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Sample records for dna restriction systems

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

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    Ramakrishnan eSitaraman

    2016-01-01

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

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

    Science.gov (United States)

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

    2006-02-01

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

  3. Comparative Genomics Reveals the Diversity of Restriction-Modification Systems and DNA Methylation Sites in Listeria monocytogenes.

    Science.gov (United States)

    Chen, Poyin; den Bakker, Henk C; Korlach, Jonas; Kong, Nguyet; Storey, Dylan B; Paxinos, Ellen E; Ashby, Meredith; Clark, Tyson; Luong, Khai; Wiedmann, Martin; Weimer, Bart C

    2017-02-01

    Listeria monocytogenes is a bacterial pathogen that is found in a wide variety of anthropogenic and natural environments. Genome sequencing technologies are rapidly becoming a powerful tool in facilitating our understanding of how genotype, classification phenotypes, and virulence phenotypes interact to predict the health risks of individual bacterial isolates. Currently, 57 closed L. monocytogenes genomes are publicly available, representing three of the four phylogenetic lineages, and they suggest that L. monocytogenes has high genomic synteny. This study contributes an additional 15 closed L. monocytogenes genomes that were used to determine the associations between the genome and methylome with host invasion magnitude. In contrast to previous findings, large chromosomal inversions and rearrangements were detected in five isolates at the chromosome terminus and within rRNA genes, including a previously undescribed inversion within rRNA-encoding regions. Each isolate's epigenome contained highly diverse methyltransferase recognition sites, even within the same serotype and methylation pattern. Eleven strains contained a single chromosomally encoded methyltransferase, one strain contained two methylation systems (one system on a plasmid), and three strains exhibited no methylation, despite the occurrence of methyltransferase genes. In three isolates a new, unknown DNA modification was observed in addition to diverse methylation patterns, accompanied by a novel methylation system. Neither chromosome rearrangement nor strain-specific patterns of epigenome modification observed within virulence genes were correlated with serotype designation, clonal complex, or in vitro infectivity. These data suggest that genome diversity is larger than previously considered in L. monocytogenes and that as more genomes are sequenced, additional structure and methylation novelty will be observed in this organism. Listeria monocytogenes is the causative agent of listeriosis, a disease

  4. 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

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

    OpenAIRE

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

    2007-01-01

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

  6. DNA replication stress restricts ribosomal DNA copy number

    Science.gov (United States)

    Salim, Devika; Bradford, William D.; Freeland, Amy; Cady, Gillian; Wang, Jianmin

    2017-01-01

    Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100–200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how “normal” copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a “normal” rDNA copy number. PMID:28915237

  7. DNA replication stress restricts ribosomal DNA copy number.

    Science.gov (United States)

    Salim, Devika; Bradford, William D; Freeland, Amy; Cady, Gillian; Wang, Jianmin; Pruitt, Steven C; Gerton, Jennifer L

    2017-09-01

    Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100-200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how "normal" copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a "normal" rDNA copy number.

  8. DNA replication stress restricts ribosomal DNA copy number.

    Directory of Open Access Journals (Sweden)

    Devika Salim

    2017-09-01

    Full Text Available Ribosomal RNAs (rRNAs in budding yeast are encoded by ~100-200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how "normal" copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a "normal" rDNA copy number.

  9. 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

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

    Science.gov (United States)

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

    2014-01-01

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

  11. Restriction-modification systems in Mycoplasma spp

    Directory of Open Access Journals (Sweden)

    Marcelo Brocchi

    2007-01-01

    Full Text Available Restriction and Modification (R-M systems are present in all Mycoplasma species sequenced so far. The presence of these genes poses barriers to gene transfer and could protect the cell against phage infections. The number and types of R-M genes between different Mycoplasma species are variable, which is characteristic of a polymorphism. The majority of the CDSs code for Type III R-M systems and particularly for methyltransferase enzymes, which suggests that functions other than the protection against the invasion of heterologous DNA may exist. A possible function of these enzymes could be the protection against the invasion of other but similar R-M systems. In Mycoplasma hyopneumoniae strain J, three of the putative methyltransferase genes were clustered in a region forming a genomic island. Many R-M CDSs were mapped in the vicinity of transposable elements suggesting an association between these genes and reinforcing the idea of R-M systems as mobile selfish DNA. Also, many R-M genes present repeats within their coding sequences, indicating that their expression is under the control of phase variation mechanisms. Altogether, these data suggest that R-M systems are a remarkable characteristic of Mycoplasma species and are probably involved in the adaptation of these bacteria to different environmental conditions.

  12. Cut-and-Paste of DNA Using an Artificial Restriction DNA Cutter

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    Makoto Komiyama

    2013-02-01

    Full Text Available DNA manipulations using a completely chemistry-based DNA cutter (ARCUT have been reviewed. This cutter, recently developed by the authors, is composed of Ce(IV/EDTA complex and two strands of pseudo-complementary peptide nucleic acid. The site-selective scission proceeds via hydrolysis of targeted phosphodiester linkages, so that the resultant scission fragments can be easily ligated with other fragments by using DNA ligase. Importantly, scission-site and site-specificity of the cutter are freely tuned in terms of the Watson–Crick rule. Thus, when one should like to manipulate DNA according to the need, he or she does not have to think about (1 whether appropriate “restriction enzyme sites” exist near the manipulation site and (2 whether the site-specificity of the restriction enzymes, if any, are sufficient to cut only the aimed position without chopping the DNA at non-targeted sites. Even the human genome can be manipulated, since ARCUT can cut the genome at only one predetermined site. Furthermore, the cutter is useful to promote homologous recombination in human cells, converting a site to desired sequence. The ARCUT-based DNA manipulation should be promising for versatile applications.

  13. Interplay between Ku and Replication Protein A in the Restriction of Exo1-mediated DNA Break End Resection*

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    Krasner, Danielle S.; Daley, James M.; Sung, Patrick; Niu, Hengyao

    2015-01-01

    DNA double-strand breaks can be eliminated via non-homologous end joining or homologous recombination. Non-homologous end joining is initiated by the association of Ku with DNA ends. In contrast, homologous recombination entails nucleolytic resection of the 5′-strands, forming 3′-ssDNA tails that become coated with replication protein A (RPA). Ku restricts end access by the resection nuclease Exo1. It is unclear how partial resection might affect Ku engagement and Exo1 restriction. Here, we addressed these questions in a reconstituted system with yeast proteins. With blunt-ended DNA, Ku protected against Exo1 in a manner that required its DNA end-binding activity. Despite binding poorly to ssDNA, Ku could nonetheless engage a 5′-recessed DNA end with a 40-nucleotide (nt) ssDNA overhang, where it localized to the ssDNA-dsDNA junction and efficiently blocked resection by Exo1. Interestingly, RPA could exclude Ku from a partially resected structure with a 22-nt ssDNA tail and thus restored processing by Exo1. However, at a 40-nt tail, Ku remained stably associated at the ssDNA-dsDNA junction, and RPA simultaneously engaged the ssDNA region. We discuss a model in which the dynamic equilibrium between Ku and RPA binding to a partially resected DNA end influences the timing and efficiency of the resection process. PMID:26067273

  14. A Mimicking-of-DNA-Methylation-Patterns Pipeline for Overcoming the Restriction Barrier of Bacteria

    Science.gov (United States)

    Zhang, Guoqiang; Wang, Wenzhao; Deng, Aihua; Sun, Zhaopeng; Zhang, Yun; Liang, Yong; Che, Yongsheng; Wen, Tingyi

    2012-01-01

    Genetic transformation of bacteria harboring multiple Restriction-Modification (R-M) systems is often difficult using conventional methods. Here, we describe a mimicking-of-DNA-methylation-patterns (MoDMP) pipeline to address this problem in three difficult-to-transform bacterial strains. Twenty-four putative DNA methyltransferases (MTases) from these difficult-to-transform strains were cloned and expressed in an Escherichia coli strain lacking all of the known R-M systems and orphan MTases. Thirteen of these MTases exhibited DNA modification activity in Southwestern dot blot or Liquid Chromatography–Mass Spectrometry (LC–MS) assays. The active MTase genes were assembled into three operons using the Saccharomyces cerevisiae DNA assembler and were co-expressed in the E. coli strain lacking known R-M systems and orphan MTases. Thereafter, results from the dot blot and restriction enzyme digestion assays indicated that the DNA methylation patterns of the difficult-to-transform strains are mimicked in these E. coli hosts. The transformation of the Gram-positive Bacillus amyloliquefaciens TA208 and B. cereus ATCC 10987 strains with the shuttle plasmids prepared from MoDMP hosts showed increased efficiencies (up to four orders of magnitude) compared to those using the plasmids prepared from the E. coli strain lacking known R-M systems and orphan MTases or its parental strain. Additionally, the gene coding for uracil phosphoribosyltransferase (upp) was directly inactivated using non-replicative plasmids prepared from the MoDMP host in B. amyloliquefaciens TA208. Moreover, the Gram-negative chemoautotrophic Nitrobacter hamburgensis strain X14 was transformed and expressed Green Fluorescent Protein (GFP). Finally, the sequence specificities of active MTases were identified by restriction enzyme digestion, making the MoDMP system potentially useful for other strains. The effectiveness of the MoDMP pipeline in different bacterial groups suggests a universal potential

  15. Fork rotation and DNA precatenation are restricted during DNA replication to prevent chromosomal instability.

    Science.gov (United States)

    Schalbetter, Stephanie A; Mansoubi, Sahar; Chambers, Anna L; Downs, Jessica A; Baxter, Jonathan

    2015-08-18

    Faithful genome duplication and inheritance require the complete resolution of all intertwines within the parental DNA duplex. This is achieved by topoisomerase action ahead of the replication fork or by fork rotation and subsequent resolution of the DNA precatenation formed. Although fork rotation predominates at replication termination, in vitro studies have suggested that it also occurs frequently during elongation. However, the factors that influence fork rotation and how rotation and precatenation may influence other replication-associated processes are unknown. Here we analyze the causes and consequences of fork rotation in budding yeast. We find that fork rotation and precatenation preferentially occur in contexts that inhibit topoisomerase action ahead of the fork, including stable protein-DNA fragile sites and termination. However, generally, fork rotation and precatenation are actively inhibited by Timeless/Tof1 and Tipin/Csm3. In the absence of Tof1/Timeless, excessive fork rotation and precatenation cause extensive DNA damage following DNA replication. With Tof1, damage related to precatenation is focused on the fragile protein-DNA sites where fork rotation is induced. We conclude that although fork rotation and precatenation facilitate unwinding in hard-to-replicate contexts, they intrinsically disrupt normal chromosome duplication and are therefore restricted by Timeless/Tipin.

  16. Partial digestion with restriction enzymes of ultraviolet-irradiated human genomic DNA: a method for identifying restriction site polymorphisms

    International Nuclear Information System (INIS)

    Nobile, C.; Romeo, G.

    1988-01-01

    A method for partial digestion of total human DNA with restriction enzymes has been developed on the basis of a principle already utilized by P.A. Whittaker and E. Southern for the analysis of phage lambda recombinants. Total human DNA irradiated with uv light of 254 nm is partially digested by restriction enzymes that recognize sequences containing adjacent thymidines because of TT dimer formation. The products resulting from partial digestion of specific genomic regions are detected in Southern blots by genomic-unique DNA probes with high reproducibility. This procedure is rapid and simple to perform because the same conditions of uv irradiation are used for different enzymes and probes. It is shown that restriction site polymorphisms occurring in the genomic regions analyzed are recognized by the allelic partial digest patterns they determine

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

    NARCIS (Netherlands)

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

    2005-01-01

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

  18. Sequence specific inhibition of DNA restriction enzyme cleavage by PNA

    DEFF Research Database (Denmark)

    Nielsen, P.E.; Egholm, M.; Berg, R.H.

    1993-01-01

    Plasmids containing double-stranded 10-mer PNA (peptide nucleic acid chimera) targets proximally flanked by two restriction enzyme sites were challenged with the complementary PNA or PNAs having one or two mismatches, and the effect on the restriction enzyme cleavage of the flanking sites was ass...

  19. 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.

  20. Restriction-modification mediated barriers to exogenous DNA uptake and incorporation employed by Prevotella intermedia.

    Science.gov (United States)

    Johnston, Christopher D; Skeete, Chelsey A; Fomenkov, Alexey; Roberts, Richard J; Rittling, Susan R

    2017-01-01

    Prevotella intermedia, a major periodontal pathogen, is increasingly implicated in human respiratory tract and cystic fibrosis lung infections. Nevertheless, the specific mechanisms employed by this pathogen remain only partially characterized and poorly understood, largely due to its total lack of genetic accessibility. Here, using Single Molecule, Real-Time (SMRT) genome and methylome sequencing, bisulfite sequencing, in addition to cloning and restriction analysis, we define the specific genetic barriers to exogenous DNA present in two of the most widespread laboratory strains, P. intermedia ATCC 25611 and P. intermedia Strain 17. We identified and characterized multiple restriction-modification (R-M) systems, some of which are considerably divergent between the two strains. We propose that these R-M systems are the root cause of the P. intermedia transformation barrier. Additionally, we note the presence of conserved Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) systems in both strains, which could provide a further barrier to exogenous DNA uptake and incorporation. This work will provide a valuable resource during the development of a genetic system for P. intermedia, which will be required for fundamental investigation of this organism's physiology, metabolism, and pathogenesis in human disease.

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

    OpenAIRE

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

    2008-01-01

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

  2. DNA Repair Systems

    Indian Academy of Sciences (India)

    DNA molecule which makes it ideal for storage and propagation of genetic information. ... of these errors are broadly referred to as DNA repair. DNA can ... changes occur in the human genome per day. ..... nails, frequent physical and mental.

  3. Enhanced resolution of DNA restriction fragments: A procedure by two-dimensional electrophoresis and double-labeling

    International Nuclear Information System (INIS)

    Yi, M.; Au, L.C.; Ichikawa, N.; Ts'o, P.O.

    1990-01-01

    A probe-free method was developed to detect DNA rearrangement in bacteria based on the electrophoretic separation of twice-digested restriction fragments of genomic DNA into a two-dimensional (2-D) pattern. The first restriction enzyme digestion was done in solution, followed by electrophoresis of the restriction fragments in one dimension. A second restriction enzyme digestion was carried out in situ in the gel, followed by electrophoresis in a second dimension perpendicular to the first electrophoresis. The 2-D pattern provides for the resolution of 300-400 spots, which are defined and indexed by an x,y coordinate system with size markers. This approach has greatly increased the resolution power over conventional one-dimensional (1-D) electrophoresis. To study DNA rearrangement, a 2-D pattern from a test strain was compared with the 2-D pattern from a reference strain. After the first digestion, genomic DNA fragments from the test strain were labeled with 35S, while those from the reference strain were labeled with 32P. This was done to utilize the difference in the energy emission of 35S and 32P isotopes for autoradiography when two x-ray films were exposed simultaneously on top of the gel after the 2-D electrophoresis. The irradiation from the decay of 35S exposed only the lower film, whereas the irradiation from the decay of 32P exposed both the lower and upper films. Different DNA fragments existed in the test DNA compared with the reference DNA can be identified unambiguously by the differential two 2-D patterns produced on two films upon exposure to the 35S and 32P fragments in the same gel. An appropriate photographic procedure further simplified the process, allowing only the difference in DNA fragments between these two patterns to be shown in the map

  4. 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

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

    NARCIS (Netherlands)

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

    2010-01-01

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

  6. Correspondence between radioactive and functional methods in the quality control of DNA restriction and modifying enzymes

    DEFF Research Database (Denmark)

    Trujillo, L E; Pupo, E; Miranda, F

    1996-01-01

    We evaluated the use of two radiolabeled lambda DNA/Hpa II substrates to detect 5'-->3', 3'-->5' single and double stranded DNA dependent exonuclease and phosphatase activities found as contaminants in restriction and modifying enzyme preparations. Looking for the meaning of the radioactive assay...

  7. Exogenous DNA internalisation by sperm cells is improved by combining lipofection and restriction enzyme mediated integration.

    Science.gov (United States)

    Churchil, R R; Gupta, J; Singh, A; Sharma, D

    2011-06-01

    1. Three types of exogenous DNA inserts, i.e. complete linearised pVIVO2-GFP/LacZ vector (9620 bp), the LacZ gene (5317 bp) and the GFP gene (2152 bp) were used to transfect chicken spermatozoa through simple incubation of sperm cells with insert. 2. PCR assay, Dot Blot hybridisation and Southern hybridisation showed the successful internalisation of exogenous DNA by chicken sperm cells. 3. Lipofection and Restriction Enzyme Mediated Integration (REMI) were used to improve the rate of internalisation of exogenous DNA by sperm cells. 4. Results from dot blot as well as Southern hybridisation were semi-quantified and improved exogenous DNA uptake by sperm cells through lipofection and REMI. Stronger signals were observed from hybridisation of LacZ as well as GFP specific probe with the DNA from lipofected exogenous DNA transfected sperm DNA in comparison with those transfected with nude exogenous DNA.

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

    Science.gov (United States)

    Rodriguez, R.J.

    1993-01-01

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

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

    International Nuclear Information System (INIS)

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

    1994-01-01

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

  10. DNA Repair Systems

    Indian Academy of Sciences (India)

    Thanks to the pioneering research work of Lindahl, Sancar, Modrich and their colleagues, we now have an holistic awareness of how DNA damage occurs and how the damage is rectified in bacteria as well as in higher organisms including human beings. A comprehensive understanding of DNA repair has proven crucial ...

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

    DEFF Research Database (Denmark)

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

    2000-01-01

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

  12. 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...

  13. 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

  14. Alignment of Escherichia coli K12 DNA sequences to a genomic restriction map.

    Science.gov (United States)

    Rudd, K E; Miller, W; Ostell, J; Benson, D A

    1990-01-25

    We use the extensive published information describing the genome of Escherichia coli and new restriction map alignment software to align DNA sequence, genetic, and physical maps. Restriction map alignment software is used which considers restriction maps as strings analogous to DNA or protein sequences except that two values, enzyme name and DNA base address, are associated with each position on the string. The resulting alignments reveal a nearly linear relationship between the physical and genetic maps of the E. coli chromosome. Physical map comparisons with the 1976, 1980, and 1983 genetic maps demonstrate a better fit with the more recent maps. The results of these alignments are genomic kilobase coordinates, orientation and rank of the alignment that best fits the genetic data. A statistical measure based on extreme value distribution is applied to the alignments. Additional computer analyses allow us to estimate the accuracy of the published E. coli genomic restriction map, simulate rearrangements of the bacterial chromosome, and search for repetitive DNA. The procedures we used are general enough to be applicable to other genome mapping projects.

  15. Differential diagnosis of genetic disease by DNA restriction fragment length polymorphisms

    NARCIS (Netherlands)

    Bolhuis, P. A.; Defesche, J. C.; van der Helm, H. J.

    1987-01-01

    DNA restriction fragment length polymorphisms (RFLPs) are used for diagnosis of genetic disease in families known to be affected by specific disorders, but RFLPs can be also useful for the differential diagnosis of hereditary disease. An RFLP pattern represents the inheritance of chromosomal markers

  16. Cloning and restriction enzyme mapping of ribosomal DNA of Giardia duodenalis, Giardia ardeae and Giardia muris.

    Science.gov (United States)

    van Keulen, H; Campbell, S R; Erlandsen, S L; Jarroll, E L

    1991-06-01

    In an attempt to study Giardia at the DNA sequence level, the rRNA genes of three species, Giardia duodenalis, Giardia ardeae and Giardia muris were cloned and restriction enzyme maps were constructed. The rDNA repeats of these Giardia show completely different restriction enzyme recognition patterns. The size of the rDNA repeat ranges from approximately 5.6 kb in G. duodenalis to 7.6 kb in both G. muris and G. ardeae. These size differences are mainly attributable to the variation in length of the spacer. Minor differences exist among these Giardia in the sizes of their small subunit rRNA and the internal transcribed spacer between small and large subunit rRNA. The genetic maps were constructed by sequence analysis of the DNA around the 5' and 3' ends of the mature rRNA genes and between the rRNA covering the 5.8S rRNA gene and internal transcribed spacer. Comparison of the 5.8S rDNA and 3' end of large subunit rDNA from these three Giardia species showed considerable sequence variation, but the rDNA sequences of G. duodenalis and G. ardeae appear more closely related to each other than to G. muris.

  17. Restricted diffusion of DNA segments within the isolated Escherichia coli nucleoid.

    NARCIS (Netherlands)

    Cunha, S.; Woldringh, C.L.; Odijk, T.

    2005-01-01

    To study the dynamics and organization of the DNA within isolated Escherichia coli nucleoids, we track the movement of a specific DNA region. Labeling of such a region is achieved using the Lac-O/Lac-I system. The Lac repressor-GFP fusion protein binds to the DNA section where tandem repeats of the

  18. 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.

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

    International Nuclear Information System (INIS)

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

    1986-01-01

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

  20. Sequence dependent DNA conformations: Raman spectroscopic studies and a model of action of restriction enzymes

    International Nuclear Information System (INIS)

    Nishimura, Y.

    1985-01-01

    Raman spectra have been examined to clarify the polymorphic forms of DNA, A, B, and Z forms. From an analysis the authors found that the guanine ring breathing vibration is sensitive to its local conformation. Examination of nine crystals of guanosine residues in which the local conformations are well established revealed that a guanosine residue with a C3'endo-anti gives a strong line at 666+-2 cm/sup -1/, O4'endo-anti at 682 cm/sup -1/, C1'exo-anti at 673 cm/sup -1/, C2'endo-anti at 677 cm/sup -1/ and syn-forms around 625 cm/sup -1/. Using this characteristic line, they were able to obtain the local conformations of guanosine moieties in poly(dG-dC). Such a sequence derived variation is suggested to be recognized by sequence specific proteins such as restriction enzymes. The authors found a correlation between sequence dependent DNA conformation and a mode of action of restriction enzymes. The cutting mode of restriction enzymes is classified into three groups. The classification of whether the products have blunt ends, two-base-long cohesive ends, or four-base-long cohesive ends depends primarily on the substrate, not on the enzyme. It is suggested that sequence dependent DNA conformation causes such a classification by the use of the Calladine-Dickerson analysis. In the recognition of restriction enzymes, the methyl group in a certain sequence is considered to play an important role by changing the local conformation of DNA

  1. Model for how type I restriction enzymes select cleavage sites in DNA

    International Nuclear Information System (INIS)

    Studier, F.W.; Bandyopadhyay, P.K.

    1988-01-01

    Under appropriate conditions, digestion of phage T7 DNA by the type I restriction enzyme EcoK produces an orderly progression of discrete DNA fragments. All details of the fragmentation pattern can be explained on the basis of the known properties of type I enzymes, together with two further assumptions: (i) in the ATP-stimulated translocation reaction, the enzyme bound at the recognition sequence translocates DNA toward itself from both directions simultaneously; and (ii) when translocation causes neighboring enzymes to meet, they cut the DNA between them. The kinetics of digestion at 37 degree C indicates that the rate of translocation of DNA from each side of a bound enzyme is about 200 base pairs per second, and the cuts are completed within 15-25 sec of the time neighboring enzymes meet. The resulting DNA fragments each contain a single recognition site with an enzyme (or subunit) remaining bound to it. At high enzyme concentrations, such fragments can bu further degraded, apparently by cooperation between the specifically bound and excess enzymes. This model is consistent with a substantial body of previous work on the nuclease activity of EcoB and EcoK, and it explains in a simple way how cleavage sites are selected

  2. Nondetectability of restriction fragments and independence of DNA fragment sizes within and between loci in RFLP typing of DNA

    Energy Technology Data Exchange (ETDEWEB)

    Chakraborty, R.; Zhong, Y.; Jin, L. (Univ. of Texas Health Science Center, Houston, TX (United States)); Budowle, B. (FBI Academy, Quantico, VA (United States))

    1994-08-01

    The authors provide experimental evidence showing that, during the restriction-enzyme digestion of DNA samples, some of the HaeIII-digested DNA fragments are small enough to prevent their reliable sizing on a Southern gel. As a result of such nondetectability of DNA fragments, individuals who show a single-band DNA profile at a VNTR locus may not necessarily be true homozygotes. In a population database, when the presence of such nondetectable alleles is ignored, they show that a pseudodependence of alleles within as well as across loci may occur. Using a known statistical method, under the hypothesis of independence of alleles within loci, they derive an efficient estimate of null allele frequency, which may be subsequently used for testing allelic independence within and across loci. The estimates of null allele frequencies, thus derived, are shown to agree with direct experimental data on the frequencies of HaeIII-null alleles. Incorporation of null alleles into the analysis of the forensic VNTR database suggests that the assumptions of allelic independence within and between loci are appropriate. In contrast, a failure to incorporate the occurrence of null alleles would provide a wrong inference regarding the independence of alleles within and between loci. 47 refs., 2 figs., 4 tabs.

  3. DNA Methylation Pattern in Overweight Women under an Energy-Restricted Diet Supplemented with Fish Oil

    Directory of Open Access Journals (Sweden)

    Cátia Lira do Amaral

    2014-01-01

    Full Text Available Dietary factors modulate gene expression and are able to alter epigenetic signatures in peripheral blood mononuclear cells (PBMC. However, there are limited studies about the effects of omega-3 polyunsaturated fatty acids (n-3 PUFA on the epigenetic mechanisms that regulate gene expression. This research investigates the effects of n-3-rich fish oil supplementation on DNA methylation profile of several genes whose expression has been reported to be downregulated by n-3 PUFA in PBMC: CD36, FFAR3, CD14, PDK4, and FADS1. Young overweight women were supplemented with fish oil or control in a randomized 8-week intervention trial following a balanced diet with 30% energy restriction. Fatty acid receptor CD36 decreased DNA methylation at CpG +477 due to energy restriction. Hypocaloric diet-induced weight loss also reduced the methylation percentages of CpG sites located in CD14, PDK4, and FADS1. The methylation patterns of these genes were only slightly affected by the fish oil supplementation, being the most relevant to the attenuation of the weight loss-induced decrease in CD36 methylation after adjusting by baseline body weight. These results suggest that the n-3 PUFA-induced changes in the expression of these genes in PBMC are not mediated by DNA methylation, although other epigenetic mechanisms cannot be discarded.

  4. 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.

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

    Science.gov (United States)

    Boyko, Alex; Kovalchuk, Igor

    2010-01-01

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

  6. Electricity system planning under the CO2 emission restriction

    International Nuclear Information System (INIS)

    Lim, Chae Young; Lee, Man Ki; Roh, Jae Hyung; Kim, Eun Hwan

    2004-01-01

    Objective of this study is to analyze how the restriction of CO 2 emission from power generation will affect the national electricity supply system. The role of nuclear power is investigated under the restriction of CO 2 emission in Korea. A simplified electricity system was modeled for the analysis. To analyze the impact of CO 2 emission restriction, 2 different scenarios were established and compared with the base scenario. The first scenario was 'CO 2 emission restriction with new nuclear power installation'. In this scenario, a CO 2 emission restriction of 0.11kg-C/kWh was imposed and there was no restriction on the nuclear power construction. While, in the second scenario, 'CO 2 emission restriction without new nuclear power installation' the same amount of CO 2 restriction was imposed with no consideration of nuclear power installation. It is found out that the current national emission target(0.11kg- C/kWh) in the electricity sector can not be achieved without nuclear and renewable(wind power) options considered

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

    International Nuclear Information System (INIS)

    Cleaver, J.E.

    1983-01-01

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

  8. maintainability of manpower system with restricted recruitment

    African Journals Online (AJOL)

    JERRY

    departmentalized manpower systems. ... homogenous k × k probability transition matrix of the manpower system; r and w be 1 .... is an advantage in manpower planning, especially in the area of manpower control, Bartholomew, et al (1991).

  9. Mapped DNA probes from Ioblolly pine can be used for restriction fragment length polymorphism mapping in other conifers

    Science.gov (United States)

    M.R. Ahuja; M.E. Devey; A.T. Groover; K.D. Jermstad; D.B Neale

    1994-01-01

    A high-density genetic map based on restriction fragment length polymorphisms (RFLPs) is being constructed for loblolly pine (Pinus taeda L.). Consequently, a large number of DNA probes from loblolly pine are potentially available for use in other species. We have used some of these DNA probes to detect RFLPs in 12 conifers and an angiosperm....

  10. Behavior of restriction-modification systems as selfish mobile elements and their impact on genome evolution.

    Science.gov (United States)

    Kobayashi, I

    2001-09-15

    Restriction-modification (RM) systems are composed of genes that encode a restriction enzyme and a modification methylase. RM systems sometimes behave as discrete units of life, like viruses and transposons. RM complexes attack invading DNA that has not been properly modified and thus may serve as a tool of defense for bacterial cells. However, any threat to their maintenance, such as a challenge by a competing genetic element (an incompatible plasmid or an allelic homologous stretch of DNA, for example) can lead to cell death through restriction breakage in the genome. This post-segregational or post-disturbance cell killing may provide the RM complexes (and any DNA linked with them) with a competitive advantage. There is evidence that they have undergone extensive horizontal transfer between genomes, as inferred from their sequence homology, codon usage bias and GC content difference. They are often linked with mobile genetic elements such as plasmids, viruses, transposons and integrons. The comparison of closely related bacterial genomes also suggests that, at times, RM genes themselves behave as mobile elements and cause genome rearrangements. Indeed some bacterial genomes that survived post-disturbance attack by an RM gene complex in the laboratory have experienced genome rearrangements. The avoidance of some restriction sites by bacterial genomes may result from selection by past restriction attacks. Both bacteriophages and bacteria also appear to use homologous recombination to cope with the selfish behavior of RM systems. RM systems compete with each other in several ways. One is competition for recognition sequences in post-segregational killing. Another is super-infection exclusion, that is, the killing of the cell carrying an RM system when it is infected with another RM system of the same regulatory specificity but of a different sequence specificity. The capacity of RM systems to act as selfish, mobile genetic elements may underlie the structure and

  11. Mapping DNA cleavage by the Type ISP restriction-modification enzymes following long-range communication between DNA sites in different orientations

    OpenAIRE

    van Aelst, Kara; Saikrishnan, Kayarat; Szczelkun, Mark D

    2015-01-01

    The prokaryotic Type ISP restriction-modification enzymes are single-chain proteins comprising an Mrr-family nuclease, a superfamily 2 helicase-like ATPase, a coupler domain, a methyltransferase, and a DNA-recognition domain. Upon recognising an unmodified DNA target site, the helicase-like domain hydrolyzes ATP to cause site release (remodeling activity) and to then drive downstream translocation consuming 1-2 ATP per base pair (motor activity). On an invading foreign DNA, double-strand brea...

  12. Creation of chimeric human/rabbit APOBEC1 with HIV-1 restriction and DNA mutation activities

    Science.gov (United States)

    Ikeda, Terumasa; Ong, Eugene Boon Beng; Watanabe, Nobumoto; Sakaguchi, Nobuo; Maeda, Kazuhiko; Koito, Atsushi

    2016-01-01

    APOBEC1 (A1) proteins from lagomorphs and rodents have deaminase-dependent restriction activity against HIV-1, whereas human A1 exerts a negligible effect. To investigate these differences in the restriction of HIV-1 by A1 proteins, a series of chimeric proteins combining rabbit and human A1s was constructed. Homology models of the A1s indicated that their activities derive from functional domains that likely act in tandem through a dimeric interface. The C-terminal region containing the leucine-rich motif and the dimerization domains of rabbit A1 is important for its anti-HIV-1 activity. The A1 chimeras with strong anti-HIV-1 activity were incorporated into virions more efficiently than those without anti-HIV-1 activity, and exhibited potent DNA-mutator activity. Therefore, the C-terminal region of rabbit A1 is involved in both its packaging into the HIV-1 virion and its deamination activity against both viral cDNA and genomic RNA. This study identifies the novel molecular mechanism underlying the target specificity of A1.

  13. Molecular discrimination of lactobacilli used as starter and probiotic cultures by amplified ribosomal DNA restriction analysis.

    Science.gov (United States)

    Roy, D; Sirois, S; Vincent, D

    2001-04-01

    Lactic acid bacteria such as Lactobacillus helveticus, L. delbrueckii subsp. delbrueckii, L. delbrueckii subsp. lactis, L. delbrueckii subsp. bulgaricus, L. acidophilus, and L. casei related taxa which are widely used as starter or probiotic cultures can be identified by amplified ribosomal DNA restriction analysis (ARDRA). The genetic discrimination of the related species belonging to these groups was first obtained by PCR amplifications by using group-specific or species-specific 16S rDNA primers. The numerical analysis of the ARDRA patterns obtained by using CfoI, HinfI, Tru9I, and ScrFI was an efficient typing tool for identification of species of the L. acidophilus and L. casei complex. ARDRA by using CfoI was a reliable method for differentiation of L. delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis. Finally, strains ATCC 393 and ATCC 15820 exhibited unique ARDRA patterns with CfoI and Tru9I restriction enzymes as compared with the other strains of L. casei, L. paracasei, and L. rhamnosus.

  14. Modified protocol for genomic DNA extraction from newly plucked feathers of lophura leucomelana hamiltoni (Galliformes) for genetic studies and its endo-restriction analysis

    International Nuclear Information System (INIS)

    Andleeb, S.; Shamim, S.; Minhas, R.A.

    2012-01-01

    A rapid and accurate protocol was used first time to isolate the high-quality genomic DNA from newly plucked feathers of Lophura leucomelana. Two different lysis protocols were used depending on the feather size and it was observed that 55 deg. C for 3 to 4 days showed better results of feathers lysis as compared with the 37 deg. C for overnight with gentle shaking. Purification of genomic DNA was also performed with phenol: chloroform: isoamyl alcohol and 100% absolute ethanol precipitation methods. By using this protocol, a significant amount of high-quality genomic DNA was obtained and the purity of DNA was analyzed through endo-restriction analysis. Genomic DNA isolated with this modified method will be used for Southern blotting and also in several polymerase chain reaction systems devoted to sex determination and paternity testing and the evolutionary relationships among the other pheasants. (author)

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

    Directory of Open Access Journals (Sweden)

    Roberts Richard J

    2008-05-01

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

  16. Restricted linking of emissions trading systems

    NARCIS (Netherlands)

    Schneider, Lambert; Lazarus, Michael; Lee, Carrie; Asselt, van Harro

    2017-01-01

    With over 17 emissions trading systems (ETSs) now in place across four continents, interest in linking ETSs is growing. Linking ETSs offers economic, political, and administrative benefits. It also faces major challenges. Linking can affect overall ambition, financial flows, and the location and

  17. Air pollution restrictions in electrical production system

    International Nuclear Information System (INIS)

    Gallizioli, G.

    1993-01-01

    A description of the principal characteristics regarding the Italian electrical power system and the evolution of standardization in air pollution control is given. Afterwards, ENEL (the Italian National Electricity Board) actions in the environmental protection field (with particular respect to thermo-electrical production) are presented. Finally, principal ENEL research programs on new air pollution control technologies are discussed

  18. Maintainability of manpower system with restricted recruitment ...

    African Journals Online (AJOL)

    The maintainability of a manpower system is studied under a Markov framework. The classical method of controlling only one factor of flow is extended to highlight the case in which two factors are under control simultaneously. One special case of this extension, where recruitment of units faces partial embargo, is given, ...

  19. Genome-wide DNA methylation analysis in jejunum of Sus scrofa with intrauterine growth restriction.

    Science.gov (United States)

    Hu, Yue; Hu, Liang; Gong, Desheng; Lu, Hanlin; Xuan, Yue; Wang, Ru; Wu, De; Chen, Daiwen; Zhang, Keying; Gao, Fei; Che, Lianqiang

    2018-02-01

    Intrauterine growth restriction (IUGR) may elicit a series of postnatal body developmental and metabolic diseases due to their impaired growth and development in the mammalian embryo/fetus during pregnancy. In the present study, we hypothesized that IUGR may lead to abnormally regulated DNA methylation in the intestine, causing intestinal dysfunctions. We applied reduced representation bisulfite sequencing (RRBS) technology to study the jejunum tissues from four newborn IUGR piglets and their normal body weight (NBW) littermates. The results revealed extensively regional DNA methylation changes between IUGR/NBW pairs from different gilts, affecting dozens of genes. Hiseq-based bisulfite sequencing PCR (Hiseq-BSP) was used for validations of 19 genes with epigenetic abnormality, confirming three genes (AIFM1, MTMR1, and TWIST2) in extra samples. Furthermore, integrated analysis of these 19 genes with proteome data indicated that there were three main genes (BCAP31, IRAK1, and AIFM1) interacting with important immunity- or metabolism-related proteins, which could explain the potential intestinal dysfunctions of IUGR piglets. We conclude that IUGR can lead to disparate DNA methylation in the intestine and these changes may affect several important biological processes such as cell apoptosis, cell differentiation, and immunity, which provides more clues linking IUGR and its long-term complications.

  20. A universal DNA-based protein detection system.

    Science.gov (United States)

    Tran, Thua N N; Cui, Jinhui; Hartman, Mark R; Peng, Songming; Funabashi, Hisakage; Duan, Faping; Yang, Dayong; March, John C; Lis, John T; Cui, Haixin; Luo, Dan

    2013-09-25

    Protein immune detection requires secondary antibodies which must be carefully selected in order to avoid interspecies cross-reactivity, and is therefore restricted by the limited availability of primary/secondary antibody pairs. Here we present a versatile DNA-based protein detection system using a universal adapter to interface between IgG antibodies and DNA-modified reporter molecules. As a demonstration of this capability, we successfully used DNA nano-barcodes, quantum dots, and horseradish peroxidase enzyme to detect multiple proteins using our DNA-based labeling system. Our system not only eliminates secondary antibodies but also serves as a novel method platform for protein detection with modularity, high capacity, and multiplexed capability.

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

    Science.gov (United States)

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

    2013-01-01

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

  2. 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.

  3. Phylogenomics of Phrynosomatid Lizards: Conflicting Signals from Sequence Capture versus Restriction Site Associated DNA Sequencing

    Science.gov (United States)

    Leaché, Adam D.; Chavez, Andreas S.; Jones, Leonard N.; Grummer, Jared A.; Gottscho, Andrew D.; Linkem, Charles W.

    2015-01-01

    Sequence capture and restriction site associated DNA sequencing (RADseq) are popular methods for obtaining large numbers of loci for phylogenetic analysis. These methods are typically used to collect data at different evolutionary timescales; sequence capture is primarily used for obtaining conserved loci, whereas RADseq is designed for discovering single nucleotide polymorphisms (SNPs) suitable for population genetic or phylogeographic analyses. Phylogenetic questions that span both “recent” and “deep” timescales could benefit from either type of data, but studies that directly compare the two approaches are lacking. We compared phylogenies estimated from sequence capture and double digest RADseq (ddRADseq) data for North American phrynosomatid lizards, a species-rich and diverse group containing nine genera that began diversifying approximately 55 Ma. Sequence capture resulted in 584 loci that provided a consistent and strong phylogeny using concatenation and species tree inference. However, the phylogeny estimated from the ddRADseq data was sensitive to the bioinformatics steps used for determining homology, detecting paralogs, and filtering missing data. The topological conflicts among the SNP trees were not restricted to any particular timescale, but instead were associated with short internal branches. Species tree analysis of the largest SNP assembly, which also included the most missing data, supported a topology that matched the sequence capture tree. This preferred phylogeny provides strong support for the paraphyly of the earless lizard genera Holbrookia and Cophosaurus, suggesting that the earless morphology either evolved twice or evolved once and was subsequently lost in Callisaurus. PMID:25663487

  4. Re-evaluating the kinetics of ATP hydrolysis during initiation of DNA sliding by Type III restriction enzymes.

    Science.gov (United States)

    Tóth, Júlia; Bollins, Jack; Szczelkun, Mark D

    2015-12-15

    DNA cleavage by the Type III restriction enzymes requires long-range protein communication between recognition sites facilitated by thermally-driven 1D diffusion. This 'DNA sliding' is initiated by hydrolysis of multiple ATPs catalysed by a helicase-like domain. Two distinct ATPase phases were observed using short oligoduplex substrates; the rapid consumption of ∼10 ATPs coupled to a protein conformation switch followed by a slower phase, the duration of which was dictated by the rate of dissociation from the recognition site. Here, we show that the second ATPase phase is both variable and only observable when DNA ends are proximal to the recognition site. On DNA with sites more distant from the ends, a single ATPase phase coupled to the conformation switch was observed and subsequent site dissociation required little or no further ATP hydrolysis. The overall DNA dissociation kinetics (encompassing site release, DNA sliding and escape via a DNA end) were not influenced by the second phase. Although the data simplifies the ATP hydrolysis scheme for Type III restriction enzymes, questions remain as to why multiple ATPs are hydrolysed to prepare for DNA sliding. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  5. Stability analysis of nonlinear systems with slope restricted nonlinearities.

