The effect of essential oil of basil (Ocimum basilicum L.) on UV-induced mutagenesis in Escherichia coli and Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Stanojević, Jasna; Berić, Tanja; Opačić, Biljana; Vuković-Gačić, Branka; Simić, Draga; Knežević-Vukčević, Jelena

2008-01-01

The antimutagenic potential of essential oil (EO) of basil (Ocimum basilicum L.) and its major constituent linalool were studied with the E. coli K12 and S. cerevisiae D7 assays. In the E. coli assay, EO and linalool inhibited UV-induced mutagenesis in a repair-proficient strain, but had no effect on spontaneous mutagenesis in repair-proficient, nucleotide excision repair-deficient, and mismatch-deficient strains. By testing participation of different mechanisms involved in antimutagenesis, it was concluded that the antimutagenic effect against UV-induced mutagenesis involved decrease of protein synthesis and cell proliferation which led to increased efficiency of nucleotide excision repair. An antimutagenic effect of basil derivatives in S. cerevisiae was not detected. (author)

Induced repair and mutagenesis in animal cells

International Nuclear Information System (INIS)

Takimoto, Koichi

1981-01-01

Induced repair and mutagenesis of animal cells against UV were studied in contrast with SOS repair of E. coli primarily by the use of viruses. Since UV-enhanced reactivation is a phenomenon similar to UV-reactivation (mutagenesis) and the presence of lesion bypass synthsis has been suggested, UV-enhanced reactivation has several common aspects with SOS reactivation of E. coli. However, correlation is not necessarily noted between increase in the viral survival rate and mutagenesis, nor do protease blockers exert any effect. Therefore, SOS repair of E. coli may have different mechansms from induced repair and mutagenesis in animal cells. (Ueda, J.)

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

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

1998-01-01

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

Crowding depression of UV-mutagenesis in E. coli

International Nuclear Information System (INIS)

Bockrath, R.; Harper, D.; Kristoff, S.; Stanford Univ., CA

1980-01-01

Strains of E. coli Br were exposed to UV radiation and assayed for reversion mutation, using a standard selection medium. If more irradiated bacteria were assayed per petri dish, a proportional increase in the number of indicated reversion mutants was oud only up to a limiting plating density. Beyond a density of about 10 8 viable bacteria per petri dish, the number of indicated revertants per viable bacteriy assayed (the mutation frequency) decreased as the plating density was increased. The crowding depression of mutagenesis was more severe for de novo and converted suppressor mutations, the mutation frequency being reduced 100-fold at a plating density of about 6 x 10 9 viable bacteria per plate. The effect on backmutation was 10 times less. Crowding depression of mutagenesis occured in excision-proficient and -deficient strains, with identical effects in the 2 strains on de novo and converted suppressor mutation, but different effects on backmutations. There were no accompanying effects on viability. Irreversible loss of potential mutants during crowded growth was indicated in wash-off experiments. The kinetics suggested a half-life of approximately 1 h. Kinetics for accumulation by the bacteria of the limiting metabolite (tyrosine) on the assay plate indicated a short period of time for protein synthesis, but direct examination of the proteins synthesized during early growth on a crowded plate demonstrated successful induction of recA protein. (orig.)

DNA polymerase III of Escherichia coli is required for UV and ethyl methanesulfonate mutagenesis

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Hagensee, M.E.; Timme, T.L.; Bryan, S.K.; Moses, R.E.

1987-06-01

Strains of Escherichia coli possessing the pcbA1 mutation, a functional DNA polymerase I, and a temperature-sensitive mutation in DNA polymerase III can survive at the restrictive temperature (43 degrees C) for DNA polymerase III. The mutation rate of the bacterial genome of such strains after exposure to either UV light or ethyl methanesulfonate was measured by its rifampicin resistance or amino acid requirements. In addition, Weigle mutagenesis of preirradiated lambda phage was also measured. In all cases, no increase in mutagenesis was noted at the restrictive temperature for DNA polymerase III. Introduction of a cloned DNA polymerase III gene returned the mutation rate of the bacterial genome as well as the Weigle mutagenesis to normal at 43 degrees C. Using a recA-lacZ fusion, the SOS response after UV irradiation was measured and found to be normal at the restrictive and permissive temperature for DNA polymerase III, as was induction of lambda prophage. Recombination was also normal at either temperature. Our studies demonstrate that a functional DNA polymerase III is strictly required for mutagenesis at a step other than SOS induction.

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

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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

Site-specific genomic (SSG and random domain-localized (RDL mutagenesis in yeast

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Honigberg Saul M

2004-04-01

Full Text Available Abstract Background A valuable weapon in the arsenal available to yeast geneticists is the ability to introduce specific mutations into yeast genome. In particular, methods have been developed to introduce deletions into the yeast genome using PCR fragments. These methods are highly efficient because they do not require cloning in plasmids. Results We have modified the existing method for introducing deletions in the yeast (S. cerevisiae genome using PCR fragments in order to target point mutations to this genome. We describe two PCR-based methods for directing point mutations into the yeast genome such that the final product contains no other disruptions. In the first method, site-specific genomic (SSG mutagenesis, a specific point mutation is targeted into the genome. In the second method, random domain-localized (RDL mutagenesis, a mutation is introduced at random within a specific domain of a gene. Both methods require two sequential transformations, the first transformation integrates the URA3 marker into the targeted locus, and the second transformation replaces URA3 with a PCR fragment containing one or a few mutations. This PCR fragment is synthesized using a primer containing a mutation (SSG mutagenesis or is synthesized by error-prone PCR (RDL mutagenesis. In SSG mutagenesis, mutations that are proximal to the URA3 site are incorporated at higher frequencies than distal mutations, however mutations can be introduced efficiently at distances of at least 500 bp from the URA3 insertion. In RDL mutagenesis, to ensure that incorporation of mutations occurs at approximately equal frequencies throughout the targeted region, this region is deleted at the same time URA3 is integrated. Conclusion SSG and RDL mutagenesis allow point mutations to be easily and efficiently incorporated into the yeast genome without disrupting the native locus.

Mutational spectrum analysis of umuC-independent and umuC-dependent γ-radiation mutagenesis in Escherichia coli

International Nuclear Information System (INIS)

Sargentini, N.J.; Smith, K.C.

1989-01-01

γ-radiation mutagenesis Escherichia coli K-12. Mutagenesis (argE3(OC) A rg + ) was blocked in a δ(recA-srlR)306 strain at the same doses that induced mutations in umuC122::Tn5 and wild-type strains, indicating that both umuC-independent and umuC-dependent mechanisms function within recA-dependent misrepair. Analyses of various suppressor and back mutations that result in argE3 and hisG4 ochre reversion and an analysis of trpE9777 reversion were performed on umuC and wild-type cells irradiated in the presence and absence of oxygen. While the umuC strain showed the γ-radiation induction of base substitution and frameshifts when irradiated in the absence of oxygen, the umuC mutation blocked all oxygen-dependent base-substitution mutagenesis, but non all oxygen-dependent frameshift mutagenesis. For anoxically irradiated cells, the yields of GC T and AT GC transitions were essentially umuC independent, while the yields of (AT or GC) TA transversions were heavily umuC dependent. These data suggest new concepts about the nature of the DNA lesions and the mutagenic mechanisms that lead to γ-radiation mutagenesis. (author). 48 refs.; 1 tab.; 6 refs

The antimutagenic effect of monoterpenes against UV-irradiation-, 4NQO- and t-BOOH-induced mutagenesis in coli

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Nikolić Biljana

2011-01-01

Full Text Available The aim of this work was to investigate the antimutagenic potential of monoterpenes from sage and basil in Escherichia coli. The mutagenic potential of monoterpenes was pre-screened with Salmonella/microsome reversion assay in strain TA100 and no mutagenic effect was detected. The antimutagenic potential against UV- 4NQO- and t-BOOH induced mutagenesis was evaluated in E. coli K12 and E. coli WP2 by reversion assays. The obtained results indicate that camphor and thujone reduce UV- and 4NQO-induced mutations; myrcene reduces t-BOOH-induced mutations, while eucalyptol and linalool reduce mutagenicity by all tested mutagens. Considering evolutionary conservation of DNA repair and antioxidative protection, the obtained results indicate that further antigenotoxicity studies should be undertaken in eukaryotes.

Plasmid (pKM101)-mediated enhancement of repair and mutagenesis: dependence on chromosomal genes in 'Escherichia coli' K-12

International Nuclear Information System (INIS)

Walker, G.C.

1977-01-01

The drug resistance plasmid pKM101 plays a major role in the Ames Salmonella/microsome carcinogen detecting system by enhancing chemical mutagenesis. It is shown that in Escherichia coli K-12 the plasmid pKM101 enhances both spontaneous and methyl methanesulfonate-caused reversion of an ochre mutation, bacterial survival after ultaviolet irradiation, and reactivation of ultraviolet-irradiated lambda in unirradiated cells. All these effects are shown to be dependent on the recA + lexA + genotype but not on the recB + recC + or recF + genotypes. The recA lexA-dependence of the plasmid-mediated repair and mutagenesis suggests an interaction with the cell's inducible error-prone repair system. The presence of pKM101 is shown to cause an additional increase in methyl methanesulfonate mutagenesis in a tif mutant beyond that caused by growth at 42 0 . The presence of the plasmid raises the level of the Weigle-reactivation curve for the reactivation of ultraviolet-irradiated lambda in E. coli and causes a shift of the maximum to a higher UV fluence. These observations suggest that pKM101 does not exert its effects by altering the regulation of the cell's error-prone repair system but rather by supplying a mechanistic component or components. (orig.) [de

Amino acid substitutions in random mutagenesis libraries: lessons from analyzing 3000 mutations.

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Zhao, Jing; Frauenkron-Machedjou, Victorine Josiane; Kardashliev, Tsvetan; Ruff, Anna Joëlle; Zhu, Leilei; Bocola, Marco; Schwaneberg, Ulrich

2017-04-01

The quality of amino acid substitution patterns in random mutagenesis libraries is decisive for the success in directed evolution campaigns. In this manuscript, we provide a detailed analysis of the amino acid substitutions by analyzing 3000 mutations of three random mutagenesis libraries (1000 mutations each; epPCR with a low-mutation and a high-mutation frequency and SeSaM-Tv P/P) employing lipase A from Bacillus subtilis (bsla). A comparison of the obtained numbers of beneficial variants in the mentioned three random mutagenesis libraries with a site saturation mutagenesis (SSM) (covering the natural diversity at each amino acid position of BSLA) concludes the diversity analysis. Seventy-six percent of the SeSaM-Tv P/P-generated substitutions yield chemically different amino acid substitutions compared to 64% (epPCR-low) and 69% (epPCR-high). Unique substitutions from one amino acid to others are termed distinct amino acid substitutions. In the SeSaM-Tv P/P library, 35% of all theoretical distinct amino acid substitutions were found in the 1000 mutation library compared to 25% (epPCR-low) and 26% (epPCR-high). Thirty-six percent of distinct amino acid substitutions found in SeSaM-Tv P/P were unobtainable by epPCR-low. Comparison with the SSM library showed that epPCR-low covers 15%, epPCR-high 18%, and SeSaM-Tv P/P 21% of obtainable beneficial amino acid positions. In essence, this study provides first insights on the quality of epPCR and SeSaM-Tv P/P libraries in terms of amino acid substitutions, their chemical differences, and the number of obtainable beneficial amino acid positions.

Uvm mutants of Escherichia coli K 12 deficient in UV mutagenesis. Pt. 1

International Nuclear Information System (INIS)

Steinborn, G.

1978-01-01

Selection for defective reversion induction, after UV treatment of E. coli K 12, yielded uvm mutants. These mutants exhibited highly reduced or no UV mutability for all loci tested although they were moderately and normally mutable by X-rays and EMS, respectively. Uvm mutations confer only a slight sensitivity to killing by UV and X-rays and no clear sensitivity to the lethal effect of HN2, EMS or MMS. Growth and viability of untreated uvm cells were normal. The properties of uvm mutants are discussed in relation to those of other relevant mutant types and to some actual problems of induced mutagenesis. (orig.) 891 AJ [de

Misrepair of overlapping daughter strand gaps as a possible mechanism for UV induced mutagenesis in uvr strains of Escherichia coli: a general model for induced mutagenesis by misrepair (SOS repair) of closely spaced DNA lesions

International Nuclear Information System (INIS)

Sedgwick, S.G.

1976-01-01

It has been previously reported that an inducible form of post-replication repair appeared to be required for UV induced mutagenesis in an uvrA strain of Escherichia coli. It is shown here that the numbers of daughter strand gaps requiring inducible repair were similar to the numbers calculated to be overlapping one another in opposite daughter chromosomes. An estimation of survival with no repair of these gaps resembled the survival predicted with mutagenesis. It is thus proposed that inducible post-replication repair causes mutagenesis by the repair of overlapping daughter strand gaps. A general model for induced mutagenesis is presented. It is proposed that (a) some DNA lesions introduced by any DNA damaging agent may be close enough to interfere with constitutive repair replication of each other, (b) these lesions induce a repair system (SOS repair) which involves the recA + . lexA + and polC + genes (c) repair, and noncomitant mutagenesis occurs during repair replication by the insertion of mismatched bases oppposite the noncoding DNA lesions

Chemical and UV Mutagenesis.

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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.

Different efficiency of UmuDC and MucAB proteins in UV light induced mutagenesis in Escherichia coli

International Nuclear Information System (INIS)

Blanco, M.; Herrera, G.; Aleixandre, V.

1986-01-01

Two multicopy plasmids carrying either the umuDC or the mucAB operon were used to compare the efficiency of UmuDC and MucAB proteins in UV mutagenesis of Escherichia coli K12. It was found that in recA + uvr + bacteria, plasmid pIC80, mucAB + mediated UV mutagenesis more efficiently than did plasmid pSE 117, umuDC + . A similar result was obtained in lex A51(Def) cells, excluding the possibility that this was due to a differential regulation by LexA of the umuDC and mucAB operons. We conclude that some structural characteristic of the UmuDC and MucAB proteins determines their different efficiency in UV mutagenesis. This characteristic could be also responsible for the observation that in the recA430 mutant, pIC80 but no pSE117 can mediate UV mutagenesis. In the recAS142 mutant pIC80 also promoted UV mutagenesis more efficiently than pSE117. In this mutant, the recombination proficiency, the protease activity toward LexA and the mutation frequency were increased by the presence of adenine in the medium. In recA + uvrB5 bacteria, plasmid pSE117, umuDC caused both an increase in UV sensitivity as well as a reduction in the mutation frequency. These negative effects resulting from the overproduction of UmuDC proteins were higher in recA142 uvrB5 than in recA + uvrB5 cells. In contrast, overproduction of MucAB proteins in excision-deficient bacteria containing pIC80 led to a large increase in the mutation frequency. We suggest that the functional differences between UmuDC and MucAB proteins might be due to their different dependence on the direct role of RecA protease in UV mutagenesis. (orig.)

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

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

2016-11-08

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.

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

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

2017-01-01

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

Single d(ApG)/cis-diamminedichloroplatinum(II) adduct-induced mutagenesis in Escherichia coli

International Nuclear Information System (INIS)

Burnouf, D.; Fuchs, R.P.P.; Gauthier, C.; Chottard, J.C.

1990-01-01

The mutation spectrum induced by the widely used antitumor drug cis-diamminedichloroplatinum(II) (cis-DDP) showed that cisDDP[d(ApG)] adducts, although they account for only 25% of the lesions formed are ∼5 times more mutagenic than the major GG adduct. The authors report the construction of vectors bearing a single cisDDP[d(ApG)] lesion and their use in mutagenesis experiments in Escherichia coli. The mutagenic processing of the lesion is found to depend strictly on induction of the SOS system of the bacterial host cells. In SOS-induced cells, mutation frequencies of 1-2% were detected. All these mutations are targeted to the 5' base of the adduct. Single A → T transversions are mainly observed (80%), whereas A → G transitions account for 10% of the total mutations. Tandem base-pair substitutions involving the adenine residue and the thymine residue immediately 5' to the adduct occur at a comparable frequency (10%). No selective loss of the strand bearing the platinum adduct was seen, suggesting that, in vivo, cisDDP[d(ApG)] adducts are not blocking lesions. The high mutation specificity of cisDDP-[d(ApG)]-induced mutagenesis is discussed in relation to structural data

FimH-mediated autoaggregation of Escherichia coli

DEFF Research Database (Denmark)

Schembri, Mark; Christiansen, G.; Klemm, Per

2001-01-01

Autoaggregation is a phenomenon thought to contribute to colonization of mammalian hosts by pathogenic bacteria. Type 1 fimbriae are surface organelles of Escherichia coli that mediate D-mannose-sensitive binding to various host surfaces. This binding is conferred by the minor fimbrial component...... FimH. In this study, we have used random mutagenesis to identify variants of the FimH adhesin that confer the ability of E. coli to autoaggregate and settle from liquid cultures. Three separate autoaggregating clones were identified, all of which contained multiple amino acid changes located within...

Directed combinatorial mutagenesis of Escherichia coli for complex phenotype engineering

Energy Technology Data Exchange (ETDEWEB)

Liu, Rongming; Liang, Liya; Garst, Andrew D.; Choudhury, Alaksh; Nogué, Violeta Sànchez i.; Beckham, Gregg T.; Gill, Ryan T.

2018-05-01

Strain engineering for industrial production requires a targeted improvement of multiple complex traits, which range from pathway flux to tolerance to mixed sugar utilization. Here, we report the use of an iterative CRISPR EnAbled Trackable genome Engineering (iCREATE) method to engineer rapid glucose and xylose co-consumption and tolerance to hydrolysate inhibitors in E. coli. Deep mutagenesis libraries were rationally designed, constructed, and screened to target ~40,000 mutations across 30 genes. These libraries included global and high-level regulators that regulate global gene expression, transcription factors that play important roles in genome-level transcription, enzymes that function in the sugar transport system, NAD(P)H metabolism, and the aldehyde reduction system. Specific mutants that conferred increased growth in mixed sugars and hydrolysate tolerance conditions were isolated, confirmed, and evaluated for changes in genome-wide expression levels. We tested the strain with positive combinatorial mutations for 3-hydroxypropionic acid (3HP) production under high furfural and high acetate hydrolysate fermentation, which demonstrated a 7- and 8-fold increase in 3HP productivity relative to the parent strain, respectively.

Enhanced vanillin production from recombinant E. coli using NTG mutagenesis and adsorbent resin.

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Yoon, Sang-Hwal; Lee, Eun-Gyeong; Das, Amitabha; Lee, Sook-Hee; Li, Cui; Ryu, Hee-Kyoung; Choi, Myung-Suk; Seo, Weon-Taek; Kim, Seon-Won

2007-01-01

Vanillin production was tested with different concentrations of added ferulic acid in E. coli harboring plasmid pTAHEF containing fcs (feruloyl-CoA synthase) and ech (enoyl-CoA hydratase/aldolase) genes cloned from Amycolatopsis sp. strain HR104. The maximum production of vanillin from E. coli DH5alpha harboring pTAHEF was found to be 1.0 g/L at 2.0 g/L of ferulic acid for 48 h of culture. To improve the vanillin production by reducing its toxicity, two approaches were followed: (1) generation of vanillin-resistant mutant of NTG-VR1 through NTG mutagenesis and (2) removal of toxic vanillin from the medium by XAD-2 resin absorption. The vanillin production of NTG-VR1 increased to three times at 5 g/L of ferulic acid when compared with its wild-type strain. When 50% (w/v) of XAD-2 resin was employed in culture with 10 g/L of ferulic acid, the vanillin production of NTG-VR1 was 2.9 g/L, which was 2-fold higher than that obtained with no use of the resin.

Himar1 Transposon for Efficient Random Mutagenesis in Aggregatibacter actinomycetemcomitans

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Qinfeng Ding

2017-09-01

Full Text Available Aggregatibacter actinomycetemcomitans is the primary etiological agent of aggressive periodontal disease. Identification of novel virulence factors at the genome-wide level is hindered by lack of efficient genetic tools to perform mutagenesis in this organism. The Himar1 mariner transposon is known to yield a random distribution of insertions in an organism’s genome with requirement for only a TA dinucleotide target and is independent of host-specific factors. However, the utility of this system in A. actinomycetemcomitans is unknown. In this study, we found that Himar1 transposon mutagenesis occurs at a high frequency (×10-4, and can be universally applied to wild-type A. actinomycetemcomitans strains of serotypes a, b, and c. The Himar1 transposon inserts were stably inherited in A. actinomycetemcomitans transconjugants in the absence of antibiotics. A library of 16,000 mutant colonies of A. actinomycetemcomitans was screened for reduced biofilm formation. Mutants with transposon inserts in genes encoding pilus, putative ion transporters, multidrug resistant proteins, transcription regulators and enzymes involved in the synthesis of extracellular polymeric substance, bacterial metabolism and stress response were discovered in this screen. Our results demonstrated the utility of the Himar1 mutagenesis system as a novel genetic tool for functional genomic analysis in A. actinomycetemcomitans.

The Glyphosate-Based Herbicide Roundup Does not Elevate Genome-Wide Mutagenesis of Escherichia coli.

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Tincher, Clayton; Long, Hongan; Behringer, Megan; Walker, Noah; Lynch, Michael

2017-10-05

Mutations induced by pollutants may promote pathogen evolution, for example by accelerating mutations conferring antibiotic resistance. Generally, evaluating the genome-wide mutagenic effects of long-term sublethal pollutant exposure at single-nucleotide resolution is extremely difficult. To overcome this technical barrier, we use the mutation accumulation/whole-genome sequencing (MA/WGS) method as a mutagenicity test, to quantitatively evaluate genome-wide mutagenesis of Escherichia coli after long-term exposure to a wide gradient of the glyphosate-based herbicide (GBH) Roundup Concentrate Plus. The genome-wide mutation rate decreases as GBH concentration increases, suggesting that even long-term GBH exposure does not compromise the genome stability of bacteria. Copyright © 2017 Tincher et al.

The Activity of Escherichia coli Dihydroorotate Dehydrogenase Is Dependent on a Conserved Loop Identified by Sequence Homology, Mutagenesis, and Limited Proteolysis

DEFF Research Database (Denmark)

Björnberg, Olof; Grüner, Anne Charlotte; Roepstorff, Peter

1999-01-01

of dihydroorotate dehydrogenases, but sedimentation in sucrose gradients suggests a dimeric structure also of the E. coli enzyme. Product inhibition showed that the E. coli enzyme, in contrast to the L. lactis enzyme, has separate binding sites for dihydroorotate and the electron acceptor. Trypsin readily cleaved...... the E. coli enzyme into two fragments of 182 and 154 residues, respectively. Cleavage reduced the activity more than 100-fold but left other molecular properties, including the heat stability, intact. The trypsin cleavage site, at R182, is positioned in a conserved region that, in the L. lactis enzyme......, forms a loop where a cysteine residue is very critical for activity. In the corresponding position, the enzyme from E. coli has a serine residue. Mutagenesis of this residue (S175) to alanine or cysteine reduced the activities 10000- and 500-fold, respectively. The S175C mutant was also defective...

Highly Efficient ENU Mutagenesis in Zebrafish.

NARCIS (Netherlands)

de Bruijn, E.; Cuppen, E.; Feitsma, H.

2009-01-01

ENU (N-ethyl-N-nitrosourea) mutagenesis is a widely accepted and proven method to introduce random point mutations in the genome. Because there are no targeted knockout strategies available for zebrafish so far, random mutagenesis is currently the preferred method in both forward and reverse genetic

Role of the RecF gene product in UV mutagenesis of lambda phage

International Nuclear Information System (INIS)

Wood, R.D.; Stein, J.

1986-01-01

E. coli recF mutants have a greatly reduced capacity for Weigle mutagenesis of ultraviolet light-irradiated lambda phage. A recF 332::Tn3 mutation was introduced into an E. coli recA441 lex A51 strain which constitutively expresses SOS functions. Weigle mutagenesis of phage lambda could occur in the resulting strain in the absence of host cell irradiation, and was increased when the recA441 (tif) allele was activated of recF strains to support Weigle mutagenesis can therefore be ascribed to a defect in expression of SOS functions after irradiation. (orig.)

Mutational specificity of SOS mutagenesis

International Nuclear Information System (INIS)

Kato, Takeshi

1986-01-01

In an approach to the isolation of mutants of E. coli unable to produce mutations by ultraviolet light, the author has found new umuC-mutants. Their properties could be explained by ''SOS hypothesis of Radman and Witkin'', which has now been justified by many investigators. Analysis of the umuC region of E. coli chromosome cloned in pSK 100 has led to the conclusion that two genes, umuD and umuC, having the capacity of mutation induction express in the same mechanism as that of SOS genes, which is known to be inhibited by LexA protein bonding to ''SOS box'' found at promotor region. Suppressor analysis for mutational specificity has revealed: (i) umuDC-independent mutagens, such as EMS and (oh) 4 Cy, induce selected base substitution alone; and (ii) umuDC-dependent mutagens, such as X-rays and gamma-rays, induce various types of base substitution simultaneously, although they have mutational specificity. In the umuDC-dependent processes of basechange mutagenesis, the spectra of base substitution were a mixture of base substitution reflecting the specific base damages induced by individual mutagens and nonspecific base substitution. In conclusion, base substitution plays the most important role in umuDC-dependent mutagenesis, although mutagenesis of umuDC proteins remains uncertain. (Namekawa, K.)

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

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Cory A. Leonard

2013-01-01

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

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

Science.gov (United States)

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

2013-01-01

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

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

Science.gov (United States)

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

2013-01-01

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

Random mutagenesis of human serine racemase reveals residues important for the enzymatic activity

Czech Academy of Sciences Publication Activity Database

Hoffman, Hillary Elizabeth; Jirásková, Jana; Zvelebil, M.; Konvalinka, Jan

2010-01-01

Roč. 75, č. 1 (2010), s. 59-79 ISSN 0010-0765 R&D Projects: GA MŠk 1M0508 Institutional research plan: CEZ:AV0Z40550506 Keywords : D-serine * serine racemase * random mutagenesis Subject RIV: CE - Biochemistry Impact factor: 0.853, year: 2010

The Roles of UmuD in Regulating Mutagenesis

Directory of Open Access Journals (Sweden)

Jaylene N. Ollivierre

2010-01-01

Full Text Available All organisms are subject to DNA damage from both endogenous and environmental sources. DNA damage that is not fully repaired can lead to mutations. Mutagenesis is now understood to be an active process, in part facilitated by lower-fidelity DNA polymerases that replicate DNA in an error-prone manner. Y-family DNA polymerases, found throughout all domains of life, are characterized by their lower fidelity on undamaged DNA and their specialized ability to copy damaged DNA. Two E. coli Y-family DNA polymerases are responsible for copying damaged DNA as well as for mutagenesis. These DNA polymerases interact with different forms of UmuD, a dynamic protein that regulates mutagenesis. The UmuD gene products, regulated by the SOS response, exist in two principal forms: UmuD2, which prevents mutagenesis, and UmuD2′, which facilitates UV-induced mutagenesis. This paper focuses on the multiple conformations of the UmuD gene products and how their protein interactions regulate mutagenesis.

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

Science.gov (United States)

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

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

Science.gov (United States)

Whitmire, Jeannette M; Merrell, D Scott

2017-01-01

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

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

OpenAIRE

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

2013-01-01

Multidrug-resistant bacteria cause severe infections in hospitals and communities. Development of new drugs to combat resistant microorganisms is needed. Natural products of microbial origin are the source of most currently available antibiotics. We hypothesized that random mutagenesis of Aspergillus oryzae would result in secretion of antibacterial compounds. To address this hypothesis, we developed a screen to identify individual A. oryzae mutants that inhibit the growth of Methicillin-res...

Mutagenesis and Teratogenesis Section

International Nuclear Information System (INIS)

Anon.

1976-01-01

Progress is reported on research with mice in the areas of radioinduced and chemical mutagenesis, cytologic studies, radiation effects on DNA synthesis, radiation effects on germ cells, mutagenicity of coal-conversion products, and others. Research on Drosophila was concerned with mutagenesis and genetics of nucleases. Studies were conducted on hamster cells with regard to cytotoxicity and mutagenicity of alkylating agents, modification of the microtubule system, protein kinase activity, and others. Research on bacteria was concerned with effects of x radiation on bacteriophage of Haemophilus influenzae, x-ray induced DNA polymerase I-directed repair synthesis in Escherichia coli, transformation by DNA polymerase II in Bacillus subtilis, and others. Research on xenopus laevis was conducted in the areas of calcium-induced cleavage of oocytes, yolk degradation in explants, and others

Random transposon mutagenesis of the Saccharopolyspora erythraea genome reveals additional genes influencing erythromycin biosynthesis

Science.gov (United States)

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

Effect of umuC mutations on targeted and untargeted ultraviolet mutagenesis in bacteriophage lambda

International Nuclear Information System (INIS)

Maenhaut-Michel, G.; Caillet-Fauquet, P.

1984-01-01

Mutagenesis of phage lambda towards clear-plaque (c + → c) results in two classes of mutants that can be distinguished genetically and morphologically. Indirect mutagenesis, i.e. mutagenesis of unirradiated phage lambdac + stimulated by the ultraviolet irradiation of the Escherichia coli host, results in mixed bursts (c/c + ) of turbid wild-type and clear=plaque mutant phages. Pure bursts of lambdac mutants are induced by irradiation of the phage genome. Irradiation of both phages and host bacteria stimulates the production of the two classes of mutant clones. It is shown that three different mutant alleles of the E. coli umuC gene only prevent the appearance of pure bursts of clear-plaque mutants, while mixed bursts are produced at least as frequently in umuC mutants as in the umuC + parent. (author)

Random mutagenesis of aspergillus niger and process optimization for enhanced production of glucose oxidase

International Nuclear Information System (INIS)

Haq, I.; Nawaz, A.; Mukhtar, A.N.H.; Mansoor, H.M.Z.; Ameer, S.M.

2014-01-01

The study deals with the improvement of wild strain Aspergillus niger IIB-31 through random mutagenesis using chemical mutagens. The main aim of the work was to enhance the glucose oxidase (GOX) yield of wild strain (24.57+-0.01 U/g of cell mass) through random mutagenesis and process optimization. The wild strain of Aspergillus niger IIB-31 was treated with chemical mutagens such as Ethyl methane sulphonate (EMS) and nitrous acid for this purpose. Mutagen treated 98 variants indicating the positive results were picked and screened for the glucose oxidase production using submerged fermentation. EMS treated E45 mutant strain gave the highest glucose oxidase production (69.47 + 0.01 U/g of cell mass), which was approximately 3-folds greater than the wild strain IIB-31. The preliminary cultural conditions for the production of glucose oxidase using submerged fermentation from strain E45 were also optimized. The highest yield of GOD was obtained using 8% glucose as carbon and 0.3% peptone as nitrogen source at a medium pH of 7.0 after an incubation period of 72 hrs at 30 degree. (author)

Comparative mutagenesis and interaction between near-ultraviolet (313- to 405-nm) and far-ultraviolet (254-nm) radiation in Escherichia coli strains with differing repair capabilities

International Nuclear Information System (INIS)

Turner, M.A.; Webb, R.B.

1981-01-01

Comparative mutagenesis and possible synergistic interaction between broad-spectrum (313- to 405-nm) near-ultraviolet (black light bulb [BLB]) radiation and 254-nm radiation were studied in Escherichia coli strains WP2 (wild type), WP2s (uvrA), WP10 (recA), WP6 (polA), WP6s (polA uvrA), WP100 (uvrA recA), and WP5 (lexA). With BLB radiation, strains WP2s and WP6s demonstrated a high level of mutagenesis, whereas strains WP2, WP5, WP6, WP10, and WP100 did not demonstrate significant mutagenesis. In contrast, 254-nm radiation was mutagenic in strains WP2, WP2s, WP6, and WP6s, but strains WP5, WP10, and WP100 were not significantly mutated. The absence of mutagenesis by BLB radiation in lexA and recA strains WP10, WP5, and WP100 suggests that lex + rec + repair may play a major role in mutagenesis by both BLB and 254-nm radiation. The hypothesis that BLB radiation selectively inhibits rec + lex + repair was tested by sequential BLB-254 nm radiation. With strain WP2, a fluence of 30 J/m 2 at 254 nm induced trp + revertants at a frequency of 15 x 10 -6 . However, when 10 5 J/m 2 or more BLB radiation preceded the 254-nm exposure, no trp + revertants could be detected. A similar inhibition of 254-nm mutagenesis was observed with strain WP6 (polA). However, strains WP2s (uvrA) and WP6s (polA uvrA) showed enhanced 254-nm mutagenesis when a prior exposure to BLB radiation was given

The relation between repair of DNA and radiation and chemical mutagenesis in Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Prakash, L.

1976-01-01

The effect of various genes involved in DNA repair functions on radiation and chemical mutagenesis in Escherichia coli is discussed and compared to similar studies done in yeast. Results of the effect of various genes conferring radiation-sensitivty on mutation induction in yeast are presented and related to current ideas of mutagenesis

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

Science.gov (United States)

Noma, Kentaro; Jin, Yishi

2016-11-14

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

Excision repair and mutagenesis in Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Kilbey, Brian

1987-01-01

This and succeeding letters discuss the James and Kilbey (1977 and 1978) model for the initiation of u.v. mutagenesis in Saccharomyces cerevisiae and its application to include a number of chemical mutagens. The Baranowska et al (1987) results indicating the role of DNA replication, the differing mechanisms in Escherichia coli, are all discussed. (UK)

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

Science.gov (United States)

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

2017-06-21

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.

Novel Random Mutagenesis Method for Directed Evolution.

Science.gov (United States)

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.

groE mutants of Escherichia coli are defective in umuDC-dependent UV mutagenesis

International Nuclear Information System (INIS)

Donnelly, C.E.; Walker, G.C.

1989-01-01

Overexpression of the SOS-inducible umuDC operon of Escherichia coli results in the inability of these cells to grow at 30 degrees C. Mutations in several heat shock genes suppress this cold sensitivity. Suppression of umuD+C+-dependent cold sensitivity appears to occur by two different mechanisms. We show that mutations in lon and dnaK heat shock genes suppress cold sensitivity in a lexA-dependent manner. In contrast, mutations in groES, groEL, and rpoH heat shock genes suppress cold sensitivity regardless of the transcriptional regulation of the umuDC genes. We have also found that mutations in groES and groEL genes are defective in umuDC-dependent UV mutagenesis. This defect can be suppressed by increased expression of the umuDC operon. The mechanism by which groE mutations affect umuDC gene product function may be related to the stability of the UmuC protein, since the half-life of this protein is shortened because of mutations at the groE locus

Laboratory of Mutagenesis and DNA Repair

International Nuclear Information System (INIS)

2000-01-01

Full text: Two main lines of research were continued: the first one concerned the mechanisms controlling the fidelity of DNA replication in Escherichia coli; the second concerned cellular responses of Saccharomyces cerevisiae to DNA damaging agents. We have been investigating the question whether during chromosomal DNA replication in Escherichia coli the two DNA strands may be replicated with differential accuracy. To address this question we set up a new system that allows the examination of mutagenesis either of the leading strand or the lagging strand. Our results suggest that the lagging strand replication of the E. coli chromosome may be more accurate than leading strand replication. More recently, we studied mutagenesis of the two strands in recA730 strains which exhibit constitutive expression of the SOS system. Our results clearly indicate that in recA730 strains there is a significant difference in the fidelity of replication between the two replicating strands. Based on our data we propose a model describing a possible mechanism of SOS mutagenesis. To get more insight into cellular responses to DNA damage we have isolated several novel genes of S. cerevisiae, the transcription of which is induced by DNA lesions. Main effort was concentrated on the characterization of the DIN7 gene. We found that Din7p specifically affects the metabolism of mitochondrial DNA (mtDNA). The elevated level of Din7p results in an increased frequency of mitochondrial petite mutants, as well as in a higher frequency of mitochondrial point mutations. Din7p affects also the stability of microsatellite sequences present in the mitochondrial genome. As expected, Din7p was found to be located in mitochondria. In another project, we found that the DIN8 gene isolated in our laboratory is identical with the UMP1 gene encoding a chaperone-like protein involved in 20S proteasome maturation. Interestingly, induction of UMP1 expression in response to DNA damage is subject to regulation

Effect of radiation-sensitive mutations and mutagens/carcinogens on bacterial recombination and mutagenesis. Progress report

Energy Technology Data Exchange (ETDEWEB)

Matney, T.S.

1978-01-01

Progress is reported on effects of temperature sensitive DNA-initiation mutation in E. coli K-12 mutants; the use of Bacillus subtilis transforming system as an in vitro mutagenesis system; characteristics of the E. coli lysogen used to test the permeability to polycyclic aromatic hydrocarbons; and the genetic toxicology of gentian violet. (PCS)

Enhancement of oxidative stability of the subtilisin nattokinase by site-directed mutagenesis expressed in Escherichia coli.

Science.gov (United States)

Weng, MeiZhi; Zheng, ZhongLiang; Bao, Wei; Cai, YongJun; Yin, Yan; Zou, GuoLin; Zou, GouLin

2009-11-01

Nattokinase (subtilisin NAT, NK) is a bacterial serine protease with strong fibrinolytic activity and it is a potent cardiovascular drug. In medical and commercial applications, however, it is susceptible to chemical oxidation, and subsequent inactivation or denaturation. Here we show that the oxidative stability of NK was substantially increased by optimizing the amino acid residues Thr(220) and Met(222), which were in the vicinity of the catalytic residue Ser(221) of the enzyme. Two nonoxidative amino acids (Ser and Ala) were introduced at these sites using site-directed mutagenesis. Active enzymes were successfully expressed in Escherichia coli with periplasmic secretion and enzymes were purified to homogeneity. The purified enzymes were analyzed with respect to oxidative stability, kinetic parameters, fibrinolytic activity and thermal stability. M222A mutant was found to have a greatly increased oxidative stability compared with wild-type enzyme and it was resistant to inactivation by more than 1 M H(2)O(2), whereas the wild-type enzyme was inactivated by 0.1 M H(2)O(2) (t(1/2) approximately 11.6 min). The other mutant (T220S) also showed an obvious increase in antioxidative ability. Molecular dynamic simulations on wild-type and T220S mutant proteins suggested that a hydrogen bond was formed between Ser(220) and Asn(155), and the spatial structure of Met(222) was changed compared with the wild-type. The present study demonstrates the feasibility of improving oxidative stability of NK by site-directed mutagenesis and shows successful protein engineering cases to improve stability of NK as a potent therapeutic agent.

Improvement of Lead Tolerance of Saccharomyces cerevisiae by Random Mutagenesis of Transcription Regulator SPT3.

Science.gov (United States)

Zhu, Liying; Gao, Shan; Zhang, Hongman; Huang, He; Jiang, Ling

2018-01-01

Bioremediation of heavy metal pollution with biomaterials such as bacteria and fungi usually suffer from limitations because of microbial sensitivity to high concentration of heavy metals. Herein, we adopted a novel random mutagenesis technique called RAISE to manipulate the transcription regulator SPT3 of Saccharomyces cerevisiae to improve cell lead tolerance. The best strain Mutant VI was selected from the random mutagenesis libraries on account of the growth performance, with higher specific growth rate than the control strain (0.068 vs. 0.040 h -1 ) at lead concentration as high as 1.8 g/L. Combined with the transcriptome analysis of S. cerevisiae, expressing the SPT3 protein was performed to make better sense of the global regulatory effects of SPT3. The data analysis revealed that 57 of S. cerevisiae genes were induced and 113 genes were suppressed, ranging from those for trehalose synthesis, carbon metabolism, and nucleotide synthesis to lead resistance. Especially, the accumulation of intracellular trehalose in S. cerevisiae under certain conditions of stress is considered important to lead resistance. The above results represented that SPT3 was acted as global transcription regulator in the exponential phase of strain and accordingly improved heavy metal tolerance in the heterologous host S. cerevisiae. The present study provides a route to complex phenotypes that are not readily accessible by traditional methods.

Chloroacetaldehyde-induced mutagenesis in Escherichia coli: The role of AlkB protein in repair of 3,N4-ethenocytosine and 3,N4-α-hydroxyethanocytosine

International Nuclear Information System (INIS)

Maciejewska, Agnieszka M.; Ruszel, Karol P.; Nieminuszczy, Jadwiga; Lewicka, Joanna; Sokolowska, Beata; Grzesiuk, Elzbieta; Kusmierek, Jaroslaw T.

2010-01-01

Etheno (ε) adducts are formed in reaction of DNA bases with various environmental carcinogens and endogenously created products of lipid peroxidation. Chloroacetaldehyde (CAA), a metabolite of carcinogen vinyl chloride, is routinely used to generate ε-adducts. We studied the role of AlkB, along with AlkA and Mug proteins, all engaged in repair of ε-adducts, in CAA-induced mutagenesis. The test system used involved pIF102 and pIF104 plasmids bearing the lactose operon of CC102 or CC104 origin (Cupples and Miller (1989) ) which allowed to monitor Lac + revertants, the latter arose by GC → AT or GC → TA substitutions, respectively, as a result of modification of guanine and cytosine. The plasmids were CAA-damaged in vitro and replicated in Escherichia coli of various genetic backgrounds. To modify the levels of AlkA and AlkB proteins, mutagenesis was studied in E. coli cells induced or not in adaptive response. Formation of εC proceeds via a relatively stable intermediate, 3,N 4 -α-hydroxyethanocytosine (HEC), which allowed to compare repair of both adducts. The results indicate that all three genes, alkA, alkB and mug, are engaged in alleviation of CAA-induced mutagenesis. The frequency of mutation was higher in AlkA-, AlkB- and Mug-deficient strains in comparison to alkA + , alkB + , and mug + controls. Considering the levels of CAA-induced Lac + revertants in strains harboring the pIF plasmids and induced or not in adaptive response, we conclude that AlkB protein is engaged in the repair of εC and HEC in vivo. Using the modified TTCTT 5-mers as substrates, we confirmed in vitro that AlkB protein repairs εC and HEC although far less efficiently than the reference adduct 3-methylcytosine. The pH optimum for repair of HEC and εC is significantly different from that for 3-methylcytosine. We propose that the protonated form of adduct interact in active site of AlkB protein.

Contribution of transcription-coupled DNA repair to MMS-induced mutagenesis in E. coli strains deficient in functional AlkB protein.

Science.gov (United States)

Wrzesiński, Michał; Nieminuszczy, Jadwiga; Sikora, Anna; Mielecki, Damian; Chojnacka, Aleksandra; Kozłowski, Marek; Krwawicz, Joanna; Grzesiuk, Elzbieta

2010-06-01

In Escherichia coli the alkylating agent methyl methanesulfonate (MMS) induces defense systems (adaptive and SOS responses), DNA repair pathways, and mutagenesis. We have previously found that AlkB protein induced as part of the adaptive (Ada) response protects cells from the genotoxic and mutagenic activity of MMS. AlkB is a non-heme iron (II), alpha-ketoglutarate-dependent dioxygenase that oxidatively demethylates 1meA and 3meC lesions in DNA, with recovery of A and C. Here, we studied the impact of transcription-coupled DNA repair (TCR) on MMS-induced mutagenesis in E. coli strain deficient in functional AlkB protein. Measuring the decline in the frequency of MMS-induced argE3-->Arg(+) revertants under transient amino acid starvation (conditions for TCR induction), we have found a less effective TCR in the BS87 (alkB(-)) strain in comparison with the AB1157 (alkB(+)) counterpart. Mutation in the mfd gene encoding the transcription-repair coupling factor Mfd, resulted in weaker TCR in MMS-treated and starved AB1157 mfd-1 cells in comparison to AB1157 mfd(+), and no repair in BS87 mfd(-) cells. Determination of specificity of Arg(+) revertants allowed to conclude that MMS-induced 1meA and 3meC lesions, unrepaired in bacteria deficient in AlkB, are the source of mutations. These include AT-->TA transversions by supL suppressor formation (1meA) and GC-->AT transitions by supB or supE(oc) formation (3meC). The repair of these lesions is partly Mfd-dependent in the AB1157 mfd-1 and totally Mfd-dependent in the BS87 mfd-1 strain. The nucleotide sequence of the mfd-1 allele shows that the mutated Mfd-1 protein, deprived of the C-terminal translocase domain, is unable to initiate TCR. It strongly enhances the SOS response in the alkB(-)mfd(-) bacteria but not in the alkB(+)mfd(-) counterpart. Copyright 2010 Elsevier B.V. All rights reserved.

Phage transposon mutagenesis.

Science.gov (United States)

Siegrist, M Sloan; Rubin, Eric J

2009-01-01

Phage transduction is an attractive method of genetic manipulation in mycobacteria. PhiMycoMarT7 is well suited for transposon mutagenesis as it is temperature sensitive for replication and contains T7 promoters that promote transcription, a highly active transposase gene, and an Escherichia coli oriR6 K origin of replication. Mycobacterial transposon mutant libraries produced by PhiMycoMarT7 transduction are amenable to both forward and reverse genetic studies. In this protocol, we detail the preparation of PhiMycoMarT7, including a description of the phage, reconstitution of the phage, purification of plaques, preparation of phage stock, and titering of phage stock. We then describe the transduction procedure and finally outline the isolation of individual transposon mutants.

Differential expression of SOS genes in an E. coli mutant producing unstable lexA protein enhances excision repair but inhibits mutagenesis

International Nuclear Information System (INIS)

Peterson, K.R.; Ganesan, A.K.; Mount, D.W.; Stanford Univ., CA)

1986-01-01

The SOS response is displayed following treatments which damage DNA or inhibit DNA replication. Two associated activities include enhanced capacity for DNA repair resulting from derepression of the recA, uvrA, uvrB and uvrD genes and increased mutagenesis due to derepression of recA, umuC and umuD. These changes are the consequence of the derepression of at least seventeen unlinked operons negatively regulated by LexA repressor. Following treatments that induce the SOS response, a signal molecule interacts with RecA protein, converting it to an activated form. Activated RecA protein facilitates the proteolytic cleavage of LexA repressor, which results in derepression of the regulon. The cell then enters a new physiological state during which time DNA repair processes are augmented. The lexA41 mutant of E. coli is a uv-resistant derivative of another mutant, lexA3, which produces a repressor that is not cleaved following inducing treatments. The resultant protein is unstable. Lac operon fusions to most of the genes in the SOS regulon were used to show that the various damage-inducible genes were derepressed to different extents. uvrA, B, and D were almost fully derepressed. Consistent with this finding, the rate of removal of T4 endonuclease V-sensitive sites was more rapid in the uv-irradiated lexA41 mutant than in normal cells, suggesting a more active excision repair system. We propose that the instability of the LexA41 protein reduces the intracellular concentration of repressor to a level that allows a high level of excision repair. The additional observation that SOS mutagenesis was only weakly induced in a lexA41 uvrA - mutant implies that the mutant protein partially represses one or more genes whose products promote SOS mutagenesis. 17 refs., 4 figs., 1 tab

Plasmid pKM101-dependent repair and mutagenesis in Escherichia coli cells with mutations lexB30 tif and zab-53 in the recA gene

International Nuclear Information System (INIS)

Blanco, M.; Rebollo, J.E.

1981-01-01

Bacterial survival after UV irradiation was increased in E. coli K12 lex B30 and tif zab-53 mutants harboring plasmid pKM101. Mutagenesis in response to UV was observed in these bacteria which, in absence of pKM101, are not UV-mutable. The mutator effect observed in unirradiated wild-type cells containing pKM101 was higher after incubation at 30 0 C with adenine than at 37 0 C. This effect was still enhanced by tif mutation, even in the tif zab-53 strain, but it was abolished by lexB30 mutation. In the tif zab-53 (pKM101) strain, repair and mutagenesis of UV-irradiated phage lambda was observed, but not in the lexB30 mutant carrying pKM101. The pKM101 mutant, pGW1, was unable to protect tif zab-53 bacteria against killing by UV, whereas the protection of lexB30 was intermediate; moreover, it did not promote the mutator effect at 30 0 C or enhance phage repair and mutagenesis in tif zab-53 cells. All UV-induced bacterial mutations in lexB30 (pKM101) strain were suppressors; in contrast, true revertants were found after UV irradiation of the tif zab-53 (pKM101) cells. (orig./AJ)

Investigation of lethal and mutagenetic effects of UV-light on Salmonella currying wild and mutant alleles of lex A gene of Escherichia coli in the Salmonella genome

International Nuclear Information System (INIS)

Andreeva, I.V.; Tiganova, I.G.; Skavronskaya, A.G.

1981-01-01

Inheritance of LexA-gene of Escherichia coli- by Salmonella takes place during intergeneric trunsduction cross of Escherichia coli and Salmonella typhimurium. The presence of LexA-E. coli gene-did not eliminate earlier revealed peculiarity consisting in the absence of UV-induced mutagenesis in most of studied salmonollosis strains. So it is shown that the absence of UV mutagenesis in Salmonella does not result from mutation in LexA-gene. Inheritance of pKM101 by LexA-hybrid provides pronounced UV mutability and protective effect. Inheritance of this plasmid by LexA-hybrid did not result in the appearance of capability for UV-induced mutagenesis and improving UV resistance of bacteria. Thus the plasmids effect on repair and mutagenesis in Salmonella, the same as in E. coli, reveals in LexA-phenotype [ru

E. coli mismatch repair enhances AT-to-GC mutagenesis caused by alkylating agents.

Science.gov (United States)

Nakano, Kota; Yamada, Yoko; Takahashi, Eizo; Arimoto, Sakae; Okamoto, Keinosuke; Negishi, Kazuo; Negishi, Tomoe

2017-03-01

Alkylating agents are known to induce the formation of O 6 -alkylguanine (O 6 -alkG) and O 4 -alkylthymine (O 4 -alkT) in DNA. These lesions have been widely investigated as major sources of mutations. We previously showed that mismatch repair (MMR) facilitates the suppression of GC-to-AT mutations caused by O 6 -methylguanine more efficiently than the suppression of GC-to-AT mutations caused by O 6 -ethylguanine. However, the manner by which O 4 -alkyT lesions are repaired remains unclear. In the present study, we investigated the repair pathway involved in the repair of O 4 -alkT. The E. coli CC106 strain, which harbors Δprolac in its genomic DNA and carries the F'CC106 episome, can be used to detect AT-to-GC reverse-mutation of the gene encoding β-galactosidase. Such AT-to-GC mutations should be induced through the formation of O 4 -alkT at AT base pairs. As expected, an O 6 -alkylguanine-DNA alkyltransferase (AGT) -deficient CC106 strain, which is defective in both ada and agt genes, exhibited elevated mutant frequencies in the presence of methylating agents and ethylating agents. However, in the UvrA-deficient strain, the methylating agents were less mutagenic than in wild-type, while ethylating agents were more mutagenic than in wild-type, as observed with agents that induce O 6 -alkylguanine modifications. Unexpectedly, the mutant frequencies decreased in a MutS-deficient strain, and a similar tendency was observed in MutL- or MutH-deficient strains. Thus, MMR appears to promote mutation at AT base pairs. Similar results were obtained in experiments employing double-mutant strains harboring defects in both MMR and AGT, or MMR and NER. E. coli MMR enhances AT-to-GC mutagenesis, such as that caused by O 4 -alkylthymine. We hypothesize that the MutS protein recognizes the O 4 -alkT:A base pair more efficiently than O 4 -alkT:G. Such a distinction would result in misincorporation of G at the O 4 -alkT site, followed by higher mutation frequencies in wild

Molecular processes as basis for plasmid-mediated bacterial UV-light resistance and mutagenesis

International Nuclear Information System (INIS)

Aleshkin, G.I.; Brukhanskij, G.V.; Skavronskaya, A.G.

1985-01-01

The increase of UV-resistance and UV-induced mutagenesis by lambda 1 pint intmid as well as molecular-genetic mechanisms of plasmid participation in reparation and DNA replication and its degradation after UV-irradiation in plasmid cells on pKM101 plasmid model have been investigated. Data testifying to the necessity of intmid integration in chromosome as obligatory stage of intmid participation in increasing UV-resistance of bacterial cells are obtained. It has been found that intmid raises UV-resistance of cells and increases respectively the UV-induced reverants efficiency. On the basis of the experiment data the conclusion is drawn that the intmid capacity to raise UV-resistance and, possibly, mutagenesis is bound not only with its integration into chromosome but also with pol A + chromosome replication by dependendent imtmid replication complex. It is shown that pKM101 plasmid ensures functioning in E coli cells of inducible, chloroamphenicol-resistant DNA replication, highly resistant to UV-light harmful effect and that the volume of excision reparation in E. coli cells carrying pKM101 plasmid is increased as compared with the volume of reparation in plasmid legs cells. The combination of the data obtained gives grounds to the authors to assume that inducible replication, inducible reparation of DNA and inducible decrease of DNA degradation determined by pKM101 plasmid may serve as recA + lexA + basis dependent increase of UV-resistance and mutagenesis and that these processes provide the possibility of functioning of integrative replication mechanism of plasmid participation in ensuring UV-resistance and mutagenesis of plants

R-prime site-directed transposon Tn7 mutagenesis of the photosynthetic apparatus in Rhodopseudomonas capsulata

Energy Technology Data Exchange (ETDEWEB)

Youvan, D C [Univ. of California, Berkeley; Elder, J T; Sandlin, D E; Zsebo, K; Alder, D P; Panopoulos, N J; Marrs, B L; Hearst, J E

1982-01-01

Site-directed mutagenesis of the photosynthetic apparatus (PSA) genes in Rhodopseudomonas capsulata is presented utilizing a transposon Tn7 mutagenized R-prime. The R-prime, pRPS404, bears most of the genes necessary for the differentiation of the photosynthetic apparatus. Mutagenesis of the R-prime with Tn7 in Escherichia coli, conjugation into R. capsulata, and homologous recombination with the wild-type alleles efficiently generates photosynthetic apparatus lesions. Wild-type alleles are lost spontaneously and the Tn7-induced lesions are revealed by subsequent intramolecular recombination between IS21 insertion elements that bracket the prime sequences in direct repeat. The molecular nature of the intermediates involved in the transposition, recombination and deletion have been investigated by Southern hybridization analysis. The spontaneous loss of wild-type alleles after homologous recombination with the chromosome may be of general use to other prokaryotic site-directed transposon mutagenesis schemes. The IS21-mediated deletion of the prime DNA is dependent on the RecA protein in E. coli, generating the parental R-factor bearing one IS21 element. A genetic-physical map exists for a portion of the prime photosynthetic apparatus DNA. When Tn7 is inserted into a bacteriochlorophyll gene in the R-prime and then crossed into R. capsulata, mutants are produced that accumulate a bacteriochlorophyll precursor, which is in excellent agreement with the existing genetic-physical map. This corroborates the mutagenesis scheme.

