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Sample records for restriction endonuclease ecorii

  1. Identification of a DNA restriction-modification system in Pectobacterium carotovorum strains isolated from Poland.

    Science.gov (United States)

    Waleron, K; Waleron, M; Osipiuk, J; Podhajska, A J; Lojkowska, E

    2006-02-01

    Polish isolates of pectinolytic bacteria from the species Pectobacterium carotovorum were screened for the presence of a DNA restriction-modification (R-M) system. Eighty-nine strains of P. carotovorum were isolated from infected potato plants. Sixty-six strains belonged to P. carotovorum ssp. atrosepticum and 23 to P. carotovorum ssp. carotovorum. The presence of restriction enzyme Pca17AI, which is an isoschizomer of EcoRII endonuclease, was observed in all isolates of P. c. atrosepticum but not in P. c. carotovorum. The biochemical properties, PCR amplification, and sequences of the Pca17AI restriction endonuclease and methyltransferase genes were compared with the prototype EcoRII R-M system genes. Only when DNA isolated from cells of P. c. atrosepticum was used as a template, amplification of a 680 bp homologous to the gene coding EcoRII endonuclease. Endonuclease Pca17AI, having a relatively low temperature optimum, was identified. PCR amplification revealed that the nucleotide sequence of genes for EcoRII and Pca17AI R-M are different. Dcm methylation was observed in all strains of Pectobacterium and other Erwinia species tested. The sequence of a DNA fragment coding Dcm methylase in P. carotovorum was different from that of Escherichia coli. Pca17AI is the first psychrophilic isoschizomer of EcoRII endonuclease. The presence of specific Dcm methylation in chromosomal DNA isolated from P. carotovorum is described for the first time. A 680 bp PCR product, unique for P. c. atrosepticum strains, could serve as a molecular marker for detection of these bacteria in environmental samples.

  2. A new restriction endonuclease from Citrobacter freundii

    OpenAIRE

    Janulaitis, A.A.; Stakenas, P.S.; Lebedenko, E.N.; Berlin, Yu.A.

    1982-01-01

    CfrI, a new restriction endonuclease of unique substrate specificity, has been isolated from a Citrobacter freundii strain. The enzyme recognizes a degenerated sequence PyGGCCPu in double-strand DNA and cleaves it between Py and G residues to yield 5′ -protruding tetranucleotide ends GGCC.

  3. Comparison of genomes of malignant catarrhal fever-associated herpesviruses by restriction endonuclease analysis.

    Science.gov (United States)

    Shih, L M; Zee, Y C; Castro, A E

    1989-01-01

    The restriction endonuclease DNA cleavage patterns of eight isolates of malignant catarrhal fever-associated herpesviruses were examined using the restriction endonucleases HindIII and EcoRI. The eight viruses could be assigned to two distinct groups. Virus isolates from a blue wildebeest, a sika deer and an ibex had restriction endonuclease DNA cleavage patterns that were in general similar to each other. The restriction pattern of these three viruses was distinct from the other five. Of these five, four were isolated from a greater kudu, a white tailed wildebeest, a white bearded wildebeest, and a cape hartebeest. The fifth isolate C500, was isolated from a domestic cow with malignant catarrhal fever. These five viruses had similar DNA cleavage patterns.

  4. A new restriction endonuclease from Citrobacter freundii

    Science.gov (United States)

    Janulaitis, A.A.; Stakenas, P.S.; Lebedenko, E.N.; Berlin, Yu.A.

    1982-01-01

    CfrI, a new restriction endonuclease of unique substrate specificity, has been isolated from a Citrobacter freundii strain. The enzyme recognizes a degenerated sequence PyGGCCPu in double-strand DNA and cleaves it between Py and G residues to yield 5′ -protruding tetranucleotide ends GGCC. Images PMID:6294607

  5. DNA Nucleotide Sequence Restricted by the RI Endonuclease

    Science.gov (United States)

    Hedgpeth, Joe; Goodman, Howard M.; Boyer, Herbert W.

    1972-01-01

    The sequence of DNA base pairs adjacent to the phosphodiester bonds cleaved by the RI restriction endonuclease in unmodified DNA from coliphage λ has been determined. The 5′-terminal nucleotide labeled with 32P and oligonucleotides up to the heptamer were analyzed from a pancreatic DNase digest. The following sequence of nucleotides adjacent to the RI break made in λ DNA was deduced from these data and from the 3′-dinucleotide sequence and nearest-neighbor analysis obtained from repair synthesis with the DNA polymerase of Rous sarcoma virus [Formula: see text] The RI endonuclease cleavage of the phosphodiester bonds (indicated by arrows) generates 5′-phosphoryls and short cohesive termini of four nucleotides, pApApTpT. The most striking feature of the sequence is its symmetry. PMID:4343974

  6. Simple and sensitive fluorescence assay of restriction endonuclease on graphene oxide

    International Nuclear Information System (INIS)

    Gang, Jong Back

    2015-01-01

    Restriction endonucleases hydrolyze internal phosphodiester bonds at specific sites in a DNA sequence. These enzymes are essential in a variety of fields, such as biotechnology and clinical diagnostics. It is of great importance and necessity for the scientific and biomedical use of enzymes to measure endonuclease activity. In this study, graphene oxide (GO) has been used as a platform to measure enzyme activity with high sensitivity. To increase the detection sensitivity of Hinf I, the endonuclease-digested reaction was treated with exonuclease III (Exo III) and a fluorescence assay was conducted to measure the emission. Results showed that Exo III treatment enhanced 2.7-fold signal-to-background ratio for the detection of Hinf I compared with that done without Exo III in the presence of GO

  7. Survival of Saccharomyces cerevisiae after treatment with the restriction endonuclease Alu I

    International Nuclear Information System (INIS)

    Winckler, K.; Bach, B.; Obe, G.

    1988-01-01

    Treatment of yeast cells proficient in the repair of radiation damage (Saccharomyces cervisiae) with the restriction endonuclease Alu I leads to a positive dose-effect relationship between inactivation level and enzyme concentration. The data suggest an uptake of the active restriction enzyme into the cells and a relationship between induction of DNA double-strand breaks and cell killing. (author)

  8. Expression and Purification of BmrI Restriction Endonuclease and Its N-terminal Cleavage Domain Variants

    OpenAIRE

    Bao, Yongming; Higgins, Lauren; Zhang, Penghua; Chan, Siu-hong; Laget, Sophie; Sweeney, Suzanne; Lunnen, Keith; Xu, Shuang-yong

    2007-01-01

    BmrI (ACTGGG N5/N4) is one of the few metal-independent restriction endonucleases (REases) found in bacteria. The BmrI restriction-modification system was cloned by the methylase selection method, inverse PCR, and PCR. BmrI REase shows significant amino acid sequence identity to BfiI and a putative endonuclease MspBNCORF3798 from the sequenced Mesorhizobium sp. BNC1 genome. The EDTA-resistant BmrI REase was successfully over-expressed in a pre-modified E. coli strain from pET21a or pBAC-expIQ...

  9. Purification, crystallization, X-ray diffraction analysis and phasing of an engineered single-chain PvuII restriction endonuclease

    International Nuclear Information System (INIS)

    Meramveliotaki, Chrysi; Kotsifaki, Dina; Androulaki, Maria; Hountas, Athanasios; Eliopoulos, Elias; Kokkinidis, Michael

    2007-01-01

    PvuII is the first type II restriction endonuclease to be converted from its wild-type homodimeric form into an enzymatically active single-chain variant. The enzyme was crystallized and phasing was successfully performed by molecular replacement. The restriction endonuclease PvuII from Proteus vulgaris has been converted from its wild-type homodimeric form into the enzymatically active single-chain variant scPvuII by tandemly joining the two subunits through the peptide linker Gly-Ser-Gly-Gly. scPvuII, which is suitable for the development of programmed restriction endonucleases for highly specific DNA cleavage, was purified and crystallized. The crystals diffract to a resolution of 2.35 Å and belong to space group P4 2 , with unit-cell parameters a = b = 101.92, c = 100.28 Å and two molecules per asymmetric unit. Phasing was successfully performed by molecular replacement

  10. Cofactor requirement of HpyAV restriction endonuclease.

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    Siu-Hong Chan

    Full Text Available BACKGROUND: Helicobacter pylori is the etiologic agent of common gastritis and a risk factor for gastric cancer. It is also one of the richest sources of Type II restriction-modification (R-M systems in microorganisms. PRINCIPAL FINDINGS: We have cloned, expressed and purified a new restriction endonuclease HpyAV from H. pylori strain 26695. We determined the HpyAV DNA recognition sequence and cleavage site as CCTTC 6/5. In addition, we found that HpyAV has a unique metal ion requirement: its cleavage activity is higher with transition metal ions than in Mg(++. The special metal ion requirement of HpyAV can be attributed to the presence of a HNH catalytic site similar to ColE9 nuclease instead of the canonical PD-X-D/EXK catalytic site found in many other REases. Site-directed mutagenesis was carried out to verify the catalytic residues of HpyAV. Mutation of the conserved metal-binding Asn311 and His320 to alanine eliminated cleavage activity. HpyAV variant H295A displayed approximately 1% of wt activity. CONCLUSIONS/SIGNIFICANCE: Some HNH-type endonucleases have unique metal ion cofactor requirement for optimal activities. Homology modeling and site-directed mutagenesis confirmed that HpyAV is a member of the HNH nuclease family. The identification of catalytic residues in HpyAV paved the way for further engineering of the metal binding site. A survey of sequenced microbial genomes uncovered 10 putative R-M systems that show high sequence similarity to the HpyAV system, suggesting lateral transfer of a prototypic HpyAV-like R-M system among these microorganisms.

  11. Cleavage and protection of locked nucleic acid-modified DNA by restriction endonucleases

    DEFF Research Database (Denmark)

    Crouzier, Lucile; Dubois, Camille; Wengel, Jesper

    2012-01-01

    Locked nucleic acid (LNA) is one of the most prominent nucleic acid analogues reported so far. We herein for the first time report cleavage by restriction endonuclease of LNA-modified DNA oligonucleotides. The experiments revealed that RsaI is an efficient enzyme capable of recognizing and cleaving...

  12. Type II restriction endonucleases : a historical perspective and more

    OpenAIRE

    Pingoud, Alfred; Wilson, Geoffrey G.; Wende, Wolfgang

    2014-01-01

    This article continues the series of Surveys and Summaries on restriction endonucleases (REases) begun this year in Nucleic Acids Research. Here we discuss ‘Type II’ REases, the kind used for DNA analysis and cloning. We focus on their biochemistry: what they are, what they do, and how they do it. Type II REases are produced by prokaryotes to combat bacteriophages. With extreme accuracy, each recognizes a particular sequence in double-stranded DNA and cleaves at a fixed position within or nea...

  13. [Restriction endonuclease digest - melting curve analysis: a new SNP genotyping and its application in traditional Chinese medicine authentication].

    Science.gov (United States)

    Jiang, Chao; Huang, Lu-Qi; Yuan, Yuan; Chen, Min; Hou, Jing-Yi; Wu, Zhi-Gang; Lin, Shu-Fang

    2014-04-01

    Single nucleotide polymorphisms (SNP) is an important molecular marker in traditional Chinese medicine research, and it is widely used in TCM authentication. The present study created a new genotyping method by combining restriction endonuclease digesting with melting curve analysis, which is a stable, rapid and easy doing SNP genotyping method. The new method analyzed SNP genotyping of two chloroplast SNP which was located in or out of the endonuclease recognition site, the results showed that when attaching a 14 bp GC-clamp (cggcgggagggcgg) to 5' end of the primer and selecting suited endonuclease to digest the amplification products, the melting curve of Lonicera japonica and Atractylodes macrocephala were all of double peaks and the adulterants Shan-yin-hua and A. lancea were of single peaks. The results indicated that the method had good stability and reproducibility for identifying authentic medicines from its adulterants. It is a potential SNP genotyping method and named restriction endonuclease digest - melting curve analysis.

  14. Transient and Switchable (Triethylsilyl)ethynyl Protection of DNA against Cleavage by Restriction Endonucleases

    Czech Academy of Sciences Publication Activity Database

    Kielkowski, Pavel; Macíčková-Cahová, Hana; Pohl, Radek; Hocek, Michal

    2011-01-01

    Roč. 50, č. 37 (2011), s. 8727-8730 ISSN 1433-7851 R&D Projects: GA ČR GA203/09/0317 Institutional research plan: CEZ:AV0Z40550506 Keywords : alkynes * DNA * protecting groups * nucleotides * restriction endonucleases Subject RIV: CC - Organic Chemistry Impact factor: 13.455, year: 2011

  15. Restriction endonuclease analysis of chloroplast DNA in interspecies somatic Hybrids of Petunia.

    Science.gov (United States)

    Kumar, A; Cocking, E C; Bovenberg, W A; Kool, A J

    1982-12-01

    Restriction endonuclease cleavage pattern analysis of chloroplast DNA (cpDNA) of three different interspecific somatic hybrid plants revealed that the cytoplasms of the hybrids contained only cpDNA of P. parodii. The somatic hybrid plants analysed were those between P. parodii (wild type) + P. hybrida (wild type); P. parodii (wild type)+P. inflata (cytoplasmic albino mutant); P. parodii (wild type) + P. parviflora (nuclear albino mutant). The presence of only P. parodii chloroplasts in the somatic hybrid of P. parodii + P. inflata is possibly due to the stringent selection used for somatic hybrid production. However, in the case of the two other somatic hybrids P. parodii + P. hybrida and P. parodii + P. parviflora it was not possible to determine whether the presence of only P. parodii chloroplasts in these somatic hybrid plants was due to the nature of the selection schemes used or simply occurred by chance. The relevance of such somatic hybrid material for the study of genomic-cytoplasmic interaction is discussed, as well as the use of restriction endonuclease fragment patterns for the analysis of taxonomic and evolutionary inter-relationships in the genus Petunia.

  16. Cleavage of DNA containing 5-fluorocytosine or 5-fluorouracil by type II restriction endonucleases

    Czech Academy of Sciences Publication Activity Database

    Olszewska, Agata; Daďová, Jitka; Mačková, Michaela; Hocek, Michal

    2015-01-01

    Roč. 23, č. 21 (2015), s. 6885-6890 ISSN 0968-0896 R&D Projects: GA ČR GA14-04289S Institutional support: RVO:61388963 Keywords : modified nucleotides * DNA * restriction endonucleases * DNA polymerase * pyrimidine nucleosides Subject RIV: CC - Organic Chemistry Impact factor: 2.923, year: 2015

  17. Restriction endonuclease analysis of Pasteurella multocida isolates from three California turkey premises.

    Science.gov (United States)

    Christiansen, K H; Carpenter, T E; Snipes, K P; Hird, D W; Ghazikhanian, G Y

    1992-01-01

    Three California turkey premises that had repeated outbreaks of fowl cholera were studied for periods of 2 to 4 years. Using biochemical, serologic, plasmid DNA, and restriction endonuclease analyses of isolates of Pasteurella multocida from turkeys and wildlife on the premises, strains of the organism were found to be enzootic on two of the premises. On the third, a variety of strains of P. multocida were isolated from fowl cholera outbreak flocks.

  18. Geographically diverse Australian isolates of Melissococcus pluton exhibit minimal genotypic diversity by restriction endonuclease analysis.

    Science.gov (United States)

    Djordjevic, S P; Smith, L A; Forbes, W A; Hornitzky, M A

    1999-04-15

    Melissococcus pluton, the causative agent of European foulbrood is an economically significant disease of honey bees (Apis mellifera) across most regions of the world and is prevalent throughout most states of Australia. 49 Isolates of M. pluton recovered from diseased colonies or honey samples in New South Wales, Queensland, South Australia, Tasmania and Victoria were compared using SDS-PAGE, Western immunoblotting and restriction endonuclease analyses. DNA profiles of all 49 geographically diverse isolates showed remarkably similar AluI profiles although four isolates (one each from Queensland, South Australia, New South Wales and Victoria) displayed minor profile variations compared to AluI patterns of all other isolates. DNA from a subset of the 49 Australian and three isolates from the United Kingdom were digested separately with the restriction endonucleases CfoI, RsaI and DraI. Restriction endonuclease fragment patterns generated using these enzymes were also similar although minor variations were noted. SDS-PAGE of whole cell proteins from 13 of the 49 isolates from different states of Australia, including the four isolates which displayed minor profile variations (AluI) produced indistinguishable patterns. Major immunoreactive proteins of approximate molecular masses of 21, 24, 28, 30, 36, 40, 44, 56, 60, 71, 79 and 95 kDa were observed in immunoblots of whole cell lysates of 22 of the 49 isolates and reacted with rabbit hyperimmune antibodies raised against M. pluton whole cells. Neither SDS-PAGE or immunoblotting was capable of distinguishing differences between geographically diverse isolates of M. pluton. Collectively these data confirm that Australian isolates of M. pluton are genetically homogeneous and that this species may be clonal. Plasmid DNA was not detected in whole cell DNA profiles of any isolate resolved using agarose gel electrophoresis.

  19. Alteration of Sequence Specificity of the Type IIS Restriction Endonuclease BtsI

    OpenAIRE

    Guan, Shengxi; Blanchard, Aine; Zhang, Penghua; Zhu, Zhenyu

    2010-01-01

    The Type IIS restriction endonuclease BtsI recognizes and digests at GCAGTG(2/0). It comprises two subunits: BtsIA and BtsIB. The BtsIB subunit contains the recognition domain, one catalytic domain for bottom strand nicking and part of the catalytic domain for the top strand nicking. BtsIA has the rest of the catalytic domain that is responsible for the DNA top strand nicking. BtsIA alone has no activity unless it mixes with BtsIB to reconstitute the BtsI activity. During characterization of ...

  20. A RecB-family nuclease motif in the Type I restriction endonuclease EcoR124I

    Czech Academy of Sciences Publication Activity Database

    Šišáková, Eva; Stanley, L. K.; Weiserová, Marie; Szczelkun, M. D.

    2008-01-01

    Roč. 36, č. 12 (2008), s. 1-11 ISSN 0305-1048 R&D Projects: GA ČR GA204/07/0325 Grant - others:XE(XE) BioNano-Switch 043288 Institutional research plan: CEZ:AV0Z50200510 Keywords : restriction endonuclease * mutagenesis * dsdna Subject RIV: EE - Microbiology, Virology Impact factor: 6.878, year: 2008

  1. Atypical myxomatosis--virus isolation, experimental infection of rabbits and restriction endonuclease analysis of the isolate.

    Science.gov (United States)

    Psikal, I; Smíd, B; Rodák, L; Valícek, L; Bendová, J

    2003-08-01

    Atypical form of myxomatosis, which caused non-lethal and clinically mild disease in domestic rabbits 1 month after immunization with a commercially available vaccine MXT, is described. The isolated myxoma virus designated as Litovel 2 (Li-2) did not induce systemic disease following subcutaneous and intradermal applications in susceptible experimental rabbits but led to the immune response demonstrated by ELISA. No severe disease was induced in those Li-2 inoculated rabbits by challenge with the virulent strains Lausanne (Lu) or Sanar (SA), while the control animals showed nodular form of myxomatosis with lethal course of the illness. Restriction fragment length polymorphism (RFLP) of genomic DNA with KpnI and BamHI endonucleases was used for genetic characterization of the Li-2 isolate, the vaccine strain MXT and both virulent strains Lu and SA, respectively. In general, RFLP analysis has shown to be informative for inferring genetic relatedness between myxoma viruses. Based on restriction endonuclease DNA fragment size distribution, it was evident that the pathogenic strain SA is genetically related to the reference strain Lu and the isolate Li-2 is more related, but not identical, to the vaccination strain MXT.

  2. Multiplex, rapid and sensitive isothermal detection of nucleic-acid sequence by endonuclease restriction-mediated real-time multiple cross displacement amplification

    Directory of Open Access Journals (Sweden)

    Yi eWang

    2016-05-01

    Full Text Available We have devised a novel isothermal amplification technology, termed endonuclease restriction-mediated real-time multiple cross displacement amplification (ET-MCDA, which facilitated multiplex, rapid, specific and sensitive detection of nucleic-acid sequences at a constant temperature. The ET-MCDA integrated multiple cross displacement amplification strategy, restriction endonuclease cleavage and real-time fluorescence detection technique. In the ET-MCDA system, the functional cross primer E-CP1 or E-CP2 was constructed by adding a short sequence at the 5’ end of CP1 or CP2, respectively, and the new E-CP1 or E-CP2 primer was labelled at the 5’ end with a fluorophore and in the middle with a dark quencher. The restriction endonuclease Nb.BsrDI specifically recognized the short sequence and digested the newly synthesized double-stranded terminal sequences (5’ end short sequences and their complementary sequences, which released the quenching, resulting on a gain of fluorescence signal. Thus, the ET-MCDA allowed real-time detection of single or multiple targets in only a single reaction, and the positive results were observed in as short as 12 minutes, detecting down to 3.125 fg of genomic DNA per tube. Moreover, the analytical specificity and the practical application of the ET-MCDA were also successfully evaluated in this study. Here we provided the details on the novel ET-MCDA technique and expounded the basic ET-MCDA amplification mechanism.

  3. Multiplex, Rapid, and Sensitive Isothermal Detection of Nucleic-Acid Sequence by Endonuclease Restriction-Mediated Real-Time Multiple Cross Displacement Amplification.

    Science.gov (United States)

    Wang, Yi; Wang, Yan; Zhang, Lu; Liu, Dongxin; Luo, Lijuan; Li, Hua; Cao, Xiaolong; Liu, Kai; Xu, Jianguo; Ye, Changyun

    2016-01-01

    We have devised a novel isothermal amplification technology, termed endonuclease restriction-mediated real-time multiple cross displacement amplification (ET-MCDA), which facilitated multiplex, rapid, specific and sensitive detection of nucleic-acid sequences at a constant temperature. The ET-MCDA integrated multiple cross displacement amplification strategy, restriction endonuclease cleavage and real-time fluorescence detection technique. In the ET-MCDA system, the functional cross primer E-CP1 or E-CP2 was constructed by adding a short sequence at the 5' end of CP1 or CP2, respectively, and the new E-CP1 or E-CP2 primer was labeled at the 5' end with a fluorophore and in the middle with a dark quencher. The restriction endonuclease Nb.BsrDI specifically recognized the short sequence and digested the newly synthesized double-stranded terminal sequences (5' end short sequences and their complementary sequences), which released the quenching, resulting on a gain of fluorescence signal. Thus, the ET-MCDA allowed real-time detection of single or multiple targets in only a single reaction, and the positive results were observed in as short as 12 min, detecting down to 3.125 fg of genomic DNA per tube. Moreover, the analytical specificity and the practical application of the ET-MCDA were also successfully evaluated in this study. Here, we provided the details on the novel ET-MCDA technique and expounded the basic ET-MCDA amplification mechanism.

  4. Endonucleases induced TRAIL-insensitive apoptosis in ovarian carcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Geel, Tessa M. [Department of Pathology and Medical Biology, Groningen University Institute for Drug Exploration (GUIDE), University Medical Center Groningen (UMCG), Hanzeplein 1, 9713 GZ, Groningen (Netherlands); Meiss, Gregor [Institute of Biochemistry, Justus-Liebig-University Giessen, D-35392 Giessen (Germany); Gun, Bernardina T. van der; Kroesen, Bart Jan; Leij, Lou F. de [Department of Pathology and Medical Biology, Groningen University Institute for Drug Exploration (GUIDE), University Medical Center Groningen (UMCG), Hanzeplein 1, 9713 GZ, Groningen (Netherlands); Zaremba, Mindaugas; Silanskas, Arunas [Institute of Biotechnology, Vilnius LT-02241 (Lithuania); Kokkinidis, Michael [IMBB/FORTH and University of Crete/Department of Biology, GR-71409 Heraklion/Crete (Greece); Pingoud, Alfred [Institute of Biochemistry, Justus-Liebig-University Giessen, D-35392 Giessen (Germany); Ruiters, Marcel H. [Department of Pathology and Medical Biology, Groningen University Institute for Drug Exploration (GUIDE), University Medical Center Groningen (UMCG), Hanzeplein 1, 9713 GZ, Groningen (Netherlands); Synvolux therapeutics, Groningen (Netherlands); McLaughlin, Pamela M. [Department of Pathology and Medical Biology, Groningen University Institute for Drug Exploration (GUIDE), University Medical Center Groningen (UMCG), Hanzeplein 1, 9713 GZ, Groningen (Netherlands); Rots, Marianne G., E-mail: m.g.rots@med.umcg.nl [Department of Pathology and Medical Biology, Groningen University Institute for Drug Exploration (GUIDE), University Medical Center Groningen (UMCG), Hanzeplein 1, 9713 GZ, Groningen (Netherlands)

    2009-09-10

    TRAIL induced apoptosis of tumor cells is currently entering phase II clinical settings, despite the fact that not all tumor types are sensitive to TRAIL. TRAIL resistance in ovarian carcinomas can be caused by a blockade upstream of the caspase 3 signaling cascade. We explored the ability of restriction endonucleases to directly digest DNA in vivo, thereby circumventing the caspase cascade. For this purpose, we delivered enzymatically active endonucleases via the cationic amphiphilic lipid SAINT-18{sup Registered-Sign }:DOPE to both TRAIL-sensitive and insensitive ovarian carcinoma cells (OVCAR and SKOV-3, respectively). Functional nuclear localization after delivery of various endonucleases (BfiI, PvuII and NucA) was indicated by confocal microscopy and genomic cleavage analysis. For PvuII, analysis of mitochondrial damage demonstrated extensive apoptosis both in SKOV-3 and OVCAR. This study clearly demonstrates that cellular delivery of restriction endonucleases holds promise to serve as a novel therapeutic tool for the treatment of resistant ovarian carcinomas.

  5. Restriction endonucleases from invasive Neisseria gonorrhoeae cause double-strand breaks and distort mitosis in epithelial cells during infection.

    Directory of Open Access Journals (Sweden)

    Linda Weyler

    Full Text Available The host epithelium is both a barrier against, and the target for microbial infections. Maintaining regulated cell growth ensures an intact protective layer towards microbial-induced cellular damage. Neisseria gonorrhoeae infections disrupt host cell cycle regulation machinery and the infection causes DNA double strand breaks that delay progression through the G2/M phase. We show that intracellular gonococci upregulate and release restriction endonucleases that enter the nucleus and damage human chromosomal DNA. Bacterial lysates containing restriction endonucleases were able to fragment genomic DNA as detected by PFGE. Lysates were also microinjected into the cytoplasm of cells in interphase and after 20 h, DNA double strand breaks were identified by 53BP1 staining. In addition, by using live-cell microscopy and NHS-ester stained live gonococci we visualized the subcellular location of the bacteria upon mitosis. Infected cells show dysregulation of the spindle assembly checkpoint proteins MAD1 and MAD2, impaired and prolonged M-phase, nuclear swelling, micronuclei formation and chromosomal instability. These data highlight basic molecular functions of how gonococcal infections affect host cell cycle regulation, cause DNA double strand breaks and predispose cellular malignancies.

  6. Restriction endonucleases from invasive Neisseria gonorrhoeae cause double-strand breaks and distort mitosis in epithelial cells during infection.

    Science.gov (United States)

    Weyler, Linda; Engelbrecht, Mattias; Mata Forsberg, Manuel; Brehwens, Karl; Vare, Daniel; Vielfort, Katarina; Wojcik, Andrzej; Aro, Helena

    2014-01-01

    The host epithelium is both a barrier against, and the target for microbial infections. Maintaining regulated cell growth ensures an intact protective layer towards microbial-induced cellular damage. Neisseria gonorrhoeae infections disrupt host cell cycle regulation machinery and the infection causes DNA double strand breaks that delay progression through the G2/M phase. We show that intracellular gonococci upregulate and release restriction endonucleases that enter the nucleus and damage human chromosomal DNA. Bacterial lysates containing restriction endonucleases were able to fragment genomic DNA as detected by PFGE. Lysates were also microinjected into the cytoplasm of cells in interphase and after 20 h, DNA double strand breaks were identified by 53BP1 staining. In addition, by using live-cell microscopy and NHS-ester stained live gonococci we visualized the subcellular location of the bacteria upon mitosis. Infected cells show dysregulation of the spindle assembly checkpoint proteins MAD1 and MAD2, impaired and prolonged M-phase, nuclear swelling, micronuclei formation and chromosomal instability. These data highlight basic molecular functions of how gonococcal infections affect host cell cycle regulation, cause DNA double strand breaks and predispose cellular malignancies.

  7. Cloning and analysis of a bifunctional methyltransferase/restriction endonuclease TspGWI, the prototype of a Thermus sp. enzyme family

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    Zylicz-Stachula Agnieszka

    2009-05-01

    Full Text Available Abstract Background Restriction-modification systems are a diverse class of enzymes. They are classified into four major types: I, II, III and IV. We have previously proposed the existence of a Thermus sp. enzyme family, which belongs to type II restriction endonucleases (REases, however, it features also some characteristics of types I and III. Members include related thermophilic endonucleases: TspGWI, TaqII, TspDTI, and Tth111II. Results Here we describe cloning, mutagenesis and analysis of the prototype TspGWI enzyme that recognises the 5'-ACGGA-3' site and cleaves 11/9 nt downstream. We cloned, expressed, and mutagenised the tspgwi gene and investigated the properties of its product, the bifunctional TspGWI restriction/modification enzyme. Since TspGWI does not cleave DNA completely, a cloning method was devised, based on amino acid sequencing of internal proteolytic fragments. The deduced amino acid sequence of the enzyme shares significant sequence similarity with another representative of the Thermus sp. family – TaqII. Interestingly, these enzymes recognise similar, yet different sequences in the DNA. Both enzymes cleave DNA at the same distance, but differ in their ability to cleave single sites and in the requirement of S-adenosylmethionine as an allosteric activator for cleavage. Both the restriction endonuclease (REase and methyltransferase (MTase activities of wild type (wt TspGWI (either recombinant or isolated from Thermus sp. are dependent on the presence of divalent cations. Conclusion TspGWI is a bifunctional protein comprising a tandem arrangement of Type I-like domains; particularly noticeable is the central HsdM-like module comprising a helical domain and a highly conserved S-adenosylmethionine-binding/catalytic MTase domain, containing DPAVGTG and NPPY motifs. TspGWI also possesses an N-terminal PD-(D/EXK nuclease domain related to the corresponding domains in HsdR subunits, but lacks the ATP-dependent translocase module

  8. Species attribution and strain typing of Oenococcus oeni (formerly Leuconostoc oenos) with restriction endonuclease fingerprints.

    Science.gov (United States)

    Viti, C; Giovannetti, L; Granchi, L; Ventura, S

    1996-10-01

    In several wines, malolactic fermentation is required to improve the organoleptic characters and to stabilize the final product. In order to establish a controlled malolactic fermentation in wine, easy identification and sensitive typing of strains of Oenococcus oeni (new name of the malolactic bacterium Leuconostoc oenos) used as starter cultures are necessary. To accomplish these tasks, several strains of Oenococcus oeni isolated from wines of the Chianti region (Italy), along with reference strains and strains of L. mesenteroides subsp. mesenteroides, L. carnosum, L. fallax, L. pseudomesenteroides, L. lactis and Weisella paramesenteroides, were studied with RFLP of ribosomal genes and ultrasensitive total DNA restriction pattern analysis performed on polyacrylamide gel. With each of four restriction endonucleases used, identical restriction profiles of ribosomal genes were obtained for all strains of O. oeni. These ribopatterns, being strongly dissimilar to profiles of the other lactic acid bacteria tested, appear to be well suited for the attribution of wine lactic acid bacteria to the species O. oeni. Cluster analysis performed on two total DNA restriction profile data sets showed that the species O. oeni possesses a good degree of genomic homogeneity. Very sensitive typing of strains of O. oeni was obtained with total DNA restriction profiles. The potential of an integrated approach using restriction profiles for species assignment and typing of selected malolactic bacteria is demonstrated.

  9. Cleavage of phosphorothioated DNA and methylated DNA by the type IV restriction endonuclease ScoMcrA.

    Directory of Open Access Journals (Sweden)

    Guang Liu

    2010-12-01

    Full Text Available Many taxonomically diverse prokaryotes enzymatically modify their DNA by replacing a non-bridging oxygen with a sulfur atom at specific sequences. The biological implications of this DNA S-modification (phosphorothioation were unknown. We observed that simultaneous expression of the dndA-E gene cluster from Streptomyces lividans 66, which is responsible for the DNA S-modification, and the putative Streptomyces coelicolor A(32 Type IV methyl-dependent restriction endonuclease ScoA3McrA (Sco4631 leads to cell death in the same host. A His-tagged derivative of ScoA3McrA cleaved S-modified DNA and also Dcm-methylated DNA in vitro near the respective modification sites. Double-strand cleavage occurred 16-28 nucleotides away from the phosphorothioate links. DNase I footprinting demonstrated binding of ScoA3McrA to the Dcm methylation site, but no clear binding could be detected at the S-modified site under cleavage conditions. This is the first report of in vitro endonuclease activity of a McrA homologue and also the first demonstration of an enzyme that specifically cleaves S-modified DNA.

  10. Type II restriction endonucleases--a historical perspective and more.

    Science.gov (United States)

    Pingoud, Alfred; Wilson, Geoffrey G; Wende, Wolfgang

    2014-07-01

    This article continues the series of Surveys and Summaries on restriction endonucleases (REases) begun this year in Nucleic Acids Research. Here we discuss 'Type II' REases, the kind used for DNA analysis and cloning. We focus on their biochemistry: what they are, what they do, and how they do it. Type II REases are produced by prokaryotes to combat bacteriophages. With extreme accuracy, each recognizes a particular sequence in double-stranded DNA and cleaves at a fixed position within or nearby. The discoveries of these enzymes in the 1970s, and of the uses to which they could be put, have since impacted every corner of the life sciences. They became the enabling tools of molecular biology, genetics and biotechnology, and made analysis at the most fundamental levels routine. Hundreds of different REases have been discovered and are available commercially. Their genes have been cloned, sequenced and overexpressed. Most have been characterized to some extent, but few have been studied in depth. Here, we describe the original discoveries in this field, and the properties of the first Type II REases investigated. We discuss the mechanisms of sequence recognition and catalysis, and the varied oligomeric modes in which Type II REases act. We describe the surprising heterogeneity revealed by comparisons of their sequences and structures. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. The mechanisms of action of E. coli endonuclease III and T4 UV endonuclease (endonuclease V) at AP sites.

    OpenAIRE

    Kim, J; Linn, S

    1988-01-01

    Treatment of DNA containing AP sites with either T4 UV endonuclease or with E. coli endonuclease III followed by a human class II AP endonuclease releases a putative beta-elimination product. This result suggests that both the T4 endonuclease and E. coli endonuclease III class I AP endonucleases catalyze phosphodiester bond cleavage via a lyase- rather than a hydrolase mechanism. Indeed, we have not detected a class I AP endonuclease which hydrolytically catalyzes phosphodiester bond cleavage...

  12. Study on detection of mutation DNA fragment in gastric cancer by restriction endonuclease fingerprinting with capillary electrophoresis.

    Science.gov (United States)

    Wang, Rong; Xie, Hua; Xu, Yue-Bing; Jia, Zheng-Ping; Meng, Xian-Dong; Zhang, Juan-Hong; Ma, Jun; Wang, Juan; Wang, Xian-Hua

    2012-03-01

    The DNA fragment detection focusing technique has further enhanced the sensitivity and information of DNA targets. The DNA fragment detection method was established by capillary electrophoresis with laser-induced fluorescence detection and restriction endonuclease chromatographic fingerprinting (CE-LIF-REF) in our experiment. The silica capillary column was coated with short linear polyarclarylamide (SLPA) using nongel sieving technology. The excision product of various restricted enzymes of DNA fragments was obtained by REF with the molecular biology software Primer Premier 5. The PBR322/BsuRI DNA marker was used to establish the optimization method. The markers were focused electrophoretically and detected by CE-LIF. The results demonstrate that the CE-LIF-REF with SLPA can improve separation, sensitivity and speed of analysis. This technique may be applied to analysis of the excision product of various restricted enzymes of prokaryotic plasmid (pIRES2), eukaryote plasmid (pcDNA3.1) and the PCR product of codon 248 region of gastric cancer tissue. The results suggest that this method could very sensitively separate the excision products of various restricted enzymes at a much better resolution than the traditional agarose electrophoresis. Copyright © 2011 John Wiley & Sons, Ltd.

  13. A new endonuclease recognizing the deoxynucleotide sequence CCNNGG from the cyanobacterium Synechocystis 6701.

    Science.gov (United States)

    Calléja, F; Tandeau de Marsac, N; Coursin, T; van Ormondt, H; de Waard, A

    1985-09-25

    A new sequence-specific endonuclease from the cyanobacterium Synechocystis species PCC 6701 has been purified and characterized. This enzyme, SecI, is unique in recognizing the nucleotide sequence: 5' -CCNNGG-3' 3' -GGNNCC-5' and cleaves it at the position indicated by the symbol. Two other restriction endonucleases, SecII and SecIII, found in this organism are isoschizomers of MspI and MstII, respectively.

  14. The Helicobacter pylori HpyAXII restriction–modification system limits exogenous DNA uptake by targeting GTAC sites but shows asymmetric conservation of the DNA methyltransferase and restriction endonuclease components

    Science.gov (United States)

    Humbert, Olivier; Salama, Nina R.

    2008-01-01

    The naturally competent organism Helicobacter pylori encodes a large number of restriction–modification (R–M) systems that consist of a restriction endonuclease and a DNA methyltransferase. R–M systems are not only believed to limit DNA exchange among bacteria but may also have other cellular functions. We report a previously uncharacterized H. pylori type II R–M system, M.HpyAXII/R.HpyAXII. We show that this system targets GTAC sites, which are rare in the H. pylori chromosome but numerous in ribosomal RNA genes. As predicted, this type II R–M system showed attributes of a selfish element. Deletion of the methyltransferase M.HpyAXII is lethal when associated with an active endonuclease R.HpyAXII unless compensated by adaptive mutation or gene amplification. R.HpyAXII effectively restricted both unmethylated plasmid and chromosomal DNA during natural transformation and was predicted to belong to the novel ‘half pipe’ structural family of endonucleases. Analysis of a panel of clinical isolates revealed that R.HpyAXII was functional in a small number of H. pylori strains (18.9%, n = 37), whereas the activity of M.HpyAXII was highly conserved (92%, n = 50), suggesting that GTAC methylation confers a selective advantage to H. pylori. However, M.HpyAXII activity did not enhance H. pylori fitness during stomach colonization of a mouse infection model. PMID:18978016

  15. Selective inhibition by harmane of the apurinic apyrimidinic endonuclease activity of phage T4-induced UV endonuclease.

    Science.gov (United States)

    Warner, H R; Persson, M L; Bensen, R J; Mosbaugh, D W; Linn, S

    1981-11-25

    1-Methyl-9H-pyrido-[3,4-b]indole (harmane) inhibits the apurinic/apyrimidinic (AP) endonuclease activity of the UV endonuclease induced by phage T4, whereas it stimulates the pyrimidine dimer-DNA glycosylase activity of that enzyme. E. coli endonuclease IV, E. coli endonuclease VI (the AP endonuclease activity associated with E. coli exonuclease III), and E. coli uracil-DNA glycosylase were not inhibited by harmane. Human fibroblast AP endonucleases I and II also were only slightly inhibited. Therefore, harmane is neither a general inhibitor of AP endonucleases, nor a general inhibitor of Class I AP endonucleases which incise DNA on the 3'-side of AP sites. However, E. coli endonuclease III and its associated dihydroxythymine-DNA glycosylase activity were both inhibited by harmane. This observation suggests that harmane may inhibit only AP endonucleases which have associated glycosylase activities.

  16. R Factor-Controlled Restriction and Modification of Deoxyribonucleic Acid: Restriction Mutants

    Science.gov (United States)

    Yoshimori, Robert; Roulland-Dussoix, Daisy; Boyer, Herbert W.

    1972-01-01

    Restriction mutants of two different R factor-controlled host specificities (RI and RII) were isolated. All of the restriction mutants examined had a normal modification phenotype. No complementation was observed between the RI and RII host specificities. It is concluded that for each host specificity no protein subunit is shared by the restriction endonuclease and modification methylase. PMID:4565538

  17. How quantum entanglement in DNA synchronizes double-strand breakage by type II restriction endonucleases.

    Science.gov (United States)

    Kurian, P; Dunston, G; Lindesay, J

    2016-02-21

    Macroscopic quantum effects in living systems have been studied widely in pursuit of fundamental explanations for biological energy transport and sensing. While it is known that type II endonucleases, the largest class of restriction enzymes, induce DNA double-strand breaks by attacking phosphodiester bonds, the mechanism by which simultaneous cutting is coordinated between the catalytic centers remains unclear. We propose a quantum mechanical model for collective electronic behavior in the DNA helix, where dipole-dipole oscillations are quantized through boundary conditions imposed by the enzyme. Zero-point modes of coherent oscillations would provide the energy required for double-strand breakage. Such quanta may be preserved in the presence of thermal noise by the enzyme's displacement of water surrounding the DNA recognition sequence. The enzyme thus serves as a decoherence shield. Palindromic mirror symmetry of the enzyme-DNA complex should conserve parity, because symmetric bond-breaking ceases when the symmetry of the complex is violated or when physiological parameters are perturbed from optima. Persistent correlations in DNA across longer spatial separations-a possible signature of quantum entanglement-may be explained by such a mechanism. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Sequence specificity of DNA cleavage by Micrococcus luteus γ endonuclease

    International Nuclear Information System (INIS)

    Hentosh, P.; Henner, W.D.; Reynolds, R.J.

    1985-01-01

    DNA fragments of defined sequence have been used to determine the sites of cleavage by γ-endonuclease activity in extracts prepared from Micrococcus luteus. End-labeled DNA restriction fragments of pBR322 DNA that had been irradiated under nitrogen in the presence of potassium iodide or t-butanol were treated with M. luteus γ endonuclease and analyzed on irradiated DNA preferentially at the positions of cytosines and thymines. DNA cleavage occurred immediately to the 3' side of pyrimidines in irradiated DNA and resulted in fragments that terminate in a 5'-phosphoryl group. These studies indicate that both altered cytosines and thymines may be important DNA lesions requiring repair after exposure to γ radiation

  19. A new restriction endonuclease-based method for highly-specific detection of DNA targets from methicillin-resistant Staphylococcus aureus.

    Directory of Open Access Journals (Sweden)

    Maria W Smith

    Full Text Available PCR multiplexing has proven to be challenging, and thus has provided limited means for pathogen genotyping. We developed a new approach for analysis of PCR amplicons based on restriction endonuclease digestion. The first stage of the restriction enzyme assay is hybridization of a target DNA to immobilized complementary oligonucleotide probes that carry a molecular marker, horseradish peroxidase (HRP. At the second stage, a target-specific restriction enzyme is added, cleaving the target-probe duplex at the corresponding restriction site and releasing the HRP marker into solution, where it is quantified colorimetrically. The assay was tested for detection of the methicillin-resistant Staphylococcus aureus (MRSA pathogen, using the mecA gene as a target. Calibration curves indicated that the limit of detection for both target oligonucleotide and PCR amplicon was approximately 1 nM. Sequences of target oligonucleotides were altered to demonstrate that (i any mutation of the restriction site reduced the signal to zero; (ii double and triple point mutations of sequences flanking the restriction site reduced restriction to 50-80% of the positive control; and (iii a minimum of a 16-bp target-probe dsDNA hybrid was required for significant cleavage. Further experiments showed that the assay could detect the mecA amplicon from an unpurified PCR mixture with detection limits similar to those with standard fluorescence-based qPCR. Furthermore, addition of a large excess of heterologous genomic DNA did not affect amplicon detection. Specificity of the assay is very high because it involves two biorecognition steps. The proposed assay is low-cost and can be completed in less than 1 hour. Thus, we have demonstrated an efficient new approach for pathogen detection and amplicon genotyping in conjunction with various end-point and qPCR applications. The restriction enzyme assay may also be used for parallel analysis of multiple different amplicons from the same

  20. One-tube loop-mediated isothermal amplification combined with restriction endonuclease digestion and ELISA for colorimetric detection of resistance to isoniazid, ethambutol and streptomycin in Mycobacterium tuberculosis isolates.

    Science.gov (United States)

    Lee, Mei-Feng; Chen, Yen-Hsu; Hsu, Hui-Jine; Peng, Chien-Fang

    2010-10-01

    In this study, we designed a simple and rapid colorimetric detection method, a one-tube loop-mediated isothermal amplification (LAMP)-PCR-hybridization-restriction endonuclease-ELISA [one-tube LAMP-PCR-HY-RE-ELISA] system, to detect resistance to isoniazid, ethambutol and streptomycin in strains of Mycobacterium tuberculosis isolated from clinical specimens. The clinical performance of this method for detecting isoniazid-resistant, ethambutol-resistant and streptomycin-resistant isolates of M. tuberculosis showed 98.9%, 94.3% and 93.8%, respectively. This assay is rapid and convenient that can be performed within one working day. One-tube LAMP-PCR-HY-RE-ELISA system was designed based on hot spot point mutations in target drug-resistant genes, using LAMP-PCR, hybridization, digestion with restriction endonuclease and colorimetric method of ELISA. In this study, LAMP assay was used to amplify DNA from drug-resistant M. tuberculosis, and ELISA was used for colorimetrical determination. This assay will be a useful tool for rapid diagnosis of mutant codons in strains of M. tuberculosis for isoniazid at katG 315 and katG 463, ethambutol at embB 306 and embB 497, and streptomycin at rpsL 43. Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.

  1. The elastic network model reveals a consistent picture on intrinsic functional dynamics of type II restriction endonucleases

    International Nuclear Information System (INIS)

    Uyar, A; Kurkcuoglu, O; Doruker, P; Nilsson, L

    2011-01-01

    The vibrational dynamics of various type II restriction endonucleases, in complex with cognate/non-cognate DNA and in the apo form, are investigated with the elastic network model in order to reveal common functional mechanisms in this enzyme family. Scissor-like and tong-like motions observed in the slowest modes of all enzymes and their complexes point to common DNA recognition and cleavage mechanisms. Normal mode analysis further points out that the scissor-like motion has an important role in differentiating between cognate and non-cognate sequences at the recognition site, thus implying its catalytic relevance. Flexible regions observed around the DNA-binding site of the enzyme usually concentrate on the highly conserved β-strands, especially after DNA binding. These β-strands may have a structurally stabilizing role in functional dynamics for target site recognition and cleavage. In addition, hot spot residues based on high-frequency modes reveal possible communication pathways between the two distant cleavage sites in the enzyme family. Some of these hot spots also exist on the shortest path between the catalytic sites and are highly conserved

  2. Functions that Protect Escherichia coli from Tightly Bound DNA-Protein Complexes Created by Mutant EcoRII Methyltransferase.

    Science.gov (United States)

    Henderson, Morgan L; Kreuzer, Kenneth N

    2015-01-01

    Expression of mutant EcoRII methyltransferase protein (M.EcoRII-C186A) in Escherichia coli leads to tightly bound DNA-protein complexes (TBCs), located sporadically on the chromosome rather than in tandem arrays. The mechanisms behind the lethality induced by such sporadic TBCs are not well studied, nor is it clear whether very tight binding but non-covalent complexes are processed in the same way as covalent DNA-protein crosslinks (DPCs). Using 2D gel electrophoresis, we found that TBCs induced by M.EcoRII-C186A block replication forks in vivo. Specific bubble molecules were detected as spots on the 2D gel, only when M.EcoRII-C186A was induced, and a mutation that eliminates a specific EcoRII methylation site led to disappearance of the corresponding spot. We also performed a candidate gene screen for mutants that are hypersensitive to TBCs induced by M.EcoRII-C186A. We found several gene products necessary for protection against these TBCs that are known to also protect against DPCs induced with wild-type M.EcoRII (after 5-azacytidine incorporation): RecA, RecBC, RecG, RuvABC, UvrD, FtsK, XerCD and SsrA (tmRNA). In contrast, the RecFOR pathway and Rep helicase are needed for protection against TBCs but not DPCs induced by M.EcoRII. We propose that stalled fork processing by RecFOR and RecA promotes release of tightly bound (but non-covalent) blocking proteins, perhaps by licensing Rep helicase-driven dissociation of the blocking M.EcoRII-C186A. Our studies also argued against the involvement of several proteins that might be expected to protect against TBCs. We took the opportunity to directly compare the sensitivity of all tested mutants to two quinolone antibiotics, which target bacterial type II topoisomerases and induce a unique form of DPC. We uncovered rep, ftsK and xerCD as novel quinolone hypersensitive mutants, and also obtained evidence against the involvement of a number of functions that might be expected to protect against quinolones.

  3. Functions that Protect Escherichia coli from Tightly Bound DNA-Protein Complexes Created by Mutant EcoRII Methyltransferase.

    Directory of Open Access Journals (Sweden)

    Morgan L Henderson

    Full Text Available Expression of mutant EcoRII methyltransferase protein (M.EcoRII-C186A in Escherichia coli leads to tightly bound DNA-protein complexes (TBCs, located sporadically on the chromosome rather than in tandem arrays. The mechanisms behind the lethality induced by such sporadic TBCs are not well studied, nor is it clear whether very tight binding but non-covalent complexes are processed in the same way as covalent DNA-protein crosslinks (DPCs. Using 2D gel electrophoresis, we found that TBCs induced by M.EcoRII-C186A block replication forks in vivo. Specific bubble molecules were detected as spots on the 2D gel, only when M.EcoRII-C186A was induced, and a mutation that eliminates a specific EcoRII methylation site led to disappearance of the corresponding spot. We also performed a candidate gene screen for mutants that are hypersensitive to TBCs induced by M.EcoRII-C186A. We found several gene products necessary for protection against these TBCs that are known to also protect against DPCs induced with wild-type M.EcoRII (after 5-azacytidine incorporation: RecA, RecBC, RecG, RuvABC, UvrD, FtsK, XerCD and SsrA (tmRNA. In contrast, the RecFOR pathway and Rep helicase are needed for protection against TBCs but not DPCs induced by M.EcoRII. We propose that stalled fork processing by RecFOR and RecA promotes release of tightly bound (but non-covalent blocking proteins, perhaps by licensing Rep helicase-driven dissociation of the blocking M.EcoRII-C186A. Our studies also argued against the involvement of several proteins that might be expected to protect against TBCs. We took the opportunity to directly compare the sensitivity of all tested mutants to two quinolone antibiotics, which target bacterial type II topoisomerases and induce a unique form of DPC. We uncovered rep, ftsK and xerCD as novel quinolone hypersensitive mutants, and also obtained evidence against the involvement of a number of functions that might be expected to protect against quinolones.

  4. Endonuclease IV Is the major apurinic/apyrimidinic endonuclease in Mycobacterium tuberculosis and is important for protection against oxidative damage.

    Directory of Open Access Journals (Sweden)

    Rupangi Verma Puri

    Full Text Available During the establishment of an infection, bacterial pathogens encounter oxidative stress resulting in the production of DNA lesions. Majority of these lesions are repaired by base excision repair (BER pathway. Amongst these, abasic sites are the most frequent lesions in DNA. Class II apurinic/apyrimidinic (AP endonucleases play a major role in BER of damaged DNA comprising of abasic sites. Mycobacterium tuberculosis, a deadly pathogen, resides in the human macrophages and is continually subjected to oxidative assaults. We have characterized for the first time two AP endonucleases namely Endonuclease IV (End and Exonuclease III (XthA that perform distinct functions in M.tuberculosis. We demonstrate that M.tuberculosis End is a typical AP endonuclease while XthA is predominantly a 3'→5' exonuclease. The AP endonuclease activity of End and XthA was stimulated by Mg(2+ and Ca(2+ and displayed a preferential recognition for abasic site paired opposite to a cytosine residue in DNA. Moreover, End exhibited metal ion independent 3'→5' exonuclease activity while in the case of XthA this activity was metal ion dependent. We demonstrate that End is not only a more efficient AP endonuclease than XthA but it also represents the major AP endonuclease activity in M.tuberculosis and plays a crucial role in defense against oxidative stress.

  5. Phage T4 endonuclease SegD that is similar to group I intron endonucleases does not initiate homing of its own gene.

    Science.gov (United States)

    Sokolov, Andrey S; Latypov, Oleg R; Kolosov, Peter M; Shlyapnikov, Michael G; Bezlepkina, Tamara A; Kholod, Natalia S; Kadyrov, Farid A; Granovsky, Igor E

    2018-02-01

    Homing endonucleases are a group of site-specific endonucleases that initiate homing, a nonreciprocal transfer of its own gene into a new allele lacking this gene. This work describes a novel phage T4 endonuclease, SegD, which is homologous to the GIY-YIG family of homing endonucleases. Like other T4 homing endonucleases SegD recognizes an extended, 16bp long, site, cleaves it asymmetrically to form 3'-protruding ends and digests both unmodified DNA and modified T-even phage DNA with similar efficiencies. Surprisingly, we revealed that SegD cleavage site was identical in the genomes of segD - and segD + phages. We found that segD gene was expressed during the T4 developmental cycle. Nevertheless, endonuclease SegD was not able to initiate homing of its own gene as well as genetic recombination between phages in its site inserted into the rII locus. Copyright © 2018 Elsevier Inc. All rights reserved.

  6. Complementary specificity of restriction endonucleases of Diplococcus pneumoniae with respect to DNA methylation. [Haemophilus influenzae, Escherichia coli, Paramecium aurelia

    Energy Technology Data Exchange (ETDEWEB)

    Lacks, S.; Greenberg, B.

    1977-01-01

    Restriction endonucleases Dpn I and Dpn II are produced by two distinct strains of Diplococcus pneumoniae. The two enzymes show complementary specificity with respect to methylation of sites in DNA. From the identity of its cleavage site with that of Mbo I, it appears that Dpn II cleaves at the unmodified sequence 5'-G-A-T-C-3'. Dpn I cleaves at the same sequence when the adenine residue is methylated. Both enzymes produce only double-strand breaks in susceptible DNA. Their susceptibility to Dpn I and not Dpn II shows that essentially all the G-A-T-C sequences are methylated in DNA from the pneumococcal strain that produces Dpn II as well as in DNA from Hemophilus influenzae and Escherichia coli. In the dam-3 mutant of E. coli none of these sequences appear to be methylated. Residual adenine methylation in the dam-3 mutant DNA most likely occurs at different sites. Different but characteristic degrees of methylation at G-A-T-C sites are found in the DNA of bacterial viruses grown in E. coli. DNAs from mammalian cells and viruses are not methylated at this sequence. Mitochondrial DNA from Paramecium aurelia is not methylated, but a small proportion of G-A-T-C sequences in the macronuclear DNA of this eukaryote appear to be methylated. Possible roles of sequence-specific methylation in the accommodation of plasmids, in the replication of DNA, in the regulation of gene function and in the restriction of viral infection are discussed.

  7. Identification of a highly conserved domain in the EcoRII methyltransferase which can be photolabeled with S-adenosyl-L-[methyl-3H]methionine. Evidence for UV-induced transmethylation of cysteine 186

    International Nuclear Information System (INIS)

    Som, S.; Friedman, S.

    1991-01-01

    DNA methyltransferases can be photolabeled with S-adenosyl-L-methionine (AdoMet). Specific incorporation of radioactivity has been demonstrated after photolabeling with either [methyl-3H]AdoMet or [35S]AdoMet. The labeling is believed to occur at the AdoMet binding site. With the purpose of localizing the site responsible for [methyl-3H]AdoMet photolabeling, we cleaved the labeled EcoRII methyltransferase by chemical and enzymatic reactions and isolated the radiolabeled peptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high pressure liquid chromatography. The labeled peptides were identified by amino-terminal sequencing. A common region was localized which accounted for 65-70% of the total label. This region includes a highly conserved core sequence present in all DNA (cytosine 5)-methyltransferases. One such fragment was digested further with chymotrypsin, and amino acid analysis of the resulting 3H-labeled peptide was consistent with the sequence Ala-Gly-Phe-Pro-(Cys)-Gln-Pro-Phe-Ser-Leu. However, the cysteine residue was not recovered as carboxymethylcysteine. The Pro-Cys bond was found to be protected from cleavage at cysteine residues after cyanylation. These results suggest that the cysteine residue is modified by the labeling reaction. The chymotryptic fragment was hydrolyzed enzymatically to single amino acids, and the labeled amino acid was identified as S-methylcysteine by thin layer chromatography. These results indicate that the cysteine residue is located at or close to the AdoMet binding site of EcoRII methyltransferase

  8. Type III restriction endonucleases are heterotrimeric: comprising one helicase–nuclease subunit and a dimeric methyltransferase that binds only one specific DNA

    Science.gov (United States)

    Butterer, Annika; Pernstich, Christian; Smith, Rachel M.; Sobott, Frank; Szczelkun, Mark D.; Tóth, Júlia

    2014-01-01

    Fundamental aspects of the biochemistry of Type III restriction endonucleases remain unresolved despite being characterized by numerous research groups in the past decades. One such feature is the subunit stoichiometry of these hetero-oligomeric enzyme complexes, which has important implications for the reaction mechanism. In this study, we present a series of results obtained by native mass spectrometry and size exclusion chromatography with multi-angle light scattering consistent with a 1:2 ratio of Res to Mod subunits in the EcoP15I, EcoPI and PstII complexes as the main holoenzyme species and a 1:1 stoichiometry of specific DNA (sDNA) binding by EcoP15I and EcoPI. Our data are also consistent with a model where ATP hydrolysis activated by recognition site binding leads to release of the enzyme from the site, dissociation from the substrate via a free DNA end and cleavage of the DNA. These results are discussed critically in the light of the published literature, aiming to resolve controversies and discuss consequences in terms of the reaction mechanism. PMID:24510100

  9. Endonuclease activities in extracts of Micrococcus luteus that act on. gamma. -irradiated DNA

    Energy Technology Data Exchange (ETDEWEB)

    Schoen-Bopp, A; Schaefer, G; Hagen, U [Kernforschungszentrum Karlsruhe (Germany, F.R.). Inst. fuer Strahlenbiologie

    1977-03-01

    Several protein fractions containing endonuclease activity against ..gamma..-irradiated DNA (..gamma..-endonuclease) were isolated from M.luteus. The crude extract was eluted on a phosphocellulose column and chromatographed on TEAE cellulose and subsequently on hydroxypatite. Five peaks of ..gamma..-endonuclease were obtained from each preparation. Repeated experiments showed comparable chromatographic behaviour of the fractions. There was no detectable activity of uv-endonuclease in the fractions with ..gamma..-endonuclease but a small contamination of endonuclease against unirradiated DNA and against DNA with apurinic sites. The ..gamma..-endonuclease was stimulated by, but was not dependent on, magnesium. Several tests for endonuclease activity have been used: the analysis of strand breaks in calf-thymus DNA or in PM2 DNA, and the determination of end-groups formed by endonuclease, either 3'OH end-groups or phosphomonoester end groups. From the results obtained it can be assumed that the strand breaks induced by the ..gamma..-endonuclease carry 3'OH and 5' phosphate end groups.

  10. Crystal structure of the apurinic/apyrimidinic endonuclease IV from Mycobacterium tuberculosis.

    Science.gov (United States)

    Zhang, Wei; Xu, Yueyang; Yan, Mengrong; Li, Shanshan; Wang, Huiying; Yang, Haitao; Zhou, Weihong; Rao, Zihe

    2018-03-25

    Endonuclease IV is a typical endonuclease of the apurinic-apyrimidinic (AP) or abasic endonuclease superfamily. It repairs damaged DNA through base excision repair by cleaving the DNA backbone immediately 5' of an AP site. In Mycobacterium tuberculosis, endonuclease IV is the major AP endonuclease. This enzyme is absent from mammalian cells, making it an attractive target for anti-tuberculosis drug development. In this study, the structure of the recombinant endonuclease IV from M. tuberculosis (MtbEndo IV) was determined at a high resolution of 1.18 Å. MtbEndo IV was found to have a classical α8β8-fold TIM barrel with loops on its surface connecting the α-helices and β-strands that constitute a groove for DNA binding. Three zinc ions were identified at the active site. A comparison between the structures of MtbEndo IV and Escherichia coli End IV suggested that Gln32 of MtbEndo IV may plays a role in regulating substrate binding. Copyright © 2018. Published by Elsevier Inc.

  11. Endonuclease IV of Escherichia coli is induced by paraquat

    Energy Technology Data Exchange (ETDEWEB)

    Chan, E.; Weiss, B.

    1987-05-01

    The addition of paraquat (methyl viologen) to a growing culture of Escherichia coli K-12 led within 1 hr to a 10- to 20-fold increase in the level of endonuclease IV, a DNase for apurinic/apyrimidinic sites. The induction was blocked by chloramphenicol. Increases of 3-fold or more were also seen with plumbagin, menadione, and phenazine methosulfate. H/sub 2/O/sub 2/ produced no more than a 2-fold increase in endonuclease IV activity. The following agents had no significant effect: streptonigrin, nitrofurantoin, tert-butyl hydroperoxide, ..gamma.. rays, 260-nm UV radiation, methyl methanesulfonate, mitomycin C, and ascorbate. Paraquat, plumbagin, menadione, and phenazine methosulfate are known to generate superoxide radical anions via redox cycling in vivo. A mutant lacking superoxide dismutase was unusually sensitive to induction by paraquat. In addition, endonuclease IV could be induced by merely growing the mutant in pure O/sub 2/. The levels of endonuclease IV in uninduced or paraquat-treated cells were unaffected by mutations of oxyR, a H/sub 2/O/sub 2/-inducible gene that governs an oxidative-stress regulon. The results indicate that endonuclease IV is an inducible DNA-repair enzyme and that its induction can be mediated via the production of superoxide radicals.

  12. Endonuclease IV of Escherichia coli is induced by paraquat

    International Nuclear Information System (INIS)

    Chan, E.; Weiss, B.

    1987-01-01

    The addition of paraquat (methyl viologen) to a growing culture of Escherichia coli K-12 led within 1 hr to a 10- to 20-fold increase in the level of endonuclease IV, a DNase for apurinic/apyrimidinic sites. The induction was blocked by chloramphenicol. Increases of 3-fold or more were also seen with plumbagin, menadione, and phenazine methosulfate. H 2 O 2 produced no more than a 2-fold increase in endonuclease IV activity. The following agents had no significant effect: streptonigrin, nitrofurantoin, tert-butyl hydroperoxide, γ rays, 260-nm UV radiation, methyl methanesulfonate, mitomycin C, and ascorbate. Paraquat, plumbagin, menadione, and phenazine methosulfate are known to generate superoxide radical anions via redox cycling in vivo. A mutant lacking superoxide dismutase was unusually sensitive to induction by paraquat. In addition, endonuclease IV could be induced by merely growing the mutant in pure O 2 . The levels of endonuclease IV in uninduced or paraquat-treated cells were unaffected by mutations of oxyR, a H 2 O 2 -inducible gene that governs an oxidative-stress regulon. The results indicate that endonuclease IV is an inducible DNA-repair enzyme and that its induction can be mediated via the production of superoxide radicals

  13. Restriction enzyme body doubles and PCR cloning: on the general use of type IIs restriction enzymes for cloning.

    Science.gov (United States)

    Tóth, Eszter; Huszár, Krisztina; Bencsura, Petra; Kulcsár, Péter István; Vodicska, Barbara; Nyeste, Antal; Welker, Zsombor; Tóth, Szilvia; Welker, Ervin

    2014-01-01

    The procedure described here allows the cloning of PCR fragments containing a recognition site of the restriction endonuclease (Type IIP) used for cloning in the sequence of the insert. A Type IIS endonuclease--a Body Double of the Type IIP enzyme--is used to generate the same protruding palindrome. Thus, the insert can be cloned to the Type IIP site of the vector without digesting the PCR product with the same Type IIP enzyme. We achieve this by incorporating the recognition site of a Type IIS restriction enzyme that cleaves the DNA outside of its recognition site in the PCR primer in such a way that the cutting positions straddle the desired overhang sequence. Digestion of the PCR product by the Body Double generates the required overhang. Hitherto the use of Type IIS restriction enzymes in cloning reactions has only been used for special applications, the approach presented here makes Type IIS enzymes as useful as Type IIP enzymes for general cloning purposes. To assist in finding Body Double enzymes, we summarised the available Type IIS enzymes which are potentially useful for Body Double cloning and created an online program (http://group.szbk.u-szeged.hu/welkergr/body_double/index.html) for the selection of suitable Body Double enzymes and the design of the appropriate primers.

  14. Identity of zinc finger nucleases with specificity to herpes simplex virus type II genomic DNA: novel HSV-2 vaccine/therapy precursors

    Directory of Open Access Journals (Sweden)

    Wayengera Misaki

    2011-06-01

    Full Text Available Abstract Background Herpes simplex type II (HSV-2 is a member of the family herpesviridae. Human infection with this double stranded linear DNA virus causes genital ulcerative disease and existing treatment options only serve to resolve the symptomatology (ulcers associated with active HSV-2 infection but do not eliminate latent virus. As a result, infection with HSV-2 follows a life-long relapsing (active versus latent course. On the basis of a primitive bacterium anti-phage DNA defense, the restriction modification (R-M system, we previously identified the Escherichia coli restriction enzyme (REase EcoRII as a novel peptide to excise or irreversibly disrupt latent HSV-2 DNA from infected cells. However, sequences of the site specificity palindrome of EcoRII 5'-CCWGG-3' (W = A or T are equally present within the human genome and are a potential source of host-genome toxicity. This feature has limited previous HSV-2 EcoRII based therapeutic models to microbicides only, and highlights the need to engineer artificial REases (zinc finger nucleases-ZFNs with specificity to HSV-2 genomic-DNA only. Herein, the therapeutic-potential of zinc finger arrays (ZFAs and ZFNs is identified and modeled, with unique specificity to the HSV-2 genome. Methods and results Using the whole genome of HSV-2 strain HG52 (Dolan A et al.,, and with the ZFN-consortium's CoDA-ZiFiT software pre-set at default, more than 28,000 ZFAs with specificity to HSV-2 DNA were identified. Using computational assembly (through in-silico linkage to the Flavobacterium okeanokoites endonuclease Fok I of the type IIS class, 684 ZFNs with specificity to the HSV-2 genome, were constructed. Graphic-analysis of the HSV-2 genome-cleavage pattern using the afore-identified ZFNs revealed that the highest cleavage-incidence occurred within the 30,950 base-pairs (~between the genomic context coordinates 0.80 and 1.00 at the 3' end of the HSV-2 genome. At approximately 3,095 bp before and after the

  15. Identification of a mismatch-specific endonuclease in hyperthermophilic Archaea.

    Science.gov (United States)

    Ishino, Sonoko; Nishi, Yuki; Oda, Soichiro; Uemori, Takashi; Sagara, Takehiro; Takatsu, Nariaki; Yamagami, Takeshi; Shirai, Tsuyoshi; Ishino, Yoshizumi

    2016-04-20

    The common mismatch repair system processed by MutS and MutL and their homologs was identified in Bacteria and Eukarya. However, no evidence of a functional MutS/L homolog has been reported for archaeal organisms, and it is not known whether the mismatch repair system is conserved in Archaea. Here, we describe an endonuclease that cleaves double-stranded DNA containing a mismatched base pair, from the hyperthermophilic archaeon Pyrococcus furiosus The corresponding gene revealed that the activity originates from PF0012, and we named this enzyme Endonuclease MS (EndoMS) as the mismatch-specific Endonuclease. The sequence similarity suggested that EndoMS is the ortholog of NucS isolated from Pyrococcus abyssi, published previously. Biochemical characterizations of the EndoMS homolog from Thermococcus kodakarensis clearly showed that EndoMS specifically cleaves both strands of double-stranded DNA into 5'-protruding forms, with the mismatched base pair in the central position. EndoMS cleaves G/T, G/G, T/T, T/C and A/G mismatches, with a more preference for G/T, G/G and T/T, but has very little or no effect on C/C, A/C and A/A mismatches. The discovery of this endonuclease suggests the existence of a novel mismatch repair process, initiated by the double-strand break generated by the EndoMS endonuclease, in Archaea and some Bacteria. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  16. Karyopherin-Mediated Nuclear Import of the Homing Endonuclease VMA1-Derived Endonuclease Is Required for Self-Propagation of the Coding Region

    OpenAIRE

    Nagai, Yuri; Nogami, Satoru; Kumagai-Sano, Fumi; Ohya, Yoshikazu

    2003-01-01

    VMA1-derived endonuclease (VDE), a site-specific endonuclease in Saccharomyces cerevisiae, enters the nucleus to generate a double-strand break in the VDE-negative allelic locus, mediating the self-propagating gene conversion called homing. Although VDE is excluded from the nucleus in mitotic cells, it relocalizes at premeiosis, becoming localized in both the nucleus and the cytoplasm in meiosis. The nuclear localization of VDE is induced by inactivation of TOR kinases, which constitute centr...

  17. Template-directed addition of nucleosides to DNA by the BfiI restriction enzyme

    OpenAIRE

    Sasnauskas, Giedrius; Connolly, Bernard A.; Halford, Stephen E.; Siksnys, Virginijus

    2008-01-01

    Restriction endonucleases catalyse DNA cleavage at specific sites. The BfiI endonuclease cuts DNA to give staggered ends with 1-nt 3′-extensions. We show here that BfiI can also fill in the staggered ends: while cleaving DNA, it can add a 2′-deoxynucleoside to the reaction product to yield directly a blunt-ended DNA. We propose that nucleoside incorporation proceeds through a two-step reaction, in which BfiI first cleaves the DNA to make a covalent enzyme–DNA intermediate and then resolves it...

  18. Endonuclease α from Saccharomyces cerevisiae shows increased activity on ultraviolet irradiated native DNA

    International Nuclear Information System (INIS)

    Bryant, D.W.; Haynes, R.H.

