unstable and stable Lac+ revertant types form in equal numbers and both types arise with no mutagenesis. Amplification and selection are central to behavior of the Cairns–Foster system, whereas mutagenesis is a system-specific side effect or artifact caused by coamplification of dinB with lac. Study of this system has revealed several broadly applicable principles. In all populations, gene duplications are frequent stable genetic polymorphisms, common near-neutral mutant alleles can gain a positive phenotype when amplified under selection, and natural selection can operate without cell division when variability is generated by overreplication of local genome subregions. PMID:26134316

  1. Rolling Circle Mutagenesis of GST-mCherry to Understand Mutation, Gene Expression, and Regulation.

    PubMed

    Cole, Jessica; Ferguson, Amanda; Segarra, Verónica A; Walsh, Susan

    2017-01-01

    Undergraduates are often familiar with textbook examples of human mutations that affect coding regions and the subsequent disorders, but they may struggle with understanding the implications of mutations in the regulatory regions of genes. We have designed a laboratory sequence that will allow students to explore the effect random mutagenesis can have on protein function, expression, and ultimately phenotype. Students design and perform a safe and time-efficient random mutagenesis experiment using error-prone rolling circular amplification of a plasmid expressing the inducible fusion protein glutathione S-transferase (GST)-mCherry. Mutagenized and wild-type control plasmid DNA, respectively, are then purified and transformed into bacteria to assess phenotypic changes. While bacteria transformed with the wild type control should be pink, some bacterial colonies transformed with mutagenized plasmids will exhibit a different color. Students attempt to identify their mutations by isolating plasmid from these mutant colonies, sequencing, and comparing their mutant sequence to the wild-type sequence. Additionally, students evaluate the potential effects of mutations on protein production by inducing GST-mCherry expression in cultures, generating cell lysates, and analyzing them using SDS-PAGE. Students who have a phenotypic difference but do not obtain a coding region mutation will be able to think critically about plasmid structure and regulation outside of the gene sequence. Students who do not obtain bacterial transformants have the chance to contemplate how mutation of antibiotic resistance genes or replication origins may have contributed to their results. Overall, this series of laboratories exposes students to basic genetic techniques and helps them conceptualize mutation beyond coding regions.

  2. Gene Deletion by Fluorescence-Reported Allelic Exchange Mutagenesis in Chlamydia trachomatis

    PubMed Central

    Mueller, Konrad E.; Wolf, Katerina

    2016-01-01

    ABSTRACT Although progress in Chlamydia genetics has been rapid, genomic modification has previously been limited to point mutations and group II intron insertions which truncate protein products. The bacterium has thus far been intractable to gene deletion or more-complex genomic integrations such as allelic exchange. Herein, we present a novel suicide vector dependent on inducible expression of a chlamydial gene that renders Chlamydia trachomatis fully genetically tractable and permits rapid reverse genetics by fluorescence-reported allelic exchange mutagenesis (FRAEM). We describe the first available system of targeting chlamydial genes for deletion or allelic exchange as well as curing plasmids from C. trachomatis serovar L2. Furthermore, this approach permits the monitoring of mutagenesis by fluorescence microscopy without disturbing bacterial growth, a significant asset when manipulating obligate intracellular organisms. As proof of principle, trpA was successfully deleted and replaced with a sequence encoding both green fluorescent protein (GFP) and β-lactamase. The trpA-deficient strain was unable to grow in indole-containing medium, and this phenotype was reversed by complementation with trpA expressed in trans. To assess reproducibility at alternate sites, FRAEM was repeated for genes encoding type III secretion effectors CTL0063, CTL0064, and CTL0065. In all four cases, stable mutants were recovered one passage after the observation of transformants, and allelic exchange was limited to the specific target gene, as confirmed by whole-genome sequencing. Deleted sequences were not detected by quantitative real-time PCR (qPCR) from isogenic mutant populations. We demonstrate that utilization of the chlamydial suicide vector with FRAEM renders C. trachomatis highly amenable to versatile and efficient genetic manipulation. PMID:26787828

  3. BAX and tumor suppressor TRP53 are important in regulating mutagenesis in spermatogenic cells in mice.

    PubMed

    Xu, Guogang; Vogel, Kristine S; McMahan, C Alex; Herbert, Damon C; Walter, Christi A

    2010-12-01

    During the first wave of spermatogenesis, and in response to ionizing radiation, elevated mutant frequencies are reduced to a low level by unidentified mechanisms. Apoptosis is occurring in the same time frame that the mutant frequency declines. We examined the role of apoptosis in regulating mutant frequency during spermatogenesis. Apoptosis and mutant frequencies were determined in spermatogenic cells obtained from Bax-null or Trp53-null mice. The results showed that spermatogenic lineage apoptosis was markedly decreased in Bax-null mice and was accompanied by a significantly increased spontaneous mutant frequency in seminiferous tubule cells compared to that of wild-type mice. Apoptosis profiles in the seminiferous tubules for Trp53-null were similar to control mice. Spontaneous mutant frequencies in pachytene spermatocytes and in round spermatids from Trp53-null mice were not significantly different from those of wild-type mice. However, epididymal spermatozoa from Trp53-null mice displayed a greater spontaneous mutant frequency compared to that from wild-type mice. A greater proportion of spontaneous transversions and a greater proportion of insertions/deletions 15 days after ionizing radiation were observed in Trp53-null mice compared to wild-type mice. Base excision repair activity in mixed germ cell nuclear extracts prepared from Trp53-null mice was significantly lower than that for wild-type controls. These data indicate that BAX-mediated apoptosis plays a significant role in regulating spontaneous mutagenesis in seminiferous tubule cells obtained from neonatal mice, whereas tumor suppressor TRP53 plays a significant role in regulating spontaneous mutagenesis between postmeiotic round spermatid and epididymal spermatozoon stages of spermiogenesis.

  4. Transposon insertional mutagenesis in mice identifies human breast cancer susceptibility genes and signatures for stratification

    PubMed Central

    Chen, Liming; Jenjaroenpun, Piroon; Pillai, Andrea Mun Ching; Ivshina, Anna V.; Ow, Ghim Siong; Efthimios, Motakis; Zhiqun, Tang; Lee, Song-Choon; Rogers, Keith; Ward, Jerrold M.; Mori, Seiichi; Adams, David J.; Jenkins, Nancy A.; Copeland, Neal G.; Ban, Kenneth Hon-Kim; Kuznetsov, Vladimir A.; Thiery, Jean Paul

    2017-01-01

    Robust prognostic gene signatures and therapeutic targets are difficult to derive from expression profiling because of the significant heterogeneity within breast cancer (BC) subtypes. Here, we performed forward genetic screening in mice using Sleeping Beauty transposon mutagenesis to identify candidate BC driver genes in an unbiased manner, using a stabilized N-terminal truncated β-catenin gene as a sensitizer. We identified 134 mouse susceptibility genes from 129 common insertion sites within 34 mammary tumors. Of these, 126 genes were orthologous to protein-coding genes in the human genome (hereafter, human BC susceptibility genes, hBCSGs), 70% of which are previously reported cancer-associated genes, and ∼16% are known BC suppressor genes. Network analysis revealed a gene hub consisting of E1A binding protein P300 (EP300), CD44 molecule (CD44), neurofibromin (NF1) and phosphatase and tensin homolog (PTEN), which are linked to a significant number of mutated hBCSGs. From our survival prediction analysis of the expression of human BC genes in 2,333 BC cases, we isolated a six-gene-pair classifier that stratifies BC patients with high confidence into prognostically distinct low-, moderate-, and high-risk subgroups. Furthermore, we proposed prognostic classifiers identifying three basal and three claudin-low tumor subgroups. Intriguingly, our hBCSGs are mostly unrelated to cell cycle/mitosis genes and are distinct from the prognostic signatures currently used for stratifying BC patients. Our findings illustrate the strength and validity of integrating functional mutagenesis screens in mice with human cancer transcriptomic data to identify highly prognostic BC subtyping biomarkers. PMID:28251929

  5. Site-Directed Mutagenesis to Improve Sensitivity of a Synthetic Two-Component Signaling System.

    PubMed

    Olshefsky, Audrey; Shehata, Laila; Kuldell, Natalie

    2016-01-01

    Two-component signaling (2CS) systems enable bacterial cells to respond to changes in their local environment, often using a membrane-bound sensor protein and a cytoplasmic responder protein to regulate gene expression. Previous work has shown that Escherichia coli's natural EnvZ/OmpR 2CS could be modified to construct a light-sensing bacterial photography system. The resulting bacterial photographs, or "coliroids," rely on a phosphotransfer reaction between Cph8, a synthetic version of EnvZ that senses red light, and OmpR. Gene expression changes can be visualized through upregulation of a LacZ reporter gene by phosphorylated OmpR. Unfortunately, basal LacZ expression leads to a detectable reporter signal even when cells are grown in the light, diminishing the contrast of the coliroids. We performed site-directed mutagenesis near the phosphotransfer site of Cph8 to isolate mutants with potentially improved image contrast. Five mutants were examined, but only one of the mutants, T541S, increased the ratio of dark/light gene expression, as measured by β-galactosidase activity. The ratio changed from 2.57 fold in the starting strain to 5.59 in the T541S mutant. The ratio decreased in the four other mutant strains we examined. The phenotype observed in the T541S mutant strain may arise because the serine sidechain is chemically similar but physically smaller than the threonine sidechain. This may minimally change the protein's local structure, but may be less sterically constrained when compared to threonine, resulting in a higher probability of a phosphotransfer event. Our initial success pairing synthetic biology and site-directed mutagenesis to optimize the bacterial photography system's performance encourages us to imagine further improvements to the performance of this and other synthetic systems, especially those based on 2CS signaling.

  6. Parameters affecting frequency of CRISPR/Cas9 mediated targeted mutagenesis in rice.

    PubMed

    Mikami, Masafumi; Toki, Seiichi; Endo, Masaki

    2015-10-01

    Frequency of CRISPR/Cas9-mediated targeted mutagenesis varies depending on Cas9 expression level and culture period of rice callus. Recent reports have demonstrated that the CRISPR/Cas9 system can function as a sequence-specific nuclease in various plant species. Induction of mutation in proliferating tissue during embryogenesis or in germline cells is a practical means of generating heritable mutations. In the case of plant species in which cultured cells are used for transformation, non-chimeric plants can be obtained when regeneration occurs from mutated cells. Since plantlets are regenerated from both mutated and non-mutated cells in a random manner, any increment in the proportion of mutated cells in Cas9- and guide RNA (gRNA)-expressing cells will help increase the number of plants containing heritable mutations. In this study, we examined factors affecting mutation frequency in rice calli. Following sequential transformation of rice calli with Cas9- and gRNA- expression constructs, the mutation frequency in independent Cas9 transgenic lines was analyzed. A positive correlation between Cas9 expression level and mutation frequency was found. This positive relationship was observed regardless of whether the transgene or an endogenous gene was used as the target for CRISPR/Cas9-mediated mutagenesis. Furthermore, we found that extending the culture period increased the proportion of mutated cells as well as the variety of mutations obtained. Because mutated and non-mutated cells might proliferate equally, these results suggest that a prolonged tissue culture period increases the chance of inducing de novo mutations in non-mutated cells. This fundamental knowledge will help improve systems for obtaining non-chimeric regenerated plants in many plant species.

  7. Cloning and rational mutagenesis of kexstatin I, a potent proteinaceous inhibitor of Kex2 proteinase.

    PubMed Central

    Oda, K; Oyama, H; Ito, S; Fukiharu, M; Miyagawa, Y; Takahashi, S; Hirose, M; Kikuchi, N; Nakayama, T; Shibano, Y

    2001-01-01

    Kexstatin I is a potent proteinaceous inhibitor of Kex2 proteinase (EC 3.4.21.61). In the present study we show the molecular cloning, primary structure determination and expression of the gene encoding kexstatin I. We also demonstrate its enhanced activity and specificity for Kex2 proteinase inhibition by rational mutagenesis. The cloned kexstatin I gene encoded a protein of 145 amino acid residues, including the 35-residue signal sequence for secretion. The amino acid sequence showed 52% identity with those of the Streptomyces subtilisin inhibitors (SSIs). Thus kexstatin I is the first SSI-family member that can inhibit Kex2 proteinase. The reactive site of the inhibitor was determined to be -Thr(69)-Lys(70) downward arrowGlu(71)-, where downward arrow indicates the reactive site. Because Kex2 proteinase generally shows the highest affinity for substrates with basic amino acid residues at the P(1) and P(2) sites, conversion of the Thr(69)-Lys(70) segment of the inhibitor into dibasic motifs was expected to result in enhanced inhibitory activities. Thus we constructed kexstatin I mutants, in which the Thr(69)-Lys(70) sequence was replaced by the Thr(69)-Arg(70), Lys(69)-Lys(70) and Lys(69)-Arg(70) sequences using PCR-based mutagenesis, and analysed them kinetically. Among these mutants, the Lys(69)-Arg(70) mutant was the most potent inhibitor. The K(i) for Kex2 proteinase was 3.2x10(-10) M, which was 140-fold lower than that of the inhibitor with the Thr(69)-Lys(70) sequence. Although kexstatin I could also inhibit subtilisin, the enhancement of inhibitory activity upon such mutations was specific for Kex2 proteinase inhibition. PMID:11284720

  8. Low-copy piggyBac transposon mutagenesis in mice identifies genes driving melanoma.

    PubMed

    Ni, Thomas K; Landrette, Sean F; Bjornson, Robert D; Bosenberg, Marcus W; Xu, Tian

    2013-09-17

    Despite considerable efforts to sequence hypermutated cancers such as melanoma, distinguishing cancer-driving genes from thousands of recurrently mutated genes remains a significant challenge. To circumvent the problematic background mutation rates and identify new melanoma driver genes, we carried out a low-copy piggyBac transposon mutagenesis screen in mice. We induced eleven melanomas with mutation burdens that were 100-fold lower relative to human melanomas. Thirty-eight implicated genes, including two known drivers of human melanoma, were classified into three groups based on high, low, or background-level mutation frequencies in human melanomas, and we further explored the functional significance of genes in each group. For two genes overlooked by prevailing discovery methods, we found that loss of membrane associated guanylate kinase, WW and PDZ domain containing 2 and protein tyrosine phosphatase, receptor type, O cooperated with the v-raf murine sarcoma viral oncogene homolog B (BRAF) recurrent V600E mutation to promote cellular transformation. Moreover, for infrequently mutated genes often disregarded by current methods, we discovered recurrent mitogen-activated protein kinase kinase kinase 1 (Map3k1)-activating insertions in our screen, mirroring recurrent MAP3K1 up-regulation in human melanomas. Aberrant expression of Map3k1 enabled growth factor-autonomous proliferation and drove BRAF-independent ERK signaling, thus shedding light on alternative means of activating this prominent signaling pathway in melanoma. In summary, our study contributes several previously undescribed genes involved in melanoma and establishes an important proof-of-principle for the utility of the low-copy transposon mutagenesis approach for identifying cancer-driving genes, especially those masked by hypermutation.

  9. 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.

  10. 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…

  11. 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...

  12. 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...

  13. 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

  14. Agrobacterium tumefaciens-mediated transformation: An efficient tool for insertional mutagenesis and targeted gene disruption in Harpophora oryzae.