Comprehensive mutagenesis of the fimS promoter regulatory switch reveals novel regulation of type 1 pili in uropathogenic Escherichia coli.

Science.gov (United States)

Zhang, Huibin; Susanto, Teodorus T; Wan, Yue; Chen, Swaine L

2016-04-12

Type 1 pili (T1P) are major virulence factors for uropathogenic Escherichia coli (UPEC), which cause both acute and recurrent urinary tract infections. T1P expression therefore is of direct relevance for disease. T1P are phase variable (both piliated and nonpiliated bacteria exist in a clonal population) and are controlled by an invertible DNA switch (fimS), which contains the promoter for the fim operon encoding T1P. Inversion of fimS is stochastic but may be biased by environmental conditions and other signals that ultimately converge at fimS itself. Previous studies of fimS sequences important for T1P phase variation have focused on laboratory-adapted E coli strains and have been limited in the number of mutations or by alteration of the fimS genomic context. We surmounted these limitations by using saturating genomic mutagenesis of fimS coupled with accurate sequencing to detect both mutations and phase status simultaneously. In addition to the sequences known to be important for biasing fimS inversion, our method also identifies a previously unknown pair of 5' UTR inverted repeats that act by altering the relative fimA levels to control phase variation. Thus we have uncovered an additional layer of T1P regulation potentially impacting virulence and the coordinate expression of multiple pilus systems.

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

Science.gov (United States)

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

2017-11-01

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

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

Science.gov (United States)

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.

Mutations at the cysteine codons of the recA gene of Escherichia coli

International Nuclear Information System (INIS)

Weisemann, J.M.; Weinstock, G.M.

1988-01-01

Each of the three cysteine residues in the Escherichia coli RecA protein was replaced with a number of other amino acids. To do this, each cysteine codon was first converted to a chain-terminating amber codon by oligonucleotide-directed mutagenesis. These amber mutants were then either assayed for function in different suppressor strains or reverted by a second round of mutagenesis with oligonucleotides that had random sequences at the amber codon. Thirty-three different amino acid substitutions were obtained. Mutants were tested for three functions of RecA: survival following UV irradiation, homologous recombination, and induction of the SOS response. It was found that although none of the cysteines is essential for activity, mutations at each of these positions can affect one or more of the activities of RecA, depending on the particular amino acid substitution. In addition, the cysteine at position 116 appears to be involved in the RecA-promoted cleavage of the LexA protein

Complementation of a pKM101 derivative that decreases resistance to UV killing but increases susceptibility to mutagenesis

International Nuclear Information System (INIS)

Langer, P.J.; Perry, K.L.; Walker, G.C.

1985-01-01

The drug resistance plasmid pKM101 makes Escherichia coli resistant to the lethal effects of ultraviolet (UV) irradiation and more susceptible to mutagenesis by a variety of agents. The plasmid operon responsible for increasing mutagenesis has been termed mucAB (Mutagenesis, UV and chemical). The authors have isolated a derivative of pKM101 called pGW1975 which makes cells more sensitive to killing by UV but which retains the ability of pKM101 to increase susceptibility to methyl methanesulfonate (MMS) mutagenesis. pGW1975 increases UV mutagenesis less than pKM101 in a uvrA + strain but more than pKM101 in a uvrA - strain. muc - point and insertion mutants of pKM101 and pGW1975 complement to restore the plasmid-mediated: (i) ability to reactivate UV-irradiated phage, (ii) resistance to killing by UV, and (iii) level of susceptibility to UV mutagenesis. They have identified a 2.0 kb region of pKM101 which is responsible for the complementation and which maps counterclockwise of mucAB. (Auth)

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

International Nuclear Information System (INIS)

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

2012-01-01

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, a mathematical model of the bacterial mismatch repair system is developed. Within this model, the key pathways of this type of repair are simulated on the basis of modern experimental data related to its mechanisms. Here we have modelled in detail five main pathways of DNA misincorporation removal with different DNA exonucleases. Using our calculations, we have tested the hypothesis that the bacterial mismatch repair system is responsible for the removal of the nucleotides misincorporated by DNA polymerase V (the UmuD' 2 C complex) during ultraviolet-induced SOS response. For the theoretical analysis of the mutation frequency, we have combined the proposed mathematical approach with the model of SOS-induced mutagenesis in the E.coli bacterial cell developed earlier. Our calculations support the hypothesis that methyl-directed mismatch repair influences the mutagenic effect of ultraviolet radiation

Formate detection by potassium permanganate for enhanced hydrogen production in Escherichia coli

Energy Technology Data Exchange (ETDEWEB)

Maeda, Toshinari [Artie McFerrin Department of Chemical Engineering, 220 Jack E. Brown Building, Texas A and M University, College Station, TX 77843-3122 (United States); Wood, Thomas K. [Artie McFerrin Department of Chemical Engineering, 220 Jack E. Brown Building, Texas A and M University, College Station, TX 77843-3122 (United States); Department of Biology, Texas A and M University, College Station, TX 77843-3258 (United States); Zachry Department of Civil and Environmental Engineering, Texas A and M University, College Station, TX 77843-3136 (United States)

2008-05-15

Mutagenesis of Escherichia coli for hydrogen production is difficult since there is no high-throughput screen. Here we describe a method for rapid detection of enhanced hydrogen production by engineered strains by detecting formate via potassium permanganate; in E. coli, hydrogen is synthesized from formate using the formate hydrogen lyase system. (author)

Mechanisms of uv mutagenesis in yeast

International Nuclear Information System (INIS)

Lawrence, C.W.; Christensen, R.; Schwartz, A.

1982-01-01

The uv mutagenesis in yeast depends on the function of the RAD6 locus, a gene that is also responsible for a substantial fraction of wild-type resistance, suggesting that this eukaryote may possess a misrepair mechanism analogous to that proposed for Escherichia coli. The molecular mechanism responsible for RAD6 repair or recovery is not yet known, but it is different from either excision or recombination-dependent repair, processes carried out by the other two main repair pathways in yeast. RAD6-dependent mutagenesis has been found to have the following characteristics. It is associated at best with only a small fraction of RAD6-dependent repair, the majority of the sensitivity of rad6 mutants being due to their lack of nonmutagenic repair. SRS2 metabolic suppressors restore a substantial fraction of uv resistance to rad6 mutants but do not restore their uv mutability. Strains containing mutations at loci (rev, umr) that are probably more directly involved in mutagenesis are only mildly sensitive, and there is a poor correlation between their sensitivity and mutational deficiency. The uv mutagenesis appears to require a large number of gene functions, perhaps ten or more. Where examined in detail, these genes have been found to be concerned in the production of only a specific range of mutational events, not all of them. Mating experiments have shown that a substantial fraction, probably 40% or more, of uv-induced mutations are untargeted, that is, occur in lesion-free regions of DNA. The uv irradiation, therefore, produces a general reduction in the normally high fidelity with which DNA is replicated on undamaged templates. It does not appear to be necessary for the causal lesion to be present in the same chromosome as the mutation it induces. The reduction in fidelity may be the consequence of the production of a diffusible factor in uv-irradiated cells, but definite evidence supporting this proposal has not yet been obtained

Repair system and mitomycin mutagenesis in Escherichia coli

Energy Technology Data Exchange (ETDEWEB)

Otsuji, N; Murayama, I [Kyushu Univ., Fukuoka (Japan). Faculty of Pharmaceutical Sciences

1974-03-01

Ultraviolet light sensitive mutants of E. coli defective at the uvrA, uvrB or uvrC locus showed increased sensitivity to the lethal effects of monofunctional mitomycin, 7-methoxymitosene (7-MMT) or decarbamoyl mitomycin C (DCMTC), as well as mitomycin C (MTC). (1) Treatment of wild type bacteria with these monofunctional mitomycins resulted in the production of single-strand breaks in DNA, which were repaired upon incubation in a growth medium. Such breaks in DNA were not produced in the UvrA and the UvrB mutants. In the UvrC mutant, single-strand breaks were produced by 7-MMT or by DCMTC, but these breaks were not repaired upon incubation. (2) Exposure of E. coli to MTC caused cross-linkage between DNA strands, which converted to a normally denaturable form during further incubation after treatment. This was not occurred in the UvrB strain. In the UvrC mutant, a portion of the cross-links was removed upon incubation. (3) 7-MMT and DCMTC induced mutation in UV sensitive UvrB and UvrC mutants with much higher frequency than in wild type bacteria, while MTC did not induce mutation in these Uvr-strains.

Repair system and mitomycin mutagenesis in Escherichia coli

International Nuclear Information System (INIS)

Otsuji, Nozomu; Murayama, Ichiko

1974-01-01

Ultraviolet light sensitive mutants of E. coli defective at the uvrA, uvrB or uvrC locus showed increased sensitivity to the lethal effects of monofunctional mitomycin, 7-methoxymitosene (7-MMT) or decarbamoyl mitomycin C (DCMTC), as well as mitomycin C (MTC). (1) Treatment of wild type bacteria with these monofunctional mitomycins resulted in the production of single-strand breaks in DNA, which were repaired upon incubation in a growth medium. Such breaks in DNA were not produced in the UvrA and the UvrB mutants. In the UvrC mutant, single-strand breaks were produced by 7-MMT or by DCMTC, but these breaks were not repaired upon incubation. (2) Exposure of E. coli to MTC caused cross-linkage between DNA strands, which converted to a normally denaturable form during further incubation after treatment. This was not occurred in the UvrB strain. In the UvrC mutant, a portion of the cross-links was removed upon incubation. (3) 7-MMT and DCMTC induced mutation in UV sensitive UvrB and UvrC mutants with much higher frequency than in wild type bacteria, while MTC did not induce mutation in these Uvr-strains. (author)

Identification of NH4+-regulated genes of Herbaspirillum seropedicae by random insertional mutagenesis.

Science.gov (United States)

Schwab, Stefan; Ramos, Humberto J; Souza, Emanuel M; Pedrosa, Fábio O; Yates, Marshall G; Chubatsu, Leda S; Rigo, Liu U

2007-05-01

Random mutagenesis using transposons with promoterless reporter genes has been widely used to examine differential gene expression patterns in bacteria. Using this approach, we have identified 26 genes of the endophytic nitrogen-fixing bacterium Herbaspirillum seropedicae regulated in response to ammonium content in the growth medium. These include nine genes involved in the transport of nitrogen compounds, such as the high-affinity ammonium transporter AmtB, and uptake systems for alternative nitrogen sources; nine genes coding for proteins responsible for restoring intracellular ammonium levels through enzymatic reactions, such as nitrogenase, amidase, and arginase; and a third group includes metabolic switch genes, coding for sensor kinases or transcription regulation factors, whose role in metabolism was previously unknown. Also, four genes identified were of unknown function. This paper describes their involvement in response to ammonium limitation. The results provide a preliminary profile of the metabolic response of Herbaspirillum seropedicae to ammonium stress.

RecA-mediated cleavage activates UmuD for mutagenesis: Mechanistic relationship between transcriptional derepression and posttranslational activation

International Nuclear Information System (INIS)

Nohmi, Takehiko; Battista, J.R.; Dodson, L.A.; Walker, G.C.

1988-01-01

The products of the SOS-regulated umuDC operon are required for most UV and chemical mutagenesis in Escherichia coli. It has been shown that the UmuD protein shares homology with LexA, the repressor of the SOS genes. In this paper the authors describe a series of genetic experiments that indicate that the purpose of RecA-mediated cleavage of UmuD at its bond between Cys-24 and Gly-25 is to activate UmuD for its role in mutagenesis and that the COOH-terminal fragment of UmuD is necessary and sufficient for the role of UmuD in UV mutagenesis. Other genetic experiments are presented that (i) support the hypothesis that the primary role of Ser-60 in UmuD function is to act as a nucleophile in the RecA-mediated cleavage reaction and (ii) raise the possibility that RecA has a third role in UV mutagenesis besides mediating the cleavage of LexA and UmuD

Study of UV-mutagenesis in Bacillus subtilis

International Nuclear Information System (INIS)

Lotareva, O.V.; Filippov, V.D.

1974-01-01

The sensitivity of Bac. subtilis to the inactivating and mutagenic effects of UV-mutants has been determined: uvr, which does not extract pyrimidine dimers from damaged DNA; recsub(x), which exhibits a reduced activity of ATP-dependent DNAase; poll, which is devoid of DNA polymerase, and wild strains (DT). The sensitivity of these strains to the inactivating effects of UV rays increases in the order: DT<= recsub(x) << uvr < poll, and UV mutability in the order: DT = rec(sub(x) < poll<< uvr. A comparison of UV mutagenesis in Bac. subtilis and E. coli suggests the hypothesis that the mechanisms of UV mutation formation are similar in these two organisms. (author)

Genetic analysis of an Escherichia coli urease locus: evidence of DNA rearrangement.

OpenAIRE

Collins, C M; Falkow, S

1988-01-01

Ureolytic Escherichia coli strains are uncommon clinical isolates. The urease phenotype in a large percentage of these isolates is unstable and lost upon storage. We examined two urease-positive uropathogenic E. coli isolates that give off urease-negative segregants and determined that the urease phenotype was chromosomally encoded. The urease phenotype was cloned from E. coli 1021 and found to be encoded on a 9.4-kilobase HindIII restriction fragment. Transposon mutagenesis indicated that at...

Theory of misrepair mutagenesis

International Nuclear Information System (INIS)

Bresler, S.E.

1975-01-01

On the basis of experimental data, a model of induced mutagenesis is proposed that takes into account the repair of DNA damage by the Rec system. The peculiar feature of the Rec system is the cleavage and resynthesis of long sequences near the recognized DNA damage. Up to 1000-2000 nucleotides are replaced in one act. Therefore a definite probability exists of finding a damaged point on the second strand serving as template. It is believed that at this point no requirements of complementarity exist and that a random substitution can take place. This is the origin of a point mutation (transversion or frameshift). From this model, a general formula for the dose-response curve of mutagenesis is deduced which also takes into account the possibility of simultaneously initiated repair on both complementary strands of DNA. The latter leads to a lethal event when the points are situated proximally. This formula fits the observations in different cases studied. Some fundamental observations such as the absence of mutants from predominant single-strand breaks of DNA chains are readily explained

Novel patterns of ultraviolet mutagenesis and Weigle reactivation in Staphylococcus aureus and phage phi II

International Nuclear Information System (INIS)

Thompson, J.K.; Hart, M.G.R.

1981-01-01

The effects of u.v. irradiation on the survival of Staphylococcus aureus and its phage phi11 were studied. The recA and uvr mutations affected their survival like synonymous mutations in Escherichia coli. Weigle reactivation (W-reactivation) of phi11 occurred in wild-type S. aureus and in a uvr mutant. Reactivation was recA-dependent and was accompanied by u.v.-induced mutagenesis in a temperature-sensitive mutant of phi11. Bacterial mutation to streptomycin resistance was induced by u.v. and was also recA-dependent. In S. aureus, as in E. coli, u.v. was a more effective mutagen in the uvr genetic background. However, a dose-squared response for u.v.-induced mutation of wild-type and uvr strains of S. aureus to streptomycin resistance, and of a trp auxotroph to tryptophan independence, was found only with u.v. doses below 1 J m -2 . In relation to the Uvr mechanism of DNA repair, u.v. mutagenesis in S. aureus may involve both repairable and non-repairable lesions. As in E. Coli, the uvr genetic background reduced the u.v. dose required for maximal W-reactivation of u.v.-irradiated phage. However, there was no enhancement of W-reactivation by post-irradiation broth incubation of S. aureus. The results are compatible with a non-inducible mechanism for this phenomenon. (author)

Step-By-Step In Vitro Mutagenesis: Lessons From Fucose-Binding Lectin PA-IIL.

Science.gov (United States)

Mrázková, Jana; Malinovská, Lenka; Wimmerová, Michaela

2017-01-01

Site-directed mutagenesis is a powerful technique which is used to understand the basis of interactions between proteins and their binding partners, as well as to modify these interactions. Methods of rational design that are based on detailed knowledge of the structure of a protein of interest are often used for preliminary investigations of the possible outcomes which can result from the practical application of site-directed mutagenesis. Also, random mutagenesis can be used in tandem with site-directed mutagenesis for an examination of amino acid "hotspots."Lectins are sugar-binding proteins which, among other functions, mediate the recognition of host cells by a pathogen and its adhesion to the host cell surface. Hence, lectins and their binding properties are studied and engineered using site-directed mutagenesis.In this chapter, we describe a site-directed mutagenesis method used for investigating the sugar binding pattern of the PA-IIL lectin from the pathogenic bacterium Pseudomonas aeruginosa. Moreover, procedures for the production and purification of PA-IIL mutants are described, and several basic methods for characterizing the mutants are discussed.

The two umuDC-like operons, samAB and umuDCST, in Salmonella typhimurium: The umuDCST operon may reduce UV-mutagenesis-promoting ability of the samAB operon

International Nuclear Information System (INIS)

Nohmi, Takehiko; Hakura, Atsushi; Watanabe, Masahiko; Yamada, Masami; Sofuni, Toshio; Nakai, Yasuharu; Murayama, Somay Y.

1993-01-01

Salmonella typhimurium, especially its derivatives containing pKM101 plasmid, has been widely used in the Ames test for the detection of environmental mutagens and carcinogens. It is known, however, that if the pKM101 plasmid is eliminated, S. typhimurium itself shows a much weaker mutagenic response to UV and some chemical mutagens than does Escherichia coli. In fact, certain potent base-change type mutagens, such as furylfuramide and aflatoxin B 1 , are nonmutagenic to S. typhimurium in the absence of pKM101, whereas they are strongly mutagenic to S. typhimurium in the presence of pKM101 plasmid as well as to E. coli. The low mutability can be restored to levels comparable to E. coli by introducing the plasmid carrying the E. coli umuDC operon or the pKM101 plasmid carrying mucAB operon. Salmonella typhimurium has an SOS regulatory system which resembles that of E. coli. Thus, it was suggested that S. typhimurium is deficient in the function of umuDC operon, which plays an essential role in UV and most chemical mutagenesis in E. coli. In order to clarify the implications of umuDC genes in mutagenesis and antimutagenesis in typhimurium, we have independently screened the umuDC-like genes of S. typhimurium TA1538. Consequently, we have cloned another umuDC-like operon which is 40% diverged from the aforementioned umuDC operon of S. typhimurium LT2 at the nucleotide level (16). We have termed the cloned DNA the samAB (Salmonella; mutagenesis) operon, and tentatively referred to the umuDC operon cloned from S. typhimurium LT2 (27,31) as the umuDC ST operon. Based on the results of the Southern hybridization experiment, we concluded that the two sets of umuDC-like operons reside in the same cells of S. typhimurium LT2 and TA1538. Our results also suggested that the umuDC ST operon reduces the UV-mutagenesis promoting ability of the samAB operon when the two operons are present on the same multi-copy number plasmid

Ultraviolet radiation-induced mutability of uvrD3 strains of Escherichia coli B/r and K-12: a problem in analyzing mutagenesis data

International Nuclear Information System (INIS)

Smith, K.C.

1976-01-01

The involvement of the uvrD gene product in UV-induced mutagenesis in Escherichia coli was studied by comparing wild-type and uvrA or uvrB strains with their uvrD derivatives in B/r and K-12(W3110) backgrounds. Mutations per survivor (reversions to prototrophy) were compared as a function of surviving fraction and of UV fluence. While recognizing that both methods are not without problems, arguments are presented for favoring the former rather than the latter method of presenting the data when survival is less than 100%. When UV-induced mutation frequencies were plotted as a function of surviving fraction, the uvrD derivatives were less mutable than the corresponding parent strains. The B/r strains exhibited higher mutation frequencies than did the K-12(W3110) strains. A uvrB mutation increased the mutation frequency of its parental K-12 strain, but a uvrA mutation only increased the mutation frequency of its parental B/r strain at UV survivals greater than approximately 80%. Both the uvrA and uvrB mutations increased the mutation frequencies of the uvrD strains in the B/r and K-12 backgrounds, respectively. Rather different conclusions would be drawn if mutagenesis were considered as a function of UV fluence rather than of survival, a situation that calls for further work and discussion. Ideally mutation efficiencies should be compared as a function of the number of repair events per survivor, a number that is currently unobtainable. (author)

The comparative investigation of gene mutation induction in Bacillus subtilis and Escherichia coli cells after irradiation by different LET radiation

International Nuclear Information System (INIS)

Borejko, A.V.; Bulah, A.P.

2005-01-01

The data of mutagenetic action of ionizing radiation with different physical characteristics on bacterial cells with various genotypes are presented. It was shown that regularities of inducible mutagenesis in Bacillus subtilis and E. coli are consimilar. The dose-response dependence for both types of cells is described by the linear-quadratic function. The RBE on LET relationship has a local maximum at 20 keV/μm. The crucial role in inducible mutagenesis in E. coli and Bacillus subtilis cells is played by the error-prone SOS-repair

Mechanism of replication of ultraviolet-irradiated single-stranded DNA by DNA polymerase III holoenzyme of Escherichia coli. Implications for SOS mutagenesis

International Nuclear Information System (INIS)

Livneh, Z.

1986-01-01

Replication of UV-irradiated oligodeoxynucleotide-primed single-stranded phi X174 DNA with Escherichia coli DNA polymerase III holoenzyme in the presence of single-stranded DNA-binding protein was investigated. The extent of initiation of replication on the primed single-stranded DNA was not altered by the presence of UV-induced lesions in the DNA. The elongation step exhibited similar kinetics when either unirradiated or UV-irradiated templates were used. Inhibition of the 3'----5' proofreading exonucleolytic activity of the polymerase by dGMP or by a mutD mutation did not increase bypass of pyrimidine photodimers, and neither did purified RecA protein influence the extent of photodimer bypass as judged by the fraction of full length DNA synthesized. Single-stranded DNA-binding protein stimulated bypass since in its absence the fraction of full length DNA decreased 5-fold. Termination of replication at putative pyrimidine dimers involved dissociation of the polymerase from the DNA, which could then reinitiate replication at other available primer templates. Based on these observations a model for SOS-induced UV mutagenesis is proposed

Transgenic Chinese hamster V79 cell lines which exhibit variable levels of gpt mutagenesis

International Nuclear Information System (INIS)

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

1990-01-01

The Escherichia coli gpt gene coding for xanthine-guanine phosphoribosyl transferase has been stably transfected into HPRT - Chinese hamster V79 cells. Several gpt - cell lines have been established, which retain the sequence(s) even after long-term culture without selection for gpt. While spontaneous mutagenesis to gpt - occurs rather frequently for most cell lines, it cannot be correlated with either the number of plasmid integration sites or deletion of the plasmid sequence(s). One transgenic cell line (g12), which continuously maintains a low spontaneous mutation frequency was used in comparative mutagenesis studies with wild-type V79 cells (gpt vs. hprt). Alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and β-propiolactone (BPL) are shown to be equally toxic and mutagenic in both g12 and V79 cells. UV and X-rays are also equally toxic to both cell lines. The data presented here suggests that g12 cells may be useful to study mammalian mutagenesis by agents which yield limited response at the hprt locus

UV irradiation alters deoxynucleoside triphosphate pools in Escherichia coli

International Nuclear Information System (INIS)

Das, S.K.; Loeb, L.A.

1984-01-01

UV irradiation of exponentially growing Escherichia coli increased intracellular concentration of dATP and dTTP without significantly changing the concentrations of dGTP and dCTP. These selective increases in dATP and dTTP pools are seen in wild-type E. coli K12 and AB1157, as well as in recA and umuC strains, and are proportional to UV dose. The possible significance of these findings with respect to induction of the SOS response and nontargeted mutagenesis are discussed. (orig.)

Improving isopropanol tolerance and production of Clostridium beijerinckii DSM 6423 by random mutagenesis and genome shuffling.

Science.gov (United States)

Gérando, H Máté de; Fayolle-Guichard, F; Rudant, L; Millah, S K; Monot, F; Ferreira, Nicolas Lopes; López-Contreras, A M

2016-06-01

Random mutagenesis and genome shuffling was applied to improve solvent tolerance and isopropanol/butanol/ethanol (IBE) production in the strictly anaerobic bacteria Clostridium beijerinckii DSM 6423. Following chemical mutagenesis with N-methyl-N-nitro-N-nitrosoguanidine (NTG), screening of putatively improved strains was done by submitting the mutants to toxic levels of inhibitory chemicals or by screening for their tolerance to isopropanol (>35 g/L). Suicide substrates, such as ethyl or methyl bromobutyrate or alcohol dehydrogenase inhibitors like allyl alcohol, were tested and, finally, 36 mutants were isolated. The fermentation profiles of these NTG mutant strains were characterized, and the best performing mutants were used for consecutive rounds of genome shuffling. Screening of strains with further enhancement in isopropanol tolerance at each recursive shuffling step was then used to spot additionally improved strains. Three highly tolerant strains were finally isolated and able to withstand up to 50 g/L isopropanol on plates. Even if increased tolerance to the desired end product was not always accompanied by higher production capabilities, some shuffled strains showed increased solvent titers compared to the parental strains and the original C. beijerinckii DSM 6423. This study confirms the efficiency of genome shuffling to generate improved strains toward a desired phenotype such as alcohol tolerance. This tool also offers the possibility of obtaining improved strains of Clostridium species for which targeted genetic engineering approaches have not been described yet.

Mutagenesis of lambda phage by tif-expression or host-irradiation functions is largely independent of damage in the phage DNA

International Nuclear Information System (INIS)

Von Wright, A.; Bridges, B.A.

1980-01-01

The survival and mutagenesis of UV-irradiated phage lambda, as well as bacterial mutagenesis, are enhanced in tif mutants of Escherichia coli when these strains are grown at 43 0 C (Castellazzi et al., 1972). This was interpreted on the basis of a hypothesis (the SOS hypothesis) according to which the UV-inducible phenomena connected with reactivation and mutagenesis of UV-irradiated bacteriophages (Weigle, 1953; Radman, 1975) are constitutively expressed in tif-bacteria at high temperature (Witkin, 1974). In unpublished experiments with phage T3 we found that the survival of UV-irradiated phage is also better at 43 0 C than at 32 0 C in tif + cells and this made us reexamine the significance and nature of tif expression and examine its effects on both unirradiated and UV-irradiated phage lambda. Our results indicate that tif-induced mutagenesis and possibly reactivation of UV-irradiated phage lambda should be reinterpreted. (orig./AJ)

Oxazolidinone resistance mutations in 23S rRNA of Escherichia coli reveal the central region of domain V as the primary site of drug action

DEFF Research Database (Denmark)

Xiong, L; Kloss, P; Douthwaite, S

2000-01-01

Oxazolidinone antibiotics inhibit bacterial protein synthesis by interacting with the large ribosomal subunit. The structure and exact location of the oxazolidinone binding site remain obscure, as does the manner in which these drugs inhibit translation. To investigate the drug-ribosome interaction......, we selected Escherichia coli oxazolidinone-resistant mutants, which contained a randomly mutagenized plasmid-borne rRNA operon. The same mutation, G2032 to A, was identified in the 23S rRNA genes of several independent resistant isolates. Engineering of this mutation by site-directed mutagenesis...

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

Science.gov (United States)

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…

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

Science.gov (United States)

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

2017-06-22

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

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

Science.gov (United States)

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.

Mapping important nucleotides in the peptidyl transferase centre of 23 S rRNA using a random mutagenesis approach

DEFF Research Database (Denmark)

Porse, B T; Garrett, R A

1995-01-01

Random mutations were generated in the lower half of the peptidyl transferase loop in domain V of 23 S rRNA from Escherichia coli using a polymerase chain reaction (PCR) approach, a rapid procedure for identifying mutants and a plasmid-based expression system. The effects of 21 single-site mutati...

The Comparative Investigation of Gene Mutation Induction in {\\it Bacillus subtilis} and {\\it Escherichia coli} Cells after Irradiation by Different LET Radiation

CERN Document Server

Boreyko, A V

2005-01-01

The data of mutagenic action of ionizing radiation with different physical characteristics on bacterial cells with various genotypes are presented. It was shown that regularities of inducible mutagenesis in {\\it Bacillus subtilis} and {\\it E.coli} are consimilar. The dose-response dependence for both types of cells is described by the linear-quadratic function. The RBE on LET relationship has a local maximum at 20 keV/$\\mu $m. The crucial role in inducible mutagenesis in {\\it E.coli} and {\\it Bacillus subtilis} cells is played by the error-prone $SOS$-repair.

Dominant negative umuD mutations decreasing RecA-mediated cleavage suggest roles for intact UmuD in modulation of SOS mutagenesis

International Nuclear Information System (INIS)

Battista, J.R.; Ohta, Toshihiro; Nohmi, Takehiko; Sun, W.; Walker, G.C.

1990-01-01

The products of the SOS-regulated umuDC operon are required for most UV and chemical mutagenesis in Escherichia coli. The UmuD protein shares homology with a family of proteins that includes LexA and several bacteriophage repressors. UmuD is posttranslationally activated for its role n mutagenesis by a RecA-mediated proteolytic cleavage that yields UmuD'. A set of missense mutants of umuD was isolated and shown to encode mutant UmuD proteins that are deficient in RecA-mediated cleavage in vivo. Most of these mutations are dominant to umuD + with respect to UV mutagenesis yet do not interfere with SOS induction. Although both UmuD and UmuD' form homodimers, the authors provide evidence that they preferentially form heterodimers. The relationship of UmuD to LexA, λ repressor, and other members of the family of proteins is discussed and possible roles intact UmuD in modulating SOS mutagenesis are discussed

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

Science.gov (United States)

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

2014-01-01

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

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

Directory of Open Access Journals (Sweden)

Anke Bill

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

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

Directory of Open Access Journals (Sweden)

Janet L Gibson

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

Structure-Function Analysis of Chloroplast Proteins via Random Mutagenesis Using Error-Prone PCR.

Science.gov (United States)

Dumas, Louis; Zito, Francesca; Auroy, Pascaline; Johnson, Xenie; Peltier, Gilles; Alric, Jean

2018-06-01

Site-directed mutagenesis of chloroplast genes was developed three decades ago and has greatly advanced the field of photosynthesis research. Here, we describe a new approach for generating random chloroplast gene mutants that combines error-prone polymerase chain reaction of a gene of interest with chloroplast complementation of the knockout Chlamydomonas reinhardtii mutant. As a proof of concept, we targeted a 300-bp sequence of the petD gene that encodes subunit IV of the thylakoid membrane-bound cytochrome b 6 f complex. By sequencing chloroplast transformants, we revealed 149 mutations in the 300-bp target petD sequence that resulted in 92 amino acid substitutions in the 100-residue target subunit IV sequence. Our results show that this method is suited to the study of highly hydrophobic, multisubunit, and chloroplast-encoded proteins containing cofactors such as hemes, iron-sulfur clusters, and chlorophyll pigments. Moreover, we show that mutant screening and sequencing can be used to study photosynthetic mechanisms or to probe the mutational robustness of chloroplast-encoded proteins, and we propose that this method is a valuable tool for the directed evolution of enzymes in the chloroplast. © 2018 American Society of Plant Biologists. All rights reserved.

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

Science.gov (United States)

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

2014-12-20

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

Improving Escherichia coli FucO for furfural tolerance by saturation mutagenesis of individual amino acid positions.

Science.gov (United States)

Zheng, Huabao; Wang, Xuan; Yomano, Lorraine P; Geddes, Ryan D; Shanmugam, Keelnatham T; Ingram, Lonnie O

2013-05-01

Furfural is an inhibitory side product formed during the depolymerization of hemicellulose with mineral acids. In Escherichia coli, furfural tolerance can be increased by expressing the native fucO gene (encoding lactaldehyde oxidoreductase). This enzyme also catalyzes the NADH-dependent reduction of furfural to the less toxic alcohol. Saturation mutagenesis was combined with growth-based selection to isolate a mutated form of fucO that confers increased furfural tolerance. The mutation responsible, L7F, is located within the interfacial region of FucO homodimers, replacing the most abundant codon for leucine with the most abundant codon for phenylalanine. Plasmid expression of the mutant gene increased FucO activity by more than 10-fold compared to the wild-type fucO gene and doubled the rate of furfural metabolism during fermentation. No inclusion bodies were evident with either the native or the mutated gene. mRNA abundance for the wild-type and mutant fucO genes differed by less than 2-fold. The Km (furfural) for the mutant enzyme was 3-fold lower than that for the native enzyme, increasing efficiency at low substrate concentrations. The L7F mutation is located near the FucO N terminus, within the ribosomal binding region associated with translational initiation. Free-energy calculations for mRNA folding in this region (nucleotides -7 to +37) were weak for the native gene (-4.1 kcal mol(-1)) but weaker still for the fucO mutant (-1.0 to -0.1 kcal mol(-1)). The beneficial L7F mutation in FucO is proposed to increase furfural tolerance by improving gene expression and increasing enzyme effectiveness at low substrate levels.

Design of cAMP-CRP-activated promoters in Escherichia coli

DEFF Research Database (Denmark)

Valentin-Hansen, P; Holst, B; Søgaard-Andersen, L

1991-01-01

We have studied the deoP2 promoter of Escherichia coli to define features that are required for optimal activation by the complex of adenosine 3',5' monophosphate (cAMP) and the cAMP receptor protein (CRP). Systematic mutagenesis of deoP2 shows that the distance between the CRP site and the -10...

Protein Engineering by Random Mutagenesis and Structure-Guided Consensus of Geobacillus stearothermophilus Lipase T6 for Enhanced Stability in Methanol

Science.gov (United States)

Dror, Adi; Shemesh, Einav; Dayan, Natali

2014-01-01

The abilities of enzymes to catalyze reactions in nonnatural environments of organic solvents have opened new opportunities for enzyme-based industrial processes. However, the main drawback of such processes is that most enzymes have a limited stability in polar organic solvents. In this study, we employed protein engineering methods to generate a lipase for enhanced stability in methanol, which is important for biodiesel production. Two protein engineering approaches, random mutagenesis (error-prone PCR) and structure-guided consensus, were applied in parallel on an unexplored lipase gene from Geobacillus stearothermophilus T6. A high-throughput colorimetric screening assay was used to evaluate lipase activity after an incubation period in high methanol concentrations. Both protein engineering approaches were successful in producing variants with elevated half-life values in 70% methanol. The best variant of the random mutagenesis library, Q185L, exhibited 23-fold-improved stability, yet its methanolysis activity was decreased by one-half compared to the wild type. The best variant from the consensus library, H86Y/A269T, exhibited 66-fold-improved stability in methanol along with elevated thermostability (+4.3°C) and a 2-fold-higher fatty acid methyl ester yield from soybean oil. Based on in silico modeling, we suggest that the Q185L substitution facilitates a closed lid conformation that limits access for both the methanol and substrate excess into the active site. The enhanced stability of H86Y/A269T was a result of formation of new hydrogen bonds. These improved characteristics make this variant a potential biocatalyst for biodiesel production. PMID:24362426

Design of Deinococcus radiodurans thioredoxin reductase with altered thioredoxin specificity using computational alanine mutagenesis

OpenAIRE

Obiero, Josiah; Sanders, David AR

2011-01-01

In this study, the X-ray crystal structure of the complex between Escherichia coli thioredoxin reductase (EC TrxR) and its substrate thioredoxin (Trx) was used as a guide to design a Deinococcus radiodurans TrxR (DR TrxR) mutant with altered Trx specificity. Previous studies have shown that TrxRs have higher affinity for cognate Trxs (same species) than that for Trxs from different species. Computational alanine scanning mutagenesis and visual inspection of the EC TrxR–Trx interface suggested...

The serum resistome of a globally disseminated multidrug resistant uropathogenic Escherichia coli clone.

Science.gov (United States)

Phan, Minh-Duy; Peters, Kate M; Sarkar, Sohinee; Lukowski, Samuel W; Allsopp, Luke P; Gomes Moriel, Danilo; Achard, Maud E S; Totsika, Makrina; Marshall, Vikki M; Upton, Mathew; Beatson, Scott A; Schembri, Mark A

2013-01-01

Escherichia coli ST131 is a globally disseminated, multidrug resistant clone responsible for a high proportion of urinary tract and bloodstream infections. The rapid emergence and successful spread of E. coli ST131 is strongly associated with antibiotic resistance; however, this phenotype alone is unlikely to explain its dominance amongst multidrug resistant uropathogens circulating worldwide in hospitals and the community. Thus, a greater understanding of the molecular mechanisms that underpin the fitness of E. coli ST131 is required. In this study, we employed hyper-saturated transposon mutagenesis in combination with multiplexed transposon directed insertion-site sequencing to define the essential genes required for in vitro growth and the serum resistome (i.e. genes required for resistance to human serum) of E. coli EC958, a representative of the predominant E. coli ST131 clonal lineage. We identified 315 essential genes in E. coli EC958, 231 (73%) of which were also essential in E. coli K-12. The serum resistome comprised 56 genes, the majority of which encode membrane proteins or factors involved in lipopolysaccharide (LPS) biosynthesis. Targeted mutagenesis confirmed a role in serum resistance for 46 (82%) of these genes. The murein lipoprotein Lpp, along with two lipid A-core biosynthesis enzymes WaaP and WaaG, were most strongly associated with serum resistance. While LPS was the main resistance mechanism defined for E. coli EC958 in serum, the enterobacterial common antigen and colanic acid also impacted on this phenotype. Our analysis also identified a novel function for two genes, hyxA and hyxR, as minor regulators of O-antigen chain length. This study offers novel insight into the genetic make-up of E. coli ST131, and provides a framework for future research on E. coli and other Gram-negative pathogens to define their essential gene repertoire and to dissect the molecular mechanisms that enable them to survive in the bloodstream and cause disease.

The serum resistome of a globally disseminated multidrug resistant uropathogenic Escherichia coli clone.

Directory of Open Access Journals (Sweden)

Minh-Duy Phan

Full Text Available Escherichia coli ST131 is a globally disseminated, multidrug resistant clone responsible for a high proportion of urinary tract and bloodstream infections. The rapid emergence and successful spread of E. coli ST131 is strongly associated with antibiotic resistance; however, this phenotype alone is unlikely to explain its dominance amongst multidrug resistant uropathogens circulating worldwide in hospitals and the community. Thus, a greater understanding of the molecular mechanisms that underpin the fitness of E. coli ST131 is required. In this study, we employed hyper-saturated transposon mutagenesis in combination with multiplexed transposon directed insertion-site sequencing to define the essential genes required for in vitro growth and the serum resistome (i.e. genes required for resistance to human serum of E. coli EC958, a representative of the predominant E. coli ST131 clonal lineage. We identified 315 essential genes in E. coli EC958, 231 (73% of which were also essential in E. coli K-12. The serum resistome comprised 56 genes, the majority of which encode membrane proteins or factors involved in lipopolysaccharide (LPS biosynthesis. Targeted mutagenesis confirmed a role in serum resistance for 46 (82% of these genes. The murein lipoprotein Lpp, along with two lipid A-core biosynthesis enzymes WaaP and WaaG, were most strongly associated with serum resistance. While LPS was the main resistance mechanism defined for E. coli EC958 in serum, the enterobacterial common antigen and colanic acid also impacted on this phenotype. Our analysis also identified a novel function for two genes, hyxA and hyxR, as minor regulators of O-antigen chain length. This study offers novel insight into the genetic make-up of E. coli ST131, and provides a framework for future research on E. coli and other Gram-negative pathogens to define their essential gene repertoire and to dissect the molecular mechanisms that enable them to survive in the bloodstream and

[Stress-induced cellular adaptive mutagenesis].

Science.gov (United States)

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.

Role of RecA protein in untargeted UV mutagenesis of bacteriophage lambda: evidence for the requirement for the dinB gene

International Nuclear Information System (INIS)

Brotcorne-Lannoye, A.; Maenhaut-Michel, G.

1986-01-01

Untargeted UV mutagenesis of bacteriophage lambda--i.e., the increased recovery of lambda mutants when unirradiated lambda infects UV-irradiated Escherichia coli--is thought to be mediated by a transient decrease in DNA replication fidelity, generating mutations in the newly synthesized strands. Using the bacteriophage lambda cI857----lambda c mutation system, we provide evidence that the RecA protein, shown previously to be required for this mutagenic pathway, is no longer needed when the LexA protein is inactivated by mutation. We suggest that the error-prone DNA replication responsible for UV-induced untargeted mutagenesis is turned on by the presence of replication-blocking lesions in the host cell DNA and that the RecA protein is required only to derepress the relevant din gene(s). This is in contrast to mutagenesis of irradiated bacteria or irradiated phage lambda, in which activated RecA protein has a second role in mutagenesis in addition to the cleavage of the LexA protein. Among the tested din genes, the dinB gene product (in addition to the uvrA and uvrB gene products) was found to be required for untargeted mutagenesis of bacteriophage lambda. To our knowledge, a phenotype associated with the dinB gene has not been reported previously

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

Science.gov (United States)

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

2016-04-18

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

Inducible error-prone repair in Escherichia coli

International Nuclear Information System (INIS)

Sedgwick, S.G.

1975-01-01

A hypothesis that ultraviolet-induced mutagenesis arises from the induction of an error-prone mode of postreplication repair that requires the exrA + recA + genotype has been tested with alkaline sucrose gradient centrifugation coupled with assays of fixation determined by loss of photoreversibility. The inhibitor of protein synthesis, chloramphenicol, added before irradiation, prevented a small amount of postreplication repair and completely eliminated mutation fixation in E. coli WP2/sub s/ uvrA. However, chloramphenicol did not affect strand joining: in uvrA bacteria allowed 20 min of growth between irradiation and antibiotic treatment; in nonmutable uvrA exrA bacteria; and in urvA tif bacteria grown at 42 0 for 70 min before irradiation. These observations indicate that an inducible product is involved in a fraction of postreplication repair and is responsible for induced mutagenesis. (auth)

Mechanisms of umuC-dependent mutagenesis

International Nuclear Information System (INIS)

Kato, Takeji; Kitagawa, Yoshinori

1985-01-01

Present status of studies on umcDC genes-induced mutagenesis is introduced. Specificity of umuCD-dependent and -independent base substitution and frameshift mutagenesis is presented. Biochemical examinations of U.V.-induced umuCD gene function are described. Previous studies suggest that umuCD genes are induced by SOS inhibitory systems, that gene products are directly responsible for mutagenesis, that base substitution is largely involved in inducible mutagenesis, and that many of frameshifts are induced irrespective of gene function. (Namekawa, K.)

Genetic separation of Escherichia coli recA functions for SOS mutagenesis and repressor cleavage

International Nuclear Information System (INIS)

Ennis, D.G.; Ossanna, N.; Mount, D.W.

1989-01-01

Evidence is presented that recA functions which promote the SOS functions of mutagenesis, LexA protein proteolysis, and lambda cI repressor proteolysis are each genetically separable from the others. This separation was observed in recombination-proficient recA mutants and rec+ (F' recA56) heterodiploids. recA430, recA433, and recA435 mutants and recA+ (F' recA56) heterodiploids were inducible for only one or two of the three functions and defective for mutagenesis. recA80 and recA432 mutants were constitutively activated for two of the three functions in that these mutants did not have to be induced to express the functions. We propose that binding of RecA protein to damaged DNA and subsequent interaction with small inducer molecules gives rise to conformational changes in RecA protein. These changes promote surface-surface interactions with other target proteins, such as cI and LexA proteins. By this model, the recA mutants are likely to have incorrect amino acids substituted as sites in the RecA protein structure which affect surface regions required for protein-protein interactions. The constitutively activated mutants could likewise insert altered amino acids at sites in RecA which are involved in the activation of RecA protein by binding small molecules or polynucleotides which metabolically regulate RecA protein

A plasmid-transposon hybrid mutagenesis system effective in a broad range of Enterobacteria

Directory of Open Access Journals (Sweden)

Rita eMonson

2015-12-01

Full Text Available 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.

Random insertion and gene disruption via transposon mutagenesis of Ureaplasma parvum using a mini-transposon plasmid.

Science.gov (United States)

Aboklaish, Ali F; Dordet-Frisoni, Emilie; Citti, Christine; Toleman, Mark A; Glass, John I; Spiller, O Brad

2014-11-01

While transposon mutagenesis has been successfully used for Mycoplasma spp. to disrupt and determine non-essential genes, previous attempts with Ureaplasma spp. have been unsuccessful. Using a polyethylene glycol-transformation enhancing protocol, we were able to transform three separate serovars of Ureaplasma parvum with a Tn4001-based mini-transposon plasmid containing a gentamicin resistance selection marker. Despite the large degree of homology between Ureaplasma parvum and Ureaplasma urealyticum, all attempts to transform the latter in parallel failed, with the exception of a single clinical U. urealyticum isolate. PCR probing and sequencing were used to confirm transposon insertion into the bacterial genome and identify disrupted genes. Transformation of prototype serovar 3 consistently resulted in transfer only of sequence between the mini-transposon inverted repeats, but some strains showed additional sequence transfer. Transposon insertion occurred randomly in the genome resulting in unique disruption of genes UU047, UU390, UU440, UU450, UU520, UU526, UU582 for single clones from a panel of screened clones. An intergenic insertion between genes UU187 and UU188 was also characterised. Two phenotypic alterations were observed in the mutated strains: Disruption of a DEAD-box RNA helicase (UU582) altered growth kinetics, while the U. urealyticum strain lost resistance to serum attack coincident with disruption of gene UUR10_137 and loss of expression of a 41 kDa protein. Transposon mutagenesis was used successfully to insert single copies of a mini-transposon into the genome and disrupt genes leading to phenotypic changes in Ureaplasma parvum strains. This method can now be used to deliver exogenous genes for expression and determine essential genes for Ureaplasma parvum replication in culture and experimental models. Copyright © 2014 Elsevier GmbH. All rights reserved.

The contribution of Nth and Nei DNA glycosylases to mutagenesis in Mycobacterium smegmatis.

Science.gov (United States)

Moolla, Nabiela; Goosens, Vivianne J; Kana, Bavesh D; Gordhan, Bhavna G

2014-01-01

The increased prevalence of drug resistant strains of Mycobacterium tuberculosis (Mtb) indicates that significant mutagenesis occurs during tuberculosis disease in humans. DNA damage by host-derived reactive oxygen/nitrogen species is hypothesized to be critical for the mutagenic process in Mtb thus, highlighting an important role for DNA repair enzymes in maintenance of genome fidelity. Formamidopyrimidine (Fpg/MutM/Fapy) and EndonucleaseVIII (Nei) constitute the Fpg/Nei family of DNA glycosylases and together with EndonucleaseIII (Nth) are central to the base excision repair pathway in bacteria. In this study we assess the contribution of Nei and Nth DNA repair enzymes in Mycobacterium smegmatis (Msm), which retains a single nth homologue and duplications of the Fpg (fpg1 and fpg2) and Nei (nei1 and nei2) homologues. Using an Escherichia coli nth deletion mutant, we confirm the functionality of the mycobacterial nth gene in the base excision repair pathway. Msm mutants lacking nei1, nei2 and nth individually or in combination did not display aberrant growth in broth culture. Deletion of nth individually results in increased UV-induced mutagenesis and combinatorial deletion with the nei homologues results in reduced survival under oxidative stress conditions and an increase in spontaneous mutagenesis to rifampicin. Deletion of nth together with the fpg homolgues did not result in any growth/survival defects or changes in mutation rate. Furthermore, no differential emergence of the common rifampicin resistance conferring genotypes were noted. Collectively, these data confirm a role for Nth in base excision repair in mycobacteria and further highlight a novel interplay between the Nth and Nei homologues in spontaneous mutagenesis. Copyright © 2013 Elsevier B.V. All rights reserved.

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

Science.gov (United States)

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.

The effects of exogenous catalase on broad-spectrum near-UV (300-400nm) treated Escherichia coli cells

International Nuclear Information System (INIS)

Sammartano, L.J.; Tuveson, R.W.

1984-01-01

Catalase incorporated into plating medium protects against inactivation and mutagenesis by broad-spectrum near-ultraviolet wavelength (300-400nm) (NUV) radiation in strains of Escherichia coli. Plating medium containing catalase does not provide protection against inactivation by wavelengths in the FUV region. Catalase added to the cell suspension during or added immediately after NUV exposure also protects against inactivation. The protection provided by catalase suggests a possible role for hydrogen peroxide in the processes of inactivation and mutagenesis by broad-spectrum NUV. (author)

Transposon mutagenesis in Mycoplasma hyopneumoniae using a novel mariner-based system for generating random mutations.

Science.gov (United States)

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.

Construction of intergeneric conjugal transfer for molecular genetic ...

African Journals Online (AJOL)

SAM

2014-03-26

Mar 26, 2014 ... The attB integration site in the S. mobaraensis genome was detected as a single attB ... present study, to promote the molecular genetic study of. S. mobaraensis .... further increase in the number of E. coli donor cells. (≥1.25 × 108) (Choi et .... rational mutagenesis and random mutagenesis. Appl. Microbiol.

Forward and reverse mutagenesis in C. elegans

Science.gov (United States)

Kutscher, Lena M.; Shaham, Shai

2014-01-01

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

X-ray-induced mutations in Escherichia coli K-12 strains with altered DNA polymerase I activities

International Nuclear Information System (INIS)

Nagata, Yuki; Kawata, Masakado; Komura, Jun-ichiro; Ono, Tetsuya; Yamamoto, Kazuo

2003-01-01

Spectra of ionizing radiation mutagenesis were determined by sequencing X-ray-induced endogenous tonB gene mutations in Escherichia coli polA strains. We used two polA alleles, the polA1 mutation, defective for Klenow domain, and the polA107 mutation, defective for flap domain. We demonstrated that irradiation of 75 and 50 Gy X-rays could induce 3.8- and 2.6-fold more of tonB mutation in polA1 and polA107 strains, respectively, than spontaneous level. The radiation induced spectrum of 51 tonB mutations in polA1 and 51 in polA107 indicated that minus frameshift, A:T→T:A transversion and G:C→T:A transversion were the types of mutations increased. Previously, we have reported essentially the same X-ray-induced tonB mutation spectra in the wild-type strain. These results indicate that (1) X-rays can induce minus frameshift, A:T→T:A transversion and G:C→T:A transversion in E. coli and (2) presence or absence of polymerase I (PolI) of E. coli does not have any effects on the process of X-ray mutagenesis

Viral Bacterial Artificial Chromosomes: Generation, Mutagenesis, and Removal of Mini-F Sequences

Directory of Open Access Journals (Sweden)

B. Karsten Tischer

2012-01-01

Full Text Available Maintenance and manipulation of large DNA and RNA virus genomes had presented an obstacle for virological research. BAC vectors provided a solution to both problems as they can harbor large DNA sequences and can efficiently be modified using well-established mutagenesis techniques in Escherichia coli. Numerous DNA virus genomes of herpesvirus and pox virus were cloned into mini-F vectors. In addition, several reverse genetic systems for RNA viruses such as members of Coronaviridae and Flaviviridae could be established based on BAC constructs. Transfection into susceptible eukaryotic cells of virus DNA cloned as a BAC allows reconstitution of recombinant viruses. In this paper, we provide an overview on the strategies that can be used for the generation of virus BAC vectors and also on systems that are currently available for various virus species. Furthermore, we address common mutagenesis techniques that allow modification of BACs from single-nucleotide substitutions to deletion of viral genes or insertion of foreign sequences. Finally, we review the reconstitution of viruses from BAC vectors and the removal of the bacterial sequences from the virus genome during this process.

DNA sequence changes in mutation induced by ultraviolet light in the gpt gene on the chromosome of Escherichia coli uvr+ und uvrA cells

International Nuclear Information System (INIS)

Sockett, H.; Romac, S.; Hutchinson, F.

1991-01-01

Sequence changes in mutations induced by ultraviolet light are reported for the chromosomal Escherichia coli gpt gene in almost isogenic E. coli uvr + and excision-deficient uvrA cells. Differences between the mutagenic spectra are ascribed to preferential removal of photoproducts in the transcribed strand by excision repair in uvr + cells. This conclusion is confirmed by analysis of published results for genes in both uvr + and uvr − cells, showing a similar selective removal of mutagenic products from the transcribed strand of the E. coli lacI gene and of the lambda phage cl repressor gene. Comparison of these data with published results for ultraviolet mutagenesis of gpt on a chromosome in Chinese hamster ovary cells showed that a mutagenic hot spot in mammalian cells is not present in E. coli; the possibility is suggested that the hot spot might arise from localized lack of excision repair. Otherwise, mutagenesis in hamster cells appeared similar to that in E. coli uvr + cells, except there appears to be a smaller fraction of single-base additions and deletions (frameshifts) in mammalian than in bacterial cells. Phenotypes of 6-thioguanine-resistant E. coli showed there is a gene (or genes) other than gpt involved in the utilization of thioguanine by bacteria

Induction of lacI- mutations in Escherichia coli cells after single and split-dose irradiation

International Nuclear Information System (INIS)

Kozubek, S.; Ryznar, L.

1992-01-01

In the lacI system of Escherichia coli, X-ray mutagenesis follows a linear-quadratic curve with suppression; the survival curve is exponential. Dose fractionation leads to nearly complete repair of premutational lesions during an incubation interval of 3.5 h. Repair starts with a delay of 1.5-2 h, suggesting the involvement of an inducible repair/mutation fixation system. The dose-dependence of mutagenesis is described by a simple model assuming two hits being required. A probable explanation might be that the premutagenic lesions consist of two closely spaced lesions on the opposite strands of the DNA molecule. (author)

Protracted radiation mutagenesis

International Nuclear Information System (INIS)

Dubinina, L.G.; Shanazarova, A.S.; Chernikova, O.P.