    1978-01-01

    Endonuclease α isolated from the nucleus of the yeast Saccharomyces cerevisiae is a DNA endonuclease which has been shown to act preferentially on denatured T7 DNA. The purified enzyme is more active with UV-irradiated native T7 DNA than with unirradiated substrate. The relation between damage, measured by pyrimidine dimer concentration, and excess endonuclease activity is most readily explained by local denaturation caused by the presence of pyrimidine dimers. When three radiation sensitive mutants of yeast were tested for the level of endonuclease α present, none were found lacking the enzyme. However, nuclei of strain rad 1-1, a mutant that may be defective in heteroduplex repair as well as excision repair, were found to contain reduced levels of the endonuclease. (orig./AJ) [de

  19. Site-specific DNA transesterification catalyzed by a restriction enzyme

    OpenAIRE

    Sasnauskas, Giedrius; Connolly, Bernard A.; Halford, Stephen E.; Siksnys, Virginijus

    2007-01-01

    Most restriction endonucleases use Mg2+ to hydrolyze phosphodiester bonds at specific DNA sites. We show here that BfiI, a metal-independent restriction enzyme from the phospholipase D superfamily, catalyzes both DNA hydrolysis and transesterification reactions at its recognition site. In the presence of alcohols such as ethanol or glycerol, it attaches the alcohol covalently to the 5′ terminus of the cleaved DNA. Under certain conditions, the terminal 3′-OH of one DNA strand can attack the t...

  20. Demonstration that Australian Pasteurella multocida isolates from sporadic outbreaks of porcine pneumonia are non-toxigenic (toxA-) and display heterogeneous DNA restriction endonuclease profiles compared with toxigenic isolates from herds with progressive atrophic rhinitis.

    Science.gov (United States)

    Djordjevic, S P; Eamens, G J; Ha, H; Walker, M J; Chin, J C

    1998-08-01

    Capsular types A and D of Pasteurella multocida cause economic losses in swine because of their association with progressive atrophic rhinitis (PAR) and enzootic pneumonia. There have been no studies comparing whole-cell DNA profiles of isolates associated with these two porcine respiratory diseases. Twenty-two isolates of P. multocida from diseased pigs in different geographic localities within Australia were characterised genotypically by restriction endonuclease analysis (REA) with the enzyme CfoI. Seven of 12 P. multocida isolates from nasal swabs from pigs in herds where PAR was either present or suspected displayed a capsular type D phenotype. These were shown to possess the toxA gene by polymerase chain reaction (PCR) and Southern hybridisation, and further substantiated by production of cytotoxin in vitro. The CfoI profile of one of these seven isolates, which was from the initial outbreak of PAR in Australia (in Western Australia, WA), was identical with profiles of all six other toxigenic isolates from sporadic episodes in New South Wales (NSW). The evidence suggests that the strain involved in the initial outbreak was responsible for the spread of PAR to the eastern states of Australia. Another 10 isolates, representing both capsular types A and D, were isolated exclusively from porcine lung lesions after sporadic outbreaks of enzootic pneumonia in NSW and WA. CfoI restriction endonuclease profiles of these isolates revealed considerable genomic heterogeneity. Furthermore, none of these possessed the toxA gene. This suggests that P. multocida strains with the toxA gene do not have a competitive survival advantage in the lower respiratory tract or that toxin production does not play a role in the pathology of pneumonic lesions, or both. REA with polyacrylamide gel electrophoresis and silver staining was found to be a practical and discriminatory tool for epidemiological tracing of P. multocida outbreaks associated with PAR or pneumonia in pigs.

  1. Engineering a Nickase on the Homing Endonuclease I-DmoI Scaffold

    DEFF Research Database (Denmark)

    Molina, Rafael; Marcaida, María José; Redondo, Pilar

    2015-01-01

    strand break could be an approach to reduce the toxicity associated with non-homologous end joining by promoting the use of homologous recombination to repair the cleavage of a single DNA break. Taking advantage of the sequential DNA cleavage mechanism of I-DmoI LAGLIDADG homing endonuclease, we have......Homing endonucleases are useful tools for genome modification because of their capability to recognize and cleave specifically large DNA targets. These endonucleases generate a DNA double strand break that can be repaired by the DNA damage response machinery. The break can be repaired by homologous...

  2. The role of DNA restriction-modification systems in the biology of Bacillus anthracis

    Directory of Open Access Journals (Sweden)

    Ramakrishnan eSitaraman

    2016-01-01

    Full Text Available Restriction-modification (R-M systems are widespread among prokaryotes and, depending on their type, may be viewed as selfish genetic elements that persist as toxin-antitoxin modules or as cellular defense systems against phage infection. Studies in the last decade have made it amply clear that these two options do not exhaust the list of possible biological roles for R-M systems. Their presence in a cell may also have a bearing on other processes such as horizontal gene transfer and gene regulation. From genome sequencing and experimental data, we know that Bacillus anthracis encodes at least three methylation-dependent (typeIV restriction endonucleases, and an orphan DNA methyltransferase. In this article, we first present an outline of our current knowledge of R-M systems in Bacillus anthracis. Based on available DNA sequence data, and on our current understanding of the functions of similar genes in other systems, we conclude with hypotheses on the possible roles of the three restriction endonucleases and the orphan DNA methyltransferase.

  3. Genetic diversity of the HpyC1I restriction modification system in Helicobacter pylori.

    Science.gov (United States)

    Lehours, Philippe; Dupouy, Sandrine; Chaineux, Julien; Ruskoné-Fourmestraux, Agnès; Delchier, Jean-Charles; Morgner, Andrea; Mégraud, Francis; Ménard, Armelle

    2007-04-01

    Helicobacter pylori is unique because of the unusually high number and diversity of its restriction modification (R-M) systems. HpyC1I R-M was recently characterized and contains an endonuclease which is an isoschizomer of the endonuclease BccI. This R-M is involved in adherence to gastric epithelial cells, a crucial step in bacterial pathogenesis. This observation illustrates the fact that R-M systems have other putative biological functions in addition to protecting the bacterial genome from external DNA. The genomic diversity of HpyC1I R-M was evaluated more precisely on a large collection of H. pylori strains by PCR, susceptibility to BccI digestion and sequencing. The results obtained support the mechanism of gain and loss of this R-M system in the H. pylori genome, and suggest that it is an ancestral system which gradually disappears during H. pylori evolution, following successive steps: (1) inactivation of the endonuclease gene, followed or accompanied by: (2) inactivation of the methyltransferase genes, and then: (3) definitive loss, leaving only short endonuclease remnant sequences.

  4. DNA-tension dependence of restriction enzyme activity reveals mechanochemical properties of the reaction pathway

    NARCIS (Netherlands)

    van den Broek, B.; Noom, M.C.; Wuite, G.J.L.

    2005-01-01

    Type II restriction endonucleases protect bacteria against phage infections by cleaving recognition sites on foreign double-stranded DNA (dsDNA) with extraordinary specificity. This capability arises primarily from large conformational changes in enzyme and/or DNA upon target sequence recognition.

  5. Binding of T4 endonuclease V to deoxyribonucleic acid irradiated with ultraviolet light

    International Nuclear Information System (INIS)

    Seawell, P.C.; Simon, T.J.; Ganesan, A.K.

    1980-01-01

    Endonuclease V of bacteriophage T4 binds to uv-irradiated deoxyribonucleic acid (DNA) but not to unirradiated DNA. We have developed an assay to detect this binding, based on the retention of enzyme - DNA complexes on nitrocellulose filters. The amount of complex retained, ascertained by using radioactive DNA, is a measure of T4 endonuclease V activity. From our data we conclude that (1) T4 endonuclease V binds to uv-irradiated DNA but not to DNA that has been previously incised by the endonuclease, (2) equilibrium between the free and complexed form of the enzyme is attained under our reaction conditions, (3) dissociation of enzyme - DNA complexes is retarded by sodium cyanide, and (4) retention of enzyme - DNA complexes on nitrocellulose filters is enhanced by high concentrations of saline-citrate

  6. Next-generation sequencing of multiple individuals per barcoded library by deconvolution of sequenced amplicons using endonuclease fragment analysis

    DEFF Research Database (Denmark)

    Andersen, Jeppe D; Pereira, Vania; Pietroni, Carlotta

    2014-01-01

    The simultaneous sequencing of samples from multiple individuals increases the efficiency of next-generation sequencing (NGS) while also reducing costs. Here we describe a novel and simple approach for sequencing DNA from multiple individuals per barcode. Our strategy relies on the endonuclease...... digestion of PCR amplicons prior to library preparation, creating a specific fragment pattern for each individual that can be resolved after sequencing. By using both barcodes and restriction fragment patterns, we demonstrate the ability to sequence the human melanocortin 1 receptor (MC1R) genes from 72...... individuals using only 24 barcoded libraries....

  7. Induction of DNA double-strand breaks by restriction enzymes in X-ray-sensitive mutant Chinese hamster ovary cells measured by pulsed-field gel electrophoresis

    International Nuclear Information System (INIS)

    Kinashi, Yuko; Nagasawa, Hatsumi; Little, J.B.; Okayasu, Ryuichi; Iliakis, G.E.

    1995-01-01

    This investigation was designed to determine whether the cytotoxic effects of different restriction endonucleases are related to the number and type of DNA double-strand breaks (DSBs) they produce. Chinese hamster ovary (CHO) K1 and xrs-5 cells, a radiosensitive mutant of CHO K1, were exposed to restriction endonucleases HaeIII, HinfI, PvuII and BamHI by electroporation. These enzymes represent both blunt and sticky end cutters with differing recognition sequence lengths. The number of DSBs was measured by pulsed-field gel electrophoresis (PFGE). Two forms of PFGE were employed: asymmetric field-inversion gel electrophoresis (AFIGE) for measuring the kinetics of DNA breaks by enzyme digestion and clamped homogeneous gel electrophoresis (CHEF) for examining the size distributions of damaged DNA. The amount of DNA damage induced by exposure to all four restriction enzymes was significantly greater in xrs-5 compared to CHO K1 cells, consistent with the reported DSB repair deficiency in these cells. Since restriction endonucleases produce DSBs alone as opposed to the various types of DNA damage induced by X rays, these results confirm that the repair defect in this mutant involves the rejoining of DSBs. Although the cutting frequency was directly related to the length of the recognition sequence for four restriction enzymes, there was no simple correlation between the cytotoxic effect and the amount of DNA damage produced by each enzyme in either cell line. This finding suggests that the type or nature of the cutting sequence itself may play a role in restriction enzyme-induced cell killing. 32 refs., 6 figs., 3 tabs

  8. Gibberellin-induced change in the structure of chromatin in wheat sprouts: decrease in the accessibility of DNA in preparations of soluble chromatin to the action of EcoRII methylase

    International Nuclear Information System (INIS)

    Noskov, V.A.; Kintsurashvili, L.N.; Smirnova, T.A.; Manamsh'yan, T.A.; Kir'yanov, G.I.; Vanyushin, B.F.

    1986-01-01

    A method has been perfected for producing soluble chromatin from whole wheat sprouts at low ionic strength. The chromatin preparations isolated possess a native structure: they have a nucleosome organization. Under identical conditions the soluble wheat chromatin undergoes more profound degradation by DNase I and staphylococcal nuclease than the chromatin from the rat liver. The DNA contained in the isolated chromatin is capable of accepting CHnumber groups from S-[methyl- 3 H]-adenosylmethionine during incubation with DNA methylase EcoRII; not all the CC A/T GG sequences in DNA are methylated in vivo. Chromatin from gibberellin A 3 -treated wheat sprout DNA accepts 40% fewer CH 3 groups than that from the control sprouts, which is probably due to the greater compactness of the chromatin. In the case of longer incubation, the level of methylation of the chromatin falls, which may be associated with the presence of DNA-demethylating activity

  9. Chemical display of pyrimidine bases flipped out by modification-dependent restriction endonucleases of MspJI and PvuRts1I families.

    Directory of Open Access Journals (Sweden)

    Evelina Zagorskaitė

    Full Text Available The epigenetic DNA modifications 5-methylcytosine (5mC and 5-hydroxymethylcytosine (5hmC in eukaryotes are recognized either in the context of double-stranded DNA (e.g., by the methyl-CpG binding domain of MeCP2, or in the flipped-out state (e.g., by the SRA domain of UHRF1. The SRA-like domains and the base-flipping mechanism for 5(hmC recognition are also shared by the recently discovered prokaryotic modification-dependent endonucleases of the MspJI and PvuRts1I families. Since the mechanism of modified cytosine recognition by many potential eukaryotic and prokaryotic 5(hmC "readers" is still unknown, a fast solution based method for the detection of extrahelical 5(hmC would be very useful. In the present study we tested base-flipping by MspJI- and PvuRts1I-like restriction enzymes using several solution-based methods, including fluorescence measurements of the cytosine analog pyrrolocytosine and chemical modification of extrahelical pyrimidines with chloroacetaldehyde and KMnO4. We find that only KMnO4 proved an efficient probe for the positive display of flipped out pyrimidines, albeit the method required either non-physiological pH (4.3 or a substitution of the target cytosine with thymine. Our results imply that DNA recognition mechanism of 5(hmC binding proteins should be tested using a combination of all available methods, as the lack of a positive signal in some assays does not exclude the base flipping mechanism.

  10. Properties of an endonuclease activity in Micrococcus luteus acting on γ-irradiated DNA and on apurinic DNA

    International Nuclear Information System (INIS)

    Schaefer, G.; Haas, P.; Coquerelle, Th.; Hagen, U.

    1980-01-01

    A protein fraction from Micrococcus luteus with endonuclease activity against γ-irradiated DNA was isolated and characterized. An additional activity on apurinic sites could not be separated, either by sucrose gradient sedimentation or by gel filtration through Sephadex G 100. From gel filtration, a molecular weight of about 25 000 was calculated for both endonuclease activities. The endonuclease activity against γ-irradiated DNA was stimulated five-fold with 5 mM Mg ++ , whereas that against apurinic sites was less dependent on the Mg ++ concentration. 100 mM KCl inhibited the γ-ray endonuclease, but not the apurinic endonuclease activity. In γ-irradiated DNA the protein recognized 1.65 endonuclease sensitive sites per radiation-induced single-strand break, among which are 0.45 alkali labile lesions in the nucleotide strand. The was evaluated resulting in a Ksub(m)-value of 73 nM. (author)

  11. Evolutionary maintenance of selfish homing endonuclease genes in the absence of horizontal transfer.

    Science.gov (United States)

    Yahara, Koji; Fukuyo, Masaki; Sasaki, Akira; Kobayashi, Ichizo

    2009-11-03

    Homing endonuclease genes are "selfish" mobile genetic elements whose endonuclease promotes the spread of its own gene by creating a break at a specific target site and using the host machinery to repair the break by copying and inserting the gene at this site. Horizontal transfer across the boundary of a species or population within which mating takes place has been thought to be necessary for their evolutionary persistence. This is based on the assumption that they will become fixed in a host population, where opportunities of homing will disappear, and become susceptible to degeneration. To test this hypothesis, we modeled behavior of a homing endonuclease gene that moves during meiosis through double-strand break repair. We mathematically explored conditions for persistence of the homing endonuclease gene and elucidated their parameter dependence as phase diagrams. We found that, if the cost of the pseudogene is lower than that of the homing endonuclease gene, the 2 forms can persist in a population through autonomous periodic oscillation. If the cost of the pseudogene is higher, 2 types of dynamics appear that enable evolutionary persistence: bistability dependent on initial frequency or fixation irrespective of initial frequency. The prediction of long persistence in the absence of horizontal transfer was confirmed by stochastic simulations in finite populations. The average time to extinction of the endonuclease gene was found to be thousands of meiotic generations or more based on realistic parameter values. These results provide a solid theoretical basis for an understanding of these and other extremely selfish elements.

  12. Substrate specificity of Micrococcus luteus uv endonuclease and its overlap with DNA photolyase activity

    International Nuclear Information System (INIS)

    Patrick, M.H.

    1975-01-01

    The action of an endonuclease from Micrococcus luteus that operates on uv damage in DNA overlaps with that of DNA photolyase from yeast: homo- and heterocyclobutane dipyrimidines in DNA are substrates for both enzymes, but pyrimidine adducts or the spore photoproduct in DNA are not. As expected from this overlap, the action of the two enzymes is mutually interfering: single-strand nicks introduced by the endonuclease effectively preclude photoreactivation; conversely, formation of a photolyase-cyclobutane dipyrimidine complex can prevent nicking by the endonuclease

  13. Properties of an endonuclease activity in Micrococcus luteus acting on. gamma. -irradiated DNA and on apurinic DNA

    Energy Technology Data Exchange (ETDEWEB)

    Schaefer, G; Haas, P; Coquerelle, Th; Hagen, U [Kernforschungszentrum Karlsruhe G.m.b.H. (Germany, F.R.). Inst. fuer Genetik und fuer Toxikologie von Spaltstoffen

    1980-01-01

    A protein fraction from Micrococcus luteus with endonuclease activity against ..gamma..-irradiated DNA was isolated and characterized. An additional activity on apurinic sites could not be separated, either by sucrose gradient sedimentation or by gel filtration through Sephadex G 100. From gel filtration, a molecular weight of about 25 000 was calculated for both endonuclease activities. The endonuclease activity against ..gamma..-irradiated DNA was stimulated five-fold with 5 mM Mg/sup + +/, whereas that against apurinic sites was less dependent on the Mg/sup + +/ concentration. 100 mM KCl inhibited the ..gamma..-ray endonuclease, but not the apurinic endonuclease activity. In ..gamma..-irradiated DNA the protein recognized 1.65 endonuclease sensitive sites per radiation-induced single-strand break, among which are 0.45 alkali labile lesions in the nucleotide strand. The was evaluated resulting in a Ksub(m)-value of 73 nM.

  14. Processive nicking activity of T4 endonuclease V on UV-irradiated chromatin

    International Nuclear Information System (INIS)

    Gruskin, E.A.; Lloyd, R.S.

    1986-01-01

    T4 endonuclease V initiates the excision repair of pyrimidine dimers in UV-irradiated T4 infected E. coli cells. The pyrimidine dimer specific nicking activity of T4 endonuclease V functions by a processive scanning on UV-irradiated DNA. Previously it has been demonstrated that introduction of endonuclease V into repair-deficient human cells causes a restoration of UV survival in these cells. This demonstrates that endonuclease V is competent to incise mammalian DNA at the site of pyrimidine dimers. In order to assess the ability of endonuclease V to act processively on DNA associated as chromatin, minichromosomes were prepared for use as a substrate. Form I DNA was reconstituted with H3, H4 +/- H1 histones by sequential dialysis steps from 2.0 M NaCl to 50 mM NaCl. Time course reactions were performed with minichromosomes containing 10 and 25 dimers per molecule. In each case the rate of disappearance of form I DNA which was associated as chromatin was decreased relative to that of naked form I DNA. Concurrent with that observation, the rate and extent of appearance of form III DNA was increased with the DNA in minichromosomes relative to naked DNA. This is diagnostic of an enhancement of processivity. The inclusion of H1 in the minichromosomes resulted in a slight additional increase in processivity relative to minichromosomes consisting only of H3 and H4

  15. The metabolic enhancer piracetam attenuates mitochondrion-specific endonuclease G translocation and oxidative DNA fragmentation.

    Science.gov (United States)

    Gupta, Sonam; Verma, Dinesh Kumar; Biswas, Joyshree; Rama Raju, K Siva; Joshi, Neeraj; Wahajuddin; Singh, Sarika

    2014-08-01

    This study was performed to investigate the involvement of mitochondrion-specific endonuclease G in piracetam (P)-induced protective mechanisms. Studies have shown the antiapoptotic effects of piracetam but the mechanism of action of piracetam is still an enigma. To assess the involvement of endonuclease G in piracetam-induced protective effects, astrocyte glial cells were treated with lipopolysaccharide (LPS) and piracetam. LPS treatment caused significantly decreased viability, mitochondrial activity, oxidative stress, chromatin condensation, and DNA fragmentation, which were attenuated by piracetam cotreatment. Cotreatment of astrocytes with piracetam showed its significantly time-dependent absorption as observed with high-performance liquid chromatography. Astrocytes treated with piracetam alone showed enhanced mitochondrial membrane potential (MMP) in comparison to control astrocytes. However, in LPS-treated cells no significant alteration in MMP was observed in comparison to control cells. Protein and mRNA levels of the terminal executor of the caspase-mediated pathway, caspase-3, were not altered significantly in LPS or LPS + piracetam-treated astrocytes, whereas endonuclease G was significantly translocated to the nucleus in LPS-treated astrocytes. Piracetam cotreatment attenuated the LPS-induced endonuclease G translocation. In conclusion this study indicates that LPS treatment of astrocytes caused decreased viability, oxidative stress, mitochondrial dysfunction, chromatin condensation, DNA damage, and translocation of endonuclease G to the nucleus, which was inhibited by piracetam cotreatment, confirming that the mitochondrion-specific endonuclease G is one of the factors involved in piracetam-induced protective mechanisms. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. A functional endonuclease Q exists in the bacterial domain: identification and characterization of endonuclease Q from Bacillus pumilus.

    Science.gov (United States)

    Shiraishi, Miyako; Ishino, Sonoko; Cann, Isaac; Ishino, Yoshizumi

    2017-05-01

    DNA base deamination occurs spontaneously under physiological conditions and is promoted by high temperature. Therefore, hyperthermophiles are expected to have efficient repair systems of the deaminated bases in their genomes. Endonuclease Q (EndoQ) was originally identified from the hyperthermophlic archaeon, Pyrococcus furiosus, as a hypoxanthine-specific endonuclease recently. Further biochemical analyses revealed that EndoQ also recognizes uracil, xanthine, and the AP site in DNA, and is probably involved in a specific repair process for damaged bases. Initial phylogenetic analysis showed that an EndoQ homolog is found only in the Thermococcales and some of the methanogens in Archaea, and is not present in most members of the domains Bacteria and Eukarya. A better understanding of the distribution of the EndoQ-mediated repair system is, therefore, of evolutionary interest. We showed here that an EndoQ-like polypeptide from Bacillus pumilus, belonging to the bacterial domain, is functional and has similar properties with the archaeal EndoQs.

  17. The BsaHI restriction-modification system: Cloning, sequencing and analysis of conserved motifs

    Directory of Open Access Journals (Sweden)

    Roberts Richard J

    2008-05-01

    Full Text Available Abstract Background Restriction and modification enzymes typically recognise short DNA sequences of between two and eight bases in length. Understanding the mechanism of this recognition represents a significant challenge that we begin to address for the BsaHI restriction-modification system, which recognises the six base sequence GRCGYC. Results The DNA sequences of the genes for the BsaHI methyltransferase, bsaHIM, and restriction endonuclease, bsaHIR, have been determined (GenBank accession #EU386360, cloned and expressed in E. coli. Both the restriction endonuclease and methyltransferase enzymes share significant similarity with a group of 6 other enzymes comprising the restriction-modification systems HgiDI and HgiGI and the putative HindVP, NlaCORFDP, NpuORFC228P and SplZORFNP restriction-modification systems. A sequence alignment of these homologues shows that their amino acid sequences are largely conserved and highlights several motifs of interest. We target one such conserved motif, reading SPERRFD, at the C-terminal end of the bsaHIR gene. A mutational analysis of these amino acids indicates that the motif is crucial for enzymatic activity. Sequence alignment of the methyltransferase gene reveals a short motif within the target recognition domain that is conserved among enzymes recognising the same sequences. Thus, this motif may be used as a diagnostic tool to define the recognition sequences of the cytosine C5 methyltransferases. Conclusion We have cloned and sequenced the BsaHI restriction and modification enzymes. We have identified a region of the R. BsaHI enzyme that is crucial for its activity. Analysis of the amino acid sequence of the BsaHI methyltransferase enzyme led us to propose two new motifs that can be used in the diagnosis of the recognition sequence of the cytosine C5-methyltransferases.

  18. Efficient fdCas9 Synthetic Endonuclease with Improved Specificity for Precise Genome Engineering

    KAUST Repository

    Aouida, Mustapha

    2015-07-30

    The Cas9 endonuclease is used for genome editing applications in diverse eukaryotic species. A high frequency of off-target activity has been reported in many cell types, limiting its applications to genome engineering, especially in genomic medicine. Here, we generated a synthetic chimeric protein between the catalytic domain of the FokI endonuclease and the catalytically inactive Cas9 protein (fdCas9). A pair of guide RNAs (gRNAs) that bind to sense and antisense strands with a defined spacer sequence range can be used to form a catalytically active dimeric fdCas9 protein and generate double-strand breaks (DSBs) within the spacer sequence. Our data demonstrate an improved catalytic activity of the fdCas9 endonuclease, with a spacer range of 15–39 nucleotides, on surrogate reporters and genomic targets. Furthermore, we observed no detectable fdCas9 activity at known Cas9 off-target sites. Taken together, our data suggest that the fdCas9 endonuclease variant is a superior platform for genome editing applications in eukaryotic systems including mammalian cells.

  19. Efficient fdCas9 Synthetic Endonuclease with Improved Specificity for Precise Genome Engineering

    KAUST Repository

    Aouida, Mustapha; Eid, Ayman; Ali, Zahir; Cradick, Thomas; Lee, Ciaran; Deshmukh, Harshavardhan; Atef, Ahmed; Abu Samra, Dina Bashir Kamil; Gadhoum, Samah Zeineb; Merzaban, Jasmeen; Bao, Gang; Mahfouz, Magdy M.

    2015-01-01

    The Cas9 endonuclease is used for genome editing applications in diverse eukaryotic species. A high frequency of off-target activity has been reported in many cell types, limiting its applications to genome engineering, especially in genomic medicine. Here, we generated a synthetic chimeric protein between the catalytic domain of the FokI endonuclease and the catalytically inactive Cas9 protein (fdCas9). A pair of guide RNAs (gRNAs) that bind to sense and antisense strands with a defined spacer sequence range can be used to form a catalytically active dimeric fdCas9 protein and generate double-strand breaks (DSBs) within the spacer sequence. Our data demonstrate an improved catalytic activity of the fdCas9 endonuclease, with a spacer range of 15–39 nucleotides, on surrogate reporters and genomic targets. Furthermore, we observed no detectable fdCas9 activity at known Cas9 off-target sites. Taken together, our data suggest that the fdCas9 endonuclease variant is a superior platform for genome editing applications in eukaryotic systems including mammalian cells.

  20. Sequence-specific protection of duplex DNA against restriction and methylation enzymes by pseudocomplementary PNAs

    DEFF Research Database (Denmark)

    Izvolsky, K I; Demidov, V V; Nielsen, P E

    2000-01-01

    I restriction endonuclease and dam methylase. The pcPNA-assisted protection against enzymatic methylation is more efficient when the PNA-binding site embodies the methylase-recognition site rather than overlaps it. We conclude that pcPNAs may provide the robust tools allowing to sequence-specifically manipulate...... DNA duplexes in a virtually sequence-unrestricted manner....

  1. Visualizing phosphodiester-bond hydrolysis by an endonuclease

    DEFF Research Database (Denmark)

    Molina, Rafael; Stella, Stefano; Redondo, Pilar

    2015-01-01

    The enzymatic hydrolysis of DNA phosphodiester bonds has been widely studied, but the chemical reaction has not yet been observed. Here we follow the generation of a DNA double-strand break (DSB) by the Desulfurococcus mobilis homing endonuclease I-DmoI, trapping sequential stages of a two-metal-...

  2. Highlights of the DNA cutters: a short history of the restriction enzymes.

    Science.gov (United States)

    Loenen, Wil A M; Dryden, David T F; Raleigh, Elisabeth A; Wilson, Geoffrey G; Murray, Noreen E

    2014-01-01

    In the early 1950's, 'host-controlled variation in bacterial viruses' was reported as a non-hereditary phenomenon: one cycle of viral growth on certain bacterial hosts affected the ability of progeny virus to grow on other hosts by either restricting or enlarging their host range. Unlike mutation, this change was reversible, and one cycle of growth in the previous host returned the virus to its original form. These simple observations heralded the discovery of the endonuclease and methyltransferase activities of what are now termed Type I, II, III and IV DNA restriction-modification systems. The Type II restriction enzymes (e.g. EcoRI) gave rise to recombinant DNA technology that has transformed molecular biology and medicine. This review traces the discovery of restriction enzymes and their continuing impact on molecular biology and medicine.

  3. Excision repair of gamma-ray-induced alkali-stable DNA lesions with the help of γ-endonuclease from Micrococcus luteus

    International Nuclear Information System (INIS)

    Tomilin, N.V.; Barenfeld, L.S.

    1979-01-01

    γ-endonuclease Y, an enzyme that hydrolyses phosphodiester bonds at alkali-stable lesions in γ-irradiated (N 2 , tris buffer) DNA, has been partially purified from Micrococcus luteus. The enzyme has a molecular weight of about 19 000, induces single-strand breaks with 3'OH-5'PO 4 termini and contains endonuclease activity towards DNA treated with 7-bromomethylbenz(a)anthracene. γ-endonuclease Y induces breaks in OsO 4 -treated poly(dA-dT) and apparently is specific towards γ-ray-induced base lesions of the t' type. The complete excision repair of γ-endonuclease Y substrate sites has been performed in vitro by γ-endonuclease Y, DNA polymerase and ligase. (author)

  4. Excision repair of gamma-ray-induced alkali-stable DNA lesions with the help of. gamma. -endonuclease from Micrococcus luteus

    Energy Technology Data Exchange (ETDEWEB)

    Tomilin, N V; Barenfeld, L S [AN SSSR, Leningrad. Inst. Tsitologii

    1979-03-01

    ..gamma..-endonuclease Y, an enzyme that hydrolyses phosphodiester bonds at alkali-stable lesions in ..gamma..-irradiated (N/sub 2/, tris buffer) DNA, has been partially purified from Micrococcus luteus. The enzyme has a molecular weight of about 19 000, induces single-strand breaks with 3'OH-5'PO/sub 4/ termini and contains endonuclease activity towards DNA treated with 7-bromomethylbenz(a)anthracene. ..gamma..-endonuclease Y induces breaks in OsO/sub 4/-treated poly(dA-dT) and apparently is specific towards ..gamma..-ray-induced base lesions of the t' type. The complete excision repair of ..gamma..-endonuclease Y substrate sites has been performed in vitro by ..gamma..-endonuclease Y, DNA polymerase and ligase.

  5. DENV gene of bacteriophage T4 codes for both pyrimidine dimer-DNA glycosylase and apyrimidinic endonuclease activities

    International Nuclear Information System (INIS)

    McMillan, S.; Edenberg, H.J.; Radany, E.H.; Friedberg, R.C.; Friedberg, E.C.

    1981-01-01

    Recent studies have shown that purified preparations of phage T4 UV DNA-incising activity (T4 UV endonuclease or endonuclease V of phase T4) contain a pyrimidine dimer-DNA glycosylase activity that catalyzes hydrolysis of the 5' glycosyl bond of dimerized pyrimidines in UV-irradiated DNA. Such enzyme preparations have also been shown to catalyze the hydrolysis of phosphodiester bonds in UV-irradiated DNA at a neutral pH, presumably reflecting the action of an apurinic/apyrimidinic endonuclease at the apyrimidinic sites created by the pyrimidine dimer-DNA glycosylase. In this study we found that preparations of T4 UV DNA-incising activity contained apurinic/apyrimidinic endonuclease activity that nicked depurinated form I simian virus 40 DNA. Apurinic/apyrimidinic endonuclease activity was also found in extracts of Escherichia coli infected with T4 denV + phage. Extracts of cells infected with T4 denV mutants contained significantly lower levels of apurinic/apyrimidinic endonuclease activity; these levels were no greater than the levels present in extracts of uninfected cells. Furthermore, the addition of DNA containing UV-irradiated DNA and T4 enzyme resulted in competition for pyrimidine dimer-DNA glycosylase activity against the UV-irradiated DNA. On the basis of these results, we concluded that apurinic/apyrimidinic endonuclease activity is encoded by the denV gene of phage T4, the same gene that codes for pyrimidine dimer-DNA glycosylase activity

  6. Structural aspects of catalytic mechanisms of endonucleases and their binding to nucleic acids

    Energy Technology Data Exchange (ETDEWEB)

    Zhukhlistova, N. E.; Balaev, V. V.; Lyashenko, A. V.; Lashkov, A. A., E-mail: alashkov83@gmail.com [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation)

    2012-05-15

    Endonucleases (EC 3.1) are enzymes of the hydrolase class that catalyze the hydrolytic cleavage of deoxyribonucleic and ribonucleic acids at any region of the polynucleotide chain. Endonucleases are widely used both in biotechnological processes and in veterinary medicine as antiviral agents. Medical applications of endonucleases in human cancer therapy hold promise. The results of X-ray diffraction studies of the spatial organization of endonucleases and their complexes and the mechanism of their action are analyzed and generalized. An analysis of the structural studies of this class of enzymes showed that the specific binding of enzymes to nucleic acids is characterized by interactions with nitrogen bases and the nucleotide backbone, whereas the nonspecific binding of enzymes is generally characterized by interactions only with the nucleic-acid backbone. It should be taken into account that the specificity can be modulated by metal ions and certain low-molecular-weight organic compounds. To test the hypotheses about specific and nonspecific nucleic-acid-binding proteins, it is necessary to perform additional studies of atomic-resolution three-dimensional structures of enzyme-nucleic-acid complexes by methods of structural biology.

  7. Characterization of DNA substrate specificities of apurinic/apyrimidinic endonucleases from Mycobacterium tuberculosis.

    Science.gov (United States)

    Abeldenov, Sailau; Talhaoui, Ibtissam; Zharkov, Dmitry O; Ishchenko, Alexander A; Ramanculov, Erlan; Saparbaev, Murat; Khassenov, Bekbolat

    2015-09-01

    Apurinic/apyrimidinic (AP) endonucleases are key enzymes involved in the repair of abasic sites and DNA strand breaks. Pathogenic bacteria Mycobacterium tuberculosis contains two AP endonucleases: MtbXthA and MtbNfo members of the exonuclease III and endonuclease IV families, which are exemplified by Escherichia coli Xth and Nfo, respectively. It has been shown that both MtbXthA and MtbNfo contain AP endonuclease and 3'→5' exonuclease activities. However, it remains unclear whether these enzymes hold 3'-repair phosphodiesterase and nucleotide incision repair (NIR) activities. Here, we report that both mycobacterial enzymes have 3'-repair phosphodiesterase and 3'-phosphatase, and MtbNfo contains in addition a very weak NIR activity. Interestingly, depending on pH, both enzymes require different concentrations of divalent cations: 0.5mM MnCl2 at pH 7.6 and 10 mM at pH 6.5. MtbXthA requires a low ionic strength and 37 °C, while MtbNfo requires high ionic strength (200 mM KCl) and has a temperature optimum at 60 °C. Point mutation analysis showed that D180 and N182 in MtbXthA and H206 and E129 in MtbNfo are critical for enzymes activities. The steady-state kinetic parameters indicate that MtbXthA removes 3'-blocking sugar-phosphate and 3'-phosphate moieties at DNA strand breaks with an extremely high efficiency (kcat/KM=440 and 1280 μM(-1)∙min(-1), respectively), while MtbNfo exhibits much lower 3'-repair activities (kcat/KM=0.26 and 0.65 μM(-1)∙min(-1), respectively). Surprisingly, both MtbXthA and MtbNfo exhibited very weak AP site cleavage activities, with kinetic parameters 100- and 300-fold lower, respectively, as compared with the results reported previously. Expression of MtbXthA and MtbNfo reduced the sensitivity of AP endonuclease-deficient E. coli xth nfo strain to methylmethanesulfonate and H2O2 to various degrees. Taken together, these data establish the DNA substrate specificity of M. tuberculosis AP endonucleases and suggest their possible role

  8. Genotyping of the fish rhabdovirus, viral haemorrhagic septicaemia virus, by restriction fragment length polymorphisms

    DEFF Research Database (Denmark)

    Einer-Jensen, Katja; Winton, J.; Lorenzen, Niels

    2005-01-01

    The aim of this study was to develop a standardized molecular assay that used limited resources and equipment for routine genotyping of isolates of the fish rhabdovirus, viral haemorrhagic septicaemia virus (VHSV). Computer generated restriction maps, based on 62 unique full-length (1524 nt....... Experimental evaluation of the method consisted of three steps: (i) RT-PCR amplification of the G-gene of VHSV isolates using purified viral RNA as template, (ii) digestion of the PCR products with a panel of restriction endonucleases and (iii) interpretation of the resulting RFLP profiles. The RFLP analysis...

  9. PMS2 endonuclease activity has distinct biological functions and is essential for genome maintenance.

    Science.gov (United States)

    van Oers, Johanna M M; Roa, Sergio; Werling, Uwe; Liu, Yiyong; Genschel, Jochen; Hou, Harry; Sellers, Rani S; Modrich, Paul; Scharff, Matthew D; Edelmann, Winfried

    2010-07-27

    The DNA mismatch repair protein PMS2 was recently found to encode a novel endonuclease activity. To determine the biological functions of this activity in mammals, we generated endonuclease-deficient Pms2E702K knock-in mice. Pms2EK/EK mice displayed increased genomic mutation rates and a strong cancer predisposition. In addition, class switch recombination, but not somatic hypermutation, was impaired in Pms2EK/EK B cells, indicating a specific role in Ig diversity. In contrast to Pms2-/- mice, Pms2EK/EK male mice were fertile, indicating that this activity is dispensable in spermatogenesis. Therefore, the PMS2 endonuclease activity has distinct biological functions and is essential for genome maintenance and tumor suppression.

  10. DNA synapsis through transient tetramerization triggers cleavage by Ecl18kI restriction enzyme

    NARCIS (Netherlands)

    Zaremba, M.; Lyubchenko, Y.L.; Laurens, N.; van den Broek, B.; Wuite, G.J.L.; Siksnys, V.

    2010-01-01

    To cut DNA at their target sites, restriction enzymes assemble into different oligomeric structures. The Ecl18kI endonuclease in the crystal is arranged as a tetramer made of two dimers each bound to a DNA copy. However, free in solution Ecl18kI is a dimer. To find out whether the Ecl18kI dimer or

  11. Adenosine triphosphate stimulates Aquifex aeolicus MutL endonuclease activity.

    Directory of Open Access Journals (Sweden)

    Jerome Mauris

    2009-09-01

    Full Text Available Human PMS2 (hPMS2 homologues act to nick 5' and 3' to misincorporated nucleotides during mismatch repair in organisms that lack MutH. Mn(++ was previously found to stimulate the endonuclease activity of these homologues. ATP was required for the nicking activity of hPMS2 and yPMS1, but was reported to inhibit bacterial MutL proteins from Thermus thermophilus and Aquifex aeolicus that displayed homology to hPMS2. Mutational analysis has identified the DQHA(X(2E(X(4E motif present in the C-terminus of PMS2 homologues as important for endonuclease activity.We examined the effect ATP had on the Mn(++ induced nicking of supercoiled pBR322 by full-length and mutant A. aeolicus MutL (Aae MutL proteins. Assays were single time point, enzyme titration experiments or reaction time courses. The maximum velocity for MutL nicking was determined to be 1.6+/-0.08x10(-5 s(-1 and 4.2+/-0.3x10(-5 s(-1 in the absence and presence of ATP, respectively. AMPPNP stimulated the nicking activity to a similar extent as ATP. A truncated Aae MutL protein composed of only the C-terminal 123 amino acid residues was found to nick supercoiled DNA. Furthermore, mutations in the conserved C-terminal DQHA(X(2E(X(4E and CPHGRP motifs were shown to abolish Aae MutL endonuclease activity.ATP stimulated the Mn(++ induced endonuclease activity of Aae MutL. Experiments utilizing AMPPNP implied that the stimulation did not require ATP hydrolysis. A mutation in the DQHA(X(2E(X(4E motif of Aae MutL further supported the role of this region in endonclease activity. For the first time, to our knowledge, we demonstrate that changing the histidine residue in the conserved CPHGRP motif abolishes endonucleolytic activity of a hPMS2 homologue. Finally, the C-terminal 123 amino acid residues of Aae MutL were sufficient to display Mn(++ induced nicking activity.

  12. Evolution of divergent DNA recognition specificities in VDE homing endonucleases from two yeast species.

    Science.gov (United States)

    Posey, Karen L; Koufopanou, Vassiliki; Burt, Austin; Gimble, Frederick S

    2004-01-01

    Homing endonuclease genes (HEGs) are mobile DNA elements that are thought to confer no benefit to their host. They encode site-specific DNA endonucleases that perpetuate the element within a species population by homing and disseminate it between species by horizontal transfer. Several yeast species contain the VMA1 HEG that encodes the intein-associated VMA1-derived endonuclease (VDE). The evolutionary state of VDEs from 12 species was assessed by assaying their endonuclease activities. Only two enzymes are active, PI-ZbaI from Zygosaccharomyces bailii and PI-ScaI from Saccharomyces cariocanus. PI-ZbaI cleaves the Z.bailii recognition sequence significantly faster than the Saccharomyces cerevisiae site, which differs at six nucleotide positions. A mutational analysis indicates that PI-ZbaI cleaves the S.cerevisiae substrate poorly due to the absence of a contact that is analogous to one made in PI-SceI between Gln-55 and nucleotides +9/+10. PI-ZbaI cleaves the Z.bailii substrate primarily due to a single base-pair substitution (A/T+5 --> T/A+5). Structural modeling of the PI-ZbaI/DNA complex suggests that Arg-331, which is absent in PI-SceI, contacts T/A+5, and the reduced activity observed in a PI-ZbaI R331A mutant provides evidence for this interaction. These data illustrate that homing endonucleases evolve altered specificity as they adapt to recognize alternative target sites.

  13. Evolution of divergent DNA recognition specificities in VDE homing endonucleases from two yeast species

    Science.gov (United States)

    Posey, Karen L.; Koufopanou, Vassiliki; Burt, Austin; Gimble, Frederick S.

    2004-01-01

    Homing endonuclease genes (HEGs) are mobile DNA elements that are thought to confer no benefit to their host. They encode site-specific DNA endonucleases that perpetuate the element within a species population by homing and disseminate it between species by horizontal transfer. Several yeast species contain the VMA1 HEG that encodes the intein-associated VMA1-derived endonuclease (VDE). The evolutionary state of VDEs from 12 species was assessed by assaying their endonuclease activities. Only two enzymes are active, PI-ZbaI from Zygosaccharomyces bailii and PI-ScaI from Saccharomyces cariocanus. PI-ZbaI cleaves the Z.bailii recognition sequence significantly faster than the Saccharomyces cerevisiae site, which differs at six nucleotide positions. A mutational analysis indicates that PI-ZbaI cleaves the S.cerevisiae substrate poorly due to the absence of a contact that is analogous to one made in PI-SceI between Gln-55 and nucleotides +9/+10. PI-ZbaI cleaves the Z.bailii substrate primarily due to a single base-pair substitution (A/T+5 → T/A+5). Structural modeling of the PI-ZbaI/DNA complex suggests that Arg-331, which is absent in PI-SceI, contacts T/A+5, and the reduced activity observed in a PI-ZbaI R331A mutant provides evidence for this interaction. These data illustrate that homing endonucleases evolve altered specificity as they adapt to recognize alternative target sites. PMID:15280510

  14. Crystal Structure of the Homing Endonuclease I-CvuI Provides a New Template for Genome Modification

    DEFF Research Database (Denmark)

    Molina, Rafael; Redondo, Pilar; López-Méndez, Blanca

    2015-01-01

    Homing endonucleases recognize and generate a DNA double-strand break, which has been used to promote gene targeting. These enzymes recognize long DNA stretches; they are highly sequence-specific enzymes and display a very low frequency of cleavage even in complete genomes. Although a large number...... of homing endonucleases have been identified, the landscape of possible target sequences is still very limited to cover the complexity of the whole eukaryotic genome. Therefore, the finding and molecular analysis of homing endonucleases identified but not yet characterized may widen the landscape...

  15. Purification, crystallization and preliminary crystallographic analysis of a thermostable endonuclease IV from Thermotoga maritima

    International Nuclear Information System (INIS)

    Hughes, Ronny C.; Tomanicek, Stephen J.; Ng, Joseph D.; Coates, Leighton

    2009-01-01

    The overexpression, purification and crystallization of endonuclease IV from T. maritima are reported. The crystals belonged to the hexagonal space group P6 1 and diffracted to 2.36 Å resolution. The DNA-repair enzyme endonuclease IV from the thermophilic bacterium Thermotoga maritima MSB8 (reference sequence NC-000853) has been expressed in Escherichia coli and crystallized for X-ray analysis. T. maritima endonuclease IV is a 287-amino-acid protein with 32% sequence identity to E. coli endonuclease IV. The protein was purified to homogeneity and was crystallized using the sitting-drop vapor-diffusion method. The protein crystallized in space group P6 1 , with one biological molecule in the asymmetric unit, corresponding to a Matthews coefficient of 2.39 Å 3 Da −1 and 47% solvent content. The unit-cell parameters of the crystals were a = b = 123.2, c = 35.6 Å. Microseeding and further optimization yielded crystals with an X-ray diffraction limit of 2.36 Å. A single 70° data set was collected and processed, resulting in an overall R merge and a completeness of 9.5% and 99.3%, respectively

  16. Expression analysis of a ''Cucurbita'' cDNA encoding endonuclease

    International Nuclear Information System (INIS)

    Szopa, J.

    1995-01-01

    The nuclear matrices of plant cell nuclei display intrinsic nuclease activity which consists in nicking supercoiled DNA. A cDNA encoding a 32 kDa endonuclease has been cloned and sequenced. The nucleotide and deduced amino-acid sequences show high homology to known 14-3-3-protein sequences from other sources. The amino-acid sequence shows agreement with consensus sequences for potential phosphorylation by protein kinase A and C and for calcium, lipid and membrane-binding sites. The nucleotide-binding site is also present within the conserved part of the sequence. By Northern blot analysis, the differential expression of the corresponding mRNA was detected; it was the strongest in sink tissues. The endonuclease activity found on DNA-polyacrylamide gel electrophoresis coincided with mRNA content and was the highest in tuber. (author). 22 refs, 6 figs

  17. Conserved structural chemistry for incision activity in structurally non-homologous apurinic/apyrimidinic endonuclease APE1 and endonuclease IV DNA repair enzymes.

    Energy Technology Data Exchange (ETDEWEB)

    Tsutakawa, Susan E.; Shin, David S.; Mol, Clifford D.; Izum, Tadahide; Arvai, Andrew S.; Mantha, Anil K.; Szczesny, Bartosz; Ivanov, Ivaylo N.; Hosfield, David J.; Maiti, Buddhadev; Pique, Mike E.; Frankel, Kenneth A.; Hitomi, Kenichi; Cunningham, Richard P.; Mitra, Sankar; Tainer, John A.

    2013-03-22

    Non-coding apurinic/apyrimidinic (AP) sites in DNA form spontaneously and as DNA base excision repair intermediates are the most common toxic and mutagenic in vivo DNA lesion. For repair, AP sites must be processed by 5' AP endonucleases in initial stages of base repair. Human APE1 and bacterial Nfo represent the two conserved 5' AP endonuclease families in the biosphere; they both recognize AP sites and incise the phosphodiester backbone 5' to the lesion, yet they lack similar structures and metal ion requirements. Here, we determined and analyzed crystal structures of a 2.4 ? resolution APE1-DNA product complex with Mg(2+) and a 0.92 Nfo with three metal ions. Structural and biochemical comparisons of these two evolutionarily distinct enzymes characterize key APE1 catalytic residues that are potentially functionally similar to Nfo active site components, as further tested and supported by computational analyses. We observe a magnesium-water cluster in the APE1 active site, with only Glu-96 forming the direct protein coordination to the Mg(2+). Despite differences in structure and metal requirements of APE1 and Nfo, comparison of their active site structures surprisingly reveals strong geometric conservation of the catalytic reaction, with APE1 catalytic side chains positioned analogously to Nfo metal positions, suggesting surprising functional equivalence between Nfo metal ions and APE1 residues. The finding that APE1 residues are positioned to substitute for Nfo metal ions is supported by the impact of mutations on activity. Collectively, the results illuminate the activities of residues, metal ions, and active site features for abasic site endonucleases.

  18. Murine leukemia virus pol gene products: analysis with antisera generated against reverse transcriptase and endonuclease fusion proteins expressed in Escherichia coli

    International Nuclear Information System (INIS)

    Hu, S.C.; Court, D.L.; Zweig, M.; Levin, J.G.

    1986-01-01

    The organization of the murine leukemia virus (MuLV) pol gene was investigated by expressing molecular clones containing AKR MuLV reverse transcriptase or endonuclease or both gene segments in Escherichia coli and generating specific antisera against the expressed bacterial proteins. Reaction of these antisera with detergent-disrupted virus precipitated and 80-kilodalton (kDa) protein, the MuLV reverse transcriptase, and a 46-kDa protein which we believe is the viral endonuclease. A third (50-kDa) protein, related to reverse transcriptase, was also precipitated. Bacterial extracts of clones expressing reverse transcriptase and endonuclease sequences competed with the viral 80- and 46-kDa proteins, respectively. These results demonstrate that the antisera are specific for viral reverse transcriptase and endonuclease. Immunoprecipitation of AKR MuLV with antisera prepared against a bacterial protein containing only endonuclease sequences led to the observation that reverse transcriptase and endonuclease can be associated as a complex involving a disulfide bond(s)

  19. Physical association of pyrimidine dimer DNA glycosylase and apurinic/apyrimidinic DNA endonuclease essential for repair of ultraviolet-damaged DNA

    International Nuclear Information System (INIS)

    Nakabeppu, Y.; Sekiguchi, M.

    1981-01-01

    T4 endonuclease, which is involved in repair of uv-damaged DNA, has been purified to apparent physical homogeneity. Incubation of uv-irradiated poly(dA).poly(dT) with the purified enzyme preparations resulted in production of alkali-labile apyrimidinic sites, followed by formation of nicks in the polymer. By performing a limited reaction with T4 endonuclease V at pH 8.5, irradiated polymer was converted to an intermediate form that carried a large number of alkali-labile sites but only a few nicks. The intermediate was used as substrate for the assay of apurinic/apyrimidinic DNA endonuclease activity. The two activities, a pyrimidine dimer DNA glycosylase and an apurinic/apyrimidinic DNA endonuclease, were copurified and found in enzyme preparations that contained only a 16,000-dalton polypeptide. These results strongly suggested that a DNA glycosylase specific for pyrimidine dimers and an apurinic/apyrimidinic DNA endonuclease reside in a single polypeptide chain coded by the denV gene of bacteriophage T4

  20. Cloning and analysis of the genes encoding the type IIS restriction-modification system HphI from Haemophilus parahaemolyticus.

    Science.gov (United States)

    Lubys, A; Lubienè, J; Kulakauskas, S; Stankevicius, K; Timinskas, A; Janulaitis, A

    1996-07-15

    The genomic region encoding the type IIS restriction-modification (R-M) system HphI (enzymes recognizing the asymmetric sequence 5'-GGTGA-3'/5'-TCACC-3') from Haemophilus parahaemolyticus were cloned into Escherichia coli and sequenced. Sequence analysis of the R-M HphI system revealed three adjacent genes aligned in the same orientation: a cytosine 5 methyltransferase (gene hphIMC), an adenine N6 methyltransferase (hphIMA) and the HphI restriction endonuclease (gene hphIR). Either methyltransferase is capable of protecting plasmid DNA in vivo against the action of the cognate restriction endonuclease. hphIMA methylation renders plasmid DNA resistant to R.Hindill at overlapping sites, suggesting that the adenine methyltransferase modifies the 3'-terminal A residue on the GGTGA strand. Strong homology was found between the N-terminal part of the m6A methyltransferasease and an unidentified reading frame interrupted by an incomplete gaIE gene of Neisseria meningitidis. The HphI R-M genes are flanked by a copy of a 56 bp direct nucleotide repeat on each side. Similar sequences have also been identified in the non-coding regions of H.influenzae Rd DNA. Possible involvement of the repeat sequences in the mobility of the HphI R-M system is discussed.

  1. Hold your horSSEs: controlling structure-selective endonucleases MUS81 and Yen1/GEN1.

    Science.gov (United States)

    Blanco, Miguel G; Matos, Joao

    2015-01-01

    Repair of DNA lesions through homologous recombination promotes the establishment of stable chromosomal interactions. Multiple helicases, topoisomerases and structure-selective endonucleases (SSEs) act upon recombining joint molecules (JMs) to disengage chromosomal connections and safeguard chromosome segregation. Recent studies on two conserved SSEs - MUS81 and Yen1/GEN1- uncovered multiple layers of regulation that operate to carefully tailor JM-processing according to specific cellular needs. Temporal restriction of SSE function imposes a hierarchy in pathway usage that ensures efficient JM-processing while minimizing reciprocal exchanges between the recombining DNAs. Whereas a conserved strategy of fine-tuning SSE functions exists in different model systems, the precise molecular mechanisms to implement it appear to be significantly different. Here, we summarize the current knowledge on the cellular switches that are in place to control MUS81 and Yen1/GEN1 functions.

  2. Expression of ultraviolet-induced restriction alleviation in Escherichia coli K-12

    International Nuclear Information System (INIS)

    Thoms, B.; Wackernagel, W.

    1983-01-01

    Ultraviolet-induced restriction alleviation is an SOS function which partially relieves the K-12-specific DNA restriction in Escherichia coli. Restriction alleviation is determined by observing elevated survival of unmodified phage lambda in cells irradiated with ultraviolet prior to infection. The authors demonstrate that restriction of lambda is also relieved when log-phase cells are irradiated as late as 50 min after adsorption of lambda. At this time more than 60% of the lambda DNA is already released as acid-soluble material from the cells. Experiments involving reextraction of lambda DNA from infected cells and a mild detergent treatment removing adsorbed phages from the cellular surface showed that only a small specific fraction of all lambda infections is destined to escape restriction due to restriction alleviation. This fraction (10-20%) has a retarded mode of DNA injection (60 min or longer) after adsorption which allows the expression of the restriction alleviation function before the phage DNA is exposed to restriction endonucleases. This behaviour of a fraction of lambda phages explains why the SOS function restriction alleviation could initially be discovered. The authors show that the retarded mode of DNA injection is not required for another SOS function acting on lambda DNA, the increased repair of ultraviolet-irradiated DNA (Weigle reactivation). (Auth.)

  3. Biochemical properties and base excision repair complex formation of apurinic/apyrimidinic endonuclease from Pyrococcus furiosus

    OpenAIRE

    Kiyonari, Shinichi; Tahara, Saki; Shirai, Tsuyoshi; Iwai, Shigenori; Ishino, Sonoko; Ishino, Yoshizumi

    2009-01-01

    Apurinic/apyrimidinic (AP) sites are the most frequently found mutagenic lesions in DNA, and they arise mainly from spontaneous base loss or modified base removal by damage-specific DNA glycosylases. AP sites are cleaved by AP endonucleases, and the resultant gaps in the DNA are repaired by DNA polymerase/DNA ligase reactions. We identified the gene product that is responsible for the AP endonuclease activity in the hyperthermophilic euryarchaeon, Pyrococcus furiosus. Furthermore, we detected...

  4. Modulation of the DNA scanning activity of the Micrococcus luteus UV endonuclease

    International Nuclear Information System (INIS)

    Hamilton, R.W.; Lloyd, R.S.

    1989-01-01

    Micrococcus luteus UV endonuclease incises DNA at the sites of ultraviolet (UV) light-induced pyrimidine dimers. The mechanism of incision has been previously shown to be a glycosylic bond cleavage at the 5'-pyrimidine of the dimer followed by an apyrimidine endonuclease activity which cleaves the phosphodiester backbone between the pyrimidines. The process by which M. luteus UV endonuclease locates pyrimidine dimers within a population of UV-irradiated plasmids was shown to occur, in vitro, by a processive or sliding mechanism on non-target DNA as opposed to a distributive or random hit mechanism. Form I plasmid DNA containing 25 dimers per molecule was incubated with M. luteus UV endonuclease in time course reactions. The three topological forms of plasmid DNA generated were analyzed by agarose gel electrophoresis. When the enzyme encounters a pyrimidine dimer, it is significantly more likely to make only the glycosylase cleavage as opposed to making both the glycosylic and phosphodiester bond cleavages. Thus, plasmids are accumulated with many alkaline-labile sites relative to single-stranded breaks. In addition, reactions were performed at both pH 8.0 and pH 6.0, in the absence of NaCl, as well as 25,100, and 250 mM NaCl. The efficiency of the DNA scanning reaction was shown to be dependent on both the ionic strength and pH of the reaction. At low ionic strengths, the reaction was shown to proceed by a processive mechanism and shifted to a distributive mechanism as the ionic strength of the reaction increased. Processivity at pH 8.0 is shown to be more sensitive to increases in ionic strength than reactions performed at pH 6.0

  5. DNA scanning mechanism of T4 endonuclease V. Effect of NaCl concentration on processive nicking activity

    International Nuclear Information System (INIS)

    Gruskin, E.A.; Lloyd, R.S.

    1986-01-01

    T4 endonuclease V is a pyrimidine dimer-specific endonuclease which generates incisions in DNA at the sites of pyrimidine dimers by a processive reaction mechanism. A model is presented in which the degree of processivity is directly related to the efficacy of the one-dimensional diffusion of endonuclease V on DNA by which the enzyme locates pyrimidine dimers. The modulation of the processive nicking activity of T4 endonuclease V on superhelical covalently closed circular DNA (form I) which contains pyrimidine dimers has been investigated as a function of the ionic strength of the reaction. Agarose gel electrophoresis was used to separate the three topological forms of the DNA which were generated in time course reactions of endonuclease V with dimer-containing form I DNA in the absence of NaCl, and in 25, 50, and 100 mM NaCl. The degree of processivity was evaluated in terms of the mass fraction of form III (linear) DNA which was produced as a function of the fraction of form I DNA remaining. Processivity is maximal in the absence of NaCl and decreases as the NaCl concentration is increased. At 100 mM NaCl, processivity is abolished and endonuclease V generates incisions in DNA at the site of dimers by a distributive reaction mechanism. The change from the distributive to a processive reaction mechanism occurs at NaCl concentrations slightly below 50 mM. The high degree of processivity which is observed in the absence of NaCl is reversible to the distributive mechanism, as demonstrated by experiments in which the NaCl concentration was increased during the time course reaction. In addition, unirradiated DNA inhibited the incision of irradiated DNA only at NaCl concentrations at which processivity was observed

  6. Karyopherin-mediated nuclear import of the homing endonuclease VMA1-derived endonuclease is required for self-propagation of the coding region.

    Science.gov (United States)

    Nagai, Yuri; Nogami, Satoru; Kumagai-Sano, Fumi; Ohya, Yoshikazu

    2003-03-01

    VMA1-derived endonuclease (VDE), a site-specific endonuclease in Saccharomyces cerevisiae, enters the nucleus to generate a double-strand break in the VDE-negative allelic locus, mediating the self-propagating gene conversion called homing. Although VDE is excluded from the nucleus in mitotic cells, it relocalizes at premeiosis, becoming localized in both the nucleus and the cytoplasm in meiosis. The nuclear localization of VDE is induced by inactivation of TOR kinases, which constitute central regulators of cell differentiation in S. cerevisiae, and by nutrient depletion. A functional genomic approach revealed that at least two karyopherins, Srp1p and Kap142p, are required for the nuclear localization pattern. Genetic and physical interactions between Srp1p and VDE imply direct involvement of karyopherin-mediated nuclear transport in this process. Inactivation of TOR signaling or acquisition of an extra nuclear localization signal in the VDE coding region leads to artificial nuclear localization of VDE and thereby induces homing even during mitosis. These results serve as evidence that VDE utilizes the host systems of nutrient signal transduction and nucleocytoplasmic transport to ensure the propagation of its coding region.

  7. Fluorescence quenching of graphene oxide combined with the site-specific cleavage of restriction endonuclease for deoxyribonucleic acid demethylase activity assay

    Energy Technology Data Exchange (ETDEWEB)

    Ji, Lijuan; Qian, Yingdan; Wu, Ping; Zhang, Hui; Cai, Chenxin, E-mail: cxcai@njnu.edu.cn

    2015-04-15

    Highlights: • An approach for sensitive and selective DNA demethylase activity assay is reported. • This assay is based on the fluorescence quenching of GO and site-specific cleavage of endonuclease. • It can determine as low as 0.05 ng mL{sup −1} of MBD2 with a linear range of 0.2–300 ng mL{sup −1}. • It has an ability to recognize MBD2 from other possibly coexisting proteins and cancer cell extracts. • It can avoid false signals, requiring no bisulfite conversion, PCR amplification, radioisotope-labeling. - Abstract: We report on the development of a sensitive and selective deoxyribonucleic acid (DNA) demethylase (using MBD2 as an example) activity assay by coupling the fluorescence quenching of graphene oxide (GO) with the site-specific cleavage of HpaII endonuclease to improve the selectivity. This approach was developed by designing a single-stranded probe (P1) that carries a binding region to facilitate the interaction with GO, which induces fluorescence quenching of the labeled fluorophore (FAM, 6-carboxyfluorescein), and a sensing region, which contains a hemi-methylated site of 5′-CmCGG-3′, to specifically recognize the target (T1, a 32-mer DNA from the promoter region of p53 gene) and hybridize with it to form a P1/T1 duplex. After demethylation with MBD2, the duplex can be specifically cleaved using HpaII, which releases the labeled FAM from the GO surface and results in the recovery of fluorescence. However, this cleavage is blocked by the hemi-methylation of this site. Thus, the magnitude of the recovered fluorescence signal is related to the MBD2 activity, which establishes the basis of the DNA demethylase activity assay. This assay can determine as low as ∼(0.05 ± 0.01) ng mL{sup −1} (at a signal/noise of 3) of MBD2 with a linear range of 0.2–300 ng mL{sup −1} and recognize MBD2 from other possibly coexisting proteins and cancer cell extracts. The advantage of this assay is its ability to avoid false signals and no

  8. Home and away- the evolutionary dynamics of homing endonucleases

    Directory of Open Access Journals (Sweden)

    Barzel Adi

    2011-11-01

    Full Text Available Abstract Background Homing endonucleases (HEases are a large and diverse group of site-specific DNAases. They reside within self-splicing introns and inteins, and promote their horizontal dissemination. In recent years, HEases have been the focus of extensive research due to their promising potential use in gene targeting procedures for the treatment of genetic diseases and for the genetic engineering of crop, animal models and cell lines. Results Using mathematical analysis and computational modeling, we present here a novel account for the evolution and population dynamics of HEase genes (HEGs. We describe HEGs as paradoxical selfish elements whose long-term persistence in a single population relies on low transmission rates and a positive correlation between transmission efficiency and toxicity. Conclusion Plausible conditions allow HEGs to sustain at high frequency through long evolutionary periods, with the endonuclease frequency being either at equilibrium or periodically oscillating. The predictions of our model may prove important not only for evolutionary theory but also for gene therapy and bio-engineering applications of HEases.

  9. Divalent metal ion differentially regulates the sequential nicking reactions of the GIY-YIG homing endonuclease I-BmoI.

    Directory of Open Access Journals (Sweden)

    Benjamin P Kleinstiver

    Full Text Available Homing endonucleases are site-specific DNA endonucleases that function as mobile genetic elements by introducing double-strand breaks or nicks at defined locations. Of the major families of homing endonucleases, the modular GIY-YIG endonucleases are least understood in terms of mechanism. The GIY-YIG homing endonuclease I-BmoI generates a double-strand break by sequential nicking reactions during which the single active site of the GIY-YIG nuclease domain must undergo a substantial reorganization. Here, we show that divalent metal ion plays a significant role in regulating the two independent nicking reactions by I-BmoI. Rate constant determination for each nicking reaction revealed that limiting divalent metal ion has a greater impact on the second strand than the first strand nicking reaction. We also show that substrate mutations within the I-BmoI cleavage site can modulate the first strand nicking reaction over a 314-fold range. Additionally, in-gel DNA footprinting with mutant substrates and modeling of an I-BmoI-substrate complex suggest that amino acid contacts to a critical GC-2 base pair are required to induce a bottom-strand distortion that likely directs conformational changes for reaction progress. Collectively, our data implies mechanistic roles for divalent metal ion and substrate bases, suggesting that divalent metal ion facilitates the re-positioning of the GIY-YIG nuclease domain between sequential nicking reactions.

  10. Sequential and Multistep Substrate Interrogation Provides the Scaffold for Specificity in Human Flap Endonuclease 1

    KAUST Repository

    Sobhy, M.; Joudeh, L.; Huang, X.; Takahashi, Masateru; Hamdan, S.

    2013-01-01

    Human flap endonuclease 1 (FEN1), one of the structure-specific 5' nucleases, is integral in replication, repair, and recombination of cellular DNA. The 5' nucleases share significant unifying features yet cleave diverse substrates at similar positions relative to 5' end junctions. Using single-molecule Förster resonance energy transfer, we find a multistep mechanism that verifies all substrate features before inducing the intermediary-DNA bending step that is believed to unify 5' nuclease mechanisms. This is achieved by coordinating threading of the 5' flap of a nick junction into the conserved capped-helical gateway, overseeing the active site, and bending by binding at the base of the junction. We propose that this sequential and multistep substrate recognition process allows different 5' nucleases to recognize different substrates and restrict the induction of DNA bending to the last common step. Such mechanisms would also ensure the protection ofDNA junctions from nonspecific bending and cleavage. 2013 The Authors.

  11. Sequential and Multistep Substrate Interrogation Provides the Scaffold for Specificity in Human Flap Endonuclease 1

    KAUST Repository

    Sobhy, M.

    2013-06-06

    Human flap endonuclease 1 (FEN1), one of the structure-specific 5\\' nucleases, is integral in replication, repair, and recombination of cellular DNA. The 5\\' nucleases share significant unifying features yet cleave diverse substrates at similar positions relative to 5\\' end junctions. Using single-molecule Förster resonance energy transfer, we find a multistep mechanism that verifies all substrate features before inducing the intermediary-DNA bending step that is believed to unify 5\\' nuclease mechanisms. This is achieved by coordinating threading of the 5\\' flap of a nick junction into the conserved capped-helical gateway, overseeing the active site, and bending by binding at the base of the junction. We propose that this sequential and multistep substrate recognition process allows different 5\\' nucleases to recognize different substrates and restrict the induction of DNA bending to the last common step. Such mechanisms would also ensure the protection ofDNA junctions from nonspecific bending and cleavage. 2013 The Authors.

  12. Presence of UV-endonuclease sensitive sites in daughter DNA of UV-irradiated mammalian cells

    International Nuclear Information System (INIS)

    D'Ambrosio, S.; Setlow, R.B.

    1978-02-01

    Asynchronous Chinese hamster cells were irradiated with 10 Jm -2 uv radiation and 0.25 to 4 hours later pulse-labeled with [ 3 H]thymidine. Cells synchronized by shaking off mitotic and G 1 cells were irradiated in either the G 1 -phase or S-phase of the cell cycle and pulse-labeled with [ 3 H]thymidine in the S-phase. After a 12 to 14 hour chase in unlabeled medium, the DNA was extracted, incubated with Micrococcus luteus uv-endonuclease and sedimented in alkaline sucrose. The number of endonuclease sensitive sites decreased as the time between uv irradiation and pulse-labeling of daughter DNA increased. Further, there were significantly less endonuclease sensitive sites in the daughter DNA from cells irradiated in the G 1 -phase than in the S-phase. These data indicate that very few, if any, dimers are transferred from parental DNA to daughter DNA and that the dimers detected in daughter DNA may be due to the irradiation of replicating daughter DNA before labeling

  13. Specific action of T4 endonuclease V on damaged DNA in xeroderma pigmentosum cells in vivo

    International Nuclear Information System (INIS)

    Tanaka, K.; Hayakawa, H.; Sekiguchi, M.; Okada, Y.

    1977-01-01

    The specific action of T4 endonuclease V on damaged DNA in xeroderma pigmentosum cells was examined using an in vivo assay system with hemagglutinating virus of Japan (Sendai virus) inactivated by uv light. A clear dose response was observed between the level of uv-induced unscheduled DNA synthesis of xeroderma pigmentosum cells and the amount of T4 endonuclease V activity added. The T4 enzyme was unstable in human cells, and its half-life was 3 hr. Fractions derived from an extract of Escherichia coli infected with T4v 1 , a mutant defective in the endonuclease V gene, showed no ability to restore the uv-induced unscheduled DNA synthesis of xeroderma pigmentosum cells. However, fractions derived from an extract of T4D-infected E. coli with endonuclease V activity were effective. The T4 enzyme was effective in xeroderma pigmentosum cells on DNA damaged by uv light but not in cells damaged by 4-nitroquinoline 1-oxide. The results of these experiments show that the T4 enzyme has a specific action on human cell DNA in vivo. Treatment with the T4 enzyme increased the survival of group A xeroderma pigmentosum cells after uv irradiation

  14. Restriction map of the single-stranded DNA genome of Kilham rat virus strain 171, a nondefective parvovirus

    International Nuclear Information System (INIS)

    Banerjee, P.T.; Rathrock, R.; Mitra, S.

    1981-01-01

    A physical map of Kilham rat virus strain 171 DNA was constructed by analyzing the sizes and locations of restriction endonuclease-generated fragments of the replicative-form viral DNA synthesized in vitro. BglI, KpnI, BamHI, SmaI, XhoI, and XorII did not appear to have any cleavage sites, whereas 11 other enzymes cleaved the genome at one to eight sites, and AluI generated more than 12 distinct fragments. The 30 restriction sites that were mapped were distributed randomly in the viral genome. A comparison of the restriction fragments of in vivo- and in vitro-replicated replicative-form DNAs showed that these DNAs were identical except in the size or configuration of the terminal fragments

  15. Molecular Recognition of DNA Damage Sites by Apurinic/Apyrimidinic Endonucleases

    Energy Technology Data Exchange (ETDEWEB)

    Braun, W. A.

    2005-07-28

    The DNA repair/redox factor AP endonuclease 1 (APE1) is a multifunctional protein which is known to to be essential for DNA repair activity in human cells. Structural/functional analyses of the APE activity is thus been an important research field to assess cellular defense mechanisms against ionizing radiation.

