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Sample records for giy-yig homing endonuclease

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

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    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.

  2. Phylogenomic analysis of the GIY-YIG nuclease superfamily

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    Bujnicki Janusz M

    2006-04-01

    Full Text Available Abstract Background The GIY-YIG domain was initially identified in homing endonucleases and later in other selfish mobile genetic elements (including restriction enzymes and non-LTR retrotransposons and in enzymes involved in DNA repair and recombination. However, to date no systematic search for novel members of the GIY-YIG superfamily or comparative analysis of these enzymes has been reported. Results We carried out database searches to identify all members of known GIY-YIG nuclease families. Multiple sequence alignments together with predicted secondary structures of identified families were represented as Hidden Markov Models (HMM and compared by the HHsearch method to the uncharacterized protein families gathered in the COG, KOG, and PFAM databases. This analysis allowed for extending the GIY-YIG superfamily to include members of COG3680 and a number of proteins not classified in COGs and to predict that these proteins may function as nucleases, potentially involved in DNA recombination and/or repair. Finally, all old and new members of the GIY-YIG superfamily were compared and analyzed to infer the phylogenetic tree. Conclusion An evolutionary classification of the GIY-YIG superfamily is presented for the very first time, along with the structural annotation of all (subfamilies. It provides a comprehensive picture of sequence-structure-function relationships in this superfamily of nucleases, which will help to design experiments to study the mechanism of action of known members (especially the uncharacterized ones and will facilitate the prediction of function for the newly discovered ones.

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

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    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.

  4. 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.

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    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.

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

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    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.

  6. 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...

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

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    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.

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

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    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.

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

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    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...

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

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    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.

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

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    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...

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

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    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

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

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    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.

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

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    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.

  15. Optimising homing endonuclease gene drive performance in a semi-refractory species: the Drosophila melanogaster experience.

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    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.

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

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    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...

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

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    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.

  19. Expression of homing endonuclease gene and insertion-like element in sea anemone mitochondrial genomes: Lesson learned from Anemonia viridis.

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    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.

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

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    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.

  1. Involvement of the VDE homing endonuclease and rapamycin in regulation of the Saccharomyces cerevisiae GSH11 gene encoding the high affinity glutathione transporter.

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    Miyake, Tsuyoshi; Hiraishi, Hiroyuki; Sammoto, Hiroyuki; Ono, Bun-Ichiro

    2003-10-10

    The Saccharomyces cerevisiae gene HGT1/GSH11 encodes the high affinity glutathione transporter and is repressed by cysteine added to the culture medium. It has been found previously that a 5'-upstream cis-element, CCGCCACAC, is responsible for regulating GSH11 expression and that several proteins bind to this element (Miyake, T., Kanayama, M., Sammoto, H., and Ono, B. (2002) Mol. Genet. Genomics 266, 1004-1011). In this report we present evidence that the most prominent of these proteins is VDE, known previously as the homing endonuclease encoded by VMA1. We show also that GSH11 is not expressed in a VDE-deleted strain and that inability to express the GSH11 of this strain is overcome by introduction of the coding region of VDE or the entire VMA1 gene. It is also found that VDE does not cut DNA in the vicinity of the GSH11 cis-element. Rapamycin, an inhibitor of the target of rapamycin (TOR) signal-transduction system, is found to enhance expression of GSH11 in a VDE-dependent manner under conditions of sulfur starvation. These results indicate that GSH11 is regulated by a system sensitive to sulfur starvation (presumably via cysteine depletion) and a more general system involving the nutritional starvation signal mediated by the TOR system. Both systems need to be operational (inhibition of TOR and sulfur starvation) for full expression of GSH11.

  2. Group I introns and associated homing endonuclease genes reveals a clinal structure for Porphyra spiralis var. amplifolia (Bangiales, Rhodophyta along the Eastern coast of South America

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    Matioli Sergio R

    2008-11-01

    Full Text Available Abstract Background Group I introns are found in the nuclear small subunit ribosomal RNA gene (SSU rDNA of some species of the genus Porphyra (Bangiales, Rhodophyta. Size polymorphisms in group I introns has been interpreted as the result of the degeneration of homing endonuclease genes (HEG inserted in peripheral loops of intron paired elements. In this study, intron size polymorphisms were characterized for different Porphyra spiralis var. amplifolia (PSA populations on the Southern Brazilian coast, and were used to infer genetic relationships and genetic structure of these PSA populations, in addition to cox2-3 and rbcL-S regions. Introns of different sizes were tested qualitatively for in vitro self-splicing. Results Five intron size polymorphisms within 17 haplotypes were obtained from 80 individuals representing eight localities along the distribution of PSA in the Eastern coast of South America. In order to infer genetic structure and genetic relationships of PSA, these polymorphisms and haplotypes were used as markers for pairwise Fst analyses, Mantel's test and median joining network. The five cox2-3 haplotypes and the unique rbcL-S haplotype were used as markers for summary statistics, neutrality tests Tajima's D and Fu's Fs and for median joining network analyses. An event of demographic expansion from a population with low effective number, followed by a pattern of isolation by distance was obtained for PSA populations with the three analyses. In vitro experiments have shown that introns of different lengths were able to self-splice from pre-RNA transcripts. Conclusion The findings indicated that degenerated HEGs are reminiscent of the presence of a full-length and functional HEG, once fixed for PSA populations. The cline of HEG degeneration determined the pattern of isolation by distance. Analyses with the other markers indicated an event of demographic expansion from a population with low effective number. The different degrees of

  3. Group I introns and associated homing endonuclease genes reveals a clinal structure for Porphyra spiralis var. amplifolia (Bangiales, Rhodophyta) along the Eastern coast of South America

    Science.gov (United States)

    2008-01-01

    Background Group I introns are found in the nuclear small subunit ribosomal RNA gene (SSU rDNA) of some species of the genus Porphyra (Bangiales, Rhodophyta). Size polymorphisms in group I introns has been interpreted as the result of the degeneration of homing endonuclease genes (HEG) inserted in peripheral loops of intron paired elements. In this study, intron size polymorphisms were characterized for different Porphyra spiralis var. amplifolia (PSA) populations on the Southern Brazilian coast, and were used to infer genetic relationships and genetic structure of these PSA populations, in addition to cox2-3 and rbcL-S regions. Introns of different sizes were tested qualitatively for in vitro self-splicing. Results Five intron size polymorphisms within 17 haplotypes were obtained from 80 individuals representing eight localities along the distribution of PSA in the Eastern coast of South America. In order to infer genetic structure and genetic relationships of PSA, these polymorphisms and haplotypes were used as markers for pairwise Fst analyses, Mantel's test and median joining network. The five cox2-3 haplotypes and the unique rbcL-S haplotype were used as markers for summary statistics, neutrality tests Tajima's D and Fu's Fs and for median joining network analyses. An event of demographic expansion from a population with low effective number, followed by a pattern of isolation by distance was obtained for PSA populations with the three analyses. In vitro experiments have shown that introns of different lengths were able to self-splice from pre-RNA transcripts. Conclusion The findings indicated that degenerated HEGs are reminiscent of the presence of a full-length and functional HEG, once fixed for PSA populations. The cline of HEG degeneration determined the pattern of isolation by distance. Analyses with the other markers indicated an event of demographic expansion from a population with low effective number. The different degrees of degeneration of the HEG

  4. 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...

  5. 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-...

  6. Whole genome sequencing of Rhodotorula mucilaginosa isolated from the chewing stick (Distemonanthus benthamianus): insights into Rhodotorula phylogeny, mitogenome dynamics and carotenoid biosynthesis.

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    Gan, Han Ming; Thomas, Bolaji N; Cavanaugh, Nicole T; Morales, Grace H; Mayers, Ashley N; Savka, Michael A; Hudson, André O

    2017-01-01

    In industry, the yeast Rhodotorula mucilaginosa is commonly used for the production of carotenoids. The production of carotenoids is important because they are used as natural colorants in food and some carotenoids are precursors of retinol (vitamin A). However, the identification and molecular characterization of the carotenoid pathway/s in species belonging to the genus Rhodotorula is scarce due to the lack of genomic information thus potentially impeding effective metabolic engineering of these yeast strains for improved carotenoid production. In this study, we report the isolation, identification, characterization and the whole nuclear genome and mitogenome sequence of the endophyte R. mucilaginosa RIT389 isolated from Distemonanthus benthamianus, a plant known for its anti-fungal and antibacterial properties and commonly used as chewing sticks. The assembled genome of R. mucilaginosa RIT389 is 19 Mbp in length with an estimated genomic heterozygosity of 9.29%. Whole genome phylogeny supports the species designation of strain RIT389 within the genus in addition to supporting the monophyly of the currently sequenced Rhodotorula species. Further, we report for the first time, the recovery of the complete mitochondrial genome of R. mucilaginosa using the genome skimming approach. The assembled mitogenome is at least 7,000 bases larger than that of Rhodotorula taiwanensis which is largely attributed to the presence of large intronic regions containing open reading frames coding for homing endonuclease from the LAGLIDADG and GIY-YIG families. Furthermore, genomic regions containing the key genes for carotenoid production were identified in R. mucilaginosa RIT389, revealing differences in gene synteny that may play a role in the regulation of the biotechnologically important carotenoid synthesis pathways in yeasts.

  7. Whole genome sequencing of Rhodotorula mucilaginosa isolated from the chewing stick (Distemonanthus benthamianus): insights into Rhodotorula phylogeny, mitogenome dynamics and carotenoid biosynthesis

    Science.gov (United States)

    Thomas, Bolaji N.; Cavanaugh, Nicole T.; Morales, Grace H.; Mayers, Ashley N.; Savka, Michael A.

    2017-01-01

    In industry, the yeast Rhodotorula mucilaginosa is commonly used for the production of carotenoids. The production of carotenoids is important because they are used as natural colorants in food and some carotenoids are precursors of retinol (vitamin A). However, the identification and molecular characterization of the carotenoid pathway/s in species belonging to the genus Rhodotorula is scarce due to the lack of genomic information thus potentially impeding effective metabolic engineering of these yeast strains for improved carotenoid production. In this study, we report the isolation, identification, characterization and the whole nuclear genome and mitogenome sequence of the endophyte R. mucilaginosa RIT389 isolated from Distemonanthus benthamianus, a plant known for its anti-fungal and antibacterial properties and commonly used as chewing sticks. The assembled genome of R. mucilaginosa RIT389 is 19 Mbp in length with an estimated genomic heterozygosity of 9.29%. Whole genome phylogeny supports the species designation of strain RIT389 within the genus in addition to supporting the monophyly of the currently sequenced Rhodotorula species. Further, we report for the first time, the recovery of the complete mitochondrial genome of R. mucilaginosa using the genome skimming approach. The assembled mitogenome is at least 7,000 bases larger than that of Rhodotorula taiwanensis which is largely attributed to the presence of large intronic regions containing open reading frames coding for homing endonuclease from the LAGLIDADG and GIY-YIG families. Furthermore, genomic regions containing the key genes for carotenoid production were identified in R. mucilaginosa RIT389, revealing differences in gene synteny that may play a role in the regulation of the biotechnologically important carotenoid synthesis pathways in yeasts. PMID:29158974

  8. 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...

  9. 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.

  10. 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

  11. 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.

  12. Home

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    Military Commission Seal VWAP Login Home Go ABOUT US Organization Overview Organizational Chart Families VWAP Login CCTV Sites Travel Media MC News CCTV Sites Travel Today at OMC Home Today at OMC Daily

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    AF Branding & Trademark Licensing Join the Air Force Home About Us The Air Force Symbol Display Resources Document Library TM Connect Search AF Branding and Trademark Licensing Program: important links Legal Documents 10 U.S.C. § 2260 15 U.S.C. § 167;167; 1114-1125 DODI 5535.12, DoD Branding and

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    intersect as Attack Wing leaders change roles The 112th COS postured as cyber shield for Pa. infrastructure 111th Attack Wing 111th Attack Wing 21st Century Guard Airmen Home News Photos Art Video Resources - The Balance Search 111th Attack Wing: COMMUNITY/ENVIRO May 16, 2018; Pa. Department of Health update

  15. 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...

  16. 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.

  17. 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.

  18. 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

  19. 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.

  20. 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.

  1. 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.

  2. 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

  3. 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

  4. 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

  5. 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.

  6. 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...

  7. 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.

  8. 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...

  9. 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...

  10. 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

  11. 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.

  12. Cofactor requirement of HpyAV restriction endonuclease.

    Directory of Open Access Journals (Sweden)

    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.

  13. 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

  14. 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

  15. 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...

  16. 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.

  17. 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.

  18. 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.

  19. 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

  20. 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.

  1. 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.

  2. 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.

  3. 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)

  4. 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.

  5. 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.

  6. Archaeal rRNA operons, intron splicing and homing endonucleases, RNA polymerase operons and phylogeny

    DEFF Research Database (Denmark)

    Garrett, Roger Antony; Aagaard, Claus Sindbjerg; Andersen, Morten

    1994-01-01

    Over the past decade our laboratory has had a strong interest in defining the phylogenetic status of the archaea. This has involved determining and analysing the sequences of operons of both rRNAs and RNA polymerases and it led to the discovery of the first archaeal rRNA intron. What follows...

  7. 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

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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

  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 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.

  16. 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...

  17. 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)

  18. 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.

  19. 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

  20. 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

  1. 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

  2. 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.

  3. 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

  4. 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

  5. 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.

  6. 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

  7. [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.

  8. 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.

  9. 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

  10. 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

  11. 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

  12. 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.

  13. 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...

  14. 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

  15. 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

  16. Nursing Homes

    Science.gov (United States)

    ... Home › Aging & Health A to Z › Nursing Homes Font size A A A Print Share Glossary Basic ... Reason For Living in A Nursing Home Some type of disability with activities of daily living (ADLs) ...

  17. Home Care

    Science.gov (United States)

    ... are part of home healthcare agencies. You may benefit from home care if you are dealing with ... it will trigger an emergency response or checkup phone call. Newer technologies ... or mobile testing technology (home diagnostics), including x-rays and ...

  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. 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. 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. 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.

  3. 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 ...

  4. 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

  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. Recruitment of RecA homologs Dmc1p and Rad51p to the double-strand break repair site initiated by meiosis-specific endonuclease VDE (PI-SceI).

    Science.gov (United States)

    Fukuda, Tomoyuki; Ohya, Yoshikazu

    2006-02-01

    During meiosis, VDE (PI-SceI), a homing endonuclease in Saccharomyces cerevisiae, introduces a double-strand break (DSB) at its recognition sequence and induces homologous recombinational repair, called homing. Meiosis-specific RecA homolog Dmc1p, as well as mitotic RecA homolog Rad51p, acts in the process of meiotic recombination, being required for strand invasion and exchange. In this study, recruitment of Dmc1p and Rad51p to the VDE-induced DSB repair site is investigated by chromatin immunoprecipitation assay. It is revealed that Dmc1p and Rad51p are loaded to the repair site in an independent manner. Association of Rad51p requires other DSB repair proteins of Rad52p, Rad55p, and Rad57p, while loading of Dmc1p is facilitated by the different protein, Sae3p. Absence of Tid1p, which can bind both RecA homologs, appears specifically to cause an abnormal distribution of Dmc1p. Lack of Hop2, Mnd1p, and Sae1p does not impair recruitment of both RecA homologs. These findings reveal the discrete functions of each strand invasion protein in VDE-initiated homing, confirm the similarity between VDE-initiated homing and Spo11p-initiated meiotic recombination, and demonstrate the availability of VDE-initiated homing for the study of meiotic recombination.

  7. 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

  8. 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

  9. 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

  10. 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...

  11. 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.

  12. 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.

  13. 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)

  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. 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.

  16. 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.

  17. 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).

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. Homing oneself

    DEFF Research Database (Denmark)

    Winther, Ida Wentzel

    2009-01-01

    What is home? A building, a physical and mental phenomenon, or a concept?  There are many homes and ways `to home oneself´. Many of us quite often dwell in other places than at home (as professional commuters between two places, as travellers staying in hotels, as children of divorced parents...

  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. Home, Smart Home

    DEFF Research Database (Denmark)

    Hansen, Ellen Kathrine; Olesen, Gitte Gylling Hammershøj; Mullins, Michael

    2013-01-01

    The article places focus on how smart technologies integrated in a one family- home and particular the window offer unique challenges and opportunities for designing buildings with the best possible environments for people and nature. Toward an interdisciplinary approach, we address the interaction...... between daylight defined in technical terms and daylight defined in aesthetic, architectural terms. Through field-tests of a Danish carbon-neutral home and an analysis of five key design parameters, we explore the contradictions and potentials in smart buildings, using the smart window as example of how...... to the energy design is central. The study illuminates an approach of the design of smart houses as living organisms by connecting technology with the needs of the occupants with the power and beauty of daylight....

  6. 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...

  7. 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

  8. 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

  9. 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

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. Home hemodialysis

    DEFF Research Database (Denmark)

    Agar, John W; Perkins, Anthony; Heaf, James G

    2015-01-01

    We describe the infrastructure that is necessary for hemodialysis in the home focusing on physical requirements, the organization of plumbing and water, and the key features that should guide the selection of machines that are suitable for home use.......We describe the infrastructure that is necessary for hemodialysis in the home focusing on physical requirements, the organization of plumbing and water, and the key features that should guide the selection of machines that are suitable for home use....

  15. Homing at an extragenic locus mediated by VDE (PI-SceI) in Saccharomyces cerevisiae.

    Science.gov (United States)

    Nogami, Satoru; Fukuda, Tomoyuki; Nagai, Yuri; Yabe, Shizu; Sugiura, Masako; Mizutani, Ryuta; Satow, Yoshinori; Anraku, Yasuhiro; Ohya, Yoshikazu

    2002-06-30

    PI-SceI (VDE), a homing endonuclease with protein splicing activity, is a genomic parasite in the VMA1 gene of Saccharomyces cerevisiae. In a heterozygous diploid of the VDE-less VMA1 allele and a VDE-containing VMA1 allele, VDE specifically cleaves its recognition sequence (VRS) in the VDE-less VMA1 allele at meiosis, followed by 'homing', i.e. a conversion to a VDE-containing allele. We found that upon VDE expression, homing of a marker gene at an extragenic locus occurs only when a 45 bp element containing the VRS is inserted at its allelic site, while mutants of VDE with no endonuclease activity lack authentic extragenic homing activity. Thus, both the VRS and VDE are required for homing. Insertion of the VRS in a homozygous diploid significantly lowered the spore germination ability, indicating that a template for gene repair at its allelic locus is essential for efficient homing and survival of yeast cells. Copyright 2002 John Wiley & Sons, Ltd.