    Science.gov (United States)

    Liu, Xian; Du, Jiajia; Gao, Qing

    2014-01-01

    The problem of absolute stability of Lur'e systems with sector and slope restricted nonlinearities is revisited. Novel time-domain and frequency-domain criteria are established by using the Lyapunov method and the well-known Kalman-Yakubovich-Popov (KYP) lemma. The criteria strengthen some existing results. Simulations are given to illustrate the efficiency of the results.

  6. Stability Analysis of Nonlinear Systems with Slope Restricted Nonlinearities

    Directory of Open Access Journals (Sweden)

    Xian Liu

    2014-01-01

    Full Text Available The problem of absolute stability of Lur’e systems with sector and slope restricted nonlinearities is revisited. Novel time-domain and frequency-domain criteria are established by using the Lyapunov method and the well-known Kalman-Yakubovich-Popov (KYP lemma. The criteria strengthen some existing results. Simulations are given to illustrate the efficiency of the results.

  7. Restrictive partially blind signature for resource-constrained information systems

    NARCIS (Netherlands)

    Qiu, Weidong; Gong, Zheng; Liu, Bozhong; Long, Yu; Chen, Kefei

    2010-01-01

    Restrictive partially blind signature, which is designed for privacy oriented information systems, allows a user to obtain a blind signature from a signer whilst the blind message must obey some certain rules. In order to reduce storage and communication costs, several public-key cryptosystems are

  8. Identification of Candida species by PCR and restriction fragment length polymorphism analysis of intergenic spacer regions of ribosomal DNA.

    OpenAIRE

    Williams, D W; Wilson, M J; Lewis, M A; Potts, A J

    1995-01-01

    The PCR was used to amplify a targeted region of the ribosomal DNA from 84 Candida isolates. Unique product sizes were obtained for Candida guilliermondii, Candida (Torulopsis) glabrata, and Candida pseudotropicalis. Isolates of Candida albicans, Candida tropicalis, Candida stellatoidea, Candida parapsilosis, and Candida krusei could be identified following restriction digestion of the PCR products.

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

    Science.gov (United States)

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

    2018-01-19

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

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

    International Nuclear Information System (INIS)

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

    1981-01-01

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

  11. Evaluation of simultaneous binding of Chromomycin A3 to the multiple sites of DNA by the new restriction enzyme assay.

    Science.gov (United States)

    Murase, Hirotaka; Noguchi, Tomoharu; Sasaki, Shigeki

    2018-06-01

    Chromomycin A3 (CMA3) is an aureolic acid-type antitumor antibiotic. CMA3 forms dimeric complexes with divalent cations, such as Mg 2+ , which strongly binds to the GC rich sequence of DNA to inhibit DNA replication and transcription. In this study, the binding property of CMA3 to the DNA sequence containing multiple GC-rich binding sites was investigated by measuring the protection from hydrolysis by the restriction enzymes, AccII and Fnu4HI, for the center of the CGCG site and the 5'-GC↓GGC site, respectively. In contrast to the standard DNase I footprinting method, the DNA substrates are fully hydrolyzed by the restriction enzymes, therefore, the full protection of DNA at all the cleavable sites indicates that CMA3 simultaneously binds to all the binding sites. The restriction enzyme assay has suggested that CMA3 has a high tendency to bind the successive CGCG sites and the CGG repeat. Copyright © 2018 Elsevier Ltd. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Jiabing Ji

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

  13. On the distinction of the mechanisms of DNA cleavage by restriction enzymes—The I-, II-, and III-type molecular motors

    Science.gov (United States)

    Pikin, S. A.

    2008-09-01

    A comparative physical description is given for the functioning of various restriction enzymes and for their processes of DNA cleavage. The previously proposed model system of kinetic equations is applied to the I-and III-type enzymes, which use ATP molecules as an energy source, while the II-type enzymes work thanks to catalytic reactions with participation of an electric field. All the enzymes achieved bending and twisting DNA, providing for either the linear motion of the II-type enzyme along the DNA chain or the DNA translocation by the I-and III-type enzymes due to moving chiral kinks. A comparative estimation of the considered linear and angular velocities is performed. The role of stalling forces for enzyme-DNA complexes, which induce the observed cutting of the DNA either inside the enzyme (II) or in some “weak” places outside enzymes I and III, which results in the supercoiling of the DNA, is shown. The role of ionic screening for the described processes is discussed.

  14. Analysis of DNA restriction fragments greater than 5.7 Mb in size from the centromeric region of human chromosomes.

    Science.gov (United States)

    Arn, P H; Li, X; Smith, C; Hsu, M; Schwartz, D C; Jabs, E W

    1991-01-01

    Pulsed electrophoresis was used to study the organization of the human centromeric region. Genomic DNA was digested with rare-cutting enzymes. DNA fragments from 0.2 to greater than 5.7 Mb were separated by electrophoresis and hybridized with alphoid and simple DNA repeats. Rare-cutting enzymes (Mlu I, Nar I, Not I, Nru I, Sal I, Sfi I, Sst II) demonstrated fewer restriction sites at centromeric regions than elsewhere in the genome. The enzyme Not I had the fewest restriction sites at centromeric regions. As much as 70% of these sequences from the centromeric region are present in Not I DNA fragments greater than 5.7 and estimated to be as large as 10 Mb in size. Other repetitive sequences such as short interspersed repeated segments (SINEs), long interspersed repeated segments (LINEs), ribosomal DNA, and mini-satellite DNA that are not enriched at the centromeric region, are not enriched in Not I fragments of greater than 5.7 Mb in size.

  15. 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.

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

    International Nuclear Information System (INIS)

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

    2010-01-01

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

  17. A survey of Type III restriction-modification systems reveals numerous, novel epigenetic regulators controlling phase-variable regulons; phasevarions

    Science.gov (United States)

    Atack, John M; Yang, Yuedong; Jennings, Michael P

    2018-01-01

    Abstract Many bacteria utilize simple DNA sequence repeats as a mechanism to randomly switch genes on and off. This process is called phase variation. Several phase-variable N6-adenine DNA-methyltransferases from Type III restriction-modification systems have been reported in bacterial pathogens. Random switching of DNA methyltransferases changes the global DNA methylation pattern, leading to changes in gene expression. These epigenetic regulatory systems are called phasevarions — phase-variable regulons. The extent of these phase-variable genes in the bacterial kingdom is unknown. Here, we interrogated a database of restriction-modification systems, REBASE, by searching for all simple DNA sequence repeats in mod genes that encode Type III N6-adenine DNA-methyltransferases. We report that 17.4% of Type III mod genes (662/3805) contain simple sequence repeats. Of these, only one-fifth have been previously identified. The newly discovered examples are widely distributed and include many examples in opportunistic pathogens as well as in environmental species. In many cases, multiple phasevarions exist in one genome, with examples of up to 4 independent phasevarions in some species. We found several new types of phase-variable mod genes, including the first example of a phase-variable methyltransferase in pathogenic Escherichia coli. Phasevarions are a common epigenetic regulation contingency strategy used by both pathogenic and non-pathogenic bacteria. PMID:29554328

  18. Hypotrochoids in conformal restriction systems and Virasoro descendants

    International Nuclear Information System (INIS)

    Doyon, Benjamin

    2013-01-01

    A conformal restriction system is a commutative, associative, unital algebra equipped with a representation of the groupoid of univalent conformal maps on connected open sets of the Riemann sphere, along with a family of linear functionals on subalgebras, satisfying a set of properties including conformal invariance and a type of restriction. This embodies some expected properties of expectation values in conformal loop ensembles CLE κ (at least for 8/3 iθ and w. We find that it has an expansion in positive powers of u and u-bar , and that the coefficients of pure u ( u-bar ) powers are holomorphic in w ( w-bar ). We identify these coefficients (the ‘hypotrochoid fields’) with certain Virasoro descendants of the identity field in conformal field theory, thereby showing that they form part of a vertex operator algebraic structure. This largely generalizes works by the author (in CLE), and the author with his collaborators Riva and Cardy (in SLE 8/3 and other restriction measures), where the case of the ellipse, at the order u 2 , led to the stress–energy tensor of CFT. The derivation uses in an essential way the Virasoro vertex operator algebra structure of conformal derivatives established recently by the author. The results suggest in particular the exact evaluation of CLE expectations of products of hypotrochoid fields as well as nontrivial relations amongst them through the vertex operator algebra, and further shed light onto the relationship between CLE and CFT. (paper)

  19. DNA - A Thermal Energy System Simulator

    DEFF Research Database (Denmark)

    2008-01-01

    DNA is a general energy system simulator for both steady-state and dynamic simulation. The program includes a * component model library * thermodynamic state models for fluids and solid fuels and * standard numerical solvers for differential and algebraic equation systems and is free and portable...... (open source, open use, standard FORTRAN77). DNA is text-based using whichever editor, you like best. It has been integerated with the emacs editor. This is usually available on unix-like systems. for windows we recommend the Installation instructions for windows: First install emacs and then run...... the DNA installer...

  20. Molecular cloning and restriction analysis of EcoRI-fragments of Vicia faba rDNA

    International Nuclear Information System (INIS)

    Yakura, Kimitaka; Tanifuji, Shigeyuki.

    1983-01-01

    EcoRI-fragments of Vicia faba rDNA were cloned in plasmid pBR325. Southern blot hybridization of BamHI-digests of these cloned plasmids and Vicia genomic DNA led to the determination of relative positions of BamHI sites in the rDNA and the physical map that had been tentatively made is corrected. (author)

  1. Transmission of the PabI family of restriction DNA glycosylase genes: mobility and long-term inheritance.

    Science.gov (United States)

    Kojima, Kenji K; Kobayashi, Ichizo

    2015-10-19

    R.PabI is an exceptional restriction enzyme that functions as a DNA glycosylase. The enzyme excises an unmethylated base from its recognition sequence to generate apurinic/apyrimidinic (AP) sites, and also displays AP lyase activity, cleaving the DNA backbone at the AP site to generate the 3'-phospho alpha, beta-unsaturated aldehyde end in addition to the 5'-phosphate end. The resulting ends are difficult to religate with DNA ligase. The enzyme was originally isolated in Pyrococcus, a hyperthermophilic archaeon, and additional homologs subsequently identified in the epsilon class of the Gram-negative bacterial phylum Proteobacteria, such as Helicobacter pylori. Systematic analysis of R.PabI homologs and their neighboring genes in sequenced genomes revealed co-occurrence of R.PabI with M.PabI homolog methyltransferase genes. R.PabI and M.PabI homolog genes are occasionally found at corresponding (orthologous) loci in different species, such as Helicobacter pylori, Helicobacter acinonychis and Helicobacter cetorum, indicating long-term maintenance of the gene pair. One R.PabI and M.PabI homolog gene pair is observed immediately after the GMP synthase gene in both Campylobacter and Helicobacter, representing orthologs beyond genera. The mobility of the PabI family of restriction-modification (RM) system between genomes is evident upon comparison of genomes of sibling strains/species. Analysis of R.PabI and M.PabI homologs in H. pylori revealed an insertion of integrative and conjugative elements (ICE), and replacement with a gene of unknown function that may specify a membrane-associated toxin (hrgC). In view of the similarity of HrgC with toxins in type I toxin-antitoxin systems, we addressed the biological significance of this substitution. Our data indicate that replacement with hrgC occurred in the common ancestor of hspAmerind and hspEAsia. Subsequently, H. pylori with and without hrgC were intermixed at this locus, leading to complex distribution of hrgC in East

  2. Leaf margin phenotype-specific restriction-site-associated DNA-derived markers for pineapple (Ananas comosus L.).

    Science.gov (United States)

    Urasaki, Naoya; Goeku, Satoko; Kaneshima, Risa; Takamine, Tomonori; Tarora, Kazuhiko; Takeuchi, Makoto; Moromizato, Chie; Yonamine, Kaname; Hosaka, Fumiko; Terakami, Shingo; Matsumura, Hideo; Yamamoto, Toshiya; Shoda, Moriyuki

    2015-06-01

    To explore genome-wide DNA polymorphisms and identify DNA markers for leaf margin phenotypes, a restriction-site-associated DNA sequencing analysis was employed to analyze three bulked DNAs of F1 progeny from a cross between a 'piping-leaf-type' cultivar, 'Yugafu', and a 'spiny-tip-leaf-type' variety, 'Yonekura'. The parents were both Ananas comosus var. comosus. From the analysis, piping-leaf and spiny-tip-leaf gene-specific restriction-site-associated DNA sequencing tags were obtained and designated as PLSTs and STLSTs, respectively. The five PLSTs and two STSLTs were successfully converted to cleaved amplified polymorphic sequence (CAPS) or simple sequence repeat (SSR) markers using the sequence differences between alleles. Based on the genotyping of the F1 with two SSR and three CAPS markers, the five PLST markers were mapped in the vicinity of the P locus, with the closest marker, PLST1_SSR, being located 1.5 cM from the P locus. The two CAPS markers from STLST1 and STLST3 perfectly assessed the 'spiny-leaf type' as homozygotes of the recessive s allele of the S gene. The recombination value between the S locus and STLST loci was 2.4, and STLSTs were located 2.2 cM from the S locus. SSR and CAPS markers are applicable to marker-assisted selection of leaf margin phenotypes in pineapple breeding.

  3. Sequence Capture versus Restriction Site Associated DNA Sequencing for Shallow Systematics.

    Science.gov (United States)

    Harvey, Michael G; Smith, Brian Tilston; Glenn, Travis C; Faircloth, Brant C; Brumfield, Robb T

    2016-09-01

    Sequence capture and restriction site associated DNA sequencing (RAD-Seq) are two genomic enrichment strategies for applying next-generation sequencing technologies to systematics studies. At shallow timescales, such as within species, RAD-Seq has been widely adopted among researchers, although there has been little discussion of the potential limitations and benefits of RAD-Seq and sequence capture. We discuss a series of issues that may impact the utility of sequence capture and RAD-Seq data for shallow systematics in non-model species. We review prior studies that used both methods, and investigate differences between the methods by re-analyzing existing RAD-Seq and sequence capture data sets from a Neotropical bird (Xenops minutus). We suggest that the strengths of RAD-Seq data sets for shallow systematics are the wide dispersion of markers across the genome, the relative ease and cost of laboratory work, the deep coverage and read overlap at recovered loci, and the high overall information that results. Sequence capture's benefits include flexibility and repeatability in the genomic regions targeted, success using low-quality samples, more straightforward read orthology assessment, and higher per-locus information content. The utility of a method in systematics, however, rests not only on its performance within a study, but on the comparability of data sets and inferences with those of prior work. In RAD-Seq data sets, comparability is compromised by low overlap of orthologous markers across species and the sensitivity of genetic diversity in a data set to an interaction between the level of natural heterozygosity in the samples examined and the parameters used for orthology assessment. In contrast, sequence capture of conserved genomic regions permits interrogation of the same loci across divergent species, which is preferable for maintaining comparability among data sets and studies for the purpose of drawing general conclusions about the impact of

  4. Mapping DNA cleavage by the Type ISP restriction-modification enzymes following long-range communication between DNA sites in different orientations

    Science.gov (United States)

    van Aelst, Kara; Saikrishnan, Kayarat; Szczelkun, Mark D.

    2015-01-01

    The prokaryotic Type ISP restriction-modification enzymes are single-chain proteins comprising an Mrr-family nuclease, a superfamily 2 helicase-like ATPase, a coupler domain, a methyltransferase, and a DNA-recognition domain. Upon recognising an unmodified DNA target site, the helicase-like domain hydrolyzes ATP to cause site release (remodeling activity) and to then drive downstream translocation consuming 1–2 ATP per base pair (motor activity). On an invading foreign DNA, double-strand breaks are introduced at random wherever two translocating enzymes form a so-called collision complex following long-range communication between a pair of target sites in inverted (head-to-head) repeat. Paradoxically, structural models for collision suggest that the nuclease domains are too far apart (>30 bp) to dimerise and produce a double-strand DNA break using just two strand-cleavage events. Here, we examined the organisation of different collision complexes and how these lead to nuclease activation. We mapped DNA cleavage when a translocating enzyme collides with a static enzyme bound to its site. By following communication between sites in both head-to-head and head-to-tail orientations, we could show that motor activity leads to activation of the nuclease domains via distant interactions of the helicase or MTase-TRD. Direct nuclease dimerization is not required. To help explain the observed cleavage patterns, we also used exonuclease footprinting to demonstrate that individual Type ISP domains can swing off the DNA. This study lends further support to a model where DNA breaks are generated by multiple random nicks due to mobility of a collision complex with an overall DNA-binding footprint of ∼30 bp. PMID:26507855

  5. DNA-Based Enzyme Reactors and Systems

    Directory of Open Access Journals (Sweden)

    Veikko Linko

    2016-07-01

    Full Text Available During recent years, the possibility to create custom biocompatible nanoshapes using DNA as a building material has rapidly emerged. Further, these rationally designed DNA structures could be exploited in positioning pivotal molecules, such as enzymes, with nanometer-level precision. This feature could be used in the fabrication of artificial biochemical machinery that is able to mimic the complex reactions found in living cells. Currently, DNA-enzyme hybrids can be used to control (multi-enzyme cascade reactions and to regulate the enzyme functions and the reaction pathways. Moreover, sophisticated DNA structures can be utilized in encapsulating active enzymes and delivering the molecular cargo into cells. In this review, we focus on the latest enzyme systems based on novel DNA nanostructures: enzyme reactors, regulatory devices and carriers that can find uses in various biotechnological and nanomedical applications.

  6. Pattern analysis approach reveals restriction enzyme cutting abnormalities and other cDNA library construction artifacts using raw EST data

    Directory of Open Access Journals (Sweden)

    Zhou Sun

    2012-05-01

    Full Text Available Abstract Background Expressed Sequence Tag (EST sequences are widely used in applications such as genome annotation, gene discovery and gene expression studies. However, some of GenBank dbEST sequences have proven to be “unclean”. Identification of cDNA termini/ends and their structures in raw ESTs not only facilitates data quality control and accurate delineation of transcription ends, but also furthers our understanding of the potential sources of data abnormalities/errors present in the wet-lab procedures for cDNA library construction. Results After analyzing a total of 309,976 raw Pinus taeda ESTs, we uncovered many distinct variations of cDNA termini, some of which prove to be good indicators of wet-lab artifacts, and characterized each raw EST by its cDNA terminus structure patterns. In contrast to the expected patterns, many ESTs displayed complex and/or abnormal patterns that represent potential wet-lab errors such as: a failure of one or both of the restriction enzymes to cut the plasmid vector; a failure of the restriction enzymes to cut the vector at the correct positions; the insertion of two cDNA inserts into a single vector; the insertion of multiple and/or concatenated adapters/linkers; the presence of 3′-end terminal structures in designated 5′-end sequences or vice versa; and so on. With a close examination of these artifacts, many problematic ESTs that have been deposited into public databases by conventional bioinformatics pipelines or tools could be cleaned or filtered by our methodology. We developed a software tool for Abnormality Filtering and Sequence Trimming for ESTs (AFST, http://code.google.com/p/afst/ using a pattern analysis approach. To compare AFST with other pipelines that submitted ESTs into dbEST, we reprocessed 230,783 Pinus taeda and 38,709 Arachis hypogaea GenBank ESTs. We found 7.4% of Pinus taeda and 29.2% of Arachis hypogaea GenBank ESTs are “unclean” or abnormal, all of which could be cleaned

  7. Iterated function systems for DNA replication

    Science.gov (United States)

    Gaspard, Pierre

    2017-10-01

    The kinetic equations of DNA replication are shown to be exactly solved in terms of iterated function systems, running along the template sequence and giving the statistical properties of the copy sequences, as well as the kinetic and thermodynamic properties of the replication process. With this method, different effects due to sequence heterogeneity can be studied, in particular, a transition between linear and sublinear growths in time of the copies, and a transition between continuous and fractal distributions of the local velocities of the DNA polymerase along the template. The method is applied to the human mitochondrial DNA polymerase γ without and with exonuclease proofreading.

  8. Analysis of Molecular Variance Inferred from Metric Distances among DNA Haplotypes: Application to Human Mitochondrial DNA Restriction Data

    OpenAIRE

    Excoffier, L.; Smouse, P. E.; Quattro, J. M.

    1992-01-01

    We present here a framework for the study of molecular variation within a single species. Information on DNA haplotype divergence is incorporated into an analysis of variance format, derived from a matrix of squared-distances among all pairs of haplotypes. This analysis of molecular variance (AMOVA) produces estimates of variance components and F-statistic analogs, designated here as φ-statistics, reflecting the correlation of haplotypic diversity at different levels of hierarchical subdivisi...

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

    Science.gov (United States)

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

    1996-07-15

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

  10. Screening of respiration deficiency mutants of yeasts (Saccharomyces cerevisiae) induced by ion irradiation and the mtDNA restriction analysis

    International Nuclear Information System (INIS)

    Mao Shuhong; Chinese Academy of Sciences, Beijing; Jin Genming; Wei Zengquan; Xie Hongmei; Ma Qiufeng; Gu Ying

    2005-01-01

    Screening of the respiration deficiency mutants of Saccharomyces cerevisiae induced by 5.19 MeV/u 22 Ne 5+ ion irradiation is reported in this paper. Some respiration deficiency mutants of white colony phenotype, in a condition of selective culture of TTC medium, were obtained. A new and simplified method based on mtDNA restriction analysis is described. The authors found that there are many obvious differences in mtDNAs between wild yeasts and the respiration deficiency mutants. The mechanism of obtaining the respiration deficiency mutants induced by heavy ion irradiation is briefly discussed. (authors)

  11. Development of Insertion and Deletion Markers for Bottle Gourd Based on Restriction Site-associated DNA Sequencing Data

    Directory of Open Access Journals (Sweden)

    Xinyi WU

    2017-01-01

    Full Text Available Bottle gourd is an important cucurbit crop worldwide. To provide more available molecular markers for this crop, a bioinformatic approach was employed to develop insertion–deletions (InDels markers in bottle gourd based on restriction site-associated DNA sequencing (RAD-Seq data. A total of 892 Indels were predicted, with the length varying from 1 bp to 167 bp. Single-nucleotide InDels were the predominant types of InDels. To validate these InDels, PCR primers were designed from 162 loci where InDels longer than 2 bp were predicated. A total of 112 InDels were found to be polymorphic among 9 bottle gourd accessions under investigation. The rate of prediction accuracy was thus at a high level of 72.7%. DNA fingerprinting for 4 cultivars were performed using 8 selected Indels markers, demonstrating the usefulness of these markers.

  12. Restriction of human papillomavirus DNA testing in primary cervical screening to women above age 30

    DEFF Research Database (Denmark)

    Rebolj, Matejka; Njor, Sisse H; Lynge, Elsebeth

    2012-01-01

    Cervical screening with human papillomavirus (HPV) testing is less specific for high-grade cervical intraepithelial neoplasia (=CIN3) than cytology. The aim of this systematic review was to determine whether a restriction of HPV testing to women aged at least 30 years would eliminate the problem...

  13. A model system for DNA repair studies

    International Nuclear Information System (INIS)

    Lange, C.S.; Perlmutter, E.

    1984-01-01

    The search for the ''lethal lesion:'' which would yield a molecular explanation of biological survival curves led to attempts to correlate unrepaired DNA lesions with loss of reproductive integrity. Such studies have shown the crucial importance of DNA repair systems. The unrepaired DSB has been sought for such correlation, but in such study the DNA was too large, polydisperse, and/or structurally complex to permit precise measurement of break induction and repair. Therefore, an analog of higher order systems but with a genome of readily measurable size, is needed. Bacteriophage T4 is such an analog. Both its biological (PFU) and molecular (DNA) survival curves are exponentials. Its aerobic /sub PFU/D/sub 37///sub DNA/D/sub 37/ ratio, (410 +- 4.5Gy/540 +- 25 Gy) indicates that 76 +- 4% of lethality at low multiplicity infection (moi 1) the survival is greater than can be explained if the assumption of no parental DSB repair were valid. Both T4 and its host have DSB repair systems which can be studied by the infectious center method. Results of such studies are discussed

  14. Updating rDNA restriction enzyme maps of Tetrahymena reveals four new intron-containing species

    DEFF Research Database (Denmark)

    Nielsen, Henrik; Simon, E M; Engberg, J

    1985-01-01

    an intron in the 26s rRNA coding region. The evolutionary relationship among the species of the T. pyriformis complex was examined on the basis of the rDNA maps with emphasis on similarities between two of the new species and the widely studied T. thermophila and T. pigmentosa. Examination of a large number...

  15. Evaluation of amplified rDNA restriction analysis (ARDRA for the identification of cultured mycobacteria in a diagnostic laboratory

    Directory of Open Access Journals (Sweden)

    Rottiers Sylvianne

    2002-03-01

    Full Text Available Abstract Background The development of DNA amplification for the direct detection of M. tuberculosis from clinical samples has been a major goal of clinical microbiology during the last ten years. However, the limited sensitivity of most DNA amplification techniques restricts their use to smear positive samples. On the other hand, the development of automated liquid culture has increased the speed and sensitivity of cultivation of mycobacteria. We have opted to combine automated culture with rapid genotypic identification (ARDRA: amplified rDNA restriction analysis for the detection resp. identification of all mycobacterial species at once, instead of attempting direct PCR based detection from clinical samples of M. tuberculosis only. Results During 1998–2000 a total of approx. 3500 clinical samples was screened for the presence of M. tuberculosis. Of the 151 culture positive samples, 61 were M. tuberculosis culture positive. Of the 30 smear positive samples, 26 were M. tuberculosis positive. All but three of these 151 mycobacterial isolates could be identified with ARDRA within on average 36 hours. The three isolates that could not be identified belonged to rare species not yet included in our ARDRA fingerprint library or were isolates with an aberrant pattern. Conclusions In our hands, automated culture in combination with ARDRA provides with accurate, practically applicable, wide range identification of mycobacterial species. The existing identification library covers most species, and can be easily updated when new species are studied or described. The drawback is that ARDRA is culture-dependent, since automated culture of M. tuberculosis takes on average 16.7 days (range 6 to 29 days. However, culture is needed after all to assess the antibiotic susceptibility of the strains.

  16. DNA polymorphism of HLA class II genes in systemic lupus erythematosus

    DEFF Research Database (Denmark)

    Cowland, J B; Andersen, V; Halberg, P

    1994-01-01

    We investigated the DNA restriction fragment length polymorphism (RFLP) of the major histocompatibility complex (MHC) genes: HLA-DRB, -DQA, -DQB, -DPB in 24 Danish patients with systemic lupus erythematosus (SLE) and in 102 healthy Danes. A highly significant increase of the frequency of the DR3...

  17. MIDAS: A Modular DNA Assembly System for Synthetic Biology.

    Science.gov (United States)

    van Dolleweerd, Craig J; Kessans, Sarah A; Van de Bittner, Kyle C; Bustamante, Leyla Y; Bundela, Rudranuj; Scott, Barry; Nicholson, Matthew J; Parker, Emily J

    2018-04-20

    A modular and hierarchical DNA assembly platform for synthetic biology based on Golden Gate (Type IIS restriction enzyme) cloning is described. This enabling technology, termed MIDAS (for Modular Idempotent DNA Assembly System), can be used to precisely assemble multiple DNA fragments in a single reaction using a standardized assembly design. It can be used to build genes from libraries of sequence-verified, reusable parts and to assemble multiple genes in a single vector, with full user control over gene order and orientation, as well as control of the direction of growth (polarity) of the multigene assembly, a feature that allows genes to be nested between other genes or genetic elements. We describe the detailed design and use of MIDAS, exemplified by the reconstruction, in the filamentous fungus Penicillium paxilli, of the metabolic pathway for production of paspaline and paxilline, key intermediates in the biosynthesis of a range of indole diterpenes-a class of secondary metabolites produced by several species of filamentous fungi. MIDAS was used to efficiently assemble a 25.2 kb plasmid from 21 different modules (seven genes, each composed of three basic parts). By using a parts library-based system for construction of complex assemblies, and a unique set of vectors, MIDAS can provide a flexible route to assembling tailored combinations of genes and other genetic elements, thereby supporting synthetic biology applications in a wide range of expression hosts.

  18. Combination of methylated-DNA precipitation and methylation-sensitive restriction enzymes (COMPARE-MS) for the rapid, sensitive and quantitative detection of DNA methylation.

    Science.gov (United States)

    Yegnasubramanian, Srinivasan; Lin, Xiaohui; Haffner, Michael C; DeMarzo, Angelo M; Nelson, William G

    2006-02-09

    Hypermethylation of CpG island (CGI) sequences is a nearly universal somatic genome alteration in cancer. Rapid and sensitive detection of DNA hypermethylation would aid in cancer diagnosis and risk stratification. We present a novel technique, called COMPARE-MS, that can rapidly and quantitatively detect CGI hypermethylation with high sensitivity and specificity in hundreds of samples simultaneously. To quantitate CGI hypermethylation, COMPARE-MS uses real-time PCR of DNA that was first digested by methylation-sensitive restriction enzymes and then precipitated by methyl-binding domain polypeptides immobilized on a magnetic solid matrix. We show that COMPARE-MS could detect five genome equivalents of methylated CGIs in a 1000- to 10,000-fold excess of unmethylated DNA. COMPARE-MS was used to rapidly quantitate hypermethylation at multiple CGIs in >155 prostate tissues, including benign and malignant prostate specimens, and prostate cell lines. This analysis showed that GSTP1, MDR1 and PTGS2 CGI hypermethylation as determined by COMPARE-MS could differentiate between malignant and benign prostate with sensitivities >95% and specificities approaching 100%. This novel technology could significantly improve our ability to detect CGI hypermethylation.

  19. Oncogenic transformation of rat lung epithelioid cells by SV40 DNA and restriction enzyme fragments

    International Nuclear Information System (INIS)

    Daya-Grosjean, L.; Lasne, C.; Nardeux, P.; Chouroulinkov, I.; Monier, R.

    1979-01-01

    Rat epithelioid lung cells were transformed with various preparations of SV40 DNA using the Ca 2+ -precipitation technique. The amount of SV40 genetic information integrated into transformed clones was evaluated by DNA-DNA renaturation kinetics. The growth properties on plastic and in soft-agar were examined, as well as the ability to induce tumors in syngeneic newborn animals or in adult nude mice. One particular transformed line, which had received the HpaII/BamHIA (59 per cent) fragment, was found to contain about 3 integrated copies of this fragment per cell and no significant amount of the HpaII/BamHIB (41 per cent fragment). This line which grew to high saturatio densities and efficiently formed clones in low serum on plastic, produced tumors in both syngeneic rats and nude mice. Thus the HpaII/BamHIA fragment, which mainly includes early viral information, was sufficient to impart these properties to rat epithelioid lung cells. (author)

  20. The N terminus of cGAS de-oligomerizes the cGAS:DNA complex and lifts the DNA size restriction of core-cGAS activity.

    Science.gov (United States)

    Lee, Arum; Park, Eun-Byeol; Lee, Janghyun; Choi, Byong-Seok; Kang, Suk-Jo

    2017-03-01

    Cyclic GMP-AMP synthase (cGAS) is a DNA-sensing enzyme in the innate immune system. Recent studies using core-cGAS lacking the N terminus investigated the mechanism for binding of double-stranded (ds) DNA and synthesis of 2',3'-cyclic GMP-AMP (cGAMP), a secondary messenger that ultimately induces type I interferons. However, the function of the N terminus of cGAS remains largely unknown. Here, we found that the N terminus enhanced the activity of core-cGAS in vivo. Importantly, the catalytic activity of core-cGAS decreased as the length of double-stranded DNA (dsDNA) increased, but the diminished activity was restored by addition of the N terminus. Furthermore, the N terminus de-oligomerized the 2 : 2 complex of core-cGAS and dsDNA into a 1 : 1 complex, suggesting that the N terminus enhanced the activity of core-cGAS by facilitating formation of a monomeric complex of cGAS and DNA. © 2017 Federation of European Biochemical Societies.

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

    Science.gov (United States)

    Szczelkun, Mark D

    2011-04-01

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

  2. DNA replication is not restricted to specific regions in young vegetative Streptomyces mycelia

    International Nuclear Information System (INIS)

    Kummer, C.; Kretschmer, S.

    1986-01-01

    In order to determine the localization of DNA-synthesis in Streptomyces granaticolor and Streptomyces hygroscopicus, mycelia (growing either on agar or in liquid medium) were pulse-labelled with 3 H-thymidine and prepared for autoradiography. The distribution of silver grains showed no regions of preferential incorporation of 3 H-thymidine in mycelia up 300 μm in length. Since mycelia grow by apical elongation of hyphae, the frequency of silver grains was quantitatively analysed along individual main hyphase. No significant difference of labelling was found within zones of different age up to a distance of 80 μm from the hyphal tip. Also, the very youngest part of the hyphae enclosing only the most apically situated nucleoid did not show any deviation from the average frequency of silver grains. (author)

  3. Quantification of DNA by Agarose Gel Electrophoresis and Analysis of the Topoisomers of Plasmid and M13 DNA Following Treatment with a Restriction Endonuclease or DNA Topoisomerase I

    Science.gov (United States)

    Tweedie, John W.; Stowell, Kathryn M.

    2005-01-01

    A two-session laboratory exercise for advanced undergraduate students in biochemistry and molecular biology is described. The first session introduces students to DNA quantification by ultraviolet absorbance and agarose gel electrophoresis followed by ethidium bromide staining. The second session involves treatment of various topological forms of…

  4. A laboratory information management system for DNA barcoding workflows

    NARCIS (Netherlands)

    Vu, D.; Eberhardt, U.; Szöke, S.; Groenewald, M.; Robert, V.

    2012-01-01

    This paper presents a laboratory information management system for DNA sequences (LIMS) created and based on the needs of a DNA barcoding project at the CBS-KNAW Fungal Biodiversity Centre (Utrecht, the Netherlands). DNA barcoding is a global initiative for species identification through simple DNA

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

    International Nuclear Information System (INIS)

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

    1995-01-01

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

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

    Science.gov (United States)

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

    2007-04-01

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

  7. Evidence for the presence of restriction/modification systems in Lactobacillus delbrueckii.

    Science.gov (United States)

    Suárez, Viviana; Zago, Miriam; Giraffa, Giorgio; Reinheimer, Jorge; Quiberoni, Andrea

    2009-11-01

    The bacteriophages Cb1/204 and Cb1/342 were obtained by induction from the commercial strain Lactobacillus delbrueckii subsp. lactis Cb1, and propagated on Lactobacillus delbrueckii subsp. lactis 204 (Lb.l 204) and Lactobacillus delbrueckii subsp. bulgaricus 342 (Lb.b 342), respectively. By cross sensitivity, it was possible to detect a delay in the lysis of Lb.l 204 with Cb1/342 phage, while the adsorption rate was high (99.5%). Modified and unmodified phages were isolated using phage Cb1/342 and strain Lb.l 204. The EOP (Efficiency of Plaquing) values for the four phages (Cb1/204, Cb1/342, Cb1/342modified and Cb1/342unmodified) suggested that an R/M system modified the original temperate phage, and the BglII-DNA restriction patterns of these phages might point out the presence of a Type II R/M system. Also, the existence of a Type I R/M system was demonstrated by PCR and nucleotide sequence, being the percentages of alignment homology with Type I R/M systems reported previously higher than 95%. In this study it was possible to demonstrate that the native phage resistant mechanisms and the occurrence of prophages in commercial host strains, contribute strongly to diversify the phage population in a factory environment.

  8. Rapid identification and classification of bacteria by 16S rDNA restriction fragment melting curve analyses (RFMCA).

    Science.gov (United States)

    Rudi, Knut; Kleiberg, Gro H; Heiberg, Ragnhild; Rosnes, Jan T

    2007-08-01

    The aim of this work was to evaluate restriction fragment melting curve analyses (RFMCA) as a novel approach for rapid classification of bacteria during food production. RFMCA was evaluated for bacteria isolated from sous vide food products, and raw materials used for sous vide production. We identified four major bacterial groups in the material analysed (cluster I-Streptococcus, cluster II-Carnobacterium/Bacillus, cluster III-Staphylococcus and cluster IV-Actinomycetales). The accuracy of RFMCA was evaluated by comparison with 16S rDNA sequencing. The strains satisfying the RFMCA quality filtering criteria (73%, n=57), with both 16S rDNA sequence information and RFMCA data (n=45) gave identical group assignments with the two methods. RFMCA enabled rapid and accurate classification of bacteria that is database compatible. Potential application of RFMCA in the food or pharmaceutical industry will include development of classification models for the bacteria expected in a given product, and then to build an RFMCA database as a part of the product quality control.

  9. A DNA-Based Assessment of the Phylogenetic Position of a Morphologically Distinct, Anchialine-Lake-Restricted Seahorse.

    Science.gov (United States)

    Rose, Emily; Masonjones, Heather D; Jones, Adam G

    2016-11-01

    Isolated populations provide special opportunities to study local adaptation and incipient speciation. In some cases, however, morphological evolution can obscure the taxonomic status of recently founded populations. Here, we use molecular markers to show that an anchialine-lake-restricted population of seahorses, originally identified as Hippocampus reidi, appears on the basis of DNA data to be Hippocampus erectus We collected seahorses from Sweetings Pond, on Eleuthera Island, Bahamas, during the summer of 2014. We measured morphological traits and sequenced 2 genes, cytochrome b and ribosomal protein S7, from 19 seahorses in our sample. On the basis of morphology, Sweetings Pond seahorses could not be assigned definitively to either of the 2 species of seahorse, H. reidi and H. erectus, that occur in marine waters surrounding the Bahamas. However, our DNA-based phylogenetic analysis showed that the Sweetings Pond fish were firmly nested within the H. erectus clade with a Bayesian posterior probability greater than 0.99. Thus, Sweetings Pond seahorses most recently shared a common ancestor with H. erectus populations from the Western Atlantic. Interestingly, the seahorses from Sweetings Pond differ morphologically from other marine populations of H. erectus in having a more even torso to tail length ratio. The substantial habitat differences between Sweetings Pond and the surrounding coastal habitat make Sweetings Pond seahorses particularly interesting from the perspectives of conservation, local adaptation, and incipient speciation. © The American Genetic Association 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  10. DNA Profiling of Convicted Offender Samples for the Combined DNA Index System

    Science.gov (United States)

    Millard, Julie T

    2011-01-01

    The cornerstone of forensic chemistry is that a perpetrator inevitably leaves trace evidence at a crime scene. One important type of evidence is DNA, which has been instrumental in both the implication and exoneration of thousands of suspects in a wide range of crimes. The Combined DNA Index System (CODIS), a network of DNA databases, provides…

  11. 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

  12. Restricted gene flow at the micro- and macro-geographical scale in marble trout based on mtDNA and microsatellite polymorphism

    Directory of Open Access Journals (Sweden)

    Patarnello Tomaso

    2011-04-01

    Full Text Available Abstract Background The genetic structure of the marble trout Salmo trutta marmoratus, an endemic salmonid of northern Italy and the Balkan peninsula, was explored at the macro- and micro-scale level using a combination of mitochondrial DNA (mtDNA and microsatellite data. Results Sequence variation in the mitochondrial control region showed the presence of nonindigenous haplotypes indicative of introgression from brown trout into marble trout. This was confirmed using microsatellite markers, which showed a higher introgression at nuclear level. Microsatellite loci revealed a strong genetic differentiation across the geographical range of marble trout, which suggests restricted gene flow both at the micro-geographic (within rivers and macro-geographic (among river systems scale. A pattern of Isolation-by-Distance was found, in which genetic samples were correlated with hydrographic distances. A general West-to-East partition of the microsatellite polymorphism was observed, which was supported by the geographic distribution of mitochondrial haplotypes. Conclusion While introgression at both mitochondrial and nuclear level is unlikely to result from natural migration and might be the consequence of current restocking practices, the pattern of genetic substructuring found at microsatellites has been likely shaped by historical colonization patterns determined by the geological evolution of the hydrographic networks.

  13. The proviral genome of radiation leukemia virus (RadLV): molecular cloning, restriction analysis and integration sites in tumor cell DNA

    International Nuclear Information System (INIS)

    Janowski, M.; Merregaert, J.; Nuyten, J.M.; Maisin, J.R.