  16. A site-specific endonuclease encoded by a typical archaeal intron

    DEFF Research Database (Denmark)

    Dalgaard, Jacob; Garrett, Roger Antony; Belfort, Malene

    1993-01-01

    The protein encoded by the archaeal intron in the 23S rRNA gene of the hyperthermophile Desulfurococcus mobilis is a double-strand DNase that, like group I intron homing endonucleases, is capable of cleaving an intronless allele of the gene. This enzyme, I-Dmo I, is unusual among the intron...

  17. Crystal structure and DNA-binding property of the ATPase domain of bacterial mismatch repair endonuclease MutL from Aquifex aeolicus.

    Science.gov (United States)

    Fukui, Kenji; Iino, Hitoshi; Baba, Seiki; Kumasaka, Takashi; Kuramitsu, Seiki; Yano, Takato

    2017-09-01

    DNA mismatch repair (MMR) system corrects mismatched bases that are generated mainly by DNA replication errors. The repair system excises the error-containing single-stranded region and enables the re-synthesis of the strand. In the early reactions of MMR, MutL endonuclease incises the newly-synthesized/error-containing strand of the duplex to initiate the downstream excision reaction. MutL endonuclease consists of the N-terminal ATPase and C-terminal endonuclease domains. In this study, we report the crystal structure of the ATPase domain of MutL endonuclease from Aquifex aeolicus. The overall structure of the domain was similar to those of human MutL homologs and Escherichia coli MutL, although E. coli MutL has no endonuclease activity. The ATPase domain was comprised of two subdomains: the N-terminal ATP-binding subdomain and the C-terminal α-β sandwich subdomain. Site-directed mutagenesis experiment identified DNA-interacting eight basic amino acid residues, which were distributed across both the two subdomains and formed a DNA-binding cleft. Docking simulation between the structures of the ATPase and endonuclease domains generated a reliable model structure for the full-length A. aeolicus MutL, which satisfies our previous result of small-angle X-ray scattering analysis. On the basis of the model structure and further experimental results, we concluded that the two separate DNA-binding sites in the full-length A. aeolicus MutL simultaneously bind a dsDNA molecule. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Analysis of mutation/rearrangement frequencies and methylation patterns at a given DNA locus using restriction fragment length polymorphism.

    Science.gov (United States)

    Boyko, Alex; Kovalchuk, Igor

    2010-01-01

    Restriction fragment length polymorphism (RFLP) is a difference in DNA sequences of organisms belonging to the same species. RFLPs are typically detected as DNA fragments of different lengths after digestion with various restriction endonucleases. The comparison of RFLPs allows investigators to analyze the frequency of occurrence of mutations, such as point mutations, deletions, insertions, and gross chromosomal rearrangements, in the progeny of stressed plants. The assay involves restriction enzyme digestion of DNA followed by hybridization of digested DNA using a radioactively or enzymatically labeled probe. Since DNA can be digested with methylation sensitive enzymes, the assay can also be used to analyze a methylation pattern of a particular locus. Here, we describe RFLP analysis using methylation-insensitive and methylation-sensitive enzymes.

  19. Measurement of M. luteus endonuclease-sensitive lesions by alkaline elution

    Energy Technology Data Exchange (ETDEWEB)

    Fornace, Jr, A J [National Cancer Inst., Bethesda, MD (USA). Lab. for Experimental Pathology

    1982-01-01

    The UV-endonuclease approach to detect DNA damage has been combined with the alkaline elution technique with a resultant marked increase in sensitivity compared to the conventional method using alkaline sedimentation. DNA from UV-irradiated cells was digested on an inert filter with an extract from Micrococcus luteus and then analyzed by alkaline elution. Endonuclease-sensitive sites (endo-sites) were measured after doses of 0.08-0.7 Jm/sup -2/ of UV-radiation. An estimate of endo-site production with UV radiation, 0.27 endo-sites/10/sup 8/ daltons of DNA/0.1 Jm/sup -2/, was similar to that usually seen at higher doses by others. With repair incubation, approx. 50% of the endo-sites were removed in 4 h by normal human fibroblasts after 0.2 or 0.4 Jm/sup -2/, no appreciable repair was seen in xeroderma pigmentosum fibroblasts from complementation group A after 24 h of repair incubation. No photoreaction of UV damage due to 0.4 Jm/sup -2/ was detected in normal human fibroblasts.

  20. Measurement of M. luteus endonuclease-sensitive lesions by alkaline elution

    International Nuclear Information System (INIS)

    Fornace, A.J. Jr.

    1982-01-01

    The UV-endonuclease approach to detect DNA damage has been combined with the alkaline elution technique with a resultant marked increase in sensitivity compared to the conventional method using alkaline sedimentation. DNA from UV-irradiated cells was digested on an inert filter with an extract from Micrococcus luteus and then analyzed by alkaline elution. Endonuclease-sensitive sites (endo-sites) were measured after doses of 0.08-0.7 Jm -2 of UV-radiation. An estimate of endo-site production with UV radiation, 0.27 endo-sites/10 8 daltons of DNA/0.1 Jm -2 , was similar to that usually seen at higher doses by others. With repair incubation, approx. 50% of the endo-sites were removed in 4 h by normal human fibroblasts after 0.2 or 0.4 Jm -2 , no appreciable repair was seen in xeroderma pigmentosum fibroblasts from complementation group A after 24 h of repair incubation. No photoreaction of UV damage due to 0.4 Jm -2 was detected in normal human fibroblasts. (orig./AJ)

  1. The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans.

    Science.gov (United States)

    Manhart, Carol M; Ni, Xiaodan; White, Martin A; Ortega, Joaquin; Surtees, Jennifer A; Alani, Eric

    2017-04-01

    Crossing over between homologs is initiated in meiotic prophase by the formation of DNA double-strand breaks that occur throughout the genome. In the major interference-responsive crossover pathway in baker's yeast, these breaks are resected to form 3' single-strand tails that participate in a homology search, ultimately forming double Holliday junctions (dHJs) that primarily include both homologs. These dHJs are resolved by endonuclease activity to form exclusively crossovers, which are critical for proper homolog segregation in Meiosis I. Recent genetic, biochemical, and molecular studies in yeast are consistent with the hypothesis of Mlh1-Mlh3 DNA mismatch repair complex acting as the major endonuclease activity that resolves dHJs into crossovers. However, the mechanism by which the Mlh1-Mlh3 endonuclease is activated is unknown. Here, we provide evidence that Mlh1-Mlh3 does not behave like a structure-specific endonuclease but forms polymers required to generate nicks in DNA. This conclusion is supported by DNA binding studies performed with different-sized substrates that contain or lack polymerization barriers and endonuclease assays performed with varying ratios of endonuclease-deficient and endonuclease-proficient Mlh1-Mlh3. In addition, Mlh1-Mlh3 can generate religatable double-strand breaks and form an active nucleoprotein complex that can nick DNA substrates in trans. Together these observations argue that Mlh1-Mlh3 may not act like a canonical, RuvC-like Holliday junction resolvase and support a novel model in which Mlh1-Mlh3 is loaded onto DNA to form an activated polymer that cleaves DNA.

  2. Yeast redoxyendonuclease, a DNA repair enzyme similar to Escherichia coli endonuclease III

    International Nuclear Information System (INIS)

    Gossett, J.; Lee, K.; Cunningham, R.P.; Doetsch, P.W.

    1988-01-01

    A DNA repair endonuclease (redoxyendonuclease) was isolated from bakers' yeast (Saccharomyces cerevisiae). The enzyme has been purified by a series of column chromatography steps and cleaves OsO 4 -damaged, double-stranded DNA at sites of thymine glycol and heavily UV-irradiated DNA at sites of cytosine, thymine, and guanine photoproducts. The base specificity and mechanism of phosphodiester bond cleavage for the yeast redoxyendonuclease appear to be identical with those of Escherichia coli endonuclease III when thymine glycol containing, end-labeled DNA fragments of defined sequence are employed as substrates. Yeast redoxyendonuclease has an apparent molecular size of 38,000-42,000 daltons and is active in the absence of divalent metal cations. The identification of such an enzyme in yeast may be of value in the elucidation of the biochemical basis for radiation sensitivity in certain yeast mutants

  3. Inroads into base excision repair I. The discovery of apurinic/apyrimidinic (AP) endonuclease. "An endonuclease for depurinated DNA in Escherichia coli B," Canadian Journal of Biochemistry, 1972.

    Science.gov (United States)

    Lindahl, Tomas; Verly, W G; Paquette Y

    2004-11-02

    DNA treated with alkylating agents is incised at sites of damage by cell extracts. A key component of this DNA repair function was shown by Verly and co-workers to be an endonuclease acting at secondary lesions, apurinic sites, rather than directly at alkylated nucleotide residues.

  4. Telomere Restriction Fragment (TRF) Analysis.

    Science.gov (United States)

    Mender, Ilgen; Shay, Jerry W

    2015-11-20

    restriction enzyme recognition sites within TTAGGG tandem telomeric repeats, therefore digestion of genomic DNA, not telomeric DNA, with a combination of 6 base restriction endonucleases reduces genomic DNA size to less than 800 bp.

  5. Enhancement of ultraviolet-DNA repair in denV gene transfectants and T4 endonuclease V-liposome recipients

    International Nuclear Information System (INIS)

    Kibitel, J.T.; Yee, V.; Yarosh, D.B.

    1991-01-01

    The phage T4 denV gene, coding for the pyrimidine-dimer specific T4 endonuclease V, was transfected into human repair-proficient fibroblasts, repair-deficient xeroderma pigmentosum fibroblasts, and wild type CHO hamster cells. Transfectants maintained denV DNA and expressed denV mRNA. Purified T4 endonuclease V encapsulated in liposomes was also used to treat repair-proficient and -deficient human cells. The denV transfected clones and liposome-treated cells showed increased unscheduled DNA synthesis and enhanced removal of pyrimidine dimers compared to controls. Both denV gene transfection and endonuclease V liposome treatment enhanced post-UV survival in xeroderma pigmentosum cells but had no effect on survival in repair-proficient human or hamster cells. The results demonstrate that an exogenous DNA repair enzyme can correct the DNA repair defect in xeroderma pigmentosum cells and enhance DNA repair in normal cells. (author)

  6. Atomic Structure and Biochemical Characterization of an RNA Endonuclease in the N Terminus of Andes Virus L Protein.

    Directory of Open Access Journals (Sweden)

    Yaiza Fernández-García

    2016-06-01

    Full Text Available Andes virus (ANDV is a human-pathogenic hantavirus. Hantaviruses presumably initiate their mRNA synthesis by using cap structures derived from host cell mRNAs, a mechanism called cap-snatching. A signature for a cap-snatching endonuclease is present in the N terminus of hantavirus L proteins. In this study, we aimed to solve the atomic structure of the ANDV endonuclease and characterize its biochemical features. However, the wild-type protein was refractory to expression in Escherichia coli, presumably due to toxic enzyme activity. To circumvent this problem, we introduced attenuating mutations in the domain that were previously shown to enhance L protein expression in mammalian cells. Using this approach, 13 mutant proteins encompassing ANDV L protein residues 1-200 were successfully expressed and purified. Protein stability and nuclease activity of the mutants was analyzed and the crystal structure of one mutant was solved to a resolution of 2.4 Å. Shape in solution was determined by small angle X-ray scattering. The ANDV endonuclease showed structural similarities to related enzymes of orthobunya-, arena-, and orthomyxoviruses, but also differences such as elongated shape and positively charged patches surrounding the active site. The enzyme was dependent on manganese, which is bound to the active site, most efficiently cleaved single-stranded RNA substrates, did not cleave DNA, and could be inhibited by known endonuclease inhibitors. The atomic structure in conjunction with stability and activity data for the 13 mutant enzymes facilitated inference of structure-function relationships in the protein. In conclusion, we solved the structure of a hantavirus cap-snatching endonuclease, elucidated its catalytic properties, and present a highly active mutant form, which allows for inhibitor screening.

  7. Structural Plasticity of PAM Recognition by Engineered Variants of the RNA-Guided Endonuclease Cas9.

    Science.gov (United States)

    Anders, Carolin; Bargsten, Katja; Jinek, Martin

    2016-03-17

    The RNA-guided endonuclease Cas9 from Streptococcus pyogenes (SpCas9) forms the core of a powerful genome editing technology. DNA cleavage by SpCas9 is dependent on the presence of a 5'-NGG-3' protospacer adjacent motif (PAM) in the target DNA, restricting the choice of targetable sequences. To address this limitation, artificial SpCas9 variants with altered PAM specificities have recently been developed. Here we report crystal structures of the VQR, EQR, and VRER SpCas9 variants bound to target DNAs containing their preferred PAM sequences. The structures reveal that the non-canonical PAMs are recognized by an induced fit mechanism. Besides mediating sequence-specific base recognition, the amino acid substitutions introduced in the SpCas9 variants facilitate conformational remodeling of the PAM region of the bound DNA. Guided by the structural data, we engineered a SpCas9 variant that specifically recognizes NAAG PAMs. Taken together, these studies inform further development of Cas9-based genome editing tools. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Early endonuclease-mediated evasion of RNA sensing ensures efficient coronavirus replication.

    Directory of Open Access Journals (Sweden)

    Eveline Kindler

    2017-02-01

    Full Text Available Coronaviruses are of veterinary and medical importance and include highly pathogenic zoonotic viruses, such as SARS-CoV and MERS-CoV. They are known to efficiently evade early innate immune responses, manifesting in almost negligible expression of type-I interferons (IFN-I. This evasion strategy suggests an evolutionary conserved viral function that has evolved to prevent RNA-based sensing of infection in vertebrate hosts. Here we show that the coronavirus endonuclease (EndoU activity is key to prevent early induction of double-stranded RNA (dsRNA host cell responses. Replication of EndoU-deficient coronaviruses is greatly attenuated in vivo and severely restricted in primary cells even during the early phase of the infection. In macrophages we found immediate induction of IFN-I expression and RNase L-mediated breakdown of ribosomal RNA. Accordingly, EndoU-deficient viruses can retain replication only in cells that are deficient in IFN-I expression or sensing, and in cells lacking both RNase L and PKR. Collectively our results demonstrate that the coronavirus EndoU efficiently prevents simultaneous activation of host cell dsRNA sensors, such as Mda5, OAS and PKR. The localization of the EndoU activity at the site of viral RNA synthesis-within the replicase complex-suggests that coronaviruses have evolved a viral RNA decay pathway to evade early innate and intrinsic antiviral host cell responses.

  9. Identification of a mismatch-specific endonuclease in hyperthermophilic Archaea

    OpenAIRE

    Ishino, Sonoko; Nishi, Yuki; Oda, Soichiro; Uemori, Takashi; Sagara, Takehiro; Takatsu, Nariaki; Yamagami, Takeshi; Shirai, Tsuyoshi; Ishino, Yoshizumi

    2016-01-01

    The common mismatch repair system processed by MutS and MutL and their homologs was identified in Bacteria and Eukarya. However, no evidence of a functional MutS/L homolog has been reported for archaeal organisms, and it is not known whether the mismatch repair system is conserved in Archaea. Here, we describe an endonuclease that cleaves double-stranded DNA containing a mismatched base pair, from the hyperthermophilic archaeon Pyrococcus furiosus. The corresponding gene revealed that the act...

  10. Creating a monomeric endonuclease TALE-I-SceI with high specificity and low genotoxicity in human cells.

    Science.gov (United States)

    Lin, Jianfei; Chen, He; Luo, Ling; Lai, Yongrong; Xie, Wei; Kee, Kehkooi

    2015-01-01

    To correct a DNA mutation in the human genome for gene therapy, homology-directed repair (HDR) needs to be specific and have the lowest off-target effects to protect the human genome from deleterious mutations. Zinc finger nucleases, transcription activator-like effector nuclease (TALEN) and CRISPR-CAS9 systems have been engineered and used extensively to recognize and modify specific DNA sequences. Although TALEN and CRISPR/CAS9 could induce high levels of HDR in human cells, their genotoxicity was significantly higher. Here, we report the creation of a monomeric endonuclease that can recognize at least 33 bp by fusing the DNA-recognizing domain of TALEN (TALE) to a re-engineered homing endonuclease I-SceI. After sequentially re-engineering I-SceI to recognize 18 bp of the human β-globin sequence, the re-engineered I-SceI induced HDR in human cells. When the re-engineered I-SceI was fused to TALE (TALE-ISVB2), the chimeric endonuclease induced the same HDR rate at the human β-globin gene locus as that induced by TALEN, but significantly reduced genotoxicity. We further demonstrated that TALE-ISVB2 specifically targeted at the β-globin sequence in human hematopoietic stem cells. Therefore, this monomeric endonuclease has the potential to be used in therapeutic gene targeting in human cells. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. Proximal Region of the Gene Encoding Cytadherence-Related Protein Permits Molecular Typing of Mycoplasma genitalium Clinical Strains by PCR-Restriction Fragment Length Polymorphism

    Science.gov (United States)

    Musatovova, Oxana; Herrera, Caleb; Baseman, Joel B.

    2006-01-01

    Restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified proximal region of the gene encoding cytadherence accessory protein P110 (MG192) revealed DNA sequence divergences among 54 Mycoplasma genitalium clinical strains isolated from the genitourinary tracts of women attending a sexually transmitted disease-related health clinic, plus one from the respiratory tract and one from synovial fluid. Seven of 56 (12.5%) strains exhibited RFLPs following digestion of the proximal region with restriction endonuclease MboI or RsaI, or both. No sequence variability was detected in the distal portion of the gene. PMID:16455921

  12. Comparison of the cleavage of pyrimidine dimers by the bacteriophage T4 and Micrococcus luteus uv-specific endonucleases

    International Nuclear Information System (INIS)

    Gordon, L.K.; Haseltine, W.A.

    1980-01-01

    A comparison was made of the activity of the uv-specific endonucleases of bacteriophage T4 (T4 endonuclease V) and of Micrococcus luteus on ultraviolet light-irradiated DNA substrates of defined sequence. The two enzyms cleave DNA at the site of pyrimidine dimers with the same frequency. The products of the cleavage reaction are the same. The pyrimidine dimer DNA-glycosylase activity of both enzymes is more active on double-stranded DNA than it is on single-stranded DNA

  13. Polyphosphate present in DNA preparations from fungal species of Collectotrichum inhibits restriction endonucleases and other enzymes

    Science.gov (United States)

    Rodriguez, R.J.

    1993-01-01

    During the development of a procedure for the isolation of total genomic DNA from filamentous fungi (Rodriguez, R. J., and Yoder, 0. C., Exp. Mycol. 15, 232-242, 1991) a cell fraction was isolated which inhibited the digestion of DNA by restriction enzymes. After elimination of DNA, RNA, proteins, and lipids, the active compound was purified by gel filtration to yield a single fraction capable of complete inhibition of restriction enzyme activity. The inhibitor did not absorb uv light above 220 nm, and was resistant to alkali and acid at 25°C and to temperatures as high as 100°C. More extensive analyses demonstrated that the inhibitor was also capable of inhibiting T4 DNA ligase and TaqI DNA polymerase, but not DNase or RNase. Chemical analyses indicated that the inhibitor was devoid of carbohydrates, proteins, lipids, and nucleic acids but rich in phosphorus. A combination of nuclear magnetic resonance, metachromatic shift of toluidine blue, and gel filtration indicated that the inhibitor was a polyphosphate (polyP) containing approximately 60 phosphate molecules. The mechanism of inhibition appeared to involve complexing of polyP to the enzymatic proteins. All species of Colletotrichum analyzed produced polyP equivalent in chain length and concentration. A modification to the original DNA extraction procedure is described which eliminates polyP and reduces the time necessary to obtain DNA of sufficient purity for restriction enzyme digestion and TaqI polymerase amplification.

  14. Cell-Autonomous Progeroid Changes in Conditional Mouse Models for Repair Endonuclease XPG Deficiency

    NARCIS (Netherlands)

    S. Barnhoorn (Sander); L.M. Uittenboogaard (Lieneke); D. Jaarsma (Dick); W.P. Vermeij (Wilbert); M. Tresini (Maria); M. Weymaere (Michael); H. Menoni (Hervé); R.M.C. Brandt (Renata); M.C. de Waard (Monique); S.M. Botter (Sander); A.H. Sarker (Altraf); N.G.J. Jaspers (Nicolaas); G.T.J. van der Horst (Gijsbertus); P.K. Cooper (Priscilla K.); J.H.J. Hoeijmakers (Jan); I. van der Pluijm (Ingrid)

    2014-01-01

    textabstractAs part of the Nucleotide Excision Repair (NER) process, the endonuclease XPG is involved in repair of helix-distorting DNA lesions, but the protein has also been implicated in several other DNA repair systems, complicating genotype-phenotype relationship in XPG patients. Defects in XPG

  15. An analysis of the repair processes in ultraviolet-irradiated Micrococcus luteus using purified ultraviolet-endonuclease

    International Nuclear Information System (INIS)

    Tomilin, N.V.; Zherebtsov, S.V.

    1982-01-01

    The measurement of the frequency of endonucleolytic incisions in ultraviolet-irradiated DNA serves as the test for the presence of pyrimidine dimers. In accordance with this approach, the lysates of three Micrococcus luteus strains containing radioactively labeled chromosomes were treated with purified M. luteus ultraviolet-endonuclease to trace segregation of dimers amongst parental and newly synthesized DNA and their removal during postreplication and excision DNA repair. A considerable proportion of the dimers in all strains tested proved to be insensitive to the action of exogenous incising enzyme. The use of chloramphenicol as an inhibitor of postirradiation protein synthesis in combination with ultraviolet-endonuclease treatment of DNA allowed to reveal at least two alternative pathways of postreplication repair: constitutively active recombinational pathway and inducible nonrecombinational one. (Auth.)

  16. A detailed experimental study of a DNA computer with two endonucleases.

    Science.gov (United States)

    Sakowski, Sebastian; Krasiński, Tadeusz; Sarnik, Joanna; Blasiak, Janusz; Waldmajer, Jacek; Poplawski, Tomasz

    2017-07-14

    Great advances in biotechnology have allowed the construction of a computer from DNA. One of the proposed solutions is a biomolecular finite automaton, a simple two-state DNA computer without memory, which was presented by Ehud Shapiro's group at the Weizmann Institute of Science. The main problem with this computer, in which biomolecules carry out logical operations, is its complexity - increasing the number of states of biomolecular automata. In this study, we constructed (in laboratory conditions) a six-state DNA computer that uses two endonucleases (e.g. AcuI and BbvI) and a ligase. We have presented a detailed experimental verification of its feasibility. We described the effect of the number of states, the length of input data, and the nondeterminism on the computing process. We also tested different automata (with three, four, and six states) running on various accepted input words of different lengths such as ab, aab, aaab, ababa, and of an unaccepted word ba. Moreover, this article presents the reaction optimization and the methods of eliminating certain biochemical problems occurring in the implementation of a biomolecular DNA automaton based on two endonucleases.

  17. Endonuclease G is a novel determinant of cardiac hypertrophy and mitochondrial function

    Czech Academy of Sciences Publication Activity Database

    McDermott-Roe, Ch.; Ye, J.; Ahmed, R.; Sun, X. M.; Serafín, A.; Ware, J.; Bottolo, L.; Muckett, P.; Caňas, X.; Zhang, J.; Rowe, G. C.; Buchan, R.; Lu, H.; Braithwaite, A.; Mancini, M.; Hauton, D.; Martí, R.; García-Arumí, E.; Hubner, N.; Jacob, H.; Serikawa, T.; Zídek, Václav; Papoušek, František; Kolář, František; Cardona, M.; Ruiz-Meana, M.; García-Dorado, D.; Comella, J. X.; Felkin, L. E.; Barton, P. J. R.; Arany, Z.; Pravenec, Michal; Petretto, E.; Sanchis, D.; Cook, S.A.

    2011-01-01

    Roč. 478, č. 7367 (2011), s. 114-118 ISSN 0028-0836 R&D Projects: GA MŠk(CZ) 1M0520; GA ČR(CZ) GA301/08/0166 Institutional research plan: CEZ:AV0Z50110509 Keywords : left ventricular hypertrophy * endonuclease G * mitochondrial dysfunction Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 36.280, year: 2011

  18. Human RECQL5beta stimulates flap endonuclease 1

    DEFF Research Database (Denmark)

    Speina, Elzbieta; Dawut, Lale; Hedayati, Mohammad

    2010-01-01

    devoid of RECQL1 and RECQL5 display increased chromosomal instability. Here, we report the physical and functional interaction of the large isomer of RECQL5, RECQL5beta, with the human flap endonuclease 1, FEN1, which plays a critical role in DNA replication, recombination and repair. RECQL5beta...... dramatically stimulates the rate of FEN1 cleavage of flap DNA substrates. Moreover, we show that RECQL5beta and FEN1 interact physically and co-localize in the nucleus in response to DNA damage. Our findings, together with the previous literature on WRN, BLM and RECQL4's stimulation of FEN1, suggests...

  19. Biological significance of facilitated diffusion in protein-DNA interactions. Applications to T4 endonuclease V-initiated DNA repair

    International Nuclear Information System (INIS)

    Dowd, D.R.; Lloyd, R.S.

    1990-01-01

    Facilitated diffusion along nontarget DNA is employed by numerous DNA-interactive proteins to locate specific targets. Until now, the biological significance of DNA scanning has remained elusive. T4 endonuclease V is a DNA repair enzyme which scans nontarget DNA and processively incises DNA at the site of pyrimidine dimers which are produced by exposure to ultraviolet (UV) light. In this study we tested the hypothesis that there exists a direct correlation between the degree of processivity of wild type and mutant endonuclease V molecules and the degree of enhanced UV resistance which is conferred to repair-deficient Eshcerichia coli. This was accomplished by first creating a series of endonuclease V mutants whose in vitro catalytic activities were shown to be very similar to that of the wild type enzyme. However, when the mechanisms by which these enzymes search nontarget DNA for its substrate were analyzed in vitro and in vivo, the mutants displayed varying degrees of nontarget DNA scanning ranging from being nearly as processive as wild type to randomly incising dimers within the DNA population. The ability of these altered endonuclease V molecules to enhance UV survival in DNA repair-deficient E. coli then was assessed. The degree of enhanced UV survival was directly correlated with the level of facilitated diffusion. This is the first conclusive evidence directly relating a reduction of in vivo facilitated diffusion with a change in an observed phenotype. These results support the assertion that the mechanisms which DNA-interactive proteins employ in locating their target sites are of biological significance

  20. Homing endonuclease genes: the rise and fall and rise again of a selfish element.

    Science.gov (United States)

    Burt, Austin; Koufopanou, Vassiliki

    2004-12-01

    Homing endonuclease genes (HEGs) are selfish genetic elements that spread by first cleaving chromosomes that do not contain them and then getting copied across to the broken chromosome as a byproduct of the repair process. The success of this strategy will depend on the opportunities for homing--in other words, the frequency with which HEG(+) and HEG(-) chromosomes come into contact--which varies widely among host taxa. HEGs are also unusual in that the selection pressure for endonuclease function disappears if they become fixed in a population, which makes them susceptible to degeneration and imposes a need for regular horizontal transmission between species. HEGs will be selected to reduce the harm done to the host organism, and this is expected to influence the evolution of their sequence specificity and maturase functions. HEGs may also be domesticated by their hosts, and are currently being put to human uses.

  1. Molecular dynamics simulations of deoxyribonucleic acids and repair enzyme T4 endonuclease V

    International Nuclear Information System (INIS)

    Pinak, Miroslav

    1999-01-01

    This report describes the results of molecular dynamics (MD) simulation of deoxyribonucleic acids (DNA) and specific repair enzyme T4 endonuclease V. Namely research described here is focused on the examination of specific recognition process, in which this repair enzyme recognizes the damaged site on the DNA molecule-thymine dimer (TD). TD is frequent DNA damage induced by UV radiation in sun light and unless properly repaired it may be mutagenic or lethal for cell, and is also considered among the major causes of skin cancer. T4 endonuclease V is a DNA specific repair enzyme from bacteriophage T4 that catalyzes the first reaction step of TD repair pathway. MD simulations of three molecules - native DNA dodecamer (12 base pairs), DNA of the same sequence of nucleotides as native one but with TD, and repair enzyme T4 endonuclease V - were performed for 1 ns individually for each molecule. Simulations were analyzed to determine the role of electrostatic interaction in the recognition process. It is found that electrostatic energies calculated for amino acids of the enzyme have positive values of around +15 kcal/mol. The electrostatic energy of TD site has negative value of approximately -9 kcal/mol, different from the nearly neutral value of the respective thymines site of the native DNA. The electrostatic interaction of TD site with surrounding water environment differs from the electrostatic interaction of other nucleotides. Differences found between TD site and respective thymines site of native DNA indicate that the electrostatic energy is an important factor contributing to proper recognition of TD site during scanning process in which enzyme scans the DNA. In addition to the electrostatic energy, the important factor in recognition process might be structural complementarity of enzyme and bent DNA with TD. There is significant kink formed around TD site, that is not observed in native DNA. (author)

  2. RPA activates the XPF‐ERCC1 endonuclease to initiate processing of DNA interstrand crosslinks

    KAUST Repository

    Abdullah, Ummi B

    2017-06-13

    During replication‐coupled DNA interstrand crosslink (ICL) repair, the XPF‐ERCC1 endonuclease is required for the incisions that release, or “unhook”, ICLs, but the mechanism of ICL unhooking remains largely unknown. Incisions are triggered when the nascent leading strand of a replication fork strikes the ICL. Here, we report that while purified XPF‐ERCC1 incises simple ICL‐containing model replication fork structures, the presence of a nascent leading strand, modelling the effects of replication arrest, inhibits this activity. Strikingly, the addition of the single‐stranded DNA (ssDNA)‐binding replication protein A (RPA) selectively restores XPF‐ERCC1 endonuclease activity on this structure. The 5′–3′ exonuclease SNM1A can load from the XPF‐ERCC1‐RPA‐induced incisions and digest past the crosslink to quantitatively complete the unhooking reaction. We postulate that these collaborative activities of XPF‐ERCC1, RPA and SNM1A might explain how ICL unhooking is achieved in vivo.

  3. RPA activates the XPF‐ERCC1 endonuclease to initiate processing of DNA interstrand crosslinks

    KAUST Repository

    Abdullah, Ummi B; McGouran, Joanna F; Brolih, Sanja; Ptchelkine, Denis; El‐Sagheer, Afaf H; Brown, Tom; McHugh, Peter J

    2017-01-01

    During replication‐coupled DNA interstrand crosslink (ICL) repair, the XPF‐ERCC1 endonuclease is required for the incisions that release, or “unhook”, ICLs, but the mechanism of ICL unhooking remains largely unknown. Incisions are triggered when the nascent leading strand of a replication fork strikes the ICL. Here, we report that while purified XPF‐ERCC1 incises simple ICL‐containing model replication fork structures, the presence of a nascent leading strand, modelling the effects of replication arrest, inhibits this activity. Strikingly, the addition of the single‐stranded DNA (ssDNA)‐binding replication protein A (RPA) selectively restores XPF‐ERCC1 endonuclease activity on this structure. The 5′–3′ exonuclease SNM1A can load from the XPF‐ERCC1‐RPA‐induced incisions and digest past the crosslink to quantitatively complete the unhooking reaction. We postulate that these collaborative activities of XPF‐ERCC1, RPA and SNM1A might explain how ICL unhooking is achieved in vivo.

  4. Permeabilization of ultraviolet-irradiated chinese hamster cells with polyethylene glycol and introduction of ultraviolet endonuclease from Micrococcus luteus

    International Nuclear Information System (INIS)

    Yarosh, D.B.; Setlow, R.B.

    1981-01-01

    Chinese hamster V-79 cells were made permeable by treatment with polyethylene glycol and then incubated with a Micrococcus luteus extract containing ultraviolet-specific endonuclease activity. This treatment introduced nicks in irradiated, but not in unirradiated, deoxyribonucleic acid. The nicks remained open for at least 3 h; there was no loss of endonuclease-sensitive sites, and no excision of dimers as measured by chromatography was detected. In addition, there was no increase in ultraviolet resistance in treated cells. This suggests that the absence of a significant amount of excision repair in rodent cells is due to the lack of both incision and excision capacity

  5. Structural studies on metal-containing enzymes: T4 endonuclease VII and D. gigas formate dehydrogenase

    NARCIS (Netherlands)

    Raaijmakers, H.C.A.

    2001-01-01

    Many biological processes require metal ions, and many of these metal-ion functions involve metalloproteins. The metal ions in metalloproteins are often critical to the protein's function, structure, or stability. This thesis focuses on two of these proteins, bacteriophage T4 endonuclease

  6. HLA-DPB1 typing with polymerase chain reaction and restriction fragment length polymorphism technique in Danes

    DEFF Research Database (Denmark)

    Hviid, Thomas Vauvert F.; Madsen, Hans O; Morling, Niels

    1992-01-01

    We have used the polymerase chain reaction (PCR) in combination with the restriction fragment length polymorphism (RFLP) technique for HLA-DBP1 typing. After PCR amplification of the polymorphic second exon of the HLA-DPB1 locus, the PCR product was digested with seven allele-specific restriction...... endonucleases: RsaI, FokI, ApaI, SacI, BstUI, EcoNI, and DdeI, and the DNA fragments were separated by electrophoresis in agarose gels. Altogether, 71 individuals were investigated and 16 different HLA-DPB1 types were observed in 26 different heterozygotic combinations, as well as five possible homozygotes....... Four heterozygotes could not be unequivocally typed with the PCR-RFLP method. The HLA-DPB1 typing results obtained with the PCR-RFLP method were compared with the typing results obtained with PCR allele-specific oligonucleotides (PCR-ASO) in 50 individuals. The results obtained with the two methods...

  7. Mlh1-Mlh3, a Meiotic Crossover and DNA Mismatch Repair Factor, Is a Msh2-Msh3-stimulated Endonuclease*

    Science.gov (United States)

    Rogacheva, Maria V.; Manhart, Carol M.; Chen, Cheng; Guarne, Alba; Surtees, Jennifer; Alani, Eric

    2014-01-01

    Crossing over between homologous chromosomes is initiated in meiotic prophase in most sexually reproducing organisms by the appearance of programmed double strand breaks throughout the genome. In Saccharomyces cerevisiae the double-strand breaks are resected to form three prime single-strand tails that primarily invade complementary sequences in unbroken homologs. These invasion intermediates are converted into double Holliday junctions and then resolved into crossovers that facilitate homolog segregation during Meiosis I. Work in yeast suggests that Msh4-Msh5 stabilizes invasion intermediates and double Holliday junctions, which are resolved into crossovers in steps requiring Sgs1 helicase, Exo1, and a putative endonuclease activity encoded by the DNA mismatch repair factor Mlh1-Mlh3. We purified Mlh1-Mlh3 and showed that it is a metal-dependent and Msh2-Msh3-stimulated endonuclease that makes single-strand breaks in supercoiled DNA. These observations support a direct role for an Mlh1-Mlh3 endonuclease activity in resolving recombination intermediates and in DNA mismatch repair. PMID:24403070

  8. The mitochondrial LSU rRNA group II intron of Ustilago maydis encodes an active homing endonuclease likely involved in intron mobility.

    Directory of Open Access Journals (Sweden)

    Anja Pfeifer

    Full Text Available BACKGROUND: The a2 mating type locus gene lga2 is critical for uniparental mitochondrial DNA inheritance during sexual development of Ustilago maydis. Specifically, the absence of lga2 results in biparental inheritance, along with efficient transfer of intronic regions in the large subunit rRNA gene between parental molecules. However, the underlying role of the predicted LAGLIDADG homing endonuclease gene I-UmaI located within the group II intron LRII1 has remained unresolved. METHODOLOGY/PRINCIPAL FINDINGS: We have investigated the enzymatic activity of I-UmaI in vitro based on expression of a tagged full-length and a naturally occurring mutant derivative, which harbors only the N-terminal LAGLIDADG domain. This confirmed Mg²⁺-dependent endonuclease activity and cleavage at the LRII1 insertion site to generate four base pair extensions with 3' overhangs. Specifically, I-UmaI recognizes an asymmetric DNA sequence with a minimum length of 14 base pairs (5'-GACGGGAAGACCCT-3' and tolerates subtle base pair substitutions within the homing site. Enzymatic analysis of the mutant variant indicated a correlation between the activity in vitro and intron homing. Bioinformatic analyses revealed that putatively functional or former functional I-UmaI homologs are confined to a few members within the Ustilaginales and Agaricales, including the phylogenetically distant species Lentinula edodes, and are linked to group II introns inserted into homologous positions in the LSU rDNA. CONCLUSIONS/SIGNIFICANCE: The present data provide strong evidence that intron homing efficiently operates under conditions of biparental inheritance in U. maydis. Conversely, uniparental inheritance may be critical to restrict the transmission of mobile introns. Bioinformatic analyses suggest that I-UmaI-associated introns have been acquired independently in distant taxa and are more widespread than anticipated from available genomic data.

  9. Selective inhibition by methoxyamine of the apurinic/apyrimidinic endonuclease activity associated with pyrimidine dimer-DNA glycosylases from Micrococcus luteus and bacteriophage T4

    International Nuclear Information System (INIS)

    Liuzzi, M.; Weinfeld, M.; Paterson, M.C.

    1987-01-01

    The UV endonucleases from Micrococcus luteus and bacteriophage T4 possess two catalytic activities specific for the site of cyclobutane pyrimidine dimers in UV-irradiated DNA: a DNA glycosylase that cleaves the 5'-glycosyl bond of the dimerized pyrimidines and an apurinic/apyrimidinic (AP) endonuclease that thereupon incises the phosphodiester bond 3' to the resulting apyrimidinic site. The authors have explored the potential use of methoxyamine, a chemical that reacts at neutral pH with AP sites in DNA, as a selective inhibitor of the AP endonuclease activities residing in the M. luteus and T4 enzymes. The presence of 50 mM methoxyamine during incubation of UV-treated, [ 3 H]thymine-labeled poly(dA) x poly(dT) with either enzyme preparation was found to protect completely the irradiated copolymer from endonucleolytic attack at dimer sites, as assayed by yield of acid-soluble radioactivity. In contrast, the dimer-DNA glycosylase activity of each enzyme remained fully functional, as monitored retrospectively by release of free thymine after either photochemical-(5 kJ/m 2 , 254 nm) or photoenzymic- (Escherichia coli photolyase plus visible light) induced reversal of pyrimidine dimers in the UV-damaged substrate. The data demonstrate that the inhibition of the strand-incision reaction arises because of chemical modification of the AP sites and is not due to inactivation of the enzyme by methoxyamine. The results, combined with earlier findings for 5'-acting AP endonucleases, strongly suggest that methoxyamine is a highly specific inhibitor of virtually all AP endonucleases, irrespective of their modes of action, and may therefore prove useful in a wide variety of DNA repair studies

  10. Spectroelectrochemical insights into structural and redox properties of immobilized endonuclease III and its catalytically inactive mutant

    Science.gov (United States)

    Moe, Elin; Rollo, Filipe; Silveira, Célia M.; Sezer, Murat; Hildebrandt, Peter; Todorovic, Smilja

    2018-01-01

    Endonuclease III is a Fe-S containing bifunctional DNA glycosylase which is involved in the repair of oxidation damaged DNA. Here we employ surface enhanced IR spectroelectrochemistry and electrochemistry to study the enzyme from the highly radiation- and desiccation-resistant bacterium Deinococcus radiodurans (DrEndoIII2). The experiments are designed to shed more light onto specific parameters that are currently proposed to govern damage search and recognition by endonucleases III. We demonstrate that electrostatic interactions required for the redox activation of DrEndoIII2 may result in high electric fields that alter its structural and thermodynamic properties. Analysis of inactive DrEndoIII2 (K132A/D150A double mutant) interacting with undamaged DNA, and the active enzyme interacting with damaged DNA also indicate that the electron transfer is modulated by subtle differences in the protein-DNA complex.

  11. Dominant mutations in S. cerevisiae PMS1 identify the Mlh1-Pms1 endonuclease active site and an exonuclease 1-independent mismatch repair pathway.

    Science.gov (United States)

    Smith, Catherine E; Mendillo, Marc L; Bowen, Nikki; Hombauer, Hans; Campbell, Christopher S; Desai, Arshad; Putnam, Christopher D; Kolodner, Richard D

    2013-10-01

    Lynch syndrome (hereditary nonpolypsis colorectal cancer or HNPCC) is a common cancer predisposition syndrome. Predisposition to cancer in this syndrome results from increased accumulation of mutations due to defective mismatch repair (MMR) caused by a mutation in one of the mismatch repair genes MLH1, MSH2, MSH6 or PMS2/scPMS1. To better understand the function of Mlh1-Pms1 in MMR, we used Saccharomyces cerevisiae to identify six pms1 mutations (pms1-G683E, pms1-C817R, pms1-C848S, pms1-H850R, pms1-H703A and pms1-E707A) that were weakly dominant in wild-type cells, which surprisingly caused a strong MMR defect when present on low copy plasmids in an exo1Δ mutant. Molecular modeling showed these mutations caused amino acid substitutions in the metal coordination pocket of the Pms1 endonuclease active site and biochemical studies showed that they inactivated the endonuclease activity. This model of Mlh1-Pms1 suggested that the Mlh1-FERC motif contributes to the endonuclease active site. Consistent with this, the mlh1-E767stp mutation caused both MMR and endonuclease defects similar to those caused by the dominant pms1 mutations whereas mutations affecting the predicted metal coordinating residue Mlh1-C769 had no effect. These studies establish that the Mlh1-Pms1 endonuclease is required for MMR in a previously uncharacterized Exo1-independent MMR pathway.

  12. Dominant mutations in S. cerevisiae PMS1 identify the Mlh1-Pms1 endonuclease active site and an exonuclease 1-independent mismatch repair pathway.

    Directory of Open Access Journals (Sweden)

    Catherine E Smith

    2013-10-01

    Full Text Available Lynch syndrome (hereditary nonpolypsis colorectal cancer or HNPCC is a common cancer predisposition syndrome. Predisposition to cancer in this syndrome results from increased accumulation of mutations due to defective mismatch repair (MMR caused by a mutation in one of the mismatch repair genes MLH1, MSH2, MSH6 or PMS2/scPMS1. To better understand the function of Mlh1-Pms1 in MMR, we used Saccharomyces cerevisiae to identify six pms1 mutations (pms1-G683E, pms1-C817R, pms1-C848S, pms1-H850R, pms1-H703A and pms1-E707A that were weakly dominant in wild-type cells, which surprisingly caused a strong MMR defect when present on low copy plasmids in an exo1Δ mutant. Molecular modeling showed these mutations caused amino acid substitutions in the metal coordination pocket of the Pms1 endonuclease active site and biochemical studies showed that they inactivated the endonuclease activity. This model of Mlh1-Pms1 suggested that the Mlh1-FERC motif contributes to the endonuclease active site. Consistent with this, the mlh1-E767stp mutation caused both MMR and endonuclease defects similar to those caused by the dominant pms1 mutations whereas mutations affecting the predicted metal coordinating residue Mlh1-C769 had no effect. These studies establish that the Mlh1-Pms1 endonuclease is required for MMR in a previously uncharacterized Exo1-independent MMR pathway.

  13. Polymerase-endonuclease amplification reaction (PEAR for large-scale enzymatic production of antisense oligonucleotides.

    Directory of Open Access Journals (Sweden)

    Xiaolong Wang

    Full Text Available Antisense oligonucleotides targeting microRNAs or their mRNA targets prove to be powerful tools for molecular biology research and may eventually emerge as new therapeutic agents. Synthetic oligonucleotides are often contaminated with highly homologous failure sequences. Synthesis of a certain oligonucleotide is difficult to scale up because it requires expensive equipment, hazardous chemicals and a tedious purification process. Here we report a novel thermocyclic reaction, polymerase-endonuclease amplification reaction (PEAR, for the amplification of oligonucleotides. A target oligonucleotide and a tandem repeated antisense probe are subjected to repeated cycles of denaturing, annealing, elongation and cleaving, in which thermostable DNA polymerase elongation and strand slipping generate duplex tandem repeats, and thermostable endonuclease (PspGI cleavage releases monomeric duplex oligonucleotides. Each round of PEAR achieves over 100-fold amplification. The product can be used in one more round of PEAR directly, and the process can be further repeated. In addition to avoiding dangerous materials and improved product purity, this reaction is easy to scale up and amenable to full automation. PEAR has the potential to be a useful tool for large-scale production of antisense oligonucleotide drugs.

  14. RPA activates the XPF-ERCC1 endonuclease to initiate processing of DNA interstrand crosslinks.

    Science.gov (United States)

    Abdullah, Ummi B; McGouran, Joanna F; Brolih, Sanja; Ptchelkine, Denis; El-Sagheer, Afaf H; Brown, Tom; McHugh, Peter J

    2017-07-14

    During replication-coupled DNA interstrand crosslink (ICL) repair, the XPF-ERCC1 endonuclease is required for the incisions that release, or "unhook", ICLs, but the mechanism of ICL unhooking remains largely unknown. Incisions are triggered when the nascent leading strand of a replication fork strikes the ICL Here, we report that while purified XPF-ERCC1 incises simple ICL-containing model replication fork structures, the presence of a nascent leading strand, modelling the effects of replication arrest, inhibits this activity. Strikingly, the addition of the single-stranded DNA (ssDNA)-binding replication protein A (RPA) selectively restores XPF-ERCC1 endonuclease activity on this structure. The 5'-3' exonuclease SNM1A can load from the XPF-ERCC1-RPA-induced incisions and digest past the crosslink to quantitatively complete the unhooking reaction. We postulate that these collaborative activities of XPF-ERCC1, RPA and SNM1A might explain how ICL unhooking is achieved in vivo . © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

  15. Interdomain communication in the endonuclease/motor subunit of type I restriction-modification enzyme EcoR124I

    Czech Academy of Sciences Publication Activity Database

    Sinha, Dhiraj; Shamayeva, Katerina; Ramasubramani, V.; Řeha, David; Bialevich, V.; Khabiri, Morteza; Guzanová, Alena; Milbar, N.; Weiserová, Marie; Cséfalvay, Eva; Carey, J.; Ettrich, Rüdiger

    2014-01-01

    Roč. 20, č. 7 (2014), s. 2334 ISSN 1610-2940 R&D Projects: GA ČR GAP207/12/2323 Institutional support: RVO:67179843 ; RVO:61388971 Keywords : DNA restriction enzymes * Molecular modeling * QM/MM calculations * principal components analysis * E. coli * Multisubunit enzyme complex * Correlated loop motions Subject RIV: EH - Ecology, Behaviour; EE - Microbiology, Virology (MBU-M) Impact factor: 1.736, year: 2014

  16. Isolation and properties of the acid site-specific endonuclease from mature eggs of the sea urchin Strongylocentrotus intermedius

    International Nuclear Information System (INIS)

    Sibirtsev, Yu.T.; Konechnyi, A.A.; Rasskazov, V.A.

    1986-01-01

    An acid site-specific endonuclease has been detected in mature sea urchin eggs and cells of embryos at early stages of differentiation. Fractionation with ammonium sulfate, followed by chromatography on columns with DEAE, phosphocellulose, and hydroxyapatite resulted in an 18,000-fold purification. The molecular weight of the enzyme was determined at ∼ 29,000, the optimum pH 5.5. The activity of the enzyme does not depend on divalent metal ions, EDTA, ATP, and tRNA, but it is modulated to a substantial degree by NaCl. The maximum rate of cleavage of the DNA supercoil (form I) is observed at 100 mM NaCl. Increasing the NaCl concentration to 350 mM only slightly lowers the rate of cleavage of form I, yielding form II, but entirely suppresses the accumulation of form III. Restriction analysis of the products of enzymatic hydrolysis of Co1E1 and pBR322 DNA showed that at the early stages of hydrolysis the enzyme exhibits pronounced specificity for definite sites, the number of which is 12 for Co1 E1 DNA and 8 sites for pBR322 DNA

  17. Ultraviolet-endonuclease activity in cell extracts of Saccharomyces cerevisiae mutants defective in excision of pyrimidine dimers

    International Nuclear Information System (INIS)

    Bekker, M.L.; Kaboev, O.K.; Akhmedov, A.T.; Luchkina, L.A.

    1980-01-01

    Cell-free extracts of ultraviolet-sensitive mutants of Saccharomyces cerevisiae defective in excision of pyrimidine dimers, rad1, rad2, rad3, rad4, rad10, and rad16, as well as the extracts of the wild-type strain RAD+, display ultraviolet-endonuclease activity

  18. The structure-specific endonuclease Ercc1-Xpf is required for targeted gene replacement in embryonic stem cells

    NARCIS (Netherlands)

    L.J. Niedernhofer (Laura); J. Essers (Jeroen); G. Weeda (Geert); H.B. Beverloo (Berna); J. de Wit (Jan); M. Muijtjens (Manja); H. Odijk (Hanny); J.H.J. Hoeijmakers (Jan); R. Kanaar (Roland)

    2001-01-01

    textabstractThe Ercc1-Xpf heterodimer, a highly conserved structure-specific endonuclease, functions in multiple DNA repair pathways that are pivotal for maintaining genome stability, including nucleotide excision repair, interstrand crosslink repair and homologous recombination. Erccl-Xpf incises

  19. Activation of Saccharomyces cerevisiae Mlh1-Pms1 Endonuclease in a Reconstituted Mismatch Repair System*

    Science.gov (United States)

    Smith, Catherine E.; Bowen, Nikki; Graham, William J.; Goellner, Eva M.; Srivatsan, Anjana; Kolodner, Richard D.

    2015-01-01

    Previous studies reported the reconstitution of an Mlh1-Pms1-independent 5′ nick-directed mismatch repair (MMR) reaction using Saccharomyces cerevisiae proteins. Here we describe the reconstitution of a mispair-dependent Mlh1-Pms1 endonuclease activation reaction requiring Msh2-Msh6 (or Msh2-Msh3), proliferating cell nuclear antigen (PCNA), and replication factor C (RFC) and a reconstituted Mlh1-Pms1-dependent 3′ nick-directed MMR reaction requiring Msh2-Msh6 (or Msh2-Msh3), exonuclease 1 (Exo1), replication protein A (RPA), RFC, PCNA, and DNA polymerase δ. Both reactions required Mg2+ and Mn2+ for optimal activity. The MMR reaction also required two reaction stages in which the first stage required incubation of Mlh1-Pms1 with substrate DNA, with or without Msh2-Msh6 (or Msh2-Msh3), PCNA, and RFC but did not require nicking of the substrate, followed by a second stage in which other proteins were added. Analysis of different mutant proteins demonstrated that both reactions required a functional Mlh1-Pms1 endonuclease active site, as well as mispair recognition and Mlh1-Pms1 recruitment by Msh2-Msh6 but not sliding clamp formation. Mutant Mlh1-Pms1 and PCNA proteins that were defective for Exo1-independent but not Exo1-dependent MMR in vivo were partially defective in the Mlh1-Pms1 endonuclease and MMR reactions, suggesting that both reactions reflect the activation of Mlh1-Pms1 seen in Exo1-independent MMR in vivo. The availability of this reconstituted MMR reaction should now make it possible to better study both Exo1-independent and Exo1-dependent MMR. PMID:26170454

  20. Activation of Saccharomyces cerevisiae Mlh1-Pms1 Endonuclease in a Reconstituted Mismatch Repair System.

    Science.gov (United States)

    Smith, Catherine E; Bowen, Nikki; Graham, William J; Goellner, Eva M; Srivatsan, Anjana; Kolodner, Richard D

    2015-08-28

    Previous studies reported the reconstitution of an Mlh1-Pms1-independent 5' nick-directed mismatch repair (MMR) reaction using Saccharomyces cerevisiae proteins. Here we describe the reconstitution of a mispair-dependent Mlh1-Pms1 endonuclease activation reaction requiring Msh2-Msh6 (or Msh2-Msh3), proliferating cell nuclear antigen (PCNA), and replication factor C (RFC) and a reconstituted Mlh1-Pms1-dependent 3' nick-directed MMR reaction requiring Msh2-Msh6 (or Msh2-Msh3), exonuclease 1 (Exo1), replication protein A (RPA), RFC, PCNA, and DNA polymerase δ. Both reactions required Mg(2+) and Mn(2+) for optimal activity. The MMR reaction also required two reaction stages in which the first stage required incubation of Mlh1-Pms1 with substrate DNA, with or without Msh2-Msh6 (or Msh2-Msh3), PCNA, and RFC but did not require nicking of the substrate, followed by a second stage in which other proteins were added. Analysis of different mutant proteins demonstrated that both reactions required a functional Mlh1-Pms1 endonuclease active site, as well as mispair recognition and Mlh1-Pms1 recruitment by Msh2-Msh6 but not sliding clamp formation. Mutant Mlh1-Pms1 and PCNA proteins that were defective for Exo1-independent but not Exo1-dependent MMR in vivo were partially defective in the Mlh1-Pms1 endonuclease and MMR reactions, suggesting that both reactions reflect the activation of Mlh1-Pms1 seen in Exo1-independent MMR in vivo. The availability of this reconstituted MMR reaction should now make it possible to better study both Exo1-independent and Exo1-dependent MMR. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Purification and characterization of VDE, a site-specific endonuclease from the yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Gimble, F S; Thorner, J

    1993-10-15

    The 119-kDa primary translation product of the VMA1 gene of Saccharomyces cerevisiae undergoes a self-catalyzed rearrangement ("protein splicing") that excises an internal 50-kDa segment of the polypeptide and joins the amino-terminal and carboxyl-terminal segments to generate the 69-kDa subunit of the vacuolar membrane-associated H(+)-ATPase. We have shown previously that the internal segment is a site-specific endonuclease (Gimble, F. S., and Thorner, J. (1992) Nature 357, 301-306). Here we describe methods for the high level expression and purification to near homogeneity of both the authentic VMA1-derived endonuclease (or VDE) from yeast (yield 18%) and a recombinant form of VDE made in bacteria (yield 29%). Detailed characterization of these preparations demonstrated that the yeast-derived and bacterially produced enzymes were indistinguishable, as judged by: (a) behavior during purification; (b) apparent native molecular mass (50 kDa); (c) immunological reactivity; and (d) catalytic properties (specific activity; cleavage site recognition; and optima for pH, temperature, divalent cation and ionic strength). The minimal site required for VDE cleavage was delimited to a 30-base pair sequence within its specific substrate (the VMA1 delta vde allele).

  2. Biochemical characterization of recombinant influenza A polymerase heterotrimer complex: Endonuclease activity and evaluation of inhibitors

    Czech Academy of Sciences Publication Activity Database

    Xing, W.; Barauskas, O.; Kirschberg, T.; Niedziela-Majka, A.; Clarke, M.; Birkuš, Gabriel; Weissburg, P.; Liu, X.; Schultz, B. E.; Sakowicz, R.; Kwon, H. J.; Feng, J. Y.

    2017-01-01

    Roč. 12, č. 8 (2017), č. článku e0181969. E-ISSN 1932-6203 Institutional support: RVO:61388963 Keywords : virus PA endonuclease * respiratory syncytial virus * RNA synthesis Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 2.806, year: 2016 http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0181969

  3. An ultrasensitive colorimeter assay strategy for p53 mutation assisted by nicking endonuclease signal amplification.

    Science.gov (United States)

    Lin, Zhenyu; Yang, Weiqiang; Zhang, Guiyun; Liu, Qida; Qiu, Bin; Cai, Zongwei; Chen, Guonan

    2011-08-28

    A novel catalytic colorimetric assay assisted by nicking endonuclease signal amplification (NESA) was developed. With the signal amplification, the detection limit of the p53 target gene can be as low as 1 pM, which is nearly 5 orders of magnitude lower than that of other previously reported colorimetric DNA detection strategies based on catalytic DNAzyme.

  4. Identification of Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) by using polymerase chain reaction amplification and restriction analysis of the mitochondrial cytochrome b gene.

    Science.gov (United States)

    Carrera, E; García, T; Céspedes, A; González, I; Sanz, B; Hernández, P E; Martín, R

    1998-04-01

    Restriction site analysis of polymerase chain reaction (PCR) products from a conserved region of the cytochrome b gene has been used for the identification of fresh and smoked samples of Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss). Digestion of the 359-bp PCR product with the endonucleases EcoRV and TaqI yielded specific banding patterns for salmon and trout. This genetic marker can be very useful for detecting fraudulent substitution of the cheaper smoked trout for the more expensive smoked salmon.

  5. Identification and characterization of inhibitors of human apurinic/apyrimidinic endonuclease APE1.

    Directory of Open Access Journals (Sweden)

    Anton Simeonov

    2009-06-01

    Full Text Available APE1 is the major nuclease for excising abasic (AP sites and particular 3'-obstructive termini from DNA, and is an integral participant in the base excision repair (BER pathway. BER capacity plays a prominent role in dictating responsiveness to agents that generate oxidative or alkylation DNA damage, as well as certain chain-terminating nucleoside analogs and 5-fluorouracil. We describe within the development of a robust, 1536-well automated screening assay that employs a deoxyoligonucleotide substrate operating in the red-shifted fluorescence spectral region to identify APE1 endonuclease inhibitors. This AP site incision assay was used in a titration-based high-throughput screen of the Library of Pharmacologically Active Compounds (LOPAC(1280, a collection of well-characterized, drug-like molecules representing all major target classes. Prioritized hits were authenticated and characterized via two high-throughput screening assays -- a Thiazole Orange fluorophore-DNA displacement test and an E. coli endonuclease IV counterscreen -- and a conventional, gel-based radiotracer incision assay. The top, validated compounds, i.e. 6-hydroxy-DL-DOPA, Reactive Blue 2 and myricetin, were shown to inhibit AP site cleavage activity of whole cell protein extracts from HEK 293T and HeLa cell lines, and to enhance the cytotoxic and genotoxic potency of the alkylating agent methylmethane sulfonate. The studies herein report on the identification of novel, small molecule APE1-targeted bioactive inhibitor probes, which represent initial chemotypes towards the development of potential pharmaceuticals.

  6. The contribution of DNA apurinic/apyrimidinic endonuclease genotype and smoking habit to Taiwan lung cancer risk.

    Science.gov (United States)

    Chen, Wei-Chun; Tsai, Chia-Wen; Hsia, Te-Chun; Chang, Wen-Shin; Lin, Liang-Yi; Liang, Shinn-Jye; Tu, Chih-Yen; Cheng, Wei-Erh; Chen, Hung-Jen; Wang, Shu-Ming; Bau, da-Tian

    2013-06-01

    To evaluate the association and interaction of genotypic polymorphism the gene for DNA-apurinic/apyrimidinic endonuclease (APEX1) with personal smoking habit and lung cancer risk in Taiwan, the polymorphic variants of APEX1, Asp(148)Glu (rs1130409), were analyzed in association with lung cancer risk, and their joint effect with personal smoking habits on lung cancer susceptibility was discussed. In this hospital-based case-control study, 358 patients with lung cancer and 716 cancer-free controls, frequency-matched by age and sex, were recruited and genotyped by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). The results showed that the percentages of TT, TG and GG APEX1 Asp(148)Glu genotypes were not significantly different at 43.0%, 41.1% and 15.9% in the lung cancer patient group and 39.9%, 46.1% and 14.0% in non-cancer control group, respectively. We further analyzed the genetic-lifestyle effects on lung cancer risk and found the contribution of APEX1 Asp(148)Glu genotypes to lung cancer susceptibility was neither enhanced in the cigarette smokers nor in the non-smokers (p=0.3550 and 0.8019, respectively). Our results provide evidence that the non-synonymous polymorphism of APEX1 Asp(148)Glu may not be directly associated with lung cancer risk, nor enhance the effects of smoking habit on lung cancer development.

  7. Stock discrimination in Great Lakes Walleye using mitochondrial DNA restriction analysis

    International Nuclear Information System (INIS)

    Billington, N.; Hebert, P.D.N.

    1986-01-01

    Over the past two years it has become evident that because of its strict maternal inheritance and rapid rate of evolutionary differentiation, mitochondrial (mt) DNA diversity offers exceptional promise in the discrimination of fish stocks. The current project aims to determine the extent of mt DNA variation among stocks of walleye (Stizostedion vitreum) from the Great Lakes. At this point, mt DNA has been isolated from 68 walleye representing the Thames River stock and a reef breeding stock from western Lake Erie, as well as from individuals of S. canadense, a species which hybridizes with S. vitreum. Mitochondrial DNA was extracted from livers of these fish, purified by CsCl density gradient centrifugation and digested using 20 endonucleases. Polymorphisms were detected with 8 of the enzymes. There was a great deal of variation among fish from both spawning populations, so much so that individual fish could be identified by this technique. No single enzyme allowed discrimination of the two stocks, but restriction pattern variation following Dde I digestion permitted separation of 50% of Lake Erie fish from Thames River stock. Comparison of mt DNA restriction patterns of walleye and sauger showed that two species are easily separable, setting the stage for a more detailed study of hybridization between the taxa

  8. Creation of targeted inversion mutations in plants using an RNA-guided endonuclease

    Institute of Scientific and Technical Information of China (English)

    Congsheng Zhang; Changlin Liu; Jianfeng Weng; Beijiu Cheng; Fang Liu; Xinhai Li; Chuanxiao Xie

    2017-01-01

    Inversions are DNA rearrangements that are essential for plant gene evolution and adaptation to environmental changes. We demonstrate the creation of targeted inversions and previously reported targeted deletion mutations via delivery of a pair of RNA-guided endonucleases (RGENs) of CRISPR/Cas9. The efficiencies of the targeted inversions were 2.6%and 2.2%in the Arabidopsis FLOWERING TIME (AtFT) and TERMINAL FLOWER 1 (AtTFL1) loci, respectively. Thus, we successfully established an approach that can potentially be used to introduce targeted DNA inversions of interest for functional studies and crop improvement.

  9. Single substitution in bacteriophage T4 RNase H alters the ratio between its exo- and endonuclease activities.

    Science.gov (United States)

    Kholod, Natalia; Sivogrivov, Dmitry; Latypov, Oleg; Mayorov, Sergey; Kuznitsyn, Rafail; Kajava, Andrey V; Shlyapnikov, Mikhail; Granovsky, Igor

    2015-11-01

    The article describes substitutions in bacteriophage T4 RNase H which provide so called das-effect. Phage T4 DNA arrest suppression (das) mutations have been described to be capable of partially suppressing the phage DNA arrest phenotype caused by a dysfunction in genes 46 and/or 47 (also known as Mre11/Rad50 complex). Genetic mapping of das13 (one of the das mutations) has shown it to be in the region of the rnh gene encoding RNase H. Here we report that Das13 mutant of RNase H has substitutions of valine 43 and leucine 242 with isoleucines. To investigate the influence of these mutations on RNase H nuclease properties we have designed a novel in vitro assay that allows us to separate and quantify exo- or endonuclease activities of flap endonuclease. The nuclease assay in vitro showed that V43I substitution increased the ratio between exonuclease/endonuclease activities of RNase H whereas L242I substitution did not affect the nuclease activity of RNase H in vitro. However, both mutations were necessary for the full das effect in vivo. Molecular modelling of the nuclease structure suggests that V43I substitution may lead to disposition of H4 helix, responsible for the interaction with the first base pairs of 5'end of branched DNA. These structural changes may affect unwinding of the first base pairs of gapped or nicked DNA generating a short flap and therefore may stabilize the DNA-enzyme complex. L242I substitution did not affect the structure of RNase H and its role in providing das-effect remains unclear. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. PI-PfuI and PI-PfuII, intein-coded homing endonucleases from Pyrococcus furiosus. II. Characterization Of the binding and cleavage abilities by site-directed mutagenesis.

    OpenAIRE

    Komori, K; Ichiyanagi, K; Morikawa, K; Ishino, Y

    1999-01-01

    PI- Pfu I and PI- Pfu II from Pyrococcus furiosus are homing endonucleases, as shown in the accompanying paper. These two endonucleases are produced by protein splicing from the precursor protein including ribonucleotide reductase (RNR). We show here that both enzymes specifically interact with their substrate DNA and distort the DNA strands by 73 degrees and 67 degrees, respectively. They have two copies of the amino acid sequence motif LAGLIDADG, which is present in the majority of homing e...

  11. DNA double-strand break measurement in mammalian cells by pulsed-field gel electrophoresis: an approach using restriction enzymes and gene probing

    International Nuclear Information System (INIS)

    Loebrich, M.; Ikpeme, S.; Kiefer, J.

    1994-01-01

    DNA samples prepared from human SP 3 cells, which had not been exposed to various doses of X-ray, were treated with NotI restriction endonuclease before being run in a contour-clamped homogeneous electrophoresis system. The restriction enzyme cuts the DNA at defined positions delivering DNA sizes which can be resolved by pulsed-field gel electrophoresis (PFGE). In order to investigate only one of the DNA fragments, a human lactoferrin cDNA, pHL-41, was hybridized to the DNA separated by PFGE. As a result, only the DNA fragment which contains the hybridized gene was detected resulting in a one-band pattern. The decrease of this band was found to be exponential with increasing radiation dose. From the slope, a double-strand break induction rate of (6.3±0.7) x 10 -3 /Mbp/Gy was deduced for 80 kV X-rays. (Author)

  12. Selective metal binding to Cys-78 within endonuclease V causes an inhibition of catalytic activities without altering nontarget and target DNA binding

    International Nuclear Information System (INIS)

    Prince, M.A.; Friedman, B.; Gruskin, E.A.; Schrock, R.D. III; Lloyd, R.S.

    1991-01-01

    T4 endonuclease V is a pyrimidine dimer-specific DNA repair enzyme which has been previously shown not to require metal ions for either of its two catalytic activities or its DNA binding function. However, we have investigated whether the single cysteine within the enzyme was able to bind metal salts and influence the various activities of this repair enzyme. A series of metals (Hg2+, Ag+, Cu+) were shown to inactivate both endonuclease Vs pyrimidine dimer-specific DNA glycosylase activity and the subsequent apurinic nicking activity. The binding of metal to endonuclease V did not interfere with nontarget DNA scanning or pyrimidine dimer-specific binding. The Cys-78 codon within the endonuclease V gene was changed by oligonucleotide site-directed mutagenesis to Thr-78 and Ser-78 in order to determine whether the native cysteine was directly involved in the enzyme's DNA catalytic activities and whether the cysteine was primarily responsible for the metal binding. The mutant enzymes were able to confer enhanced ultraviolet light (UV) resistance to DNA repair-deficient Escherichia coli at levels equal to that conferred by the wild type enzyme. The C78T mutant enzyme was purified to homogeneity and shown to be catalytically active on pyrimidine dimer-containing DNA. The catalytic activities of the C78T mutant enzyme were demonstrated to be unaffected by the addition of Hg2+ or Ag+ at concentrations 1000-fold greater than that required to inhibit the wild type enzyme. These data suggest that the cysteine is not required for enzyme activity but that the binding of certain metals to that amino acid block DNA incision by either preventing a conformational change in the enzyme after it has bound to a pyrimidine dimer or sterically interfering with the active site residue's accessibility to the pyrimidine dimer

  13. Apurinic/apyrimidinic endonucleases of Mycobacterium tuberculosis protect against DNA damage but are dispensable for the growth of the pathogen in guinea pigs.

    Science.gov (United States)

    Puri, Rupangi Verma; Reddy, P Vineel; Tyagi, Anil K

    2014-01-01

    In host cells, Mycobacterium tuberculosis encounters an array of reactive molecules capable of damaging its genome. Non-bulky DNA lesions are the most common damages produced on the exposure of the pathogen to reactive species and base excision repair (BER) pathway is involved in the repair of such damage. During BER, apurinic/apyrimidinic (AP) endonuclease enzymes repair the abasic sites that are generated after spontaneous DNA base loss or by the action of DNA glycosylases, which if left unrepaired lead to inhibition of replication and transcription. However, the role of AP endonucleases in imparting protection against DNA damage and in the growth and pathogenesis of M.tuberculosis has not yet been elucidated. To demonstrate the biological significance of these enzymes in M.tuberculosis, it would be desirable to disrupt the relevant genes and evaluate the resulting mutants for their ability to grow in the host and cause disease. In this study, we have generated M.tuberculosis mutants of the base excision repair (BER) system, disrupted in either one (MtbΔend or MtbΔxthA) or both the AP endonucleases (MtbΔendΔxthA). We demonstrate that these genes are crucial for bacteria to withstand alkylation and oxidative stress in vitro. In addition, the mutant disrupted in both the AP endonucleases (MtbΔendΔxthA) exhibited a significant reduction in its ability to survive inside human macrophages. However, infection of guinea pigs with either MtbΔend or MtbΔxthA or MtbΔendΔxthA resulted in the similar bacillary load and pathological damage in the organs as observed in the case of infection with wild-type M.tuberculosis. The implications of these observations are discussed.

  14. Apurinic/apyrimidinic endonucleases of Mycobacterium tuberculosis protect against DNA damage but are dispensable for the growth of the pathogen in guinea pigs.

    Directory of Open Access Journals (Sweden)

    Rupangi Verma Puri

    Full Text Available In host cells, Mycobacterium tuberculosis encounters an array of reactive molecules capable of damaging its genome. Non-bulky DNA lesions are the most common damages produced on the exposure of the pathogen to reactive species and base excision repair (BER pathway is involved in the repair of such damage. During BER, apurinic/apyrimidinic (AP endonuclease enzymes repair the abasic sites that are generated after spontaneous DNA base loss or by the action of DNA glycosylases, which if left unrepaired lead to inhibition of replication and transcription. However, the role of AP endonucleases in imparting protection against DNA damage and in the growth and pathogenesis of M.tuberculosis has not yet been elucidated. To demonstrate the biological significance of these enzymes in M.tuberculosis, it would be desirable to disrupt the relevant genes and evaluate the resulting mutants for their ability to grow in the host and cause disease. In this study, we have generated M.tuberculosis mutants of the base excision repair (BER system, disrupted in either one (MtbΔend or MtbΔxthA or both the AP endonucleases (MtbΔendΔxthA. We demonstrate that these genes are crucial for bacteria to withstand alkylation and oxidative stress in vitro. In addition, the mutant disrupted in both the AP endonucleases (MtbΔendΔxthA exhibited a significant reduction in its ability to survive inside human macrophages. However, infection of guinea pigs with either MtbΔend or MtbΔxthA or MtbΔendΔxthA resulted in the similar bacillary load and pathological damage in the organs as observed in the case of infection with wild-type M.tuberculosis. The implications of these observations are discussed.