  16. 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.)

  17. Unimaginable homes

    DEFF Research Database (Denmark)

    Møller, Kristian; Klausen, Maja

    2018-01-01

    The chapter draw from critical mediatization theory, critical intimacy theory, and cultural gerontology and asks: How do elderly people practice their mediatized homes? Which roles do media play in constituting and disturbing the flows of bodies into the home? Moreover: how do dominant...... in the making of the mediatized home space. We conclude by returning to the research questions and making explicit how researching flows of bodies that in many ways inhabit homes of the in-between contributes to both gerontological and geomediatization research agendas....

  18. [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

  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. 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)

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. 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....

  6. 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...

  7. [Home births].

    Science.gov (United States)

    Welffens, K; Kirkpatrick, C; Daelemans, C; Derisbourg, S

    In Belgium, very few women give birth outside the delivery room. In the United Kingdom and in the Netherlands, they are more numerous. Several studies evaluated obstetric and neonatal outcomes of home births compared with hospital births. We selected seven recent and large studies (with cohorts of more than 5.000 women) using PubMed, Science Direct and Cochrane Database of Systematic Reviews. Several questions were examined. Is there any difference in maternal and neonatal outcomes depending on the intended place of birth? Does parity affect outcomes ? What are the characteristics of women who choose to deliver at home ? We conclude that giving birth at home improves obstetric outcomes but is riskier for the baby, especially for the first one. The women delivering at home are mainly white Europeans, between 25 and 35 years old, in a relationship, multiparous and wealthier. In order to avoid this increased risk for the baby while preserving the obstetric advantages, alongside birth centers offer an intermediate solution. They combine the reassuring home-like atmosphere with the safety of the hospital. In Belgium, the first alongside birth center " Le Cocon " (a low technicity unit distinct from the delivery room) offers now this type of alternative place of birth for women in Hôpital Erasme in Brussels.

  8. Returning home

    DEFF Research Database (Denmark)

    Agergaard, Jytte; Brøgger, Ditte

    2016-01-01

    flows. By focusing on these educational migrants, this paper explores how they connect to their rural homes. Guided by a critical reading of the migration-development scholarship, the paper examines how migrants and their relatives make sense of educational migrants’ remitting and returning practices......, and by comparing three groups of educational migrants, the migrants’ reasons for staying connected and sending remittances are scrutinized. The paper finds that although educational migrants do not generate extensive economic remittances for local development in Nepal, they stay connected to their rural homes...

  9. 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

  10. Expression of a LINE-1 endonuclease variant in gastric cancer: its association with clinicopathological parameters

    International Nuclear Information System (INIS)

    Wang, Gangshi; Wu, Benyan; Wang, Mengwei; Gao, Jie; Huang, Haili; Tian, Yu; Xue, Liyan; Wang, Weihua; You, Weidi; Lian, Hongwei; Duan, Xiaojian

    2013-01-01

    -specific PCR. BLASTP program analysis revealed that GCRG213p peptide shared 83.0% alignment with the C-terminal region of L1 endonuclease (L1-EN). GCRG213p sequence possesses the important residues that compose the conserved features of L1-EN. GCRG213p could be a variant of L1-EN, a functional member of L1-EN family. Overexpression of GCRG213p is common in both primary gastric cancer and lymph node metastasis. These findings provide evidence of somatic L1 expression in gastric cancer, and its potential consequences in the form of tumor

  11. 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

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. 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)

  17. 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)

  18. 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.

  19. 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.

  20. 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.

  1. Fermilab | Home

    Science.gov (United States)

    Industry Students and teachers Media ... Five (more) fascinating facts about DUNE Engineering the Mathematics in Music June 2 10 a.m. Get to Know the Lederman Science Center June 3 1 p.m. Ask a Scientist Security, Privacy, Legal Use of Cookies Quick Links Home Contact Phone Book Fermilab at Work For Industry

  2. Home Automation

    OpenAIRE

    Ahmed, Zeeshan

    2010-01-01

    In this paper I briefly discuss the importance of home automation system. Going in to the details I briefly present a real time designed and implemented software and hardware oriented house automation research project, capable of automating house's electricity and providing a security system to detect the presence of unexpected behavior.

  3. Molecular mechanism of VDE-initiated intein homing in yeast nuclear genome.

    Science.gov (United States)

    Fukuda, Tomoyuki; Nagai, Yuri; Ohya, Yoshikazu

    2004-01-01

    In Saccharomyces cerevisiae, VMA1 intein encodes a homing endonuclease termed VDE which is produced by an autocatalytic protein splicing reaction. VDE introduces a DSB at its recognition sequence on intein-minus allele, resulting in the lateral transfer of VMA1 intein. In this review, we summarize a decade of in vitro study on VDE and describe our recent study on the in vivo behavior of both VDE and host proteins involved in intein mobility. Meiotic DSBs caused by VDE are repaired in the similar pathway to that working in meiotic recombination induced by Spo11p-mediated DSBs. Meiosis-specific DNA cleavage and homing is shown to be guaranteed by the two distinct mechanisms, the subcellular localization of VDE and a requirement of premeiotic DNA replication. Based on these lines of evidence, we present the whole picture of molecular mechanism of VDE-initiated homing in yeast cells.

  4. Bringing Your Baby Home

    Science.gov (United States)

    ... for Educators Search English Español Bringing Your Baby Home KidsHealth / For Parents / Bringing Your Baby Home What's ... recall your baby's seemingly endless crying episodes. The Home Front Introducing your baby to others at home ...

  5. The Medical Home

    Science.gov (United States)

    ... Videos for Educators Search English Español The Medical Home KidsHealth / For Parents / The Medical Home What's in ... for your child. What Does the Term "Medical Home" Mean? A medical home isn't a place ...

  6. 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

  7. 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

  8. A Home Away from Home

    DEFF Research Database (Denmark)

    McIlvenny, Paul

    2008-01-01

    The House of Tiny Tearaways (HTT) first appeared on British television in May 2005. Over a six-day period, three families are invited to reside in a specially designed house together with a resident clinical psychologist. The house is to be “a home away from home” for the resident families...... in order to analyze excerpts from the program and to explore how the affordances and constraints of the specially designed house—its architecture and spatial configuration, as well as the surveillance technology embedded within its walls—are assembled within particular familial activities, and how...... the relationships between family members are reshaped as a result. The analysis focuses on several key phenomena: 1) practices of video observation in relation to the domestic sphere; 2) use of inscription devices, such as video displays, to capture and visualize behavior and action in the “home;” 3) practicing...

  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. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. Exercise at Home

    Science.gov (United States)

    ... Home Health Insights Exercise & Weight Exercise at Home Exercise at Home Make an Appointment Ask a Question ... with the movement and contact your provider. Posture Exercises Better posture means better breathing and movement. Axial ...

  18. Respiratory Home Health Care

    Science.gov (United States)

    ... Us Home > Healthy Living > Living With Lung Disease > Respiratory Home Health Care Font: Aerosol Delivery Oxygen Resources ... Teenagers Living With Lung Disease Articles written by Respiratory Experts Respiratory Home Health Care Respiratory care at ...

  19. Asthma Home Environment Checklist

    Science.gov (United States)

    This checklist guides home care visitors in identifying environmental asthma triggers most commonly found in homes. It includes sections on the building, home interior and room interior and provides low-cost action steps for remediation.

  20. Home Care Services

    Science.gov (United States)

    Home care is care that allows a person with special needs stay in their home. It might be for people who are getting ... are chronically ill, recovering from surgery, or disabled. Home care services include Personal care, such as help ...

  1. HOME Grantee Areas

    Data.gov (United States)

    Department of Housing and Urban Development — The HOME Investment Partnership Program (HOME) is authorized under Title II of the Cranston-Gonzalez National Affordable Housing Act. HOME provides formula grants to...

  2. Eldercare at Home: Choosing a Nursing Home

    Science.gov (United States)

    ... required, these services can be provided by a separate home health agency as directed by a doctor or ... complaints made by or on behalf of nursing home residents and work to resolve the problems. If they are unable ...

  3. Home safe home: Evaluation of a childhood home safety program.

    Science.gov (United States)

    Stewart, Tanya Charyk; Clark, Andrew; Gilliland, Jason; Miller, Michael R; Edwards, Jane; Haidar, Tania; Batey, Brandon; Vogt, Kelly N; Parry, Neil G; Fraser, Douglas D; Merritt, Neil

    2016-09-01

    The London Health Sciences Centre Home Safety Program (HSP) provides safety devices, education, a safety video, and home safety checklist to all first-time parents for the reduction of childhood home injuries. The objective of this study was to evaluate the HSP for the prevention of home injuries in children up to 2 years of age. A program evaluation was performed with follow-up survey, along with an interrupted time series analysis of emergency department (ED) visits for home injuries 5 years before (2007-2013) and 2 years after (2013-2015) implementation. Spatial analysis of ED visits was undertaken to assess differences in home injury rates by dissemination areas controlling differences in socioeconomic status (i.e., income, education, and lone-parent status) at the neighborhood level. A total of 3,458 first-time parents participated in the HSP (a 74% compliance rate). Of these, 20% (n = 696) of parents responded to our questionnaire, with 94% reporting the program to be useful (median, 6; interquartile range, 2 on a 7-point Likert scale) and 81% learning new strategies for preventing home injuries. The median age of the respondent's babies were 12 months (interquartile range, 1). The home safety check list was used by 87% of respondents to identify hazards in their home, with 95% taking action to minimize the risk. The time series analysis demonstrated a significant decline in ED visits for home injuries in toddlers younger than2 years of age after HSP implementation. The declines in ED visits for home injuries remained significant over and above each socioeconomic status covariate. Removing hazards, supervision, and installing safety devices are key facilitators in the reduction of home injuries. Parents found the HSP useful to identify hazards, learn new strategies, build confidence, and provide safety products. Initial finding suggests that the program is effective in reducing home injuries in children up to 2 years of age. Therapeutic/care management study

  4. 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.

  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. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. 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.

  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

    2012-01-01

    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

  13. 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.

  14. 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.

  15. 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

  16. 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.

  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. 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

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. [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.

  5. 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.

  6. 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.

  7. HIGLE is a bifunctional homing endonuclease that directly interacts with HYL1 and SERRATE in Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Cho, Seok Keun; Ryu, Moon Young; Poulsen, Christian

    2017-01-01

    A highly coordinated complex known as the microprocessor precisely processes primary transcripts of MIRNA genes into mature miRNAs. In plants, the microprocessor minimally consists of three components: Dicer-like protein 1 (DCL1), HYPONASTIC LEAF 1 (HYL1), and SERRATE (SE). To precisely modulate ...

  8. Smart Home Hacking

    OpenAIRE

    Kodra, Suela

    2016-01-01

    Smart Home is an intelligent home equipped with devices and communications systems that enables the residents to connect and control their home appliances and systems. This technology has changed the way a consumer interacts with his home, enabling more control and convenience. Another advantage of this technology is the positive impact it has on savings on energy and other resources. However, despite the consumer's excitement about smart home, security and privacy have shown to be the strong...

  9. Meals in nursing homes

    DEFF Research Database (Denmark)

    Kofod, Jens Erik; Birkemose, A.

    2004-01-01

    Undernutrition is present among 33% of nursing home residents in Denmark. Hence, it is relevant to examine the meal situation at nursing homes to single out factors that may increase or reduce the residents' food intake. in the ongoing Danish nursing home debate it is claimed that a new type...... of nursing home improves the residents' meal situation with a positive effect on nutrition. The aim of this work is to test the general hypothesis that (i) residents appreciate the meal situation in these nursing homes and (ii) nutritional status of the residents is improved in this type of nursing home....... This study was carried out in four Danish nursing homes at various locations in Denmark. The methods used are qualitative interviews and observations at four nursing homes in combination with measurement of body mass index (BMI) at two of the four nursing homes. Undernutrition is defined as a BMI below 20...

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. TRAVEL AND HOME LEAVE

    CERN Multimedia

    Human Resources Division

    2002-01-01

    Administrative procedures for : Travel to the home station and home leave (hl) Additional travel to the home station (at) Travel to the home station and home leave for family reasons (hlf) As part of the process of simplifying administrative procedures, HR and AS Divisions have devised a new, virtually automatic procedure for payment of travel expenses to the home station. The changes are aimed at rationalising administrative procedures and not at reducing benefits. The conditions of eligibility are unchanged. The new procedure, which will be operational with effect from 1st June 2002, will greatly simplify the administrative processing of claims for travel expenses and the recording of home leaves. Currently, requests for payment are introduced manually into the Advances and Claims system (AVCL) by divisional secretariats. All travel to the home station starting prior to 1st June 2002 will be processed according to the existing system whereas that starting on 1st June and after will be processed accordi...

  15. Home blood sugar testing

    Science.gov (United States)

    Diabetes - home glucose testing; Diabetes - home blood sugar testing ... Usual times to test your blood sugar are before meals and at bedtime. Your provider may ask you to check your blood sugar 2 hours after a meal or ...

  16. Nursing Home Quality Initiative

    Data.gov (United States)

    U.S. Department of Health & Human Services — This Nursing Home Quality Initiative (NHQI) website provides consumer and provider information regarding the quality of care in nursing homes. NHQI discusses quality...

  17. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... Overview The skills kit contains: A booklet with information on the operation, home skills such as emptying and changing a pouch, problem solving, and home management. A DVD with demonstration of each skill Stoma ...

  18. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... JACS Jobs Events Find a Surgeon Patients and Family Contact My Profile Shop ( 0 ) Cart Donate American College of Surgeons Education Patients and Family Skills Programs Ostomy Home Skills Program Ostomy Home ...

  19. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... kit contains: A booklet with information on the operation, home skills such as emptying and changing a pouch, problem solving, and home management. A DVD with demonstration of each skill Stoma ...

  20. Home Health Compare

    Data.gov (United States)

    U.S. Department of Health & Human Services — Home Health Compare has information about the quality of care provided by Medicare-certified home health agencies throughout the nation. Medicare-certified means the...

  1. Using oxygen at home

    Science.gov (United States)

    ... at Home Tell your local fire department, electric company, and telephone company that you use oxygen in your home. They ... M. is also a founding member of Hi-Ethics and subscribes to the principles of the Health ...

  2. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... Careers at ACS Careers at ACS About ACS Career Types Working at ACS ... American College of Surgeons Education Patients and Family Skills Programs Ostomy Home Skills Program Ostomy Home Skills ...

  3. HOME Rent Limits

    Data.gov (United States)

    Department of Housing and Urban Development — In accordance with 24 CFR Part 92.252, HUD provides maximum HOME rent limits. The maximum HOME rents are the lesser of: The fair market rent for existing housing for...

  4. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... ACS Careers at ACS About ACS Career Types Working at ACS ... Education Patients and Family Skills Programs Ostomy Home Skills Program Ostomy Home Skills Program Adult Ostomy ...

  5. Community Nursing Home (CNH)

    Data.gov (United States)

    Department of Veterans Affairs — The Community Nursing Home (CNH) database contains a list of all Community Nursing Home facilities under local contract to Veterans Health Administration (VHA). CNH...

  6. Home Improvements Prevent Falls

    Science.gov (United States)

    ... turn JavaScript on. Feature: Falls and Older Adults Home Improvements Prevent Falls Past Issues / Winter 2014 Table ... and ensure your safety. "Safe-ty-fy" Your Home Some Questions for Your Provider Will my medicines ...

  7. Home Canning and Botulism

    Science.gov (United States)

    ... this? Submit What's this? Submit Button Past Emails Home Canning and Botulism Language: English (US) Español (Spanish) ... myself and others safe when it comes to home-canned foods? Many cases of foodborne botulism have ...

  8. 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

  9. 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.

  10. 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.

  11. 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.

  12. 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

  13. 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 ...

  14. 6. Home deliveries

    African Journals Online (AJOL)

    Sitwala

    determine factors associated with home deliveries. Main outcome ... deliver at home than a health facility compared to those who .... regression analysis, women who had four years of schooling or .... by report bias, the burden of home deliveries is a real challenge .... Journal of Econometrics 1987; 36: 185-204. 14. Michelo ...

  15. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... Ostomy Home Skills Program Ostomy Home Skills Program Adult Ostomy Pediatric Ostomy Programa de Destrezas para manejo Doméstico de Ostomía Ostomy Home Skills Program Adult Ostomy Pediatric Ostomy Programa de Destrezas para manejo ...

  16. Home area networks

    NARCIS (Netherlands)

    Koonen, A.M.J.

    2013-01-01

    This article consists of a collection of slides from the author's conference presentation. Some of the specific areas/topics discussed include: Convergence in home networks, home service scenarios; Home wired network architectures, CapEx and OpEx; Residential Gateway; Optical fiber types;

  17. Home in the Making

    DEFF Research Database (Denmark)

    Kreuzer, Maria; von Wallpach, Sylvia; Muehlbacher, Hans

    2016-01-01

    In a context of unprecedented migration home reaches high relevance. This study aims at understanding the (re-)construction of home by first generation consumer migrants. The findings provide insights into consumers’ (re-)construction of various dimensions of home and identify “inner home” as a n...

  18. Home Energy Saver

    Science.gov (United States)

    release announcing Home Energy Saver and a Q-and-A. The "About" page should tell you everything you need to know about using Home Energy Saver. If you have any questions, please email the project leader, Dr. Evan Mills. News Releases Microsoft Licenses Berkeley Lab's Home Energy Saver Code for Its

  19. Strategy Guideline: Demonstration Home

    Energy Technology Data Exchange (ETDEWEB)

    Savage, C.; Hunt, A.