    PubMed

    Liu, Ning; Chen, Guo-Qing; Ning, Guo-Ao; Shi, Huan-Bin; Zhang, Chu-Long; Lu, Jian-Ping; Mao, Li-Juan; Feng, Xiao-Xiao; Liu, Xiao-Hong; Su, Zhen-Zhu; Lin, Fu-Cheng

    2016-01-01

    The endophytic filamentous fungus Harpophora oryzae is a beneficial endosymbiont isolated from the wild rice. H. oryzae could not only effectively improve growth rate and biomass yield of rice crops, but also induce systemic resistance against the rice blast fungus, Magnaporthe oryzae. In this study, Agrobacterium tumefaciens-mediated transformation (ATMT) was employed and optimized to modify the H. oryzae genes by either random DNA fragment integration or targeted gene replacement. Our results showed that co-cultivation of H. oryzae conidia with A. tumefaciens in the presence of acetosyringone for 48 h at 22 °C could lead to a relatively highest frequency of transformation, and 200 μM acetosyringone (AS) pre-cultivation of A. tumefaciens is also suggested. ATMT-mediated knockout mutagenesis was accomplished with the gene-deletion cassettes using a yeast homologous recombination method with a yeast-Escherichia-Agrobacterium shuttle vector pKOHo. Using the ATMT-mediated knockout mutagenesis, we successfully deleted three genes of H. oryzae (HoATG5, HoATG7, and HoATG8), and then got the null mutants ΔHoatg5, ΔHoatg7, and ΔHoatg8. These results suggest that ATMT is an efficient tool for gene modification including randomly insertional mutagenesis and gene deletion mutagenesis in H. oryzae. Copyright © 2015 Elsevier GmbH. All rights reserved.

  15. Stationary phase mutagenesis in B. subtilis: a paradigm to study genetic diversity programs in cells under stress.

    PubMed

    Robleto, Eduardo A; Yasbin, Ronald; Ross, Christian; Pedraza-Reyes, Mario

    2007-01-01

    One of the experimental platforms to study programs increasing genetic diversity in cells under stressful or nondividing conditions is adaptive mutagenesis, also called stationary phase mutagenesis or stress-induced mutagenesis. In some model systems, there is evidence that mutagenesis occurs in genes that are actively transcribed. Some of those genes may be actively transcribed as a result of environmental stress giving the appearance of directed mutation. That is, cells under conditions of starvation or other stresses accumulate mutations in transcribed genes, including those transcribed because of the selective pressure. An important question concerns how, within the context of stochastic processes, a cell biases mutation to genes under selection pressure? Because the mechanisms underlying DNA transactions in prokaryotic cells are well conserved among the three domains of life, these studies are likely to apply to the examination of genetic programs in eukaryotes. In eukaryotes, increasing genetic diversity in differentiated cells has been implicated in neoplasia and cell aging. Historically, Escherichia coli has been the paradigm used to discern the cellular processes driving the generation of adaptive mutations; however, examining adaptive mutation in Bacillus subtilis has contributed new insights. One noteworthy contribution is that the B. subtilis' ability to accumulate chromosomal mutations under conditions of starvation is influenced by cell differentiation and transcriptional derepression, as well as by proteins homologous to transcription and repair factors. Here we revise and discuss concepts pertaining to genetic programs that increase diversity in B. subtilis cells under nutritional stress.

  16. A combination of site-directed mutagenesis and chemical modification to improve diastereopreference of Pseudomonas alcaligenes lipase.

    PubMed

    Chen, Hui; Wu, Jianping; Yang, Lirong; Xu, Gang

    2013-12-01

    A combination of site-directed mutagenesis and chemical modification was employed to alter protein structure with the objective of improving diastereopreference over that achieved by simple site-directed mutagenesis. Conformational analysis using molecular dynamic (MD) simulation of Pseudomonas alcaligenes lipase (PAL) indicated that stronger steric exclusion and structural rigidity facilitated diastereopreference. A cysteine (Cys) residue was introduced using site-directed mutagenesis to construct variant A272C. The modifier 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) was then reacted with the introduced Cys residue to provide stronger steric exclusion and structural rigidity. The modification was verified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Diastereopreference was improved significantly. The diastereomeric excess (dep) of l-menthol increased from 35% with wild type PAL to 90% with A272C-DTNB modified PAL when the conversion ratio of l-menthyl propionate was nearly 100%. Conformation and kinetic parameter analysis showed that A272C-DTNB modified PAL exhibited stronger steric exclusion and increased structural rigidity around the modification site that inhibited the hydrolysis of non-targeted substrates. The combination of site-directed mutagenesis and chemical modification could be an effective method to alter protein properties and enhance diastereopreference through the combined effect of steric exclusion and structural rigidity.

  17. Targeted mutagenesis of multiple and paralogous genes in Xenopus laevis using two pairs of transcription activator-like effector nucleases.

    PubMed

    Sakane, Yuto; Sakuma, Tetsushi; Kashiwagi, Keiko; Kashiwagi, Akihiko; Yamamoto, Takashi; Suzuki, Ken-Ichi T

    2014-01-01

    Transcription activator-like effector nucleases (TALENs) have been extensively used in genome editing in various organisms. In some cases, however, it is difficult to efficiently disrupt both paralogous genes using a single pair of TALENs in Xenopus laevis because of its polyploidy. Here, we report targeted mutagenesis of multiple and paralogous genes using two pairs of TALENs in X. laevis. First, we show simultaneous targeted mutagenesis of three genes, tyrosinase paralogues (tyra and tyrb) and enhanced green fluorescent protein (egfp) by injection of two TALENs pairs in transgenic embryos carrying egfp. Consistent with the high frequency of both severe phenotypic traits, albinism and loss of GFP fluorescence, frameshift mutation rates of tyr paralogues and egfp reached 40-80%. Next, we show early introduction of TALEN-mediated mutagenesis of these target loci during embryogenesis. Finally, we also demonstrate that two different pairs of TALENs can simultaneously introduce mutations to both paralogues encoding histone chaperone with high efficiency. Our results suggest that targeted mutagenesis of multiple genes using TALENs can be applied to analyze the functions of paralogous genes with redundancy in X. laevis.

  18. [Measurement of mutagenesis to study the effects of chemical agents]. Final report, August 1, 1993--July 31, 1994

    SciTech Connect

    Puck, T.T.

    1994-12-31

    This is the final report of a study conducted at the Eleanor Roosevelt Institute for Cancer Research, Inc. This study looked at mutagenesis as a measurement of the effects of chemical agents. Topics discussed in this report include: development of a new theory for the role of lipids and lipoproteins in the interactions of macromolecules; the action of caffeine in synergizing mutagenesis of agents like ionizing radiation by inhibition of cellular repair processes which was incorporated into a rapid procedure for detection of mutagenicity with high sensitivity; quantitative theoretical analysis of the mutagenesis process in cells exposed to physical and chemical mutagenic agents; theoretical analysis was developed leading to the conclusion that the visible chromosomal lesions described will also include a significant proportion of point mutations; application of this methodology for meaningful measurement of mutagenesis to study the effects of chemical agents was begun; and investigation of the cell cytoskeleton`s effect of genome exposure operating in the course of the differentiation process.

  19. 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…

  20. 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...

  1. Production and Screening of High Yield Avermectin B1b Mutant of Streptomyces avermitilis 41445 Through Mutagenesis

    PubMed Central

    Siddique, Samia; Syed, Quratulain; Adnan, Ahmad; Qureshi, Fahim Ashraf

    2014-01-01

    Background: Secondary metabolite production from wild strains is very low for economical purpose therefore certain strain improvement strategies are required to achieve hundred times greater yield of metabolites. Most important strain improvement techniques include physical and chemical mutagenesis. Broad spectrum mutagenesis through UV irradiation is the most important and convenient physical method. Objectives: The present study was conducted for enhanced production of avermectin B1b from Streptomyces avermitilis 41445 by mutagenesis using ultraviolet (UV) radiation, ethidium bromide (EB), and ethyl methanesulfonate (EMS) as mutagens. Materials and Methods: S. avermitilis DSM 41445 maintained on yeast extract malt extract glucose medium (YMG) was used as inoculum for SM2 fermentation medium. Spores of S. avermitilis DSM 41445 were exposed to UV radiation for physical broad spectrum mutagenesis and to EMS and EB for chemical mutagenesis. For each mutagen, the lethality rate and mutation rate were calculated along with positive mutation rate. Results: Avermectin B1b-hyper-producing mutant, produced using these three different methods, was selected according to the HPLC results. The mutant obtained after 45 minutes of UV radiation to the spores of S. avermitilis 41445, was found to be the best mutant for the enhanced production of avermectin B1b component (254.14 mg/L). Other avermectin B1b-hyper-producing mutants, were obtained from EMS (1 µL/mL) and EB (30 µL/mL) treatments, and yielded 202.63 mg/L and 199.30 mg/L of B1b, respectively. Conclusions: The hereditary stability analysis of the UV mentioning 45 minutes revealed the UV exposure time for mutants and 3 represented the colony taken from the plate irradiated for 45 minutes mutant showed that the production of avermectin B1b remained constant and no reverse mutation occurred after 15 generations. PMID:25147669

  2. Role of copper and ceruloplasmin in oxidative mutagenesis induced by the gluthathione-{gamma}-glutamyl transpeptidase system and by other thiols

    SciTech Connect

    Stark, A.A.; Glass, G.A.

    1997-10-01

    Glutathione is activated to a mutagen by {gamma}-glutamyl transpeptidase. Other thiols, such as cysteine, penicillamine, cysteine ethylester, and cysteinylglycine, are direct mutagens in the Ames Salmonella mutagenicity test. Thiol mutagenesis is oxidative in nature and involves H{sub 2}O{sub 2} and possibly hydroxyl radicals. Transition metals are crucial for thiol autoxidation. The role of copper and ceruloplasmin (CP) in thiol-dependent mutagenesis was studied in Salmonella typhimurium strain TA 102. Cu and CP at low concentrations enhanced thiol-dependent mutagenesis in the presence, but not in the absence, and added Fe. The degree of enhancement depended on the type of thiol. At high Cu or CP concentrations, thiol mutagenesis was inhibited. Cu also decreased the mutagenicity of H{sub 2}O{sub 2}. Cu- and CP-enhanced mutagenesis were inhibited by radical scavengers, catalase, and peroxidase but not by superoxide dismutase. The effects of Cu and CP on thiol-dependent mutagenesis were similar to their effects on thiol-driven lipid peroxidation. The results indicate that the role of Cu and CP in the enhancement of thiol mutagenesis is the facilitation of the transfer of electrons from a thiol to iron, rather than in catalysis of the Fenton reaction. 34 refs., 7 figs., 2 tabs.

  3. Role of copper and ceruloplasmin in oxidative mutagenesis induced by the glutathione-gamma-glutamyl transpeptidase system and by other thiols.

    PubMed

    Stark, A A; Glass, G A

    1997-01-01

    Glutathione is activated to a mutagen by gamma-glutamyl transpeptidase. Other thiols, such as cysteine, penicillamine, cysteine ethylester, and cysteinylglycine, are direct mutagens in the Ames Salmonella mutagenicity test. Thiol mutagenesis is oxidative in nature and involves H2O2 and possibly hydroxyl radicals. Transition metals are crucial for thiol autoxidation. The role of copper and ceruloplasmin (CP) in thiol-dependent mutagenesis was studied in Salmonella typhimurium strain TA102. Cu and CP at low concentrations enhanced thiol-dependent mutagenesis in the presence, but not in the absence, of added Fe. The degree of enhancement depended on the type of thiol. At high Cu or CP concentrations, thiol mutagenesis was inhibited. Cu also decreased the mutagenicity of H2O2. Cu- and CP-enhanced mutagenesis were inhibited by radical scavengers, catalase, and peroxidase but not by superoxide dismutase. The effects of Cu and CP on thiol-dependent mutagenesis were similar to their effects on thiol-driven lipid peroxidation. The results indicate that the role of Cu and CP in the enhancement of thiol mutagenesis is the facilitation of the transfer of electrons from a thiol to iron, rather than in catalysis of the Fenton reaction.

  4. Transposon and deletion mutagenesis of genes involved in perchlorate reduction in Azospira suillum PS.

    PubMed

    Melnyk, Ryan A; Clark, Iain C; Liao, Annette; Coates, John D

    2013-12-31

    Although much work on the biochemistry of the key enzymes of bacterial perchlorate reduction, chlorite dismutase, and perchlorate reductase has been published, understanding of the molecular mechanisms of this metabolism has been somewhat hampered by the lack of a clear model system amenable to genetic manipulation. Using transposon mutagenesis and clean deletions, genes important for perchlorate reduction in Azospira suillum PS have been identified both inside and outside the previously described perchlorate reduction genomic island (PRI). Transposon mutagenesis identified 18 insertions in 11 genes that completely abrogate growth via reduction of perchlorate but have no phenotype during denitrification. Of the mutants deficient in perchlorate reduction, 14 had insertions that were mapped to eight different genes within the PRI, highlighting its importance in this metabolism. To further explore the role of these genes, we also developed systems for constructing unmarked deletions and for complementing these deletions. Using these tools, every core gene in the PRI was systematically deleted; 8 of the 17 genes conserved in the PRI are essential for perchlorate respiration, including 3 genes that comprise a unique histidine kinase system. Interestingly, the other 9 genes in the PRI are not essential for perchlorate reduction and may thus have unknown functions during this metabolism. We present a model detailing our current understanding of perchlorate reduction that incorporates new concepts about this metabolism. Although perchlorate is generated naturally in the environment, groundwater contamination is largely a result of industrial activity. Bacteria capable of respiring perchlorate and remediating contaminated water have been isolated, but relatively little is known about the biochemistry and genetics of this process. Here we used two complementary approaches to identify genes involved in perchlorate reduction. Most of these genes are located on a genomic island

  5. Detecting Statistically Significant Common Insertion Sites in Retroviral Insertional Mutagenesis Screens

    PubMed Central

    de Ridder, Jeroen; Uren, Anthony; Kool, Jaap; Reinders, Marcel; Wessels, Lodewyk

    2006-01-01

    Retroviral insertional mutagenesis screens, which identify genes involved in tumor development in mice, have yielded a substantial number of retroviral integration sites, and this number is expected to grow substantially due to the introduction of high-throughput screening techniques. The data of various retroviral insertional mutagenesis screens are compiled in the publicly available Retroviral Tagged Cancer Gene Database (RTCGD). Integrally analyzing these screens for the presence of common insertion sites (CISs, i.e., regions in the genome that have been hit by viral insertions in multiple independent tumors significantly more than expected by chance) requires an approach that corrects for the increased probability of finding false CISs as the amount of available data increases. Moreover, significance estimates of CISs should be established taking into account both the noise, arising from the random nature of the insertion process, as well as the bias, stemming from preferential insertion sites present in the genome and the data retrieval methodology. We introduce a framework, the kernel convolution (KC) framework, to find CISs in a noisy and biased environment using a predefined significance level while controlling the family-wise error (FWE) (the probability of detecting false CISs). Where previous methods use one, two, or three predetermined fixed scales, our method is capable of operating at any biologically relevant scale. This creates the possibility to analyze the CISs in a scale space by varying the width of the CISs, providing new insights in the behavior of CISs across multiple scales. Our method also features the possibility of including models for background bias. Using simulated data, we evaluate the KC framework using three kernel functions, the Gaussian, triangular, and rectangular kernel function. We applied the Gaussian KC to the data from the combined set of screens in the RTCGD and found that 53% of the CISs do not reach the significance

  6. N-Nitrosocarbaryl-induced mutagenesis in Haemophilus influenzae strains deficient in repair and recombination.

    PubMed

    Beattie, K L

    1975-02-01

    Mutagenesis was studied in repair- and recombination-deficient strains of Haemophilus influenzae after treatment with N-nitrosocarbaryl (NC). Three different strains of H. influenzae carrying mutations affecting excision-repair of UV-induced pyrimidine dimers exhibited normal repair of premutational lesions (as detected by decreased mutation yield resulting from post-treatment DNA synthesis delay) and normal nonreplicative mutation fixation. This indicated that neither of these phenomena are caused by the smae repair mechanism that removes UV-induced pyrimidine dimers from the DNA. The recombination-deficient mutant recI is apparently deficient in the replication-dependent mode of NC-induced mutation fixation. This conclusion is based on the following results: (I) NC-induced mutagenesis is lower in the recI strain than in rec+ cells. (2) Repair of premutational lesions (which depends on the existence of replication-dependent mutation fixation for its detection) was not detected in the recI strain. (3) When nonreplicative mutation fixation and final mutation frequency were measured in the same experiment, about I/4 to I/3 of the final mutation yield could be accounted for by nonreplicative mutation fixation in the rec+ strain, whereas all of the mutation could be accounted for in the recI strain by the nonreplicative mutation fixation. (4) When mutation fixation in strain dna9 recI was followed at the permissive (36 degrees) and nonpermissive (41 degrees) temperatures, it became apparent that in the recI strain replication-dependent mutation fixation occurs at early times, but these newly fixed mutations are unstable and disappear at later times, leaving only the mutations fixed by the nonreplicative process. The recI strain exhibits normal repair of NC-induced single-strand breaks or alkali-labile bonds in the DNA labeled before treatment, but is slow in joining discontinuties present in DNA synthesized after treatment. The results are consistent with the idea that

  7. Improvement of multiple stress tolerance in yeast strain by sequential mutagenesis for enhanced bioethanol production.