  15. A newly discovered Bordetella species carries a transcriptionally active CRISPR-Cas with a small Cas9 endonuclease

    Science.gov (United States)

    The Cas9 endonuclease of the Type II-a clustered regularly interspersed short palindromic repeats (CRISPR), of Streptococcus pyogenes (SpCas9) has been adapted as a widely used tool for genome editing and genome engineering. Herein, we describe a gene encoding a novel Cas9 ortholog (BpsuCas9) and th...

  16. Analysis of Endonuclease R·EcoRI Fragments of DNA from Lambdoid Bacteriophages and Other Viruses by Agarose-Gel Electrophoresis

    Science.gov (United States)

    Helling, Robert B.; Goodman, Howard M.; Boyer, Herbert W.

    1974-01-01

    By means of agarose-gel electrophoresis, endonuclease R·EcoRI-generated fragments of DNA from various viruses were separated, their molecular weights were determined, and complete or partial fragment maps for lambda, φ80, and hybrid phages were constructed. Images PMID:4372397

  17. Key Players in I-DmoI Endonuclease Catalysis Revealed from Structure and Dynamics

    DEFF Research Database (Denmark)

    Molina, Rafael; Besker, Neva; Marcaida, Maria Jose

    2016-01-01

    . The cleavage mechanism was related both to key structural effects, such as the position of water molecules and ions participating in the cleavage reaction, and to dynamical effects related to protein behavior. In particular, we found that the protein perturbation pattern significantly changes between cleaved......Homing endonucleases, such as I-DmoI, specifically recognize and cleave long DNA target sequences (∼20 bp) and are potentially powerful tools for genome manipulation. However, inefficient and off-target DNA cleavage seriously limits specific editing in complex genomes. One approach to overcome...

  18. The use of the hypervariable P8 region of trnL(UAA intron for identification of orchid species: Evidence from restriction site polymorphism analysis.

    Directory of Open Access Journals (Sweden)

    Rajkumar Kishor

    Full Text Available The P8 stem-loop region of the trnL intron, which is known to be hypervariable in size with multiple repeat motifs and created difficulties in alignment, is always excluded in phylogenetic as well as barcode analyses. This region was investigated for species discrimination in 98 taxa of orchids belonging to the tribe Vandeae using in silico mapping of restriction site polymorphism. The length of the P8 regions varied from 200 nucleotides in Aerides rosea to 669 nucleotides in Dendrophylax sallei. Forty two taxa had unique lengths, while as many as eight shared a common length of 521 nucleotides. Of the 35 restriction endonucleases producing digestions in the P8 regions, three, viz., AgsI, ApoI and TspDTI turned out to have recognition sites across all the 98 taxa being studied. When their restriction data were combined, 92 taxa could be discriminated leaving three taxon pairs. However, Acampe papillosa and Aeranthes arachnites despite having similar restriction sites differed in their P8 lengths. This is the first report on thorough investigation of the P8 region of trnL intron for search of species specific restriction sites and hence its use as a potential plant DNA barcode.

  19. Blot hybridization analysis of TCR genes of T cells for five people exposed in a radiation accident

    International Nuclear Information System (INIS)

    Min Rui; Liu Benti; Cheng Tianmin; Yang Rujun; Meng Xiangshun; Xiao Jinsong

    1996-01-01

    Human lymphocyte total DNA was prepared in agarose plug by mixing cells with low melting agarose, and two restriction endonucleases were used for digestion of the total DNA with human α and β TCR cDNA probes. The total digested DNA from five people who were whole body exposed to 2.0-2.5 Gy ionizing radiation in an accident 4.5 years ago was hybridized by Southern blot method. The results showed that no obvious difference in hybridization bands was found between controls and the five victims when hybridizations were fulfilled in the total DNA which was digested by Hind III restriction endonuclease with both α and β probes. However, when the total DNA was digested with restriction endonuclease EcoR I and was hybridized with TCR α probe, four of the five exposed people showed a different hybridizing band pattern compared with the controls. The results are also discussed

  20. Microinjection of Micrococcus luteus UV-endonuclease restores UV-induced unscheduled DNA synthesis in cells of 9 xeroderma pigmentosum complementation groups.

    NARCIS (Netherlands)

    A.J.R. de Jonge; W. Vermeulen (Wim); W. Keijzer; J.H.J. Hoeijmakers (Jan); D. Bootsma (Dirk)

    1985-01-01

    textabstractThe UV-induced unscheduled DNA synthesis (UDS) in cultured cells of excision-deficient xeroderma pigmentosum (XP) complementation groups A through I was assayed after injection of Micrococcus luteus UV-endonuclease using glass microneedles. In all complementation groups a restoration of

  1. Identification of flap structure-specific endonuclease 1 as a factor involved in long-term memory formation of aversive learning.

    Science.gov (United States)

    Saavedra-Rodríguez, Lorena; Vázquez, Adrinel; Ortiz-Zuazaga, Humberto G; Chorna, Nataliya E; González, Fernando A; Andrés, Lissette; Rodríguez, Karen; Ramírez, Fernando; Rodríguez, Alan; Peña de Ortiz, Sandra

    2009-05-06

    We previously proposed that DNA recombination/repair processes play a role in memory formation. Here, we examined the possible role of the fen-1 gene, encoding a flap structure-specific endonuclease, in memory consolidation of conditioned taste aversion (CTA). Quantitative real-time PCR showed that amygdalar fen-1 mRNA induction was associated to the central processing of the illness experience related to CTA and to CTA itself, but not to the central processing resulting from the presentation of a novel flavor. CTA also increased expression of the Fen-1 protein in the amygdala, but not the insular cortex. In addition, double immunofluorescence analyses showed that amygdalar Fen-1 expression is mostly localized within neurons. Importantly, functional studies demonstrated that amygdalar antisense knockdown of fen-1 expression impaired consolidation, but not short-term memory, of CTA. Overall, these studies define the fen-1 endonuclease as a new DNA recombination/repair factor involved in the formation of long-term memories.

  2. Spectrophotometric, colorimetric and visually detection of Pseudomonas aeruginosa ETA gene based gold nanoparticles DNA probe and endonuclease enzyme

    Science.gov (United States)

    Amini, Bahram; Kamali, Mehdi; Salouti, Mojtaba; Yaghmaei, Parichehreh

    2018-06-01

    Colorimetric DNA detection is preferred over other methods for clinical molecular diagnosis because it does not require expensive equipment. In the present study, the colorimetric method based on gold nanoparticles (GNPs) and endonuclease enzyme was used for the detection of P. aeruginosa ETA gene. Firstly, the primers and probe for P. aeruginosa exotoxin A (ETA) gene were designed and checked for specificity by the PCR method. Then, GNPs were synthesized using the citrate reduction method and conjugated with the prepared probe to develop the new nano-biosensor. Next, the extracted target DNA of the bacteria was added to GNP-probe complex to check its efficacy for P. aeruginosa ETA gene diagnosis. A decrease in absorbance was seen when GNP-probe-target DNA cleaved into the small fragments of BamHI endonuclease due to the weakened electrostatic interaction between GNPs and the shortened DNA. The right shift of the absorbance peak from 530 to 562 nm occurred after adding the endonuclease. It was measured using a UV-VIS absorption spectroscopy that indicates the existence of the P. aeruginosa ETA gene. Sensitivity was determined in the presence of different concentrations of target DNA of P. aeruginosa. The results obtained from the optimized conditions showed that the absorbance value has linear correlation with concentration of target DNA (R: 0.9850) in the range of 10-50 ng mL-1 with the limit detection of 9.899 ng mL-1. Thus, the specificity of the new method for detection of P. aeruginosa was established in comparison with other bacteria. Additionally, the designed assay was quantitatively applied to detect the P. aeruginosa ETA gene from 103 to 108 CFU mL-1 in real samples with a detection limit of 320 CFU mL-1.

  3. Molecular mechanisms involved in the production of chromosomal aberrations. I. Utilization of Neurospora endonuclease for the study of aberration production in G2 stage of the cell cycle

    Energy Technology Data Exchange (ETDEWEB)

    Natarajan, A T; Obe, G [Rijksuniversiteit Leiden (Netherlands). J.A. Cohen Inst. voor Radiopathologie en Stralingsbescherming

    1978-10-01

    Chinese hamster ovary cells (CHO) were X-irradiated in G2 stage of the cell cycle and immediately treated, in the presence of inactivated Sendai virus, with Neurospora endonuclease (E.C. 3.1.4.), an enzyme which is specific for cleaving single-stranded DNA. With this treatment, the frequencies of all types of chromosome aberrations increased when compared to X-irradiated controls. These results are interpreted as due to the conversion of some of the X-ray induced single-stranded DNA breaks into double-strand breaks by this enzyme. Similar enhancement due to this enzyme was found following treatment with methyl methanesulfonate (MMS) and bleomycin, but not following UV and mitomycin C. Addition of Micrococcus endonuclease and Neurospora endonuclease to the cells did not alter the frequencies of aberrations induced by UV. The introduction of enzymes with specific DNA-repair function offers possibilities to probe into the molecular events involved in the formation of structural chromosome aberrations induced by different classes of physical and chemical mutagens.

  4. Specificity of binding to four-way junctions in DNA by bacteriophage T7 endonuclease I.

    OpenAIRE

    Parsons, C A; West, S C

    1990-01-01

    T7 endonuclease I binds specifically to four-way junctions in duplex DNA and promotes their resolution into linear duplexes. Under conditions in which the nuclease activity is blocked by the absence of divalent cations, the enzyme forms a distinct protein-DNA complex with the junction, as detected by gel retardation and filter binding assays. The formation of this complex is structure-specific and contrasts with the short-lived binding complexes formed on linear duplex DNA. The binding comple...

  5. An ultra-sensitive colorimetric Hg(2+)-sensing assay based on DNAzyme-modified Au NP aggregation, MNPs and an endonuclease.

    Science.gov (United States)

    Li, Chao; Dai, Peiqing; Rao, Xinyi; Shao, Lin; Cheng, Guifang; He, Pingang; Fang, Yuzhi

    2015-01-01

    This paper reports the development of an ultra-sensitive colorimetric method for the detection of trace mercury ions involving DNAzymes, Au nanoparticle aggregation, magnetic nanoparticles and an endonuclease. DNAzyme-sensing elements are conjugated to the surface of Au nanoparticle-2, which can crosslink with the T-rich strands coated on Au nanoparticle-1 to form Au nanoparticle aggregation. Other T-rich stands are immobilized on the surface of MNPs. The specific hybridization of these two T-rich strands depends on the presence of Hg(2+), resulting in the formation of a T-Hg(2+)-T structure. Added endonuclease then digests the hybridized strands, and DNAzyme-modified Au NP aggregation is released, catalysing the conversion of the colourless ABTS into a blue-green product by H2O2-mediated oxidation. The increase in the adsorption spectrum of ABTS(+) at 421 nm is related to the concentration of Hg(2+). This assay was validated by detecting mercury ion concentrations in river water. The colorimetric responses were not significantly altered in the presence of 100-fold excesses of other metal ions such as Zn(2+), Pb(2+), Cd(2+), Mn(2+), Ca(2+) and Ni(2+). The inclusion of both Au NP aggregation and an endonuclease enables the assay to eliminate interference from the magnetic nanoparticles with colorimetric detection, decrease the background and improve the detection sensitivity. The calibration curve of the assay was linear over the range of Hg(2+) concentrations from 1 to 30 nM, and the detection limit was 0.8 nM, which is far lower than the 10 nM US EPA limit for drinking water. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. Restriction Cascade Exponential Amplification (RCEA) assay with an attomolar detection limit: a novel, highly specific, isothermal alternative to qPCR.

    Science.gov (United States)

    Ghindilis, Andrey L; Smith, Maria W; Simon, Holly M; Seoudi, Ihab A; Yazvenko, Nina S; Murray, Iain A; Fu, Xiaoqing; Smith, Kenneth; Jen-Jacobson, Linda; Xu, Shuang-Yong

    2015-01-13

    An alternative to qPCR was developed for nucleic acid assays, involving signal rather than target amplification. The new technology, Restriction Cascade Exponential Amplification (RCEA), relies on specific cleavage of probe-target hybrids by restriction endonucleases (REase). Two mutant REases for amplification (Ramp), S17C BamHI and K249C EcoRI, were conjugated to oligonucleotides, and immobilized on a solid surface. The signal generation was based on: (i) hybridization of a target DNA to a Ramp-oligonucleotide probe conjugate, followed by (ii) specific cleavage of the probe-target hybrid using a non-immobilized recognition REase. The amount of Ramp released into solution upon cleavage was proportionate to the DNA target amount. Signal amplification was achieved through catalysis, by the free Ramp, of a restriction cascade containing additional oligonucleotide-conjugated Ramp and horseradish peroxidase (HRP). Colorimetric quantification of free HRP indicated that the RCEA achieved a detection limit of 10 aM (10(-17) M) target concentration, or approximately 200 molecules, comparable to the sensitivity of qPCR-based assays. The RCEA assay had high specificity, it was insensitive to non-specific binding, and detected target sequences in the presence of foreign DNA. RCEA is an inexpensive isothermal assay that allows coupling of the restriction cascade signal amplification with any DNA target of interest.

  7. Restoration of ultraviolet-induced unscheduled DNA synthesis of xeroderma pigmentosum cells by the concomitant treatment with bacteriophage T4 endonuclease V and HVJ (Sendai virus)

    International Nuclear Information System (INIS)

    Tanaka, K.; Sekiguchi, M.; Okada, Y.

    1975-01-01

    Ultraviolet (uv)-induced unscheduled DNA synthesis of xeroderma pigmentosum cells, belonging to complementation groups, A, B, C, D, and E, was restored to the normal level by concomitant treatment of the cells with T4 endonuclease V and uv-inactivated HVJ (Sendai virus). The present results suggest that T4 endonuclease molecules were inserted effectively into the cells by the interaction of HVJ with the cell membranes, the enzyme was functional on human chromosomal DNA which had been damaged by uv irradiation in the viable cells, all the studied groups of xeroderma pigmentosum (variant was not tested) were defective in the first step (incision) of excision repair

  8. A Mismatch EndoNuclease Array-Based Methodology (MENA) for Identifying Known SNPs or Novel Point Mutations.

    Science.gov (United States)

    Comeron, Josep M; Reed, Jordan; Christie, Matthew; Jacobs, Julia S; Dierdorff, Jason; Eberl, Daniel F; Manak, J Robert

    2016-04-05

    Accurate and rapid identification or confirmation of single nucleotide polymorphisms (SNPs), point mutations and other human genomic variation facilitates understanding the genetic basis of disease. We have developed a new methodology (called MENA (Mismatch EndoNuclease Array)) pairing DNA mismatch endonuclease enzymology with tiling microarray hybridization in order to genotype both known point mutations (such as SNPs) as well as identify previously undiscovered point mutations and small indels. We show that our assay can rapidly genotype known SNPs in a human genomic DNA sample with 99% accuracy, in addition to identifying novel point mutations and small indels with a false discovery rate as low as 10%. Our technology provides a platform for a variety of applications, including: (1) genotyping known SNPs as well as confirming newly discovered SNPs from whole genome sequencing analyses; (2) identifying novel point mutations and indels in any genomic region from any organism for which genome sequence information is available; and (3) screening panels of genes associated with particular diseases and disorders in patient samples to identify causative mutations. As a proof of principle for using MENA to discover novel mutations, we report identification of a novel allele of the beethoven (btv) gene in Drosophila, which encodes a ciliary cytoplasmic dynein motor protein important for auditory mechanosensation.

  9. A Novel Tool for Microbial Genome Editing Using the Restriction-Modification System.

    Science.gov (United States)

    Bai, Hua; Deng, Aihua; Liu, Shuwen; Cui, Di; Qiu, Qidi; Wang, Laiyou; Yang, Zhao; Wu, Jie; Shang, Xiuling; Zhang, Yun; Wen, Tingyi

    2018-01-19

    Scarless genetic manipulation of genomes is an essential tool for biological research. The restriction-modification (R-M) system is a defense system in bacteria that protects against invading genomes on the basis of its ability to distinguish foreign DNA from self DNA. Here, we designed an R-M system-mediated genome editing (RMGE) technique for scarless genetic manipulation in different microorganisms. For bacteria with Type IV REase, an RMGE technique using the inducible DNA methyltransferase gene, bceSIIM (RMGE-bceSIIM), as the counter-selection cassette was developed to edit the genome of Escherichia coli. For bacteria without Type IV REase, an RMGE technique based on a restriction endonuclease (RMGE-mcrA) was established in Bacillus subtilis. These techniques were successfully used for gene deletion and replacement with nearly 100% counter-selection efficiencies, which were higher and more stable compared to conventional methods. Furthermore, precise point mutation without limiting sites was achieved in E. coli using RMGE-bceSIIM to introduce a single base mutation of A128C into the rpsL gene. In addition, the RMGE-mcrA technique was applied to delete the CAN1 gene in Saccharomyces cerevisiae DAY414 with 100% counter-selection efficiency. The effectiveness of the RMGE technique in E. coli, B. subtilis, and S. cerevisiae suggests the potential universal usefulness of this technique for microbial genome manipulation.

  10. A putative Type IIS restriction endonuclease GeoICI

    Indian Academy of Sciences (India)

    As opposed to the unstable prototype, which cleaves DNA at 30°C, GeoICI is highly active at elevated temperatures, up to 73°C and over a very wide salt concentration range. Recognition/cleavage sites were determined by: (i) digestion of plasmid and bacteriophage lambda DNA (λ); (ii) cleavage of custom PCR substrates, ...

  11. Random Tagging Genotyping by Sequencing (rtGBS, an Unbiased Approach to Locate Restriction Enzyme Sites across the Target Genome.

    Directory of Open Access Journals (Sweden)

    Elena Hilario

    Full Text Available Genotyping by sequencing (GBS is a restriction enzyme based targeted approach developed to reduce the genome complexity and discover genetic markers when a priori sequence information is unavailable. Sufficient coverage at each locus is essential to distinguish heterozygous from homozygous sites accurately. The number of GBS samples able to be pooled in one sequencing lane is limited by the number of restriction sites present in the genome and the read depth required at each site per sample for accurate calling of single-nucleotide polymorphisms. Loci bias was observed using a slight modification of the Elshire et al.some restriction enzyme sites were represented in higher proportions while others were poorly represented or absent. This bias could be due to the quality of genomic DNA, the endonuclease and ligase reaction efficiency, the distance between restriction sites, the preferential amplification of small library restriction fragments, or bias towards cluster formation of small amplicons during the sequencing process. To overcome these issues, we have developed a GBS method based on randomly tagging genomic DNA (rtGBS. By randomly landing on the genome, we can, with less bias, find restriction sites that are far apart, and undetected by the standard GBS (stdGBS method. The study comprises two types of biological replicates: six different kiwifruit plants and two independent DNA extractions per plant; and three types of technical replicates: four samples of each DNA extraction, stdGBS vs. rtGBS methods, and two independent library amplifications, each sequenced in separate lanes. A statistically significant unbiased distribution of restriction fragment size by rtGBS showed that this method targeted 49% (39,145 of BamH I sites shared with the reference genome, compared to only 14% (11,513 by stdGBS.

  12. Construction of physical and genetic maps of Chlamydia trachomatis serovar L2 by pulsed-field gel electrophoresis

    DEFF Research Database (Denmark)

    Birkelund, Svend; Stephens, RS

    1992-01-01

    We constructed the physical map of Chlamydia trachomatis serovar L2 by using three restriction endonucleases, NotI (GC[GGCCGC), SgrAI (C(A/G)[CCGG(T/G)G), and Sse8387I (CCTGCA[GG), and we analyzed the fragments by pulsed-field gel electrophoresis. A total of 25 restriction endonuclease sites and 13...... genes and/or operons were located on the map. The genome size was determined to be 1,045 kb. Neither highly transcribed chlamydia genes nor developmental cycle-specific genes were clustered on the genome....

  13. Lactobacillus strain diversity based on partial hsp60 gene sequences and design of PCR-restriction fragment length polymorphism assays for species identification and differentiation.

    Science.gov (United States)

    Blaiotta, Giuseppe; Fusco, Vincenzina; Ercolini, Danilo; Aponte, Maria; Pepe, Olimpia; Villani, Francesco

    2008-01-01

    A phylogenetic tree showing diversities among 116 partial (499-bp) Lactobacillus hsp60 (groEL, encoding a 60-kDa heat shock protein) nucleotide sequences was obtained and compared to those previously described for 16S rRNA and tuf gene sequences. The topology of the tree produced in this study showed a Lactobacillus species distribution similar, but not identical, to those previously reported. However, according to the most recent systematic studies, a clear differentiation of 43 single-species clusters was detected/identified among the sequences analyzed. The slightly higher variability of the hsp60 nucleotide sequences than of the 16S rRNA sequences offers better opportunities to design or develop molecular assays allowing identification and differentiation of either distant or very closely related Lactobacillus species. Therefore, our results suggest that hsp60 can be considered an excellent molecular marker for inferring the taxonomy and phylogeny of members of the genus Lactobacillus and that the chosen primers can be used in a simple PCR procedure allowing the direct sequencing of the hsp60 fragments. Moreover, in this study we performed a computer-aided restriction endonuclease analysis of all 499-bp hsp60 partial sequences and we showed that the PCR-restriction fragment length polymorphism (RFLP) patterns obtainable by using both endonucleases AluI and TacI (in separate reactions) can allow identification and differentiation of all 43 Lactobacillus species considered, with the exception of the pair L. plantarum/L. pentosus. However, the latter species can be differentiated by further analysis with Sau3AI or MseI. The hsp60 PCR-RFLP approach was efficiently applied to identify and to differentiate a total of 110 wild Lactobacillus strains (including closely related species, such as L. casei and L. rhamnosus or L. plantarum and L. pentosus) isolated from cheese and dry-fermented sausages.

  14. Restriction enzyme cleavage of ultraviolet-damaged Simian virus 40 and pBR322 DNA

    International Nuclear Information System (INIS)

    Cleaver, J.E.

    1983-01-01

    Cleavage of specific DNA sequences by the restriction enzymes EcoRI, HindIII and TaqI was prevented when the DNA was irradiated with ultraviolet light. Most of the effects were attributed to cyclobutane pyrimidine dimers in the recognition sequences; the effectiveness of irradiation was directly proportional to the number of potential dimer sites in the DNA. Combining EcoRI with dimer-specific endonuclease digestion revealed that pyrimidine dimers blocked cleavage within one base-pair on the strand opposite to the dimer but did not block cleavage three to four base-pairs away on the same strand. These are the probable limits for the range of influence of pyrimidine dimers along the DNA, at least for this enzyme. The effect of irradiation on cleavage by TaqI seemed far greater than expected for the cyclobutane dimer yield, possibly because of effects from photoproducts flanking the tetranucleotide recognition sequence and the effect of non-cyclobutane (6-4)pyrimidine photoproducts involving adjacent T and C bases. (author)

  15. Phage T4 SegB protein is a homing endonuclease required for the preferred inheritance of T4 tRNA gene region occurring in co-infection with a related phage.

    Science.gov (United States)

    Brok-Volchanskaya, Vera S; Kadyrov, Farid A; Sivogrivov, Dmitry E; Kolosov, Peter M; Sokolov, Andrey S; Shlyapnikov, Michael G; Kryukov, Valentine M; Granovsky, Igor E

    2008-04-01

    Homing endonucleases initiate nonreciprocal transfer of DNA segments containing their own genes and the flanking sequences by cleaving the recipient DNA. Bacteriophage T4 segB gene, which is located in a cluster of tRNA genes, encodes a protein of unknown function, homologous to homing endonucleases of the GIY-YIG family. We demonstrate that SegB protein is a site-specific endonuclease, which produces mostly 3' 2-nt protruding ends at its DNA cleavage site. Analysis of SegB cleavage sites suggests that SegB recognizes a 27-bp sequence. It contains 11-bp conserved sequence, which corresponds to a conserved motif of tRNA TpsiC stem-loop, whereas the remainder of the recognition site is rather degenerate. T4-related phages T2L, RB1 and RB3 contain tRNA gene regions that are homologous to that of phage T4 but lack segB gene and several tRNA genes. In co-infections of phages T4 and T2L, segB gene is inherited with nearly 100% of efficiency. The preferred inheritance depends absolutely on the segB gene integrity and is accompanied by the loss of the T2L tRNA gene region markers. We suggest that SegB is a homing endonuclease that functions to ensure spreading of its own gene and the surrounding tRNA genes among T4-related phages.

  16. Complex group-I introns in nuclear SSU rDNA of red and green algae: evidence of homing-endonuclease pseudogenes in the Bangiophyceae

    DEFF Research Database (Denmark)

    Haugen, P; Huss, V A; Nielsen, Henrik

    1999-01-01

    on the complementary strand. A comparison between related group-I introns in the Bangiophyceae revealed homing-endonuclease-like pseudogenes due to frame-shifts and deletions in Porphyra and Bangia. The Scenedesmus and Porphyra introns provide new insights into the evolution and possible novel functions of nuclear...

  17. Translocation, switching and gating: potential roles for ATP in long-range communication on DNA by Type III restriction endonucleases.

    Science.gov (United States)

    Szczelkun, Mark D

    2011-04-01

    To cleave DNA, the Type III RM (restriction-modification) enzymes must communicate the relative orientation of two recognition sequences, which may be separated by many thousands of base pairs. This long-range interaction requires ATP hydrolysis by a helicase domain, and both active (DNA translocation) and passive (DNA sliding) modes of motion along DNA have been proposed. Potential roles for ATP binding and hydrolysis by the helicase domains are discussed, with a focus on bipartite ATPases that act as molecular switches.

  18. Apoptotic DNA Degradation into Oligonucleosomal Fragments, but Not Apoptotic Nuclear Morphology, Relies on a Cytosolic Pool of DFF40/CAD Endonuclease*

    Science.gov (United States)

    Iglesias-Guimarais, Victoria; Gil-Guiñon, Estel; Gabernet, Gisela; García-Belinchón, Mercè; Sánchez-Osuna, María; Casanelles, Elisenda; Comella, Joan X.; Yuste, Victor J.

    2012-01-01

    Apoptotic cell death is characterized by nuclear fragmentation and oligonucleosomal DNA degradation, mediated by the caspase-dependent specific activation of DFF40/CAD endonuclease. Here, we describe how, upon apoptotic stimuli, SK-N-AS human neuroblastoma-derived cells show apoptotic nuclear morphology without displaying concomitant internucleosomal DNA fragmentation. Cytotoxicity afforded after staurosporine treatment is comparable with that obtained in SH-SY5Y cells, which exhibit a complete apoptotic phenotype. SK-N-AS cell death is a caspase-dependent process that can be impaired by the pan-caspase inhibitor q-VD-OPh. The endogenous inhibitor of DFF40/CAD, ICAD, is correctly processed, and dff40/cad cDNA sequence does not reveal mutations altering its amino acid composition. Biochemical approaches show that both SH-SY5Y and SK-N-AS resting cells express comparable levels of DFF40/CAD. However, the endonuclease is poorly expressed in the cytosolic fraction of healthy SK-N-AS cells. Despite this differential subcellular distribution of DFF40/CAD, we find no differences in the subcellular localization of both pro-caspase-3 and ICAD between the analyzed cell lines. After staurosporine treatment, the preferential processing of ICAD in the cytosolic fraction allows the translocation of DFF40/CAD from this fraction to a chromatin-enriched one. Therefore, the low levels of cytosolic DFF40/CAD detected in SK-N-AS cells determine the absence of DNA laddering after staurosporine treatment. In these cells DFF40/CAD cytosolic levels can be restored by the overexpression of their own endonuclease, which is sufficient to make them proficient at degrading their chromatin into oligonucleosome-size fragments after staurosporine treatment. Altogether, the cytosolic levels of DFF40/CAD are determinants in achieving a complete apoptotic phenotype, including oligonucleosomal DNA degradation. PMID:22253444

  19. Distinguishing Heterodera filipjevi and H. avenae using polymerase chain reaction-restriction fragment length polymorphism and cyst morphology.

    Science.gov (United States)

    Yan, Guiping; Smiley, Richard W

    2010-03-01

    The cereal cyst nematodes Heterodera filipjevi and H. avenae impede wheat production in the Pacific Northwest (PNW). Accurate identification of cyst nematode species and awareness of high population density in affected fields are essential for designing effective control measures. Morphological methods for differentiating these species are laborious. These species were differentiated using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) of internal transcribed spacer (ITS)-ribosomal (r)DNA with up to six restriction endonucleases (TaqI, HinfI, PstI, HaeIII, RsaI, and AluI). The method was validated by inspecting underbridge structures of cyst vulval cones. Grid soil sampling of an Oregon field infested by both species revealed that H. filipjevi was present at most of the infested grid sites but mixtures of H. avenae and H. filipjevi also occurred. These procedures also detected and differentiated H. filipjevi and H. avenae in soil samples from nearby fields in Oregon and H. avenae in samples from Idaho and Washington. Intraspecific polymorphism was not observed within H. filipjevi or PNW H. avenae populations based on the ITS-rDNA. However, intraspecific variation was observed between H. avenae populations occurring in the PNW and France. Methods described here will improve detection and identification efficiencies for cereal cyst nematodes in wheat fields.

  20. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9.

    Science.gov (United States)

    Sternberg, Samuel H; Redding, Sy; Jinek, Martin; Greene, Eric C; Doudna, Jennifer A

    2014-03-06

    The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA-DNA base-pairing to target foreign DNA in bacteria. Cas9-guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9-RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9-RNA. Competition assays provide evidence that DNA strand separation and RNA-DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA.

  1. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9

    Science.gov (United States)

    Sternberg, Samuel H.; Redding, Sy; Jinek, Martin; Greene, Eric C.; Doudna, Jennifer A.

    2014-03-01

    The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA-DNA base-pairing to target foreign DNA in bacteria. Cas9-guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9-RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9-RNA. Competition assays provide evidence that DNA strand separation and RNA-DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA.

  2. Germline excision of transgenes in Aedes aegypti by homing endonucleases.

    Science.gov (United States)

    Aryan, Azadeh; Anderson, Michelle A E; Myles, Kevin M; Adelman, Zach N

    2013-01-01

    Aedes (Ae.) aegypti is the primary vector for dengue viruses (serotypes1-4) and chikungunya virus. Homing endonucleases (HEs) are ancient selfish elements that catalyze double-stranded DNA breaks (DSB) in a highly specific manner. In this report, we show that the HEs Y2-I-AniI, I-CreI and I-SceI are all capable of catalyzing the excision of genomic segments from the Ae. aegypti genome in a heritable manner. Y2-I-AniI demonstrated the highest efficiency at two independent genomic targets, with 20-40% of Y2-I-AniI-treated individuals producing offspring that had lost the target transgene. HE-induced DSBs were found to be repaired via the single-strand annealing (SSA) and non-homologous end-joining (NHEJ) pathways in a manner dependent on the availability of direct repeat sequences in the transgene. These results support the development of HE-based gene editing and gene drive strategies in Ae. aegypti, and confirm the utility of HEs in the manipulation and modification of transgenes in this important vector.

  3. Effects of Dimerization of Serratia marcescens Endonuclease on Water Dynamics.

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Chuanying; Beck, Brian W.; Krause, Kurt; Weksberg, Tiffany E.; Pettitt, Bernard M.

    2007-02-15

    The research described in this product was performed in part in the Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by the Department of Energy's Office of Biological and Environmental Research and located at Pacific Northwest National Laboratory. The dynamics and structure of Serratia marcescens endonuclease and its neighboring solvent are investigated by molecular dynamics (MD). Comparisons are made with structural and biochemical experiments. The dimer form is physiologic and functions more processively than the monomer. We previously found a channel formed by connected clusters of waters from the active site to the dimer interface. Here, we show that dimerization clearly changes correlations in the water structure and dynamics in the active site not seen in the monomer. Our results indicate that water at the active sites of the dimer is less affected compared with bulk solvent than in the monomer where it has much slower characteristic relaxation times. Given that water is a required participant in the reaction, this gives a clear advantage to dimerization in the absence of an apparent ability to use both active sites simultaneously.

  4. Computational study of hydration at the TD damaged site of DNA in complex with repair enzyme T4 endonuclease V

    International Nuclear Information System (INIS)

    Pinak, Miroslav

    2000-02-01

    An analysis of the distribution of water around DNA surface focusing on the role of the distribution of water molecules in the proper recognition of damaged site by repair enzyme T4 Endonuclease V was performed. The native DNA dodecamer, dodecamer with the thymine dimer (TD) and complex of DNA and part of repair enzyme T4 Endonuclease V were examined throughout the 500 ps of molecular dynamics simulation. During simulation the number of water molecules close to the DNA atoms and the residence time were calculated. There is an increase in number of water molecules lying in the close vicinity to TD if compared with those lying close to two native thymines (TT). Densely populated area with water molecules around TD is one of the factors detected by enzyme during scanning process. The residence time was found higher for molecule of the complex and the six water molecules were found occupying the stabile positions between the TD and catalytic center close to atoms P, C3' and N3. These molecules originate water mediated hydrogen bond network that contribute to the stability of complex required for the onset of repair process. (author)

  5. Computational study of hydration at the TD damaged site of DNA in complex with repair enzyme T4 endonuclease V

    Energy Technology Data Exchange (ETDEWEB)

    Pinak, Miroslav [Japan Atomic Energy Research Inst., Tokai, Ibaraki (Japan). Tokai Research Establishment

    2000-02-01

    An analysis of the distribution of water around DNA surface focusing on the role of the distribution of water molecules in the proper recognition of damaged site by repair enzyme T4 Endonuclease V was performed. The native DNA dodecamer, dodecamer with the thymine dimer (TD) and complex of DNA and part of repair enzyme T4 Endonuclease V were examined throughout the 500 ps of molecular dynamics simulation. During simulation the number of water molecules close to the DNA atoms and the residence time were calculated. There is an increase in number of water molecules lying in the close vicinity to TD if compared with those lying close to two native thymines (TT). Densely populated area with water molecules around TD is one of the factors detected by enzyme during scanning process. The residence time was found higher for molecule of the complex and the six water molecules were found occupying the stabile positions between the TD and catalytic center close to atoms P, C3' and N3. These molecules originate water mediated hydrogen bond network that contribute to the stability of complex required for the onset of repair process. (author)

  6. Simultaneous and rapid differential diagnosis of Mycoplasma genitalium and Ureaplasma urealyticum based on a polymerase chain reaction-restriction fragment length polymorphism

    Directory of Open Access Journals (Sweden)

    R Mirnejad

    2011-01-01

    Full Text Available Objectives: The aim of this investigation was to simultaneously detect and differentiate Mycoplasma genitalium and Ureaplasma urealyticum in female patients suffering from genital complications by polymerase chain reaction (PCR-restriction fragment length polymorphism (RFLP. Materials and Methods : Genital swabs were taken from 210 patients. They were transported to the laboratory in phosphate-buffered saline. For PCR, samples were analysed with genus-specific MyUu-R and MyUu-F primers. This primer set, which was originally designed in our laboratory, amplified a 465 bp fragment (M. genitalium and a 559 bp fragment (U. urealyticum. Samples containing a band of the expected sizes for the Mycoplasma strains were subjected to digestion with a restriction endonuclease enzyme of TaqI and Cac8I. Results: Of the 210 samples, a total of 100 (47.6% samples were found to be positive for Mycoplasmas (seven M. genitalium isolates, 3.3%; and 89 U. urealyticum isolates, 42.4%, and coinfections with both species were detected in four samples (1.9%. The PCR-RFLP results showed that M. genitalium and U. urealyticum are different by enzyme patterns. Conclusion: PCR-RFLP offers a rapid and easily applicable protocol to simultaneous detection and differentiation of M. genitalium and U. urealyticum from clinical samples when specific primers and restriction enzymes are used.

  7. ALIS-FLP: Amplified ligation selected fragment-length polymorphism method for microbial genotyping

    DEFF Research Database (Denmark)

    Brillowska-Dabrowska, A.; Wianecka, M.; Dabrowski, Slawomir

    2008-01-01

    A DNA fingerprinting method known as ALIS-FLP (amplified ligation selected fragment-length polymorphism) has been developed for selective and specific amplification of restriction fragments from TspRI restriction endonuclease digested genomic DNA. The method is similar to AFLP, but differs...

  8. Molecular analysis of Leptospira spp. isolated from humans by restriction fragment length polymorphism, real-time PCR and pulsed-field gel electrophoresis.

    Science.gov (United States)

    Turk, Nenad; Milas, Zoran; Mojcec, Vesna; Ruzic-Sabljic, Eva; Staresina, Vilim; Stritof, Zrinka; Habus, Josipa; Postic, Daniele

    2009-11-01

    A total of 17 Leptospira clinical strains isolated from humans in Croatia were serologically and genetically analysed. For serovar identification, the microscopic agglutination test (MAT) and pulsed-field gel electrophoresis (PFGE) were used. To identify isolates on genomic species level, PCR-based restriction fragment length polymorphism (RFLP) and real-time PCR were performed. MAT revealed the following serogroup affinities: Grippotyphosa (seven isolates), Icterohaemorrhagiae (eight isolates) and Javanica (two isolates). RFLP of PCR products from a 331-bp-long fragment of rrs (16S rRNA gene) digested with endonucleases MnlI and DdeI and real-time PCR revealed three Leptospira genomic species. Grippotyphosa isolates belonged to Leptospira kirschneri, Icterohaemorrhagiae isolates to Leptospira interrogans and Javanica isolates to Leptospira borgpetersenii. Genomic DNA from 17 leptospiral isolates was digested with NotI and SgrAI restriction enzymes and analysed by PFGE. Results showed that seven isolates have the same binding pattern to serovar Grippotyphosa, eight isolates to serovar Icterohaemorrhagiae and two isolates to serovar Poi. Results demonstrate the diversity of leptospires circulating in Croatia. We point out the usefulness of a combination of PFGE, RFLP and real-time PCR as appropriate molecular methods in molecular analysis of leptospires.

  9. Bacterial CRISPR/Cas DNA endonucleases: A revolutionary technology that could dramatically impact viral research and treatment

    International Nuclear Information System (INIS)

    Kennedy, Edward M.; Cullen, Bryan R.

    2015-01-01

    CRISPR/Cas systems mediate bacterial adaptive immune responses that evolved to protect bacteria from bacteriophage and other horizontally transmitted genetic elements. Several CRISPR/Cas systems exist but the simplest variant, referred to as Type II, has a single effector DNA endonuclease, called Cas9, which is guided to its viral DNA target by two small RNAs, the crRNA and the tracrRNA. Initial efforts to adapt the CRISPR/Cas system for DNA editing in mammalian cells, which focused on the Cas9 protein from Streptococcus pyogenes (Spy), demonstrated that Spy Cas9 can be directed to DNA targets in mammalian cells by tracrRNA:crRNA fusion transcripts called single guide RNAs (sgRNA). Upon binding, Cas9 induces DNA cleavage leading to mutagenesis as a result of error prone non-homologous end joining (NHEJ). Recently, the Spy Cas9 system has been adapted for high throughput screening of genes in human cells for their relevance to a particular phenotype and, more generally, for the targeted inactivation of specific genes, in cell lines and in vivo in a number of model organisms. The latter aim seems likely to be greatly enhanced by the recent development of Cas9 proteins from bacterial species such as Neisseria meningitidis and Staphyloccus aureus that are small enough to be expressed using adeno-associated (AAV)-based vectors that can be readily prepared at very high titers. The evolving Cas9-based DNA editing systems therefore appear likely to not only impact virology by allowing researchers to screen for human genes that affect the replication of pathogenic human viruses of all types but also to derive clonal human cell lines that lack individual gene products that either facilitate or restrict viral replication. Moreover, high titer AAV-based vectors offer the possibility of directly targeting DNA viruses that infect discrete sites in the human body, such as herpes simplex virus and hepatitis B virus, with the hope that the entire population of viral DNA genomes

  10. Bacterial CRISPR/Cas DNA endonucleases: A revolutionary technology that could dramatically impact viral research and treatment

    Energy Technology Data Exchange (ETDEWEB)

    Kennedy, Edward M.; Cullen, Bryan R., E-mail: bryan.cullen@duke.edu

    2015-05-15

    CRISPR/Cas systems mediate bacterial adaptive immune responses that evolved to protect bacteria from bacteriophage and other horizontally transmitted genetic elements. Several CRISPR/Cas systems exist but the simplest variant, referred to as Type II, has a single effector DNA endonuclease, called Cas9, which is guided to its viral DNA target by two small RNAs, the crRNA and the tracrRNA. Initial efforts to adapt the CRISPR/Cas system for DNA editing in mammalian cells, which focused on the Cas9 protein from Streptococcus pyogenes (Spy), demonstrated that Spy Cas9 can be directed to DNA targets in mammalian cells by tracrRNA:crRNA fusion transcripts called single guide RNAs (sgRNA). Upon binding, Cas9 induces DNA cleavage leading to mutagenesis as a result of error prone non-homologous end joining (NHEJ). Recently, the Spy Cas9 system has been adapted for high throughput screening of genes in human cells for their relevance to a particular phenotype and, more generally, for the targeted inactivation of specific genes, in cell lines and in vivo in a number of model organisms. The latter aim seems likely to be greatly enhanced by the recent development of Cas9 proteins from bacterial species such as Neisseria meningitidis and Staphyloccus aureus that are small enough to be expressed using adeno-associated (AAV)-based vectors that can be readily prepared at very high titers. The evolving Cas9-based DNA editing systems therefore appear likely to not only impact virology by allowing researchers to screen for human genes that affect the replication of pathogenic human viruses of all types but also to derive clonal human cell lines that lack individual gene products that either facilitate or restrict viral replication. Moreover, high titer AAV-based vectors offer the possibility of directly targeting DNA viruses that infect discrete sites in the human body, such as herpes simplex virus and hepatitis B virus, with the hope that the entire population of viral DNA genomes

  11. Small-angle X-ray scattering analysis reveals the ATP-bound monomeric state of the ATPase domain from the homodimeric MutL endonuclease, a GHKL phosphotransferase superfamily protein.

    Science.gov (United States)

    Iino, Hitoshi; Hikima, Takaaki; Nishida, Yuya; Yamamoto, Masaki; Kuramitsu, Seiki; Fukui, Kenji

    2015-05-01

    DNA mismatch repair is an excision system that removes mismatched bases chiefly generated by replication errors. In this system, MutL endonucleases direct the excision reaction to the error-containing strand of the duplex by specifically incising the newly synthesized strand. Both bacterial homodimeric and eukaryotic heterodimeric MutL proteins belong to the GHKL ATPase/kinase superfamily that comprises the N-terminal ATPase and C-terminal dimerization regions. Generally, the GHKL proteins show large ATPase cycle-dependent conformational changes, including dimerization-coupled ATP binding of the N-terminal domain. Interestingly, the ATPase domain of human PMS2, a subunit of the MutL heterodimer, binds ATP without dimerization. The monomeric ATP-bound state of the domain has been thought to be characteristic of heterodimeric GHKL proteins. In this study, we characterized the ATP-bound state of the ATPase domain from the Aquifex aeolicus MutL endonuclease, which is a homodimeric GHKL protein unlike the eukaryotic MutL. Gel filtration, dynamic light scattering, and small-angle X-ray scattering analyses clearly showed that the domain binds ATP in a monomeric form despite its homodimeric nature. This indicates that the uncoupling of dimerization and ATP binding is a common feature among bacterial and eukaryotic MutL endonucleases, which we suggest is closely related to the molecular mechanisms underlying mismatch repair.

  12. Genetic typing in a cluster of Legionella pneumophila infections

    NARCIS (Netherlands)

    van Ketel, R. J.; de Wever, B.

    1989-01-01

    Legionella pneumophila strains isolated from six patients, three air-conditioning- and cooling tower-derived strains, and three hot water supply-derived strains were analyzed by three genetic typing methods. The results of the whole-cell DNA restriction endonuclease analysis and the restriction

  13. A putative Type IIS restriction endonuclease GeoICI from ...

    Indian Academy of Sciences (India)

    2016-02-15

    Feb 15, 2016 ... 41(1), 27–38 * Indian Academy of Sciences. 27. Keywords. ... tis (Subang Jaya, Malaysia), DEAE-cellulose and Phosphocel- lulose P11 were from ... conditions at 67.5°C, subsequently the culture was chilled down and centrifuged. ..... influence of ionic strength on GeoICI REase activity. 0.3 μg PCR.

  14. Comparative studies of the endonucleases from two related Xenopus laevis retrotransposons, Tx1L and Tx2L: target site specificity and evolutionary implications.

    Science.gov (United States)

    Christensen, S; Pont-Kingdon, G; Carroll, D

    2000-01-01

    In the genome of the South African frog, Xenopus laevis, there are two complex families of transposable elements, Tx1 and Tx2, that have identical overall structures, but distinct sequences. In each family there are approximately 1500 copies of an apparent DNA-based element (Tx1D and Tx2D). Roughly 10% of these elements in each family are interrupted by a non-LTR retrotransposon (Tx1L and Tx2L). Each retrotransposon is flanked by a 23-bp target duplication of a specific D element sequence. In earlier work, we showed that the endonuclease domain (Tx1L EN) located in the second open reading frame (ORF2) of Tx1L encodes a protein that makes a single-strand cut precisely at the expected site within its target sequence, supporting the idea that Tx1L is a site-specific retrotransposon. In this study, we express the endonuclease domain of Tx2L (Tx2L EN) and compare the target preferences of the two enzymes. Each endonuclease shows some preference for its cognate target, on the order of 5-fold over the non-cognate target. The observed discrimination is not sufficient, however, to explain the observation that no cross-occupancy is observed - that is, L elements of one family have never been found within D elements of the other family. Possible sources of additional specificity are discussed. We also compare two hypotheses regarding the genome duplication event that led to the contemporary pseudotetraploid character of Xenopus laevis in light of the Tx1L and Tx2L data.

  15. Type II restriction endonucleases—a historical perspective and more

    Science.gov (United States)

    Pingoud, Alfred; Wilson, Geoffrey G.; Wende, Wolfgang

    2014-01-01

    This article continues the series of Surveys and Summaries on restriction endonucleases (REases) begun this year in Nucleic Acids Research. Here we discuss ‘Type II’ REases, the kind used for DNA analysis and cloning. We focus on their biochemistry: what they are, what they do, and how they do it. Type II REases are produced by prokaryotes to combat bacteriophages. With extreme accuracy, each recognizes a particular sequence in double-stranded DNA and cleaves at a fixed position within or nearby. The discoveries of these enzymes in the 1970s, and of the uses to which they could be put, have since impacted every corner of the life sciences. They became the enabling tools of molecular biology, genetics and biotechnology, and made analysis at the most fundamental levels routine. Hundreds of different REases have been discovered and are available commercially. Their genes have been cloned, sequenced and overexpressed. Most have been characterized to some extent, but few have been studied in depth. Here, we describe the original discoveries in this field, and the properties of the first Type II REases investigated. We discuss the mechanisms of sequence recognition and catalysis, and the varied oligomeric modes in which Type II REases act. We describe the surprising heterogeneity revealed by comparisons of their sequences and structures. PMID:24878924

  16. Restriction Fragment Length Polymorphisms of Virulence Plasmids in Rhodococcus equi

    Science.gov (United States)

    Takai, Shinji; Shoda, Masato; Sasaki, Yukako; Tsubaki, Shiro; Fortier, Guillaume; Pronost, Stephane; Rahal, Karim; Becu, Teotimo; Begg, Angela; Browning, Glenn; Nicholson, Vivian M.; Prescott, John F.

    1999-01-01

    Virulent Rhodococcus equi, which is a well-known cause of pyogranulomatous pneumonia in foals, possesses a large plasmid encoding virulence-associated 15- to 17-kDa antigens. Foal and soil isolates from five countries—Argentina, Australia, Canada, France, and Japan—were investigated for the presence of 15- to 17-kDa antigens by colony blotting, using the monoclonal antibody 10G5, and the gene coding for 15- to 17-kDa antigens by PCR. Plasmid DNAs extracted from positive isolates were digested with restriction endonucleases BamHI, EcoRI, EcoT22I, and HindIII, and the digestion patterns that resulted divided the plasmids of virulent isolates into five closely related types. Three of the five types had already been reported in Canadian and Japanese isolates, and the two new types had been found in French and Japanese isolates. Therefore, we tentatively designated these five types 85-kb type I (pREAT701), 85-kb type II (a new type), 87-kb type I (EcoRI and BamHI type 2 [V. M. Nicholson and J. F. Prescott, J. Clin. Microbiol. 35:738–740, 1997]), 87-kb type II (a new type), and 90-kb (pREL1) plasmids. The 85-kb type I plasmid was found in isolates from Argentina, Australia, Canada, and France. Plasmid 87-kb type I was isolated in specimens from Argentina, Canada, and France. The 85-kb type II plasmid appeared in isolates from France. On the other hand, plasmids 87-kb type II and 90-kb were found only in isolates from Japan. These results revealed geographic differences in the distribution of the virulence plasmids found in the five countries and suggested that the restriction fragment length polymorphism of virulence plasmids might be useful to elucidate the molecular epidemiology of virulent R. equi in the world. PMID:10488224

  17. Flap Endonuclease 1 Limits Telomere Fragility on the Leading Strand*

    Science.gov (United States)

    Teasley, Daniel C.; Parajuli, Shankar; Nguyen, Mai; Moore, Hayley R.; Alspach, Elise; Lock, Ying Jie; Honaker, Yuchi; Saharia, Abhishek; Piwnica-Worms, Helen; Stewart, Sheila A.

    2015-01-01

    The existence of redundant replication and repair systems that ensure genome stability underscores the importance of faithful DNA replication. Nowhere is this complexity more evident than in challenging DNA templates, including highly repetitive or transcribed sequences. Here, we demonstrate that flap endonuclease 1 (FEN1), a canonical lagging strand DNA replication protein, is required for normal, complete leading strand replication at telomeres. We find that the loss of FEN1 nuclease activity, but not DNA repair activities, results in leading strand-specific telomere fragility. Furthermore, we show that FEN1 depletion-induced telomere fragility is increased by RNA polymerase II inhibition and is rescued by ectopic RNase H1 expression. These data suggest that FEN1 limits leading strand-specific telomere fragility by processing RNA:DNA hybrid/flap intermediates that arise from co-directional collisions occurring between the replisome and RNA polymerase. Our data reveal the first molecular mechanism for leading strand-specific telomere fragility and the first known role for FEN1 in leading strand DNA replication. Because FEN1 mutations have been identified in human cancers, our findings raise the possibility that unresolved RNA:DNA hybrid structures contribute to the genomic instability associated with cancer. PMID:25922071

  18. The DNA repair endonuclease XPG interacts directly and functionally with the WRN helicase defective in Werner syndrome

    Energy Technology Data Exchange (ETDEWEB)

    Trego, Kelly S.; Chernikova, Sophia B.; Davalos, Albert R.; Perry, J. Jefferson P.; Finger, L. David; Ng, Cliff; Tsai, Miaw-Sheue; Yannone, Steven M.; Tainer, John A.; Campisi, Judith; Cooper, Priscilla K.

    2011-04-20

    XPG is a structure-specific endonuclease required for nucleotide excision repair (NER). XPG incision defects result in the cancer-prone syndrome xeroderma pigmentosum, whereas truncating mutations of XPG cause the severe postnatal progeroid developmental disorder Cockayne syndrome. We show that XPG interacts directly with WRN protein, which is defective in the premature aging disorder Werner syndrome, and that the two proteins undergo similar sub-nuclear redistribution in S-phase and co-localize in nuclear foci. The co-localization was observed in mid- to late-S-phase, when WRN moves from nucleoli to nuclear foci that have been shown to contain protein markers of both stalled replication forks and telomeric proteins. We mapped the interaction between XPG and WRN to the C-terminal domains of each and show that interaction with the C-terminal domain of XPG strongly stimulates WRN helicase activity. WRN also possesses a competing DNA single-strand annealing activity that, combined with unwinding, has been shown to coordinate regression of model replication forks to form Holliday junction/chicken foot intermediate structures. We tested whether XPG stimulated WRN annealing activity and found that XPG itself has intrinsic strand annealing activity that requires the unstructured R- and C-terminal domains, but not the conserved catalytic core or endonuclease activity. Annealing by XPG is cooperative, rather than additive, with WRN annealing. Taken together, our results suggest a novel function for XPG in S-phase that is at least in part carried out coordinately with WRN, and which may contribute to the severity of the phenotypes that occur upon loss of XPG.

  19. Structural insights of the ssDNA binding site in the multifunctional endonuclease AtBFN2 from Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Tsung-Fu Yu

    Full Text Available The multi S1/P1 nuclease AtBFN2 (EC 3.1.30.1 encoded by the Arabidopsis thaliana At1g68290 gene is a glycoprotein that digests RNA, ssDNA, and dsDNA. AtBFN2 depends on three zinc ions for cleaving DNA and RNA at 3'-OH to yield 5'-nucleotides. In addition, AtBFN2's enzymatic activity is strongly glycan dependent. Plant Zn(2+-dependent endonucleases present a unique fold, and belong to the Phospholipase C (PLC/P1 nuclease superfamily. In this work, we present the first complete, ligand-free, AtBFN2 crystal structure, along with sulfate, phosphate and ssDNA co-crystal structures. With these, we were able to provide better insight into the glycan structure and possible enzymatic mechanism. In comparison with other nucleases, the AtBFN2/ligand-free and AtBFN2/PO4 models suggest a similar, previously proposed, catalytic mechanism. Our data also confirm that the phosphate and vanadate can inhibit the enzyme activity by occupying the active site. More importantly, the AtBFN2/A5T structure reveals a novel and conserved secondary binding site, which seems to be important for plant Zn(2+-dependent endonucleases. Based on these findings, we propose a rational ssDNA binding model, in which the ssDNA wraps itself around the protein and the attached surface glycan, in turn, reinforces the binding complex.

  20. Rapid differentiation of closely related isolates of two plant viruses by polymerase chain reaction and restriction fragment length polymorphism analysis.

    Science.gov (United States)

    Barbara, D J; Morton, A; Spence, N J; Miller, A

    1995-09-01

    Immunocapture reverse transcriptase-polymerase chain reaction (RT-PCR) followed by restriction fragment length polymorphism (RFLP) analysis of the product has been shown to be an effective procedure for discriminating serologically indistinguishable isolates of two plant viruses, raspberry bushy dwarf (RBDV) and zucchini yellow mosaic (ZYMV). For both viruses, only limited sequence information was available at the time of primer design, but most of the isolates which were tested could be amplified (the one exception being a serologically quite distinct isolate of ZYMV). Restriction endonucleases revealing diagnostic RFLPs were readily identified. Each of two isolates of ZYMV could be detected in the presence of the other and the relative proportions approximately quantified by visual estimation of the relative intensity of the appropriate bands. A range of isolates of different RBDV pathotypes were compared; isolates were grouped in ways that accorded with their known history. Computer analysis of the published sequence from which the primers had been derived showed the sequenced isolate to be identical with an isolate imported from the USSR. The PCR/RFLP procedure is rapid (it can be completed in less than 2 days), effective and will probably be generally applicable to distinguishing closely related virus isolates, even where little sequence information is available.

  1. Differential distribution of a SINE element in the Entamoeba histolytica and Entamoeba dispar genomes: Role of the LINE-encoded endonuclease

    Directory of Open Access Journals (Sweden)

    Gupta Abhishek K

    2011-05-01

    Full Text Available Abstract Background Entamoeba histolytica and Entamoeba dispar are closely related protistan parasites but while E. histolytica can be invasive, E. dispar is completely non pathogenic. Transposable elements constitute a significant portion of the genome in these species; there being three families of LINEs and SINEs. These elements can profoundly influence the expression of neighboring genes. Thus their genomic location can have important phenotypic consequences. A genome-wide comparison of the location of these elements in the E. histolytica and E. dispar genomes has not been carried out. It is also not known whether the retrotransposition machinery works similarly in both species. The present study was undertaken to address these issues. Results Here we extracted all genomic occurrences of full-length copies of EhSINE1 in the E. histolytica genome and matched them with the homologous regions in E. dispar, and vice versa, wherever it was possible to establish synteny. We found that only about 20% of syntenic sites were occupied by SINE1 in both species. We checked whether the different genomic location in the two species was due to differences in the activity of the LINE-encoded endonuclease which is required for nicking the target site. We found that the endonucleases of both species were essentially very similar, both in their kinetic properties and in their substrate sequence specificity. Hence the differential distribution of SINEs in these species is not likely to be influenced by the endonuclease. Further we found that the physical properties of the DNA sequences adjoining the insertion sites were similar in both species. Conclusions Our data shows that the basic retrotransposition machinery is conserved in these sibling species. SINEs may indeed have occupied all of the insertion sites in the genome of the common ancestor of E. histolytica and E. dispar but these may have been subsequently lost from some locations. Alternatively, SINE

  2. DNA polymorphism of butyrophilin gene by PCR-RFLP technique ...

    African Journals Online (AJOL)

    We used the polymerase chain reaction-restriction fragment length polymorphism (PCRRFLP) technique to screen for DNA polymorphism in 109 cattle. In all cattle, we amplified an 863 fragment consisting of part of exon 8. The amplified fragment digested with HaeIII restriction endonuclease and subjected to electrophoretic ...

  3. Coupling of the nucleotide incision and 3' {yields} 5' exonuclease activities in Escherichia coli endonuclease IV: Structural and genetic evidences

    Energy Technology Data Exchange (ETDEWEB)

    Golan, Gali [Hebrew University of Jerusalem, Jerusalem 91904 (Israel); Ishchenko, Alexander A. [Groupe Reparation de l' ADN, CNRS UMR 8126, Univ. Paris-Sud, Institut de Cancerologie Gustave Roussy, 39, rue Camille Desmoulins, F-94805 Villejuif Cedex (France); Khassenov, Bekbolat [National Center for Biotechnology, Astana (Kazakhstan); Shoham, Gil, E-mail: gil2@vms.huji.ac.il [Hebrew University of Jerusalem, Jerusalem 91904 (Israel); Saparbaev, Murat K., E-mail: smurat@igr.fr [Groupe Reparation de l' ADN, CNRS UMR 8126, Univ. Paris-Sud, Institut de Cancerologie Gustave Roussy, 39, rue Camille Desmoulins, F-94805 Villejuif Cedex (France)

    2010-03-01

    Aerobic respiration generates reactive oxygen species (ROS) as a by-product of cellular metabolism which can damage DNA. The complex nature of oxidative DNA damage requires actions of several repair pathways. Oxidized DNA bases are substrates for two overlapping pathways: base excision repair (BER) and nucleotide incision repair (NIR). In the BER pathway a DNA glycosylase cleaves the N-glycosylic bond between the abnormal base and deoxyribose, leaving either an abasic site or single-stranded DNA break. Alternatively, in the NIR pathway, an apurinic/apyrimidinic (AP) endonuclease incises duplex DNA 5' next to oxidatively damaged nucleotide. The multifunctional Escherichia coli endonuclease IV (Nfo) is involved in both BER and NIR pathways. Nfo incises duplex DNA 5' of a damaged residue but also possesses an intrinsic 3' {yields} 5' exonuclease activity. Herein, we demonstrate that Nfo-catalyzed NIR and exonuclease activities can generate a single-strand gap at the 5' side of 5,6-dihydrouracil residue. Furthermore, we show that Nfo mutants carrying amino acid substitutions H69A and G149D are deficient in both NIR and exonuclease activities, suggesting that these two functions are genetically linked and governed by the same amino acid residues. The crystal structure of Nfo-H69A mutant reveals the loss of one of the active site zinc atoms (Zn1) and rearrangements of the catalytic site, but no gross changes in the overall enzyme conformation. We hypothesize that these minor changes strongly affect the DNA binding of Nfo. Decreased affinity may lead to a different kinking angle of the DNA helix and this in turn thwart nucleotide incision and exonuclease activities of Nfo mutants but to lesser extent of their AP endonuclease function. Based on the biochemical and genetic data we propose a model where nucleotide incision coupled to 3' {yields} 5' exonuclease activity prevents formation of lethal double-strand breaks when repairing bi

  4. Modification and restriction of T-even bacteriophages. In vitro degradation of deoxyribonucleic acid containing 5-hydroxymethylctosine.

    Science.gov (United States)

    Fleischman, R A; Cambell, J L; Richardson, C C

    1976-03-25

    Using the single-stranded circular DNA of bacteriophage fd as template, double-stranded circular DNA has been prepared in vitro with either 5-hydroxymethylcytosine ([hmdC]DNA) or cytosine ([dC]DNA) in the product strand. Extracts prepared from Escherichia coli cells restrictive to T-even phage containing nonglucosylated DNA degrade [hmdC]DNA to acid-soluble material in vitro, but do not degrade [dC]dna. In contrast, extracts prepared from E. coli K12 rglA- rglB-, a strain permissive to T-even phage containing nonglucosylated DNA, do not degrade [hmdC]DNA or [dC]DNA. In addition, glucosylation of the [hmdC]DNA renders it resistant to degradation by extracts from restrictive strains. The conversion of [hmdC]DNA to acid-soluble material in vitro consists of an HmCyt-specific endonucleolytic cleavage requiring the presence of the RglB gene product to form a linear molecule, followed by a non-HmCyt-specific hydrolysis of the linear DNA to acid-soluble fragments, catalyzed in part by exonuclease V. The RglB protein present in extracts of E. coli K12 rglA- rglB+ has been purified 200-fold by complementation with extracts from E. coli K12 rglA- rglB-. The purified RglB protein does not contain detectable HmCyt-specific endonuclease or exonuclease activity. In vitro endonucleolytic cleavage of [hmdC]DNA thus requires additional factors present in cell extracts.

  5. A Qualitative and Quantitative Assay to Study DNA/Drug Interaction ...

    African Journals Online (AJOL)

    Purpose: To explore the use of restriction inhibition assay (RIA) to study the binding specificity of some anticancer drugs. Methods: A 448 bp DNA fragment derived from pBCKS+ plasmid (harboring the polylinker region with multiple restriction endonuclease sites) was used as a template for sequence selective inhibition of ...

  6. Variation in Ribosomal DNA among Isolates of the Mycorrhizal Fungus Cenococcum Geophilum FR.

    Science.gov (United States)

    Lobuglio, Katherine Frances

    1990-01-01

    Cenococcum geophilum Fr., a cosmopolitan mycorrhizal fungus, is well-known for its extremely wide host and habitat range. The ecological diversity of C. geophilum sharply contrasts its present taxonomic status as a monotypic form -genus. Restriction fragment length polymorphisms (RFLPs) in nuclear ribosomal DNA (rDNA) was used to assess the degree of genetic variation among 72 isolates of C. geophilum. The probe used in this study was the rDNA repeat cloned from C. geophilum isolate A145 (pCG15). Length of the rDNA repeat was approximately 9 kb. The rDNA clone was mapped for 5 restriction endonucleases. Hybridization with cloned Saccharomyces cerevisiae rDNA (pSR118, and pSR125 containing the 18S, and 5.8-25S rRNA genes respectively), and alignment of restriction endonuclease sites conserved in the rDNA genes of other fungi, were used to position the corresponding rDNAs of C. geophilum. Southern hybridizations with EcoRI, HindIII, XhoI, and PstI digested DNAs indicated extensive variation among the C. geophilum isolates, greater than has been previously reported to occur within a fungal species. Most of the rDNA polymorphisms occurred in the IGS region. Restriction endonuclease site and length polymorphisms were also observed in the 5.8S-26S genic regions. Sixteen size categories of length mutations, 6 restriction endonuclease site additions, and 4 restriction endonuclease site deletions were determined using isolate A145 as a reference. The rDNA repeat length among the isolates varied from approximately 8.5 to 10.2 kb. RFLPs were also observed in the mitochondrial (mt) 24S rRNA gene and flanking regions of HindIII digested DNAs of C. geophilum isolates representing both geographically distinct and similar origins. Among the C. geophilum isolates analyzed there were fewer RFLPs in mt-DNA than in nuclear rDNA. EcoRI rDNA phenotypes between C. geophilum and Elaphomyces anthracinus, its proposed teleomorph or sexual state, did not correspond. In addition, the four

  7. Micrococcus luteus correndonucleases. II. Mechanism of action of two endonucleases specific for DNA containing pyrimidine dimers

    International Nuclear Information System (INIS)

    Riazuddin, S.; Grossman, L.

    1977-01-01

    Py--Py correndonucleases I and II from Micrococcus luteus act exclusively on thymine-thymine, cytosine-cytosine, and thymine-cytosine cyclobutyl dimers in DNA, catalyzing incision 5' to the damage and generating 3'-hydroxyl and 5'-phosphoryl termini. Both enzymes initiate excision of pyrimidine dimers in vitro by correxonucleases and DNA polymerase I. The respective incised DNAs, however, differ in their ability to act as substrate for phage T4 polynucleotide ligase or bacterial alkaline phosphatase, suggesting that each endonuclease is specific for a conformationally unique site. The possibility that their respective action generates termini which represent different degrees of single strandedness is suggested by the unequal protection by Escherichia coli binding protein from the hydrolytic action of exonuclease VII

  8. Pre-steady-state fluorescence analysis of damaged DNA transfer from human DNA glycosylases to AP endonuclease APE1.

    Science.gov (United States)

    Kuznetsova, Alexandra A; Kuznetsov, Nikita A; Ishchenko, Alexander A; Saparbaev, Murat K; Fedorova, Olga S

    2014-10-01

    DNA glycosylases remove the modified, damaged or mismatched bases from the DNA by hydrolyzing the N-glycosidic bonds. Some enzymes can further catalyze the incision of a resulting abasic (apurinic/apyrimidinic, AP) site through β- or β,δ-elimination mechanisms. In most cases, the incision reaction of the AP-site is catalyzed by special enzymes called AP-endonucleases. Here, we report the kinetic analysis of the mechanisms of modified DNA transfer from some DNA glycosylases to the AP endonuclease, APE1. The modified DNA contained the tetrahydrofurane residue (F), the analogue of the AP-site. DNA glycosylases AAG, OGG1, NEIL1, MBD4(cat) and UNG from different structural superfamilies were used. We found that all DNA glycosylases may utilise direct protein-protein interactions in the transient ternary complex for the transfer of the AP-containing DNA strand to APE1. We hypothesize a fast "flip-flop" exchange mechanism of damaged and undamaged DNA strands within this complex for monofunctional DNA glycosylases like MBD4(cat), AAG and UNG. Bifunctional DNA glycosylase NEIL1 creates tightly specific complex with DNA containing F-site thereby efficiently competing with APE1. Whereas APE1 fast displaces other bifunctional DNA glycosylase OGG1 on F-site thereby induces its shifts to undamaged DNA regions. Kinetic analysis of the transfer of DNA between human DNA glycosylases and APE1 allows us to elucidate the critical step in the base excision repair pathway. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Flap Endonuclease 1 Limits Telomere Fragility on the Leading Strand.

    Science.gov (United States)

    Teasley, Daniel C; Parajuli, Shankar; Nguyen, Mai; Moore, Hayley R; Alspach, Elise; Lock, Ying Jie; Honaker, Yuchi; Saharia, Abhishek; Piwnica-Worms, Helen; Stewart, Sheila A

    2015-06-12

    The existence of redundant replication and repair systems that ensure genome stability underscores the importance of faithful DNA replication. Nowhere is this complexity more evident than in challenging DNA templates, including highly repetitive or transcribed sequences. Here, we demonstrate that flap endonuclease 1 (FEN1), a canonical lagging strand DNA replication protein, is required for normal, complete leading strand replication at telomeres. We find that the loss of FEN1 nuclease activity, but not DNA repair activities, results in leading strand-specific telomere fragility. Furthermore, we show that FEN1 depletion-induced telomere fragility is increased by RNA polymerase II inhibition and is rescued by ectopic RNase H1 expression. These data suggest that FEN1 limits leading strand-specific telomere fragility by processing RNA:DNA hybrid/flap intermediates that arise from co-directional collisions occurring between the replisome and RNA polymerase. Our data reveal the first molecular mechanism for leading strand-specific telomere fragility and the first known role for FEN1 in leading strand DNA replication. Because FEN1 mutations have been identified in human cancers, our findings raise the possibility that unresolved RNA:DNA hybrid structures contribute to the genomic instability associated with cancer. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. RECQ5 Helicase Cooperates with MUS81 Endonuclease in Processing Stalled Replication Forks at Common Fragile Sites during Mitosis

    DEFF Research Database (Denmark)

    Di Marco, Stefano; Hasanova, Zdenka; Kanagaraj, Radhakrishnan

    2017-01-01

    The MUS81-EME1 endonuclease cleaves late replication intermediates at common fragile sites (CFSs) during early mitosis to trigger DNA-repair synthesis that ensures faithful chromosome segregation. Here, we show that these DNA transactions are promoted by RECQ5 DNA helicase in a manner dependent...... on its Ser727 phosphorylation by CDK1. Upon replication stress, RECQ5 associates with CFSs in early mitosis through its physical interaction with MUS81 and promotes MUS81-dependent mitotic DNA synthesis. RECQ5 depletion or mutational inactivation of its ATP-binding site, RAD51-interacting domain...

  11. Association of transforming growth factor-ß3 gene polymorphism ...

    African Journals Online (AJOL)

    Genotyping for the TGF-β3 gene using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method and BslI restriction endonuclease showed a mutation in 294-bp fragment located on the fourth intron of chromosome 5. Polymorphism in TGF-β3 gene was significantly (P < 0.1) associated with ...