    2012-12-01

    This guideline will provide a general overview of the different kinds of demonstration home projects, a basic understanding of the different roles and responsibilities involved in the successful completion of a demonstration home, and an introduction into some of the lessons learned from actual demonstration home projects. Also, this guideline will specifically look at the communication methods employed during demonstration home projects. And lastly, we will focus on how to best create a communication plan for including an energy efficient message in a demonstration home project and carry that message to successful completion.

  20. Strategy Guideline. Demonstration Home

    Energy Technology Data Exchange (ETDEWEB)

    Hunt, A.; Savage, C.

    2012-12-01

    This guideline will provide a general overview of the different kinds of demonstration home projects, a basic understanding of the different roles and responsibilities involved in the successful completion of a demonstration home, and an introduction into some of the lessons learned from actual demonstration home projects. Also, this guideline will specifically look at the communication methods employed during demonstration home projects. And lastly, we will focus on how to best create a communication plan for including an energy efficient message in a demonstration home project and carry that message to successful completion.

  1. Digital Living at Home

    DEFF Research Database (Denmark)

    Andersen, Pernille Viktoria Kathja; Christiansen, Ellen Tove

    2013-01-01

    of these user voices has directed us towards a ‘home-keeping’ design discourse, which opens new horizons for design of digital home control systems by allowing users to perform as self-determined controllers and groomers of their habitat. The paper concludes by outlining the implications of a ‘home......Does living with digital technology inevitably lead to digital living? Users talking about a digital home control system, they have had in their homes for eight years, indicate that there is more to living with digital technology than a functional-operational grip on regulation. Our analysis......-keeping’ design discourse....

  2. 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

  3. 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

  4. Leaving home in Denmark

    DEFF Research Database (Denmark)

    Nielsen, Rikke Skovgaard

    2015-01-01

    The paper focuses on ethnic differences in the timing and patterns of leaving the parental home. Leaving home is a key transition in the life course of the individual, and extensive research has been conducted on the timing and patterns of leaving it. However, ethnic differences in these patterns...... of leaving home. Results showed that while some differences disappeared when controlling for covariates, others persisted, thus indicating ethnic differences in home-leaving patterns. A strong link between leaving home and marriage was substantiated for Turks, but not for Somalis. The home-leaving patterns...... of Somalis were much more similar to those of Danes. Overall, Turkish descendants were similar to Turkish immigrants but with some differentiation. The analyses identified the existence of ethnic differences in home-leaving patterns but also found evidence of a shift towards less traditional patterns, i...

  5. LHC@home gets new home

    CERN Multimedia

    Oates, John

    2007-01-01

    "The distributed computing project LHC@home is moving to London from Cern in Switzerland. Researchers at Qeen Mary University have been trialling the system since June, but are now ready for the offical launch" (1 page)

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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

  11. Home range and travels

    Science.gov (United States)

    Stickel, L.F.; King, John A.

    1968-01-01

    The concept of home range was expressed by Seton (1909) in the term 'home region,' which Burr (1940, 1943) clarified with a definition of home range and exemplified in a definitive study of Peromyscus in the field. Burt pointed out the ever-changing characteristics of home-range area and the consequent absence of boundaries in the usual sense--a finding verified by investigators thereafter. In the studies summarized in this paper, sizes of home ranges of Peromyscus varied within two magnitudes, approximately from 0.1 acre to ten acres, in 34 studies conducted in a variety of habitats from the seaside dunes of Florida to the Alaskan forests. Variation in sizes of home ranges was correlated with both environmental and physiological factors; with habitat it was conspicuous, both in the same and different regions. Food supply also was related to size of home range, both seasonally and in relation to habitat. Home ranges generally were smallest in winter and largest in spring, at the onset of the breeding season. Activity and size also were affected by changes in weather. Activity was least when temperatures were low and nights were bright. Effects of rainfall were variable. Sizes varied according to sex and age; young mice remained in the parents' range until they approached maturity, when they began to travel more widely. Adult males commonly had larger home ranges than females, although there were a number of exceptions. An inverse relationship between population density and size of home range was shown in several studies and probably is the usual relationship. A basic need for activity and exploration also appeared to influence size of home range. Behavior within the home range was discussed in terms of travel patterns, travels in relation to home sites and refuges, territory, and stability of size of home range. Travels within the home range consisted of repeated use of well-worn trails to sites of food, shelter, and refuge, plus more random exploratory travels

  12. Structure-specific endonucleases

    DEFF Research Database (Denmark)

    Minocherhomji, Sheroy; Hickson, Ian D

    2014-01-01

    Fragile sites are conserved loci predisposed to form breaks in metaphase chromosomes. The inherent instability of these loci is associated with chromosomal rearrangements in cancers and is a feature of cells from patients with chromosomal instability syndromes. One class of fragile sites, the com...

  13. HomePort

    DEFF Research Database (Denmark)

    Madsen, Per Printz

    2009-01-01

    In the last couple of year's computer based home control systems are getting more and more common in modern homes. For instance these systems take care of light control, heat control and security systems.  The latest trend is to use wireless communication like Z-Wave and ZigBee to interconnect...... different components in these systems. One of the characteristics is that each system, like for instance heat and light, has their own specific way of using the communication system.   This paper describes a way to connect different home control systems through an intelligent gateway, called a Home......Port. The HomePort consists of a number of Subsystem communication drivers, a virtual communication layer, an interpreter and a PC- based compiler for a high level control language, called GIL (Gateway intelligence language). The focus in this paper will be on the upper two layers in the Home...

  14. Facilitating home birth.

    Science.gov (United States)

    Finigan, Valerie; Chadderton, Diane

    2015-06-01

    The birth of a baby is a family experience. However, in the United Kingdom birth often occurs outside the family environment, in hospital. Both home and hospital births have risks and benefits, but research shows that, for most women, it is as safe to give birth at home as it is in hospital. Women report home-birth to be satisfying with lowered risks of intervention and less likelihood of being separated from their family. It is also more cost effective for the National Health Service. Yet, whilst midwives are working hard to promote home birth as an option, it remains controversial. The aim of this paper is to raise awareness of the safety of home birth and the needs of women and midwives when a home birth is chosen. It provides an overview of care required and the role of the midwife in the ensuring care is woman-centred and personalised.

  15. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... Advocate at Home Program State Legislative Action Center Leadership & Advocacy Summit Webinars Practice Management Practice Management Practice Management CPT Coding Bulletin Articles ...

  16. 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

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

    Directory of Open Access Journals (Sweden)

    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

  18. VDE-initiated intein homing in Saccharomyces cerevisiae proceeds in a meiotic recombination-like manner.

    Science.gov (United States)

    Fukuda, Tomoyuki; Nogami, Satoru; Ohya, Yoshikazu

    2003-07-01

    Inteins and group I introns found in prokaryotic and eukaryotic organisms occasionally behave as mobile genetic elements. During meiosis of the yeast Saccharomyces cerevisiae, the site-specific endonuclease encoded by VMA1 intein, VDE, triggers a single double-strand break (DSB) at an inteinless allele, leading to VMA1 intein homing. Besides the accumulating information on the in vitro activity of VDE, very little has been known about the molecular mechanism of intein homing in yeast nucleus. We developed an assay to detect the product of VMA1 intein homing in yeast genome. We analysed mutant phenotypes of RecA homologs, Rad51p and Dmc1p, and their interacting proteins, Rad54p and Tid1p, and found that they all play critical roles in intein inheritance. The absence of DSB end processing proteins, Sae2p and those in the Mre11-Rad50-Xrs2 complex, also causes partial reduction in homing efficiency. As with meiotic recombination, crossover events are frequently observed during intein homing. We also observed that the absence of premeiotic DNA replication caused by hydroxyurea (HU) or clb5delta clb6delta mutation reduces VDE-mediated DSBs. The repairing system working in intein homing shares molecular machinery with meiotic recombination induced by Spo11p. Moreover, like Spo11p-induced DNA cleavage, premeiotic DNA replication is a prerequisite for a VDE-induced DSB. VMA1 intein thus utilizes several host factors involved in meiotic and recombinational processes to spread its genetic information and guarantee its progeny through establishment of a parasitic relationship with the organism.

  19. Sex Away from Home

    Science.gov (United States)

    Greenwald, Harold

    1971-01-01

    The reasons why people who are normally truthful to their spouses engage in sex away from home are discussed. These reasons can include loneliness, ego building or the opportunity to have homosexual relations. Sex away from home is likely to increase since the number of people traveling is increasing. (Author/CG)

  20. SETI@home

    Science.gov (United States)

    Project Help Donate Porting Graphics Add-ons Science About SETI@home About Astropulse Science Community Message boards Questions and Answers Teams Profiles User search Web sites Pictures and music User University of California SETI@home and Astropulse are funded by grants from the National Science Foundation

  1. Home | SREL Herpetology Program

    Science.gov (United States)

    Savannah River Ecology Laboratory Herpetology Program Herp Home Research Publications Herps of SC /GA P.A.R.C. Outreach SREL Home powered by Google Search Herpetology at SREL The University of SREL herpetology research programs have always included faculty of the University of Georgia, post

  2. Home Teaching and Herbart.

    Science.gov (United States)

    Rust, Val D.; Reed, Frances

    1979-01-01

    Viewing the growing disenchantment with state-controlled schooling, the authors predict that home teaching will become an established educational alternative within a short time, and they reflect on the teachings and writings of Johann Friedrich Herbart, an eighteenth-century advocate of educating children at home. (Editor/SJL)

  3. Creating a new home

    DEFF Research Database (Denmark)

    Gram-Hanssen, Kirsten; Bech-Danielsen, Claus

    2012-01-01

    Housing research is increasingly focusing on how different groups of residents use their dwelling and transform it into a home. In this article, we look at the homes of immigrants in Danish social housing. The article is based on qualitative interviews with Somali, Iraqi and Turkish immigrants, a...

  4. European Home Energy

    DEFF Research Database (Denmark)

    Tommerup, Henrik M.

    2009-01-01

    An important aim of the european energy performance of buildings directive is to improve the overall energy efficiency of new homes......An important aim of the european energy performance of buildings directive is to improve the overall energy efficiency of new homes...

  5. Health Begins at Home

    Centers for Disease Control (CDC) Podcasts

    2009-03-30

    Clean and well-maintained homes can prevent many illnesses and injuries. This podcast discusses how good health begins at home.  Created: 3/30/2009 by Coordinating Center for Environmental Health and Injury Prevention (CCEHIP).   Date Released: 3/30/2009.

  6. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... de Destrezas para manejo Doméstico de Ostomía Ostomy Home Skills Program Adult Ostomy Pediatric Ostomy Programa de Destrezas para manejo Doméstico de Ostomía The Ostomy Home Skills Kit supports patients with educational and simulation ...

  7. Technologies for Home Networking

    DEFF Research Database (Denmark)

    A broad overview of the home networking field, ranging from wireless technologies to practical applications. In the future, it is expected that private networks (e.g. home networks) will become part of the global network ecosystem, participating in sharing their own content, running IP...

  8. Genetics Home Reference

    Science.gov (United States)

    ... Page Search Home Health Conditions Genes Chromosomes & mtDNA Resources Help Me Understand Genetics Share: Email Facebook Twitter Genetics Home Reference provides consumer-friendly information about the effects of genetic variation on human health. Health Conditions More than 1,200 health ...

  9. 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)

  10. Biometrics for home networks security

    KAUST Repository

    Ansari, Imran Shafique

    2009-01-01

    Hacking crimes committed to the home networks are increasing. Advanced network protection is not always possible for the home networks. In this paper we will study the ability of using biometric systems for authentication in home networks. ©2009 IEEE.

  11. Biometrics for home networks security

    KAUST Repository

    Ansari, Imran Shafique; Ahmad, Qutbuddin S.

    2009-01-01

    Hacking crimes committed to the home networks are increasing. Advanced network protection is not always possible for the home networks. In this paper we will study the ability of using biometric systems for authentication in home networks. ©2009

  12. 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.

  13. 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.

  14. 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.

  15. Evolution of Home Automation Technology

    OpenAIRE

    Mohd. Rihan; M. Salim Beg

    2009-01-01

    In modern society home and office automation has becomeincreasingly important, providing ways to interconnectvarious home appliances. This interconnection results infaster transfer of information within home/offices leading tobetter home management and improved user experience.Home Automation, in essence, is a technology thatintegrates various electrical systems of a home to provideenhanced comfort and security. Users are grantedconvenient and complete control over all the electrical homeappl...

  16. Home Within Me

    DEFF Research Database (Denmark)

    Kreuzer, Maria; Mühlbacher, Hans; von Wallpach, Sylvia

    2017-01-01

    In an increasingly globalized, digitalized and perceived unmanageable world, consumers strive for belongingness, identification and security and re-discover the importance of home. Home is central to peoples’ individual as well as collective identities and their self-development (McCracken, 1989...... in Austria and the sample consisted of 15 locals (study 1) and 17 first generation immigrants (study 2) to identify possible commonalities and differences. This research adds to existing literature by 1) empirically confirming the existence of dimensions of home (e.g., physical, social, temporary...

  17. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... Medical Student Core Curriculum ACS/ASE Medical Student Simulation-Based Surgical Skills Curriculum Cancer Education Cancer Education ... Home Skills Kit supports patients with educational and simulation materials to learn and practice the skills needed ...

  18. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... of Surgeons Professional Association Advocate at Home Program State Legislative Action Center Leadership & Advocacy Summit Webinars Practice Management Practice Management Practice Management CPT Coding Bulletin Articles ...

  19. Nursing Home Data Compendium

    Data.gov (United States)

    U.S. Department of Health & Human Services — The compendium contains figures and tables presenting data on all Medicare- and Medicaid-certified nursing homes in the United States as well as the residents in...

  20. HOME Income Limits

    Data.gov (United States)

    Department of Housing and Urban Development — HOME Income Limits are calculated using the same methodology that HUD uses for calculating the income limits for the Section 8 program. These limits are based on HUD...

  1. Pervasive Home Environments

    Science.gov (United States)

    Bull, P.; Limb, R.; Payne, R.

    An increasing number of computers and other equipment, such as games consoles and multimedia appliances for the home, have networking capability. The rapid growth of broadband in the home is also fuelling the demand for people to network their homes. In the near future we will see a number of market sectors trying to 'own' the home by providing gateways either from the traditional ISP or from games and other service providers. The consumer is bombarded with attractive advertising to acquire the latest technological advances, but is left with a plethora of different appliances, which have a bewildering range of requirements and features in terms of networking, user interface, and higher-level communications protocols. In many cases, these are proprietary, preventing interworking. Such technical and usability anarchy confuses the consumer and could ultimately suppress market adoption.

  2. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... for Hospitals Ostomy Home Skills Hospital Quality Improvement Package The standardized interactive program has been developed by the American College of Surgeons ... and Associates Medical Students International Surgeons ...

  3. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... login or create account first) Skills Kits Broadcast Rights for Hospitals Ostomy Home Skills Hospital Quality Improvement Package The standardized interactive program has been developed by the ...

  4. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... at ACS ACS and Veterans Diversity at ACS Benefits ... Profile Shop ( 0 ) Cart Donate American College of Surgeons Education Patients and Family Skills Programs Ostomy Home Skills ...

  5. Genetics Home Reference: achondroplasia

    Science.gov (United States)

    ... Share: Email Facebook Twitter Home Health Conditions Achondroplasia Achondroplasia Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Achondroplasia is a form of short-limbed dwarfism. The ...

  6. Home Health Quality Initiative

    Data.gov (United States)

    U.S. Department of Health & Human Services — The instrument-data collection tool used to collect and report performance data by home health agencies is called the Outcome and Assessment Information Set (OASIS)....

  7. Genetics Home Reference: phenylketonuria

    Science.gov (United States)

    ... Share: Email Facebook Twitter Home Health Conditions Phenylketonuria Phenylketonuria Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Phenylketonuria (commonly known as PKU) is an inherited disorder ...

  8. Home Health Care Agencies

    Data.gov (United States)

    U.S. Department of Health & Human Services — A list of all Home Health Agencies that have been registered with Medicare. The list includes addresses, phone numbers, and quality measure ratings for each agency.

  9. Genetics Home Reference: schizophrenia

    Science.gov (United States)

    ... Share: Email Facebook Twitter Home Health Conditions Schizophrenia Schizophrenia Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Schizophrenia is a brain disorder classified as a psychosis, ...

  10. Nursing Home Compare Data

    Data.gov (United States)

    U.S. Department of Health & Human Services — These are the official datasets used on the Medicare.gov Nursing Home Compare Website provided by the Centers for Medicare and Medicaid Services. These data allow...

  11. Building Homes, Building Careers.

    Science.gov (United States)

    Cohn, Meredith

    1987-01-01

    The Construction Trades Foundation, a nonprofit corporation of business, industry, and school leaders, provides high school students in Montgomery County, Maryland, with unique hands-on experiences in construction, home design, marketing, public relations, and other fields. (SK)

  12. Home Electrical Safety Checklist

    Science.gov (United States)

    ... Interrupter Protection for Pools, Spas and Hot Tubs Metal Ladders and Electricity Don’t Mix Electrocution Hazard with Do-It-Yourself Repairs of Microwave Ovens Preventing Home Fires: Arc Fault Circuit Interrupters (AFCIs) Power up with ...

  13. Genetics Home Reference: depression

    Science.gov (United States)

    ... Share: Email Facebook Twitter Home Health Conditions Depression Depression Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Depression (also known as major depression or major depressive ...

  14. Genetics Home Reference: alkaptonuria

    Science.gov (United States)

    ... Share: Email Facebook Twitter Home Health Conditions Alkaptonuria Alkaptonuria Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Alkaptonuria is an inherited condition that causes urine to ...

  15. Home Health PPS - Reports

    Data.gov (United States)

    U.S. Department of Health & Human Services — Abt Associates July 21, 2010 Analysis of 2000-2008 Home Health Case-mix Change Report estimates the extent to which the observed increases in average case-mix were...