    1984-01-01

    An infectious clone of the linear, unintegrated RadLV provirus was obtained by insertion in the plasmid pBR322. Its restriction map was indistinguishable from that of the majority of the multiple proviral copies, which are found apparently at random sites in the DNA of RadLV-induced rat thymic lymphomas [fr

  14. Construction of a high-density genetic map for grape using next generation restriction-site associated DNA sequencing

    Directory of Open Access Journals (Sweden)

    Wang Nian

    2012-08-01

    Full Text Available Abstract Background Genetic mapping and QTL detection are powerful methodologies in plant improvement and breeding. Construction of a high-density and high-quality genetic map would be of great benefit in the production of superior grapes to meet human demand. High throughput and low cost of the recently developed next generation sequencing (NGS technology have resulted in its wide application in genome research. Sequencing restriction-site associated DNA (RAD might be an efficient strategy to simplify genotyping. Combining NGS with RAD has proven to be powerful for single nucleotide polymorphism (SNP marker development. Results An F1 population of 100 individual plants was developed. In-silico digestion-site prediction was used to select an appropriate restriction enzyme for construction of a RAD sequencing library. Next generation RAD sequencing was applied to genotype the F1 population and its parents. Applying a cluster strategy for SNP modulation, a total of 1,814 high-quality SNP markers were developed: 1,121 of these were mapped to the female genetic map, 759 to the male map, and 1,646 to the integrated map. A comparison of the genetic maps to the published Vitis vinifera genome revealed both conservation and variations. Conclusions The applicability of next generation RAD sequencing for genotyping a grape F1 population was demonstrated, leading to the successful development of a genetic map with high density and quality using our designed SNP markers. Detailed analysis revealed that this newly developed genetic map can be used for a variety of genome investigations, such as QTL detection, sequence assembly and genome comparison.

  15. DNA-Enabled Integrated Molecular Systems for Computation and Sensing

    Science.gov (United States)

    2014-05-21

    Computational devices can be chemically conjugated to different strands of DNA that are then self-assembled according to strict Watson − Crick binding rules... DNA -Enabled Integrated Molecular Systems for Computation and Sensing Craig LaBoda,† Heather Duschl,† and Chris L. Dwyer*,†,‡ †Department of...guided folding of DNA , inspired by nature, allows designs to manipulate molecular-scale processes unlike any other material system. Thus, DNA can be

  16. Genomic-based restriction enzyme selection for specific detection of Piscirickettsia salmonis by 16S rDNA PCR-RFLP

    Directory of Open Access Journals (Sweden)

    Dinka eMandakovic

    2016-05-01

    Full Text Available The gram negative facultative bacterium P. salmonis is the etiological agent of Salmonid Rickettsial Septicaemia (SRS, a severe disease that causes important economic losses in the global salmon farmer industry. Despite efforts to control this disease, the high frequency of new epizootic events indicate that the vaccine and antibiotics treatments have limited effectiveness, therefore the preventive and diagnostic approaches must be improved. A comparison of several methodologies for SRS diagnostic indicate differences in their specificity and its capacity to detect other bacteria coexisting with P. salmonis in culture media (contamination and fish samples (coinfection, aspects relevant for research, vaccine development and clinical diagnostic. By computer-simulation analyses, we identified a group of restriction enzymes that generate unique P. salmonis 16S rDNA band patterns, distinguishable from all other bacteria. From this information, we designed and developed a PCR-RFLP (Polymerase Chain Reaction - Restriction Fragment Length Polymorphism assay, which was validated using 16S rDNA universal primers and restriction enzyme PmaCI for the amplification and digestion, respectively. Experimental validation was performed by comparing the restriction pattern of P. salmonis with the restriction patterns generated by bacteria that cohabit with P. salmonis (fish bacterial isolates and culture media contaminants. Our results indicate that the restriction enzyme selection pipeline was suitable to design a more specific, sensible, faster and cheaper assay than the currently used P. salmonis detection methodologies.

  17. Intrauterine growth restriction increases circulating mitochondrial DNA and Toll-like receptor 9 expression in adult offspring: could aerobic training counteract these adaptations?

    Science.gov (United States)

    Oliveira, V; Silva Junior, S D; de Carvalho, M H C; Akamine, E H; Michelini, L C; Franco, M C

    2017-04-01

    It has been demonstrated that intrauterine growth restriction (IUGR) can program increase cardiometabolic risk. There are also evidences of the correlation between IUGR with low-grade inflammation and, thus can contribute to development of several cardiometabolic comorbidities. Therefore, we investigated the influence of IUGR on circulating mitochondrial DNA (mtDNA)/Toll-like receptor 9 (TLR9) and TNF-α expression in adult offspring. Considering that the aerobic training has anti-inflammatory actions, we also investigated whether aerobic training would improve these inflammatory factors. Pregnant Wistar rats received ad libitum or 50% of ad libitum diet throughout gestation. At 8 weeks of age, male offspring from both groups were randomly assigned to control, trained control, restricted and trained restricted. Aerobic training protocol was performed on a treadmill and after that, we evaluated circulating mtDNA, cardiac protein expression of TLR9, plasma and cardiac TNF-α levels, and left ventricle (LV) mass. We found that IUGR promoted an increase in the circulating mtDNA, TLR9 expression and plasma TNF-α levels. Further, our results revealed that aerobic training can restore mtDNA/TLR9 content and plasma levels of TNF-α among restricted rats. The cardiac TNF-α content and LV mass were not influenced either by IUGR or aerobic training. In conclusion, IUGR can program mtDNA/TLR9 content, which may lead to high levels of TNF-α. However, aerobic training was able to normalize these alterations. These findings evidenced that the association of IUGR and aerobic training seems to exert an important interaction effect regarding pro-inflammatory condition and, aerobic training may be used as a strategy to reduce deleterious adaptations in IUGR offspring.

  18. Genetic Evaluation of Natural Populations of the Endangered Conifer Thuja koraiensis Using Microsatellite Markers by Restriction-Associated DNA Sequencing

    Directory of Open Access Journals (Sweden)

    Lu Hou

    2018-04-01

    Full Text Available Thuja koraiensis Nakai is an endangered conifer of high economic and ecological value in Jilin Province, China. However, studies on its population structure and conservation genetics have been limited by the lack of genomic data. Here, 37,761 microsatellites (simple sequence repeat, SSR were detected based on 875,792 de novo-assembled contigs using a restriction-associated DNA (RAD approach. Among these SSRs, 300 were randomly selected to test for polymorphisms and 96 obtained loci were able to amplify a fragment of expected size. Twelve polymorphic SSR markers were developed to analyze the genetic diversity and population structure of three natural populations. High genetic diversity (mean NA = 5.481, HE = 0.548 and moderate population differentiation (pairwise Fst = 0.048–0.078, Nm = 2.940–4.958 were found in this species. Molecular variance analysis suggested that most of the variation (83% existed within populations. Combining the results of STRUCTURE, principal coordinate, and neighbor-joining analysis, the 232 individuals were divided into three genetic clusters that generally correlated with their geographical distributions. Finally, appropriate conservation strategies were proposed to protect this species. This study provides genetic information for the natural resource conservation and utilization of T. koraiensis and will facilitate further studies of the evolution and phylogeography of the species.

  19. Reference-free SNP discovery for the Eurasian beaver from restriction site-associated DNA paired-end data.

    Science.gov (United States)

    Senn, Helen; Ogden, Rob; Cezard, Timothee; Gharbi, Karim; Iqbal, Zamin; Johnson, Eric; Kamps-Hughes, Nick; Rosell, Frank; McEwing, Ross

    2013-06-01

    In this study, we used restriction site-associated DNA (RAD) sequencing to discover SNP markers suitable for population genetic and parentage analysis with the aim of using them for monitoring the reintroduction of the Eurasian beaver (Castor fibre) to Scotland. In the absence of a reference genome for beaver, we built contigs and discovered SNPs within them using paired-end RAD data, so as to have sufficient flanking region around the SNPs to conduct marker design. To do this, we used a simple pipeline which catalogued the Read 1 data in stacks and then used the assembler cortex_var to conduct de novo assembly and genotyping of multiple samples using the Read 2 data. The analysis of around 1.1 billion short reads of sequence data was reduced to a set of 2579 high-quality candidate SNP markers that were polymorphic in Norwegian and Bavarian beaver. Both laboratory validation of a subset of eight of the SNPs (1.3% error) and internal validation by confirming patterns of Mendelian inheritance in a family group (0.9% error) confirmed the success of this approach. © 2013 John Wiley & Sons Ltd.

  20. The highly heterogeneous methylated genomes and diverse restriction-modification systems of bloom-forming Microcystis.

    Science.gov (United States)

    Zhao, Liang; Song, Yulong; Li, Lin; Gan, Nanqin; Brand, Jerry J; Song, Lirong

    2018-05-01

    The occurrence of harmful Microcystis blooms is increasing in frequency in a myriad of freshwater ecosystems. Despite considerable research pertaining to the cause and nature of these blooms, the molecular mechanisms behind the cosmopolitan distribution and phenotypic diversity in Microcystis are still unclear. We compared the patterns and extent of DNA methylation in three strains of Microcystis, PCC 7806SL, NIES-2549 and FACHB-1757, using Single Molecule Real-Time (SMRT) sequencing technology. Intact restriction-modification (R-M) systems were identified from the genomes of these strains, and from two previously sequenced strains of Microcystis, NIES-843 and TAIHU98. A large number of methylation motifs and R-M genes were identified in these strains, which differ substantially among different strains. Of the 35 motifs identified, eighteen had not previously been reported. Strain NIES-843 contains a larger number of total putative methyltransferase genes than have been reported previously from any bacterial genome. Genomic comparisons reveal that methyltransferases (some partial) may have been acquired from the environment through horizontal gene transfer. Copyright © 2018 Elsevier B.V. All rights reserved.

  1. SIRT3 restricts HBV transcription and replication via epigenetic regulation of cccDNA involving SUV39H1 and SETD1A histone methyltransferases.

    Science.gov (United States)

    Ren, Ji-Hua; Hu, Jie-Li; Cheng, Sheng-Tao; Yu, Hai-Bo; Wong, Vincent Kam Wai; Law, Betty Yuen Kwan; Yang, Yong-Feng; Huang, Ying; Liu, Yi; Chen, Wei-Xian; Cai, Xue-Fei; Tang, Hua; Hu, Yuan; Zhang, Wen-Lu; Liu, Xiang; Long, Quan-Xin; Zhou, Li; Tao, Na-Na; Zhou, Hong-Zhong; Yang, Qiu-Xia; Ren, Fang; He, Lin; Gong, Rui; Huang, Ai-Long; Chen, Juan

    2018-04-06

    Hepatitis B virus (HBV) infection remains a major health problem worldwide. Maintenance of the covalently closed circular DNA (cccDNA) which serves as a template for HBV RNA transcription is responsible for the failure of eradicating chronic HBV during current antiviral therapy. cccDNA is assembled with cellular histone proteins into chromatin, but little is known about the regulation of HBV chromatin by histone posttranslational modifications. In this study, we identified SIRT3 as a host factor restricting HBV transcription and replication by screening seven members of Sirtuin family which is the class III histone deacetylase. Ectopic SIRT3 expression significantly reduced total HBV RNAs, 3.5-kb RNA as well as replicative intermediate DNA in HBV-infected HepG2-NTCP cells and PHH. In contrast, gene silencing of SIRT3 promoted HBV transcription and replication. Mechanistic study found nuclear SIRT3 was recruited to the HBV cccDNA, where it deacetylated histone 3 lysine 9 (H3K9). Importantly, occupancy of SIRT3 onto cccDNA could increase the recruitment of histone methyltransferase SUV39H1 to cccDNA and decrease recruitment of SETD1A, leading to a marked increase of H3K9me3 and a decrease of H3K4me3 on cccDNA. Moreover, SIRT3-mediated HBV cccDNA transcriptional repression involved decreased binding of host RNA polymerase II and transcription factor YY1 to cccDNA. Finally, viral protein HBx could relieve SIRT3-mediated cccDNA transcriptional repression by inhibiting both SIRT3 expression and its recruitment to cccDNA. SIRT3 is a novel host factor epigenetically restricting HBV cccDNA transcription by acting cooperatively with histone methyltransferase. These data provided a rational for the use of SIRT3 activators in the prevention or treatment of HBV infection. This article is protected by copyright. All rights reserved. © 2018 by the American Association for the Study of Liver Diseases.

  2. SNP discovery in common bean by restriction-associated DNA (RAD) sequencing for genetic diversity and population structure analysis.

    Science.gov (United States)

    Valdisser, Paula Arielle M R; Pappas, Georgios J; de Menezes, Ivandilson P P; Müller, Bárbara S F; Pereira, Wendell J; Narciso, Marcelo G; Brondani, Claudio; Souza, Thiago L P O; Borba, Tereza C O; Vianello, Rosana P

    2016-06-01

    Researchers have made great advances into the development and application of genomic approaches for common beans, creating opportunities to driving more real and applicable strategies for sustainable management of the genetic resource towards plant breeding. This work provides useful polymorphic single-nucleotide polymorphisms (SNPs) for high-throughput common bean genotyping developed by RAD (restriction site-associated DNA) sequencing. The RAD tags were generated from DNA pooled from 12 common bean genotypes, including breeding lines of different gene pools and market classes. The aligned sequences identified 23,748 putative RAD-SNPs, of which 3357 were adequate for genotyping; 1032 RAD-SNPs with the highest ADT (assay design tool) score are presented in this article. The RAD-SNPs were structurally annotated in different coding (47.00 %) and non-coding (53.00 %) sequence components of genes. A subset of 384 RAD-SNPs with broad genome distribution was used to genotype a diverse panel of 95 common bean germplasms and revealed a successful amplification rate of 96.6 %, showing 73 % of polymorphic SNPs within the Andean group and 83 % in the Mesoamerican group. A slightly increased He (0.161, n = 21) value was estimated for the Andean gene pool, compared to the Mesoamerican group (0.156, n = 74). For the linkage disequilibrium (LD) analysis, from a group of 580 SNPs (289 RAD-SNPs and 291 BARC-SNPs) genotyped for the same set of genotypes, 70.2 % were in LD, decreasing to 0.10 %in the Andean group and 0.77 % in the Mesoamerican group. Haplotype patterns spanning 310 Mb of the genome (60 %) were characterized in samples from different origins. However, the haplotype frameworks were under-represented for the Andean (7.85 %) and Mesoamerican (5.55 %) gene pools separately. In conclusion, RAD sequencing allowed the discovery of hundreds of useful SNPs for broad genetic analysis of common bean germplasm. From now, this approach provides an excellent panel

  3. Genotypic diversity of oscillatoriacean strains belonging to the genera Geitlerinema and Spirulina determined by 16S rDNA restriction analysis.

    Science.gov (United States)

    Margheri, Maria C; Piccardi, Raffaella; Ventura, Stefano; Viti, Carlo; Giovannetti, Luciana

    2003-05-01

    Genotypic diversity of several cyanobacterial strains mostly isolated from marine or brackish waters, belonging to the genera Geitlerinema and Spirulina, was investigated by amplified 16S ribosomal DNA restriction analysis and compared with morphological features and response to salinity. Cluster analysis was performed on amplified 16S rDNA restriction profiles of these strains along with profiles obtained from sequence data of five Spirulina-like strains, including three representatives of the new genus Halospirulina. Our strains with tightly coiled trichomes from hypersaline waters could be assigned to the Halospirulina genus. Among the uncoiled strains, the two strains of hypersaline origin clustered together and were found to be distant from their counterparts of marine and freshwater habitat. Moreover, another cluster, formed by alkali-tolerant strains with tightly coiled trichomes, was well delineated.

  4. Isolation and characterization of DNA probes from a flow-sorted human chromosome 8 library that detect restriction fragment length polymorphism (RFLP).

    Science.gov (United States)

    Wood, S; Starr, T V; Shukin, R J

    1986-01-01

    We have used a recombinant DNA library constructed from flow-sorted human chromosome 8 as a source of single-copy human probes. These probes have been screened for restriction fragment length polymorphism (RFLP) by hybridization to Southern transfers of genomic DNA from five unrelated individuals. We have detected six RFLPs distributed among four probes after screening 741 base pairs for restriction site variation. These RFLPs all behave as codominant Mendelian alleles. Two of the probes detect rare variants, while the other two detect RFLPs with PIC values of .36 and .16. Informative probes will be useful for the construction of a linkage map for chromosome 8 and for the localization of mutant alleles to this chromosome. Images Fig. 1 PMID:2879441

  5. Dietary restriction but not angiotensin II type 1 receptor blockade improves DNA damage-related vasodilator dysfunction in rapidly aging Ercc1Δ/- mice.

    Science.gov (United States)

    Wu, Haiyan; van Thiel, Bibi S; Bautista-Niño, Paula K; Reiling, Erwin; Durik, Matej; Leijten, Frank P J; Ridwan, Yanto; Brandt, Renata M C; van Steeg, Harry; Dollé, Martijn E T; Vermeij, Wilbert P; Hoeijmakers, Jan H J; Essers, Jeroen; van der Pluijm, Ingrid; Danser, A H Jan; Roks, Anton J M

    2017-08-01

    DNA damage is an important contributor to endothelial dysfunction and age-related vascular disease. Recently, we demonstrated in a DNA repair-deficient, prematurely aging mouse model ( Ercc1 Δ/- mice) that dietary restriction (DR) strongly increases life- and health span, including ameliorating endothelial dysfunction, by preserving genomic integrity. In this mouse mutant displaying prominent accelerated, age-dependent endothelial dysfunction we investigated the signaling pathways involved in improved endothelium-mediated vasodilation by DR, and explore the potential role of the renin-angiotensin system (RAS). Ercc1 Δ/- mice showed increased blood pressure and decreased aortic relaxations to acetylcholine (ACh) in organ bath experiments. Nitric oxide (NO) signaling and phospho-Ser 1177 -eNOS were compromised in Ercc1 Δ / - DR improved relaxations by increasing prostaglandin-mediated responses. Increase of cyclo-oxygenase 2 and decrease of phosphodiesterase 4B were identified as potential mechanisms. DR also prevented loss of NO signaling in vascular smooth muscle cells and normalized angiotensin II (Ang II) vasoconstrictions, which were increased in Ercc1 Δ/- mice. Ercc1 Δ/ - mutants showed a loss of Ang II type 2 receptor-mediated counter-regulation of Ang II type 1 receptor-induced vasoconstrictions. Chronic losartan treatment effectively decreased blood pressure, but did not improve endothelium-dependent relaxations. This result might relate to the aging-associated loss of treatment efficacy of RAS blockade with respect to endothelial function improvement. In summary, DR effectively prevents endothelium-dependent vasodilator dysfunction by augmenting prostaglandin-mediated responses, whereas chronic Ang II type 1 receptor blockade is ineffective. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  6. Genome-wide placental DNA methylation analysis of severely growth-discordant monochorionic twins reveals novel epigenetic targets for intrauterine growth restriction.

    Science.gov (United States)

    Roifman, Maian; Choufani, Sanaa; Turinsky, Andrei L; Drewlo, Sascha; Keating, Sarah; Brudno, Michael; Kingdom, John; Weksberg, Rosanna

    2016-01-01

    Intrauterine growth restriction (IUGR), which refers to reduced fetal growth in the context of placental insufficiency, is etiologically heterogeneous. IUGR is associated not only with perinatal morbidity and mortality but also with adult-onset disorders, such as cardiovascular disease and diabetes, posing a major health burden. Placental epigenetic dysregulation has been proposed as one mechanism that causes IUGR; however, the spectrum of epigenetic pathophysiological mechanisms leading to IUGR remains to be elucidated. Monozygotic monochorionic twins are particularly affected by IUGR, in the setting of severe discordant growth. Because monozygotic twins have the same genotype at conception and a shared maternal environment, they provide an ideal model system for studying epigenetic dysregulation of the placenta. We compared genome-wide placental DNA methylation patterns of severely growth-discordant twins to identify novel candidate genes for IUGR. Snap-frozen placental samples for eight severely growth-discordant monozygotic monochorionic twin pairs were obtained at delivery from each twin. A high-resolution DNA methylation array platform was used to identify methylation differences between IUGR and normal twins. Our analysis revealed differentially methylated regions in the promoters of eight genes: DECR1, ZNF300, DNAJA4, CCL28, LEPR, HSPA1A/L, GSTO1, and GNE. The largest methylation differences between the two groups were in the promoters of DECR1 and ZNF300. The significance of these group differences was independently validated by bisulfite pyrosequencing, implicating aberrations in fatty acid beta oxidation and transcriptional regulation, respectively. Further analysis of the array data identified methylation changes most prominently affecting the Wnt and cadherin pathways in the IUGR cohort. Our results suggest that IUGR in monozygotic twins is associated with impairments in lipid metabolism and transcriptional regulation as well as cadherin and Wnt

  7. EBR-II argon cooling system restricted fuel handling I and C upgrade

    International Nuclear Information System (INIS)

    Start, S.E.; Carlson, R.B.; Gehrman, R.L.

    1995-01-01

    The instrumentation and control of the Argon Cooling System (ACS) restricted fuel handling control system at Experimental Breeder Reactor II (EBR-II) is being upgraded from a system comprised of many discrete components and controllers to a computerized system with a graphical user interface (GUI). This paper describes the aspects of the upgrade including reasons for the upgrade, the old control system, upgrade goals, design decisions, philosophies and rationale, and the new control system hardware and software

  8. Restricted and disrupted sleep : Effects on autonomic function, neuroendocrine stress systems and stress responsivity

    NARCIS (Netherlands)

    Meerlo, Peter; Sgoifo, Andrea; Suchecki, Deborah

    2008-01-01

    Frequently disrupted and restricted sleep is a common problem for many people in our modern around-the-clock society. In this context, it is an important question how sleep loss affects the stress systems in our bodies since these systems enable us to deal with everyday challenges. Altered activity

  9. Analytical workflow of double-digest restriction site-associated DNA sequencing based on empirical and in silico optimization in tomato.

    Science.gov (United States)

    Shirasawa, Kenta; Hirakawa, Hideki; Isobe, Sachiko

    2016-04-01

    Double-digest restriction site-associated DNA sequencing (ddRAD-Seq) enables high-throughput genome-wide genotyping with next-generation sequencing technology. Consequently, this method has become popular in plant genetics and breeding. Although computational in silico prediction of restriction sites from the genome sequence is recognized as an effective approach for choosing the restriction enzymes to be used, few reports have evaluated the in silico predictions in actual experimental data. In this study, we designed and demonstrated a workflow for in silico and empirical ddRAD-Seq analysis in tomato, as follows: (i)in silico prediction of optimum restriction enzymes from the reference genome, (ii) verification of the prediction by actual ddRAD-Seq data of four restriction enzyme combinations, (iii) establishment of a computational data processing pipeline for high-confidence single nucleotide polymorphism (SNP) calling, and (iv) validation of SNP accuracy by construction of genetic linkage maps. The quality of SNPs based on de novo assembly reference of the ddRAD-Seq reads was comparable with that of SNPs obtained using the published reference genome of tomato. Comparisons of SNP calls in diverse tomato lines revealed that SNP density in the genome influenced the detectability of SNPs by ddRAD-Seq. In silico prediction prior to actual analysis contributed to optimization of the experimental conditions for ddRAD-Seq, e.g. choices of enzymes and plant materials. Following optimization, this ddRAD-Seq pipeline could help accelerate genetics, genomics, and molecular breeding in both model and non-model plants, including crops. © The Author 2016. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

  10. Dietary restriction delays the secretion of senescence associated secretory phenotype by reducing DNA damage response in the process of renal aging.

    Science.gov (United States)

    Wang, Wenjuan; Cai, Guangyan; Chen, Xiangmei

    2017-09-13

    Dietary restriction (DR) has multiple and essential effects in protecting against DNA damage in model organisms. Persistent DNA damage plays a central role in the process of aging. Senescence-associated secretory phenotype (SASP), as a product of cellular aging, can accelerate the process of cellular senescence as a feedback. In this study, we directly observed whether a DR of 30% for 6months in aged rats could retard SASP by delaying the progression of DNA damage and also found the specific mechanism. The results revealed that a 30% DR could significantly improve renal pathology and some metabolic characteristics. The biomarkers and products of DNA damage were decreased in the process of renal aging on a 30% DR. A series of SASP, notably cytokine, chemokine, and growth factor, were obviously reduced by DR during renal aging. The phosphorylation levels of NF-κB and IκBα in aged kidneys of DR group were markedly reduced. These findings suggest that a 30% DR for 6months can delay renal aging and reduce the accumulation of SASP by retarding the progression of DNA damage and decreasing the transcription activity of NF-κB, thus providing a target to delay renal aging. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Quantitative Analysis Method of Output Loss due to Restriction for Grid-connected PV Systems

    Science.gov (United States)

    Ueda, Yuzuru; Oozeki, Takashi; Kurokawa, Kosuke; Itou, Takamitsu; Kitamura, Kiyoyuki; Miyamoto, Yusuke; Yokota, Masaharu; Sugihara, Hiroyuki

    Voltage of power distribution line will be increased due to reverse power flow from grid-connected PV systems. In the case of high density grid connection, amount of voltage increasing will be higher than the stand-alone grid connection system. To prevent the over voltage of power distribution line, PV system's output will be restricted if the voltage of power distribution line is close to the upper limit of the control range. Because of this interaction, amount of output loss will be larger in high density case. This research developed a quantitative analysis method for PV systems output and losses to clarify the behavior of grid connected PV systems. All the measured data are classified into the loss factors using 1 minute average of 1 second data instead of typical 1 hour average. Operation point on the I-V curve is estimated to quantify the loss due to the output restriction using module temperature, array output voltage, array output current and solar irradiance. As a result, loss due to output restriction is successfully quantified and behavior of output restriction is clarified.

  12. PRMT5 restricts hepatitis B virus replication through epigenetic repression of covalently closed circular DNA transcription and interference with pregenomic RNA encapsidation.

    Science.gov (United States)

    Zhang, Wen; Chen, Jieliang; Wu, Min; Zhang, Xiaonan; Zhang, Min; Yue, Lei; Li, Yaming; Liu, Jiangxia; Li, Baocun; Shen, Fang; Wang, Yang; Bai, Lu; Protzer, Ulrike; Levrero, Massimo; Yuan, Zhenghong

    2017-08-01

    Chronic hepatitis B virus (HBV) infection remains a major health problem worldwide. The covalently closed circular DNA (cccDNA) minichromosome, which serves as the template for the transcription of viral RNAs, plays a key role in viral persistence. While accumulating evidence suggests that cccDNA transcription is regulated by epigenetic machinery, particularly the acetylation of cccDNA-bound histone 3 (H3) and H4, the potential contributions of histone methylation and related host factors remain obscure. Here, by screening a series of methyltransferases and demethylases, we identified protein arginine methyltransferase 5 (PRMT5) as an effective restrictor of HBV transcription and replication. In cell culture-based models for HBV infection and in liver tissues of patients with chronic HBV infection, we found that symmetric dimethylation of arginine 3 on H4 on cccDNA was a repressive marker of cccDNA transcription and was regulated by PRMT5 depending on its methyltransferase domain. Moreover, PRMT5-triggered symmetric dimethylation of arginine 3 on H4 on the cccDNA minichromosome involved an interaction with the HBV core protein and the Brg1-based human SWI/SNF chromatin remodeler, which resulted in down-regulation of the binding of RNA polymerase II to cccDNA. In addition to the inhibitory effect on cccDNA transcription, PRMT5 inhibited HBV core particle DNA production independently of its methyltransferase activity. Further study revealed that PRMT5 interfered with pregenomic RNA encapsidation by preventing its interaction with viral polymerase protein through binding to the reverse transcriptase-ribonuclease H region of polymerase, which is crucial for the polymerase-pregenomic RNA interaction. PRMT5 restricts HBV replication through a two-part mechanism including epigenetic suppression of cccDNA transcription and interference with pregenomic RNA encapsidation; these findings improve the understanding of epigenetic regulation of HBV transcription and host

  13. DNA – A General Energy System Simulation Tool

    DEFF Research Database (Denmark)

    Elmegaard, Brian; Houbak, Niels

    2005-01-01

    The paper reviews the development of the energy system simulation tool DNA (Dynamic Network Analysis). DNA has been developed since 1989 to be able to handle models of any kind of energy system based on the control volume approach, usually systems of lumped parameter components. DNA has proven...... to be a useful tool in the analysis and optimization of several types of thermal systems: Steam turbines, gas turbines, fuels cells, gasification, refrigeration and heat pumps for both conventional fossil fuels and different types of biomass. DNA is applicable for models of both steady state and dynamic...... operation. The program decides at runtime to apply the DAE solver if the system contains differential equations. This makes it easy to extend an existing steady state model to simulate dynamic operation of the plant. The use of the program is illustrated by examples of gas turbine models. The paper also...

  14. On-Chip Evaluation of DNA Methylation with Electrochemical Combined Bisulfite Restriction Analysis Utilizing a Carbon Film Containing a Nanocrystalline Structure.

    Science.gov (United States)

    Kurita, Ryoji; Yanagisawa, Hiroyuki; Kamata, Tomoyuki; Kato, Dai; Niwa, Osamu

    2017-06-06

    This paper reports an on-chip electrochemical assessment of the DNA methylation status in genomic DNA on a conductive nanocarbon film electrode realized with combined bisulfite restriction analysis (COBRA). The film electrode consists of sp 2 and sp 3 hybrid bonds and is fabricated with an unbalanced magnetron (UBM) sputtering method. First, we studied the effect of the sp 2 /sp 3 ratio of the UBM nanocarbon film electrode with p-aminophenol, which is a major electro-active product of the labeling enzyme from p-aminophenol phosphate. The signal current for p-aminophenol increases as the sp 2 content in the UBM nanocarbon film electrode increases because of the π-π interaction between aromatic p-aminophenol and the graphene-like sp 2 structure. Furthermore, the capacitative current at the UBM nanocarbon film electrode was successfully reduced by about 1 order of magnitude thanks to the angstrom-level surface flatness. Therefore, a high signal-to-noise ratio was achieved compared with that of conventional electrodes. Then, after performing an ELISA-like hybridization assay with a restriction enzyme, we undertook an electrochemical evaluation of the cytosine methylation status in DNA by measuring the oxidation current derived from p-aminophenol. When the target cytosine in the analyte sequence is methylated (unmethylated), the restriction enzyme of HpyCH4IV is able (unable) to cleave the sequence, that is, the detection probe cannot (can) hybridize. We succeeded in estimating the methylation ratio at a site-specific CpG site from the peak current of a cyclic voltammogram obtained from a PCR product solution ranging from 0.01 to 1 nM.

  15. BOLDMirror: a global mirror system of DNA barcode data.

    Science.gov (United States)

    Liu, D; Liu, L; Guo, G; Wang, W; Sun, Q; Parani, M; Ma, J

    2013-11-01

    DNA barcoding is a novel concept for taxonomic identification using short, specific genetic markers and has been applied to study a large number of eukaryotes. The huge amount of data output generated by DNA barcoding requires well-organized information systems. Besides the Barcode of Life Data system (BOLD) established in Canada, the mirror system is also important for the international barcode of life project (iBOL). For this purpose, we developed the BOLDMirror, a global mirror system of DNA barcode data. It is open-sourced and can run on the LAMP (Linux + Apache + MySQL + PHP) environment. BOLDMirror has data synchronization, data representation and statistics modules, and also provides spaces to store user operation history. BOLDMirror can be accessed at http://www.boldmirror.net and several countries have used it to setup their site of DNA barcoding. © 2012 John Wiley & Sons Ltd.

  16. Is DNA a nonlinear dynamical system where solitary conformational ...

    Indian Academy of Sciences (India)

    Unknown

    DNA is considered as a nonlinear dynamical system in which solitary conformational waves can be excited. The ... nonlinear differential equations and their soliton-like solu- .... structure and dynamics can be added till the most accurate.

  17. Development of a defined-sequence DNA system for use in DNA misrepair studies

    International Nuclear Information System (INIS)

    Sutton, S.; Tobias, C.A.

    1984-01-01

    The authors have developed a system that allows them to study cellular DNA repair processes at the molecular level. In particular, the authors are using this system to examine the consequences of a misrepair of radiation-induced DNA damage, as a function of dose. The cells being used are specially engineered haploid yeast cells. Maintained in the cells, at one copy per cell, is a cen plasmid, a plasmid that behaves like a functional chromosome. This plasmid carries a small defined sequence of DNA from the E. coli lac z gene. It is this lac z region (called the alpha region) that serves as the target for radiation damage. Two copies of the complimentary portion of the lac z gene are integrated into the yeast genome. Irradiated cells are screened for possible mutation in the alpha region by testing the cells' ability to hydrolyze xgal, a lactose substrate. The DNA of interest is then extracted from the cells, sequenced, and the sequence is compared to that of the control. Unlike the usual defined-sequence DNA systems, theirs is an in vivo system. A disadvantage is the relatively high background mutation rate. Results achieved with this system, as well as future applications, are discussed

  18. DNA repair systems as targets of cadmium toxicity

    International Nuclear Information System (INIS)

    Giaginis, Constantinos; Gatzidou, Elisavet; Theocharis, Stamatios

    2006-01-01

    Cadmium (Cd) is a heavy metal and a potent carcinogen implicated in tumor development through occupational and environmental exposure. Recent evidence suggests that proteins participating in the DNA repair systems, especially in excision and mismatch repair, are sensitive targets of Cd toxicity. Cd by interfering and inhibiting these DNA repair processes might contribute to increased risk for tumor formation in humans. In the present review, the information available on the interference of Cd with DNA repair systems and their inhibition is summarized. These actions could possibly explain the indirect contribution of Cd to mutagenic effects and/or carcinogenicity

  19. Progressive multifocal leukoencephalopathy restricted to the posterior fossa in a patient with systemic lupus erythematosus

    Energy Technology Data Exchange (ETDEWEB)

    Goncalves, Fabricio Guimaraes; Lamb, Leslie; Del Carpio-O' Donovan, Raquel, E-mail: goncalves.neuroradio@gmail.com [McGill University Health Center Montreal General Hospital (Canada)

    2011-11-15

    Progressive multifocal leukoencephalopathy is a neurological infectious disease caused by the John Cunningham polyoma virus (JCV), an opportunistic agent with worldwide distribution. This disease is frequently seen in immunosuppressed patients and rarely associated with systemic lupus erythematosus. In the central nervous system PML demyelinating lesions occur in the supratentorial compartment. The authors describe a rare case of PML secondary to SLE treatment with atypical presentation restricted to the posterior fossa (author)

  20. Progressive multifocal leukoencephalopathy restricted to the posterior fossa in a patient with systemic lupus erythematosus

    International Nuclear Information System (INIS)

    Goncalves, Fabricio Guimaraes; Lamb, Leslie; Del Carpio-O'Donovan, Raquel

    2011-01-01

    Progressive multifocal leukoencephalopathy is a neurological infectious disease caused by the John Cunningham polyoma virus (JCV), an opportunistic agent with worldwide distribution. This disease is frequently seen in immunosuppressed patients and rarely associated with systemic lupus erythematosus. In the central nervous system PML demyelinating lesions occur in the supratentorial compartment. The authors describe a rare case of PML secondary to SLE treatment with atypical presentation restricted to the posterior fossa (author)

  1. Level-2 Milestone 6007: Sierra Early Delivery System Deployed to Secret Restricted Network

    Energy Technology Data Exchange (ETDEWEB)

    Bertsch, A. D. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

    2017-09-06

    This report documents the delivery and installation of Shark, a CORAL Sierra early delivery system deployed on the LLNL SRD network. Early ASC program users have run codes on the machine in support of application porting for the final Sierra system which will be deployed at LLNL in CY2018. In addition to the SRD resource, Shark, unclassified resources, Rzmanta and Ray, have been deployed on the LLNL Restricted Zone and Collaboration Zone networks in support of application readiness for the Sierra platform.

  2. 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

  3. Charge transfer in pi-stacked systems including DNA

    International Nuclear Information System (INIS)

    Siebbeles, L.D.A.

    2003-01-01

    Charge migration in DNA is a subject of intense current study motivated by long-range detection of DNA damage and the potential application of DNA as a molecular wire in nanoscale electronic devices. A key structural element, which makes DNA a medium for long-range charge transfer, is the array of stacked base pairs in the interior of the double helix. The overlapping pi-orbitals of the nucleobases provide a pathway for motion of charge carriers generated on the stack. This 'pi-pathway' resembles the columnarly stacked macrocyclic cores in discotic materials such as triphenylenes. The structure of these pi-stacked systems is highly disordered with dynamic fluctuations occurring on picosecond to nanosecond time scales. Theoretical calculations, concerning the effects of structural disorder and nucleobase sequence in DNA, on the dynamics of charge carriers are presented. Electronic couplings and localization energies of charge carriers were calculated using density functional theory (DFT). Results for columnarly stacked triphenylenes and DNA nucleobases are compared. The results are used to provide insight into the factors that control the mobility of charge carriers. Further, experimental results on the site-selective oxidation of guanine nucleobases in DNA (hot spots for DNA damage) are analyzed on basis of the theoretical results

  4. Chronic grouped social restriction triggers long-lasting immune system adaptations.

    Science.gov (United States)

    Tian, Rui; Hou, Gonglin; Song, Liuwei; Zhang, Jianming; Yuan, Ti-Fei

    2017-05-16

    Chronic stress triggers rigorous psychological and physiological changes, including immunological system adaptations. However, the effects of long-term social restriction on human immune system have not been investigated. The present study is to investigate the effect of chronic stress on immune changes in human blood, with the stress stimuli controlled.10 male volunteers were group isolated from the modern society in a 50-meter-square room for 150 days, with enriched nutrition and good living conditions provided. Serum examination of immune system markers demonstrated numerous changes in different aspects of the immune functions. The changes were observed as early as 30 days and could last for another 150 days after the termination of the restriction period (300 days' time point). The results strongly argued for the adaptation of immunological system under chronic social restriction stress in adult human, preceding a clear change in psychological conditions. The changes of these immune system factors could as well act as the serum biomarkers in clinical early-diagnosis of stress-related disorders.

  5. DNA in the Criminal Justice System: The DNA Success Story in Perspective.

    Science.gov (United States)

    Mapes, Anna A; Kloosterman, Ate D; de Poot, Christianne J

    2015-07-01

    Current figures on the efficiency of DNA as an investigative tool in criminal investigations only tell part of the story. To get the DNA success story in the right perspective, we examined all forensic reports from serious (N = 116) and high-volume crime cases (N = 2791) over the year 2011 from one police region in the Netherlands. These data show that 38% of analyzed serious crime traces (N = 384) and 17% of analyzed high-volume crime traces (N = 386) did not result in a DNA profile. Turnaround times (from crime scene to DNA report) were 66 days for traces from serious crimes and 44 days for traces from high-volume crimes. Suspects were truly identified through a match with the Offender DNA database of the Netherlands in 3% of the serious crime cases and in 1% of the high-volume crime cases. These data are important for both the forensic laboratory and the professionals in the criminal justice system to further optimize forensic DNA testing as an investigative tool. © 2015 American Academy of Forensic Sciences.

  6. DNA barcode-based molecular identification system for fish species.

    Science.gov (United States)

    Kim, Sungmin; Eo, Hae-Seok; Koo, Hyeyoung; Choi, Jun-Kil; Kim, Won

    2010-12-01

    In this study, we applied DNA barcoding to identify species using short DNA sequence analysis. We examined the utility of DNA barcoding by identifying 53 Korean freshwater fish species, 233 other freshwater fish species, and 1339 saltwater fish species. We successfully developed a web-based molecular identification system for fish (MISF) using a profile hidden Markov model. MISF facilitates efficient and reliable species identification, overcoming the limitations of conventional taxonomic approaches. MISF is freely accessible at http://bioinfosys.snu.ac.kr:8080/MISF/misf.jsp .

  7. A laboratory information management system for DNA barcoding workflows.

    Science.gov (United States)

    Vu, Thuy Duong; Eberhardt, Ursula; Szöke, Szániszló; Groenewald, Marizeth; Robert, Vincent

    2012-07-01

    This paper presents a laboratory information management system for DNA sequences (LIMS) created and based on the needs of a DNA barcoding project at the CBS-KNAW Fungal Biodiversity Centre (Utrecht, the Netherlands). DNA barcoding is a global initiative for species identification through simple DNA sequence markers. We aim at generating barcode data for all strains (or specimens) included in the collection (currently ca. 80 k). The LIMS has been developed to better manage large amounts of sequence data and to keep track of the whole experimental procedure. The system has allowed us to classify strains more efficiently as the quality of sequence data has improved, and as a result, up-to-date taxonomic names have been given to strains and more accurate correlation analyses have been carried out.

  8. 31 CFR 205.17 - Are funds transfers delayed by automated payment systems restrictions based on the size and...

    Science.gov (United States)

    2010-07-01

    ... automated payment systems restrictions based on the size and timing of the drawdown request subject to this... Treasury-State Agreement § 205.17 Are funds transfers delayed by automated payment systems restrictions... to payment processes that automatically reject drawdown requests that fall outside a pre-determined...