    PubMed

    Kumari, Rajni; Pramanik, Krishna

    2012-12-01

    The present work deals with the improvement of multiple stress tolerance in a glucose-xylose co-fermenting hybrid yeast strain RPR39 by sequential mutagenesis using ethyl methane sulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, near and far ultraviolet radiations. The mutants were evaluated for their tolerance to ethanol, temperature and fermentation inhibitors. Among these mutants, mutant RPRT90 exhibited highest tolerance to 10% initial ethanol concentration, 2 g L(-1) furfural and 8 g L(-1) acetic acid. The mutant also showed good growth at high temperature (39-40°C). A study on the combined effect of multiple stresses during fermentation of glucose-xylose mixture (3:1 ratio) was performed using mutant RPRT90. Under the combined effect of thermal (39°C) and inhibitor stress (0.25 g L(-1) vanillin, 0.5 g L(-1) furfural and 4 g L(-1) acetic acid), the mutant produced ethanol with a yield of 0.379 g g(-1), while under combined effect of ethanol (7% v/v) and inhibitor stress the ethanol yield obtained was 0.43 g g(-1). Further, under the synergistic effect of sugar (250 g L(-1)), thermal (39°C), ethanol (7% v/v) and inhibitors stress, the strain produced a maximum of 47.93 g L(-1) ethanol by utilizing 162.42 g L(-1) of glucose-xylose mixture giving an ethanol yield of 0.295 g g(-1) and productivity of 0.57 g L(-1) h(-1). Under same condition the fusant RPR39 produced a maximum of 30.0 g L(-1) ethanol giving a yield and productivity of 0.21 g g(-1) and 0.42 g L(-1) h(-1) respectively. The molecular characterization of mutant showed considerable difference in its genetic profile from hybrid RPR39. Thus, sequential mutagenesis was found to be effective to improve the stress tolerance properties in yeast.

  8. Engineering of Harobin for enhanced fibrinolytic activity obtained by random and site-directed mutagenesis.

    PubMed

    Li, Zhuojian; Chen, Xiaojia; Guo, Shujun; Zhang, Huihua; Dong, Haojun; Wu, Guoyi; Hong, An; Gu, Jun

    2017-01-01

    We have previously published a report on the cloning and characterization of Harobin, a fibrinolytic serine protease. However, the broad application of this fibrinolytic enzyme is limited by its low expression level that was achieved in Pichia pastoris. To counteract this shortcoming, random and site-directed mutagenesis have been combined in order to improve functional expression and activity of Harobin. By screening 400 clones from random mutant libraries for enhanced fibrinolytic activity, two mutants were obtained: N111R, R230G. By performing site-directed mutagenesis, a Harobin double mutant, N111R/R230G, was constructed and can be functionally expressed at higher level than the wild type enzyme. In addition, it possessed much higher fibrinolytic and amidolytic activity than the wild type enzyme and other single mutants. The N111R/R230G expressed in basal salts medium was purified by a three step purification procedure. At pH of 6.0-9.0, and the temperature range of 40-90 °C, N111R/R230G was more active and more heat resistant. The fibrinolytic activities of Harobin mutants were completely inhibited by PMSF and SBTI, but not by EDTA, EGTA, DTT, indicating that Harobin is a serine protease. N111R/R230G showed much better anti-thrombosis effect than wild type Harobin and single mutants, and could significantly increase bleeding and clotting time. Intravenous injection of N111R/R230G in spontaneous hypertensive rats (SHR) led to a significant reduction in systolic blood pressure (SBP), diastolic blood pressure (DBP) and mean arterial pressure (MAP) (p < 0.01), while heart rate (HR) was not affected. The in vitro and in vivo results of the present study revealed that Harobin double mutant N111R/R230G is an appropriate candidate for biotechnological applications due to its high expression level and high activity in area of thrombosis and hypertension. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Structural insights from random mutagenesis of Campylobacter jejuni oligosaccharyltransferase PglB.

    PubMed

    Ihssen, Julian; Kowarik, Michael; Wiesli, Luzia; Reiss, Renate; Wacker, Michael; Thöny-Meyer, Linda

    2012-09-24

    Protein glycosylation is of fundamental importance in many biological systems. The discovery of N-glycosylation in bacteria and the functional expression of the N-oligosaccharyltransferase PglB of Campylobacter jejuni in Escherichia coli enabled the production of engineered glycoproteins and the study of the underlying molecular mechanisms. A particularly promising application for protein glycosylation in recombinant bacteria is the production of potent conjugate vaccines where polysaccharide antigens of pathogenic bacteria are covalently bound to immunogenic carrier proteins. In this study capsular polysaccharides of the clinically relevant pathogen Staphylococcus aureus serotype 5 (CP5) were expressed in Escherichia coli and linked in vivo to a detoxified version of Pseudomonas aeruginosa exotoxin (EPA). We investigated which amino acids of the periplasmic domain of PglB are crucial for the glycosylation reaction using a newly established 96-well screening system enabling the relative quantification of glycoproteins by enzyme-linked immunosorbent assay. A random mutant library was generated by error-prone PCR and screened for inactivating amino acid substitutions. In addition to 15 inactive variants with amino acid changes within the previously known, strictly conserved WWDYG motif of N-oligosaccharyltransferases, 8 inactivating mutations mapped to a flexible loop in close vicinity of the amide nitrogen atom of the acceptor asparagine as revealed in the crystal structure of the homologous enzyme C. lari PglB. The importance of the conserved loop residue H479 for glycosylation was confirmed by site directed mutagenesis, while a change to alanine of the adjacent, non-conserved L480 had no effect. In addition, we investigated functional requirements in the so-called MIV motif of bacterial N-oligosaccharyltransferases. Amino acid residues I571 and V575, which had been postulated to interact with the acceptor peptide, were subjected to cassette saturation mutagenesis

  10. Structural insights from random mutagenesis of Campylobacter jejuni oligosaccharyltransferase PglB

    PubMed Central

    2012-01-01

    Background Protein glycosylation is of fundamental importance in many biological systems. The discovery of N-glycosylation in bacteria and the functional expression of the N-oligosaccharyltransferase PglB of Campylobacter jejuni in Escherichia coli enabled the production of engineered glycoproteins and the study of the underlying molecular mechanisms. A particularly promising application for protein glycosylation in recombinant bacteria is the production of potent conjugate vaccines where polysaccharide antigens of pathogenic bacteria are covalently bound to immunogenic carrier proteins. Results In this study capsular polysaccharides of the clinically relevant pathogen Staphylococcus aureus serotype 5 (CP5) were expressed in Escherichia coli and linked in vivo to a detoxified version of Pseudomonas aeruginosa exotoxin (EPA). We investigated which amino acids of the periplasmic domain of PglB are crucial for the glycosylation reaction using a newly established 96-well screening system enabling the relative quantification of glycoproteins by enzyme-linked immunosorbent assay. A random mutant library was generated by error-prone PCR and screened for inactivating amino acid substitutions. In addition to 15 inactive variants with amino acid changes within the previously known, strictly conserved WWDYG motif of N-oligosaccharyltransferases, 8 inactivating mutations mapped to a flexible loop in close vicinity of the amide nitrogen atom of the acceptor asparagine as revealed in the crystal structure of the homologous enzyme C. lari PglB. The importance of the conserved loop residue H479 for glycosylation was confirmed by site directed mutagenesis, while a change to alanine of the adjacent, non-conserved L480 had no effect. In addition, we investigated functional requirements in the so-called MIV motif of bacterial N-oligosaccharyltransferases. Amino acid residues I571 and V575, which had been postulated to interact with the acceptor peptide, were subjected to cassette

  11. Large-Scale Transposition Mutagenesis of Streptomyces coelicolor Identifies Hundreds of Genes Influencing Antibiotic Biosynthesis.

    PubMed

    Xu, Zhong; Wang, Yemin; Chater, Keith F; Ou, Hong-Yu; Xu, H Howard; Deng, Zixin; Tao, Meifeng

    2017-03-15

    Gram-positive Streptomyces bacteria produce thousands of bioactive secondary metabolites, including antibiotics. To systematically investigate genes affecting secondary metabolism, we developed a hyperactive transposase-based Tn5 transposition system and employed it to mutagenize the model species Streptomyces coelicolor, leading to the identification of 51,443 transposition insertions. These insertions were distributed randomly along the chromosome except for some preferred regions associated with relatively low GC content in the chromosomal core. The base composition of the insertion site and its flanking sequences compiled from the 51,443 insertions implied a 19-bp expanded target site surrounding the insertion site, with a slight nucleic acid base preference in some positions, suggesting a relative randomness of Tn5 transposition targeting in the high-GC Streptomyces genome. From the mutagenesis library, 724 mutants involving 365 genes had altered levels of production of the tripyrrole antibiotic undecylprodigiosin (RED), including 17 genes in the RED biosynthetic gene cluster. Genetic complementation revealed that most of the insertions (more than two-thirds) were responsible for the changed antibiotic production. Genes associated with branched-chain amino acid biosynthesis, DNA metabolism, and protein modification affected RED production, and genes involved in signaling, stress, and transcriptional regulation were overrepresented. Some insertions caused dramatic changes in RED production, identifying future targets for strain improvement.IMPORTANCE High-GC Gram-positive streptomycetes and related actinomycetes have provided more than 100 clinical drugs used as antibiotics, immunosuppressants, and antitumor drugs. Their genomes harbor biosynthetic genes for many more unknown compounds with potential as future drugs. Here we developed a useful genome-wide mutagenesis tool based on the transposon Tn5 for the study of secondary metabolism and its regulation

  12. Optimizing glycosyltransferase specificity via ‘hot spot’ saturation mutagenesis presents a new catalyst for novobiocin glycorandomization

    PubMed Central

    Williams, Gavin J.; Goff, Randal D.; Zhang, Changsheng; Thorson, Jon S.

    2010-01-01

    Summary A comprehensive two phase ‘hot spot’ saturation mutagenesis strategy to rapidly evolve glycosyltransferase specificity for non-natural acceptors is described. Specifically, the application of a high throughput screen (based upon the fluorescent acceptor umbelliferone) was used to identify key amino acid ‘hot spots’ that contribute to GT proficiency and/or promiscuity. Saturation mutagenesis of the corresponding hot spots facilitated the utilization of a lower throughput screen to provide OleD prodigy capable of efficiently glycosylating the non-natural acceptor novobiocic acid with an array of unique sugars. Incredibly, even in the absence of a high-throughput screen for novobiocic acid glycosylation, this approach rapidly led to improvements in the desired catalytic activity of several hundred-fold. PMID:18420146

  13. Genome-wide analysis of mutagenesis bias and context sensitivity of N-methyl-N'-nitro-N-nitrosoguanidine (NTG).

    PubMed

    Harper, Marc; Lee, Christopher J

    2012-03-01

    We have analyzed the mutation spectrum of N-methyl-N'-nitro-N-nitrosoguanidine (NTG) from a set of 4099 mutations identified from whole-genome sequencing of 32 E. coli strains mutagenized with NTG. These data permit precise measurement of NTG's bias for G/C to A/T transitions (96.6% of all mutations) and also show that NTG mutagenesis is strongly sensitive to context, favoring guanine residues preceded by purines by five-fold over those preceded by pyrimidines. These data give confident estimates for the GC bias and transition/transversion ratios of NTG mutagenesis, which could not be estimated confidently from previous, much smaller datasets. Published by Elsevier B.V.

  14. Space mutagenesis of genetically engineered bacteria expressing recombinant human interferon α1b and screening of higher yielding strains.

    PubMed

    Wang, Junfeng; Liu, Changting; Liu, Jinyi; Fang, Xiangqun; Xu, Chen; Guo, Yinghua; Chang, De; Su, Longxiang

    2014-03-01

    The aim of this study was to investigate the space mutagenesis of genetically engineered bacteria expressing recombinant human interferon α1b. The genetically engineered bacteria expressing the recombinant interferon α1b were sent into outer space on the Chinese Shenzhou VIII spacecraft. After the 17 day space flight, mutant strains that highly expressed the target gene were identified. After a series of screening of spaceflight-treated bacteria and the quantitative comparison of the mutant strains and original strain, we found five strains that showed a significantly higher production of target proteins, compared with the original strain. Our results support the notion that the outer space environment has unique effects on the mutation breeding of microorganisms, including genetically engineered strains. Mutant strains that highly express the target protein could be obtained through spaceflight-induced mutagenesis.

  15. 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.

  16. Crystallization of a fragment of human fibronectin: introduction of methionine by site-directed mutagenesis to allow phasing via selenomethionine.

    PubMed

    Leahy, D J; Erickson, H P; Aukhil, I; Joshi, P; Hendrickson, W A

    1994-05-01

    Crystals of a fragment of human fibronectin encompassing the 7th through the RGD-containing 10th type III repeats (FN7-10) have been produced with protein expressed in E. coli. The crystals are monoclinic with one molecule in the asymmetric unit and diffract to beyond 2.0 A Bragg spacings. A mutant FN7-10 was produced in which three methionines, in addition to the single native methionine already present, have been introduced by site-directed mutagenesis. Diffraction-quality crystals of this mutant protein have been grown in which methionine was replaced with selenomethionine. The introduction of methionine by site-directed mutagenesis to allow phasing from selenomethionyl-substituted crystals is shown to be feasible by this example and is proposed as a general approach to solving the crystallographic phase problem. Strategies for selecting propitious sites for methionine mutations are discussed.

  17. Random mutagenesis and media optimisation for hyperproduction of cellulase from Bacillus species using proximally analysed Saccharum spontaneum in submerged fermentation.

    PubMed

    Abdullah, Roheena; Zafar, Wajeeha; Nadeem, Muhammad; Iqtedar, Mehwish; Naz, Shagufta; Syed, Quratulain; Butt, Zahid Ali

    2015-01-01

    This study deals with the isolation of novel mutant of Bacillus and optimisation of media for the hyperproduction of cellulase. Cellulase-producing Bacillus PC-BC6 was subjected to physical and chemical mutagenesis to enhance the cellulolytic potential. Later, mutagenesis isolates were screened both qualitatively and quantitatively. Among all the tested isolates, Bacillus N3 yielded maximum (CMCase 1250 IU/mL/min and FPase 629 IU/mL/min) activity. The Bacillus N3 strain exhibited 1.7-fold more enzyme production as compared with the parental strain. Proximate analysis of untreated and pretreated Saccharum spontaneum was carried out to improve cellulase production. Three different media were tested for the production of cellulase, among which M2 medium containing MgSO4, pretreated S. spontaneum, K2HPO4, (NH4)2SO4 and peptone was found to be the best for maximum enzyme production by mutant Bacillus N3.

  18. 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

  19. Selection of Zygosaccharomyces rouxii strains resistant to cadmium with improved removal abilities through ultraviolet-diethyl sulfate cooperative mutagenesis.

    PubMed

    Liu, Yu; Xu, Ying; Wang, Dongfeng; Jiang, Wei

    2017-06-25

    Cd(2+) resistance and bioaccumulation capacity were selected from parental Zygosaccharomyces rouxii (CRZ-0) while maintaining NaCl tolerance using protoplast mutagenesis technology. Ultraviolet-diethyl sulfate (UV-DES) cooperative mutagenesis, followed by preliminary screening and rescreening, was used to select the mutant strain CRZ-9. CRZ-9 grew better than CRZ-0 in YPD medium with 20 or 50 mg L(-1) of Cd(2+). Scanning electron microscopy observations and flow cytometry tests indicated that CRZ-9 was more effective at eliminating reactive oxygen species (ROS) generated by Cd(2+), which led to less cellular structural damage and lower lethality. Furthermore, compared with CRZ-0, CRZ-9 exhibited increased potential for application with higher Cd(2+) removal ratio, wider working pH range, and lower biomass dosage in Cd(2+) bioaccumulation. The mutant strain CRZ-9 possessed improved Cd(2+) resistance and bioaccumulation capacity and therefore is a promising strain to remove Cd(2+) from wastewater.