  16. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... Resources Educational Resources E-Learning Entering Resident Readiness Assessment Evidence-Based Decisions in Surgery Medical Student Resources ... checklist Evaluation (Complete the Ostomy Patient Survey . We need your opinion!) Program outcomes The ACS Ostomy Home ...

  17. Genetics Home Reference: preeclampsia

    Science.gov (United States)

    ... Share: Email Facebook Twitter Home Health Conditions Preeclampsia Preeclampsia Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Preeclampsia is a complication of pregnancy in which affected ...

  18. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... Stay Up to Date with ACS Association Management Jobs Events Find a Surgeon Patients and Family Contact My Profile Shop ( 0 ) Cart Donate American College of Surgeons Education Patients and Family Skills Programs Ostomy Home Skills ...

  19. Home Health Compare Data

    Data.gov (United States)

    U.S. Department of Health & Human Services — These are the official datasets used on the Medicare.gov Home Health Compare Website provided by the Centers for Medicare and Medicaid Services. These data allow you...

  20. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... Centers National Cancer Database National Accreditation Program for Rectal Cancer Oncology Medical Home Accreditation Program Stereotactic Breast ... collaboration with the American Society of Colon and Rectal Surgeons (ASCRS), American Urological Association (AUA), Certified Enterostomal ...

  1. Nursing Home Compare

    Data.gov (United States)

    U.S. Department of Health & Human Services — The data that is used by the Nursing Home Compare tool can be downloaded for public use. This functionality is primarily used by health policy researchers and the...

  2. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... Trauma and EMS Cancer and Research Health Information Technology Scope of Practice Pediatric Issues Other Federal Legislative ... The Ostomy Home Skills Kit supports patients with educational and simulation materials to learn and practice the ...

  3. Heart failure - home monitoring

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/patientinstructions/000113.htm Heart failure - home monitoring To use the sharing features on ... your high blood pressure Fast food tips Heart failure - discharge Heart failure - fluids and diuretics Heart failure - what to ...

  4. Home garden plums

    Science.gov (United States)

    The paper is to provide extension information on plums for home owners in Georgia and other Southeastern states. It includes seven sections: introduction, varieties, planting, pruning, fertilization, pests/diseases, and long term care....

  5. Home Health PPS

    Data.gov (United States)

    U.S. Department of Health & Human Services — Under prospective payment, Medicare pays home health agencies (HHAs) a predetermined base payment. The payment is adjusted for the health condition and care needs of...

  6. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... Specific Registry Surgeon Specific Registry News and Updates Account Setup Resources and FAQs Features of the SSR ... Today Ostomy Home Skills Kit (login or create account first) Skills Kits Broadcast Rights for Hospitals Ostomy ...

  7. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... Up to Date with ACS Association Management JACS Jobs Events Find a Surgeon Patients and Family Contact My Profile Shop ( 0 ) Cart Donate American College of Surgeons Education Patients and Family Skills Programs Ostomy Home Skills ...

  8. Eye Injuries at Home

    Science.gov (United States)

    ... by the Numbers — Infographic Five Steps to Safer Champagne Celebrations Eye Injuries at Home Leer en Español: ... that can splatter hot grease or oil. Opening champagne bottles during a celebration. Drilling or hammering screws ...

  9. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... ACS ACS and Veterans Diversity at ACS Benefits ... Profile Shop ( 0 ) Cart Donate American College of Surgeons Education Patients and Family Skills Programs Ostomy Home Skills ...

  10. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... skills such as emptying and changing a pouch, problem solving, and home management. A DVD with demonstration of each skill Stoma Practice Model Stoma supplies (measurement guide, marking ...

  11. High performance homes

    DEFF Research Database (Denmark)

    Beim, Anne; Vibæk, Kasper Sánchez

    2014-01-01

    Can prefabrication contribute to the development of high performance homes? To answer this question, this chapter defines high performance in more broadly inclusive terms, acknowledging the technical, architectural, social and economic conditions under which energy consumption and production occur....... Consideration of all these factors is a precondition for a truly integrated practice and as this chapter demonstrates, innovative project delivery methods founded on the manufacturing of prefabricated buildings contribute to the production of high performance homes that are cost effective to construct, energy...

  12. Australia's Bond Home Bias

    OpenAIRE

    Anil V. Mishra; Umaru B. Conteh

    2014-01-01

    This paper constructs the float adjusted measure of home bias and explores the determinants of bond home bias by employing the International Monetary Fund's high quality dataset (2001 to 2009) on cross-border bond investment. The paper finds that Australian investors' prefer investing in countries with higher economic development and more developed bond markets. Exchange rate volatility appears to be an impediment for cross-border bond investment. Investors prefer investing in countries with ...

  13. Smart assistants for smart homes

    OpenAIRE

    Rasch, Katharina

    2013-01-01

    The smarter homes of tomorrow promise to increase comfort, aid elderly and disabled people, and help inhabitants save energy. Unfortunately, smart homes today are far from this vision – people who already live in such a home struggle with complicated user interfaces, inflexible home configurations, and difficult installation procedures. Under these circumstances, smart homes are not ready for mass adoption. This dissertation addresses these issues by proposing two smart assistants for smart h...

  14. 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

  15. Reflections: Volunteering at Home.

    Science.gov (United States)

    Hu, Amanda

    2016-08-01

    Many young people look forward to volunteering abroad and overlook the ample volunteer opportunities at home. There are several advantages to volunteering at home: you help people in your own community; you can make a long-term commitment; and you have continuity of care for your patients. There are >1200 free clinics in the United States whose main goal is to provide care to the indigent population. These free clinics are always looking for volunteers with specialized medical training. This article reviews the medically related and unrelated volunteer opportunities available in the United States. Volunteering at home is a worthwhile experience, and I encourage the otolaryngology community to explore these opportunities. © American Academy of Otolaryngology—Head and Neck Surgery Foundation 2016.

  16. 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

  17. 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.

  18. Mining the Home Environment

    Science.gov (United States)

    Cook, Diane J.; Krishnan, Narayanan

    2014-01-01

    Individuals spend a majority of their time in their home or workplace and for many, these places are our sanctuaries. As society and technology advance there is a growing interest in improving the intelligence of the environments in which we live and work. By filling home environments with sensors and collecting data during daily routines, researchers can gain insights on human daily behavior and the impact of behavior on the residents and their environments. In this article we provide an overview of the data mining opportunities and challenges that smart environments provide for researchers and offer some suggestions for future work in this area. PMID:25506128

  19. Mining the Home Environment.

    Science.gov (United States)

    Cook, Diane J; Krishnan, Narayanan

    2014-12-01

    Individuals spend a majority of their time in their home or workplace and for many, these places are our sanctuaries. As society and technology advance there is a growing interest in improving the intelligence of the environments in which we live and work. By filling home environments with sensors and collecting data during daily routines, researchers can gain insights on human daily behavior and the impact of behavior on the residents and their environments. In this article we provide an overview of the data mining opportunities and challenges that smart environments provide for researchers and offer some suggestions for future work in this area.

  20. HomePort

    DEFF Research Database (Denmark)

    Le Guilly, Thibaut; Olsen, Petur; Ravn, Anders Peter

    2013-01-01

    Ambient Intelligence systems use many sensors and actuators, with a diversity of networks, protocols and technologies which makes it impossible to access the devices in a common manner. This paper presents the HomePort software, which provides an open source RESTful interface to heterogeneous...... sensor networks, allowing a simple unified access to virtually any kind of protocol using well known standards. HomePort includes means to provide event notification, as well as a tracing mechanism. The software is implemented and we report on initial experiments and provide an evaluation that shows...

  1. Windows Home Server users guide

    CERN Document Server

    Edney, Andrew

    2008-01-01

    Windows Home Server brings the idea of centralized storage, backup and computer management out of the enterprise and into the home. Windows Home Server is built for people with multiple computers at home and helps to synchronize them, keep them updated, stream media between them, and back them up centrally. Built on a similar foundation as the Microsoft server operating products, it's essentially Small Business Server for the home.This book details how to install, configure, and use Windows Home Server and explains how to connect to and manage different clients such as Windows XP, Windows Vist

  2. Home Health Compare: Find a Home Health Agency

    Science.gov (United States)

    ... page could not be loaded. The Medicare.gov Home page currently does not fully support browsers with " ... widget - Select to show Back to top Footer Home A federal government website managed and paid for ...

  3. Mycobacterium tuberculosis class II apurinic/apyrimidinic-endonuclease/3'-5' exonuclease III exhibits DNA regulated modes of interaction with the sliding DNA β-clamp.

    Science.gov (United States)

    Khanam, Taran; Rai, Niyati; Ramachandran, Ravishankar

    2015-10-01

    The class-II AP-endonuclease (XthA) acts on abasic sites of damaged DNA in bacterial base excision repair. We identified that the sliding DNA β-clamp forms in vivo and in vitro complexes with XthA in Mycobacterium tuberculosis. A novel 239 QLRFPKK245 motif in the DNA-binding domain of XthA was found to be important for the interactions. Likewise, the peptide binding-groove (PBG) and the C-terminal of β-clamp located on different domains interact with XthA. The β-clamp-XthA complex can be disrupted by clamp binding peptides and also by a specific bacterial clamp inhibitor that binds at the PBG. We also identified that β-clamp stimulates the activities of XthA primarily by increasing its affinity for the substrate and its processivity. Additionally, loading of the β-clamp onto DNA is required for activity stimulation. A reduction in XthA activity stimulation was observed in the presence of β-clamp binding peptides supporting that direct interactions between the proteins are necessary to cause stimulation. Finally, we found that in the absence of DNA, the PBG located on the second domain of the β-clamp is important for interactions with XthA, while the C-terminal domain predominantly mediates functional interactions in the substrate's presence. © 2015 John Wiley & Sons Ltd.

  4. Increased sensitivity of UV-repair-deficient human cells to DNA bound platinum products which unlike thymine dimers are not recognised by an endonuclease extracted from Micrococcus luteus

    Energy Technology Data Exchange (ETDEWEB)

    Fraval, H N.A.; Rawlings, C J; Roberts, J J [Institute of Cancer Research, Royal Cancer Hospital, Pollards Wood Research Station, Chalfont St. Giles, Bucks, UK

    1978-07-01

    The response of human cells in culture to cis platinum (II) diammine dichloride (cis Pt(II)) induced DNA damage has been studied. The survival data, measured as a function of cis Pt(II) dose were similar in a normal cell line (Human foetal lung) compared to a UV-sensitive, thymine dimer excision repair-deficient cell line (Xeroderma pigmentatosum). However, there was a marked difference between the two cell lines when binding to DNA was plotted against dose of cis Pt(II) given for 1 h. When these findings were expressed as cell survival versus binding to DNA, a 4.1-fold difference between the slopes of the survival curves for the two cell lines was obtained. These findings are consistent with the notion that normal cells are able to excise cis Pt(II) induced damage from their genome and thus increase their ability to survive as compared to excision deficient cells. An endonuclease preparation from Micrococcus luteus is able to recognise UV damage in DNA, but did not recognise cis Pt(II) induced damage. These results possibly indicate differences in the pathways of repair of damage caused by the two agents.

  5. 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

  6. 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.

  7. 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

  8. 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.

  9. DNA Polymerase α (swi7) and the Flap Endonuclease Fen1 (rad2) Act Together in the S-Phase Alkylation Damage Response in S. pombe

    Science.gov (United States)

    Koulintchenko, Milana; Vengrova, Sonya; Eydmann, Trevor; Arumugam, Prakash; Dalgaard, Jacob Z.

    2012-01-01

    Polymerase α is an essential enzyme mainly mediating Okazaki fragment synthesis during lagging strand replication. A specific point mutation in Schizosaccharomyces pombe polymerase α named swi7-1, abolishes imprinting required for mating-type switching. Here we investigate whether this mutation confers any genome-wide defects. We show that the swi7-1 mutation renders cells hypersensitive to the DNA damaging agents methyl methansulfonate (MMS), hydroxyurea (HU) and UV and incapacitates activation of the intra-S checkpoint in response to DNA damage. In addition we show that, in the swi7-1 background, cells are characterized by an elevated level of repair foci and recombination, indicative of increased genetic instability. Furthermore, we detect novel Swi1-, -Swi3- and Pol α- dependent alkylation damage repair intermediates with mobility on 2D-gel that suggests presence of single-stranded regions. Genetic interaction studies showed that the flap endonuclease Fen1 works in the same pathway as Pol α in terms of alkylation damage response. Fen1 was also required for formation of alkylation- damage specific repair intermediates. We propose a model to explain how Pol α, Swi1, Swi3 and Fen1 might act together to detect and repair alkylation damage during S-phase. PMID:23071723

  10. AKT2 Blocks Nucleus Translocation of Apoptosis-Inducing Factor (AIF and Endonuclease G (EndoG While Promoting Caspase Activation during Cardiac Ischemia

    Directory of Open Access Journals (Sweden)

    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.

  11. 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.

  12. 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)

  13. 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

  14. 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.

  15. 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.

  16. 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

  17. ThinkHome Energy Efficiency in Future Smart Homes

    OpenAIRE

    Christian Reinisch; Mario J. Kofler; Félix Iglesias; Wolfgang Kastner

    2011-01-01

    Abstract Smart homes have been viewed with increasing interest by both home owners and the research community in the past few years. One reason for this development is that the use of modern automation technology in the home or building promises considerable savings of energy, therefore, simultaneously reducing the operational costs of the building over its whole lifecycle. However, the full potential of smart homes still lies fallow, due to the complexity and diversity of the systems, badly ...

  18. Home Automation : Smart home technology and template house design

    OpenAIRE

    Zheng, Zeya

    2013-01-01

    In this thesis, home automation’s general knowledge, technology information and each component will be introduced to the reader in the first half of the whole thesis. In the second half, thesis includes the Home Automation template design and market competitiveness analysis. The author assumes that the reader is going to spend lots of money to have a smart home. In this situation, the author introduces the home automation to the reader at each component. So the reader in this thesis actu...

  19. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... Workforce Trauma and EMS Cancer and Research Health Information Technology Scope of Practice Pediatric Issues Other Federal Legislative ... Overview The skills kit contains: A booklet with information on the ... and home management. A DVD with demonstration of each skill Stoma ...

  20. Future Home Network Requirements

    DEFF Research Database (Denmark)

    Charbonnier, Benoit; Wessing, Henrik; Lannoo, Bart

    This paper presents the requirements for future Home Area Networks (HAN). Firstly, we discuss the applications and services as well as their requirements. Then, usage scenarios are devised to establish a first specification for the HAN. The main requirements are an increased bandwidth (towards 1...

  1. Home Ventilator Guide

    Science.gov (United States)

    ... lower cost, and compensation for leaks from masks. Disadvantages include lack of internal batteries, no or few ... www.gerespiratorycarecentral.com/home_care.php Löwenstein Medical Technology GmbH + Co. KG (Weinmann.de) https://loewensteinmedical.de/ ...

  2. Kinunigmi (At Home).

    Science.gov (United States)

    Pulu, Tupou L.; And Others

    This first level social studies text, designed for children in bilingual Inupiat-English programs in the Alaskan villages of Ambler, Kobuk, Kiana, Noorvik, Selawik, and Shungnak, is a story about a little girl's activities in her home. Each page of text is illustrated with a black-and-white drawing. (CFM)

  3. Home, Hearth and Computing.

    Science.gov (United States)

    Seelig, Anita

    1982-01-01

    Advantages of having children use microcomputers at school and home include learning about sophisticated concepts early in life without a great deal of prodding, playing games that expand knowledge, and becoming literate in computer knowledge needed later in life. Includes comments from parents on their experiences with microcomputers and…

  4. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... Workforce Trauma and EMS Cancer and Research Health Information Technology Scope of Practice Pediatric Issues Other Federal Legislative ... supports the entire surgical team with quality, comprehensive education. The ... A booklet with information on the operation, home skills such as emptying ...

  5. Home vision tests

    Science.gov (United States)

    ... missing areas. If any lines appear distorted or broken, note their location on the grid using a pen or pencil. DISTANCE VISION This is the standard eye chart doctors use, which has been adapted for home use. The chart is attached to a wall ...

  6. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... emptying and changing a pouch, problem solving, and home management. A DVD with demonstration of each skill Stoma Practice Model Stoma supplies (measurement guide, marking pen, scissors, sample pouch) Ostomy self-care checklist Evaluation (Complete the Ostomy Patient Survey . We ...

  7. Solar Electricity for Homes

    Science.gov (United States)

    Roman, Harry T.

    2012-01-01

    Every day, the sun showers the Earth with millions of times more energy than its people use. The only problem is that energy is spread out over the entire Earth's surface and must be harvested. Engineers are learning to capture and use some of this energy to make electricity for homes. Solar panels make up the heart of a solar system. They can be…

  8. At Home with Students

    DEFF Research Database (Denmark)

    Lyngsø, Anita

    2015-01-01

    This article reflects the methodological challenges presented in the research process, where the principle of 'following the field’ means that the researcher must also follow students engaged in online activities in their own homes. The ethnographic studies are a part of a PhD project...

  9. Depression in nursing homes.

    Science.gov (United States)

    Snowdon, John

    2010-11-01

    Although studies have shown the prevalence of depression in nursing homes to be high, under-recognition of depression in these facilities is widespread. Use of screening tests to enhance detection of depressive symptoms has been recommended. This paper aims to provoke discussion about optimal management of depression in nursing homes. The utility of the Cornell Scale for Depression in Dementia (CSDD) is considered. CSDD data relating to residents assessed in 2008-2009 were collected from three Sydney nursing homes. CSDD scores were available from 162 residents, though raters stated they were unable to score participants on at least one item in 47 cases. Scores of 13 or more were recorded for 23% of residents in these facilities, but in most of these cases little was documented in case files to show that the results had been discussed by staff, or that they led to interventions, or that follow-up testing was arranged. Results of CSDD testing should prompt care staff (including doctors) to consider causation of depression in cases where residents are identified as possibly depressed. In particular, there needs to be discussion of how to help residents to cope with disability, losses, and feelings of powerlessness. Research is needed, examining factors that might predict response to antidepressants, and what else helps. Accreditation of nursing homes could be made to depend partly on evidence that staff regularly search for, and (if found) ensure appropriate responses to, depression.