  9. DNA polymorphism of HLA class II genes in systemic lupus erythematosus

    DEFF Research Database (Denmark)

    Cowland, J B; Andersen, V; Halberg, P

    1994-01-01

    We investigated the DNA restriction fragment length polymorphism (RFLP) of the major histocompatibility complex (MHC) genes: HLA-DRB, -DQA, -DQB, -DPB in 24 Danish patients with systemic lupus erythematosus (SLE) and in 102 healthy Danes. A highly significant increase of the frequency of the DR3......- and DRw6-associated 7.00 kb DRB TaqI DNA fragment was found in SLE patients compared to normal controls (83.3% vs 35.5%; RR = 9.1, p 1*0501-associated 4.56 kb DQA TaqI fragment and the DRB3*01/03-associated 9.79 kb TaqI fragment were also found to be significantly...... increased in SLE patients (70.8% vs 29.7%; RR = 5.8, p 1%; RR = 4.3, p

  10. New polymorphic mtDNA restriction site in the 12S rRNA gene detected in Tunisian patients with non-syndromic hearing loss

    International Nuclear Information System (INIS)

    Mkaouar-Rebai, Emna; Tlili, Abdelaziz; Masmoudi, Saber; Charfeddine, Ilhem; Fakhfakh, Faiza

    2008-01-01

    The 12S rRNA gene was shown to be a hot spot for aminoglycoside-induced and non-syndromic hearing loss since several deafness-associated mtDNA mutations were identified in this gene. Among them, we distinguished the A1555G, the C1494T and the T1095C mutations and C-insertion or deletion at position 961. One hundred Tunisian patients with non-syndromic hearing loss and 100 hearing individuals were analysed in this study. A PCR-RFLP analysis with HaeIII restriction enzyme showed the presence of the A1555G mutation in the 12S rRNA gene in only one out of the 100 patients. In addition, PCR-RFLP and radioactive PCR revealed the presence of a new HaeIII polymorphic restriction site in the same gene of 12S rRNA site in 4 patients with non-syndromic hearing loss. UVIDOC-008-XD analyses showed the presence of this new polymorphic restriction site with a variable heteroplasmic rates at position +1517 of the human mitochondrial genome. On the other hand, direct sequencing of the entire mitochondrial 12S rRNA gene in the 100 patients and in 100 hearing individuals revealed the presence of the A750G and A1438G polymorphisms and the absence of the C1494T, T1095C and 961insC mutations in all the tested individuals. Sequencing of the whole mitochondrial genome in the 4 patients showing the new HaeIII polymorphic restriction site revealed only the presence of the A8860G transition in the MT-ATP6 gene and the A4769G polymorphism in the ND2 gene

  11. Restriction enzyme analysis of the chloroplast DNA of Phaseolus vulgaris L. vr. Rio Negro Análise de restrição do DNA cloroplástico de Phaseolus vulgaris vr. Rio Negro

    Directory of Open Access Journals (Sweden)

    Sergio Echeverrigaray

    1996-12-01

    Full Text Available The chloroplast DNA of Phaseolus vulgaris L. vr. Rio Negro was isola ted from chloroplasts obtained by descontiuous sucrose gradient centrifugation. The restriction analysis with the enzymes HindIII, EcoRI and BamHI and their combination, allowed to identified more than 20 fragments of 18 to 0.65kb. The size of Phaseolus vulgaris L. cp DNA was estimated in 140kb with the presence of a repeat sequence of about 22kb.O DNA cloroplástico do cultivar Rio Negro (Phaseolus vulgaris L. foi isolado a partir de cloroplastos obtidos por gradiente descontínuo de sacarose. A análise de restrição com as enzimas HindIII, EcoRI e BamHI e a combinação destas, permitiu a identificação de mais de 20 fragmentos na faixa de 18 a 0.65kb. O tamanho do cp DNA de Phaseolus vulgaris L. foi estimado em 140kb com a existência de sequências repetidas de aproximadamente 22kb.

  12. A Rewritable, Random-Access DNA-Based Storage System.

    Science.gov (United States)

    Yazdi, S M Hossein Tabatabaei; Yuan, Yongbo; Ma, Jian; Zhao, Huimin; Milenkovic, Olgica

    2015-09-18

    We describe the first DNA-based storage architecture that enables random access to data blocks and rewriting of information stored at arbitrary locations within the blocks. The newly developed architecture overcomes drawbacks of existing read-only methods that require decoding the whole file in order to read one data fragment. Our system is based on new constrained coding techniques and accompanying DNA editing methods that ensure data reliability, specificity and sensitivity of access, and at the same time provide exceptionally high data storage capacity. As a proof of concept, we encoded parts of the Wikipedia pages of six universities in the USA, and selected and edited parts of the text written in DNA corresponding to three of these schools. The results suggest that DNA is a versatile media suitable for both ultrahigh density archival and rewritable storage applications.

  13. Restrictive Cardiomyopathy Associated With Long-Term Use of Hydroxychloroquine for Systemic Lupus Erythematosus.

    Science.gov (United States)

    Sabato, Leah A; Mendes, Lisa A; Cox, Zachary L

    2017-10-01

    Hydroxychloroquine (HQ) is commonly prescribed for autoimmune diseases such as systemic lupus erythematosus. We report a case of a 75-year-old female presenting with de novo decompensated heart failure and restrictive cardiomyopathy (left ventricular ejection fraction: 40%-45%) after treatment with HQ for more than 11 years. Hydroxychloroquine was discontinued, and follow-up echocardiogram 57 days after discontinuation showed normalization of her left ventricular ejection fraction. A score of 7 on the Naranjo Adverse Drug Reaction Probability Scale indicates that HQ is a probable cause of this patient's cardiomyopathy. An adverse drug effect due to HQ should be considered in treated patients who present with restrictive cardiomyopathy. Discontinuation may allow for partial or complete reversal of the cardiomyopathy.

  14. H∞ control of Lur'e systems with sector and slope restricted nonlinearities

    International Nuclear Information System (INIS)

    Park, Ju H.; Ji, D.H.; Won, S.C.; Lee, S.M.; Choi, S.J.

    2009-01-01

    This Letter considers H ∞ controller design scheme for Lur'e systems with sector/slope restrictions and external disturbance. Based on Lyapunov theory and linear matrix inequality (LMI) formulation, a state feedback controller is designed to not only guarantee stability of systems but also reduce the effect of external disturbance to an H ∞ norm constraint. The nonlinearities are expressed as convex combinations of sector and slope bounds so that equality constraints are converted into inequality constraints using convex properties of the nonlinear function. Then, the stabilizing feedback gain matrix is derived through LMI formulation. Finally, a numerical example shows the effectiveness of the proposed method.

  15. The effect of 1-week feed restriction on performance, digestibility of nutrients and digestive system development in the growing rabbit.

    Science.gov (United States)

    Tůmová, E; Volek, Z; Chodová, D; Härtlová, H; Makovický, P; Svobodová, J; Ebeid, T A; Uhlířová, L

    2016-01-01

    A 3 to 4 week feed restriction of about 20% to 25% of the free intake is widely applied in rabbit breeding systems to reduce post-weaning digestive disorders. However, a short intensive feed restriction is described in few studies and can be beneficial for growing rabbits due to a longer re-alimentation period. The aim of this study was to evaluate the effect of ad libitum (AL) and two restriction levels of feeding (50 and 65 g/rabbit per day) applied for 1 week on performance, gastrointestinal morphology and physiological parameters during the restriction and during the re-alimentation period. Rabbits were divided into three experimental groups: AL rabbits were fed AL, R1 rabbits were restricted from 42 to 49 days of age and received 50 g daily (29% of AL) and R2 rabbits were restricted at the same age and were fed 65 g of feed daily (37% of AL). In the 1(st) week after weaning and in the weeks after restriction, all the groups were fed AL. During the restriction period, daily weight gain (DWG) in R1 significantly dropped to 11% (experiment 1) and 5% (experiment 2) compared with rabbits in the AL group, although they were fed 29% of AL, whereas in the R2 group it decreased to 20% (experiment 1) and 10% (experiment 2). In the week following feed restriction, DWG in the restricted groups increased (Pdepth of crypts, which might be involved in the compensatory growth and defence mechanism.

  16. EDNA-An expert software system for comparison and evaluation of DNA profiles in forensic casework

    DEFF Research Database (Denmark)

    Haldemann, B.; Dornseifer, S.; Heylen, T.

    2015-01-01

    eDNA is an expert software system for DNA profile comparison, match interpretation and automated report generation in forensic DNA casework. Process automation and intelligent graphical representation maximise reliability of DNA evidence, while facilitating and accelerating the work of DNA experts....

  17. Restrictive Cardiomyopathy

    Science.gov (United States)

    ... up in the circulatory system. In time, the heart fails. What causes it? Restrictive cardiomyopathy is often caused by diseases in other parts of the body. One known cause is cardiac ... build up in the heart tissue, making the tissue stiff and thickened. Cardiac ...

  18. Comparing the effects of food restriction and overeating on brain reward systems.

    Science.gov (United States)

    Avena, Nicole M; Murray, Susan; Gold, Mark S

    2013-10-01

    Both caloric restriction and overeating have been shown to affect neural processes associated with reinforcement. Both preclinical and some clinical studies have provided evidence that food restriction may increase reward sensitivity, and while there are mixed findings regarding the effects of overeating on reward sensitivity, there is strong evidence linking this behavior with changes in reward-related brain regions. Evidence of these changes comes in part from findings that show that such eating patterns are associated with increased drug use. The data discussed here regarding the differential effects of various eating patterns on reward systems may be particularly relevant to the aging population, as this population has been shown to exhibit altered reward sensitivity and decreased caloric consumption. Moreover, members of this population appear to be increasingly affected by the current obesity epidemic. Food, like alcohol or drugs, can stimulate its own consumption and produce similar neurochemical changes in the brain. Age-related loss of appetite, decreased eating, and caloric restriction are hypothesized to be associated with changes in the prevalence of substance misuse, abuse, and dependence seen in this cohort. Copyright © 2013 Elsevier Inc. All rights reserved.

  19. Diversity of the DNA Replication System in the Archaea Domain

    Directory of Open Access Journals (Sweden)

    Felipe Sarmiento

    2014-01-01

    Full Text Available The precise and timely duplication of the genome is essential for cellular life. It is achieved by DNA replication, a complex process that is conserved among the three domains of life. Even though the cellular structure of archaea closely resembles that of bacteria, the information processing machinery of archaea is evolutionarily more closely related to the eukaryotic system, especially for the proteins involved in the DNA replication process. While the general DNA replication mechanism is conserved among the different domains of life, modifications in functionality and in some of the specialized replication proteins are observed. Indeed, Archaea possess specific features unique to this domain. Moreover, even though the general pattern of the replicative system is the same in all archaea, a great deal of variation exists between specific groups.

  20. DNA-assisted swarm control in a biomolecular motor system.

    Science.gov (United States)

    Keya, Jakia Jannat; Suzuki, Ryuhei; Kabir, Arif Md Rashedul; Inoue, Daisuke; Asanuma, Hiroyuki; Sada, Kazuki; Hess, Henry; Kuzuya, Akinori; Kakugo, Akira

    2018-01-31

    In nature, swarming behavior has evolved repeatedly among motile organisms because it confers a variety of beneficial emergent properties. These include improved information gathering, protection from predators, and resource utilization. Some organisms, e.g., locusts, switch between solitary and swarm behavior in response to external stimuli. Aspects of swarming behavior have been demonstrated for motile supramolecular systems composed of biomolecular motors and cytoskeletal filaments, where cross-linkers induce large scale organization. The capabilities of such supramolecular systems may be further extended if the swarming behavior can be programmed and controlled. Here, we demonstrate that the swarming of DNA-functionalized microtubules (MTs) propelled by surface-adhered kinesin motors can be programmed and reversibly regulated by DNA signals. Emergent swarm behavior, such as translational and circular motion, can be selected by tuning the MT stiffness. Photoresponsive DNA containing azobenzene groups enables switching between solitary and swarm behavior in response to stimulation with visible or ultraviolet light.

  1. Quantum entanglement and quantum information in biological systems (DNA)

    Science.gov (United States)

    Hubač, Ivan; Švec, Miloslav; Wilson, Stephen

    2017-12-01

    Recent studies of DNA show that the hydrogen bonds between given base pairs can be treated as diabatic systems with spin-orbit coupling. For solid state systems strong diabaticity and spin-orbit coupling the possibility of forming Majorana fermions has been discussed. We analyze the hydrogen bonds in the base pairs in DNA from this perspective. Our analysis is based on a quasiparticle supersymmetric transformation which couples electronic and vibrational motion and includes normal coordinates and the corresponding momenta. We define qubits formed by Majorana fermions in the hydrogen bonds and also discuss the entangled states in base pairs. Quantum information and quantum entropy are introduced. In addition to the well-known classical information connected with the DNA base pairs, we also consider quantum information and show that the classical and quantum information are closely connected.

  2. Mitochondrial DNA polymerase editing mutation, PolgD257A, disturbs stem-progenitor cell cycling in the small intestine and restricts excess fat absorption.

    Science.gov (United States)

    Fox, Raymond G; Magness, Scott; Kujoth, Gregory C; Prolla, Tomas A; Maeda, Nobuyo

    2012-05-01

    Changes in intestinal absorption of nutrients are important aspects of the aging process. To address this issue, we investigated the impact of accelerated mitochondrial DNA mutations on the stem/progenitor cells in the crypts of Lieberkühn in mice homozygous for a mitochondrial DNA polymerase gamma mutation, Polg(D257A), that exhibit accelerated aging phenotype. As early as 3-7 mo of age, the small intestine was significantly enlarged in the PolgD257A mice. The crypts of the PolgD257A mice contained 20% more cells than those of their wild-type littermates and exhibited a 10-fold increase in cellular apoptosis primarily in the stem/progenitor cell zones. Actively dividing cells were proportionally increased, yet a significantly smaller proportion of cells was in the S phase of the cell cycle. Stem cell-derived organoids from PolgD257A mice failed to develop fully in culture and exhibited fewer crypt units, indicating an impact of the mutation on the intestinal epithelial stem/progenitor cell maintenance. In addition, epithelial cell migration along the crypt-villus axis was slowed and less organized, and the ATP content in the villi was significantly reduced. On a high-fat, high-carbohydrate diet, PolgD257A mice showed significantly restricted absorption of excess lipids accompanied by an increase in fecal steatocrits. We conclude that the PolgD257A mutation causes cell cycle dysregulation in the crypts leading to the age-associated changes in the morphology of the small intestine and contributes to the restricted absorption of dietary lipids.

  3. Aberrant septin 9 DNA methylation in colorectal cancer is restricted to a single CpG island

    International Nuclear Information System (INIS)

    Wasserkort, Reinhold; Kalmar, Alexandra; Valcz, Gabor; Spisak, Sandor; Krispin, Manuel; Toth, Kinga; Tulassay, Zsolt; Sledziewski, Andrew Z; Molnar, Bela

    2013-01-01

    The septin 9 gene (SEPT9) codes for a GTP-binding protein associated with filamentous structures and cytoskeleton formation. SEPT9 plays a role in multiple cancers as either an oncogene or a tumor suppressor gene. Regulation of SEPT9 expression is complex and not well understood; however, hypermethylation of the gene was recently introduced as a biomarker for early detection of colorectal cancer (CRC) and has been linked to cancer of the breast and of the head and neck. Because the DNA methylation landscape of different regions of SEPT9 is poorly understood in cancer, we analyzed the methylation patterns of this gene in distinct cell populations from normal and diseased colon mucosa. Laser capture microdissection was performed to obtain homogeneous populations of epithelial and stromal cells from normal, adenomatous, and tumorous colon mucosa. Microdissected samples were analyzed using direct bisulfite sequencing to determine the DNA methylation status of eight regions within and near the SEPT9 gene. Septin-9 protein expression was assessed using immunohistochemistry (IHC). Regions analyzed in SEPT9 were unmethylated in normal tissue except for a methylation boundary detected downstream of the largest CpG island. In adenoma and tumor tissues, epithelial cells displayed markedly increased DNA methylation levels (>80%, p <0.0001) in only one of the CpG islands investigated. SEPT9 methylation in stromal cells increased in adenomatous and tumor tissues (≤50%, p <0.0001); however, methylation did not increase in stromal cells of normal tissue close to the tumor. IHC data indicated a significant decrease (p <0.01) in Septin-9 protein levels in epithelial cells derived from adenoma and tumor tissues; Septin-9 protein levels in stromal cells were low in all tissues. Hypermethylation of SEPT9 in adenoma and CRC specimens is confined to one of several CpG islands of this gene. Tumor-associated aberrant methylation originates in epithelial cells; stromal cells appear to

  4. DNA Damage Repair System in Plants: A Worldwide Research Update.

    Science.gov (United States)

    Gimenez, Estela; Manzano-Agugliaro, Francisco

    2017-10-30

    Living organisms are usually exposed to various DNA damaging agents so the mechanisms to detect and repair diverse DNA lesions have developed in all organisms with the result of maintaining genome integrity. Defects in DNA repair machinery contribute to cancer, certain diseases, and aging. Therefore, conserving the genomic sequence in organisms is key for the perpetuation of life. The machinery of DNA damage repair (DDR) in prokaryotes and eukaryotes is similar. Plants also share mechanisms for DNA repair with animals, although they differ in other important details. Plants have, surprisingly, been less investigated than other living organisms in this context, despite the fact that numerous lethal mutations in animals are viable in plants. In this manuscript, a worldwide bibliometric analysis of DDR systems and DDR research in plants was made. A comparison between both subjects was accomplished. The bibliometric analyses prove that the first study about DDR systems in plants (1987) was published thirteen years later than that for other living organisms (1975). Despite the increase in the number of papers about DDR mechanisms in plants in recent decades, nowadays the number of articles published each year about DDR systems in plants only represents 10% of the total number of articles about DDR. The DDR research field was done by 74 countries while the number of countries involved in the DDR & Plant field is 44. This indicates the great influence that DDR research in the plant field currently has, worldwide. As expected, the percentage of studies published about DDR systems in plants has increased in the subject area of agricultural and biological sciences and has diminished in medicine with respect to DDR studies in other living organisms. In short, bibliometric results highlight the current interest in DDR research in plants among DDR studies and can open new perspectives in the research field of DNA damage repair.

  5. High Efficiency Hydrodynamic DNA Fragmentation in a Bubbling System.

    Science.gov (United States)

    Li, Lanhui; Jin, Mingliang; Sun, Chenglong; Wang, Xiaoxue; Xie, Shuting; Zhou, Guofu; van den Berg, Albert; Eijkel, Jan C T; Shui, Lingling

    2017-01-18

    DNA fragmentation down to a precise fragment size is important for biomedical applications, disease determination, gene therapy and shotgun sequencing. In this work, a cheap, easy to operate and high efficiency DNA fragmentation method is demonstrated based on hydrodynamic shearing in a bubbling system. We expect that hydrodynamic forces generated during the bubbling process shear the DNA molecules, extending and breaking them at the points where shearing forces are larger than the strength of the phosphate backbone. Factors of applied pressure, bubbling time and temperature have been investigated. Genomic DNA could be fragmented down to controllable 1-10 Kbp fragment lengths with a yield of 75.30-91.60%. We demonstrate that the ends of the genomic DNAs generated from hydrodynamic shearing can be ligated by T4 ligase and the fragmented DNAs can be used as templates for polymerase chain reaction. Therefore, in the bubbling system, DNAs could be hydrodynamically sheared to achieve smaller pieces in dsDNAs available for further processes. It could potentially serve as a DNA sample pretreatment technique in the future.

  6. Sistemas multi–modales de profundidad restringida Multi-modal systems of restricted depth

    Directory of Open Access Journals (Sweden)

    Manuel Sierra A.

    2008-12-01

    Full Text Available Se presentan como extensiones del cálculo proposicional clásico, la jerarquíade sistemas deductivos SMM–n con n > 1. SMM–n es el sistema multi–modalde profundidad–n. El sistema SMM–1 es el cálculo proposicional clásico. Elsistema SMM–(n + 1 puede ser visto como el resultado de aplicar la regla denecesariedad, asociada a los razonadores con suficiente capacidad de razona-miento, una vez a los teoremas del sistema SMM–n. El sistema SMM resultade la reunión de los sistemas de la jerarquía, y puede ser visto como el sis-tema de lógica multi–modal Km con restricciones. Los sistemas SMM–n soncaracterizados con una semántica al estilo Kripke, en la cual, la longitud delas cadenas de mundos posibles se encuentra restringida.They are presented as extensions of the classical propositional logic, the hierarchy of deductive systems SMM–n with n > 1. SMM–n is the multi–modal system of depth–n. The system SMM–1 is the classical propositional logic. The system SMM–(n + 1 it can be seen as the result of applying the necesariedad rule, associated to the reasoners with enough reasoning capacity, once to the theorems of the system SMM–n. The system SMM is of the union of the systems of the hierarchy, and it can be seen as the system of logic multimodal Km with restrictions. The systems SMM–n are characterized with a semantics to the style Kripke, in the one which, the longitude of the chains of possible worlds is restricted.

  7. Increased systemic oxidatively generated DNA and RNA damage in schizophrenia

    DEFF Research Database (Denmark)

    Jørgensen, Anders; Brødbæk, Kasper; Fink-Jensen, Anders

    2013-01-01

    such as cardiovascular disease, type 2 diabetes and dementia. We determined the urinary excretion of markers of systemic Deoxyribonucleic Acid (DNA) and Ribonucleic Acid (RNA) oxidation, 8-oxo-7,8-dihydro-2'-deoxyguanosine and 8-oxo-7,8-dihydroguanosine, respectively, in 40 schizophrenia patients and 40 age- and sex...

  8. KERAGAMAN GENETIK BENIH IKAN KERAPU SUNU, Plectrophomus leopardus TURUNAN PERTAMA (F1 DENGAN ANALISIS RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP MT-DNA

    Directory of Open Access Journals (Sweden)

    Gusti Ngurah Permana

    2016-11-01

    The variability of differences size was occurred on every culture period of coral trout. The aimed of this study was to know genetics variability and evaluated of which are expressed on large, medium, and small size fry on total of length sizes and different weight. Amplification of single fragment using set primer 16 SrDNA (F5’CGCCTG TTTAACAAAAACAT-3’ and reverse (R: 5’-CCGGTCTGAACTCAGATCATGT-3’. Result showed that PCR amplification of mt-DNA was 625 bp. Restriction digestion processed with Mnl I enzyme showed that polymorphism in large size and monomorphic in both medium and small sizes. Two types of haplotype were found in large size (ABABB and ABAAB while one haplotype observed in medium and small sizes ABABB. The heterozygosities value of large, medium and small sizes from Bali location were 0.480, 0.000, and 0.000 restectively. Heterozygosities value of samples from East Java were 0.211, 0.000, and 0.000 restectively. Samples from Lampung were monomorphic (0.000.

  9. Failure to detect circulating DNA-anti-DNA complexes by four radioimmunological methods in patients with systemic lupus erythematosus

    International Nuclear Information System (INIS)

    Izui, S.; Lambert, P.H.; Miescher, P.A.

    1977-01-01

    The presence of DNA-anti-DNA complexes in sera from patients with systemic lupus erythematosus (SLE) was investigated by two new radioimmunoassays (RIA) developed for this purpose and by measuring the CLq and DNA binding activity of serum before and after treatment with DNAse. Two direct RIA developed in this study were based on the reactivity of [ 3 H]actinomycin D ([ 3 H]ACT-D) or solid-phase methylated bovine serum albumin (mBSA) with DNA-anti-DNA complexes. DNA-anti-DNA complexes prepared in vitro could be efficiently detected at various antigen-antibody ratios by these two RIA. Increased levels of circulating immune complexes as indicated by the CLq binding test were found in 52% of SLE sera. However, the frequency of specific DNA-anti-DNA complexes detected in SLE sera was very low. Only 6% of sera exhibited an increased value deviating by more than three s.d. from the normal mean when tested with the [ 3 H]ACT-D binding RIA or the solid-phase mBSA RIA. On the other hand, there was no significant difference in the serum CLq or DNA binding activity after treatment with DNAse. These results suggest that DNA-anti-DNA complexes do not occur frequently in circulating blood and represent only a very small portion of the immune complexes detected in serum from patients with SLE. (author)

  10. Failure to detect circulating DNA-anti-DNA complexes by four radioimmunological methods in patients with systemic lupus erythematosus

    Energy Technology Data Exchange (ETDEWEB)

    Izui, S; Lambert, P H; Miescher, P A [Hopital Cantonal Geneve (Switzerland)

    1977-12-01

    The presence of The DNA-anti-DNA complexes in sera from patients with systemic lupus erythematosus (SLE) was investigated by two new radioimmunoassays (RIA) developed for this purpose and by measuring the CLq and DNA binding activity of serum before and after treatment with DNAse. Two direct RIA developed in this study were based on the reactivity of (/sup 3/H)actinomycin D ((/sup 3/H)ACT-D) or solid-phase methylated bovine serum albumin (mBSA) with DNA-anti-DNA complexes. DNA-anti-DNA complexes prepared in vitro could be efficiently detected at various antigen-antibody ratios by these two RIA. Increased levels of circulating immune complexes as indicated by the CLq binding test were found in 52% of the SLE sera. However, the frequency of specific DNA-anti-DNA complexes detected in the SLE sera was very low. Only 6% of the sera exhibited an increased value deviating by more than three s.d. from the normal mean when tested with the (/sup 3/H)ACT-D binding RIA or the solid-phase mBSA RIA. On the other hand, there was no significant difference in the serum CLq or DNA binding activity after treatment with DNAse. These results suggest that DNA-anti-DNA complexes do not occur frequently in circulating blood and represent only a very small portion of the immune complexes detected in serum from patients with SLE.

  11. The GOAT-ghrelin system is not essential for hypoglycemia prevention during prolonged calorie restriction.

    Directory of Open Access Journals (Sweden)

    Chun-Xia Yi

    Full Text Available Ghrelin acylation by ghrelin O-acyltransferase (GOAT has recently been reported to be essential for the prevention of hypoglycemia during prolonged negative energy balance. Using a unique set of four different genetic loss-of-function models for the GOAT/ghrelin/growth hormone secretagogue receptor (GHSR system, we thoroughly tested the hypothesis that lack-of-ghrelin activation or signaling would lead to hypoglycemia during caloric deprivation.Male and female knockout (KO mice for GOAT, ghrelin, GHSR, or both ghrelin and GHSR (dKO were subjected to prolonged calorie restriction (40% of ad libitum chow intake. Body weight, fat mass, and glucose levels were recorded daily and compared to wildtype (WT controls. Forty-eight hour blood glucose profiles were generated for each individual mouse when 2% or less body fat mass was reached. Blood samples were obtained for analysis of circulating levels of acyl- and desacyl-ghrelin, IGF-1, and insulin.Chronic calorie restriction progressively decreased body weight and body fat mass in all mice regardless of genotype. When fat mass was depleted to 2% or less of body weight for 2 consecutive days, random hypoglycemic events occurred in some mice across all genotypes. There was no increase in the incidence of hypoglycemia in any of the four loss-of-function models for ghrelin signaling including GOAT KO mice. Furthermore, no differences in insulin or IGF-1 levels were observed between genotypes.The endogenous GOAT-ghrelin-GHSR system is not essential for the maintenance of euglycemia during prolonged calorie restriction.

  12. Repair of uv damaged DNA in systemic lupus erythematosus. [Mice

    Energy Technology Data Exchange (ETDEWEB)

    Beighlie, D J; Teplitz, R L

    1975-06-01

    The NZB NZW hybrid mouse is an animal model of human systemic lupus erythematosus (SLE). Two breeding schemes were devised using NZB, NZW, B/W, and CBA mice, which permit definitive decisions regarding genetic and/or viral origin of the disease. It is proposed that at least two factors must be involved: a genetic abnormality producing hyper-responsiveness to nucleic acid antigens, and a DNA repair defect which results in liberation of DNA and RNA when cells are lethally injured. Evidence is presented for a DNA repair deficit in human SLE lymphocytes following in vitro irradiation with ultraviolet (uv) light. Lymphocytes from adult New Zealand and control mice were found to lack normal amounts of endonuclease necessary for repairing uv damage.

  13. Arduino-based automation of a DNA extraction system.

    Science.gov (United States)

    Kim, Kyung-Won; Lee, Mi-So; Ryu, Mun-Ho; Kim, Jong-Won

    2015-01-01

    There have been many studies to detect infectious diseases with the molecular genetic method. This study presents an automation process for a DNA extraction system based on microfluidics and magnetic bead, which is part of a portable molecular genetic test system. This DNA extraction system consists of a cartridge with chambers, syringes, four linear stepper actuators, and a rotary stepper actuator. The actuators provide a sequence of steps in the DNA extraction process, such as transporting, mixing, and washing for the gene specimen, magnetic bead, and reagent solutions. The proposed automation system consists of a PC-based host application and an Arduino-based controller. The host application compiles a G code sequence file and interfaces with the controller to execute the compiled sequence. The controller executes stepper motor axis motion, time delay, and input-output manipulation. It drives the stepper motor with an open library, which provides a smooth linear acceleration profile. The controller also provides a homing sequence to establish the motor's reference position, and hard limit checking to prevent any over-travelling. The proposed system was implemented and its functionality was investigated, especially regarding positioning accuracy and velocity profile.

  14. Characterization of primary biogenic aerosol particles in urban, rural, and high-alpine air by DNA sequence and restriction fragment analysis of ribosomal RNA genes

    Directory of Open Access Journals (Sweden)

    V. R. Després

    2007-12-01

    Full Text Available This study explores the applicability of DNA analyses for the characterization of primary biogenic aerosol (PBA particles in the atmosphere. Samples of fine particulate matter (PM2.5 and total suspended particulates (TSP have been collected on different types of filter materials at urban, rural, and high-alpine locations along an altitude transect in the south of Germany (Munich, Hohenpeissenberg, Mt. Zugspitze.

    From filter segments loaded with about one milligram of air particulate matter, DNA could be extracted and DNA sequences could be determined for bacteria, fungi, plants and animals. Sequence analyses were used to determine the identity of biological organisms, and terminal restriction fragment length polymorphism analyses (T-RFLP were applied to estimate diversities and relative abundances of bacteria. Investigations of blank and background samples showed that filter materials have to be decontaminated prior to use, and that the sampling and handling procedures have to be carefully controlled to avoid artifacts in the analyses.

    Mass fractions of DNA in PM2.5 were found to be around 0.05% in urban, rural, and high-alpine aerosols. The average concentration of DNA determined for urban air was on the order of ~7 ng m−3, indicating that human adults may inhale about one microgram of DNA per day (corresponding to ~108 haploid bacterial genomes or ~105 haploid human genomes, respectively.

    Most of the bacterial sequences found in PM2.5 were from Proteobacteria (42 and some from Actinobacteria (10 and Firmicutes (1. The fungal sequences were characteristic for Ascomycota (3 and Basidiomycota (1, which are known to actively discharge spores into the atmosphere. The plant sequences could be attributed to green plants (2 and moss spores (2, while animal DNA was found only for one unicellular eukaryote (protist.

  15. Is the Evolution of Salmonella enterica subsp. enterica Linked to Restriction-Modification Systems?

    DEFF Research Database (Denmark)

    Roer, Louise; Hendriksen, Rene S.; Leekitcharoenphon, Pimlapas

    2016-01-01

    Salmonella enterica subsp. enterica bacteria are highly diverse foodborne pathogens that are subdivided into more than 1,500 serovars. The diversity is believed to result from mutational evolution, as well as intra- and interspecies recombination that potentially could be influenced by restriction...... to the conjugational mode of horizontal gene transfer in Salmonella. Thus, we conclude that other factors must be involved in shaping the evolution of bacteria.......-modification (RM) systems. The aim of this study was to investigate whether RM systems were linked to the evolution of Salmonella enterica subsp. enterica. The study included 221 Salmonella enterica genomes, of which 68 were de novo sequenced and 153 were public available genomes from ENA. The data set covered 97...

  16. Specialty pharmacies and other restricted drug distribution systems: financial and safety considerations for patients and health-system pharmacists.

    Science.gov (United States)

    Kirschenbaum, Bonnie E

    2009-12-15

    To discuss the role of restricted drug distribution systems in the implementation of risk evaluation and mitigation strategies (REMS), health-system pharmacists' concerns associated with the use of specialty pharmacies and other restricted drug distribution systems, reimbursement policies for high-cost specialty drugs, supply chain models for traditional and specialty drugs, and emerging trends in the management of and reimbursement for specialty pharmaceuticals. Restricted drug distribution systems established by pharmaceutical manufacturers, specialty pharmacies, or other specialty suppliers may be a component of REMS, which are required by the Food and Drug Administration for the management of known or potential serious risks from certain drugs. Concerns of health-system pharmacists using specialty suppliers include access to pharmaceuticals, operational challenges, product integrity, financial implications, continuity of care, and patient safety. An ambulatory care patient taking a specialty drug product from home to a hospital outpatient clinic or inpatient setting for administration, a practice known as "brown bagging," raises concerns about product integrity and institutional liability. An institution's finances, tolerance for liability, and ability to skillfully manage the processes involved often determine its choice between an approach that prohibits brown bagging but is costly and one that permits the practice under certain conditions and is less costly. The recent shift from a traditional supply chain model to a specialty pharmacy supply chain model for high-cost pharmaceuticals has the potential to increase pharmaceutical costs for health systems. A dialogue is needed between health-system pharmacists and group purchasing organizations to address the latter's role in mitigating the financial implications of this change and to help clarify the safety issues. Some health plans have shifted part of the cost of expensive drugs to patients by establishing a

  17. Genotypic Characterization of Bradyrhizobium Strains Nodulating Endemic Woody Legumes of the Canary Islands by PCR-Restriction Fragment Length Polymorphism Analysis of Genes Encoding 16S rRNA (16S rDNA) and 16S-23S rDNA Intergenic Spacers, Repetitive Extragenic Palindromic PCR Genomic Fingerprinting, and Partial 16S rDNA Sequencing

    Science.gov (United States)

    Vinuesa, Pablo; Rademaker, Jan L. W.; de Bruijn, Frans J.; Werner, Dietrich

    1998-01-01

    We present a phylogenetic analysis of nine strains of symbiotic nitrogen-fixing bacteria isolated from nodules of tagasaste (Chamaecytisus proliferus) and other endemic woody legumes of the Canary Islands, Spain. These and several reference strains were characterized genotypically at different levels of taxonomic resolution by computer-assisted analysis of 16S ribosomal DNA (rDNA) PCR-restriction fragment length polymorphisms (PCR-RFLPs), 16S-23S rDNA intergenic spacer (IGS) RFLPs, and repetitive extragenic palindromic PCR (rep-PCR) genomic fingerprints with BOX, ERIC, and REP primers. Cluster analysis of 16S rDNA restriction patterns with four tetrameric endonucleases grouped the Canarian isolates with the two reference strains, Bradyrhizobium japonicum USDA 110spc4 and Bradyrhizobium sp. strain (Centrosema) CIAT 3101, resolving three genotypes within these bradyrhizobia. In the analysis of IGS RFLPs with three enzymes, six groups were found, whereas rep-PCR fingerprinting revealed an even greater genotypic diversity, with only two of the Canarian strains having similar fingerprints. Furthermore, we show that IGS RFLPs and even very dissimilar rep-PCR fingerprints can be clustered into phylogenetically sound groupings by combining them with 16S rDNA RFLPs in computer-assisted cluster analysis of electrophoretic patterns. The DNA sequence analysis of a highly variable 264-bp segment of the 16S rRNA genes of these strains was found to be consistent with the fingerprint-based classification. Three different DNA sequences were obtained, one of which was not previously described, and all belonged to the B. japonicum/Rhodopseudomonas rDNA cluster. Nodulation assays revealed that none of the Canarian isolates nodulated Glycine max or Leucaena leucocephala, but all nodulated Acacia pendula, C. proliferus, Macroptilium atropurpureum, and Vigna unguiculata. PMID:9603820

  18. Genotyping of major histocompatibility complex Class II DRB gene in Rohilkhandi goats by polymerase chain reaction-restriction fragment length polymorphism and DNA sequencing

    Directory of Open Access Journals (Sweden)

    Kush Shrivastava

    2015-10-01

    Full Text Available Aim: To study the major histocompatibility complex (MHC Class II DRB1 gene polymorphism in Rohilkhandi goat using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP and nucleotide sequencing techniques. Materials and Methods: DNA was isolated from 127 Rohilkhandi goats maintained at sheep and goat farm, Indian Veterinary Research Institute, Izatnagar, Bareilly. A 284 bp fragment of exon 2 of DRB1 gene was amplified and digested using BsaI and TaqI restriction enzymes. Population genetic parameters were calculated using Popgene v 1.32 and SAS 9.0. The genotypes were then sequenced using Sanger dideoxy chain termination method and were compared with related breeds/species using MEGA 6.0 and Megalign (DNASTAR software. Results: TaqI locus showed three and BsaI locus showed two genotypes. Both the loci were found to be in Hardy–Weinberg equilibrium (HWE, however, population genetic parameters suggest that heterozygosity is still maintained in the population at both loci. Percent diversity and divergence matrix, as well as phylogenetic analysis revealed that the MHC Class II DRB1 gene of Rohilkhandi goats was found to be in close cluster with Garole and Scottish blackface sheep breeds as compared to other goat breeds included in the sequence comparison. Conclusion: The PCR-RFLP patterns showed population to be in HWE and absence of one genotype at one locus (BsaI, both the loci showed excess of one or the other homozygote genotype, however, effective number of alleles showed that allelic diversity is present in the population. Sequence comparison of DRB1 gene of Rohilkhandi goat with other sheep and goat breed assigned Rohilkhandi goat in divergence with Jamanupari and Angora goats.

  19. ATR-p53 restricts homologous recombination in response to replicative stress but does not limit DNA interstrand crosslink repair in lung cancer cells.

    Directory of Open Access Journals (Sweden)

    Bianca M Sirbu

    Full Text Available Homologous recombination (HR is required for the restart of collapsed DNA replication forks and error-free repair of DNA double-strand breaks (DSB. However, unscheduled or hyperactive HR may lead to genomic instability and promote cancer development. The cellular factors that restrict HR processes in mammalian cells are only beginning to be elucidated. The tumor suppressor p53 has been implicated in the suppression of HR though it has remained unclear why p53, as the guardian of the genome, would impair an error-free repair process. Here, we show for the first time that p53 downregulates foci formation of the RAD51 recombinase in response to replicative stress in H1299 lung cancer cells in a manner that is independent of its role as a transcription factor. We find that this downregulation of HR is not only completely dependent on the binding site of p53 with replication protein A but also the ATR/ATM serine 15 phosphorylation site. Genetic analysis suggests that ATR but not ATM kinase modulates p53's function in HR. The suppression of HR by p53 can be bypassed under experimental conditions that cause DSB either directly or indirectly, in line with p53's role as a guardian of the genome. As a result, transactivation-inactive p53 does not compromise the resistance of H1299 cells to the interstrand crosslinking agent mitomycin C. Altogether, our data support a model in which p53 plays an anti-recombinogenic role in the ATR-dependent mammalian replication checkpoint but does not impair a cell's ability to use HR for the removal of DSB induced by cytotoxic agents.

  20. 21 CFR 809.40 - Restrictions on the sale, distribution, and use of OTC test sample collection systems for drugs...

    Science.gov (United States)

    2010-04-01

    ... OTC test sample collection systems for drugs of abuse testing. 809.40 Section 809.40 Food and Drugs... Restrictions on the sale, distribution, and use of OTC test sample collection systems for drugs of abuse testing. (a) Over-the-counter (OTC) test sample collection systems for drugs of abuse testing (§ 864.3260...

  1. Sign restriction approach to macro stress-testing of the Croatian banking system

    Directory of Open Access Journals (Sweden)

    Nataša Erjavec

    2012-12-01

    Full Text Available The paper employs Uhlig’s sign restriction approach to stress-testing of the Croatian banking system. The analysis is based on a standard monetary VAR comprising real economic activity, inflation and short-term interest rates augmented by the ratio of non-performing loans or return on average equity, both measures representing the aggregate banking sector. In spite of the selected indicator, the results suggest a strong sensitivity of the Croatian banking sector to macroeconomic shocks. The effects are the strongest for contractionary monetary policy shocks, followed by negative demand shocks while the effects of supply shocks turned out to be statistically insignificant. Since Croatia is a small open economy with banking the dominant financial sector, the results obtained could be interesting for policy makers in Croatia and other transition economies with similar characteristics.