  12. Primary processing of CRISPR RNA by the endonuclease Cas6 in Staphylococcus epidermidis.

    Science.gov (United States)

    Wakefield, Noelle; Rajan, Rakhi; Sontheimer, Erik J

    2015-10-07

    In many bacteria and archaea, an adaptive immune system (CRISPR-Cas) provides immunity against foreign genetic elements. This system uses CRISPR RNAs (crRNAs) derived from the CRISPR array, along with CRISPR-associated (Cas) proteins, to target foreign nucleic acids. In most CRISPR systems, endonucleolytic processing of crRNA precursors (pre-crRNAs) is essential for the pathway. Here we study the Cas6 endonuclease responsible for crRNA processing in the Type III-A CRISPR-Cas system from Staphylococcus epidermidis RP62a, a model for Type III-A CRISPR-Cas systems, and define substrate requirements for SeCas6 activity. We find that SeCas6 is necessary and sufficient for full-length crRNA biogenesis in vitro, and that it relies on both sequence and stem-loop structure in the 3' half of the CRISPR repeat for recognition and processing. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  13. Computational Characterization of Small Molecules Binding to the Human XPF Active Site and Virtual Screening to Identify Potential New DNA Repair Inhibitors Targeting the ERCC1-XPF Endonuclease

    Directory of Open Access Journals (Sweden)

    Francesco Gentile

    2018-04-01

    Full Text Available The DNA excision repair protein ERCC-1-DNA repair endonuclease XPF (ERCC1-XPF is a heterodimeric endonuclease essential for the nucleotide excision repair (NER DNA repair pathway. Although its activity is required to maintain genome integrity in healthy cells, ERCC1-XPF can counteract the effect of DNA-damaging therapies such as platinum-based chemotherapy in cancer cells. Therefore, a promising approach to enhance the effect of these therapies is to combine their use with small molecules, which can inhibit the repair mechanisms in cancer cells. Currently, there are no structures available for the catalytic site of the human ERCC1-XPF, which performs the metal-mediated cleavage of a DNA damaged strand at 5′. We adopted a homology modeling strategy to build a structural model of the human XPF nuclease domain which contained the active site and to extract dominant conformations of the domain using molecular dynamics simulations followed by clustering of the trajectory. We investigated the binding modes of known small molecule inhibitors targeting the active site to build a pharmacophore model. We then performed a virtual screening of the ZINC Is Not Commercial 15 (ZINC15 database to identify new ERCC1-XPF endonuclease inhibitors. Our work provides structural insights regarding the binding mode of small molecules targeting the ERCC1-XPF active site that can be used to rationally optimize such compounds. We also propose a set of new potential DNA repair inhibitors to be considered for combination cancer therapy strategies.

  14. Effect of ionizing radiation on the activity of restriction nucleases PvuII and HindIII

    International Nuclear Information System (INIS)

    Luzova, M.; Michaelidesova, A.; Davidkova, M.

    2014-01-01

    The research is focused on the influence of the ionizing radiation on the activity of the restriction enzymes PvuII and HindIII. Enzymes PvuII and HindIII are restriction endonucleases of type II. These enzymes can be found in bacteria and they have a significant role in defense mechanisms of bacteria against viruses. They cleave DNA double helix at specific recognition palindromic sequences in the presence of cofactor Mg 2+ . PvuII cleaves the sequence CAG↓CTG and HindIII cleaves the sequence A↓AGCTT in marked places. Plasmid pcDNA3 has been used as the DNA substrate for the whole experimental study. It is 5446 base pairs (bp) long, circular DNA molecule and it contains three recognition sites for enzyme PvuII and one recognition site for enzyme HindIII. After the correct interaction of pcDNA3 with PvuII, we thus have three plasmid fragments with lengths 1069, 1097 and 3280 bp. When HindIII is incubated with this plasmid, we shall obtain the linear form of the DNA plasmid.The method for processing the cleaved DNA samples is the agarose gel electrophoresis. The activity of the irradiated enzymes decreases with increasing dose of radiation, because a part of the enzymes is deactivated due to induced radiation damage. To determine effect of radiation quality, samples were irradiated using proton and gamma sources. The results of our experimental study will be presented and discussed with respect to molecular structure of both enzymes and particular sites of radical damage influencing their function. (authors)

  15. Generation of a restriction minus enteropathogenic Escherichia coli E2348/69 strain that is efficiently transformed with large, low copy plasmids

    Directory of Open Access Journals (Sweden)

    Ward Jordan D

    2008-08-01

    Full Text Available Abstract Background Many microbes possess restriction-modification systems that protect them from parasitic DNA molecules. Unfortunately, the presence of a restriction-modification system in a given microbe also hampers genetic analysis. Although plasmids can be successfully conjugated into the enteropathogenic Escherichia coli strain E2348/69 and optimized protocols for competent cell preparation have been developed, we found that a large, low copy (~15 bioluminescent reporter plasmid, pJW15, that we modified for use in EPEC, was exceedingly difficult to transform into E2348/69. We reasoned that a restriction-modification system could be responsible for the low transformation efficiency of E2348/69 and sought to identify and inactivate the responsible gene(s, with the goal of creating an easily transformable strain of EPEC that could complement existing protocols for genetic manipulation of this important pathogen. Results Using bioinformatics, we identified genes in the unfinished enteropathogenic Escherichia coli (EPEC strain E2348/69 genome whose predicted products bear homology to the HsdM methyltransferases, HsdS specificity subunits, and HsdR restriction endonucleases of type I restriction-modification systems. We constructed a strain carrying a deletion of the conserved enzymatic domain of the EPEC HsdR homologue, NH4, and showed that its transformation efficiency was up to four orders of magnitude higher than that of the parent strain. Further, the modification capacity of NH4 remained intact, since plasmids that were normally recalcitrant to transformation into E2348/69 could be transformed upon passage through NH4. NH4 was unaffected in virulence factor production, since bundle forming pilus (BFP subunits and type III secreted (T3S proteins were present at equivalent levels to those seen in E2348/69. Further, NH4 was indistinguishable from E2348/69 in tissue culture infection model assays of localized adherence and T3S. Conclusion We

  16. Activation of apurinic/apyrimidinic endonuclease in human cells by reactive oxygen species and its correlation with their adaptive response to genotoxicity of free radicals

    Science.gov (United States)

    Ramana, Chilakamarti V.; Boldogh, Istvan; Izumi, Tadahide; Mitra, Sankar

    1998-01-01

    Apurinic/apyrimidinic (AP) endonuclease (APE; EC 4.2.99.18) plays a central role in repair of DNA damage due to reactive oxygen species (ROS) because its DNA 3′-phosphoesterase activity removes 3′ blocking groups in DNA that are generated by DNA glycosylase/AP-lyases during removal of oxidized bases and by direct ROS reaction with DNA. The major human APE (APE-1) gene is activated selectively by sublethal levels of a variety of ROS and ROS generators, including ionizing radiation, but not by other genotoxicants—e.g., UV light and alkylating agents. Increased expression of APE mRNA and protein was observed both in the HeLa S3 tumor line and in WI 38 primary fibroblasts, and it was accompanied by translocation of the endonuclease to the nucleus. ROS-treated cells showed a significant increase in resistance to the cytotoxicity of such ROS generators as H2O2 and bleomycin, but not to UV light. This “adaptive response” appears to result from enhanced repair of cytotoxic DNA lesions due to an increased activity of APE-1, which may be limiting in the base excision repair process for ROS-induced toxic lesions. PMID:9560228

  17. [Endonuclease modified comet assay for oxidative DNA damage induced by detection of genetic toxicants].

    Science.gov (United States)

    Zhao, Jian; Li, Hongli; Zhai, Qingfeng; Qiu, Yugang; Niu, Yong; Dai, Yufei; Zheng, Yuxin; Duan, Huawei

    2014-03-01

    The aim of this study was to investigate the use of the lesion-specific endonucleases-modified comet assay for analysis of DNA oxidation in cell lines. DNA breaks and oxidative damage were evaluated by normal alkaline and formamidopyrimidine-DNA-glycosylase (FPG) modified comet assays. Cytotoxicity were assessed by MTT method. The human bronchial epithelial cell (16HBE) were treated with benzo (a) pyrene (B(a)P), methyl methanesulfonate (MMS), colchicine (COL) and vincristine (VCR) respectively, and the dose is 20 µmol/L, 25 mg/ml, 5 mg/L and 0.5 mg/L for 24 h, respectively. Oxidative damage was also detected by levels of reactive oxygen species in treated cells. Four genotoxicants give higher cytotoxicity and no significant changes on parameters of comet assay treated by enzyme buffer. Cell survival rate were (59.69 ± 2.60) %, (54.33 ± 2.81) %, (53.11 ± 4.00) %, (51.43 ± 3.92) % in four groups, respectively. There was the direct DNA damage induced by test genotoxicants presented by tail length, Olive tail moment (TM) and tail DNA (%) in the comet assay. The presence of FPG in the assays increased DNA migration in treated groups when compared to those without it, and the difference was statistically significant which indicated that the clastogen and aneugen could induce oxidative damage in DNA strand. In the three parameters, the Olive TM was changed most obviously after genotoxicants treatment. In the contrast group, the Olive TM of B(a) P,MMS, COL,VCR in the contrast groups were 22.99 ± 17.33, 31.65 ± 18.86, 19.86 ± 9.56 and 17.02 ± 9.39, respectively, after dealing with the FPG, the Olive TM were 34.50 ± 17.29, 43.80 ± 10.06, 33.10 ± 12.38, 28.60 ± 10.53, increased by 58.94%, 38.48%, 66.86% and 68.21%, respectively (t value was 3.91, 3.89, 6.66 and 3.87, respectively, and all P comet assay appears more specific for detecting oxidative DNA damage induced by genotoxicants exposure, and the application of comet assay will be expanded. The endonuclease

  18. Molecular Characterization of Yeast Strains Isolated from Different Sources by Restriction Fragment Length Polymorphism

    International Nuclear Information System (INIS)

    Ali, M. S.; Latif, Z.

    2016-01-01

    Various molecular techniques like analysis of the amplified rDNA internal transcribed spacers (ITS), intragenic spacers and total ITS region analysis by restriction fragment length polymorphism (RFLP) has been introduced for yeast identification but there are limited databases to identify yeast species on the basis of 5.8S rDNA. In this study, twenty nine yeast strains from various sources including spoiled fruits, vegetables, foodstuffs, and concentrated juices were characterized by PCR-RFLP. PCR-RFLP has been used to characterize yeasts present in different spoiled food samples after isolation of the yeasts. By using this technique, the isolated yeast strains were characterized by direct 5.8S-ITS rDNA region amplification. RFLP analysis was applied to each of the amplification products (varied from 400bp to 800bp) detected, and the corresponding yeast identifications were made according to each specific restriction patterns obtained after treatment with two endonucleases TaqI and HaeIII which yielded a specific banding pattern for each species. For further confirmation amplified products of eleven selected isolates were sequenced and blast on NCBI. Both RFLP and sequence analyses of the strains with accession nos. KF472163, KF472164, KF472165, KF472166, KF472167, KF472168, KF472169, KF472170, KF472171, KF472172, KF472173 gave significantly similar results. The isolates were found to belong five different yeast species including; Candida spp., Pichia spp., Kluyveromyces spp., Clavispora spp. and Hanseniaspora spp. This method provides a fast, easy, reliable and authentic way for determining yeast population present in different type of samples, as compared to traditional characterization technique. (author)

  19. Fusion of GFP to the M.EcoKI DNA methyltransferase produces a new probe of Type I DNA restriction and modification enzymes

    International Nuclear Information System (INIS)

    Chen, Kai; Roberts, Gareth A.; Stephanou, Augoustinos S.; Cooper, Laurie P.; White, John H.; Dryden, David T.F.

    2010-01-01

    Research highlights: → Successful fusion of GFP to M.EcoKI DNA methyltransferase. → GFP located at C-terminal of sequence specificity subunit does not later enzyme activity. → FRET confirms structural model of M.EcoKI bound to DNA. -- Abstract: We describe the fusion of enhanced green fluorescent protein to the C-terminus of the HsdS DNA sequence-specificity subunit of the Type I DNA modification methyltransferase M.EcoKI. The fusion expresses well in vivo and assembles with the two HsdM modification subunits. The fusion protein functions as a sequence-specific DNA methyltransferase protecting DNA against digestion by the EcoKI restriction endonuclease. The purified enzyme shows Foerster resonance energy transfer to fluorescently-labelled DNA duplexes containing the target sequence and to fluorescently-labelled ocr protein, a DNA mimic that binds to the M.EcoKI enzyme. Distances determined from the energy transfer experiments corroborate the structural model of M.EcoKI.

  20. Construction of Biologically Functional Bacterial Plasmids In Vitro

    Science.gov (United States)

    Cohen, Stanley N.; Chang, Annie C. Y.; Boyer, Herbert W.; Helling, Robert B.

    1973-01-01

    The construction of new plasmid DNA species by in vitro joining of restriction endonuclease-generated fragments of separate plasmids is described. Newly constructed plasmids that are inserted into Escherichia coli by transformation are shown to be biologically functional replicons that possess genetic properties and nucleotide base sequences from both of the parent DNA molecules. Functional plasmids can be obtained by reassociation of endonuclease-generated fragments of larger replicons, as well as by joining of plasmid DNA molecules of entirely different origins. Images PMID:4594039

  1. Rapid differentiation of Staphylococcus aureus isolates harbouring egc loci with pseudogenes psient1 and psient2 and the selu or seluv gene using PCR-RFLP.

    Science.gov (United States)

    Collery, Mark M; Smyth, Cyril J

    2007-02-01

    The egc locus of Staphylococus aureus harbours two enterotoxin genes (seg and sei) and three enterotoxin-like genes (selm, seln and selo). Between the sei and seln genes are located two pseudogenes, psient1 and psient2, or the selu or seluv gene. While these two alternative sei-seln intergenic regions can be distinguished by PCR, to date, DNA sequencing has been the only confirmatory option because of the very high degree of sequence similarity between egc loci bearing the pseudogenes and the selu or seluv gene. In silico restriction enzyme digestion of genomic regions encompassing the egc locus from the 3' end of the sei gene through the 5' first quarter of the seln gene allowed pseudogene- and selu- or seluv-bearing egc loci to be distinguished by PCR-RFLP. Experimental application of these findings demonstrated that endonuclease HindIII cleaved PCR amplimers bearing pseudogenes but not those with a selu or seluv gene, while selu- or seluv-bearing amplimers were susceptible to cleavage by endonuclease HphI, but not by endonuclease HindIII. The restriction enzyme BccI cleaved selu- or seluv-harbouring amplimers at a unique restriction site created by their signature 15 bp insertion compared with pseudogene-bearing amplimers, thereby allowing distinction of these egc loci. PCR-RFLP analysis using these restriction enzymes provides a rapid, easy to interpret alternative to DNA sequencing for verification of PCR findings on the nature of an egc locus type, and can also be used for the primary identification of the intergenic sei-seln egc locus type.

  2. Restriction site extension PCR: a novel method for high-throughput characterization of tagged DNA fragments and genome walking.

    Directory of Open Access Journals (Sweden)

    Jiabing Ji

    Full Text Available BACKGROUND: Insertion mutant isolation and characterization are extremely valuable for linking genes to physiological function. Once an insertion mutant phenotype is identified, the challenge is to isolate the responsible gene. Multiple strategies have been employed to isolate unknown genomic DNA that flanks mutagenic insertions, however, all these methods suffer from limitations due to inefficient ligation steps, inclusion of restriction sites within the target DNA, and non-specific product generation. These limitations become close to insurmountable when the goal is to identify insertion sites in a high throughput manner. METHODOLOGY/PRINCIPAL FINDINGS: We designed a novel strategy called Restriction Site Extension PCR (RSE-PCR to efficiently conduct large-scale isolation of unknown genomic DNA fragments linked to DNA insertions. The strategy is a modified adaptor-mediated PCR without ligation. An adapter, with complementarity to the 3' overhang of the endonuclease (KpnI, NsiI, PstI, or SacI restricted DNA fragments, extends the 3' end of the DNA fragments in the first cycle of the primary RSE-PCR. During subsequent PCR cycles and a second semi-nested PCR (secondary RSE-PCR, touchdown and two-step PCR are combined to increase the amplification specificity of target fragments. The efficiency and specificity was demonstrated in our characterization of 37 tex mutants of Arabidopsis. All the steps of RSE-PCR can be executed in a 96 well PCR plate. Finally, RSE-PCR serves as a successful alternative to Genome Walker as demonstrated by gene isolation from maize, a plant with a more complex genome than Arabidopsis. CONCLUSIONS/SIGNIFICANCE: RSE-PCR has high potential application in identifying tagged (T-DNA or transposon sequence or walking from known DNA toward unknown regions in large-genome plants, with likely application in other organisms as well.

  3. A novel approach to the generation of seamless constructs for plant transformation

    DEFF Research Database (Denmark)

    Kronbak, Remy; Ingvardsen, Christina R.; Madsen, Claus K.

    2014-01-01

    Background: When creating plant transformation vectors, full control of nucleotides flanking the insert in the final construct may be desirable. Modern ligase-independent methods for DNA-recombination are based on linearization by classical type II restriction endonucleases (REs) alone or in comb......Background: When creating plant transformation vectors, full control of nucleotides flanking the insert in the final construct may be desirable. Modern ligase-independent methods for DNA-recombination are based on linearization by classical type II restriction endonucleases (REs) alone...... on wheat and barley endosperm cells for transient gfp expression.Conclusions: All nucleotides flanking an insert in a biolistic plant transformation vector can be customized by means of SRL in combination with SLIC. Especially type IIS REs promote an efficient cloning result. Based on our findings, we...

  4. Single-molecule FRET unveils induced-fit mechanism for substrate selectivity in flap endonuclease 1

    KAUST Repository

    Rashid, Fahad

    2017-02-23

    Human flap endonuclease 1 (FEN1) and related structure-specific 5\\'nucleases precisely identify and incise aberrant DNA structures during replication, repair and recombination to avoid genomic instability. Yet, it is unclear how the 5\\'nuclease mechanisms of DNA distortion and protein ordering robustly mediate efficient and accurate substrate recognition and catalytic selectivity. Here, single-molecule sub-millisecond and millisecond analyses of FEN1 reveal a protein-DNA induced-fit mechanism that efficiently verifies substrate and suppresses off-target cleavage. FEN1 sculpts DNA with diffusion-limited kinetics to test DNA substrate. This DNA distortion mutually \\'locks\\' protein and DNA conformation and enables substrate verification with extreme precision. Strikingly, FEN1 never misses cleavage of its cognate substrate while blocking probable formation of catalytically competent interactions with noncognate substrates and fostering their pre-incision dissociation. These findings establish FEN1 has practically perfect precision and that separate control of induced-fit substrate recognition sets up the catalytic selectivity of the nuclease active site for genome stability.

  5. Structure of the motor subunit of type I restriction-modification complex EcoR124I

    Czech Academy of Sciences Publication Activity Database

    Lapkouski, Mikalai

    2009-01-01

    Roč. 1, č. 16 (2009), s. 94-95 ISSN 1545-9985 Institutional research plan: CEZ:AV0Z60870520 Keywords : SWI2/SNF2 ATPASE * DNA * ENDONUCLEASE Subject RIV: CE - Biochemistry Impact factor: 12.273, year: 2009

  6. Small molecule inhibitors uncover synthetic genetic interactions of human flap endonuclease 1 (FEN1 with DNA damage response genes.

    Directory of Open Access Journals (Sweden)

    Thomas A Ward

    Full Text Available Flap endonuclease 1 (FEN1 is a structure selective endonuclease required for proficient DNA replication and the repair of DNA damage. Cellularly active inhibitors of this enzyme have previously been shown to induce a DNA damage response and, ultimately, cell death. High-throughput screens of human cancer cell-lines identify colorectal and gastric cell-lines with microsatellite instability (MSI as enriched for cellular sensitivity to N-hydroxyurea series inhibitors of FEN1, but not the PARP inhibitor olaparib or other inhibitors of the DNA damage response. This sensitivity is due to a synthetic lethal interaction between FEN1 and MRE11A, which is often mutated in MSI cancers through instabilities at a poly(T microsatellite repeat. Disruption of ATM is similarly synthetic lethal with FEN1 inhibition, suggesting that disruption of FEN1 function leads to the accumulation of DNA double-strand breaks. These are likely a result of the accumulation of aberrant replication forks, that accumulate as a consequence of a failure in Okazaki fragment maturation, as inhibition of FEN1 is toxic in cells disrupted for the Fanconi anemia pathway and post-replication repair. Furthermore, RAD51 foci accumulate as a consequence of FEN1 inhibition and the toxicity of FEN1 inhibitors increases in cells disrupted for the homologous recombination pathway, suggesting a role for homologous recombination in the resolution of damage induced by FEN1 inhibition. Finally, FEN1 appears to be required for the repair of damage induced by olaparib and cisplatin within the Fanconi anemia pathway, and may play a role in the repair of damage associated with its own disruption.

  7. Evaluation of an Internally Controlled Multiplex Tth Endonuclease Cleavage Loop-Mediated Isothermal Amplification (TEC-LAMP Assay for the Detection of Bacterial Meningitis Pathogens

    Directory of Open Access Journals (Sweden)

    Owen Higgins

    2018-02-01

    Full Text Available Bacterial meningitis infection is a leading global health concern for which rapid and accurate diagnosis is essential to reduce associated morbidity and mortality. Loop-mediated isothermal amplification (LAMP offers an effective low-cost diagnostic approach; however, multiplex LAMP is difficult to achieve, limiting its application. We have developed novel real-time multiplex LAMP technology, TEC-LAMP, using Tth endonuclease IV and a unique LAMP primer/probe. This study evaluates the analytical specificity, limit of detection (LOD and clinical application of an internally controlled multiplex TEC-LAMP assay for detection of leading bacterial meningitis pathogens: Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae. Analytical specificities were established by testing 168 bacterial strains, and LODs were determined using Probit analysis. The TEC-LAMP assay was 100% specific, with LODs for S. pneumoniae, N. meningitidis and H. influenzae of 39.5, 17.3 and 25.9 genome copies per reaction, respectively. Clinical performance was evaluated by testing 65 archived PCR-positive samples. Compared to singleplex real-time PCR, the multiplex TEC-LAMP assay demonstrated diagnostic sensitivity and specificity of 92.3% and 100%, respectively. This is the first report of a single-tube internally controlled multiplex LAMP assay for bacterial meningitis pathogen detection, and the first report of Tth endonuclease IV incorporation into nucleic acid amplification diagnostic technology.

  8. Evaluation of an Internally Controlled Multiplex Tth Endonuclease Cleavage Loop-Mediated Isothermal Amplification (TEC-LAMP) Assay for the Detection of Bacterial Meningitis Pathogens

    Science.gov (United States)

    Clancy, Eoin; Cormican, Martin; Boo, Teck Wee; Cunney, Robert

    2018-01-01

    Bacterial meningitis infection is a leading global health concern for which rapid and accurate diagnosis is essential to reduce associated morbidity and mortality. Loop-mediated isothermal amplification (LAMP) offers an effective low-cost diagnostic approach; however, multiplex LAMP is difficult to achieve, limiting its application. We have developed novel real-time multiplex LAMP technology, TEC-LAMP, using Tth endonuclease IV and a unique LAMP primer/probe. This study evaluates the analytical specificity, limit of detection (LOD) and clinical application of an internally controlled multiplex TEC-LAMP assay for detection of leading bacterial meningitis pathogens: Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae. Analytical specificities were established by testing 168 bacterial strains, and LODs were determined using Probit analysis. The TEC-LAMP assay was 100% specific, with LODs for S. pneumoniae, N. meningitidis and H. influenzae of 39.5, 17.3 and 25.9 genome copies per reaction, respectively. Clinical performance was evaluated by testing 65 archived PCR-positive samples. Compared to singleplex real-time PCR, the multiplex TEC-LAMP assay demonstrated diagnostic sensitivity and specificity of 92.3% and 100%, respectively. This is the first report of a single-tube internally controlled multiplex LAMP assay for bacterial meningitis pathogen detection, and the first report of Tth endonuclease IV incorporation into nucleic acid amplification diagnostic technology. PMID:29425124

  9. Three-dimensional solution structure of a DNA duplex containing the BclI restriction sequence: Two-dimensional NMR studies, distance geometry calculations, and refinement by back-calculation of the NOESY spectrum

    International Nuclear Information System (INIS)

    Banks, K.M.; Hare, D.R.; Reid, B.R.

    1989-01-01

    A three-dimensional solution structure for the self-complementary dodecanucleotide [(d-GCCTGATCAGGC)] 2 has been determined by distance geometry with further refinements being performed after back-calculation of the NOESY spectrum. This DNA dodecamer contains the hexamer [d(TGATCA)] 2 recognized and cut by the restriction endonuclease BclI, and its structure was determined in hopes of obtaining a better understanding of the sequence-specific interactions which occur between proteins and DNA. Preliminary examination of the structure indicates the structure is underwound with respect to idealized B-form DNA though some of the local structural parameters (glycosyl torsion angle and pseudorotation angle) suggest a B-family type of structure is present. This research demonstrates the requirements (resonance assignments, interproton distance measurements, distance geometry calculations, and NOESY spectra back-calculation) to generate experimentally self-consistent solution structures for short DNA sequences

  10. Repair of potentially lethal damage by introduction of T4 DNA ligase in eucaryotic cells

    International Nuclear Information System (INIS)

    Durante, M.; Grossi, G.F.; Napolitano, M.; Gialanella, G.

    1991-01-01

    The bacterial enzyme PvuII, which generates blunt-ended DNA double-strand breaks, and T4 DNA ligase, which seals adjacent DNA fragments in coupling to ATP cleavage, were introduced in mouse C3H10T1/2 fibroblasts using osmolytic shock of pinocytic vesicles. Cells were then assayed for their clonogenic ability. In agreement with previous studies by others, the authors found that PvuII restriction endonuclease simulates ionizing radiation effects by causing a dose-dependent loss of reproductive capacity. They show that concomitant treatment with DNA ligase considerably increases cell survival. Survival curves were shown to be dependent on ligase enzyme dose and on ATP concentration in the hypertonic medium. They conclude that T4 DNA ligase is able to repair some potentially lethal damage produced by restriction endonucleases in eucaryotic cells. (author)

  11. Positioning the 5'-flap junction in the active site controls the rate of flap endonuclease-1-catalyzed DNA cleavage

    KAUST Repository

    Song, Bo

    2018-02-09

    Flap endonucleases catalyze cleavage of single-stranded DNA flaps formed during replication, repair and recombination, and are therefore essential for genome processing and stability. Recent crystal structures of DNA-bound human flap endonuclease (hFEN1) offer new insights into how conformational changes in the DNA and hFEN1 may facilitate the reaction mechanism. For example, previous biochemical studies of DNA conformation performed under non-catalytic conditions with Ca2+ have suggested that base unpairing at the 5\\'-flap:template junction is an important step in the reaction, but the new structural data suggest otherwise. To clarify the role of DNA changes in the kinetic mechanism, we measured a series of transient steps - from substrate binding to product release - during the hFEN1-catalyzed reaction in the presence of Mg2+. We found that while hFEN1 binds and bends DNA at a fast, diffusion-limited rate, much slower Mg2+-dependent conformational changes in DNA around the active site are subsequently necessary and rate-limiting for 5\\'-flap cleavage. These changes are reported overall by fluorescence of 2-aminopurine at the 5\\'-flap:template junction, indicating that local DNA distortion (e.g., disruption of base stacking observed in structures), associated with positioning the 5\\'-flap scissile phosphodiester bond in the hFEN1 active site, controls catalysis. hFEN1 residues with distinct roles in the catalytic mechanism, including those binding metal ions (Asp-34, Asp-181), steering the 5\\'-flap through the active site and binding the scissile phosphate (Lys-93, Arg-100), and stacking against the base 5\\' to the scissile phosphate (Tyr-40), all contribute to these rate-limiting conformational changes, ensuring efficient and specific cleavage of 5\\'-flaps.

  12. Haplotype-based case-control study on human apurinic/apyrimidinic endonuclease 1/redox effector factor-1 gene and essential hypertension.

    Science.gov (United States)

    Naganuma, Takahiro; Nakayama, Tomohiro; Sato, Naoyuki; Fu, Zhenyan; Soma, Masayoshi; Yamaguchi, Mai; Shimodaira, Masanori; Aoi, Noriko; Usami, Ron

    2010-02-01

    Oxidative DNA damage is involved in the pathophysiology of essential hypertension (EH), which is a multifactorial disorder. Apurinic/apyrimidinic endonuclease 1/redox effector factor-1 (APE1/REF-1) is an essential endonuclease in the base excision repair pathway of oxidatively damaged DNA, in addition to having reducing properties that promote the binding of redox-sensitive transcription factors. Blood pressure in APE1/REF-1-knockout mice is reported to be significantly higher than in wild-type mice. The aim of this study was to investigate the relationship between EH and the human APE1/REF-1 gene through a haplotype-based case-control study using single-nucleotide polymorphisms (SNPs). We selected five SNPs in the human APE1/REF-1 gene (rs1760944, rs3136814, rs17111967, rs3136817, and rs1130409), and performed case-control studies in 265 EH patients and 266 age-matched normotensive (NT) subjects. rs17111967 was found to show nonheterogeneity among Japanese subjects. There were no significant differences in the overall distribution of genotypes or alleles for each SNP between EH and NT groups. In the overall distribution of the haplotype-based case-control study constructed based on rs1760944, rs3136817, and rs1130409, the frequency of the G-T-T haplotype was significantly higher in the EH group than in the NT group (2.1% vs. 0.0%, P = 0.001). Multiple logistic regression analysis also revealed significant differences for the G-T-T haplotype, even after adjustment for confounding factors (OR = 8.600, 95% CI: 1.073-68.951, P = 0.043). Based on the present results, the G-T-T haplotype appears to be a genetic marker of EH, and the APE1/REF-1 gene appears to be a susceptibility gene for EH.

  13. Positioning the 5'-flap junction in the active site controls the rate of flap endonuclease-1-catalyzed DNA cleavage

    KAUST Repository

    Song, Bo; Hamdan, Samir; Hingorani, Manju M

    2018-01-01

    Flap endonucleases catalyze cleavage of single-stranded DNA flaps formed during replication, repair and recombination, and are therefore essential for genome processing and stability. Recent crystal structures of DNA-bound human flap endonuclease (hFEN1) offer new insights into how conformational changes in the DNA and hFEN1 may facilitate the reaction mechanism. For example, previous biochemical studies of DNA conformation performed under non-catalytic conditions with Ca2+ have suggested that base unpairing at the 5'-flap:template junction is an important step in the reaction, but the new structural data suggest otherwise. To clarify the role of DNA changes in the kinetic mechanism, we measured a series of transient steps - from substrate binding to product release - during the hFEN1-catalyzed reaction in the presence of Mg2+. We found that while hFEN1 binds and bends DNA at a fast, diffusion-limited rate, much slower Mg2+-dependent conformational changes in DNA around the active site are subsequently necessary and rate-limiting for 5'-flap cleavage. These changes are reported overall by fluorescence of 2-aminopurine at the 5'-flap:template junction, indicating that local DNA distortion (e.g., disruption of base stacking observed in structures), associated with positioning the 5'-flap scissile phosphodiester bond in the hFEN1 active site, controls catalysis. hFEN1 residues with distinct roles in the catalytic mechanism, including those binding metal ions (Asp-34, Asp-181), steering the 5'-flap through the active site and binding the scissile phosphate (Lys-93, Arg-100), and stacking against the base 5' to the scissile phosphate (Tyr-40), all contribute to these rate-limiting conformational changes, ensuring efficient and specific cleavage of 5'-flaps.

  14. Problem-Solving Test: Conditional Gene Targeting Using the Cre/loxP Recombination System

    Science.gov (United States)

    Szeberényi, József

    2013-01-01

    Terms to be familiar with before you start to solve the test: gene targeting, knock-out mutation, bacteriophage, complementary base-pairing, homologous recombination, deletion, transgenic organisms, promoter, polyadenylation element, transgene, DNA replication, RNA polymerase, Shine-Dalgarno sequence, restriction endonuclease, polymerase chain…

  15. Host Factors Influencing the Retrohoming Pathway of Group II Intron RmInt1, Which Has an Intron-Encoded Protein Naturally Devoid of Endonuclease Activity.

    Directory of Open Access Journals (Sweden)

    Rafael Nisa-Martínez

    Full Text Available Bacterial group II introns are self-splicing catalytic RNAs and mobile retroelements that have an open reading frame encoding an intron-encoded protein (IEP with reverse transcriptase (RT and RNA splicing or maturase activity. Some IEPs carry a DNA endonuclease (En domain, which is required to cleave the bottom strand downstream from the intron-insertion site for target DNA-primed reverse transcription (TPRT of the inserted intron RNA. Host factors complete the insertion of the intron. By contrast, the major retrohoming pathway of introns with IEPs naturally lacking endonuclease activity, like the Sinorhizobium meliloti intron RmInt1, is thought to involve insertion of the intron RNA into the template for lagging strand DNA synthesis ahead of the replication fork, with possible use of the nascent strand to prime reverse transcription of the intron RNA. The host factors influencing the retrohoming pathway of such introns have not yet been described. Here, we identify key candidates likely to be involved in early and late steps of RmInt1 retrohoming. Some of these host factors are common to En+ group II intron retrohoming, but some have different functions. Our results also suggest that the retrohoming process of RmInt1 may be less dependent on the intracellular free Mg2+ concentration than those of other group II introns.

  16. Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease.

    Science.gov (United States)

    Anders, Carolin; Niewoehner, Ole; Duerst, Alessia; Jinek, Martin

    2014-09-25

    The CRISPR-associated protein Cas9 is an RNA-guided endonuclease that cleaves double-stranded DNA bearing sequences complementary to a 20-nucleotide segment in the guide RNA. Cas9 has emerged as a versatile molecular tool for genome editing and gene expression control. RNA-guided DNA recognition and cleavage strictly require the presence of a protospacer adjacent motif (PAM) in the target DNA. Here we report a crystal structure of Streptococcus pyogenes Cas9 in complex with a single-molecule guide RNA and a target DNA containing a canonical 5'-NGG-3' PAM. The structure reveals that the PAM motif resides in a base-paired DNA duplex. The non-complementary strand GG dinucleotide is read out via major-groove interactions with conserved arginine residues from the carboxy-terminal domain of Cas9. Interactions with the minor groove of the PAM duplex and the phosphodiester group at the +1 position in the target DNA strand contribute to local strand separation immediately upstream of the PAM. These observations suggest a mechanism for PAM-dependent target DNA melting and RNA-DNA hybrid formation. Furthermore, this study establishes a framework for the rational engineering of Cas9 enzymes with novel PAM specificities.

  17. Restricted Interval Valued Neutrosophic Sets and Restricted Interval Valued Neutrosophic Topological Spaces

    Directory of Open Access Journals (Sweden)

    Anjan Mukherjee

    2016-08-01

    Full Text Available In this paper we introduce the concept of restricted interval valued neutrosophic sets (RIVNS in short. Some basic operations and properties of RIVNS are discussed. The concept of restricted interval valued neutrosophic topology is also introduced together with restricted interval valued neutrosophic finer and restricted interval valued neutrosophic coarser topology. We also define restricted interval valued neutrosophic interior and closer of a restricted interval valued neutrosophic set. Some theorems and examples are cites. Restricted interval valued neutrosophic subspace topology is also studied.

  18. Interaction of the E. coli DNA G:T-mismatch endonuclease (vsr protein) with oligonucleotides containing its target sequence.

    Science.gov (United States)

    Turner, D P; Connolly, B A

    2000-12-15

    The Escherichia coli vsr endonuclease recognises G:T base-pair mismatches in double-stranded DNA and initiates a repair pathway by hydrolysing the phosphate group 5' to the incorrectly paired T. The enzyme shows a preference for G:T mismatches within a particular sequence context, derived from the recognition site of the E. coli dcm DNA-methyltransferase (CC[A/T]GG). Thus, the preferred substrate for the vsr protein is (CT[A/T]GG), where the underlined T is opposed by a dG base. This paper provides quantitative data for the interaction of the vsr protein with a number of oligonucleotides containing G:T mismatches. Evaluation of specificity constant (k(st)/K(D); k(st)=rate constant for single turnover, K(D)=equilibrium dissociation constant) confirms vsr's preference for a G:T mismatch within a hemi-methylated dcm sequence, i.e. the best substrate is a duplex (both strands written in the 5'-3' orientation) composed of CT[A/T]GG and C(5Me)C[T/A]GG. Conversion of the mispaired T (underlined) to dU or the d(5Me)C to dC gave poorer substrates. No interaction was observed with oligonucleotides that lacked a G:T mismatch or did not possess a dcm sequence. An analysis of the fraction of active protein, by "reverse-titration" (i.e. adding increasing amounts of DNA to a fixed amount of protein followed by gel-mobility shift analysis) showed that less than 1% of the vsr endonuclease was able to bind to the substrate. This was confirmed using "competitive titrations" (where competitor oligonucleotides are used to displace a (32)P-labelled nucleic acid from the vsr protein) and burst kinetic analysis. This result is discussed in the light of previous in vitro and in vivo data which indicate that the MutL protein may be needed for full vsr activity. Copyright 2000 Academic Press.

  19. Analysis of rRNA gene methylation in Arabidopsis thaliana by CHEF-Conventional 2D gel electrophoresis

    Science.gov (United States)

    Mohannath, Gireesha; Pikaard, Craig S.

    2017-01-01

    Summary Contour-clamped homogenous electric field (CHEF) gel electrophoresis, a variant of Pulsed-field gel electrophoresis (PFGE), is a powerful technique for resolving large fragments of DNA (10 kb to 9 Mb). CHEF has many applications including the physical mapping of chromosomes, artificial chromosomes and sub-chromosomal DNA fragments, etc. Here we describe the use of CHEF and two-dimensional gel electrophoresis to analyze rRNA gene methylation patterns within the two ~ 4 million base pair nucleolus organizer regions (NORs) of Arabidopsis thaliana. The method involves CHEF gel electrophoresis of agarose-embedded DNA following restriction endonuclease digestion to cut the NORs into large but resolvable segments, followed by digestion with methylation-sensitive restriction endonucleases and conventional (or CHEF) gel electrophoresis, in a second dimension. Resulting products are then detected by Southern blotting or PCR analyses capable of discriminating rRNA gene subtypes. PMID:27576719

  20. Activation of apurinic/apyrimidinic endonuclease in human cells by reactive oxygen species and its correlation with their adaptive response to genotoxicity of free radicals

    OpenAIRE

    Ramana, Chilakamarti V.; Boldogh, Istvan; Izumi, Tadahide; Mitra, Sankar

    1998-01-01

    Apurinic/apyrimidinic (AP) endonuclease (APE; EC 4.2.99.18) plays a central role in repair of DNA damage due to reactive oxygen species (ROS) because its DNA 3′-phosphoesterase activity removes 3′ blocking groups in DNA that are generated by DNA glycosylase/AP-lyases during removal of oxidized bases and by direct ROS reaction with DNA. The major human APE (APE-1) gene is activated selectively by sublethal levels of a variety of ROS and ROS generators, including ionizing radiation, but not by ...

  1. Identification of restriction endonuclease with potential ability to cleave the HSV-2 genome: Inherent potential for biosynthetic versus live recombinant microbicides

    Directory of Open Access Journals (Sweden)

    Wayengera Misaki

    2008-08-01

    Full Text Available Abstract Background Herpes Simplex virus types 1 and 2 are enveloped viruses with a linear dsDNA genome of ~120–200 kb. Genital infection with HSV-2 has been denoted as a major risk factor for acquisition and transmission of HIV-1. Developing biomedical strategies for HSV-2 prevention is thus a central strategy in reducing global HIV-1 prevalence. This paper details the protocol for the isolation of restriction endunucleases (REases with potent activity against the HSV-2 genome and models two biomedical interventions for preventing HSV-2. Methods and Results Using the whole genome of HSV-2, 289 REases and the bioinformatics software Webcutter2; we searched for potential recognition sites by way of genome wide palindromics. REase application in HSV-2 biomedical therapy was modeled concomitantly. Of the 289 enzymes analyzed; 77(26.6% had potential to cleave the HSV-2 genome in > 100 but 400 but Conclusion Viral genome slicing by way of these bacterially- derived R-M enzymatic peptides may have therapeutic potential in HSV-2 infection; a cofactor for HIV-1 acquisition and transmission.

  2. Organization of the BcgI restriction-modification protein for the cleavage of eight phosphodiester bonds in DNA

    Science.gov (United States)

    Smith, Rachel M.; Marshall, Jacqueline J. T.; Jacklin, Alistair J.; Retter, Susan E.; Halford, Stephen E.; Sobott, Frank

    2013-01-01

    Type IIB restriction-modification systems, such as BcgI, feature a single protein with both endonuclease and methyltransferase activities. Type IIB nucleases require two recognition sites and cut both strands on both sides of their unmodified sites. BcgI cuts all eight target phosphodiester bonds before dissociation. The BcgI protein contains A and B polypeptides in a 2:1 ratio: A has one catalytic centre for each activity; B recognizes the DNA. We show here that BcgI is organized as A2B protomers, with B at its centre, but that these protomers self-associate to assemblies containing several A2B units. Moreover, like the well known FokI nuclease, BcgI bound to its site has to recruit additional protomers before it can cut DNA. DNA-bound BcgI can alternatively be activated by excess A subunits, much like the activation of FokI by its catalytic domain. Eight A subunits, each with one centre for nuclease activity, are presumably needed to cut the eight bonds cleaved by BcgI. Its nuclease reaction may thus involve two A2B units, each bound to a recognition site, with two more A2B units bridging the complexes by protein–protein interactions between the nuclease domains. PMID:23147005

  3. Tropical Journal of Pharmaceutical Research - Vol 11, No 5 (2012)

    African Journals Online (AJOL)

    A Qualitative and Quantitative Assay to Study DNA/Drug Interaction Based on Sequence Selective Inhibition of Restriction Endonucleases · EMAIL FREE FULL ... Relaxant Activity of the Methanol Extract of Acanthus Montanus (Nees) T Anderson (Acanthaceae) on Isolated Guinea Pig Trachea · EMAIL FREE FULL TEXT ...

  4. The entire β-globin gene cluster is deleted in a form of τδβ-thalassemia.

    NARCIS (Netherlands)

    E.R. Fearon; H.H.Jr. Kazazian; P.G. Waber (Pamela); J.I. Lee (Joseph); S.E. Antonarakis; S.H. Orkin (Stuart); E.F. Vanin; P.S. Henthorn; F.G. Grosveld (Frank); A.F. Scott; G.R. Buchanan

    1983-01-01

    textabstractWe have used restriction endonuclease mapping to study a deletion involving the beta-globin gene cluster in a Mexican-American family with gamma delta beta-thalassemia. Analysis of DNA polymorphisms demonstrated deletion of the beta-globin gene from the affected chromosome. Using a DNA

  5. Structure of the motor subunit of type I restriction-modification complex EcoR124I

    Czech Academy of Sciences Publication Activity Database

    Lapkouski, Mikalai; Panjikar, S.; Janscak, P.; Kutá-Smatanová, Ivana; Carrey, J.; Ettrich, Rüdiger; Cséfalvay, Eva

    2009-01-01

    Roč. 16, č. 1 (2009), s. 94-95 ISSN 1545-9985 R&D Projects: GA MŠk LC06073 Institutional research plan: CEZ:AV0Z60870520 Keywords : SWI2/SNF2 ATPASE * DNA * endonuclease * protein * translocation * helicase * treckings * enzymes * core * time Subject RIV: CE - Biochemistry Impact factor: 12.273, year: 2009

  6. A simple, rapid and efficient method for the extraction of genomic ...

    African Journals Online (AJOL)

    The isolation of intact, high-molecular-mass genomic DNA is essential for many molecular biology applications including long range PCR, endonuclease restriction digestion, southern blot analysis, and genomic library construction. Many protocols are available for the extraction of DNA from plant material, but obtain it is ...

  7. Nucleobase protection strategy for gene cloning and expression

    Czech Academy of Sciences Publication Activity Database

    Kielkowski, Pavel; Brock, N. L.; Dickschat, J. S.; Hocek, Michal

    2013-01-01

    Roč. 14, č. 7 (2013), s. 801-804 ISSN 1439-4227 R&D Projects: GA ČR GA203/09/0317 Institutional support: RVO:61388963 Keywords : DNA * polymerase chain reaction * protecting groups * protein production * restriction endonucleases Subject RIV: CE - Biochemistry Impact factor: 3.060, year: 2013

  8. "Sickle Cell Anemia: Tracking down a Mutation": An Interactive Learning Laboratory That Communicates Basic Principles of Genetics and Cellular Biology

    Science.gov (United States)

    Jarrett, Kevin; Williams, Mary; Horn, Spencer; Radford, David; Wyss, J. Michael

    2016-01-01

    "Sickle cell anemia: tracking down a mutation" is a full-day, inquiry-based, biology experience for high school students enrolled in genetics or advanced biology courses. In the experience, students use restriction endonuclease digestion, cellulose acetate gel electrophoresis, and microscopy to discover which of three putative patients…

  9. Functional Coupling of Duplex Translocation to DNA Cleavage in a Type I Restriction Enzyme

    Czech Academy of Sciences Publication Activity Database

    Cséfalvay, Eva; Lapkouski, Mikalai; Guzanová, Alena; Cséfalvay, Ladislav; Baikova, T.; Shevelev, Igor; Bialevich, V.; Shamayeva, Katerina; Janščák, Pavel; Kutá-Smatanová, Ivana; Panjikar, S.; Carey, J.; Weiserová, Marie; Ettrich, Rüdiger

    2015-01-01

    Roč. 10, č. 6 (2015), e0128700 E-ISSN 1932-6203 R&D Projects: GA ČR GAP207/12/2323; GA ČR GAP305/10/0281 Institutional support: RVO:67179843 ; RVO:61388971 ; RVO:68378050 Keywords : Escherichia-Coli * Endonuclease ecor1241 * HSDR subunit * RECBCD enzyme * proteins * genes * helicase * sequence * family * domain Subject RIV: CE - Biochemistry Impact factor: 3.057, year: 2015

  10. Restrictions and Proportionality

    DEFF Research Database (Denmark)

    Werlauff, Erik

    2009-01-01

    The article discusses three central aspects of the freedoms under European Community law, namely 1) the prohibition against restrictions as an important extension of the prohibition against discrimination, 2) a prohibition against exit restrictions which is just as important as the prohibition...... against host country restrictions, but which is often not recognised to the same extent by national law, and 3) the importance of also identifying and recognising an exit restriction, so that it is possible to achieve the required test of appropriateness and proportionality in relation to the rule...

  11. HLA-DPB1 typing with polymerase chain reaction and restriction fragment length polymorphism technique in Danes

    DEFF Research Database (Denmark)

    Hviid, T V; Madsen, H O; Morling, N

    1992-01-01

    endonucleases: RsaI, FokI, ApaI, SacI, BstUI, EcoNI, and DdeI, and the DNA fragments were separated by electrophoresis in agarose gels. Altogether, 71 individuals were investigated and 16 different HLA-DPB1 types were observed in 26 different heterozygotic combinations, as well as five possible homozygotes...

  12. Modulation of the Pyrococcus abyssi NucS endonuclease activity by replication clamp at functional and structural levels.

    Science.gov (United States)

    Creze, Christophe; Ligabue, Alessio; Laurent, Sébastien; Lestini, Roxane; Laptenok, Sergey P; Khun, Joelle; Vos, Marten H; Czjzek, Mirjam; Myllykallio, Hannu; Flament, Didier

    2012-05-04

    Pyrococcus abyssi NucS is the founding member of a new family of structure-specific DNA endonucleases that interact with the replication clamp proliferating cell nuclear antigen (PCNA). Using a combination of small angle x-ray scattering and surface plasmon resonance analyses, we demonstrate the formation of a stable complex in solution, in which one molecule of the PabNucS homodimer binds to the outside surface of the PabPCNA homotrimer. Using fluorescent labels, PCNA is shown to increase the binding affinity of NucS toward single-strand/double-strand junctions on 5' and 3' flaps, as well as to modulate the cleavage specificity on the branched DNA structures. Our results indicate that the presence of a single major contact between the PabNucS and PabPCNA proteins, together with the complex-induced DNA bending, facilitate conformational flexibility required for specific cleavage at the single-strand/double-strand DNA junction.

  13. Modulation of the Pyrococcus abyssi NucS Endonuclease Activity by Replication Clamp at Functional and Structural Levels*

    Science.gov (United States)

    Creze, Christophe; Ligabue, Alessio; Laurent, Sébastien; Lestini, Roxane; Laptenok, Sergey P.; Khun, Joelle; Vos, Marten H.; Czjzek, Mirjam; Myllykallio, Hannu; Flament, Didier

    2012-01-01

    Pyrococcus abyssi NucS is the founding member of a new family of structure-specific DNA endonucleases that interact with the replication clamp proliferating cell nuclear antigen (PCNA). Using a combination of small angle x-ray scattering and surface plasmon resonance analyses, we demonstrate the formation of a stable complex in solution, in which one molecule of the PabNucS homodimer binds to the outside surface of the PabPCNA homotrimer. Using fluorescent labels, PCNA is shown to increase the binding affinity of NucS toward single-strand/double-strand junctions on 5′ and 3′ flaps, as well as to modulate the cleavage specificity on the branched DNA structures. Our results indicate that the presence of a single major contact between the PabNucS and PabPCNA proteins, together with the complex-induced DNA bending, facilitate conformational flexibility required for specific cleavage at the single-strand/double-strand DNA junction. PMID:22431731

  14. [Effect of endonuclease G depletion on plasmid DNA uptake and levels of homologous recombination in hela cells].

    Science.gov (United States)

    Misic, V; El-Mogy, M; Geng, S; Haj-Ahmad, Y

    2016-01-01

    Endonuclease G (EndoG) is a mitochondrial apoptosis regulator that also has roles outside of programmed cell death. It has been implicated as a defence DNase involved in the degradation of exogenous DNA after transfection of mammalian cells and in homologous recombination of viral and endogenous DNA. In this study, we looked at the effect of EndoG depletion on plasmid DNA uptake and the levels of homologous recombination in HeLa cells. We show that the proposed defence role of EndoG against uptake of non-viral DNA vectors does not extend to the cervical carcinoma HeLa cells, as targeting of EndoG expression by RNA interference failed to increase intracellular plasmid DNA levels. However, reducing EndoG levels in HeLa cells resulted in a statistically significant reduction of homologous recombination between two plasmid DNA substrates. These findings suggest that non-viral DNA vectors are also substrates for EndoG in its role in homologous recombination.

  15. Molecular phylogeny of Fusarium species by AFLP fingerprint ...

    African Journals Online (AJOL)

    The high-resolution genotyping method of amplified fragment length polymorphism (AFLP) analysis was used to study the genetic relationships within and between natural populations of five Fusarium spp. AFLP templates were prepared by the digestion of Fusarium DNA with EcoRI and MseI restriction endonucleases and ...

  16. Characterization of rat lines with normotensive and hypertensive status using genomic fingerprinting

    NARCIS (Netherlands)

    Adarichev, V A; Korokhov, N P; Ostapchuk, Ia V; Dymshits, G M; Markel', A L; Ostaptchouk, Jana

    1996-01-01

    The properties of normotensive and hypertensive rat lines were investigated by the DNA fingerprinting method using a multilocus micro-satellite (CAC)5 probe. The HaeIII and HinfI restriction endonucleases were found to be the most informative enzymes in this case. The high genetic homogeneity of the

  17. Preparation of short cytosine-modified oligonucleotides by nicking enzyme amplification reaction

    Czech Academy of Sciences Publication Activity Database

    Ménová, Petra; Hocek, Michal

    2012-01-01

    Roč. 48, č. 55 (2012), s. 6921-6923 ISSN 1359-7345 R&D Projects: GA ČR GA203/09/0317 Institutional support: RVO:61388963 Keywords : cross - coupling reactions * nucleoside triphosphates * functionalized DNA * restriction endonucleases * polymerase incorporation Subject RIV: CC - Organic Chemistry Impact factor: 6.378, year: 2012

  18. A Qualitative and Quantitative Assay to Study DNA/Drug Interaction ...

    African Journals Online (AJOL)

    Research Article. A Qualitative and Quantitative Assay to Study. DNA/Drug Interaction Based on Sequence Selective. Inhibition of Restriction Endonucleases. Syed A Hassan1*, Lata Chauhan2, Ritu Barthwal2 and Aparna Dixit3. 1 Faculty of Computing and Information Technology, King Abdul Aziz University, Rabigh-21911 ...

  19. Multipronged regulatory functions of a novel endonuclease (TieA) from Helicobacter pylori.

    Science.gov (United States)

    Devi, Savita; Ansari, Suhail A; Tenguria, Shivendra; Kumar, Naveen; Ahmed, Niyaz

    2016-11-02

    Helicobacter pylori portrays a classical paradigm of persistent bacterial infections. A well balanced homeostasis of bacterial effector functions and host responses is purported to be the key in achieving long term colonization in specific hosts. H. pylori nucleases have been shown to assist in natural transformation, but their role in virulence and colonization remains elusive. Therefore, it is imperative to understand the involvement of these nucleases in the pathogenesis of H. pylori Here, we report the multifaceted role of a TNFR-1 interacting endonuclease A (TieA) from H. pylori. tieA expression is differentially regulated in response to environmental stress and post adherence to gastric epithelial cells. Studies with isogenic knockouts of tieA revealed it to be a secretory protein which translocates into the host gastric epithelial cells independent of a type IV secretion system, gets phosphorylated by DNA-PK kinase and auto-phosphorylates as serine kinase. Furthermore, TieA binds to and cleaves DNA in a non-specific manner and promotes Fas mediated apoptosis in AGS cells. Additionally, TieA induced pro-inflammatory cytokine secretion via activation of transcription factor AP-1 and signaled through MAP kinase pathway. Collectively, TieA with its multipronged and moonlighting functions could facilitate H. pylori in maintaining a balance of bacterial adaptation, and elimination by the host responses. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  20. Hemolytic disease of the newborn caused by a new deletion of the entire beta-globin cluster.

    OpenAIRE

    Pirastu, M; Kan, Y W; Lin, C C; Baine, R M; Holbrook, C T

    1983-01-01

    We describe a new type of gamma delta beta-thalassemia in four generations of a family of Scotch-Irish descent. The proposita presented with hemolytic disease of the newborn, which was characterized by a microcytic anemia. Initial restriction endonuclease analysis of the DNA showed no grossly abnormal patterns, but studies of polymorphic restriction sites and gene dosage revealed an extensive deletion that removed all the beta- and beta-like globin genes from the affected chromosome. In situ ...

  1. Sequencing by ligation variation with endonuclease V digestion and deoxyinosine-containing query oligonucleotides

    Directory of Open Access Journals (Sweden)

    Ho Antoine

    2011-12-01

    Full Text Available Abstract Background Sequencing-by-ligation (SBL is one of several next-generation sequencing methods that has been developed for massive sequencing of DNA immobilized on arrayed beads (or other clonal amplicons. SBL has the advantage of being easy to implement and accessible to all because it can be performed with off-the-shelf reagents. However, SBL has the limitation of very short read lengths. Results To overcome the read length limitation, research groups have developed complex library preparation processes, which can be time-consuming, difficult, and result in low complexity libraries. Herein we describe a variation on traditional SBL protocols that extends the number of sequential bases that can be sequenced by using Endonuclease V to nick a query primer, thus leaving a ligatable end extended into the unknown sequence for further SBL cycles. To demonstrate the protocol, we constructed a known DNA sequence and utilized our SBL variation, cyclic SBL (cSBL, to resequence this region. Using our method, we were able to read thirteen contiguous bases in the 3' - 5' direction. Conclusions Combining this read length with sequencing in the 5' - 3' direction would allow a read length of over twenty bases on a single tage. Implementing mate-paired tags and this SBL variation could enable > 95% coverage of the genome.

  2. Efficient genome editing in hematopoietic stem cells with helper-dependent Ad5/35 vectors expressing site-specific endonucleases under microRNA regulation

    Directory of Open Access Journals (Sweden)

    Kamola Saydaminova

    Full Text Available Genome editing with site-specific endonucleases has implications for basic biomedical research as well as for gene therapy. We generated helper-dependent, capsid-modified adenovirus (HD-Ad5/35 vectors for zinc-finger nuclease (ZFN– or transcription activator-like effector nuclease (TALEN–mediated genome editing in human CD34+ hematopoietic stem cells (HSCs from mobilized adult donors. The production of these vectors required that ZFN and TALEN expression in HD-Ad5/35 producer 293-Cre cells was suppressed. To do this, we developed a microRNA (miRNA-based system for regulation of gene expression based on miRNA expression profiling of 293-Cre and CD34+ cells. Using miR-183-5p and miR-218-5p based regulation of transgene gene expression, we first produced an HD-Ad5/35 vector expressing a ZFN specific to the HIV coreceptor gene ccr5. We demonstrated that HD-Ad5/35.ZFNmiR vector conferred ccr5 knock out in primitive HSC (i.e., long-term culture initiating cells and NOD/SCID repopulating cells. The ccr5 gene disruption frequency achieved in engrafted HSCs found in the bone marrow of transplanted mice is clinically relevant for HIV therapy considering that these cells can give rise to multiple lineages, including all the lineages that represent targets and reservoirs for HIV. We produced a second HD-Ad5/35 vector expressing a TALEN targeting the DNase hypersensitivity region 2 (HS2 within the globin locus control region. This vector has potential for targeted gene correction in hemoglobinopathies. The miRNA regulated HD-Ad5/35 vector platform for expression of site-specific endonucleases has numerous advantages over currently used vectors as a tool for genome engineering of HSCs for therapeutic purposes.

  3. PAM-Dependent Target DNA Recognition and Cleavage by C2c1 CRISPR-Cas Endonuclease

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Hui; Gao, Pu; Rajashankar, Kanagalaghatta R.; Patel, Dinshaw J. (MSKCC); (Cornell); (Chinese Aca. Sci.)

    2016-12-01

    C2c1 is a newly identified guide RNA-mediated type V-B CRISPR-Cas endonuclease that site-specifically targets and cleaves both strands of target DNA. We have determined crystal structures of Alicyclobacillus acidoterrestris C2c1 (AacC2c1) bound to sgRNA as a binary complex and to target DNAs as ternary complexes, thereby capturing catalytically competent conformations of AacC2c1 with both target and non-target DNA strands independently positioned within a single RuvC catalytic pocket. Moreover, C2c1-mediated cleavage results in a staggered seven-nucleotide break of target DNA. crRNA adopts a pre-ordered five-nucleotide A-form seed sequence in the binary complex, with release of an inserted tryptophan, facilitating zippering up of 20-bp guide RNA:target DNA heteroduplex on ternary complex formation. Notably, the PAM-interacting cleft adopts a “locked” conformation on ternary complex formation. Structural comparison of C2c1 ternary complexes with their Cas9 and Cpf1 counterparts highlights the diverse mechanisms adopted by these distinct CRISPR-Cas systems, thereby broadening and enhancing their applicability as genome editing tools.

  4. Problem-Solving Test: Analysis of DNA Damage Recognizing Proteins in Yeast and Human Cells

    Science.gov (United States)

    Szeberenyi, Jozsef

    2013-01-01

    The experiment described in this test was aimed at identifying DNA repair proteins in human and yeast cells. Terms to be familiar with before you start to solve the test: DNA repair, germline mutation, somatic mutation, inherited disease, cancer, restriction endonuclease, radioactive labeling, [alpha-[superscript 32]P]ATP, [gamma-[superscript…

  5. Two mutant alleles of the human cytochrome P-450dbl gene (P450C2D1) associated with genetically deficient metabolism of debrisoquine and other drugs

    International Nuclear Information System (INIS)

    Skoda, R.C.; Gonzalez, F.L.; Demierre, A.; Meyer, R.A.

    1988-01-01

    The debrisoquine polymorphism is a clinically important genetic defect of drug metabolism affecting 5-10% of individuals in Caucasian populations. It is inherited as an autosomal recessive trait. A full-length cDNA for human cytochrome P-450db1, the deficient enzyme (also designated P450IID1 for P450 family II subfamily D isozyme 1), has recently been cloned. Leukocyte DNA from extensive metabolizers (EMs) or poor metabolizers (PMs) of debrisoquine was examined by Southern analysis. Two polymorphic restriction fragments were associated with the PM phenotype when DNAs from 24 unrelated PM and 29 unrelated EM individuals were probed with P-450db1 cDNA after digestion with Xba I restriction endonuclease and Southern blotting. Seventy-five percent of PMs had either the 44-kb or the 11.5-kb fragment or both. Segregation of these restriction fragment length polymorphisms in the families of six PM probands demonstrated that each of the two fragments is allelic with the 29-kb fragment present in all EM individuals and suggests that they identify two independent mutated alleles of the P-450db1 gene (designated P450C2D1). The Xba I 44-kb fragment and 11.5-kb fragment were in linkage disequilibrium with restriction fragment length polymorphisms generated by four and five additional restriction endonucleases, respectively, which can be used to identify the same mutant alleles for the P-450db1 gene

  6. Total sequence decomposition distinguishes functional modules, "molegos" in apurinic/apyrimidinic endonucleases

    Directory of Open Access Journals (Sweden)

    Braun Werner

    2002-11-01

    Full Text Available Abstract Background Total sequence decomposition, using the web-based MASIA tool, identifies areas of conservation in aligned protein sequences. By structurally annotating these motifs, the sequence can be parsed into individual building blocks, molecular legos ("molegos", that can eventually be related to function. Here, the approach is applied to the apurinic/apyrimidinic endonuclease (APE DNA repair proteins, essential enzymes that have been highly conserved throughout evolution. The APEs, DNase-1 and inositol 5'-polyphosphate phosphatases (IPP form a superfamily that catalyze metal ion based phosphorolysis, but recognize different substrates. Results MASIA decomposition of APE yielded 12 sequence motifs, 10 of which are also structurally conserved within the family and are designated as molegos. The 12 motifs include all the residues known to be essential for DNA cleavage by APE. Five of these molegos are sequentially and structurally conserved in DNase-1 and the IPP family. Correcting the sequence alignment to match the residues at the ends of two of the molegos that are absolutely conserved in each of the three families greatly improved the local structural alignment of APEs, DNase-1 and synaptojanin. Comparing substrate/product binding of molegos common to DNase-1 showed that those distinctive for APEs are not directly involved in cleavage, but establish protein-DNA interactions 3' to the abasic site. These additional bonds enhance both specific binding to damaged DNA and the processivity of APE1. Conclusion A modular approach can improve structurally predictive alignments of homologous proteins with low sequence identity and reveal residues peripheral to the traditional "active site" that control the specificity of enzymatic activity.

  7. Measuring the Restrictiveness of Living Environments for Children and Youth: Reconceptualizing Restriction

    Science.gov (United States)

    Rauktis, Mary E.; Huefner, Jonathan C.; O'Brien, Kirk; Pecora, Peter J.; Doucette, Ann; Thompson, Ronald W.

    2009-01-01

    The "Restrictiveness of Living Environment Scale" has long been the primary way to conceptualize the "restrictiveness" of a child's living situation. However, changes in systems of care and other factors have created a need to revisit how restrictiveness is conceptualized and measured. A measure was created to assess an environment's level of…

  8. Problem-Based Test: Functional Analysis of Mutant 16S rRNAs

    Science.gov (United States)

    Szeberenyi, Jozsef

    2010-01-01

    Terms to be familiar with before you start to solve the test: ribosome, ribosomal subunits, antibiotics, point mutation, 16S, 5S, and 23S rRNA, Shine-Dalgarno sequence, mRNA, tRNA, palindrome, hairpin, restriction endonuclease, fMet-tRNA, peptidyl transferase, initiation, elongation, termination of translation, expression plasmid, transformation,…

  9. Genetic variability of cultivated cowpea in Benin assessed by ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-12-17

    Dec 17, 2008 ... Res. Crop Evol. 48: 559-566. Mignouna HD, Ng NQ, Ikea J, Thottapilly G (1998). Genetic diversity in cowpea as revealed by random amplified polymorphic DNA. J. Gen. Breed. 52: 151-159. Nei M, Li WH (1979). Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc.

  10. Detection and RFLP Analysis of Canine Parvovirus (CPV) DNA by Polymerase Chain Reaction (PCR) in a Dog

    OpenAIRE

    ÖZKUL, Aykut

    2002-01-01

    In this study, the detection of canine parvovirus (CPV) in a fecal sample from a dog with enteritis was performed for the first time using the polymerase chain reaction (PCR) in Turkey. The final PCR product was analyzed using the restriction fragment length polymorphysm (RFLP) technique. RFLP analysis using Apa LI and Eco RV restriction endonucleases revealed homology in the nucleotide sequence in at least the VP2 coding region of the virus DNAs detected in the fecal specimen and prepared fr...

  11. Aromatic residues located close to the active center are essential for the catalytic reaction of flap endonuclease-1 from hyperthermophilic archaeon Pyrococcus horikoshii.

    Science.gov (United States)

    Matsui, Eriko; Abe, Junko; Yokoyama, Hideshi; Matsui, Ikuo

    2004-04-16

    Flap endonuclease-1 (FEN-1) possessing 5'-flap endonuclease and 5'-->3' exonuclease activity plays important roles in DNA replication and repair. In this study, the kinetic parameters of mutants at highly conserved aromatic residues, Tyr33, Phe35, Phe79, and Phe278-Phe279, in the vicinity of the catalytic centers of FEN-1 were examined. The substitution of these aromatic residues with alanine led to a large reduction in kcat values, although these mutants retained Km values similar to that of the wild-type enzyme. Notably, the kcat of Y33A and F79A decreased 333-fold and 71-fold, respectively, compared with that of the wild-type enzyme. The aromatic residues Tyr33 and Phe79, and the aromatic cluster Phe278-Phe279 mainly contributed to the recognition of the substrates without the 3' projection of the upstream strand (the nick, 5'-recess-end, single-flap, and pseudo-Y substrates) for the both exo- and endo-activities, but played minor roles in recognizing the substrates with the 3' projection (the double flap substrate and the nick substrate with the 3' projection). The replacement of Tyr33, Phe79, and Phe278-Phe279, with non-charged aromatic residues, but not with aliphatic hydrophobic residues, recovered the kcat values almost fully for the substrates without the 3' projection of the upstream strand, suggesting that the aromatic groups of Tyr33, Phe79, and Phe278-Phe279 might be involved in the catalytic reaction, probably via multiple stacking interactions with nucleotide bases. The stacking interactions of Tyr33 and Phe79 might play important roles in fixing the template strand and the downstream strand, respectively, in close proximity to the active center to achieve the productive transient state leading to the hydrolysis.

  12. Identification of four families of yCCR4- and Mg2+-dependent endonuclease-related proteins in higher eukaryotes, and characterization of orthologs of yCCR4 with a conserved leucine-rich repeat essential for hCAF1/hPOP2 binding

    Directory of Open Access Journals (Sweden)

    Corbo Laura

    2001-11-01

    Full Text Available Abstract Background The yeast yCCR4 factor belongs to the CCR4-NOT transcriptional regulatory complex, in which it interacts, through its leucine-rich repeat (LRR motif with yPOP2. Recently, yCCR4 was shown to be a component of the major cytoplasmic mRNA deadenylase complex, and to contain a fold related to the Mg2+-dependent endonuclease core. Results Here, we report the identification of nineteen yCCR4-related proteins in eukaryotes (including yeast, plants and animals, which all contain the yCCR4 endonuclease-like fold, with highly conserved CCR4-specific residues. Phylogenetic and genomic analyses show that they form four distinct families, one of which contains the yCCR4 orthologs. The orthologs in animals possess a leucine-rich repeat domain. We show, using two-hybrid and far-Western assays, that the human member binds to the human yPOP2 homologs, i.e. hCAF1 and hPOP2, in a LRR-dependent manner. Conclusions We have identified the mammalian orthologs of yCCR4 and have shown that the human member binds to the human yPOP2 homologs, thus strongly suggesting conservation of the CCR4-NOT complex from yeast to human. All members of the four identified yCCR4-related protein families show stricking conservation of the endonuclease-like catalytic motifs of the yCCR4 C-terminal domain and therefore constitute a new family of potential deadenylases in mammals.

  13. Phosphate steering by Flap Endonuclease 1 promotes 5′-flap specificity and incision to prevent genome instability

    KAUST Repository

    Tsutakawa, Susan E.

    2017-06-27

    DNA replication and repair enzyme Flap Endonuclease 1 (FEN1) is vital for genome integrity, and FEN1 mutations arise in multiple cancers. FEN1 precisely cleaves single-stranded (ss) 5\\'-flaps one nucleotide into duplex (ds) DNA. Yet, how FEN1 selects for but does not incise the ss 5\\'-flap was enigmatic. Here we combine crystallographic, biochemical and genetic analyses to show that two dsDNA binding sites set the 5\\'polarity and to reveal unexpected control of the DNA phosphodiester backbone by electrostatic interactions. Via phosphate steering\\', basic residues energetically steer an inverted ss 5\\'-flap through a gateway over FEN1\\'s active site and shift dsDNA for catalysis. Mutations of these residues cause an 18,000-fold reduction in catalytic rate in vitro and large-scale trinucleotide (GAA) repeat expansions in vivo, implying failed phosphate-steering promotes an unanticipated lagging-strand template-switch mechanism during replication. Thus, phosphate steering is an unappreciated FEN1 function that enforces 5\\'-flap specificity and catalysis, preventing genomic instability.

  14. Using Group II Introns for Attenuating the In Vitro and In Vivo Expression of a Homing Endonuclease.

    Directory of Open Access Journals (Sweden)

    Tuhin Kumar Guha

    Full Text Available In Chaetomium thermophilum (DSM 1495 within the mitochondrial DNA (mtDNA small ribosomal subunit (rns gene a group IIA1 intron interrupts an open reading frame (ORF encoded within a group I intron (mS1247. This arrangement offers the opportunity to examine if the nested group II intron could be utilized as a regulatory element for the expression of the homing endonuclease (HEase. Constructs were generated where the codon-optimized ORF was interrupted with either the native group IIA1 intron or a group IIB type intron. This study showed that the expression of the HEase (in vivo in Escherichia coli can be regulated by manipulating the splicing efficiency of the HEase ORF-embedded group II introns. Exogenous magnesium chloride (MgCl2 stimulated the expression of a functional HEase but the addition of cobalt chloride (CoCl2 to growth media antagonized the expression of HEase activity. Ultimately the ability to attenuate HEase activity might be useful in precision genome engineering, minimizing off target activities, or where pathways have to be altered during a specific growth phase.