  10. Home Network Security

    NARCIS (Netherlands)

    Scholten, Hans; van Dijk, Hylke

    2008-01-01

    Service discovery and secure and safe service usage are essential elements in the deployment of home and personal networks. Because no system administrator is present, setup and daily operation of such a network has to be automated as much as possible with a high degree of user friendliness. To

  11. Personalized Home-Networks

    DEFF Research Database (Denmark)

    Soler, José; Gandy, Michael

    2007-01-01

    The paper provides details of a home-networking architecture based on an enhanced residential gateway. Initially the need for mechanisms allowing user-dependent network behavior is described and afterwards details of an initial implementation are provided in terms of architectural description...

  12. Home-based care

    African Journals Online (AJOL)

    Mrs. Patience Edoho Samson-Akpan

    study was to ascertain the relationship between home-based care and quality of life of PLWHA in support groups in. Calabar South Local Government Area. A correlational design was utilized and a purposive sample of 74 PLWHA participated in the study. A self developed and well validated questionnaire was used for data ...

  13. Doing Home Works

    DEFF Research Database (Denmark)

    Nelund, Sidsel

    2013-01-01

    of aesthetic analysis of artworks and ethnographic fieldwork (Georgina Born); and 3) the use of generative ethnographic stories as a writing tool (Helen Verran). The latter two, especially, are then employed in analysing the Beirut-based extended exhibition, ‘Home Works: A Forum on Cultural Practices...

  14. Protokoller til Home Automation

    DEFF Research Database (Denmark)

    Kjær, Kristian Ellebæk

    2008-01-01

    computer, der kan skifte mellem foruddefinerede indstillinger. Nogle gange kan computeren fjernstyres over internettet, så man kan se hjemmets status fra en computer eller måske endda fra en mobiltelefon. Mens nævnte anvendelser er klassiske indenfor home automation, er yderligere funktionalitet dukket op...

  15. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... The Ostomy Home Skills Kit supports patients with educational and simulation materials to learn and practice the skills needed for optimal postoperative recovery. The kit supports the entire surgical team with quality, comprehensive education. The standardized interactive program has been ...

  16. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... Contact My Profile Shop ( 0 ) Cart Donate American College of Surgeons Education Patients and Family Skills Programs Ostomy Home Skills ... facs.org Copyright © 1996-2018 by the American College of Surgeons, Chicago, IL 60611-3295 | Privacy Policy | Terms of Use

  17. Composting Begins at Home.

    Science.gov (United States)

    Dreckman, George P.

    1994-01-01

    Reports the results of a year-long home composting pilot program run by the city of Madison, Wisconsin. The study was designed to gather data on the amount and type of materials composted by 300 volunteer households and to determine the feasibility of a full-scale program. (LZ)

  18. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... Use Patient Opioid Use Position Statements and Task Force Patient Education Initiatives Advocacy and Health Policy Updates Selected Research ... at ACS ACS and Veterans Diversity at ACS Benefits ... Profile Shop ( 0 ) Cart Donate American College of Surgeons Education Patients and Family Skills Programs Ostomy Home Skills ...

  19. High performance homes

    DEFF Research Database (Denmark)

    Beim, Anne; Vibæk, Kasper Sánchez

    2014-01-01

    . Consideration of all these factors is a precondition for a truly integrated practice and as this chapter demonstrates, innovative project delivery methods founded on the manufacturing of prefabricated buildings contribute to the production of high performance homes that are cost effective to construct, energy...

  20. Eldercare at Home: Mobility Problems

    Science.gov (United States)

    ... Community Home › Resources › Eldercare at Home: Mobility Problems Font size A A A Print Share Glossary previous ... doctor or physical therapist to find out what type of cane or walker the older person needs. ...

  1. Blood pressure monitors for home

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/007482.htm Blood pressure monitors for home To use the sharing features ... may ask you to keep track of your blood pressure at home. To do this, you will need ...

  2. Dental plaque identification at home

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/003426.htm Dental plaque identification at home To use the sharing ... a sticky substance that collects around and between teeth. The home dental plaque identification test shows where ...

  3. Home-Use Tests - Cholesterol

    Science.gov (United States)

    ... Medical Procedures In Vitro Diagnostics Home Use Tests Cholesterol Share Tweet Linkedin Pin it More sharing options ... a home-use test kit to measure total cholesterol. What cholesterol is: Cholesterol is a fat (lipid) ...

  4. Genetics Home Reference: PURA syndrome

    Science.gov (United States)

    ... TJ, Vreeburg M, Rouhl RPW, Stevens SJC, Stegmann APA, Schieving J, Pfundt R, van Dijk K, Smeets ... article on PubMed Central More from Genetics Home Reference Bulletins Genetics Home Reference Celebrates Its 15th Anniversary ...

  5. Home apnea monitor use - infants

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/patientinstructions/000755.htm Home apnea monitor use - infants To use the sharing ... portable. Why is an Apnea Monitor Used at Home? A monitor may be needed when: Your baby ...

  6. Telemedicine in Neonatal Home Care

    DEFF Research Database (Denmark)

    Holm, Kristina Garne; Brødsgaard, Anne; Zachariassen, Gitte

    2016-01-01

    participatory design and qualitative methods. Data were collected from observational studies, individual interviews, and focus group interviews. Two neonatal units participated. One unit was experienced in providing neonatal home care with home visits, and the other planned to offer neonatal home care......BACKGROUND: For the majority of preterm infants, the last weeks of hospital admission mainly concerns tube feeding and establishment of breastfeeding. Neonatal home care (NH) was developed to allow infants to remain at home for tube feeding and establishment of breastfeeding with regular home...... visits from neonatal nurses. For hospitals covering large regions, home visits may be challenging, time consuming, and expensive and alternative approaches must be explored. OBJECTIVE: To identify parental needs when wanting to provide neonatal home care supported by telemedicine. METHODS: The study used...

  7. Home Automation and Security System

    OpenAIRE

    Surinder Kaur,; Rashmi Singh; Neha Khairwal; Pratyk Jain

    2016-01-01

    Easy Home or Home automation plays a very important role in modern era because of its flexibility in using it at different places with high precision which will save money and time by decreasing human hard work. Prime focus of this technology is to control the household equipment’s like light, fan, door, AC etc. automatically. This research paper has detailed information on Home Automation and Security System using Arduino, GSM and how we can control home appliances using Android application....

  8. [Maintaining patients' autonomy at home].

    Science.gov (United States)

    Niang, Bénédicte; Coudre, Jean Pierre

    2015-01-01

    To maintain the flow of hospital discharges, the patient's return home with support from a home nursing service is important. If any difficulties are identified, there are various programmes or good practices which can be put into place. The future law on adapting society to ageing also comprises a scheme combining home assistance and nursing care.

  9. Ensuring Quality Nursing Home Care

    Science.gov (United States)

    ... cases, you can also call the Department of Health. Nursing homes are required to post information on how you ... nursing homes in your area, go to Medicare’s Nursing Home Compare website at ... information is not intended to diagnose health problems or to take the place of medical ...

  10. Home advantage in professional tennis

    NARCIS (Netherlands)

    Koning, Ruud H.

    2011-01-01

    Home advantage is a pervasive phenomenon in sport. It has been established in team sports such as basketball, baseball, American football, and European soccer. Attention to home advantage in individual sports has so far been limited. The aim of this study was to examine home advantage in

  11. When Globalization Hits Home

    DEFF Research Database (Denmark)

    Borrás, Susana; Haakonsson, Stine Jessen

    Lead firms are increasingly reorganizing their innovation activities into global innovation networks. Such reorganization has potentially major impact on their existing home-based innovation networks. Based on 31 interviews in four case studies of lead firms in the Danish food industry, the paper...... analyzes the dynamics of five key features of home-based innovation networks: 1) size of the national networks, 2) type of organization, 3) content of collaboration within the network, 4) concurrent globalization of organizations in the network, and 5) degree of formalization of network interactions....... The dynamics are generally differentiated according to the type of lead firm strategy, i.e. knowledge augmenting or knowledge exploiting. The qualitative and exploratory findings point towards some effects on national innovation networks. Hence, the paper concludes by hypothesizing that the globalization...

  12. The @Home project

    DEFF Research Database (Denmark)

    Coxon, Ian Robert

    2014-01-01

    of being overshadowed by what is increasingly seen as new (and substantial) pharmacological and health equipment business market opportunities in what is now viewed as a health 'industry'. The Centre for Innovation at Mayo Clinic (CFI) in Rochester USA, has conducted many projects which border on and have...... investigated various aspects of care provision and patient experience within a patient's home environment. Considerable work by other researchers inside and outside the health field has also contributed insights and platforms for moving healthcare in this direction. In most areas of the western world......, the healthcare sector is struggling to cope with the scale of strain that shifting demographics, rising costs and increasing chronic/complex care is placing on the health system. The shift towards home based care and personal health self-management is seen as offering some possibilities to alleviate...

  13. Energy comes home

    International Nuclear Information System (INIS)

    Aune, Margrethe

    2007-01-01

    The growth in private energy consumption is an increasing problem in western countries. From an environmental point of view, this consumption has to be reduced. On the basis of two Norwegian case studies, this article discusses private energy consumption and possibilities for reduction, with a special focus on the home. It argues against a rational economic view of the consumer and emphasizes the significance of a more subtle understanding of private energy use. The article approaches the challenges of reducing private energy consumption by analysing the domestication of the home and discusses everyday life activities as well as the phenomenon of rebuilding and redecorating. By using the concept of domestication, the article challenges the linear understanding of technological as well as behavioural change. Private energy consumption is part of a complex network and it is necessary to understand this network in order to achieve a more permanent reduction

  14. The Home of Man

    DEFF Research Database (Denmark)

    Hauberg, Jørgen; Bjerrum, Peter

    2018-01-01

    Abstract: Accordingly, Le Corbusier in “Towards a New Architecture”, 1922, the question was: “Architecture or revolution” – also stated in “The Home of Man”, 1942: “… people live in poor conditions, this is the real, the most profound reason for the battles and upheavals of our time.” The ninetee......Abstract: Accordingly, Le Corbusier in “Towards a New Architecture”, 1922, the question was: “Architecture or revolution” – also stated in “The Home of Man”, 1942: “… people live in poor conditions, this is the real, the most profound reason for the battles and upheavals of our time...

  15. Palliative Sedation at Home

    Science.gov (United States)

    Barathi, B

    2012-01-01

    Patients with advanced cancer often suffer from multiple intractable physical symptoms. Though majority of the symptoms can be controlled, in some of the patients these symptoms remain refractory and uncontrolled till the end. Palliative sedation (PS) is one of the ways to relieve intractable suffering of the dying cancer patients. The main concern while using PS is its life-shortening effect. This case report describes the feasibility of administering PS in Indian home settings. PMID:22837615

  16. Palliative sedation at home

    Directory of Open Access Journals (Sweden)

    B Barathi

    2015-04-01

    Full Text Available Patients with advanced cancer often suffer from multiple intractable physical symptoms. Though majority of the symptoms can be controlled, in some of the patients these symptoms remain refractory and uncontrolled till the end. Palliative sedation (PS is one of the ways to relieve intractable suffering of the dying cancer patients. The main concern while using PS is its life-shortening effect. This case report describes the feasibility of administering PS in Indian home settings.

  17. [Aromatherapy in nursing homes].

    Science.gov (United States)

    Barré, Lucile

    2015-01-01

    Pierre Delaroche de Clisson hospital uses essential oils as part of its daily organisation for the treatment of pain and the development of palliative care. The setting up of this project, in nursing homes and long-term care units, is the fruit of a complex mission carried out by a multidisciplinary team, which had to take into account the risks involved and overcome a certain amount of reluctance. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  18. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... Workforce Trauma and EMS Cancer and Research Health Information Technology Scope of Practice Pediatric Issues Other Federal Legislative Issues Regulatory Issues Regulatory Issues Regulatory Issues Stop Overregulating My OR ... Rectal Surgeons (ASCRS), American Urological Association (AUA), Certified Enterostomal Therapy Nurses (CETN), and the United Ostomy Associations of America (UOAA). The skills kit contains: A booklet with information on the operation, home skills such as emptying ...

  19. Ostomy Home Skills Program

    Medline Plus

    Full Text Available ... Workforce Trauma and EMS Cancer and Research Health Information Technology Scope of Practice Pediatric Issues Other Federal Legislative Issues Regulatory Issues Regulatory Issues Regulatory Issues Stop Overregulating My OR ... American Urological Association (AUA), Certified Enterostomal Therapy Nurses (CETN), and the United Ostomy Associations of America (UOAA). Program Overview The skills kit contains: A booklet with information on the operation, home skills such as emptying ...

  20. PLANNED HOME BIRTH: A REVIEW

    Directory of Open Access Journals (Sweden)

    Tamara Serdinšek

    2016-05-01

    Full Text Available Background: Home birth is as old as humanity, but still most middle- and high-income countries consider hospitals as the safest birth settings, as complications regarding birth are highly unpredictable. Despite this there are a few countries in which home birth in integrated into official healthcare system (the Netherlands, United Kingdom, Canada etc.. Home births can be divided into unplanned and planned, and the latter can be further categorized by the presence of the birth attendants. This review focuses on planned home births, which are differently represented throughout the world. In the United States 0.6-1.0% of all children are born at home, in the United Kingdom 2-3%, in Canada 1.6% and in the Netherlands 20-30%. For Slovenia, the number of planned home births is unknown; however, in 2010 0.1% of children were born outside medical facilities.Conclusions: The safety of home birth in still under the debate. While research confirms smaller number of obstetric interventions and some complications in mothers who give birth at home, the data regarding the neonatal and perinatal mortality and morbidity is still conflicting. This confirms the need for large multicentric trials in this field. Current home birth guidelines emphasize that women should be well informed regarding the possible advantages and disadvantages of home births. In addition, the emphasis is on definition of selection criteria for home birth, indications for intrapartal transfer to the hospital and appropriate education of birth attendants. 

  1. 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

  2. Tactics for homing in mobile life

    DEFF Research Database (Denmark)

    Petersen, Marianne Graves; Lynggard, Aviaja Borup; Krogh, Peter Gall

    2010-01-01

    For many people home making is an activity, which extends beyond a single house. We introduce the terminology of Homing as the act of home making, when in a primary home, secondary home or more temporary spaces. By point of departure in existing literature on home making and through ethnographic...

  3. 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.

  4. Detection of endonuclease III- and 8-oxoguanine glycosylase-sensitive base modifications in γ-irradiated DNA and cells by the aldehyde reactive probe (ARP) assay

    International Nuclear Information System (INIS)

    Mohsin Ali, M.; Kurisu, Satofumi; Yoshioka, Yoshihiro; Terato, Hiroaki; Ohyama, Yoshihiko; Ide Hiroshi; Kubo, Kihei

    2004-01-01

    Ionizing radiation generates diverse DNA lesions that differentially induce cell death and mutations. In the present study, calf thymus DNA (400 μg/ml) and HeLa cells were irradiated by 60 Co γ-rays, and abasic (AP) sites and endonuclease (Endo) III- and 8-oxoguanine glycosylase (hOGG1)-sensitive base modifications in DNA were quantitated by the aldehyde reactive probe (ARP) assay. The irradiation of calf thymus DNA in phosphate buffer generated 91 Endo III- and 100 hOGG1-sensitive base modifications and 110 AP sites per 10 6 base pairs (bp) per Gy. The yield of the lesions in Tris buffer was 41- to 91-fold lower than that in phosphate, demonstrating a radioprotective effect of Tris. The HeLa cell chromosomal DNA contained 12 Endo III- and 3.8 hOGG1-sensitive base modifications and less than 1 AP sites per 10 6 bp as endogenous damage, and their level was increased by irradiation. The yields of the damage at 1 Gy (roughly equivalent to the lethal dose of HeLa cells [1.6-1.8 Gy]) were 0.13 Endo III, 0.091 hOGG1, and 0.065 AP sites per 10 6 bp, showing that irradiation with a lethal dose brought about only a marginal increase in base damage relative to an endogenous one. A comparison of the present data with those reported for DNA strand breaks supports the primary importance of double-strand breaks and clustered lesions as lethal damages formed by ionizing radiation. (author)

  5. 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

  6. A unique dual recognition hairpin probe mediated fluorescence amplification method for sensitive detection of uracil-DNA glycosylase and endonuclease IV activities.

    Science.gov (United States)

    Wu, Yushu; Yan, Ping; Xu, Xiaowen; Jiang, Wei

    2016-03-07

    Uracil-DNA glycosylase (UDG) and endonuclease IV (Endo IV) play cooperative roles in uracil base-excision repair (UBER) and inactivity of either will interrupt the UBER to cause disease. Detection of UDG and Endo IV activities is crucial to evaluate the UBER process in fundamental research and diagnostic application. Here, a unique dual recognition hairpin probe mediated fluorescence amplification method was developed for sensitively and selectively detecting UDG and Endo IV activities. For detecting UDG activity, the uracil base in the probe was excised by the target enzyme to generate an apurinic/apyrimidinic (AP) site, achieving the UDG recognition. Then, the AP site was cleaved by a tool enzyme Endo IV, releasing a primer to trigger rolling circle amplification (RCA) reaction. Finally, the RCA reaction produced numerous repeated G-quadruplex sequences, which interacted with N-methyl-mesoporphyrin IX to generate an enhanced fluorescence signal. Alternatively, for detecting Endo IV activity, the uracil base in the probe was first converted into an AP site by a tool enzyme UDG. Next, the AP site was cleaved by the target enzyme, achieving the Endo IV recognition. The signal was then generated and amplified in the same way as those in the UDG activity assay. The detection limits were as low as 0.00017 U mL(-1) for UDG and 0.11 U mL(-1) for Endo IV, respectively. Moreover, UDG and Endo IV can be well distinguished from their analogs. This method is beneficial for properly evaluating the UBER process in function studies and disease prognoses.

  7. Molecular cloning of Kuruma shrimp Marsupenaeus japonicus endonuclease-reverse transcriptase and its positive role in white spot syndrome virus and Vibrio alginolyticus infection.