  2. 21 CFR 864.7280 - Factor V Leiden DNA mutation detection systems.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Factor V Leiden DNA mutation detection systems....7280 Factor V Leiden DNA mutation detection systems. (a) Identification. Factor V Leiden deoxyribonucleic acid (DNA) mutation detection systems are devices that consist of different reagents and...

  3. Using DNA nanotechnology to produce a drug delivery system

    International Nuclear Information System (INIS)

    La, Thi Huyen; Nguyen, Thi Thu Thuy; Pham, Van Phuc; Nguyen, Thi Minh Huyen; Le, Quang Huan

    2013-01-01

    Drug delivery to cancer cells in chemotherapy is one of the most advanced research topics. The effectiveness of the current cancer treatment drugs is limited because they are not capable of distinguishing between cancer cells and normal cells so that they kill not only cancer cells but also normal ones. To overcome this disadvantage by profiting from the differences in physical and chemical properties between cancer and normal cells, nanoparticles (NPs) delivering a drug are designed in a specific manner such that they can distinguish the cancer cells from the normal ones and are targeted only to the cancer cells. Currently, there are various drug delivery systems with many advantages, but sharing some common disadvantages such as difficulty with controlling the size, low encapsulation capacity and low stability. With the development and success of DNA nanotechnology, DNA strands are used to create effective drug delivery NPs with precisely controlled size and structure, safety and high stability. This article presents our study on drug encapsulation in DNA nanostructure which loaded docetaxel and curcumin in a desire to create a new and effective drug delivery system with high biological compatibility. (paper)

  4. Using DNA nanotechnology to produce a drug delivery system

    Science.gov (United States)

    Huyen La, Thi; Thu Thuy Nguyen, Thi; Phuc Pham, Van; Huyen Nguyen, Thi Minh; Huan Le, Quang

    2013-03-01

    Drug delivery to cancer cells in chemotherapy is one of the most advanced research topics. The effectiveness of the current cancer treatment drugs is limited because they are not capable of distinguishing between cancer cells and normal cells so that they kill not only cancer cells but also normal ones. To overcome this disadvantage by profiting from the differences in physical and chemical properties between cancer and normal cells, nanoparticles (NPs) delivering a drug are designed in a specific manner such that they can distinguish the cancer cells from the normal ones and are targeted only to the cancer cells. Currently, there are various drug delivery systems with many advantages, but sharing some common disadvantages such as difficulty with controlling the size, low encapsulation capacity and low stability. With the development and success of DNA nanotechnology, DNA strands are used to create effective drug delivery NPs with precisely controlled size and structure, safety and high stability. This article presents our study on drug encapsulation in DNA nanostructure which loaded docetaxel and curcumin in a desire to create a new and effective drug delivery system with high biological compatibility. Invited talk at the 6th International Workshop on Advanced Materials Science and Nanotechnology, 30 October-2 November, 2012, Ha Long, Vietnam.

  5. Brain renin-angiotensin system: fetal epigenetic programming by maternal protein restriction during pregnancy.

    Science.gov (United States)

    Goyal, Ravi; Goyal, Dipali; Leitzke, Arthur; Gheorghe, Ciprian P; Longo, Lawrence D

    2010-03-01

    Maternal protein malnutrition during pregnancy can lead to significant alterations in the systemic renin-angiotensin system (RAS) in the fetus. All components of the RAS are present in brain and may be altered in many disease states. Importantly, these disorders are reported to be of higher incidence in prenatally malnourished individuals. In the current study, we tested the hypothesis that antenatal maternal low protein diet (MLPD) leads to epigenetic changes and alterations in gene expression of brain RAS of the mouse fetus. Mice dams were given control and 50% MLPD during second half of the gestation. We analyzed messenger RNA (mRNA), microRNA (miRNA), promoter DNA methylation, and protein expression of various RAS genes in the fetal offspring. As a consequence of 50% MLPD, fetal brains showed increased mRNA expression of angiotensinogen and angiotensin converting enzyme-1 (ACE-1), with a decrease in mRNA levels of angiotensin II type-2 (AT2) receptors. In contrast, while angiotensinogen protein expression was unaltered, the protein levels of ACE-1 and AT2 receptor genes were significantly reduced in the fetal brain from the MLPD dams. Our results also demonstrated hypomethylation of the CpG islands in the promoter regions of ACE-1 gene, and upregulation of the miRNAs, mmu-mir-27a and 27b, which regulate ACE-1 mRNA translation. Furthermore, our study showed reduced expression of the miRNA mmu-mir-330, which putatively regulates AT2 translation. For the developing fetal brain RAS, MLPD leads to significant alterations in the mRNA and protein expression, with changes in DNA methylation and miRNA, key regulators of hypertension in adults.

  6. A positioning system with no line-of-sight restrictions for cluttered environments

    Science.gov (United States)

    Prigge, Eric A.

    Accurate sensing of vehicle location and attitude is a fundamental requirement in many mobile-robot applications, but is a very challenging problem in the cluttered and unstructured environment of the real world. Many existing indoor positioning systems are limited in workspace and robustness because they require clear lines of sight or do not provide absolute, drift-free measurements. Examples include overhead vision systems, where an unobstructed view must be maintained between robot and camera, and inertial systems, where the measurements drift over time. The research presented in this dissertation provides a new location- and attitude-sensing system designed specifically to meet the challenges of operation in a realistic, cluttered indoor environment, such as that of an office building or warehouse. The system is not limited by line-of-sight restrictions and produces drift-free measurements throughout a three-dimensional operating volume that can span a large building. Accuracy of several centimeters and a few degrees is delivered at 10 Hz, and any number of the small sensor units can be in operation, all providing estimates in a common reference frame. This positioning system is based on extremely-low-frequency magnetic fields, which have excellent characteristics for penetrating line-of-sight obstructions. Beacons located throughout the workspace create the low-level fields. A sensor unit on the mobile robot samples the local magnetic field and processes the measurements to determine its location and attitude. This research overcomes limitations in existing magnetic-based systems. The design of the signal structure, based on pseudorandom codes, enables the use of multiple, distributed L-beacons and greatly expands coverage volume. The development of real-time identification and correction methods mitigates the impact of distortions caused by materials in the environment. A novel solution algorithm combats both challenges, providing increased coverage volume

  7. Tumor transfection after systemic injection of DNA lipid nanocapsules.

    Science.gov (United States)

    Morille, Marie; Passirani, Catherine; Dufort, Sandrine; Bastiat, Guillaume; Pitard, Bruno; Coll, Jean-Luc; Benoit, Jean-Pierre

    2011-03-01

    With the goal of generating an efficient vector for systemic gene delivery, a new kind of nanocarrier consisting of lipid nanocapsules encapsulating DOTAP/DOPE lipoplexes (DNA LNCs) was pegylated by the post-insertion of amphiphilic and flexible polymers. The aim of this surface modification was to create a long-circulating vector, able to circulate in the blood stream and efficient in transfecting tumoral cells after passive targeting by enhanced permeability and retention effect (EPR effect). PEG conformation, electrostatic features, and hydrophylicity are known to be important factors able to influence the pharmacokinetic behaviour of vectors. In this context, the surface structure characteristics of the newly pegylated DNA LNCs were studied by measuring electrophoretic mobility as a function of ionic strength in order to establish a correlation between surface properties and in vivo performance of the vector. Finally, thanks to this PEGylation, gene expression was measured up to 84-fold higher in tumor compared to other tested organs after intravenous injection. The present results indicate that PEGylated DNA LNCs are promising carriers for an efficient cancer gene therapy. Copyright © 2010 Elsevier Ltd. All rights reserved.

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

    International Nuclear Information System (INIS)

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

    1989-01-01

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

  9. Computational method and system for modeling, analyzing, and optimizing DNA amplification and synthesis

    Science.gov (United States)

    Vandersall, Jennifer A.; Gardner, Shea N.; Clague, David S.

    2010-05-04

    A computational method and computer-based system of modeling DNA synthesis for the design and interpretation of PCR amplification, parallel DNA synthesis, and microarray chip analysis. The method and system include modules that address the bioinformatics, kinetics, and thermodynamics of DNA amplification and synthesis. Specifically, the steps of DNA selection, as well as the kinetics and thermodynamics of DNA hybridization and extensions, are addressed, which enable the optimization of the processing and the prediction of the products as a function of DNA sequence, mixing protocol, time, temperature and concentration of species.

  10. A first linkage map and downy mildew resistance QTL discovery for sweet basil (Ocimum basilicum) facilitated by double digestion restriction site associated DNA sequencing (ddRADseq).

    Science.gov (United States)

    Pyne, Robert; Honig, Josh; Vaiciunas, Jennifer; Koroch, Adolfina; Wyenandt, Christian; Bonos, Stacy; Simon, James

    2017-01-01

    Limited understanding of sweet basil (Ocimum basilicum L.) genetics and genome structure has reduced efficiency of breeding strategies. This is evidenced by the rapid, worldwide dissemination of basil downy mildew (Peronospora belbahrii) in the absence of resistant cultivars. In an effort to improve available genetic resources, expressed sequence tag simple sequence repeat (EST-SSR) and single nucleotide polymorphism (SNP) markers were developed and used to genotype the MRI x SB22 F2 mapping population, which segregates for response to downy mildew. SNP markers were generated from genomic sequences derived from double digestion restriction site associated DNA sequencing (ddRADseq). Disomic segregation was observed in both SNP and EST-SSR markers providing evidence of an O. basilicum allotetraploid genome structure and allowing for subsequent analysis of the mapping population as a diploid intercross. A dense linkage map was constructed using 42 EST-SSR and 1,847 SNP markers spanning 3,030.9 cM. Multiple quantitative trait loci (QTL) model (MQM) analysis identified three QTL that explained 37-55% of phenotypic variance associated with downy mildew response across three environments. A single major QTL, dm11.1 explained 21-28% of phenotypic variance and demonstrated dominant gene action. Two minor QTL dm9.1 and dm14.1 explained 5-16% and 4-18% of phenotypic variance, respectively. Evidence is provided for an additive effect between the two minor QTL and the major QTL dm11.1 increasing downy mildew susceptibility. Results indicate that ddRADseq-facilitated SNP and SSR marker genotyping is an effective approach for mapping the sweet basil genome.

  11. Determination of antimicrobial resistance of Enterococcus strains isolated from pigs and their genotypic characterization by method of amplification of DNA fragments surrounding rare restriction sites (ADSRRS fingerprinting).

    Science.gov (United States)

    Nowakiewicz, Aneta; Ziółkowska, Grażyna; Trościańczyk, Aleksandra; Zięba, Przemysław; Gnat, Sebastian

    2017-03-01

    In this study, we analysed phenotypic resistance profiles and their reflection in the genomic profiles of Enterococcus spp. strains isolated from pigs raised on different farms. Samples were collected from five pig farms (n=90 animals) and tested for Enterococcus. MICs of 12 antimicrobials were determined using the broth microdilution method, and epidemiological molecular analysis of strains belonging to selected species (faecalis, faecium and hirae) was performed using the ADSRRS-fingerprinting (amplification of DNA fragments surrounding rare restriction sites) method with a few modifications. The highest percentage of strains was resistant to tetracycline (73.4 %), erythromycin and tylosin (42.5 %) and rifampin (25.2 %), and a large number of strains exhibited high-level resistance to both kanamycin (25.2 %) and streptomycin (27.6 %). The strains of E. faecalis, E. faecium and E. hirae (n=184) revealed varied phenotypic resistance profiles, among which as many as seven met the criteria for multidrug resistance (30.4 % of strains tested). ADSRRS-fingerprinting analysis produced 17 genotypic profiles of individual strains which were correlated with their phenotypic resistance profiles. Only E. hirae strains susceptible to all of the chemotherapeutics tested had two different ADSRRS profiles. Moreover, eight animals were carriers of more than one genotype belonging to the same Enterococcus spp., mainly E. faecalis. Given the possibility of transmission to humans of the high-resistance/multidrug resistance enterococci and the significant role of pigs as food animals in this process, it is necessary to introduce a multilevel control strategy by carrying out research on the resistance and molecular characteristics of indicator bacterial strains isolated from animals on individual farms.

  12. Unveiling the Identity of Wenwan Walnuts and Phylogenetic Relationships of Asian Juglans Species Using Restriction Site-Associated DNA-Sequencing

    Directory of Open Access Journals (Sweden)

    Xian-Yun Mu

    2017-10-01

    Full Text Available Juglans species have considerable ecological and economic value worldwide. In China, Wenwan walnuts have been collected by aristocrats and noblemen for more than 2000 years. As a diversity center of Asian Juglans, five species are widely distributed in China. The most famous of these is Mahetao (J. hopeiensis, which is an uncharacterized species that is mostly cultivated. Wild J. hopeiensis individuals are very rare and are endemic to Hebei Province. Because of the minimal variations in previously used molecular markers and the heterogeneity between chloroplast and nuclear genomes, determining the phylogenetic relationships among the Juglans species has been challenging, and has hindered subsequent evolutionary inferences. In this study, we collected enough materials for both cultivated and wild Mahetao to construct well-resolved phylogenetic trees for Asian Juglans species. We used a high-throughput genome-wide restriction site-associated DNA sequencing method. Consequently, the identity of J. hopeiensis has been clearly resolved. Our results indicate that J. hopeiensis is a hybrid of J. regia and J. mandshurica. However, J. hopeiensis, J. regia and J. sigillata should be considered as a single species from section Juglans. Additionally, J. ailantifolia, J. cathayensis, and J. mandshurica likely represent one species from section Cardiocaryon according to morphological and molecular studies. These results are supported by population structure analysis and morphological comparison. We propose that J. hopeiensis trees growing in the wild should be conserved because of the economic value of their nuts. These trees may be of particular importance to impoverished communities. Furthermore, they may serve as a valuable genetic resource relevant for enhancing the production of edible walnuts. The 2b-RAD method is a viable option for future phylogenetic studies of Juglans species as well as other plant species.

  13. DNA methylation-based classification of central nervous system tumours

    DEFF Research Database (Denmark)

    Capper, David; Jones, David T.W.; Sill, Martin

    2018-01-01

    Accurate pathological diagnosis is crucial for optimal management of patients with cancer. For the approximately 100 known tumour types of the central nervous system, standardization of the diagnostic process has been shown to be particularly challenging - with substantial inter-observer variabil......Accurate pathological diagnosis is crucial for optimal management of patients with cancer. For the approximately 100 known tumour types of the central nervous system, standardization of the diagnostic process has been shown to be particularly challenging - with substantial inter......-observer variability in the histopathological diagnosis of many tumour types. Here we present a comprehensive approach for the DNA methylation-based classification of central nervous system tumours across all entities and age groups, and demonstrate its application in a routine diagnostic setting. We show...

  14. Use of sperm plasmid DNA lipofection combined with REMI (restriction enzyme-mediated insertion) for production of transgenic chickens expressing eGFP (enhanced green fluorescent protein) or human follicle-stimulating hormone.

    Science.gov (United States)

    Harel-Markowitz, Eliane; Gurevich, Michael; Shore, Laurence S; Katz, Adi; Stram, Yehuda; Shemesh, Mordechai

    2009-05-01

    Linearized p-eGFP (plasmid-enhanced green fluorescent protein) or p-hFSH (plasmid human FSH) sequences with the corresponding restriction enzyme were lipofected into sperm genomic DNA. Sperm transfected with p-eGFP were used for artificial insemination in hens, and in 17 out of 19 of the resultant chicks, the exogenous DNA was detected in their lymphocytes as determined by PCR and expressed in tissues as determined by (a) PCR, (b) specific emission of green fluorescence by the eGFP, and (c) Southern blot analysis. A complete homology was found between the Aequorea Victoria eGFP DNA and a 313-bp PCR product of extracted DNA from chick blood cells. Following insemination with sperm lipofected with p-hFSH, transgenic offspring were obtained for two generations as determined by detection of the transgene for human FSH (PCR) and expression of the gene (RT-PCR and quantitative real-time PCR) and the presence of the protein in blood (radioimmunoassay). Data demonstrate that lipofection of plasmid DNA with restriction enzyme is a highly efficient method for the production of transfected sperm to produce transgenic offspring by direct artificial insemination.

  15. A Novel Rrm3 Function in Restricting DNA Replication via an Orc5-Binding Domain Is Genetically Separable from Rrm3 Function as an ATPase/Helicase in Facilitating Fork Progression

    Science.gov (United States)

    Syed, Salahuddin; Desler, Claus; Rasmussen, Lene J.; Schmidt, Kristina H.

    2016-01-01

    In response to replication stress cells activate the intra-S checkpoint, induce DNA repair pathways, increase nucleotide levels, and inhibit origin firing. Here, we report that Rrm3 associates with a subset of replication origins and controls DNA synthesis during replication stress. The N-terminal domain required for control of DNA synthesis maps to residues 186–212 that are also critical for binding Orc5 of the origin recognition complex. Deletion of this domain is lethal to cells lacking the replication checkpoint mediator Mrc1 and leads to mutations upon exposure to the replication stressor hydroxyurea. This novel Rrm3 function is independent of its established role as an ATPase/helicase in facilitating replication fork progression through polymerase blocking obstacles. Using quantitative mass spectrometry and genetic analyses, we find that the homologous recombination factor Rdh54 and Rad5-dependent error-free DNA damage bypass act as independent mechanisms on DNA lesions that arise when Rrm3 catalytic activity is disrupted whereas these mechanisms are dispensable for DNA damage tolerance when the replication function is disrupted, indicating that the DNA lesions generated by the loss of each Rrm3 function are distinct. Although both lesion types activate the DNA-damage checkpoint, we find that the resultant increase in nucleotide levels is not sufficient for continued DNA synthesis under replication stress. Together, our findings suggest a role of Rrm3, via its Orc5-binding domain, in restricting DNA synthesis that is genetically and physically separable from its established catalytic role in facilitating fork progression through replication blocks. PMID:27923055

  16. A Novel Rrm3 Function in Restricting DNA Replication via an Orc5-Binding Domain Is Genetically Separable from Rrm3 Function as an ATPase/Helicase in Facilitating Fork Progression.

    Science.gov (United States)

    Syed, Salahuddin; Desler, Claus; Rasmussen, Lene J; Schmidt, Kristina H

    2016-12-01

    In response to replication stress cells activate the intra-S checkpoint, induce DNA repair pathways, increase nucleotide levels, and inhibit origin firing. Here, we report that Rrm3 associates with a subset of replication origins and controls DNA synthesis during replication stress. The N-terminal domain required for control of DNA synthesis maps to residues 186-212 that are also critical for binding Orc5 of the origin recognition complex. Deletion of this domain is lethal to cells lacking the replication checkpoint mediator Mrc1 and leads to mutations upon exposure to the replication stressor hydroxyurea. This novel Rrm3 function is independent of its established role as an ATPase/helicase in facilitating replication fork progression through polymerase blocking obstacles. Using quantitative mass spectrometry and genetic analyses, we find that the homologous recombination factor Rdh54 and Rad5-dependent error-free DNA damage bypass act as independent mechanisms on DNA lesions that arise when Rrm3 catalytic activity is disrupted whereas these mechanisms are dispensable for DNA damage tolerance when the replication function is disrupted, indicating that the DNA lesions generated by the loss of each Rrm3 function are distinct. Although both lesion types activate the DNA-damage checkpoint, we find that the resultant increase in nucleotide levels is not sufficient for continued DNA synthesis under replication stress. Together, our findings suggest a role of Rrm3, via its Orc5-binding domain, in restricting DNA synthesis that is genetically and physically separable from its established catalytic role in facilitating fork progression through replication blocks.

  17. SMA Diagnosis: Detection of SMN1 Deletion with Real-Time mCOP-PCR System Using Fresh Blood DNA.

    Science.gov (United States)

    Niba, Emma Tabe Eko; Ar Rochmah, Mawaddah; Harahap, Nur Imma Fatimah; Awano, Hiroyuki; Morioka, Ichiro; Iijima, Kazumoto; Saito, Toshio; Saito, Kayoko; Takeuchi, Atsuko; Lai, Poh San; Bouike, Yoshihiro; Nishio, Hisahide; Shinohara, Masakazu

    2017-12-18

    Spinal muscular atrophy (SMA) is one of the most common autosomal recessive disorders. The symptoms are caused by defects of lower motor neurons in the spinal cord. More than 95% of SMA patients are homozygous for survival motor neuron 1 (SMN1) deletion. We previously developed a screening system for SMN1 deletion based on a modified competitive oligonucleotide priming-PCR (mCOP-PCR) technique using dried blood spot (DBS) on filter paper. This system is convenient for mass screening in the large population and/or first-tier diagnostic method of the patients in the remote areas. However, this system was still time-consuming and effort-taking, because it required pre-amplification procedure to avoid non-specific amplification and gel-electrophoresis to detect the presence or absence of SMN1 deletion. When the fresh blood samples are used instead of DBS, or when the gel-electrophoresis is replaced by real-time PCR, we may have a simpler and more rapid diagnostic method for SMA. To establish a simpler and more rapid diagnostic method of SMN1 deletion using fresh blood DNA. DNA samples extracted from fresh blood and stored at 4 ℃ for 1 month. The samples were assayed using a real-time mCOP-PCR system without pre-amplification procedures. DNA samples had already been genotyped by PCR-restriction fragment length polymorphism (PCR-RFLP), showing the presence or absence of SMN1 exon 7. The DNA samples were directly subjected to the mCOP-PCR step. The amplification of mCOP-PCR was monitored in a real-time PCR apparatus. The genotyping results of the real-time mCOP-PCR system using fresh blood DNA were completely matched with those of PCR-RFLP. In this real-time mCOP-PCR system using fresh blood-DNA, it took only four hours from extraction of DNA to detection of the presence or absence of SMN1 deletion, while it took more than 12 hours in PCR-RFLP. Our real-time mCOP-PCR system using fresh blood DNA was rapid and accurate, suggesting it may be useful for the first

  18. DNA nanocarriers for systemic administration: characterization and in vivo bioimaging in healthy mice.

    Science.gov (United States)

    David, Stephanie; Passirani, Catherine; Carmoy, Nathalie; Morille, Marie; Mevel, Mathieu; Chatin, Benoit; Benoit, Jean-Pierre; Montier, Tristan; Pitard, Bruno

    2013-01-08

    We hereby present different DNA nanocarriers consisting of new multimodular systems (MMS), containing the cationic lipid dioleylaminesuccinylparomomycin (DNA MMS DOSP), or bis (guanidinium)-tren-cholesterol (DNA MMS BGTC), and DNA lipid nanocapsules (DNA LNCs). Active targeting of the asialoglycoprotein receptor (ASGP-R) using galactose as a ligand for DNA MMS (GAL DNA MMS) and passive targeting using a polyethylene glycol coating for DNA LNCs (PEG DNA LNCs) should improve the properties of these DNA nanocarriers. All systems were characterized via physicochemical methods and the DNA payload of DNA LNCs was quantified for the first time. Afterwards, their biodistribution in healthy mice was analyzed after encapsulation of a fluorescent dye via in vivo biofluorescence imaging (BFI), revealing various distribution profiles depending on the cationic lipid used and their surface characteristics. Furthermore, the two vectors with the best prolonged circulation profile were administered twice in healthy mice revealing that the new DNA MMS DOSP vectors showed no toxicity and the same distribution profile for both injections, contrary to PEG DNA LNCs which showed a rapid clearance after the second injection, certainly due to the accelerated blood clearance phenomenon.Molecular Therapy - Nucleic Acids (2013) 2, e64; doi:10.1038/mtna.2012.56; published online 8 January 2013.

  19. DNA Nanocarriers for Systemic Administration: Characterization and In Vivo Bioimaging in Healthy Mice

    Directory of Open Access Journals (Sweden)

    Stephanie David

    2013-01-01

    Full Text Available We hereby present different DNA nanocarriers consisting of new multimodular systems (MMS, containing the cationic lipid dioleylaminesuccinylparomomycin (DNA MMS DOSP, or bis (guanidinium-tren-cholesterol (DNA MMS BGTC, and DNA lipid nanocapsules (DNA LNCs. Active targeting of the asialoglycoprotein receptor (ASGP-R using galactose as a ligand for DNA MMS (GAL DNA MMS and passive targeting using a polyethylene glycol coating for DNA LNCs (PEG DNA LNCs should improve the properties of these DNA nanocarriers. All systems were characterized via physicochemical methods and the DNA payload of DNA LNCs was quantified for the first time. Afterwards, their biodistribution in healthy mice was analyzed after encapsulation of a fluorescent dye via in vivo biofluorescence imaging (BFI, revealing various distribution profiles depending on the cationic lipid used and their surface characteristics. Furthermore, the two vectors with the best prolonged circulation profile were administered twice in healthy mice revealing that the new DNA MMS DOSP vectors showed no toxicity and the same distribution profile for both injections, contrary to PEG DNA LNCs which showed a rapid clearance after the second injection, certainly due to the accelerated blood clearance phenomenon.

  20. Restricted Mobilities

    DEFF Research Database (Denmark)

    Nielsen, Mette; Lassen, Claus

    2012-01-01

    communities and shopping centres through mobility lenses. The article shows how different mobility systems enable and restrict the public access to private-public spaces, and it points out that proprietary communities create an unequal potential for human movement and access in the city. The main argument......Privatisation of public spaces in the contemporary city has increased during the last decades but only few studies have approached this field from a mobility perspective. Therefore the article seeks to rectify this by exploring two Australian examples of private spaces in the city; gated...... and stratification mechanisms. In conclusion the article therefore suggests that future urban research and planning also needs a mobile understanding of spaces in the cities and how different mobility systems play an important role to sustain the exclusiveness that often characterises the private/public spaces...

  1. Effects of microbial DNA on human DNA profiles generated using the PowerPlex® 16 HS system.

    Science.gov (United States)

    Dembinski, Gina M; Picard, Christine J

    2017-11-01

    Most crime scenes are not sterile and therefore may be contaminated with environmental DNA, especially if a decomposing body is found. Collecting biological evidence from this individual will yield DNA samples mixed with microbial DNA. This also becomes important if postmortem swabs are collected from sexually assaulted victims. Although genotyping kits undergo validation tests, including bacterial screens, they do not account for the diverse microbial load during decomposition. We investigated the effect of spiking human DNA samples with known concentrations of DNA from 17 microbe species associated with decomposition on DNA profiles produced using the Promega PowerPlex ® HS system. Two species, Bacillus subtilis and Mycobacterium smegmatis, produced an extraneous allele at the TPOX locus. When repeated with the PowerPlex ® Fusion kit, the extra allele no longer amplified with these two species. This experiment demonstrates that caution should be exhibited if microbial load is high and the PowerPlex ® 16HS system is used. Copyright © 2017 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

  2. Human DNA quantification and sample quality assessment: Developmental validation of the PowerQuant(®) system.

    Science.gov (United States)

    Ewing, Margaret M; Thompson, Jonelle M; McLaren, Robert S; Purpero, Vincent M; Thomas, Kelli J; Dobrowski, Patricia A; DeGroot, Gretchen A; Romsos, Erica L; Storts, Douglas R

    2016-07-01

    Quantification of the total amount of human DNA isolated from a forensic evidence item is crucial for DNA normalization prior to short tandem repeat (STR) DNA analysis and a federal quality assurance standard requirement. Previous commercial quantification methods determine the total human DNA and total human male DNA concentrations, but provide limited information about the condition of the DNA sample. The PowerQuant(®) System includes targets for quantification of total human and total human male DNA as well as targets for evaluating whether the human DNA is degraded and/or PCR inhibitors are present in the sample. A developmental validation of the PowerQuant(®) System was completed, following SWGDAM Validation Guidelines, to evaluate the assay's specificity, sensitivity, precision and accuracy, as well as the ability to detect degraded DNA or PCR inhibitors. In addition to the total human DNA and total human male DNA concentrations in a sample, data from the degradation target and internal PCR control (IPC) provide a forensic DNA analyst meaningful information about the quality of the isolated human DNA and the presence of PCR inhibitors in the sample that can be used to determine the most effective workflow and assist downstream interpretation. Copyright © 2016 The Author(s). Published by Elsevier Ireland Ltd.. All rights reserved.

  3. Nuclear-electronic orbital reduced explicitly correlated Hartree-Fock approach: Restricted basis sets and open-shell systems

    International Nuclear Information System (INIS)

    Brorsen, Kurt R.; Sirjoosingh, Andrew; Pak, Michael V.; Hammes-Schiffer, Sharon

    2015-01-01

    The nuclear electronic orbital (NEO) reduced explicitly correlated Hartree-Fock (RXCHF) approach couples select electronic orbitals to the nuclear orbital via Gaussian-type geminal functions. This approach is extended to enable the use of a restricted basis set for the explicitly correlated electronic orbitals and an open-shell treatment for the other electronic orbitals. The working equations are derived and the implementation is discussed for both extensions. The RXCHF method with a restricted basis set is applied to HCN and FHF − and is shown to agree quantitatively with results from RXCHF calculations with a full basis set. The number of many-particle integrals that must be calculated for these two molecules is reduced by over an order of magnitude with essentially no loss in accuracy, and the reduction factor will increase substantially for larger systems. Typically, the computational cost of RXCHF calculations with restricted basis sets will scale in terms of the number of basis functions centered on the quantum nucleus and the covalently bonded neighbor(s). In addition, the RXCHF method with an odd number of electrons that are not explicitly correlated to the nuclear orbital is implemented using a restricted open-shell formalism for these electrons. This method is applied to HCN + , and the nuclear densities are in qualitative agreement with grid-based calculations. Future work will focus on the significance of nonadiabatic effects in molecular systems and the further enhancement of the NEO-RXCHF approach to accurately describe such effects

  4. Nuclear-electronic orbital reduced explicitly correlated Hartree-Fock approach: Restricted basis sets and open-shell systems

    Energy Technology Data Exchange (ETDEWEB)

    Brorsen, Kurt R.; Sirjoosingh, Andrew; Pak, Michael V.; Hammes-Schiffer, Sharon, E-mail: shs3@illinois.edu [Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Ave., Urbana, Illinois 61801 (United States)

    2015-06-07

    The nuclear electronic orbital (NEO) reduced explicitly correlated Hartree-Fock (RXCHF) approach couples select electronic orbitals to the nuclear orbital via Gaussian-type geminal functions. This approach is extended to enable the use of a restricted basis set for the explicitly correlated electronic orbitals and an open-shell treatment for the other electronic orbitals. The working equations are derived and the implementation is discussed for both extensions. The RXCHF method with a restricted basis set is applied to HCN and FHF{sup −} and is shown to agree quantitatively with results from RXCHF calculations with a full basis set. The number of many-particle integrals that must be calculated for these two molecules is reduced by over an order of magnitude with essentially no loss in accuracy, and the reduction factor will increase substantially for larger systems. Typically, the computational cost of RXCHF calculations with restricted basis sets will scale in terms of the number of basis functions centered on the quantum nucleus and the covalently bonded neighbor(s). In addition, the RXCHF method with an odd number of electrons that are not explicitly correlated to the nuclear orbital is implemented using a restricted open-shell formalism for these electrons. This method is applied to HCN{sup +}, and the nuclear densities are in qualitative agreement with grid-based calculations. Future work will focus on the significance of nonadiabatic effects in molecular systems and the further enhancement of the NEO-RXCHF approach to accurately describe such effects.

  5. Time-dependent restricted-active-space self-consistent-field theory for bosonic many-body systems

    International Nuclear Information System (INIS)

    Lévêque, Camille; Madsen, Lars Bojer

    2017-01-01

    We develop an ab initio time-dependent wavefunction based theory for the description of a many-body system of cold interacting bosons. Like the multi-configurational time-dependent Hartree method for bosons (MCTDHB), the theory is based on a configurational interaction Ansatz for the many-body wavefunction with time-dependent self-consistent-field orbitals. The theory generalizes the MCTDHB method by incorporating restrictions on the active space of the orbital excitations. The restrictions are specified based on the physical situation at hand. The equations of motion of this time-dependent restricted-active-space self-consistent-field (TD-RASSCF) theory are derived. The similarity between the formal development of the theory for bosons and fermions is discussed. The restrictions on the active space allow the theory to be evaluated under conditions where other wavefunction based methods due to exponential scaling in the numerical effort cannot, and to clearly identify the excitations that are important for an accurate description, significantly beyond the mean-field approach. For ground state calculations we find it to be important to allow a few particles to have the freedom to move in many orbitals, an insight facilitated by the flexibility of the restricted-active-space Ansatz . Moreover, we find that a high accuracy can be obtained by including only even excitations in the many-body self-consistent-field wavefunction. Time-dependent simulations of harmonically trapped bosons subject to a quenching of their noncontact interaction, show failure of the mean-field Gross-Pitaevskii approach within a fraction of a harmonic oscillation period. The TD-RASSCF theory remains accurate at much reduced computational cost compared to the MCTDHB method. Exploring the effect of changes of the restricted-active-space allows us to identify that even self-consistent-field excitations are mainly responsible for the accuracy of the method. (paper)

  6. Impact of Nutrient Restriction on the Structure of Listeria monocytogenes Biofilm Grown in a Microfluidic System

    Science.gov (United States)

    Cherifi, Tamazight; Jacques, Mario; Quessy, Sylvain; Fravalo, Philippe

    2017-01-01

    Biofilm formation by the pathogen Listeria monocytogenes is a major concern in food industries. The aim of this work was to elucidate the effect of nutrient limitation on both biofilm architecture and on the viability of the bacteria in microfluidic growth conditions. Biofilm formation by two L. monocytogenes strains was performed in a rich medium (BHI) and in a 10-fold diluted BHI (BHI/10) at 30°C for 24 h by using both static conditions and the microfluidic system Bioflux. In dynamic conditions, biofilms grown in rich and poor medium showed significant differences as well in structure and in the resulting biovolume. In BHI/10, biofilm was organized in a knitted network where cells formed long chains, whereas in the rich medium, the observed structure was homogeneous cellular multilayers. Biofilm biovolume production in BHI/10 was significantly higher than in BHI in these dynamic conditions. Interestingly, biovolume of dead cells in biofilms formed under limited nutrient conditions (BHI/10) was significantly higher than in biofilms formed in the BHI medium. In the other hand, in static conditions, biofilm is organized in a multilayer cells and dispersed cells in a rich medium BHI and poor medium BHI/10 respectively. There was significantly more biomass in the rich medium compared to BHI/10 but no difference was noted in the dead/damaged subpopulation showing how L. monocytogenes biofilm could be affected by the growth conditions. This work demonstrated that nutrient concentration affects biofilm structure and the proportion of dead cells in biofilms under microfluidic condition. Our study also showed that limited nutrients play an important role in the structural stability of L. monocytogenes biofilm by enhancing cell death and liberating extracellular DNA. PMID:28567031

  7. Impact of Nutrient Restriction on the Structure of Listeria monocytogenes Biofilm Grown in a Microfluidic System

    Directory of Open Access Journals (Sweden)

    Tamazight Cherifi

    2017-05-01

    Full Text Available Biofilm formation by the pathogen Listeria monocytogenes is a major concern in food industries. The aim of this work was to elucidate the effect of nutrient limitation on both biofilm architecture and on the viability of the bacteria in microfluidic growth conditions. Biofilm formation by two L. monocytogenes strains was performed in a rich medium (BHI and in a 10-fold diluted BHI (BHI/10 at 30°C for 24 h by using both static conditions and the microfluidic system Bioflux. In dynamic conditions, biofilms grown in rich and poor medium showed significant differences as well in structure and in the resulting biovolume. In BHI/10, biofilm was organized in a knitted network where cells formed long chains, whereas in the rich medium, the observed structure was homogeneous cellular multilayers. Biofilm biovolume production in BHI/10 was significantly higher than in BHI in these dynamic conditions. Interestingly, biovolume of dead cells in biofilms formed under limited nutrient conditions (BHI/10 was significantly higher than in biofilms formed in the BHI medium. In the other hand, in static conditions, biofilm is organized in a multilayer cells and dispersed cells in a rich medium BHI and poor medium BHI/10 respectively. There was significantly more biomass in the rich medium compared to BHI/10 but no difference was noted in the dead/damaged subpopulation showing how L. monocytogenes biofilm could be affected by the growth conditions. This work demonstrated that nutrient concentration affects biofilm structure and the proportion of dead cells in biofilms under microfluidic condition. Our study also showed that limited nutrients play an important role in the structural stability of L. monocytogenes biofilm by enhancing cell death and liberating extracellular DNA.

  8. Inducible DNA-repair systems in yeast: competition for lesions.

    Science.gov (United States)

    Mitchel, R E; Morrison, D P

    1987-03-01

    DNA lesions may be recognized and repaired by more than one DNA-repair process. If two repair systems with different error frequencies have overlapping lesion specificity and one or both is inducible, the resulting variable competition for the lesions can change the biological consequences of these lesions. This concept was demonstrated by observing mutation in yeast cells (Saccharomyces cerevisiae) exposed to combinations of mutagens under conditions which influenced the induction of error-free recombinational repair or error-prone repair. Total mutation frequency was reduced in a manner proportional to the dose of 60Co-gamma- or 254 nm UV radiation delivered prior to or subsequent to an MNNG exposure. Suppression was greater per unit radiation dose in cells gamma-irradiated in O2 as compared to N2. A rad3 (excision-repair) mutant gave results similar to wild-type but mutation in a rad52 (rec-) mutant exposed to MNNG was not suppressed by radiation. Protein-synthesis inhibition with heat shock or cycloheximide indicated that it was the mutation due to MNNG and not that due to radiation which had changed. These results indicate that MNNG lesions are recognized by both the recombinational repair system and the inducible error-prone system, but that gamma-radiation induction of error-free recombinational repair resulted in increased competition for the lesions, thereby reducing mutation. Similarly, gamma-radiation exposure resulted in a radiation dose-dependent reduction in mutation due to MNU, EMS, ENU and 8-MOP + UVA, but no reduction in mutation due to MMS. These results suggest that the number of mutational MMS lesions recognizable by the recombinational repair system must be very small relative to those produced by the other agents. MNNG induction of the inducible error-prone systems however, did not alter mutation frequencies due to ENU or MMS exposure but, in contrast to radiation, increased the mutagenic effectiveness of EMS. These experiments demonstrate

  9. Fundamental study of the radiation monitoring system based on evaluation of DNA lesions

    International Nuclear Information System (INIS)

    Shimizu, K.; Matuo, Y.; Izumi, Y.; Ikeda, T.

    2011-01-01

    The biological dosemeter that measures biological responses to ionising radiation is useful for radiation protection. This paper presents the development and characterisation of a gamma ray irradiation dosimetry system based on real-time PCR (polymerase chain reaction) methodology. Real-time PCR is used to amplify and simultaneously quantify a targeted DNA molecule. If there are no limitations due to limiting substrates or reagents, at each extension step, the amount of DNA target is doubled, leading to exponential (geometric) amplification of the specific DNA fragment. The essential point of this assay is that DNA lesions caused by ionising radiation block DNA synthesis by DNA polymerase, resulting in a decrease in the amplification of a damaged DNA template compared with that of non-damaged DNA templates. (authors)

  10. Technical note: Validation of an automated system for monitoring and restricting water intake in group-housed beef steers.

    Science.gov (United States)

    Allwardt, K; Ahlberg, C; Broocks, A; Bruno, K; Taylor, A; Place, S; Richards, C; Krehbiel, C; Calvo-Lorenzo, M; DeSilva, U; VanOverbeke, D; Mateescu, R; Goad, C; Rolf, M M

    2017-09-01

    The Insentec Roughage Intake Control (RIC) system has been validated for the collection of water intake; however, this system has not been validated for water restriction. The objective of this validation was to evaluate the agreement between direct observations and automated intakes collected by the RIC system under both ad libitum and restricted water conditions. A total of 239 crossbred steers were used in a 3-d validation trial, which assessed intake values generated by the RIC electronic intake monitoring system for both ad libitum water intake ( = 122; BASE) and restricted water intake ( = 117; RES). Direct human observations were collected on 4 Insentec water bins for three 24-h periods and three 12-h periods for BASE and RES, respectively. An intake event was noted by the observer when the electronic identification of the animal was read by the transponder and the gate lowered, and starting and ending bin weights were recorded for each intake event. Data from direct observations across each validation period were compared to automated observations generated from the RIC system. Missing beginning or ending weight values for visual observations occasionally occurred due to the observer being unable to capture the value before the monitor changed when bin activity was high. To estimate the impact of these missing values, analyses denoted as OBS were completed with the incomplete record coded as missing data. These analyses were contrasted with analyses where observations with a single missing beginning or end weight (but not both) were assumed to be identical to that which was recorded by the Insentec system (OBS). Difference in mean total intake across BASE steers was 0.60 ± 2.06 kg OBS (0.54 ± 1.99 kg OBS) greater for system observations than visual observations. The comparison of mean total intake across the 3 RES validation days was 0.53 ± 2.30 kg OBS (0.13 ± 1.83 kg OBS) greater for system observations than direct observations. Day was not a

  11. Genomic DNA fingerprinting of clinical Haemophilus influenzae isolates by polymerase chain reaction amplification: comparison with major outer-membrane protein and restriction fragment length polymorphism analysis

    NARCIS (Netherlands)

    van Belkum, A.; Duim, B.; Regelink, A.; Möller, L.; Quint, W.; van Alphen, L.