1976-01-01

The aim of the work is investigation of the dynamics of structural mutations of Cr.capillaris chromosomes induced by irradiation of seeds at different stages of the cell cycle with subsequent storage. The results obtained show that irradiation is followed by mutagenesis wave kinetics under such conditions. The level and the character of this phenomenon depends on the functional state of the nucleus or on the relationship between this state and the amount of water in the seeds. Studies of this phenomenon will bring better understanding to the mechanism of radiation mutagenesis [ru

Ethyl methanesulfonate mutagenesis in Schizosaccharomyces pombe

DEFF Research Database (Denmark)

Ekwall, Karl; Thon, Genevieve

2017-01-01

Here we provide an ethyl methanesulfonate (EMS) mutagenesis protocol for Schizosaccharomyces pombe cells.......Here we provide an ethyl methanesulfonate (EMS) mutagenesis protocol for Schizosaccharomyces pombe cells....

Optogenetic mutagenesis in Caenorhabditis elegans.

Science.gov (United States)

Noma, Kentaro; Jin, Yishi

2015-12-03

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

Role of leader peptide synthesis in tryptophanase operon expression in Escherichia coli K-12.

OpenAIRE

Stewart, V; Yanofsky, C

1986-01-01

We used site-directed mutagenesis to replace the Escherichia coli tryptophanase (tna) operon leader peptide start codon with AUC. This change greatly decreased the uninduced rate of tna operon expression, and it also lowered the response to inducer. We conclude that leader peptide synthesis plays an essential role in tna operon expression.

Mutagenesis: Interactions with a parallel universe.

Science.gov (United States)

Miller, Jeffrey H

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

Receptor mutagenesis strategies for examination of structure-function relationships

NARCIS (Netherlands)

Blomenröhr, Marion; Vischer, Henry F; Bogerd, Jan

2004-01-01

This chapter describes three different strategies of receptor mutagenesis with their advantages, disadvantages, and limitations. Oligonucleotide-directed mutagenesis using either the Altered Sites II in vitro mutagenesis system or the GeneTailor site-directed mutagenesis system can generate base

Mutagenesis and cytotoxicity in human epithelial cells by far- and near-ultraviolet radiations: action spectra

International Nuclear Information System (INIS)

Jones, C.A.; Huberman, E.; Cunningham, M.L.; Peak, M.J.

1987-01-01

Action spectra were determined for cell killing and mutation by monochromatic ultraviolet and visible radiations (254-434 nm) in cultured human epithelial P3 cells. Cell killing was more efficient following radiation at the shorter wavelengths (254-434 nm) than at longer wavelengths (365-434 nm). At 254 nm, for example, a fluence of 11 Jm-2 gave 37% cell survival, while at 365 nm, 17 X 10(5) Jm-2 gave equivalent survival. At 434 nm little killing was observed with fluences up to 3 X 10(6) Jm-2. Mutant induction, determined at the hypoxanthine-guanine phosphoribosyltransferase locus, was caused by radiation at 254, 313, and 365 nm. There was no mutant induction at 334 nm although this wavelength was highly cytotoxic. Mutagenesis was not induced by 434 nm radiation, either. There was a weak response at 405 nm; the mutant frequencies were only slightly increased above background levels. For the mutagenic wavelengths, log-log plots of the mutation frequency against fluence showed linear regressions with positive slopes of 2.5, consistent with data from a previous study using Escherichia coli. The data points of the action spectra for lethality and mutagenesis were similar to the spectrum for DNA damage at wavelengths shorter than 313 nm, whereas at longer wavelengths the lethality spectrum had a shoulder, and the mutagenesis spectrum had a secondary peak at 365 nm. No correlation was observed for the P3 cells between the spectra for cell killing and mutagenesis caused by wavelengths longer than 313 nm and the induction of DNA breakage or the formation of DNA-to-protein covalent bonds in these cells

Respiration shutoff in Escherichia coli after far-uv irradiation

International Nuclear Information System (INIS)

Swenson, P.A.; Norton, I.L.

1984-01-01

Damage to DNA of Escherichia coli by uv, ionizing radiation and chemicals causes a number of responses that require the recA + and lexA + gene products. The responses include error prone repair (as indicated by mutagenesis), filamentation and induction of prophage lambda. Another important rec/lex response, shutoff of respiration, which occurs 60 min after exposure to uv, is studied. Objective is to understand the genetic and biochemical bases of the shutoff process and its control

Phenomenology of an inducible mutagenic DNA repair pathway in Escherichia coli: SOS repair hypothesis

International Nuclear Information System (INIS)

Radman, M.

1974-01-01

A hypothesis is proposed according to which E. coli possesses an inducible DNA repair system. This hypothetical repair, which we call SOS repair, is manifested only following damage to DNA, and requires de novo protein synthesis. SOS repair in E. coli requires some known genetic elements: recA + , lex + and probably zab + . Mutagenesis by ultraviolet light is observed only under conditions of functional SOS repair: we therefore suspect that this is a mutation-prone repair. A number of phenomena and experiments is reviewed which at this point can best be interpreted in terms of an inducible mutagenic DNA repair system. Two recently discovered phenomena support the proposed hypothesis: existence of a mutant (tif) which, after a shift to elevated temperature, mimicks the effect of uv irradiation in regard to repair of phage lambda and uv mutagenesis, apparent activation of SOS repair by introduction into the recipient cell of damaged plasmid or Hfr DNA. Several specific predictions based on SOS repair hypothesis are presented in order to stimulate further experimental tests. (U.S.)

Complexity of the ultraviolet mutation frequency response curve in Escherichia coli B/r: SOS induction, one-lesion and two-lesion mutagenesis

International Nuclear Information System (INIS)

Doudney, C.O.

1976-01-01

Three distinct sections of the ultraviolet mutation frequency response (MFR) curve toward tryptophan prototrophy have been demonstrated in Escherichia coli B/r WP2 trp thy and its uvrA derivative in log-phase growth in minimal medium. The initial section, which appears fluence-squared, may reflect the necessity, if mutation is to result, for induction of two lesions, one located within the potentially mutated genetic locus and the other damaging deoxyribonucleic acid replication and resulting in induction of the error-prone SOS repair function. A second linear section is ascribed to the continued induction, after exposure above that sufficient for complete SOS expression, of isolated lesions which lead to mutation in potentially mutated loci. The third section demonstrates an increased rate of mutagenesis and suggests the induction of two lesions in proximity which result in additional mutations. Split-exposure studies support the inducible nature of the SOS function and suggest that mutation frequency decline (MFD) is due to excision resulting from or related to the prevention of SOS induction by inhibition of protein synthesis. Preirradiation tryptophan starvation of the uvr + strain for 30 min decreases MFR in the first and second sections of the curve. Reduction of MFR in the third section requires more prestarvation time and is blocked by nalidixic acid. The decreased MFR of the first and second sections is ascribed to promotion of postirradiation MFD based on excision and that of the third section to completion of the chromosome during the prestarvation period

Application of In Vitro Transposon Mutagenesis to Erythromycin Strain Improvement in Saccharopolyspora erythraea.

Science.gov (United States)

Weber, J Mark; Reeves, Andrew; Cernota, William H; Wesley, Roy K

2017-01-01

Transposon mutagenesis is an invaluable technique in molecular biology for the creation of random mutations that can be easily identified and mapped. However, in the field of microbial strain improvement, transposon mutagenesis has scarcely been used; instead, chemical and physical mutagenic methods have been traditionally favored. Transposons have the advantage of creating single mutations in the genome, making phenotype to genotype assignments less challenging than with traditional mutagens which commonly create multiple mutations in the genome. The site of a transposon mutation can also be readily mapped using DNA sequencing primer sites engineered into the transposon termini. In this chapter an in vitro method for transposon mutagenesis of Saccharopolyspora erythraea is presented. Since in vivo transposon tools are not available for most actinomycetes including S. erythraea, an in vitro method was developed. The in vitro method involves a significant investment in time and effort to create the mutants, but once the mutants are made and screened, a large number of highly relevant mutations of direct interest to erythromycin production can be found.

Enhancing succinic acid biosynthesis in Escherichia coli by engineering its global transcription factor, catabolite repressor/activator (Cra).

Science.gov (United States)

Zhu, Li-Wen; Xia, Shi-Tao; Wei, Li-Na; Li, Hong-Mei; Yuan, Zhan-Peng; Tang, Ya-Jie

2016-11-04

This study was initiated to improve E. coli succinate production by engineering the E. coli global transcription factor, Cra (catabolite repressor/activator). Random mutagenesis libraries were generated through error-prone PCR of cra. After re-screening and mutation site integration, the best mutant strain was Tang1541, which provided a final succinate concentration of 79.8 ± 3.1 g/L: i.e., 22.8% greater than that obtained using an empty vector control. The genes and enzymes involved in phosphoenolpyruvate (PEP) carboxylation and the glyoxylate pathway were activated, either directly or indirectly, through the mutation of Cra. The parameters for interaction of Cra and DNA indicated that the Cra mutant was bound to aceBAK, thereby activating the genes involved in glyoxylate pathway and further improving succinate production even in the presence of its effector fructose-1,6-bisphosphate (FBP). It suggested that some of the negative effect of FBP on Cra might have been counteracted through the enhanced binding affinity of the Cra mutant for FBP or the change of Cra structure. This work provides useful information about understanding the transcriptional regulation of succinate biosynthesis.

Antimutagenic effect of isocyanates and related compounds in escherichia coli

International Nuclear Information System (INIS)

Kawazoe, Yutaka; Kato, Masanari

1982-01-01

Isocyanates and isothiocyanates have been suggested to inactivate enzymes involved in the metabolic activation of chemical carcinogens and the repair of DNA damage. These compounds decrease the mutability of a tester strain of Escherichia coli B under UV irradiation. This paper deals with the antimutagenicity of acylating agents, including isocyanates and isothiocyanates, and some anti-oxidants which are suspected to be anticarcinogenic. The results can be summarized as follows. (1) The antimutagenic effect observed in the present study operates on UV-induced mutagenesis but not on X-ray-induced mutagenesis. (2) This effect operates only on the wild-type strain, H/r30R, but not on Hs30R deficient in the excision repair system. (3) This effect may function through giving the irradiated cells a greater chance to carry out excision repair by prolonging the lag-period before entry into the S-phase. (4) The carbamoylating ability of isocyanates and isothiocyanates may be responsible for the antimutagenicity, but other type of reactivities may also be involved. These antimutagens also participate in inactivating enzymes relevant to the metabolic activation of mutagens, resulting in a decrease in the frequency of chemically induced mutagenesis. (author)

Mutagenesis

International Nuclear Information System (INIS)

Dubinin, N.P.

1986-01-01

Problems on radiation mutagenesis, in particular, data on general factors of genetic radiation effects, dependences of mutation frequencies on radiation dose and threshold in genetic radiation effects, problems of low doses, modification of genetic radiation effects, repauir of injuries of genetic material, photoreactivation, causing structure chromosomal mutations under radiation action, on relative genetic efficiency of different types of radiation are considered besides others

Absence of ultraviolet-inducible DNA polymerase I-like activity in Escherichia coli strains harbouring R plasmids

International Nuclear Information System (INIS)

Upton, C.; Pinney, R.J.

1981-01-01

No DNA polymerase I-like activity was found associated with the ultraviolet (u.v.)-protecting plasmids R205, R46 or pKM101 in either uninduced or u.v.-induced wild-type or DNA polymerase I-deficient strains of Escherichia coli. Nor was any plasmid-associated polymerase activity detectable in similar systems containing u.v.-irradiated DNA as template. However, plasmids R205, R46 and pKM 101 still increased survival and mutagenesis of the polymerase I-deficient E. coli strain after u.v. irradiation. (author)

Activated RecA protein may induce expression of a gene that is not controlled by the LexA repressor and whose function is required for mutagenesis and repair of UV-irradiated bacteriophage lambda

International Nuclear Information System (INIS)

Calsou, P.; Villaverde, A.; Defais, M.

1987-01-01

The activated form of the RecA protein (RecA) is known to be involved in the reactivation and mutagenesis of UV-irradiated bacteriophage lambda and in the expression of the SOS response in Escherichia coli K-12. The expression of the SOS response requires cleavage of the LexA repressor by RecA and the subsequent expression of LexA-controlled genes. The evidence presented here suggests that RecA induces the expression of a gene(s) that is not under LexA control and that is also necessary for maximal repair and mutagenesis of damaged phage. This conclusion is based on the chloramphenicol sensitivity of RecA -dependent repair and mutagenesis of damaged bacteriophage lambda in lexA(Def) hosts

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

Science.gov (United States)

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

2013-08-30

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

Cellular components required for mutagenesis

International Nuclear Information System (INIS)

Elledge, S.J.; Perry, K.L.; Krueger, J.H.; Mitchell, B.B.; Walker, G.C.

1983-01-01

We have cloned the umuD and umuC genes of Escherichia coli and have shown that they code for two proteins of 16,000 and 45,000 daltons respectively; the two genes are organized in an operon that is repressed by the LexA protein. Similarly, we have shown that the mucA and mucB genes of the mutagenesis-enhancing plasmid pKM101 code for proteins of 16,000 and 45,000 daltons respectively and, like umuD/C, the genes are organized in an operon. Preliminary sequencing studies have indicated that the umuD/C and mucA/B loci are approximately 50% homologous at both the nucleic acid and deduced protein sequence levels and that the umuD gene is preceeded by two putative LexA binding sites separated by 4 basepairs. Like umuD/C, the mucA/B genes of pKM101 are induced by DNA damage and are repressed by LexA. In addition to inducing recA + lexA + -regulated din genes, DNA damaging agents such as uv and nalidixic acid also induce the heat shock proteins GroEL and DnaK in an htpR-dependent fashion. 22 references, 1 figure, 1 table

2004 Mutagenesis Gordon Conference

Energy Technology Data Exchange (ETDEWEB)

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.

Inducible pathway is required for mutagenesis in Salmonella typhimurium LT2

International Nuclear Information System (INIS)

Orrego, C.; Eisenstadt, E.

1987-01-01

UV mutability of Salmonella typhimurium LT2 was eliminated in the presence of a multicopy plasmid carrying the Escherichia coli lexA + 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

Characterization of the rec-1 gene of Haemophilus influenzae and behavior of the gene in Escherichia coli

Energy Technology Data Exchange (ETDEWEB)

Setlow, J.K.; Spikes, D.; Griffin, K.

1988-09-01

The rec-1 gene of Haemophilus influenzae was cloned into a shuttle vector that replicates in Escherichia coli as well as in H. influenzae. The plasmid, called pRec1, complemented the defects of a rec-1 mutant in repair of UV damage, transformation, and ability of prophage to be induced by UV radiation. Although UV resistance and recombination were caused by pRec1 in E. coli recA mutants, UV induction of lambda and UV mutagenesis were not. We suggest that the ability of the H. influenzae Rec-1 protein to cause cleavage of repressors but not the recombinase function differs from that of the E. coli RecA protein.

Characterization of the rec-1 gene of Haemophilus influenzae and behavior of the gene in Escherichia coli

International Nuclear Information System (INIS)

Setlow, J.K.; Spikes, D.; Griffin, K.

1988-01-01

The rec-1 gene of Haemophilus influenzae was cloned into a shuttle vector that replicates in Escherichia coli as well as in H. influenzae. The plasmid, called pRec1, complemented the defects of a rec-1 mutant in repair of UV damage, transformation, and ability of prophage to be induced by UV radiation. Although UV resistance and recombination were caused by pRec1 in E. coli recA mutants, UV induction of lambda and UV mutagenesis were not. We suggest that the ability of the H. influenzae Rec-1 protein to cause cleavage of repressors but not the recombinase function differs from that of the E. coli RecA protein

Mutagenic DNA repair in Escherichia coli. VII

International Nuclear Information System (INIS)

Bridges, B.A.; Mottershead, R.P.

1978-01-01

Incubation of E. coli WP2 in the presence of chloramphenicol (CAP) for 90 min before and 60 min after γ-irradiation had no effect on the induction of Trp + mutations. Bacteria that had been treated with CAP for 90 min prior to UV irradiation showed normal or near normal yields of induced mutations to streptomycin or colicin E2 resistance. Most of these mutations lost their photoreversibility (indicating 'fixation') during continued incubation with CAP for a further 60 min after irradiation, during which time neither protein nor DNA synthesis was detectable. It is suggested that CAP-sensitive protein synthesis is not required for mutagenic (error-prone) repair of lesions in pre-existing DNA, arguing against an inducible component in this repair. In contrast the frequency of UV-induced mutations to Trp + (largely at suppressor loci) was drastically reduced by CAP pretreatment, confirming the need for an active replication fork for UV-mutagenesis at these loci. It is known from the work of others that CAP given after UV abolishes mutagenesis at these loci. It is concluded that CAP-sensitive protein synthesis (consistent with a requirement for an inducible function) is necessary for mutagenic repair only in newly-replicated DNA (presumably at daughter strand gaps) and not in pre-existing DNA. The data are consistent with but do not prove the hypothesis that CAP-sensitive and insensitive modes of mutagenesis reflect minor differences in the operation of a single basic mutagenic repair system. (Auth.)

Combining rational and random strategies in β-glucosidase Zm-p60.1 protein library construction.

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Dušan Turek

Full Text Available Saturation mutagenesis is a cornerstone technique in protein engineering because of its utility (in conjunction with appropriate analytical techniques for assessing effects of varying residues at selected positions on proteins' structures and functions. Site-directed mutagenesis with degenerate primers is the simplest and most rapid saturation mutagenesis technique. Thus, it is highly appropriate for assessing whether or not variation at certain sites is permissible, but not necessarily the most time- and cost-effective technique for detailed assessment of variations' effects. Thus, in the presented study we applied the technique to randomize position W373 in β-glucosidase Zm-p60.1, which is highly conserved among β-glucosidases. Unexpectedly, β-glucosidase activity screening of the generated variants showed that most variants were active, although they generally had significantly lower activity than the wild type enzyme. Further characterization of the library led us to conclude that a carefully selected combination of randomized codon-based saturation mutagenesis and site-directed mutagenesis may be most efficient, particularly when constructing and investigating randomized libraries with high fractions of positive hits.

Combining rational and random strategies in β-glucosidase Zm-p60.1 protein library construction.

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Turek, Dušan; Klimeš, Pavel; Mazura, Pavel; Brzobohatý, Břetislav

2014-01-01

Saturation mutagenesis is a cornerstone technique in protein engineering because of its utility (in conjunction with appropriate analytical techniques) for assessing effects of varying residues at selected positions on proteins' structures and functions. Site-directed mutagenesis with degenerate primers is the simplest and most rapid saturation mutagenesis technique. Thus, it is highly appropriate for assessing whether or not variation at certain sites is permissible, but not necessarily the most time- and cost-effective technique for detailed assessment of variations' effects. Thus, in the presented study we applied the technique to randomize position W373 in β-glucosidase Zm-p60.1, which is highly conserved among β-glucosidases. Unexpectedly, β-glucosidase activity screening of the generated variants showed that most variants were active, although they generally had significantly lower activity than the wild type enzyme. Further characterization of the library led us to conclude that a carefully selected combination of randomized codon-based saturation mutagenesis and site-directed mutagenesis may be most efficient, particularly when constructing and investigating randomized libraries with high fractions of positive hits.

Mutagenesis and carcinogenesis resulting from environment pollution

International Nuclear Information System (INIS)

Dimitrov, B.

2001-01-01

The paper reviews different ways of environmental contamination with natural and artificial harmful substances (chemical and radioactive) and their role in the processes of mutagenesis and carcinogenesis. The recent studies of the mechanism of mutagenesis and carcinogenesis due to environmental pollution are discussed

A Review on Microbial Mutagenesis through Gamma Irradiation for Agricultural Applications

International Nuclear Information System (INIS)

Hoe, P.C.K.; Khairuddin Abdul Rahim

2016-01-01

Gamma irradiation is widely used in sterilization and mutagenesis, especially for plant breeding and crop protection. Microbial mutagenesis through gamma irradiation is mainly applied in fermentation industry. In agriculture, gamma irradiation is mostly applied in crop improvement. Microbial mutagenesis is mainly applied against fungus and spore-forming bacteria, which are resistant to gamma irradiation. Response of microbes to gamma irradiation varies and depends on various factors. Review of previous works on gamma irradiation for microbial mutagenesis in agriculture may provide some information for the use of this method. The general view on gamma irradiation, its application, and mutagenesis are discussed in this paper. Further investigation on microbial mutagenesis should consider molecular changes, information on which is lacking in previous works. Moreover, studies on microbial mutagenesis are still lacking in Malaysia despite having several gamma irradiation facilities. Therefore, further studies on microbial mutagenesis should be conducted. (author)

Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes.

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Burger, Alexa; Lindsay, Helen; Felker, Anastasia; Hess, Christopher; Anders, Carolin; Chiavacci, Elena; Zaugg, Jonas; Weber, Lukas M; Catena, Raul; Jinek, Martin; Robinson, Mark D; Mosimann, Christian

2016-06-01

CRISPR-Cas9 enables efficient sequence-specific mutagenesis for creating somatic or germline mutants of model organisms. Key constraints in vivo remain the expression and delivery of active Cas9-sgRNA ribonucleoprotein complexes (RNPs) with minimal toxicity, variable mutagenesis efficiencies depending on targeting sequence, and high mutation mosaicism. Here, we apply in vitro assembled, fluorescent Cas9-sgRNA RNPs in solubilizing salt solution to achieve maximal mutagenesis efficiency in zebrafish embryos. MiSeq-based sequence analysis of targeted loci in individual embryos using CrispRVariants, a customized software tool for mutagenesis quantification and visualization, reveals efficient bi-allelic mutagenesis that reaches saturation at several tested gene loci. Such virtually complete mutagenesis exposes loss-of-function phenotypes for candidate genes in somatic mutant embryos for subsequent generation of stable germline mutants. We further show that targeting of non-coding elements in gene regulatory regions using saturating mutagenesis uncovers functional control elements in transgenic reporters and endogenous genes in injected embryos. Our results establish that optimally solubilized, in vitro assembled fluorescent Cas9-sgRNA RNPs provide a reproducible reagent for direct and scalable loss-of-function studies and applications beyond zebrafish experiments that require maximal DNA cutting efficiency in vivo. © 2016. Published by The Company of Biologists Ltd.

A highly efficient transposon mutagenesis system for the tomato pathogen Clavibacter michiganensis subsp. michiganensis.

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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.

Comparing Different Strategies in Directed Evolution of Enzyme Stereoselectivity: Single- versus Double-Code Saturation Mutagenesis.

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Sun, Zhoutong; Lonsdale, Richard; Li, Guangyue; Reetz, Manfred T

2016-10-04

Saturation mutagenesis at sites lining the binding pockets of enzymes constitutes a viable protein engineering technique for enhancing or inverting stereoselectivity. Statistical analysis shows that oversampling in the screening step (the bottleneck) increases astronomically as the number of residues in the randomization site increases, which is the reason why reduced amino acid alphabets have been employed, in addition to splitting large sites into smaller ones. Limonene epoxide hydrolase (LEH) has previously served as the experimental platform in these methodological efforts, enabling comparisons between single-code saturation mutagenesis (SCSM) and triple-code saturation mutagenesis (TCSM); these employ either only one or three amino acids, respectively, as building blocks. In this study the comparative platform is extended by exploring the efficacy of double-code saturation mutagenesis (DCSM), in which the reduced amino acid alphabet consists of two members, chosen according to the principles of rational design on the basis of structural information. The hydrolytic desymmetrization of cyclohexene oxide is used as the model reaction, with formation of either (R,R)- or (S,S)-cyclohexane-1,2-diol. DCSM proves to be clearly superior to the likewise tested SCSM, affording both R,R- and S,S-selective mutants. These variants are also good catalysts in reactions of further substrates. Docking computations reveal the basis of enantioselectivity. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

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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.

Multiple Site-Directed and Saturation Mutagenesis by the Patch Cloning Method.

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Taniguchi, Naohiro; Murakami, Hiroshi

2017-01-01

Constructing protein-coding genes with desired mutations is a basic step for protein engineering. Herein, we describe a multiple site-directed and saturation mutagenesis method, termed MUPAC. This method has been used to introduce multiple site-directed mutations in the green fluorescent protein gene and in the moloney murine leukemia virus reverse transcriptase gene. Moreover, this method was also successfully used to introduce randomized codons at five desired positions in the green fluorescent protein gene, and for simple DNA assembly for cloning.

Adenosine diphosphate sugar pyrophosphatase prevents glycogen biosynthesis in Escherichia coli

Science.gov (United States)

Moreno-Bruna, Beatriz; Baroja-Fernández, Edurne; Muñoz, Francisco José; Bastarrica-Berasategui, Ainara; Zandueta-Criado, Aitor; Rodríguez-López, Milagros; Lasa, Iñigo; Akazawa, Takashi; Pozueta-Romero, Javier

2001-01-01

An adenosine diphosphate sugar pyrophosphatase (ASPPase, EC 3.6.1.21) has been characterized by using Escherichia coli. This enzyme, whose activities in the cell are inversely correlated with the intracellular glycogen content and the glucose concentration in the culture medium, hydrolyzes ADP-glucose, the precursor molecule of glycogen biosynthesis. ASPPase was purified to apparent homogeneity (over 3,000-fold), and sequence analyses revealed that it is a member of the ubiquitously distributed group of nucleotide pyrophosphatases designated as “nudix” hydrolases. Insertional mutagenesis experiments leading to the inactivation of the ASPPase encoding gene, aspP, produced cells with marginally low enzymatic activities and higher glycogen content than wild-type bacteria. aspP was cloned into an expression vector and introduced into E. coli. Transformed cells were shown to contain a dramatically reduced amount of glycogen, as compared with the untransformed bacteria. No pleiotropic changes in the bacterial growth occurred in both the aspP-overexpressing and aspP-deficient strains. The overall results pinpoint the reaction catalyzed by ASPPase as a potential step of regulating glycogen biosynthesis in E. coli. PMID:11416161

Evidence for a Rad18-independent frameshift mutagenesis pathway in human cell-free extracts.

Directory of Open Access Journals (Sweden)

Régine Janel-Bintz

Full Text Available Bypass of replication blocks by specialized DNA polymerases is crucial for cell survival but may promote mutagenesis and genome instability. To gain insight into mutagenic sub-pathways that coexist in mammalian cells, we examined N-2-acetylaminofluorene (AAF-induced frameshift mutagenesis by means of SV40-based shuttle vectors containing a single adduct. We found that in mammalian cells, as previously observed in E. coli, modification of the third guanine of two target sequences, 5'-GGG-3' (3G and 5'-GGCGCC-3' (NarI site, induces -1 and -2 frameshift mutations, respectively. Using an in vitro assay for translesion synthesis, we investigated the biochemical control of these events. We showed that Pol eta, but neither Pol iota nor Pol zeta, plays a major role in the frameshift bypass of the AAF adduct located in the 3G sequence. By complementing PCNA-depleted extracts with either a wild-type or a non-ubiquitinatable form of PCNA, we found that this Pol eta-mediated pathway requires Rad18 and ubiquitination of PCNA. In contrast, when the AAF adduct is located within the NarI site, TLS is only partially dependent upon Pol eta and Rad18, unravelling the existence of alternative pathways that concurrently bypass this lesion.

Structural and mutational analysis of Escherichia coli AlkB provides insight into substrate specificity and DNA damage searching.

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Paul J Holland

Full Text Available BACKGROUND: In Escherichia coli, cytotoxic DNA methyl lesions on the N1 position of purines and N3 position of pyrimidines are primarily repaired by the 2-oxoglutarate (2-OG iron(II dependent dioxygenase, AlkB. AlkB repairs 1-methyladenine (1-meA and 3-methylcytosine (3-meC lesions, but it also repairs 1-methylguanine (1-meG and 3-methylthymine (3-meT at a much less efficient rate. How the AlkB enzyme is able to locate and identify methylated bases in ssDNA has remained an open question. METHODOLOGY/PRINCIPAL FINDINGS: We determined the crystal structures of the E. coli AlkB protein holoenzyme and the AlkB-ssDNA complex containing a 1-meG lesion. We coupled this to site-directed mutagenesis of amino acids in and around the active site, and tested the effects of these mutations on the ability of the protein to bind both damaged and undamaged DNA, as well as catalyze repair of a methylated substrate. CONCLUSIONS/SIGNIFICANCE: A comparison of our substrate-bound AlkB-ssDNA complex with our unliganded holoenzyme reveals conformational changes of residues within the active site that are important for binding damaged bases. Site-directed mutagenesis of these residues reveals novel insight into their roles in DNA damage recognition and repair. Our data support a model that the AlkB protein utilizes at least two distinct conformations in searching and binding methylated bases within DNA: a "searching" mode and "repair" mode. Moreover, we are able to functionally separate these modes through mutagenesis of residues that affect one or the other binding state. Finally, our mutagenesis experiments show that amino acid D135 of AlkB participates in both substrate specificity and catalysis.

Identification of genes important for growth of asymptomatic bacteriuria Escherichia coli in urine.

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Vejborg, Rebecca M; de Evgrafov, Mari R; Phan, Minh Duy; Totsika, Makrina; Schembri, Mark A; Hancock, Viktoria

2012-09-01

Escherichia coli is the most important etiological agent of urinary tract infections (UTIs). Unlike uropathogenic E. coli, which causes symptomatic infections, asymptomatic bacteriuria (ABU) E. coli strains typically lack essential virulence factors and colonize the bladder in the absence of symptoms. While ABU E. coli can persist in the bladder for long periods of time, little is known about the genetic determinants required for its growth and fitness in urine. To identify such genes, we have employed a transposon mutagenesis approach using the prototypic ABU E. coli strain 83972 and the clinical ABU E. coli strain VR89. Six genes involved in the biosynthesis of various amino acids and nucleobases were identified (carB, argE, argC, purA, metE, and ilvC), and site-specific mutants were subsequently constructed in E. coli 83972 and E. coli VR89 for each of these genes. In all cases, these mutants exhibited reduced growth rates and final cell densities in human urine. The growth defects could be complemented in trans as well as by supplementation with the appropriate amino acid or nucleobase. When assessed in vivo in a mouse model, E. coli 83972carAB and 83972argC showed a significantly reduced competitive advantage in the bladder and/or kidney during coinoculation experiments with the parent strain, whereas 83972metE and 83972ilvC did not. Taken together, our data have identified several biosynthesis pathways as new important fitness factors associated with the growth of ABU E. coli in human urine.

Recent advances of microbial breeding via heavy-ion mutagenesis at IMP.

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Hu, W; Li, W; Chen, J

2017-10-01

Nowadays, the value of heavy-ion mutagenesis has been accepted as a novel powerful mutagen technique to generate new microbial mutants due to its high linear energy transfer and high relative biological effectiveness. This paper briefly reviews recent progress in developing a more efficient mutagenesis technique for microbial breeding using heavy-ion mutagenesis, and also presents the outline of the beam line for microbial breeding in Heavy Ion Research Facility of Lanzhou. Then, new insights into microbial biotechnology via heavy-ion mutagenesis are also further explored. We hope that our concerns will give deep insight into microbial breeding biotechnology via heavy-ion mutagenesis. We also believe that heavy-ion mutagenesis breeding will greatly contribute to the progress of a comprehensive study industrial strain engineering for bioindustry in the future. There is currently a great interest in developing rapid and diverse microbial mutation tool for strain modification. Heavy-ion mutagenesis has been proved as a powerful technology for microbial breeding due to its broad spectrum of mutation phenotypes with high efficiency. In order to deeply understand heavy-ion mutagenesis technology, this paper briefly reviews recent progress in microbial breeding using heavy-ion mutagenesis at IMP, and also presents the outline of the beam line for microbial breeding in Heavy Ion Research Facility of Lanzhou (HIRFL) as well as new insights into microbial biotechnology via heavy-ion mutagenesis. Thus, this work can provide the guidelines to promote the development of novel microbial biotechnology cross-linking heavy-ion mutagenesis breeding that could make breeding process more efficiently in the future. © 2017 The Society for Applied Microbiology.

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

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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. Copyright © 2015 Elsevier Inc. All rights reserved.

Environmental stress induces trinucleotide repeat mutagenesis in human cells.

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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.

An Agrobacterium-delivered CRISPR/Cas9 system for high-frequency targeted mutagenesis in maize.

Science.gov (United States)

Char, Si Nian; Neelakandan, Anjanasree K; Nahampun, Hartinio; Frame, Bronwyn; Main, Marcy; Spalding, Martin H; Becraft, Philip W; Meyers, Blake C; Walbot, Virginia; Wang, Kan; Yang, Bing

2017-02-01

CRISPR/Cas9 is a powerful genome editing tool in many organisms, including a number of monocots and dicots. Although the design and application of CRISPR/Cas9 is simpler compared to other nuclease-based genome editing tools, optimization requires the consideration of the DNA delivery and tissue regeneration methods for a particular species to achieve accuracy and efficiency. Here, we describe a public sector system, ISU Maize CRISPR, utilizing Agrobacterium-delivered CRISPR/Cas9 for high-frequency targeted mutagenesis in maize. This system consists of an Escherichia coli cloning vector and an Agrobacterium binary vector. It can be used to clone up to four guide RNAs for single or multiplex gene targeting. We evaluated this system for its mutagenesis frequency and heritability using four maize genes in two duplicated pairs: Argonaute 18 (ZmAgo18a and ZmAgo18b) and dihydroflavonol 4-reductase or anthocyaninless genes (a1 and a4). T 0 transgenic events carrying mono- or diallelic mutations of one locus and various combinations of allelic mutations of two loci occurred at rates over 70% mutants per transgenic events in both Hi-II and B104 genotypes. Through genetic segregation, null segregants carrying only the desired mutant alleles without the CRISPR transgene could be generated in T 1 progeny. Inheritance of an active CRISPR/Cas9 transgene leads to additional target-specific mutations in subsequent generations. Duplex infection of immature embryos by mixing two individual Agrobacterium strains harbouring different Cas9/gRNA modules can be performed for improved cost efficiency. Together, the findings demonstrate that the ISU Maize CRISPR platform is an effective and robust tool to targeted mutagenesis in maize. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Modification of Antibody Function by Mutagenesis.

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Dasch, James R; Dasch, Amy L

2017-09-01

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

Combinatorial effect of mutagenesis and medium component optimization on Bacillus amyloliquefaciens antifungal activity and efficacy in eradicating Botrytis cinerea.

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Masmoudi, Fatma; Ben Khedher, Saoussen; Kamoun, Amel; Zouari, Nabil; Tounsi, Slim; Trigui, Mohamed

2017-04-01

This work is directed towards Bacillus amyloliquefaciens strain BLB371 metabolite production for biocontrol of fungal phytopathogens. In order to maximise antifungal metabolite production by this strain, two approaches were combined: random mutagenesis and medium component optimization. After three rounds of mutagenesis, a hyper active mutant, named M3-7, was obtained. It produces 7 fold more antifungal metabolites (1800AU/mL) than the wild strain in MC medium. A hybrid design was applied to optimise a new medium to enhance antifungal metabolite production by M3-7. The new optimized medium (35g/L of peptone, 32.5g/L of sucrose, 10.5g/L of yeast extract, 2.4g/L of KH 2 PO 4 , 1.3g/L of MgSO 4 and 23mg/L of MnSO 4 ) achieved 1.62 fold enhancement in antifungal compound production (3000AU/mL) by this mutant, compared to that achieved in MC medium. Therefore, combinatory effect of these two approaches (mutagenesis and medium component optimization) allowed 12 fold improvement in antifungal activity (from 250UA/mL to 3000UA/mL). This improvement was confirmed against several phytopathogenic fungi with an increase of MIC and MFC over than 50%. More interestingly, a total eradication of gray mold was obtained on tomato fruits infected by Botrytis cinerea and treated by M3-7, compared to those treated by BLB371. From the practical point of view, combining random mutagenesis and medium optimization could be considered as an excellent tool for obtaining promising biological products useful against phytopathogenic fungi. Copyright © 2017 Elsevier GmbH. All rights reserved.

Cloning and properties of the Salmonella typhimurium tricarboxylate transport operon in Escherichia coli

International Nuclear Information System (INIS)

Widenhorn, K.A.; Boos, W.; Somers, J.M.; Kay, W.W.

1988-01-01

The tricarboxylate transport operon (tctI) was cloned in Escherichia coli as a 12-kilobase (kb) fragment from an EcoRI library of the Salmonella typhimurium chromosome in λgtWES. It was further subcloned as a 12-kb fragment into pACYC184 and as an 8-kb fragment into pBR322. By insertional mutagenesis mediated by λTn5, restriction mapping, and phenotypic testing, the tctI operon was localized to a 4.5-kb region. The tctC gene which encodes a periplasmic binding protein (C-protein) was located near the center of the insert. E. coli/tctI clones on either multicopy or single-copy vectors grew on the same tricarboxylates as S. typhimurium, although unusually long growth lags were observed. E. coli/tctI clones exhibited similar [ 14 C] fluorocitrate transport kinetics to those of S. typhimurium, whereas E. coli alone was virtually impermeable to [ 14 C] fluorocitrate. The periplasmic C proteins (C1 and C2 isoelectric forms) were produced in prodigious quantities from the cloned strains. Motile E. coli/tctI clones were not chemotactic toward citrate, whereas tctI deletion mutants of S. typhimurium were. Taken together, these observations indicate that tctI is not an operon involved in chemotaxis

Modification of γ-induced mutagenesis in Ames test-strains

International Nuclear Information System (INIS)

Basha, S.G.; Krasavin, E.A.; Kozubek, S.; Amirtaev, K.G.

1990-01-01

Glycerine and cysteamine protective effect on mutagenesis was studied in 3 strains of Salmonella typhimurium under γ-radiation. Glycerine modifying effect was shown to be not similar for various test-strains and depended on DNA injury nature. DNA complex injuries were shown to play significant role in mutagenesis of TA100 and TA102 strains. Absence of cysteamine modifying effect on γ-induced mutagenesis testified to cysteamine effect on enzyme balance. 20 refs.; 2 figs.; 1 tab

Untargeted viral mutagenesis is not found in X-irradiated monkey cells

International Nuclear Information System (INIS)

Lytle, C.D.; Carney, P.G.; Lee, W.; Bushar, H.F.

1988-01-01

The existence of untargeted viral mutagenesis in X-irradiated cells was investigated in a mammalian virus/cell system, where a low level of such viral mutagenesis can be demonstrated in UV-irradiated cells. In the positive control experiment UV-elicited mutagenesis was shown with cell exposures of 5, 10 and 15 J/m 2 and a delay of 24 h between cell irradiation and infection with unirradiated herpes simplex virus. Although X-ray doses of 1, 3 and 10 Gy elicit enhanced reactivation of UV-irradiated virus, no untargeted mutagenesis for any X-ray dose at post-irradiation infection times of 0, 24 or 72 h was observed in this study. Thus untargeted mutagenesis of herpes simplex virus was not demonstrated in X-irradiated monkey cells, under conditions where X-ray-enhanced reactivation occurs and where untargeted mutagenesis in UV-irradiated cells occurs. (author)

The Yeast Environmental Stress Response Regulates Mutagenesis Induced by Proteotoxic Stress

Science.gov (United States)

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

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

Science.gov (United States)

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

2017-01-01

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

Mass spectrometry and site-directed mutagenesis identify several autophosphorylated residues required for the activity of PrkC, a Ser/Thr kinase from Bacillus subtilis

DEFF Research Database (Denmark)

Madec, Edwige; Stensballe, Allan; Kjellström, Sven

2003-01-01

We have shown recently that PrkC, which is involved in developmental processes in Bacillus subtilis, is a Ser/Thr kinase with features of the receptor kinase family of eukaryotic Hanks kinases. In this study, we expressed and purified from Escherichia coli the cytoplasmic domain of PrkC containing...... the kinase and a short juxtamembrane region. This fragment, which we designate PrkCc, undergoes autophosphorylation in E.coli. PrkCc is further autophosphorylated in vitro, apparently through a trans-kinase, intermolecular reaction. PrkC also displays kinase activity with myelin basic protein. Using high...... mass accuracy electrospray tandem mass spectrometry (LC-MS/MS) and nanoelectrospray tandem mass spectrometry, we identified seven phosphorylated threonine and one serine residue in PrkCc. All the corresponding residues were replaced by systematic site-directed mutagenesis and the purified mutant...

p53 Mutagenesis by Benzo[a]pyrene derived Radical Cations

Science.gov (United States)

Sen, Sushmita; Bhojnagarwala, Pratik; Francey, Lauren; Lu, Ding; Jeffrey Field, Trevor M. Penning

2013-01-01

Benzo[a]pyrene (B[a]P), a major human carcinogen in combustion products such as cigarette smoke and diesel exhaust, is metabolically activated into DNA-reactive metabolites via three different enzymatic pathways. The pathways are the anti-(+)-benzo[a]pyrene 7,8-diol 9, 10-epoxide pathway (P450/ epoxide hydrolase catalyzed) (B[a]PDE), the benzo[a]pyrene o-quinone pathway (aldo ketose reductase (AKR) catalyzed) and the B[a]P radical cation pathway (P450 peroxidase catalyzed). We used a yeast p53 mutagenesis system to assess mutagenesis by B[a]P radical cations. Because radical cations are short-lived, they were generated in situ by reacting B[a]P with cumene hydroperoxide (CuOOH) and horse radish peroxidase (HRP) and then monitoring the generation of the more stable downstream products, B[a]P-1,6-dione and B[a]P-3,6-dione. Based on the B[a]P-1,6 and 3,6-dione formation, approximately 4µM of radical cation was generated. In the mutagenesis assays, the radical cations produced in situ showed a dose-dependent increase in mutagenicity from 0.25 µM to 10 µM B[a]P with no significant increase seen with further escalation to 50 µM B[a]P. However, mutagenesis was 200-fold less than with the AKR pathway derived B[a]P, 7–8 dione. Mutant p53 plasmids, which yield red colonies, were recovered from the yeast to study the pattern and spectrum of mutations. The mutation pattern observed was G to T (31%) > G to C (29%) > G to A (14%). The frequency of codons mutated by the B[a]P radical cations was essentially random and not enriched at known cancer hotspots. The quinone products of radical cations, B[a]P-1,6-dione and B[a]P-3,6-dione were more mutagenic than the radical cation reactions, but still less mutagenic than AKR derived B[a]P-7,8-dione. We conclude that B[a]P radical cations and their quinone products are weakly mutagenic in this yeast-based system compared to redox cycling PAH o-quinones. PMID:22768918

Functional studies of elongation factor Tu from Escherichia coli : Site-directed mutagenesis and antibiotic action

NARCIS (Netherlands)

Krab, Ivo Maarten

2001-01-01

This PhD thesis describes several studies into the structure and function of Escherichia coli Elongation Factor Tu (EF-Tu). EF-Tu plays a central role in the bacterial protein synthesis machinery as the carrier of "coded building blocks" for protein synthesis, aminoacylated tRNA (aa-tRNA). Without

Photodynamic action of the methylene blue: mutagenesis and sinergism

International Nuclear Information System (INIS)

Capella, M.A.M.

1988-01-01

Two aspects of photodynamic therapy were studied: the associated mutagenesis and the interactions with physical agents, in order to increase its biological effects. The photodynamic action with methylene blue in the mutagenesis and sinergism is studied. (L.M.J.)

Computer Simulation of Mutagenesis.

Science.gov (United States)

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)

Photoreactivation of conversion and de novo suppressor mutation in Escherichia coli

Energy Technology Data Exchange (ETDEWEB)

Bockrath, R C; Plamer, J E [Indiana Univ., Indianapolis (USA). Dept. of Microbiology

1977-04-01

Studies of mutagenesis and photoreactivation in various E.coli strains have shown that conversion mutation of a mutant containing an amber suppressor to one containing an ochre suppressor is sensitive to photoreactivation. Direct photoreactivation by photoreactivating light (PRL) after uv mutagenesis reduced mutation frequencies by a factor of about 2 for each minute of exposure during the first 5 to 8 min of exposure for cells with normal repair capacity. Conversion and potential de novo suppressor mutations were about equally sensitive. For conversion, the sensitivities to PRL were identical in the repair-normal and excisions-repair-deficient strains. For de novo suppressor mutation, the rate of mutation frequency reduction by PRL in the repair-deficient strain was about one-half that in the other strains. The results suggest that ultraviolet radiation produces both de novo suppressor mutation and conversion at the sup(E,B) locus by photoreversible pyrimidine dimers in the DNA. The causative dimers could be Thy()Cyt dimers in the transcribed strand or the non-transcribed strand, respectively.

A report on 36 years practical work on crop improvement through induced mutagenesis

Energy Technology Data Exchange (ETDEWEB)

Datta, S.K. [CSIR, Madhyamgram Experimental Farm, Bose Institute, Kolkata (India)], E-mail: subodhskdatta@rediffmail.com

2008-07-01

Physical and/or chemical mutagens cause random changes in the nuclear DNA or cytoplasmic organelles, resulting in gene, chromosomal or genomic mutations. The author will share his life time experience and achievement on induced mutagenesis. The author initiated induced mutagenesis work in 1971 till July 2007 and used both physical (X-ray and Gamma rays) and chemical (EMS, MMS, Colchicine) mutagens for improvement of vegetables (Trichosanthes anguina L, T. cucumarina , Cucurbita maxima L, Cephalandra indica, Luffa acutangula Roxb., Lagenaria ciceraria), medicinal (Trigonella foenum-graecum L, Mentha citrate Ehrh), pulse (Winged Bean (Psophocarpus tetragonolobus L. D.C.), oil bearing (Jatropha curcas L, Rosa damascene, Cymbopogon flexuosus (Nees) Wats) and ornamental (Bougainvillea, Chrysanthemum, Dahlia, Gladiolus, Hibiscus, Lantana depressa Naud, Rose, Tuberose, Narcissus etc.) crops. All classical and advanced mutagenesis methods have been extensively used for the development of new and novel cultivars of economic importance. Early flowering, late flowering, dwarf, yellow fruit color, crinkled leaf, short thick fruit, increased branching, increased pod and seed number, seed size, seed color (green, brown, chocolate color) high fruit-, seed-, oil- and punicic acidyielding mutants have been developed in T. anguina, T. fornum-graecum, Winged Bean and in J.curcas containing 'curcas oil', an efficient substitute fuel for diesel engines. Induction of flower color and chlorophyll variegated mutants in L. depressa proved the efficiency of mutation technique for domestication of wild relatives. Author was deeply engaged for the last 30 years for improvement of ornamentals and has been most successful to produce quite a large number of new promising mutant varieties in different ornamentals. Colchicine has been successfully used to develop new flower color in chrysanthemum and rose and high yielding strains in T. anguina. A novel direct in vitro regeneration technique has

Empirical complexities in the genetic foundations of lethal mutagenesis.

Science.gov (United States)

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.

Favipiravir elicits antiviral mutagenesis during virus replication in vivo.

Science.gov (United States)

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.

Effective mutagenesis of Arabidopsis by heavy ion beam-irradiation

International Nuclear Information System (INIS)

Yamamoto, Y.Y.; Saito, H.; Ryuto, H.; Fukunishi, N.; Yoshida, S.; Abe, T.

2005-01-01

Full text: Arabidopsis researches frequently include the genetic approach, so efficient, convenient, and safe methods for mutagenesis are required. Currently, the most popular method for in house mutagenesis is application of EMS. Although this method is very effective, its base substitution-type mutations often gives leaky mutants with residual gene functions, leading some difficulty in understanding the corresponding gene functions. Heavy ion beam generated by accelerators gives highest energy transfer rates among known radiation-based mutagenesis methods including X ray, gamma ray, fast neutron, electron and proton irradiation. This feature is thought to give high frequency of the double strand break of genomic DNA and resultant short deletions, resulting frame shift-type mutations. At RIKEN Accelerator Research Facility (RARF, http://www.rarf.riken.go.jp/index-e.html), we have optimized conditions for effective mutagenesis of Arabidopsis regarding to ion species and irradiation dose, and achieved comparable mutation rates to the method with EMS. (author)

Enhancement of Lutein Production in Chlorella sorokiniana (Chorophyta by Improvement of Culture Conditions and Random Mutagenesis

Directory of Open Access Journals (Sweden)

Maria Angeles Vargas

2011-09-01

Full Text Available Chlorella sorokiniana has been selected for lutein production, after a screening of thirteen species of microalgae, since it showed both a high content in this carotenoid and a high growth rate. The effects of several nutritional and environmental factors on cell growth and lutein accumulation have been studied. Maximal specific growth rate and lutein content were attained at 690 µmol photons m−2 s−1, 28 °C, 2 mM NaCl, 40 mM nitrate and under mixotrophic conditions. In general, optimal conditions for the growth of this strain also lead to maximal lutein productivity. High lutein yielding mutants of C. sorokiniana have been obtained by random mutagenesis, using N-methyl-N′-nitro-nitrosoguanidine (MNNG as a mutagen and selecting mutants by their resistance to the inhibitors of the carotenogenic pathway nicotine and norflurazon. Among the mutants resistant to the herbicides, those exhibiting both high content in lutein and high growth rate were chosen. Several mutants exhibited higher contents in this carotenoid than the wild type, showing, in addition, either a similar or higher growth rate than the latter strain. The mutant MR-16 exhibited a 2.0-fold higher volumetric lutein content than that of the wild type, attaining values of 42.0 mg L−1 and mutants DMR-5 and DMR-8 attained a lutein cellular content of 7.0 mg g−1 dry weight. The high lutein yield exhibited by C. sorokiniana makes this microalga an excellent candidate for the production of this commercially interesting pigment.

UV-C mutagenesis of Kluyveromyces marxianus NRRL Y-1109 strain for improved anaerobic growth at elevated temperature on pentose and hexose sugars

Science.gov (United States)

More robust industrial yeast strains from Kluyveromyces marxianus NRRL Y-1109 and have been produced using UV-C irradiation specifically for anaerobic conversion of lignocellulosic sugar streams to fuel ethanol at elevated temperature (45°C). This type of random mutagenesis offers the possibility o...

Comments on mutagenesis risk estimation

International Nuclear Information System (INIS)

Russell, W.L.

1976-01-01

Several hypotheses and concepts have tended to oversimplify the problem of mutagenesis and can be misleading when used for genetic risk estimation. These include: the hypothesis that radiation-induced mutation frequency depends primarily on the DNA content per haploid genome, the extension of this concept to chemical mutagenesis, the view that, since DNA is DNA, mutational effects can be expected to be qualitatively similar in all organisms, the REC unit, and the view that mutation rates from chronic irradiation can be theoretically and accurately predicted from acute irradiation data. Therefore, direct determination of frequencies of transmitted mutations in mammals continues to be important for risk estimation, and the specific-locus method in mice is shown to be not as expensive as is commonly supposed for many of the chemical testing requirements

Molecular fundamentals of chromosomal mutagenesis

International Nuclear Information System (INIS)

Ganassi, E.Eh.; Zaichkina, S.I.; Malakhova, L.V.

1987-01-01

Precise quantitative correlation between the yield of chromosome structure damages and the yield of DNA damages is shown when comparing data on molecular and cytogenetic investigations carried out in cultural Mammalia cells. As the chromosome structure damage is to be connected with the damage of its carcass structure, then it is natural that DNA damage in loop regions is not to affect considerably the structure, while DNA damage lying on the loop base and connected with the chromosome carcass is to play a determining role in chromosomal mutagenesis. This DNA constitutes 1-2% from the total quantity of nuclear DNA. If one accepts that damages of these regions of DNA are ''hot'' points of chromosomal mutagenesis, then it becomes clear why 1-2% of preparation damages in a cell are realized in chromosome structural damages

Processing closely spaced lesions during Nucleotide Excision Repair triggers mutagenesis in E. coli

Science.gov (United States)

Isogawa, Asako; Fujii, Shingo

2017-01-01

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. PMID:28686598

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

Science.gov (United States)

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.

Gain-of-function mutagenesis approaches in rice for functional genomics and improvement of crop productivity.

Science.gov (United States)

Moin, Mazahar; Bakshi, Achala; Saha, Anusree; Dutta, Mouboni; Kirti, P B

2017-07-01

The epitome of any genome research is to identify all the existing genes in a genome and investigate their roles. Various techniques have been applied to unveil the functions either by silencing or over-expressing the genes by targeted expression or random mutagenesis. Rice is the most appropriate model crop for generating a mutant resource for functional genomic studies because of the availability of high-quality genome sequence and relatively smaller genome size. Rice has syntenic relationships with members of other cereals. Hence, characterization of functionally unknown genes in rice will possibly provide key genetic insights and can lead to comparative genomics involving other cereals. The current review attempts to discuss the available gain-of-function mutagenesis techniques for functional genomics, emphasizing the contemporary approach, activation tagging and alterations to this method for the enhancement of yield and productivity of rice. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

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

Science.gov (United States)

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

2016-01-01

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

Heat shock and herpes virus: enhanced reactivation without untargeted mutagenesis

International Nuclear Information System (INIS)

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

Mutagenesis and repair of DNA

International Nuclear Information System (INIS)

Janion, C.; Grzesiuk, E.; Fabisiewicz, A.; Tudek, B.; Ciesla, J.; Graziewicz, M.; Wojcik, A.; Speina, E.

1998-01-01

Full text. The discovery that the mfd gene codes for a transcription-coupling repair factor (TRCF) prompted us to re-investigate the MFD (mutation frequency decline) phenomenon in E.coli K-12 strain when mutations were induced by ultraviolet light, halogen light or MMS-treatment. These studies revealed that: (i) the process of MFD involves the proofreading activity of DNA pol III and the mismatch repair system, as well as, TRCF and the UvrABC-excinuclease (ii) a semi-rich plate test may be replaced by a rich liquid medium, (iii) the T-T pyrimidine dimers are the lesions excised with the highest activity, and (iv) overproduction of UmuD(D'C) proteins leads to a great increase in mutant frequency in irradiated and MMS-treated cells. The role of mismatch repair (MR) in MMS-induced mutagenesis is obscured by the fact that the spectra of mutational specificity are different in bacteria proficient and deficient in MR. It has been found that transposons Tn10 (and Tn5) when inserted into chromosomal DNA of E. coli influence the phenotype lowering the survival and frequency of mutations induced by UV or halogen light irradiation. This is connected with a deficiency of UmuD(D') and UmuC proteins. Transformation of bacteria with plasmids bearing the umuD(D')C genes, suppresses the effects of the transposon insertion, a phenomenon which has not been described before. Single-stranded DNA of M13mp18 phage was oxidized in vitro by a hydroxyl radical generating system including hypoxanthine/xanthine oxidase/Fe3+/EDTA, and it was found that Fapy-Ade, Fapy-Gua, 8-oxyAde and thymine glycol were the main products formed. Replication of the oxidized template by T7 phage DNA polymerase, Klenow fragment of polymerase I, or polymerase beta from bovine thymus has revealed that oxidized pyrimidines are stronger blockers than oxidized purines for T7 phage and Klenow fragment polymerases and the blocking potency depends on the neighboring bases and on the type of polymerase. Studies of

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

Science.gov (United States)

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

2017-01-01

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

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

Science.gov (United States)

Xia, Yongzhen; Xun, Luying

2017-01-01

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

Recovery during radiation and chemical mutagenesis

International Nuclear Information System (INIS)

Deen, D.F.