  15. Epidemiologic analysis of sporadic Salmonella typhi isolates and those from outbreaks by pulsed-field gel electrophoresis.

    Science.gov (United States)

    Thong, K L; Cheong, Y M; Puthucheary, S; Koh, C L; Pang, T

    1994-05-01

    Pulsed-field gel electrophoresis (PFGE) was used to compare and analyze 158 isolates of Salmonella typhi from five well-defined outbreaks of typhoid fever in Malaysia and also isolates involved in sporadic cases of typhoid fever occurring during the same period. Digestion of chromosomal DNAs from these S. typhi isolates with the restriction endonucleases XbaI (5'-TCTAGA-3'), SpeI (5'-ACTAGT-3'), and AvrII (5'-CCTAGG-3') and then PFGE produced restriction endonuclease analysis (REA) patterns consisting of 11 to 24 DNA fragments ranging in size from 20 to 630 kbp. Analysis of the REA patterns generated by PFGE after digestion with XbaI and SpeI indicated that the S. typhi isolates obtained from sporadic cases of infection were much more heterogeneous (at least 13 different REA patterns were detected; Dice coefficient, between 0.73 and 1.0) than those obtained during outbreaks of typhoid fever. The clonal nature and the close genetic identities of isolates from outbreaks in Alor Setar, Penang, Kota Kinabalu, Johor Bahru, and Kota Bahru were suggested by the fact that only a limited number of REA patterns, which mostly differed by only a single band, were detected (one to four patterns; Dice coefficient, between 0.82 and 1.0), although a different pattern was associated with each of these outbreaks. Comparison of REA patterns with ribotyping for 18 S. typhi isolates involved in sporadic cases of infection showed a good correlation, in that 72% of the isolates were in the same group. There was no clear correlation of phage types with a specific REA pattern. We conclude that PFGE of s. typhi chromosomal DNA digested with infrequently cutting restriction endonucleases is a useful method for comparing and differentiating S. typhi isolates for epidemiological purposes.

  16. RFLP of analyses of an intergenic spacer region of chloroplast DNA ...

    African Journals Online (AJOL)

    user

    2006-11-16

    Nov 16, 2006 ... amplified with PCR and digested with 6 restriction endonucleases (Hpa II, Alu I, Hinc II, Ava III, Nde I and. Hae III). According to results ... the environment, but DNA based techniques represent reliable tools and do not have many of ... existence of some variations in gene content (Downie and Palmer, 1992).

  17. Terminal Restriction Fragment Length Polymorphism for the Identification of Spirorchiid Ova in Tissues from the Green Sea Turtle, Chelonia mydas.

    Directory of Open Access Journals (Sweden)

    Phoebe A Chapman

    Full Text Available Blood flukes are among the most common disease causing pathogens infecting vertebrates, including humans and some of the world's most globally endangered fauna. Spirorchiid blood flukes are parasites of marine turtles, and are associated with pathology, strandings and mortalities worldwide. Their ova embolize in tissues and incite significant inflammatory responses, however attempts to draw correlations between species and lesions are frustrated by difficulties in identifying ova beyond the genus level. In this study, a newly developed terminal restriction fragment length polymorphism (T-RFLP method was validated as a tool for differentiating between mixed spirorchiid ova in turtle tissue. Initially, a multiplex PCR was used to differentiate between the five genera of spirorchiid flukes. Following this, PCR was performed using genus/genera-specific fluorescently tagged primer pairs and PCR products digested analysis using restriction endonucleases. Using capillary electrophoresis, this T-RFLP method could differentiate between twelve species and genotypes of spirorchiid flukes in turtles. It was applied to 151 tissue samples and successfully identified the spirorchiid species present. It was found to be more sensitive than visual diagnosis, detecting infections in 28 of 32 tissues that were negative on histology. Spirorchiids were present in 96.7% of tissues tested, with Neospirorchis genotype 2 being the most prevalent, present in 93% of samples. Mixed infections were common, being present in 60.7% of samples tested. The method described here is, to our knowledge, the first use of the T-RFLP technique on host tissues or in an animal ecology context, and describes a significant advancement in the clinical capacity to diagnose a common cause of illness in our environment. It is proven as a sensitive, specific and cost-efficient means of identifying spirorchiid flukes and ova in turtles, with the potential to contribute valuable information to

  18. Mapping of the human APOB gene to chromosome 2p and demonstration of a two-allele restriction fragment length polymorphism

    International Nuclear Information System (INIS)

    Huang, L.; Miller, D.A.; Bruns, G.A.P.; Breslow, J.L.

    1986-01-01

    ApoB is a large glycoprotein with an apparent molecular mass of 550 kDa on NaDodSO 4 /PAGE. Recently, apoB cDNA clones have been isolated from an expression library made with mRNA from a human hepatoma cell line. These clones, which were all 1.5-1.6 kilobases (kb) long and corresponded to the 3' end of apoB mRNA, were used to demonstrate that hepatic apoB mRNA is ≅ 22 kb long. In the current report, a probe derived from one of these cDNA clones, pB8, was used for in situ hybridization experiments to map the human gene for apoB, APOB, to the distal half of the short arm of chromosome 2. This probe was also used to analyze somatic cell hybrids and, in agreement with the in situ hybridization studies, concordancy was demonstrated with chromosome 2. In addition, two hybrids with chromosome 2 translocations that contain only the short arm reacted with the pB8 probe. A third hybrid with a complex rearrangement of chromosome 2, which deleted an interstitial region and the tip of the short arm of chromosome 2, did not react. These data indicate that APOB maps to either 2p21-p23 or 2p24-pter. In further studies, DNA from normal individuals, digested with the restriction endonuclease EcoRI and subjected to Southern blot analysis with the pB8 probe, revealed a two-allele restriction fragment length polymorphism (RFLP). The mapping studies provide the means for understanding the relationship of the APOB locus to others in the human genome, whereas the demonstration of an APOB RFLP increases their ability to assess the role of this locus in determining plasma lipoprotein levels

  19. Human AP Endonuclease 1: A Potential Marker for the Prediction of Environmental Carcinogenesis Risk

    Directory of Open Access Journals (Sweden)

    Jae Sung Park

    2014-01-01

    Full Text Available Human apurinic/apyrimidinic endonuclease 1 (APE1 functions mainly in DNA repair as an enzyme removing AP sites and in redox signaling as a coactivator of various transcription factors. Based on these multifunctions of APE1 within cells, numerous studies have reported that the alteration of APE1 could be a crucial factor in development of human diseases such as cancer and neurodegeneration. In fact, the study on the combination of an individual’s genetic make-up with environmental factors (gene-environment interaction is of great importance to understand the development of diseases, especially lethal diseases including cancer. Recent reports have suggested that the human carcinogenic risk following exposure to environmental toxicants is affected by APE1 alterations in terms of gene-environment interactions. In this review, we initially outline the critical APE1 functions in the various intracellular mechanisms including DNA repair and redox regulation and its roles in human diseases. Several findings demonstrate that the change in expression and activity as well as genetic variability of APE1 caused by environmental chemical (e.g., heavy metals and cigarette smoke and physical carcinogens (ultraviolet and ionizing radiation is likely associated with various cancers. These enable us to ultimately suggest APE1 as a vital marker for the prediction of environmental carcinogenesis risk.

  20. Bacteriophages use hypermodified nucleosides to evade host's defence systems

    DEFF Research Database (Denmark)

    Kot, Witold; Olsen, Nikoline S.; Carstens, Alexander Byth

    developed several strategies to evade these defence mechanisms. Ultimately, this led to the oldest and still running arms race - microorganisms vs. their molecular parasites. We here describe a remarkable new strategy used by the recently isolated Escherichia coli phage CAjan belonging to...... to investigate this mechanism in detail we have used several methods including direct plaque sequencing, restriction endonuclease analysis and CRISPR-Cas genome editing. Through generation of specific mutants, we were able to introduce a restriction sensitive phenotype in the CAjan bacteriophage providing new...

  1. Evaluation of the use of amplified 16S rRNA gene-restriction fragment length polymorphism analysis to detect enterobacter cloacae and bacillus licheniformis for microbial enhanced oil recovery field pilot

    Energy Technology Data Exchange (ETDEWEB)

    Fujiwara, Kazuhiro; Tanaka, Shinji; Otsuka, Makiko; Ichimura, Naoya [Lansai Research Institute, Kyoto (Japan); Yonebayashi, Hideharu [Japan National Oil Corp., Chiba (Japan); Hong, Chengxie; Enomoto, Heiji [Tohoku University, Miyagi (Japan)

    1999-09-01

    Evaluation of effectiveness of restriction fragment length polymorphism (RFLP) analysis of the 16S rRNA gene of microorganisms injected into an oil reservoir, for monitoring their levels over time, was conducted. Two microorganisms, enterobacter cloacae TRC-322 and Bacillus licheniformis TRC-18-2-a, were focused in this paper among the microorganisms selected for injection, and gene fragments of the 16S rRNA gene of these microorganisms were amplified by polymerase chain reaction (PCP), using one set of universal primers. Samples of the reservoir brine and reservoir rock were obtained; the microorganisms inhabiting in the reservoir were isolated from these samples, and the 16S rRNA gene of these microorganisms was amplified, condition remaining the same. RFLP analysis was performed on the 16S rRNA gene of each of these microorganisms, using restriction endonucleases HhaI, MspI, AluI and TaqI as necessary. Comparison of the resultant rRNA gene fragments, demonstrated that closely-related species displaying RFLP profile similar to that of E. cloacae TRC-322 or B. licheniformis TRC-18-2-a were not among the microorganisms isolated from the reservoir. PCR-RFLP analysis of the 16S rRNA gene, using the protocol; presented in this paper, is effective to detect the presence appropriate injecting microorganisms. This method was also effective for studying microorganisms isolated from the reservoir, which have the ability to grow on a molasses. (author)

  2. Site- and strand-specific nicking of DNA by fusion proteins derived from MutH and I-SceI or TALE repeats.

    Science.gov (United States)

    Gabsalilow, Lilia; Schierling, Benno; Friedhoff, Peter; Pingoud, Alfred; Wende, Wolfgang

    2013-04-01

    Targeted genome engineering requires nucleases that introduce a highly specific double-strand break in the genome that is either processed by homology-directed repair in the presence of a homologous repair template or by non-homologous end-joining (NHEJ) that usually results in insertions or deletions. The error-prone NHEJ can be efficiently suppressed by 'nickases' that produce a single-strand break rather than a double-strand break. Highly specific nickases have been produced by engineering of homing endonucleases and more recently by modifying zinc finger nucleases (ZFNs) composed of a zinc finger array and the catalytic domain of the restriction endonuclease FokI. These ZF-nickases work as heterodimers in which one subunit has a catalytically inactive FokI domain. We present two different approaches to engineer highly specific nickases; both rely on the sequence-specific nicking activity of the DNA mismatch repair endonuclease MutH which we fused to a DNA-binding module, either a catalytically inactive variant of the homing endonuclease I-SceI or the DNA-binding domain of the TALE protein AvrBs4. The fusion proteins nick strand specifically a bipartite recognition sequence consisting of the MutH and the I-SceI or TALE recognition sequences, respectively, with a more than 1000-fold preference over a stand-alone MutH site. TALE-MutH is a programmable nickase.

  3. The restriction-modification genes of Escherichia coli K-12 may not be selfish: they do not resist loss and are readily replaced by alleles conferring different specificities.

    Science.gov (United States)

    O'Neill, M; Chen, A; Murray, N E

    1997-12-23

    Type II restriction and modification (R-M) genes have been described as selfish because they have been shown to impose selection for the maintenance of the plasmid that encodes them. In our experiments, the type I R-M system EcoKI does not behave in the same way. The genes specifying EcoKI are, however, normally residents of the chromosome and therefore our analyses were extended to monitor the deletion of chromosomal genes rather than loss of plasmid vector. If EcoKI were to behave in the same way as the plasmid-encoded type II R-M systems, the loss of the relevant chromosomal genes by mutation or recombination should lead to cell death because the cell would become deficient in modification enzyme and the bacterial chromosome would be vulnerable to the restriction endonuclease. Our data contradict this prediction; they reveal that functional type I R-M genes in the chromosome are readily replaced by mutant alleles and by alleles encoding a type I R-M system of different specificity. The acquisition of allelic genes conferring a new sequence specificity, but not the loss of the resident genes, is dependent on the product of an unlinked gene, one predicted [Prakash-Cheng, A., Chung, S. S. & Ryu, J. (1993) Mol. Gen. Genet. 241, 491-496] to be relevant to control of expression of the genes that encode EcoKI. Our evidence suggests that not all R-M systems are evolving as "selfish" units; rather, the diversity and distribution of the family of type I enzymes we have investigated require an alternative selective pressure.

  4. Aging, adiposity, and calorie restriction.

    Science.gov (United States)

    Fontana, Luigi; Klein, Samuel

    2007-03-07

    Excessive calorie intake and subsequent obesity increases the risk of developing chronic disease and decreases life expectancy. In rodent models, calorie restriction with adequate nutrient intake decreases the risk of developing chronic disease and extends maximum life span. To evaluate the physiological and clinical implications of calorie restriction with adequate nutrient intake. Search of PubMed (1966-December 2006) using terms encompassing various aspects of calorie restriction, dietary restriction, aging, longevity, life span, adiposity, and obesity; hand search of journals that focus on obesity, geriatrics, or aging; and search of reference lists of pertinent research and review articles and books. Reviewed reports (both basic science and clinical) included epidemiologic studies, case-control studies, and randomized controlled trials, with quality of data assessed by taking into account publication in a peer-reviewed journal, number of animals or individuals studied, objectivity of measurements, and techniques used to minimize bias. It is not known whether calorie restriction extends maximum life span or life expectancy in lean humans. However, calorie restriction in adult men and women causes many of the same metabolic adaptations that occur in calorie-restricted rodents and monkeys, including decreased metabolic, hormonal, and inflammatory risk factors for diabetes, cardiovascular disease, and possibly cancer. Excessive calorie restriction causes malnutrition and has adverse clinical effects. Calorie restriction in adult men and women causes beneficial metabolic, hormonal, and functional changes, but the precise amount of calorie intake or body fat mass associated with optimal health and maximum longevity in humans is not known. In addition, it is possible that even moderate calorie restriction may be harmful in specific patient populations, such as lean persons who have minimal amounts of body fat.

  5. The GAN Exonuclease or the Flap Endonuclease Fen1 and RNase HII Are Necessary for Viability of Thermococcus kodakarensis.

    Science.gov (United States)

    Burkhart, Brett W; Cubonova, Lubomira; Heider, Margaret R; Kelman, Zvi; Reeve, John N; Santangelo, Thomas J

    2017-07-01

    Many aspects of and factors required for DNA replication are conserved across all three domains of life, but there are some significant differences surrounding lagging-strand synthesis. In Archaea , a 5'-to-3' exonuclease, related to both bacterial RecJ and eukaryotic Cdc45, that associates with the replisome specifically through interactions with GINS was identified and designated GAN (for G INS- a ssociated n uclease). Despite the presence of a well-characterized flap endonuclease (Fen1), it was hypothesized that GAN might participate in primer removal during Okazaki fragment maturation, and as a Cdc45 homologue, GAN might also be a structural component of an archaeal CMG (Cdc45, MCM, and GINS) replication complex. We demonstrate here that, individually, either Fen1 or GAN can be deleted, with no discernible effects on viability and growth. However, deletion of both Fen1 and GAN was not possible, consistent with both enzymes catalyzing the same step in primer removal from Okazaki fragments in vivo RNase HII has also been proposed to participate in primer processing during Okazaki fragment maturation. Strains with both Fen1 and RNase HII deleted grew well. GAN activity is therefore sufficient for viability in the absence of both RNase HII and Fen1, but it was not possible to construct a strain with both RNase HII and GAN deleted. Fen1 alone is therefore insufficient for viability in the absence of both RNase HII and GAN. The ability to delete GAN demonstrates that GAN is not required for the activation or stability of the archaeal MCM replicative helicase. IMPORTANCE The mechanisms used to remove primer sequences from Okazaki fragments during lagging-strand DNA replication differ in the biological domains. Bacteria use the exonuclease activity of DNA polymerase I, whereas eukaryotes and archaea encode a flap endonuclease (Fen1) that cleaves displaced primer sequences. RNase HII and the GINS-associated exonuclease GAN have also been hypothesized to assist in primer

  6. Dietary restriction with and without caloric restriction for healthy aging [version 1; referees: 3 approved

    Directory of Open Access Journals (Sweden)

    Changhan Lee

    2016-01-01

    Full Text Available Caloric restriction is the most effective and reproducible dietary intervention known to regulate aging and increase the healthy lifespan in various model organisms, ranging from the unicellular yeast to worms, flies, rodents, and primates. However, caloric restriction, which in most cases entails a 20–40% reduction of food consumption relative to normal intake, is a severe intervention that results in both beneficial and detrimental effects. Specific types of chronic, intermittent, or periodic dietary restrictions without chronic caloric restriction have instead the potential to provide a significant healthspan increase while minimizing adverse effects. Improved periodic or targeted dietary restriction regimens that uncouple the challenge of food deprivation from the beneficial effects will allow a safe intervention feasible for a major portion of the population. Here we focus on healthspan interventions that are not chronic or do not require calorie restriction.

  7. Mitochondrial Targeted Endonuclease III DNA Repair Enzyme Protects against Ventilator Induced Lung Injury in Mice

    Directory of Open Access Journals (Sweden)

    Masahiro Hashizume

    2014-08-01

    Full Text Available The mitochondrial targeted DNA repair enzyme, 8-oxoguanine DNA glycosylase 1, was previously reported to protect against mitochondrial DNA (mtDNA damage and ventilator induced lung injury (VILI. In the present study we determined whether mitochondrial targeted endonuclease III (EndoIII which cleaves oxidized pyrimidines rather than purines from damaged DNA would also protect the lung. Minimal injury from 1 h ventilation at 40 cmH2O peak inflation pressure (PIP was reversed by EndoIII pretreatment. Moderate lung injury due to ventilation for 2 h at 40 cmH2O PIP produced a 25-fold increase in total extravascular albumin space, a 60% increase in W/D weight ratio, and marked increases in MIP-2 and IL-6. Oxidative mtDNA damage and decreases in the total tissue glutathione (GSH and the GSH/GSSH ratio also occurred. All of these indices of injury were attenuated by mitochondrial targeted EndoIII. Massive lung injury caused by 2 h ventilation at 50 cmH2O PIP was not attenuated by EndoIII pretreatment, but all untreated mice died prior to completing the two hour ventilation protocol, whereas all EndoIII-treated mice lived for the duration of ventilation. Thus, mitochondrial targeted DNA repair enzymes were protective against mild and moderate lung damage and they enhanced survival in the most severely injured group.

  8. Influence of major-groove chemical modifications of DNA on transcription by bacterial RNA polymerases

    Czech Academy of Sciences Publication Activity Database

    Raindlová, Veronika; Janoušková, Martina; Slavíčková, Michaela; Perlíková, Pavla; Boháčová, Soňa; Milisavljevič, Nemanja; Šanderová, Hana; Benda, Martin; Barvík, I.; Krásný, Libor; Hocek, Michal

    2016-01-01

    Roč. 44, č. 7 (2016), s. 3000-3012 ISSN 0305-1048 R&D Projects: GA ČR GA14-04289S Institutional support: RVO:61388963 ; RVO:61388971 Keywords : Escherichia coli RNA * Bacillus subtilis * restriction endonucleases Subject RIV: CC - Organic Chemistry; EE - Microbiology, Virology (MBU-M) Impact factor: 10.162, year: 2016 http://nar.oxfordjournals.org/content/44/7/3000.full

  9. 49 CFR 215.203 - Restricted cars.

    Science.gov (United States)

    2010-10-01

    ... 49 Transportation 4 2010-10-01 2010-10-01 false Restricted cars. 215.203 Section 215.203..., DEPARTMENT OF TRANSPORTATION RAILROAD FREIGHT CAR SAFETY STANDARDS Restricted Equipment § 215.203 Restricted cars. (a) This section restricts the operation of any railroad freight car that is— (1) More than 50...

  10. 49 CFR 383.95 - Restrictions.

    Science.gov (United States)

    2010-10-01

    ... the skills test and the restriction, air brakes shall include any braking system operating fully or...; REQUIREMENTS AND PENALTIES Vehicle Groups and Endorsements § 383.95 Restrictions. (a) Air brake restrictions... skills test in a vehicle not equipped with air brakes, the State must indicate on the CDL, if issued...

  11. Ribotyping of strains of Moraxella (Branhamella) catarrhalis cultured from the nasopharynx and middle ear of children with otitis media

    DEFF Research Database (Denmark)

    Brygge, K; Sørensen, C H; Colding, H

    1998-01-01

    Moraxella (Branhaomella) catarrhalis is frequently present in the nasopharyngeal microflora of small children, especially during episodes of acute otitis media . By means of ribotyping (restriction endonuclease analysis of chromosomal DNA combined with rRNA probing), we studied the genetic...... heterogeneity of 78 cultures of M. catarrhalis obtained from different localities in the nasopharynx of nine young children with secretory otitis media. Using HindIII and PstI as endonucleases, five different ribotypes were recognized, representing at least five different genotypes of M. catarrhalis....... The distribution of these types was found to be almost identical to the distribution among 16 M. catarrhalis strains cultured from middle ear exudates of 16 children with acute otitis media. Ribotype HAPA was found in two-thirds of all the cultures investigated, and 44% of the children harboured more than one...

  12. Expression of homing endonuclease gene and insertion-like element in sea anemone mitochondrial genomes: Lesson learned from Anemonia viridis.

    Science.gov (United States)

    Chi, Sylvia Ighem; Urbarova, Ilona; Johansen, Steinar D

    2018-04-30

    The mitochondrial genomes of sea anemones are dynamic in structure. Invasion by genetic elements, such as self-catalytic group I introns or insertion-like sequences, contribute to sea anemone mitochondrial genome expansion and complexity. By using next generation sequencing we investigated the complete mtDNAs and corresponding transcriptomes of the temperate sea anemone Anemonia viridis and its closer tropical relative Anemonia majano. Two versions of fused homing endonuclease gene (HEG) organization were observed among the Actiniidae sea anemones; in-frame gene fusion and pseudo-gene fusion. We provided support for the pseudo-gene fusion organization in Anemonia species, resulting in a repressed HEG from the COI-884 group I intron. orfA, a putative protein-coding gene with insertion-like features, was present in both Anemonia species. Interestingly, orfA and COI expression were significantly up-regulated upon long-term environmental stress corresponding to low seawater pH conditions. This study provides new insights to the dynamics of sea anemone mitochondrial genome structure and function. Copyright © 2018 Elsevier B.V. All rights reserved.

  13. Haplotype-based case-control study between human apurinic/apyrimidinic endonuclease 1/redox effector factor-1 gene and cerebral infarction.

    Science.gov (United States)

    Naganuma, Takahiro; Nakayama, Tomohiro; Sato, Naoyuki; Fu, Zhenyan; Yamaguchi, Mai; Soma, Masayoshi; Aoi, Noriko; Usami, Ron; Doba, Nobutaka; Hinohara, Shigeaki

    2009-10-01

    The aim of this study was to investigate the relationship between cerebral infarction (CI) and the human apurinic/apyrimidinic endonuclease 1/redox effector factor-1 (APE1/REF-1) gene using single-nucleotide polymorphisms (SNPs) and a haplotype-based case-control study. We selected 5 SNPs in the human APE1/REF1 gene (rs1760944, rs3136814, rs17111967, rs3136817 and rs1130409), and performed case-control studies in 177 CI patients and 309 control subjects. rs17111967 was found to have no heterogeneity in Japanese. The overall distribution of the haplotype-based case-control study constructed by rs1760944, rs3136814 and rs1130409 showed a significant difference. The frequency of the G-C-T haplotype was significantly higher in the CI group than in the control group (2.5% vs. 0.0%, p>0.001). Based on the results of the haplotype-based case-control-study, the G-C-T haplotype may be a genetic marker of CI, and the APE1/REF-1 gene may be a CI susceptibility gene.

  14. Deoxyribonuclease probing of sea urchin embryo chromatin

    International Nuclear Information System (INIS)

    Landsman, D.

    1983-01-01

    The role that the sea urchin, Parechinus angulosus, embryo and sperm histone variants plays in chromatin structure has been investigated. Chromatin structure has been determined at different levels of resolution in sperm and in developing embryos using micrococcal nuclease, pancreatic deoxyribonuclease (DNase I) and restriction endonucleases. Micrococcal nuclease and restriction endonuclease digestions of sea urchin gastrula chromatin have been analysed and it is shown that it is not possible to isolate large polynucleosomal chromatin complexes which are soluble in low ionic strength buffers. The repeat length for sperm is significantly larger than blastula and gastrula repeat lengths whereas blastula and gastrula repeat lengths are not significantly different. Nucleosomal core particles have been isolated from early blastula, gastrula and sperm of sea urchins. After DNase I digestion of 5'-labelled core particles the rate constants of cutting of the DNA at the susceptible sites on these core particles have been determined. The DNase I digestion kinetics of blastula and gastrula core particles are similar whereas sperm core particles are digested at a slower rate, mainly at the sites which are closest to the ends of the core particle DNA

  15. Whole-genome shotgun optical mapping of Rhodospirillum rubrum

    Energy Technology Data Exchange (ETDEWEB)

    Reslewic, S. [Univ. Wisc.-Madison; Zhou, S. [Univ. Wisc.-Madison; Place, M. [Univ. Wisc.-Madison; Zhang, Y. [Univ. Wisc.-Madison; Briska, A. [Univ. Wisc.-Madison; Goldstein, S. [Univ. Wisc.-Madison; Churas, C. [Univ. Wisc.-Madison; Runnheim, R. [Univ. Wisc.-Madison; Forrest, D. [Univ. Wisc.-Madison; Lim, A. [Univ. Wisc.-Madison; Lapidus, A. [Univ. Wisc.-Madison; Han, C. S. [Univ. Wisc.-Madison; Roberts, G. P. [Univ. Wisc.-Madison; Schwartz, D. C. [Univ. Wisc.-Madison

    2005-09-01

    Rhodospirillum rubrum is a phototrophic purple nonsulfur bacterium known for its unique and well-studied nitrogen fixation and carbon monoxide oxidation systems and as a source of hydrogen and biodegradable plastic production. To better understand this organism and to facilitate assembly of its sequence, three whole-genome restriction endonuclease maps (XbaI, NheI, and HindIII) of R. rubrum strain ATCC 11170 were created by optical mapping. Optical mapping is a system for creating whole-genome ordered restriction endonuclease maps from randomly sheared genomic DNA molecules extracted from cells. During the sequence finishing process, all three optical maps confirmed a putative error in sequence assembly, while the HindIII map acted as a scaffold for high-resolution alignment with sequence contigs spanning the whole genome. In addition to highlighting optical mapping's role in the assembly and confirmation of genome sequence, this work underscores the unique niche in resolution occupied by the optical mapping system. With a resolution ranging from 6.5 kb (previously published) to 45 kb (reported here), optical mapping advances a "molecular cytogenetics" approach to solving problems in genomic analysis.

  16. Urban water restrictions: Attitudes and avoidance

    Science.gov (United States)

    Cooper, Bethany; Burton, Michael; Crase, Lin

    2011-12-01

    In most urban cities across Australia, water restrictions remain the dominant policy mechanism to restrict urban water consumption. The extensive adoption of water restrictions as a means to limit demand, over several years, means that Australian urban water prices have consistently not reflected the opportunity cost of water. Given the generally strong political support for water restrictions and the likelihood that they will persist for some time, there is value in understanding households' attitudes in this context. More specifically, identifying the welfare gains associated with avoiding urban water restrictions entirely would be a nontrivial contribution to our knowledge and offer insights into the benefits of alternative policy responses. This paper describes the results from a contingent valuation study that investigates consumers' willingness to pay to avoid urban water restrictions. Importantly, the research also investigates the influence of cognitive and exogenous dimensions on the utility gain associated with avoiding water restrictions. The results provide insights into the impact of the current policy mechanism on economic welfare.

  17. Double-stranded endonuclease activity in Bacillus halodurans clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas2 protein.

    Science.gov (United States)

    Nam, Ki Hyun; Ding, Fran; Haitjema, Charles; Huang, Qingqiu; DeLisa, Matthew P; Ke, Ailong

    2012-10-19

    The CRISPR (clustered regularly interspaced short palindromic repeats) system is a prokaryotic RNA-based adaptive immune system against extrachromosomal genetic elements. Cas2 is a universally conserved core CRISPR-associated protein required for the acquisition of new spacers for CRISPR adaptation. It was previously characterized as an endoribonuclease with preference for single-stranded (ss)RNA. Here, we show using crystallography, mutagenesis, and isothermal titration calorimetry that the Bacillus halodurans Cas2 (Bha_Cas2) from the subtype I-C/Dvulg CRISPR instead possesses metal-dependent endonuclease activity against double-stranded (ds)DNA. This activity is consistent with its putative function in producing new spacers for insertion into the 5'-end of the CRISPR locus. Mutagenesis and isothermal titration calorimetry studies revealed that a single divalent metal ion (Mg(2+) or Mn(2+)), coordinated by a symmetric Asp pair in the Bha_Cas2 dimer, is involved in the catalysis. We envision that a pH-dependent conformational change switches Cas2 into a metal-binding competent conformation for catalysis. We further propose that the distinct substrate preferences among Cas2 proteins may be determined by the sequence and structure in the β1-α1 loop.

  18. Ultrasensitive electrochemical biosensor for detection of DNA from Bacillus subtilis by coupling target-induced strand displacement and nicking endonuclease signal amplification.

    Science.gov (United States)

    Hu, Yuhua; Xu, Xueqin; Liu, Qionghua; Wang, Ling; Lin, Zhenyu; Chen, Guonan

    2014-09-02

    A simple, ultrasensitive, and specific electrochemical biosensor was designed to determine the given DNA sequence of Bacillus subtilis by coupling target-induced strand displacement and nicking endonuclease signal amplification. The target DNA (TD, the DNA sequence from the hypervarient region of 16S rDNA of Bacillus subtilis) could be detected by the differential pulse voltammetry (DPV) in a range from 0.1 fM to 20 fM with the detection limit down to 0.08 fM at the 3s(blank) level. This electrochemical biosensor exhibits high distinction ability to single-base mismatch, double-bases mismatch, and noncomplementary DNA sequence, which may be expected to detect single-base mismatch and single nucleotide polymorphisms (SNPs). Moreover, the applicability of the designed biosensor for detecting the given DNA sequence from Bacillus subtilis was investigated. The result obtained by electrochemical method is approximately consistent with that by a real-time quantitative polymerase chain reaction detecting system (QPCR) with SYBR Green.

  19. Role of alkaline endonucleases in the release of soluble chromatin from thymus, spleen and liver nuclei of normal and irradiated mice

    International Nuclear Information System (INIS)

    Suciu, D.

    1979-01-01

    Thymus, spleen and liver nuclei released a large fraction of soluble chromatin in vitro when incubation was carried out in sucrose media containing low concentrations of CaCl 2 and/or MgCl 2 . A significant fraction of deoxyribopolynucleotides (DPN) was also extracted from nuclei. After 30 min of incubation at 37 0 C, the maximum release of soluble chromatin was observed near a pH of 8, which corresponds to the optimum pH of the alkaline endonuclease activity from thymus, spleen and liver. The soluble chromatin and DPN were precipitated by increasing the bivalent ion concentration of the medium. The protein/DNA ratio and the molecular weight of DNA suggest that the soluble chromatin and DPN represent nucleosome-like particles. The release of soluble chromatin in the first 4 hours of incubation was significantly increased if the nuclear fraction was isolated from the thymus and spleen of whole-body irradiated mice (1000 rad). This effect was absent in the liver nuclei. (author)

  20. 5-Substituted Pyrimidine and 7-Substituted 7-Deazapurine dNTPs as Substrates for DNA Polymerases in Competitive Primer Extension in the Presence of Natural dNTPs

    Czech Academy of Sciences Publication Activity Database

    Cahová, Hana; Panattoni, Alessandro; Kielkowski, Pavel; Fanfrlík, Jindřich; Hocek, Michal

    2016-01-01

    Roč. 11, č. 11 (2016), s. 3165-3171 ISSN 1554-8929 R&D Projects: GA ČR GA14-04289S EU Projects: European Commission(XE) 642023 - ClickGene Institutional support: RVO:61388963 Keywords : enzymatic synthesis * 2'-deoxyribonucleoside triphosphates * restriction endonucleases Subject RIV: CC - Organic Chemistry Impact factor: 4.995, year: 2016 http://pubs.acs.org/doi/full/10.1021/acschembio.6b00714

  1. Recruitment and positioning determine the specific role of the XPF-ERCC1 endonuclease in interstrand crosslink repair.

    Science.gov (United States)

    Klein Douwel, Daisy; Hoogenboom, Wouter S; Boonen, Rick Acm; Knipscheer, Puck

    2017-07-14

    XPF-ERCC1 is a structure-specific endonuclease pivotal for several DNA repair pathways and, when mutated, can cause multiple diseases. Although the disease-specific mutations are thought to affect different DNA repair pathways, the molecular basis for this is unknown. Here we examine the function of XPF-ERCC1 in DNA interstrand crosslink (ICL) repair. We used Xenopus egg extracts to measure both ICL and nucleotide excision repair, and we identified mutations that are specifically defective in ICL repair. One of these separation-of-function mutations resides in the helicase-like domain of XPF and disrupts binding to SLX4 and recruitment to the ICL A small deletion in the same domain supports recruitment of XPF to the ICL, but inhibited the unhooking incisions most likely by disrupting a second, transient interaction with SLX4. Finally, mutation of residues in the nuclease domain did not affect localization of XPF-ERCC1 to the ICL but did prevent incisions on the ICL substrate. Our data support a model in which the ICL repair-specific function of XPF-ERCC1 is dependent on recruitment, positioning and substrate recognition. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

  2. A comparative study of cold- and warm-adapted Endonucleases A using sequence analyses and molecular dynamics simulations.

    Directory of Open Access Journals (Sweden)

    Davide Michetti

    Full Text Available The psychrophilic and mesophilic endonucleases A (EndA from Aliivibrio salmonicida (VsEndA and Vibrio cholera (VcEndA have been studied experimentally in terms of the biophysical properties related to thermal adaptation. The analyses of their static X-ray structures was no sufficient to rationalize the determinants of their adaptive traits at the molecular level. Thus, we used Molecular Dynamics (MD simulations to compare the two proteins and unveil their structural and dynamical differences. Our simulations did not show a substantial increase in flexibility in the cold-adapted variant on the nanosecond time scale. The only exception is a more rigid C-terminal region in VcEndA, which is ascribable to a cluster of electrostatic interactions and hydrogen bonds, as also supported by MD simulations of the VsEndA mutant variant where the cluster of interactions was introduced. Moreover, we identified three additional amino acidic substitutions through multiple sequence alignment and the analyses of MD-based protein structure networks. In particular, T120V occurs in the proximity of the catalytic residue H80 and alters the interaction with the residue Y43, which belongs to the second coordination sphere of the Mg2+ ion. This makes T120V an amenable candidate for future experimental mutagenesis.

  3. Cloning vector

    Science.gov (United States)

    Guilfoyle, Richard A.; Smith, Lloyd M.

    1994-01-01

    A vector comprising a filamentous phage sequence containing a first copy of filamentous phage gene X and other sequences necessary for the phage to propagate is disclosed. The vector also contains a second copy of filamentous phage gene X downstream from a promoter capable of promoting transcription in a bacterial host. In a preferred form of the present invention, the filamentous phage is M13 and the vector additionally includes a restriction endonuclease site located in such a manner as to substantially inactivate the second gene X when a DNA sequence is inserted into the restriction site.

  4. Cloning vector

    Science.gov (United States)

    Guilfoyle, R.A.; Smith, L.M.

    1994-12-27

    A vector comprising a filamentous phage sequence containing a first copy of filamentous phage gene X and other sequences necessary for the phage to propagate is disclosed. The vector also contains a second copy of filamentous phage gene X downstream from a promoter capable of promoting transcription in a bacterial host. In a preferred form of the present invention, the filamentous phage is M13 and the vector additionally includes a restriction endonuclease site located in such a manner as to substantially inactivate the second gene X when a DNA sequence is inserted into the restriction site. 2 figures.

  5. Placental weight and birth weight to placental weight ratio in monochorionic and dichorionic growth-restricted and non-growth-restricted twins

    Directory of Open Access Journals (Sweden)

    Mariângela Alves Souza

    Full Text Available OBJECTIVE: The aim of the present study was to compare the placental weight and birth weight/placental weight ratio for intrauterine growth-restricted and non-intrauterine growth-restricted monochorionic and dichorionic twins. METHODS: This was a retrospective analysis of placentas from twin pregnancies. Placental weight and the birth weight/placental weight ratio were compared in intrauterine growth-restricted and non-intrauterine growth-restricted monochorionic and dichorionic twins. The association between cord insertion type and placental lesions in intrauterine growth-restricted and non-intrauterine growth-restricted monochorionic and dichorionic twins was also investigated. RESULTS: A total of 105 monochorionic (intrauterine growth restriction=40; non-intrauterine growth restriction=65 and 219 dichorionic (intrauterine growth restriction=57; non-intrauterine growth restriction=162 placentas were analyzed. A significantly lower placental weight was observed in intrauterine growth-restricted monochorionic (p=0.022 and dichorionic (p<0.001 twins compared to non-intrauterine growth-restricted twins. There was no difference in the birth weight/placental weight ratio between the intrauterine growth restriction and non-intrauterine growth restriction groups for either monochorionic (p=0.36 or dichorionic (p=0.68 twins. Placental weight and the birth weight/placental weight ratio were not associated with cord insertion type or with placental lesions. CONCLUSION: Low placental weight, and consequently reduced functional mass, appears to be involved in fetal growth restriction in monochorionic and dichorionic twins. The mechanism by which low placental weight influences the birth weight/placental weight ratio in intrauterine growth-restricted monochorionic and dichorionic twins needs to be determined in larger prospective studies.

  6. Cell-autonomous progeroid changes in conditional mouse models for repair endonuclease XPG deficiency.

    Directory of Open Access Journals (Sweden)

    Sander Barnhoorn

    2014-10-01

    Full Text Available As part of the Nucleotide Excision Repair (NER process, the endonuclease XPG is involved in repair of helix-distorting DNA lesions, but the protein has also been implicated in several other DNA repair systems, complicating genotype-phenotype relationship in XPG patients. Defects in XPG can cause either the cancer-prone condition xeroderma pigmentosum (XP alone, or XP combined with the severe neurodevelopmental disorder Cockayne Syndrome (CS, or the infantile lethal cerebro-oculo-facio-skeletal (COFS syndrome, characterized by dramatic growth failure, progressive neurodevelopmental abnormalities and greatly reduced life expectancy. Here, we present a novel (conditional Xpg-/- mouse model which -in a C57BL6/FVB F1 hybrid genetic background- displays many progeroid features, including cessation of growth, loss of subcutaneous fat, kyphosis, osteoporosis, retinal photoreceptor loss, liver aging, extensive neurodegeneration, and a short lifespan of 4-5 months. We show that deletion of XPG specifically in the liver reproduces the progeroid features in the liver, yet abolishes the effect on growth or lifespan. In addition, specific XPG deletion in neurons and glia of the forebrain creates a progressive neurodegenerative phenotype that shows many characteristics of human XPG deficiency. Our findings therefore exclude that both the liver as well as the neurological phenotype are a secondary consequence of derailment in other cell types, organs or tissues (e.g. vascular abnormalities and support a cell-autonomous origin caused by the DNA repair defect itself. In addition they allow the dissection of the complex aging process in tissue- and cell-type-specific components. Moreover, our data highlight the critical importance of genetic background in mouse aging studies, establish the Xpg-/- mouse as a valid model for the severe form of human XPG patients and segmental accelerated aging, and strengthen the link between DNA damage and aging.

  7. Restrictive annuloplasty to treat functional mitral regurgitation: optimize the restriction to improve the results?

    Science.gov (United States)

    Totaro, Pasquale; Adragna, Nicola; Argano, Vincenzo

    2008-03-01

    Today, the 'gold standard' treatment of functional mitral regurgitation (MR) is the subject of much discussion. Although restrictive annuloplasty is currently considered the most reproducible technique, the means by which the degree of annular restriction is optimized remains problematic. The study was designed in order to identify whether the degree of restriction of the mitral annulus could influence early and midterm results following the treatment of functional MR using restrictive annuloplasty. A total of 32 consecutive patients with functional MR grade > or = 3+ was enrolled, among whom the mean anterior-posterior (AP) mitral annulus diameter was 39 +/- 3 mm. Restrictive mitral annuloplasty (combined with coronary artery bypass grafting) was performed in all patients using a Carpentier-Edwards Classic or Physio ring (size 26 or 28). The degree of AP annular restriction was calculated for each patient, and correlated with early and mid-term residual MR and left ventricular (LV) reverse remodeling (in terms of LV end-diastolic diameter (LVEDD) and LV end-diastolic volume (LVEDV) reduction). All surviving patients were examined at a one-year follow up. The mean AP mitral annulus restriction achieved was 48 +/- 4%. Intraoperatively, transesophageal echocardiography showed no residual MR in any patient. Before discharge from hospital, transthoracic echocardiography confirmed an absence of residual MR and showed significant LV reverse remodeling (LVEDV from 121 +/- 25 ml to 97 +/- 26 ml; LVEDD from 55 +/- 6 mm to 47 +/- 8 mm). A significant correlation (r = 0.57, p 40% of preoperative) appears to have a favorable influence on early postoperative LV reverse remodeling, and also allows for complete resolution of functional MR. In addition, 'no tolerance' of early residual MR seems to have a favorable influence on mid-term results, leading to a reduction in the one-year recurrence of significant MR.

  8. Protein restriction and cancer.

    Science.gov (United States)

    Yin, Jie; Ren, Wenkai; Huang, Xingguo; Li, Tiejun; Yin, Yulong

    2018-03-26

    Protein restriction without malnutrition is currently an effective nutritional intervention known to prevent diseases and promote health span from yeast to human. Recently, low protein diets are reported to be associated with lowered cancer incidence and mortality risk of cancers in human. In murine models, protein restriction inhibits tumor growth via mTOR signaling pathway. IGF-1, amino acid metabolic programing, FGF21, and autophagy may also serve as potential mechanisms of protein restriction mediated cancer prevention. Together, dietary intervention aimed at reducing protein intake can be beneficial and has the potential to be widely adopted and effective in preventing and treating cancers. Copyright © 2018 Elsevier B.V. All rights reserved.

  9. Property Rights, Restrictions and Responsibilities

    DEFF Research Database (Denmark)

    Enemark, Stig

    more to a social, ethical commitment or attitude to environmental sustainability and good husbandry. This paper provides an overall understanding of the concept of land administration systems for dealing with rights, restrictions and responsibilities in future spatially enabled government. Finally......Land Administration Systems are the basis for conceptualizing rights, restrictions and responsibilities related to people, policies and places. Property rights are normally concerned with ownership and tenure whereas restrictions usually control use and activities on land. Responsibilities relate...

  10. Restricting wolves risks escape

    Science.gov (United States)

    Mech, L. David; Ballard, Warren; Bangs, Ed; Ream, Bob

    2010-01-01

    Implementing the proposal set forth by Licht and colleagues (BioScience 60: 147–153) requires restricting wolves to tiny "islands," areas that are magnitudes smaller than the ranges of most wolf populations. Wolves naturally have large ranges; restricting their spatial needs increases the risk of wolves escaping, exacerbating public relations and political and legal problems.

  11. 9 CFR 92.3 - Movement restrictions.

    Science.gov (United States)

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Movement restrictions. 92.3 Section 92... ANIMAL PRODUCTS: PROCEDURES FOR REQUESTING RECOGNITION OF REGIONS § 92.3 Movement restrictions. Whenever... exist and the EC imposes prohibitions or other restrictions on the movement of animals or animal...

  12. About 'restriction', 'justified' and 'necessary'

    DEFF Research Database (Denmark)

    Werlauff, Erik

    2016-01-01

    The article is an academic fairy tale about why and how all national corporate tax protection legislation should undergo a 3-part test to ensure its consistency with EU law. Each Member State introduce a compulsory 3-step test for each new (corporate) tax provision. The test is simple: (1) Does...... the tax provision constitute a restriction in the sense of EU law? (2) If the answer is yes: Is the restriction justified? (3) If the answer is yes: Is the restriction necessary?"...

  13. Epitopes associated with MHC restriction site of T cells. III. I-J epitope on MHC-restricted T helper cells

    International Nuclear Information System (INIS)

    Asano, Y.; Nakayama, T.; Kubo, M.; Yagi, J.; Tada, T.

    1987-01-01

    I-J epitopes were found to be associated with the functional site of the class II MHC-restricted helper T (Th) cells: Virtually all of the H-2k-restricted Th cell function of H-2kxbF1 T cells was inhibited by the anti-I-Jk mAb, leaving the H-2b-restricted function unaffected. The I-Jk epitope was inducible in Th cells of different genotype origin according to the environmental class II antigens present in the early ontogeny of T cells. Although above results suggested that I-J is the structure reflecting the inducible MHC restriction specificity, further studies revealed some interesting controversies: First, the I-J phenotype did not always correlate with the class II restriction specificity, e.g., I-Ab-restricted Th from 5R was I-Jk-positive, whereas I-Ak-restricted Th of 4R was not. Second, there was no trans expression of parental I-J phenotypes and restriction specificities in F1 Th, e.g., the I-J phenotype was detected only on I-Ab-restricted Th of (4R X 5R)F1, whereas it was absent on I-Ak-restricted Th. This strict linkage between the restriction specificity and I-J phenotype was also found on Th cells developed in bone marrow chimera constructed with intra-H-2-recombinant mice. The expression of I-Jk was always associated with the restriction specificity of the relevant host. Thus, the restriction specificity of Th cells followed the host type, and the I-J expression on Th was exactly the same as that expressed by the host haplotype. These results indicate that I-J is an isomorphic structure adaptively expressed on Th cells that is involved in the unidirectional regulatory cell interactions, and that the polymorphism cannot be explained merely by the restriction specificity of the conventional T cell receptor heterodimer

  14. Altered endoribonuclease activity of apurinic/apyrimidinic endonuclease 1 variants identified in the human population.

    Directory of Open Access Journals (Sweden)

    Wan Cheol Kim

    Full Text Available Apurinic/apyrimidinic endonuclease 1 (APE1 is the major mammalian enzyme in the DNA base excision repair pathway and cleaves the DNA phosphodiester backbone immediately 5' to abasic sites. APE1 also has 3'-5' DNA exonuclease and 3' DNA phosphodiesterase activities, and regulates transcription factor DNA binding through its redox regulatory function. The human APE1 has recently been shown to endonucleolytically cleave single-stranded regions of RNA. Towards understanding the biological significance of the endoribonuclease activity of APE1, we examined eight different amino acid substitution variants of APE1 previously identified in the human population. Our study shows that six APE1 variants, D148E, Q51H, I64V, G241R, R237A, and G306A, exhibit a 76-85% reduction in endoribonuclease activity against a specific coding region of the c-myc RNA, yet fully retain the ability to cleave apurinic/apyrimidinic DNA. We found that two APE1 variants, L104R and E126D, exhibit a unique RNase inhibitor-resistant endoribonuclease activity, where the proteins cleave c-myc RNA 3' of specific single-stranded guanosine residues. Expression of L104R and E126D APE1 variants in bacterial Origami cells leads to a 60-80% reduction in colony formation and a 1.5-fold increase in cell doubling time, whereas the other variants, which exhibit diminished endoribonuclease activity, had no effect. These data indicate that two human APE1 variants exhibit a unique endoribonuclease activity, which correlates with their ability to induce cytotoxicity or slow down growth in bacterial cells and supports the notion of their biological functionality.

  15. Restrictive cardiomyopathy

    Science.gov (United States)

    ... People with restrictive cardiomyopathy may be heart transplant candidates. The outlook depends on the cause of the ... www.urac.org). URAC's accreditation program is an independent audit to verify that A.D.A.M. ...

  16. 21 CFR 203.20 - Sales restrictions.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 4 2010-04-01 2010-04-01 false Sales restrictions. 203.20 Section 203.20 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS: GENERAL PRESCRIPTION DRUG MARKETING Sales Restrictions § 203.20 Sales restrictions. Except as provided in § 203.22 or...

  17. The possibility of discriminating within the Bacillus cereus group using gyrB sequencing and PCR-RFLP

    DEFF Research Database (Denmark)

    Jensen, Gert B; Fisker, Niels; Sparsø, Thomas

    2005-01-01

    Based on a combination of PCR and restriction endonuclease (RE) digestion (PCR-RE digestion), we have examined the possibility of differentiating members of the Bacillus cereus group. Fragments of the gyrB gene (362 bp) from pure cultures of 12 B. cereus, 25 B. thuringiensis, 25 B. mycoides and two......, it was not possible to discriminate between the B. cereus and the B. thuringiensis strains using the methods described....

  18. A Traffic Restriction Scheme for Enhancing Carpooling

    Directory of Open Access Journals (Sweden)

    Dong Ding

    2017-01-01

    Full Text Available For the purpose of alleviating traffic congestion, this paper proposes a scheme to encourage travelers to carpool by traffic restriction. By a variational inequity we describe travelers’ mode (solo driving and carpooling and route choice under user equilibrium principle in the context of fixed demand and detect the performance of a simple network with various restriction links, restriction proportions, and carpooling costs. Then the optimal traffic restriction scheme aiming at minimal total travel cost is designed through a bilevel program and applied to a Sioux Fall network example with genetic algorithm. According to various requirements, optimal restriction regions and proportions for restricted automobiles are captured. From the results it is found that traffic restriction scheme is possible to enhance carpooling and alleviate congestion. However, higher carpooling demand is not always helpful to the whole network. The topology of network, OD demand, and carpooling cost are included in the factors influencing the performance of the traffic system.

  19. Molecular motion in restricted geometries

    Indian Academy of Sciences (India)

    Molecular dynamics in restricted geometries is known to exhibit anomalous behaviour. Diffusion, translational or rotational, of molecules is altered significantly on confinement in restricted geometries. Quasielastic neutron scattering (QENS) offers a unique possibility of studying molecular motion in such systems. Both time ...

  20. Rapid Genome-wide Single Nucleotide Polymorphism Discovery in Soybean and Rice via Deep Resequencing of Reduced Representation Libraries with the Illumina Genome Analyzer

    Directory of Open Access Journals (Sweden)

    Stéphane Deschamps

    2010-07-01

    Full Text Available Massively parallel sequencing platforms have allowed for the rapid discovery of single nucleotide polymorphisms (SNPs among related genotypes within a species. We describe the creation of reduced representation libraries (RRLs using an initial digestion of nuclear genomic DNA with a methylation-sensitive restriction endonuclease followed by a secondary digestion with the 4bp-restriction endonuclease This strategy allows for the enrichment of hypomethylated genomic DNA, which has been shown to be rich in genic sequences, and the digestion with serves to increase the number of common loci resequenced between individuals. Deep resequencing of these RRLs performed with the Illumina Genome Analyzer led to the identification of 2618 SNPs in rice and 1682 SNPs in soybean for two representative genotypes in each of the species. A subset of these SNPs was validated via Sanger sequencing, exhibiting validation rates of 96.4 and 97.0%, in rice ( and soybean (, respectively. Comparative analysis of the read distribution relative to annotated genes in the reference genome assemblies indicated that the RRL strategy was primarily sampling within genic regions for both species. The massively parallel sequencing of methylation-sensitive RRLs for genome-wide SNP discovery can be applied across a wide range of plant species having sufficient reference genomic sequence.

  1. 28 CFR 804.3 - Restrictions.

    Science.gov (United States)

    2010-07-01

    ... 28 Judicial Administration 2 2010-07-01 2010-07-01 false Restrictions. 804.3 Section 804.3 Judicial Administration COURT SERVICES AND OFFENDER SUPERVISION AGENCY FOR THE DISTRICT OF COLUMBIA ACCEPTANCE OF GIFTS § 804.3 Restrictions. (a) The Agency is not authorized to accept gifts of money, stock...

  2. 5 CFR 1304.4605 - Post-employment restrictions.

    Science.gov (United States)

    2010-01-01

    ... 5 Administrative Personnel 3 2010-01-01 2010-01-01 false Post-employment restrictions. 1304.4605 Section 1304.4605 Administrative Personnel OFFICE OF MANAGEMENT AND BUDGET ADMINISTRATIVE PROCEDURES POST EMPLOYMENT CONFLICT OF INTEREST § 1304.4605 Post-employment restrictions. (a) General Restrictions Applicable...

  3. Restrictive Cardiomyopathy

    Science.gov (United States)

    ... up in the circulatory system. In time, the heart fails. What causes it? Restrictive cardiomyopathy is often caused by diseases in other parts of the body. One known cause is cardiac ... build up in the heart tissue, making the tissue stiff and thickened. Cardiac ...

  4. Identification and typing of herpes simplex viruses with monoclonal antibodies.

    OpenAIRE

    Balachandran, N; Frame, B; Chernesky, M; Kraiselburd, E; Kouri, Y; Garcia, D; Lavery, C; Rawls, W E

    1982-01-01

    Monoclonal antibodies which reacted with type-specific antigens of herpes simplex virus type 2 or with antigens shared by herpes simplex virus types 1 and 2 were used in an indirect immunofluorescence assay to type virus isolates and to detect viral antigens in cells obtained from herpetic lesions. Complete concordance was obtained for 42 isolates typed by endonuclease restriction analysis of viral DNA and by indirect immunofluorescence with monoclonal antibodies. Examination of a limited num...

  5. Infección con el virus de la inmunodeficiencia felina: desarrollo y aplicación de una técnica de doble reacción en cadena de la polimerasa (PRC) para el diagnóstico

    OpenAIRE

    Oliva, Graciela A.; Vila Roza, M. V.; Teodoroff, T.; Castellano, María Cecilia; Pecoraro, Marcelo R.; Arias, Daniel Osvaldo; González, E. T.; Etcheverrigaray, María Elisa; Galosi, Cecilia Mónica

    2000-01-01

    A Nested PCR technique for direct identification of Feline Immunodeficiency Virus (FIV) was developed. Two methods for proviral DNA extraction and two pairs of primers were evaluated. The method for amplification was standardized and its specificity was determined by the restriction endonucleases digestion of PCR products. Antibodies specific to FIV were determined using a commercial kit. Thirty heparinized blood samples obtained from cats with and without clinical signs consistent with FIV i...

  6. 7 CFR 322.28 - General requirements; restricted articles.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 5 2010-01-01 2010-01-01 false General requirements; restricted articles. 322.28... EQUIPMENT Importation and Transit of Restricted Articles § 322.28 General requirements; restricted articles. (a) The following articles from any region are restricted articles: (1) Dead bees of any genus; (2...

  7. Assessing restrictiveness of national alcohol marketing policies.

    Science.gov (United States)

    Esser, Marissa B; Jernigan, David H

    2014-01-01

    To develop an approach for monitoring national alcohol marketing policies globally, an area of the World Health Organization's (WHO) Global Alcohol Strategy. Data on restrictiveness of alcohol marketing policies came from the 2002 and 2008 WHO Global Surveys on Alcohol and Health. We included four scales in a sensitivity analysis to determine optimal weights to score countries on their marketing policies and applied the selected scale to assess national marketing policy restrictiveness. Nearly, 36% of countries had no marketing restrictions. The overall restrictiveness levels were not significantly different between 2002 and 2008. The number of countries with strict marketing regulations did not differ across years. This method of monitoring alcohol marketing restrictiveness helps track progress towards implementing WHO'S Global Alcohol Strategy. Findings indicate a consistent lack of restrictive policies over time, making this a priority area for national and global action. © The Author 2014. Medical Council on Alcohol and Oxford University Press. All rights reserved.

  8. 7 CFR 319.75-2 - Restricted articles. 1

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 5 2010-01-01 2010-01-01 false Restricted articles. 1 319.75-2 Section 319.75-2... SERVICE, DEPARTMENT OF AGRICULTURE FOREIGN QUARANTINE NOTICES Khapra Beetle § 319.75-2 Restricted articles. 1 1 The importation of restricted articles may be subject to prohibitions or restrictions under...

  9. Optimising homing endonuclease gene drive performance in a semi-refractory species: the Drosophila melanogaster experience.

    Directory of Open Access Journals (Sweden)

    Yuk-Sang Chan

    Full Text Available Homing endonuclease gene (HEG drive is a promising insect population control technique that employs meganucleases to impair the fitness of pest populations. Our previous studies showed that HEG drive was more difficult to achieve in Drosophila melanogaster than Anopheles gambiae and we therefore investigated ways of improving homing performance in Drosophila. We show that homing in Drosophila responds to increased expression of HEGs specifically during the spermatogonia stage and this could be achieved through improved construct design. We found that 3'-UTR choice was important to maximise expression levels, with HEG activity increasing as we employed Hsp70, SV40, vasa and βTub56D derived UTRs. We also searched for spermatogonium-specific promoters and found that the Rcd-1r promoter was able to drive specific expression at this stage. Since Rcd-1 is a regulator of differentiation in other species, it suggests that Rcd-1r may serve a similar role during spermatogonial differentiation in Drosophila. Contrary to expectations, a fragment containing the entire region between the TBPH gene and the bgcn translational start drove strong HEG expression only during late spermatogenesis rather than in the germline stem cells and spermatogonia as expected. We also observed that the fraction of targets undergoing homing was temperature-sensitive, falling nearly four-fold when the temperature was lowered to 18°C. Taken together, this study demonstrates how a few simple measures can lead to substantial improvements in the HEG-based gene drive strategy and reinforce the idea that the HEG approach may be widely applicable to a variety of insect control programs.

  10. Restricted gravity: Abelian projection of Einstein's theory

    International Nuclear Information System (INIS)

    Cho, Y.M.

    2013-01-01

    Treating Einstein's theory as a gauge theory of Lorentz group, we decompose the gravitational connection Γμ into the restricted connection made of the potential of the maximal Abelian subgroup H of Lorentz group G and the valence connection made of G/H part of the potential which transforms covariantly under Lorentz gauge transformation. With this we show that Einstein's theory can be decomposed into the restricted gravity made of the restricted connection which has the full Lorentz gauge invariance which has the valence connection as gravitational source. The decomposition shows the existence of a restricted theory of gravitation which has the full general invariance but is much simpler than Einstein's theory. Moreover, it tells that the restricted gravity can be written as an Abelian gauge theory,

  11. 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.

  12. Curves of restricted type in euclidean spaces

    Directory of Open Access Journals (Sweden)

    Bengü Kılıç Bayram

    2014-01-01

    Full Text Available Submanifolds of restricted type were introduced in [7]. In the present study we consider restricted type of curves in Em. We give some special examples. We also show that spherical curve in S2(r C E3 is of restricted type if and only if either ƒ(s is constant or a linear function of s of the form ƒ(s = ±s + b and every closed W - curve of rank k and of length 2(r in E2k is of restricted type.

  13. 36 CFR 13.1174 - Whale water restrictions.

    Science.gov (United States)

    2010-07-01

    ... 36 Parks, Forests, and Public Property 1 2010-07-01 2010-07-01 false Whale water restrictions. 13... Vessel Operating Restrictions § 13.1174 Whale water restrictions. (a) May 15 through September 30, the following waters are designated as whale waters. (1) Waters north of a line drawn from Point Carolus to...

  14. Identification of human flap endonuclease 1 (FEN1) inhibitors using a machine learning based consensus virtual screening.

    Science.gov (United States)

    Deshmukh, Amit Laxmikant; Chandra, Sharat; Singh, Deependra Kumar; Siddiqi, Mohammad Imran; Banerjee, Dibyendu

    2017-07-25

    Human Flap endonuclease1 (FEN1) is an enzyme that is indispensable for DNA replication and repair processes and inhibition of its Flap cleavage activity results in increased cellular sensitivity to DNA damaging agents (cisplatin, temozolomide, MMS, etc.), with the potential to improve cancer prognosis. Reports of the high expression levels of FEN1 in several cancer cells support the idea that FEN1 inhibitors may target cancer cells with minimum side effects to normal cells. In this study, we used large publicly available, high-throughput screening data of small molecule compounds targeted against FEN1. Two machine learning algorithms, Support Vector Machine (SVM) and Random Forest (RF), were utilized to generate four classification models from huge PubChem bioassay data containing probable FEN1 inhibitors and non-inhibitors. We also investigated the influence of randomly selected Zinc-database compounds as negative data on the outcome of classification modelling. The results show that the SVM model with inactive compounds was superior to RF with Matthews's correlation coefficient (MCC) of 0.67 for the test set. A Maybridge database containing approximately 53 000 compounds was screened and top ranking 5 compounds were selected for enzyme and cell-based in vitro screening. The compound JFD00950 was identified as a novel FEN1 inhibitor with in vitro inhibition of flap cleavage activity as well as cytotoxic activity against a colon cancer cell line, DLD-1.

  15. 50 CFR 697.23 - Restricted gear areas.

    Science.gov (United States)

    2010-10-01

    ... 50 Wildlife and Fisheries 9 2010-10-01 2010-10-01 false Restricted gear areas. 697.23 Section 697... Measures § 697.23 Restricted gear areas. (a) Resolution of lobster gear conflicts with fisheries managed...) Restricted Gear Area I—(1) Duration—(i) Mobile Gear. From October 1 through June 15 of each fishing year, no...

  16. Two models of distribution of sites sensitive to the endonuclease from Micrococcus luteus in the DNA of UV irradiated Escherichia coli B/r Hcr-

    International Nuclear Information System (INIS)

    Kleibl, K.; Sedliakova, M.

    1984-01-01

    Cells prelabelled with 14 C-thymine and irradiated with 5 J/m 2 were at various intervals after UV labelled with 3 H-thymidine then treated with the extract from M. luteus and DNA was analyzed in alkaline sucrose gradients. Loss of endonuclease sensitive sites (Es sites) from the parental DNA and their occurrence in the daughter DNA were followed for at least three replication cycles. Data obtained indicate that about 50% of Es sites were lost during the first replication cycle but no additional loss was observed during subsequent cycles. Thus our data do not support a hypothesis that a half of the dimers are transferred from the parental into the daughter strands at each replication cycle. They rather indicate that dimers remain in situ and distortions accompanying dimers are distinguished either on the side of the parental or on the side of the daughter strands with an equal probability. (author)

  17. Parenting and restrictions in childhood epilepsy

    NARCIS (Netherlands)

    Rodenburg, R.; Meijer, A.M.; Scherphof, C.; Carpay, J.A.; Augustijn, P.; Aldenkamp, A.P.; Deković, M.

    2013-01-01

    Purpose: From the overprotection literature, the predictive and interactional (moderation) effects of controlling and indulgent parenting on restrictions in children with epilepsy were examined. Methods: Parents of 73 children with epilepsy completed questionnaires on parenting, restrictions, and

  18. Calorie for Calorie, Dietary Fat Restriction Results in More Body Fat Loss than Carbohydrate Restriction in People with Obesity.

    Science.gov (United States)

    Hall, Kevin D; Bemis, Thomas; Brychta, Robert; Chen, Kong Y; Courville, Amber; Crayner, Emma J; Goodwin, Stephanie; Guo, Juen; Howard, Lilian; Knuth, Nicolas D; Miller, Bernard V; Prado, Carla M; Siervo, Mario; Skarulis, Monica C; Walter, Mary; Walter, Peter J; Yannai, Laura

    2015-09-01

    Dietary carbohydrate restriction has been purported to cause endocrine adaptations that promote body fat loss more than dietary fat restriction. We selectively restricted dietary carbohydrate versus fat for 6 days following a 5-day baseline diet in 19 adults with obesity confined to a metabolic ward where they exercised daily. Subjects received both isocaloric diets in random order during each of two inpatient stays. Body fat loss was calculated as the difference between daily fat intake and net fat oxidation measured while residing in a metabolic chamber. Whereas carbohydrate restriction led to sustained increases in fat oxidation and loss of 53 ± 6 g/day of body fat, fat oxidation was unchanged by fat restriction, leading to 89 ± 6 g/day of fat loss, and was significantly greater than carbohydrate restriction (p = 0.002). Mathematical model simulations agreed with these data, but predicted that the body acts to minimize body fat differences with prolonged isocaloric diets varying in carbohydrate and fat. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. 5-(Hydroxymethyl) uracil and -cytosine as potential epigenetic marks enhancing or inhibiting transcription with bacterial RNA polymerase

    Czech Academy of Sciences Publication Activity Database

    Janoušková, Martina; Vaníková, Zuzana; Nici, Fabrizia; Boháčová, Soňa; Vítovská, Dragana; Šanderová, Hana; Hocek, Michal; Krásný, Libor

    2017-01-01

    Roč. 53, č. 99 (2017), s. 13253-13255 ISSN 1359-7345 R&D Projects: GA ČR(CZ) GA17-03419S Grant - others:AV ČR(CZ) AP1501 Program:Akademická prémie - Praemium Academiae Institutional support: RVO:61388971 ; RVO:61388963 Keywords : STEM-CELL DNA * GENE-EXPRESSION * RESTRICTION ENDONUCLEASES Subject RIV: EE - Microbiology, Virology; CE - Biochemistry (UOCHB-X) OBOR OECD: Microbiology; Biochemistry and molecular biology (UOCHB-X) Impact factor: 6.319, year: 2016

  20. Evolutionary genomics and HIV restriction factors.

    Science.gov (United States)

    Pyndiah, Nitisha; Telenti, Amalio; Rausell, Antonio

    2015-03-01

    To provide updated insights into innate antiviral immunity and highlight prototypical evolutionary features of well characterized HIV restriction factors. Recently, a new HIV restriction factor, Myxovirus resistance 2, has been discovered and the region/residue responsible for its activity identified using an evolutionary approach. Furthermore, IFI16, an innate immunity protein known to sense several viruses, has been shown to contribute to the defense to HIV-1 by causing cell death upon sensing HIV-1 DNA. Restriction factors against HIV show characteristic signatures of positive selection. Different patterns of accelerated sequence evolution can distinguish antiviral strategies--offense or defence--as well as the level of specificity of the antiviral properties. Sequence analysis of primate orthologs of restriction factors serves to localize functional domains and sites responsible for antiviral action. We use recent discoveries to illustrate how evolutionary genomic analyses help identify new antiviral genes and their mechanisms of action.

  1. Activity restriction among women with a short cervix.

    Science.gov (United States)

    Grobman, William A; Gilbert, Sharon A; Iams, Jay D; Spong, Catherine Y; Saade, George; Mercer, Brian M; Tita, Alan T N; Rouse, Dwight J; Sorokin, Yoram; Leveno, Kenneth J; Tolosa, Jorge E; Thorp, John M; Caritis, Steve N; Van Dorsten, J Peter

    2013-06-01

    To estimate determinants of and outcomes associated with activity restriction among women with a short cervix. This was a secondary analysis of a randomized trial of 17-α hydroxyprogesterone caproate for prevention of preterm birth among nulliparous women with singleton gestations and cervices less than 30 mm by midtrimester ultrasonography. Women were asked weekly whether they had been placed on pelvic, work, or nonwork rest. "Any activity restriction" was defined as being placed on any type of rest. Factors associated with any activity restriction were determined and the association between preterm birth and activity restriction was estimated with multivariable logistic regression. Of the 657 women in the trial, 646 (98%) responded to questions regarding activity restriction. Two hundred fifty-two (39.0%) were placed on any activity restriction at a median of 23.9 weeks (interquartile range 22.6-27.9 weeks). Women on activity restriction were older, more likely to have private insurance, less likely to be Hispanic, had a shorter cervical length, and were more likely to have funneling and intra-amniotic debris. Preterm birth at less than 37 weeks of gestation was more common among women placed on activity restriction (37% compared with 17%, Prestriction (adjusted odds ratio 2.37, 95% confidence interval 1.60-3.53). Results were similar for preterm birth at less than 34 weeks of gestation. Activity restriction did not reduce the rate of preterm birth in asymptomatic nulliparous women with a short cervix.

  2. Differences in the second internal transcribed spacer of four species of Nematodirus (Nematoda: Molineidae).

    Science.gov (United States)

    Newton, L A; Chilton, N B; Beveridge, I; Gasser, R B

    1998-02-01

    Genetic differences among Nematodirus spathiger, Nematodirus filicollis, Nematodirus helvetianus and Nematodirus battus in the nucleotide sequence of the second internal transcribed spacer (ITS-2) of ribosomal DNA ranged from 3.9 to 24.7%. Pairwise comparisons of their ITS-2 sequences indicated that the most genetically similar species were N. spathiger and N. helvetianus. N. battus was the most genetically distinct species, with differences ranging from 22.8 to 24.7% with respect to the other three species. Some of the nucleotide differences among species provided different endonuclease restriction sites that could be used in restriction fragment length polymorphism studies. The ITS-2 sequence data may prove useful in studies of the systematics of molineid nematodes.

  3. Meat species identification and Halal authentication analysis using mitochondrial DNA.

    Science.gov (United States)

    Murugaiah, Chandrika; Noor, Zainon Mohd; Mastakim, Maimunah; Bilung, Lesley Maurice; Selamat, Jinap; Radu, Son

    2009-09-01

    A method utilizing PCR-restriction fragment length polymorphism (RFLP) in the mitochondrial genes was developed for beef (Bos taurus), pork (Sus scrofa), buffalo (Bubalus bubali), quail (Coturnix coturnix), chicken (Gallus gallus), goat (Capra hircus), rabbit (Oryctolagus cuniculus) species identification and Halal authentication. PCR products of 359-bp were successfully obtained from the cyt b gene of these six meats. AluI, BsaJI, RsaI, MseI, and BstUI enzymes were identified as potential restriction endonucleases to differentiate the meats. The genetic differences within the cyt b gene among the meat were successfully confirmed by PCR-RFLP. A reliable typing scheme of species which revealed the genetic differences among the species was developed.