    1994-01-01

    Non-capsulate strains of Haemophilus influenzae were genotyped by analysis of variable DNA segments obtained by amplification of genomic DNA with the polymerase chain reaction (PCR fingerprinting). Discrete fragments of 100-2000 bp were obtained. The reproducibility of the procedure was assessed by

  12. Modified-cytosine restriction-system-induced recombinant cloning artefacts in Escherichia coli

    DEFF Research Database (Denmark)

    Williamson, M R; Doherty, J P; Woodcock, D M

    1993-01-01

    .HpaII plus M.HhaI) or (2) methylated with M.SssI which methylates at all CpG dinucleotides. These two protocols generated theoretical levels of DNA methylation in the central fragment of 10.5% and 33%, respectively. The construct was transformed into a series of isogenic (recA+) bacterial strains that were...

  13. DNA methylation-based classification of central nervous system tumours.

    Science.gov (United States)

    Capper, David; Jones, David T W; Sill, Martin; Hovestadt, Volker; Schrimpf, Daniel; Sturm, Dominik; Koelsche, Christian; Sahm, Felix; Chavez, Lukas; Reuss, David E; Kratz, Annekathrin; Wefers, Annika K; Huang, Kristin; Pajtler, Kristian W; Schweizer, Leonille; Stichel, Damian; Olar, Adriana; Engel, Nils W; Lindenberg, Kerstin; Harter, Patrick N; Braczynski, Anne K; Plate, Karl H; Dohmen, Hildegard; Garvalov, Boyan K; Coras, Roland; Hölsken, Annett; Hewer, Ekkehard; Bewerunge-Hudler, Melanie; Schick, Matthias; Fischer, Roger; Beschorner, Rudi; Schittenhelm, Jens; Staszewski, Ori; Wani, Khalida; Varlet, Pascale; Pages, Melanie; Temming, Petra; Lohmann, Dietmar; Selt, Florian; Witt, Hendrik; Milde, Till; Witt, Olaf; Aronica, Eleonora; Giangaspero, Felice; Rushing, Elisabeth; Scheurlen, Wolfram; Geisenberger, Christoph; Rodriguez, Fausto J; Becker, Albert; Preusser, Matthias; Haberler, Christine; Bjerkvig, Rolf; Cryan, Jane; Farrell, Michael; Deckert, Martina; Hench, Jürgen; Frank, Stephan; Serrano, Jonathan; Kannan, Kasthuri; Tsirigos, Aristotelis; Brück, Wolfgang; Hofer, Silvia; Brehmer, Stefanie; Seiz-Rosenhagen, Marcel; Hänggi, Daniel; Hans, Volkmar; Rozsnoki, Stephanie; Hansford, Jordan R; Kohlhof, Patricia; Kristensen, Bjarne W; Lechner, Matt; Lopes, Beatriz; Mawrin, Christian; Ketter, Ralf; Kulozik, Andreas; Khatib, Ziad; Heppner, Frank; Koch, Arend; Jouvet, Anne; Keohane, Catherine; Mühleisen, Helmut; Mueller, Wolf; Pohl, Ute; Prinz, Marco; Benner, Axel; Zapatka, Marc; Gottardo, Nicholas G; Driever, Pablo Hernáiz; Kramm, Christof M; Müller, Hermann L; Rutkowski, Stefan; von Hoff, Katja; Frühwald, Michael C; Gnekow, Astrid; Fleischhack, Gudrun; Tippelt, Stephan; Calaminus, Gabriele; Monoranu, Camelia-Maria; Perry, Arie; Jones, Chris; Jacques, Thomas S; Radlwimmer, Bernhard; Gessi, Marco; Pietsch, Torsten; Schramm, Johannes; Schackert, Gabriele; Westphal, Manfred; Reifenberger, Guido; Wesseling, Pieter; Weller, Michael; Collins, Vincent Peter; Blümcke, Ingmar; Bendszus, Martin; Debus, Jürgen; Huang, Annie; Jabado, Nada; Northcott, Paul A; Paulus, Werner; Gajjar, Amar; Robinson, Giles W; Taylor, Michael D; Jaunmuktane, Zane; Ryzhova, Marina; Platten, Michael; Unterberg, Andreas; Wick, Wolfgang; Karajannis, Matthias A; Mittelbronn, Michel; Acker, Till; Hartmann, Christian; Aldape, Kenneth; Schüller, Ulrich; Buslei, Rolf; Lichter, Peter; Kool, Marcel; Herold-Mende, Christel; Ellison, David W; Hasselblatt, Martin; Snuderl, Matija; Brandner, Sebastian; Korshunov, Andrey; von Deimling, Andreas; Pfister, Stefan M

    2018-03-22

    Accurate pathological diagnosis is crucial for optimal management of patients with cancer. For the approximately 100 known tumour types of the central nervous system, standardization of the diagnostic process has been shown to be particularly challenging-with substantial inter-observer variability in the histopathological diagnosis of many tumour types. Here we present a comprehensive approach for the DNA methylation-based classification of central nervous system tumours across all entities and age groups, and demonstrate its application in a routine diagnostic setting. We show that the availability of this method may have a substantial impact on diagnostic precision compared to standard methods, resulting in a change of diagnosis in up to 12% of prospective cases. For broader accessibility, we have designed a free online classifier tool, the use of which does not require any additional onsite data processing. Our results provide a blueprint for the generation of machine-learning-based tumour classifiers across other cancer entities, with the potential to fundamentally transform tumour pathology.

  14. Effect of a Health Care System Respiratory Fluoroquinolone Restriction Program To Alter Utilization and Impact Rates of Clostridium difficile Infection.

    Science.gov (United States)

    Shea, Katherine M; Hobbs, Athena L V; Jaso, Theresa C; Bissett, Jack D; Cruz, Christopher M; Douglass, Elizabeth T; Garey, Kevin W

    2017-06-01

    Fluoroquinolones are one of the most commonly prescribed antibiotic classes in the United States despite their association with adverse consequences, including Clostridium difficile infection (CDI). We sought to evaluate the impact of a health care system antimicrobial stewardship-initiated respiratory fluoroquinolone restriction program on utilization, appropriateness of quinolone-based therapy based on institutional guidelines, and CDI rates. After implementation, respiratory fluoroquinolone utilization decreased from a monthly mean and standard deviation (SD) of 41.0 (SD = 4.4) days of therapy (DOT) per 1,000 patient days (PD) preintervention to 21.5 (SD = 6.4) DOT/1,000 PD and 4.8 (SD = 3.6) DOT/1,000 PD posteducation and postrestriction, respectively. Using segmented regression analysis, both education (14.5 DOT/1,000 PD per month decrease; P = 0.023) and restriction (24.5 DOT/1,000 PD per month decrease; P cost of moxifloxacin, the formulary respiratory fluoroquinolone, was observed postrestriction compared to preintervention within the health care system ($123,882 versus $12,273; P = 0.002). Implementation of a stewardship-initiated respiratory fluoroquinolone restriction program can increase appropriate use while reducing overall utilization, acquisition cost, and CDI rates within a health care system. Copyright © 2017 American Society for Microbiology.

  15. High efficiency hydrodynamic DNA fragmentation in a bubbling system

    NARCIS (Netherlands)

    Li, Lanhui; Jin, Mingliang; Sun, Chenglong; Wang, Xiaoxue; Xie, Shuting; Zhou, Guofu; Van Den Berg, Albert; Eijkel, Jan C.T.; Shui, Lingling

    2017-01-01

    DNA fragmentation down to a precise fragment size is important for biomedical applications, disease determination, gene therapy and shotgun sequencing. In this work, a cheap, easy to operate and high efficiency DNA fragmentation method is demonstrated based on hydrodynamic shearing in a bubbling

  16. Assessing environmental DNA detection in controlled lentic systems.

    Science.gov (United States)

    Moyer, Gregory R; Díaz-Ferguson, Edgardo; Hill, Jeffrey E; Shea, Colin

    2014-01-01

    Little consideration has been given to environmental DNA (eDNA) sampling strategies for rare species. The certainty of species detection relies on understanding false positive and false negative error rates. We used artificial ponds together with logistic regression models to assess the detection of African jewelfish eDNA at varying fish densities (0, 0.32, 1.75, and 5.25 fish/m3). Our objectives were to determine the most effective water stratum for eDNA detection, estimate true and false positive eDNA detection rates, and assess the number of water samples necessary to minimize the risk of false negatives. There were 28 eDNA detections in 324, 1-L, water samples collected from four experimental ponds. The best-approximating model indicated that the per-L-sample probability of eDNA detection was 4.86 times more likely for every 2.53 fish/m3 (1 SD) increase in fish density and 1.67 times less likely for every 1.02 C (1 SD) increase in water temperature. The best section of the water column to detect eDNA was the surface and to a lesser extent the bottom. Although no false positives were detected, the estimated likely number of false positives in samples from ponds that contained fish averaged 3.62. At high densities of African jewelfish, 3-5 L of water provided a >95% probability for the presence/absence of its eDNA. Conversely, at moderate and low densities, the number of water samples necessary to achieve a >95% probability of eDNA detection approximated 42-73 and >100 L, respectively. Potential biases associated with incomplete detection of eDNA could be alleviated via formal estimation of eDNA detection probabilities under an occupancy modeling framework; alternatively, the filtration of hundreds of liters of water may be required to achieve a high (e.g., 95%) level of certainty that African jewelfish eDNA will be detected at low densities (i.e., <0.32 fish/m3 or 1.75 g/m3).

  17. DNA fragments assembly based on nicking enzyme system.

    Directory of Open Access Journals (Sweden)

    Rui-Yan Wang

    Full Text Available A couple of DNA ligation-independent cloning (LIC methods have been reported to meet various requirements in metabolic engineering and synthetic biology. The principle of LIC is the assembly of multiple overlapping DNA fragments by single-stranded (ss DNA overlaps annealing. Here we present a method to generate single-stranded DNA overlaps based on Nicking Endonucleases (NEases for LIC, the method was termed NE-LIC. Factors related to cloning efficiency were optimized in this study. This NE-LIC allows generating 3'-end or 5'-end ss DNA overlaps of various lengths for fragments assembly. We demonstrated that the 10 bp/15 bp overlaps had the highest DNA fragments assembling efficiency, while 5 bp/10 bp overlaps showed the highest efficiency when T4 DNA ligase was added. Its advantage over Sequence and Ligation Independent Cloning (SLIC and Uracil-Specific Excision Reagent (USER was obvious. The mechanism can be applied to many other LIC strategies. Finally, the NEases based LIC (NE-LIC was successfully applied to assemble a pathway of six gene fragments responsible for synthesizing microbial poly-3-hydroxybutyrate (PHB.

  18. An assay system for factors involved in mammalian DNA replication

    International Nuclear Information System (INIS)

    Reinhard, P.; Maillart, P.; Schluchter, M.; Gautschi, J.R.; Schindler, R.

    1979-01-01

    An assay for cellular factors stimulating DNA synthesis by partially lysed CHO cells is presented. The assay is based on the observation that in highly lysed cells, DNA synthesis, as determined by [ 3 H]dTTP incorporation, was only 2-5% of that in gently lysed cells, and that this low level of DNA synthesis could be increased by a factor of approx. 50 by the addition of CHO cell extract (i.e. supernatant of a cell homogenate subjected to high-speed centrifugation.) (Auth.)

  19. Electronic imaging systems for quantitative electrophoresis of DNA

    International Nuclear Information System (INIS)

    Sutherland, J.C.

    1989-01-01

    Gel electrophoresis is one of the most powerful and widely used methods for the separation of DNA. During the last decade, instruments have been developed that accurately quantitate in digital form the distribution of materials in a gel or on a blot prepared from a gel. In this paper, I review the various physical properties that can be used to quantitate the distribution of DNA on gels or blots and the instrumentation that has been developed to perform these tasks. The emphasis here is on DNA, but much of what is said also applies to RNA, proteins and other molecules. 36 refs

  20. Restrictive cardiomyopathy

    Science.gov (United States)

    ... People with restrictive cardiomyopathy may be heart transplant candidates. The outlook depends on the cause of the ... www.urac.org). URAC's accreditation program is an independent audit to verify that A.D.A.M. ...

  1. Compliance to antihypertensive drugs, salt restriction, exercise and control of systemic hypertension in hypertensive patients at abbottabad

    International Nuclear Information System (INIS)

    Ahmed, N.; Waqas, A.; Khaliq, M.A.

    2008-01-01

    Hypertension is one of the most important cardiovascular risk factor but its control is still a challenge for physicians all around the world. Control of blood pressure can reduce cardiovascular morbidity and mortality, so the compliance to antihypertensive drugs and life style modification play an important role for the control of hypertension. This analytical (cross-sectional) study was conducted to assess prevalence of control of hypertension among hypertensive patients and to assess the relationship of control of hypertension with factors like compliance to antihypertensive drugs, salt restriction and exercise among the hypertensive patients. This study was conducted at outpatient clinic of medicine at Shahina Jamil Hospital Abbottabad from April 2007 to September 2007. Eighty-nine patients seen in the outpatient clinic of medicine were enrolled in the study. All the patients with age 15 years or above, diagnosed as a case of systemic hypertension were included. Among eighty nine patients, 67 were female and 22 were male with mean age of 55.8+-13.4 years, mean systolic and diastolic blood pressure of 160+-28.6 and 97.8+-14.1 mm Hg respectively, and pulse rate of 85.9+-11.4 per minutes. Out of 89 patients, 25.8% were having controlled hypertension, 48.3% were compliant and 51.7% were not compliant to antihypertensive drugs, 55.1% were having salt restriction and 44.9% were having no salt restriction and 23.6% were used to do physical activity while 76.4% were not used to do physical activity. In group A consisted of patients with controlled hypertension, 95.7% patients were compliant to antihypertensive patients, 95.7% were having salt restriction and 43.5% were used to do physical activity. In group B consisted of patients with uncontrolled hypertension, only 31.8% were compliant to antihypertensive drugs, 40.9% were having salt restriction, 16.7% were used to do physical activity. Hypertension can be controlled if the hypertensive patients have good compliance

  2. Avatar DNA Nanohybrid System in Chip-on-a-Phone

    Science.gov (United States)

    Park, Dae-Hwan; Han, Chang Jo; Shul, Yong-Gun; Choy, Jin-Ho

    2014-05-01

    Long admired for informational role and recognition function in multidisciplinary science, DNA nanohybrids have been emerging as ideal materials for molecular nanotechnology and genetic information code. Here, we designed an optical machine-readable DNA icon on microarray, Avatar DNA, for automatic identification and data capture such as Quick Response and ColorZip codes. Avatar icon is made of telepathic DNA-DNA hybrids inscribed on chips, which can be identified by camera of smartphone with application software. Information encoded in base-sequences can be accessed by connecting an off-line icon to an on-line web-server network to provide message, index, or URL from database library. Avatar DNA is then converged with nano-bio-info-cogno science: each building block stands for inorganic nanosheets, nucleotides, digits, and pixels. This convergence could address item-level identification that strengthens supply-chain security for drug counterfeits. It can, therefore, provide molecular-level vision through mobile network to coordinate and integrate data management channels for visual detection and recording.

  3. DNA accumulation on ventilation system filters in university buildings in Singapore.

    Science.gov (United States)

    Luhung, Irvan; Wu, Yan; Xu, Siyu; Yamamoto, Naomichi; Chang, Victor Wei-Chung; Nazaroff, William W

    2017-01-01

    Biological particles deposit on air handling system filters as they process air. This study reports and interprets abundance and diversity information regarding biomass accumulation on ordinarily used filters acquired from several locations in a university environment. DNA-based analysis was applied both to quantify (via DNA fluorometry and qPCR) and to characterize (via high-throughput sequencing) the microbial material on filters, which mainly processed recirculated indoor air. Results were interpreted in relation to building occupancy and ventilation system operational parameters. Based on accumulated biomass, average DNA concentrations per AHU filter surface area across nine indoor locations after twelve weeks of filter use were in the respective ranges 1.1 to 41 ng per cm2 for total DNA, 0.02 to 3.3 ng per cm2 for bacterial DNA and 0.2 to 2.0 ng DNA per cm2 for fungal DNA. The most abundant genera detected on the AHU filter samples were Clostridium, Streptophyta, Bacillus, Acinetobacter and Ktedonobacter for bacteria and Aspergillus, Cladosporium, Nigrospora, Rigidoporus and Lentinus for fungi. Conditional indoor airborne DNA concentrations (median (range)) were estimated to be 13 (2.6-107) pg/m3 for total DNA, 0.4 (0.05-8.4) pg/m3 for bacterial DNA and 2.3 (1.0-5.1) pg/m3 for fungal DNA. Conditional airborne concentrations and the relative abundances of selected groups of genera correlate well with occupancy level. Bacterial DNA was found to be more responsive than fungal DNA to differences in occupancy level and indoor environmental conditions.

  4. Multiple regulatory systems coordinate DNA replication with cell growth in Bacillus subtilis.

    Directory of Open Access Journals (Sweden)

    Heath Murray

    2014-10-01

    Full Text Available In many bacteria the rate of DNA replication is linked with cellular physiology to ensure that genome duplication is coordinated with growth. Nutrient-mediated growth rate control of DNA replication initiation has been appreciated for decades, however the mechanism(s that connects these cell cycle activities has eluded understanding. In order to help address this fundamental question we have investigated regulation of DNA replication in the model organism Bacillus subtilis. Contrary to the prevailing view we find that changes in DnaA protein level are not sufficient to account for nutrient-mediated growth rate control of DNA replication initiation, although this regulation does require both DnaA and the endogenous replication origin. We go on to report connections between DNA replication and several essential cellular activities required for rapid bacterial growth, including respiration, central carbon metabolism, fatty acid synthesis, phospholipid synthesis, and protein synthesis. Unexpectedly, the results indicate that multiple regulatory systems are involved in coordinating DNA replication with cell physiology, with some of the regulatory systems targeting oriC while others act in a oriC-independent manner. We propose that distinct regulatory systems are utilized to control DNA replication in response to diverse physiological and chemical changes.

  5. Development of a recombinant DNA assay system for the detection of genetic change in astronauts' cells

    International Nuclear Information System (INIS)

    Atchley, S.V.; Chen, D.J.C.; Strniste, G.F.; Walters, R.A.; Moyzis, R.K.

    1984-01-01

    We are developing a new recombinant DNA system for the detection and measurement of genetic change in humans caused by exposure to low level ionizing radiation. A unique feature of the method is the use of cloned repetitive DNA probes to assay human DNA for structural changes during or after irradiation. Repetitive sequences exist in different families. Collectively they constitute over 25% of the DNA in a human cell. Repeat families have between 10 and 500,000 members. We have constructed repetitive DNA sequence libraries using recombinant DNA techniques. From these libraries we have isolated and characterized individual repeats comprising 75 to 90% of the mass of human repetitive DNA. Repeats used in our assay system exist in tandem arrays in the genome. Perturbation of these sequences in a cell, followed by detection with a repeat probe, produces a new, multimeric ''ladder'' pattern on an autoradiogram. The repeat probe used in our initial study is complementary to 1% of human DNA. Therefore, the sensitivity of this method is several orders of magnitude better than existing assays. Preliminary evidence from human skin cells exposed to acute, low-dose x-ray treatments indicates that DNA is affected at a dose as low as 5R. The radiation doses used in this system are well within the range of doses received by astronauts during spaceflight missions. Due to its small material requirements, this technique could easily be adapted for use in space. 16 refs., 1 fig

  6. Multiple regulatory systems coordinate DNA replication with cell growth in Bacillus subtilis.

    Science.gov (United States)

    Murray, Heath; Koh, Alan

    2014-10-01

    In many bacteria the rate of DNA replication is linked with cellular physiology to ensure that genome duplication is coordinated with growth. Nutrient-mediated growth rate control of DNA replication initiation has been appreciated for decades, however the mechanism(s) that connects these cell cycle activities has eluded understanding. In order to help address this fundamental question we have investigated regulation of DNA replication in the model organism Bacillus subtilis. Contrary to the prevailing view we find that changes in DnaA protein level are not sufficient to account for nutrient-mediated growth rate control of DNA replication initiation, although this regulation does require both DnaA and the endogenous replication origin. We go on to report connections between DNA replication and several essential cellular activities required for rapid bacterial growth, including respiration, central carbon metabolism, fatty acid synthesis, phospholipid synthesis, and protein synthesis. Unexpectedly, the results indicate that multiple regulatory systems are involved in coordinating DNA replication with cell physiology, with some of the regulatory systems targeting oriC while others act in a oriC-independent manner. We propose that distinct regulatory systems are utilized to control DNA replication in response to diverse physiological and chemical changes.

  7. Multiple Regulatory Systems Coordinate DNA Replication with Cell Growth in Bacillus subtilis

    Science.gov (United States)

    Murray, Heath; Koh, Alan

    2014-01-01

    In many bacteria the rate of DNA replication is linked with cellular physiology to ensure that genome duplication is coordinated with growth. Nutrient-mediated growth rate control of DNA replication initiation has been appreciated for decades, however the mechanism(s) that connects these cell cycle activities has eluded understanding. In order to help address this fundamental question we have investigated regulation of DNA replication in the model organism Bacillus subtilis. Contrary to the prevailing view we find that changes in DnaA protein level are not sufficient to account for nutrient-mediated growth rate control of DNA replication initiation, although this regulation does require both DnaA and the endogenous replication origin. We go on to report connections between DNA replication and several essential cellular activities required for rapid bacterial growth, including respiration, central carbon metabolism, fatty acid synthesis, phospholipid synthesis, and protein synthesis. Unexpectedly, the results indicate that multiple regulatory systems are involved in coordinating DNA replication with cell physiology, with some of the regulatory systems targeting oriC while others act in a oriC-independent manner. We propose that distinct regulatory systems are utilized to control DNA replication in response to diverse physiological and chemical changes. PMID:25340815

  8. Disruption of the Hepcidin/Ferroportin Regulatory System Causes Pulmonary Iron Overload and Restrictive Lung Disease

    Directory of Open Access Journals (Sweden)

    Joana Neves

    2017-06-01

    Full Text Available Emerging evidence suggests that pulmonary iron accumulation is implicated in a spectrum of chronic lung diseases. However, the mechanism(s involved in pulmonary iron deposition and its role in the in vivo pathogenesis of lung diseases remains unknown. Here we show that a point mutation in the murine ferroportin gene, which causes hereditary hemochromatosis type 4 (Slc40a1C326S, increases iron levels in alveolar macrophages, epithelial cells lining the conducting airways and lung parenchyma, and in vascular smooth muscle cells. Pulmonary iron overload is associated with oxidative stress, restrictive lung disease with decreased total lung capacity and reduced blood oxygen saturation in homozygous Slc40a1C326S/C326S mice compared to wild-type controls. These findings implicate iron in lung pathology, which is so far not considered a classical iron-related disorder.

  9. Effects of benzo[a]pyrene-DNA adducts on a reconstituted replication system

    International Nuclear Information System (INIS)

    Brown, W.C.; Romano, L.J.

    1991-01-01

    The authors have used a partially reconstituted replication system consisting of T7 DNA polymerase and T7 gene 4 protein to examine the effect of benzo[a]pyrene (B[a]P) adducts on DNA synthesis and gene 4 protein activities. The gene 4 protein is required for T7 DNA replication because of its ability to act as both a primase and helicase. They show here that total synthesis decreases as the level of adducts per molecule of DNA increases, suggesting that the B[a]P adducts are blocking an aspect of the replication process. By challenging synthesis on oligonucleotide-primed B[a]P-modified DNA with unmodified DNA, they present evidence that the T7 DNA polymerase freely dissociates after encountering an adduct. Prior studies have shown that the gene 4 protein alone does not dissociate from the template during translocation upon encountering an adduct. However, when gene 4 protein primed DNA synthesis is challenged, they observe an increase in synthesis but to a lesser extent than observed on oligonucleotide-primed synthesis. Finally, they have examined DNA synthesis on duplex templates and show the B[a]P adducts inhibit synthesis by the T7 DNA polymerase and gene 4 protein to the same extent regardless of whether the adducts are positioned in the leading or lagging strand, while synthesis by the polymerase alone is inhibited only when the adducts are in the template strand

  10. Targeting DNA repair systems in antitubercular drug development.

    Science.gov (United States)

    Minias, Alina; Brzostek, Anna; Dziadek, Jaroslaw

    2018-01-28

    Infections with Mycobacterium tuberculosis, the causative agent of tuberculosis, are difficult to treat using currently available chemotherapeutics. Clinicians agree on the urgent need for novel drugs to treat tuberculosis. In this mini review, we summarize data that prompts the consideration of DNA repair-associated proteins as targets for the development of new antitubercular compounds. We discuss data, including gene expression data, that highlight the importance of DNA repair genes during the pathogenic cycle as well as after exposure to antimicrobials currently in use. Specifically, we report experiments on determining the essentiality of DNA repair-related genes. We report the availability of protein crystal structures and summarize discovered protein inhibitors. Further, we describe phenotypes of available gene mutants of M. tuberculosis and model organisms Mycobacterium bovis and Mycobacterium smegmatis. We summarize experiments regarding the role of DNA repair-related proteins in pathogenesis and virulence performed both in vitro and in vivo during the infection of macrophages and animals. We detail the role of DNA repair genes in acquiring mutations, which influence the rate of drug resistance acquisition. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  11. Development and production of Lab-on-Chip systems for DNA mapping

    DEFF Research Database (Denmark)

    Østergaard, Peter Friis

    as low as 1:200. The developed polymer systems are tested by conducting two different experiments on DNA. Since such experiments are highly sensitive, efforts have been taken in order to lower the autofluorescence of the devices, resulting in a decrease of the background signal to roughly half...... several nanochannels can be placed parallel to each other, a large number of DNA molecules can be investigated. In the second experiment, mapping is performed on human DNA in nanoslit devices. A fluorescent profile is created by heating the sample up to a temperature, where the DNA is partially denatured....... The fluorescent dye will diffuse away from the denatured regions, and by analysing these black areas, the DNA molecule can be identified and potential mutations can be found. In the nanoslits, the DNA is stretched out via a shear flow, resulting in a stretching of more than 95% of the contour length meaning...

  12. Integration of the Reconfigurable Self-Healing eDNA Architecture in an Embedded System

    Science.gov (United States)

    Boesen, Michael Reibel; Keymeulen, Didier; Madsen, Jan; Lu, Thomas; Chao, Tien-Hsin

    2011-01-01

    In this work we describe the first real world case study for the self-healing eDNA (electronic DNA) architecture by implementing the control and data processing of a Fourier Transform Spectrometer (FTS) on an eDNA prototype. For this purpose the eDNA prototype has been ported from a Xilinx Virtex 5 FPGA to an embedded system consisting of a PowerPC and a Xilinx Virtex 5 FPGA. The FTS instrument features a novel liquid crystal waveguide, which consequently eliminates all moving parts from the instrument. The addition of the eDNA architecture to do the control and data processing has resulted in a highly fault-tolerant FTS instrument. The case study has shown that the early stage prototype of the autonomous self-healing eDNA architecture is expensive in terms of execution time.

  13. Feasibility of using DNA-immobilized nanocellulose-based immunoadsorbent for systemic lupus erythematosus plasmapheresis.

    Science.gov (United States)

    Xu, Changgang; Carlsson, Daniel O; Mihranyan, Albert

    2016-07-01

    The goal of this project was to study the feasibility of using a DNA-immobilized nanocellulose-based immunoadsorbent for possible application in medical apheresis such as systemic lupus erythematosus (SLE) treatment. Calf thymus DNA was bound to high surface area nanocellulose membrane at varying concentrations using UV-irradiation. The DNA-immobilized samples were characterized with scanning electron microscopy, atomic force microscopy, and phosphorus elemental analysis. The anti-ds-DNA IgG binding was tested in vitro using ELISA. The produced sample showed high affinity in vitro to bind anti-ds-DNA-antibodies from mice, as much as 80% of added IgG was bound by the membrane. Furthermore, the binding efficiency was quantitatively dependent on the amount of immobilized DNA onto nanocellulose membrane. The described nanocellulose membranes are interesting immunoadsorbents for continued clinical studies. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. A DNA Mini-Barcoding System for Authentication of Processed Fish Products.

    Science.gov (United States)

    Shokralla, Shadi; Hellberg, Rosalee S; Handy, Sara M; King, Ian; Hajibabaei, Mehrdad

    2015-10-30

    Species substitution is a form of seafood fraud for the purpose of economic gain. DNA barcoding utilizes species-specific DNA sequence information for specimen identification. Previous work has established the usability of short DNA sequences-mini-barcodes-for identification of specimens harboring degraded DNA. This study aims at establishing a DNA mini-barcoding system for all fish species commonly used in processed fish products in North America. Six mini-barcode primer pairs targeting short (127-314 bp) fragments of the cytochrome c oxidase I (CO1) DNA barcode region were developed by examining over 8,000 DNA barcodes from species in the U.S. Food and Drug Administration (FDA) Seafood List. The mini-barcode primer pairs were then tested against 44 processed fish products representing a range of species and product types. Of the 44 products, 41 (93.2%) could be identified at the species or genus level. The greatest mini-barcoding success rate found with an individual primer pair was 88.6% compared to 20.5% success rate achieved by the full-length DNA barcode primers. Overall, this study presents a mini-barcoding system that can be used to identify a wide range of fish species in commercial products and may be utilized in high throughput DNA sequencing for authentication of heavily processed fish products.

  15. Effects of DNA Extraction Procedures on Bacteroides Profiles in Fecal Samples From Various Animals Determined by Terminal Restriction Fragment Length Polymorphism Analysis

    Science.gov (United States)

    A major assumption in microbial source tracking is that some fecal bacteria are specific to a host animal, and thus provide unique microbial fingerprints that can be used to differentiate hosts. However, the DNA information obtained from a particular sample may be biased dependi...

  16. Comparative genome and methylome analysis reveals restriction/modification system diversity in the gut commensal Bifidobacterium breve

    Science.gov (United States)

    Bottacini, Francesca; Morrissey, Ruth; Roberts, Richard John; James, Kieran; van Breen, Justin; Egan, Muireann; Lambert, Jolanda; van Limpt, Kees; Knol, Jan; Motherway, Mary O’Connell; van Sinderen, Douwe

    2018-01-01

    Abstract Bifidobacterium breve represents one of the most abundant bifidobacterial species in the gastro-intestinal tract of breast-fed infants, where their presence is believed to exert beneficial effects. In the present study whole genome sequencing, employing the PacBio Single Molecule, Real-Time (SMRT) sequencing platform, combined with comparative genome analysis allowed the most extensive genetic investigation of this taxon. Our findings demonstrate that genes encoding Restriction/Modification (R/M) systems constitute a substantial part of the B. breve variable gene content (or variome). Using the methylome data generated by SMRT sequencing, combined with targeted Illumina bisulfite sequencing (BS-seq) and comparative genome analysis, we were able to detect methylation recognition motifs and assign these to identified B. breve R/M systems, where in several cases such assignments were confirmed by restriction analysis. Furthermore, we show that R/M systems typically impose a very significant barrier to genetic accessibility of B. breve strains, and that cloning of a methyltransferase-encoding gene may overcome such a barrier, thus allowing future functional investigations of members of this species. PMID:29294107

  17. Characterization of Erwinia amylovora strains from different host plants using repetitive-sequences PCR analysis, and restriction fragment length polymorphism and short-sequence DNA repeats of plasmid pEA29.

    Science.gov (United States)

    Barionovi, D; Giorgi, S; Stoeger, A R; Ruppitsch, W; Scortichini, M

    2006-05-01

    The three main aims of the study were the assessment of the genetic relationship between a deviating Erwinia amylovora strain isolated from Amelanchier sp. (Maloideae) grown in Canada and other strains from Maloideae and Rosoideae, the investigation of the variability of the PstI fragment of the pEA29 plasmid using restriction fragment length polymorphism (RFLP) analysis and the determination of the number of short-sequence DNA repeats (SSR) by DNA sequence analysis in representative strains. Ninety-three strains obtained from 12 plant genera and different geographical locations were examined by repetitive-sequences PCR using Enterobacterial Repetitive Intergenic Consensus, BOX and Repetitive Extragenic Palindromic primer sets. Upon the unweighted pair group method with arithmetic mean analysis, a deviating strain from Amelanchier sp. was analysed using amplified ribosomal DNA restriction analysis (ARDRA) analysis and the sequencing of the 16S rDNA gene. This strain showed 99% similarity to other E. amylovora strains in the 16S gene and the same banding pattern with ARDRA. The RFLP analysis of pEA29 plasmid using MspI and Sau3A restriction enzymes showed a higher variability than that previously observed and no clear-cut grouping of the strains was possible. The number of SSR units reiterated two to 12 times. The strains obtained from pear orchards showing for the first time symptoms of fire blight had a low number of SSR units. The strains from Maloideae exhibit a wider genetic variability than previously thought. The RFLP analysis of a fragment of the pEA29 plasmid would not seem a reliable method for typing E. amylovora strains. A low number of SSR units was observed with first epidemics of fire blight. The current detection techniques are mainly based on the genetic similarities observed within the strains from the cultivated tree-fruit crops. For a more reliable detection of the fire blight pathogen also in wild and ornamentals Rosaceous plants the genetic

  18. Robe's Restricted Problem of 2 + 2 Bodies with a Roche Ellipsoid - Triaxial System

    Science.gov (United States)

    Aggarwal, Rajiv; Kaur, Bhavneet; Yadav, Sushil

    2018-03-01

    This paper investigates the motion of two infinitesimal masses on the location and stability of the equilibrium points in Robe's restricted problem of 2 + 2 bodies with the bigger primary a Roche ellipsoid and the smaller a triaxial body. We suppose the bigger primary of mass m 1 to be filled with a homogeneous incompressible fluid of density ρ 1. The third and the fourth bodies (of mass m 3 and m 4 respectively) are small solid spheres of density ρ 3 and ρ 4 respectively inside the ellipsoid, with the assumption that the mass and the radius of the third and the fourth body are infinitesimal. We assume that m 2 is describing a circle around m 1. The masses m 3 and m 4 mutually attract each other, do not influence the motion of m 1 and m 2 but are influenced by them. We have taken into consideration all the three components of the pressure field in deriving the expression for the buoyancy force viz (i) due to the own gravitational field of the fluid (ii) that originating in the attraction of m 2 (iii) that arising from the centrifugal force. In this paper, equilibrium solutions of m 3 and m 4 and their linear stability are analyzed.

  19. New applications of CRISPR/Cas9 system on mutant DNA detection.

    Science.gov (United States)

    Jia, Chenqiang; Huai, Cong; Ding, Jiaqi; Hu, Lingna; Su, Bo; Chen, Hongyan; Lu, Daru

    2018-01-30

    The detection of mutant DNA is critical for precision medicine, but low-frequency DNA mutation is very hard to be determined. CRISPR/Cas9 is a robust tool for in vivo gene editing, and shows the potential for precise in vitro DNA cleavage. Here we developed a DNA mutation detection system based on CRISPR/Cas9 that can detect gene mutation efficiently even in a low-frequency condition. The system of CRISPR/Cas9 cleavage in vitro showed a high accuracy similar to traditional T7 endonuclease I (T7E1) assay in estimating mutant DNA proportion in the condition of normal frequency. The technology was further used for low-frequency mutant DNA detection of EGFR and HBB somatic mutations. To the end, Cas9 was employed to cleave the wild-type (WT) DNA and to enrich the mutant DNA. Using amplified fragment length polymorphism analysis (AFLPA) and Sanger sequencing, we assessed the sensitivity of CRISPR/Cas9 cleavage-based PCR, in which mutations at 1%-10% could be enriched and detected. When combined with blocker PCR, its sensitivity reached up to 0.1%. Our results suggested that this new application of CRISPR/Cas9 system is a robust and potential method for heterogeneous specimens in the clinical diagnosis and treatment management. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Diet as a system: an observational study investigating a multi-choice system of moderately restricted low-protein diets.

    Science.gov (United States)

    Piccoli, Giorgina Barbara; Nazha, Marta; Capizzi, Irene; Vigotti, Federica Neve; Scognamiglio, Stefania; Consiglio, Valentina; Mongilardi, Elena; Bilocati, Marilisa; Avagnina, Paolo; Versino, Elisabetta

    2016-12-07

    There is no single, gold-standard, low-protein diet (LPD) for CKD patients; the best compliance is probably obtained by personalization. This study tests the hypothesis that a multiple choice diet network allows patients to attain a good compliance level, and that, in an open-choice system, overall results are not dependent upon the specific diet, but upon the clinical characteristics of the patients. Observational study: Three LPD options were offered to all patients with severe or rapidly progressive CKD: vegan diets supplemented with alpha-ketoacids and essential aminoacids; protein-free food in substitution of normal bread and pasta; other (traditional, vegan non supplemented and tailored). Dialysis-free follow-up and survival were analyzed by Kaplan Meier curves according to diet, comorbidity and age. Compliance and metabolic control were estimated in 147 subjects on diet at March 2015, with recent complete data, prescribed protein intake 0.6 g/Kg/day. Protein intake was assessed by Maroni Mitch formula. Four hundreds and forty nine patients followed a LPD in December, 2007- March, 2015 (90% moderately restricted LPDs, 0.6 g/Kg/day of protein, 10% at lower targets); age (median 70 (19-97)) and comorbidity (Charlson index: 7) characterized our population as being in line with the usual CKD European population. Median e-GFR at start of the diet was 20 mL/min, 33.2% of the patients were diabetics. Baseline data differ significantly across diets: protein-free schemas are preferred by older, high-comorbidity patients (median age 76 years, Charlson index 8, GFR 20.5 mL/min, Proteinuria: 0.3 g/day), supplemented vegan diets by younger patients with lower GFR and higher proteinuria (median age 65 years, Charlson index 6, GFR 18.9 mL/min; Proteinuria: 1.2 g/day); other diets are chosen by an intermediate population (median age 71 years, Charlson index 6; GFR 22.5 mL/min; Proteinuria: 0.9 g/day); (p <0.001 for age, Charlson index, proteinuria, GFR

  1. Horizontal gene transfer of a chloroplast DnaJ-Fer protein to Thaumarchaeota and the evolutionary history of the DnaK chaperone system in Archaea.

    Science.gov (United States)

    Petitjean, Céline; Moreira, David; López-García, Purificación; Brochier-Armanet, Céline

    2012-11-26

    In 2004, we discovered an atypical protein in metagenomic data from marine thaumarchaeotal species. This protein, referred as DnaJ-Fer, is composed of a J domain fused to a Ferredoxin (Fer) domain. Surprisingly, the same protein was also found in Viridiplantae (green algae and land plants). Because J domain-containing proteins are known to interact with the major chaperone DnaK/Hsp70, this suggested that a DnaK protein was present in Thaumarchaeota. DnaK/Hsp70, its co-chaperone DnaJ and the nucleotide exchange factor GrpE are involved, among others, in heat shocks and heavy metal cellular stress responses. Using phylogenomic approaches we have investigated the evolutionary history of the DnaJ-Fer protein and of interacting proteins DnaK, DnaJ and GrpE in Thaumarchaeota. These proteins have very complex histories, involving several inter-domain horizontal gene transfers (HGTs) to explain the contemporary distribution of these proteins in archaea. These transfers include one from Cyanobacteria to Viridiplantae and one from Viridiplantae to Thaumarchaeota for the DnaJ-Fer protein, as well as independent HGTs from Bacteria to mesophilic archaea for the DnaK/DnaJ/GrpE system, followed by HGTs among mesophilic and thermophilic archaea. We highlight the chimerical origin of the set of proteins DnaK, DnaJ, GrpE and DnaJ-Fer in Thaumarchaeota and suggest that the HGT of these proteins has played an important role in the adaptation of several archaeal groups to mesophilic and thermophilic environments from hyperthermophilic ancestors. Finally, the evolutionary history of DnaJ-Fer provides information useful for the relative dating of the diversification of Archaeplastida and Thaumarchaeota.