  20. Insertional mutagenesis reveals progression genes and checkpoints in MYC/Runx2 lymphomas

    PubMed Central

    2008-01-01

    In this study we have exploited the power of insertional mutagenesis to elucidate tumor progression pathways in mice carrying two oncogenes (MYC/Runx2) that collaborate to drive early lymphoma development. Neonatal infection of these mice with Moloney murine leukemia virus (MLV) resulted in accelerated tumor onset with associated increases in clonal complexity and lymphoid dissemination. Large-scale analysis of retroviral integration sites in these tumors revealed a profound bias towards a narrow range of target genes including Jdp2 (Jundm2), D cyclin and Pim family genes. Remarkably, direct PCR analysis of integration hot-spots revealed that every progressing tumor consisted of multiple clones harbouring hits at these loci, giving access to large numbers of independent insertion events and uncovering the contrasting mutagenic mechanisms operating at each target gene. Direct PCR analysis showed that high frequency targeting occurs only in the tumor environment in vivo and is specific for the progression gene set. These results indicate that early lymphomas in MYC/Runx2 mice remain dependent on exogenous growth signals and that progression can be achieved by constitutive activation of pathways converging on a cell cycle checkpoint that acts as the major rate-limiting step for lymphoma outgrowth. PMID:17545590

  1. Mutagenesis and functional analysis of the pore-forming toxin HALT-1 from Hydra magnipapillata.

    PubMed

    Liew, Yvonne Jing Mei; Soh, Wai Tuck; Jiemy, William Febry; Hwang, Jung Shan

    2015-02-03

    Actinoporins are small 18.5 kDa pore-forming toxins. A family of six actinoporin genes has been identified in the genome of Hydra magnipapillata, and HALT-1 (Hydra actinoporin-like toxin-1) has been shown to have haemolytic activity. In this study, we have used site-directed mutagenesis to investigate the role of amino acids in the pore-forming N-terminal region and the conserved aromatic cluster required for cell membrane binding. A total of 10 mutants of HALT-1 were constructed and tested for their haemolytic and cytolytic activity on human erythrocytes and HeLa cells, respectively. Insertion of 1-4 negatively charged residues in the N-terminal region of HALT-1 strongly reduced haemolytic and cytolytic activity, suggesting that the length or charge of the N-terminal region is critical for pore-forming activity. Moreover, substitution of amino acids in the conserved aromatic cluster reduced haemolytic and cytolytic activity by more than 80%, suggesting that these aromatic amino acids are important for attachment to the lipid membrane as shown for other actinoporins. The results suggest that HALT-1 and other actinoporins share similar mechanisms of pore formation and that it is critical for HALT-1 to maintain an amphipathic helix at the N-terminus and an aromatic amino acid-rich segment at the site of membrane binding.

  2. Towards Understanding the Catalytic Mechanism of Human Paraoxonase 1: Experimental and In Silico Mutagenesis Studies.

    PubMed

    Tripathy, Rajan K; Aggarwal, Geetika; Bajaj, Priyanka; Kathuria, Deepika; Bharatam, Prasad V; Pande, Abhay H

    2017-02-04

    Human paraoxonase 1 (h-PON1) is a ~45-kDa serum enzyme that can hydrolyze a variety of substrates, including organophosphate (OP) compounds. It is a potential candidate for the development of antidote against OP poisoning in humans. However, insufficient OP-hydrolyzing activity of native enzyme affirms the urgent need to develop improved variant(s) having enhanced OP-hydrolyzing activity. The crystal structure of h-PON1 remains unsolved, and the molecular details of how the enzyme catalyses hydrolysis of different types of substrates are also not clear. Understanding the molecular details of the catalytic mechanism of h-PON1 is essential to engineer better variant(s) of enzyme. In this study, we have used a random mutagenesis approach to increase the OP-hydrolyzing activity of recombinant h-PON1. The mutants not only showed a 10-340-fold increased OP-hydrolyzing activity against different OP substrates but also exhibited differential lactonase and arylesterase activities. In order to investigate the mechanistic details of the effect of observed mutations on the hydrolytic activities of enzyme, molecular docking studies were performed with selected mutants. The results suggested that the observed mutations permit differential binding of substrate/inhibitor into the enzyme's active site. This may explain differential hydrolytic activities of the enzyme towards different substrates.

  3. Comprehensive Essentiality Analysis of the Mycobacterium tuberculosis Genome via Saturating Transposon Mutagenesis

    PubMed Central

    DeJesus, Michael A.; Gerrick, Elias R.; Xu, Weizhen; Park, Sae Woong; Long, Jarukit E.; Boutte, Cara C.; Rubin, Eric J.; Schnappinger, Dirk; Ehrt, Sabine; Fortune, Sarah M.; Sassetti, Christopher M.

    2017-01-01

    ABSTRACT   For decades, identifying the regions of a bacterial chromosome that are necessary for viability has relied on mapping integration sites in libraries of random transposon mutants to find loci that are unable to sustain insertion. To date, these studies have analyzed subsaturated libraries, necessitating the application of statistical methods to estimate the likelihood that a gap in transposon coverage is the result of biological selection and not the stochasticity of insertion. As a result, the essentiality of many genomic features, particularly small ones, could not be reliably assessed. We sought to overcome this limitation by creating a completely saturated transposon library in Mycobacterium tuberculosis. In assessing the composition of this highly saturated library by deep sequencing, we discovered that a previously unknown sequence bias of the Himar1 element rendered approximately 9% of potential TA dinucleotide insertion sites less permissible for insertion. We used a hidden Markov model of essentiality that accounted for this unanticipated bias, allowing us to confidently evaluate the essentiality of features that contained as few as 2 TA sites, including open reading frames (ORF), experimentally identified noncoding RNAs, methylation sites, and promoters. In addition, several essential regions that did not correspond to known features were identified, suggesting uncharacterized functions that are necessary for growth. This work provides an authoritative catalog of essential regions of the M. tuberculosis genome and a statistical framework for applying saturating mutagenesis to other bacteria. PMID:28096490

  4. Dopamine D1 receptor-agonist interactions: A mutagenesis and homology modeling study.

    PubMed

    Mente, Scot; Guilmette, Edward; Salafia, Michelle; Gray, David

    2015-01-01

    The dopamine D1 receptor is a G protein-coupled receptor that regulates intracellular signaling via agonist activation. Although the number of solved GPCR X-ray structures has been steadily increasing, still no structure of the D1 receptor exists. We have used site-directed mutagenesis of 12 orthosteric vicinity residues of possible importance to G protein-coupled activation to examine the function of prototypical orthosteric D1 agonists and partial agonists. We find that residues from four different regions of the D1 receptor make significant contributions to agonist function. All compounds studied, which are catechol-amines, are found to interact with the previously identified residues: the conserved D103(3.32), as well as the trans-membrane V serine residues. Additional key interactions are found for trans-membrane VI residues F288(6.51), F289(6.52) and N292(6.55), as well as the extra-cellular loop residue L190(ECL2). Molecular dynamics simulations of a D1 homology model have been used to help put the ligand-residue interactions into context. Finally, we considered the rescaling of fold-shift data as a method to account for the change in the size of the mutated side-chain and found that this rescaling helps to relate the calculated ligand-residue energies with observed experimental fold-shifts.

  5. Site Directed Mutagenesis of Schizosaccharomyces pombe Glutathione Synthetase Produces an Enzyme with Homoglutathione Synthetase Activity

    PubMed Central

    Dworeck, Tamara; Zimmermann, Martin

    2012-01-01

    Three different His-tagged, mutant forms of the fission yeast glutathione synthetase (GSH2) were derived by site-directed mutagenesis. The mutant and wild-type enzymes were expressed in E. coli DH5α and affinity purified in a two-step procedure. Analysis of enzyme activity showed that it was possible to shift the substrate specificity of GSH2 from Gly (km 0,19; wild-type) to β-Ala or Ser. One mutation (substitution of Ile471, Cy472 to Met and Val and Ala 485 and Thr486 to Leu and Pro) increased the affinity of GSH2 for β-Ala (km 0,07) and lowered the affinity for Gly (km 0,83), which is a characteristic of the enzyme homoglutathione synthetase found in plants. Substitution of Ala485 and Thr486 to Leu and Pro only, increased instead the affinity of GSH2 for Ser (km 0,23) as a substrate, while affinity to Gly was preserved (km 0,12). This provides a new biosynthetic pathway for hydroxymethyl glutathione, which is known to be synthesized from glutathione and Ser in a reaction catalysed by carboxypeptidase Y. The reported findings provide further insight into how specific amino acids positioned in the GSH2 active site facilitate the recognition of different amino acid substrates, furthermore they support the evolutionary theory that homoglutathione synthetase evolved from glutathione synthetase by a single gene duplication event. PMID:23091597

  6. SDM-Assist software to design site-directed mutagenesis primers introducing "silent" restriction sites.

    PubMed

    Karnik, Abhijit; Karnik, Rucha; Grefen, Christopher

    2013-03-22

    Over the past decades site-directed mutagenesis (SDM) has become an indispensable tool for biological structure-function studies. In principle, SDM uses modified primer pairs in a PCR reaction to introduce a mutation in a cDNA insert. DpnI digestion of the reaction mixture is used to eliminate template copies before amplification in E. coli; however, this process is inefficient resulting in un-mutated clones which can only be distinguished from mutant clones by sequencing. We have developed a program - 'SDM-Assist' which creates SDM primers adding a specific identifier: through additional silent mutations a restriction site is included or a previous one removed which allows for highly efficient identification of 'mutated clones' by a simple restriction digest. The direct identification of SDM clones will save time and money for researchers. SDM-Assist also scores the primers based on factors such as Tm, GC content and secondary structure allowing for simplified selection of optimal primer pairs.

  7. Structure and mutagenesis of the DNA modification-dependent restriction endonuclease AspBHI.

    PubMed

    Horton, John R; Nugent, Rebecca L; Li, Andrew; Mabuchi, Megumu Yamada; Fomenkov, Alexey; Cohen-Karni, Devora; Griggs, Rose M; Zhang, Xing; Wilson, Geoffrey G; Zheng, Yu; Xu, Shuang-yong; Cheng, Xiaodong

    2014-03-07

    The modification-dependent restriction endonuclease AspBHI recognizes 5-methylcytosine (5mC) in the double-strand DNA sequence context of (C/T)(C/G)(5mC)N(C/G) (N = any nucleotide) and cleaves the two strands a fixed distance (N12/N16) 3' to the modified cytosine. We determined the crystal structure of the homo-tetrameric AspBHI. Each subunit of the protein comprises two domains: an N-terminal DNA-recognition domain and a C-terminal DNA cleavage domain. The N-terminal domain is structurally similar to the eukaryotic SET and RING-associated (SRA) domain, which is known to bind to a hemi-methylated CpG dinucleotide. The C-terminal domain is structurally similar to classic Type II restriction enzymes and contains the endonuclease catalytic-site motif of DX20EAK. To understand how specific amino acids affect AspBHI recognition preference, we generated a homology model of the AspBHI-DNA complex, and probed the importance of individual amino acids by mutagenesis. Ser41 and Arg42 are predicted to be located in the DNA minor groove 5' to the modified cytosine. Substitution of Ser41 with alanine (S41A) and cysteine (S41C) resulted in mutants with altered cleavage activity. All 19 Arg42 variants resulted in loss of endonuclease activity.

  8. Thermosensitivity of a barosensitive Saccharomyces cerevisiae mutant obtained by UV mutagenesis

    NASA Astrophysics Data System (ADS)

    Shigematsu, Toru; Nomura, Kazuki; Nasuhara, Yusuke; Ikarashi, Kenta; Nagai, Gen; Hirayama, Masao; Hayashi, Mayumi; Ueno, Shigeaki; Fujii, Tomoyuki

    2010-12-01

    Using UV mutagenesis, a high pressure (HP)-sensitive (barosensitive) mutant of Saccharomyces cerevisiae was obtained. The mutant strain a924E1 showed a significant loss of viability at HP levels of 175 to 250 MPa at 20 °C compared with the parent strain. This strain also showed a significant loss of viability following heat treatment at 50-58 °C at 0.1 MPa. These results showed that the mutation caused a significant thermosensitivity as well as barosensitivity. The activation volume and activation energy values for the inactivation of strain a924E1 were equivalent to those of the parent strain. This suggested that the mechanism for the HP and thermal inactivation reaction of strain a924E1 was basically the same as that of the parent strain. Strain a924E1 showed no deficiency in growth and fermentation ability as well as auxotrophic property. Although the identification of the genetic sites of mutation introduced is underway, these phenotypes are favorable for the application of HP treatment and heat-assisted HP treatment on fermentation control.

  9. Improved inhibitor tolerance in xylose-fermenting yeast Spathaspora passalidarum by mutagenesis and protoplast fusion.

    PubMed

    Hou, Xiaoru; Yao, Shuo

    2012-03-01

    The xylose-fermenting yeast Spathaspora passalidarum showed excellent fermentation performance utilizing glucose and xylose under anaerobic conditions. But this yeast is highly sensitive to the inhibitors such as furfural present in the pretreated lignocellulosic biomass. In order to improve the inhibitor tolerance of this yeast, a combination of UV mutagenesis and protoplast fusion was used to construct strains with improved performance. Firstly, UV-induced mutants were screened and selected for improved tolerance towards furfural. The most promised mutant, S. passalidarum M7, produced 50% more final ethanol than the wild-type strain in a synthetic xylose medium containing 2 g/l furfural. However, this mutant was unable to grow in a medium containing 75% liquid fraction of pretreated wheat straw (WSLQ), in which furfural and many other inhibitors were present. Hybrid yeast strains, obtained from fusion of the protoplasts of S. passalidarum M7 and a robust yeast, Saccharomyces cerevisiae ATCC 96581, were able to grow in 75% WSLQ and produce around 0.4 g ethanol/g consumed xylose. Among the selected hybrid strains, the hybrid FS22 showed the best fermentation capacity in 75% WSLQ. Phenotypic and partial molecular analysis indicated that S. passalidarum M7 was the dominant parental contributor to the hybrid. In summary, the hybrids are characterized by desired phenotypes derived from both parents, namely the ability to ferment xylose from S. passalidarum and an increased tolerance to inhibitors from S. cerevisiae ATCC 96581.

  10. Study of substrate specificity of human aromatase by site directed mutagenesis.

    PubMed

    Auvray, P; Nativelle, C; Bureau, R; Dallemagne, P; Séralini, G-E; Sourdaine, P

    2002-03-01

    Human aromatase is responsible for estrogen biosynthesis and is implicated, in particular, in reproduction and estrogen-dependent tumor proliferation. The molecular structure model is largely derived from the X-ray structure of bacterial cytochromes sharing only 15-20% identities with hP-450arom. In the present study, site directed mutagenesis experiments were performed to examine the role of K119, C124, I125, K130, E302, F320, D309, H475, D476, S470, I471 and I474 of aromatase in catalysis and for substrate binding. The catalytic properties of mutants, transfected in 293 cells, were evaluated using androstenedione, testosterone or nor-testosterone as substrates. In addition, inhibition profiles for these mutants with indane or indolizinone derivatives were obtained. Our results, together with computer modeling, show that catalytic properties of mutants vary in accordance with the substrate used, suggesting possible differences in substrates positioning within the active site. In this respect, importance of residues H475, D476 and K130 was discussed. These results allow us to hypothesize that E302 could be involved in the aromatization mechanism with nor-androgens, whereas D309 remains involved in androgen aromatization. This study highlights the flexibility of the substrate-enzyme complex conformation, and thus sheds new light on residues that may be responsible for substrate specificity between species or aromatase isoforms.