1975-01-01

These investigations were directed toward the study of recovery in radiation and chemical mutagenesis in cultured mammalian cells. A mutagenesis system was established in which mutation of V79-17lb Chinese hamster cells to 8-azaguanine resistance was tested. The effects of split dose and postirradiation treatments upon both x-ray and EMS induced mutagenesis were determined. Increasing the cell inoculum by a factor of 5 (from 10 5 to 5 x 10 5 ) decreased both the spontaneous and x-ray induced mutation frequencies by two orders of magnitude. The x-ray induced mutation frequency was found to be higher for those cells allowed to attach for 5 hours before irradiation, in comparison to those allowed to attach for 2 hours. The uv spectrum of 8-azaguanine changes as a function of storage time at low temperature, but not when diluted to either 10 μg/ml or 30 μg/ml and maintained at 37 0 C. The optimal expression time required after irradiation is dose dependent and can be determined from the relationship: E.T. = 1.93(10 -2 )D + 15.5. (E.T. = hours; D = rads). The duration of the optimal expression time can be estimated by summing the cell cycle time and the radiation induced lag time

Mutant fatty acid desaturase and methods for directed mutagenesis

Science.gov (United States)

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.

Multiplex Conditional Mutagenesis Using Transgenic Expression of Cas9 and sgRNAs.

Science.gov (United States)

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

2015-06-01

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

Enhancement of the catalytic activity of ferulic acid decarboxylase from Enterobacter sp. Px6-4 through random and site-directed mutagenesis.

Science.gov (United States)

Lee, Hyunji; Park, Jiyoung; Jung, Chaewon; Han, Dongfei; Seo, Jiyoung; Ahn, Joong-Hoon; Chong, Youhoon; Hur, Hor-Gil

2015-11-01

The enzyme ferulic acid decarboxylase (FADase) from Enterobacter sp. Px6-4 catalyzes the decarboxylation reaction of lignin monomers and phenolic compounds such as p-coumaric acid, caffeic acid, and ferulic acid into their corresponding 4-vinyl derivatives, that is, 4-vinylphenol, 4-vinylcatechol, and 4-vinylguaiacol, respectively. Among various ferulic acid decarboxylase enzymes, we chose the FADase from Enterobacter sp. Px6-4, whose crystal structure is known, and produced mutants to enhance its catalytic activity by random and site-directed mutagenesis. After three rounds of sequential mutations, FADase(F95L/D112N/V151I) showed approximately 34-fold higher catalytic activity than wild-type for the production of 4-vinylguaiacol from ferulic acid. Docking analyses suggested that the increased activity of FADase(F95L/D112N/V151I) could be due to formation of compact active site compared with that of the wild-type FADase. Considering the amount of phenolic compounds such as lignin monomers in the biomass components, successfully bioengineered FADase(F95L/D112N/V151I) from Enterobacter sp. Px6-4 could provide an ecofriendly biocatalytic tool for producing diverse styrene derivatives from biomass.

Probing the ubiquinol-binding site of recombinant Sauromatum guttatum alternative oxidase expressed in E. coli membranes through site-directed mutagenesis.

Science.gov (United States)

Young, Luke; May, Benjamin; Pendlebury-Watt, Alice; Shearman, Julia; Elliott, Catherine; Albury, Mary S; Shiba, Tomoo; Inaoka, Daniel Ken; Harada, Shigeharu; Kita, Kiyoshi; Moore, Anthony L

2014-07-01

In the present paper we have investigated the effect of mutagenesis of a number of highly conserved residues (R159, D163, L177 and L267) which we have recently shown to line the hydrophobic inhibitor/substrate cavity in the alternative oxidases (AOXs). Measurements of respiratory activity in rSgAOX expressed in Escherichia coli FN102 membranes indicate that all mutants result in a decrease in maximum activity of AOX and in some cases (D163 and L177) a decrease in the apparent Km (O2). Of particular importance was the finding that when the L177 and L267 residues, which appear to cause a bottleneck in the hydrophobic cavity, are mutated to alanine the sensitivity to AOX antagonists is reduced. When non-AOX anti-malarial inhibitors were also tested against these mutants widening the bottleneck through removal of isobutyl side chain allowed access of these bulkier inhibitors to the active-site and resulted in inhibition. Results are discussed in terms of how these mutations have altered the way in which the AOX's catalytic cycle is controlled and since maximum activity is decreased we predict that such mutations result in an increase in the steady state level of at least one O2-derived AOX intermediate. Such mutations should therefore prove to be useful in future stopped-flow and electron paramagnetic resonance experiments in attempts to understand the catalytic cycle of the alternative oxidase which may prove to be important in future rational drug design to treat diseases such as trypanosomiasis. Furthermore since single amino acid mutations in inhibitor/substrate pockets have been found to be the cause of multi-drug resistant strains of malaria, the decrease in sensitivity to main AOX antagonists observed in the L-mutants studied in this report suggests that an emergence of drug resistance to trypanosomiasis may also be possible. Therefore we suggest that the design of future AOX inhibitors should have structures that are less reliant on the orientation by the two

Tif-stimulated deoxyribonucleic acid repair in Escherichia coli K-12

International Nuclear Information System (INIS)

Castellazzi, M.; Jacques, M.; George, J.

1980-01-01

Bacterial survival is significantly increased after ultraviolet irradiation in tif sfi cells, provided that the thermosensitive tif mutation has been expressed at 41 0 C before irradiation. This tif-mediated reactivation of ultraviolet irradiated bacteria needs de novo protein synthesis, as is the case for the tif-mediated reactivation of ultraviolet-irradiated phage lambda. However, in striking contrast to the phage reactivation process, this tif-mediated reactivation is no longer associated with mutagenesis. It also requires the presence of the uvrA + excision function. These results strongly suggest the existence in Escherichia coli K-12 of a repair pathway acting on bacterial deoxyribonucleic acid which is inducible, error free, and uvr dependent

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

Science.gov (United States)

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.

Mutagenesis by alkylating agents: coding properties for DNA polymerase of poly (dC) template containing 3-methylcytosine

Energy Technology Data Exchange (ETDEWEB)

Boiteux, S.; Laval, J. (Institut Gustave-Roussy, 94 - Villejuif (France))

After treatment of poly(dC) by the simple alkylating agent (/sup 3/H)dimethylsulfate, 90 percent of the radioactivity cochromatographed with 3-methylcytosine and 10 percent with 5-methylcytosine which is the normally occurring methylated base. In order to study the influence of 3-methylcytosine on DNA replication, untreated and MDS-treated poly(dC) were used as templates for E. coli DNA polymerase I. The alkylation of poly(dC) inhibits DNA chain elongation, and does not induce any mispairing under high fidelity conditions. The alteration of DNA polymerase I fidelity by manganese ions allows some replication of 3-methylcytosine which mispairs with either dAMP or dTMP. Our results suggest that 3-methylcytosine could be responsible, at least partially, for killing and the mutagenesis observed after cell treatment by alkylating agents.

ENU mutagenesis to generate genetically modified rat models.

Science.gov (United States)

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.

Radiation-induced base substitution mutagenesis in single-stranded DNA phage M13

International Nuclear Information System (INIS)

Brandenburger, A.; Godson, G.N.; Glickman, B.W.; Sluis, C.A. van

1981-01-01

To elucidate the relative contributions of targeted and untargeted mutations to γ and UV radiation mutagenesis, the DNA sequences of 174 M13 revertant phages isolated from stocks of irradiated or unirradiated amber mutants grown in irradiated (SOS-induced) or unirradiated (non-induced) host bacteria, have been determined. Differences in the spectra of base change mutations induced in the various conditions were apparent, but no obvious specificity of mutagenesis was detected. In particular, under the present conditions, pyrimidine dimers did not seem to be the principal sites of UV-induced base substitution mutagenesis, suggesting that such mutagenesis occurs at the sites of lesions other than pyrimidine dimers, or is untargeted. (U.K.)

Genetic modifications of established varieties of potato through mutagenesis

International Nuclear Information System (INIS)

Brown, C.R.

1984-01-01

Owing to the high intercrossability of improved clones with primitive cultivars and many wild species there is little justification for use of induced mutations in potato to increase variability per se. Modification of certain traits while leaving the genotype basically intact is a promising use of mutagenesis in potato. The successful curing of defects in clones will depend on the establishment a priori of three principles. First, the clones undergoing mutagenesis should be well established varieties tolerant or resistant to the major biotic and abiotic stresses in the area of cultivation. The yield and culinary quality should also be considered high. Second, there should exist some indication that the variation desired is induceable, either through reports of natural intra-clone variation or previous mutagenesis studies. Third, initial screening should be done in virus-free materials

Mutagenicity and co-mutagenicity of static magnetic field in SOD-deficient Escherichia coli

International Nuclear Information System (INIS)

Yoshie, Sachiko; Ikehata, Masateru; Hayakawa, Toshio; Hirota, Noriyuki; Takemura, Taro; Minowa, Takashi; Hanagata, Nobutaka

2008-01-01

The effects of strong static magnetic fields (SMFs) on mutagenesis related to reactive oxygen species were investigated. To estimate mutagenicity of SMFs, superoxide dismutase (SOD)-deficient Escherichia coli QC774 and its parental strain GC4468 were employed. Tester strains were exposed to 5, 10 and 13 T SMFs for 24 hr at 37 C degrees in LB medium. After exposure, mutation frequency on thymine synthesis genes was determined for evaluation of mutagenicity of SMFs exposure. In the result, no statistically significant difference in mutation frequency on thymine synthesis genes was observed between SMF-exposed cells and unexposed cells in all of magnetic flux densities. Furthermore, SMFs up to 13 T did not affect mutagenicity of plumbagine under its presence of 25 μM, respectively. It suggests that SMF did not have either mutagenicity or co-mutagenicity in SOD-deficient and its parental E. coli strains under the condition in this study. (author)

Direct Mutagenesis of Thousands of Genomic Targets using Microarray-derived Oligonucleotides

DEFF Research Database (Denmark)

Bonde, Mads; Kosuri, Sriram; Genee, Hans Jasper

2015-01-01

Multiplex Automated Genome Engineering (MAGE) allows simultaneous mutagenesis of multiple target sites in bacterial genomes using short oligonucleotides. However, large-scale mutagenesis requires hundreds to thousands of unique oligos, which are costly to synthesize and impossible to scale-up by ...

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

Science.gov (United States)

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

2016-01-01

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

A Simple Combinatorial Codon Mutagenesis Method for Targeted Protein Engineering.

Science.gov (United States)

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

2017-03-17

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

CRISPR/Cas9 mediates efficient conditional mutagenesis in Drosophila.

Science.gov (United States)

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

2014-09-05

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

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

Energy Technology Data Exchange (ETDEWEB)

Demple, Bruce [Stony Brook Univ., NY (United States). School of Medicine

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.

Implication of the E. coli K12 uvrA and recA genes in the repair of 8-methoxypsoralen-induced mono adducts and crosslinks on plasmid DNA; Implicacion de los genes uvrA de E. coli K12 en la reparacion de monoaductos y entrecruzamien tos inducidos en DNA plasmidico por 8-metoxipso raleno mas luz ultravioleta A

Energy Technology Data Exchange (ETDEWEB)

Paramio, J M; Bauluz, C; Vidania, R de

1986-07-01

Genotoxicity of psoralen damages on plasmid DNA has been studied. pBR322 DNA was randomly modified with several concentrations of 8-methoxypsoralen plus 365 nm-UV light. After transformation into E. coli strains (wild-type, uvrA and recA) plasmid survival and mutagenesis were analyzed. To study the influence of the SOS response on plasmid recovery, preirradiation of the cells was performed. In absence of cell preirradiation, crosslinks were not repaired in any strain. Mono adducts were also lethal but in part removed by the excision-repair pathway. Preirradiation of the cells significantly. increased plasmid recovery in recA+ celia. In uvrA- only the mutagenic pathway seemed to be involved in the repair of the damaged DNA. Wild type strain showed the highest increase in plasmid survival, involving the repair of mono adducts and some fraction of crosslinks mainly through an error-free repair pathway. This suggests an enhancement of the excision repair promoted by the induction of SOS functions. (Author) 32 refs.

Effective lethal mutagenesis of influenza virus by three nucleoside analogs.

Science.gov (United States)

Pauly, Matthew D; Lauring, Adam S

2015-04-01

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

Ultraviolet radiation-induced mutability of isogenic uvrA and uvrB strains of Escherichia coli K-12 W3110

International Nuclear Information System (INIS)

Barfknecht, T.R.; Smith, K.C.

1977-01-01

E. coli K-12 W3110 uvrB5 strain has been shown to have a higher UV induced reversion frequency than its wild-type parent when plotted on the basis of mutation frequency versus survival. However for the E. coli B/r WP2s uvrA strain this higher mutability has been observed only at survival levels of 80-100%. A study was undertaken to determine if these differences in UV mutability were due primarily to the uvrA and uvrB mutations, or to other genetic background differences. Isogenic strains of E. coli K-12 W3110 carrying uvrA6, uvrB5, uvrA6 and uvrB5, and the uvrA allele from E.coli B/r WP2s were used. Results indicate that the enrichment of minimal medium with a small amount of nutrient broth is sufficient to inhibit minimal medium recovery (MMR) and to enhance leu + reversion of the leu B missense mutation in these uvr - strains. This suggests that there may be a relationship between MMR and error-free postreplication repair. Further research is in progress to clarify the relationship between MMR and broth enhancement of UV-induced mutagenesis in uvr - strains of E. Coli K-12 W3110. (author)

Ultraviolet radiation-induced mutability of isogenic uvrA and uvrB strains of Escherichia coli K-12 W3110

Energy Technology Data Exchange (ETDEWEB)

Barfknecht, T R; Smith, K C [Stanford Univ., Calif. (USA). Dept. of Radiology

1977-12-01

Escherichia coli K-12 W3110 uvrB5 strain has been shown to have a higher uv-induced reversion frequency than its wild-type parent when plotted on the basis of mutation frequency versus survival. However for the E. coli B/r WP2s uvrA strain this higher mutability has been observed only at survival levels of 80 to 100%. A study was undertaken to determine if ly to the uvrA and uvrB mutations, or to other genetic background differences. Isogenic strains of E. coli K-12 W3110 carrying uvrA6, uvrB5, uvrA6, and uvrB5, and the uvrA allele from E.coli B/r WP2s were used. Results indicate that the enrichment of minimal medium with a small amount of nutrient broth is sufficient to inhibit minimal medium recovery (MMR) and to enhance leu/sup +/ reversion of the leu B missense mutation in these uvr/sup -/ strains. This suggests that there may be a relationship between MMR and error-free postreplication repair. Further research is in progress to clarify the relationship between MMR and broth enhancement of uv-induced mutagenesis in uvr/sup -/ strains of E. Coli K-12 W3110.

Suppression of radiation mutagenesis by dactinomycin in Chinese hamster cells

International Nuclear Information System (INIS)

Tokita, N.; Capenter, S.G.; Chen, D.J.; MacInnes, M.A.; Raju, M.R.

1985-01-01

Dactinomycin (AMD) suppression of radiation mutagenesis was investigated using an in vitro mutation assay (6-thioguanine resistance) in Chinese hamster ovary cells. Cells were exposed to acute single doses of x rays followed by 1 hr-treatment with 0.1 or 1 μg/ml AMD. The cell survival curves plotted as a function of x-ray doses were similar for radiation alone and radiation plus AMD. The results suggest that AMD treatment was only slightly mutagenic, however, when given immediately after irradiation, it suppressed radiatiion mutagenesis at higher x-ray dose regions (below 10% survival levels). Higher AMD concentrations appeared more suppressive than lower concentrations. Dose-response data analyzed based on Poisson distribution models suggest the stochastic dependence of x-ray mutagenesis and AMD cytotoxity

Phage annealing proteins promote oligonucleotide-directed mutagenesis in Escherichia coli and mouse ES cells

Directory of Open Access Journals (Sweden)

Muyrers Joep PP

2003-01-01

Full Text Available Abstract Background The phage protein pairs, RecE/RecT from Rac or Redα/Redβ from λ, initiate efficient double strand break repair (DSBR in Escherichia coli that has proven very useful for DNA engineering. These phage pairs initiate DSBR either by annealing or by another mechanism that is not defined. Results Here we report that these proteins also mediate single strand oligonucleotide repair (ssOR at high efficiencies. The ssOR activity, unlike DSBR, does not require a phage exonuclease (RecE or Redα but only requires a phage annealing protein (RecT or Redβ. Notably, the P22 phage annealing protein Erf, which does not mediate the same DSBR reactions, also delivers ssOR activity. By altering aspects of the oligonucleotides, we document length and design parameters that affect ssOR efficiency to show a simple relationship to homologies either side of the repair site. Notably, ssOR shows strand bias. Oligonucleotides that can prime lagging strand replication deliver more ssOR than their leading complements. This suggests a model in which the annealing proteins hybridize the oligonucleotides to single stranded regions near the replication fork. We also show that ssOR is a highly efficient way to engineer BACs and can be detected in a eukaryotic cell upon expression of a phage annealing protein. Conclusion Phage annealing proteins can initiate the recombination of single stranded oligonucleotides into endogenous targets in Escherichia coli at very high efficiencies. This expands the repertoire of useful DNA engineering strategies, shows promise for applications in eukaryotic cells, and has implications for the unanswered questions regarding DSBR mediated by RecE/RecT and Redα/Redβ.

Radiation biophysical study of biological molecules. Progress report, July 1, 1976--August 31, 1977

International Nuclear Information System (INIS)

Fluke, D.J.

1977-01-01

Progress is reported on the following research projects: x-ray induction of uv mutagenesis enhancement in lambda-phage; action spectrum for uv mutagenesis in Escherichia coli; survival of E. coli colonies after uv damage; repair of radiation damage to lambda-phage by the W-reactivation system; experiments on the Weigle-reactivation of irradiated lambda-phage; and studies on the wavelength dependence of uv mutagenesis

Faecal Escherichia coli from patients with E. coli urinary tract infection and healthy controls who have never had a urinary tract infection

DEFF Research Database (Denmark)

Nielsen, Karen L; Dynesen, Pia; Larsen, Preben

2014-01-01

Urinary tract infections (UTIs) are primarily caused by Escherichia coli with the patient's own faecal flora acting as a reservoir for the infecting E. coli. Here we sought to characterize the E. coli faecal flora of UTI patients and healthy controls who had never had a UTI. Up to 20 E. coli...... colonies from each rectal swab were random amplified polymorphic DNA (RAPD) typed for clonality, dominance in the sample and correlation to the infecting UTI isolate in patients. Each distinct clone was phylotyped and tested for antimicrobial susceptibility. Eighty-seven per cent of the UTI patients...... carried the infecting strain in their faecal flora, and faecal clones causing UTI were more often dominant in the faecal flora. Patients had a larger diversity of E. coli in their gut flora by carrying more unique E. coli clones compared to controls, and patient faecal clones were more often associated...

Overexpression of SOS genes in ciprofloxacin resistant Escherichia coli mutants.

Science.gov (United States)

Pourahmad Jaktaji, Razieh; Pasand, Shirin

2016-01-15

Fluoroquinolones are important antibiotics for the treatment of urinary tract infections caused by Escherichia coli. Mutational studies have shown that ciprofloxacin, a member of fluoroquinolones induces SOS response and mutagenesis in pathogenic bacteria which in turn develop antibiotic resistance. However, inhibition of SOS response can increase recombination activity which in turn leads to genetic variation. The aim of this study was to measure 5 SOS genes expressions in nine E. coli mutants with different MICs for ciprofloxacin following exposure to ciprofloxacin. Gene expression was assessed by quantitative real time PCR. Gene alteration assessment was conducted by PCR amplification and DNA sequencing. Results showed that the expression of recA was increased in 5 mutants. This overexpression is not related to gene alteration, and enhances the expression of polB and umuCD genes encoding nonmutagenic and mutagenic polymerases, respectively. The direct relationship between the level of SOS expression and the level of resistance to ciprofloxacin was also indicated. It was concluded that novel therapeutic strategy that inhibits RecA activity would enhance the efficiency of common antibiotics against pathogenic bacteria. Copyright © 2015 Elsevier B.V. All rights reserved.

Implication of the E. coli K12 uvrA and recA genes in the repair of 8-methoxypsoralen-induced mono adducts and crosslinks on plasmid DNA

International Nuclear Information System (INIS)

Paramio, J.M.; Bauluz, C.; Vidania, R. de

1986-01-01

Genotoxicity of psoralen damages on plasmid DNA has been studied. pBR322 DNA was randomly modified with several concentrations of 8-methoxypsoralen plus 365 nm-UV light. After transformation into E. coli strains (wild-type, uvrA and recA) plasmid survival and mutagenesis were analyzed. To study the influence of the SOS response on plasmid recovery, preirradiation of the cells was performed. In absence of cell preirradiation, crosslinks were not repaired in any strain. Mono adducts were also lethal but in part removed by the excision-repair pathway. Preirradiation of the cells significantly. increased plasmid recovery in recA+ celia. In uvrA- only the mutagenic pathway seemed to be involved in the repair of the damaged DNA. Wild type strain showed the highest increase in plasmid survival, involving the repair of mono adducts and some fraction of crosslinks mainly through an error-free repair pathway. This suggests an enhancement of the excision repair promoted by the induction of SOS functions. (Author) 32 refs

Engineering the biosynthesis of novel rhamnolipids in Escherichia coli for enhanced oil recovery.

Science.gov (United States)

Han, L; Liu, P; Peng, Y; Lin, J; Wang, Q; Ma, Y

2014-07-01

The interfacial tension of rhamnolipids and their applications in enhanced oil recovery are dependent on their chemical structures and compositions. To improve their performances of interfacial tension and enhanced oil recovery, the engineered strategies were applied to produce novel rhamnolipids with different chemical structures and compositions. By introducing different key genes for rhamnolipid biosynthesis, Escherichia coli was firstly constructed to produce rhamnolipids that showed different performances in interfacial tension from those from Pseudomonas aeruginosa due to the different fatty acyl compositions. Then, the mutant RhlBs were created by directed evolution and subsequent site-directed mutagenesis and resulted in the production of the novel rhamnolipids with the different performances in interfacial tension as well as enhanced oil recovery. Lastly, computational modelling elucidates that the single amino acid mutation at the position 168 in RhlB would change the volume of binding pocket for substrate and thus affect the selectivity of rhamnolipid formation in E. coli. The novel rhamnolipids that showed the improved performances of interfacial tension and the potential different applications in enhanced oil recovery were successfully produced by engineered E. coli. This study proved that the combination of metabolic engineering and protein engineering is an important engineered strategy to produce many novel metabolites in micro-organisms. © 2014 The Society for Applied Microbiology.

The Origin of Mutants Under Selection: How Natural Selection Mimics Mutagenesis (Adaptive Mutation)

Science.gov (United States)

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

Characterization of new radiation-sensitive mutant, Escherichia coli K-12 radC102

International Nuclear Information System (INIS)

Felzenszwalb, I.; Sargentini, N.J.; Smith, K.C.

1984-01-01

A new radiation-sensitive mutant, radC, has been isolated. The radC gene is located at 81.0 min on the Escherichia coli K-12 linkage map. The radC mutation sensitized cells to uv radiation, but unlike most DNA repair mutations, sensitization to X rays was observed only for rich medium-grown cells. For cells grown in rich medium, the radC mutant was normal for γ radiation mutagenesis, but showed less uv-radiation mutagenesis than the wild-type strain; it showed normal amount of X- and uv-radiation-induced DNA degradation, and it wasapprox. =60% deficient in recombination ability. The radC strain was normal for host cell reactivation of γ and uv-irradiated bacteriophage the radC mutation did not sensitize a recA strain, but did sensitize a radA and a polA strain to X and uv radiation and a uvrA strain to uv radiation. Therefore, it is suggested that the radC gene product plays a role in the growth medium-dependent, recA gene-dependent repair of DNA single-strand breaks after X irradiation, and in postreplication repair after uv irradiation

Development of a counterselectable seamless mutagenesis system in lactic acid bacteria.

Science.gov (United States)

Xin, Yongping; Guo, Tingting; Mu, Yingli; Kong, Jian

2017-07-05

Lactic acid bacteria (LAB) are receiving more attention to act as cell factories for the production of high-value metabolites. However, the molecular tools for genetic modifying these strains are mainly vector-based double-crossover strategies, which are laborious and inefficient. To address this problem, several counterselectable markers have been developed, while few of them could be used in the wild-type host cells without pretreatment. The pheS gene encoding phenylalanyl-tRNA synthetase alpha subunit was identified in Lactococcus lactis NZ9000 genome. When mutant pheS gene (pheS*) under the control of the Lc. lactis NZ9000 L-lactate dehydrogenase promoter (P ldh ) was expressed from a plasmid, the resulted PheS* with an A312G substitution rendered cells sensitive to the phenylalanine analog p-chloro-phenylalanine (p-Cl-Phe). This result suggested pheS* was suitable to be used as a counterselectable marker in Lc. lactis. However, the expression level of pheS* from a chromosomal copy was too low to confer p-Cl-Phe sensitivity. Therefore, a strategy of cascading promoters was attempted for strengthening the expression level of pheS*. Expectedly, a cassette 5Pldh-pheS* with five tandem repetitive promoters P ldh resulted in a sensitivity to 15 mM p-Cl-Phe. Subsequently, a counterselectable seamless mutagenesis system PheS*/pG + host9 based on a temperature-sensitive plasmid pG + host9 harboring a 5Pldh-pheS* cassette was developed in Lc. lactis. We also demonstrated the possibility of applying pheS* to be a counterselectable marker in Lactobacillus casei BL23. As reported in E. coli, pheS* as a counterselectable marker has been demonstrated to be functional in targeted gene(s) deletion in Lc. lactis as well as in L. casei. Moreover, the efficiency and timesaving counterselectable seamless mutagenesis system PheS*/pG + host9 could be used in the wild-type host cells without pretreatment.

Back to the future: revisiting HIV-1 lethal mutagenesis

Science.gov (United States)

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

2012-01-01

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

Complex epidemiological approach to human mutagenesis

International Nuclear Information System (INIS)

Czeizel, A.

1980-01-01

The main characteristics of the epidemiological approach are summarised and the criteria discussed for the adoption of this approach for the detection of human mutagenesis. Mutation monitoring systems are described and results of epidemiological studies of higher risk populations are presented. (C.F.)

Genetic improvement of soybean through induced mutagenesis

International Nuclear Information System (INIS)

Manjaya, J.G.; Nandanwar, R.S.; Thengane, R.J.; Muthiah, A.R.

2009-01-01

Soybean (Glycine max (L.) Merril) is one of the important oilseed crops of India. The country produces more than 9.00 million tonnes of soybean per annum and has acquired first place amongst oilseed crops grown in India. Narrow genetic base of cultivated varieties in soybean is of global concern. Efficient mutant production systems, through physical or chemical mutagenesis, have been well established in soybean. A vast amount of genetic variability, of both quantitative and qualitative traits, has been generated through experimental mutagenesis. Two soybean varieties TAMS-38 and TAMS 98-21 have been developed and released for commercial cultivation by Bhabha Atomic Research Centre (BARC). In this paper the role of mutation breeding in soybean improvement has been discussed. (author)

Lethal mutagenesis: targeting the mutator phenotype in cancer.

Science.gov (United States)

Fox, Edward J; Loeb, Lawrence A

2010-10-01

The evolution of cancer and RNA viruses share many similarities. Both exploit high levels of genotypic diversity to enable extensive phenotypic plasticity and thereby facilitate rapid adaptation. In order to accumulate large numbers of mutations, we have proposed that cancers express a mutator phenotype. Similar to cancer cells, many viral populations, by replicating their genomes with low fidelity, carry a substantial mutational load. As high levels of mutation are potentially deleterious, the viral mutation frequency is thresholded at a level below which viral populations equilibrate in a traditional mutation-selection balance, and above which the population is no longer viable, i.e., the population undergoes an error catastrophe. Because their mutation frequencies are fine-tuned just below this error threshold, viral populations are susceptible to further increases in mutational load and, recently this phenomenon has been exploited therapeutically by a concept that has been termed lethal mutagenesis. Here we review the application of lethal mutagenesis to the treatment of HIV and discuss how lethal mutagenesis may represent a novel therapeutic approach for the treatment of solid cancers. Copyright © 2010 Elsevier Ltd. All rights reserved.

A function of mutagenesis on rhodotorula RY strain irradiated by heavy ion

International Nuclear Information System (INIS)

Li Hongyu; Li Chenghua; Ding Xinchun; Wang Jufang; Zhou Guangming; Xie Hongmei; Li Qiang; Dang bingrong; Wen Xiaoqiong; Li Wenjian; Wei Zengquan

2004-01-01

In this paper, red yeast (Rhodotorula RY Strain) that produces carotene is irradiated by 50 MeV/u 12 C 6+ heavy ion from Heavy Ion Accelerator in IMP. Fermentation tests show that 50 MeV/u 12 C 6+ heavy ion has a mutagenesis effect on the red yeast. Some strains of red yeast with changed production of carotene were found by screening. Meanwhile, by RFLP and RAPD analysis, authors have a further evidence that heavy ion can cause mutagenesis in Rhodotorula RY Strain. This presents a new prospect for the mutagenesis breeding by heavy ion in industry

Efficacy of Probiotic Escherichia coli Nissle 1917 in Patients with Irritable Bowel Syndrome: a Double Blind Placebocontrolled Randomized Trial

Directory of Open Access Journals (Sweden)

Amir H Faghihi

2015-07-01

Full Text Available Aim: to evaluate potential improvement effect for probiotic E. coliNissle 1917 in the management of refractory IBS in an Iranian population. Methods: a double blind placebo controlled approach as been used in the current clinical trial. 139 confirmed IBS patients were included into the study, and were given probiotic E.coli Nissle 1917 for 6 weeks. 11 items Birmingham IBS Symptom Questionnairehas been used for evaluation of changes in the symptoms every 2 weeks. Results: sixty eight subjects (49% were males. Mean±SD age of the participants was 38±13.3 years. 49(35.3% of the patients were diarrhea-predominant. The total scores showed no significant difference between the intervention vs. control group(-6.7±6.8 vs. -6.7±6.5, respectively; p=0.95; neither did any of the questionnaire items any significant alterations in the two groups. After stratification of patients based on their IBS type, diarrhea-predominant patients showed a positive response to the probiotic improving their sleep (p=0.05&0.03 at weeks 2&6, respectively. Patients with constipation-predominant IBS showed no response to the probiotic; while patients with diarrhea-constipation mixed IBS showed unfavorable response to the probiotic in the need for strain to pass a motion compared to the placebo (p=0.03&0.02 at weeks 4&6, respectively. Conclusion: probiotic therapy with E.coliNissle 1917 was not able to induce significant improvement in the symptoms of patients with non-categorized IBS. Nevertheless, when IBS patients were recategorized to subgroups according to their main symptoms, evaluation of the efficacy of the probiotic on some individual items in the symptom list reached the significance level. Prospective clinical trials are recommended to confirm our findings. Key words: probiotic Escherichia Coli Nissle 1917, irritable bowel syndrome, double blind randomized controlled trial.

Mutagenesis of Trichoderma Viride by Ultraviolet and Plasma

International Nuclear Information System (INIS)

Yao Risheng; Li Manman; Deng Shengsong; Hu Huajia; Wang Huai; Li Fenghe

2012-01-01

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.

Mutagenesis of Trichoderma Viride by Ultraviolet and Plasma

Science.gov (United States)

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.

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

Science.gov (United States)

Lee, Raymond Teck Ho; 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.

DNA base sequence changes induced by ultraviolet light mutagenesis of a gene on a chromosome in Chinese hamster ovary cells

Energy Technology Data Exchange (ETDEWEB)

Romac, S; Leong, P; Sockett, H; Hutchinson, F [Yale Univ., New Haven, CT (USA). Dept. of Molecular Biophysics and Biochemistry

1989-09-20

The DNA base sequence changes induced by mutagenesis with ultraviolet light have been determined in a gene on a chromosome of cultured Chinese hamster ovary (CHO) cells. The gene was the Excherichia coli gpt gene, of which a single copy was stably incorporated and expressed in the CHO cell genome. The cells were irradiated with ultraviolet light and gpt{sup -} colonies were selected by resistance to 6-thioguanine. The gpt gene was amplified from chromosomal DNA by use of the polymerase chain reaction (PCR) and the amplified DNA sequenced directly by the dideoxy method. Of the 58 sequenced mutants of independent origin 53 were base change mutations. Forty-one base substitutions were single base changes, ten had two adjacent (or tandem) base changes, and one had two base changes separated by a single base-pair. Only one mutant had a multiple base change mutation with two or more well separated base changes. In contrast much higher levels of such mutations were reported in ultraviolet mutagenesis of genes on a shuttle vector in primate cells. Two deletions of a single base-pair were observed and three deletions ranging from 6 to 37 base-pairs. The mutation spectrum in the gpt gene had similarities to the ultraviolet mutation spectra for several genes in prokaryotes, which suggests similarities in mutational mechanisms in prokaryotes and eukaryotes. (author).

Development of a high-frequency in vivo transposon mutagenesis system for Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942.

Science.gov (United States)

Watabe, Kazuyuki; Mimuro, Mamoru; Tsuchiya, Tohru

2014-11-01

Synechocystis sp. PCC 6803 (Synechocystis) is the first sequenced photosynthetic organism and has two advantages: natural transformation and light-activated heterotrophic growth. Such characteristics have mainly promoted reverse genetic analysis in this organism, however, to date approximately 50% of genes are still annotated as 'unknown protein' or 'hypothetical protein'. Therefore, forward genetic analysis is required for the identification of significant genes responsible for photosynthesis and other physiological phenomena among the genes of unknown function. The in vivo transposon mutagenesis system is one of the major methods for random mutagenesis. However, present in vivo transposon mutagenesis systems for cyanobacteria face problems such as relatively low frequency of transposition and repeated transposition in the host cells. In this study, we constructed vectors based on a mini-Tn5-derived vector that was designed to prevent repeated transposition. Our vectors carry a hyperactive transposase and optimized recognition sequence of transposase, which were reported to enhance frequency of transposition. Using the vector, we succeeded in highly frequent transposition (9×10(-3) per recipient cell) in Synechocystis. Transposon insertion sites of 10 randomly selected mutants indicated that the insertion sites spread throughout the genome with low sequence dependency. Furthermore, one of the 10 mutants exhibited the slow-growing phenotype, and the mutant was functionally complemented by using our expression vector. Our system also worked with another model cyanobacterium, Synechococcus elongatus PCC 7942, with high frequency. These results indicate that the developed system can be applied to the forward genetic analysis of a broad range of cyanobacteria. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Blocking by the carcinogen, L-ethionine, of SOS functions in a tif-1 mutant of Escherichia coli B/r

International Nuclear Information System (INIS)

Wiesner, R.; Troll, W.

1981-01-01

In Escherichia coli, DNA damage by carcinogenic agents results in the coordinate expression of a diversity of functions (SOS functions), many of which are thermally inducible without any damage to DNA in a tif-1 mutant. These include prophage induction, filamentous growth, and an error-prone DNA repair activity, which is responsible for ultraviolet-induced mutagenesis. Ethionine causes hepatic carcinoma in rats after prolonged feeding but is not a mutagen in the Ames test. The present study shows that 10 mM ethionine prevents the thermal induction of lambda-prophage in a tif-1 derivative of E. coli. The enhancement of mutation, which normally occurs at high temperature after a low dose of ultraviolet light, is also blocked by ethionine. Ethionine does not block, to any appreciable extent, the incorporation of radioactive precursors into RNA, DNA, or protein

Target-selected mutagenesis of the rat

NARCIS (Netherlands)

Smits, B.M.; Mudde, J.B.; Plasterk, R.; Cuppen, E.

2004-01-01

The rat is one of the most extensively studied model organisms, and with its genome being sequenced, tools to manipulate gene function in vivo have become increasingly important. We here report proof of principle for target-selected mutagenesis as a reverse genetic or knockout approach for the rat.

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

Science.gov (United States)

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

2016-09-01

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

Antibiotic stress-induced modulation of the endoribonucleolytic activity of RNase III and RNase G confers resistance to aminoglycoside antibiotics in Escherichia coli.

Science.gov (United States)

Song, Wooseok; Kim, Yong-Hak; Sim, Se-Hoon; Hwang, Soonhye; Lee, Jung-Hyun; Lee, Younghoon; Bae, Jeehyeon; Hwang, Jihwan; Lee, Kangseok

2014-04-01

Here, we report a resistance mechanism that is induced through the modulation of 16S ribosomal RNA (rRNA) processing on the exposure of Escherichia coli cells to aminoglycoside antibiotics. We observed decreased expression levels of RNase G associated with increased RNase III activity on rng mRNA in a subgroup of E. coli isolates that transiently acquired resistance to low levels of kanamycin or streptomycin. Analyses of 16S rRNA from the aminoglycoside-resistant E. coli cells, in addition to mutagenesis studies, demonstrated that the accumulation of 16S rRNA precursors containing 3-8 extra nucleotides at the 5' terminus, which results from incomplete processing by RNase G, is responsible for the observed aminoglycoside resistance. Chemical protection, mass spectrometry analysis and cell-free translation assays revealed that the ribosomes from rng-deleted E. coli have decreased binding capacity for, and diminished sensitivity to, streptomycin and neomycin, compared with wild-type cells. It was observed that the deletion of rng had similar effects in Salmonella enterica serovar Typhimurium strain SL1344. Our findings suggest that modulation of the endoribonucleolytic activity of RNase III and RNase G constitutes a previously uncharacterized regulatory pathway for adaptive resistance in E. coli and related gram-negative bacteria to aminoglycoside antibiotics.

Persistence and decay of thermoinducible error-prone repair activity in nonfilamentous derivatives of tif-1 Escherichia coli B/r

International Nuclear Information System (INIS)

Witkin, E.M.

1975-01-01

Ultraviolet mutagenesis in E. coli is associated with a UV-inducible type of error-prone postreplication repair ('SOS' repair) which, in tif-1 strains, is thermoinducible in coordination with other recA + lexA + -dependent inducible functions, including filamentous growth. Mutants of E. coli B/r tif-1 strains have been isolated which retain thermoinducibility of SOS repair activity, but lack the thermosensitivity caused by filamentous growth at 42 0 C. These strains have been used to determine the kinetics of decay at 30 0 C of thermally induced ability to enhance UV mutagenesis, the kinetics of thermal enhancement of spontaneous and UV-induced mutability at 42 0 C, and the kinetics of decay at 30 0 C of susceptibility to thermal enhancement of spontaneous and UV-induced mutability. Mutations from tryptophane requirement to prototrophy (Trp - to Trp + ) were scored. UV doses were 0.2 J/m 2 for excision repair-deficient (Uvr - ) and 2 J/m 2 for Uvr + strains. The results support the following conclusions. Thermally induced SOS repair activity decays at 30 0 C to about 25% of its maximum level in 45 min, and is no longer detectable after 90 min. Thermal enhancement of UV mutability occurs at sites produced primarily (perhaps exclusively) before completion of the first post-irradiation cell division. UV-induced sites susceptible to thermally induced SOS repair are stable at 30 0 C in cells not containing the error-prone repair system, and are refractory to constitutive error-free repair for at least 2 - 3 hours. UV produces a potentially mutagenic type of photoproduct in DNA which can, without interacting with another UV lesion, provide a site susceptible to SOS repair, but which is not a sufficient signal for SOS induction. 50 - 70% of the SOS-mutable SOS-noninducing UV photoproducts are photoreversible pyrimidine dimers. The results are discussed in relation to current models of UV mutagenesis and induction of UV-inducible functions. (orig.) [de

The use of plant tissue culture system in the mutagenesis of Secale cereale L

International Nuclear Information System (INIS)

Rybczynski, J.J.; KozIowska, W.; Turzynski, D.

1990-01-01

Full text: Among cereals, Secale cereale L. is the worst species for 'in vitro' mutagenesis. In the case of seed mutagenesis of rye each seed is expected to be a different genotype and only somatic embryogenesis assures propagation towards numerous individuals possessing the same genotype. Therefore, another system of in-vitro mutagenesis is explored. Immature embryos were isolated from spikes of field growing plants. The established cultures were irradiated with 0.5; 1.0 and 1.5 kR gamma rays on the first day of the culture and after 6 weeks in culture. After irradiation all cultures were subcultured. For mutagenesis in general uniformity of the original material is very important. Therefore, in rye, irradiation of regenerated somatic embryos may be a good approach. (author)

Comparative study of SOS response induced by hydrogen peroxide in the absence or presence of iron ions, in Escherichia coli

International Nuclear Information System (INIS)

Almeida, Carlos Eduardo Bonacossa de

1994-01-01

The H 2 O 2 is an reactive oxygen specie that arises from cell respiration process. It may cause deleterious effects on cell, by reacting with transition metals like iron. In this way it yields free radicals that are able to damage organic molecules, mainly DNA. Recent works have suggested that in the absence of Fe ions H 2 O 2 still damages Escherichia coli DNA. This work presents a comparative analysis of cell SOS responses to DNA damage in Escherichia coli and Salmonella typhimurium mutants pretreated or not with a Fe 2+ ion chelator (dipyridyl) and then treated with H 2 O 2 . The systems analysed were the lysogenic induction, Weigle reactivation, mutagenesis and cell inactivation curves. The cell inactivation curves were themselves distinct, in relation to both treatments. The increased sensitivity found in the lexA1 and recA13 mutants, when treated with dipyridyl and H 2 O 2 , suggests an important role of SOS response in repairing the lesions caused by this treatment. The profiles of the lysogenic induction and mutagenesis curves were also distinct in both treatments. The results of Weigle reactivation suggest that the products of uvrA and lexA genes have an important role in UV-damaged bacteriophage DNA repair, when dipyridyl-pretreated cells are treated with H 2 O 2 . All the results suggest that Fe-independent lesions produced by H 2 O 2 are different from the ones produced in the presence of this ion. (author)

Comparative study of SOS response induced by hydrogen peroxide in the absence or presence of iron ions, in Escherichia coli; Estudo comparativo da resposta SOS induzida pelo peroxido de hidrogenio em presenca e ausencia de ions ferro, em Escherichia coli

Energy Technology Data Exchange (ETDEWEB)

Almeida, Carlos Eduardo Bonacossa de

1994-07-01

The H{sub 2}O{sub 2} is an reactive oxygen specie that arises from cell respiration process. It may cause deleterious effects on cell, by reacting with transition metals like iron. In this way it yields free radicals that are able to damage organic molecules, mainly DNA. Recent works have suggested that in the absence of Fe ions H{sub 2}O{sub 2} still damages Escherichia coli DNA. This work presents a comparative analysis of cell SOS responses to DNA damage in Escherichia coli and Salmonella typhimurium mutants pretreated or not with a Fe{sup 2+} ion chelator (dipyridyl) and then treated with H{sub 2}O{sub 2}. The systems analysed were the lysogenic induction, Weigle reactivation, mutagenesis and cell inactivation curves. The cell inactivation curves were themselves distinct, in relation to both treatments. The increased sensitivity found in the lexA1 and recA13 mutants, when treated with dipyridyl and H{sub 2}O{sub 2}, suggests an important role of SOS response in repairing the lesions caused by this treatment. The profiles of the lysogenic induction and mutagenesis curves were also distinct in both treatments. The results of Weigle reactivation suggest that the products of uvrA and lexA genes have an important role in UV-damaged bacteriophage DNA repair, when dipyridyl-pretreated cells are treated with H{sub 2}O{sub 2}. All the results suggest that Fe-independent lesions produced by H{sub 2}O{sub 2} are different from the ones produced in the presence of this ion. (author)

Systems Biology-Based Investigation of Cellular Antiviral Drug Targets Identified by Gene-Trap Insertional Mutagenesis.

Directory of Open Access Journals (Sweden)

Feixiong Cheng

2016-09-01

Full Text Available Viruses require host cellular factors for successful replication. A comprehensive systems-level investigation of the virus-host interactome is critical for understanding the roles of host factors with the end goal of discovering new druggable antiviral targets. Gene-trap insertional mutagenesis is a high-throughput forward genetics approach to randomly disrupt (trap host genes and discover host genes that are essential for viral replication, but not for host cell survival. In this study, we used libraries of randomly mutagenized cells to discover cellular genes that are essential for the replication of 10 distinct cytotoxic mammalian viruses, 1 gram-negative bacterium, and 5 toxins. We herein reported 712 candidate cellular genes, characterizing distinct topological network and evolutionary signatures, and occupying central hubs in the human interactome. Cell cycle phase-specific network analysis showed that host cell cycle programs played critical roles during viral replication (e.g. MYC and TAF4 regulating G0/1 phase. Moreover, the viral perturbation of host cellular networks reflected disease etiology in that host genes (e.g. CTCF, RHOA, and CDKN1B identified were frequently essential and significantly associated with Mendelian and orphan diseases, or somatic mutations in cancer. Computational drug repositioning framework via incorporating drug-gene signatures from the Connectivity Map into the virus-host interactome identified 110 putative druggable antiviral targets and prioritized several existing drugs (e.g. ajmaline that may be potential for antiviral indication (e.g. anti-Ebola. In summary, this work provides a powerful methodology with a tight integration of gene-trap insertional mutagenesis testing and systems biology to identify new antiviral targets and drugs for the development of broadly acting and targeted clinical antiviral therapeutics.

Symposium on molecular and cellular mechanisms of mutagenesis

International Nuclear Information System (INIS)

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

Seed mutagenesis in Portulaca grandiflora (Hook)

International Nuclear Information System (INIS)

Bennani, F.; Rossi-Hassani, B.D.

2001-01-01

Betalain pigments have been used as natural additives. Despite their importance, the biochemistry and genetics of betalain synthesis remain relatively undetermined. Portulaca grandiflora represents an ideal material for genetic analysis. In the present work, seed mutagenesis was examined with a view to enhance the chance of detection of new genetic markers in this species

Symposium on molecular and cellular mechanisms of mutagenesis

Energy Technology Data Exchange (ETDEWEB)

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)

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

Science.gov (United States)

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

2015-06-01

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

Towards controlled mutagenesis with transposons Ac and Tam3

Energy Technology Data Exchange (ETDEWEB)

Haring, M; Veken, J; Windrich, R; Kneppers, T; Rommens, C; Nijkamp, H J.J.; Hille, J [Department of Genetics, Free University, Amsterdam (Netherlands)

1990-01-01

Full text: The discovery of mobile genetic elements in plants has permitted the use of these transposons for insertional mutagenesis. This applies so far only to Zea mays and Antirrhinum majus, because other plant transposable elements have not been characterised so thoroughly at the genetic and the molecular level. To establish whether transposons (Ac from maize and Tam3 from Antirrhinum) remain mobile in heterologous hosts, either in somatic tissue or after meiosis, a phenotypic assay system for transposition was developed. The separation of the two transposition functions will allow controlled mutagenesis of plant genes. Our results indicate that both transposable elements remain active in heterologous hosts. (author)

Tissue culture regeneration and radiation induced mutagenesis in banana

International Nuclear Information System (INIS)

Kulkarni, V.M.; Ganapathi, T.R.

2009-01-01

Radiation induced mutagenesis is an important tool for banana genetic improvement. At BARC, protocols for shoo-tip multiplication of commercial banana varieties have been developed and transferred to user agencies for commercial production. Excellent embryogenic cell suspensions were established in banana cvs. Rasthali and Rajeli, and were maintained at low temperatures for long-term storage. Normal plantlets were successfully regenerated from these cell suspensions. The cell suspensions and shoot-tip cultures were gamma-irradiated for mutagenesis. The mutagenized populations were field screened and a few interesting mutants have been isolated. The existence of genetic variation was confirmed using DNA markers. Further evaluation of these mutants is in progress. (author)

Identification of the Serratia marcescens hemolysin determinant by cloning into Escherichia coli

International Nuclear Information System (INIS)

Braun, V.; Neuss, B.; Ruan, Y.; Schiebel, E.; Schoeffler, H.; Jander, G.

1987-01-01

A cosmid bank of Serratia marcescens was established from which DNA fragments were cloned into the plasmid pBR322, which conferred the chromosomally encoded hemolytic activity to Escherichia coli K-12. By transposon mutagenesis with Tn1000 and Tn5 IS50/sub L/::phoA (TnphoA), the coding region was assigned to a DNA fragment, designated hyl, comprising approximately 7 kilobases. Two proteins with molecular weights of 61,000 (61K protein) and 160,000 (160K protein) were expressed by the pBR322 derivatives and by a plasmid which contained the hly genes under the control of a phage T7 promoter and the T7 RNA polymerase. When strongly overexpressed the 160K protein was released by E. coli cells into the extracellular medium concomitant with hemolytic activity. The genes encoding the 61K and the 160K proteins were transcribed in the same direction. Mutants expressing a 160K protein truncated at the carboxyl-terminal end were partially hemolytic. Hemolysis was progressively inhibited by saccharides with increasing molecular weights from maltotriose (M/sub r/ 504) to maltoheptaose (M/sub r/ 1152) and as totally abolished by dextran 4 (M/sub r/ 4000). This result and the observed influx of [ 14 C]sucrose into erythrocytes in the presence of hemolytic E. coli transformants under osmotically protective conditions suggest the formation of defined transmembrane channels by the hemolysin

Genetic and physiological factors affecting repair and mutagenesis in yeast

International Nuclear Information System (INIS)

Lemontt, J.F.

1979-01-01

Current views of DNA repair and mutagenesis in the yeast Saccharomyces cerevisiae are discussed in the light of recent data, and with emphasis on the isolation and characterization of genetically well-defined mutations that affect DNA metabolism in general (including replication and recombination). Various pathways of repair are described particularly in relation to their involvement in mutagenic mechanisms. In addition to genetic control, certain physiological factors such as cell age, DNA replication, and the regulatory state of the mating-type locus, are shown to also play a role in repair and mutagenesis

Genetic and physiological factors affecting repair and mutagenesis in yeast

International Nuclear Information System (INIS)

Lemontt, J.F.

1979-01-01

Current views of DNA repair and mutagenesis in the yeast Saccharomyces cerevisiae are discussed in the light of recent data and with emphasis on the isolation and characterization of genetically well-defined mutations that affect DNA metabolism in general (including replication and recombination). Various pathways of repair are described, particularly in relation to their imvolvement in mutagenic mechanisms. In addition to genetic control, certain physiological factors such as cell age, DNA replication, and the regulatory state of the mating-type locus are shown to also play a role in repair and mutagenesis

Genetic and physiological factors affecting repair and mutagenesis in yeast

Energy Technology Data Exchange (ETDEWEB)

Lemontt, J F

1979-01-01

Current views of DNA repair and mutagenesis in the yeast Saccharomyces cerevisiae are discussed in the light of recent data, and with emphasis on the isolation and characterization of genetically well-defined mutations that affect DNA metabolism in general (including replication and recombination). Various pathways of repair are described particularly in relation to their involvement in mutagenic mechanisms. In addition to genetic control, certain physiological factors such as cell age, DNA replication, and the regulatory state of the mating-type locus, are shown to also play a role in repair and mutagenesis.

Genetic and physiological factors affecting repair and mutagenesis in yeast

Energy Technology Data Exchange (ETDEWEB)

Lemontt, J F

1979-01-01

Current views of DNA repair and mutagenesis in the yeast Saccharomyces cerevisiae are discussed in the light of recent data and with emphasis on the isolation and characterization of genetically well-defined mutations that affect DNA metabolism in general (including replication and recombination). Various pathways of repair are described, particularly in relation to their imvolvement in mutagenic mechanisms. In addition to genetic control, certain physiological factors such as cell age, DNA replication, and the regulatory state of the mating-type locus are shown to also play a role in repair and mutagenesis.

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

Science.gov (United States)

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.

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

Science.gov (United States)

Mikami, Masafumi; Toki, Seiichi; Endo, Masaki

2015-08-01

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

Targeted mutagenesis in sea urchin embryos using TALENs.

Science.gov (United States)

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.

Quantitative Missense Variant Effect Prediction Using Large-Scale Mutagenesis Data.

Science.gov (United States)

Gray, Vanessa E; Hause, Ronald J; Luebeck, Jens; Shendure, Jay; Fowler, Douglas M

2018-01-24

Large datasets describing the quantitative effects of mutations on protein function are becoming increasingly available. Here, we leverage these datasets to develop Envision, which predicts the magnitude of a missense variant's molecular effect. Envision combines 21,026 variant effect measurements from nine large-scale experimental mutagenesis datasets, a hitherto untapped training resource, with a supervised, stochastic gradient boosting learning algorithm. Envision outperforms other missense variant effect predictors both on large-scale mutagenesis data and on an independent test dataset comprising 2,312 TP53 variants whose effects were measured using a low-throughput approach. This dataset was never used for hyperparameter tuning or model training and thus serves as an independent validation set. Envision prediction accuracy is also more consistent across amino acids than other predictors. Finally, we demonstrate that Envision's performance improves as more large-scale mutagenesis data are incorporated. We precompute Envision predictions for every possible single amino acid variant in human, mouse, frog, zebrafish, fruit fly, worm, and yeast proteomes (https://envision.gs.washington.edu/). Copyright © 2017 Elsevier Inc. All rights reserved.