  4. Cellular Restriction Factors of Feline Immunodeficiency Virus

    Science.gov (United States)

    Zielonka, Jörg; Münk, Carsten

    2011-01-01

    Lentiviruses are known for their narrow cell- and species-tropisms, which are determined by cellular proteins whose absence or presence either support viral replication (dependency factors, cofactors) or inhibit viral replication (restriction factors). Similar to Human immunodeficiency virus type 1 (HIV-1), the cat lentivirus Feline immunodeficiency virus (FIV) is sensitive to recently discovered cellular restriction factors from non-host species that are able to stop viruses from replicating. Of particular importance are the cellular proteins APOBEC3, TRIM5α and tetherin/BST-2. In general, lentiviruses counteract or escape their species’ own variant of the restriction factor, but are targeted by the orthologous proteins of distantly related species. Most of the knowledge regarding lentiviral restriction factors has been obtained in the HIV-1 system; however, much less is known about their effects on other lentiviruses. We describe here the molecular mechanisms that explain how FIV maintains its replication in feline cells, but is largely prevented from cross-species infections by cellular restriction factors. PMID:22069525

  5. HIV-related travel restrictions: trends and country characteristics.

    Science.gov (United States)

    Chang, Felicia; Prytherch, Helen; Nesbitt, Robin C; Wilder-Smith, Annelies

    2013-06-03

    Increasingly, HIV-seropositive individuals cross international borders. HIV-related restrictions on entry, stay, and residence imposed by countries have important consequences for this mobile population. Our aim was to describe the geographical distribution of countries with travel restrictions and to examine the trends and characteristics of countries with such restrictions. In 2011, data presented to UNAIDS were used to establish a list of countries with and without HIV restrictions on entry, stay, and residence and to describe their geographical distribution. The following indicators were investigated to describe the country characteristics: population at mid-year, international migrants as a percentage of the population, Human Development Index, estimated HIV prevalence (age: 15-49), presence of a policy prohibiting HIV screening for general employment purposes, government and civil society responses to having non-discrimination laws/regulations which specify migrants/mobile populations, government and civil society responses to having laws/regulations/policies that present obstacles to effective HIV prevention, treatment, care, and support for migrants/mobile populations, Corruption Perception Index, and gross national income per capita. HIV-related restrictions exist in 45 out of 193 WHO countries (23%) in all regions of the world. We found that the Eastern Mediterranean and Western Pacific Regions have the highest proportions of countries with these restrictions. Our analyses showed that countries that have opted for restrictions have the following characteristics: smaller populations, higher proportions of migrants in the population, lower HIV prevalence rates, and lack of legislation protecting people living with HIV from screening for employment purposes, compared with countries without restrictions. Countries with a high proportion of international migrants tend to have travel restrictions - a finding that is relevant to migrant populations and travel

  6. Eclampsia despite strict dietary sodium restriction.

    NARCIS (Netherlands)

    Delemarre, F.M.C.; Steegers, E.A.P.; Berendes, J.N.

    2001-01-01

    The classic indication for prescribing dietary sodium restriction in pregnancy has been the prevention of eclampsia. We describe a case of intrapartum eclampsia in a 24-year-old nulliparous woman. A strongly sodium restricted diet was prescribed because of pre-eclampsia. Compliance to the diet was

  7. 7 CFR 3430.205 - Funding restrictions.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 15 2010-01-01 2010-01-01 false Funding restrictions. 3430.205 Section 3430.205... Funding restrictions. (a) Prohibition against construction. Funds made available under this subpart shall not be used for the construction of a new building or facility or the acquisition, expansion...

  8. High-resolution crystal structure reveals a HEPN domain at the C-terminal region of S. cerevisiae RNA endonuclease Swt1

    International Nuclear Information System (INIS)

    Peng, Shuxia; Zhou, Ke; Wang, Wenjia; Gao, Zengqiang; Dong, Yuhui; Liu, Quansheng

    2014-01-01

    Highlights: • Crystal structure of the C-terminal (CT) domain of Swt1 was determined at 2.3 Å. • Structure of the CT domain was identified as HEPN domain superfamily member. • Low-resolution envelope of Swt1 full-length in solution was analyzed by SAXS. • The middle and CT domains gave good fit to SAXS structural model. - Abstract: Swt1 is an RNA endonuclease that plays an important role in quality control of nuclear messenger ribonucleoprotein particles (mRNPs) in eukaryotes; however, its structural details remain to be elucidated. Here, we report the crystal structure of the C-terminal (CT) domain of Swt1 from Saccharomyces cerevisiae, which shares common characteristics of higher eukaryotes and prokaryotes nucleotide binding (HEPN) domain superfamily. To study in detail the full-length protein structure, we analyzed the low-resolution architecture of Swt1 in solution using small angle X-ray scattering (SAXS) method. Both the CT domain and middle domain exhibited a good fit upon superimposing onto the molecular envelope of Swt1. Our study provides the necessary structural information for detailed analysis of the functional role of Swt1, and its importance in the process of nuclear mRNP surveillance

  9. Rate-determining Step of Flap Endonuclease 1 (FEN1) Reflects a Kinetic Bias against Long Flaps and Trinucleotide Repeat Sequences.

    Science.gov (United States)

    Tarantino, Mary E; Bilotti, Katharina; Huang, Ji; Delaney, Sarah

    2015-08-21

    Flap endonuclease 1 (FEN1) is a structure-specific nuclease responsible for removing 5'-flaps formed during Okazaki fragment maturation and long patch base excision repair. In this work, we use rapid quench flow techniques to examine the rates of 5'-flap removal on DNA substrates of varying length and sequence. Of particular interest are flaps containing trinucleotide repeats (TNR), which have been proposed to affect FEN1 activity and cause genetic instability. We report that FEN1 processes substrates containing flaps of 30 nucleotides or fewer at comparable single-turnover rates. However, for flaps longer than 30 nucleotides, FEN1 kinetically discriminates substrates based on flap length and flap sequence. In particular, FEN1 removes flaps containing TNR sequences at a rate slower than mixed sequence flaps of the same length. Furthermore, multiple-turnover kinetic analysis reveals that the rate-determining step of FEN1 switches as a function of flap length from product release to chemistry (or a step prior to chemistry). These results provide a kinetic perspective on the role of FEN1 in DNA replication and repair and contribute to our understanding of FEN1 in mediating genetic instability of TNR sequences. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. A statistical mechanical approach to restricted integer partition functions

    Science.gov (United States)

    Zhou, Chi-Chun; Dai, Wu-Sheng

    2018-05-01

    The main aim of this paper is twofold: (1) suggesting a statistical mechanical approach to the calculation of the generating function of restricted integer partition functions which count the number of partitions—a way of writing an integer as a sum of other integers under certain restrictions. In this approach, the generating function of restricted integer partition functions is constructed from the canonical partition functions of various quantum gases. (2) Introducing a new type of restricted integer partition functions corresponding to general statistics which is a generalization of Gentile statistics in statistical mechanics; many kinds of restricted integer partition functions are special cases of this restricted integer partition function. Moreover, with statistical mechanics as a bridge, we reveal a mathematical fact: the generating function of restricted integer partition function is just the symmetric function which is a class of functions being invariant under the action of permutation groups. Using this approach, we provide some expressions of restricted integer partition functions as examples.

  11. How Harmful are Adaptation Restrictions

    OpenAIRE

    Bruin, de, K.C.; Dellink, R.B.

    2009-01-01

    The dominant assumption in economic models of climate policy remains that adaptation will be implemented in an optimal manner. There are, however, several reasons why optimal levels of adaptation may not be attainable. This paper investigates the effects of suboptimal levels of adaptation, i.e. adaptation restrictions, on the composition and level of climate change costs and on welfare. Several adaptation restrictions are identified and then simulated in a revised DICE model, extended with ad...

  12. DNA and Protein Requirements for Substrate Conformational Changes Necessary for Human Flap Endonuclease-1-catalyzed Reaction*

    Science.gov (United States)

    Algasaier, Sana I.; Exell, Jack C.; Bennet, Ian A.; Thompson, Mark J.; Gotham, Victoria J. B.; Shaw, Steven J.; Craggs, Timothy D.; Finger, L. David; Grasby, Jane A.

    2016-01-01

    Human flap endonuclease-1 (hFEN1) catalyzes the essential removal of single-stranded flaps arising at DNA junctions during replication and repair processes. hFEN1 biological function must be precisely controlled, and consequently, the protein relies on a combination of protein and substrate conformational changes as a prerequisite for reaction. These include substrate bending at the duplex-duplex junction and transfer of unpaired reacting duplex end into the active site. When present, 5′-flaps are thought to thread under the helical cap, limiting reaction to flaps with free 5′-termini in vivo. Here we monitored DNA bending by FRET and DNA unpairing using 2-aminopurine exciton pair CD to determine the DNA and protein requirements for these substrate conformational changes. Binding of DNA to hFEN1 in a bent conformation occurred independently of 5′-flap accommodation and did not require active site metal ions or the presence of conserved active site residues. More stringent requirements exist for transfer of the substrate to the active site. Placement of the scissile phosphate diester in the active site required the presence of divalent metal ions, a free 5′-flap (if present), a Watson-Crick base pair at the terminus of the reacting duplex, and the intact secondary structure of the enzyme helical cap. Optimal positioning of the scissile phosphate additionally required active site conserved residues Tyr40, Asp181, and Arg100 and a reacting duplex 5′-phosphate. These studies suggest a FEN1 reaction mechanism where junctions are bound and 5′-flaps are threaded (when present), and finally the substrate is transferred onto active site metals initiating cleavage. PMID:26884332

  13. TALE-PvuII Fusion Proteins – Novel Tools for Gene Targeting

    Science.gov (United States)

    Yanik, Mert; Alzubi, Jamal; Lahaye, Thomas; Cathomen, Toni; Pingoud, Alfred; Wende, Wolfgang

    2013-01-01

    Zinc finger nucleases (ZFNs) consist of zinc fingers as DNA-binding module and the non-specific DNA-cleavage domain of the restriction endonuclease FokI as DNA-cleavage module. This architecture is also used by TALE nucleases (TALENs), in which the DNA-binding modules of the ZFNs have been replaced by DNA-binding domains based on transcription activator like effector (TALE) proteins. Both TALENs and ZFNs are programmable nucleases which rely on the dimerization of FokI to induce double-strand DNA cleavage at the target site after recognition of the target DNA by the respective DNA-binding module. TALENs seem to have an advantage over ZFNs, as the assembly of TALE proteins is easier than that of ZFNs. Here, we present evidence that variant TALENs can be produced by replacing the catalytic domain of FokI with the restriction endonuclease PvuII. These fusion proteins recognize only the composite recognition site consisting of the target site of the TALE protein and the PvuII recognition sequence (addressed site), but not isolated TALE or PvuII recognition sites (unaddressed sites), even at high excess of protein over DNA and long incubation times. In vitro, their preference for an addressed over an unaddressed site is > 34,000-fold. Moreover, TALE-PvuII fusion proteins are active in cellula with minimal cytotoxicity. PMID:24349308

  14. TALE-PvuII fusion proteins--novel tools for gene targeting.

    Science.gov (United States)

    Yanik, Mert; Alzubi, Jamal; Lahaye, Thomas; Cathomen, Toni; Pingoud, Alfred; Wende, Wolfgang

    2013-01-01

    Zinc finger nucleases (ZFNs) consist of zinc fingers as DNA-binding module and the non-specific DNA-cleavage domain of the restriction endonuclease FokI as DNA-cleavage module. This architecture is also used by TALE nucleases (TALENs), in which the DNA-binding modules of the ZFNs have been replaced by DNA-binding domains based on transcription activator like effector (TALE) proteins. Both TALENs and ZFNs are programmable nucleases which rely on the dimerization of FokI to induce double-strand DNA cleavage at the target site after recognition of the target DNA by the respective DNA-binding module. TALENs seem to have an advantage over ZFNs, as the assembly of TALE proteins is easier than that of ZFNs. Here, we present evidence that variant TALENs can be produced by replacing the catalytic domain of FokI with the restriction endonuclease PvuII. These fusion proteins recognize only the composite recognition site consisting of the target site of the TALE protein and the PvuII recognition sequence (addressed site), but not isolated TALE or PvuII recognition sites (unaddressed sites), even at high excess of protein over DNA and long incubation times. In vitro, their preference for an addressed over an unaddressed site is > 34,000-fold. Moreover, TALE-PvuII fusion proteins are active in cellula with minimal cytotoxicity.

  15. Ochratoxigenic Black Species of Aspergilli in Grape Fruits of Northern Italy Identified by an Improved PCR-RFLP Procedure

    Directory of Open Access Journals (Sweden)

    Maria Lodovica Gullino

    2012-01-01

    Full Text Available A collection of 356 isolates of Aspergillus spp. collected during 2006 and 2007 from grapevines in northern Italy were identified through Internal Transcribed Spacer based Restriction Fragment Length Polymorphism (ITS-RFLP and tested for ochratoxin A (OTA production. Restriction endonuclease digestion of the ITS products using the endonucleases HhaI, HinfI and RsaI, distinguished five different RFLPs. From each pattern, three samples were sequenced and the nucleotide sequences showed different species corresponding to Aspergillus niger, A. carbonarius, A. tubingensis, A. japonicus and A. aculeatus. By comparing the sequences of the ITS regions, also the uniseriate species A. japonicus and A. aculeatus could be differentiated by HinfI digestion of the ITS products. Among the aspergilli, A. niger was the major species associated with grapes during 2006 (57.4%, while A. carbonarius was the major species during 2007 (46.6%. All the strains of Aspergillus were tested for their ability to produce OTA on Yeast extract sucrose medium (YES, as it was tested as an optimal substrate for the evaluation of OTA production by black aspergilli. Out of 356 isolates, 63 (17.7% isolates produced OTA ranging from 0.05 to 3.0 µg mL−1. Most of the ochratoxigenic isolates were A. carbonarius (46 in both years, but also some strains of A. tubingensis (11 and A. japonicus (6 produced lower amounts of OTA.

  16. 48 CFR 552.203-71 - Restriction on Advertising.

    Science.gov (United States)

    2010-10-01

    ... 48 Federal Acquisition Regulations System 4 2010-10-01 2010-10-01 false Restriction on Advertising... Restriction on Advertising. As prescribed in 503.570-2, insert the following clause: Restriction on Advertising (SEP 1999) The Contractor shall not refer to this contract in commercial advertising or similar...

  17. Cardiomyocyte hypertrophy induced by Endonuclease G deficiency requires reactive oxygen radicals accumulation and is inhibitable by the micropeptide humanin.

    Science.gov (United States)

    Blasco, Natividad; Cámara, Yolanda; Núñez, Estefanía; Beà, Aida; Barés, Gisel; Forné, Carles; Ruíz-Meana, Marisol; Girón, Cristina; Barba, Ignasi; García-Arumí, Elena; García-Dorado, David; Vázquez, Jesús; Martí, Ramon; Llovera, Marta; Sanchis, Daniel

    2018-06-01

    The endonuclease G gene (Endog), which codes for a mitochondrial nuclease, was identified as a determinant of cardiac hypertrophy. How ENDOG controls cardiomyocyte growth is still unknown. Thus, we aimed at finding the link between ENDOG activity and cardiomyocyte growth. Endog deficiency induced reactive oxygen species (ROS) accumulation and abnormal growth in neonatal rodent cardiomyocytes, altering the AKT-GSK3β and Class-II histone deacethylases (HDAC) signal transduction pathways. These effects were blocked by ROS scavengers. Lack of ENDOG reduced mitochondrial DNA (mtDNA) replication independently of ROS accumulation. Because mtDNA encodes several subunits of the mitochondrial electron transport chain, whose activity is an important source of cellular ROS, we investigated whether Endog deficiency compromised the expression and activity of the respiratory chain complexes but found no changes in these parameters nor in ATP content. MtDNA also codes for humanin, a micropeptide with possible metabolic functions. Nanomolar concentrations of synthetic humanin restored normal ROS levels and cell size in Endog-deficient cardiomyocytes. These results support the involvement of redox signaling in the control of cardiomyocyte growth by ENDOG and suggest a pathway relating mtDNA content to the regulation of cell growth probably involving humanin, which prevents reactive oxygen radicals accumulation and hypertrophy induced by Endog deficiency. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

  18. Inhibitors of nuclease and redox activity of apurinic/apyrimidinic endonuclease 1/redox effector factor 1 (APE1/Ref-1).

    Science.gov (United States)

    Laev, Sergey S; Salakhutdinov, Nariman F; Lavrik, Olga I

    2017-05-01

    Human apurinic/apyrimidinic endonuclease 1/redox effector factor 1 (APE1/Ref-1) is a multifunctional protein which is essential in the base excision repair (BER) pathway of DNA lesions caused by oxidation and alkylation. This protein hydrolyzes DNA adjacent to the 5'-end of an apurinic/apyrimidinic (AP) site to produce a nick with a 3'-hydroxyl group and a 5'-deoxyribose phosphate moiety or activates the DNA-binding activity of certain transcription factors through its redox function. Studies have indicated a role for APE1/Ref-1 in the pathogenesis of cancer and in resistance to DNA-interactive drugs. Thus, this protein has potential as a target in cancer treatment. As a result, major efforts have been directed to identify small molecule inhibitors against APE1/Ref-1 activities. These agents have the potential to become anticancer drugs. The aim of this review is to present recent progress in studies of all published small molecule APE1/Ref-1 inhibitors. The structures and activities of APE1/Ref-1 inhibitors, that target both DNA repair and redox activities, are presented and discussed. To date, there is an urgent need for further development of the design and synthesis of APE1/Ref-1 inhibitors due to high importance of this protein target. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Investigation of restricted baby Skyrme models

    International Nuclear Information System (INIS)

    Adam, C.; Romanczukiewicz, T.; Wereszczynski, A.; Sanchez-Guillen, J.

    2010-01-01

    A restriction of the baby Skyrme model consisting of the quartic and potential terms only is investigated in detail for a wide range of potentials. Further, its properties are compared with those of the corresponding full baby Skyrme models. We find that topological (charge) as well as geometrical (nucleus/shell shape) features of baby Skyrmions are captured already by the soliton solutions of the restricted model. Further, we find a coincidence between the compact or noncompact nature of solitons in the restricted model, on the one hand, and the existence or nonexistence of multi-Skyrmions in the full baby Skyrme model, on the other hand.

  20. Prognostic value of human apurinic/apyrimidinic endonuclease 1 (APE1 expression in breast cancer.

    Directory of Open Access Journals (Sweden)

    Joohyun Woo

    Full Text Available Human apurinic/apyrimidinic endonuclease 1 (APE1 is an essential protein for DNA base excision repair (BER and redox regulation. The ability of cancer cells to recognize DNA damage and initiate DNA repair is an important mechanism for therapeutic resistance. Several recent studies have suggested that APE1 expression levels and/or subcellular dysregulation may be used to indicate the sensitivity of tumors to radiotherapy or chemotherapy. In this study, we assessed the prognostic significance of APE1 and differences in APE1 expression levels according to breast cancer molecular subtypes. We analyzed formalin-fixed, paraffin-embedded tumor tissue sections from 243 cases diagnosed as invasive breast cancer at Ewha Womans University Medical Center between January 2003 and December 2008. Immunohistochemistry was performed and the nuclear level of APE1 was scored by taking into account the percentage of positive cells. Medical records were reviewed to investigate clinicopathologic characteristics. We found that nuclear APE1 high-level expression (proportion ≥50% in breast cancer showed a tendency towards unfavorable prognosis regarding disease-free survival (p = 0.093. However, there was no significant difference in overall survival between low and high-level expression groups (p = 0.294. Interestingly, within the Ki-67 low-level expression group, APE1 low-level expression was significantly associated with poor overall survival (p = 0.007. A significant positive correlation was observed between APE1 nuclear expression and estrogen receptor status (75.7% vs. 59.7%, p = 0.022. Also, the luminal A subtype was the most commonly observed breast cancer subtype in the APE1 high-level expression group (61.6% vs. 45.2%, p = 0.000. This study suggests that APE1 expression may be associated with breast cancer prognosis. In particular, its role as a prognostic factor would be significant for breast cancers with a low Ki-67 proliferation index

  1. Apurinic/apyrimidinic endonuclease 1 regulates angiogenesis in a transforming growth factor β-dependent manner in human osteosarcoma.

    Science.gov (United States)

    Jiang, Xuan; Shan, Jinlu; Dai, Nan; Zhong, Zhaoyang; Qing, Yi; Yang, Yuxing; Zhang, Shiheng; Li, Chongyi; Sui, Jiangdong; Ren, Tao; Li, Mengxia; Wang, Dong

    2015-10-01

    Angiogenesis plays an important role in tumor growth and metastasis and has been reported to be inversely correlated with overall survival of osteosarcoma patients. It has been shown that apurinic/apyrimidinic endonuclease 1 (APE1), a dually functional protein possessing both base excision repair and redox activities, is involved in tumor angiogenesis, although these mechanisms are not fully understood. Our previous study showed that the expression of transforming growth factor β (TGFβ) was significantly reduced in APE1-deficient osteosarcoma cells. Transforming growth factor β promotes cancer metastasis through various mechanisms including immunosuppression, angiogenesis, and invasion. In the current study, we initially revealed that APE1, TGFβ, and microvessel density (MVD) have pairwise correlation in osteosarcoma tissue samples, whereas TGFβ, tumor size, and MVD were inversely related to the prognosis of the cohort. We found that knocking down APE1 in osteosarcoma cells resulted in TGFβ downregulation. In addition, APE1-siRNA led to suppression of angiogenesis in vitro based on HUVECs in Transwell and Matrigel tube formation assays. Reduced secretory protein level of TGFβ of culture medium also resulted in decreased phosphorylation of Smad3 of HUVECs. In a mouse xenograft model, siRNA-mediated silencing of APE1 downregulated TGFβ expression, tumor size, and MVD. Collectively, the current evidence indicates that APE1 regulates angiogenesis in osteosarcoma by controlling the TGFβ pathway, suggesting a novel target for anti-angiogenesis therapy in human osteosarcoma. © 2015 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

  2. Electricity system planning under the CO2 emission restriction

    International Nuclear Information System (INIS)

    Lim, Chae Young; Lee, Man Ki; Roh, Jae Hyung; Kim, Eun Hwan

    2004-01-01

    Objective of this study is to analyze how the restriction of CO 2 emission from power generation will affect the national electricity supply system. The role of nuclear power is investigated under the restriction of CO 2 emission in Korea. A simplified electricity system was modeled for the analysis. To analyze the impact of CO 2 emission restriction, 2 different scenarios were established and compared with the base scenario. The first scenario was 'CO 2 emission restriction with new nuclear power installation'. In this scenario, a CO 2 emission restriction of 0.11kg-C/kWh was imposed and there was no restriction on the nuclear power construction. While, in the second scenario, 'CO 2 emission restriction without new nuclear power installation' the same amount of CO 2 restriction was imposed with no consideration of nuclear power installation. It is found out that the current national emission target(0.11kg- C/kWh) in the electricity sector can not be achieved without nuclear and renewable(wind power) options considered

  3. Protein restriction in chronic renal failure

    NARCIS (Netherlands)

    ECHTEN, JEKT; NAUTA, J; HOP, WCJ; de Jong, MCJ; REITSMABIERENS, WCC; VANAMSTEL, SLBP; VANACKER, KJ; NOORDZIJ, CM; WOLFF, ED

    The aim of the study was to investigate the effect of a protein restricted diet on renal function and growth of children with chronic renal failure. In a multicentre prospective study 56 children (aged 2-18 years) with chronic renal failure were randomly assigned to the protein restricted (0.8-1.1

  4. 49 CFR 215.303 - Stenciling of restricted cars.

    Science.gov (United States)

    2010-10-01

    ... 49 Transportation 4 2010-10-01 2010-10-01 false Stenciling of restricted cars. 215.303 Section 215... ADMINISTRATION, DEPARTMENT OF TRANSPORTATION RAILROAD FREIGHT CAR SAFETY STANDARDS Stenciling § 215.303 Stenciling of restricted cars. (a) Each restricted railroad freight car that is described in § 215.205(a) of...

  5. Cellular Restriction Factors of Feline Immunodeficiency Virus

    Directory of Open Access Journals (Sweden)

    Carsten Münk

    2011-10-01

    Full Text Available Lentiviruses are known for their narrow cell- and species-tropisms, which are determined by cellular proteins whose absence or presence either support viral replication (dependency factors, cofactors or inhibit viral replication (restriction factors. Similar to Human immunodeficiency virus type 1 (HIV-1, the cat lentivirus Feline immunodeficiency virus (FIV is sensitive to recently discovered cellular restriction factors from non-host species that are able to stop viruses from replicating. Of particular importance are the cellular proteins APOBEC3, TRIM5α and tetherin/BST-2. In general, lentiviruses counteract or escape their species’ own variant of the restriction factor, but are targeted by the orthologous proteins of distantly related species. Most of the knowledge regarding lentiviral restriction factors has been obtained in the HIV-1 system; however, much less is known about their effects on other lentiviruses. We describe here the molecular mechanisms that explain how FIV maintains its replication in feline cells, but is largely prevented from cross-species infections by cellular restriction factors.

  6. An AP endonuclease 1-DNA polymerase beta complex: theoretical prediction of interacting surfaces.

    Directory of Open Access Journals (Sweden)

    Alexej Abyzov

    2008-04-01

    Full Text Available Abasic (AP sites in DNA arise through both endogenous and exogenous mechanisms. Since AP sites can prevent replication and transcription, the cell contains systems for their identification and repair. AP endonuclease (APEX1 cleaves the phosphodiester backbone 5' to the AP site. The cleavage, a key step in the base excision repair pathway, is followed by nucleotide insertion and removal of the downstream deoxyribose moiety, performed most often by DNA polymerase beta (pol-beta. While yeast two-hybrid studies and electrophoretic mobility shift assays provide evidence for interaction of APEX1 and pol-beta, the specifics remain obscure. We describe a theoretical study designed to predict detailed interacting surfaces between APEX1 and pol-beta based on published co-crystal structures of each enzyme bound to DNA. Several potentially interacting complexes were identified by sliding the protein molecules along DNA: two with pol-beta located downstream of APEX1 (3' to the damaged site and three with pol-beta located upstream of APEX1 (5' to the damaged site. Molecular dynamics (MD simulations, ensuring geometrical complementarity of interfaces, enabled us to predict interacting residues and calculate binding energies, which in two cases were sufficient (approximately -10.0 kcal/mol to form a stable complex and in one case a weakly interacting complex. Analysis of interface behavior during MD simulation and visual inspection of interfaces allowed us to conclude that complexes with pol-beta at the 3'-side of APEX1 are those most likely to occur in vivo. Additional multiple sequence analyses of APEX1 and pol-beta in related organisms identified a set of correlated mutations of specific residues at the predicted interfaces. Based on these results, we propose that pol-beta in the open or closed conformation interacts and makes a stable interface with APEX1 bound to a cleaved abasic site on the 3' side. The method described here can be used for analysis in

  7. Characterization of Virulence Plasmids and Serotyping of Rhodococcus equi Isolates from Submaxillary Lymph Nodes of Pigs in Hungary

    OpenAIRE

    Makrai, László; Takayama, Saki; Dénes, Béla; Hajtós, István; Sasaki, Yukako; Kakuda, Tsutomu; Tsubaki, Shiro; Major, Andrea; Fodor, László; Varga, János; Takai, Shinji

    2005-01-01

    The plasmid types and serotypes of 164 Rhodococcus equi strains obtained from submaxillary lymph nodes of swine from different piggeries in 28 villages and towns located throughout the country were examined. The strains were tested by PCR for the presence of 15- to 17-kDa virulence-associated protein antigen (VapA) and 20-kDa virulence-associated protein antigen (VapB) genes. Plasmid DNAs were isolated and analyzed by digestion with restriction endonucleases to estimate size and compare their...

  8. Replication and Transcription of Eukaryotic DNA in Esherichia coli

    Science.gov (United States)

    Morrow, John F.; Cohen, Stanley N.; Chang, Annie C. Y.; Boyer, Herbert W.; Goodman, Howard M.; Helling, Robert B.

    1974-01-01

    Fragments of amplified Xenopus laevis DNA, coding for 18S and 28S ribosomal RNA and generated by EcoRI restriction endonuclease, have been linked in vitro to the bacterial plasmid pSC101; and the recombinant molecular species have been introduced into E. coli by transformation. These recombinant plasmids, containing both eukaryotic and prokaryotic DNA, replicate stably in E. coli. RNA isolated from E. coli minicells harboring the plasmids hybridizes to amplified X. laevis rDNA. Images PMID:4600264

  9. 36 CFR 1275.58 - Deletion of restricted portions.

    Science.gov (United States)

    2010-07-01

    ... 36 Parks, Forests, and Public Property 3 2010-07-01 2010-07-01 false Deletion of restricted... HISTORICAL MATERIALS OF THE NIXON ADMINISTRATION Access by the Public § 1275.58 Deletion of restricted... materials after the deletion of the portions which are restricted under this § 1275.50 or § 1275.52. ...

  10. Freedom and Restrictions in Language Use.

    Science.gov (United States)

    O'Donnell, Roy C.

    Since freedom of thought and expression is essential in a democracy, censorship of language is rightly regarded as a threat to all other freedoms. Still, it is inevitable that certain restrictions will occasionally be imposed on language in America and in other societies. Restrictions on language date back to the Ten Commandments, which condemned…

  11. Review: Neuroinflammation in intrauterine growth restriction.

    Science.gov (United States)

    Wixey, Julie A; Chand, Kirat K; Colditz, Paul B; Bjorkman, S Tracey

    2017-06-01

    Disruption to the maternal environment during pregnancy from events such as hypoxia, stress, toxins, inflammation, and reduced placental blood flow can affect fetal development. Intrauterine growth restriction (IUGR) is commonly caused by chronic placental insufficiency, interrupting supply of oxygen and nutrients to the fetus resulting in abnormal fetal growth. IUGR is a major cause of perinatal morbidity and mortality, occurring in approximately 5-10% of pregnancies. The fetal brain is particularly vulnerable in IUGR and there is an increased risk of long-term neurological disorders including cerebral palsy, epilepsy, learning difficulties, behavioural difficulties and psychiatric diagnoses. Few studies have focused on how growth restriction interferes with normal brain development in the IUGR neonate but recent studies in growth restricted animal models demonstrate increased neuroinflammation. This review describes the role of neuroinflammation in the progression of brain injury in growth restricted neonates. Identifying the mediators responsible for alterations in brain development in the IUGR infant is key to prevention and treatment of brain injury in these infants. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Nutrigenetics and nutrigenomics of caloric restriction.

    Science.gov (United States)

    Abete, Itziar; Navas-Carretero, Santiago; Marti, Amelia; Martinez, J Alfredo

    2012-01-01

    Obesity is a complex disease resulting from a chronic and long-term positive energy balance in which both genetic and environmental factors are involved. Weight-reduction methods are mainly focused on dietary changes and increased physical activity. However, responses to nutritional intervention programs show a wide range of interindividual variation, which is importantly influenced by genetic determinants. In this sense, subjects carrying several obesity-related single-nucleotide polymorphisms (SNPs) show differences in the response to calorie-restriction programs. Furthermore, there is evidence indicating that dietary components not only fuel the body but also participate in the modulation of gene expression. Thus, the expression pattern and nutritional regulation of several obesity-related genes have been studied, as well as those that are differentially expressed by caloric restriction. The responses to caloric restriction linked to the presence of SNPs in obesity-related genes are reviewed in this chapter. Also, the influence of energy restriction on gene expression pattern in different tissues is addressed. Copyright © 2012 Elsevier Inc. All rights reserved.

  13. Sight Restrictions in Maghrib Muslim Architecture

    Directory of Open Access Journals (Sweden)

    Mustapha Ben Hamouche

    1999-12-01

    Full Text Available Sight in Islamic culture is subject to legal restrictions that aim at preserving moral consciousness in Muslim societies. These restrictions have a direct impact on architecture in traditional Muslim cities. Details such as placement of doors and windows, the use of balconies and rooftops, and building heights were shaped by legal reasoning based on sight restrictions. The present study aims at highlighting this legal reasoning system by analyzing legal opinions that were continuously advocated by jurists in response to daily practices, and the legal principles on which these opinions were based. This is expected to contribute in developing a new intellectual discourse on Muslim architecture that could go beyond the present design theories.

  14. Recommendations of activity restriction in high-risk pregnancy scenarios

    DEFF Research Database (Denmark)

    Bendix, Jane; Hegaard, Hanne Kristine; Bergholt, Thomas

    2015-01-01

    activity restriction more often than obstetricians in five of the nine scenarios, in women with preterm premature rupture of membranes, preterm labour, cervical ripening, total placenta praevia, and intrauterine growth restriction, whereas no differences were found in the remaining scenarios. Compared...... to the obstetricians, the midwives also reported that they expected the recommendation to be more effective. Most midwives and obstetricians reported that they thought strict activity restriction was associated with severe or moderate adverse effect, and recommended antithrombotic prophylaxis. Conclusions: Danish...... obstetricians and midwives prescribe activity restriction in most high-risk pregnancies. The degree of activity restriction and the presumed effect vary between clinicians. This may reflect different attitudes and lack of guidelines based on clinical studies of a possible benefit of activity restriction....

  15. Health Benefits of Fasting and Caloric Restriction.

    Science.gov (United States)

    Golbidi, Saeid; Daiber, Andreas; Korac, Bato; Li, Huige; Essop, M Faadiel; Laher, Ismail

    2017-10-23

    Obesity and obesity-related diseases, largely resulting from urbanization and behavioral changes, are now of global importance. Energy restriction, though, is associated with health improvements and increased longevity. We review some important mechanisms related to calorie limitation aimed at controlling of metabolic diseases, particularly diabetes. Calorie restriction triggers a complex series of intricate events, including activation of cellular stress response elements, improved autophagy, modification of apoptosis, and alteration in hormonal balance. Intermittent fasting is not only more acceptable to patients, but it also prevents some of the adverse effects of chronic calorie restriction, especially malnutrition. There are many somatic and potentially psychologic benefits of fasting or intermittent calorie restriction. However, some behavioral modifications related to abstinence of binge eating following a fasting period are crucial in maintaining the desired favorable outcomes.

  16. Relationship Between Calorie Restriction, Lipid Peroxidation ...

    African Journals Online (AJOL)

    In the brain of the caloric restricted rats, there was little or no change in the tGSH and GSH, although the GSSG and GSSG/GSH% ratio were increased significantly. These results suggest that aging of rats had been decelerated by caloric restriction due to the decrease in the peroxidative damage in the lungs and brain.

  17. 78 FR 40958 - Amendment of Restricted Areas R-2504A & R-2504B; Camp Roberts, CA, and Restricted Area R-2530...

    Science.gov (United States)

    2013-07-09

    ...-0515; Airspace Docket No. 13-AWP-8] RIN 2120-AA66 Amendment of Restricted Areas R-2504A & R-2504B; Camp Roberts, CA, and Restricted Area R-2530; Sierra Army Depot, CA AGENCY: Federal Aviation Administration (FAA), DOT. ACTION: Final rule. SUMMARY: This action amends the descriptions of restricted areas R...

  18. Curcumin and EGCG Suppress Apurinic/Apyrimidinic Endonuclease 1 and Induce Complete Remission in B-cell Non-Hodgkin's lymphoma Patients

    Directory of Open Access Journals (Sweden)

    Hashem M. Neenaa

    2011-12-01

    Full Text Available ABSTRACT:Background: Follicular lymphoma (FL is the most common subtype of indolent lymphoma. FL is still considered to be an incurable disease and palliation of symptoms is an acceptable approach to the expected pattern of repeated relapses due to developing resistance to chemotherapy agents. Apurinic/apyrimidinic endonuclease/redox factor-1 (APE1/Ref-1 is a multifunctional protein involved in DNA base excision repair (BER of oxidative DNA damage and in redox regulation of a number of transcription factors. It was observed that cytoplasmic APE1 induced COX-2 expression through NF-êB activation. It has been shown that chemopreventive agents potentiate the efficacy of chemotherapy through the regulation of multiple signaling pathways, including NF-êB, c-Myc, cyclooxygenase-2, apoptosis, and others, suggesting a multitargeted nature of chemopreventive agents. We hypothesized that curcumin, a polyphenolic antioxidant derived from the spice turmeric, and epigallocatechin gallate (EGCG from green tea would potentiate the effect of chemotherapy in B-cell lymphoma.Objective: We examined the role of human apurinic/apyrimidinic endonuclease 1 (APE1 in resistance and prognosis in patients with FL. Our major objective was to update the safety and efficacy results of the antitumor effect of combination of curcumin and EGCG therapy in relapsed or resistant indolent or transformed non-Hodgkin follicular lymphoma patients and their peripheral blood mononuclear cells (PBMCs compared with healthy donors’ controls.Methods: Thirty patients with FL with over-expression of constitutive active NF-êB in their PBMCs received regular CHOP and consumed capsules compatible with curcumin doses between 0.9 and 5.4 g daily for up to 9 months and 9.0 g/day green tea whole extract "1000 mg tablets of green tea whole extract containing 200 mg EGCG. We designed a dose-escalation Functional Foods in Health and Disease 2011, 1(12:525-544 study to explore the efficacy of CHOP

  19. Restrictive Dermopathy: Molecular diagnosis of restrictive dermopathy in a stillborn fetus from a consanguineous Iranian family

    International Nuclear Information System (INIS)

    Karaminejad, A.; Goodarzi, P.; Huong, Le Thi Thanh; Wehnert, Manfred S.

    2009-01-01

    Restrictive dermopathy (RD) is an autosomal recessive lethal human genetic disorder. It is characterized by intrauterine growth retardation, tight and rigid skin with erosions, multiple joint contractures, lung hypoplasia, prominent superficial vasculature and epidermal hyperkeratosis. In the present report, we describe the first case of restrictive dermopathy in a stillborn fetus of Iranian origin, confirmed by molecular genetic diagnosis. In the index case (G-30159), a homozygous one base insertion in ZMPSTE24exon 9 (c.1085-1086insT) was identified. We believe that by increasing awareness of this disease in clinicians, gynecologists and pathologists, we may be able to help families who have had suspected cases of restrictive dermopathy be diagnosed and offer molecular testing in carriers and prenatal diagnosis to prevent the occurrence of further affected cases. (author)

  20. Challenges in nourishing the intrauterine growth-restricted foetus - Lessons learned from studies in the intrauterine growth-restricted foetal sheep.

    Science.gov (United States)

    Hay, William W; Brown, Laura D; Rozance, Paul J; Wesolowski, Stephanie R; Limesand, Sean W

    2016-08-01

    Previous attempts to improve growth and development of the intrauterine growth-restricted (IUGR) foetus during pregnancy have not worked or caused harm. Our research identifies tissue-specific mechanisms underlying foetal growth restriction and then tests strategies to improve growth and ameliorate many of the metabolic problems before the infant is born. The goal of our studies is to reduce the impact of foetal growth restriction at critical stages of development on the lifelong complications of IUGR offspring. Defining specific mechanisms that cause growth restriction in the foetus might identify specific nutrients and hormones that could be given to the mother to improve foetal growth and reduce metabolic complications, using strategies first tested in our IUGR animal model. ©2016 Foundation Acta Paediatrica. Published by John Wiley & Sons Ltd.

  1. Restrictions in Availability of Drugs Used for Suicide

    DEFF Research Database (Denmark)

    Nordentoft, Merete

    2007-01-01

    Availability of drugs with high lethality has been hypothesized to increase the risk of self-poisoning suicides. A literature search concerning deliberate self-poisoning and the effect of restricting access to drugs was conducted, and the effect of restrictions in availability of barbiturates, tr...... in availability of drugs with high case fatality should be a part of suicide prevention strategies.......Availability of drugs with high lethality has been hypothesized to increase the risk of self-poisoning suicides. A literature search concerning deliberate self-poisoning and the effect of restricting access to drugs was conducted, and the effect of restrictions in availability of barbiturates......, tricyclic antidepressants, dextropropoxyphene, and weak analgesics was reviewed. The correlations between method-specific and overall suicide rates and sales figures for barbiturates, dextropropoxyphene, weak analgesics, and tricyclic antidepressants were reviewed. It is concluded that restriction...

  2. Newer antidiabetic drugs and calorie restriction mimicry

    Directory of Open Access Journals (Sweden)

    Sanjay Kalra

    2016-01-01

    Full Text Available De-acceleration of aging and delayed development of age-related morbidity accompanies the restriction of calories (without malnutrition in laboratory mice, nematodes, yeast, fish, and dogs. Recent results from long-term longitudinal studies conducted on primates have suggested longevity benefits of a 30% restriction of calories in rhesus monkeys as well. Among calorie restricted rhesus monkeys one of the mechanisms for the improvement in lifespan was the reduction in the development of glucose intolerance and cardiovascular disease. Although there are no comparable human studies, it is likely that metabolic and longevity benefits will accompany a reduction in calories in humans as well. However, considering the difficulties in getting healthy adults to limit food intake science has focused on understanding the biochemical processes that accompany calorie restriction (CR to formulate drugs that would mimic the effects of CR without the need to actually restrict calories. Drugs in this emerging therapeutic field are called CR mimetics. Some of the currently used anti-diabetic agents may have some CR mimetic like effects. This review focuses on the CR mimetic properties of the currently available anti-diabetic agents.

  3. Compensatory mechanisms activated with intermittent energy restriction

    DEFF Research Database (Denmark)

    Coutinho, Sílvia Ribeiro; Halset, Eline Holli; Gåsbakk, Sigrid

    2018-01-01

    Background & aims: Strong compensatory responses, with reduced resting metabolic rate (RMR), increased exercise efficiency (ExEff) and appetite, are activated when weight loss (WL) is achieved with continuous energy restriction (CER), which try to restore energy balance. Intermittent energy...... restriction (IER), where short spells of energy restriction are interspaced by periods of habitual energy intake, may offer some protection in minimizing those responses. We aimed to compare the effect of IER versus CER on body composition and the compensatory responses induced by WL. Methods: 35 adults (age......: 39 ± 9 y) with obesity (BMI: 36 ± 4 kg/m2) were randomized to lose a similar weight with an IER (N = 18) or a CER (N = 17) diet over a 12 week period. Macronutrient composition and overall energy restriction (33% reduction) were similar between groups. Body weight/composition, RMR, fasting...

  4. Decoding restricted participation in sequential electricity markets

    Energy Technology Data Exchange (ETDEWEB)

    Knaut, Andreas; Paschmann, Martin

    2017-06-15

    Restricted participation in sequential markets may cause high price volatility and welfare losses. In this paper we therefore analyze the drivers of restricted participation in the German intraday auction which is a short-term electricity market with quarter-hourly products. Applying a fundamental electricity market model with 15-minute temporal resolution, we identify the lack of sub-hourly market coupling being the most relevant driver of restricted participation. We derive a proxy for price volatility and find that full market coupling may trigger quarter-hourly price volatility to decrease by a factor close to four.

  5. Superimposition of postnatal calorie restriction protects the aging male intrauterine growth- restricted offspring from metabolic maladaptations.

    Science.gov (United States)

    Dai, Yun; Thamotharan, Shanthie; Garg, Meena; Shin, Bo-Chul; Devaskar, Sherin U

    2012-09-01

    Intrauterine growth restriction (IUGR) results in dysregulated glucose homeostasis and adiposity in the adult. We hypothesized that with aging, these perturbations will wane, and superimposition of postnatal growth restriction (PNGR) on IUGR [intrauterine and postnatal growth restriction (IPGR)] will reverse the residual IUGR phenotype. We therefore undertook hyperinsulinemic-euglycemic clamp, energy balance, and physical activity studies during fed, fasted, and refed states, in light and dark cycles, on postweaned chow diet-fed more than 17-month aging male IUGR, PNGR, and IPGR vs. control (CON) rat offspring. Hyperinsulinemic-euglycemic clamp revealed similar whole-body insulin sensitivity and physical activity in the nonobese IUGR vs. CON, despite reduced heat production and energy expenditure. Compared with CON and IUGR, IPGR mimicking PNGR was lean and growth restricted with increased physical activity, O(2) consumption (VO(2)), energy intake, and expenditure. Although insulin sensitivity was no different in IPGR and PNGR, skeletal muscle insulin-induced glucose uptake was enhanced. This presentation proved protective against the chronologically earlier (5.5 months) development of obesity and dysregulated energy homeostasis after 19 wk on a postweaned high-fat diet. This protective role of PNGR on the metabolic IUGR phenotype needs future fine tuning aimed at minimizing unintended consequences.

  6. 9 CFR 590.905 - Importation of restricted eggs or eggs containing more restricted eggs than permitted in the...

    Science.gov (United States)

    2010-01-01

    ... 9 Animals and Animal Products 2 2010-01-01 2010-01-01 false Importation of restricted eggs or eggs containing more restricted eggs than permitted in the official standards for U.S. Consumer Grade B. 590.905... EGG PRODUCTS INSPECTION INSPECTION OF EGGS AND EGG PRODUCTS (EGG PRODUCTS INSPECTION ACT) Imports...

  7. Lundep, a sand fly salivary endonuclease increases Leishmania parasite survival in neutrophils and inhibits XIIa contact activation in human plasma.

    Directory of Open Access Journals (Sweden)

    Andrezza C Chagas

    2014-02-01

    Full Text Available Neutrophils are the host's first line of defense against infections, and their extracellular traps (NET were recently shown to kill Leishmania parasites. Here we report a NET-destroying molecule (Lundep from the salivary glands of Lutzomyia longipalpis. Previous analysis of the sialotranscriptome of Lu. longipalpis showed the potential presence of an endonuclease. Indeed, not only was the cloned cDNA (Lundep shown to encode a highly active ss- and dsDNAse, but also the same activity was demonstrated to be secreted by salivary glands of female Lu. longipalpis. Lundep hydrolyzes both ss- and dsDNA with little sequence specificity with a calculated DNase activity of 300000 Kunitz units per mg of protein. Disruption of PMA (phorbol 12 myristate 13 acetate- or parasite-induced NETs by treatment with recombinant Lundep or salivary gland homogenates increases parasite survival in neutrophils. Furthermore, co-injection of recombinant Lundep with metacyclic promastigotes significantly exacerbates Leishmania infection in mice when compared with PBS alone or inactive (mutagenized Lundep. We hypothesize that Lundep helps the parasite to establish an infection by allowing it to escape from the leishmanicidal activity of NETs early after inoculation. Lundep may also assist blood meal intake by lowering the local viscosity caused by the release of host DNA and as an anticoagulant by inhibiting the intrinsic pathway of coagulation.

  8. Intracerebral metastasis showing restricted diffusion: Correlation with histopathologic findings

    Energy Technology Data Exchange (ETDEWEB)

    Duygulu, G. [Radiology Department, Ege University Medicine School, Izmir (Turkey); Ovali, G. Yilmaz [Radiology Department, Celal Bayar University Medicine School, Manisa (Turkey)], E-mail: gulgun.yilmaz@bayar.edu.tr; Calli, C.; Kitis, O.; Yuenten, N. [Radiology Department, Ege University Medicine School, Izmir (Turkey); Akalin, T. [Pathology Department, Ege University Medicine School, Izmir (Turkey); Islekel, S. [Neurosurgery Department, Ege University Medicine School, Izmir (Turkey)

    2010-04-15

    Objective: We aimed to detect the frequency of restricted diffusion in intracerebral metastases and to find whether there is correlation between the primary tumor pathology and diffusion-weighted MR imaging (DWI) findings of these metastases. Material and methods: 87 patients with intracerebral metastases were examined with routine MR imaging and DWI. 11 hemorrhagic metastatic lesions were excluded. The routine MR imaging included three plans before and after contrast enhancement. The DWI was performed with spin-echo EPI sequence with three b values (0, 500 and 1000), and ADC maps were calculated. 76 patients with metastases were grouped according to primary tumor histology and the ratios of restricted diffusion were calculated according to these groups. ADCmin values were measured within the solid components of the tumors and the ratio of metastases with restricted diffusion to that which do not show restricted diffusion were calculated. Fisher's exact and Mann-Whitney U tests were used for the statistical analysis. Results: Restricted diffusion was observed in a total of 15 metastatic lesions (19, 7%). Primary malignancy was lung carcinoma in 10 of these cases (66, 6%) (5 small cell carcinoma, 5 non-small cell carcinoma), and breast carcinoma in three cases (20%). Colon carcinoma and testicular teratocarcinoma were the other two primary tumors in which restricted diffusion in metastasis was detected. There was no statistical significant difference between the primary pathology groups which showed restricted diffusion (p > 0.05). ADCmin values of solid components of the metastasis with restricted diffusion and other metastasis without restricted diffusion also showed no significant statistical difference (0.72 {+-} 0.16 x 10{sup -3} mm{sup 2}/s and 0.78 {+-} 21 x 10{sup -3} mm{sup 2}/s respectively) (p = 0.325). Conclusion: Detection of restricted diffusion on DWI in intracerebral metastasis is not rare, particularly if the primary tumor is lung or breast

  9. Origins and Consequences of Religious Restrictions: A Global Overview

    Science.gov (United States)

    Finke, Roger

    2014-01-01

    Despite the international controversies surrounding religious restrictions and freedoms, the topic has only recently received substantial research attention. Drawing on this new body of research, and multiple research projects in progress, this address explores both the origins and consequences of religious restrictions in the global arena. To understand the motives for restrictions, I propose hypotheses in three areas: the relationship or lack of relationship between institutional religion and the state, the willingness and capacity of the state to ensure freedoms, and the larger social and cultural pressures restricting freedoms, including social and political movements targeting minority religions. Turning to the consequences of religious restrictions, I explore how and why restrictions alter the religious economy (i.e., formation, supply and operation of religions) and are associated with higher levels of religious persecution, religious violence and intrastate conflict in general. Finally, I review additional areas where research is needed. PMID:25364225

  10. 10 CFR 1045.33 - Appointment of restricted data management official.

    Science.gov (United States)

    2010-01-01

    ... 10 Energy 4 2010-01-01 2010-01-01 false Appointment of restricted data management official. 1045... DECLASSIFICATION Generation and Review of Documents Containing Restricted Data and Formerly Restricted Data § 1045.33 Appointment of restricted data management official. (a) Each agency with access to RD or FRD shall...

  11. A newly discovered Bordetella species carries a transcriptionally active CRISPR-Cas with a small Cas9 endonuclease.

    Science.gov (United States)

    Ivanov, Yury V; Shariat, Nikki; Register, Karen B; Linz, Bodo; Rivera, Israel; Hu, Kai; Dudley, Edward G; Harvill, Eric T

    2015-10-26

    Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (cas) are widely distributed among bacteria. These systems provide adaptive immunity against mobile genetic elements specified by the spacer sequences stored within the CRISPR. The CRISPR-Cas system has been identified using Basic Local Alignment Search Tool (BLAST) against other sequenced and annotated genomes and confirmed via CRISPRfinder program. Using Polymerase Chain Reactions (PCR) and Sanger DNA sequencing, we discovered CRISPRs in additional bacterial isolates of the same species of Bordetella. Transcriptional activity and processing of the CRISPR have been assessed via RT-PCR. Here we describe a novel Type II-C CRISPR and its associated genes-cas1, cas2, and cas9-in several isolates of a newly discovered Bordetella species. The CRISPR-cas locus, which is absent in all other Bordetella species, has a significantly lower GC-content than the genome-wide average, suggesting acquisition of this locus via horizontal gene transfer from a currently unknown source. The CRISPR array is transcribed and processed into mature CRISPR RNAs (crRNA), some of which have homology to prophages found in closely related species B. hinzii. Expression of the CRISPR-Cas system and processing of crRNAs with perfect homology to prophages present in closely related species, but absent in that containing this CRISPR-Cas system, suggest it provides protection against phage predation. The 3,117-bp cas9 endonuclease gene from this novel CRISPR-Cas system is 990 bp smaller than that of Streptococcus pyogenes, the 4,017-bp allele currently used for genome editing, and which may make it a useful tool in various CRISPR-Cas technologies.

  12. Synthesis of conformationally restricted beta-turn mimics

    NARCIS (Netherlands)

    IJsselstijn, M.

    2006-01-01

    This thesis aims at developing methods for introducing conformational restriction in Beta-turns, the turn elements present in Beta-sheets. A conformationally restricted peptide might either be formed via incorporation of a bridging diamino acids in a growing peptide chain, or via covalent bond

  13. 32 CFR 228.15 - Restriction regarding animals.

    Science.gov (United States)

    2010-07-01

    ... 32 National Defense 2 2010-07-01 2010-07-01 false Restriction regarding animals. 228.15 Section 228.15 National Defense Department of Defense (Continued) OFFICE OF THE SECRETARY OF DEFENSE (CONTINUED) MISCELLANEOUS SECURITY PROTECTIVE FORCE § 228.15 Restriction regarding animals. No animals except...

  14. Restrictions on Software for Personal and Professional Use

    CERN Multimedia

    2004-01-01

    A growing number of computer security incidents detected at CERN are due to additional software installed for personal and professional use. As a consequence, the smooth operation of CERN is put at risk and often many hours are lost solving the problems. To reduce this security risk, installation and/or use of software on CERN's computing and network infrastructure needs to be restricted. Therefore: Do NOT install software for personal use Do NOT install 'free' or other software unless you have the expertise to configure and maintain it securely. Please comply to these rules to keep our computer systems safe. Further explanation of these restrictions is at http://cern.ch/security/software-restrictions Restricted software, known to cause security and/or network problems (e.g. KaZaA and other P2P/Peer-to-Peer file sharing applications, Skype P2P telephony software, ICQ, VNC, ...), is listed at: http://cern.ch/security/software-restrictions/list

  15. Can early protein restriction induce the development of binge eating?

    Science.gov (United States)

    Fechine, Madge Farias; Borba, Tássia Karin; Cabral-Filho, José Eulálio; Bolaños-Jiménez, Francisco; Lopes-de-Souza, Sandra; Manhães-de-Castro, Raul

    2016-04-01

    We tested the hypothesis that perinatal undernourishment is a factor for binge eating. At 52 days rats born from dams fed on 17% protein (Control) or 8% protein (Undernourished) were distributed into four groups, two of which continued to be fed ad libitum chow and two were submitted to three consecutive Restricted/Refeeding (R/R) cycles. According to the following schedule: Control Naïve (from mothers fed 17% protein/no restriction phase); Control Restricted (from mothers fed 17% protein/restriction phase); Undernourished Naïve (from mothers fed 8% protein/no restriction phase); and Undernourished Restricted (from mothers fed 8% protein/restriction phase). Each cycle consisted of a restriction phase (in the first four days 40% of the mean daily individual chow intake was offered for consumption), followed by a refeeding phase (4 days of chow ad libitum). After the three cycles, all animals were subjected to a feeding test (chow diet and palatable food ad libitum for 24h). During the feeding test, the Undernourished Restricted demonstrated rebound hyperphagia during 2, 4 and 6h. These results suggest the perinatal undernourishment cannot contribute to a binge eating phenotype. Copyright © 2016. Published by Elsevier B.V.

  16. 7 CFR 57.905 - Importation of restricted eggs or eggs containing more restricted eggs than permitted in the...

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 3 2010-01-01 2010-01-01 false Importation of restricted eggs or eggs containing more restricted eggs than permitted in the official standards for U.S. Consumer Grade B. 57.905 Section 57.905... AGRICULTURAL MARKETING ACT OF 1946 AND THE EGG PRODUCTS INSPECTION ACT (CONTINUED) INSPECTION OF EGGS (EGG...

  17. Consequences in Infants That Were Intrauterine Growth Restricted

    Directory of Open Access Journals (Sweden)

    Erich Cosmi

    2011-01-01

    Full Text Available Intrauterine growth restriction is a condition fetus does not reach its growth potential and associated with perinatal mobility and mortality. Intrauterine growth restriction is caused by placental insufficiency, which determines cardiovascular abnormalities in the fetus. This condition, moreover, should prompt intensive antenatal surveillance of the fetus as well as follow-up of infants that had intrauterine growth restriction as short and long-term sequele should be considered.

  18. Fasting or caloric restriction for healthy aging.

    Science.gov (United States)

    Anton, Stephen; Leeuwenburgh, Christiaan

    2013-10-01

    Aging is associated with a host of biological changes that contribute to a progressive decline in cognitive and physical function, ultimately leading to a loss of independence, and increased risk of mortality. To date, prolonged caloric restriction (i.e., a reduction in caloric intake without malnutrition) is the only non-genetic intervention that has consistently been found to extend both mean and maximal life span across a variety of species. Most individuals have difficulty sustaining prolonged caloric restriction, which has led to a search for alternative approaches that can produce similar to benefits as caloric restriction. A growing body of evidence indicates that fasting periods and intermittent fasting regimens in particular can trigger similar biological pathways as caloric restriction. For this reason, there is increasing scientific interest in further exploring the biological and metabolic effects of intermittent fasting periods, as well as whether long-term compliance may be improved by this type of dietary approach. This special will highlight the latest scientific findings related to the effects of both caloric restriction and intermittent fasting across various species including yeast, fruit flies, worms, rodents, primates, and humans. A specific emphasis is placed on translational research with findings from basic bench to bedside reviewed and practical clinical implications discussed. Copyright © 2013 Elsevier Inc. All rights reserved.

  19. 10 CFR 1016.34 - Accountability for Secret Restricted Data.

    Science.gov (United States)

    2010-01-01

    ... 10 Energy 4 2010-01-01 2010-01-01 false Accountability for Secret Restricted Data. 1016.34 Section... Information § 1016.34 Accountability for Secret Restricted Data. Each permittee possessing documents containing Secret Restricted Data shall establish a document accountability procedure and shall maintain...

  20. Cardiovascular adaptation to extrauterine life after intrauterine growth restriction.

    Science.gov (United States)

    Rodriguez-Guerineau, Luciana; Perez-Cruz, Miriam; Gomez Roig, María D; Cambra, Francisco J; Carretero, Juan; Prada, Fredy; Gómez, Olga; Crispi, Fátima; Bartrons, Joaquim

    2018-02-01

    Introduction The adaptive changes of the foetal heart in intrauterine growth restriction can persist postnatally. Data regarding its consequences for early circulatory adaptation to extrauterine life are scarce. The aim of this study was to assess cardiac morphometry and function in newborns with late-onset intrauterine growth restriction to test the hypothesis that intrauterine growth restriction causes cardiac shape and functional changes at birth. A comprehensive echocardiographic study was performed in 25 neonates with intrauterine growth restriction and 25 adequate-for-gestational-age neonates. Compared with controls, neonates with intrauterine growth restriction had more globular ventricles, lower longitudinal tricuspid annular motion, and higher left stroke volume without differences in the heart rate. Neonates with intrauterine growth restriction also showed subclinical signs of diastolic dysfunction in the tissue Doppler imaging with lower values of early (e') diastolic annular peak velocities in the septal annulus. Finally, the Tei index in the tricuspid annulus was higher in the intrauterine growth restriction group. Neonates with history of intrauterine growth restriction showed cardiac remodelling and signs of systolic and diastolic dysfunction. Overall, there was a significant tendency to worse cardiac function results in the right heart. The adaptation to extrauterine life occurred with more globular hearts, higher stroke volumes but a similar heart rate compared to adequate-for-gestational-age neonates.

  1. Supporting several levels of restriction in the UML

    DEFF Research Database (Denmark)

    Hansen, Klaus Marius; Damm, Christian Heide; Thomsen, Michael

    2000-01-01

    The emergence of the Unified Modeling Language (UML) has provided software developers with an effective and efficient shared language. However, UML is often too restrictive in initial, informal, and creative modelling, and it is in some cases not restrictive enough, e.g., for code generation. Bas....... This approach potentially increases the usability of the UML, and thus ultimately leads to greater quality and adoption of UML models.......The emergence of the Unified Modeling Language (UML) has provided software developers with an effective and efficient shared language. However, UML is often too restrictive in initial, informal, and creative modelling, and it is in some cases not restrictive enough, e.g., for code generation. Based...

  2. Intrauterine growth restriction

    Directory of Open Access Journals (Sweden)

    Bernardita Donoso Bernales

    2012-07-01

    Full Text Available It is estimated that the true prevalence of intrauterine growth restriction is 3-10% of all pregnancies, making this fetal condition one of the most frequent obstetric problems, together with premature labor and premature rupture of membranes. The article stresses the importance of early diagnosis because of the associated risks.

  3. Restricted mass energy absorption coefficients for use in dosimetry

    International Nuclear Information System (INIS)

    Brahme, A.

    1977-02-01

    When matter is irradiated by a photon beam the fraction of energy absorbed locally in some region Rsub(Δ) (where the size of the region Rsub(Δ) is related to the range of secondary electrons of some restriction energy Δ) is expressed by the restricted mass energy absorption coefficient. In this paper an example is given of how restricted mass energy absorption coefficients can be calculated from existing differential photon interaction cross sections. Some applications of restricted mass absorption coefficients in dosimetry are also given. (B.D.)

  4. Universal Cycles of Restricted Classes of Words

    OpenAIRE

    Leitner, Arielle; Godbole, Anant

    2008-01-01

    It is well known that Universal Cycles of $k$-letter words on an $n$-letter alphabet exist for all $k$ and $n$. In this paper, we prove that Universal Cycles exist for restricted classes of words, including: non-bijections, equitable words (under suitable restrictions), ranked permutations, and "passwords".

  5. 32 CFR 234.12 - Restriction on animals.

    Science.gov (United States)

    2010-07-01

    ... 32 National Defense 2 2010-07-01 2010-07-01 false Restriction on animals. 234.12 Section 234.12 National Defense Department of Defense (Continued) OFFICE OF THE SECRETARY OF DEFENSE (CONTINUED) MISCELLANEOUS CONDUCT ON THE PENTAGON RESERVATION § 234.12 Restriction on animals. Animals, except guide dogs...

  6. Genetic polymorphism of toll-like receptors 4 gene by polymerase chain reaction-restriction fragment length polymorphisms, polymerase chain reaction-single-strand conformational polymorphism to correlate with mastitic cows

    Directory of Open Access Journals (Sweden)

    Pooja H. Gupta

    2015-05-01

    Full Text Available Aim: An attempt has been made to study the toll-like receptors 4 (TLR4 gene polymorphism from cattle DNA to correlate with mastitis cows. Materials and Methods: In present investigation, two fragments of TLR4 gene named T4CRBR1 and T4CRBR2 of a 316 bp and 382 bp were amplified by polymerase chain reaction (PCR, respectively from Kankrej (22 and Triple cross (24 cattle. The genetic polymorphisms in the two populations were detected by a single-strand conformational polymorphism in the first locus and by digesting the fragments with restriction endonuclease Alu I in the second one. Results: Results showed that both alleles (A and B of two loci were found in all the two populations and the value of polymorphism information content indicated that these were highly polymorphic. Statistical results of χ2 test indicated that two polymorphism sites in the two populations fit with Hardy–Weinberg equilibrium (p˂0.05. Meanwhile, the effect of polymorphism of TLR4 gene on the somatic cell score (SCS indicated the cattle with allele a in T4CRBR1 showed lower SCS than that of allele B (p<0.05. Thus, the allele A might play an important role in mastitis resistance in cows. Conclusion: The relationship between the bovine mastitis trait and the polymorphism of TLR4 gene indicated that the bovine TLR4 gene may play an important role in mastitis resistance.

  7. Optimizing Restriction Site Placement for Synthetic Genomes

    Science.gov (United States)

    Montes, Pablo; Memelli, Heraldo; Ward, Charles; Kim, Joondong; Mitchell, Joseph S. B.; Skiena, Steven

    Restriction enzymes are the workhorses of molecular biology. We introduce a new problem that arises in the course of our project to design virus variants to serve as potential vaccines: we wish to modify virus-length genomes to introduce large numbers of unique restriction enzyme recognition sites while preserving wild-type function by substitution of synonymous codons. We show that the resulting problem is NP-Complete, give an exponential-time algorithm, and propose effective heuristics, which we show give excellent results for five sample viral genomes. Our resulting modified genomes have several times more unique restriction sites and reduce the maximum gap between adjacent sites by three to nine-fold.

  8. 32 CFR 1903.16 - Restriction on animals.

    Science.gov (United States)

    2010-07-01

    ... 32 National Defense 6 2010-07-01 2010-07-01 false Restriction on animals. 1903.16 Section 1903.16... AGENCY INSTALLATIONS § 1903.16 Restriction on animals. Animals, except for those animals used for the assistance of persons with disabilities, or animals under the charge and control of the Central Intelligence...

  9. 37 CFR 1.142 - Requirement for restriction.

    Science.gov (United States)

    2010-07-01

    ... at any time before final action. (b) Claims to the invention or inventions not elected, if not... Inventions in One Application; Restriction § 1.142 Requirement for restriction. (a) If two or more independent and distinct inventions are claimed in a single application, the examiner in an Office action will...

  10. [Restricted motion after total knee arthroplasty].

    Science.gov (United States)

    Kucera, T; Urban, K; Karpas, K; Sponer, P

    2007-10-01

    The aim of the study was to ascertain what proportion of patients undergoing total knee arthroplasty (TKA) complain of restricted knee joint motion, and to investigate options for improvement of this situation. Our evaluation included a group of 796 patients treated with TKA at our department in the period from January 1, 1990, to December 31, 2004. In all cases, a condylar implant with preservation of the posterior cruciate ligaments was used. In addition to medical history, the range of motion, knee joint malalignment and radiological findings were assessed before surgery. After THA, the type of implant and complications, if any, were recorded, and improvement in joint motion was followed up. Based on the results of Kim et al., flexion contracture equal to or higher than 15 degrees and/or flexion less than 75 degrees were made the criteria of stiffness after THA. Patients with restricted THA motion who had aseptic or septic implant loosening were not included. Of the 796 evaluated patients, 32 (4.14 %) showed restricted motion after total knee arthroplasty, as assessed by the established criteria. In 16 patients, stiffness defined by these criteria had existed before surgery, and three patients showed an excessive production of adhesions and heterotopic ossifications. In three patients, the implantation procedure resulted in an elevated level of the original joint line and subsequent development of patella infera and increased tension of the posterior cruciate ligament. Four patients declined physical therapy and, in six, the main cause of stiffness could not be found. Seventeen patients did not require surgical therapy for restricted motion; TKA provided significant pain relief and they considered the range of motion achieved to be sufficient. One patient underwent redress 3 months after surgery, but with no success. Repeated releases of adhesions, replacement of a polyethylene liner and revision surgery of the extensor knee structures were performed in 15

  11. Intermittent′ restrictive ventricular septal defect in Tetralogy of Fallot

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    Sudhir S Shetkar

    2015-01-01

    Full Text Available Ventricular septal defect (VSD in Tetralogy of Fallot (TOF is usually large and non-restrictive with equalization of right and left ventricular pressures. Restrictive VSD in TOF is rare. We present an unusual case of TOF with restriction to VSD caused by accessory tricuspid valve tissue that varied with respiration.

  12. Fragmentation of chromatin DNA in mouse thymus cells after whole body γ-irradiation

    International Nuclear Information System (INIS)

    Wei Kang; Liu Xueying; Zhu Xuefen

    1984-01-01

    The characteristics of soluble chromatin in mouse thymus nuclei after whole body γ-irradiation were investigated by means of polyacrylamide gel electrophoresis. After deproteinization and electrophoresis eight regular DNA bands were revealed. The molecular weights of these bands were estimated by comparing their migration rates with those of the standard fragments obtained from PBR 322 digested completely by restrictive endonuclease Hae III. The molecular weight of the first band was calculated to be 186 base pairs corresponding approximately to the size of DNA fragment from a single nucleosome, and those of other bands appeared to be its multiples. The results suggested that the disintegration of chromatin DNA after γ-irradiation might have occurred at the linkage regions of chromatin. The autolysis product of normal thymus chromatin under sterile condition were also analyzed and its electrophoretic pattern was found to be just the same as that of the postirradiation product. It seems, therefore, that the endonuclease existing in normal tissues might be responsible for the postirradiation chromatin degradation. The mechanism of this kind of enzymatic digestion remains to be elucidated in further investigation. (author)

  13. Natural Endophytic Occurrence of Acetobacter diazotrophicus in Pineapple Plants.

    Science.gov (United States)

    Tapia-Hernández; Bustillos-Cristales; Jiménez-Salgado; Caballero-Mellado; Fuentes-Ramírez

    2000-01-01

    The presence of endophytic Acetobacter diazotrophicus was tested for pineapple plants (Ananas comosus [L.] Merr.) grown in the field. Diazotrophic bacteria were isolated from the inner tissues of surface sterilized roots, stems, and leaves of pineapple plants. Phenotypic tests permitted the selection of presumptive nitrogen-fixing A. diazotrophicus isolates. Restriction fragment length polymorphisms (RFLPs) of small subunit (SSU) rDNA using total DNA digested with endonuclease SphI and with endonuclease NcoI, hybridizations of RNA with an A. diazotrophicus large subunit (LSU) rRNA specific probe, as well as patterns in denaturing protein electrophoresis (SDS-PAGE) and multilocus enzyme tests allowed the identification of A. diazotrophicus isolates. High frequencies of isolation were obtained from propagative buds that had not been nitrogen-fertilized, and lower frequencies from 3-month-old plants that had been nitrogen-fertilized. No isolates were recovered from 5- to 7-month-old nitrogen-fertilized plants. All the A. diazotrophicus isolates recovered from pineapple plants belonged to the multilocus genotype which shows the most extensive distribution among all host species previously analyzed.