    Science.gov (United States)

    Ma, Xiongchao; Sun, Baozhen; Zhu, Fei

    2018-02-01

    This study investigated the function of endonuclease-reverse transcriptase (mjERT) in Marsupenaeus japonicus. The 1129 bp cDNA sequence of mjERT was cloned from M. japonicus using rapid amplification of cDNA ends (RACE) PCR, and RT-qPCR analysis indicated that mjERT was highly expressed in the gills and hepatopancreas of M. japonicus. We also found that white spot syndrome virus (WSSV) or Vibrio alginolyticus challenge could enhance the expression of mjERT. When mjERT was inhibited, immune genes such as toll, p53, hemocyanin and tumor necrosis factor-α (TNF-α) were significantly down-regulated (P shrimp, while myosin was significantly up-regulated (P shrimps was significantly increased following mjERT RNA interfere (RNAi). Apoptosis data provided information to suggest that mjERT-dsRNA challenge caused less apoptosis in hemocytes in both the disease-free and viral group. We also revealed that mjERT-dsRNA treatment resulted in a lower phagocytosis rate in the hemocytes of V. alginolyticus-challenged shrimp. Finally, we found that the absence of mjERT had an significantly negative impact upon shrimp phenoloxidase (PO) activity, superoxide dismutase (SOD) activity and total hemocyte count (THC) following WSSV or V. alginolyticus infection, indicating a regulative role for mjERT in the innate immunity of shrimp in response to pathogenic infection. In summary, we concluded that mjERT might promote the anti-WSSV immune response of shrimp by regulating apoptosis, PO activity, THC and SOD activity, and also exert a positive role in the immune response against V. alginolyticus by regulating phagocytosis, SOD activity, PO activity and THC. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Second-home electricity consumption

    Energy Technology Data Exchange (ETDEWEB)

    Andersen, Frits M.; Morthorst, Poul Erik [Risoe, Systems Analysis Department, Technical University of Denmark, P.O. Box 49, DK-4000 Roskilde (Denmark); Christensen, Morten S.; Kofoed, Niels-Ulrik [Esbensen Consulting Engineers, Carl Jacobsens Vej 25D, DK-2500 Valby (Denmark); Jensen, Ole Michael [Danish Building Research Institute, Department of Energy and Environment, University of Aalborg, P.O. Box 119, DK-2970 Horsholm (Denmark)

    2008-01-15

    In Denmark, electricity consumption in first and second homes has developed quite differently. Since 1990, electricity consumption in ordinary residences has grown moderately, while consumption in weekend and second homes has increased considerably. In turn, this development has been blamed on a growing number of luxury cottages, new legislation permitting senior citizens to have their permanent address in their second home and a growing number of electric appliances. In order to examine the growing electricity consumption in second homes and to estimate future demand, a multidisciplinary study combining top-down and bottom-up analyses was conducted, i.e., combining models using aggregated economic parameters and feasibility studies using technical parameters, respectively. The top-down estimation showed that changes in electricity consumption in second homes correlate to changes in income. The bottom-up estimation showed that consumption was mainly affected by the frequency with which second homes were used in the winter time. This indicates that additional second homes, increased full-time use and intensified use of electric appliances are the main reasons for the observed increases in electricity consumption. Luxury tourism use and senior citizens' that use a few per cent of the second homes as their home contribute to a minor degree to the overall increase of electricity consumption. Scenarios show that this development may accelerate with increased leisure time, increased use and more permanent settlement in second homes. (author)

  9. Vascular access for home haemodialysis.

    Science.gov (United States)

    Al Shakarchi, Julien; Day, C; Inston, N

    2018-03-01

    Home haemodialysis has been advocated due to improved quality of life. However, there are very little data on the optimum vascular access for it. A retrospective cohort study was carried on all patients who initiated home haemodialysis between 2011 and 2016 at a large university hospital. Access-related hospital admissions and interventions were used as primary outcome measures. Our cohort consisted of 74 patients. On initiation of home haemodialysis, 62 individuals were using an arteriovenous fistula as vascular access, while the remaining were on a tunnelled dialysis catheter. Of the 12 patients who started on a tunnelled dialysis catheter, 5 were subsequently converted to either an arteriovenous fistula ( n = 4) or an arteriovenous graft ( n = 1). During the period of home haemodialysis use, four arteriovenous fistula failed or thrombosed with patients continuing on home haemodialysis using an arteriovenous graft ( n = 3) or a tunnelled dialysis catheter ( n = 1). To maintain uninterrupted home haemodialysis, interventional rates were 0.32 per arteriovenous fistula/arteriovenous graft access-year and 0.4 per tunnelled dialysis catheter access-year. Hospital admission rates for patients on home haemodialysis were 0.33 per patient-year. Our study has shown that home haemodialysis can be safely and independently performed at home within a closely managed home haemodialysis programme. The authors also advocate the use of arteriovenous fistulas for this cohort of patients due to both low complication and intervention rates.

  10. Second-home electricity consumption

    International Nuclear Information System (INIS)

    Andersen, Frits M.; Christensen, Morten S.; Jensen, Ole Michael; Kofoed, Niels-Ulrik; Morthorst, Poul Erik

    2008-01-01

    In Denmark, electricity consumption in first and second homes has developed quite differently. Since 1990, electricity consumption in ordinary residences has grown moderately, while consumption in weekend and second homes has increased considerably. In turn, this development has been blamed on a growing number of luxury cottages, new legislation permitting senior citizens to have their permanent address in their second home and a growing number of electric appliances. In order to examine the growing electricity consumption in second homes and to estimate future demand, a multidisciplinary study combining top-down and bottom-up analyses was conducted, i.e., combining models using aggregated economic parameters and feasibility studies using technical parameters, respectively. The top-down estimation showed that changes in electricity consumption in second homes correlate to changes in income. The bottom-up estimation showed that consumption was mainly affected by the frequency with which second homes were used in the winter time. This indicates that additional second homes, increased full-time use and intensified use of electric appliances are the main reasons for the observed increases in electricity consumption. Luxury tourism use and senior citizens' that use a few per cent of the second homes as their home contribute to a minor degree to the overall increase of electricity consumption. Scenarios show that this development may accelerate with increased leisure time, increased use and more permanent settlement in second homes

  11. Alert management for home healthcare based on home automation analysis.

    Science.gov (United States)

    Truong, T T; de Lamotte, F; Diguet, J-Ph; Said-Hocine, F

    2010-01-01

    Rising healthcare for elder and disabled people can be controlled by offering people autonomy at home by means of information technology. In this paper, we present an original and sensorless alert management solution which performs multimedia and home automation service discrimination and extracts highly regular home activities as sensors for alert management. The results of simulation data, based on real context, allow us to evaluate our approach before application to real data.

  12. Family and home characteristics correlate with mold in homes.

    Science.gov (United States)

    Reponen, Tiina; Levin, Linda; Zheng, Shu; Vesper, Stephen; Ryan, Patrick; Grinshpun, Sergey A; LeMasters, Grace

    2013-07-01

    Previously, we demonstrated that infants residing in homes with higher Environmental Relative Moldiness Index were at greater risk for developing asthma by age seven. The purpose of this analysis was to identify the family and home characteristics associated with higher moldiness index values in infants' homes at age one. Univariate linear regression of each characteristic determined that family factors associated with moldiness index were race and income. Home characteristics associated with the moldiness index values were: air conditioning, carpet, age of the home, season of home assessment, and house dust mite allergen. Parental history of asthma, use of dehumidifier, visible mold, dog and cat allergen levels were not associated with moldiness index. Results of multiple linear regression showed that older homes had 2.9 units higher moldiness index (95% confidence interval [CI]=0.4, 5.4), whereas homes with central air conditioning had 2.5 units lower moldiness index (95% CI=-4.7, -0.4). In addition, higher dust mite allergen levels and carpeting were positively and negatively associated with higher moldiness index, respectively. Because older homes and lack of air conditioning were also correlated with race and lower income, whereas carpeting was associated with newer homes, the multivariate analyses suggests that lower overall socioeconomic position is associated with higher moldiness index values. This may lead to increased asthma risk in homes inhabited by susceptible, vulnerable population subgroups. Further, age of the home was a surrogate of income, race and carpeting in our population; thus the use of these factors should carefully be evaluated in future studies. Copyright © 2013 Elsevier Inc. All rights reserved.

  13. ConductHome: Gesture Interface Control of Home Automation Boxes

    OpenAIRE

    J. Branstett; V. Gagneux; A. Leleu; B. Levadoux; J. Pascale

    2015-01-01

    This paper presents the interface ConductHome which controls home automation systems with a Leap Motion using "invariant gesture protocols". This interface is meant to simplify the interaction of the user with its environment. A hardware part allows the Leap Motion to be carried around the house. A software part interacts with the home automation box and displays the useful information for the user. An objective of this work is the development of a natural/invariant/simpl...

  14. Smart Cities - Smart Homes and Smart Home Technology

    OpenAIRE

    Faanes, Erlend Kydland

    2014-01-01

    This master’s thesis consists of two articles where the first article is theoretical and the second is the empirical study. Article I The purpose with this paper is to explore and illuminate how smart home and smart home technology can contribute to enhance health and Quality of Life in elderly citizens and allow them to live longer in their home. The paper provides a brief introduction to health promotion and highlights the thesis theoretical framework and foundation of Aaron Antonov...

  15. Home Rx: The Health Benefits of Home Performance

    Energy Technology Data Exchange (ETDEWEB)

    Wilson, Jonathan [National Center for Healthy Housing (NCHH), Columbia, MD (United States); Jacobs, David [National Center for Healthy Housing (NCHH), Columbia, MD (United States); Reddy, Amanda [National Center for Healthy Housing (NCHH), Columbia, MD (United States); Tohn, Ellen [Tohn Environmental Strategies, Wayland, MA (United States); Cohen, Jonathan [Dept. of Energy (DOE), Washington DC (United States); Jacobsohn, Ely [Dept. of Energy (DOE), Washington DC (United States)

    2016-12-01

    Evidence in a new, groundbreaking U.S. Department of Energy report, Home Rx: The Health Benefits of Home Performance, shows that home performance upgrades can improve the quality of a home’s indoor environment by reducing the prevalence of harmful indoor air pollutants and contaminants. Until recently, no systematic review of this evidence had been conducted, limiting full understanding of the link between home performance and health. This new report summarizes current knowledge and identifies research gaps. The design characteristics and results of each of the 40 studies considered in the report are summarized in a searchable matrix.

  16. Lighting Options for Homes.

    Energy Technology Data Exchange (ETDEWEB)

    Baker, W.S.

    1991-04-01

    This report covers many aspects of various lighting options for homes. Types of light sources described include natural light, artificial light, incandescent lamps, fluorescent lamps, and high intensity discharge lamps. A light source selection guide gives the physical characteristics of these, design considerations, and common applications. Color, strategies for efficient lighting, and types of lighting are discussed. There is one section giving tips for various situations in specific rooms. Rooms and types of fixtures are shown on a matrix with watts saved by using the recommended type lighting for that room and room location. A major emphasis of this report is saving energy by utilizing the most suitable, recommended lighting option. (BN)

  17. PLANNED HOME BIRTH: A REVIEW

    OpenAIRE

    Tamara Serdinšek; Iztok Takač

    2016-01-01

    Background: Home birth is as old as humanity, but still most middle- and high-income countries consider hospitals as the safest birth settings, as complications regarding birth are highly unpredictable. Despite this there are a few countries in which home birth in integrated into official healthcare system (the Netherlands, United Kingdom, Canada etc.). Home births can be divided into unplanned and planned, and the latter can be further categorized by the presence of the birth attendants. Thi...

  18. The Science of Home Automation

    Science.gov (United States)

    Thomas, Brian Louis

    Smart home technologies and the concept of home automation have become more popular in recent years. This popularity has been accompanied by social acceptance of passive sensors installed throughout the home. The subsequent increase in smart homes facilitates the creation of home automation strategies. We believe that home automation strategies can be generated intelligently by utilizing smart home sensors and activity learning. In this dissertation, we hypothesize that home automation can benefit from activity awareness. To test this, we develop our activity-aware smart automation system, CARL (CASAS Activity-aware Resource Learning). CARL learns the associations between activities and device usage from historical data and utilizes the activity-aware capabilities to control the devices. To help validate CARL we deploy and test three different versions of the automation system in a real-world smart environment. To provide a foundation of activity learning, we integrate existing activity recognition and activity forecasting into CARL home automation. We also explore two alternatives to using human-labeled data to train the activity learning models. The first unsupervised method is Activity Detection, and the second is a modified DBSCAN algorithm that utilizes Dynamic Time Warping (DTW) as a distance metric. We compare the performance of activity learning with human-defined labels and with automatically-discovered activity categories. To provide evidence in support of our hypothesis, we evaluate CARL automation in a smart home testbed. Our results indicate that home automation can be boosted through activity awareness. We also find that the resulting automation has a high degree of usability and comfort for the smart home resident.

  19. Towards a smart home framework

    OpenAIRE

    Alam, Muddasser; Alan, Alper; Rogers, Alex; Ramchurn, Sarvapali D.

    2013-01-01

    We present our Smart Home Framework (SHF) which simplifies the modelling, prototyping and simulation of smart infrastructure (i.e., smart home and smart communities). It provides the buildings blocks (e.g., home appliances) that can be extended and assembled together to build a smart infrastructure model to which appropriate AI techniques can be applied. This approach enables rapid modelling where new research initiatives can build on existing work.

  20. Associations Between Home Death and the Use and Type of Care at Home.

    Science.gov (United States)

    McEwen, Rebecca; Asada, Yukiko; Burge, Frederick; Lawson, Beverley

    2018-01-01

    Despite wishes for and benefits of home deaths, a discrepancy between preferred and actual location of death persists. Provision of home care may be an effective policy response to support home deaths. Using the population-based mortality follow-back study conducted in Nova Scotia, we investigated the associations between home death and formal care at home and between home death and the type of formal care at home. We found (1) the use of formal care at home at the end of life was associated with home death and (2) the use of formal home support services at home was associated with home death among those whose symptoms were well managed.

  1. The Nest Home

    Energy Technology Data Exchange (ETDEWEB)

    Pickerill, Heath [Missouri Univ. of Science and Technology, Rolla, MO (United States)

    2016-07-11

    The purpose of the project was to build a competitive solar-powered house for the U.S. Department of Energy Solar Decathlon 2015 held in Irvine, California. The house, named the Nest Home, was an innovative design that works with the environment to meet the needs of the occupants, identified as a growing family. Reused materials were instrumental in the design. Three refurbished shipping containers composed the primary structure of the house, creating an open floor plan that defies common architecture for container homes. The exterior siding was made of deconstructed shipping pallets collected locally. Other recycled products included carpet composed of discarded fishing nets, denim batting made of recycled blue jeans that outperform traditional fiberglass insulation in sound proofing and thermal resistance, and kitchen cabinets that were purchased used and refinished. Collectively these elements formed a well-balanced blend of modern design, comfort, and sustainability. The house was Missouri University of Science and Technology’s sixth entry in the DOE Solar Decathlon. Missouri S&T has been invited to compete in six of the seven decathlons held, more than any other university worldwide. The house was brought back to Rolla after the Decathlon in California where it has been placed in its permanent location on the S&T campus.

  2. Research on the model of home networking

    Science.gov (United States)

    Yun, Xiang; Feng, Xiancheng

    2007-11-01

    It is the research hotspot of current broadband network to combine voice service, data service and broadband audio-video service by IP protocol to transport various real time and mutual services to terminal users (home). Home Networking is a new kind of network and application technology which can provide various services. Home networking is called as Digital Home Network. It means that PC, home entertainment equipment, home appliances, Home wirings, security, illumination system were communicated with each other by some composing network technology, constitute a networking internal home, and connect with WAN by home gateway. It is a new network technology and application technology, and can provide many kinds of services inside home or between homes. Currently, home networking can be divided into three kinds: Information equipment, Home appliances, Communication equipment. Equipment inside home networking can exchange information with outer networking by home gateway, this information communication is bidirectional, user can get information and service which provided by public networking by using home networking internal equipment through home gateway connecting public network, meantime, also can get information and resource to control the internal equipment which provided by home networking internal equipment. Based on the general network model of home networking, there are four functional entities inside home networking: HA, HB, HC, and HD. (1) HA (Home Access) - home networking connects function entity; (2) HB (Home Bridge) Home networking bridge connects function entity; (3) HC (Home Client) - Home networking client function entity; (4) HD (Home Device) - decoder function entity. There are many physical ways to implement four function entities. Based on theses four functional entities, there are reference model of physical layer, reference model of link layer, reference model of IP layer and application reference model of high layer. In the future home network

  3. Home-based Healthcare Technology

    DEFF Research Database (Denmark)

    Verdezoto, Nervo

    of these systems target a specific treatment or condition and might not be sufficient to support the care management work at home. Based on a case study approach, my research investigates home-based healthcare practices and how they can inform future design of home-based healthcare technology that better account......Sustaining daily, unsupervised healthcare activities in non-clinical settings such as the private home can challenge, among others, older adults. To support such unsupervised care activities, an increasingly number of reminders and monitoring systems are being designed. However, most...

  4. Optimal taxation with home production

    OpenAIRE

    Olovsson, Conny

    2014-01-01

    Optimal taxes for Europe and the U.S. are derived in a realistically calibrated model in which agents buy consumption goods and services and use home capital and labor to produce household services. The optimal tax rate on services is substantially lower than the tax rate on goods. Specifically, the planner cannot tax home production directly and instead lowers the tax rate on market services to increase the relative price of home production. The optimal tax rate on the return to home capital...