Faux Mutagenesis: Teaching Troubleshooting through Controlled Failure

Science.gov (United States)

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…

Longitudinal study of transmission of Escherichia coli from broiler breeders to broilers

DEFF Research Database (Denmark)

Poulsen, Louise Ladefoged; Thoefner, Ida; Bisgaard, Magne

2017-01-01

of E. coli, isolated from broiler breeders with salpingitis, to the progeny and the possibility of subsequent first week mortality. Four parent flocks were followed during the whole production period (20-60 weeks) by post mortem and bacteriological examination of randomly selected dead birds. Newly...... selected for Pulsed-Field-Gel-Electrophoresis (PFGE) and Multi-Locus-Sequence-Typing (MLST) to determine their clonal relationships. E. coli was the main cause of both salpingitis in parents and first week mortality in broilers, and E. coli dominated the bacterial flora of the cloaca of newly hatched...... chickens. PFGE of E. coli showed identical band patterns in isolates from the three different sources indicating a transmission of E. coli from parent birds to chickens. In conclusion, E. coli isolated from salpingitis in broiler parents were found to be transmitted to broilers in which some sequence types...

Effects of radiations on DNA and repair of the damage. Progress report, May 1, 1974--June 30, 1977

International Nuclear Information System (INIS)

Hutchinson, F.

1977-01-01

Repair of DNA double-strand breaks produced by gamma rays takes place in E. coli. Such repair requires recA function and the presence of another DNA molecule of the same base sequence, so it may involve a recombination-like event. Ultraviolet light acting on DNA containing bromouracil produces doublestrand breaks by single photochemical events, and a simple model can explain this, as well as other results. Bromouracil mutagenesis of either E. coli or lambda phage does not involve the recA or red functions. Bromouracil mutagenesis is greatly increased in E. coli mutants such as uvrE, mutL, mutR and mutS, which are defective in mismatch repair. This, and other results, suggest that bromouracil mutagenesis occurs when cell enzymes fail to remove mismatched bases. Ultraviolet mutagenesis of lambda phage may be a useful model for the study of mutagenesis in cells, because the effects of lesions in the gene mutated (i.e. in the phage) and changes in enzyme systems (by treating the host cells) can be examined separately. Quantitative data support this approach

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

Directory of Open Access Journals (Sweden)

Janire Mingo

Full Text Available 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.

Primer Extension Mutagenesis Powered by Selective Rolling Circle Amplification

Science.gov (United States)

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

2012-01-01

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

Directed mutagenesis affects recombination in Azospirillum brasilense nif genes

Directory of Open Access Journals (Sweden)

C.P. Nunes

2000-12-01

Full Text Available In order to improve the gene transfer/mutagenesis system for Azospirillum brasilense, gene-cartridge mutagenesis was used to replace the nifD gene with the Tn5 kanamycin resistance gene. The construct was transferred to A. brasilense by electrotransformation. Of the 12 colonies isolated using the suicide plasmid pSUP202 as vector, only four did not show vector integration into the chromosome. Nevertheless, all 12 colonies were deficient in acetylene reduction, indicating an Nif- phenotype. Four Nif- mutants were analyzed by Southern blot, using six different probes spanning the nif and Km r genes and the plasmid vector. Apparently, several recombination events occurred in the mutant genomes, probably caused mainly by gene disruption owing to the mutagenesis technique used: resistance gene-cartridge mutagenesis combined with electrotransformation.Com o objetivo de melhorar os sistemas de transferência gênica e mutagênese para Azospirillum brasilense, a técnica de mutagênese através do uso de um gene marcador ("gene-cartridge mutagenesis" foi utilizada para substituir a região genômica de A. brasilense correspondente ao gene nifD por um segmento de DNA do transposon Tn5 contendo o gene que confere resistência ao antibiótico canamicina. A construção foi transferida para a linhagem de A. brasilense por eletrotransformação. Doze colônias transformantes foram isoladas com o plasmídeo suicida pSUP202 servindo como vetor. Dessas, somente quatro não possuíam o vetor integrado no cromossomo da bactéria. Independentemente da integração ou não do vetor, as 12 colônias foram deficientes na redução do gás acetileno, evidenciando o fenótipo Nif -. Quatro mutantes Nif - foram analisados através da técnica de Southern blot, utilizando-se seis diferentes fragmentos contendo genes nif, de resistência à canamicina e do vetor como sondas. Os resultados sugerem a ocorrência de eventos recombinacionais variados no genoma dos mutantes. A

Study of UV-induced mutagenesis in Bacillus subtilis

International Nuclear Information System (INIS)

Filippov, V.D.; Lotareva, O.V.

1978-01-01

The mechanism of UV-induced mutagenesis was studied in Bacillus subtilis departing from the assumption that a lower yield of UV-induced mutations should be found in mutants deficient in the recombination if production of mutations is coupled with the recombination process. Three recombination-deficient strains were used: two (recA and recF) with defects in different recombination pathways and the third (recB) has a block at a stage common for both of them. UV light induced reversions to prototrophy in recB cells and did not in recA and recF strains. Direct mutations, which confer to the cell additional growth requirements, were induced by UV light in recA and recF mutants. It is concluded that UV-induced mutagenesis in B subtilis is independent of the two known recombination mechanisms

Molecular techniques as complementary tools in orchid mutagenesis

Energy Technology Data Exchange (ETDEWEB)

Mohd Nazir Basiran; Sakinah Ariffin [Malaysian Institute for Nuclear Technology Research (MINT), Bangi (Malaysia)

2002-02-01

Orchid breeders have always been dependent on hybridization technology to produce new orchid hybrids and varieties. The technology has proven very reliable and easy to use and has produced wide range of successful cultivars with attractive combinations of spray length, bud number, flower colour and form, vase life, fragrance, seasonality, and compactness. By introducing mutagenesis however, wide variations of flower colours, form and size can still be obtained in addition to overcoming the problem of sexual incompatibility and sterility. In addition, complementary use of molecular techniques will allow breeders to target more specific characteristic changes and cut short breeding time. PCR-based techniques used to analyse the DNA of mutagenic clones found polymorphic fragments that can be developed as molecular markers. This paper describes how mutagenesis and molecular techniques can be used to enhance orchid breeding efforts. (author)

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

Science.gov (United States)

Davidson, Edgar; Doranz, Benjamin J

2014-09-01

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

Agrobacterium tumefaciens-mediated transformation as an efficient tool for insertional mutagenesis of Cercospora zeae-maydis.

Science.gov (United States)

Lu, Yuanyuan; Xiao, Shuqin; Wang, Fen; Sun, Jiaying; Zhao, Likun; Yan, Libin; Xue, Chunsheng

2017-02-01

An efficient Agrobacterium tumefaciens-mediated transformation (ATMT) approach was developed for the plant pathogenic fungus, Cercospora zeae-maydis, which is the causative agent of gray leaf spot in maize. The transformation was evaluated with five parameters to test the efficiencies of transformation. Results showed that spore germination time, co-cultivation temperature and time were the significant influencing factors in all parameters. Randomly selected transformants were confirmed and the transformants were found to be mitotically stable, with single-copy T-DNA integration in the genome. T-DNA flanking sequences were cloned by thermal asymmetric interlaced PCR. Thus, the ATMT approach is an efficient tool for insertional mutagenesis of C. zeae-maydis. Copyright © 2016 Elsevier B.V. All rights reserved.

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

Science.gov (United States)

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

2016-01-01

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

DNA fingerprinting approaches to trace Escherichia coli sharing between dogs and owners.

Science.gov (United States)

Naziri, Z; Derakhshandeh, A; Firouzi, R; Motamedifar, M; Shojaee Tabrizi, A

2016-02-01

To determine the prevalence of cross-species sharing of Escherichia coli between healthy dogs and humans living in the same household. Two faecal E. coli isolates from 25 healthy dog-owner pairs and 16 healthy control humans were tested using three fingerprinting methods. The prevalence of within-household sharing of E. coli was 4, 8 and 8% using pulsed-field gel electrophoresis, randomly amplified polymorphic DNA and enterobacterial repetitive intergenic consensus-PCR analyses respectively. Within-household bacterial sharing was more prevalent than across-household sharing (P fingerprinting techniques will help to find ways for reducing the economic impact of E. coli infections. This study support claims that public health concerns regarding the cross-species sharing of E. coli are warranted but this risk is minimal. © 2015 The Society for Applied Microbiology.

New function for Escherichia coli xanthosine phophorylase (xapA): genetic and biochemical evidences on its participation in NAD+ salvage from nicotinamide

Science.gov (United States)

2014-01-01

Background In an effort to reconstitute the NAD+ synthetic pathway in Escherichia coli (E. coli), we produced a set of gene knockout mutants with deficiencies in previously well-defined NAD+de novo and salvage pathways. Unexpectedly, the mutant deficient in NAD+de novo and salvage pathway I could grow in M9/nicotinamide medium, which was contradictory to the proposed classic NAD+ metabolism of E. coli. Such E. coli mutagenesis assay suggested the presence of an undefined machinery to feed nicotinamide into the NAD+ biosynthesis. We wanted to verify whether xanthosine phophorylase (xapA) contributed to a new NAD+ salvage pathway from nicotinamide. Results Additional knockout of xapA further slowed down the bacterial growth in M9/nicotinamide medium, whereas the complementation of xapA restored the growth phenotype. To further validate the new function of xapA, we cloned and expressed E. coli xapA as a recombinant soluble protein. Biochemical assay confirmed that xapA was capable of using nicotinamide as a substrate for nicotinamide riboside formation. Conclusions Both the genetic and biochemical evidences indicated that xapA could convert nicotinamide to nicotinamide riboside in E. coli, albeit with relatively weak activity, indicating that xapA may contribute to a second NAD+ salvage pathway from nicotinamide. We speculate that this xapA-mediated NAD+ salvage pathway might be significant in some bacteria lacking NAD+de novo and NAD+ salvage pathway I or II, to not only use nicotinamide riboside, but also nicotinamide as precursors to synthesize NAD+. However, this speculation needs to be experimentally tested. PMID:24506841

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

Science.gov (United States)

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

2014-01-01

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

ANALISIS CEMARAN BAKTERI Escherichia coli ANALISIS CEMARAN BAKTERI Escherichia coli ANALISIS CEMARAN BAKTERI Escherichia coli

OpenAIRE

ANGGREINI, RAHAYU

2015-01-01

2015 RAHAYU ANGGREINI coli Penelitian ini bertujuan untuk melakukan identifikasi cemaran bakteri E. coli O157:H7 pada daging sapi di kota Makassar. Sampel pada penelitian ini sebanyak 72 sampel Kata Kunci : Daging sapi, pasar tradisional, E. coli, E. coli O157:H7, kontaminasi bakteri, identifikasi E. coli O157:H7.

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

Science.gov (United States)

Yonemoto, Isaac T; Weyman, Philip D

2017-01-01

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

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

Science.gov (United States)

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

High dietary zinc feeding promotes persistence of multi-resistant E. coli in the swine gut.

Science.gov (United States)

Ciesinski, Lisa; Guenther, Sebastian; Pieper, Robert; Kalisch, Martin; Bednorz, Carmen; Wieler, Lothar H

2018-01-01

High levels of zinc oxide are used frequently as feed additive in pigs to improve gut health and growth performance and are still suggested as an alternative to antimicrobial growth promoters. However, we have recently described an increase of multi-resistant E. coli in association to zinc feeding in piglets. This previous study focused on clonal diversity of E. coli, observing the effect on multi-resistant strains by chance. To shed further light into this highly important topic and falsify our previous findings, we performed a zinc pig feeding trial where we specifically focused on in-depth analysis of antimicrobial resistant E. coli. Under controlled experimental conditions, piglets were randomly allocated to a high dietary zinc (zinc group) and a background zinc feeding group (control group). At different ages samples were taken from feces, digesta, and mucosa and absolute E. coli numbers were determined. A total of 2665 E. coli isolates were than phenotypically tested for antimicrobial resistance and results were confirmed by minimum inhibitory concentration testing for random samples. In piglets fed with high dietary zinc, we detected a substantial increase of multi-resistant E. coli in all gut habitats tested, ranging from 28.9-30.2% multi-resistant E. coli compared to 5.8-14.0% in the control group. This increase was independent of the total number of E. coli. Interestingly, the total amount of the E. coli population decreased over time. Thus, the increase of the multi-resistant E. coli populations seems to be linked with persistence of the resistant population, caused by the influence of high dietary zinc feeding. In conclusion, these findings corroborate our previous report linking high dietary zinc feeding of piglets with the occurrence of antimicrobial resistant E. coli and therefore question the feeding of high dietary zinc oxide as alternative to antimicrobial growth promoters.

Stimulation of Escherichia coli F-18Col- Type-1 fimbriae synthesis by leuX

DEFF Research Database (Denmark)

Newman, Joseph V.; Burghoff, Robert L.; Pallesen, Lars

1994-01-01

Escherichia coli F-18, a normal human fecal isolate, is an excellent colonizer of the streptomycin-treated mouse large intestine. E. coli F-18Col-, a derivative of E. coli F-18 which no longer makes the E. coli F-18 colicin, colonizes the large intestine as well as E. coli F-18 when fed to mice...... alone but is eliminated when fed together with E. coli F-18. Recently we randomly cloned E. coli F-18 DNA into E. coli F-18Col- and let the mouse intestine select the best colonizer. In this way, we isolated a 6.5-kb E. coli F-18 DNA sequence that simultaneously stimulated synthesis of type 1 fimbriae...... and enhanced E. coli F-18Col- colonizing ability. In the present investigation we show that the gene responsible for stimulation of type 1 fimbriae synthesis appears to be leuX, which encodes a tRNA specific for the rare leucine codon UUG. Moreover, it appears that expression of leuX may be regulated by two...

Antibiotic resistance of Verotoxigenic Escherichia coli isolated from vegetables

Directory of Open Access Journals (Sweden)

mojtaba boniadian

2017-01-01

Full Text Available Introduction: Human gastrointestinal disease caused by verotoxigenic Escherichia coli has been diagnosed for recent decades. Escherichia coli O157:H7 is the most important serotype of verotoxigenic Escherichia coli that cause hemolytic uremic syndrome and hemorrhagic colitis in humans. This study was conducted to determine the occurrence of verotoxigenic E. coli and antibiotic resistance of the isolates from vegetables. Materials and methods: A total of 500 fresh vegetable samples were collected randomly from retail shops in Shahrekord, Iran. E. coli was isolated and identified using bacteriological and biochemical tests. PCR method was used to identify the rbfE, stx1, stx2 and eae genes. Also, antibiotic resistance of the isolates was determined by disk diffusion method. Results: The results represented that among 25 isolates possess virulence genes, 40, 12 and 4% of the isolates contained eaeA, STx2, and both genes, respectively. But none of them contained H7, STx1, and rfbE genes. The antibiotic resistance pattern demonstrated that the isolates were highly resistant to Gentamycin and cefotoxime. Discussion and conclusion: The results of this study showed that the presence of verotoxigenic E.coli in vegetables; and high resistance of the isolates to antibiotics could be hazardous for public health.

Antibiotic resistant Salmonella and Escherichia coli isolated from ...

African Journals Online (AJOL)

Objective: To characterise and investigate antimicrobial resistance of Esherichia coli and salmonella strains isolated from indigenous Gallus gallus in a leading slaughterhouse/market outlet in Nairobi-Kenya. Design: A repeated cross sectional study and based on random sampling was used. Setting: The study was carried ...

Tolerance of a knotted near infrared fluorescent protein to random circular permutation

Science.gov (United States)

Pandey, Naresh; Kuypers, Brianna E.; Nassif, Barbara; Thomas, Emily E.; Alnahhas, Razan N.; Segatori, Laura; Silberg, Jonathan J.

2016-01-01

Bacteriophytochrome photoreceptors (BphP) are knotted proteins that have been developed as near-infrared fluorescent protein (iRFP) reporters of gene expression. To explore how rearrangements in the peptides that interlace into the knot within the BphP photosensory core affect folding, we subjected iRFP to random circular permutation using an improved transposase mutagenesis strategy and screened for variants that fluoresce. We identified twenty seven circularly permuted iRFP that display biliverdin-dependent fluorescence in Escherichia coli. The variants with the brightest whole cell fluorescence initiated translation at residues near the domain linker and knot tails, although fluorescent variants were discovered that initiated translation within the PAS and GAF domains. Circularly permuted iRFP retained sufficient cofactor affinity to fluoresce in tissue culture without the addition of biliverdin, and one variant displayed enhanced fluorescence when expressed in bacteria and tissue culture. This variant displayed a similar quantum yield as iRFP, but exhibited increased resistance to chemical denaturation, suggesting that the observed signal increase arose from more efficient protein maturation. These results show how the contact order of a knotted BphP can be altered without disrupting chromophore binding and fluorescence, an important step towards the creation of near-infrared biosensors with expanded chemical-sensing functions for in vivo imaging. PMID:27304983

Genome-wide LORE1 retrotransposon mutagenesis and high-throughput insertion detection in Lotus japonicus

DEFF Research Database (Denmark)

Urbanski, Dorian Fabian; Malolepszy, Anna; Stougaard, Jens

2012-01-01

Insertion mutants facilitate functional analysis of genes, but for most plant species it has been difficult to identify a suitable mutagen and to establish large populations for reverse genetics. The main challenge is developing efficient high-throughput procedures for both mutagenesis and insert......Insertion mutants facilitate functional analysis of genes, but for most plant species it has been difficult to identify a suitable mutagen and to establish large populations for reverse genetics. The main challenge is developing efficient high-throughput procedures for both mutagenesis...... plants. The identified insertions showed that the endogenous LORE1 retrotransposon is well suited for insertion mutagenesis due to its homogenous gene targeting and exonic insertion preference. Since LORE1 transposition occurs in the germline, harvesting seeds from a single founder line and cultivating...... progeny generates a complete mutant population. This ease of LORE1 mutagenesis combined with the efficient FSTpoolit protocol, which exploits 2D pooling, Illumina sequencing, and automated data analysis, allows highly cost-efficient development of a comprehensive reverse genetic resource....

Parameters affecting frequency of CRISPR/Cas9 mediated targeted mutagenesis in rice.

Science.gov (United States)

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.

Mutagenesis breeding research of Lactobacillus brevis of nitrite reduction

Directory of Open Access Journals (Sweden)

LI Zeli

2015-10-01

Full Text Available The pollution of nitrite in food became one of the focus of food safety issues,the use of biotechnology methods degrading nitrite became hotspot.The primitive strain was Lactobacillus brevis C2,preserved in our laboratory,had the ability to degrade nitrite,through composite mutagenesis of 15 W,254 nm,20 cm ultraviolet mutagenesis (UV for 120 s and 0.8% diethyl sulfate(DES in 37℃ mutation for 40 min,after screening,we successfully obtained high efficient strain of nitrite degradation,named UV6-DS2,relative to the starting strain,under the condition of 400 mg/L nitrite,after 12 h degradation,nitrite degradation rate increased from 92.8% to 97.8%,to explore its application in food was able to effectively reduce concentration of nitrite in food.

Site-specifically modified oligodeoxyribonucleotides as templates for Escherichia coli DNA polymerase I

International Nuclear Information System (INIS)

O'Connor, D.; Stoehrer, G.

1985-01-01

Oligodeoxyribonucleotides with site-specific modifications have been used as substrates for Escherichia coli DNA polymerase I holoenzyme and Klenow fragment. Modifications included the bulky guanine-8-aminofluorene adduct and a guanine oxidation product resembling the product of photosensitized DNA oxidation. By a combination of primers and nick-mers, conditions of single-strand-directed DNA synthesis and nick-translation could be created. The results show that the polymerase can bypass both types of lesions. Bypass occurs on a single-stranded template but is facilitated on a nicked, double-stranded template. Only purines, with guanine more favored than adenine, are incorporated across both lesions. The results indicate that site-specifically modified oligonucleotides can be sensitive probes for the action of polymerases on damaged templates. They also suggest a function for polymerase I, in its nick-translation capacity, during DNA repair and mutagenesis

Site-directed Mutagenesis of Cysteine Residues in Phi-class Glutathione S-transferase F3 from Oryza sativa

Energy Technology Data Exchange (ETDEWEB)

Jo, Hyunjoo; Lee, Juwon; Noh, Jinseok; Kong, Kwanghoon [Chung-Ang Univ., Seoul (Korea, Republic of)

2012-12-15

To elucidate the roles of cysteine residues in rice Phi-class GST F3, in this study, all three cysteine residues were replaced with alanine by site-directed mutagenesis in order to obtain mutants C22A, C73A and C77A. Three mutant enzymes were expressed in Escherichia coli and purified to electrophoretic homogeneity by affinity chromatography on immobilized GSH. The substitutions of Cys73 and Cys77 residues in OsGSTF3 with alanine did not affect the glutathione conjugation activities, showing non-essentiality of these residues. On the other hand, the substitution of Cys22 residue with alanine resulted in approximately a 60% loss of specific activity toward ethacrynic acid. Moreover, the K{sub m}{sup CDNB} value of the mutant C22A was approximately 2.2 fold larger than that of the wild type. From these results, the evolutionally conserved cysteine 22 residue seems to participate rather in the structural stability of the active site in OsGSTF3 by stabilizing the electrophilic substrates-binding site's conformation than in the substrate binding directly.

Random mutagenesis in Corynebacterium glutamicum ATCC 13032 using an IS6100-based transposon vector identified the last unknown gene in the histidine biosynthesis pathway

Directory of Open Access Journals (Sweden)

Gaigalat Lars

2006-08-01

essential L-histidinol-phosphate phosphatase (EC 3.1.3.15 in C. glutamicum. The cg0910 gene, renamed hisN, and its encoded enzyme have putative orthologs in almost all Actinobacteria, including mycobacteria and streptomycetes. Conclusion The absence of regional and sequence preferences of IS6100-transposition demonstrate that the established system is suitable for efficient genome-scale random mutagenesis in the sequenced type strain C.glutamicum ATCC 13032. The identification of the hisN gene encoding histidinol-phosphate phosphatase in C. glutamicum closed the last gap in histidine synthesis in the Actinobacteria. The system might be a valuable genetic tool also in other bacteria due to the broad host-spectrum of IS6100.

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

Science.gov (United States)

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.

Methods for targetted mutagenesis in gram-positive bacteria

Science.gov (United States)

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.

Insertional mutagenesis using Tnt1 retrotransposon in potato

Science.gov (United States)

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...

The European dimension for the mouse genome mutagenesis

Czech Academy of Sciences Publication Activity Database

Auwerx, J.; Avner, P.; Baldock, R.; Ballabio, A.; Balling, R.; Barbacid, M.; Berns, A.; Bradley, A.; Brown, S.; Carmeliet, P.; Chambon, P.; Cox, R.; Davidson, D.; Davies, K.; Duboule, D.; Forejt, Jiří; Granucci, F.; Hastie, N.; Angelis, M. H. de; Jackson, I.; Kioussis, D.; Kollias, G.; Lathrop, M.; Lendahl, U.; Malumbres, M.; von Melchner, H.; Müller, W.; Partanen, J.; Ricciardi-Castagnoli, P.; Rigby, P.; Rosen, B.; Rosenthal, N.; Skarnes, B.; Stewart, A. F.; Thornton, J.; Tocchini-Valentini, G.; Wagner, E.; Wahli, W.; Wurst, W.

2004-01-01

Roč. 16, - (2004), s. 925-927 ISSN 1061-4036 R&D Projects: GA MŠk(CZ) LN00A079 Institutional research plan: CEZ:AV0Z5052915 Keywords : The European Mouse Mutagenesis Consortium Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 24.695, year: 2004

Effects of 32 P incorporated in plasmid DNA: strand breaks and mutagenesis

International Nuclear Information System (INIS)

Fonseca, Adenilson de S. da; Felzenszwalb, Israel

1996-01-01

In order to study the 32 P decay effects in DNA, bacterial plasmid were labeled with different activities of the radioisotope in vivo: 1,2 and 6 x 10 5 Bk/ml of bacterial culture, leading to 1,2 and 6 x 10 3 Bk/μg of nucleic acid or in vitro: 0.7, 1.5 and 3.5 x 10 3 Bk/μg of nucleic acid, stored at -20 deg C and its electroforetic profiles, transformation capacity of wild type and DNA repair. E. coli mutants cells and mutagenesis, were followed during three months. The results achieved in this work suggest that: the decay of the incorporated 32 P in vivo is able to change the pBR322 electroforetic profile, we detected a decrease on the form III (super coiled) and increase on the form II (circular), indicating single strands breaks; the decay incorporated 32 in vitro does not modify the electrophoretic profile of pBR322, suggesting that in some way the effects of the radioactive decay of incorporated 32 P is dependent of the DNA topology, the damages induced by 32 P decay increase mutation frequency in pAC189 plasmids. MRF is increased by a factor of three after 6 t 1/2 of storage, indicating direct or indirect action through mismatch DNA repair pathway. (author)

Design and optimization of N-acylhydrazone pyrimidine derivatives as E. coli PDHc E1 inhibitors: Structure-activity relationship analysis, biological evaluation and molecular docking study.

Science.gov (United States)

He, Haifeng; Xia, Hongying; Xia, Qin; Ren, Yanliang; He, Hongwu

2017-10-15

By targeting the thiamin diphosphate (ThDP) binding site of Escherichia coli (E. coli) pyruvate dehydrogenase multienzyme complex E1 (PDHc E1), a series of novel 'open-chain' classes of ThDP analogs A, B, and C with N-acylhydrazone moieties was designed and synthesized to explore their activities against E. coli PHDc E1 in vitro and their inhibitory activity against microbial diseases were further evaluated in vivo. As a result, A1-23 exhibited moderate to potent inhibitory activities against E. coli PDHc E1 (IC 50 =0.15-23.55μM). The potent inhibitors A13, A14, A15, C2, had strong inhibitory activities with IC 50 values of 0.60, 0.15, 0.39 and 0.34μM against E. coli PDHc E1 and with good enzyme-selective inhibition between microorganisms and mammals. Especially, the most powerful inhibitor A14 could 99.37% control Xanthimonas oryzae pv. Oryzae. Furthermore, the binding features of compound A14 within E. coli PDHc E1 were investigated to provide useful insights for the further construction of new inhibitor by molecular docking, site-directed mutagenesis, and enzymatic assays. The results indicated that A14 had most powerful inhibition against E. coli PDHc E1 due to the establishment of stronger interaction with Glu571, Met194, Glu522, Leu264 and Phe602 at active site of E.coli PDHc E1. It could be used as a lead compound for further optimization, and may have potential as a new microbicide. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

Science.gov (United States)

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

2008-07-01

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

Codon cassette mutagenesis: a general method to insert or replace individual codons by using universal mutagenic cassettes.

OpenAIRE

Kegler-Ebo, D M; Docktor, C M; DiMaio, D

1994-01-01

We describe codon cassette mutagenesis, a simple method of mutagenesis that uses universal mutagenic cassettes to deposit single codons at specific sites in double-stranded DNA. A target molecule is first constructed that contains a blunt, double-strand break at the site targeted for mutagenesis. A double-stranded mutagenic codon cassette is then inserted at the target site. Each mutagenic codon cassette contains a three base pair direct terminal repeat and two head-to-head recognition sequen...

Targeted mutagenesis using CRISPR/Cas in inbred potatoes

Science.gov (United States)

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...

Minimizing off-Target Mutagenesis Risks Caused by Programmable Nucleases.

Science.gov (United States)

Ishida, Kentaro; Gee, Peter; Hotta, Akitsu

2015-10-16

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.

The role of misrepair in experimental mutagenesis

International Nuclear Information System (INIS)

Yamaguchi, Hikoyuki

1983-01-01

Mutagenesis is classified as being either mispairing occuring at the time of chromosome replication or as being misrepair occuring when damaged nucleotides are converted to the paired ones. In the cell population of the root meristem of barley which is considered to be steadystate, the possibility of the selective segregation of the newly synthesized and the older template strands of DNA at mitosis was studied by the incorporation of 3 H-thymidine. Stochastic removal of de novo synthesized DNA strand to a zone of non-dividing cell population was unlikely. Thus, it has been concluded that there is special mechanisms for protecting the integrity of the DNA by removing the mispairing lesions. Barly seeds first exposed to a low level γ-radiation before treating with ethylmethane sulfonate. Survival rate of M 1 plants as well as mutation frequency of M 2 were higher for the combined treatment than for single treatment of chemical mutagen. A mutational response of barley cell to DNA damaging agent was much affected by a previous treatment with mutagens. It is suggested that in the experimental mutagenesis misrepair plays rather an important role than mispairing. (author)

DNA damage and mutagenesis of lambda phage induced by gamma-rays

International Nuclear Information System (INIS)

Bertram, Heidi

1988-01-01

Lambda phage DNA was gamma irradiated in aqueous solution and strand breakage determined. Twice as much minor structural damage per lethal hit was found in this DNA compared with DNA from irradiated phage suspensions. The in vitro irradiated DNA was repackaged into infectious particles. Induction of mutations in the cI or cII cistron was scored using SOS-induced host cells. In vitro prepared particles were found to have second-order kinetics for mutagenesis induced by gamma rays indicating two pre-mutational events were necessary to produce a mutation, but bacteria-free phage suspensions ('lys-phage') showed single hit kinetics for mutagenesis after irradiation. Increase in the mutation rate in the phage particles was mainly due to minor lesions, i.e. ssb, als and unidentified base damage. In lys-phage, mutagenesis might be enhanced by clustered DNA damage - configuration not existing in pack-phage. Loss of infectivity was analysed in comparison with structural damage. All lesions contributed to biological inactivation. Minor lesions were tolerated by lambda phage to a limited extent. Major lesions (e.g. dsb) contributed most to infectivity loss and were considered lethal events. (U.K.)

Radiation mutagenesis of subtropic plants

International Nuclear Information System (INIS)

Kerkadze, I.G.

1987-01-01

Possibilities of expansion of subtropic plant changeability and development of new gene bank for future selection-genetic studies are detected. New trends of radiation mutagenesis of subtropic plants are formulated as results of studies during many years. A lot of mutants is subjected to sufficient tests, and concrete results are obtained with the help of these tests for definite species. Summing genetic and selection estimations of the results, it is possible to make the conclusion that mutant selection represents one of the powerful methods of preparation of productive and qualitative species of subtropic plants, which are successfully introduced into practice

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

Science.gov (United States)

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.

Biofilm formation in Escherichia coli cra mutants is impaired due to down-regulation of curli biosynthesis.

Science.gov (United States)

Reshamwala, Shamlan M S; Noronha, Santosh B

2011-10-01

Cra is a pleiotropic regulatory protein that controls carbon and energy flux in enteric bacteria. Recent studies have shown that Cra also regulates other cell processes and influences biofilm formation. The purpose of the present study was to investigate the role of Cra in biofilm formation in Escherichia coli. Congo red-binding studies suggested that curli biosynthesis is impaired in cra mutants. Microarray analysis of wild-type and mutant E. coli cultivated in conditions promoting biofilm formation revealed that the curli biosynthesis genes, csgBAC and csgDEFG, are poorly expressed in the mutant, suggesting that transcription of genes required for curli production is regulated by Cra. Four putative Cra-binding sites were identified in the curli intergenic region, which were experimentally validated by performing electromobility shift assays. Site-directed mutagenesis of three Cra-binding sites in the promoter region of the csgDEFG operon suggests that Cra activates transcription of this operon upon binding to operator regions both downstream and upstream of the transcription start site. Based on the Cra-binding sites identified in this and other studies, the Cra consensus sequence is refined.

Contamination of Ethiopian paper currency notes from various food handlers with E. coli.

Science.gov (United States)

Hiko, Adem; Abdata, Kasahun; Muktar, Yimer; Woyesa, Mezene; Mohammed, Abdela

2016-01-01

Contamination rate of Ethiopian paper currency notes handled by various food handlers with Escherichia coli and antimicrobial susceptibility of the isolates was assessed. A total of 384 Ethiopian Birr (ETB) notes were randomly sampled from meat handlers at butchers, bread and the related food handlers at cafeteria, fruit and vegetables handlers at supermarket, and milk sellers both at open market and dairy station. Fifty control new currencies were also sampled from Commercial Bank of Ethiopia. Both surfaces of the currency were swabbed using wet sterile cotton. The swab was overnight incubated in buffered peptone water. A loop full was streaked on eosin methylene blue agar and followed by biochemical test on presumptive E. coli colonies. Randomly selected isolates were exposed to chloramphenicol (C-30 µg), neomycin (N-30 µg), oxytetracycline (OT-30 µg), polymyxin-B (PB-300 IU) and trimethoprim-sulfamethoxazole (SXT-1.25/23.75/µg) susceptibility using disc diffusion techniques. E. coli was not isolated from currency used as control. A total of 288 (75 %) currency notes were found carrying E. coli. E. coli prevalence was ranges from 67.2 % at open market milk sellers to 87.2 % at dairy station milk sellers; from 64.8 % on ETB 100 to 82.9 % on ETB 1. Differences were not observed in E. coli prevalence on currency notes from among almost all food handlers (P > 0.05). Susceptibility of tested isolates to each chloramphenicol, oxytetracycline and trimethoprim-sulfamethoxazole was 100 %, and to polymyxin-B was 97.3 %. High resistance (83.7 %) was observed to neomycin. The finding indicates, contaminated food can be a source of E. coli for further contamination of currency which again transfer through various foods ready for consumption.

Repair and mutagenesis of herpes simplex virus in UV-irradiated monkey cells

International Nuclear Information System (INIS)

Lytle, C.D.; Goddard, J.G.; Lin, C.H.

1980-01-01

Mutagenic repair in mammalian cells was investigated by determining the mutagenesis of UV-irradiated or unirradiated herpes simplex virus in UV-irradiated CV-1 monkey kidney cells. These results were compared with the results for UV-enhanced virus reactivation (UVER) in the same experimental situation. High and low multiplicities of infection were used to determine the effects of multiplicity reactivation (MR). UVER and MR were readily demonstrable and were approximately equal in amount in an infectious center assay. For this study, a forward-mutation assay was developed to detect virus mutants resistant to iododeoxycytidine (ICdR), probably an indication of the mutant virus being defective at its thymidine kinase locus. ICdR-resistant mutants did not have a growth advantage over wild-type virus in irradiated or unirradiated cells. Thus, higher fractions of mutant virus indicated greater mutagenesis during virus repair and/or replication. The data showed that: (1) unirradiated virus was mutated in unirradiated cells, providing a background level of mutagenesis; (2) unirradiated virus was mutated about 40% more in irradiated cells, indicating that virus replication (DNA synthesis) became more mutagenic as a result of cell irradiation; (3) irradiated virus was mutated much more (about 6-fold) than unirradiated virus, even in unirradiated cells; (4) cell irradiation did not change the mutagenesis of irradiated virus except at high multiplicity of infection. High multiplicity of infection did not demonstrate UVER or MR alone to be either error-free or error-prone. When the two processes were present simultaneously, they were mutagenic. (orig.)

Expression and purification of recombinant hemoglobin in Escherichia coli.

Directory of Open Access Journals (Sweden)

Chandrasekhar Natarajan

Full Text Available Recombinant DNA technologies have played a pivotal role in the elucidation of structure-function relationships in hemoglobin (Hb and other globin proteins. Here we describe the development of a plasmid expression system to synthesize recombinant Hbs in Escherichia coli, and we describe a protocol for expressing Hbs with low intrinsic solubilities. Since the α- and β-chain Hbs of different species span a broad range of solubilities, experimental protocols that have been optimized for expressing recombinant human HbA may often prove unsuitable for the recombinant expression of wildtype and mutant Hbs of other species.As a test case for our expression system, we produced recombinant Hbs of the deer mouse (Peromyscus maniculatus, a species that has been the subject of research on mechanisms of Hb adaptation to hypoxia. By experimentally assessing the combined effects of induction temperature, induction time and E. coli expression strain on the solubility of recombinant deer mouse Hbs, we identified combinations of expression conditions that greatly enhanced the yield of recombinant protein and which also increased the efficiency of post-translational modifications.Our protocol should prove useful for the experimental study of recombinant Hbs in many non-human animals. One of the chief advantages of our protocol is that we can express soluble recombinant Hb without co-expressing molecular chaperones, and without the need for additional reconstitution or heme-incorporation steps. Moreover, our plasmid construct contains a combination of unique restriction sites that allows us to produce recombinant Hbs with different α- and β-chain subunit combinations by means of cassette mutagenesis.

Expression and purification of recombinant hemoglobin in Escherichia coli.

Science.gov (United States)

Natarajan, Chandrasekhar; Jiang, Xiaoben; Fago, Angela; Weber, Roy E; Moriyama, Hideaki; Storz, Jay F

2011-01-01

Recombinant DNA technologies have played a pivotal role in the elucidation of structure-function relationships in hemoglobin (Hb) and other globin proteins. Here we describe the development of a plasmid expression system to synthesize recombinant Hbs in Escherichia coli, and we describe a protocol for expressing Hbs with low intrinsic solubilities. Since the α- and β-chain Hbs of different species span a broad range of solubilities, experimental protocols that have been optimized for expressing recombinant human HbA may often prove unsuitable for the recombinant expression of wildtype and mutant Hbs of other species. As a test case for our expression system, we produced recombinant Hbs of the deer mouse (Peromyscus maniculatus), a species that has been the subject of research on mechanisms of Hb adaptation to hypoxia. By experimentally assessing the combined effects of induction temperature, induction time and E. coli expression strain on the solubility of recombinant deer mouse Hbs, we identified combinations of expression conditions that greatly enhanced the yield of recombinant protein and which also increased the efficiency of post-translational modifications. Our protocol should prove useful for the experimental study of recombinant Hbs in many non-human animals. One of the chief advantages of our protocol is that we can express soluble recombinant Hb without co-expressing molecular chaperones, and without the need for additional reconstitution or heme-incorporation steps. Moreover, our plasmid construct contains a combination of unique restriction sites that allows us to produce recombinant Hbs with different α- and β-chain subunit combinations by means of cassette mutagenesis.

Genetic analysis of the SOS response of Escherichia coli

International Nuclear Information System (INIS)

Mount, D.W.; Wertman, K.F.; Ennis, D.G.; Peterson, K.R.; Fisher, B.L.; Lyons, G.

1983-01-01

In the SOS response, a large number of E. coli genes having different functions are derepressed when the cellular DNA is damaged. This derepression occurs through inactivation of a repressor, the product of the lexA gene, by a protease activity of the recA gene product. The protease is thought to be activated in response to changes in DNA metabolism which follow the damage. After the SOS functions have acted, the protease activity declines and repression is again established. Because the DNA sequence of both lexA and recA have been determined, it is possible to induce many mutations in their regulatory and structural regions in order to analyze further the control of the SOS response. We are studying the effects of mutations in both the lexA and recA regulatory regions, and mutations which affect the protease activity or the sensitivity of repressor to the protease. Finally, we are using genetic methods to analyze a newly identified requirement for recA protein, induced mutagenesis in cells lacking repressor. 16 references, 3 figures

A source of artifact in the lacZ reversion assay in Escherichia coli.

Science.gov (United States)

Hoffmann, George R; Gray, Carol L; Lange, Paulina B; Marando, Christie I

2015-06-01

The lacZ reversion assay in Escherichia coli measures point mutations that occur by specific base substitutions and frameshift mutations. The tester strains cannot use lactose as a carbon source (Lac(-)), and revertants are easily detected by growth on lactose medium (Lac(+)). Six strains identify the six possible base substitutions, and five strains measure +G, -G, -CG, +A and -A frameshifts. Strong mutagens give dose-dependent increases in numbers of revertants per plate and revertant frequencies. Testing compounds that are arguably nonmutagens or weakly mutagenic, we often noted statistically significant dose-dependent increases in revertant frequency that were not accompanied by an absolute increase in numbers of revertants. The increase in frequency was wholly ascribable to a declining number of viable cells owing to toxicity. Analysis of the conditions revealed that the frequency of spontaneous revertants is higher when there are fewer viable cells per plate. The phenomenon resembles "adaptive" or "stress" mutagenesis, whereby lactose revertants accumulate in Lac(-) bacteria under starvation conditions in the absence of catabolite repression. Adaptive mutation is observed after long incubation and might be expected to be irrelevant in a standard assay using 48-h incubation. However, we found that elevated revertant frequencies occur under typical assay conditions when the bacterial lawn is thin, and this can cause increases in revertant frequency that mimic chemical mutagenesis when treatments are toxic but not mutagenic. Responses that resemble chemical mutagenesis were observed in the absence of mutagenic treatment in strains that revert by different frameshift mutations. The magnitude of the artifact is affected by cell density, dilution, culture age, incubation time, catabolite repression and the age and composition of media. Although the specific reversion assay is effective for quickly distinguishing classes of mutations induced by potent mutagens, its

Model building of a thermolysin-like protease by mutagenesis

NARCIS (Netherlands)

Frigerio, F; Margarit, [No Value; Nogarotto, R; Grandi, G; Vriend, G; Hardy, F; Veltman, OR; Venema, G; Eijsink, VGH

The present study concerns the use of site-directed mutagenesis experiments to optimize a three-dimensional model of the neutral protease of Bacillus subtilis (NP-sub), An initial model of NP-sub was constructed using the crystal structures of the homologous neutral proteases of Bacillus

Effects of mutation on the downfield proton nuclear magnetic resonance spectrum of the 5S RNA of Escherichia coli

International Nuclear Information System (INIS)

Gewirth, D.T.; Moore, P.B.

1987-01-01

The imino proton spectra of several mutants of the 5S RNA of Escherichia coli are compared with that of the wild type. Three of the variants discussed are point mutations, and the fourth is a deletion mutant lacking bases 11-69 of the parent sequence, all obtained by site-directed mutagenesis techniques. The spectroscopic effects of mutation are limited in all cases, and the differences between normal and mutant spectra can be used to make or confirm the assignments of resonances. Several new assignments in the 5S spectrum are reported. Spectroscopic differences due to sequence differences permit the products of single genes within the 5S gene family to be distinguished and their fates followed by NMR

Fluorometric method of quantitative cell mutagenesis

Science.gov (United States)

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.

Mutagenesis of metal compounds in bacteria

Energy Technology Data Exchange (ETDEWEB)

Nishioka, H

1974-01-01

The mutagenic activity of 41 metal compounds was examined by applying the Rec-assay method with Bacillus subtilis H17 (rec/sup +/) and M45 (rec/sup -/) strains. Among these compounds, Na/sub 2/HAsO/sub 4/, CdCl/sub 2/, K/sub 2/CrO/sub 4/, K/sub 2/Cr/sub 2/O/sub 7/, CH/sub 3/HgCl, C/sub 2/H/sub 5/HgCl, CH/sub 3/COOHgC/sub 6/H/sub 5/, MnCl/sub 2/, MnNO/sub 3/, MnSO/sub 4/, Mn(CH/sub 3/COO)/sub 2/, (NH/sub 4/)/sub 2/MoO/sub 4/ and KMoO/sub 4/ showed positive results. The reactions of K/sub 2/Cr/sub 2/O/sub 7/ and (NH/sub 4/)/sub 2/MoO/sub 4/ were especially strong in the assay. Therefore, mutation induction to reversion (try/sup +/) and streptomycin resistance (SM/sup r/) of E. coli B/r WP2 try/sup -/ (hcl/sup +/ and hcr/sup -/) by the two compounds were examined by the following two experimental procedures. Stationary phase bacteria were exposed to the compounds at high concentrations (6.9 x 10/sup -3/ approx. 3.44 x 10/sup -2/M) in M9 buffer for 15 min at 37/sup -/ with shaking. After incubation at 37/sup 0/ for 48 h visible colonies on the plates were scored. Bacteria in M9 buffer were plated in media supplemented with low concentrations (1.7 x 10/sup -5/ approx. 3.4 x 10/sup -5/M) of the compounds. K/sub 2/Cr/sub 2/O/sub 7/ and (NH/sub 4/)/sub 2/MoO/sub 4/ increased the mutation rate of SM/sup r/ and try/sup +/ in both strains treated with either procedure. No marked differences in mutation rate were found between hcr/sup +/ and hcr/sup -/. After treatment with high concentrations of compounds one can imagine that a peroxidation state produced by these peroxides in the media might affect the killing and mutation induction. These results suggest the possibility that the mutagenesis of the metals relate to their atomic values, rather than the peroxidation state as far as these two compounds are concerned.

Ionizing radiation-induced bystander mutagenesis and adaptation: Quantitative and temporal aspects

International Nuclear Information System (INIS)

Zhang Ying; Zhou Junqing; Baldwin, Joseph; Held, Kathryn D.; Prise, Kevin M.; Redmond, Robert W.; Liber, Howard L.

2009-01-01

This work explores several quantitative aspects of radiation-induced bystander mutagenesis in WTK1 human lymphoblast cells. Gamma-irradiation of cells was used to generate conditioned medium containing bystander signals, and that medium was transferred onto naive recipient cells. Kinetic studies revealed that it required up to 1 h to generate sufficient signal to induce the maximal level of mutations at the thymidine kinase locus in the bystander cells receiving the conditioned medium. Furthermore, it required at least 1 h of exposure to the signal in the bystander cells to induce mutations. Bystander signal was fairly stable in the medium, requiring 12-24 h to diminish. Medium that contained bystander signal was rendered ineffective by a 4-fold dilution; in contrast a greater than 20-fold decrease in the cell number irradiated to generate a bystander signal was needed to eliminate bystander-induced mutagenesis. This suggested some sort of feedback inhibition by bystander signal that prevented the signaling cells from releasing more signal. Finally, an ionizing radiation-induced adaptive response was shown to be effective in reducing bystander mutagenesis; in addition, low levels of exposure to bystander signal in the transferred medium induced adaptation that was effective in reducing mutations induced by subsequent γ-ray exposures.

Temporal variations of Escherichia coli concentrations in a large Midwestern river

Science.gov (United States)

Schilling, K.E.; Zhang, Y.-K.; Hill, D.R.; Jones, C.S.; Wolter, C.F.

2009-01-01

The Raccoon River used by the Des Moines Water Works to serve more than 400,000 people in central Iowa is threatened by contamination from Escherichia coli bacteria from point and nonpoint sources. The 9389 km2 watershed is highly agricultural, with 73% of the land in row crop production and widespread animal production. Results from 2155 grab samples from 1997 to 2005 for E. coli analysis were examined for temporal variations. E. coli concentrations were found to vary across years, seasons, and flow conditions, with a 9-year mean value of 1156 most probable number (MPN)/100 ml. Monthly concentrations exhibited clear seasonality with highest values in May through July. Although E. coli concentrations were higher during periods of greater discharge, the relation of log E. coli to log discharge was not particularly strong (r2 = 0.35). The variogram of E. coli concentrations showed temporal correlation within a span of 4 days suggesting that concentrations measured on 1 day may be related in time to concentrations measured up to 3 days later and beyond 4 days the concentrations vary randomly. The spectral analysis of the time series of E. coli was also carried out and was fitted well with the spectrum of an exponential covariance function. Deciphering temporal patterns and correlation of E. coli bacteria in streams may be useful for developing future monitoring strategies to track concentration patterns and loads. ?? 2008 Elsevier B.V. All rights reserved.

Characteristics of Escherichia coli causing persistence or relapse of urinary tract infections

DEFF Research Database (Denmark)

Ejrnæs, Karen; Stegger, Marc; Reisner, Andreas

2011-01-01

Recurrent urinary tract infections (RUTIs) pose a major problem but little is known about characteristics of Escherichia coli associated with RUTI. This study includes E. coli from 155 women with community-acquired lower urinary tract infections (UTIs) randomized to one of three dosing regiments...... of persistence or relapse of UTI compared with three days. In vitro biofilm formation was not higher among E. coli causing persistence or relapse. The presence of agn43a(CFT073) or agn43b(CFT073) was associated with biofilm forming capacity. In conclusion, our results show potential targets for prevention...... and treatment of persistence/relapse of UTI and potential markers for selecting treatment lengths and warrant studies of these and new VFGs....

New mutations affecting induced mutagenesis in yeast.

Science.gov (United States)

Lawrence, C W; Krauss, B R; Christensen, R B

1985-01-01

Previously isolated mutations in baker's yeast, Saccharomyces cerevisiae, that impair induced mutagenesis were all identified with the aid of tests that either exclusively or predominantly detect base-pair substitutions. To avoid this bias, we have screened 11 366 potentially mutant clones for UV-induced reversion of the frameshift allele, his4-38, and have identified 10 mutants that give much reduced yields of revertants. Complementation and recombination tests show that 6 of these carry mutations at the previously known REV1, REV1 and REV3 loci, while the remaining 4 define 3 new genes, REV4 (2 mutations), REV5 and REV6. The rev4 mutations are readily suppressed in many genetic backgrounds and, like the rev5 mutation, impart only a limited deficiency for induced mutagenesis: it is likely, therefore that the REV4+ and REV5+ gene functions are only remotely concerned with this process. The rev6 mutants have a more general deficiency, however, as well as marked sensitivity to UV and an increased spontaneous mutation rate, properties that suggest the REV6 gene is directly involved in mutation induction. The REV5 gene is located about 1 cM proximal to CYC1 on chromosome X.

Effect of an aminothiol (WR-1065) on radiation-induced mutagenesis and cytotoxicity in two repair-deficient mammalian cell lines

International Nuclear Information System (INIS)

Grdina, D.J.; Nagy, B.; Meechan, P.J.

1991-01-01

WR-2721 and its free thiol WR-1065 have been found to effectively protect against radiation- and/or chemotherapy-induced mutagenesis, transformation and carcinogenesis. With respect to the antimutagenic effect, WR-1065 significantly reduced the frequency of HGPRT mutants even when it was administered up to three hours following exposure of cells to radiation. The mechanisms of action most often attributed to these agents include their ability to scavenge free radicals, enter into chemical repair processes through the donation of hydrogen atoms, and induce intracellular hypoxia by means of auto-oxidative processes. Although evidence exists for each of these processes, none is sufficiently satisfactory to account for the post-irradiation protection of WR-1065 against mutation induction in mammalian cells. The most elegant work describing the role of aminothiols on cellular enzymatic repair processes has focused on well-characterized repair-proficient and -deficient bacterial and yeast cell systems. Protection against radiation-induced cytotoxicity by the aminothiol cysteamine was absent in E. coli cell lines that were characterized as having genetically defective repair systems. Until recently, such studies could not be effectively performed with mammalian cells. However, with the isolation and characterization of rodent cell lines deficient in their ability to repair DNA damage, it is now possible to investigate the role of cell-mediated repair systems on aminothiol radioprotection. Specifically, the authors have investigated the effects of WR-1065 on radiation-induced mutagenesis and cytotoxicity in cell lines EM9 and xrs-5, which are defective in DNA single-strand break (SSB) and double-strand break (DSB) rejoining, respectively. Corresponding parental repair-proficient cell lines, AA8 and K1, were also studied for comparative purposes. 26 refs., 5 figs., 2 tabs

Moderate repetitions (mobile elements) and hot points of chromosomal mutagenesis in Drozophila melanogaster

International Nuclear Information System (INIS)

Aleksandrova, M.V.; Sevan'kaev, A.V.; Aleksandrov, I.D.

1989-01-01

The results of experimental examination of hypothesis on mobile elements (ME) as target for radiation-induced chromosomal mutagenesis do not confirm it in this direct variant though indicate on the presence of nonincidental connection between sites of ME localization of certain type and points of chromosomal mutagenesis are presented. The presented regularity is explained by assumption that settling of the ME by genome is going along postulated static chromomer-binding-DNA complexes, playing an important role in space organization of interphase chromatin. 11 refs.; 2 figs.; 2 tabs

Forsythia improvement by mutagenesis

International Nuclear Information System (INIS)

Cadic, A.; Martin, Denise; Renoux, A.

1980-01-01

Mutagenesis is a method used by selectors to modify the genetic heritage of a species. Since about twenty years ago the list of varieties obtained has lengthened steadily. For various reasons, plants which propagate vegetatively, and amongst these a large number of decorative plants, have been especially improved by this method. Of the mutagenic agents known at present a favourite choice has often been the gamma radiations emitted by radioactive cobalt ( 60 Co). Several clones of forsythia, very irregular in decorative value, were exposed to gamma radiation for the purpose of judging the breadth of the easily identifiable mutation range and creating new varieties. From the results it is hoped very soon to release compact varieties with short internodes and varieties better suited to forcing because of their earlier flowering season [fr

What Can a Micronucleus Teach? Learning about Environmental Mutagenesis

Science.gov (United States)

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…

Functional mapping of the fission yeast DNA polymerase δ B-subunit Cdc1 by site-directed and random pentapeptide insertion mutagenesis

Directory of Open Access Journals (Sweden)

Gray Fiona C

2009-08-01

Full Text Available Abstract Background DNA polymerase δ plays an essential role in chromosomal DNA replication in eukaryotic cells, being responsible for synthesising the bulk of the lagging strand. In fission yeast, Pol δ is a heterotetrameric enzyme comprising four evolutionarily well-conserved proteins: the catalytic subunit Pol3 and three smaller subunits Cdc1, Cdc27 and Cdm1. Pol3 binds directly to the B-subunit, Cdc1, which in turn binds the C-subunit, Cdc27. Human Pol δ comprises the same four subunits, and the crystal structure was recently reported of a complex of human p50 and the N-terminal domain of p66, the human orthologues of Cdc1 and Cdc27, respectively. Results To gain insights into the structure and function of Cdc1, random and directed mutagenesis techniques were used to create a collection of thirty alleles encoding mutant Cdc1 proteins. Each allele was tested for function in fission yeast and for binding of the altered protein to Pol3 and Cdc27 using the two-hybrid system. Additionally, the locations of the amino acid changes in each protein were mapped onto the three-dimensional structure of human p50. The results obtained from these studies identify amino acid residues and regions within the Cdc1 protein that are essential for interaction with Pol3 and Cdc27 and for in vivo function. Mutations specifically defective in Pol3-Cdc1 interactions allow the identification of a possible Pol3 binding surface on Cdc1. Conclusion In the absence of a three-dimensional structure of the entire Pol δ complex, the results of this study highlight regions in Cdc1 that are vital for protein function in vivo and provide valuable clues to possible protein-protein interaction surfaces on the Cdc1 protein that will be important targets for further study.

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

Science.gov (United States)

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.

Tolerance of a Knotted Near-Infrared Fluorescent Protein to Random Circular Permutation.