  14. Pulsed-field gel electrophoresis of bacterial chromosomes.

    Science.gov (United States)

    Mawer, Julia S P; Leach, David R F

    2013-01-01

    The separation of fragments of DNA by agarose gel electrophoresis is integral to laboratory life. Nevertheless, standard agarose gel electrophoresis cannot resolve fragments bigger than 50 kb. Pulsed-field gel electrophoresis is a technique that has been developed to overcome the limitations of standard agarose gel electrophoresis. Entire linear eukaryotic chromosomes, or large fragments of a chromosome that have been generated by the action of rare-cutting restriction endonucleases, can be separated using this technique. As a result, pulsed-field gel electrophoresis has many applications, from karyotype analysis of microbial genomes, to the analysis of chromosomal strand breaks and their repair intermediates, to the study of DNA replication and the identification of origins of replication. This chapter presents a detailed protocol for the preparation of Escherichia coli chromosomal DNA that has been embedded in agarose plugs, digested with the rare-cutting endonuclease NotI, and separated by contour-clamped homogeneous field electrophoresis. The principles in this protocol can be applied to the separation of all fragments of DNA whose size range is between 40 kb and 1 Mb.

  15. AKT2 Blocks Nucleus Translocation of Apoptosis-Inducing Factor (AIF and Endonuclease G (EndoG While Promoting Caspase Activation during Cardiac Ischemia

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    Shuai Yang

    2017-03-01

    Full Text Available The AKT (protein kinase B, PKB family has been shown to participate in diverse cellular processes, including apoptosis. Previous studies demonstrated that protein kinase B2 (AKT2−/− mice heart was sensitized to apoptosis in response to ischemic injury. However, little is known about the mechanism and apoptotic signaling pathway. Here, we show that AKT2 inhibition does not affect the development of cardiomyocytes but increases cell death during cardiomyocyte ischemia. Caspase-dependent apoptosis of both the extrinsic and intrinsic pathway was inactivated in cardiomyocytes with AKT2 inhibition during ischemia, while significant mitochondrial disruption was observed as well as intracytosolic translocation of cytochrome C (Cyto C together with apoptosis-inducing factor (AIF and endonuclease G (EndoG, both of which are proven to conduct DNA degradation in a range of cell death stimuli. Therefore, mitochondria-dependent cell death was investigated and the results suggested that AIF and EndoG nucleus translocation causes cardiomyocyte DNA degradation during ischemia when AKT2 is blocked. These data are the first to show a previous unrecognized function and mechanism of AKT2 in regulating cardiomyocyte survival during ischemia by inducing a unique mitochondrial-dependent DNA degradation pathway when it is inhibited.

  16. Extrauterine growth restriction: Universal problem among premature infants

    Directory of Open Access Journals (Sweden)

    Brunnella Alcantara Chagas de FREITAS

    2016-02-01

    Full Text Available ABSTRACT Objective: To analyze the growth rate of premature infants in the first weeks of life and factors associated with extrauterine growth restriction. Methods: This is a cross-sectional study of 254 premature infants in a neonatal intensive care unit conducted from January 1, 2008 to December 31, 2010. Infants who died or had malformations incompatible with life were excluded. Median weight curves according to gestational age were constructed for the first four weeks of life. The Fenton growth chart calculations provided the weight Z-scores. Extrauterine growth restriction was defined as corrected weight-for-age Z-score ≤-2. Perinatal, morbidity, and health care variables were analyzed. The Poisson regression model yielded the prevalenceratios . Associations between extrauterine growth restriction and the perinatal, morbidity, and care variables were investigated. Poisson regression controlled possible confounding factors. Results: The frequency of extrauterine growth restriction was 24.0%. Most (85.0% small-for-gestational-age infants developed extrauterine growth restriction; 55.3% of extrauterine growth restriction cases involved small-for-gestational-age infants. Premature infants with gestational age >32 weeks did not recover the median birth weight until the third week of life and had a higher frequency of small-for-gestational-age. The Z-scores of non-small-for-gestational-age infants decreased more after birth than those of small-for-gestational-age infants. extrauterine growth restriction was associated with small-for-gestational-age (PR=6.14; 95%CI=3.33-11.33;p <0.001 and time without enteral diet (PR=1.08; 95%CI=1.04-1.13; p =0.010. Conclusion: Extrauterine growth restriction occurs in premature infants of all gestational age. The participation of small-for-gestational-age and nutritional practices in its genesis is noteworthy. We suggest prospective studies of all premature infants. The implementation of best care practices

  17. Epigenetic regulation of caloric restriction in aging

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    Daniel Michael

    2011-08-01

    Full Text Available Abstract The molecular mechanisms of aging are the subject of much research and have facilitated potential interventions to delay aging and aging-related degenerative diseases in humans. The aging process is frequently affected by environmental factors, and caloric restriction is by far the most effective and established environmental manipulation for extending lifespan in various animal models. However, the precise mechanisms by which caloric restriction affects lifespan are still not clear. Epigenetic mechanisms have recently been recognized as major contributors to nutrition-related longevity and aging control. Two primary epigenetic codes, DNA methylation and histone modification, are believed to dynamically influence chromatin structure, resulting in expression changes of relevant genes. In this review, we assess the current advances in epigenetic regulation in response to caloric restriction and how this affects cellular senescence, aging and potential extension of a healthy lifespan in humans. Enhanced understanding of the important role of epigenetics in the control of the aging process through caloric restriction may lead to clinical advances in the prevention and therapy of human aging-associated diseases.

  18. Metabolic Regulation of Methionine Restriction in Diabetes.

    Science.gov (United States)

    Yin, Jie; Ren, Wenkai; Chen, Shuai; Li, Yuying; Han, Hui; Gao, Jing; Liu, Gang; Wu, Xin; Li, Tiejun; Kim, Sung Woo; Yin, Yulong

    2018-03-30

    Although the effects of dietary methionine restriction have been investigated in the physiology of aging and diseases related to oxidative stress, the relationship between methionine restriction and the development of metabolic disorders has not been explored extensively. This review summarizes studies of the possible involvement of dietary methionine restriction in improving insulin resistance, glucose homeostasis, oxidative stress, lipid metabolism, the pentose phosphate pathway, and inflammation, with an emphasis on the fibroblast growth factor 21 and protein phosphatase 2A signals and autophagy in diabetes. Diets deficient in methionine may be a useful nutritional strategy in patients with diabetes. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  19. Retroviral restriction and dependency factors in primates and carnivores

    Science.gov (United States)

    Fadel, Hind J.; Poeschla, Eric M.

    2014-01-01

    Recent studies have extended the rapidly developing retroviral restriction factor field to cells of carnivore species. Carnivoran genomes, and the domestic cat genome in particular, are revealing intriguing properties vis-à;-vis the primate and feline lentiviruses, not only with respect to their repertoires of virus-blocking restriction factors but also replication-enabling dependency factors. Therapeutic application of restriction factors is envisioned for human immunodeficiency virus (HIV) disease and the feline immunodeficiency virus (FIV) model has promise for testing important hypotheses at the basic and translational level. Feline cell-tropic HIV-1 clones have also been generated by a strategy of restriction factor evasion. We review progress in this area in the context of what is known about retroviral restriction factors such as TRIM5alpha, TRIMCyp, APOBEC3 proteins and BST-2/Tetherin. PMID:21715018

  20. 48 CFR 1403.570 - Restrictions on contractor advertising.

    Science.gov (United States)

    2010-10-01

    ... 48 Federal Acquisition Regulations System 5 2010-10-01 2010-10-01 false Restrictions on contractor advertising. 1403.570 Section 1403.570 Federal Acquisition Regulations System DEPARTMENT OF THE INTERIOR... 1403.570 Restrictions on contractor advertising. ...

  1. HIV restriction by APOBEC3 in humanized mice.

    Directory of Open Access Journals (Sweden)

    John F Krisko

    2013-03-01

    Full Text Available Innate immune restriction factors represent important specialized barriers to zoonotic transmission of viruses. Significant consideration has been given to their possible use for therapeutic benefit. The apolipoprotein B mRNA editing enzyme catalytic polypeptide 3 (APOBEC3 family of cytidine deaminases are potent immune defense molecules capable of efficiently restricting endogenous retroelements as well as a broad range of viruses including Human Immunodeficiency virus (HIV, Hepatitis B virus (HBV, Human Papilloma virus (HPV, and Human T Cell Leukemia virus (HTLV. The best characterized members of this family are APOBEC3G (A3G and APOBEC3F (A3F and their restriction of HIV. HIV has evolved to counteract these powerful restriction factors by encoding an accessory gene designated viral infectivity factor (vif. Here we demonstrate that APOBEC3 efficiently restricts CCR5-tropic HIV in the absence of Vif. However, our results also show that CXCR4-tropic HIV can escape from APOBEC3 restriction and replicate in vivo independent of Vif. Molecular analysis identified thymocytes as cells with reduced A3G and A3F expression. Direct injection of vif-defective HIV into the thymus resulted in viral replication and dissemination detected by plasma viral load analysis; however, vif-defective viruses remained sensitive to APOBEC3 restriction as extensive G to A mutation was observed in proviral DNA recovered from other organs. Remarkably, HIV replication persisted despite the inability of HIV to develop resistance to APOBEC3 in the absence of Vif. Our results provide novel insight into a highly specific subset of cells that potentially circumvent the action of APOBEC3; however our results also demonstrate the massive inactivation of CCR5-tropic HIV in the absence of Vif.

  2. Partial digestion with restriction enzymes of ultraviolet-irradiated human genomic DNA: a method for identifying restriction site polymorphisms

    International Nuclear Information System (INIS)

    Nobile, C.; Romeo, G.

    1988-01-01

    A method for partial digestion of total human DNA with restriction enzymes has been developed on the basis of a principle already utilized by P.A. Whittaker and E. Southern for the analysis of phage lambda recombinants. Total human DNA irradiated with uv light of 254 nm is partially digested by restriction enzymes that recognize sequences containing adjacent thymidines because of TT dimer formation. The products resulting from partial digestion of specific genomic regions are detected in Southern blots by genomic-unique DNA probes with high reproducibility. This procedure is rapid and simple to perform because the same conditions of uv irradiation are used for different enzymes and probes. It is shown that restriction site polymorphisms occurring in the genomic regions analyzed are recognized by the allelic partial digest patterns they determine

  3. Analysis of cytoplasmic genomes in somatic hybrids between navel orange (Citrus sinensis Osb.) and 'Murcott' tangor.

    Science.gov (United States)

    Kobayashi, S; Ohgawara, T; Fujiwara, K; Oiyama, I

    1991-07-01

    Somatic hybrid plants were produced by protoplast fusion of navel orange and 'Murcott' tangor. Hybridity of the plants was confirmed by the restriction endonuclease analysis of nuclear ribosomal DNA. All of the plants (16 clones) were normal, uniform, and had the amphidiploid chromosome number of 36 (2n=2x=18 for each parent). The cpDNA analysis showed that each of the 16 somatic hybrids contained either one parental chloroplast genome or the other. In all cases, the mitochondrial genomes of the regenerated somatic hybrids were of the navel orange type.

  4. Screening, diagnosis, and management of intrauterine growth restriction.

    Science.gov (United States)

    Lausman, Andrea; McCarthy, Fergus P; Walker, Melissa; Kingdom, John

    2012-01-01

    To provide comprehensive background knowledge relevant to the SOGC Maternal-Fetal Medicine Committee-approved guideline entitled "Intrauterine Growth Restriction: Screening, Diagnosis, and Management." Publications in English were retrieved through searches of PubMed or Medline, CINAHL, and the Cochrane Library in January 2011 using appropriate controlled vocabulary via MeSH terms (fetal growth restriction and small for gestational age) and any key words (fetal growth, restriction, growth retardation, intrauterine growth restriction [IUGR], low birth weight, small for gestational age). Results were restricted to systematic reviews, randomized controlled trials or controlled clinical trials, and high-quality prospective and retrospective observational studies. Grey (unpublished) literature was identified through searching the websites of health technology assessment and health technology assessment-related agencies, clinical practice guideline collections, clinical trial registries, and national and international medical specialty societies. Evidence obtained from at least one properly randomized controlled trial, Cochrane Reviews, and high quality cohort data have been combined to provide clinicians with evidence to optimize their practice for screening, diagnosis, and management of intrauterine growth restriction. Considerable advances have been made to improve clinicians' ability to screen, diagnose, and manage pregnancies with suspected IUGR more effectively, including several properly randomized controlled trials. Pregnancies with late-onset IUGR may be managed equally effectively by early delivery or delayed delivery (with increased surveillance) anticipating favourable outcomes. By contrast, many aspects of the management of early-onset IUGR require further clinical trials.

  5. Maternal protein restriction induces alterations in insulin signaling and ATP sensitive potassium channel protein in hypothalami of intrauterine growth restriction fetal rats.

    Science.gov (United States)

    Liu, Xiaomei; Qi, Ying; Gao, Hong; Jiao, Yisheng; Gu, Hui; Miao, Jianing; Yuan, Zhengwei

    2013-01-01

    It is well recognized that intrauterine growth restriction leads to the development of insulin resistance and type 2 diabetes mellitus in adulthood. To investigate the mechanisms behind this "metabolic imprinting" phenomenon, we examined the impact of maternal undernutrition on insulin signaling pathway and the ATP sensitive potassium channel expression in the hypothalamus of intrauterine growth restriction fetus. Intrauterine growth restriction rat model was developed through maternal low protein diet. The expression and activated levels of insulin signaling molecules and K(ATP) protein in the hypothalami which were dissected at 20 days of gestation, were analyzed by western blot and real time PCR. The tyrosine phosphorylation levels of the insulin receptor substrate 2 and phosphatidylinositol 3'-kinase p85α in the hypothalami of intrauterine growth restriction fetus were markedly reduced. There was also a downregulation of the hypothalamic ATP sensitive potassium channel subunit, sulfonylurea receptor 1, which conveys the insulin signaling. Moreover, the abundances of gluconeogenesis enzymes were increased in the intrauterine growth restriction livers, though no correlation was observed between sulfonylurea receptor 1 and gluconeogenesis enzymes. Our data suggested that aberrant intrauterine milieu impaired insulin signaling in the hypothalamus, and these alterations early in life might contribute to the predisposition of the intrauterine growth restriction fetus toward the adult metabolic disorders.

  6. BpuAmI: a novel SacI neoschizomer from Bacillus pumilus discovered in an isolate from Amazon Basin, recognizing 5'-GAG¯CTC-3' BpuAmI: um novo neoesquisomero de SacI de Bacillus pumilus descoberta em um isolado oriundo da Bacia Amazônica, reconhecendo 5'-GAG¯CTC-3'

    Directory of Open Access Journals (Sweden)

    Jocelei M. Chies

    2006-03-01

    Full Text Available A strain of Bacillus pumilus was isolated and identified from water samples collected from a small affluent of the Amazon River. Type II restriction endonuclease activity was detected in these bacteria. The enzyme was purified and the molecular weight of the native protein estimated by gel filtration and SDS-PAGE. The optimum pH, temperature and salt requirements were determined. Quality control assays showed the complete absence of "nonspecific nucleases." Restriction cleavage analysis and DNA sequencing of restriction fragments allowed the unequivocal demonstration of 5´GAG¯CTC3´ as the recognition sequence. This enzyme was named BpuAmI and is apparently a neoschizomer of the prototype restriction endonuclease SacI. This is the first report of an isoschizomer and/or neoschizomer of the prototype SacI identified in the genus Bacillus.Uma linhagem de Bacillus pumilus foi isolada e identificada de amostras de águas coletadas em um pequeno Igarapé do Rio Amazonas. Foi detectada atividade de restrição do tipo II nesta bactéria. A enzima foi purificada e o peso molecular da proteína nativa foi estimado por gel filtração e por eletroforese em gel de poliacrilamida. Foram determinados, o pH e temperatura ótimos e as necessidades de sais. Os ensaios do controle de qualidade mostraram uma ausência completa de "nucleases não específicas". As analises das clivagens e o seqüenciamento do DNA dos fragmentos de restrição permitiram uma demonstração inequívoca de que 5´GAG¯CTC 3´ é a seqüência de reconhecimento da enzima. Esta enzima foi denominada de BpuAmI e aparentemente é um neoesquisômero da enzima protótipo SacI. Este é o primeiro relato de um isoesquisômero e/ou neoesquisômero da enzima protótipo SacI identificada no gênero Bacillus.

  7. Novel host restriction factors implicated in HIV-1 replication.

    Science.gov (United States)

    Ghimire, Dibya; Rai, Madhu; Gaur, Ritu

    2018-04-01

    Human immunodeficiency virus-1 (HIV-1) is known to interact with multiple host cellular proteins during its replication in the target cell. While many of these host cellular proteins facilitate viral replication, a number of them are reported to inhibit HIV-1 replication at various stages of its life cycle. These host cellular proteins, which are known as restriction factors, constitute an integral part of the host's first line of defence against the viral pathogen. Since the discovery of apolipoprotein B mRNA-editing enzyme 3G (APOBEC3G) as an HIV-1 restriction factor, several human proteins have been identified that exhibit anti-HIV-1 restriction. While each restriction factor employs a distinct mechanism of inhibition, the HIV-1 virus has equally evolved complex counter strategies to neutralize their inhibitory effect. APOBEC3G, tetherin, sterile alpha motif and histidine-aspartate domain 1 (SAMHD1), and trim-5α are some of the best known HIV-1 restriction factors that have been studied in great detail. Recently, six novel restriction factors were discovered that exhibit significant antiviral activity: endoplasmic reticulum α1,2-mannosidase I (ERManI), translocator protein (TSPO), guanylate-binding protein 5 (GBP5), serine incorporator (SERINC3/5) and zinc-finger antiviral protein (ZAP). The focus of this review is to discuss the antiviral mechanism of action of these six restriction factors and provide insights into the probable counter-evasion strategies employed by the HIV-1 virus. The recent discovery of new restriction factors substantiates the complex host-pathogen interactions occurring during HIV-1 pathogenesis and makes it imperative that further investigations are conducted to elucidate the molecular basis of HIV-1 replication.

  8. Host Cell Restriction Factors that Limit Influenza A Infection

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    Fernando Villalón-Letelier

    2017-12-01

    Full Text Available Viral infection of different cell types induces a unique spectrum of host defence genes, including interferon-stimulated genes (ISGs and genes encoding other proteins with antiviral potential. Although hundreds of ISGs have been described, the vast majority have not been functionally characterised. Cellular proteins with putative antiviral activity (hereafter referred to as “restriction factors” can target various steps in the virus life-cycle. In the context of influenza virus infection, restriction factors have been described that target virus entry, genomic replication, translation and virus release. Genome wide analyses, in combination with ectopic overexpression and/or gene silencing studies, have accelerated the identification of restriction factors that are active against influenza and other viruses, as well as providing important insights regarding mechanisms of antiviral activity. Herein, we review current knowledge regarding restriction factors that mediate anti-influenza virus activity and consider the viral countermeasures that are known to limit their impact. Moreover, we consider the strengths and limitations of experimental approaches to study restriction factors, discrepancies between in vitro and in vivo studies, and the potential to exploit restriction factors to limit disease caused by influenza and other respiratory viruses.

  9. The feasibility of age-specific travel restrictions during influenza pandemics.

    Science.gov (United States)

    Lam, Elson H Y; Cowling, Benjamin J; Cook, Alex R; Wong, Jessica Y T; Lau, Max S Y; Nishiura, Hiroshi

    2011-11-11

    Epidemiological studies have shown that imposing travel restrictions to prevent or delay an influenza pandemic may not be feasible. To delay an epidemic substantially, an extremely high proportion of trips (~99%) would have to be restricted in a homogeneously mixing population. Influenza is, however, strongly influenced by age-dependent transmission dynamics, and the effectiveness of age-specific travel restrictions, such as the selective restriction of travel by children, has yet to be examined. A simple stochastic model was developed to describe the importation of infectious cases into a population and to model local chains of transmission seeded by imported cases. The probability of a local epidemic, and the time period until a major epidemic takes off, were used as outcome measures, and travel restriction policies in which children or adults were preferentially restricted were compared to age-blind restriction policies using an age-dependent next generation matrix parameterized for influenza H1N1-2009. Restricting children from travelling would yield greater reductions to the short-term risk of the epidemic being established locally than other policy options considered, and potentially could delay an epidemic for a few weeks. However, given a scenario with a total of 500 imported cases over a period of a few months, a substantial reduction in the probability of an epidemic in this time period is possible only if the transmission potential were low and assortativity (i.e. the proportion of contacts within-group) were unrealistically high. In all other scenarios considered, age-structured travel restrictions would not prevent an epidemic and would not delay the epidemic for longer than a few weeks. Selectively restricting children from traveling overseas during a pandemic may potentially delay its arrival for a few weeks, depending on the characteristics of the pandemic strain, but could have less of an impact on the economy compared to restricting adult

  10. The feasibility of age-specific travel restrictions during influenza pandemics

    Directory of Open Access Journals (Sweden)

    Lam Elson HY

    2011-11-01

    Full Text Available Abstract Background Epidemiological studies have shown that imposing travel restrictions to prevent or delay an influenza pandemic may not be feasible. To delay an epidemic substantially, an extremely high proportion of trips (~99% would have to be restricted in a homogeneously mixing population. Influenza is, however, strongly influenced by age-dependent transmission dynamics, and the effectiveness of age-specific travel restrictions, such as the selective restriction of travel by children, has yet to be examined. Methods A simple stochastic model was developed to describe the importation of infectious cases into a population and to model local chains of transmission seeded by imported cases. The probability of a local epidemic, and the time period until a major epidemic takes off, were used as outcome measures, and travel restriction policies in which children or adults were preferentially restricted were compared to age-blind restriction policies using an age-dependent next generation matrix parameterized for influenza H1N1-2009. Results Restricting children from travelling would yield greater reductions to the short-term risk of the epidemic being established locally than other policy options considered, and potentially could delay an epidemic for a few weeks. However, given a scenario with a total of 500 imported cases over a period of a few months, a substantial reduction in the probability of an epidemic in this time period is possible only if the transmission potential were low and assortativity (i.e. the proportion of contacts within-group were unrealistically high. In all other scenarios considered, age-structured travel restrictions would not prevent an epidemic and would not delay the epidemic for longer than a few weeks. Conclusions Selectively restricting children from traveling overseas during a pandemic may potentially delay its arrival for a few weeks, depending on the characteristics of the pandemic strain, but could have less

  11. Incremental Effect of the Addition of Prescriber Restrictions on a State Medicaid's Pharmacy-Only Patient Review and Restriction Program.

    Science.gov (United States)

    Keast, Shellie L; Pham, Timothy; Teel, Ashley; Nesser, Nancy J

    2017-08-01

    Patient review and restriction programs (PRRPs), used by state Medicaid programs to limit potential abuse and misuse of opioids and related controlled medications, often restrict members to a single pharmacy for controlled medications. While most states use a restricted pharmacy access model, not all states include restricted prescriber access. Oklahoma Medicaid (MOK) added a restricted prescriber access feature to its PRRP in July 2014. To evaluate the incremental effect that the addition of a prescriber restriction to MOK's pharmacy-only PRRP had on the pharmacy and resource utilization of the enrolled members. MOK members with at least 6 months of enrollment in the pharmacy-only PRRP were restricted to a maximum of 3 prescribers for controlled substances in July 2014 and were identified as "cases." Using a propensity score method, cases were matched to controls from the MOK non-PRRP enrolled population based on demographics and baseline health care utilization. Data from January 1, 2014, through December 31, 2014, were evaluated. Each member's monthly health care resource utilization, defined in terms of medical and pharmacy costs, prescription counts, and opioid use per member per month (PMPM), was analyzed. A difference-indifferences (DID) regression estimated the change in resource utilization following the July 2014 policy change. This study included 378 controls and 126 cases after propensity matching. No differences were noted for daily morphine equivalents, benzodiazepine prescriptions, or maintenance prescriptions. There were decreases in mean PMPM use for both groups for short-acting opioid (SAO) claims (P evidence that overall opioid claims were affected, the addition of prescriber restrictions may have resulted in an incremental change to SAO, prescriber, and pharmacy use in the PRPP population. Use of PRRPs may be an effective tool in reducing inappropriate use of prescription opioids within payer systems. The question remains whether these changes

  12. Role of HIV-2 envelope in Lv2-mediated restriction

    International Nuclear Information System (INIS)

    Reuter, Sandra; Kaumanns, Patrick; Buschhorn, Sabine B.; Dittmar, Matthias T.

    2005-01-01

    We have characterized envelope protein pseudotyped HIV-2 particles derived from two HIV-2 isolates termed prCBL23 and CBL23 in order to define the role of the envelope protein for the Lv2-mediated restriction to infection. Previously, it has been described that the primary isolate prCBL23 is restricted to infection of several human cell types, whereas the T cell line adapted isolate CBL23 is not restricted in these cell types. Molecular cloning of the two isolates revealed that the env and the gag gene are responsible for the observed phenotype and that this restriction is mediated by Lv2, which is distinct from Ref1/Lv1 (Schmitz, C., Marchant, D., Neil, S.J., Aubin, K., Reuter, S., Dittmar, M.T., McKnight, A., Kizhatil, K., Albritton, L.M., 2004. Lv2, a novel postentry restriction, is mediated by both capsid and envelope. J. Virol. 78 (4), 2006-2016). We generated pseudotyped viruses consisting of HIV-2 (ROD-AΔenv-GFP, ROD-AΔenv-RFP, or ROD-AΔenv-REN) and the prCBL23 or CBL23 envelope proteins as well as chimeric proteins between these envelopes. We demonstrate that a single amino acid exchange at position 74 in the surface unit of CBL23-Env confers restriction to infection. This single point mutation causes tighter CD4 binding, resulting in a less efficient fusion into the cytosol of the restricted cell line. Prevention of endosome formation and prevention of endosome acidification enhance infectivity of the restricted particles for GHOST/X4 cells indicating a degradative lysosomal pathway as a cause for the reduced cytosolic entry. The described restriction to infection of the primary isolate prCBL23 is therefore largely caused by an entry defect. A remaining restriction to infection (19-fold) is preserved when endosomal acidification is prevented. This restriction to infection is also dependent on the presence of the point mutation at position 74 (G74E)

  13. Effects of restricted and free suckling

    OpenAIRE

    Fröberg, Sofie

    2008-01-01

    The aim of this thesis was to study the effects of restricted and free suckling in comparison with non-suckling on production and behaviour of cow and calf in dairy production systems. In the first and second study cows of Zebu × Holstein (n=24) and Holstein breed (n=27) and their calves were allocated to two treatments, restricted suckling (RS) and artificial rearing (AR) and studied during eight weeks. In the first study calves were present during milking and RS calves suckled after milking...

  14. Host Specificity of Salmonella typhimurium Deoxyribonucleic Acid Restriction and Modification

    Science.gov (United States)

    Slocum, Harvey; Boyer, Herbert W.

    1973-01-01

    The restriction and modification genes of Salmonella typhimurium which lie near the thr locus were transferred to a restrictionless mutant of Escherichia coli. These genes were found to be allelic to the E. coli K, B, and A restriction and modification genes. E. coli recombinants with the restriction and modification host specificity of S. typhimurium restricted phage λ that had been modified by each of the seven known host specificities of E. coli at efficiency of plating levels of about 10−2. Phage λ modified with the S. typhimurium host specificity was restricted by six of the seven E. coli host specificities but not by the RII (fi− R-factor controlled) host specificity. It is proposed that the restriction and modification enzymes of this S. typhimurium host specificity have two substrates, one of which is a substrate for the RII host specificity enzymes. PMID:4570605

  15. Effect of dietary protein restriction on renal ammonia metabolism

    Science.gov (United States)

    Lee, Hyun-Wook; Osis, Gunars; Handlogten, Mary E.; Guo, Hui; Verlander, Jill W.

    2015-01-01

    Dietary protein restriction has multiple benefits in kidney disease. Because protein intake is a major determinant of endogenous acid production, it is important that net acid excretion change in parallel during protein restriction. Ammonia is the primary component of net acid excretion, and inappropriate ammonia excretion can lead to negative nitrogen balance. Accordingly, we examined ammonia excretion in response to protein restriction and then we determined the molecular mechanism of the changes observed. Wild-type C57Bl/6 mice fed a 20% protein diet and then changed to 6% protein developed an 85% reduction in ammonia excretion within 2 days, which persisted during a 10-day study. The expression of multiple proteins involved in renal ammonia metabolism was altered, including the ammonia-generating enzymes phosphate-dependent glutaminase (PDG) and phosphoenolpyruvate carboxykinase (PEPCK) and the ammonia-metabolizing enzyme glutamine synthetase. Rhbg, an ammonia transporter, increased in expression in the inner stripe of outer medullary collecting duct intercalated cell (OMCDis-IC). However, collecting duct-specific Rhbg deletion did not alter the response to protein restriction. Rhcg deletion did not alter ammonia excretion in response to dietary protein restriction. These results indicate 1) dietary protein restriction decreases renal ammonia excretion through coordinated regulation of multiple components of ammonia metabolism; 2) increased Rhbg expression in the OMCDis-IC may indicate a biological role in addition to ammonia transport; and 3) Rhcg expression is not necessary to decrease ammonia excretion during dietary protein restriction. PMID:25925252

  16. Consequences of intrauterine growth restriction for the kidney

    NARCIS (Netherlands)

    M.F. Schreuder; A. van Wijk (Ans); H.A. Delemarre-van de Waal (Henriette)

    2006-01-01

    textabstractLow birth weight due to intrauterine growth restriction is associated with various diseases in adulthood, such as hypertension, cardiovascular disease, insulin resistance and end-stage renal disease. The purpose of this review is to describe the effects of intrauterine growth restriction

  17. Restricted Variance Interaction Effects

    DEFF Research Database (Denmark)

    Cortina, Jose M.; Köhler, Tine; Keeler, Kathleen R.

    2018-01-01

    Although interaction hypotheses are increasingly common in our field, many recent articles point out that authors often have difficulty justifying them. The purpose of this article is to describe a particular type of interaction: the restricted variance (RV) interaction. The essence of the RV int...

  18. Late gestational nutrient restriction

    DEFF Research Database (Denmark)

    Tygesen, Malin Plumhoff; Nielsen, Mette Olaf; Nørgaard, Peder

    2008-01-01

    We investigated the effect of 50% nutrient restriction during the last 6 weeks of gestation on twin-pregnant ewes' plasma glucose, non-esterified fatty acid, ß-hydroxybutyrate, insulin, IGF-1 and leptin concentrations and the effects on lamb birth weight and ewes' lactation performance. Plasma...

  19. Comparative Study of Genome Divergence in Salmonids with Various Rates of Genetic Isolation

    Directory of Open Access Journals (Sweden)

    Elena A. Shubina

    2013-01-01

    Full Text Available The aim of the study is a comparative investigation of changes that certain genome parts undergo during speciation. The research was focused on divergence of coding and noncoding sequences in different groups of salmonid fishes of the Salmonidae (Salmo, Parasalmo, Oncorhynchus, and Salvelinus genera and the Coregonidae families under different levels of reproductive isolation. Two basic approaches were used: (1 PCR-RAPD with a 20–22 nt primer design with subsequent cloning and sequencing of the products and (2 a modified endonuclease restriction analysis. The restriction fragments were shown with sequencing to represent satellite DNA. Effects of speciation are found in repetitive sequences. The revelation of expressed sequences in the majority of the employed anonymous loci allows for assuming the adaptive selection during allopatric speciation in isolated char forms.

  20. 7 CFR 319.37-12 - Prohibited articles accompanying restricted articles.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 5 2010-01-01 2010-01-01 false Prohibited articles accompanying restricted articles... Stock, Plants, Roots, Bulbs, Seeds, and Other Plant Products 1,2 § 319.37-12 Prohibited articles accompanying restricted articles. A restricted article for importation into the United States shall not be...

  1. Qualitative versus quantitative feed restriction in Pekin breeder ...

    African Journals Online (AJOL)

    titative (60% of a group fed ad lib) feed restriction during the rearing period with a ... restriction to reduce growth rate and delay sexual maturity. Since the amount of .... ing in the cost of feed when compared to the more expensive ad lib diet.

  2. Impact of Total Vending Machine Restrictions on US Young Adult Smoking

    Science.gov (United States)

    Kelly, Brian C.; Kadowaki, Joy

    2016-01-01

    Introduction: In an analysis of smoking using a longitudinal sample of US young adults, we extend research on tobacco vending machine restrictions beyond its prior focus on minors by examining the influence of total vending machine restrictions, which apply to adult-only facilities and represents the only remaining vending machine exemption since the enactment of the Family Smoking Prevention and Tobacco Control Act. We identify whether the passage of a restriction influences an individual’s smoking on repeated observations, and if the propensity is lower among those who live in locations with a restriction. Methods: Combining a repository of US tobacco policies at all geographic levels with the nationally-representative geocoded National Longitudinal Survey of Youth 1997 and Census data, we use multilevel logistic regression to examine the impact of total vending machine restrictions on any past 30-day smoking and past 30-day smoking of one pack per day among young adults (ages 19–31), while accounting for other tobacco control policy, community, and individual covariates. Results: We find that total vending machine restrictions decrease any recent smoking (OR = 0.451; p vending machine restrictions on smoking a pack daily. Conclusions: Total vending machine restrictions appear to be an effective, yet highly underutilized, means of tobacco control. Implications: Past scientific inquiries examining vending machine restrictions have focused upon minor access, adolescent perceptions of availability, and subsequent smoking. The potential for total vending machine restrictions, which extend to adult-only facilities, to influence patterns of smoking among those of legal age, remains significant. Those who are subject to total vending machine restrictions for longer periods are less likely to have recently smoked, but individuals do not change their smoking behavior in response to the passage of a restriction. These restrictions do not affect heavy smokers. Such

  3. Company Law as a Restriction to Free Movement

    DEFF Research Database (Denmark)

    Sørensen, Karsten Engsig

    2014-01-01

    . There is evidence that non-discriminatory obstacles to free movement found in company law can be restrictions. However, recent cases show that the Court may be willing to apply a more restrictive approach where only non-discriminatory measures that have a qualified deterrent effect may constitute restrictions.......Focusing on recent judgments from the Court of Justice of the European Union, this article investigates how the notion of ‘restriction’ – which is the first step in examining whether there is an infringement of the free movement rights – is used in cases involving company law measures...

  4. 49 CFR 236.813a - State, most restrictive.

    Science.gov (United States)

    2010-10-01

    ... 49 Transportation 4 2010-10-01 2010-10-01 false State, most restrictive. 236.813a Section 236.813a Transportation Other Regulations Relating to Transportation (Continued) FEDERAL RAILROAD ADMINISTRATION... State, most restrictive. The mode of an electric or electronic device that is equivalent to a track...

  5. Avoidant/Restrictive Food Intake Disorder

    Science.gov (United States)

    ... Eating Disorder Bulimia Nervosa Pica Rumination Disorder Avoidant/restrictive food intake disorder is characterized by eating very little food and/or avoiding eating certain foods. People with this disorder eat ...

  6. 5 CFR 332.407 - Restriction of consideration to one sex.

    Science.gov (United States)

    2010-01-01

    ... 5 Administrative Personnel 1 2010-01-01 2010-01-01 false Restriction of consideration to one sex... Restriction of consideration to one sex. An appointing officer may not restrict his consideration of eligibles... competitive service to one sex, except in unusual circumstances when OPM finds the action justified. [34 FR...

  7. Work-hour restrictions and orthopaedic resident education: a systematic review.

    Science.gov (United States)

    Mauser, Nathan S; Michelson, James D; Gissel, Hannah; Henderson, Corey; Mauffrey, Cyril

    2016-05-01

    The ACGME (US) and The European Working Time Directive (UK) placed work-hour restrictions on medical trainees with the goal of improved patient safety. However, there has been concern over a potential decrease in medical education. Orthopaedic training is the focus of this study. We examined previously published subjective and objective data regarding education and work-hour restrictions and developed the questions: Do specific perceptions emerge within the subjective studies examined? Are there objective differences in educational measures before and after work-hour restrictions? Is there a difference between the subjective and objective data? A systematic review was conducted via MedLine, regarding orthopaedic studies in the USA and UK, with reference to work-hour restrictions and education. Subjective survey studies demonstrate that residents and attending physicians have a negative response to work-hour restrictions because of the perceived impact on their overall education and operating room experience. Conversely, limited objective studies demonstrated no change in operative volume before or after implementation of restrictions. This review highlights the need for more objective studies on the educational implications of work-hour restrictions. Studies to date have not demonstrated a measurable difference based on case logs or training scores. Opinion-based surveys demonstrate an overall negative perception by both residents and attending physicians, on the impact of work-hour restrictions on orthopaedic education. Current published data is limited and stronger evidence-based data are needed before definitive conclusions can be reached.

  8. Calorie Restriction Attenuates Terminal Differentiation of Immune Cells.

    Science.gov (United States)

    White, Matthew J; Beaver, Charlotte M; Goodier, Martin R; Bottomley, Christian; Nielsen, Carolyn M; Wolf, Asia-Sophia F M; Boldrin, Luisa; Whitmore, Charlotte; Morgan, Jennifer; Pearce, Daniel J; Riley, Eleanor M

    2016-01-01

    Immune senescence is a natural consequence of aging and may contribute to frailty and loss of homeostasis in later life. Calorie restriction increases healthy life-span in C57BL/6J (but not DBA/2J) mice, but whether this is related to preservation of immune function, and how it interacts with aging, is unclear. We compared phenotypic and functional characteristics of natural killer (NK) cells and T cells, across the lifespan, of calorie-restricted (CR) and control C57BL/6 and DBA/2 mice. Calorie restriction preserves a naïve T cell phenotype and an immature NK cell phenotype as mice age. The splenic T cell populations of CR mice had higher proportions of CD11a - CD44 lo cells, lower expression of TRAIL, KLRG1, and CXCR3, and higher expression of CD127, compared to control mice. Similarly, splenic NK cells from CR mice had higher proportions of less differentiated CD11b - CD27 + cells and correspondingly lower proportions of highly differentiated CD11b + CD27 - NK cells. Within each of these subsets, cells from CR mice had higher expression of CD127, CD25, TRAIL, NKG2A/C/E, and CXCR3 and lower expression of KLRG1 and Ly49 receptors compared to controls. The effects of calorie restriction on lymphoid cell populations in lung, liver, and lymph nodes were identical to those seen in the spleen, indicating that this is a system-wide effect. The impact of calorie restriction on NK cell and T cell maturation is much more profound than the effect of aging and, indeed, calorie restriction attenuates these age-associated changes. Importantly, the effects of calorie restriction on lymphocyte maturation were more marked in C57BL/6 than in DBA/2J mice indicating that delayed lymphocyte maturation correlates with extended lifespan. These findings have implications for understanding the interaction between nutritional status, immunity, and healthy lifespan in aging populations.

  9. Emergency building temperature restrictions. Final evaluation

    Energy Technology Data Exchange (ETDEWEB)

    None

    1980-11-01

    On July 5, 1979, DOE promulgated final regulations of the Emergency Building Temperature Restrictions program, placing emergency restrictions on thermostat settings for space heating, space cooling, and hot water in commercial, industrial, and nonresidential public buildings. The final regulations restricted space heating to a maximum of 65/sup 0/F, hot water temperature to a maximum of 105/sup 0/F, and cooling temperature to a minimum of 78/sup 0/F. A comprehensive evaluation of the entire EBTF program for a nine-month period from July 16, 1979 is presented. In Chapter 1, an estimate of the population of buildings covered by EBTR is presented. In Chapter 2, EBTR compliance by building type and region is reported. Exemptions are also discussed. In Chapter 3, the simulations of building energy use are explained and the relative impact of various building characteristics and effectiveness of different control strategies are estimated. Finally, in Chapter 4, the methodology for scaling the individual building energy savings to the national level is described, and estimated national energy savings are presented.

  10. 7 CFR 301.92 - Restrictions on interstate movement.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 5 2010-01-01 2010-01-01 false Restrictions on interstate movement. 301.92 Section... Restrictions on interstate movement. (a) No person may move interstate from any quarantined area any regulated... plant that was found infected with Phytophthora ramorum, unless such movement is in accordance with part...

  11. Resource implications of adopting a restrictive neonatal blood ...

    African Journals Online (AJOL)

    Resource implications of adopting a restrictive neonatal blood transfusion policy. ... Objective. To determine whether adopting a restrictive BTF policy results in fewer transfusions. Methods. Data were retrospectively collected on all infants who received BTFs in the GSH nursery over a 6-month period following adoption of a ...

  12. 30 CFR 57.4461 - Gasoline use restrictions underground.

    Science.gov (United States)

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Gasoline use restrictions underground. 57.4461... Prevention and Control Flammable and Combustible Liquids and Gases § 57.4461 Gasoline use restrictions underground. If gasoline is used underground to power internal combustion engines— (a) The mine shall be...

  13. 9 CFR 590.800 - Identification of restricted eggs.

    Science.gov (United States)

    2010-01-01

    ... 9 Animals and Animal Products 2 2010-01-01 2010-01-01 false Identification of restricted eggs. 590... AGRICULTURE EGG PRODUCTS INSPECTION INSPECTION OF EGGS AND EGG PRODUCTS (EGG PRODUCTS INSPECTION ACT) Identification of Restricted Eggs Or Egg Products Not Intended for Human Consumption § 590.800 Identification of...

  14. Growth throughout childhood of children born growth restricted

    NARCIS (Netherlands)

    Beukers, Fenny; Rotteveel, Joost; van Weissenbruch, Mirjam M.; Ganzevoort, Wessel; van Goudoever, Johannes B.; van Wassenaer-Leemhuis, Aleid G.

    2017-01-01

    Many studies that examine growth in growth-restricted children at birth do not discriminate between fetal growth restriction (FGR) and small for gestational age (SGA). These terms however are not synonymous. In SGA, stunting and increased weight gain have been reported. We do not know if this holds

  15. Intrauterine growth restriction: screening, diagnosis, and management.

    Science.gov (United States)

    Lausman, Andrea; Kingdom, John

    2013-08-01

    Intrauterine growth restriction (IUGR) is an obstetrical complication, which by definition would screen in 10% of fetuses in the general population. The challenge is to identify the subset of pregnancies affected with pathological growth restriction in order to allow intervention that would decrease morbidity and mortality. The purpose of this guideline is to provide summary statements and recommendations and to establish a framework for screening, diagnosis, and management of pregnancies affected with IUGR. Affected pregnancies are compared with pregnancies in which the fetus is at an appropriate weight for its gestational age. History, physical examination, and laboratory investigations including biochemical markers and ultrasound characteristics of IUGR are reviewed, and a management strategy is suggested. Published literature in English was retrieved through searches of PubMed or MEDLINE, CINAHL, and The Cochrane Library in January 2013 using appropriate controlled vocabulary via MeSH terms (fetal growth restriction and small for gestational age) and key words (fetal growth, restriction, growth retardation, IUGR, low birth weight, small for gestational age). Results were restricted to systematic reviews, randomized control trials/controlled clinical trials, and observational studies. Grey (unpublished) literature was identified through searching the websites of health technology assessment and health technology-related agencies, clinical practice guideline collections, clinical trial registries, and national and international medical specialty societies. The quality of evidence in this document was rated using the criteria described in the Report of the Canadian Task Force on Preventive Health Care (Table). Implementation of the recommendations in this guideline should increase clinician recognition of IUGR and guide intervention where appropriate. Optimal long-term follow-up of neonates diagnosed as IUGR may improve their long-term health.

  16. Predicting support for restricting food marketing to youth.

    Science.gov (United States)

    Goren, Amir; Harris, Jennifer L; Schwartz, Marlene B; Brownell, Kelly D

    2010-01-01

    To address the obesity crisis, public health experts recommend major reductions in the marketing of unhealthy food to youth. However, policies to restrict food marketing are not currently viewed as politically feasible. This paper examines attitudes and knowledge about food marketing and support for restricting unhealthy food marketing [corrected] among one group of constituents: parents. A survey of 807 parents found that those most likely to support food marketing restrictions were also more likely to have negative views of current food practices. [corrected] These findings suggest that increased public education about the harm caused by food marketing may increase public support for policy interventions.

  17. 25 CFR 141.14 - Trade in livestock restricted.

    Science.gov (United States)

    2010-04-01

    ... 25 Indians 1 2010-04-01 2010-04-01 false Trade in livestock restricted. 141.14 Section 141.14... livestock restricted. (a) No person other than an enrolled member of the tribe or any association... livestock from tribal members without a special permit issued by the Commissioner. (b) The Commissioner...

  18. 21 CFR 1314.20 - Restrictions on sales quantity.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 9 2010-04-01 2010-04-01 false Restrictions on sales quantity. 1314.20 Section 1314.20 Food and Drugs DRUG ENFORCEMENT ADMINISTRATION, DEPARTMENT OF JUSTICE RETAIL SALE OF SCHEDULED LISTED CHEMICAL PRODUCTS Sales by Regulated Sellers § 1314.20 Restrictions on sales quantity. (a) Without...

  19. Public Investment, Revenue Shocks, and Borrowing Restrictions

    OpenAIRE

    Büttner, Thiess; Wildasin, David E.

    2010-01-01

    This paper lays out a theory of taxation and public investment in an intertemporal setting under conditions of revenue shocks. Without borrowing restrictions, the optimal policy is characterized by smooth time paths of taxes and public investment. While the introduction of formal borrowing restrictions leads to some precautionary savings, it gives rise to fluctuations in public investment in response to adverse but also favorable revenue shocks. This theoretical result is tested empirically u...

  20. Food restriction reduces neurogenesis in the avian hippocampal formation.

    Directory of Open Access Journals (Sweden)

    Barbara-Anne Robertson

    Full Text Available The mammalian hippocampus is particularly vulnerable to chronic stress. Adult neurogenesis in the dentate gyrus is suppressed by chronic stress and by administration of glucocorticoid hormones. Post-natal and adult neurogenesis are present in the avian hippocampal formation as well, but much less is known about its sensitivity to chronic stressors. In this study, we investigate this question in a commercial bird model: the broiler breeder chicken. Commercial broiler breeders are food restricted during development to manipulate their growth curve and to avoid negative health outcomes, including obesity and poor reproductive performance. Beyond knowing that these chickens are healthier than fully-fed birds and that they have a high motivation to eat, little is known about how food restriction impacts the animals' physiology. Chickens were kept on a commercial food-restricted diet during the first 12 weeks of life, or released from this restriction by feeding them ad libitum from weeks 7-12 of life. To test the hypothesis that chronic food restriction decreases the production of new neurons (neurogenesis in the hippocampal formation, the cell proliferation marker bromodeoxyuridine was injected one week prior to tissue collection. Corticosterone levels in blood plasma were elevated during food restriction, even though molecular markers of hypothalamic-pituitary-adrenal axis activation did not differ between the treatments. The density of new hippocampal neurons was significantly reduced in the food-restricted condition, as compared to chickens fed ad libitum, similar to findings in rats at a similar developmental stage. Food restriction did not affect hippocampal volume or the total number of neurons. These findings indicate that in birds, like in mammals, reduction in hippocampal neurogenesis is associated with chronically elevated corticosterone levels, and therefore potentially with chronic stress in general. This finding is consistent with the

  1. Identification of a residue critical for the excision of 3′-blocking ends in apurinic/apyrimidinic endonucleases of the Xth family

    Science.gov (United States)

    Castillo-Acosta, Víctor M.; Ruiz-Pérez, Luis M.; Yang, Wei; González-Pacanowska, Dolores; Vidal, Antonio E.

    2009-01-01

    DNA single-strand breaks containing 3′-blocking groups are generated from attack of the sugar backbone by reactive oxygen species or after base excision by DNA glycosylase/apurinic/apyrimidinic (AP) lyases. In human cells, APE1 excises sugar fragments that block the 3′-ends thus facilitating DNA repair synthesis. In Leishmania major, the causal agent of leishmaniasis, the APE1 homolog is the class II AP endonuclease LMAP. Expression of LMAP but not of APE1 reverts the hypersensitivity of a xth nfo repair-deficient Escherichia coli strain to the oxidative compound hydrogen peroxide (H2O2). To identify the residues specifically involved in the repair of oxidative DNA damage, we generated random mutations in the ape1 gene and selected those variants that conferred protection against H2O2. Among the resistant clones, we isolated a mutant in the nuclease domain of APE1 (D70A) with an increased capacity to remove 3′-blocking ends in vitro. D70 of APE1 aligns with A138 of LMAP and mutation of the latter to aspartate significantly reduces its 3′-phosphodiesterase activity. Kinetic analysis shows a novel role of residue D70 in the excision rate of 3′-blocking ends. The functional and structural differences between the parasite and human enzymes probably reflect a divergent molecular evolution of their DNA repair responses to oxidative damage. PMID:19181704

  2. Enrichment of G2/M cell cycle phase in human pluripotent stem cells enhances HDR-mediated gene repair with customizable endonucleases.

    Science.gov (United States)

    Yang, Diane; Scavuzzo, Marissa A; Chmielowiec, Jolanta; Sharp, Robert; Bajic, Aleksandar; Borowiak, Malgorzata

    2016-02-18

    Efficient gene editing is essential to fully utilize human pluripotent stem cells (hPSCs) in regenerative medicine. Custom endonuclease-based gene targeting involves two mechanisms of DNA repair: homology directed repair (HDR) and non-homologous end joining (NHEJ). HDR is the preferred mechanism for common applications such knock-in, knock-out or precise mutagenesis, but remains inefficient in hPSCs. Here, we demonstrate that synchronizing synchronizing hPSCs in G2/M with ABT phase increases on-target gene editing, defined as correct targeting cassette integration, 3 to 6 fold. We observed improved efficiency using ZFNs, TALENs, two CRISPR/Cas9, and CRISPR/Cas9 nickase to target five genes in three hPSC lines: three human embryonic stem cell lines, neural progenitors and diabetic iPSCs. neural progenitors and diabetic iPSCs. Reversible synchronization has no effect on pluripotency or differentiation. The increase in on-target gene editing is locus-independent and specific to the cell cycle phase as G2/M phase enriched cells show a 6-fold increase in targeting efficiency compared to cells in G1 phase. Concurrently inhibiting NHEJ with SCR7 does not increase HDR or improve gene targeting efficiency further, indicating that HR is the major DNA repair mechanism after G2/M phase arrest. The approach outlined here makes gene editing in hPSCs a more viable tool for disease modeling, regenerative medicine and cell-based therapies.

  3. Real-time observation of DNA target interrogation and product release by the RNA-guided endonuclease CRISPR Cpf1 (Cas12a).

    Science.gov (United States)

    Singh, Digvijay; Mallon, John; Poddar, Anustup; Wang, Yanbo; Tippana, Ramreddy; Yang, Olivia; Bailey, Scott; Ha, Taekjip

    2018-05-22

    CRISPR-Cas9, which imparts adaptive immunity against foreign genomic invaders in certain prokaryotes, has been repurposed for genome-engineering applications. More recently, another RNA-guided CRISPR endonuclease called Cpf1 (also known as Cas12a) was identified and is also being repurposed. Little is known about the kinetics and mechanism of Cpf1 DNA interaction and how sequence mismatches between the DNA target and guide-RNA influence this interaction. We used single-molecule fluorescence analysis and biochemical assays to characterize DNA interrogation, cleavage, and product release by three Cpf1 orthologs. Our Cpf1 data are consistent with the DNA interrogation mechanism proposed for Cas9. They both bind any DNA in search of protospacer-adjacent motif (PAM) sequences, verify the target sequence directionally from the PAM-proximal end, and rapidly reject any targets that lack a PAM or that are poorly matched with the guide-RNA. Unlike Cas9, which requires 9 bp for stable binding and ∼16 bp for cleavage, Cpf1 requires an ∼17-bp sequence match for both stable binding and cleavage. Unlike Cas9, which does not release the DNA cleavage products, Cpf1 rapidly releases the PAM-distal cleavage product, but not the PAM-proximal product. Solution pH, reducing conditions, and 5' guanine in guide-RNA differentially affected different Cpf1 orthologs. Our findings have important implications on Cpf1-based genome engineering and manipulation applications.

  4. Phage T4 endonuclease V stimulates DNA repair replication in isolated nuclei from ultraviolet-irradiated human cells, including xeroderma pigmentosum fibroblasts

    International Nuclear Information System (INIS)

    Smith, C.A.; Hanawalt, P.C.

    1978-01-01

    The repair mode of DNA replication has been demonstrated in isolated nuclei from uv-irradiated human cells. Nuclei are incubated in a mixture containing [ 3 H]thymidine triphosphate and bromodeoxyuridine triphosphate in a 1:5 ratio. The 3 H at the density of parental DNA in alkaline CsCl density gradients is then a measure of repair. In nuclei prepared from WI38 cells 30 min after irradiation, repair replication is uv-dependent and proceeds at approximately the in vivo rate for 5 min. Repair replication is reduced in irradiated nuclei or in nuclei prepared immediately after irradiation. It is Mg 2+ -dependent and stimulated by added ATP and deoxyribonucleoside triphosphates. No repair replication is observed in nuclei from xeroderma pigmentosum (complementation group A) cells. However, upon addition of coliphage T4 endonuclease V, which specifically nicks DNA containing pyrimidine dimers, repair replication is observed in nuclei from irradiated xeroderma pigmentosum cells and is stimulated in WI38 nuclei. The reaction then persists for an hour and is dependent upon added ATP and deoxyribonucleoside triphosphates. The repair label is in stretches of roughly 35 nucleotides, as it is in intact cells. Added pancreatic DNase does not promote uv-dependent repair synthesis. Our results support the view that xeroderma pigmentosum (group A) cells are defective in the incision step of the DNA excision repair pathway, and demonstrate the utility of this system for probing DNA repair mechanisms

  5. Sales Restriction, Quality Selection and the Mode of Competition

    OpenAIRE

    Boccard, Nicolas; Wauthy, Xavier

    2003-01-01

    A regulator imposing "sales restrictions" on firms competing in oligopolistic markets may enhance quality provision by the firms. Moreover, for most restrictions levels, the impact on quality selection is invariant to the mode of competition.

  6. Sleep restriction during simulated wildfire suppression: effect on physical task performance.

    Science.gov (United States)

    Vincent, Grace; Ferguson, Sally A; Tran, Jacqueline; Larsen, Brianna; Wolkow, Alexander; Aisbett, Brad

    2015-01-01

    To examine the effects of sleep restriction on firefighters' physical task performance during simulated wildfire suppression. Thirty-five firefighters were matched and randomly allocated to either a control condition (8-hour sleep opportunity, n = 18) or a sleep restricted condition (4-hour sleep opportunity, n = 17). Performance on physical work tasks was evaluated across three days. In addition, heart rate, core temperature, and worker activity were measured continuously. Rate of perceived and exertion and effort sensation were evaluated during the physical work periods. There were no differences between the sleep-restricted and control groups in firefighters' task performance, heart rate, core temperature, or perceptual responses during self-paced simulated firefighting work tasks. However, the sleep-restricted group were less active during periods of non-physical work compared to the control group. Under self-paced work conditions, 4 h of sleep restriction did not adversely affect firefighters' performance on physical work tasks. However, the sleep-restricted group were less physically active throughout the simulation. This may indicate that sleep-restricted participants adapted their behaviour to conserve effort during rest periods, to subsequently ensure they were able to maintain performance during the firefighter work tasks. This work contributes new knowledge to inform fire agencies of firefighters' operational capabilities when their sleep is restricted during multi-day wildfire events. The work also highlights the need for further research to explore how sleep restriction affects physical performance during tasks of varying duration, intensity, and complexity.

  7. 50 CFR 640.22 - Gear and diving restrictions.

    Science.gov (United States)

    2010-10-01

    ... 50 Wildlife and Fisheries 8 2010-10-01 2010-10-01 false Gear and diving restrictions. 640.22... ATLANTIC Management Measures § 640.22 Gear and diving restrictions. (a) Prohibited gear and methods. (1) A spiny lobster may not be taken in the EEZ with a spear, hook, or similar device, or gear containing such...

  8. 10 CFR 2.903 - Protection of restricted data and national security information.

    Science.gov (United States)

    2010-01-01

    ... 10 Energy 1 2010-01-01 2010-01-01 false Protection of restricted data and national security... Restricted Data and/or National Security Information § 2.903 Protection of restricted data and national security information. Nothing in this subpart shall relieve any person from safeguarding Restricted Data or...

  9. Nuclear depletion of apurinic/apyrimidinic endonuclease 1 (Ape1/Ref-1) is an indicator of energy disruption in neurons.

    Science.gov (United States)

    Singh, Shilpee; Englander, Ella W

    2012-11-01

    Apurinic/apyrimidinic endonuclease 1 (Ape1/Ref-1) is a multifunctional protein critical for cellular survival. Its involvement in adaptive survival responses includes key roles in redox sensing, transcriptional regulation, and repair of DNA damage via the base excision repair (BER) pathway. Ape1 is abundant in most cell types and central in integrating the first BER step catalyzed by different DNA glycosylases. BER is the main process for removal of oxidative DNA lesions in postmitotic brain cells, and after ischemic brain injury preservation of Ape1 coincides with neuronal survival, while its loss has been associated with neuronal death. Here, we report that in cultured primary neurons, diminution of cellular ATP by either oligomycin or H(2)O(2) is accompanied by depletion of nuclear Ape1, while other BER proteins are unaffected and retain their nuclear localization under these conditions. Importantly, while H(2)O(2) induces γH2AX phosphorylation, indicative of chromatin rearrangements in response to DNA damage, oligomycin does not. Furthermore, despite comparable diminution of ATP content, H(2)O(2) and oligomycin differentially affect critical parameters of mitochondrial respiration that ultimately determine cellular ATP content. Taken together, our findings demonstrate that in neurons, nuclear compartmentalization of Ape1 depends on ATP and loss of nuclear Ape1 reflects disruption of neuronal energy homeostasis. Energy crisis is a hallmark of stroke and other ischemic/hypoxic brain injuries. In vivo studies have shown that Ape1 deficit precedes neuronal loss in injured brain regions. Thus, our findings bring to light the possibility that energy failure-induced Ape1 depletion triggers neuronal death in ischemic brain injuries. Copyright © 2012 Elsevier Inc. All rights reserved.

  10. ECO-generation for some restricted classes of compositions

    Directory of Open Access Journals (Sweden)

    Jean-Luc Baril

    2013-04-01

    Full Text Available We study several restricted classes of compositions by giving one-to-one maps between them and different classes of restricted binary strings or pattern avoiding permutations. Inspired by the ECO method \\cite{BDPP99}, new succession rules for these classes are presented. Finally, we obtain generating algorithms in Constant Amortized Time (CAT for theses classes.

  11. Parental perceptions of teen driving: Restrictions, worry and influence.

    Science.gov (United States)

    Jewett, Amy; Shults, Ruth A; Bhat, Geeta

    2016-12-01

    Parents play a critical role in preventing crashes among teens. Research of parental perceptions and concerns regarding teen driving safety is limited. We examined results from the 2013 Summer ConsumerStyles survey that queried parents about restrictions placed on their teen drivers, their perceived level of "worry" about their teen driver's safety, and influence of parental restrictions regarding their teen's driving. We produced frequency distributions for the number of restrictions imposed, parental "worry," and influence of rules regarding their teen's driving, reported by teen's driving license status (learning to drive or obtained a driver's license). Response categories were dichotomized because of small cell sizes, and we ran separate log-linear regression models to explore whether imposing all four restrictions on teen drivers was associated with either worry intensity ("a lot" versus "somewhat, not very much or not at all") or perceived influence of parental rules ("a lot" versus "somewhat, not very much or not at all"). Among the 456 parent respondents, 80% reported having restrictions for their teen driver regarding use of safety belts, drinking and driving, cell phones, and text messaging while driving. However, among the 188 parents of licensed teens, only 9% reported having a written parent-teen driving agreement, either currently or in the past. Worrying "a lot" was reported less frequently by parents of newly licensed teens (36%) compared with parents of learning teens (61%). Parents report having rules and restrictions for their teen drivers, but only a small percentage formalize the rules and restrictions in a written parent-teen driving agreement. Parents worry less about their teen driver's safety during the newly licensed phase, when crash risk is high as compared to the learning phase. Further research is needed into how to effectively support parents in supervising and monitoring their teen driver. Published by Elsevier Ltd.

  12. Immediate metabolic consequences of intrauterine growth restriction and low birthweight.

    Science.gov (United States)

    Bhatia, Jatinder; Gates, Amy

    2013-01-01

    Optimal fetal growth resulting in a 'normally grown' term infant is of paramount importance for assuring a healthy start for postnatal growth and development. Fetal, infant and childhood growth restriction is an important clinical problem for obstetricians, neonatologists, pediatricians and globally, for public health. Worldwide, an estimated 20 million infants are born with low birthweight and a substantial proportion are small for gestational age. Many advances have been made in defining growth restriction by prenatal techniques, thus allowing the recognition of intrauterine growth restriction. Distinguishing infants who are small but have appropriate growth potential from those with growth restriction is important in order to apply obstetric surveillance, anticipate neonatal problems and plan for postneonatal guidance. It is clear that the fetus in growth-restricted pregnancies has limited supply of nutrients and oxygen. The resultant changes, if involving the placenta as well, can lead to circulatory and metabolic changes affecting both short- and long-term survival and development. In this paper, the causes and immediate consequence of being born with low birthweight, intrauterine growth restriction or small for gestational age will be discussed. Copyright © 2013 Nestec Ltd., Vevey/S. Karger AG, Basel.

  13. Napping reverses increased pain sensitivity due to sleep restriction.

    Science.gov (United States)

    Faraut, Brice; Léger, Damien; Medkour, Terkia; Dubois, Alexandre; Bayon, Virginie; Chennaoui, Mounir; Perrot, Serge

    2015-01-01

    To investigate pain sensitivity after sleep restriction and the restorative effect of napping. A strictly controlled randomized crossover study with continuous polysomnography monitoring was performed. Laboratory-based study. 11 healthy male volunteers. Volunteers attended two three-day sessions: "sleep restriction" alone and "sleep restriction and nap". Each session involved a baseline night of normal sleep, a night of sleep deprivation and a night of free recovery sleep. Participants were allowed to sleep only from 02:00 to 04:00 during the sleep deprivation night. During the "sleep restriction and nap" session, volunteers took two 30-minute naps, one in the morning and one in the afternoon. Quantitative sensory testing was performed with heat, cold and pressure, at 10:00 and 16:00, on three areas: the supraspinatus, lower back and thigh. After sleep restriction, quantitative sensory testing revealed differential changes in pain stimuli thresholds, but not in thermal threshold detection: lower back heat pain threshold decreased, pressure pain threshold increased in the supraspinatus area and no change was observed for the thigh. Napping restored responses to heat pain stimuli in the lower back and to pressure stimuli in the supraspinatus area. Sleep restriction induces different types of hypersensitivity to pain stimuli in different body areas, consistent with multilevel mechanisms, these changes being reversed by napping. The napping restorative effect on pain thresholds result principally from effects on pain mechanisms, since it was independent of vigilance status.

  14. Napping reverses increased pain sensitivity due to sleep restriction.

    Directory of Open Access Journals (Sweden)

    Brice Faraut

    Full Text Available To investigate pain sensitivity after sleep restriction and the restorative effect of napping.A strictly controlled randomized crossover study with continuous polysomnography monitoring was performed.Laboratory-based study.11 healthy male volunteers.Volunteers attended two three-day sessions: "sleep restriction" alone and "sleep restriction and nap". Each session involved a baseline night of normal sleep, a night of sleep deprivation and a night of free recovery sleep. Participants were allowed to sleep only from 02:00 to 04:00 during the sleep deprivation night. During the "sleep restriction and nap" session, volunteers took two 30-minute naps, one in the morning and one in the afternoon.Quantitative sensory testing was performed with heat, cold and pressure, at 10:00 and 16:00, on three areas: the supraspinatus, lower back and thigh. After sleep restriction, quantitative sensory testing revealed differential changes in pain stimuli thresholds, but not in thermal threshold detection: lower back heat pain threshold decreased, pressure pain threshold increased in the supraspinatus area and no change was observed for the thigh. Napping restored responses to heat pain stimuli in the lower back and to pressure stimuli in the supraspinatus area.Sleep restriction induces different types of hypersensitivity to pain stimuli in different body areas, consistent with multilevel mechanisms, these changes being reversed by napping. The napping restorative effect on pain thresholds result principally from effects on pain mechanisms, since it was independent of vigilance status.

  15. Restricted Social Engagement among Adults Living with Chronic Conditions

    Directory of Open Access Journals (Sweden)

    Kayla P. Meek

    2018-01-01

    Full Text Available Background: Social engagement is key to health and quality of life. Little is known about social engagement patterns of middle-aged and older adults who live with one or more chronic illnesses. This study investigated social engagement restrictions among middle-aged and older adults with chronic conditions and factors associated with these restrictions. Methods: Cross-sectional representative data from the National Council on Aging Chronic Care Survey were examined for relationships between social engagement restrictions and chronic conditions, health status, support, quality of life implications, self-care barriers, caregiving, and demographics. Associations were tested using bivariate analyses and binary logistic regression. Results: Participants were 793 middle-aged (age 44–64 and older adults (age 65+ with one or more chronic conditions. Factors associated with social engagement restrictions included having higher education, receiving care, having more physician visits and hospitalizations, being disabled, being unemployed, and having higher Emotional and Physical Problems Scale scores. Conclusions: Findings reveal the prevalence of social engagement restrictions among middle-aged and older adults with chronic conditions. Results highlight the importance of promoting research, assessments, and interventions to increase social engagement among this aging population.

  16. Human cellular restriction factors that target HIV-1 replication

    Directory of Open Access Journals (Sweden)

    Jeang Kuan-Teh

    2009-09-01

    Full Text Available Abstract Recent findings have highlighted roles played by innate cellular factors in restricting intracellular viral replication. In this review, we discuss in brief the activities of apolipoprotein B mRNA-editing enzyme 3G (APOBEC3G, bone marrow stromal cell antigen 2 (BST-2, cyclophilin A, tripartite motif protein 5 alpha (Trim5α, and cellular microRNAs as examples of host restriction factors that target HIV-1. We point to countermeasures encoded by HIV-1 for moderating the potency of these cellular restriction functions.

  17. Predictive factors for intrauterine growth restriction.

    Science.gov (United States)

    Albu, A R; Anca, A F; Horhoianu, V V; Horhoianu, I A

    2014-06-15

    Reduced fetal growth is seen in about 10% of the pregnancies but only a minority has a pathological background and is known as intrauterine growth restriction or fetal growth restriction (IUGR / FGR). Increased fetal and neonatal mortality and morbidity as well as adult pathologic conditions are often associated to IUGR. Risk factors for IUGR are easy to assess but have poor predictive value. For the diagnostic purpose, biochemical serum markers, ultrasound and Doppler study of uterine and spiral arteries, placental volume and vascularization, first trimester growth pattern are object of assessment today. Modern evaluations propose combined algorithms using these strategies, all with the goal of a better prediction of risk pregnancies.

  18. Optimal Policy under Restricted Government Spending

    DEFF Research Database (Denmark)

    Sørensen, Anders

    2006-01-01

    Welfare ranking of policy instruments is addressed in a two-sector Ramsey model with monopoly pricing in one sector as the only distortion. When government spending is restricted, i.e. when a government is unable or unwilling to finance the required costs for implementing the optimum policy...... effectiveness canexceed the welfare loss from introducing new distortions. Moreover, it is found that the investment subsidy is gradually phased out of the welfare maximizing policy, which may be a policy combining the two subsidies, when the level of government spending is increased.Keywords: welfare ranking......, indirect and direct policy instruments, restricted government spending JEL: E61, O21, O41...

  19. Sleep restriction during simulated wildfire suppression: effect on physical task performance.

    Directory of Open Access Journals (Sweden)

    Grace Vincent

    Full Text Available OBJECTIVES: To examine the effects of sleep restriction on firefighters' physical task performance during simulated wildfire suppression. METHODS: Thirty-five firefighters were matched and randomly allocated to either a control condition (8-hour sleep opportunity, n = 18 or a sleep restricted condition (4-hour sleep opportunity, n = 17. Performance on physical work tasks was evaluated across three days. In addition, heart rate, core temperature, and worker activity were measured continuously. Rate of perceived and exertion and effort sensation were evaluated during the physical work periods. RESULTS: There were no differences between the sleep-restricted and control groups in firefighters' task performance, heart rate, core temperature, or perceptual responses during self-paced simulated firefighting work tasks. However, the sleep-restricted group were less active during periods of non-physical work compared to the control group. CONCLUSIONS: Under self-paced work conditions, 4 h of sleep restriction did not adversely affect firefighters' performance on physical work tasks. However, the sleep-restricted group were less physically active throughout the simulation. This may indicate that sleep-restricted participants adapted their behaviour to conserve effort during rest periods, to subsequently ensure they were able to maintain performance during the firefighter work tasks. This work contributes new knowledge to inform fire agencies of firefighters' operational capabilities when their sleep is restricted during multi-day wildfire events. The work also highlights the need for further research to explore how sleep restriction affects physical performance during tasks of varying duration, intensity, and complexity.

  20. Controlling pandemic flu: the value of international air travel restrictions.

    Directory of Open Access Journals (Sweden)

    Joshua M Epstein

    2007-05-01

    Full Text Available Planning for a possible influenza pandemic is an extremely high priority, as social and economic effects of an unmitigated pandemic would be devastating. Mathematical models can be used to explore different scenarios and provide insight into potential costs, benefits, and effectiveness of prevention and control strategies under consideration.A stochastic, equation-based epidemic model is used to study global transmission of pandemic flu, including the effects of travel restrictions and vaccination. Economic costs of intervention are also considered. The distribution of First Passage Times (FPT to the United States and the numbers of infected persons in metropolitan areas worldwide are studied assuming various times and locations of the initial outbreak. International air travel restrictions alone provide a small delay in FPT to the U.S. When other containment measures are applied at the source in conjunction with travel restrictions, delays could be much longer. If in addition, control measures are instituted worldwide, there is a significant reduction in cases worldwide and specifically in the U.S. However, if travel restrictions are not combined with other measures, local epidemic severity may increase, because restriction-induced delays can push local outbreaks into high epidemic season. The per annum cost to the U.S. economy of international and major domestic air passenger travel restrictions is minimal: on the order of 0.8% of Gross National Product.International air travel restrictions may provide a small but important delay in the spread of a pandemic, especially if other disease control measures are implemented during the afforded time. However, if other measures are not instituted, delays may worsen regional epidemics by pushing the outbreak into high epidemic season. This important interaction between policy and seasonality is only evident with a global-scale model. Since the benefit of travel restrictions can be substantial while