  5. Home financing for new and existing energy efficient homes

    International Nuclear Information System (INIS)

    1994-01-01

    The video describes how the home federal savings and loan association in Rockford, IL, has developed lending standards for new conventional and solar homes. The nine standards are illustrated. The video follows the customer through the loan process, including an appraisal of energy efficient items, loan closing, and continuing education. The primary audience is savings and loan management personnel

  6. A reagentless electrochemiluminescent immunosensor for apurinic/apyrimidinic endonuclease 1 detection based on the new Ru(bpy){sub 3}{sup 2+}/bi-arginine system

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Min; Chai, Xi Deng Ya-Qin; Han, Jing; Gui, Guo-Feng; Yuan, Ruo, E-mail: yuanruo@swu.edu.cn; Zhuo, Ying, E-mail: yingzhuo@swu.edu.cn

    2014-10-10

    Highlights: • A reagentless ECL biosensor based on the new Ru(bpy){sub 3}{sup 2+}/bi-arginine system. • The successful preparation of bi-Arg/Au@Fe3O4–rGO as enhancer. • Using the APE-1 as target by the sandwich-type immunoassay format. - Abstract: Apurinic/apyrimidinic endonuclease 1 (APE-1), a kind of multifunctional protein widely-distributed in the body, plays an essential role in the DNA base excision repair and serves as multiple possible roles in the response of human cancer to radiotherapy and chemotherapy. In this work, an ultrasensitive solid-state electrochemiluminescence (ECL) immunosensor is designed to determine APE-1 based on the new Ru(bpy){sub 3}{sup 2+}/bi-arginine system. The bi-arginine (bi-Arg) is decorated on the Au nanoparticles functionalized magnetic Fe{sub 3}O{sub 4}/reduced graphene oxide (bi-Arg/Au@Fe{sub 3}O{sub 4}–rGO) according to the self-assembling and covalent cross-linking interaction to obtain the functionalized nanocomposite of bi-Arg/Au@Fe{sub 3}O{sub 4}–rGO. Herein, the bi-Arg/Au@Fe{sub 3}O{sub 4}–rGO plays not only an amplification label to enhance the ECL signal of Ru(bpy){sub 3}{sup 2+} due to the coreactant of bi-Arg but also an ideal nanocarrier to load numerous secondary antibody. Based on sandwich-type immunoassay format, this proposed method offers a linear range of 1.0 fg mL{sup −1}–5.0 pg mL{sup −1} and an estimated detection limit of 0.3 fg mL{sup −1} for the APE-1. Moreover, the reagentless ECL immunosensor also exhibits high sensitivity, excellent selectivity and good stability, which has greatly potential development and application in clinical diagnostics, immunology and biomedical research.

  7. The safe home project.

    Science.gov (United States)

    Arphorn, Sara; Jiraniratisai, Sopaphan; Rungtakul, Rungsri; Phutta, Nikom

    2011-12-01

    The Thai Health Promotion Foundation supported the Improvement of Quality of Life of Informal Workers project in Ban Luang District, Amphur Photaram, Ratchaburi Province. There were many informal workers in Ban Luang District. Sweet-crispy fish producers in Ban Luang were the largest group among the sweet-crispy fish producers in Thailand. This project was aimed at improving living and working conditions of informal workers, with a focus on the sweet-crispy fish group. Good practices of improved living and working conditions were used to help informal workers build safe, healthy and productive work environments. These informal workers often worked in substandard conditions and were exposed to various hazards in the working area. These hazards included risk of exposure to hot work environment, ergonomics-related injuries, chemical hazards, electrical hazards etc. Ergonomics problems were commonly in the sweet-crispy fish group. Unnatural postures such as prolonged sitting were performed dominantly. One hundred and fifty informal workers participated in this project. Occupational health volunteers were selected to encourage occupational health and safety in four groups of informal workers in 2009. The occupational health volunteers trained in 2008 were farmers, beauty salon workers and doll makers. The occupational health and safety knowledge is extended to a new informal worker group: sweet-crispy fish producer, in 2009. The occupational health and safety training for sweet-crispy fish group is conducted by occupational health volunteers. The occupational health volunteers increased their skills and knowledge assist in to make safe home and safe community through participatory oriented training. The improvement of living and working condition is conducted by using a modified WISH, Work Improvement for Safe Home, checklist. The plans of improvement were recorded. The informal workers showed improvement mostly on material handling and storage. The safe uses and safe

  8. Home geriatric physiological measurements.

    Science.gov (United States)

    Tamura, Toshiyo

    2012-10-01

    In an ageing society, the elderly can be monitored with numerous physiological, physical and passive devices. Sensors can be installed in the home for continuous mobility assistance and unobtrusive disease prevention. This review presents several modern sensors, which improve the quality of life and assist the elderly, disabled people and their caregivers. The main concept of geriatric sensors is that they are capable of providing assistance without limiting or disturbing the subject's daily routine, giving him or her greater comfort, pleasure and well-being. Furthermore, this review includes associated technologies of wearable/implantable monitoring systems and the 'smart-house' project. This review concludes by discussing future challenges of the future aged society.

  9. Avatars@Home

    Science.gov (United States)

    Morandell, Martin M.; Hochgatterer, Andreas; Wöckl, Bernhard; Dittenberger, Sandra; Fagel, Sascha

    Avatars are a common field of research for interfacing smart homes, especially for elderly people. The present study focuses on the usage of photo-realistic faces with different levels of movements (video, avatar and photo) as components of the graphical user interface (GUI) for Ambient Assisted Living (AAL) environments. Within a usability test, using the "Wizard of Oz" technique, these presentation modes were compared with a text and a voice only interface with users of the target groups: elderly people with (nMCI=12) and without (nMCI=12) Mild Cognitive Impairment (MCI). Results show that faces on the GUI were liked by both, elderly with and without cognitive restrictions. However, users' performance on executing tasks did not differ much between the different presentation modes.

  10. Home geriatric physiological measurements

    International Nuclear Information System (INIS)

    Tamura, Toshiyo

    2012-01-01

    In an ageing society, the elderly can be monitored with numerous physiological, physical and passive devices. Sensors can be installed in the home for continuous mobility assistance and unobtrusive disease prevention. This review presents several modern sensors, which improve the quality of life and assist the elderly, disabled people and their caregivers. The main concept of geriatric sensors is that they are capable of providing assistance without limiting or disturbing the subject's daily routine, giving him or her greater comfort, pleasure and well-being. Furthermore, this review includes associated technologies of wearable/implantable monitoring systems and the ‘smart-house’ project. This review concludes by discussing future challenges of the future aged society. (topical review)

  11. Broken Homes: Impact on Adolescents.

    Science.gov (United States)

    Koziey, Paul W.; Davies, Leigh

    1982-01-01

    Tends to support assertion that children from homes broken by separation, divorce, or death are less well-adjusted in terms of California Personality Inventory scales of self-control, socialization, femininity, and good impression, than children from intact homes. Age and sex were not found to be linked to the degree of maladjustment. (AH)

  12. Home parenteral nutrition in children

    International Nuclear Information System (INIS)

    Kalousova, J.; Rouskova, B.; Styblova, J.

    2011-01-01

    Parenteral nutrition delivered at home presents a major improvement in the quality of life of children dependent on long term parenteral nutrition. Indications, technical conditions, logistics, complications, prognosis of home parenteral nutrition as well as some health-care issues to be addressed by pediatric practitioner are summarized. (author)

  13. The HomePort System

    DEFF Research Database (Denmark)

    Brønsted, Jeppe Rørbæk; Skou, Arne Joachim; Madsen, Per Printz

    2010-01-01

    Residential gateways for home automation are prerequisites to obtain optimal exploitation of energy resources, and they also have the potential to provide a unified operation of various home devices and appliances. Although a number of protocol standards have been proposed, the number...

  14. Home Education: The Social Motivation

    Science.gov (United States)

    Beck, Christian W.

    2010-01-01

    Data from a Norwegian survey show correlation between a student's socially related problems at school and the parent's social motivation for home education. I argue that more time spent at school by a student could result in more socially related problems at school, which can explain an increase in social motivation for home education.

  15. Mapping standards for home networking

    NARCIS (Netherlands)

    Kaa, G. van de; Hartog, F.T.H. den; Vries, H.J. de

    2009-01-01

    In this study, we apply a step-by-step approach for the identification of standards for home networking. We develop a classification and we use this classification to categorize sixty-four (sets of) standards. By developing this categorization, we have brought order to the chaos of home networking

  16. Second-home electricity consumption

    DEFF Research Database (Denmark)

    Møller Andersen, Frits; Christensen, M.S.; Jensen, O.M.

    2008-01-01

    in electricity consumption. Luxury tourism use and senior citizens' that use a few per cent of the second homes as their home contribute to a minor degree to the overall increase of electricity consumption. Scenarios show that this development may accelerate with increased leisure time, increased use and more...

  17. Financing home care in Europe.

    NARCIS (Netherlands)

    Genet, N.; Gulácsi, L.; Boerma, W.; Hutchinson, A.; Garms-Homolova, V.; Naiditch, M.

    2010-01-01

    Introduction: Financial incentives are widely used to get better value for money. Incentives can be applied to authorities responsible for home care, or to agencies that provide services or to clients who receive care. Details of the financing system of home care services very much determine the

  18. 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.

  19. Acquiring taste in home economics?

    DEFF Research Database (Denmark)

    Stenbak Larsen, Christian

    Objective: To explore how home economics was taught in Denmark before the recent Danish school reform, which also revised the objectives and content of home economics, naming it Food Knowledge (Madkundskab) Methods: Participant observation was done in home economic lessons in two case schools...... appreciated by the group of boys, and others again learned to stick with their idiosyncrasies when pressured by the teacher. Conclusions: Children were acquiring taste in the home economic lessons, but not only the kind of tastes that the teacher had planned for. This leads to reflections on the very complex...... process of taste acquiring and to a call for further research into taste acquiring in complex real life contexts as home economics lessons....

  20. Cost-effectiveness implications based on a comparison of nursing home and home health case mix.

    OpenAIRE

    Kramer, A M; Shaughnessy, P W; Pettigrew, M L

    1985-01-01

    Case-mix differences between 653 home health care patients and 650 nursing home patients, and between 455 Medicare home health patients and 447 Medicare nursing home patients were assessed using random samples selected from 20 home health agencies and 46 nursing homes in 12 states in 1982 and 1983. Home health patients were younger, had shorter lengths of stay, and were less functionally disabled than nursing home patients. Traditional long-term care problems requiring personal care were more...

  1. Foraging optimally for home ranges

    Science.gov (United States)

    Mitchell, Michael S.; Powell, Roger A.

    2012-01-01

    Economic models predict behavior of animals based on the presumption that natural selection has shaped behaviors important to an animal's fitness to maximize benefits over costs. Economic analyses have shown that territories of animals are structured by trade-offs between benefits gained from resources and costs of defending them. Intuitively, home ranges should be similarly structured, but trade-offs are difficult to assess because there are no costs of defense, thus economic models of home-range behavior are rare. We present economic models that predict how home ranges can be efficient with respect to spatially distributed resources, discounted for travel costs, under 2 strategies of optimization, resource maximization and area minimization. We show how constraints such as competitors can influence structure of homes ranges through resource depression, ultimately structuring density of animals within a population and their distribution on a landscape. We present simulations based on these models to show how they can be generally predictive of home-range behavior and the mechanisms that structure the spatial distribution of animals. We also show how contiguous home ranges estimated statistically from location data can be misleading for animals that optimize home ranges on landscapes with patchily distributed resources. We conclude with a summary of how we applied our models to nonterritorial black bears (Ursus americanus) living in the mountains of North Carolina, where we found their home ranges were best predicted by an area-minimization strategy constrained by intraspecific competition within a social hierarchy. Economic models can provide strong inference about home-range behavior and the resources that structure home ranges by offering falsifiable, a priori hypotheses that can be tested with field observations.

  2. ThinkHome Energy Efficiency in Future Smart Homes

    Directory of Open Access Journals (Sweden)

    Reinisch Christian

    2011-01-01

    Full Text Available Abstract Smart homes have been viewed with increasing interest by both home owners and the research community in the past few years. One reason for this development is that the use of modern automation technology in the home or building promises considerable savings of energy, therefore, simultaneously reducing the operational costs of the building over its whole lifecycle. However, the full potential of smart homes still lies fallow, due to the complexity and diversity of the systems, badly engineered and configured installations, as well as the frequent problem of suboptimal control strategies. Summarized, these problems converge to two undesirable conditions in the "not-so-smart" home: energy consumption is still higher than actually necessary and users are unable to yield full comfort in their automated homes. This work puts its focus on alleviating the current problems by proposing a comprehensive system concept, that shall ensure that smart homes can keep their promise in the future. The system operates on an extensive knowledge base that stores all information needed to fulfill the goals of energy efficiency and user comfort. Its intelligence is implemented as and within a multiagent system that also caters for the system's openness to the outside world. As a first evaluation, a profile-based control strategy for thermal comfort is developed and verified by means of simulation.

  3. 24 CFR 982.612 - Group home: State approval of group home.

    Science.gov (United States)

    2010-04-01

    ... 24 Housing and Urban Development 4 2010-04-01 2010-04-01 false Group home: State approval of group... Types Group Home § 982.612 Group home: State approval of group home. A group home must be licensed..., Retardation, or Social Services) as a group home for elderly persons or persons with disabilities. ...

  4. FastStats: Home Health Care

    Science.gov (United States)

    ... this? Submit What's this? Submit Button NCHS Home Home Health Care Recommend on Facebook Tweet Share Compartir Data are ... National Study of Long-Term Care Providers Nursing Home Care Residential Care Communities Centers for Medicare and Medicaid ...

  5. FastStats: Nursing Home Care

    Science.gov (United States)

    ... Submit What's this? Submit Button NCHS Home Nursing Home Care Recommend on Facebook Tweet Share Compartir Data are ... Person’s Health Related Links Adult Day Services Centers Home Health Care Hospice Care National Study of Long-Term Care ...

  6. Beyond Home Automation: Designing More Effective Smart Home Systems

    OpenAIRE

    Carner, Paolo

    2009-01-01

    This paper outlines a Smart Home Proof-of-Concept system that uses a Bayesian Network to predict the likelihood of a monitored event to occur. Firstly, this paper will provide an introduction to the concept of a smart home system; then it will outline how Artificial Intelligence concepts can be used to make such systems more effective. Finally, it will detail the implementation of a smart home system, which uses an inference engine to determine the likelihood of a fire. The system prototype h...

  7. "Home is always home" : (former) street youth in Blantyre, Malawi, and the fluidity of constructing home

    NARCIS (Netherlands)

    Hendriks, T.D.

    2016-01-01

    For many Malawians the concept of home is strongly associated with the rural areas and one's (supposedly rural) place of birth. This 'grand narrative about home', though often reiterated, doesn't necessarily depict lived reality. Malawi's history of movement and labor migration coupled with

  8. "Home is always home" : (former) street youth in Blantyre, Malawi, and the fluidity of constructing home

    NARCIS (Netherlands)

    Hendriks, T.D.

    2017-01-01

    For many Malawians the concept of home is strongly associated with the rural areas and one's (supposedly rural) place of birth. This 'grand narrative about home', though often reiterated, doesn't necessarily depict lived reality. Malawi's history of movement and labor migration coupled with

  9. Achieving Improvement Through Nursing Home Quality Measurement

    OpenAIRE

    Harris, Yael; Clauser, Steven B.

    2002-01-01

    CMS has initiated the Nursing Home Quality Initiative (NHQI) to improve the quality of nursing home care. Central to the NHQI is the public reporting of nursing home quality measures that serve as the basis for the Initiative's communication and quality improvement program. This article provides an overview of the NHQI, focusing on the role of nursing home quality measures in achieving improvements in nursing home care. We also describe the evolution of quality measurement in nursing homes, a...

  10. SMS security system for smart home detectors

    OpenAIRE

    Cekova, Katerina; Gelev, Saso

    2016-01-01

    Security has always been an important problem everywhere. Home security has been a major issue where crime is increasing and everybody wants home security to protect the home. Safety from theft and flame are the most important requirements of a home security system for people. A remote home security system offers many benefits from keeping homeowners, and their property safe. This paper present controlling of the home security system remotely from Android Application. Owners can turn on or...

  11. The Home as aural Haven

    Directory of Open Access Journals (Sweden)

    Rowland Atkinson

    2008-12-01

    Full Text Available The home is not only a place of physical refuge from the elements and unwanted intruders. It is also a refuge from unwanted sound, a place where we can enjoy calm spaces that permit quiet reflection. Our homes, as discrete and personally modifiable micro-soundscapes, offer a kind of environmental nourishment to us as social beings. Noise in the home – from neighbours, roads, airports, air conditioners, parties – unsettles our sense of control, autonomy and authorship over what is our primary territorial domain.

  12. Bandwidth Reservations in Home Networks

    DEFF Research Database (Denmark)

    Nelis, Jelle; Verslype, Dieter; Develder, Chris

    2010-01-01

    In order for service providers to provide their users high quality services in the home network, Quality of Service (QoS) provisioning is needed to protect premium services. In this paper, we describe how a Universal Plug-and-Play (UPnP) based home network architecture solves this problem...... in a heterogeneous home network. We outline how it both relieves the end user from troublesome configuration and still offers control to the service provider. We particularly present performance assessment results for UPnP-QoS v3, based on a fully operational experimental implementation. The quantitative measurement...

  13. Genetics Home Reference: Parkinson disease

    Science.gov (United States)

    ... Email Facebook Twitter Home Health Conditions Parkinson disease Parkinson disease Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Parkinson disease is a progressive disorder of the nervous ...

  14. Fall prevention in nursing homes

    DEFF Research Database (Denmark)

    Andresen, Mette; Hauge, Johnny

    2014-01-01

    that the number of hospitalization after a fall injury will become an even greater task for the Danish hospitals, The aim of the study was to show if there is a relationship between physically frail elderly nursing home resident’s subjective evaluation of fall-risk and an objective evaluation of their balance....... Further, to suggest tools for fall prevention in nursing home settings on the basis of the results of this study and the literature. A quantitative method inspired by the survey method was used to give an overview of fall patterns, subjective and objective evaluations of fallrisk. Participants were 16...... physically frail elderly nursing home residents from three different nursing homes. Measures: a small staff-questionnaire about incidences and places where the participants had falling-episodes during a 12 month period, The Falls Effi cacy Scale Swedish version (FES(S)) and Berg Balance Scale (BBS) Results...