Science.gov (United States)

Pandey, Naresh; Kuypers, Brianna E; Nassif, Barbara; Thomas, Emily E; Alnahhas, Razan N; Segatori, Laura; Silberg, Jonathan J

2016-07-12

Bacteriophytochrome photoreceptors (BphP) are knotted proteins that have been developed as near-infrared fluorescent protein (iRFP) reporters of gene expression. To explore how rearrangements in the peptides that interlace into the knot within the BphP photosensory core affect folding, we subjected iRFPs to random circular permutation using an improved transposase mutagenesis strategy and screened for variants that fluoresce. We identified 27 circularly permuted iRFPs that display biliverdin-dependent fluorescence in Escherichia coli. The variants with the brightest whole cell fluorescence initiated translation at residues near the domain linker and knot tails, although fluorescent variants that initiated translation within the PAS and GAF domains were discovered. Circularly permuted iRFPs retained sufficient cofactor affinity to fluoresce in tissue culture without the addition of biliverdin, and one variant displayed enhanced fluorescence when expressed in bacteria and tissue culture. This variant displayed a quantum yield similar to that of iRFPs but exhibited increased resistance to chemical denaturation, suggesting that the observed increase in the magnitude of the signal arose from more efficient protein maturation. These results show how the contact order of a knotted BphP can be altered without disrupting chromophore binding and fluorescence, an important step toward the creation of near-infrared biosensors with expanded chemical sensing functions for in vivo imaging.

Isolation of temperature-sensitive mutants of 16 S rRNA in Escherichia coli

DEFF Research Database (Denmark)

Triman, K; Becker, E; Dammel, C

1989-01-01

Temperature-sensitive mutants have been isolated following hydroxylamine mutagenesis of a plasmid containing Escherichia coli rRNA genes carrying selectable markers for spectinomycin resistance (U1192 in 16 S rRNA) and erythromycin resistance (G2058 in 23 S rRNA). These antibiotic resistance....... The mutations were localized by in vitro restriction fragment replacement followed by in vivo marker rescue and were identified by DNA sequence analysis. We report here seven single-base alterations in 16 S rRNA (A146, U153, A350, A359, A538, A1292 and U1293), five of which produce temperature......-sensitive spectinomycin resistance and two that produce unconditional loss of resistance. In each case, loss of ribosomal function can be accounted for by disruption of base-pairing in the secondary structure of 16 S rRNA. For the temperature-sensitive mutants, there is a lag period of about two generations between...

The influence of glycerol on γ-induced mutagenesis in Salmonella typhimurium cells

International Nuclear Information System (INIS)

Basha, S.G.; Krasavin, E.A.; Kozubek, S.; Amirtaev, K.G.

1990-01-01

A study was made of the modifying effect of glycerol on the survival rate and γ-radiation-induced mutagenesis of Salmonella typhimurium cells TA98, TA100 and TA102. The DMF value, with respect to the survival rate, was 2.05-0.20. The dependence of the yield of γ-radiation-induced mutants on radiation dose was described by the curve with a maximum; the mutation frequency M(D) was well described by a gradual function M(D)=kD x . DMF values of the induced mutagenesis amounted to 2 for strains TA100 and TA102, and 1.5 for strain TA98

Study on mutagenesis of Signal grass (Brachiaria decumbens) by gamma irradiation

International Nuclear Information System (INIS)

Abdul Rahim Harun; Shuhami Shamsuddin; Ghazali Hussin

2000-01-01

Before starting experiments on induced mutation breeding of crops it is essential to carry out radiosensitivity test when determining the optimum doses of every plant genotype. Factors such as moisture content and oxsigen availability could influence mutagenesis process through ionizing radiation. Many methodologies could be used for determining the effect of radiation on seed of plants such as the 'Sandwich blotter technique' and 'Sand bed technique'. Different species have different sensitivity to mutagenic agents. Even varieties of the same species show variations in sensitivity to irradiation. Brachiaria clecumbens was found to be less sensitive to gamma radiation. At a dose of 800 Gy, the percentage reduction in growth was around 40%. Early result has shown that the optimum doses for mutagenesis was between 700 to 900 Gy

CYTOTOXICITY AND MUTAGENESIS METHODS FOR EVALUATING TOXICITY REMOVAL FROM WASTEWATERS

Science.gov (United States)

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

Integrating structural and mutagenesis data to elucidate GPCR ligand binding

DEFF Research Database (Denmark)

Munk, Christian; Harpsøe, Kasper; Hauser, Alexander S

2016-01-01

is reported that exhibit activity through multiple receptors, binding in allosteric sites, and bias towards different intracellular signalling pathways. Furthermore, a wealth of single point mutants has accumulated in literature and public databases. Integrating these structural and mutagenesis data will help...

Escherichia coli pathotypes

Science.gov (United States)

Escherichia coli strains are important commensals of the intestinal tract of humans and animals; however, pathogenic strains, including diarrhea-inducing E. coli and extraintestinal pathogenic E. coli. Intestinal E. coli pathotypes may cause a dehydrating watery diarrhea, or more severe diseases su...

Visualization of tandem repeat mutagenesis in Bacillus subtilis.

Science.gov (United States)

Dormeyer, Miriam; Lentes, Sabine; Ballin, Patrick; Wilkens, Markus; Klumpp, Stefan; Kohlheyer, Dietrich; Stannek, Lorena; Grünberger, Alexander; Commichau, Fabian M

2018-03-01

Mutations are crucial for the emergence and evolution of proteins with novel functions, and thus for the diversity of life. Tandem repeats (TRs) are mutational hot spots that are present in the genomes of all organisms. Understanding the molecular mechanism underlying TR mutagenesis at the level of single cells requires the development of mutation reporter systems. Here, we present a mutation reporter system that is suitable to visualize mutagenesis of TRs occurring in single cells of the Gram-positive model bacterium Bacillus subtilis using microfluidic single-cell cultivation. The system allows measuring the elimination of TR units due to growth rate recovery. The cultivation of bacteria carrying the mutation reporter system in microfluidic chambers allowed us for the first time to visualize the emergence of a specific mutation at the level of single cells. The application of the mutation reporter system in combination with microfluidics might be helpful to elucidate the molecular mechanism underlying TR (in)stability in bacteria. Moreover, the mutation reporter system might be useful to assess whether mutations occur in response to nutrient starvation. Copyright © 2018 Elsevier B.V. All rights reserved.

Drug Resistance Patterns of Escherichia coli in Ethiopia: A Meta-Analysis.

Science.gov (United States)

Tuem, Kald Beshir; Gebre, Abadi Kahsu; Atey, Tesfay Mehari; Bitew, Helen; Yimer, Ebrahim M; Berhe, Derbew Fikadu

2018-01-01

Antimicrobial drug resistance is a global threat for treatment of infectious diseases and costs life and money and threatens health delivery system's effectiveness. The resistance of E. coli to frequently utilized antimicrobial drugs is becoming a major challenge in Ethiopia. However, there is no inclusive countrywide study. Therefore, this study intended to assess the prevalence of E. coli resistance and antimicrobial-specific resistance pattern among E. coli clinical isolates in Ethiopia. Articles were retrieved from PubMed, Embase, and grey literature from 2007 to 2017. The main outcome measures were overall E. coli and drug-specific resistance patterns. A random-effects model was used to determine pooled prevalence with 95% confidence interval (CI), using DerSimonian and Laird method. In addition, subgroup analysis was conducted to improve the outcome. The study bias was assessed by Begg's funnel plot. This study was registered in PROSPERO as follows: PROSPERO 2017: CRD42017070106. Of 164 articles retrieved, 35 articles were included. A total of 19,235 study samples participated in the studies and 2,635 E. coli strains were isolated. Overall, E. coli antibacterial resistance was 45.38% (95% confidence interval (CI): 33.50 to 57.27). The resistance pattern ranges from 62.55% in Addis Ababa to 27.51% in Tigray region. The highest resistance of E. coli reported was to ampicillin (83.81%) and amoxicillin (75.79%), whereas only 13.55% of E. coli isolates showed resistance to nitrofurantoin. E. coli antimicrobial resistance remains high with disparities observed among regions. The bacterium was found to be highly resistant to aminopenicillins. The finding implies the need for effective prevention strategies for the E. coli drug resistance and calls for multifaceted approaches with full involvement of all stakeholders.

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

Science.gov (United States)

Stumpf, Jeffrey D; Copeland, William C

2014-10-01

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

Genetic analysis of gamma-ray mutagenesis in yeast. I. Reversion in radiation-sensitive strains

International Nuclear Information System (INIS)

McKee, R.H.; Lawrence, C.W.

1979-01-01

The frequency of revertants induced by 60 Co γ rays of the ochre allele, cyc1-9, has been measured in radiation-sensitive strains carrying one of 19 nonallelic mutations and in wild-type strains. The results indicate that ionizing radiation mutagenesis depends on the activity of the RAD6 group of genes and that the gene functions employed are very similar, but probably not identical, to those that mediate uv mutagenesis. Repair activities dependent on the functions of the RAD50 through RAD57 loci, the major pathway for the repair of damage caused by ionizing radiation, do not appear to play any part in mutagenesis. A comparison between the γ-ray data and those obtained previously with uv and chemical mutagens suggests that the RAD6 mutagenic pathway is in fact composed of a set of processes, some of which are concerned with error-prone, and some with error-free, recovery activities

Lack of mutational hot spots during decitabine-mediated HIV-1 mutagenesis.

Science.gov (United States)

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

2015-11-01

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. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Chromosomal damages and mutagenesis in mammalian and human cells induced by ionizing radiations with different LET

International Nuclear Information System (INIS)

Govorun, R.D.

1997-01-01

On the basis of literature and proper data the inference was made about essential role of structural chromosomal (and gene) damages in spontaneous and radiation-induced mutagenesis of mammalian and human cells on HPRT-loci. The evidences of increasing role of these damages in the mutagenesis after the influence of ionizing radiations with high LET are adduced. The consequences of HPRT-gene damages have been examined hypothetically. The geterogeneity of mutant subclones on their cytogenetical properties were revealed experimentally. The data reflect a phenomenon of the reproductive chromosomal instability in many generations of mutant cell. The mutagenesis of mammalian cells is also accompanied by the impairment of chromosome integrity with high probability as a stage of appropriate genome reorganization because of changed vital conditions

Frequency distributions of Escherichia coli in the confectionery products offered in retail market in Isfahan

Directory of Open Access Journals (Sweden)

Rasoul Rezaei

2016-06-01

Full Text Available Introduction: Raw ingredients used in confectionary carry high risk of infection with Escherichia coli. Since confectionaries are offered in the market in quite varied forms and types and there is a great difference in the sanitary status of the confectionaries, this study aimed at evaluation of E.coli   frequency distribution in different types of confectionery products in Isfahan market. In addition, the effect of moisture content, products category and the sanitary level of the confectionaries as well as product types (Industrially or traditionally produced on the contamination level were studied. Materials and methods: A total of 200 samples were randomly collected from confectioneries in Isfahan city through simple random sampling method. Preparation and dilution procedures were conducted under sterile conditions. Samples were cultured on EMB agar medium. Later, some of the positive isolates were randomly selected and confirmed by TSI and IMVIC test. Data analysis was performed using descriptive indices. Also, one way ANOVA and LSD test or independent t test were applied to determine the statistically significant difference between the mean E.coli cell numbers in the categorized groups of sweets. Results: It was found that 19% of the total tested samples were contaminated with E.coli strains. The mean, median and maximum of contamination were (35±.5, (0 and (3.4 CFU/gr, respectively. Moisture content, products category and being traditionally or industrially produced have significant effects on the level of contamination; while, the sanitary status of the traditional confectionaries as graded in this study has no impact on the average E.coli cell count. Discussion and conclusion: Regarding the microbial quality, at least about 25% of the sweets in the market do not meet the national standards of confectionary products. Implementation of strict hygiene regulation in the traditional confectionaries is in need to provide the public with

Genetic analysis of gamma-ray mutagenesis in yeast. II. Allele-specific control of mutagenesis

International Nuclear Information System (INIS)

McKee, R.H.; Lawrence, C.W.

1979-01-01

We find that partially different sets of gene functions are required for the production of different kinds of mutations induced by 60 Co γ rays in Saccharomyces cerevisiae. This observation is very similar to others made previously with respect to uv mutagenesis and confirms the conclusion that such distinctive patterns of genetic control reflect properties of the test alleles and their genetic locations, rather than the kinds of lesions required to revert them. The data also support the model of mutagenic repair outlined in the first paper of this series in which partially different sets of gene functions are required for the production of different kinds of mutations, the formation of mutations at different genetic sites and the induction of mutations by different mutagens

Mutagenesis of the lac promoter region in M13 mp10 phage DNA by 4'-hydroxymethyl-4,5',8-trimethylpsoralen

International Nuclear Information System (INIS)

Piette, J.; Decuyper-Debergh, D.; Gamper, H.

1985-01-01

Double-stranded M13 phage DNA (M13 mp10 replicative form) was photoreacted with 4'-hydroxymethyl-4,5',8-trimethylpsoralen, using light of wavelength greater than 320 nm or greater than 390 nm to generate predominantly crosslinks or monoadducts, respectively. The damaged DNAs were scored for inactivation and mutagenesis after transfection into Escherichia coli. The appearance of light-blue or colorless plaques on indicator medium showed that mutation had occurred in the lac insert of the viral DNA. A comparison of the consequences of the two phototreatments with psoralen supports the idea that crosslinks are both more lethal and more mutagenic than monoadducts. Numerous mutant clones partially or totally deficient in beta-galactosidase were plaque-purified and amplified. The viral DNA of each clone was sequenced by the dideoxy chain-terminating procedure. All of the observed base-pair changes were mapped to the lac promoter region and consisted of 3 transition, 14 transversion, and 6 single base-pair frame-shift mutations. The predominant mutation was a T.A----G.C transversion

Membrane Topology and Biochemical Characterization of the Escherichia coli BacA Undecaprenyl-Pyrophosphate Phosphatase.

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Guillaume Manat

Full Text Available Several integral membrane proteins exhibiting undecaprenyl-pyrophosphate (C55-PP phosphatase activity were previously identified in Escherichia coli that belonged to two distinct protein families: the BacA protein, which accounts for 75% of the C55-PP phosphatase activity detected in E. coli cell membranes, and three members of the PAP2 phosphatidic acid phosphatase family, namely PgpB, YbjG and LpxT. This dephosphorylation step is required to provide the C55-P carrier lipid which plays a central role in the biosynthesis of various cell wall polymers. We here report detailed investigations of the biochemical properties and membrane topology of the BacA protein. Optimal activity conditions were determined and a narrow-range substrate specificity with a clear preference for C55-PP was observed for this enzyme. Alignments of BacA protein sequences revealed two particularly well-conserved regions and several invariant residues whose role in enzyme activity was questioned by using a site-directed mutagenesis approach and complementary in vitro and in vivo activity assays. Three essential residues Glu21, Ser27, and Arg174 were identified, allowing us to propose a catalytic mechanism for this enzyme. The membrane topology of the BacA protein determined here experimentally did not validate previous program-based predicted models. It comprises seven transmembrane segments and contains in particular two large periplasmic loops carrying the highly-conserved active site residues. Our data thus provide evidence that all the different E. coli C55-PP phosphatases identified to date (BacA and PAP2 catalyze the dephosphorylation of C55-PP molecules on the same (outer side of the plasma membrane.

Apoptosis-like death, an extreme SOS response in Escherichia coli.

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Erental, Ariel; Kalderon, Ziva; Saada, Ann; Smith, Yoav; Engelberg-Kulka, Hanna

2014-07-15

In bacteria, SOS is a global response to DNA damage, mediated by the recA-lexA genes, resulting in cell cycle arrest, DNA repair, and mutagenesis. Previously, we reported that Escherichia coli responds to DNA damage via another recA-lexA-mediated pathway resulting in programmed cell death (PCD). We called it apoptosis-like death (ALD) because it is characterized by membrane depolarization and DNA fragmentation, which are hallmarks of eukaryotic mitochondrial apoptosis. Here, we show that ALD is an extreme SOS response that occurs only under conditions of severe DNA damage. Furthermore, we found that ALD is characterized by additional hallmarks of eukaryotic mitochondrial apoptosis, including (i) rRNA degradation by the endoribonuclease YbeY, (ii) upregulation of a unique set of genes that we called extensive-damage-induced (Edin) genes, (iii) a decrease in the activities of complexes I and II of the electron transport chain, and (iv) the formation of high levels of OH˙ through the Fenton reaction, eventually resulting in cell death. Our genetic and molecular studies on ALD provide additional insight for the evolution of mitochondria and the apoptotic pathway in eukaryotes. Importance: The SOS response is the first described and the most studied bacterial response to DNA damage. It is mediated by a set of two genes, recA-lexA, and it results in DNA repair and thereby in the survival of the bacterial culture. We have shown that Escherichia coli responds to DNA damage by an additional recA-lexA-mediated pathway resulting in an apoptosis-like death (ALD). Apoptosis is a mode of cell death that has previously been reported only in eukaryotes. We found that E. coli ALD is characterized by several hallmarks of eukaryotic mitochondrial apoptosis. Altogether, our results revealed that recA-lexA is a DNA damage response coordinator that permits two opposite responses: life, mediated by the SOS, and death, mediated by the ALD. The choice seems to be a function of the degree

Lambda Red-mediated mutagenesis and efficient large scale affinity purification of the Escherichia coli NADH:ubiquinone oxidoreductase (complex I).

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Pohl, Thomas; Uhlmann, Mareike; Kaufenstein, Miriam; Friedrich, Thorsten

2007-09-18

The proton-pumping NADH:ubiquinone oxidoreductase, the respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. The Escherichia coli complex I consists of 13 different subunits named NuoA-N (from NADH:ubiquinone oxidoreductase), that are coded by the genes of the nuo-operon. Genetic manipulation of the operon is difficult due to its enormous size. The enzymatic activity of variants is obscured by an alternative NADH dehydrogenase, and purification of the variants is hampered by their instability. To overcome these problems the entire E. coli nuo-operon was cloned and placed under control of the l-arabinose inducible promoter ParaBAD. The exposed N-terminus of subunit NuoF was chosen for engineering the complex with a hexahistidine-tag by lambda-Red-mediated recombineering. Overproduction of the complex from this construct in a strain which is devoid of any membrane-bound NADH dehydrogenase led to the assembly of a catalytically active complex causing the entire NADH oxidase activity of the cytoplasmic membranes. After solubilization with dodecyl maltoside the engineered complex binds to a Ni2+-iminodiacetic acid matrix allowing the purification of approximately 11 mg of complex I from 25 g of cells. The preparation is pure and monodisperse and comprises all known subunits and cofactors. It contains more lipids than earlier preparations due to the gentle and fast purification procedure. After reconstitution in proteoliposomes it couples the electron transfer with proton translocation in an inhibitor sensitive manner, thus meeting all prerequisites for structural and functional studies.

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

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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.

The mutagenesis and breeding of high productive strains of streptomyces jingyangensis '5406'

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Qi Hongyan; Yin Xinyun

1988-03-01

The purpose of these experiments is to explore the mutagenesis rhythm and breed high productive strains of actinomycete '5406'. The single colony agar pieces of strain F 358 were treated with fast neutron and 60 Co-γ ray irradiation Two mutants have been selected from 20025 treated single colonies. The output of cytokinins from them is higher than from strain F 358 . The original strain 'Mu-Tan-al' rejuvenated by freezing was treated with several physical and chemical mutagens. The mutagenesis rhythm has been summed up tentatively. Eight mutants obtained from 93014 treated single colonies produced more '5406' antibiotics than that of strain 'Mu-Tan-al,. The effect of mutant 'N2-10-Ra3' was the best

Use of a Chimeric Hsp70 to Enhance the Quality of Recombinant Plasmodium falciparum S-Adenosylmethionine Decarboxylase Protein Produced in Escherichia coli

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Makhoba, Xolani Henry; Burger, Adélle; Coertzen, Dina; Zininga, Tawanda; Birkholtz, Lyn-Marie; Shonhai, Addmore

2016-01-01

S-adenosylmethionine decarboxylase (PfAdoMetDC) from Plasmodium falciparum is a prospective antimalarial drug target. The production of recombinant PfAdoMetDC for biochemical validation as a drug target is important. The production of PfAdoMetDC in Escherichia coli has been reported to result in unsatisfactory yields and poor quality product. The co-expression of recombinant proteins with molecular chaperones has been proposed as one way to improve the production of the former in E. coli. E. coli heat shock proteins DnaK, GroEL-GroES and DnaJ have previously been used to enhance production of some recombinant proteins. However, the outcomes were inconsistent. An Hsp70 chimeric protein, KPf, which is made up of the ATPase domain of E. coli DnaK and the substrate binding domain of P. falciparum Hsp70 (PfHsp70) has been previously shown to exhibit chaperone function when it was expressed in E. coli cells whose resident Hsp70 (DnaK) function was impaired. We proposed that because of its domain constitution, KPf would most likely be recognised by E. coli Hsp70 co-chaperones. Furthermore, because it possesses a substrate binding domain of plasmodial origin, KPf would be primed to recognise recombinant PfAdoMetDC expressed in E. coli. First, using site-directed mutagenesis, followed by complementation assays, we established that KPf with a mutation in the hydrophobic residue located in its substrate binding cavity was functionally compromised. We further co-expressed PfAdoMetDC with KPf, PfHsp70 and DnaK in E. coli cells either in the absence or presence of over-expressed GroEL-GroES chaperonin. The folded and functional status of the produced PfAdoMetDC was assessed using limited proteolysis and enzyme assays. PfAdoMetDC co-expressed with KPf and PfHsp70 exhibited improved activity compared to protein co-expressed with over-expressed DnaK. Our findings suggest that chimeric KPf may be an ideal Hsp70 co-expression partner for the production of recombinant plasmodial

A multicopy phr-plasmid increases the ultraviolet resistance of a recA strain of Escherichia coli

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Yamamoto, K.; Satake, M.; Shinagawa, H.

1984-01-01

It has been previously reported that the ultraviolet sensitivity of recA strains of Escherichia coli in the dark is suppressed by a plasmid pKY1 which carries the phr gene, suggesting that this is due to a novel effect of photoreactivating enzyme (PRE) of E. coli in the dark. In this work, it is observed that an increase of UV-resistance by pKY1 in the dark is not apparent in strains with a mutation in either uvrA, uvrB, uvrC, lexA, recBC or recF. The sensitivity of recA lexA and recA recBC multiple mutants to UV is suppressed by the plasmid but that of recA uvrA, recA uvrB and recA uvrC is not. Host-cell reactivation of UV-irradiated lambda phage is slightly more efficient in the recA/pKY1 strain compared with the parental recA strain. On the other hand, the recA and recA/pKY1 strains do not differ significantly in the following properties: Hfr recombination, induction of lambda by UV, and mutagenesis. It is suggested that dark repair of PRE is correlated with its capacity of excision repair. (Auth.)

Evaluation of genotoxicity induced by hydrogen peroxide in the presence of ions chelator Fe2+ (2,2'-dipyridyl) and of Cu2+(neocuproine), in Escherichia coli: involvement of DNA repair mechanisms in the bacteria survival

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Almeida, Carlos Eduardo Bonacossa de

1998-01-01

Prior incubation of the E. coli cultures with the iron ions chelator 2,2'-dipyridyl (1 mM) caused an intensification of the lethality and the mutagenesis induced by the hydrogen peroxide, mainly at high concentrations (20 mM). It was also detected an enhancement of DNA strand breaks in this condition. The addition of the copper ions chelator neocuproine blocked partially this phenomenon. The enzymes XthA and Nfo act alternatively in the repair of the lesions induced by H 2 O 2 in the presence of 2,2'-dipyridyl. H 2 O 2 can act synergistically with neocuproine in killing E. coli, causing an enhancement in DNA strand breaks. The recombinational repair, the UvrABC excinuclease and Fpg function appeared to participate in the repair of the synergistic lesions. (author)

Improved Biosurfactant Production by Bacillus subtilis SPB1 Mutant Obtained by Random Mutagenesis and Its Application in Enhanced Oil Recovery in a Sand System.

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Bouassida, Mouna; Ghazala, Imen; Ellouze-Chaabouni, Semia; Ghribi, Dhouha

2018-01-28

Biosurfactants or microbial surfactants are surface-active biomolecules that are produced by a variety of microorganisms. Biodegradability and low toxicity have led to the intensification of scientific studies on a wide range of industrial applications for biosurfactants in the field of environmental bioremediation as well as the petroleum industry and enhanced oil recovery. However, the major issues in biosurfactant production are high production cost and low yield. Improving the bioindustrial production processes relies on many strategies, such as the use of cheap raw materials, the optimization of medium-culture conditions, and selecting hyperproducing strains. The present work aims to obtain a mutant with higher biosurfactant production through applying mutagenesis on Bacillus subtilis SPB1 using a combination of UV irradiation and nitrous acid treatment. Following mutagenesis and screening on blood agar and subsequent formation of halos, the mutated strains were examined for emulsifying activity of their culture broth. A mutant designated B. subtilis M2 was selected as it produced biosurfactant at twice higher concentration than the parent strain. The potential of this biosurfactant for industrial uses was shown by studying its stability to environmental stresses such as pH and temperature and its applicability in the oil recovery process. It was practically stable at high temperature and at a wide range of pH, and it recovered above 90% of motor oil adsorbed to a sand sample.

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

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

1994-11-01

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

Escherichia coli in broiler chickens with airsacculitis

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Leandro S. Machado

2014-09-01

Full Text Available ABSTRACT. Machado L.S., do Nascimento E.R., Pereira V.L.A., Abreu D.L.C., Gouvea R. & Santos L.M.M. 2014. [Escherichia coli in broiler chickens with airsacculitis.] Escherichia coli em frangos de corte com aerossaculite. Revista Brasileira de Medicina Veterinária, 36(3:261-265, 2014. Departamento de Medicina Veterinária Preventiva e Saúde Pública, Faculdade de Veterinária, Universidade Federal Fluminense, Rua Dr. Vital Brazil Filho 64, Vital Brazil, Niterói, RJ 24230-340, Brazil. E-mail: leandromachadovet@yahoo.com.br The Brazilian poultry industry grows each year and becomes increasingly representative in the production and export of products. The health care with poultry have accompanied and favored this evolution, however, respiratory agents that affect the weight and carcass quality, continue to cause great damage to the poultry industry. Airsacculitis is considered the main cause of total and partial condemnation of carcasses of broilers, and has been attributed to Mycoplasmosis mostly caused by Mycoplasma gallisepticum (MG and Mycoplasma synoviae (MS and Escherichia coli. The aim of this study was to relate the positivity of MG / MS and E. coli detected by PCR as a risk factor for airsacculitis in condemnation of broilers in Health Inspection Service. We studied 30 broiler poultry slaughtered in a slaughterhouse under Federal Sanitary Inspection, located in the State of Rio de Janeiro. 30 chickens were randomly collected from different lots and tracheas obtained in each PCR. DNA was extracted by phenol-chloroform method and amplified using pairs of “primer”specific for MG, MS and E. coli. Of the 30 chickens analyzed by PCR, 30% (9/30 had lesions in air sacs. None of the birds showed infection with MG and/or MS PCR, however 33.3% (3/9 birds were positive for airsacculitis iss gene from E.coli. E.coli found in broiler chickens that were negative for mycoplasma airsacculitis, implying the presence of such bacteria may be sufficient

piggyBac transposon somatic mutagenesis with an activated reporter and tracker (PB-SMART for genetic screens in mice.

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Sean F Landrette

Full Text Available Somatic forward genetic screens have the power to interrogate thousands of genes in a single animal. Retroviral and transposon mutagenesis systems in mice have been designed and deployed in somatic tissues for surveying hematopoietic and solid tumor formation. In the context of cancer, the ability to visually mark mutant cells would present tremendous advantages for identifying tumor formation, monitoring tumor growth over time, and tracking tumor infiltrations and metastases into wild-type tissues. Furthermore, locating mutant clones is a prerequisite for screening and analyzing most other somatic phenotypes. For this purpose, we developed a system using the piggyBac (PB transposon for somatic mutagenesis with an activated reporter and tracker, called PB-SMART. The PB-SMART mouse genetic screening system can simultaneously induce somatic mutations and mark mutated cells using bioluminescence or fluorescence. The marking of mutant cells enable analyses that are not possible with current somatic mutagenesis systems, such as tracking cell proliferation and tumor growth, detecting tumor cell infiltrations, and reporting tissue mutagenesis levels by a simple ex vivo visual readout. We demonstrate that PB-SMART is highly mutagenic, capable of tumor induction with low copy transposons, which facilitates the mapping and identification of causative insertions. We further integrated a conditional transposase with the PB-SMART system, permitting tissue-specific mutagenesis with a single cross to any available Cre line. Targeting the germline, the system could also be used to conduct F1 screens. With these features, PB-SMART provides an integrated platform for individual investigators to harness the power of somatic mutagenesis and phenotypic screens to decipher the genetic basis of mammalian biology and disease.

The Mechanism of Nucleotide Excision Repair-Mediated UV-Induced Mutagenesis in Nonproliferating Cells

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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

Yeasts acquire resistance secondary to antifungal drug treatment by adaptive mutagenesis.

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David Quinto-Alemany

Full Text Available Acquisition of resistance secondary to treatment both by microorganisms and by tumor cells is a major public health concern. Several species of bacteria acquire resistance to various antibiotics through stress-induced responses that have an adaptive mutagenesis effect. So far, adaptive mutagenesis in yeast has only been described when the stress is nutrient deprivation. Here, we hypothesized that adaptive mutagenesis in yeast (Saccharomyces cerevisiae and Candida albicans as model organisms would also take place in response to antifungal agents (5-fluorocytosine or flucytosine, 5-FC, and caspofungin, CSP, giving rise to resistance secondary to treatment with these agents. We have developed a clinically relevant model where both yeasts acquire resistance when exposed to these agents. Stressful lifestyle associated mutation (SLAM experiments show that the adaptive mutation frequencies are 20 (S. cerevisiae -5-FC, 600 (C. albicans -5-FC or 1000 (S. cerevisiae--CSP fold higher than the spontaneous mutation frequency, the experimental data for C. albicans -5-FC being in agreement with the clinical data of acquisition of resistance secondary to treatment. The spectrum of mutations in the S. cerevisiae -5-FC model differs between spontaneous and acquired, indicating that the molecular mechanisms that generate them are different. Remarkably, in the acquired mutations, an ectopic intrachromosomal recombination with an 87% homologous gene takes place with a high frequency. In conclusion, we present here a clinically relevant adaptive mutation model that fulfils the conditions reported previously.

E coli enteritis

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... coli; Food poisoning - E. coli; E. coli diarrhea; Hamburger disease ... coleslaw or potato salad) that have been out of the refrigerator too ... reheated Fish or oysters Raw fruits or vegetables that have ...

Agrobacterium-mediated insertional mutagenesis in the mycorrhizal fungus Laccaria bicolor.

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Stephan, B I; Alvarez Crespo, M C; Kemppainen, M J; Pardo, A G

2017-05-01

Agrobacterium-mediated gene transfer (AMT) is extensively employed as a tool in fungal functional genomics and accordingly, in previous studies we used AMT on a dikaryotic strain of the ectomycorrhizal basidiomycete Laccaria bicolor. The interest in this fungus derives from its capacity to establish a symbiosis with tree roots, thereby playing a major role in nutrient cycling of forest ecosystems. The ectomycorrhizal symbiosis is a highly complex interaction involving many genes from both partners. To advance in the functional characterization of fungal genes, AMT was used on a monokaryotic L. bicolor. A collection of over 1200 transgenic strains was produced, of which 200 randomly selected strains were analyzed for their genomic T-DNA insertion patterns. By means of insertional mutagenesis, a number of transgenic strains were obtained displaying differential growth features. Moreover, mating with a compatible strain resulted in dikaryons that retained altered phenotypic features of the transgenic monokaryon. The analysis of the T-DNA integration pattern revealed mostly similar results to those reported in earlier studies, confirming the usefulness of AMT on different genetic backgrounds of L. bicolor. Taken together, our studies display the great versatility and potentiality of AMT as a tool for the genetic characterization of L. bicolor.

Tissue culture and mutagenesis of rain lily (zephyranthes)

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Mohd Nazir Basiran; Zaiton Ahmad; Shakinah Salleh; Shuhaimi Shamsudin; Aiza Shaliha Jamaludin

2004-01-01

There are three varieties of Zephyranthes used widely in landscaping due to their robust growth and attractive flowers in pink, yellow and white. Both in vivo and in vitro mutagenesis are an effective approach to increase the flower colour variations of Zephyranthes. In vitro propagation for the three varieties was attempted by using the induction medium developed by Sachar and Kapoor in 1959. The medium contains I ma of each indole 3-acetic acid (IAA), indole 3-butyric acid (IBA) and kinetin. Following surface sterilization of bulb scales, 17.8%, 10.5% and 10.7% of pink, white and yellow varieties respectively, were able to form small bulblets on the induction media. Further development of these bulblets into plantlets was also achieved on the same medium. Work is now being carried out to improve the efficiency of bulblet regeneration. Mutagenesis of Zephyranthes was initiated from bulbs of the pink varieties to develop new varieties with attractive combinations of flower colour and forms, shelf life and growth habits. These bulbs were irradiated using a gamma cell with a 60 Co source. Three variants with different flower colour and morphology have been achieved so far and are now being propagated in the nursery. (Author)

Mechanisms of Base Substitution Mutagenesis in Cancer Genomes

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Albino Bacolla

2014-03-01

Full Text Available 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.

Mechanisms of base substitution mutagenesis in cancer genomes.

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

2014-03-05

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.

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

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

2016-11-01

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

Regulation of mutagenesis by exogenous biological factors in the eukaryotic cell systems

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Lukash L. L.

2013-07-01

Full Text Available The representations of the mutations and the nature of spontaneous mutation process and mutagenesis induced by exogenous oncoviruses, DNAs and proteins-mitogens are analysed. Exogenous biological factors induce DNA damages in regulatory-informational way, acting on the cellular systems for maintenance of genetical stability. Molecular mechanisms are the same as at spontaneous mutagenesis but they are realized with the participation of alien genetical material. Among biological mutagens, the oncoviruses and mobile genetic elements (MGEs are distinguished as the strongest destabilizing factors which direct tumor transformation of somatic mammalian cells. Genetical reprogramming or changing the programs of gene expression at the differentiation of stem and progenitor cells under growth factors and citokines is probably followed by mutations and recombinations as well.

Scoring function to predict solubility mutagenesis

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Deutsch Christopher

2010-10-01

Full Text Available Abstract Background Mutagenesis is commonly used to engineer proteins with desirable properties not present in the wild type (WT protein, such as increased or decreased stability, reactivity, or solubility. Experimentalists often have to choose a small subset of mutations from a large number of candidates to obtain the desired change, and computational techniques are invaluable to make the choices. While several such methods have been proposed to predict stability and reactivity mutagenesis, solubility has not received much attention. Results We use concepts from computational geometry to define a three body scoring function that predicts the change in protein solubility due to mutations. The scoring function captures both sequence and structure information. By exploring the literature, we have assembled a substantial database of 137 single- and multiple-point solubility mutations. Our database is the largest such collection with structural information known so far. We optimize the scoring function using linear programming (LP methods to derive its weights based on training. Starting with default values of 1, we find weights in the range [0,2] so that predictions of increase or decrease in solubility are optimized. We compare the LP method to the standard machine learning techniques of support vector machines (SVM and the Lasso. Using statistics for leave-one-out (LOO, 10-fold, and 3-fold cross validations (CV for training and prediction, we demonstrate that the LP method performs the best overall. For the LOOCV, the LP method has an overall accuracy of 81%. Availability Executables of programs, tables of weights, and datasets of mutants are available from the following web page: http://www.wsu.edu/~kbala/OptSolMut.html.

Age-related susceptibility to infection with diarrheagenic E. coli in infants from peri-urban areas of Lima, Peru

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Ochoa, Theresa J.; Ecker, Lucie; Barletta, Francesca; Mispireta, Mónica L.; Gil, Ana I.; Contreras, Carmen; Molina, Margarita; Amemiya, Isabel; Verastegui, Hector; Hall, Eric R.; Cleary, Thomas G.; Lanata, Claudio F.

2009-01-01

Background Diarrheagenic E. coli are being recognized as important pediatric enteropathogens worldwide. However, it is unclear whether there are differences in age-related susceptibility to specific agents, especially among infants. Methods We conducted a passive surveillance diarrhea cohort study of 1034 children from 2 to 12 months of age in Lima, Perú. Control stool samples were collected from randomly selected children without diarrhea. All samples were analyzed for common enteric pathogens and for the diarrheagenic E. coli by a multiplex real-time PCR. Results The most commonly isolated pathogens from 1065 diarrheal episodes were the diarrheagenic E. coli (31%), including enteroaggregative (15.1%) and enteropathogenic E. coli (EPEC) (7.6%). Diarrheagenic E. coli, Campylobacter and rotavirus were more frequently isolated from infants ≥ 6m. Diffusely adherent E. coli and enterotoxigenic E. coli (ETEC) were more frequently isolated in diarrheal samples than in controls in older infants (pfactors and with increased exposure to contaminated food and water. PMID:19857163

Transcriptional mutagenesis: causes and involvement in tumor development

Science.gov (United States)

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

Photodynamic action of methylene blue: mutagenesis and synergism

International Nuclear Information System (INIS)

Capella, M.A.M.

1988-01-01

The associated mutagenesis and the interactions with physical agents in order to potencialize its biological effects are studied. The induction of mutation in bacterias due to photodynamic action of methylene blue is presented as well as the induction of single breaks in bacterial DNA and the relationship between the repair systems, especially the SOS one. The interaction of the photodynamic therapy with low intensity electric current is discussed. (M.A.C.) [pt

History of the science of mutagenesis from a personal perspective.

Science.gov (United States)

Malling, Heinrich V

2004-01-01

A career in the study of mutagenesis spanning 50 years is a gift few scientists have been bestowed. My tenure in the field started in 1953, the year the structure of DNA became known (Watson and Crick [1953]: Nature 171:737). Before that time, it was suspected that DNA was the genetic material based on the research of Oswald T. Avery (Avery et al. [1944]: J Exp Med 79:137), but many scientists still believed that proteins or polysaccharides could be the genetic material. The present article describes a lifetime of personal experience in the field of chemical mutagenesis. The methods used to treat viruses with chemical mutagens were well developed in the 1950s. Here I review the early use of nitrous acid and hydroxylamine as mutagens in eukaryotes, the development of methods for the metabolic activation of mutagens by microsomal preparations, and the selection of a mutant tester set for the qualitative characterization of the mutagenic activity of chemicals. These studies provided critical background information that was used by Bruce Ames in the development of his Salmonella/microsome assay, widely known as the Ames test (Ames et al. [1973]: Proc Nat Acad Sci USA 70:2281-2285). This article also describes how a set of diagnostic chemical mutagens was selected and used to identify the molecular nature of gene mutations. Today, DNA sequencing has replaced the use of diagnostic mutagens, but studies of this kind formed the foundation of modern mutation research. They also helped set the stage for the organization of the Environmental Mutagen Society and the Environmental Mutagen Information Center, which are described. The article ends with the development of mammalian single-cell mutation assays, the first system for studying in vivo mutagenesis using recoverable vectors in transgenic animals, other mutation assays in intact mammals, and my thoughts on the critically important area of germ cell mutagenesis. This narrative is not a complete autobiographical account

p21-ras effector domain mutants constructed by "cassette" mutagenesis

DEFF Research Database (Denmark)

Stone, J C; Vass, W C; Willumsen, B M

1988-01-01

A series of mutations encoding single-amino-acid substitutions within the v-rasH effector domain were constructed, and the ability of the mutants to induce focal transformation of NIH 3T3 cells was studied. The mutations, which spanned codons 32 to 40, were made by a "cassette" mutagenesis...

Using codon optimization, chaperone co-expression, and rational mutagenesis for production and NMR assignments of human eIF2α

International Nuclear Information System (INIS)

Ito, Takuhiro; Wagner, Gerhard

2004-01-01

Producing a well behaved sample at high concentration is one of the main hurdles when starting a new project on an interesting protein. Especially when one attempts to overexpress a eukaryotic protein in bacteria, some difficulties are encountered, such as low expression level, low solubility, or even lack of folded structure. Overexpression in prokaryotic systems is highly desirable for cost-effective production of different isotope-labeled samples needed for NMR studies. Here we describe generally applicable methods for obtaining highly concentrated protein samples efficiently. This approach was developed as we tried to produce a NMR-suitable sample of the 35 kDa human translation initiation factor eIF2α, a protein that expresses poorly in E. coli and has very low solubility. First, an E. coli codon-optimized gene was synthesized on a thermal cycler, which increased the expression level by a factor of two. Second, we used co-expression of bacterial chaperone proteins, which largely increased the fraction of correctly folded protein found in the soluble phase. Third, we used rational mutagenesis guided by both the sequence alignment among homologues and the homology of one domain to a known fold for improving solubility and stability of the target protein by tenfold. Combining all these methods made it possible to produce from a one-liter preparation a 0.5 mM sample of human eIF2α that showed well-resolved NMR spectra and enabled nearly complete assignment of the protein. These methods may be generally useful for studies of other eukaryotic proteins that are otherwise difficult to express and exhibit poor solubility

Cell-mediated mutagenesis and cell transformation of mammalian cells by chemical carcinogens

International Nuclear Information System (INIS)

Huberman, E.; Langenbach, R.

1977-01-01

We have developed a cell-mediated mutagenesis assay in which cells with the appropriate markers for mutagenesis are co-cultivated with either lethally irradiated rodent embryonic cells that can metabolize carcinogenic hydrocarbons or with primary rat liver cells that can metabolize chemicals carcinogenic to the liver. During co-cultivation, the reactive metabolites of the procarcinogen appear to be transmitted to the mutable cells and induce mutations in them. Assays of this type make it possible to demonstrate a relationship between carcinogenic potency of the chemicals and their ability to induce mutations in mammalian cells. In addition, by simultaneously comparing the frequencies of transformation and mutation induced in normal diploid hamster cells by benzo(a)pyrene (BP) and one of its metabolites, it is possible to estimate the genetic target size for cell transformation in vitro

Software-Supported USER Cloning Strategies for Site-Directed Mutagenesis and DNA Assembly

DEFF Research Database (Denmark)

Genee, Hans Jasper; Bonde, Mads Tvillinggaard; Bagger, Frederik Otzen

2015-01-01

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 ....

Improvement of lipid production by the oleaginous yeast Rhodosporidium toruloides through UV mutagenesis.

Science.gov (United States)

Yamada, Ryosuke; Kashihara, Tomomi; Ogino, Hiroyasu

2017-05-01

Oleaginous yeasts are considered a promising alternative lipid source for biodiesel fuel production. In this study, we attempted to improve the lipid productivity of the oleaginous yeast Rhodosporidium toruloides through UV irradiation mutagenesis and selection based on ethanol and H 2 O 2 tolerance or cerulenin, a fatty acid synthetase inhibitor. Glucose consumption, cell growth, and lipid production of mutants were evaluated. The transcription level of genes involved in lipid production was also evaluated in mutants. The ethanol and H 2 O 2 tolerant strain 8766 2-31M and the cerulenin resistant strain 8766 3-11C were generated by UV mutagenesis. The 8766 2-31M mutant showed a higher lipid production rate, and the 8766 3-11C mutant produced a larger amount of lipid and had a higher lipid production rate than the wild type strain. Transcriptional analysis revealed that, similar to the wild type strain, the ACL1 and GND1 genes were expressed at significantly low levels, whereas IDP1 and ME1 were highly expressed. In conclusion, lipid productivity in the oleaginous yeast R. toruloides was successfully improved via UV mutagenesis and selection. The study also identified target genes for improving lipid productivity through gene recombination.

Generation of Modified Pestiviruses by Targeted Recombination

DEFF Research Database (Denmark)

Rasmussen, Thomas Bruun; Friis, Martin Barfred; Risager, Peter Christian

involves targeted modification of viral cDNA genomes, cloned within BACs, by Red/ET recombination-mediated mutagenesis in E.coli DH10B cells. Using recombination-mediated mutagenesis for the targeted design, the work can be expedited and focused in principal on any sequence within the viral genome...

Uji in Vitro Penghambatan Aktivitas Escherichia coli dengan Tepung Cacing Tanah (Lumbricus rubellus

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H. Julendra

2007-04-01

Full Text Available This research was conducted to study the inhibition growth of E. coli by using earthworm (Lumbricus rubellus meal. The earthworm meal was used in various concentrations, i.e. 0, 25, 50, 75 and 100 mg of earthworm meal in 100 ml DMSO for 0%, 25%, 50%, 75% and 100% (w/v as treatments respectively. Data were analyzed by ANOVA in Randomized Complete Block Design. Duncan’s multiple range test and polynomials orthogonal were used. Inhibition effects were measured through agar well diffusion test. Results showed that earthworm meal contain antibacterial compound which inhibit E. coli activity. There was a significant difference (P0.05 with 75% (w/v. It is concluded that earthworm meal is capable to inhibit E. coli in-vitro at the optimum level of 50% (w/v.

E. Coli Infections

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E. coli is the name of a type of bacteria that lives in your intestines. Most types of E. coli are harmless. However, some types can make you ... type causes travelers' diarrhea. The worst type of E. coli causes bloody diarrhea, and can sometimes cause kidney ...

Investigations into the design principles in the chemotactic behavior of Escherichia coli.

Science.gov (United States)

Kim, Tae-Hwan; Jung, Sung Hoon; Cho, Kwang-Hyun

2008-01-01

Inspired by the recent studies on the analysis of biased random walk behavior of Escherichia coli[Passino, K.M., 2002. Biomimicry of bacterial foraging for distributed optimization and control. IEEE Control Syst. Mag. 22 (3), 52-67; Passino, K.M., 2005. Biomimicry for Optimization, Control and Automation. Springer-Verlag, pp. 768-798; Liu, Y., Passino, K.M., 2002. Biomimicry of social foraging bacteria for distributed optimization: models, principles, and emergent behaviors. J. Optim. Theory Appl. 115 (3), 603-628], we have developed a model describing the motile behavior of E. coli by specifying some simple rules on the chemotaxis. Based on this model, we have analyzed the role of some key parameters involved in the chemotactic behavior to unravel the underlying design principles. By investigating the target tracking capability of E. coli in a maze through computer simulations, we found that E. coli clusters can be controlled as target trackers in a complex micro-scale-environment. In addition, we have explored the dynamical characteristics of this target tracking mechanism through perturbation of parameters under noisy environments. It turns out that the E. coli chemotaxis mechanism might be designed such that it is sensitive enough to efficiently track the target and also robust enough to overcome environmental noises.

Is radiation an appropriate model for chemical mutagenesis and carcinogenesis

International Nuclear Information System (INIS)

Bond, V.P.

1982-01-01

This chapter attempts to show why the quadratic, or ''linear quadratic,'' relationship holds for organ dose-single cell radiation effects, and to explore the extension of this relationship to chemical exposures in general. Demonstrates that although the ''αD + βD 2 relationship'' may be unexpected for normal pharmacologicalmedical dose-response relationships, a linear, no-threshold curve of this kind is expected for all stochastic-type (accidental or risk) situations with health consequences (e.g. all common accidents) including exposure to ''low-level radiation'' (LLR). Discusses the stochastic or risk approach, relevant radiobiology, and the stochastic for chemicals. Assumes that even though actual mutational rates cannot be expected to apply to the relevance of Tradescantia or any other single cell system as a predictor for mutagenesis and carcinogenesis in animals and man, the cardinal principles of genetics largely transcend species and the particular environment in which the cell is located. Concludes that with regard to LLR, the curve shapes and other relationships developed for Tradescantia would be expected to apply in principle to animal and human mutagenesis and carcinogenesis

Genetic analysis of γ-ray mutagenesis in yeast. Vol. 3

International Nuclear Information System (INIS)

McKee, R.H.; Lawrence, C.W.

1980-01-01

Comparisons between the 60 Co γ-ray survival curves of diploid strains of the yeast Saccharomyces cerevisiae that are homozygous for two non-allelic radiation-sensitive mutations and the corresponding single-mutant diploids suggest that there are two main types of repair of ionizing radiation damage in this organism. The first, which is defined by the rad52 epistasis group, depends on the activities of the RAD50 through RAD57 genes and is responsible for repairing the larger amount of lethal damage. Previous work [22] shows that this type of repair is essentially error-free. The second, defined by the rad6 epistasis group, depends on the activities of the RAD6, RAD9, RAD18, REV1 and REV3 genes and repairs a smaller, though still substantial, amount of lethal damage. It is also responsible for induced mutagenesis [22,23]. Data for survival and mutation induction after irradiation in air and partial anoxia show that oxygen-dependent damage can be repaired by either of these two pathways. They also show similar oxygen-enhancement ratios for survival and mutagenesis. (orig.)

Evaluation of genotoxicity induced by hydrogen peroxide in the presence of ions chelator Fe{sup 2+} (2,2'-dipyridyl) and of Cu{sup 2+}(neocuproine), in Escherichia coli: involvement of DNA repair mechanisms in the bacteria survival; Avaliacao da genotoxicidade induzida pelo peroxido de hidrogenio na presenca dos quelantes de ions Fe{sup 2+} (2,2'-dipiridil) e de ions Cu{sup 2+} (neocuproina), em Escherichia coli: envolvimento de mecanismos de reparo de DNA na sobrevivencia bacteriana

Energy Technology Data Exchange (ETDEWEB)

Almeida, Carlos Eduardo Bonacossa de

1998-07-01

Prior incubation of the E. coli cultures with the iron ions chelator 2,2'-dipyridyl (1 mM) caused an intensification of the lethality and the mutagenesis induced by the hydrogen peroxide, mainly at high concentrations (20 mM). It was also detected an enhancement of DNA strand breaks in this condition. The addition of the copper ions chelator neocuproine blocked partially this phenomenon. The enzymes XthA and Nfo act alternatively in the repair of the lesions induced by H{sub 2}O{sub 2} in the presence of 2,2'-dipyridyl. H{sub 2}O{sub 2} can act synergistically with neocuproine in killing E. coli, causing an enhancement in DNA strand breaks. The recombinational repair, the UvrABC excinuclease and Fpg function appeared to participate in the repair of the synergistic lesions. (author)

Evaluation of genotoxicity induced by hydrogen peroxide in the presence of ions chelator Fe{sup 2+} (2,2'-dipyridyl) and of Cu{sup 2+}(neocuproine), in Escherichia coli: involvement of DNA repair mechanisms in the bacteria survival; Avaliacao da genotoxicidade induzida pelo peroxido de hidrogenio na presenca dos quelantes de ions Fe{sup 2+} (2,2'-dipiridil) e de ions Cu{sup 2+} (neocuproina), em Escherichia coli: envolvimento de mecanismos de reparo de DNA na sobrevivencia bacteriana

Energy Technology Data Exchange (ETDEWEB)

Almeida, Carlos Eduardo Bonacossa de

1998-07-01

Prior incubation of the E. coli cultures with the iron ions chelator 2,2'-dipyridyl (1 mM) caused an intensification of the lethality and the mutagenesis induced by the hydrogen peroxide, mainly at high concentrations (20 mM). It was also detected an enhancement of DNA strand breaks in this condition. The addition of the copper ions chelator neocuproine blocked partially this phenomenon. The enzymes XthA and Nfo act alternatively in the repair of the lesions induced by H{sub 2}O{sub 2} in the presence of 2,2'-dipyridyl. H{sub 2}O{sub 2} can act synergistically with neocuproine in killing E. coli, causing an enhancement in DNA strand breaks. The recombinational repair, the UvrABC excinuclease and Fpg function appeared to participate in the repair of the synergistic lesions. (author)

Dietary N-Carbamylglutamate Supplementation Boosts Intestinal Mucosal Immunity in Escherichia coli Challenged Piglets.

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Fengrui Zhang

Full Text Available N-carbamylglutamate (NCG has been shown to enhance performance in neonatal piglets. However, few studies have demonstrated the effect of NCG on the intestinal mucosal barrier. This study was conducted to determine the effects of dietary NCG supplementation on intestinal mucosal immunity in neonatal piglets after an Escherichia coli (E. coli challenge. New-born piglets (4 d old were assigned randomly to one of four treatments (n = 7, including (I sham challenge, (II sham challenge +50 mg/kg NCG, (III E. coli challenge, and (IV E. coli challenge +50 mg/kg NCG. On d 8, pigs in the E. coli challenge groups (III and IV were orally challenged with 5 mL of E. coli K88 (10(8 CFU/mL, whereas pigs in the sham challenge groups (I and II were orally dosed with an equal volume of water. On d 13, all piglets were sacrificed, and samples were collected and examined. The results show that average daily gain in the E. coli challenged piglets (III and IV was decreased (PE.coli<0.05. However, it tended to be higher in the NCG treated piglets (II and IV. Ileum secretory IgA, as well as IFN-γ, IL-2, IL-4 and IL-10 in ileal homogenates, were increased in E. coli challenged piglets (III and IV. Similarly, ileum SIgA and IL-10 levels, and CD4(+ percentage in NCG treated piglets (II and IV were higher than no-NCG treated piglets (PNCG<0.05. However, the IL-2 level was only decreased in the piglets of E. coli challenge + NCG group (IV compared with E. coli challenge group (III (P<0.05. No change in the IL-2 level of the sham challenged piglets (III was observed. In conclusion, dietary NCG supplementation has some beneficial effects on intestinal mucosal immunity in E. coli challenged piglets, which might be associated with stimulated lymphocyte proliferation and cytokine synthesis. Our findings have an important implication that NCG may be used to reduce diarrhea in neonatal piglets.

Mutagenesis of mNeptune Red-Shifts Emission Spectrum to 681-685 nm.