  11. Yellow fluorescent protein phiYFPv (Phialidium): structure and structure-based mutagenesis

    SciTech Connect

    Pletneva, Nadya V.; Pletnev, Vladimir Z. Souslova, Ekaterina; Chudakov, Dmitry M.; Lukyanov, Sergey; Martynov, Vladimir I.; Arhipova, Svetlena; Artemyev, Igor; Wlodawer, Alexander; Dauter, Zbigniew; Pletnev, Sergei

    2013-06-01

    The yellow fluorescent protein phiYFPv with improved folding has been developed from the spectrally identical wild-type phiYFP found in the marine jellyfish Phialidium. The yellow fluorescent protein phiYFPv (λ{sub em}{sup max} ≃ 537 nm) with improved folding has been developed from the spectrally identical wild-type phiYFP found in the marine jellyfish Phialidium. The latter fluorescent protein is one of only two known cases of naturally occurring proteins that exhibit emission spectra in the yellow–orange range (535–555 nm). Here, the crystal structure of phiYFPv has been determined at 2.05 Å resolution. The ‘yellow’ chromophore formed from the sequence triad Thr65-Tyr66-Gly67 adopts the bicyclic structure typical of fluorophores emitting in the green spectral range. It was demonstrated that perfect antiparallel π-stacking of chromophore Tyr66 and the proximal Tyr203, as well as Val205, facing the chromophore phenolic ring are chiefly responsible for the observed yellow emission of phiYFPv at 537 nm. Structure-based site-directed mutagenesis has been used to identify the key functional residues in the chromophore environment. The obtained results have been utilized to improve the properties of phiYFPv and its homologous monomeric biomarker tagYFP.

  12. US132 Cyclodextrin Glucanotransferase Engineering by Random Mutagenesis for an Anti-Staling Purpose.

    PubMed

    Jemli, Sonia; Jaoua, Mouna; Bejar, Samir

    2016-09-01

    The use of the cyclodextrin glucanotransferase (CGTase) of the US132 strain, which is an effective anti-staling agent, has been hampered by its high cyclization activity. Since that random mutagenesis using error-prone PCR is nowadays a method of choice for enzymes engineering, we have optimized this method by adjusting manganese concentration in order to obtain a high percentage of active CGTase mutants. Therefore, the amplification of the gene encoding the US132 CGTase was performed using a MnCl2 concentration ranging between 0 and 0.5 mM. The finding showed that a manganese concentration of 0.04 mM allowed for 90 % of active mutants. A simple method to rapidly screen the obtained mutants was also developed. After the examination of a small library (of less than 1000 clones), the active mutant named MJ13 was selected for a significant decrease in the cyclization activity, thereby showing a remarkable change in the enzyme specificity towards starch dextrinizing. Sequence analysis showed that MJ13 is a triple mutant with two mutations in the catalytic domain (K47E and S382P) and one substitution in the starch binding domain (N655S).

  13. Role of electrostatic potential in the in silico prediction of molecular bioactivation and mutagenesis.

    PubMed

    Ford, Kevin A

    2013-04-01

    Electrostatic potential (ESP) is a useful physicochemical property of a molecule that provides insights into inter- and intramolecular associations, as well as prediction of likely sites of electrophilic and nucleophilic metabolic attack. Knowledge of sites of metabolic attack is of paramount importance in DMPK research since drugs frequently fail in clinical trials due to the formation of bioactivated metabolites which are often difficult to measure experimentally due to their reactive nature and relatively short half-lives. Computational chemistry methods have proven invaluable in recent years as a means to predict and study bioactivated metabolites without the need for chemical syntheses, or testing on experimental animals. Additional molecular properties (heat of formation, heat of solvation and E(LUMO) - E(HOMO)) are discussed in this paper as complementary indicators of the behavior of metabolites in vivo. Five diverse examples are presented (acetaminophen, aniline/phenylamines, imidacloprid, nefazodone and vinyl chloride) which illustrate the utility of this multidimensional approach in predicting bioactivation, and in each case the predicted data agreed with experimental data described in the scientific literature. A further example of the usefulness of calculating ESP, in combination with the molecular properties mentioned above, is provided by an examination of the use of these parameters in providing an explanation for the sites of nucleophilic attack of the nucleic acid cytosine. Exploration of sites of nucleophilic attack of nucleic acids is important as adducts of DNA have the potential to result in mutagenesis.

  14. Rapid cloning of disease-resistance genes in plants using mutagenesis and sequence capture.

    PubMed

    Steuernagel, Burkhard; Periyannan, Sambasivam K; Hernández-Pinzón, Inmaculada; Witek, Kamil; Rouse, Matthew N; Yu, Guotai; Hatta, Asyraf; Ayliffe, Mick; Bariana, Harbans; Jones, Jonathan D G; Lagudah, Evans S; Wulff, Brande B H

    2016-06-01

    Wild relatives of domesticated crop species harbor multiple, diverse, disease resistance (R) genes that could be used to engineer sustainable disease control. However, breeding R genes into crop lines often requires long breeding timelines of 5-15 years to break linkage between R genes and deleterious alleles (linkage drag). Further, when R genes are bred one at a time into crop lines, the protection that they confer is often overcome within a few seasons by pathogen evolution. If several cloned R genes were available, it would be possible to pyramid R genes in a crop, which might provide more durable resistance. We describe a three-step method (MutRenSeq)-that combines chemical mutagenesis with exome capture and sequencing for rapid R gene cloning. We applied MutRenSeq to clone stem rust resistance genes Sr22 and Sr45 from hexaploid bread wheat. MutRenSeq can be applied to other commercially relevant crops and their relatives, including, for example, pea, bean, barley, oat, rye, rice and maize.

  15. Mechanisms of mutagenesis in human cells exposed to 55 MeV protons

    NASA Technical Reports Server (NTRS)

    Gauny, S.; Wiese, C.; Kronenberg, A.

    2001-01-01

    Protons represent the major type of charged particle radiation in spaceflight environments. The purpose of this study was to assess mutations arising in human lymphoid cells exposed to protons. Mutations were quantitated at the thymidine kinase (TK1) locus in cell lines derived from the same donor: TK6 cells (wt TP53) and WTK1 cells (mutant TP53). WTK1 cells were much more susceptible to mutagenesis following proton exposure than TK6 cells. Intragenic deletions were observed among early-arising TK1 mutants in TK6 cells, but not in WTK1 cells where all of the mutants arose by LOH. Deletion was the predominant mode of LOH in TK6 cells, while allelic recombination was the major mode of LOH in WTK1 cells. Deletions were of variable lengths, from <1 cM to 64 cM, while mutations that arose by allelic recombination often extended to the telomere. In summary, proton exposures elicited many types of mutations at an autosomal locus in human cells. Most involved large scale loss of genetic information, either through deletion or by recombination.

  16. Ligand Promiscuity of Aryl Hydrocarbon Receptor Agonists and Antagonists Revealed by Site-Directed Mutagenesis

    PubMed Central

    Soshilov, Anatoly A.

    2014-01-01

    The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that can be activated by structurally diverse chemicals. To examine the mechanisms responsible for the promiscuity in AhR ligand binding, we determined the effects of mutations within the AhR ligand-binding domain (LBD) on the activity of diverse AhR ligands. Site-directed mutagenesis identified Ile319 of the mouse AhR and, to a lesser extent, Phe318 as residues involved in ligand-selective modulation of AhR transformation using a panel of 12 AhR ligands. These ligands could be categorized into four distinct structurally related groups based on their ability to activate AhR mutants at position 319 in vitro. The mutation I319K was selectively activated by FICZ and not by other examined ligands in vitro and in cell culture. F318L and F318A mutations resulted in the conversion of AhR agonists β-naphthoflavone and 3-methylcholanthrene, respectively, into partial agonists/antagonists. Hsp90 binding to the AhR was decreased with several mutations and was inversely correlated with AhR ligand-binding promiscuity. Together, these data define overlapping amino acid residues within the AhR LBD involved in the selectivity of ligand binding, the agonist or antagonist mode of ligand binding, and hsp90 binding and provide insights into the ligand diversity of AhR activators. PMID:24591650

  17. Mutagenesis of aspartic acid-116 enhances the ribonucleolytic activity and angiogenic potency of angiogenin.

    PubMed Central

    Harper, J W; Vallee, B L

    1988-01-01

    Site-specific mutagenesis of the blood vessel-inducing protein angiogenin has been used to further explore both its homology to pancreatic ribonuclease and the functional roles of particular residues. Replacement of Asp-116 in angiogenin by either asparagine (D116N), alanine (D116A), or histidine (D116H) markedly enhances both its ribonucleolytic activity and angiogenic potency. Activity toward tRNA is 8-, 15-, and 18-fold greater than native angiogenin for D116N-, D116A-, and D116H-angiogenin, respectively. The enzymatic specificity of angiogenin, however, has been maintained. Thus, cleavage of 18S and 28S rRNA by the most active His-116 mutant yields the same pattern of polynucleotide products as from angiogenin, whereas there are only minor alterations in activity with cytidylyl(3',5')adenosine and uridylyl(3',5')-adenosine. Extensive biological assays on the chicken embryo chorioallantoic membrane demonstrate that D116H-angiogenin is one to two orders of magnitude more potent in inducing neovascularization than native angiogenin, which correlates well with enhanced enzymatic action. These results support the proposition that the enzymatic and angiogenic activities on angiogenin are interrelated. PMID:2459697

  18. Transposon mutagenesis in Mycoplasma hyopneumoniae using a novel mariner-based system for generating random mutations.

    PubMed

    Maglennon, Gareth A; Cook, Beth S; Deeney, Alannah S; Bossé, Janine T; Peters, Sarah E; Langford, Paul R; Maskell, Duncan J; Tucker, Alexander W; Wren, Brendan W; Rycroft, Andrew N

    2013-12-21

    Mycoplasma hyopneumoniae is the cause of enzootic pneumonia in pigs, a chronic respiratory disease associated with significant economic losses to swine producers worldwide. The molecular pathogenesis of infection is poorly understood due to the lack of genetic tools to allow manipulation of the organism and more generally for the Mycoplasma genus. The objective of this study was to develop a system for generating random transposon insertion mutants in M. hyopneumoniae that could prove a powerful tool in enabling the pathogenesis of infection to be unraveled. A novel delivery vector was constructed containing a hyperactive C9 mutant of the Himar1 transposase along with a mini transposon containing the tetracycline resistance cassette, tetM. M. hyopneumoniae strain 232 was electroporated with the construct and tetM-expressing transformants selected on agar containing tetracycline. Individual transformants contained single transposon insertions that were stable upon serial passages in broth medium. The insertion sites of 44 individual transformants were determined and confirmed disruption of several M. hyopneumoniae genes. A large pool of over 10 000 mutants was generated that should allow saturation of the M. hyopneumoniae strain 232 genome. This is the first time that transposon mutagenesis has been demonstrated in this important pathogen and could be generally applied for other Mycoplasma species that are intractable to genetic manipulation. The ability to generate random mutant libraries is a powerful tool in the further study of the pathogenesis of this important swine pathogen.

  19. Chemical Mutagenesis of Vaccinia DNA Topoisomerase Lysine-167 Provides Insights to Catalysis of DNA Transesterification

    PubMed Central

    Yakovleva, Lyudmila; Shuman, Stewart

    2013-01-01

    Vaccinia DNA topoisomerase IB (TopIB) relaxes supercoils by forming and resealing a covalent DNA-(3′-phosphotyrosyl274)-enzyme intermediate. Conserved active site side chains promote the attack of Tyr274 on the scissile phosphodiester via transition state stabilization and general acid catalysis. Two essential side chains, Lys167 and Arg130, act in concert to protonate and expel the 5′-O leaving group. Here we gained new insights to catalysis through chemical mutagenesis of Lys167. Changing Lys167 to cysteine crippled the DNA cleavage and religation transesterification steps (kcl = 4.3 × 10−4 s−1; krel = 9 × 10−4 s−1). The transesterification activities of the K167C enzyme were revived by in vitro alkylation with 2-bromoethylamine (kcl = 0.031 s−1; krel ≥ 0.4 s−1) and 3-bromopropylamine (kcl = 0.013 s−1; krel = 0.22 s−1), which convert the cysteine to γ-thialysine and γ-thiahomolysine, respectively. These chemically installed lysine analogs were more effective than a genetically programmed arginine-167 substitution characterized previously. The modest differences in the transesterification rates of the 2-bromoethylamine and 3-bromopropylamine-treated enzymes highlights that TopIB is tolerant of a longer homolysine side chain for assembly of the active site and formation of the transition state. PMID:23317114

  20. The inhibitory mechanism of Hal3 on the yeast Ppz1 phosphatase: A mutagenesis analysis.

    PubMed

    Molero, Cristina; Casado, Carlos; Ariño, Joaquín

    2017-08-18

    The Ser/Thr protein phosphatase (PPase) Ppz1 is an enzyme related to the ubiquitous type-1 PPases (PP1c) but found only in fungi. It is regulated by an inhibitory subunit, Hal3, which binds to its catalytic domain. Overexpression of Ppz1 is highly toxic for yeast cells, so its de-regulation has been proposed as a target for novel antifungal therapies. While modulation of PP1c by its many regulatory subunits has been extensively characterized, the manner by which Hal3 controls Ppz1 remains unknown. We have used error-prone PCR mutagenesis to construct a library of Ppz1 variants and developed a functional assay to identify mutations affecting the binding or/and the inhibitory capacity of Hal3. We have characterized diverse Ppz1 mutated versions in vivo and in vitro and found that, although they were clearly refractory to Hal3 inhibition, none of them exhibited significant reduction in Hal3 binding. Mapping the mutations strengthened the notion that Hal3 does not interact with Ppz1 through its RVxF-like motif (found in most PP1c regulators). In contrast, the most relevant mutations mapped to a conserved α-helix region used by mammalian Inhibitor-2 to regulate PP1c. Therefore, modulation of PP1c and Ppz1 by their subunits likely differs, but could share some structural features.

  1. [Enhancing glutamate decarboxylase activity by site-directed mutagenesis: an insight from Ramachandran plot].

    PubMed

    Ke, Piyu; Huang, Jun; Hu, Sheng; Zhao, Weirui; Lü, Changjiang; Yu, Kai; Lei, Yinlin; Wang, Jinbo; Mei, Lehe

    2016-01-01

    Glutamate decarboxylase (GAD) can catalyze the decarboxylation of glutamate into γ-aminobutyrate (GABA) and is the only enzyme of GABA biosynthesis. Improving GAD activity and thermostability will be helpful for the highly efficient biosynthesis of GABA. According to the Ramachandran plot information of GAD 1407 three-dimensional structure from Lactobacillus brevis CGMCC No. 1306, we identified the unstable site K413 as the mutation target, constructed the mutant GAD by site-directed mutagenesis and measured the thermostability and activity of the wide type and mutant GAD. Mutant K413A led to a remarkably slower inactivation rate, and its half-life at 50 °C reached 105 min which was 2.1-fold higher than the wild type GAD1407. Moreover, mutant K413I exhibited 1.6-fold higher activity in comparison with the wide type GAD1407, although it had little improvement in thermostability of GAD. Ramachandran plot can be considered as a potential approach to increase GAD thermostability and activity.

  2. [KIL-d] Protein Element Confers Antiviral Activity via Catastrophic Viral Mutagenesis.

    PubMed

    Suzuki, Genjiro; Weissman, Jonathan S; Tanaka, Motomasa

    2015-11-19

    Eukaryotic cells are targeted by pathogenic viruses and have developed cell defense mechanisms against viral infection. In yeast, the cellular extrachromosomal genetic element [KIL-d] alters killer activity of M double-stranded RNA killer virus and confers cell resistance against the killer virus. However, its underlying mechanism and the molecular nature of [KIL-d] are unknown. Here, we demonstrate that [KIL-d] is a proteinaceous prion-like aggregate with non-Mendelian cytoplasmic transmission. Deep sequencing analyses revealed that [KIL-d] selectively increases the rate of de novo mutation in the killer toxin gene of the viral genome, producing yeast harboring a defective mutant killer virus with a selective growth advantage over those with WT killer virus. These results suggest that a prion-like [KIL-d] element reprograms the viral replication machinery to induce mutagenesis and genomic inactivation via the long-hypothesized mechanism of "error catastrophe." The findings also support a role for prion-like protein aggregates in cellular defense and adaptation. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. 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

  4. Effects of protein engineering and rational mutagenesis on crystal lattice of single chain antibody fragments

    PubMed Central

    Kalyoncu, Sibel; Hyun, Jeongmin; Pai, Jennifer C.; Johnson, Jennifer L.; Entzminger, Kevin; Jain, Avni; Heaner, David P.; Morales, Ivan A.; Truskett, Thomas M.; Maynard, Jennifer A.; Lieberman, Raquel L.