  2. Horizontal gene transfer of a chloroplast DnaJ-Fer protein to Thaumarchaeota and the evolutionary history of the DnaK chaperone system in Archaea

    Directory of Open Access Journals (Sweden)

    Petitjean Céline

    2012-11-01

    Full Text Available Abstract Background In 2004, we discovered an atypical protein in metagenomic data from marine thaumarchaeotal species. This protein, referred as DnaJ-Fer, is composed of a J domain fused to a Ferredoxin (Fer domain. Surprisingly, the same protein was also found in Viridiplantae (green algae and land plants. Because J domain-containing proteins are known to interact with the major chaperone DnaK/Hsp70, this suggested that a DnaK protein was present in Thaumarchaeota. DnaK/Hsp70, its co-chaperone DnaJ and the nucleotide exchange factor GrpE are involved, among others, in heat shocks and heavy metal cellular stress responses. Results Using phylogenomic approaches we have investigated the evolutionary history of the DnaJ-Fer protein and of interacting proteins DnaK, DnaJ and GrpE in Thaumarchaeota. These proteins have very complex histories, involving several inter-domain horizontal gene transfers (HGTs to explain the contemporary distribution of these proteins in archaea. These transfers include one from Cyanobacteria to Viridiplantae and one from Viridiplantae to Thaumarchaeota for the DnaJ-Fer protein, as well as independent HGTs from Bacteria to mesophilic archaea for the DnaK/DnaJ/GrpE system, followed by HGTs among mesophilic and thermophilic archaea. Conclusions We highlight the chimerical origin of the set of proteins DnaK, DnaJ, GrpE and DnaJ-Fer in Thaumarchaeota and suggest that the HGT of these proteins has played an important role in the adaptation of several archaeal groups to mesophilic and thermophilic environments from hyperthermophilic ancestors. Finally, the evolutionary history of DnaJ-Fer provides information useful for the relative dating of the diversification of Archaeplastida and Thaumarchaeota.

  3. Bacillus subtilis DNA polymerases, PolC and DnaE, are required for both leading and lagging strand synthesis in SPP1 origin-dependent DNA replication

    Science.gov (United States)

    Seco, Elena M.

    2017-01-01

    Abstract Firmicutes have two distinct replicative DNA polymerases, the PolC leading strand polymerase, and PolC and DnaE synthesizing the lagging strand. We have reconstituted in vitro Bacillus subtilis bacteriophage SPP1 θ-type DNA replication, which initiates unidirectionally at oriL. With this system we show that DnaE is not only restricted to lagging strand synthesis as previously suggested. DnaG primase and DnaE polymerase are required for initiation of DNA replication on both strands. DnaE and DnaG synthesize in concert a hybrid RNA/DNA ‘initiation primer’ on both leading and lagging strands at the SPP1 oriL region, as it does the eukaryotic Pol α complex. DnaE, as a RNA-primed DNA polymerase, extends this initial primer in a reaction modulated by DnaG and one single-strand binding protein (SSB, SsbA or G36P), and hands off the initiation primer to PolC, a DNA-primed DNA polymerase. Then, PolC, stimulated by DnaG and the SSBs, performs the bulk of DNA chain elongation at both leading and lagging strands. Overall, these modulations by the SSBs and DnaG may contribute to the mechanism of polymerase switch at Firmicutes replisomes. PMID:28575448

  4. The validation of forensic DNA extraction systems to utilize soil contaminated biological evidence.

    Science.gov (United States)

    Kasu, Mohaimin; Shires, Karen

    2015-07-01

    The production of full DNA profiles from biological evidence found in soil has a high failure rate due largely to the inhibitory substance humic acid (HA). Abundant in various natural soils, HA co-extracts with DNA during extraction and inhibits DNA profiling by binding to the molecular components of the genotyping assay. To successfully utilize traces of soil contaminated evidence, such as that found at many murder and rape crime scenes in South Africa, a reliable HA removal extraction system would often be selected based on previous validation studies. However, for many standard forensic DNA extraction systems, peer-reviewed publications detailing the efficacy on soil evidence is either lacking or is incomplete. Consequently, these sample types are often not collected or fail to yield suitable DNA material due to the use of unsuitable methodology. The aim of this study was to validate the common forensic DNA collection and extraction systems used in South Africa, namely DNA IQ, FTA elute and Nucleosave for processing blood and saliva contaminated with HA. A forensic appropriate volume of biological evidence was spiked with HA (0, 0.5, 1.5 and 2.5 mg/ml) and processed through each extraction protocol for the evaluation of HA removal using QPCR and STR-genotyping. The DNA IQ magnetic bead system effectively removed HA from highly contaminated blood and saliva, and generated consistently acceptable STR profiles from both artificially spiked samples and crude soil samples. This system is highly recommended for use on soil-contaminated evidence over the cellulose card-based systems currently being preferentially used for DNA sample collection. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  5. MiniX-STR multiplex system population study in Japan and application to degraded DNA analysis.

    Science.gov (United States)

    Asamura, H; Sakai, H; Kobayashi, K; Ota, M; Fukushima, H

    2006-05-01

    We sought to evaluate a more effective system for analyzing X-chromosomal short tandem repeats (X-STRs) in highly degraded DNA. To generate smaller amplicon lengths, we designed new polymerase chain reaction (PCR) primers for DXS7423, DXS6789, DXS101, GATA31E08, DXS8378, DXS7133, DXS7424, and GATA165B12 at X-linked short tandem repeat (STR) loci, devising two miniX-multiplex PCR systems. Among 333 Japanese individuals, these X-linked loci were detected in amplification products ranging in length from 76 to 169 bp, and statistical analyses of the eight loci indicated a high usefulness for the Japanese forensic practice. Results of tests on highly degraded DNA indicated the miniX-STR multiplex strategies to be an effective system for analyzing degraded DNA. We conclude that analysis by the current miniX-STR multiplex systems offers high effectiveness for personal identification from degraded DNA samples.

  6. DNA repair systems and the pathogenesis of Mycobacterium tuberculosis: varying activities at different stages of infection.

    Science.gov (United States)

    Gorna, Alina E; Bowater, Richard P; Dziadek, Jaroslaw

    2010-05-25

    Mycobacteria, including most of all MTB (Mycobacterium tuberculosis), cause pathogenic infections in humans and, during the infectious process, are exposed to a range of environmental insults, including the host's immune response. From the moment MTB is exhaled by infected individuals, through an active and latent phase in the body of the new host, until the time they reach the reactivation stage, MTB is exposed to many types of DNA-damaging agents. Like all cellular organisms, MTB has efficient DNA repair systems, and these are believed to play essential roles in mycobacterial pathogenesis. As different stages of infection have great variation in the conditions in which mycobacteria reside, it is possible that different repair systems are essential for progression to specific phases of infection. MTB possesses homologues of DNA repair systems that are found widely in other species of bacteria, such as nucleotide excision repair, base excision repair and repair by homologous recombination. MTB also possesses a system for non-homologous end-joining of DNA breaks, which appears to be widespread in prokaryotes, although its presence is sporadic within different species within a genus. However, MTB does not possess homologues of the typical mismatch repair system that is found in most bacteria. Recent studies have demonstrated that DNA repair genes are expressed differentially at each stage of infection. In the present review, we focus on different DNA repair systems from mycobacteria and identify questions that remain in our understanding of how these systems have an impact upon the infection processes of these important pathogens.

  7. Flow-injection chemiluminescence assay for ultra-trace determination of DNA using rhodamine B-Ce(IV)-DNA ternary system in sulfuric acid media

    International Nuclear Information System (INIS)

    Ma Yongjun; Zhou Min; Jin Xiaoyong; Zhang Ziyu; Teng Xiulan; Chen Hui

    2004-01-01

    A novel flow-injection chemiluminescence method for the determination of DNA at ultra-trace level has been established. In 0.8 M sulfuric acid media, the chemiluminescence of the rhodamine B-cerium (IV) or Ce(IV) system is enhanced by DNA, activated previously by imidazole-HCl buffer solution (pH 7.0). The enhanced intensity of chemiluminescence is in proportion to log DNA concentration 1.0x10 -8 to 0.1 μg ml -1 for herring sperm DNA and 2.0x10 -6 to 0.2 μg ml -1 for calf thymus DNA with 3σ detection limits of 8.3x10 -9 μg ml -1 for herring sperm DNA and 3.5x10 -7 μg ml -1 for calf thymus DNA, respectively. The relative standard deviation for 1.0x10 -4 μg ml -1 herring sperm DNA was 0.99% and 2.0x10 -3 μg ml -1 for calf thymus DNA was 1.1% (n=11). Using the optimized system, DNA contents in six synthetic samples has been determined with recoveries of 99.5-109.0%. The possible mechanism has also been studied in this paper

  8. Flow-injection chemiluminescence assay for ultra-trace determination of DNA using rhodamine B-Ce(IV)-DNA ternary system in sulfuric acid media

    Energy Technology Data Exchange (ETDEWEB)

    Ma Yongjun; Zhou Min; Jin Xiaoyong; Zhang Ziyu; Teng Xiulan; Chen Hui

    2004-01-09

    A novel flow-injection chemiluminescence method for the determination of DNA at ultra-trace level has been established. In 0.8 M sulfuric acid media, the chemiluminescence of the rhodamine B-cerium (IV) or Ce(IV) system is enhanced by DNA, activated previously by imidazole-HCl buffer solution (pH 7.0). The enhanced intensity of chemiluminescence is in proportion to log DNA concentration 1.0x10{sup -8} to 0.1 {mu}g ml{sup -1} for herring sperm DNA and 2.0x10{sup -6} to 0.2 {mu}g ml{sup -1} for calf thymus DNA with 3{sigma} detection limits of 8.3x10{sup -9} {mu}g ml{sup -1} for herring sperm DNA and 3.5x10{sup -7} {mu}g ml{sup -1} for calf thymus DNA, respectively. The relative standard deviation for 1.0x10{sup -4} {mu}g ml{sup -1} herring sperm DNA was 0.99% and 2.0x10{sup -3} {mu}g ml{sup -1} for calf thymus DNA was 1.1% (n=11). Using the optimized system, DNA contents in six synthetic samples has been determined with recoveries of 99.5-109.0%. The possible mechanism has also been studied in this paper.

  9. A personal tourism navigation system to support traveling multiple destinations with time restrictions

    OpenAIRE

    Maruyama, Atsushi; Shibata, Naoki; Murata, Yoshihiro; Yasumoto, Keiichi; Ito, Minoru

    2004-01-01

    We propose a personal navigation system (called PNS) which navigates a tourist through multiple destinations efficiently. In our PNS, a tourist can specify multiple destinations with desired arrival/stay time and preference degree. The system calculates the route including part of the destinations satisfying tourist's requirements and navigates him/her. For the above route search problem, we have developed an efficient route search algorithm using a genetic algorithm. We have designed and imp...

  10. Principal Typings in a Restricted Intersection Type System for Beta Normal Forms with De Bruijn Indices

    Directory of Open Access Journals (Sweden)

    Daniel Ventura

    2010-01-01

    Full Text Available The lambda-calculus with de Bruijn indices assembles each alpha-class of lambda-terms in a unique term, using indices instead of variable names. Intersection types provide finitary type polymorphism and can characterise normalisable lambda-terms through the property that a term is normalisable if and only if it is typeable. To be closer to computations and to simplify the formalisation of the atomic operations involved in beta-contractions, several calculi of explicit substitution were developed mostly with de Bruijn indices. Versions of explicit substitutions calculi without types and with simple type systems are well investigated in contrast to versions with more elaborate type systems such as intersection types. In previous work, we introduced a de Bruijn version of the lambda-calculus with an intersection type system and proved that it preserves subject reduction, a basic property of type systems. In this paper a version with de Bruijn indices of an intersection type system originally introduced to characterise principal typings for beta-normal forms is presented. We present the characterisation in this new system and the corresponding versions for the type inference and the reconstruction of normal forms from principal typings algorithms. We briefly discuss the failure of the subject reduction property and some possible solutions for it.

  11. OPTSDNA: Performance evaluation of an efficient distributed bioinformatics system for DNA sequence analysis.

    Science.gov (United States)

    Khan, Mohammad Ibrahim; Sheel, Chotan

    2013-01-01

    Storage of sequence data is a big concern as the amount of data generated is exponential in nature at several locations. Therefore, there is a need to develop techniques to store data using compression algorithm. Here we describe optimal storage algorithm (OPTSDNA) for storing large amount of DNA sequences of varying length. This paper provides performance analysis of optimal storage algorithm (OPTSDNA) of a distributed bioinformatics computing system for analysis of DNA sequences. OPTSDNA algorithm is used for storing various sizes of DNA sequences into database. DNA sequences of different lengths were stored by using this algorithm. These input DNA sequences are varied in size from very small to very large. Storage size is calculated by this algorithm. Response time is also calculated in this work. The efficiency and performance of the algorithm is high (in size calculation with percentage) when compared with other known with sequential approach.

  12. [DNA Extraction from Old Bones by AutoMate Express™ System].

    Science.gov (United States)

    Li, B; Lü, Z

    2017-08-01

    To establish a method for extracting DNA from old bones by AutoMate Express™ system. Bones were grinded into powder by freeze-mill. After extraction by AutoMate Express™, DNA were amplified and genotyped by Identifiler®Plus and MinFiler™ kits. DNA were extracted from 10 old bone samples, which kept in different environments with the postmortem interval from 10 to 20 years, in 3 hours by AutoMate Express™ system. Complete STR typing results were obtained from 8 samples. AutoMate Express™ system can quickly and efficiently extract DNA from old bones, which can be applied in forensic practice. Copyright© by the Editorial Department of Journal of Forensic Medicine

  13. Optimizing man-machine performance of a personnel access restriction security system

    International Nuclear Information System (INIS)

    Banks, W.W.; Moore, J.W.

    1988-01-01

    This paper describes a human engineering design and analysis effort for a major security system upgrade at a DOE facility. This upgrade was accomplished by replacing an obsolete and poorly human engineered security screening both the with a new, user oriented, semiautomated, computer-based access control system. Human factors engineers assisted the designer staff in specifying a security access interface to physically and cognitively accommodate all employees which included handicapped individuals in wheel chairs, and several employees who were severely disabled, both visually and aurally. The new access system was intended to control entry into sensitive exclusion areas by requiring personnel to enter a security screening booth and interact with card reader devices and a-simple-to-operate access control panel system. Extensive man-machine testing with prototype mock-ups was conducted to assess human engineered design features and to illuminate potentially confusing or difficult-to-operated hardware placement, layout, and operation sequencing. These evaluations, along with the prototype mock-ups, provided input which resulted in a prototype which was easy to enter, operate, and understand by end users. This prototype later served as the design basis for the final systems design

  14. PAMTRAK: A system to monitor high value objects and personnel in restricted areas

    International Nuclear Information System (INIS)

    Anspach, D.A.; Walters, B.G.; Anspach, J.P.; Crain, B. Jr.

    1996-01-01

    The US Department of Energy (DOE) uses sensitive or classified parts and material that must be protected and accounted for. The authors believe there is a need for an automated system that can help protect and monitor these parts and material. In response to this need Sandia National Laboratories (SNL) has developed a real-time personnel and material tracking system called PAMTRAK that has been installed at selected DOE facilities. PAMTRAK safeguards sensitive parts and material by tracking tags worn by personnel and by monitoring sensors attached to the parts or material. This paper describes the goals when designing PAMTRAK, the PAMTRAK system components, the current installations, and the benefits a site can expect when using PAMTRAK

  15. Guardians of the mycobacterial genome: A review on DNA repair systems in Mycobacterium tuberculosis.

    Science.gov (United States)

    Singh, Amandeep

    2017-12-01

    The genomic integrity of Mycobacterium tuberculosis is continuously threatened by the harsh survival conditions inside host macrophages, due to immune and antibiotic stresses. Faithful genome maintenance and repair must be accomplished under stress for the bacillus to survive in the host, necessitating a robust DNA repair system. The importance of DNA repair systems in pathogenesis is well established. Previous examination of the M. tuberculosis genome revealed homologues of almost all the major DNA repair systems, i.e. nucleotide excision repair (NER), base excision repair (BER), homologous recombination (HR) and non-homologous end joining (NHEJ). However, recent developments in the field have pointed to the presence of novel proteins and pathways in mycobacteria. Homologues of archeal mismatch repair proteins were recently reported in mycobacteria, a pathway previously thought to be absent. RecBCD, the major nuclease-helicase enzymes involved in HR in E. coli, were implicated in the single-strand annealing (SSA) pathway. Novel roles of archeo-eukaryotic primase (AEP) polymerases, previously thought to be exclusive to NHEJ, have been reported in BER. Many new proteins with a probable role in DNA repair have also been discovered. It is now realized that the DNA repair systems in M. tuberculosis are highly evolved and have redundant backup mechanisms to mend the damage. This review is an attempt to summarize our current understanding of the DNA repair systems in M. tuberculosis.

  16. Systemic Glucose Level Changes with a Carbohydrate-Restricted and Higher Protein Diet Combined with Exercise

    Science.gov (United States)

    Bowden, Rodney G.; Lanning, Beth A.; Doyle, Eva I.; Slonaker, Becky; Johnston, Holly M.; Scanes, Georgene

    2007-01-01

    Objective: The authors' purpose in this study was to compare the effects of macronutrient intake on systemic glucose levels in previously sedentary participants who followed 1 of 4 diets that were either higher protein or high carbohydrate, while initiating an exercise program. Participants and Methods: The authors randomly assigned 94 sedentary…

  17. Media rating systems: do they work? Shop floor compliance with age restrictions in the Netherlands

    NARCIS (Netherlands)

    Gosselt, Jordi Franciscus; van Hoof, Joris Jasper; de Jong, Menno D.T.

    2012-01-01

    Media rating systems have been introduced in many countries to protect minors from being exposed to harmful media content. This study examines whether retailers comply with the guidelines of media ratings in the Netherlands. In a mystery shopping study, minors tried to buy or rent media products for

  18. A Hybrid Approach to Processing Big Data Graphs on Memory-Restricted Systems

    KAUST Repository

    Harshvardhan,; West, Brandon; Fidel, Adam; Amato, Nancy M.; Rauchwerger, Lawrence

    2015-01-01

    that sacrifice performance. In this work, we propose a novel RAM-Disk hybrid approach to graph processing that can scale well from a single shared-memory node to large distributed-memory systems. It works by partitioning the graph into sub graphs that fit in RAM

  19. Systemic oxidatively generated DNA/RNA damage in clinical depression

    DEFF Research Database (Denmark)

    Jorgensen, Anders; Krogh, Jesper; Miskowiak, Kamilla

    2013-01-01

    oxidatively generated DNA and RNA damage, 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo), respectively, were determined in healthy controls (N=28), moderately depressed, non-medicated patients (N=26) and severely depressed patients eligible for electroconvulsive therapy...... for trend=0.004). The 8-oxoGuo excretion was further increased after clinically effective ECT compared with pre-ECT values (P=0.006). There were no differences in 8-oxodG excretion between the groups or pre- vs. post-ECT. LIMITATIONS: Small sample size and the inclusion of both unipolar and bipolar patients...

  20. A Hybrid Approach to Processing Big Data Graphs on Memory-Restricted Systems

    KAUST Repository

    Harshvardhan,

    2015-05-01

    With the advent of big-data, processing large graphs quickly has become increasingly important. Most existing approaches either utilize in-memory processing techniques that can only process graphs that fit completely in RAM, or disk-based techniques that sacrifice performance. In this work, we propose a novel RAM-Disk hybrid approach to graph processing that can scale well from a single shared-memory node to large distributed-memory systems. It works by partitioning the graph into sub graphs that fit in RAM and uses a paging-like technique to load sub graphs. We show that without modifying the algorithms, this approach can scale from small memory-constrained systems (such as tablets) to large-scale distributed machines with 16, 000+ cores.

  1. Eigenfunctions of the invariant differential operators on symmetric spaces having A2 as a restricted root system

    International Nuclear Information System (INIS)

    Prati, M.C.

    1986-01-01

    The eigenfunctions psub(nm)sup(μ) (z, z-bar), n,m are elements of N, μ is an element of (-1/3, + infinity), z is an element of C, of two differential operators, which for some particular values of μ are the generators of the algebra of invariant differential operators on symmetric spaces, having A 2 as a restricted root system, are studied. The group-theoretic interpretation and the explicit form of these functions as polynomials of z , z-bar are given in the following cases: when μ = 0, 1 for every n, m belonging to N; when m = 0, for every n belonging to N and when μ is an element of (-1/3, +infinity). Furthermore, all solutions psub(nm)sup(μ) (z, z-bar) for every μ belonging to (-1/3, +infinity) and n + m <= 5 are explicitly written. This research has applications in quantum mechanics and in quantum field theory

  2. Study of DNA damage with a new system for irradiation of samples in a nuclear reactor

    Energy Technology Data Exchange (ETDEWEB)

    Gual, Maritza R., E-mail: mrgual@instec.c [Instituto Superior de Tecnologias y Ciencias Aplicadas, InSTEC, Avenida Salvador Allende y Luaces, Quinta de Los Molinos, Plaza de la Revolucion, Havana, AP 6163 (Cuba); Milian, Felix M. [Universidade Estadual de Santa Cruz, UESC (Brazil); Deppman, Airton [Instituto de Fisica, Universidad de Sao Paulo, IF-USP, Rua do Matao, Travessa R, no. 187, Ciudade Universitaria, Butanta, CEP 05508-900, Sao Paulo (Brazil); Coelho, Paulo R.P. [Instituto de Pesquisas Energeticas e Nucleares, IPEN-CNEN/SP (Brazil)

    2011-02-15

    In this paper, we report results of a quantitative analysis of the effects of neutrons on DNA, and, specifically, the production of simple and double breaks of plasmid DNA in aqueous solutions with different concentrations of free-radical scavengers. The radiation damage to DNA was evaluated by electrophoresis through agarose gels. The neutron and gamma doses were measured separately with thermoluminescent detectors. In this work, we have also demonstrated usefulness of a new system for positioning and removing samples in channel BH3 of the IEA-R1 reactor at the Instituto de Pesquisas Energeticas e Nucleares (Brazil) without necessity of interrupting the reactor operation.

  3. Purpose-restricted Anonymous Mobile Communications Using Anonymous Signatures in Online Credential Systems

    DEFF Research Database (Denmark)

    Fathi, Hanane; Shin, SeongHan; Kobara, Kazukuni

    2010-01-01

    To avoid the risk of long-term storage of secrets on a portable device, an online credential system supports the roaming user in retrieving securely at various locations his private key and other material to generate anonymous signatures. The protocol proposed here allows a roaming mobile user...... to access anonymously services such as whistle blowing and net-counselling. Our approach: (1) allows a mobile user, remembering a short password, to anonymously and securely retrieve the credentials necessary for his anonymous communication without assuming a pre-established anonymous channel...

  4. DNA restriction fragment length polymorphism analysis of Mycobacterium tuberculosis isolates from HIV-seropositive and HIV-seronegative patients in Kampala, Uganda

    Directory of Open Access Journals (Sweden)

    Katabazi Fred A

    2009-02-01

    Full Text Available Abstract Background The identification and differentiation of strains of Mycobacterium tuberculosis by DNA fingerprinting has provided a better understanding of the epidemiology and tracing the transmission of tuberculosis. We set out to determine if there was a relationship between the risk of belonging to a group of tuberculosis patients with identical mycobacterial DNA fingerprint patterns and the HIV sero-status of the individuals in a high TB incidence peri-urban setting of Kampala, Uganda. Methods One hundred eighty three isolates of Mycobacterium tuberculosis from 80 HIV seropositive and 103 HIV seronegative patients were fingerprinted by standard IS6110-RFLP. Using the BioNumerics software, strains were considered to be clustered if at least one other patient had an isolate with identical RFLP pattern. Results One hundred and eighteen different fingerprint patterns were obtained from the 183 isolates. There were 34 clusters containing 54% (99/183 of the patients (average cluster size of 2.9, and a majority (96.2% of the strains possessed a high copy number (≥ 5 copies of the IS6110 element. When strains with P = 0.615, patients aged P = 0.100, and sex (aOR 1.12, 95%CI 0.60–2.06, P = 0.715. Conclusion The sample showed evidence of a high prevalence of recent transmission with a high average cluster size, but infection with an isolate with a fingerprint found to be part of a cluster was not associated with any demographic or clinical characteristics, including HIV status.

  5. Hepatic deficiency of the pioneer transcription factor FoxA restricts hepatitis B virus biosynthesis by the developmental regulation of viral DNA methylation.

    Directory of Open Access Journals (Sweden)

    Vanessa C McFadden

    2017-02-01

    Full Text Available The FoxA family of pioneer transcription factors regulates hepatitis B virus (HBV transcription, and hence viral replication. Hepatocyte-specific FoxA-deficiency in the HBV transgenic mouse model of chronic infection prevents the transcription of the viral DNA genome as a result of the failure of the developmentally controlled conversion of 5-methylcytosine residues to cytosine during postnatal hepatic maturation. These observations suggest that pioneer transcription factors such as FoxA, which mark genes for expression at subsequent developmental steps in the cellular differentiation program, mediate their effects by reversing the DNA methylation status of their target genes to permit their ensuing expression when the appropriate tissue-specific transcription factor combinations arise during development. Furthermore, as the FoxA-deficient HBV transgenic mice are viable, the specific developmental timing, abundance and isoform type of pioneer factor expression must permit all essential liver gene expression to occur at a level sufficient to support adequate liver function. This implies that pioneer transcription factors can recognize and mark their target genes in distinct developmental manners dependent upon, at least in part, the concentration and affinity of FoxA for its binding sites within enhancer and promoter regulatory sequence elements. This selective marking of cellular genes for expression by the FoxA pioneer factor compared to HBV may offer the opportunity for the specific silencing of HBV gene expression and hence the resolution of chronic HBV infections which are responsible for approximately one million deaths worldwide annually due to liver cirrhosis and hepatocellular carcinoma.

  6. A performance study on three qPCR quantification kits and their compatibilities with the 6-dye DNA profiling systems.

    Science.gov (United States)

    Lin, Sze-Wah; Li, Christina; Ip, Stephen C Y

    2018-03-01

    DNA quantification plays an integral role in forensic DNA profiling. Not only does it estimate the total amount of amplifiable human autosomal and male DNA to ensure optimal amplification of target DNA for subsequent analysis, but also assesses the extraction efficiency and purity of the DNA extract. Latest DNA quantification systems even offer an estimate for the degree of DNA degradation in a sample. Here, we report the performance of three new generation qPCR kits, namely Investigator ® Quantiplex HYres Kit from QIAGEN, Quantifiler ® Trio DNA Quantification Kit from Applied Biosystems™, and PowerQuant ® System from Promega, and their compatibilities with three 6-dye DNA profiling systems. Our results have demonstrated that all three kits generate standard curves with satisfactory consistency and reproducibility, and are capable of screening out traces of male DNA in the presence of 30-fold excess of female DNA. They also exhibit a higher tolerance to PCR inhibition than Quantifiler ® Human DNA Quantification Kit from Applied Biosystems™ in autosomal DNA quantification. PowerQuant ® , as compared to Quantiplex HYres and Quantifiler ® Trio, shows a better precision for both autosomal and male DNA quantifications. Quantifiler ® Trio and PowerQuant ® in contrast to Quantiplex HYres offer better correlations with lower discrepancies between autosomal and male DNA quantification, and their additional degradation index features provide a detection platform for inhibited and/or degraded DNA template. Regarding the compatibility between these quantification and profiling systems: (1) both Quantifiler ® Trio and PowerQuant ® work well with GlobalFiler and Fusion 6C, allowing a fairly accurate prediction of their DNA typing results based on the quantification values; (2) Quantiplex HYres offers a fairly reliable IPC system for detecting any potential inhibitions on Investigator 24plex, whereas Quantifiler ® Trio and PowerQuant ® suit better for Global

  7. Differential Genetic Associations for Systemic Lupus Erythematosus Based on Anti–dsDNA Autoantibody Production

    Science.gov (United States)

    Chung, Sharon A.; Taylor, Kimberly E.; Graham, Robert R.; Nititham, Joanne; Lee, Annette T.; Ortmann, Ward A.; Jacob, Chaim O.; Alarcón-Riquelme, Marta E.; Tsao, Betty P.; Harley, John B.; Gaffney, Patrick M.; Moser, Kathy L.; Petri, Michelle; Demirci, F. Yesim; Kamboh, M. Ilyas; Manzi, Susan; Gregersen, Peter K.; Langefeld, Carl D.; Behrens, Timothy W.; Criswell, Lindsey A.

    2011-01-01

    Systemic lupus erythematosus (SLE) is a clinically heterogeneous, systemic autoimmune disease characterized by autoantibody formation. Previously published genome-wide association studies (GWAS) have investigated SLE as a single phenotype. Therefore, we conducted a GWAS to identify genetic factors associated with anti–dsDNA autoantibody production, a SLE–related autoantibody with diagnostic and clinical importance. Using two independent datasets, over 400,000 single nucleotide polymorphisms (SNPs) were studied in a total of 1,717 SLE cases and 4,813 healthy controls. Anti–dsDNA autoantibody positive (anti–dsDNA +, n = 811) and anti–dsDNA autoantibody negative (anti–dsDNA –, n = 906) SLE cases were compared to healthy controls and to each other to identify SNPs associated specifically with these SLE subtypes. SNPs in the previously identified SLE susceptibility loci STAT4, IRF5, ITGAM, and the major histocompatibility complex were strongly associated with anti–dsDNA + SLE. Far fewer and weaker associations were observed for anti–dsDNA – SLE. For example, rs7574865 in STAT4 had an OR for anti–dsDNA + SLE of 1.77 (95% CI 1.57–1.99, p = 2.0E-20) compared to an OR for anti–dsDNA – SLE of 1.26 (95% CI 1.12–1.41, p = 2.4E-04), with pheterogeneity<0.0005. SNPs in the SLE susceptibility loci BANK1, KIAA1542, and UBE2L3 showed evidence of association with anti–dsDNA + SLE and were not associated with anti–dsDNA – SLE. In conclusion, we identified differential genetic associations with SLE based on anti–dsDNA autoantibody production. Many previously identified SLE susceptibility loci may confer disease risk through their role in autoantibody production and be more accurately described as autoantibody propensity loci. Lack of strong SNP associations may suggest that other types of genetic variation or non-genetic factors such as environmental exposures have a greater impact on susceptibility to anti–dsDNA – SLE. PMID

  8. Differential genetic associations for systemic lupus erythematosus based on anti-dsDNA autoantibody production.

    Directory of Open Access Journals (Sweden)

    Sharon A Chung

    2011-03-01

    Full Text Available Systemic lupus erythematosus (SLE is a clinically heterogeneous, systemic autoimmune disease characterized by autoantibody formation. Previously published genome-wide association studies (GWAS have investigated SLE as a single phenotype. Therefore, we conducted a GWAS to identify genetic factors associated with anti-dsDNA autoantibody production, a SLE-related autoantibody with diagnostic and clinical importance. Using two independent datasets, over 400,000 single nucleotide polymorphisms (SNPs were studied in a total of 1,717 SLE cases and 4,813 healthy controls. Anti-dsDNA autoantibody positive (anti-dsDNA +, n = 811 and anti-dsDNA autoantibody negative (anti-dsDNA -, n = 906 SLE cases were compared to healthy controls and to each other to identify SNPs associated specifically with these SLE subtypes. SNPs in the previously identified SLE susceptibility loci STAT4, IRF5, ITGAM, and the major histocompatibility complex were strongly associated with anti-dsDNA + SLE. Far fewer and weaker associations were observed for anti-dsDNA - SLE. For example, rs7574865 in STAT4 had an OR for anti-dsDNA + SLE of 1.77 (95% CI 1.57-1.99, p = 2.0E-20 compared to an OR for anti-dsDNA - SLE of 1.26 (95% CI 1.12-1.41, p = 2.4E-04, with p(heterogeneity<0.0005. SNPs in the SLE susceptibility loci BANK1, KIAA1542, and UBE2L3 showed evidence of association with anti-dsDNA + SLE and were not associated with anti-dsDNA - SLE. In conclusion, we identified differential genetic associations with SLE based on anti-dsDNA autoantibody production. Many previously identified SLE susceptibility loci may confer disease risk through their role in autoantibody production and be more accurately described as autoantibody propensity loci. Lack of strong SNP associations may suggest that other types of genetic variation or non-genetic factors such as environmental exposures have a greater impact on susceptibility to anti-dsDNA - SLE.

  9. Influence of molecular weight of DNA on the determination of anti-DNA antibodies in systemic lupus erythematosus (SLE) sera by radioimmunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Geisert, M; Heicke, B; Metzmann, E; Zahn, R K

    1975-04-01

    Using a radioimmunoassay (RIA) based on the Farr technique with radioactively labeled /sup 3/H-DNA for quantitative measurements of anti-DNA antibodies in sera of patients with systemic lupus erythematosus (SLE), the influence of molecular weight of DNA (ranging from 0.1 x 10/sup 6/ to 22.0 x 10/sup 6/ daltons) on binding and precipitation in this system has been investigated. Comparing our results with mathematical models it follows that one antibody molecule is fixed on the average to a statistical DNA segment of 2 x 10/sup 6/ to 4 x 10/sup 6/ daltons. Furthermore binding capacity of the DNA was found to be independent of the molecular weight, as demonstrated in a double label experiment using /sup 14/C and /sup 3/H-labeled DNA of different size. However, the amount of radioactivity precipitated was found to depend on the molecular weight of the labeled DNA following a non-linear function. It was calculated that a minimal ratio of fixed antibody molecules per a certain size of DNA was necessary for precipitation. The mathematical treatment of the observed non-linear precipitation dependence will be discussed using various statistical models. The results indicate that the quantitative measurements of anti-DNA antibodies with the Farr technique e.g., for diagnosis and control of SLE in clinical immunology is highly dependent on the molecular weight of the labeled DNA used in the assay system and reliable results are only obtained with DNA of a sufficiently high molecular weight. (auth)

  10. Restrictive Factors and Output Forecast of Green Development of Agricultural Industry Based on Gray System

    Science.gov (United States)

    Sun, Fengru

    2018-01-01

    This paper analyzes the characteristics of agricultural products from the perspective of agricultural production, farmers’ income, adjustment of agricultural structure and environmental improvement, and analyzes the characteristics of agricultural products in LanZhou area. Through data mining and empirical analysis, the regional agriculture (1) forecasting model of gray system with dynamic data processing, combined with the output data of lily in 2004-2003, the yield prediction is predicted and the fitting state is good and the error is small. Finally, combined with the relevant characteristics of the local characteristics of the agricultural industry to make reference, by changing the characteristics of agricultural production as the center of the mindset, and agricultural industrialization and organic combination, take the characteristics of efficient industrialization of agricultural products.

  11. DNA index determination with Automated Cellular Imaging System (ACIS in Barrett's esophagus: Comparison with CAS 200

    Directory of Open Access Journals (Sweden)

    Klein Michael

    2005-08-01

    Full Text Available Abstract Background For solid tumors, image cytometry has been shown to be more sensitive for diagnosing DNA content abnormalities (aneuploidy than flow cytometry. Image cytometry has often been performed using the semi-automated CAS 200 system. Recently, an Automated Cellular Imaging System (ACIS was introduced to determine DNA content (DNA index, but it has not been validated. Methods Using the CAS 200 system and ACIS, we compared the DNA index (DI obtained from the same archived formalin-fixed and paraffin embedded tissue samples from Barrett's esophagus related lesions, including samples with specialized intestinal metaplasia without dysplasia, low-grade dysplasia, high-grade dysplasia and adenocarcinoma. Results Although there was a very good correlation between the DI values determined by ACIS and CAS 200, the former was 25% more sensitive in detecting aneuploidy. ACIS yielded a mean DI value 18% higher than that obtained by CAS 200 (p t test. In addition, the average time required to perform a DNA ploidy analysis was shorter with the ACIS (30–40 min than with the CAS 200 (40–70 min. Results obtained by ACIS gave excellent inter-and intra-observer variability (coefficient of correlation >0.9 for both, p Conclusion Compared with the CAS 200, the ACIS is a more sensitive and less time consuming technique for determining DNA ploidy. Results obtained by ACIS are also highly reproducible.

  12. CRISPR-Cas systems exploit viral DNA injection to establish and maintain adaptive immunity.

    Science.gov (United States)

    Modell, Joshua W; Jiang, Wenyan; Marraffini, Luciano A

    2017-04-06

    Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems provide protection against viral and plasmid infection by capturing short DNA sequences from these invaders and integrating them into the CRISPR locus of the prokaryotic host. These sequences, known as spacers, are transcribed into short CRISPR RNA guides that specify the cleavage site of Cas nucleases in the genome of the invader. It is not known when spacer sequences are acquired during viral infection. Here, to investigate this, we tracked spacer acquisition in Staphylococcus aureus cells harbouring a type II CRISPR-Cas9 system after infection with the staphylococcal bacteriophage ϕ12. We found that new spacers were acquired immediately after infection preferentially from the cos site, the viral free DNA end that is first injected into the cell. Analysis of spacer acquisition after infection with mutant phages demonstrated that most spacers are acquired during DNA injection, but not during other stages of the viral cycle that produce free DNA ends, such as DNA replication or packaging. Finally, we showed that spacers acquired from early-injected genomic regions, which direct Cas9 cleavage of the viral DNA immediately after infection, provide better immunity than spacers acquired from late-injected regions. Our results reveal that CRISPR-Cas systems exploit the phage life cycle to generate a pattern of spacer acquisition that ensures a successful CRISPR immune response.

  13. Restrictions on the detection of the Super-Earth in Solar system

    Science.gov (United States)

    Vidmachenko, A. P.

    2018-05-01

    For assessment of visibility of possible 9th planet, we use the infrared observations obtained by the "Wide-Field Infrared Survey Explorer". It turned out that the telescope could not see an analog of the planet of the giant Saturn at a distance of up to 30000 AU. This circumstance allowed us to estimate that at distances up to 1000 AU it would be clearly visible planetary body with a radius of more than 11,000 km; that is, a planet with mass of about 10 Earth masses and "earth" density (5520 kg/m3). If we take into account that the density of the "average" TNO differs little from 2000 kg/m3, that the radius of such "Super-Earth" with a mass of about 10 Earth masses - will increase to 19200 km. Then the limit of detection of a possible 9th planet will increase by almost 4 times: up to 4000 AU. And since the "WISE" telescope did not "see" even Saturn, that our estimates unequivocally suggest that there is no "Super-Earth" at a distance up to 1000 AU in the Solar system.

  14. Detection of DNA via the fluorescence quenching of Mn-doped ZnSe D-dots/doxorubicin/DNA ternary complexes system.

    Science.gov (United States)

    Gao, Xue; Niu, Lu; Su, Xingguang

    2012-01-01

    This manuscript reports a method for the detection of double-stranded DNA, based on Mn:ZnSe d-dots and intercalating agent doxorubicin (DOX). DOX can quench the photoluminescence (PL) of Mn:ZnSe d-dots through photoinduced electron transfer process, after binding with Mn:ZnSe d-dots. The addition of DNA can result in the formation of the Mn:ZnSe d-dots-DOX-DNA ternary complexes, the fluorescence of the Mn:ZnSe d-dots-DOX complexes would be further quenched by the addition of DNA, thus allowing the detection of DNA. The formation mechanism of the Mn:ZnSe d-dots-DOX-DNA ternary complexes was studied in detail in this paper. Under optimal conditions, the quenched fluorescence intensity of Mn:ZnSe d-dots-DOX system are perfectly described by Stern-Volmer equation with the concentration of hsDNA ranging from 0.006 μg mL(-1) to 6.4 μg mL(-1). The detection limit (S/N = 3) for hsDNA is 0.5 ng mL(-1). The proposed method was successfully applied to the detection of DNA in synthetic samples and the results were satisfactory.

  15. DNA Investigations.

    Science.gov (United States)

    Mayo, Ellen S.; Bertino, Anthony J.

    1991-01-01

    Presents a simulation activity that allow students to work through the exercise of DNA profiling and to grapple with some analytical and ethical questions involving a couple arranging with a surrogate mother to have a baby. Can be used to teach the principles of restriction enzyme digestion, gel electrophoresis, and probe hybridization. (MDH)

  16. DNA repair in B. subtilis: an inducible dimer-specific W-reactivation system

    International Nuclear Information System (INIS)

    Fields, P.I.; Yasbin, R.E.

    1982-01-01

    The W-reactivation system of Bacillus subtilis can repair pyrimidine dimers in bacteriophage DNA. This inducible repair system can be activated by treatment of the bacteria with uv, alkylating agents, cross-linking agents and gamma irradiation. However, bacteriophage treated with agents other than those that cause pyrimidine dimers to be produced was not repaired by this unique form of W-reactivation. In contrast, the W-reactivation system of Escherichia coli can repair a variety of damages placed in the bacteriophage DNA

  17. Property Rights, Restrictions and Responsibilities

    DEFF Research Database (Denmark)

    Enemark, Stig

    more to a social, ethical commitment or attitude to environmental sustainability and good husbandry. This paper provides an overall understanding of the concept of land administration systems for dealing with rights, restrictions and responsibilities in future spatially enabled government. Finally......Land Administration Systems are the basis for conceptualizing rights, restrictions and responsibilities related to people, policies and places. Property rights are normally concerned with ownership and tenure whereas restrictions usually control use and activities on land. Responsibilities relate...