Science.gov (United States)

Li, ZhaoYang; Zhang, ZhiPing; Bi, LiJun; Cui, ZongQiang; Deng, JiaoYu; Wang, DianBing; Zhang, Xian-En

2016-01-01

GFP-like fluorescent proteins with diverse emission wavelengths have been developed through mutagenesis, offering many possible choices in cellular and tissue imaging, such as multi-targets imaging, deep tissue imaging that require longer emission wavelength. Here, we utilized a combined approach of random mutation and structure-based rational design to develop new NIR fluorescent proteins on the basis of a far-red fluorescent protein, mNeptune (Ex/Em: 600/650 nm). We created a number of new monomeric NIR fluorescent proteins with the emission range of 681-685 nm, which exhibit the largest Stocks shifts (77-80 nm) compared to other fluorescent proteins. Among them, mNeptune681 and mNeptune684 exhibit more than 30 nm redshift in emission relative to mNeptune, owing to the major role of the extensive hydrogen-bond network around the chromophore and contributions of individual mutations to the observed redshift. Furthermore, the two variants still maintain monomeric state in solution, which is a trait crucial for their use as protein tags. In conclusion, our results suggest that there is untapped potential for developing fluorescent proteins with desired properties.

Risk factors for ceftiofur resistance in Escherichia coli from Belgian broilers.

Science.gov (United States)

Persoons, D; Haesebrouck, F; Smet, A; Herman, L; Heyndrickx, M; Martel, A; Catry, B; Berge, A C; Butaye, P; Dewulf, J

2011-05-01

A cross-sectional study on 32 different Belgian broiler farms was performed in 2007 and 2008 to identify risk factors for ceftiofur resistance in Escherichia coli. On each farm, one E. coli colony was isolated from 30 random birds. Following susceptibility testing of 14 antimicrobials, an on-farm questionnaire was used to obtain information on risk factors. Using a multilevel logistic regression model two factors were identified at the animal level: resistance to amoxicillin and to trimethoprim-sulfonamide. On the farm level, besides antimicrobial use, seven management factors were found to be associated with the occurrence of ceftiofur resistance in E. coli from broilers: poor hygienic condition of the medicinal treatment reservoir, no acidification of drinking water, more than three feed changes during the production cycle, hatchery of origin, breed, litter material used, and treatment with amoxicillin. This study confirms that not only on-farm antimicrobial therapy, but also management- and hatchery-related factors influence the occurrence of antimicrobial resistance.

E. Coli

Science.gov (United States)

... for the bacteria's medical name Escherichia coli . The strange thing about these bacteria — and lots of other ... In some cases, E. coli poisoning can cause life-threatening kidney problems. What Can Kids Do? Adults ...

[SOS-repair--60 years].

Science.gov (United States)

Zavil'gel'skiĭ, G B

2013-01-01

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

Functional studies of human intestinal alkaline sphingomyelinase by deglycosylation and mutagenesis

DEFF Research Database (Denmark)

Wu, Jun; Hansen, Gert H; Nilsson, Ake

2005-01-01

in NPP are conserved, and those of the active core are modified. We examined the functional changes of the enzyme induced by deglycosylation and mutagenesis. Treating alk-SMase cDNA-transfected COS-7 cells with tunicamycin rendered the expressed enzyme completely inactive. Mutations of the five potential...

Effect of oral cranberry extract (standardized proanthocyanidin-A) in patients with recurrent UTI by pathogenic E. coli: a randomized placebo-controlled clinical research study.

Science.gov (United States)

Singh, Iqbal; Gautam, Lokesh Kumar; Kaur, Iqbal R

2016-09-01

To evaluate the effect of cranberry extract (PAC-A ~ proanthocyanidin-A) on the in vitro bacterial properties of uropathogenic (E. coli) and its efficacy/tolerability in patients with subclinical or uncomplicated recurrent UTI (r-UTI). After obtaining clearance from the ethics committee and administering a written informed consent, 72 patients with r-UTI were enrolled as per protocol (November 2011 to March 2013) in this prospective study, to randomly receive (PAC-A: group I, 36) or (placebo: group II, 36), for 12 weeks. Any change/reduction in the incidence of r-UTI at 12 weeks was construed to be the primary endpoint of this study. After 12 weeks, bacterial adhesion scoring decreased (0.28)/(2.14) in group I/II (p UTI decreased to 33.33 versus 88.89 % in group I/II (p UTI (dysuria, bacteriuria and pyuria). Larger randomized controlled trials are needed to elucidate the precise role, exact dose and optimal duration of PAC-A therapy in patients at risk of r-UTI.

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

Science.gov (United States)

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

2017-04-01

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

UV mutagenesis in E. coli with excision repair initiated by uvrABC or denV gene products

Energy Technology Data Exchange (ETDEWEB)

Bockrath, R; Hodes, M Z; Mosbaugh, P; Valerie, K; de Riel, J K

1988-03-01

Mutation frequency responses produced by ultraviolet light are compared in 4 closely related strains of E.coli B/r having the same tyr(Oc) allele and different excision-repair capabilities. The production of Tyr/sup +/ prototrophic mutants is classified into back-mutations and de novo or converted glutamine tRNA suppressor mutations to indicate different mutation events. Cells transformed with the plasmid pdenV-7 require larger exposures than the parent strains to produce comparable mutation frequency responses, indicating that DenV activity can repair mutatagenic photoproducts. When damage reduction by UvrABC or DenV is compared for each of the specific categories of mutation, the results are consistent with the idea that pyrimidine dimers infrequently or never target back-mutations of this allele, frequently target the de novo suppressor mutations, and extensively or exclusively target the converted suppressor mutations. This analysis is based on the distinction that UvrABC-initiated excision repair recognizes dimer and non-dimer photoproducts but that DenV-initiated repair recognizes only pyrimidine dimers. 44 refs.; 3 figs.; 2 tabs.

Pyridoxal 5'-phosphate is a slow tight binding inhibitor of E. coli pyridoxal kinase.

Directory of Open Access Journals (Sweden)

Mohini S Ghatge

Full Text Available Pyridoxal 5'-phosphate (PLP is a cofactor for dozens of B(6 requiring enzymes. PLP reacts with apo-B(6 enzymes by forming an aldimine linkage with the ε-amino group of an active site lysine residue, thus yielding the catalytically active holo-B(6 enzyme. During protein turnover, the PLP is salvaged by first converting it to pyridoxal by a phosphatase and then back to PLP by pyridoxal kinase. Nonetheless, PLP poses a potential toxicity problem for the cell since its reactive 4'-aldehyde moiety forms covalent adducts with other compounds and non-B(6 proteins containing thiol or amino groups. The regulation of PLP homeostasis in the cell is thus an important, yet unresolved issue. In this report, using site-directed mutagenesis, kinetic, spectroscopic and chromatographic studies we show that pyridoxal kinase from E. coli forms a complex with the product PLP to form an inactive enzyme complex. Evidence is presented that, in the inhibited complex, PLP has formed an aldimine bond with an active site lysine residue during catalytic turnover. The rate of dissociation of PLP from the complex is very slow, being only partially released after a 2-hour incubation with PLP phosphatase. Interestingly, the inactive pyridoxal kinase•PLP complex can be partially reactivated by transferring the tightly bound PLP to an apo-B(6 enzyme. These results open new perspectives on the mechanism of regulation and role of pyridoxal kinase in the Escherichia coli cell.

Modification of a deoxynivalenol-antigen-mimicking nanobody to improve immunoassay sensitivity by site-saturation mutagenesis.

Science.gov (United States)

Qiu, Yu-Lou; He, Qing-Hua; Xu, Yang; Wang, Wei; Liu, Yuan-Yuan

2016-01-01

A nanobody (N-28) which can act as a deoxynivalenol (DON) antigen has been generated, and its residues Thr102-Ser106 were identified to bind with anti-DON monoclonal antibody by alanine-scanning mutagenesis. Site-saturation mutagenesis was used to analyze the plasticity of five residues and to improve the sensitivity of the N-28-based immunoassay. After mutagenesis, three mutants were selected by phage immunoassay and were sequenced. The half-maximal inhibitory concentrations of the immunoassay based on mutants N-28-T102Y, N-28-V103L, and N-28-Y105F were 24.49 ± 1.0, 51.83 ± 2.5, and 35.65 ± 1.6 ng/mL, respectively, showing the assay was, respectively, 3.2, 1.5, and 2.2 times more sensitive than the wild-type-based assay. The best mutant, N-28-T102Y, was used to develop a competitive phage ELISA to detect DON in cereals with high specificity and accuracy. In addition, the structural properties of N-28-T102Y and N-28 were investigated, revealing that the affinity of N-28-T102Y decreased because of increased steric hindrance with the large side chain. The lower-binding-affinity antigen mimetic may contribute to the improvement of the sensitivity of competitive immunoassays. These results demonstrate that nanobodies would be a favorable tool for engineering. Moreover, our results have laid a solid foundation for site-saturation mutagenesis of antigen-mimicking nanobodies to improve immunoassay sensitivity for small molecules.

DNA MUTAGENESIS IN PANAX GINSENG CELL CULTURES

Directory of Open Access Journals (Sweden)

Kiselev K.V.

2012-08-01

Full Text Available At the present time, it is well documented that plant tissue culture induces a number of mutations and chromosome rearrangements termed “somaclonal variations”. However, little is known about the nature and the molecular mechanisms of the tissue culture-induced mutagenesis and the effects of long-term subculturing on the rate and specific features of the mutagenesis. The aim of the present study was to investigate and compare DNA mutagenesis in different genes of Panax ginseng callus cultures of different age. It has previously been shown that the nucleotide sequences of the Agrobacterium rhizogenes rolC locus and the selective marker nptII developed mutations during long-term cultivation of transgenic cell cultures of P. ginseng. In the present work, we analyzed nucleotide sequences of selected plant gene families in a 2-year-old and 20-year-old P. ginseng 1c cell culture and in leaves of cultivated P. ginseng plants. We analysed sequence variability between the Actin genes, which are a family of house-keeping genes; the phenylalanine ammonia-lyase (PAL and dammarenediol synthase (DDS genes, which actively participate in the biosynthesis of ginsenosides; and the somatic embryogenesis receptor kinase (SERK genes, which control plant development. The frequency of point mutations in the Actin, PAL, DDS, and SERK genes in the 2-year-old callus culture was markedly higher than that in cultivated plants but lower than that in the 20-year-old callus culture of P. ginseng. Most of the mutations in the 2- and 20-year-old P. ginseng calli were A↔G and T↔C transitions. The number of nonsynonymous mutations was higher in the 2- and 20-year-old callus cultures than the number of nonsynonymous mutations in the cultivated plants of P. ginseng. Interestingly, the total number of N→G or N→C substitutions in the analyzed genes was 1.6 times higher than the total number of N→A or N→T substitutions. Using methylation-sensitive DNA fragmentation

Advances in knowledge of mutagenesis at the molecular level in plants

International Nuclear Information System (INIS)

Nilan, R.A.; Owais, W.M.; Rosichan, J.L.; Kleinhofs, A.

1979-01-01

The mechanism of action of the densely and sparsely ionizing types of radiation in plant cells has been investigated. The indirect actions of physical mutagens in plant cells through the modifying influences of oxygen, water content, and temperature are well known. In terms of the nature of the mutations induced by physical mutagens in plants, a vast array of mutational events can be induced by neutrons, X-ray, gamma-ray, etc. It was determined that Na azides have been highly efficient mutagen in barley, peas, soybean, maize, Chlamydomonas, rice, yeast, Chinese hamster cells and certain strains of S. typhimurium and E. coli. Na azides were used as respiration inhibitor to investigate how chromosomes break and mutations are induced and/or repaired in the cells of irradiated barley seeds. Azides alone in the presence of O 2 induced about 6% mutation of chlorophyll-deficient seedlings which could be increased to 20% when the seeds had been treated in azide solutions at the pH values below the pKa of azides. When the seeds were presoaked for 15 hours at 1 deg C and 12 - 16 hrs at 20 deg C in aerated solution prior to azide treatment, 75% mutation was obtained. This mutation frequency is compared to 17% and 40 - 45% induced in barley by X-ray and ethyl methanesulfonate, respectively. It was shown that both L-cysteine and L-cysteine inhibited azide mutagenesis, and the inhibition seemed to be specific to azides. The waxy locus of barley controlling the starch composition is used to measure forward and reverse mutations and mutant recombination frequency on the grain per million pollen basis, and provides high genetic resolution and a detailed map of the genes. (Yamashita, S.)

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

Energy Technology Data Exchange (ETDEWEB)

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.

An evolved xylose transporter from Zymomonas mobilis enhances sugar transport in Escherichia coli

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Zhang Jingqing

2009-12-01

Full Text Available Abstract Background Xylose is a second most abundant sugar component of lignocellulose besides glucose. Efficient fermentation of xylose is important for the economics of biomass-based biorefineries. However, sugar mixtures are sequentially consumed in xylose co-fermentation with glucose due to carbon catabolite repression (CCR in microorganisms. As xylose transmembrance transport is one of the steps repressed by CCR, it is therefore of interest to develop a transporter that is less sensitive to the glucose inhibition or CCR. Results The glucose facilitator protein Glf transporter from Zymomonas mobilis, also an efficient transporter for xylose, was chosen as the target transporter for engineering to eliminate glucose inhibition on xylose uptake. The evolution of Glf transporter was carried out with a mixture of glucose and xylose in E. coli. Error-prone PCR and random deletion were employed respectively in two rounds of evolution. Aided by a high-throughput screening assay using xylose analog p-nitrophenyl-β-D-xylopyranoside (pNPX in 96-well plates, a best mutant 2-RD5 was obtained that contains several mutations, and a deletion of 134 residues (about 28% of total residues, or three fewer transmembrane sections (TMSs. It showed a 10.8-fold improvement in terms of pNPX transport activity in the presence of glucose. The fermentation performance results showed that this mutant improved xylose consumption by 42% with M9 minimal medium containing 20 g L-1 xylose only, while with the mixture sugar of xylose and glucose, 28% more glucose was consumed, but no obvious co-utilization of xylose was observed. Further glucose fed-batch experiments suggested that the intracellular metabolism of xylose was repressed by glucose. Conclusions Through random mutagenesis and partial deletion coupled with high-throughput screening, a mutant of the Glf transporter (2-RD5 was obtained that relieved the inhibition of xylose transport by glucose. The fermentation

Chromosomal features of Escherichia coli serotype O2:K2, an avian pathogenic E. coli

DEFF Research Database (Denmark)

Jørgensen, Steffen L; Kudirkiene, Egle; Li, Lili

2017-01-01

Escherichia coli causing infection outside the gastrointestinal system are referred to as extra-intestinal pathogenic E. coli. Avian pathogenic E. coli is a subgroup of extra-intestinal pathogenic E. coli and infections due to avian pathogenic E. coli have major impact on poultry production econo...

Escherichia coli challenge and one type of smectite alter intestinal barrier of pigs.

Science.gov (United States)

Almeida, Juliana Abranches Soares; Liu, Yanhong; Song, Minho; Lee, Jeong Jae; Gaskins, H Rex; Maddox, Carol Wolfgang; Osuna, Orlando; Pettigrew, James Eugene

2013-12-20

An experiment was conducted to determine how an E. coli challenge and dietary clays affect the intestinal barrier of pigs. Two groups of 32 pigs (initial BW: 6.9 ± 1.0 kg) were distributed in a 2 × 4 factorial arrangement of a randomized complete block design (2 challenge treatments: sham or E. coli, and 4 dietary treatments: control, 0.3% smectite A, 0.3% smectite B and 0.3% zeolite), with 8 replicates total. Diarrhea score, growth performance, goblet cell size and number, bacterial translocation from intestinal lumen to lymph nodes, intestinal morphology, and relative amounts of sulfo and sialo mucins were measured. The E. coli challenge reduced performance, increased goblet cell size and number in the ileum, increased bacterial translocation from the intestinal lumen to the lymph nodes, and increased ileal crypt depth. One of the clays (smectite A) tended to increase goblet cell size in ileum, which may indicate enhanced protection. In conclusion, E. coli infection degrades intestinal barrier integrity but smectite A may enhance it.

Characterization of two new CTX-M-25-group extended-spectrum β-lactamase variants identified in Escherichia coli isolates from Israel.

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Jascha Vervoort

Full Text Available OBJECTIVES: We characterized two new CTX-M-type extended-spectrum β-lactamase (ESBL variants in Escherichia coli isolates from stool samples of two elderly patients admitted at the Tel Aviv Sourasky Medical Center, Israel. Both patients underwent treatment with cephalosporins prior to isolation of the E. coli strains. METHODS: ESBLs were detected by the double-disk synergy test and PCR-sequencing of β-lactamase genes. The bla(CTX-M genes were cloned into the pCR-BluntII-TOPO vector in E. coli TOP10. The role of amino-acid substitutions V77A and D240G was analyzed by site-directed mutagenesis of the bla(CTX-M-94 and bla(CTX-M-100 genes and comparative characterization of the resulting E. coli recombinants. MICs of β-lactams were determined by Etest. Plasmid profiling, mating experiments, replicon typing and sequencing of bla(CTX-M flanking regions were performed to identify the genetic background of the new CTX-M variants. RESULTS: The novel CTX-M β-lactamases, CTX-M-94 and -100, belonged to the CTX-M-25-group. Both variants differed from CTX-M-25 by the substitution V77A, and from CTX-M-39 by D240G. CTX-M-94 differed from all CTX-M-25-group enzymes by the substitution F119L. Glycine-240 was associated with reduced susceptibility to ceftazidime and leucine-119 with increased resistance to ceftriaxone. bla(CTX-M-94 and bla(CTX-M-100 were located within ISEcp1 transposition units inserted into ∼93 kb non-conjugative IncFI and ∼130 kb conjugative IncA/C plasmids, respectively. The plasmids carried also different class 1 integrons. CONCLUSIONS: This is the first report on CTX-M-94 and -100 ESBLs, novel members of the CTX-M-25-group.

Molecular characterization of Escherichia coli recovered from traditional milk products in Kashan, Iran

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Farhad Sharafati Chaleshtori

2017-10-01

Full Text Available Aim: Shiga toxigenic Escherichia coli (STEC strains as emerging groups of foodborne pathogens are responsible for most foodborne illnesses. The aim of this study was to determine the antibiotic resistance pattern in STEC isolated from traditional milk products and their molecular characterization. Materials and Methods: A total of 116 samples were randomly purchased from local markets in Kashan, Iran, and evaluated for the occurrence of STEC by culturing and molecular methods. The antibiotic resistance of obtained isolates was determined by Kirby Bauer method. Furthermore, isolates were assayed for the presence of Shiga toxins (stx1 and stx2 and intimin gene (eae. Results: The incidence of E. coli in 60 ice cream, 30 yoghurt, and 26 cheese samples was 8.33%, 10%, and 11.54%, respectively. The findings showed that 11 out of 11 (100% E. coli had both stx1 and stx2 while eae gene was not found in E. coli isolated of traditional milk products. For E. coli strains carrying stx1 and stx2, highest antibiotic sensitive levels were related to trimethoprim/sulfamethoxazole, norfloxacin, chloramphenicol, and ciprofloxacin, respectively. Conclusion: The results showed relationship between the presence of virulence factors and antimicrobial resistance. These results can be used for further studies on STEC as an emerging foodborne pathogen.

E. Coli and Pregnancy

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... chat Live Help Fact Sheets Share Escherichia coli (E. coli) Friday, 01 September 2017 In every pregnancy, a ... risk. This sheet talks about whether exposure to E. coli may increase the risk for birth defects over ...

Systematic mutagenesis method for enhanced production of bacitracin by Bacillus licheniformis mutant strain UV-MN-HN-6

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Muhammad Nauman Aftab

2012-03-01

Full Text Available The purpose of the current study was intended to obtain the enhanced production of bacitracin by Bacillus licheniformis through random mutagenesis and optimization of various parameters. Several isolates of Bacillus licheniformis were isolated from local habitat and isolate designated as GP-35 produced maximum bacitracin production (14±0.72 IU ml-1. Bacitracin production of Bacillus licheniformis GP-35 was increased to 23±0.69 IU ml-1 after treatment with ultraviolet (UV radiations. Similarly, treatment of vegetative cells of GP-35 with chemicals like N-methyl N'-nitro N-nitroso guanidine (MNNG and Nitrous acid (HNO2 increased the bacitracin production to a level of 31±1.35 IU ml-1 and 27±0.89 IU ml-1 respectively. Treatment of isolate GP-35 with combined effect of UV and chemical treatment yield significantly higher titers of bacitracin with maximum bacitracin production of 41.6±0.92 IU ml-1. Production of bacitracin was further enhanced (59.1±1.35 IU ml-1 by optimization of different parameters like phosphate sources, organic acids as well as temperature and pH. An increase of 4.22 fold in the production of bacitracin after mutagenesis and optimization of various parameters was achieved in comparison to wild type. Mutant strain was highly stable and produced consistent yield of bacitracin even after 15 generations. On the basis of kinetic variables, notably Yp/s (IU/g substrate, Yp/x (IU/g cells, Yx/s (g/g, Yp/s, mutant strain B. licheniformis UV-MN-HN-6 was found to be a hyperproducer of bacitracin.

Loss of BRCA1 or BRCA2 markedly increases the rate of base substitution mutagenesis and has distinct effects on genomic deletions

DEFF Research Database (Denmark)

Zamborszky, J.; Szikriszt, B.; Gervai, J. Z.

2017-01-01

-genome sequencing of multiple isogenic chicken DT40 cell clones to precisely determine the consequences of BRCA1/2 loss on all types of genomic mutagenesis. Spontaneous base substitution mutation rates increased sevenfold upon the disruption of either BRCA1 or BRCA2, and the arising mutation spectra showed strong...... of stalled replication forks as the cause of increased mutagenesis. The high rate of base substitution mutagenesis demonstrated by our experiments is likely to significantly contribute to the oncogenic effect of the inactivation of BRCA1 or BRCA2....

Chromosomal features of Escherichia coli serotype O2:K2, an avian pathogenic E. coli.

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Jørgensen, Steffen L; Kudirkiene, Egle; Li, Lili; Christensen, Jens P; Olsen, John E; Nolan, Lisa; Olsen, Rikke H

2017-01-01

Escherichia coli causing infection outside the gastrointestinal system are referred to as extra-intestinal pathogenic E. coli. Avian pathogenic E. coli is a subgroup of extra-intestinal pathogenic E. coli and infections due to avian pathogenic E. coli have major impact on poultry production economy and welfare worldwide. An almost defining characteristic of avian pathogenic E. coli is the carriage of plasmids, which may encode virulence factors and antibiotic resistance determinates. For the same reason, plasmids of avian pathogenic E. coli have been intensively studied. However, genes encoded by the chromosome may also be important for disease manifestation and antimicrobial resistance. For the E. coli strain APEC_O2 the plasmids have been sequenced and analyzed in several studies, and E. coli APEC_O2 may therefore serve as a reference strain in future studies. Here we describe the chromosomal features of E. coli APEC_O2. E. coli APEC_O2 is a sequence type ST135, has a chromosome of 4,908,820 bp (plasmid removed), comprising 4672 protein-coding genes, 110 RNA genes, and 156 pseudogenes, with an average G + C content of 50.69%. We identified 82 insertion sequences as well as 4672 protein coding sequences, 12 predicated genomic islands, three prophage-related sequences, and two clustered regularly interspaced short palindromic repeats regions on the chromosome, suggesting the possible occurrence of horizontal gene transfer in this strain. The wildtype strain of E. coli APEC_O2 is resistant towards multiple antimicrobials, however, no (complete) antibiotic resistance genes were present on the chromosome, but a number of genes associated with extra-intestinal disease were identified. Together, the information provided here on E. coli APEC_O2 will assist in future studies of avian pathogenic E. coli strains, in particular regarding strain of E. coli APEC_O2, and aid in the general understanding of the pathogenesis of avian pathogenic E. coli .

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

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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

Improved thermostability and enzyme activity of a recombinant phyA mutant phytase from Aspergillus niger N25 by directed evolution and site-directed mutagenesis.

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Tang, Zizhong; Jin, Weiqiong; Sun, Rong; Liao, Yan; Zhen, Tianrun; Chen, Hui; Wu, Qi; Gou, Lin; Li, Chenlei

2018-01-01

We previously constructed three recombinant phyA mutant strains (PP-NP m -8, PP-NP ep -6A and I44E/T252R-PhyA), showing improved catalytic efficiency or thermostability of Aspergillus niger N25 phytase, by error-prone PCR or site-directed mutagenesis. In this study, directed evolution and site-directed mutagenesis were further applied to improve the modified phytase properties. After one-round error-prone PCR for phytase gene of PP-NP ep -6A, a single transformant, T195L/Q368E/F376Y, was obtained with the significant improvements in catalytic efficiency and thermostability. The phytase gene of T195L/Q368E/F376Y, combined with the previous mutant phytase genes of PP-NP ep -6A, PP-NP m -8 and I44E/T252R-PhyA, was then sequentially modified by DNA shuffling. Three genetically engineered strains with desirable properties were then obtained, namedQ172R, Q172R/K432R andQ368E/K432R. Among them, Q172R/K432R showed the highest thermostability with the longest half-life and the greatest remaining phytase activity after heat treatment, while Q368E/K432R showed the highest catalytic activity. Five substitutions (Q172R, T195L, Q368E, F376Y, K432R) identified from random mutagenesis were added sequentially to the phytase gene of PP-NP ep -6A to investigate how the mutant sites influence the properties of phytase. Characterization and structural analysis demonstrated that these mutations could produce cumulative or synergistic improvements in thermostability or catalytic efficiency of phytase. Copyright © 2017 Elsevier Inc. All rights reserved.

Human Meningitis-Associated Escherichia coli

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KIM, KWANG SIK

2016-01-01

E. coli is the most common Gram-negative bacillary organism causing meningitis and E. coli meningitis continues to be an important cause of mortality and morbidity throughout the world. Our incomplete knowledge of its pathogenesis contributes to such mortality and morbidity. Recent reports of E. coli strains producing CTX-M-type or TEM-type extended-spectrum β-lactamases create a challenge. Studies using in vitro and in vivo models of the blood-brain barrier have shown that E. coli meningitis follows a high-degree of bacteremia and invasion of the blood-brain barrier. E. coli invasion of the blood-brain barrier, the essentials step in the development of E. coli meningitis, requires specific microbial and host factors as well as microbe- and host-specific signaling molecules. Blockade of such microbial and host factors contributing to E. coli invasion of the blood-brain barrier is shown to be efficient in preventing E. coli penetration into the brain. The basis for requiring a high-degree of bacteremia for E. coli penetration of the blood-brain barrier, however, remains unclear. Continued investigation on the microbial and host factors contributing to a high-degree of bacteremia and E. coli invasion of the blood-brain barrier is likely to identify new targets for prevention and therapy of E. coli meningitis. PMID:27223820

Diarrheagenic Escherichia coli Markers and Phenotypes among Fecal E. coli Isolates Collected from Nicaraguan Infants ▿

OpenAIRE

Reyes, Daniel; Vilchez, Samuel; Paniagua, Margarita; Colque-Navarro, Patricia; Weintraub, Andrej; Möllby, Roland; Kühn, Inger

2010-01-01

We analyzed the prevalence of diarrheagenic Escherichia coli (DEC) markers and common phenotypes in 2,164 E. coli isolates from 282 DEC-positive samples. Enteropathogenic E. coli (EPEC) and enteroaggregative E. coli (EAEC) were very diverse and were not correlated with diarrhea. Enterotoxigenic E. coli (ETEC) estA and enterohemorrhagic E. coli (EHEC) belonged to a few phenotypes and were significantly correlated with diarrhea.

Enhancing activity and thermostability of lipase A from Serratia marcescens by site-directed mutagenesis.

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Mohammadi, Mohsen; Sepehrizadeh, Zargham; Ebrahim-Habibi, Azadeh; Shahverdi, Ahmad Reza; Faramarzi, Mohammad Ali; Setayesh, Neda

2016-11-01

Lipases as significant biocatalysts had been widely employed to catalyze various chemical reactions such as ester hydrolysis, ester synthesis, and transesterification. Improving the activity and thermostability of enzymes is desirable for industrial applications. The lipase of Serratia marcescens belonging to family I.3 lipase has a very important pharmaceutical application in production of chiral precursors. In the present study, to achieve improved lipase activity and thermostability, using computational predictions of protein, four mutant lipases of SML (MutG2P, MutG59P, Mut H279K and MutL613WA614P) were constructed by site-directed mutagenesis. The recombinant mutant proteins were over-expressed in E. coli and purified by affinity chromatography on the Ni-NTA system. Circular dichroism spectroscopy, differential scanning calorimetry and kinetic parameters (Km and kcat) were determined. Our results have shown that the secondary structure of all lipases was approximately similar to one another. The MutG2P and MutG59P were more stable than wild type by approximately 2.3 and 2.9 in T 1/2 , respectively. The catalytic efficiency (kcat/Km) of MutH279K was enhanced by 2-fold as compared with the wild type (p<0.05). These results indicate that using protein modeling program and creating mutation, can enhance lipase activity and/or thermostability of SML and it also could be used for improving other properties of enzyme to the desired requirements as well as further mutations. Copyright © 2016 Elsevier Inc. All rights reserved.

Characterization of highly efficient heavy-ion mutagenesis in Arabidopsis thaliana

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Kazama Yusuke

2011-11-01

Full Text Available Abstract 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

Effects of chloramphenicol and caffeine on postreplication repair in uvrA-umuC- and uvrA-recF- strains of Escherichia coli K-12

International Nuclear Information System (INIS)

Kato, T.

1977-01-01

Postreplication repair and its inhibition by chloramphenicol and caffeine, as seen in alkaline sucrose gradients, were compared between a UV nonmutable strain uvrA - umuC - and normally mutable strains uvrA - recF - and uvrA - umu + rec + of Escherichia coli K-12. The uvrA - umuC - strain performed postreplication repair as efficiently as the parental strain, while the repair in uvrA - recF - strain was dependent on UV dose. Both chloramphenicol and caffeine inhibited postreplication repair to an equal extent of about 25%, and 10%, respectively, in all three uvrA strains of umuC36, recF and umu + rec + . These observations suggest that postreplication repair is largely not responsible for UV mutagenesis. (orig.) [de

Effect of bile on growth, peritoneal absorption, and blood clearance of Escherichia coli in E coli peritonitis

International Nuclear Information System (INIS)

Andersson, R.; Schalen, C.; Tranberg, K.G.

1991-01-01

The effect of intraperitoneal bile on growth, peritoneal absorption, and clearance of Escherichia coli was determined in E coli peritonitis in the rat. In E coli peritonitis, intraperitoneal bacterial counts gradually decreased, whereas they increased (after 2 hours) with subsequent development of bacteremia in E coli plus bile peritonitis. After an intraperitoneal injection of labeled bacteria, blood radioactivity was only initially lower in E coli plus bile peritonitis compared with E coli peritonitis. Clearance from blood was lower in E coli plus bile peritonitis than in E coli peritonitis. Organ localization was similar in E coli peritonitis and E coli plus bile peritonitis with decreased splenic, increased pulmonary, and unchanged hepatic uptakes compared with controls. Impaired peritoneal absorption of bacteria, together with impaired local host defense, is likely to enhance the noxious effect of bile in E coli peritonitis

Conjugation in Escherichia coli

Science.gov (United States)

Boyer, Herbert

1966-01-01

Boyer, Herbert (Yale University, New Haven, Conn.). Conjugation in Escherichia coli. J. Bacteriol. 91:1767–1772. 1966.—The sex factor of Escherichia coli K-12 was introduced into an E. coli B/r strain by circumventing the host-controlled modification and restriction incompatibilities known to exist between these closely related strains. The sexual properties of the constructed F+ B strain and its Hfr derivatives were examined. These studies showed that the E. coli strain B/r F+ and Hfr derivatives are similar to the E. coli strain K-12 F+ and Hfr derivatives. However, the site of sex factor integration was found to be dependent on the host genome. PMID:5327905

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

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Grégory Caignard

2014-09-01

Full Text Available 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.

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

Science.gov (United States)

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.

A mouse model of hereditary coproporphyria identified in an ENU mutagenesis screen

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Ashlee J. Conway

2017-08-01

Full Text Available A genome-wide ethyl-N-nitrosourea (ENU mutagenesis screen in mice was performed to identify novel regulators of erythropoiesis. Here, we describe a mouse line, RBC16, which harbours a dominantly inherited mutation in the Cpox gene, responsible for production of the haem biosynthesis enzyme, coproporphyrinogen III oxidase (CPOX. A premature stop codon in place of a tryptophan at amino acid 373 results in reduced mRNA expression and diminished protein levels, yielding a microcytic red blood cell phenotype in heterozygous mice. Urinary and faecal porphyrins in female RBC16 heterozygotes were significantly elevated compared with that of wild-type littermates, particularly coproporphyrinogen III, whereas males were biochemically normal. Attempts to induce acute porphyric crises were made using fasting and phenobarbital treatment on females. While fasting had no biochemical effect on RBC16 mice, phenobarbital caused significant elevation of faecal coproporphyrinogen III in heterozygous mice. This is the first known investigation of a mutagenesis mouse model with genetic and biochemical parallels to hereditary coproporphyria.

Efficient Mutagenesis Independent of Ligation (EMILI).

Science.gov (United States)

Füzik, Tibor; Ulbrich, Pavel; Ruml, Tomáš

2014-11-01

Site-directed mutagenesis is one of the most widely used techniques in life sciences. Here we describe an improved and simplified method for introducing mutations at desired sites. It consists of an inverse PCR using a plasmid template and two partially complementary primers. The synthesis step is followed by annealing of the PCR product's sticky ends, which are generated by exonuclease digestion. This method is fast, extremely efficient and cost-effective. It can be used to introduce large insertions and deletions, but also for multiple point mutations in a single step. To show the principle and to prove the efficiency of the method, we present a series of basic mutations (insertions, deletions, point mutations) on pUC19 plasmid DNA. Copyright © 2014 Elsevier B.V. All rights reserved.

Mutagenic DNA repair in Escherichia coli: Pt. 17

International Nuclear Information System (INIS)

Sharif, F.; Bridges, B.A.

1990-01-01

In contrast to the dnaE485 mutation, which is nearly 'dead-stop', dnaE1026 allows DNA synthesis for some time at restrictive temperatures. When bacteria carrying the dnaE486 or dnaE1026 temperature-sensitive mutations were incubated at restrictive temperature after exposure to UV light they showed little or no fixation of mutations as determined by loss of photoreversibility and the mutation frequency fell progressively. These results confirm a role for DnaE protein in UV mutagenesis. A derivative of dnaE1026 carrying the umuC122 allele which blocks normal UV mutagenesis showed induction of mutations when photoreversing light was given to UV-irradiated bacteria after a period of incubation at either permissive or restrictive temperature. The defective DnaE1026 protein is therefore able to carry out the misincorporations postulated to be the first step in the mutagenic process. Its inability to give rise to mutations in umu + bacteria may therefore be attributed to its inability to participate in a later step. In contrast, UV-irradiated dnaE486 umuC122 bacteria did not show mutagenesis when incubated at restrictive temperature before photoreversal, suggesting that the altered DnaE486 protein was not able to carry out the postulate misincorporation step at 43 0 C. DNA polymerase III α-subunit therefore appears to be required for both the misincorporation and bypass steps in the two-step model for UV mutagenesis. (Author)

Spontaneous mutability and light-induced mutagenesis in Salmonella typhimurium: effects of an R-plasmid

International Nuclear Information System (INIS)

Valdivia, L.

1979-01-01

The UV-protecting plasmid R46 was transferred by conjugation to a genetically marked mouse-virulent Salmonella typhimurium strain, not derived from LT2; in this host the plasmid conferred UV protection and enhanced UV mutagenesis just as it does in LT2 lines. Tra - derivatives of R46 encountered during transduction retained UV-protecting and mutagenesis-enhancing ability. Stored strains carrying the R46-derived plasmids with strong mutator effect but not UV-protecting had lost most of their original streptomycin resistance but were slightly resistant to spectinomycin; attempts to transfer such plasmids failed. R46 enhanced the weak mutagenic effect of visible light on several his and trp mutants of strain LT2, including some whose frequency of spontaneous reversion was not increased by the plasmid. A mutagenic effect was produced by visible-light irradiation of hisG46(R46), either growing cells or nonmultiplying (histidine-deprived cells at 10 0 C). Presence of catalase or cyanide during irradiation did not prevent mutagenesis, which excludes some hypothetical mechanisms. Visible-light irradiation of hisG46 or hisG46(R46) under strict anaerobiosis had little or no mutagenic effect (controls showed that revertants if produced would have been detected). This is as expected if visible-light irradiation in air causes photodynamic damage to DNA and mutations are produced during error-prone, plasmid-enhanced repair

Mutagenesis of the somaclones in vitro of cut roses by 60Co γ-rays irradiation

International Nuclear Information System (INIS)

Qu Suping; Su Yan; Wang Lihua; Tang Kaixue; Wang Jihua; Zhang Hao

2009-01-01

Mutagenesis of cut rose in vitro irradiated by 60 Co γ-rays was studied. The callus of leaves and regenerations adventitious bud was used as the explants for mutagenesis. Effect of 60 Co γ-rays irradiation on the callus's regeneration rate, adventitious bud's multiplication rate and vegetal status were studied. The results showed that the regeneration frequency of callus was decreased by 60 Co γ-rays irradiation. The regenerations adventitious bud was the better experimental materials compared with the callus of leaves. The lethal dose was 122 Gy and the semi-lethal dose was 76 Gy according to the regression equation. The appropriate dose on adventitious bud by irradiation rays was 50-60 Gy. (authors)

Mutagenesis of Jatropha curcas - Exploring new traits in the breeding of a biofuel plant

International Nuclear Information System (INIS)

Azhar Mohamad; Sobri Hussein; Abdul Rahim Harun

2010-01-01

Mutagenesis in plant species is considered effective in recovering and producing useful mutants as it leads to a high degree of chimerism and produces high degree of somaclonal variations for further selection in breeding programmes. Jatropha curcas is a species with many attributes and considerable potential, especially as bio diesel. Narrow genetic background of Jatropha spp. gives less selection to growers for better quality plant materials. In this study, a new method through nuclear technology was used to increase the genetic variability of Jatropha towards novel superior potential mutant lines. The objective of the study is to generate new mutant varieties of Jatropha curcas through the mutagenesis approach in getting new sustainable mutants for high oil yield and improved plant characteristics. Seeds of a Jatropha cultivar were from selected materials from Lembaga Kenaf and Tembakau Negara, Kelantan. Radiosensitivity test was done by irradiating a total of each 60 seeds at multiple doses (0 Gy, 20 Gy, 40 Gy, 60 Gy, 80 Gy, 100 Gy, 200 Gy, 300 Gy, 400 Gy, 600 Gy and 700 Gy). After getting the LD 50 , three doses i.e. 250 Gy, 300 Gy and 350 Gy were selected for mutagenesis, where a total of 1000 seeds were exposed to gamma radiation. The seeds were hardened and field planted at close distance of 1 m x 1 m. Pruning was conducted three times at two months interval prior to screening for early flowering, short stature and high branching mutant lines. Radiosensitivity of seeds to acute gamma irradiation revealed that the LD 50 was at 320 Gy. At nursery stage, somatic mutations related to chlorophyll changes were observed on leaves with certain shapes. Screening of Jatropha via seed mutagenesis bore 6 early flowering mutants, 7 dwarf mutants and, 17 high branching plants. In narrowing the mutant lines, cuttings from each selected trait were collected and re-planted for further evaluation. (author)

Radiation mutagenesis in selection of apple trees

International Nuclear Information System (INIS)

Kolontaev, V.M.; Kolontaev, Yu.V.

1977-01-01

After X-radiation of grafts of antonovka apple trees, three groups of morphological mutants, namely, weak-, average- and violently-growing, have been revealed. Although the mutation spectrum has some indefinite character a dose of 6 kR causes, more frequently and in a greater number, the weak-growing mutants, and a dose of 2 kR, the violently-growing ones. Mutants of each group differ in the precociousness (precocious and latefruiting), type of fruiting (nospur and spur) and yield (high- and low-yielding). Using the method of radiation mutagenesis it is possible to rise the frequency and spectrum of somatic mutability of antonovka apple trees and to induce forms having valuable features

Genetic relatedness of commensal Escherichia coli from nursery pigs in intensive pig production in Denmark and molecular characterization of genetically different strains

DEFF Research Database (Denmark)

Herrero Fresno, Ana; Larsen, Inge; Olsen, John Elmerdahl

2015-01-01

AIMS: To determine the genetic relatedness and the presence of virulence and antibiotic resistance genes in commensal Escherichia coli from nursery pigs in Danish intensive production. METHODS AND RESULTS: The genetic diversity of 1000 E. coli strains randomly picked (N = 50 isolates) from cultured...... in depth the genetic variability of commensal E. coli from pigs in Danish intensive pig production. A tendency for higher diversity was observed with in nursery pigs that were treated with zinc oxide only, in absence of other antimicrobials. Strains with potential to disseminate virulence and antibiotic...

Alleles conferring improved fiber quality from EMS mutagenesis of elite cotton genotypes

Science.gov (United States)

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...

A Novel Indirect Sequence Readout Component in the E. coli Cyclic AMP Receptor Protein Operator

DEFF Research Database (Denmark)

Lindemose, Søren; Nielsen, Peter Eigil; Valentin-Hansen, Poul

2014-01-01

binding sites in the E. coli genome, but the exact role of the N6 region in CRP interaction has not previously been systematic examined. Here we employ an in vitro selection system based on a randomized N6 spacer region to demonstrate that CRP binding to the lacP1 site may be enhanced up to 14-fold......The cyclic AMP receptor protein (CRP) from Escherichia coli has been extensively studied for several decades. In particular, a detailed characterization of CRP interaction with DNA has been obtained. The CRP dimer recognizes a consensus sequence AANTGTGANNNNNNTCACANTT through direct amino acid...

Atomic resolution structure of the E. coli YajR transporter YAM domain

Energy Technology Data Exchange (ETDEWEB)

Jiang, Daohua [National Laboratory of Macromolecules, National Center of Protein Science-Beijing, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101 (China); School of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei 430074 (China); Zhao, Yan [National Laboratory of Macromolecules, National Center of Protein Science-Beijing, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101 (China); School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027 (China); Fan, Junping; Liu, Xuehui; Wu, Yan; Feng, Wei [National Laboratory of Macromolecules, National Center of Protein Science-Beijing, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101 (China); Zhang, Xuejun C., E-mail: zhangc@ibp.ac.cn [National Laboratory of Macromolecules, National Center of Protein Science-Beijing, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101 (China)

2014-07-25

Highlights: • We report the crystal structure of the YAM domain of YajR transporter at 1.07 Å. • The YAM dimerization is related to the halogen-dependent high thermal stability. • A belt of poly-pentagonal water molecules was observed in the dimer interface. - Abstract: YajR is an Escherichia coli transporter that belongs to the major facilitator superfamily. Unlike most MFS transporters, YajR contains a carboxyl terminal, cytosolic domain of 67 amino acid residues termed YAM domain. Although it is speculated that the function of this small soluble domain is to regulate the conformational change of the 12-helix transmembrane domain, its precise regulatory role remains unclear. Here, we report the crystal structure of the YAM domain at 1.07-Å resolution, along with its structure determined using nuclear magnetic resonance. Detailed analysis of the high resolution structure revealed a symmetrical dimer in which a belt of well-ordered poly-pentagonal water molecules is embedded. A mutagenesis experiment and a thermal stability assay were used to analyze the putative role of this dimerization in response to changes in halogen concentration.

Fluorescence Spectral Properties of All4261 Binding with Phycocyanobilin in E.Coli

Science.gov (United States)

Ma, Q.; Zheng, X. J.; Zhou, Z.; Zhou, N.; Zhao, K. H.; Zhou, M.

2014-07-01

Cyanobacteriochromes (CBCRs) are chromophorylated proteins that acting as sensory photoreceptors in cyanobacteria. Based on the bioinformatics of All4261 in Nostoc sp. PCC7120, All4261 is a CBCR apoprotein composed of GAF domains in the N-terminal region. Via polymerase chain reaction with specific primers, All4261 was amplified with genome DNA of Nostoc sp. PCC7120 as template and then subcloned into the expression vector pET30(a+). To survey the fluorescence spectral properties, All4261 was coexpressed with the plasmid that catalyzes phycocyanobilin (PCB) biosynthesis, pACYC-ho1-pcyA, in E.coli BL21. Fluorescence emission spectra and excitation spectra showed that chromophorylated cells containing All4261-PCB had a fluorescence emission peak at 645 nm and a fluorescence excitation peak at 550 nm, but no reversible photoconversion. In order to identify the binding site of PCB in All4261, we obtained three variants All4261(C296L), All4261(C328A), and All4261(C339L), via sitedirected mutagenesis. The binding site was identified as C339 based on the lack of PCB binding of All4261(C339L).

ANTIMICROBIAL DRUG RESISTANCE IN STRAINS OF Escherichia coli ISOLATED FROM FOOD SOURCES

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Mohammed Uddin Rasheed

2014-07-01

Full Text Available A variety of foods and environmental sources harbor bacteria that are resistant to one or more antimicrobial drugs used in medicine and agriculture. Antibiotic resistance in Escherichia coli is of particular concern because it is the most common Gram-negative pathogen in humans. Hence this study was conducted to determine the antibiotic sensitivity pattern of E. coli isolated from different types of food items collected randomly from twelve localities of Hyderabad, India. A total of 150 samples comprising; vegetable salad, raw egg-surface, raw chicken, unpasteurized milk, and raw meat were processed microbiologically to isolate E. coli and to study their antibiotic susceptibility pattern by the Kirby-Bauer method. The highest percentages of drug resistance in isolates of E. coli were detected from raw chicken (23.3% followed by vegetable salad (20%, raw meat (13.3%, raw egg-surface (10% and unpasteurized milk (6.7%. The overall incidence of drug resistant E. coli was 14.7%. A total of six (4% Extended Spectrum β-Lactamase (ESBL producers were detected, two each from vegetable salads and raw chicken, and one each from raw egg-surface and raw meat. Multidrug resistant strains of E. coli are a matter of concern as resistance genes are easily transferable to other strains. Pathogen cycling through food is very common and might pose a potential health risk to the consumer. Therefore, in order to avoid this, good hygienic practices are necessary in the abattoirs to prevent contamination of cattle and poultry products with intestinal content as well as forbidding the use of untreated sewage in irrigating vegetables.

Effects of chlortetracycline and copper supplementation on antimicrobial resistance of fecal Escherichia coli from weaned pigs.

Science.gov (United States)

Agga, G E; Scott, H M; Amachawadi, R G; Nagaraja, T G; Vinasco, J; Bai, J; Norby, B; Renter, D G; Dritz, S S; Nelssen, J L; Tokach, M D

2014-06-01

Feed-grade chlortetracycline (CTC) and copper are both widely utilized in U.S. pig production. Cluster randomized experiment was conducted to evaluate the effects of CTC and copper supplementation in weaned pigs on antimicrobial resistance (AMR) among fecal Escherichia coli. Four treatment groups: control, copper, CTC, or copper plus CTC were randomly allocated to 32 pens with five pigs per pen. Fecal samples were collected weekly from three pigs per pen for six weeks. Two E. coli isolates per fecal sample were tested for phenotypic and genotypic resistance against antibiotics and copper. Data were analyzed with multilevel mixed effects logistic regression, multivariate probit analysis and discrete time survival analysis. CTC-supplementation was significantly (99% [95% CI=98-100%]) associated with increased tetracycline resistance compared to the control group (95% [95% CI=94-97%]). Copper supplementation was associated with decreased resistance to most of the antibiotics tested, including cephalosporins, over the treatment period. Overall, 91% of the E. coli isolates were multidrug resistant (MDR) (resistant to ≥3 antimicrobial classes). tetA and blaCMY-2 genes were positively associated (PpcoD were negatively associated with MDR. tetA and blaCMY-2 were positively associated with each other and in turn, these were negatively associated with both tetB and pcoD genes; which were also positively associated with one another. Copper minimum inhibitory concentration was not affected by copper supplementation or by pcoD gene carriage. CTC supplementation was significantly associated with increased susceptibilities of E. coli to copper (HR=7 [95% CI=2.5-19.5]) during treatment period. In conclusion, E. coli isolates from the nursery pigs exhibited high levels of antibiotic resistance, with diverse multi-resistant phenotypic profiles. The roles of copper supplementation in pig production, and pco-mediated copper resistance among E. coli in particular, need to be further

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

Science.gov (United States)

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

1989-01-01

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

Differences in mutagenic and recombinational DNA repair in enterobacteria

International Nuclear Information System (INIS)

Sedgwick, S.G.; Goodwin, P.A.

1985-01-01

The incidence of recombinational DNA repair and inducible mutagenic DNA repair has been examined in Escherichia coli and 11 related species of enterobacteria. Recombinational repair was found to be a common feature of the DNA repair repertoire of at least 6 genera of enterobacteria. This conclusion is based on observations of (i) damage-induced synthesis of RecA-like proteins, (ii) nucleotide hybridization between E. coli recA sequences and some chromosomal DNAs, and (iii) recA-negative complementation by plasmids showing SOS-inducible expression of truncated E. coli recA genes. The mechanism of DNA damage-induced gene expression is therefore sufficiently conserved to allow non-E. coli regulatory elements to govern expression of these cloned truncated E. coli recA genes. In contrast, the process of mutagenic repair, which uses umuC+ umuD+ gene products in E. coli, appeared less widespread. Little ultraviolet light-induced mutagenesis to rifampicin resistance was detected outside the genus Escherichia, and even within the genus induced mutagenesis was detected in only 3 out of 6 species. Nucleotide hybridization showed that sequences like the E. coli umuCD+ gene are not found in these poorly mutable organisms. Evolutionary questions raised by the sporadic incidence of inducible mutagenic repair are discussed

Effects of the umuC36 mutation on ultraviolet-radiation-induced base-change and frameshift mutations in Escherichia coli

International Nuclear Information System (INIS)

Kato, T.; Nakano, E.

1981-01-01

The effects of the umuC36 mutation on the induction of base-change and frameshift mutations were studied. An active umuC gene was necessary in either the uvr + or uvr - strains of Escherichia coli K12 for UV- and X-ray-induced mutations to His + , ColE and Spc, which are presumably base-change mutations, but it was not essential for ethyl methanesulphonate or N-methyl-N'-nitro-N-nitrosoguanidine-induced His + mutations. In contrast, only 1 out of 13 trp - frameshift mutations examined was UV reversible, and the process of mutagenesis was umuC + -dependent, whereas a potent frameshift mutagen, ICR191, effectively induced Trp + mutations in most of the strains regardless of the umu + or umuC genetic background. These results suggest that base substitutions are a major mutational type derived from the umuC + -dependent pathway of error-prone repair. (orig.)

A Genetic Algorithms Based Approach for Identification of Escherichia coli Fed-batch Fermentation

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Olympia Roeva

2004-10-01

Full Text Available This paper presents the use of genetic algorithms for identification of Escherichia coli fed-batch fermentation process. Genetic algorithms are a directed random search technique, based on the mechanics of natural selection and natural genetics, which can find the global optimal solution in complex multidimensional search space. The dynamic behavior of considered process has known nonlinear structure, described with a system of deterministic nonlinear differential equations according to the mass balance. The parameters of the model are estimated using genetic algorithms. Simulation examples for demonstration of the effectiveness and robustness of the proposed identification scheme are included. As a result, the model accurately predicts the process of cultivation of E. coli.

Codon cassette mutagenesis: a general method to insert or replace individual codons by using universal mutagenic cassettes.

Science.gov (United States)

Kegler-Ebo, D M; Docktor, C M; DiMaio, D

1994-05-11

We describe codon cassette mutagenesis, a simple method of mutagenesis that uses universal mutagenic cassettes to deposit single codons at specific sites in double-stranded DNA. A target molecule is first constructed that contains a blunt, double-strand break at the site targeted for mutagenesis. A double-stranded mutagenic codon cassette is then inserted at the target site. Each mutagenic codon cassette contains a three base pair direct terminal repeat and two head-to-head recognition sequences for the restriction endonuclease Sapl, an enzyme that cleaves outside of its recognition sequence. The intermediate molecule containing the mutagenic cassette is then digested with Sapl, thereby removing most of the mutagenic cassette, leaving only a three base cohesive overhang that is ligated to generate the final insertion or substitution mutation. A general method for constructing blunt-end target molecules suitable for this approach is also described. Because the mutagenic cassette is excised during this procedure and alters the target only by introducing the desired mutation, the same cassette can be used to introduce a particular codon at all target sites. Each cassette can deposit two different codons, depending on the orientation in which it is inserted into the target molecule. Therefore, a series of eleven cassettes is sufficient to insert all possible amino acids at any constructed target site. Thus codon cassettes are 'off-the-shelf' reagents, and this methodology should be a particularly useful and inexpensive approach for subjecting multiple different positions in a protein sequence to saturation mutagenesis.

Pecularities of mutagenesis of T4Br bacteriophage under the direct and indirect radiation effects

International Nuclear Information System (INIS)

Yurov, S.S.

1975-01-01

Different lethal and mutagenic effects were shown when bacteriophage T4Br + (470 r/min) was irradiated in broth (direct effect) and a buffer solution (direct and indirect action). The survival rate of the bacteriophage in the buffer solution was 0.1 percent for a dose rate of 60 kr; in the broth it was 10 percent. The frequency of mutation of the bacteriophage also showed the greater effect of the irradiation in the buffer solution than in the broth (25 and 5 r-mutants respectively at a dose rate of 10 kr). An analysis of the ratio of the r-groups when the bacteriophage was treated in various ways revealed differences between mutagenesis produced in the broth and the buffer, and spontaneous mutagenesis. (V.A.P.)

Cloacael Carriage and Multidrug Resistance Escherichia coli O157:H7 from Poultry Farms, Eastern Ethiopia.