    2014-01-01

    Protein crystallization is dependent upon, and sensitive to, the intermolecular contacts that assist in ordering proteins into a three dimensional lattice. Here we used protein engineering and mutagenesis to affect the crystallization of single chain antibody fragments (scFvs) that recognize the EE epitope (EYMPME) with high affinity. These hypercrystallizable scFvs are under development to assist difficult proteins, such as membrane proteins, in forming crystals, by acting as crystallization chaperones. Guided by analyses of intermolecular crystal lattice contacts, two second-generation anti-EE scFvs were produced, which bind to proteins with installed EE tags. Surprisingly, although non-complementarity determining region (CDR) lattice residues from the parent scFv framework remained unchanged through the processes of protein engineering and rational design, crystal lattices of the derivative scFvs differ. Comparison of energy calculations and the experimentally-determined lattice interactions for this basis set provides insight into the complexity of the forces driving crystal lattice choice and demonstrates the availability of multiple well-ordered surface features in our scFvs capable of forming versatile crystal contacts. PMID:24615866

  5. Rates and Mechanisms of Bacterial Mutagenesis from Maximum-Depth Sequencing

    PubMed Central

    Jee, Justin; Rasouly, Aviram; Shamovsky, Ilya; Akivis, Yonatan; Steinman, Susan; Mishra, Bud; Nudler, Evgeny

    2016-01-01

    In 1943, Luria and Delbrück used a phage resistance assay to establish spontaneous mutation as a driving force of microbial diversity1. Mutation rates are still studied using such assays, but these can only examine the small minority of mutations conferring survival in a particular condition. Newer approaches, such as long-term evolution followed by whole-genome sequencing 2, 3, may be skewed by mutational “hot” or “cold” spots 3, 4. Both approaches are affected by numerous caveats 5, 6, 7 (see Supplemental Information). We devise a method, Maximum-Depth Sequencing (MDS), to detect extremely rare variants in a population of cells through error-corrected, high-throughput sequencing. We directly measure locus-specific mutation rates in E. coli and show that they vary across the genome by at least an order of magnitude. Our data suggest that certain types of nucleotide misincorporation occur 104-fold more frequently than the basal rate of mutations, but are repaired in vivo. Our data also suggest specific mechanisms of antibiotic-induced mutagenesis, including downregulation of mismatch repair via oxidative stress; transcription-replication conflicts; and in the case of fluoroquinolones, direct damage to DNA. PMID:27338792

  6. 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

  7. Allosteric Features of KCNQ1 Gating Revealed by Alanine Scanning Mutagenesis

    PubMed Central

    Ma, Li-Juan; Ohmert, Iris; Vardanyan, Vitya

    2011-01-01

    Controlled opening and closing of an ion-selective pathway in response to changes of membrane potential is a fundamental feature of voltage-gated ion channels. In recent decades, various details of this process have been revealed with unprecedented precision based on studies of prototypic potassium channels. Though current scientific efforts are focused more on a thorough description of voltage-sensor movement, much less is known about the similarities and differences of the gating mechanisms among potassium channels. Here, we describe the peculiarities of the KCNQ1 gating process in parallel comparison to Shaker. We applied alanine scanning mutagenesis to the S4-S5 linker and pore region and followed the regularities of gating perturbations in KCNQ1. We found a fractional constitutive conductance for wild-type KCNQ1. This component increased significantly in mutants with considerably leftward-shifted steady-state activation curves. In contrast to Shaker, no correlation between V1/2 and Z parameters was observed for the voltage-dependent fraction of KCNQ1. Our experimental findings are explained by a simple allosteric gating scheme with voltage-driven and voltage-independent transitions. Allosteric features are discussed in the context of extreme gating adaptability of KCNQ1 upon interaction with KCNE β-subunits. PMID:21320432

  8. Increase in UV mutagenesis by heat stress on UV-irradiated E. coli cells.

    PubMed

    Saha, Swati; Basu, Tarakdas

    2012-06-01

    When leu- auxotrophs of Escherichia coli, after UV irradiation, were grown at temperatures between 30 and 47°C, the frequency of UV-induced mutation from leu- to leu+ revertant increased as the UV dose and the temperature increased. For cells exposed to a UV dose of 45 J/m2, the mutation frequency at 47°C was 1.9 times that at 30°C; for a dose of 90 J/m2, it was 3.25 times; and for 135 J/m2, it was 4.8 times. Similar enhancement of reversion frequency was observed when the irradiated cells were grown at 30°C in the presence of a heat shock inducer, ethanol (8% v/v). Heat shock-mediated enhancement of UV mutagenesis did not occur in an E. coli mutant sigma 32 (heat shock regulator protein), but sigma 32 overexpression in the mutant strain (transformed with a sigma 32-bearing plasmid) increased the UV-induced mutation frequency. These results suggest that heat stress alone has no mutagenic property, but when applied to UV-damaged cells, it enhances the UV-induced frequency of cell mutation.

  9. Damage, Repair, and Mutagenesis in Nuclear Genes after Mouse Forebrain Ischemia–Reperfusion

    PubMed Central

    Liu, Philip K.; Hsu, Chung Y.; Dizdaroglu, Miral; Floyd, Robert A.; Kow, Yoke W.; Karakaya, Asuman; Rabow, Lois E.; Cui, Jian-K.

    2009-01-01

    To determine whether oxidative stress after cerebral ischemia–reperfusion affects genetic stability in the brain, we studied mutagenesis after forebrain ischemia–reperfusion in Big Blue transgenic mice (male C57BL/6 strain) containing a reporter lacI gene, which allows detection of mutation frequency. The frequency of mutation in this reporter lacI gene increased from 1.5 to 7.7 (per 100,000) in cortical DNA after 30 min of forebrain ischemia and 8 hr of reperfusion and remained elevated at 24 hr reperfusion. Eight DNA lesions that are characteristic of DNA damage mediated by free radicals were detected. Four mutagenic lesions (2,6-diamino-4-hydroxy-5-formamidopyrimidine, 8-hydroxyadenine, 5-hydroxycytosine, and 8-hydroxyguanine) examined by gas chromatography/mass spectrometry and one corresponding 8-hydroxy-2′-deoxyguanosine by a method of HPLC with electrochemical detection increased in cortical DNA two- to fourfold (p < 0.05) during 10–20 min of reperfusion. The damage to γ-actin and DNA polymerase-β genes was detected within 20 min of reperfusion based on the presence of formamidopyrimidine DNA N-glycosylase-sensitive sites. These genes became resistant to the glycosylase within 4–6 hr of reperfusion, suggesting a reduction in DNA damage and presence of DNA repair in nuclear genes. These results suggest that nuclear genes could be targets of free radicals. PMID:8824320

  10. New mouse models for metabolic bone diseases generated by genome-wide ENU mutagenesis.

    PubMed

    Sabrautzki, Sibylle; Rubio-Aliaga, Isabel; Hans, Wolfgang; Fuchs, Helmut; Rathkolb, Birgit; Calzada-Wack, Julia; Cohrs, Christian M; Klaften, Matthias; Seedorf, Hartwig; Eck, Sebastian; Benet-Pagès, Ana; Favor, Jack; Esposito, Irene; Strom, Tim M; Wolf, Eckhard; Lorenz-Depiereux, Bettina; Hrabě de Angelis, Martin

    2012-08-01

    Metabolic bone disorders arise as primary diseases or may be secondary due to a multitude of organ malfunctions. Animal models are required to understand the molecular mechanisms responsible for the imbalances of bone metabolism in disturbed bone mineralization diseases. Here we present the isolation of mutant mouse models for metabolic bone diseases by phenotyping blood parameters that target bone turnover within the large-scale genome-wide Munich ENU Mutagenesis Project. A screening panel of three clinical parameters, also commonly used as biochemical markers in patients with metabolic bone diseases, was chosen. Total alkaline phosphatase activity and total calcium and inorganic phosphate levels in plasma samples of F1 offspring produced from ENU-mutagenized C3HeB/FeJ male mice were measured. Screening of 9,540 mice led to the identification of 257 phenodeviants of which 190 were tested by genetic confirmation crosses. Seventy-one new dominant mutant lines showing alterations of at least one of the biochemical parameters of interest were confirmed. Fifteen mutations among three genes (Phex, Casr, and Alpl) have been identified by positional-candidate gene approaches and one mutation of the Asgr1 gene, which was identified by next-generation sequencing. All new mutant mouse lines are offered as a resource for the scientific community.

  11. Posttranslational arginylation enzyme Ate1 affects DNA mutagenesis by regulating stress response

    PubMed Central

    Kumar, Akhilesh; Birnbaum, Michael D; Patel, Devang M; Morgan, William M; Singh, Jayanti; Barrientos, Antoni; Zhang, Fangliang

    2016-01-01

    Arginyltransferase 1 (Ate1) mediates protein arginylation, a poorly understood protein posttranslational modification (PTM) in eukaryotic cells. Previous evidence suggest a potential involvement of arginylation in stress response and this PTM was traditionally considered anti-apoptotic based on the studies of individual substrates. However, here we found that arginylation promotes cell death and/or growth arrest, depending on the nature and intensity of the stressing factor. Specifically, in yeast, mouse and human cells, deletion or downregulation of the ATE1 gene disrupts typical stress responses by bypassing growth arrest and suppressing cell death events in the presence of disease-related stressing factors, including oxidative, heat, and osmotic stresses, as well as the exposure to heavy metals or radiation. Conversely, in wild-type cells responding to stress, there is an increase of cellular Ate1 protein level and arginylation activity. Furthermore, the increase of Ate1 protein directly promotes cell death in a manner dependent on its arginylation activity. Finally, we found Ate1 to be required to suppress mutation frequency in yeast and mammalian cells during DNA-damaging conditions such as ultraviolet irradiation. Our study clarifies the role of Ate1/arginylation in stress response and provides a new mechanism to explain the link between Ate1 and a variety of diseases including cancer. This is also the first example that the modulation of the global level of a PTM is capable of affecting DNA mutagenesis. PMID:27685622

  12. Viable transmembrane region mutants of bacteriophage M13 coat protein prepared by site-directed mutagenesis.

    PubMed

    Li, Z; Deber, C M

    1991-10-31

    Bacteriophage M13 coat protein - a 50-residue protein located at the E. coli host membrane during phage reproduction - is subjected to cytoplasmic, membrane-bound, and DNA-interactive environments during the phage life cycle. In research to examine the specific features of primary/secondary structure in the effective transmembrane (TM) region of the protein (residues 21-39: YIGYAWAMVVVIVGATIGI) which modulate its capacity to respond conformationally to the progressive influences of these varying environments, we have prepared over two dozen viable mutant phages with alterations in their coat protein TM regions. Mutants were obtained through use of site-directed mutagenesis techniques in combination with three "randomized" oligonucleotides which spanned the TM region. No subcloning was required. Among mutations observed were those in which each of the four TM Val residues was changed to Ala, and several with increased Ser or Thr content, including one double Ser mutant (G23S-A25S). Polar substitutions arising at Gly23 and Tyr24-including G23D, Y24H, Y24D and Y24N-suggested that this local segment resides external to the host membrane. Milligram quantities of mutant coat proteins are obtained by growing M13 mutant phages in liter preparations, with isotopic (e.g., 13C) labelling at desired sites, for subsequent characterization and conformational analysis in membrane-mimetic media.

  13. Improving Erythritol Production of Aureobasidium pullulans from Xylose by Mutagenesis and Medium Optimization.

    PubMed

    Guo, Jian; Li, Junxia; Chen, Yefu; Guo, Xuewu; Xiao, Dongguang

    2016-10-01

    The biotransformation of xylose, the second most abundant sugar, has been a hot topic in recent years. In this work, Aureobasidium pullulans CGMCC3.0837 was subjected to UV mutagenesis to improve its erythritol production from xylose. The erythritol production of the obtained mutant ER35 was 50.92 % (17.28 g/L) higher than that of the parent strain. Response surface methodology was applied to optimize the medium composition. Yeast extract, KH2PO4, and citric acid were the key factors influencing erythritol synthesis, and the optimal concentrations were 17.82, 0.76, and 6.60 g/L, respectively. Under the optimized conditions, 31.75 and 31.42 g/L erythritol were obtained in shake flasks and in a 5-L fermentor, respectively. ER35 also showed a good consuming ability on xylose mother liquor with a final erythritol production of 26.35 g/L. This report provided insights into the potential of A. pullulans for the production of erythritol using xylose as a carbon source.

  14. The significance of disulfide bonding in biological activity of HB-EGF, a mutagenesis approach

    SciTech Connect

    Hoskins, J.T.; Zhou, Z.; Harding, P.A.

    2008-10-31

    A site-directed mutagenesis approach was taken to disrupt each of 3 disulfide bonds within human HB-EGF by substituting serine for both cysteine residues that contribute to disulfide bonding. Each HB-EGF disulfide analogue (HB-EGF-Cys/Ser{sub 108/121}, HB-EGF-Cys/Ser{sub 116/132}, and HB-EGF-Cys/Ser{sub 134/143}) was cloned under the regulation of the mouse metallothionein (MT) promoter and stably expressed in mouse fibroblasts. HB-EGF immunoreactive proteins with M{sub r} of 6.5, 21 and 24 kDa were observed from lysates of HB-EGF and each HB-EGF disulfide analogue. HB-EGF immunohistochemical analyses of each HB-EGF stable cell line demonstrated ubiquitous protein expression except HB-EGF-Cys/Ser{sub 108/121} and HB-EGF-Cys/Ser{sub 116/132} stable cell lines which exhibited accumulated expression immediately outside the nucleus. rHB-EGF, HB-EGF, and HB-EGF{sub 134/143} proteins competed with {sup 125}I-EGF in an A431 competitive binding assay, whereas HB-EGF-Cys/Ser{sub 108/121} and HB-EGF-Cys/Ser{sub 116/132} failed to compete. Each HB-EGF disulfide analogue lacked the ability to stimulate tyrosine phosphorylation of the 170 kDa EGFR. These results suggest that HB-EGF-Cys/Ser{sub 134/143} antagonizes EGFRs.

  15. Identification of virulence determinants for endocarditis in Streptococcus sanguinis by signature-tagged mutagenesis.

    PubMed

    Paik, Sehmi; Senty, Lauren; Das, Sankar; Noe, Jody C; Munro, Cindy L; Kitten, Todd

    2005-09-01

    Streptococcus sanguinis is a gram-positive, facultative anaerobe and a normal inhabitant of the human oral cavity. It is also one of the most common agents of infective endocarditis, a serious endovascular infection. To identify virulence factors for infective endocarditis, signature-tagged mutagenesis (STM) was applied to the SK36 strain of S. sanguinis, whose genome is being sequenced. STM allows the large-scale creation, in vivo screening, and recovery of a series of mutants with altered virulence. Screening of 800 mutants by STM identified 38 putative avirulent and 5 putative hypervirulent mutants. Subsequent molecular analysis of a subset of these mutants identified genes encoding undecaprenol kinase, homoserine kinase, anaerobic ribonucleotide reductase, adenylosuccinate lyase, and a hypothetical protein. Virulence reductions ranging from 2-to 150-fold were confirmed by competitive index assays. One putatively hypervirulent strain with a transposon insertion in an intergenic region was identified, though increased virulence was not confirmed in competitive index assays. All mutants grew comparably to SK36 in aerobic broth culture except for the homoserine kinase mutant. Growth of this mutant was restored by the addition of threonine to the medium. Mutants containing an insertion or in-frame deletion in the anaerobic ribonucleotide reductase gene failed to grow under strictly anaerobic conditions. The results suggest that housekeeping functions such as cell wall synthesis, amino acid and nucleic acid synthesis, and the ability to survive under anaerobic conditions are important virulence factors in S. sanguinis endocarditis.