  15. Eldercare at Home: Communication Problems

    Science.gov (United States)

    ... Join our e-newsletter! Resources Eldercare at Home: Communication Problems Caregiving How Tos Understanding the Problem Communication ... when the older person is most alert. Talk face-to-face and maintain eye contact. A good way to ...

  16. Genetics Home Reference: Waardenburg syndrome

    Science.gov (United States)

    ... Email Facebook Twitter Home Health Conditions Waardenburg syndrome Waardenburg syndrome Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Waardenburg syndrome is a group of genetic conditions that can ...

  17. Genetics Home Reference: Cole disease

    Science.gov (United States)

    ... Email Facebook Twitter Home Health Conditions Cole disease Cole disease Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Cole disease is a disorder that affects the skin. People ...

  18. Home childbirth: progress or retrocession?

    Directory of Open Access Journals (Sweden)

    Clara Fróes de Oliveira SANFELICE

    Full Text Available Giving birth at home represents a rising modality of delivery care in the Brazilian society, although in unrepresentative proportion when compared to the number of hospital childbirths. In Brazil, the topic has been broadly discussed by different professional categories, highlighting the safety issue involved in the process. The aim of this theoretical and reflective study was to present a brief overview of the overall care related to home childbirth, also questioning the reality of the contemporary Brazilian obstetric scenario. The scientific literature presents both obstetric and neonatal outcomes as favorable to home childbirth; similar risks when compared to hospital childbirth and higher rates of maternal satisfaction, and these both factors justify its practice. Therefore, a movement of women who are deeply unhappy with the current model of obstetric care is currently observed and they have been opting for home childbirth as a response to institutional violence, fragmentation and depersonalization of hospital care.

  19. Genetics Home Reference: lung cancer

    Science.gov (United States)

    ... Share: Email Facebook Twitter Home Health Conditions Lung cancer Lung cancer Printable PDF Open All Close All Enable Javascript ... cancer, childhood Additional NIH Resources (3 links) National Cancer Institute: Lung Cancer Overview National Cancer Institute: Lung Cancer Prevention ...

  20. Genetics Home Reference: boomerang dysplasia

    Science.gov (United States)

    ... Email Facebook Twitter Home Health Conditions Boomerang dysplasia Boomerang dysplasia Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Boomerang dysplasia is a disorder that affects the development ...

  1. Home automation with Intel Galileo

    CERN Document Server

    Dundar, Onur

    2015-01-01

    This book is for anyone who wants to learn Intel Galileo for home automation and cross-platform software development. No knowledge of programming with Intel Galileo is assumed, but knowledge of the C programming language is essential.

  2. Genetics Home Reference: hereditary pancreatitis

    Science.gov (United States)

    ... Facebook Twitter Home Health Conditions Hereditary pancreatitis Hereditary pancreatitis Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Hereditary pancreatitis is a genetic condition characterized by recurrent episodes ...

  3. Genetics Home Reference: bipolar disorder

    Science.gov (United States)

    ... Email Facebook Twitter Home Health Conditions Bipolar disorder Bipolar disorder Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Bipolar disorder is a mental health condition that causes extreme ...

  4. Genetics Home Reference: rheumatoid arthritis

    Science.gov (United States)

    ... Share: Email Facebook Twitter Home Health Conditions Rheumatoid arthritis Rheumatoid arthritis Printable PDF Open All Close All Enable ... in my area? Other Names for This Condition arthritis, rheumatoid RA Related Information How are genetic conditions and ...

  5. Genetics Home Reference: clopidogrel resistance

    Science.gov (United States)

    ... Facebook Twitter Home Health Conditions Clopidogrel resistance Clopidogrel resistance Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Clopidogrel resistance is a condition in which the drug clopidogrel ...

  6. Laffer Curves and Home Production

    Directory of Open Access Journals (Sweden)

    Kotamäki Mauri

    2017-06-01

    Full Text Available In the earlier related literature, consumption tax rate Laffer curve is found to be strictly increasing (see Trabandt and Uhlig (2011. In this paper, a general equilibrium macro model is augmented by introducing a substitute for private consumption in the form of home production. The introduction of home production brings about an additional margin of adjustment – an increase in consumption tax rate not only decreases labor supply and reduces the consumption tax base but also allows a substitution of market goods with home-produced goods. The main objective of this paper is to show that, after the introduction of home production, the consumption tax Laffer curve exhibits an inverse U-shape. Also the income tax Laffer curves are significantly altered. The result shown in this paper casts doubt on some of the earlier results in the literature.

  7. Drinking Water in your Home

    Science.gov (United States)

    Many people choose to filter or test the drinking water that comes out of their tap or from their private well for a variety of reasons. And whether at home, at work or while traveling, many Americans drink bottled water.

  8. Genetics Home Reference: familial candidiasis

    Science.gov (United States)

    ... Facebook Twitter Home Health Conditions Familial candidiasis Familial candidiasis Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Familial candidiasis is an inherited tendency to develop infections caused ...

  9. Preventing Burns in Your Home

    Science.gov (United States)

    ... clothing when you handle chemicals. Store chemicals, including gasoline, out of the reach of children. To prevent ... mild burn? What is the treatment for smoke inhalation? Resources American Red Cross, Home Fire Safety Centers ...

  10. Genetics Home Reference: Turner syndrome

    Science.gov (United States)

    ... Email Facebook Twitter Home Health Conditions Turner syndrome Turner syndrome Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Turner syndrome is a chromosomal condition that affects development in ...

  11. [Home childbirth: progress or retrocession?].

    Science.gov (United States)

    de Sanfelice, Clara Fróes Oliveira; Shimo, Antonieta Keiko Kakuda

    2014-03-01

    Giving birth at home represents a rising modality of delivery care in the Brazilian society, although in unrepresentative proportion when compared to the number of hospital childbirths. In Brazil, the topic has been broadly discussed by different professional categories, highlighting the safety issue involved in the process. The aim of this theoretical and reflective study was to present a brief overview of the overall care related to home childbirth, also questioning the reality of the contemporary Brazilian obstetric scenario. The scientific literature presents both obstetric and neonatal outcomes as favorable to home childbirth; similar risks when compared to hospital childbirth and higher rates of maternal satisfaction, and these both factors justify its practice. Therefore, a movement of women who are deeply unhappy with the current model of obstetric care is currently observed and they have been opting for home childbirth as a response to institutional violence,fragmentation and depersonalization of hospital care.

  12. Genetics Home Reference: essential tremor

    Science.gov (United States)

    ... Facebook Twitter Home Health Conditions Essential tremor Essential tremor Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Essential tremor is a movement disorder that causes involuntary, rhythmic ...

  13. Genetics Home Reference: gestational diabetes

    Science.gov (United States)

    ... Email Facebook Twitter Home Health Conditions Gestational diabetes Gestational diabetes Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Gestational diabetes is a disorder characterized by abnormally high blood ...

  14. Genetics Home Reference: beta thalassemia

    Science.gov (United States)

    ... Facebook Twitter Home Health Conditions Beta thalassemia Beta thalassemia Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Beta thalassemia is a blood disorder that reduces the production ...

  15. Genetics Home Reference: alpha thalassemia

    Science.gov (United States)

    ... Facebook Twitter Home Health Conditions Alpha thalassemia Alpha thalassemia Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Alpha thalassemia is a blood disorder that reduces the production ...

  16. Genetics Home Reference: antiphospholipid syndrome

    Science.gov (United States)

    ... Share: Email Facebook Twitter Home Health Conditions Antiphospholipid syndrome Antiphospholipid syndrome Printable PDF Open All Close All Enable ... area? Other Names for This Condition anti-phospholipid syndrome antiphospholipid antibody syndrome Hughes syndrome Related Information How are ...

  17. Advances in Home Photo Printing

    Institute of Scientific and Technical Information of China (English)

    Qian Lin; Brian Atkins; Huitao Luo

    2004-01-01

    With digital camera adoptions going main stream, consumers capture a record number of photos.Currently, the majority of the digital photos are printed at home. One of the key enablers of this transformation is the advancement of home photo printing technologies. In the past few years, inkjet printing technologies have continued to deliver smaller drop size, larger number of inks, and longer-lasting prints. In the mean time, advanced image processing automatically enhances captured digital photos while being printed. The combination of the above two forces has closed the gap between the home photo prints and AgX prints. It will give an overview of the home photo printing market and technology trends, and discuss major advancements in automatic image processing.

  18. Genetics Home Reference: opioid addiction

    Science.gov (United States)

    ... Facebook Twitter Home Health Conditions Opioid addiction Opioid addiction Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Opioid addiction is a long-lasting (chronic) disease that can ...

  19. Genetics Home Reference: systemic scleroderma

    Science.gov (United States)

    ... Facebook Twitter Home Health Conditions Systemic scleroderma Systemic scleroderma Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Systemic scleroderma is an autoimmune disorder that affects the skin ...

  20. Genetics Home Reference: polycythemia vera

    Science.gov (United States)

    ... Email Facebook Twitter Home Health Conditions Polycythemia vera Polycythemia vera Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Polycythemia vera is a condition characterized by an increased ...

  1. Genetics Home Reference: Usher syndrome

    Science.gov (United States)

    ... Email Facebook Twitter Home Health Conditions Usher syndrome Usher syndrome Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Usher syndrome is a condition characterized by partial or total ...

  2. Genetics Home Reference: Alzheimer disease

    Science.gov (United States)

    ... Email Facebook Twitter Home Health Conditions Alzheimer disease Alzheimer disease Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Alzheimer disease is a degenerative disease of the brain ...

  3. Genetics Home Reference: myasthenia gravis

    Science.gov (United States)

    ... increase the risk of myasthenia gravis , but the identity of these genes is unknown. Many factors likely ... Myasthenia Gravis Fact Sheet MalaCards: myasthenia gravis Merck Manual Home Health Handbook for Patients and Caregivers Myasthenia ...

  4. Genetics Home Reference: congenital hypothyroidism

    Science.gov (United States)

    ... Facebook Twitter Home Health Conditions Congenital hypothyroidism Congenital hypothyroidism Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Congenital hypothyroidism is a partial or complete loss of function ...

  5. Genetics Home Reference: breast cancer

    Science.gov (United States)

    ... Email Facebook Twitter Home Health Conditions Breast cancer Breast cancer Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Breast cancer is a disease in which certain cells in ...

  6. Genetics Home Reference: Norrie disease

    Science.gov (United States)

    ... Email Facebook Twitter Home Health Conditions Norrie disease Norrie disease Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Norrie disease is an inherited eye disorder that leads to ...

  7. Genetics Home Reference: Bartter syndrome

    Science.gov (United States)

    ... Email Facebook Twitter Home Health Conditions Bartter syndrome Bartter syndrome Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Bartter syndrome is a group of very similar kidney disorders ...

  8. Genetics Home Reference: Hashimoto thyroiditis

    Science.gov (United States)

    ... Facebook Twitter Home Health Conditions Hashimoto thyroiditis Hashimoto thyroiditis Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Hashimoto thyroiditis is a condition that affects the function of ...

  9. Genetics Home Reference: warfarin sensitivity

    Science.gov (United States)

    ... Email Facebook Twitter Home Health Conditions Warfarin sensitivity Warfarin sensitivity Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Warfarin sensitivity is a condition in which individuals have ...

  10. Genetics Home Reference: warfarin resistance

    Science.gov (United States)

    ... Email Facebook Twitter Home Health Conditions Warfarin resistance Warfarin resistance Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Warfarin resistance is a condition in which individuals have ...

  11. Genetics Home Reference: amelogenesis imperfecta

    Science.gov (United States)

    ... Email Facebook Twitter Home Health Conditions Amelogenesis imperfecta Amelogenesis imperfecta Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Amelogenesis imperfecta is a disorder of tooth development. This ...

  12. Genetics Home Reference: Graves disease

    Science.gov (United States)

    ... Email Facebook Twitter Home Health Conditions Graves disease Graves disease Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Graves disease is a condition that affects the function of ...

  13. Asteroids@Home

    Science.gov (United States)

    Durech, Josef; Hanus, J.; Vanco, R.

    2012-10-01

    We present a new project called Asteroids@home (http://asteroidsathome.net/boinc). It is a volunteer-computing project that uses an open-source BOINC (Berkeley Open Infrastructure for Network Computing) software to distribute tasks to volunteers, who provide their computing resources. The project was created at the Astronomical Institute, Charles University in Prague, in cooperation with the Czech National Team. The scientific aim of the project is to solve a time-consuming inverse problem of shape reconstruction of asteroids from sparse-in-time photometry. The time-demanding nature of the problem comes from the fact that with sparse-in-time photometry the rotation period of an asteroid is not apriori known and a huge parameter space must be densely scanned for the best solution. The nature of the problem makes it an ideal task to be solved by distributed computing - the period parameter space can be divided into small bins that can be scanned separately and then joined together to give the globally best solution. In the framework of the the project, we process asteroid photometric data from surveys together with asteroid lightcurves and we derive asteroid shapes and spin states. The algorithm is based on the lightcurve inversion method developed by Kaasalainen et al. (Icarus 153, 37, 2001). The enormous potential of distributed computing will enable us to effectively process also the data from future surveys (Large Synoptic Survey Telescope, Gaia mission, etc.). We also plan to process data of a synthetic asteroid population to reveal biases of the method. In our presentation, we will describe the project, show the first results (new models of asteroids), and discuss the possibilities of its further development. This work has been supported by the grant GACR P209/10/0537 of the Czech Science Foundation and by the Research Program MSM0021620860 of the Ministry of Education of the Czech Republic.

  14. Coming Home at Paranal

    Science.gov (United States)

    2002-02-01

    Unique "Residencia" Opens at the VLT Observatory Summary The Paranal Residencia at the ESO VLT Observatory is now ready and the staff and visitors have moved into their new home. This major architectural project has the form of a unique subterranean construction with a facade opening towards the Pacific Ocean , far below at a distance of about 12 km. Natural daylight is brought into the building through a 35-m wide glass-covered dome, a rectangular courtyard roof and various skylight hatches. Located in the middle of the Atacama Desert, one of the driest areas on Earth, the Residencia incorporates a small garden and a swimming pool, allowing the inhabitants to retreat from time to time from the harsh outside environment. Returning from long shifts at the VLT and other installations on the mountain, here they can breathe moist air and receive invigorating sensory impressions. With great originality of the design, it has been possible to create an interior with a feeling of open space - this is a true "home in the desert" . Moreover, with strict ecological power, air and water management , the Paranal Residencia has already become a symbol of innovative architecture in its own right. Constructed with robust, but inexpensive materials, it is an impressively elegant and utilitarian counterpart to the VLT high-tech facilities poised some two hundred meters above, on the top of the mountain. PR Photo 05a/02 : Aerial view of the Paranal Observatory area. PR Photo 05b/02 : Aerial view of the Paranal Residencia . PR Photo 05c/02 : Outside view of the Paranal Residencia . PR Photo 05d/02 : The Entry Hall (fisheye view). PR Photo 05e/02 : The Entry Hall with garden and pool. PR Photo 05f/02 : The Reception Area . PR Photo 05g/02 : The Reception Area - decoration. PR Photo 05h/02 : The Reception Area - decoration. PR Photo 05i/02 : The Reception Area - decoration. PR Photo 05j/02 : View towards the Cantine . PR Photo 05k/02 : View towards the Kitchen . PR Photo 05l/02 : View

  15. DOE Zero Energy Ready Home Case Study: Sterling Brook Custom Homes — Village Park Eco Home, Double Park, TX

    Energy Technology Data Exchange (ETDEWEB)

    none,

    2014-09-01

    This builder won a Custom Builder honor in the 2014 Housing Innovation Awards for this showcase home that serves as an energy-efficient model home for the custom home builder: 1,300 visitors toured the home, thousands more learned about the home’s advanced construction via the webpage, YouTube, Twitter, Facebook, Instagram, and Pinterest.

  16. Religious Attitudes and Home Bias

    OpenAIRE

    C. Reggiani; G. Rossini

    2008-01-01

    Home bias affects trade in goods, services and financial assets. It is mostly generated by "natural" trade barriers. Among these dividers we may list many behavioral and sociological factors, such as status quo biases and a few kind of ‘embeddedness’. Unfortunately these factors are difficult to measure. An important part of ‘embeddedness’ may be related to religious attitudes. Is there any relation between economic home bias and religious attitudes at the individual tier? Our aim is to provi...

  17. The energy aware smart home

    OpenAIRE

    Jahn, M.; Jentsch, M.; Prause, C.R.; Pramudianto, F.; Al-Akkad, A.; Reiners, R.

    2010-01-01

    In this paper, we present a novel smart home system integrating energy efficiency features. The smart home application is built on top of Hydra, a middleware framework that facilitates the intelligent communication of heterogeneous embedded devices through an overlay P2P network. We interconnect common devices available in private households and integrate wireless power metering plugs to gain access to energy consumption data. These data are used for monitoring and analyzing consumed energy o...

  18. [Organising a successful return home].

    Science.gov (United States)

    Mézière, Anthony

    Discharge from hospital is a major component of the quality and efficiency of the healthcare system. The failures of the return home of elderly people testify to the difficulties of applying guidelines in the area of hospital discharge. The action plan decided in the hospital for a successful return home can be jeopardised for personal, relational, functional and structural reasons originating from the different players involved in the hospital discharge. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  19. Home automation in the workplace.

    Science.gov (United States)

    McCormack, J E; Tello, S F

    1994-01-01

    Environmental control units and home automation devices contribute to the independence and potential of individuals with disabilities, both at work and at home. Devices currently exist that can assist people with physical, cognitive, and sensory disabilities to control lighting, appliances, temperature, security, and telephone communications. This article highlights several possible applications for these technologies and discusses emerging technologies that will increase the benefits these devices offer people with disabilities.

  20. Informatization of home care processes

    OpenAIRE

    Vidmar, Miha

    2014-01-01

    This thesis describes the complete server side system for computerisation of home care service. The solution is built as an intermediate layer between the mobile application and the existing backend of the health institution. It consists of three main applications: custody application, web service for integration with the mobile application, and web service for integration with the backend of the health institution. It supports working with the users (nurses in home care) and work orders whic...