Science.gov (United States)

Shecho, Mude; Thomas, Naod; Kemal, Jelalu; Muktar, Yimer

2017-01-01

A cross-sectional study was carried out to determine antimicrobial drug resistance patterns of E. coli O157:H7 isolates and estimate the level of the pathogen. A total of 194 cloacae swab samples were collected randomly in two poultry farms. Standard cultural, biochemical, and serological (latex agglutination) methods were used to isolate E. coli O157:H7. The isolates were subjected to antimicrobial susceptibility testing using disc diffusion method. Out of 194 cloacae samples examined, 13.4% ( n = 26) were found to be positive for E. coli O157:H7. The finding indicated differences in E. coli O157:H7 infection among the different risk factors. Chicken from Adele Poultry Farm showed higher E. coli O157:H7 infection (OR = 3.89) than Haramaya University poultry farm and young birds had more infection (OR = 4.62) than adult birds. Of the total 14 antimicrobials included in the panel of study, the susceptibility results were varied with 96.15% and 0% E. coli O157:H7 isolates expressing resistance to erythromycin, clindamycin, spectinomycin, and ciprofloxacin, respectively. Multidrug resistance to more than two antimicrobial agents was detected in 24 (92.30%) of the isolates. The study showed high presence of antimicrobial resistant isolates of E. coli O157:H7. Further study is required to better understand the ecology and evolution of bacterial resistance to antimicrobial agents.

Cloacael Carriage and Multidrug Resistance Escherichia coli O157:H7 from Poultry Farms, Eastern Ethiopia

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Mude Shecho

2017-01-01

Full Text Available A cross-sectional study was carried out to determine antimicrobial drug resistance patterns of E. coli O157:H7 isolates and estimate the level of the pathogen. A total of 194 cloacae swab samples were collected randomly in two poultry farms. Standard cultural, biochemical, and serological (latex agglutination methods were used to isolate E. coli O157:H7. The isolates were subjected to antimicrobial susceptibility testing using disc diffusion method. Out of 194 cloacae samples examined, 13.4% (n=26 were found to be positive for E. coli O157:H7. The finding indicated differences in E. coli O157:H7 infection among the different risk factors. Chicken from Adele Poultry Farm showed higher E. coli O157:H7 infection (OR = 3.89 than Haramaya University poultry farm and young birds had more infection (OR = 4.62 than adult birds. Of the total 14 antimicrobials included in the panel of study, the susceptibility results were varied with 96.15% and 0% E. coli O157:H7 isolates expressing resistance to erythromycin, clindamycin, spectinomycin, and ciprofloxacin, respectively. Multidrug resistance to more than two antimicrobial agents was detected in 24 (92.30% of the isolates. The study showed high presence of antimicrobial resistant isolates of E. coli O157:H7. Further study is required to better understand the ecology and evolution of bacterial resistance to antimicrobial agents.

Targeted mutagenesis in tetraploid switchgrass (Panicum virgatum L.) using CRISPR/Cas9.

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Liu, Yang; Merrick, Paul; Zhang, Zhengzhi; Ji, Chonghui; Yang, Bing; Fei, Shui-Zhang

2018-02-01

The CRISPR/Cas9 system has become a powerful tool for targeted mutagenesis. Switchgrass (Panicum virgatum L.) is a high yielding perennial grass species that has been designated as a model biomass crop by the U.S. Department of Energy. The self-infertility and high ploidy level make it difficult to study gene function or improve germplasm. To overcome these constraints, we explored the feasibility of using CRISPR/Cas9 for targeted mutagenesis in a tetraploid cultivar 'Alamo' switchgrass. We first developed a transient assay by which a non-functional green-fluorescent protein gene containing a 1-bp frameshift insertion in its 5' coding region was successfully mutated by a Cas9/sgRNA complex resulting in its restored function. Agrobacterium-mediated stable transformation of embryogenic calli derived from mature caryopses averaged a 3.0% transformation efficiency targeting the genes of teosinte branched 1(tb1)a and b and phosphoglycerate mutase (PGM). With a single construct containing two sgRNAs targeting different regions of tb1a and tb1b genes, primary transformants (T0) containing CRISPR/Cas9-induced mutations were obtained at frequencies of 95.5% (tb1a) and 11% (tb1b), respectively, with T0 mutants exhibiting increased tiller production. Meanwhile, a mutation frequency of 13.7% was obtained for the PGM gene with a CRISPR/Cas9 construct containing a single sgRNA. Among the PGM T0 mutants, six are heterozygous and one is homozygous for a 1-bp deletion in the target region with no apparent phenotypical alterations. We show that CRISPR/Cas9 system can generate targeted mutagenesis effectively and obtain targeted homozygous mutants in T0 generation in switchgrass, circumventing the need of inbreeding. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Virulence, resistance, and genetic relatedness of Escherichia coli and Klebsiella sp. isolated from mule foals

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V.C. Carneiro

Full Text Available ABSTRACT Respiratory diseases are common in young horses but little is known about such infections in mule foals. This study aimed to characterize Escherichia coli and Klebsiella sp. isolated from tracheal wash (TW and fecal samples (FS of mule foals, with or without cytological evidence of respiratory disease. Strains were analyzed against 13 antimicrobials, for presence of Extended spectrum beta-lactamase (ESBL, and virulence genes. Phylogrouping and Randomic (RAPD-PCR profiles were used to evaluate their genetic relatedness. E. coli strains from TW and FS showed greatest resistance to tetracycline, while Klebsiella strains were mainly resistant to ampicillin; multidrug resistance and ESBL production were also detected. The blaCTX gene prevailed among the E. coli isolates, while the blaSHV gene was more frequently found in K. pneumoniae. The fimH gene was detected in most of the isolates and multiple virulence factors were identified in three E. coli isolates. Most of the E. coli isolates belonged to the B1 phylogroup, but B2 strains displayed more virulence genes. The RAPD assay revealed genetic diversity among strains and was able to distinguish FS isolates from TW isolates. Knowledge of the bacteria associated with the respiratory tract of mule foals is important in the treatment of sick animals.

Annual Surveillance Summary: Escherichia coli (E. coli) Infections in the Military Health System (MHS), 2015

Science.gov (United States)

2017-03-01

Annual Surveillance Summary: Escherichia coli ( E . coli ) Infections in the Military Health System (MHS...or position of the Department of the Navy, Department of Defense, nor the U.S. Government. i i E . coli in the MHS: Annual Summary 2015 Prepared...March 2017 EpiData Center Department NMCPHC-EDC-TR-187-2017 ii ii E . coli in the MHS: Annual Summary 2015 Prepared March 2017 EpiData

Prevalence and Characterization of Cephalosporin Resistance in Nonpathogenic Escherichia coli from Food-Producing Animals Slaughtered in Poland

DEFF Research Database (Denmark)

Wasyl, Dariusz; Hasman, Henrik; Cavaco, Lina

2012-01-01

The prevalence of Escherichia coli with putative extended-spectrum cephalosporin resistance was assessed in cattle, pigs, broilers, layers, and turkey slaughtered in Poland. The occurrence of random E. coli isolates recovered from MacConkey agar plates with non–wild-type minimal inhibitory...... concentrations for cefotaxime and ceftazidime reached 0.6% in layers, 2.3% in turkey, and 4.7% in broilers, whereas all cattle and pigs isolates fell into the wild-type subpopulation. The use of MacConkey agar supplemented with cefotaxime (2 mg/L) increased the recovery of resistant strains up to 33...

Protective effects of indigenous Escherichia coli against a pathogenic E. coli challenge strain in pigs.

Science.gov (United States)

Vahjen, W; Cuisiniere, T; Zentek, J

2017-10-13

To investigate the inhibitory effect of indigenous enterobacteria on pathogenic Escherichia coli, a challenge trial with postweaning pigs was conducted. A pathogenic E. coli strain was administered to all animals and their health was closely monitored thereafter. Faecal samples were taken from three healthy and three diarrhoeic animals. Samples were cultivated on MacConkey agar and isolates were subcultured. A soft agar overlay assay was used to determine the inhibitory activity of the isolates. A total of 1,173 enterobacterial isolates were screened for their ability to inhibit the E. coli challenge strain. Colony forming units of enterobacteria on MacConkey agar were not different between healthy and diarrhoeic animals in the original samples. Furthermore, numbers of isolates per animal were also not significantly different between healthy (482 isolates) and diarrhoeic animals (691 isolates). A total of 43 isolates (3.7%) with inhibitory activity against the pathogenic E. coli challenge strain were detected. All inhibitory isolates were identified as E. coli via MALDI-TOF. The isolates belonged to the phylotypes A, C and E. Many isolates (67.4%) were commensal E. coli without relevant porcine pathogenic factors, but toxin- and fimbrial genes (stx2e, fae, estIb, elt1a, fas, fan) were detected in 14 inhibitory isolates. Healthy animals showed significantly (P=0.003) more inhibitory isolates (36 of 482 isolates; 7.5%) than diseased animals (7 of 691 isolates; 1.0%). There were no significant correlations regarding phylotype or pathogenic factors between healthy and diseased animals. This study has shown that a small proportion of indigenous E. coli is able to inhibit in vitro growth of a pathogenic E. coli strain in pigs. Furthermore, healthy animals possess significantly more inhibitory E. coli strains than diarrhoeic animals. The inhibition of pathogenic E. coli by specific indigenous E. coli strains may be an underlying principle for the containment of pathogenic

Campylobacter, Salmonella, Listeria monocytogenes, verotoxigenic Escherichia coli, and Escherichia coli prevalence, enumeration, and subtypes on retail chicken breasts with and without skin.

Science.gov (United States)

Cook, Angela; Odumeru, Joseph; Lee, Susan; Pollari, Frank

2012-01-01

This study examined the prevalence, counts, and subtypes of Campylobacter, Salmonella, Listeria monocytogenes, verotoxigenic Escherichia coli (VTEC), and E. coli on raw retail chicken breast with the skin on versus the skin off. From January to December 2007, 187 raw skin-on chicken breasts and 131 skin-off chicken breasts were collected from randomly selected retail grocery stores in the Region of Waterloo, Ontario, Canada. Campylobacter isolates were recovered from a higher proportion of the skin-off chicken breasts, 55 (42%) of 131, than of the skin-on chicken breasts tested, 55 (29%) of 187 (P = 0.023). There was no difference in the proportion of Salmonella isolates recovered from the two meat types (P = 0.715): 40 (31%) of 131 skin-off chicken breasts versus 61 (33%) of 187 skin-on chicken breasts. L. monocytogenes isolates were recovered from a statistically lower proportion of the skin-off chicken breasts, 15 (15%) of 99, than of the skin-on chicken breasts, 64 (34%) of 187 (P = 0.001). There was no difference in the proportion of E. coli isolates recovered from the skin-off chicken breasts, 33 (33%) of 99, than from the skin-on chicken breasts, 77 (41%) of 187 (P = 0.204). VTEC was detected on a single skin-off chicken breast. Campylobacter jejuni was the most frequent species isolated on both types of chicken meat: skin-on, 48 (87%) of 55, and skin-off, 51 (94%) of 54. Salmonella serotypes Kentucky and Heidelberg and L. monocytogenes serotype 1/2a were the most frequently detected serotypes from both skin-off and skin-on chicken breasts. Although there appeared to be a trend toward higher enumeration values of these pathogens and E. coli on the skin-on chicken, the differences did not exceed 1 log. This study suggested that skin-off chicken breast may represent a higher risk of consumer exposure to Campylobacter, a similar risk for Salmonella, VTEC, and E. coli, and a lower risk for L. monocytogenes than skin-on chicken breast.

Enterohemorrhagic Escherichia coli (EHEC

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Abdullah Kilic

2011-08-01

Full Text Available Escherichia coli is a bacterium that is commonly found in the gut of humans and warm-blooded animals. Most strains of E. coli are harmless for human. E. coli O157:H7 is the most common member of a group of pathogenic E. coli strains known variously as enterohaemorrhagic, verocytotoxin-producing, or Shiga-toxin-producing organisms. EHEC bacterium is the major cause of haemorrhagic colitis and haemolytic uraemic syndrome. The reservoir of this pathogen appears to be mainly cattle and other ruminants such as camels. It is transmitted to humans primarily through consumption of contaminated foods. [TAF Prev Med Bull 2011; 10(4.000: 387-388

Identification of a novel streptococcal gene cassette mediating SOS mutagenesis in Streptococcus uberis

NARCIS (Netherlands)

Varhimo, Emilia; Savijoki, Kirsi; Jalava, Jari; Kuipers, Oscar P.; Varmanen, Pekka

Streptococci have been considered to lack the classical SOS response, defined by increased mutation after UV exposure and regulation by LexA. Here we report the identification of a potential self-regulated SOS mutagenesis gene cassette in the Streptococcaceae family. Exposure to UV light was found

Escherichia coli ArgR mutants defective in cer/Xer recombination, but not in DNA binding.

Science.gov (United States)

Sénéchal, Hélène; Delesques, Jérémy; Szatmari, George

2010-04-01

The Escherichia coli arginine repressor (ArgR) is an L-arginine-dependent DNA-binding protein that controls the expression of the arginine biosynthetic genes and is required as an accessory factor for Xer site-specific recombination at cer and related recombination sites in plasmids. We used the technique of pentapeptide scanning mutagenesis to isolate a series of ArgR mutants that were considerably reduced in cer recombination, but were still able to repress an argA::lacZ fusion. DNA sequence analysis showed that all of the mutants mapped to the same nucleotide, resulting in a five amino acid insertion between residues 149 and 150 of ArgR, corresponding to the end of the alpha6 helix. A truncated ArgR containing a stop codon at residue 150 displayed the same phenotype as the protein with the five amino acid insertion, and both mutants displayed sequence-specific DNA-binding activity that was L-arginine dependent. These results show that the C-terminus of ArgR is more important in cer/Xer site-specific recombination than in DNA binding.

Impact of dry chilling on the genetic diversity of Escherichia coli on beef carcasses and on the survival of E. coli and E. coli O157.

Science.gov (United States)

Visvalingam, Jeyachchandran; Liu, Yang; Yang, Xianqin

2017-03-06

The objective of this study was to examine the effect of dry chilling on the genetic diversity of naturally occurring Escherichia coli on beef carcasses, and to examine whether two populations of E. coli recovered from carcasses during chilling and E. coli O157 differed in their response to desiccation. Isolates of E. coli were obtained from beef carcasses during a 67h dry chilling process and were genotyped using multiple-locus variable-number tandem-repeat analysis (MLVA). Ten E. coli genotypes found only at 0h (group A) and found more than once (group B), as well as five strains of E. coli O157 (group C) were inoculated on stainless steel coupons and their survival was examined after exposure to 75 and 100% relative humidity (RH) at 0 or 35°C for 67h. A total of 450 E. coli isolates were obtained, with 254, 49, 49, 51, 23, 20, and 4 from 0, 1, 2, 4, 6, 8 and 24h of chilling, respectively. No E. coli were recovered at 67h. MLVA of the isolates revealed 173 distinct genotypes. Genetic diversity of E. coli isolates, defined as ratio of the number of isolates to the number of genotypes, remained between 2.3 and 1.3 during the 24h of chilling. All strains inoculated on stainless steel coupons and exposed to 75% RH at 35°C were completely inactivated, irrespective of their groups. Inactivation of E. coli of the three groups was not significantly (P>0.05) different by exposure to 75% RH at 0°C. The findings indicate that the genetic diversity of E. coli on beef carcasses was not affected by dry chilling. In addition, inactivation of E. coli genotypes and E. coli O157 by desiccation on stainless steel simulating dry chilling conditions did not differ significantly (P>0.05). Thus, dry chilling may be used as an effective antimicrobial intervention for beef carcasses. Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.

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

Science.gov (United States)

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.

Cancerous hyper-mutagenesis in p53 genes is possibly associated with transcriptional bypass of DNA lesions

International Nuclear Information System (INIS)

Rodin, S.N.; Rodin, A.S.; Juhasz, A.; Holmquist, G.P.

2002-01-01

The database of tumor-associated p53 base substitutions includes about 5% of tumors with two or more base substitutions. These multiplet base substitutions in one tumor are evidence for hyper-mutagenesis. Our retrospective analysis of this database indicates that most multiplets arise from a single transient hyper-mutagenic event in one cell that subsequently proliferated into a clonal tumor. The hyper-mutagenesis, 1.8x10 -4 substitutions per base pair, is detected as multiple mutations in p53 genes of tumors. It requires one strongly tumorigenic p53 substitution, usually missense, called the driver mutation. The occurrence frequencies of ancillary base substitutions, those that hitch-hike along with the driver mutation, are independent of their amino acid coding properties. In this respect, they act like neutral mutations. In support of this neutrality, we find that the frequency distribution of hitch-hiking CpG transitions along the p53 exons, their mutational spectrum, approximates the spontaneous pre-selection mutational spectrum of most human tissues and is correlated with the mutational spectrum of p53 pseudogenes in mammalian germ cells. The driver substitutions of multiplets predominantly originate along the transcribed strand while the ancillary substitutions tend to originate along the non-transcribed strand. This data is consistent with a model of time-dependent mutagenesis in non-dividing stem cells for generating multiple strand-asymmetric p53 mutations in tumors. By transcriptional bypass of DNA lesions with concomitant misincorporation, transcriptional mutagenesis generates a transient mutant p53 mRNA. The associated mutant p53 protein could allow the host cell a growth advantage, release from G 1 -arrest. Then, during subsequent DNA replication and misreading of the same lesion, the damaged base along the transcribed DNA strand would serve as the origin of the p53 base substitution that drives the hyper-mutagenic event leading to tumors with

Pyrimidine dimers are not the principal pre-mutagenic lesions induced in lambda phage DNA by ultraviolet light

International Nuclear Information System (INIS)

Wood, R.D.

1985-01-01

Experiments were performed to examine the role of cyclobutyl pyrimidine dimers in the process of mutagenesis by ultraviolet light. Lambda phage DNA was irradiated with u.v. and then incubated with an Escherichia coli photoreactivating enzyme, which monomerizes cyclobutyl pyrimidine dimers upon exposure to visible light. The photoreactivated DNA was packaged into lambda phage particles, which were used to infect E. coli uvr - host cells that had been induced for SOS functions by ultraviolet irradiation. Photoreactivation removed most toxic lesions from irradiated phage, but did not change the frequency of induction of mutations to the clear-plaque phenotype. This implies that cyclobutyl pyrimidine dimers can be lethal, but usually do not serve as sites of mutations in the phage. The DNA sequences of mutants, derived from photoreactivated DNA showed that almost two-thirds (16/28) were transitions, the same fraction found for u.v. mutagenesis without photoreactivation. Thus the lesion inducing transitions is not the cyclobutyl pyrimidine dimer. Photoreactivation of SOS-induced host cells before infection with u.v.-irradiated phage reduced mutagenesis substantially. In this case, photoreversal of cyclobutyl dimers serves to reduce expression of the SOS functions that are required in targeted u.v. mutagenesis. (author)

Site-directed mutagenesis of Arg58 and Asp86 of elongation factor Tu from Escherichia coli: effects on the GTPase reaction and aminoacyl-tRNA binding

DEFF Research Database (Denmark)

Knudsen, Charlotte Rohde; Clark, Brian F. C.

1996-01-01

Elongation factor Tu from Escherichia coli was mutated separately at positions Asp86 and Arg58, in order to shed light both on the GTPase mechanism of elongation factor Tu and on the binding of aminoacyl-tRNA. In addition, the binding of guanine nucleotides was investigated by determination...

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

International Nuclear Information System (INIS)

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

A protocol for chemical mutagenesis in Strongyloides ratti.

Science.gov (United States)

Guo, Li; Chang, Zisong; Dieterich, Christoph; Streit, Adrian

2015-11-01

Genetic analysis using experimentally induced mutations has been a most valuable tool in the analysis of various organisms. However, genetic analysis of endoparasitic organisms tends to be difficult because of the limited accessibility of the sexually reproducing adults, which are normally located within the host. Nematodes of the genera Strogyloides and Parastrongyloides represent an exception to this because they can form facultative free-living sexually reproducing generations in between parasitic generations. Here we present a protocol for the chemical mutagenesis of Strongyloides ratti. Further we evaluate the feasibility of identifying the induced mutations by whole genome re-sequencing. Copyright © 2015 Elsevier Inc. All rights reserved.

Site directed mutagenesis of amino acid residues at the active site of mouse aldehyde oxidase AOX1.

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Silvia Schumann

Full Text Available Mouse aldehyde oxidase (mAOX1 forms a homodimer and belongs to the xanthine oxidase family of molybdoenzymes which are characterized by an essential equatorial sulfur ligand coordinated to the molybdenum atom. In general, mammalian AOs are characterized by broad substrate specificity and an yet obscure physiological function. To define the physiological substrates and the enzymatic characteristics of mAOX1, we established a system for the heterologous expression of the enzyme in Escherichia coli. The recombinant protein showed spectral features and a range of substrate specificity similar to the native protein purified from mouse liver. The EPR data of recombinant mAOX1 were similar to those of AO from rabbit liver, but differed from the homologous xanthine oxidoreductase enzymes. Site-directed mutagenesis of amino acids Val806, Met884 and Glu1265 at the active site resulted in a drastic decrease in the oxidation of aldehydes with no increase in the oxidation of purine substrates. The double mutant V806E/M884R and the single mutant E1265Q were catalytically inactive enzymes regardless of the aldehyde or purine substrates tested. Our results show that only Glu1265 is essential for the catalytic activity by initiating the base-catalyzed mechanism of substrate oxidation. In addition, it is concluded that the substrate specificity of molybdo-flavoenzymes is more complex and not only defined by the three characterized amino acids in the active site.

Characterization of Multidrug Resistant ESBL-Producing Escherichia coli Isolates from Hospitals in Malaysia

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King-Ting Lim

2009-01-01

Full Text Available The emergence of Escherichia coli that produce extended spectrum β-lactamases (ESBLs and are multidrug resistant (MDR poses antibiotic management problems. Forty-seven E. coli isolates from various public hospitals in Malaysia were studied. All isolates were sensitive to imipenem whereas 36 were MDR (resistant to 2 or more classes of antibiotics. PCR detection using gene-specific primers showed that 87.5% of the ESBL-producing E. coli harbored the blaTEM gene. Other ESBL-encoding genes detected were blaOXA, blaSHV, and blaCTX-M. Integron-encoded integrases were detected in 55.3% of isolates, with class 1 integron-encoded intI1 integrase being the majority. Amplification and sequence analysis of the 5′CS region of the integrons showed known antibiotic resistance-encoding gene cassettes of various sizes that were inserted within the respective integrons. Conjugation and transformation experiments indicated that some of the antibiotic resistance genes were likely plasmid-encoded and transmissible. All 47 isolates were subtyped by PFGE and PCR-based fingerprinting using random amplified polymorphic DNA (RAPD, repetitive extragenic palindromes (REPs, and enterobacterial repetitive intergenic consensus (ERIC. These isolates were very diverse and heterogeneous. PFGE, ERIC, and REP-PCR methods were more discriminative than RAPD in subtyping the E. coli isolates.

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

Science.gov (United States)

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...

Prevalence of Campylobacter Jejuni and Coli in Sheep Carcasses by Using

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Reza Shahrokhabadi

2013-11-01

Full Text Available Background: Campylobacter species are common bacterial pathogens causing gastroenteritis in humans worldwide. Materials and Methods: A total of 148 randomly sheep carcasses were sampled by surface section of neck meat taken immediately after slaughter analyzed using microbiological examinations. Results: Campylobacter spp. was isolated from 10.13% meat cultures samples examined. Among these 80% sample were C. jejuni and 20% sample were C. coli. Using PCR assays, the number of positive campylobacters increased to 11.48%. Of these positive samples, 82.35% were C. jejuni and 17.65% were C. coli. Significantly higher prevalence rates of Campylobacter spp. (p<0.05 were found in the meat samples taken in summer (47.05%. Conclusion: The PCR is a reliable and sensitive method which can be used as a diagnostic technique for the detection of campylobacter in lamb samples.

Building on the Past, Shaping the Future: The Environmental Mutagenesis and Genomics Society

Science.gov (United States)

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...

Biological effects of dyes on bacteria. VI. Mutation induction by acridine orange and methylene blue in the dark with special reference to Escherichia coli WP6 (polA1)

Energy Technology Data Exchange (ETDEWEB)

Webb, R.B.; Hass, B.S.

1984-01-01

Acridine orange (AO) and methylene blue (MB) in the dark were shown to be weak to moderate mutagens (induction of resistance to T5 phage) in repair-deficient strains of Escherichia coli B/r. However, strain WP2, (wild-type) was not mutated by AO in the dark, in confirmation of earlier data. The presence of 2 ..mu..M AO reduced by 41% the spontaneous mutation rate in strain WP2, from 4.1 to 2.4 mutants/10/sup 8/ cells/generation. In the polymerase I-deficient strain WP6 (polA1), 2 ..mu..M AO increased the mutation rate in the dark 14-fold. It is proposed that both spontaneous and AO-induced mutagenesis in the absence of light occur at the site of semiconservative DNA replication. If the intercalation mechanism for the effects in the absence of light is valid, the wild-type strain (WP2) may be resistant to frameshift mutagenesis induced by intercalated compounds, while the polymerase I-deficient strain (WP6) may be highly susceptible to the presence of an intercalated dye such as AO at the DNA-replication fork. MB and AO likely act through different mechanisms since MB is only a moderate mutagen in strain WP6 and the other repair-deficient strains tested.

Mutagenic DNA repair in Escherichia coli. Pt. 2

International Nuclear Information System (INIS)

Doubleday, O.P.; Bridges, B.A.; Green, M.H.L.

1975-01-01

The photoreversibility of UV-induced mutations to Trp + in strain Escherichia coli WP2 uvr A trp (unable to excise pyrimidine dimers) was lost at different rates during incubation in different media. In Casamino acids medium after a short initial lag, photoreversibility was lost over about one generation time; in minimal medium with tryptophan, photoreversibility persisted for more than two generations; in Casamino acids medium with pantoyl lactone photoreversibility was lost extremely slowly. The rate of loss of photoreversibility was unaffected by UV dose in either Casamino acids medium or in minimal medium. The same eventual number of induced mutants was obtained when cells were incubated for two generations in any of the three media before being transferred to selective plates supplemented with Casamino acids. Thus in each the proportion of cells capable of giving rise to a mutant was the same and only the rate at which these cells did so during post-irradiation growth varied, suggesting that there might be a specific fraction of pyrimidine dimers at a given site capable of initiating a mutagenic repair event, and that the size of this fraction is dose dependent. Segregation experiments have shown that error-prone repair appears to occur once only and is not repeated in subsequent replication cycles, in contrast to (presumed error-free) recombination repair. The results are discussed in the light of current models of UV mutagenesis. (orig.) [de

[E75, R78 and D82 of Escherichia coli FtsZ are key residues for FtsZ cellular self-assembly and FtsZ-MreB interaction].

Science.gov (United States)

Huo, Yujia; Lu, Qiaonan; Zheng, Xiaowei; Ma, Yuanfang; Lu, Feng

2016-02-04

To explore effects of FtsZ mutants FtsZ(E75A), FtsZ(R78G) and FtsZ(D82A) on FtsZ self-assembly and interaction of FtsZ with MreB in Escherichia coli strains. METHODS) We constructed FtsZ and its mutant's plasmids by molecular clone and site-directed mutagenesis methods, and purified targeted proteins by affinity chromatography. QN6(ftsZ::yfp-cat), QN7(tsZ::yfp-cat), QN8(ftsZ(R78G)::yfp-cat) and QN9 (ftsZ(D82A):.:yfp-cat) strains were constructed by linear DNA homologous recombination. We observed cellular localization pattern of FtsZ and its mutants in E. coli by living cell imaging experiments, examined interaction of FtsZ/FtsZ*-FtsZ* and FtsZ/FtsZ*-MreB by Coimmunoprecipitation and bacteria two hybrid, and analyzed assembly characteristics of FtsZ mutants by Light scattering. RESULTS) The Yfp-labeled FtsZ(E75A), FtsZ(R78G) and FtsZ(D82A) mutant proteins failed to assemble into functional Z-ring structure and localize correctly in E. coli strains. Interaction of FtsZ with its mutants, or FtsZ*-FtsZ* and FtsZ*-MreB interaction were weakened or completely disappeared. In addition, in vitro experiments show that E75A, R78G and D82A mutations decreased the polymerization efficiency of FtsZ monomer. FtsZ E75, R78 and D82 are critical amino acids in the assembly, function of FtsZ protein and FtsZ-MreB interaction in E. coli strains.

Modeling insertional mutagenesis using gene length and expression in murine embryonic stem cells.

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Alex S Nord

2007-07-01

Full Text Available High-throughput mutagenesis of the mammalian genome is a powerful means to facilitate analysis of gene function. Gene trapping in embryonic stem cells (ESCs is the most widely used form of insertional mutagenesis in mammals. However, the rules governing its efficiency are not fully understood, and the effects of vector design on the likelihood of gene-trapping events have not been tested on a genome-wide scale.In this study, we used public gene-trap data to model gene-trap likelihood. Using the association of gene length and gene expression with gene-trap likelihood, we constructed spline-based regression models that characterize which genes are susceptible and which genes are resistant to gene-trapping techniques. We report results for three classes of gene-trap vectors, showing that both length and expression are significant determinants of trap likelihood for all vectors. Using our models, we also quantitatively identified hotspots of gene-trap activity, which represent loci where the high likelihood of vector insertion is controlled by factors other than length and expression. These formalized statistical models describe a high proportion of the variance in the likelihood of a gene being trapped by expression-dependent vectors and a lower, but still significant, proportion of the variance for vectors that are predicted to be independent of endogenous gene expression.The findings of significant expression and length effects reported here further the understanding of the determinants of vector insertion. Results from this analysis can be applied to help identify other important determinants of this important biological phenomenon and could assist planning of large-scale mutagenesis efforts.

In vitro mutagenesis of commercial fern, Asplenium nidus from spores

International Nuclear Information System (INIS)

Norazlina Noordin

2004-01-01

Asplenium is a largest, most diverse fern genera. One of the common species is Asplenium nidus, well known as Bird's-nest fern, a medium to large fern with erect, stout, unbranched rhizomes. In creating variability of ferns for the benefit of the ornamental plant industry, in vitro mutagenesis is used. In this study, spores of Asplenium nidus were collected from frond bearing mature sporangia. Spores were cultured in modified 1/2 MS basal medium supplemented with various combinations of 6-Benzylaminopurine (BAP) and Naphtalene Acetic Acid (NAA). Spore cultures were incubated in incubation room at 24 degree C with 16 hours photoperiod (3500 lux). It was found that, the most effective combinations were 1 mg/1 BAP + 0. 1 mg/1 NAA and 2mg/1 BAP + 0. 1 mg/1 NAA. Prothallus was formed after 10 days of cultures and gametophytes were formed 1 month later. These gametophytes were irradiated with Gamma ray at doses of 0, 20, 90, 120, 150 and 180 Gy. From the preliminary result obtained from this study, for generating variations and desired phenotypic expression for Asplenium nidus, recommended doses for in vitro mutagenesis using spores are between 90 Gy to 150 Gy. Gametophytes were subcultured at monthly interval to ensure further development and propagation. Frequent monitoring for any changes in the morphology of the irradiated Asplenium nidus plants were carried out. (Author)

HTP-OligoDesigner: An Online Primer Design Tool for High-Throughput Gene Cloning and Site-Directed Mutagenesis.

Science.gov (United States)

Camilo, Cesar M; Lima, Gustavo M A; Maluf, Fernando V; Guido, Rafael V C; Polikarpov, Igor

2016-01-01

Following burgeoning genomic and transcriptomic sequencing data, biochemical and molecular biology groups worldwide are implementing high-throughput cloning and mutagenesis facilities in order to obtain a large number of soluble proteins for structural and functional characterization. Since manual primer design can be a time-consuming and error-generating step, particularly when working with hundreds of targets, the automation of primer design process becomes highly desirable. HTP-OligoDesigner was created to provide the scientific community with a simple and intuitive online primer design tool for both laboratory-scale and high-throughput projects of sequence-independent gene cloning and site-directed mutagenesis and a Tm calculator for quick queries.

The Escherichia coli BolA Protein IbaG Forms a Histidine-Ligated [2Fe-2S]-Bridged Complex with Grx4.

Science.gov (United States)

Dlouhy, Adrienne C; Li, Haoran; Albetel, Angela-Nadia; Zhang, Bo; Mapolelo, Daphne T; Randeniya, Sajini; Holland, Ashley A; Johnson, Michael K; Outten, Caryn E

2016-12-13

Two ubiquitous protein families have emerged as key players in iron metabolism, the CGFS-type monothiol glutaredoxins (Grxs) and the BolA proteins. Monothiol Grxs and BolA proteins form heterocomplexes that have been implicated in Fe-S cluster assembly and trafficking. The Escherichia coli genome encodes members of both of these proteins families, namely, the monothiol glutaredoxin Grx4 and two BolA family proteins, BolA and IbaG. Previous work has demonstrated that E. coli Grx4 and BolA interact as both apo and [2Fe-2S]-bridged heterodimers that are spectroscopically distinct from [2Fe-2S]-bridged Grx4 homodimers. However, the physical and functional interactions between Grx4 and IbaG are uncharacterized. Here we show that co-expression of Grx4 with IbaG yields a [2Fe-2S]-bridged Grx4-IbaG heterodimer. In vitro interaction studies indicate that IbaG binds the [2Fe-2S] Grx4 homodimer to form apo Grx4-IbaG heterodimer as well as the [2Fe-2S] Grx4-IbaG heterodimer, altering the cluster stability and coordination environment. Additionally, spectroscopic and mutagenesis studies provide evidence that IbaG ligates the Fe-S cluster via the conserved histidine that is present in all BolA proteins and by a second conserved histidine that is present in the H/C loop of two of the four classes of BolA proteins. These results suggest that IbaG may function in Fe-S cluster assembly and trafficking in E. coli as demonstrated for other BolA homologues that interact with monothiol Grxs.

Site-directed mutagenesis in Petunia × hybrida protoplast system using direct delivery of purified recombinant Cas9 ribonucleoproteins.

Science.gov (United States)

Subburaj, Saminathan; Chung, Sung Jin; Lee, Choongil; Ryu, Seuk-Min; Kim, Duk Hyoung; Kim, Jin-Soo; Bae, Sangsu; Lee, Geung-Joo

2016-07-01

Site-directed mutagenesis of nitrate reductase genes using direct delivery of purified Cas9 protein preassembled with guide RNA produces mutations efficiently in Petunia × hybrida protoplast system. The clustered, regularly interspaced, short palindromic repeat (CRISPR)-CRISPR associated endonuclease 9 (CRISPR/Cas9) system has been recently announced as a powerful molecular breeding tool for site-directed mutagenesis in higher plants. Here, we report a site-directed mutagenesis method targeting Petunia nitrate reductase (NR) gene locus. This method could create mutations efficiently using direct delivery of purified Cas9 protein and single guide RNA (sgRNA) into protoplast cells. After transient introduction of RNA-guided endonuclease (RGEN) ribonucleoproteins (RNPs) with different sgRNAs targeting NR genes, mutagenesis at the targeted loci was detected by T7E1 assay and confirmed by targeted deep sequencing. T7E1 assay showed that RGEN RNPs induced site-specific mutations at frequencies ranging from 2.4 to 21 % at four different sites (NR1, 2, 4 and 6) in the PhNR gene locus with average mutation efficiency of 14.9 ± 2.2 %. Targeted deep DNA sequencing revealed mutation rates of 5.3-17.8 % with average mutation rate of 11.5 ± 2 % at the same NR gene target sites in DNA fragments of analyzed protoplast transfectants. Further analysis from targeted deep sequencing showed that the average ratio of deletion to insertion produced collectively by the four NR-RGEN target sites (NR1, 2, 4, and 6) was about 63:37. Our results demonstrated that direct delivery of RGEN RNPs into protoplast cells of Petunia can be exploited as an efficient tool for site-directed mutagenesis of genes or genome editing in plant systems.

Can E. coli fly?

DEFF Research Database (Denmark)

Lindeberg, Yrja Lisa; Egedal, Karen; Hossain, Zenat Zebin

2018-01-01

, and the numbers of flies landing on the exposed rice were counted. Following exposure, the surface of the rice was microbiologically and molecularly analysed for the presence of E. coli and genes of diarrheagenic E. coli and Shigella strains. RESULTS: Rice was at greater risk (p ... with E. coli if flies landed on the rice than if no flies landed on the rice (odds ratio 5·4 (p ...-landings, the average CFU per fly-landing was > 0·6 x 103 CFU. Genes of diarrheagenic E. coli and Shigella species were detected in 39 of 60 (65%) of exposed rice samples. Two fly species were identified; the common housefly (Musca domestica) and the oriental latrine fly (Chrysomya megacephala). CONCLUSION: Flies may...

The Saccharomyces cerevisiae RAD30 gene, a homologue of Escherichia coli dinB and umuC, is DNA damage inducible and functions in a novel error-free postreplication repair mechanism

Energy Technology Data Exchange (ETDEWEB)

McDonald, J. P. [NIH, Bethesda, MD. (United States); Levine, A. S.; Woodgate, R.

1997-12-15

Damage-inducible mutagenesis in prokaryotes is largely dependent upon the activity of the UmuD'C-like proteins. Since many DNA repair processes are structurally and/or functionally conserved between prokaryotes and eukaryotes, we investigated the role of RAD30, a previously uncharacterized Saccharomyces cerevisiae DNA repair gene related to the Escherichia coli dinB, umuC and S. cerevisiae REV1 genes, in UV resistance and UV-induced mutagenesis. Similar to its prokaryotic homologues, RAD30 was found to be damage inducible. Like many S. cerevisiae genes involved in error-prone DNA repair, epistasis analysis clearly places RAD30 in the RAD6 group and rad30 mutants display moderate UV sensitivity reminiscent of rev mutants. However, unlike rev mutants, no defect in UV-induced reversion was seen in rad30 strains. While rad6 and rad18 are both epistatic to rad30, no epistasis was observed with rev1, rev3, rev7 or rad5, all of which are members of the RAD6 epistasis group. These findings suggest that RD30 participates in a novel error-free repair pathway dependent on RAD6 and RAD18, but independent of REV1, REV3, REV7 and RAD5. (author)

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

Science.gov (United States)

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

2018-01-01

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

The DNA relaxation activity and covalent complex accumulation of Mycobacterium tuberculosis topoisomerase I can be assayed in Escherichia coli: application for identification of potential FRET-dye labeling sites

Directory of Open Access Journals (Sweden)

Abrenica Maria V

2010-09-01

Full Text Available Abstract Background Mycobacterium tuberculosis topoisomerase I (MtTOP1 and Escherichia coli topoisomerase I have highly homologous transesterification domains, but the two enzymes have distinctly different C-terminal domains. To investigate the structure-function of MtTOP1 and to target its activity for development of new TB therapy, it is desirable to have a rapid genetic assay for its catalytic activity, and potential bactericidal consequence from accumulation of its covalent complex. Results We show that plasmid-encoded recombinant MtTOP1 can complement the temperature sensitive topA function of E. coli strain AS17. Moreover, expression of MtTOP1-G116 S enzyme with the TOPRIM mutation that inhibits DNA religation results in SOS induction and loss of viability in E. coli. The absence of cysteine residues in the MtTOP1 enzyme makes it an attractive system for introduction of potentially informative chemical or spectroscopic probes at specific positions via cysteine mutagenesis. Such probes could be useful for development of high throughput screening (HTS assays. We employed the AS17 complementation system to screen for sites in MtTOP1 that can tolerate cysteine substitution without loss of complementation function. These cysteine substitution mutants were confirmed to have retained the relaxation activity. One such mutant of MtTOP1 was utilized for fluorescence probe incorporation and fluorescence resonance energy transfer measurement with fluorophore-labeled oligonucleotide substrate. Conclusions The DNA relaxation and cleavage complex accumulation of M. tuberculosis topoisomerase I can be measured with genetic assays in E. coli, facilitating rapid analysis of its activities, and discovery of new TB therapy targeting this essential enzyme.

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

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Sofya A. Medvedeva

2013-01-01

Full Text Available 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.

Chromosome mutagenesis in populations of aquatic biota in the Black Sea, Aegean Sea and Danube and Dnieper rivers, 1986-1989

International Nuclear Information System (INIS)

Tsytsugina, V.G.

1991-01-01

We studied the level of structural mutagenesis in the reproductive and somatic cells of aquatic biota of various taxa from natural populations of neustic and benthic communities in the Black and Aegean Seas and the Dnieper and Danube rivers between 1986 and 1989. The cytogenetic research covered embryos, larvae and adult worms of Nereidae, Naididae, Tubificidae and Turbellaria, adult Sagitta setosa, young Bivalvia molluscs, embryos of Mysidacea, and growing roe of Engraulis encrasicholus, Sprattus sprattus, Diplodus annularis, Mullus barbatus, Trachurus trachurus, Scophthalmus maeoticus, Abramis brama, Blicca bjoerkna, Rutilus rutilus and Stizostedion lucioperca. It was established that aquatic biota in the open waters of the Black and Aegean Seas had a lower level of chromosome mutagenesis than representatives of the fluvial communities. The intensity of mutagenesis was compared with the data published in the literature on radioactive contamination/chemical pollution of the aqueous medium in these areas. The paper sets out statistical regularities in chromosome mutagenesis (inter-individual variability in the chromosome aberration rate and distribution of chromosome damage in cells), noting different patterns of chromosome aberration distribution among cells. On the basis of a large quantity on our own data from field and experimental cytogenetic studies involving aquatic biota, the paper considers the possibility of using - for the purposes of radiochemical-ecological monitoring - chromosome damage distribution in cells as an indicator of whether mutagens are radiation-related or not. (author)

Pulsed-field gel electrophoresis typing of Escherichia coli strains from samples collected before and after pivmecillinam or placebo treatment of uncomplicated community-acquired urinary tract infection in women

DEFF Research Database (Denmark)

Ejrnaes, Karen; Sandvang, Dorthe; Lundgren, Bettina

2006-01-01

The primary infecting Escherichia coli strains from 156 women with community-acquired uncomplicated urinary tract infection (UTI) randomized to pivmecillinam or placebo and the E. coli strains causing UTI at two follow-up visits were typed using pulsed-field gel electrophoresis (PFGE......). The finding that the majority of UTIs at follow-up are caused by the primary infecting E. coli strain supports the theory of a vaginal and rectal reservoir but could also support the recent discovery that E. coli strains are able to persist in the bladder epithelium despite appropriate antibiotic treatment......, constituting a reservoir for recurrent UTI....

Pulsed-field gel electrophoresis typing of Eschericia coli strains from samples collected before and after pivmecillinam or placebo treatment of uncomplicated community-acquired urinary tract infection in women

DEFF Research Database (Denmark)

Ejrnæs, K; Sandvang, D; Lundgren, Bettina

2006-01-01

The primary infecting Escherichia coli strains from 156 women with community-acquired uncomplicated urinary tract infection (UTI) randomized to pivmecillinam or placebo and the E. coli strains causing UTI at two follow-up visits were typed using pulsed-field gel electrophoresis (PFGE......). The finding that the majority of UTIs at follow-up are caused by the primary infecting E. coli strain supports the theory of a vaginal and rectal reservoir but could also support the recent discovery that E. coli strains are able to persist in the bladder epithelium despite appropriate antibiotic treatment......, constituting a reservoir for recurrent UTI....

Enhanced mutagenesis of UV-irradiated simian virus 40 occurs in mitomycin C-treated host cells only at a low multiplicity of infection

International Nuclear Information System (INIS)

Sarasin, A.; Benoit, A.

1986-01-01

Treatment of monkey kidney cells with mitomycin C (MMC) 24 h prior to infection with UV-irradiated simian virus 40 (SV40) enhanced both virus survival and virus mutagenesis. The use of SV40 as a biological probe has been taken as an easy method to analyse SOS response of mammalian cells to the stress caused by DNA damage or inhibition of DNA replication. The mutation assay we used was based on the reversion from a temperature-sensitive phenotype (tsA58 mutant) to a wild-type phenotype. The optimal conditions for producing enhanced survival and mutagenesis in the virus progeny were determined with regard to the multiplicity of infection (MOI). Results showed that the level of enhanced mutagenesis observed for UV-irradiated virus grown in MMC-treated cells was an inverse function of the MOI, while enhanced survival was observed at nearly the same level regardless of the MOI. For the unirradiated virus, almost no increase in the mutation of virus progeny issued from MMC-treated cells was observed, while a small amount of enhanced virus survival was obtained. These results show that enhanced virus mutagenesis and enhanced virus survival can be dissociated under some experimental conditions. Enhanced virus mutagenesis, analogous to the error-prone replication of phages in SOS-induced bacteria, was observed, at least for SV40, only when DNA of both virus and host cells was damaged and when infection occurred with a small number of viral particles. We therefore hypothesize that an error-prone replication mode of UV-damaged templates is observed in induced monkey kidney cells

99mTechnetium labelled Escherichia coli

International Nuclear Information System (INIS)

Diniz, S.O.F.; Cardoso, V.N.; Resende, B.M.; Nunan, E.A.; Simal, C.J.R.

1999-01-01

Samples of a culture of unlabeled Escherichia coli were incubated with different concentrations of stannous chloride for various time periods. 99m Tc (26.0 MBq) was added to each preparation and the results showed a labelling yield of 98% for E. coli. Since the bacterial viability of 99m Tc-E. coli and E. coli did not show any statistical differences, these results demonstrate that labelling of E. coli with 99m Tc does not modify the bacterial viability, and the radiolabelled bacteria may be a good model to study bacterial translocation

Production and Purification Immunoglobulin against E. coli in Egg Yolk

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Mohammadreza Nassiri

2016-08-01

presence of antibody IgY was confirmed using SDS-PAGE. Purification of IgY was carried out by polyethylene glycol precipitation method using PEG 6000 powder (Merck, Germany based on method of Polson. The purified IgY against E. coli was separated using 10% SDS-PAGE. In order to investigate the effect of the specific anti-E. Coli antibody, mice (Razi, Institute of Iran were randomly distributed into five experimental groups (6mice/group. The mice were kept in conventional animal facilities and received water and food ad libitum. All animal care and procedures were in accordance with institutional policies for animal health and well-being. Experimental groups were including group 1 (mice received IgY orally in drinking water 72 hours before intraperitoneal injection of bacteria and then injected intraperitoneally with 0.5 ml of bacteria E. coli O157: H7, group 2 (mice received IgY orally in drinking water 72 hours before the injection and then injected intraperitoneally with 0.5 ml of deionized water, group 3 (0.5 ml of E. coli O157: H7 incubated with 0.5 ml of the specific anti-E. coli IgY and then 0.5 ml of the incubated solution injected to mice intraperitoneally, group 4 (mice injected with 0.5 ml of IgY and group 5 (mice received 0.5 ml of E. coli O157: H7. Results and Discussion We obtained specific egg yolk antibody against E. coli O157: H7 by immunizing hens with the killed E. coli O157: H7 antigen. The results showed that the IgY was successfully purified from egg yolk. SDS-PAGE analysis showed presence of protein bands 27kDa and 67 kDa of IgY, which correspond to IgY light and heavy chains. Effects of IgY on mice showed that mice received IgY orally in drinking water 72 hours before intraperitoneal injection were protected against bacteria. Also, when specific anti-E. coli IgY was incubated with E.coli O157: H7 for 24 hours and then it was injected to mice led to mice protected against bacteria. The results of our study were agreement with the results of Chae et al

Rapid Integration of Multi-copy Transgenes Using Optogenetic Mutagenesis in Caenorhabditis elegans

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Kentaro Noma

2018-06-01

Full Text Available Stably transmitted transgenes are indispensable for labeling cellular components and manipulating cellular functions. In Caenorhabditis elegans, transgenes are generally generated as inheritable multi-copy extrachromosomal arrays, which can be stabilized in the genome through a mutagenesis-mediated integration process. Standard methods to integrate extrachromosomal arrays primarily use protocols involving ultraviolet light plus trimethylpsoralen or gamma- or X-ray irradiation, which are laborious and time-consuming. Here, we describe a one-step integration method, following germline-mutagenesis induced by mini Singlet Oxygen Generator (miniSOG. Upon blue light treatment, miniSOG tagged to histone (Histone-miniSOG generates reactive oxygen species (ROS and induces heritable mutations, including DNA double-stranded breaks. We demonstrate that we can bypass the need to first establish extrachromosomal transgenic lines by coupling microinjection of desired plasmids with blue light illumination on Histone-miniSOG worms to obtain integrants in the F3 progeny. We consistently obtained more than one integrant from 12 injected animals in two weeks. This optogenetic approach significantly reduces the amount of time and labor for transgene integration. Moreover, it enables to generate stably expressed transgenes that cause toxicity in animal growth.

Potensi Antibakteri Ekstrak Bunga Soka (Ixora coccinea L terhadap Staphylococcus aureus dan Escherichia coli

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Munira Munira

2016-11-01

Full Text Available Soka plants (Ixora coccineal L. often used to treat diarrhea, dysentery and wounds. This study was conducted to determine the effect of soka flower ethanol extract in inhibiting the growth of Staphylococcus aureus and Escherichia coli. This study is experimentally using completely randomized design (CRD with 4 treatments and 6 repetitions which is distilled water as a control, the ethanol extract of soka flowers concentration are 100%, 75%, and 50%. Microbiological test using the diffusion method of Kirby-Baueur. Phytochemical test results soka ethanol extracts of flowers contain alkaloids, flavonoids, tannins, saponins, and triterpenoid. ANOVA test results ethanol extracts of soka flowers greatly affect the growth of S. aureus and E. coli (P=0.000. Duncan test results an average diameter of inhibition zone for the ethanol extract of soka flowers against S. aureus at a concentration of 100% (14.50 mm was significantly different from the concentration of 75% (10.33 mm and concentration of 50% (10.67 mm, while the average diameter of inhibition zone for the ethanol extract of soka flowers against E. coli at 100% (11.00 mm was not significantly different from the 75% (7.83 mm and 50% (7.50 mm. The ability of ethanol extract of soka flower in inhibition zone against S. aureus greater than E. coli. Keywords:               Ixora coccinea L., inhibition zone, Staphylococcus aureus and Escherichia coli.

The oxygen effect in E. coli cells

International Nuclear Information System (INIS)

Myasnik, M.N.; Skvortsov, V.G.; Sokolov, V.A.

1982-01-01

In experiments on E. coli strains deficient in some stages of DNA repair from radiation damages, it was demonstrated that the value of the oxygen effect, under optimal conditions for manifestation thereof, decreases in the following order: E. coli WP2 (the wild type) → E. coli WP2 exr - and E. coli B → E. coli WP2 uvr A6 → E. coli WP2 rec Al and E. coli WP2 hcr - exr - . It was detected that 0.14 M NaCl solution sensitizes the anoxic cells of some E. coli strains to the effect of γ-radiation. It was established that mutation of the uvr A-gene increases sharply the sensitivity of cells to iradiation under the anoxic conditions in the presence of NaCl, the reverse'' oxygen effect being observed

Hemolytic porcine intestinal Escherichia coli without virulence-associated genes typical of intestinal pathogenic E. coli.

Science.gov (United States)

Schierack, Peter; Weinreich, Joerg; Ewers, Christa; Tachu, Babila; Nicholson, Bryon; Barth, Stefanie

2011-12-01

Testing 1,666 fecal or intestinal samples from healthy and diarrheic pigs, we obtained hemolytic Escherichia coli isolates from 593 samples. Focusing on hemolytic E. coli isolates without virulence-associated genes (VAGs) typical for enteropathogens, we found that such isolates carried a broad variety of VAGs typical for extraintestinal pathogenic E. coli.

Radioprotective action of glycerol and cysteamine on inactivation and mutagenesis in Salmonella tester strains after gamma and heavy ion irradiation

International Nuclear Information System (INIS)

Basha, S.G.; Krasavin, E.A.; Kozubek, S.

1991-01-01

Inactivation and mutagenesis were studied in Salmonella tester strains after γ-irradiation and after heavy ion irradiation in the presence of glycerol and cysteamine. Bacterial cells were irradiated at Dubna, JINR. Ions from deuterons to carbon were used with residual energies 2-9 MeV/u. The protective effect of glycerol was found both for γ-radiation and for heavy ions up to 50 keV/μm for both cell inactivation and mutagenesis in Salmonella tester strains with different mutation events. Cell sensitivity slightly increased with LET before falling down. The maximum was shifted in the presence of glycerol to the left and was less pronounced. The radioprotective effect of glycerol diminished gradually with LET from 2.0 for γ-radiation to 1.1 for carbon ions. Mutagenesis increases with LET in TA100 strain; in TA98 strain no marked increase could be detected. 13 refs.; 4 figs.; 5 tabs

escherichia coli serotypes confirmed in experimental mammary ...

African Journals Online (AJOL)

DJFLEX

VARIATIONS IN VIRULENCE OF THREE (3) ESCHERICHIA COLI. SEROTYPES CONFIRMED IN ... ows are susceptible to E. coli infection because. E. coli exist in the .... Coli infections in mice: A laboratory animal model for research in.

Genome-wide maps of alkylation damage, repair, and mutagenesis in yeast reveal mechanisms of mutational heterogeneity.

Science.gov (United States)

Mao, Peng; Brown, Alexander J; Malc, Ewa P; Mieczkowski, Piotr A; Smerdon, Michael J; Roberts, Steven A; Wyrick, John J

2017-10-01

DNA base damage is an important contributor to genome instability, but how the formation and repair of these lesions is affected by the genomic landscape and contributes to mutagenesis is unknown. Here, we describe genome-wide maps of DNA base damage, repair, and mutagenesis at single nucleotide resolution in yeast treated with the alkylating agent methyl methanesulfonate (MMS). Analysis of these maps revealed that base excision repair (BER) of alkylation damage is significantly modulated by chromatin, with faster repair in nucleosome-depleted regions, and slower repair and higher mutation density within strongly positioned nucleosomes. Both the translational and rotational settings of lesions within nucleosomes significantly influence BER efficiency; moreover, this effect is asymmetric relative to the nucleosome dyad axis and is regulated by histone modifications. Our data also indicate that MMS-induced mutations at adenine nucleotides are significantly enriched on the nontranscribed strand (NTS) of yeast genes, particularly in BER-deficient strains, due to higher damage formation on the NTS and transcription-coupled repair of the transcribed strand (TS). These findings reveal the influence of chromatin on repair and mutagenesis of base lesions on a genome-wide scale and suggest a novel mechanism for transcription-associated mutation asymmetry, which is frequently observed in human cancers. © 2017 Mao et al.; Published by Cold Spring Harbor Laboratory Press.

Prevalence and behavior of multidrug-resistant shiga toxin-producing Escherichia coli, enteropathogenic E. coli and enterotoxigenic E. coli on coriander.