  16. Targeted mutagenesis of dengue virus type 2 replicon RNA by yeast in vivo recombination.

    PubMed

    Manzano, Mark; Padmanabhan, Radhakrishnan

    2014-01-01

    The use of cDNA infectious clones or subgenomic replicons is indispensable in studying flavivirus biology. Mutating nucleotides or amino acid residues gives important clues to their function in the viral life cycle. However, a major challenge to the establishment of a reverse genetics system for flaviviruses is the instability of their nucleotide sequences in Escherichia coli. Thus, direct cloning using conventional restriction enzyme-based procedures usually leads to unwanted rearrangements of the construct. In this chapter, we discuss a cloning strategy that bypasses traditional cloning procedures. We take advantage of the observations from previous studies that (1) unstable sequences in bacteria can be cloned in eukaryotic systems and (2) Saccharomyces cerevisiae has a well-studied genetics system to introduce sequences using homologous recombination. We describe a protocol to perform targeted mutagenesis in a subgenomic dengue virus 2 replicon. Our method makes use of homologous recombination in yeast using a linearized replicon and a PCR product containing the desired mutation. Constructs derived from this method can be propagated in E. coli with improved stability. Thus, yeast in vivo recombination provides an excellent strategy to genetically engineer flavivirus infectious clones or replicons because this system is compatible with inherently unstable sequences of flaviviruses and is not restricted by the limitations of traditional cloning procedures.

  17. Mutagenesis and redox partners analysis of the P450 fatty acid decarboxylase OleTJE.

    PubMed

    Fang, Bo; Xu, Huifang; Liu, Yi; Qi, Fengxia; Zhang, Wei; Chen, Hui; Wang, Cong; Wang, Yilin; Yang, Wenxia; Li, Shengying

    2017-03-09

    The cytochrome P450 enzyme OleTJE from Jeotgalicoccus sp. ATCC 8456 is capable of converting free long-chain fatty acids into α-alkenes via one-step oxidative decarboxylation in presence of H2O2 as cofactor or using redox partner systems. This enzyme has attracted much attention due to its intriguing but unclear catalytic mechanism and potential application in biofuel production. Here, we investigated the functionality of a select group of residues (Arg245, Cys365, His85, and Ile170) in the active site of OleTJE through extensive mutagenesis analysis. The key roles of these residues for catalytic activity and reaction type selectivity were identified. In addition, a range of heterologous redox partners were found to be able to efficiently support the decarboxylation activity of OleTJE. The best combination turned out to be SeFdx-6 (ferredoxin) from Synechococcus elongatus PCC 7942 and CgFdR-2 (ferredoxin reductase) from Corynebacterium glutamicum ATCC 13032, which gave the highest myristic acid conversion rate of 94.4%. Moreover, Michaelis-Menton kinetic parameters of OleTJE towards myristic acid were determined.

  18. Mammalian mutagenesis using a highly mobile somatic Sleeping Beauty transposon system.

    PubMed

    Dupuy, Adam J; Akagi, Keiko; Largaespada, David A; Copeland, Neal G; Jenkins, Nancy A

    2005-07-14

    Transposons have provided important genetic tools for functional genomic screens in lower eukaryotes but have proven less useful in higher eukaryotes because of their low transposition frequency. Here we show that Sleeping Beauty (SB), a member of the Tc1/mariner class of transposons, can be mobilized in mouse somatic cells at frequencies high enough to induce embryonic death and cancer in wild-type mice. Tumours are aggressive, with some animals developing two or even three different types of cancer within a few months of birth. The tumours result from SB insertional mutagenesis of cancer genes, thus facilitating the identification of genes and pathways that induce disease. SB transposition can easily be controlled to mutagenize any target tissue and can therefore, in principle, be used to induce many of the cancers affecting humans, including those for which little is known about the aetiology. The uses of SB are also not restricted to the mouse and could potentially be used for forward genetic screens in any higher eukaryote in which transgenesis is possible.

  19. BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis

    PubMed Central

    Canver, Matthew C.; Smith, Elenoe C.; Sher, Falak; Pinello, Luca; Sanjana, Neville E.; Shalem, Ophir; Chen, Diane D.; Schupp, Patrick G.; Vinjamur, Divya S.; Garcia, Sara P.; Luc, Sidinh; Kurita, Ryo; Nakamura, Yukio; Fujiwara, Yuko; Maeda, Takahiro; Yuan, Guo-Cheng; Feng, Zhang; Orkin, Stuart H.; Bauer, Daniel E.

    2015-01-01

    Summary Enhancers, critical determinants of cellular identity, are commonly identified by correlative chromatin marks and gain-of-function potential, though only loss-of-function studies can demonstrate their requirement in the native genomic context. Previously we identified an erythroid enhancer of BCL11A, subject to common genetic variation associated with fetal hemoglobin (HbF) level, whose mouse ortholog is necessary for erythroid BCL11A expression. Here we develop pooled CRISPR-Cas9 guide RNA libraries to perform in situ saturating mutagenesis of the human and mouse enhancers. This approach reveals critical minimal features and discrete vulnerabilities of these enhancers. Despite conserved function of the composite enhancers, their architecture diverges. The crucial human sequences appear primate-specific. Through editing of primary human progenitors and mouse transgenesis, we validate the BCL11A erythroid enhancer as a target for HbF reinduction. The detailed enhancer map will inform therapeutic genome editing. The screening approach described here is generally applicable to functional interrogation of noncoding genomic elements. PMID:26375006

  20. Effects of site-specific mutagenesis of tyrosine 105 in a class A beta-lactamase.

    PubMed Central

    Escobar, W A; Miller, J; Fink, A L

    1994-01-01

    Tyr-105 is a conserved residue in the Class A beta-lactamases and is in close proximity to the active-site. Tyr-105 in beta-lactamase from Bacillus licheniformis was converted into Phe by site-directed mutagenesis. This mutation caused no significant effect on the structure of the enzyme and had only small effects on the catalytic properties. In particular, in comparison to the wild-type, kcat. for benzylpenicillin was increased slightly, whereas it was decreased slightly for several other substrates. For each substrate examined, Km increased 3-4-fold in the mutant compared with the wild-type enzyme. Examination of the effect of pH on the catalytic reaction revealed only small perturbations in the pK values for the acidic and basic limbs of the kcat./Km pH profiles due to the mutation. Overall effects of the Y105F substitution on the catalytic efficiency for different penicillin and cephalosporin substrates ranged from 14% to 56% compared with the wild-type activity. We conclude that Tyr-105 is not an essential residue for beta-lactamase catalysis, but does contribute to substrate binding. PMID:7980417

  1. Sleeping Beauty insertional mutagenesis in mice identifies drivers of steatosis-associated hepatic tumor.

    PubMed

    Tschida, Barbara R; Temiz, Nuri A; Kuka, Timothy P; Lee, Lindsey A; Riordan, Jesse D; Tierrablanca, Carlos A; Hullsiek, Robert; Wagner, Sandra; Hudson, Wendy A; Linden, Michael A; Amin, Khalid; Beckmann, Pauline J; Heuer, Rachel A; Sarver, Aaron L; Yang, Ju Dong; Roberts, Lewis R; Nadeau, Joseph H; Dupuy, Adam J; Keng, Vincent W; Largaespada, David

    2017-10-09

    Hepatic steatosis is a strong risk factor for the development of hepatocellular carcinoma (HCC), yet little is known about the molecular pathology associated with this factor. In this study, we performed a forward genetic screen using Sleeping Beauty (SB) transposon insertional mutagenesis in mice treated to induce hepatic steatosis, and compared the results to human HCC data. In humans, we determined that steatosis increased the proportion of female HCC patients, a pattern also reflected in mice. Our genetic screen identified 203 candidate steatosis-associated HCC genes, many of which are altered in human HCC and are members of established HCC-driving signaling pathways. The protein kinase A/cyclic AMP signaling pathway was altered frequently in mouse and human steatosis-associated HCC. We found that activated PKA expression drove steatosis-specific liver tumorigenesis in a mouse model. Another candidate HCC driver, the N-acetyltransferase NAT10, which we found to be overexpressed in human steatosis-associated HCC and associated with decreased survival in human HCC, also drove liver tumorigenesis in a steatotic mouse model. This study identifies genes and pathways promoting HCC that may represent novel targets for prevention and treatment in the context of hepatic steatosis, an area of rapidly growing clinical significance. Copyright ©2017, American Association for Cancer Research.

  2. Mouse models of cancer: Sleeping Beauty transposons for insertional mutagenesis screens and reverse genetic studies.

    PubMed

    Tschida, Barbara R; Largaespada, David A; Keng, Vincent W

    2014-03-01

    The genetic complexity and heterogeneity of cancer has posed a problem in designing rationally targeted therapies effective in a large proportion of human cancer. Genomic characterization of many cancer types has provided a staggering amount of data that needs to be interpreted to further our understanding of this disease. Forward genetic screening in mice using Sleeping Beauty (SB) based insertional mutagenesis is an effective method for candidate cancer gene discovery that can aid in distinguishing driver from passenger mutations in human cancer. This system has been adapted for unbiased screens to identify drivers of multiple cancer types. These screens have already identified hundreds of candidate cancer-promoting mutations. These can be used to develop new mouse models for further study, which may prove useful for therapeutic testing. SB technology may also hold the key for rapid generation of reverse genetic mouse models of cancer, and has already been used to model glioblastoma and liver cancer. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. Enhancement of Biomass and Lipid Productivities of Water Surface-Floating Microalgae by Chemical Mutagenesis.

    PubMed

    Nojima, Daisuke; Ishizuka, Yuki; Muto, Masaki; Ujiro, Asuka; Kodama, Fumito; Yoshino, Tomoko; Maeda, Yoshiaki; Matsunaga, Tadashi; Tanaka, Tsuyoshi

    2017-05-27

    Water surface-floating microalgae have great potential for biofuel applications due to the ease of the harvesting process, which is one of the most problematic steps in conventional microalgal biofuel production. We have collected promising water surface-floating microalgae and characterized their capacity for biomass and lipid production. In this study, we performed chemical mutagenesis of two water surface-floating microalgae to elevate productivity. Floating microalgal strains AVFF007 and FFG039 (tentatively identified as Botryosphaerella sp. and Chlorococcum sp., respectively) were exposed to ethyl methane sulfonate (EMS) or 1-methyl-3-nitro-1-nitrosoguanidine (MNNG), and pale green mutants (PMs) were obtained. The most promising FFG039 PM formed robust biofilms on the surface of the culture medium, similar to those formed by wild type strains, and it exhibited 1.7-fold and 1.9-fold higher biomass and lipid productivities than those of the wild type. This study indicates that the chemical mutation strategy improves the lipid productivity of water surface-floating microalgae without inhibiting biofilm formation and floating ability.

  4. Enhancement of Biomass and Lipid Productivities of Water Surface-Floating Microalgae by Chemical Mutagenesis

    PubMed Central

    Nojima, Daisuke; Ishizuka, Yuki; Muto, Masaki; Ujiro, Asuka; Kodama, Fumito; Yoshino, Tomoko; Maeda, Yoshiaki; Matsunaga, Tadashi; Tanaka, Tsuyoshi

    2017-01-01

    Water surface-floating microalgae have great potential for biofuel applications due to the ease of the harvesting process, which is one of the most problematic steps in conventional microalgal biofuel production. We have collected promising water surface-floating microalgae and characterized their capacity for biomass and lipid production. In this study, we performed chemical mutagenesis of two water surface-floating microalgae to elevate productivity. Floating microalgal strains AVFF007 and FFG039 (tentatively identified as Botryosphaerella sp. and Chlorococcum sp., respectively) were exposed to ethyl methane sulfonate (EMS) or 1-methyl-3-nitro-1-nitrosoguanidine (MNNG), and pale green mutants (PMs) were obtained. The most promising FFG039 PM formed robust biofilms on the surface of the culture medium, similar to those formed by wild type strains, and it exhibited 1.7-fold and 1.9-fold higher biomass and lipid productivities than those of the wild type. This study indicates that the chemical mutation strategy improves the lipid productivity of water surface-floating microalgae without inhibiting biofilm formation and floating ability. PMID:28555001

  5. Mutagenesis of the downstream region of the Escherichia coli hns promoter.

    PubMed

    Giangrossi, M; Gualerzi, C O; Pon, C L

    2001-02-01

    The promoter of hns, the structural gene for the abundant nucleoid-associated protein H-NS of Escherichia coli, contains, downstream of the initiation site, two four bp-long 'CG clamps', one of which overlaps the potential target sequence (CCAAT) of CspA, the cold-shock transcriptional enhancer of this gene. To establish the role of these potential regulatory signals during the cold-shock activation of hns, the CCCCAAT sequence has been subjected to mutagenesis, weakening the strength of the CG clamp and scrambling or inverting the CCAAT sequence. The resulting mutated hns promoters were placed in front of a reporter gene (cat) and their activity was studied in cells subjected to cold-shock under conditions where the increase in the concentration of CspA is either large or small. Our results allow us to conclude that although not essential, the CCCCAAT sequence, mainly due to the presence of the CG clamp, may play an important role in the CspA-mediated regulation of hns expression at both transcriptional and translational levels.

  6. BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis.

    PubMed

    Canver, Matthew C; Smith, Elenoe C; Sher, Falak; Pinello, Luca; Sanjana, Neville E; Shalem, Ophir; Chen, Diane D; Schupp, Patrick G; Vinjamur, Divya S; Garcia, Sara P; Luc, Sidinh; Kurita, Ryo; Nakamura, Yukio; Fujiwara, Yuko; Maeda, Takahiro; Yuan, Guo-Cheng; Zhang, Feng; Orkin, Stuart H; Bauer, Daniel E

    2015-11-12

    Enhancers, critical determinants of cellular identity, are commonly recognized by correlative chromatin marks and gain-of-function potential, although only loss-of-function studies can demonstrate their requirement in the native genomic context. Previously, we identified an erythroid enhancer of human BCL11A, subject to common genetic variation associated with the fetal haemoglobin level, the mouse orthologue of which is necessary for erythroid BCL11A expression. Here we develop pooled clustered regularly interspaced palindromic repeat (CRISPR)-Cas9 guide RNA libraries to perform in situ saturating mutagenesis of the human and mouse enhancers. This approach reveals critical minimal features and discrete vulnerabilities of these enhancers. Despite conserved function of the composite enhancers, their architecture diverges. The crucial human sequences appear to be primate-specific. Through editing of primary human progenitors and mouse transgenesis, we validate the BCL11A erythroid enhancer as a target for fetal haemoglobin reinduction. The detailed enhancer map will inform therapeutic genome editing, and the screening approach described here is generally applicable to functional interrogation of non-coding genomic elements.

  7. Improving the neutral phytase activity from Bacillus amyloliquefaciens DSM 1061 by site-directed mutagenesis.

    PubMed

    Xu, Wei; Shao, Rong; Wang, Zupeng; Yan, Xiuhua

    2015-03-01

    Neutral phytase is used as a feed additive for degradation of anti-nutritional phytate in aquatic feed industry. Site-directed mutagenesis of Bacillus amyloliquefaciens DSM 1061 phytase was performed with an aim to increase its activity. Mutation residues were chosen based on multiple sequence alignments and structure analysis of neutral phytsaes from different microorganisms. The mutation sites on surface (D148E, S197E and N156E) and around the active site (D52E) of phytase were selected. Analysis of the phytase variants showed that the specific activities of mutants D148E and S197E remarkably increased by about 35 and 13% over a temperature range of 40-75 °C at pH 7.0, respectively. The k cat of mutants D148E and S197E were 1.50 and 1.25 times than that of the wild-type phytase, respectively. Both D148E and S197E showed much higher thermostability than that of the wild-type phytase. However, mutants N156E and D52E led to significant loss of specific activity of the enzyme. Structural analysis revealed that these mutations may affect conformation of the active site of phytase. The present mutant phytases D148E and S197E with increased activities and thermostabilities have application potential as additives in aquaculture feed.

  8. Site-directed mutagenesis of lysine-329 of ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

    SciTech Connect

    Soper, T.S.; Larimer, F.W.; Mural, R.J.; Machanoff, R.; Foote, R.S.; Lee, E.H.; Hartman, F.C.

    1987-05-01

    A variety of data suggest that Lys-329 of the title enzyme serves a catalytic function. To test this postulate, Lys-329 has been replaced with Gly, Ala, Ser, Cys, Arg, or Glu by a technique of site-directed mutagenesis that utilizes a suitably gapped plasmid carrying the target gene. The mutant proteins were produced in E. coli JM107 and purified to near homogeneity by immunoaffinity chromatography; they were shown to be dimers, like the wild-type enzyme, by gel electrophoresis. Hence, these amino acid substitutions are compatible with proper folding and association of subunits. The purified mutant proteins do not exhibit detectable enzyme activity nor do they form a stable quaternary complex with Mg/sup 2 +/, CO/sub 2/, and a transition-state analog as observed for wild-type enzyme. However, based on ligand-selective elution of the mutant proteins from an affinity matrix (green A agarose) they do retain the ability to bind substrate analogs. These results support an absolute essentiality of Lys-329; its precise function may be illuminated by further characterization of the mutant proteins.

  9. 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.

  10. Stress-induced mutagenesis: Stress diversity facilitates the persistence of mutator genes.

    PubMed

    Lukačišinová, Marta; Novak, Sebastian; Paixão, Tiago

    2017-07-01

    Mutator strains are expected to evolve when the availability and effect of beneficial mutations are high enough to counteract the disadvantage from deleterious mutations that will inevitably accumulate. As the population becomes more adapted to its environment, both availability and effect of beneficial mutations necessarily decrease and mutation rates are predicted to decrease. It has been shown that certain molecular mechanisms can lead to increased mutation rates when the organism finds itself in a stressful environment. While this may be a correlated response to other functions, it could also be an adaptive mechanism, raising mutation rates only when it is most advantageous. Here, we use a mathematical model to investigate the plausibility of the adaptive hypothesis. We show that such a mechanism can be mantained if the population is subjected to diverse stresses. By simulating various antibiotic treatment schemes, we find that combination treatments can reduce the effectiveness of second-order selection on stress-induced mutagenesis. We discuss the implications of our results to strategies of antibiotic therapy.

  11. Processing closely spaced lesions during Nucleotide Excision Repair triggers mutagenesis in E. coli.

    PubMed

    Janel-Bintz, Régine; Napolitano, Rita L; Isogawa, Asako; Fujii, Shingo; Fuchs, Robert P

    2017-07-01

    It is generally assumed that most point mutations are fixed when damage containing template DNA undergoes replication, either right at the fork or behind the fork during gap filling. Here we provide genetic evidence for a pathway, dependent on Nucleotide Excision Repair, that induces mutations when processing closely spaced lesions. This pathway, referred to as Nucleotide Excision Repair-induced Mutagenesis (NERiM), exhibits several characteristics distinct from mutations that occur within the course of replication: i) following UV irradiation, NER-induced mutations are fixed much more rapidly (t ½ ≈ 30 min) than replication dependent mutations (t ½ ≈ 80-100 min) ii) NERiM specifically requires DNA Pol IV in addition to Pol V iii) NERiM exhibits a two-hit dose-response curve that suggests processing of closely spaced lesions. A mathematical model let us define the geometry (infer the structure) of the toxic intermediate as being formed when NER incises a lesion that resides in close proximity of another lesion in the complementary strand. This critical NER intermediate requires Pol IV / Pol II for repair, it is either lethal if left unrepaired or mutation-prone when repaired. Finally, NERiM is found to operate in stationary phase cells providing an intriguing possibility for ongoing evolution in the absence of replication.

  12. Essential genes in the infection model of Pseudomonas aeruginosa-PCR-based signature-tagged mutagenesis.

    PubMed

    Kukavica-Ibrulj, Irena; Levesque, Roger C

    2015-01-01

    PCR-based signature tagged mutagenesis is an "en masse" screening technique based upon unique oligonucleotide tags (molecular barcodes) for identification of genes that will diminish or enhance maintenance of an organism in a specific ecological niche or environment. PCR-based STM applied to Pseudomonas aeruginosa permitted the identification of genes essential for in vivo maintenance by transposon insertion and negative selection in a mixed population of bacterial mutants. The innovative adaptations and refinement of the technology presented here with P. aeruginosa STM mutants selected in the rat model of chronic lung infection have given critical information about genes essential for causing a chronic infection and a wealth of information about biological processes in vivo. The additional use of competitive index analysis for measurement of the level of virulence in vivo, microarray-based screening of selected prioritized STM mutants coupled to metabolomics analysis can now be attempted systematically on a genomic scale. PCR-based STM and combined whole-genome methods can also be applied to any organism having selectable phenotypes for screening.

  13. Lentiviral Vector-based Insertional Mutagenesis Identifies Genes Involved in the Resistance to Targeted Anticancer Therapies

    PubMed Central

    Ranzani, Marco; Annunziato, Stefano; Calabria, Andrea; Brasca, Stefano; Benedicenti, Fabrizio; Gallina, Pierangela; Naldini, Luigi; Montini, Eugenio

    2014-01-01

    The high transduction efficiency of lentiviral vectors in a wide variety of cells makes them an ideal tool for forward genetics screenings addressing issues of cancer research. Although molecular targeted therapies have provided significant advances in tumor treatment, relapses often occur by the expansion of tumor cell clones carrying mutations that confer resistance. Identification of the culprits of anticancer drug resistance is fundamental for the achievement of long-term response. Here, we developed a new lentiviral vector-based insertional mutagenesis screening to identify genes that confer resistance to clinically relevant targeted anticancer therapies. By applying this genome-wide approach to cell lines representing two subtypes of HER2+ breast cancer, we identified 62 candidate lapatinib resistance genes. We validated the top ranking genes, i.e., PIK3CA and PIK3CB, by showing that their forced expression confers resistance to lapatinib in vitro and found that their mutation/overexpression is associated to poor prognosis in human breast tumors. Then, we successfully applied this approach to the identification of erlotinib resistance genes in pancreatic cancer, thus showing the intrinsic versatility of the approach. The acquired knowledge can help identifying combinations of targeted drugs to overcome the occurrence of resistance, thus opening new horizons for more effective treatment of tumors. PMID:25195596

  14. Identification of mutations from phenotype-driven ENU mutagenesis in mouse chromosome 7.

    PubMed

    Culiat, Cymbeline T; Klebig, Mitchell L; Liu, Zhaowei; Monroe, Heidi; Stanford, Beverly; Desai, Jayashree; Tandan, Samvit; Hughes, Lori; Kerley, Marilyn K; Carpenter, Donald A; Johnson, Dabney K; Rinchik, Eugene M; Li, Qingbo

    2005-08-01

    We have used the new high-throughput mutation-scanning technique temperature-gradient capillary electrophoresis (TGCE) for the identification of point mutations induced by N-ethyl-N-nitrosourea (ENU) in the mouse genome. TGCE detects the presence of heteroduplex molecules formed between a wild-type gene segment and the corresponding homologous segment containing an induced mutation or a naturally occurring single nucleotide polymorphism (SNP). Partially denatured heteroduplex molecules are resolved from homoduplexes by virtue of their differential mobilities during capillary electrophoresis conducted in a finely controlled temperature gradient. Simultaneous heteroduplex analysis of 96 amplicons ranging from 150 to 600 bp in size is achieved in approximately 45 min without the need for predetermining the melting profile of each fragment. Initially, we exploited known mouse mutations to develop TGCE protocols for analyzing unpurified PCR samples amplified from crude tail-DNA preparations. TGCE was then applied to the rapid identification of three new ENU-induced mutations recovered from regional mutagenesis screens of a segment of mouse Chromosome 7. Enzyme assays and quantitative reverse transcription-PCR (qRT-PCR) methods validated these new mutations. Our data demonstrate that rapid mutation scanning with TGCE, followed by sequence verification only of detected positives, is an efficient approach to the identification of point mutations in the mouse genome.

  15. Single chain human interleukin 5 and its asymmetric mutagenesis for mapping receptor binding sites.

    PubMed

    Li, J; Cook, R; Dede, K; Chaiken, I

    1996-01-26

    Wild type human (h) interleukin 5 (wt IL5) is composed of two identical peptide chains linked by disulfide bonds. A gene encoding a single chain form of hIL5 dimer was constructed by linking the two hIL5 chain coding regions with Gly-Gly linker. Expression of this gene in COS cells yielded a single chain IL5 protein (sc IL5) having biological activity similar to that of wt IL5, as judged by stimulation of human cell proliferation. Single chain and wt IL5 also had similar binding affinity for soluble IL5 receptor alpha chain, the specificity subunit of the IL5 receptor, as measured kinetically with an optical biosensor. The design of functionally active sc IL5 molecule. Such mutagenesis was exemplified by changes at residues Glu-13, Arg-91, Glu-110, and Trp-111. The receptor binding and bioactivity data obtained are consistent with a model in which residues from both IL5 monomers interact with the receptor alpha chain, while the interaction likely is asymmetric due to the intrinsic asymmetry of folded receptor. The results demonstrate a general route to the further mapping of receptor and other binding sites on the surface of human IL5.

  16. Probing RNA recognition by human ADAR2 using a high-throughput mutagenesis method

    PubMed Central

    Wang, Yuru; Beal, Peter A.

    2016-01-01

    Adenosine deamination is one of the most prevalent post-transcriptional modifications in mRNA. In humans, ADAR1 and ADAR2 catalyze this modification and their malfunction correlates with disease. Recently our laboratory reported crystal structures of the human ADAR2 deaminase domain bound to duplex RNA revealing a protein loop that binds the RNA on the 5′ side of the modification site. This 5′ binding loop appears to be one contributor to substrate specificity differences between ADAR family members. In this study, we endeavored to reveal detailed structure–activity relationships in this loop to advance our understanding of RNA recognition by ADAR2. To achieve this goal, we established a high-throughput mutagenesis approach which allows rapid screening of ADAR variants in single yeast cells and provides quantitative evaluation for enzymatic activity. Using this approach, we determined the importance of specific amino acids at 19 different positions in the ADAR2 5′ binding loop and revealed six residues that provide essential structural elements supporting the fold of the loop and key RNA-binding functional groups. This work provided new insight into RNA recognition by ADAR2 and established a new tool for defining structure–function relationships in ADAR reactions. PMID:27614075

  17. Isolation of Rhodospirillum centenum Mutants Defective in Phototactic Colony Motility by Transposon Mutagenesis

    PubMed Central

    Jiang, Ze-Yu; Rushing, Brenda G.; Bai, Yong; Gest, Howard; Bauer, Carl E.

    1998-01-01

    The purple photosynthetic bacterium Rhodospirillum centenum is capable of forming swarm colonies that rapidly migrate toward or away from light, depending on the wavelength of excitation. To identify components specific for photoperception, we conducted mini-Tn5-mediated mutagenesis and screened approximately 23,000 transposition events for mutants that failed to respond to either continuous illumination or to a step down in light intensity. A majority of the ca. 250 mutants identified lost the ability to form motile swarm cells on an agar surface. These cells appeared to contain defects in the synthesis or assembly of surface-induced lateral flagella. Another large fraction of mutants that were unresponsive to light were shown to be defective in the formation of a functional photosynthetic apparatus. Several photosensory mutants also were obtained with defects in the perception and transmission of light signals. Twelve mutants in this class were shown to contain disruptions in a chemotaxis operon, and five mutants contained disruptions of components unique to photoperception. It was shown that screening for photosensory defective R. centenum swarm colonies is an effective method for genetic dissection of the mechanism of light sensing in eubacteria. PMID:9495765

  18. Synthesis of Recombinant P48 of Mycoplasma agalactiae by Site Directed Mutagenesis and its Immunological Characterization.

    PubMed

    Cheema, Pawanjit Singh; Singh, Satparkash; Kathiresan, S; Kumar, Ramesh; Bhanot, Vandna; Singh, Vijendra Pal

    2017-01-02

    Contagious agalactia caused by Mycoplasma agalactiae is an economically important disease of sheep and goats and has been prevalent worldwide including India. The present study was undertaken to evaluate the membrane protein P48 of M. agalactiae for specific diagnosis of disease. For this, p48 gene of the organism was amplified by PCR and subjected to site directed mutagenesis to convert three TGA codons to TGG's and, subsequently, cloned into prokaryotic expression vector pPRO EX HTb. Purified recombinant P48 protein reacted to anti-P48 serum in western blotting, which confirmed its immunogenic nature. Furthermore, the immune-blotting of the cell lysates from various Indian isolates of M. agalactiae against anti-P48 serum resulted in a single band at ∼ 48 kDa among all isolates, indicating the conserved nature of P48 antigen in M. agalactiae. Also, the cross reactivity of P48 antigen among various Mycoplasma spp. was checked by western blotting which revealed reactivity only with M. agalactiae and M. bovis. Hence, this antigen could be exploited to differentiate M. agalactiae from other pathogenic Mycoplasma species except M. bovis. However, the inability of P48 to distinguish M. agalactiae from M. bovis does not downgrade the significance of P48 as the two species are usually host specific.

  19. An N-ethyl-N-nitrosourea mutagenesis screen for epigenetic mutations in the mouse.

    PubMed Central

    Percec, Ivona; Thorvaldsen, Joanne L; Plenge, Robert M; Krapp, Christopher J; Nadeau, Joseph H; Willard, Huntington F; Bartolomei, Marisa S

    2003-01-01

    The mammalian epigenetic phenomena of X inactivation and genomic imprinting are incompletely understood. X inactivation equalizes X-linked expression between males and females by silencing genes on one X chromosome during female embryogenesis. Genomic imprinting functionally distinguishes the parental genomes, resulting in parent-specific monoallelic expression of particular genes. N-ethyl-N-nitrosourea (ENU) mutagenesis was used in the mouse to screen for mutations in novel factors involved in X inactivation. Previously, we reported mutant pedigrees identified through this screen that segregate aberrant X-inactivation phenotypes and we mapped the mutation in one pedigree to chromosome 15. We now have mapped two additional mutations to the distal chromosome 5 and the proximal chromosome 10 in a second pedigree and show that each of the mutations is sufficient to induce the mutant phenotype. We further show that the roles of these factors are specific to embryonic X inactivation as neither genomic imprinting of multiple genes nor imprinted X inactivation is perturbed. Finally, we used mice bearing selected X-linked alleles that regulate X chromosome choice to demonstrate that the phenotypes of all three mutations are consistent with models in which the mutations have affected molecules involved specifically in the choice or the initiation of X inactivation. PMID:12930754

  20. Modular mutagenesis of human placental ribonuclease inhibitor, a protein with leucine-rich repeats.

    PubMed Central

    Lee, F S; Vallee, B L

    1990-01-01

    Human placental ribonuclease inhibitor (PRI) is a potent protein inhibitor of pancreatic ribonucleases and the homologous blood vessel-inducing protein angiogenin. Although inhibition by PRI occurs with a 1:1 stoichiometry, its primary structure is composed predominantly of seven internal leucine-rich repeats. These internal repeats were systematically deleted either singly or in combination by "modular" mutagenesis. Deletion of repeat units 3 plus 4 or repeat unit 6 results in mutants that both bind to and inhibit ribonuclease A. Therefore, the angiogenin/ribonuclease binding site in PRI must reside primarily or entirely in repeats 1, 2, 5, or 7, the short N- or C-terminal segments, or a combination of these. Deletion of repeat units 3-5, 5-6, or 5 alone results in mutants that exhibit only binding activity. Hence, the binding site cannot reside exclusively in repeat 5. Other internal deletions or N- or C-terminal deletions of 6-86% of the protein all abolish activity. These results suggest that PRI has a modular structure, with one primary structural repeat constituting one module. The approach taken may be applicable to other proteins with repeat structures. Images PMID:2408043