  18. Seasonal variability in the persistence of dissolved environmental DNA (eDNA in a marine system: The role of microbial nutrient limitation.

    Directory of Open Access Journals (Sweden)

    Ian Salter

    Full Text Available Environmental DNA (eDNA can be defined as the DNA pool recovered from an environmental sample that includes both extracellular and intracellular DNA. There has been a significant increase in the number of recent studies that have demonstrated the possibility to detect macroorganisms using eDNA. Despite the enormous potential of eDNA to serve as a biomonitoring and conservation tool in aquatic systems, there remain some important limitations concerning its application. One significant factor is the variable persistence of eDNA over natural environmental gradients, which imposes a critical constraint on the temporal and spatial scales of species detection. In the present study, a radiotracer bioassay approach was used to quantify the kinetic parameters of dissolved eDNA (d-eDNA, a component of extracellular DNA, over an annual cycle in the coastal Northwest Mediterranean. Significant seasonal variability in the biological uptake and turnover of d-eDNA was observed, the latter ranging from several hours to over one month. Maximum uptake rates of d-eDNA occurred in summer during a period of intense phosphate limitation (turnover <5 hrs. Corresponding increases in bacterial production and uptake of adenosine triphosphate (ATP demonstrated the microbial utilization of d-eDNA as an organic phosphorus substrate. Higher temperatures during summer may amplify this effect through a general enhancement of microbial metabolism. A partial least squares regression (PLSR model was able to reproduce the seasonal cycle in d-eDNA persistence and explained 60% of the variance in the observations. Rapid phosphate turnover and low concentrations of bioavailable phosphate, both indicative of phosphate limitation, were the most important parameters in the model. Abiotic factors such as pH, salinity and oxygen exerted minimal influence. The present study demonstrates significant seasonal variability in the persistence of d-eDNA in a natural marine environment that can

  19. Genetic defects in DNA repair system and enhancement of intergenote transformation efficiency in Bacillus subtilis Marburg

    International Nuclear Information System (INIS)

    Matsumoto, K.; Takahashi, H.; Saito, H.; Ikeda, Y.

    1978-01-01

    Mechanisms of inefficiency in heterospecies transformation were studied with a transformation system consisting of Bacillus subtilis 168TI (trpC2thy) as recipient and of DNA prepared from partially hybrid strains of B. subtilis which had incorporated trp + DNA of B. amyloliquefaciens 203 (formerly, B. megaterium 203) in the chromosome (termed intergenote). The intergenote transformation was not so efficient as the corresponding homospecies transformation and the efficiency appeared to relate inversely with the length of heterologous portion in the intergenote. When a variety of ultraviolet light (UV) sensitive mutants, deficient in host-cell reactivation capacity, were used as recipients for the intergenote transformation, 2 out of 16 mutants exhibited significantly enhanced transformation efficiency of the trpC marker. Genetic studies by transformation showed that the trait relating to the enhancement of intergenote-transformation efficiency was always associated with the UV sensitivity, suggesting that these two traits are determined by a single gene. The efficiency of intergenote transformation was highly affected also by DNA concentration; the lower the concentration, the less the efficiency. When, however, the UV sensitive mutant was used as recipient, the effect of DNA concentration was largely diminished, suggesting the reduction of DNA-inactivating activity in the UV sensitive recipient. These results were discussed in relation to a possible excision-repair system selectively correcting the mismatched DNA in the course of intergenote transformation. (orig.) [de

  20. Development of an Automated Microfluidic System for DNA Collection, Amplification, and Detection of Pathogens

    Energy Technology Data Exchange (ETDEWEB)

    Hagan, Bethany S.; Bruckner-Lea, Cynthia J.

    2002-12-01

    This project was focused on developing and testing automated routines for a microfluidic Pathogen Detection System. The basic pathogen detection routine has three primary components; cell concentration, DNA amplification, and detection. In cell concentration, magnetic beads are held in a flow cell by an electromagnet. Sample liquid is passed through the flow cell and bacterial cells attach to the beads. These beads are then released into a small volume of fluid and delivered to the peltier device for cell lysis and DNA amplification. The cells are lysed during initial heating in the peltier device, and the released DNA is amplified using polymerase chain reaction (PCR) or strand displacement amplification (SDA). Once amplified, the DNA is then delivered to a laser induced fluorescence detection unit in which the sample is detected. These three components create a flexible platform that can be used for pathogen detection in liquid and sediment samples. Future developments of the system will include on-line DNA detection during DNA amplification and improved capture and release methods for the magnetic beads during cell concentration.

  1. A study of the blood flow restriction pressure of a tourniquet system to facilitate development of a system that can prevent musculoskeletal complications.

    Science.gov (United States)

    Maeda, Hiroyuki; Iwase, Hideaki; Kanda, Akio; Morohashi, Itaru; Kaneko, Kazuo; Maeda, Mutsuhiro; Kakinuma, Yuki; Takei, Yusuke; Amemiya, Shota; Mitsui, Kazuyuki

    2017-01-01

    After an emergency or disaster, subsequent trauma can cause severe bleeding and this can often prove fatal, so promptly stopping that bleeding is crucial to preventing avoidable trauma deaths. A tourniquet is often used to restrict blood flow to an extremity. In operation and hospital, the tourniquet systems currently in use are pneumatically actuated by an air compressor, so they must have a steady power supply. These devices have several drawbacks: they vibrate and are noisy since they are pneumatically actuated and they are far from portable since they are large and heavy. Presumably, the drawbacks of pneumatic tourniquets could be overcome by developing a small, lightweight, vibration-free, quiet, and battery-powered tourniquet system. The current study built a small, vibration-free electrohydrodynamic (EHD) pump and then used that pump to restrict blood flow to the leg of rats in an experiment. This study explored the optimal conditions for effective restriction of blood flow by assessing biochemical and musculoskeletal complications following the restriction of blood flow, and this study also examined whether or not an EHD pump could be used to actuate a tourniquet system. A tourniquet cuff (width 12 mm × length 150 mm, material: polyolefin) was placed on the thigh of Wistar rats and pressure was applied for 2 hours by a device that uses EHD phenomena to generate pressure (an EHD pump). Animals were divided into four groups based on how much compressive pressure was applied with a tourniquet: 40 kPa (300 mm Hg, n = 13), 30 kPa (225 mm Hg, n = 12), 20 kPa (150 mm Hg, n = 15), or 0 kPa (controls, n = 25). Tissue oxygen saturation (regional oxygen saturation, denoted here as rSO 2 ) was measured to assess the restriction of blood flow. To assess behavior once blood flow resumed, animal activity was monitored for third day and the amount of movement was counted with digital counters. Body weight was measured before and after the behavioral experiment, and

  2. distribution, abundance and properties of restriction enzymes

    African Journals Online (AJOL)

    DNA of granule-bound starch synthase (GBSS) I and II with a view to ... properties for manipulation of the genes for production of modified starch. .... procurement, storage and handling of the ..... been made on restriction enzymes of potato,.

  3. Endurance exercise rescues progeroid aging and induces systemic mitochondrial rejuvenation in mtDNA mutator mice

    Science.gov (United States)

    Safdar, Adeel; Bourgeois, Jacqueline M.; Ogborn, Daniel I.; Little, Jonathan P.; Hettinga, Bart P.; Akhtar, Mahmood; Thompson, James E.; Melov, Simon; Mocellin, Nicholas J.; Kujoth, Gregory C.; Prolla, Tomas A.; Tarnopolsky, Mark A.

    2011-01-01

    A causal role for mitochondrial DNA (mtDNA) mutagenesis in mammalian aging is supported by recent studies demonstrating that the mtDNA mutator mouse, harboring a defect in the proofreading-exonuclease activity of mitochondrial polymerase gamma, exhibits accelerated aging phenotypes characteristic of human aging, systemic mitochondrial dysfunction, multisystem pathology, and reduced lifespan. Epidemiologic studies in humans have demonstrated that endurance training reduces the risk of chronic diseases and extends life expectancy. Whether endurance exercise can attenuate the cumulative systemic decline observed in aging remains elusive. Here we show that 5 mo of endurance exercise induced systemic mitochondrial biogenesis, prevented mtDNA depletion and mutations, increased mitochondrial oxidative capacity and respiratory chain assembly, restored mitochondrial morphology, and blunted pathological levels of apoptosis in multiple tissues of mtDNA mutator mice. These adaptations conferred complete phenotypic protection, reduced multisystem pathology, and prevented premature mortality in these mice. The systemic mitochondrial rejuvenation through endurance exercise promises to be an effective therapeutic approach to mitigating mitochondrial dysfunction in aging and related comorbidities. PMID:21368114

  4. A new triple system DNA-Nanosilver-Berberine for cancer therapy

    Science.gov (United States)

    Grebinyk, Anna; Yashchuk, Valeriy; Bashmakova, Nataliya; Gryn, Dmytro; Hagemann, Tobias; Naumenko, Antonina; Kutsevol, Nataliya; Dandekar, Thomas; Frohme, Marcus

    2018-03-01

    The isoquinoline quaternary alkaloid Berberine possesses a variety of pharmacological properties that suggests its promising application for an anticancer delivery system design utilizing its ability to intercalate DNA. In the current work, we have investigated the effects of Berberine on the human T cell leukemia cell line in vitro. Fluorescent microscopy of leukemic cells revealed Berberine nuclear localization. The results showed that Berberine inhibited leukemic cell growth in a time- and dose-dependent manner, that was associated with reactive oxygen species production intensification and caspase 3/7 activity increase with followed apoptosis induction. Berberine was used as a toxic and phototoxic agent for triple system synthesis along with DNA as a carrier and nanosilver as a plasmonic accelerator of Berberine electronic transitions and high energy emission absorbent centers. The proposed method allows to obtain the complex of DNA with Berberine molecules and silver nanoparticles. The optical properties of free components as well as their various combinations, including the final triple system DNA-Nanosilver-Berberine, were investigated. Obtained results support the possibility to use the triple system DNA-Nanosilver-Berberine as an alternative therapeutic agent for cancer treatment.

  5. Effects of intrauterine growth restriction on sleep and the cardiovascular system: The use of melatonin as a potential therapy?

    Science.gov (United States)

    Yiallourou, Stephanie R; Wallace, Euan M; Miller, Suzanne L; Horne, Rosemary S C

    2016-04-01

    Intrauterine growth restriction (IUGR) complicates 5-10% of pregnancies and is associated with increased risk of preterm birth, mortality and neurodevelopmental delay. The development of sleep and cardiovascular control are closely coupled and IUGR is known to alter this development. In the long-term, IUGR is associated with altered sleep and an increased risk of hypertension in adulthood. Melatonin plays an important role in the sleep-wake cycle. Experimental animal studies have shown that melatonin therapy has neuroprotective and cardioprotective effects in the IUGR fetus. Consequently, clinical trials are currently underway to assess the short and long term effects of antenatal melatonin therapy in IUGR pregnancies. Given melatonin's role in sleep regulation, this hormone could affect the developing infants' sleep-wake cycle and cardiovascular function after birth. In this review, we will 1) examine the role of melatonin as a therapy for IUGR pregnancies and the potential implications on sleep and the cardiovascular system; 2) examine the development of sleep-wake cycle in fetal and neonatal life; 3) discuss the development of cardiovascular control during sleep; 4) discuss the effect of IUGR on sleep and the cardiovascular system and 5) discuss the future implications of melatonin therapy in IUGR pregnancies. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. 15 CFR 744.3 - Restrictions on Certain Rocket Systems (including ballistic missile systems and space launch...

    Science.gov (United States)

    2010-01-01

    ... Vehicles (including cruise missile systems, target drones and reconnaissance drones) End-Uses. 744.3... missile systems, target drones and reconnaissance drones) End-Uses. (a) General prohibition. In addition..., anywhere in the world except by governmental programs for nuclear weapons delivery of NPT Nuclear Weapons...

  7. DNA Mismatch Repair System: Repercussions in Cellular Homeostasis and Relationship with Aging

    Directory of Open Access Journals (Sweden)

    Juan Cristóbal Conde-Pérezprina

    2012-01-01

    Full Text Available The mechanisms that concern DNA repair have been studied in the last years due to their consequences in cellular homeostasis. The diverse and damaging stimuli that affect DNA integrity, such as changes in the genetic sequence and modifications in gene expression, can disrupt the steady state of the cell and have serious repercussions to pathways that regulate apoptosis, senescence, and cancer. These altered pathways not only modify cellular and organism longevity, but quality of life (“health-span”. The DNA mismatch repair system (MMR is highly conserved between species; its role is paramount in the preservation of DNA integrity, placing it as a necessary focal point in the study of pathways that prolong lifespan, aging, and disease. Here, we review different insights concerning the malfunction or absence of the DNA-MMR and its impact on cellular homeostasis. In particular, we will focus on DNA-MMR mechanisms regulated by known repair proteins MSH2, MSH6, PMS2, and MHL1, among others.

  8. A CCD-based system for the detection of DNA in electrophoresis gels by UV absorption

    International Nuclear Information System (INIS)

    Mahon, A.R.; MacDonald, J.H.; Mainwood, A.; Ott, R.J.

    1999-01-01

    A method and apparatus for the detection and quantification of large fragments of unlabelled nucleic acids in agarose gels is presented. The technique is based on ultraviolet (UV) absorption by nucleotides. A deuterium source illuminates individual sample lanes of an electrophoresis gel via an array of optical fibres. As DNA bands pass through the illuminated region of the gel the amount of UV light transmitted is reduced because of absorption by the DNA. During electrophoresis the regions of DNA are detected on-line using a UV-sensitive charge coupled device (CCD). As the absorption coefficient is proportional to the mass of DNA the technique is inherently quantitative. The mass of DNA in a region of the gel is approximately proportional to the integrated signal in the corresponding section of the CCD image. This system currently has a detection limit of less than 1.25 ng compared with 2-10 ng for the most popular conventional technique, ethidium bromide (EtBr) staining. In addition the DNA sample remains in its native state. The removal of the carcinogenic dye from the detection procedure greatly reduces associated biological hazards. (author)

  9. Antibodies to uv light denatured DNA in systemic lupus erythematosus: detection by filter radioimmunoassay and clinical correlations

    Energy Technology Data Exchange (ETDEWEB)

    Davis, P; Russell, A S; Percy, J S

    1976-12-01

    Antibodies to ultraviolet light denatured DNA (UV DNA) have been measured in patients with systemic lupus erythematosus (SLE) and normal subjects, using a millipore filter radioimmunoassay. High levels of UV DNA binding were only found in patients with SLE. The presence of UV DNA antibodies correlated well with the presence of native DNA antibodies, although immunodiffusion studies and inhibition techniques showed these antibodies to be immunologically distinct in many cases. Forty-one percent of the SLE patients had had photosensitivity at some stage of their disease, but there was a poor correlation between this symptom and the presence of UV DNA antibodies. Although UV DNA is known to be a potent immunogen, none of the results from this study suggests that antibodies to UV DNA are more than another example of the broad spectrum of antinuclear antibodies seen in SLE.

  10. Parallel Evolution of a Type IV Secretion System in Radiating Lineages of the Host-Restricted Bacterial Pathogen Bartonella

    Science.gov (United States)

    Engel, Philipp; Salzburger, Walter; Liesch, Marius; Chang, Chao-Chin; Maruyama, Soichi; Lanz, Christa; Calteau, Alexandra; Lajus, Aurélie; Médigue, Claudine; Schuster, Stephan C.; Dehio, Christoph

    2011-01-01

    Adaptive radiation is the rapid origination of multiple species from a single ancestor as the result of concurrent adaptation to disparate environments. This fundamental evolutionary process is considered to be responsible for the genesis of a great portion of the diversity of life. Bacteria have evolved enormous biological diversity by exploiting an exceptional range of environments, yet diversification of bacteria via adaptive radiation has been documented in a few cases only and the underlying molecular mechanisms are largely unknown. Here we show a compelling example of adaptive radiation in pathogenic bacteria and reveal their genetic basis. Our evolutionary genomic analyses of the α-proteobacterial genus Bartonella uncover two parallel adaptive radiations within these host-restricted mammalian pathogens. We identify a horizontally-acquired protein secretion system, which has evolved to target specific bacterial effector proteins into host cells as the evolutionary key innovation triggering these parallel adaptive radiations. We show that the functional versatility and adaptive potential of the VirB type IV secretion system (T4SS), and thereby translocated Bartonella effector proteins (Beps), evolved in parallel in the two lineages prior to their radiations. Independent chromosomal fixation of the virB operon and consecutive rounds of lineage-specific bep gene duplications followed by their functional diversification characterize these parallel evolutionary trajectories. Whereas most Beps maintained their ancestral domain constitution, strikingly, a novel type of effector protein emerged convergently in both lineages. This resulted in similar arrays of host cell-targeted effector proteins in the two lineages of Bartonella as the basis of their independent radiation. The parallel molecular evolution of the VirB/Bep system displays a striking example of a key innovation involved in independent adaptive processes and the emergence of bacterial pathogens

  11. Parallel evolution of a type IV secretion system in radiating lineages of the host-restricted bacterial pathogen Bartonella.

    Directory of Open Access Journals (Sweden)

    Philipp Engel

    2011-02-01

    Full Text Available Adaptive radiation is the rapid origination of multiple species from a single ancestor as the result of concurrent adaptation to disparate environments. This fundamental evolutionary process is considered to be responsible for the genesis of a great portion of the diversity of life. Bacteria have evolved enormous biological diversity by exploiting an exceptional range of environments, yet diversification of bacteria via adaptive radiation has been documented in a few cases only and the underlying molecular mechanisms are largely unknown. Here we show a compelling example of adaptive radiation in pathogenic bacteria and reveal their genetic basis. Our evolutionary genomic analyses of the α-proteobacterial genus Bartonella uncover two parallel adaptive radiations within these host-restricted mammalian pathogens. We identify a horizontally-acquired protein secretion system, which has evolved to target specific bacterial effector proteins into host cells as the evolutionary key innovation triggering these parallel adaptive radiations. We show that the functional versatility and adaptive potential of the VirB type IV secretion system (T4SS, and thereby translocated Bartonella effector proteins (Beps, evolved in parallel in the two lineages prior to their radiations. Independent chromosomal fixation of the virB operon and consecutive rounds of lineage-specific bep gene duplications followed by their functional diversification characterize these parallel evolutionary trajectories. Whereas most Beps maintained their ancestral domain constitution, strikingly, a novel type of effector protein emerged convergently in both lineages. This resulted in similar arrays of host cell-targeted effector proteins in the two lineages of Bartonella as the basis of their independent radiation. The parallel molecular evolution of the VirB/Bep system displays a striking example of a key innovation involved in independent adaptive processes and the emergence of bacterial

  12. Parallel evolution of a type IV secretion system in radiating lineages of the host-restricted bacterial pathogen Bartonella.

    Science.gov (United States)

    Engel, Philipp; Salzburger, Walter; Liesch, Marius; Chang, Chao-Chin; Maruyama, Soichi; Lanz, Christa; Calteau, Alexandra; Lajus, Aurélie; Médigue, Claudine; Schuster, Stephan C; Dehio, Christoph

    2011-02-10

    Adaptive radiation is the rapid origination of multiple species from a single ancestor as the result of concurrent adaptation to disparate environments. This fundamental evolutionary process is considered to be responsible for the genesis of a great portion of the diversity of life. Bacteria have evolved enormous biological diversity by exploiting an exceptional range of environments, yet diversification of bacteria via adaptive radiation has been documented in a few cases only and the underlying molecular mechanisms are largely unknown. Here we show a compelling example of adaptive radiation in pathogenic bacteria and reveal their genetic basis. Our evolutionary genomic analyses of the α-proteobacterial genus Bartonella uncover two parallel adaptive radiations within these host-restricted mammalian pathogens. We identify a horizontally-acquired protein secretion system, which has evolved to target specific bacterial effector proteins into host cells as the evolutionary key innovation triggering these parallel adaptive radiations. We show that the functional versatility and adaptive potential of the VirB type IV secretion system (T4SS), and thereby translocated Bartonella effector proteins (Beps), evolved in parallel in the two lineages prior to their radiations. Independent chromosomal fixation of the virB operon and consecutive rounds of lineage-specific bep gene duplications followed by their functional diversification characterize these parallel evolutionary trajectories. Whereas most Beps maintained their ancestral domain constitution, strikingly, a novel type of effector protein emerged convergently in both lineages. This resulted in similar arrays of host cell-targeted effector proteins in the two lineages of Bartonella as the basis of their independent radiation. The parallel molecular evolution of the VirB/Bep system displays a striking example of a key innovation involved in independent adaptive processes and the emergence of bacterial pathogens

  13. A mechanical mechanism for translocation of ring-shaped helicases on DNA and its demonstration in a macroscopic simulation system

    Science.gov (United States)

    Chou, Y. C.

    2018-04-01

    The asymmetry in the two-layered ring structure of helicases and the random thermal fluctuations of the helicase and DNA molecules are considered as the bases for the generation of the force required for translocation of the ring-shaped helicase on DNA. The helicase comprises a channel at its center with two unequal ends, through which strands of DNA can pass. The random collisions between the portion of the DNA strand in the central channel and the wall of the channel generate an impulsive force toward the small end. This impulsive force is the starting point for the helicase to translocate along the DNA with the small end in front. Such a physical mechanism may serve as a complementary for the chemomechanical mechanism of the translocation of helicase on DNA. When the helicase arrives at the junction of ssDNA and dsDNA (a fork), the collision between the helicase and the closest base pair may produce a sufficient impulsive force to break the weak hydrogen bond of the base pair. Thus, the helicase may advance and repeat the process of unwinding the dsDNA strand. This mechanism was tested in a macroscopic simulation system where the helicase was simulated using a truncated-cone structure and DNA was simulated with bead chains. Many features of translocation and unwinding such as translocation on ssDNA and dsDNA, unwinding of dsDNA, rewinding, strand switching, and Holliday junction resolution were reproduced.

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

    Science.gov (United States)

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

    1998-08-01

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

  15. Cardiac Sirt1 mediates the cardioprotective effect of caloric restriction by suppressing local complement system activation after ischemia-reperfusion.

    Science.gov (United States)

    Yamamoto, Tsunehisa; Tamaki, Kayoko; Shirakawa, Kohsuke; Ito, Kentaro; Yan, Xiaoxiang; Katsumata, Yoshinori; Anzai, Atsushi; Matsuhashi, Tomohiro; Endo, Jin; Inaba, Takaaki; Tsubota, Kazuo; Sano, Motoaki; Fukuda, Keiichi; Shinmura, Ken

    2016-04-15

    Caloric restriction (CR) confers cardioprotection against ischemia-reperfusion (I/R) injury. We previously found the essential roles of endothelial nitric oxide synthase in the development of CR-induced cardioprotection and Sirt1 activation during CR (Shinmura K, Tamaki K, Ito K, Yan X, Yamamoto T, Katsumata Y, Matsuhashi T, Sano M, Fukuda K, Suematsu M, Ishii I. Indispensable role of endothelial nitric oxide synthase in caloric restriction-induced cardioprotection against ischemia-reperfusion injury.Am J Physiol Heart Circ Physiol 308: H894-H903, 2015). However, the exact mechanism by which Sirt1 in cardiomyocytes mediates the cardioprotective effect of CR remains undetermined. We subjected cardiomyocyte-specific Sirt1 knockout (CM-Sirt1(-/-)) mice and the corresponding control mice to either 3-mo ad libitum feeding or CR (-40%). Isolated perfused hearts were subjected to 25-min global ischemia, followed by 60-min reperfusion. The recovery of left ventricle function after I/R was improved, and total lactate dehydrogenase release into the perfusate during reperfusion was attenuated in the control mice treated with CR, but a similar cardioprotective effect of CR was not observed in the CM-Sirt1(-/-)mice. The expression levels of cardiac complement component 3 (C3) at baseline and the accumulation of C3 and its fragments in the ischemia-reperfused myocardium were attenuated by CR in the control mice, but not in the CM-Sirt1(-/-)mice. Resveratrol treatment also attenuated the expression levels of C3 protein in cultured neonatal rat ventricular cardiomyocytes. Moreover, the degree of myocardial I/R injury in conventional C3 knockout (C3(-/-)) mice treated with CR was similar to that in the ad libitum-fed C3(-/-)mice, although the expression levels of Sirt1 were enhanced by CR. These results demonstrate that cardiac Sirt1 plays an essential role in CR-induced cardioprotection against I/R injury by suppressing cardiac C3 expression. This is the first report suggesting

  16. Characterization of DNA antigens from immune complexes deposited in the skin of patients with systemic lupus erythematosus

    Institute of Scientific and Technical Information of China (English)

    曾凡钦; 尹若菲; 谭国珍; 郭庆; 许德清

    2004-01-01

    Background Skin lesions are common manifestations in systemic lupus erythematosus (SLE). It is still unknown what the definite pathogenesis of skin involvement was and whether DNA participated in it. Our study was designed to explore the pathogenetic role and nature of nuclear antigen (DNA) deposited in the skin lesions of patients with SLE.Methods Thirty skin samples from patients with SLE and 2 normal skin samples were studied. Extracellular DNA was evaluated by indirect immunofluorescence methods. The deposited immune complexes were extracted by cryoprecipitation, and DNA was then isolated with phenol and chloroform. DNA fragment sizes were detected by agarose gel electrophoresis. Finally, 8 different probes were used to analyze the origin of these DNA molecules using Dot hybridization.Results Extracellular DNA staining was found only in skin lesions, mainly those located in the basement membrane zone, vascular wall, and hair follicle wall. Normal skin and non-lesion SLE skin showed no fluorescence at locations outside the nuclei. There were no differences in the rate and intensity of extracellular DNA staining when comparing active phase to remission phase patients. No relationship was found between extracellular DNA and circulating anti-dsDNA antibodies. Deposited DNA fragments clustered into four bands of somewhat discrete sizes: 20 000 bp, 1300 bp, 800-900 bp, 100-200 bp. Small sized fragments (100-200 bp) were positively correlated with disease activity (P<0.05, r=0.407). Dot hybridization showed significant homology of the various extracellular DNA fragments examined with human genomic DNA, but not with DNA from the microorganisms and viruses we examined. There were also homologies between DNA samples from different individuals.Conclusions DNA and its immune complexes may contribute to the pathogenesis of skin lesions in SLE. These DNA molecules range in size from 100 bp to 20 kb and may be endogenous in origin.

  17. Design of a Clinical Information Management System to Support DNA Analysis Laboratory Operation

    OpenAIRE

    Dubay, Christopher J.; Zimmerman, David; Popovich, Bradley

    1995-01-01

    The LabDirector system has been developed at the Oregon Health Sciences University to support the operation of our clinical DNA analysis laboratory. Through an iterative design process which has spanned two years, we have produced a system that is both highly tailored to a clinical genetics production laboratory and flexible in its implementation, to support the rapid growth and change of protocols and methodologies in use in the field. The administrative aspects of the system are integrated ...

  18. An integrated multiple capillary array electrophoresis system for high-throughput DNA sequencing

    Energy Technology Data Exchange (ETDEWEB)

    Lu, X.

    1998-03-27

    A capillary array electrophoresis system was chosen to perform DNA sequencing because of several advantages such as rapid heat dissipation, multiplexing capabilities, gel matrix filling simplicity, and the mature nature of the associated manufacturing technologies. There are two major concerns for the multiple capillary systems. One concern is inter-capillary cross-talk, and the other concern is excitation and detection efficiency. Cross-talk is eliminated through proper optical coupling, good focusing and immersing capillary array into index matching fluid. A side-entry excitation scheme with orthogonal detection was established for large capillary array. Two 100 capillary array formats were used for DNA sequencing. One format is cylindrical capillary with 150 {micro}m o.d., 75 {micro}m i.d and the other format is square capillary with 300 {micro}m out edge and 75 {micro}m inner edge. This project is focused on the development of excitation and detection of DNA as well as performing DNA sequencing. The DNA injection schemes are discussed for the cases of single and bundled capillaries. An individual sampling device was designed. The base-calling was performed for a capillary from the capillary array with the accuracy of 98%.

  19. Circulating cell free DNA as a predictor of systemic lupus ...

    African Journals Online (AJOL)

    Olfat M. Hendy

    2015-07-29

    Jul 29, 2015 ... as a potential tool to predict disease activity and treatment follow up. Subjects and ... control group. Thorough clinical examination stressing on the central nervous system, vascular, ... sis/necrosis of blood and tissue cells) and, second, active metabolic ..... alternative biomarkers should be tested. There was ...

  20. Telomere Restriction Fragment (TRF) Analysis.

    Science.gov (United States)

    Mender, Ilgen; Shay, Jerry W

    2015-11-20

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

  1. Effects of Dietary Sodium Restriction in Kidney Transplant Recipients Treated With Renin-Angiotensin-Aldosterone System Blockade: A Randomized Clinical Trial

    NARCIS (Netherlands)

    de Vries, Laura V.; Dobrowolski, Linn C.; van den Bosch, Jacqueline J. O. N.; Riphagen, Ineke J.; Krediet, C. T. Paul; Bemelman, Frederike J.; Bakker, Stephan J. L.; Navis, Gerjan

    2016-01-01

    In patients with chronic kidney disease receiving renin-angiotensin-aldosterone system (RAAS) blockade, dietary sodium restriction is an often-used treatment strategy to reduce blood pressure (BP) and albuminuria. Whether these effects extend to kidney transplant recipients is unknown. We therefore

  2. Effects of Dietary Sodium Restriction in Kidney Transplant Recipients Treated With Renin-Angiotensin-Aldosterone System Blockade : A Randomized Clinical Trial

    NARCIS (Netherlands)

    de Vries, Laura V; Dobrowolski, Linn C; van den Bosch, Jacqueline J O N; Riphagen, Ineke J; Krediet, C T Paul; Bemelman, Frederike J; Bakker, Stephan J L; Navis, Gerjan

    BACKGROUND: In patients with chronic kidney disease receiving renin-angiotensin-aldosterone system (RAAS) blockade, dietary sodium restriction is an often-used treatment strategy to reduce blood pressure (BP) and albuminuria. Whether these effects extend to kidney transplant recipients is unknown.

  3. Mathematical model of reproductive death of irradiated eukaryotic cells, which considers saturation of DNA reparation system

    International Nuclear Information System (INIS)

    Knyigavko, V.G.; Ponomarenko, N.S.; Meshcheryakova, O.P.; Protasenya, S.Yu.

    2009-01-01

    A mathematical model of the processes determining reproductive death of the exposed cells was built. The model takes into account the phenomenon of saturation of the system of DNA radiation lesion reparation and structural functional peculiarities of chromatin structure in eukaryotes. The problem of assessment of the model parameters using experimental data was discussed.

  4. [Studies on the repair of damaged DNA in bacteriophage, bacterial and mammalian systems]: Final report

    International Nuclear Information System (INIS)

    Friedberg, E.C.

    1987-08-01

    This study sought to exploit the use of uv radiation as a source of genomic damage. We explored the molecular mechanism of the repair of DNA damage at a number of different levels of biological organization, by investigating bacteriophage, bacterial, yeast and mammalian cells. Not only have observations obtained in one biological system suggested specific experimental approaches in others, but we have also learned that some biochemical pathways for DNA repair are unique to specific organisms. Our studies are summarized in terms of 4 major areas of research activity that span the past 16 years. 86 refs

  5. Involvement of the DNA mismatch repair system in cisplatin sensitivity of testicular germ cell tumours

    DEFF Research Database (Denmark)

    Rudolph, Christiane; Melau, Cecilie; Nielsen, John E.

    2017-01-01

    in the majority of tumours, although the underlying mechanism largely remains to be elucidated. The aim of this study was to investigate the role of the DNA mismatch repair (MMR) system in the cisplatin sensitivity of TGCT. MethodsThe expression pattern of key MMR proteins, including MSH2, MSH6, MLH1 and PMS2...... proteins, in particular MSH2 and MLH1, which are involved in the recognition of cisplatin adducts and in activation of the DNA damage response pathway to initiate apoptosis....

  6. Transcriptional blockages in a cell-free system by sequence-selective DNA alkylating agents.

    Science.gov (United States)

    Ferguson, L R; Liu, A P; Denny, W A; Cullinane, C; Talarico, T; Phillips, D R

    2000-04-14

    There is considerable interest in DNA sequence-selective DNA-binding drugs as potential inhibitors of gene expression. Five compounds with distinctly different base pair specificities were compared in their effects on the formation and elongation of the transcription complex from the lac UV5 promoter in a cell-free system. All were tested at drug levels which killed 90% of cells in a clonogenic survival assay. Cisplatin, a selective alkylator at purine residues, inhibited transcription, decreasing the full-length transcript, and causing blockage at a number of GG or AG sequences, making it probable that intrastrand crosslinks are the blocking lesions. A cyclopropylindoline known to be an A-specific alkylator also inhibited transcription, with blocks at adenines. The aniline mustard chlorambucil, that targets primarily G but also A sequences, was also effective in blocking the formation of full-length transcripts. It produced transcription blocks either at, or one base prior to, AA or GG sequences, suggesting that intrastrand crosslinks could again be involved. The non-alkylating DNA minor groove binder Hoechst 33342 (a bisbenzimidazole) blocked formation of the full-length transcript, but without creating specific blockage sites. A bisbenzimidazole-linked aniline mustard analogue was a more effective transcription inhibitor than either chlorambucil or Hoechst 33342, with different blockage sites occurring immediately as compared with 2 h after incubation. The blockages were either immediately prior to AA or GG residues, or four to five base pairs prior to such sites, a pattern not predicted from in vitro DNA-binding studies. Minor groove DNA-binding ligands are of particular interest as inhibitors of gene expression, since they have the potential ability to bind selectively to long sequences of DNA. The results suggest that the bisbenzimidazole-linked mustard does cause alkylation and transcription blockage at novel DNA sites. in addition to sites characteristic of

  7. Genome-Wide SNP Discovery and Analysis of Genetic Diversity in Farmed Sika Deer (Cervus nippon in Northeast China Using Double-Digest Restriction Site-Associated DNA Sequencing

    Directory of Open Access Journals (Sweden)

    Hengxing Ba

    2017-09-01

    Full Text Available Sika deer are an economically valuable species owing to their use in traditional Chinese medicine, particularly their velvet antlers. Sika deer in northeast China are mostly farmed in enclosure. Therefore, genetic management of farmed sika deer would benefit from detailed knowledge of their genetic diversity. In this study, we generated over 1.45 billion high-quality paired-end reads (288 Gbp across 42 unrelated individuals using double-digest restriction site-associated DNA sequencing (ddRAD-seq. A total of 96,188 (29.63% putative biallelic SNP loci were identified with an average sequencing depth of 23×. Based on the analysis, we found that the majority of the loci had a deficit of heterozygotes (FIS >0 and low values of Hobs, which could be due to inbreeding and Wahlund effects. We also developed a collection of high-quality SNP probes that will likely be useful in a variety of applications in genotyping for cervid species in the future.

  8. Genome-Wide SNP Discovery and Analysis of Genetic Diversity in Farmed Sika Deer (Cervus nippon) in Northeast China Using Double-Digest Restriction Site-Associated DNA Sequencing.

    Science.gov (United States)

    Ba, Hengxing; Jia, Boyin; Wang, Guiwu; Yang, Yifeng; Kedem, Gilead; Li, Chunyi

    2017-09-07

    Sika deer are an economically valuable species owing to their use in traditional Chinese medicine, particularly their velvet antlers. Sika deer in northeast China are mostly farmed in enclosure. Therefore, genetic management of farmed sika deer would benefit from detailed knowledge of their genetic diversity. In this study, we generated over 1.45 billion high-quality paired-end reads (288 Gbp) across 42 unrelated individuals using double-digest restriction site-associated DNA sequencing (ddRAD-seq). A total of 96,188 (29.63%) putative biallelic SNP loci were identified with an average sequencing depth of 23×. Based on the analysis, we found that the majority of the loci had a deficit of heterozygotes (F IS >0) and low values of H obs , which could be due to inbreeding and Wahlund effects. We also developed a collection of high-quality SNP probes that will likely be useful in a variety of applications in genotyping for cervid species in the future. Copyright © 2017 Ba et al.

  9. Genome-Wide Mapping of Growth-Related Quantitative Trait Loci in Orange-Spotted Grouper (Epinephelus coioides) Using Double Digest Restriction-Site Associated DNA Sequencing (ddRADseq).

    Science.gov (United States)

    Yu, Hui; You, Xinxin; Li, Jia; Liu, Hankui; Meng, Zining; Xiao, Ling; Zhang, Haifa; Lin, Hao-Ran; Zhang, Yong; Shi, Qiong

    2016-04-06

    Mapping of quantitative trait loci (QTL) is essential for the discovery of genetic structures that related to complex quantitative traits. In this study, we identified 264,072 raw SNPs (single-nucleotide polymorphisms) by double digest restriction site associated DNA sequencing (ddRADseq), and utilized 3029 of these SNPs to construct a genetic linkage map in orange-spotted grouper (Epinephelus coioides) using a regression mapping algorithm. The genetic map contained 24 linkage groups (LGs) spanning a total genetic distance of 1231.98 cM. Twenty-seven significant growth-related QTLs were identified. Furthermore, we identified 17 genes (fez2, alg3, ece2, arvcf, sla27a4, sgk223, camk2, prrc2b, mchr1, sardh, pappa, syk, tert, wdrcp91, ftz-f1, mate1 and notch1) including three (tert, ftz-f1 and notch1) that have been reported to be involved in fish growth. To summarize, we mapped growth-related QTLs in the orange-spotted grouper. These QTLs will be useful in marker-assisted selection (MAS) efforts to improve growth-related traits in this economically important fish.

  10. PIMS sequencing extension: a laboratory information management system for DNA sequencing facilities

    Directory of Open Access Journals (Sweden)

    Baldwin Stephen A

    2011-03-01

    Full Text Available Abstract Background Facilities that provide a service for DNA sequencing typically support large numbers of users and experiment types. The cost of services is often reduced by the use of liquid handling robots but the efficiency of such facilities is hampered because the software for such robots does not usually integrate well with the systems that run the sequencing machines. Accordingly, there is a need for software systems capable of integrating different robotic systems and managing sample information for DNA sequencing services. In this paper, we describe an extension to the Protein Information Management System (PIMS that is designed for DNA sequencing facilities. The new version of PIMS has a user-friendly web interface and integrates all aspects of the sequencing process, including sample submission, handling and tracking, together with capture and management of the data. Results The PIMS sequencing extension has been in production since July 2009 at the University of Leeds DNA Sequencing Facility. It has completely replaced manual data handling and simplified the tasks of data management and user communication. Samples from 45 groups have been processed with an average throughput of 10000 samples per month. The current version of the PIMS sequencing extension works with Applied Biosystems 3130XL 96-well plate sequencer and MWG 4204 or Aviso Theonyx liquid handling robots, but is readily adaptable for use with other combinations of robots. Conclusions PIMS has been extended to provide a user-friendly and integrated data management solution for DNA sequencing facilities that is accessed through a normal web browser and allows simultaneous access by multiple users as well as facility managers. The system integrates sequencing and liquid handling robots, manages the data flow, and provides remote access to the sequencing results. The software is freely available, for academic users, from http://www.pims-lims.org/.

  11. PIMS sequencing extension: a laboratory information management system for DNA sequencing facilities.

    Science.gov (United States)

    Troshin, Peter V; Postis, Vincent Lg; Ashworth, Denise; Baldwin, Stephen A; McPherson, Michael J; Barton, Geoffrey J

    2011-03-07

    Facilities that provide a service for DNA sequencing typically support large numbers of users and experiment types. The cost of services is often reduced by the use of liquid handling robots but the efficiency of such facilities is hampered because the software for such robots does not usually integrate well with the systems that run the sequencing machines. Accordingly, there is a need for software systems capable of integrating different robotic systems and managing sample information for DNA sequencing services. In this paper, we describe an extension to the Protein Information Management System (PIMS) that is designed for DNA sequencing facilities. The new version of PIMS has a user-friendly web interface and integrates all aspects of the sequencing process, including sample submission, handling and tracking, together with capture and management of the data. The PIMS sequencing extension has been in production since July 2009 at the University of Leeds DNA Sequencing Facility. It has completely replaced manual data handling and simplified the tasks of data management and user communication. Samples from 45 groups have been processed with an average throughput of 10000 samples per month. The current version of the PIMS sequencing extension works with Applied Biosystems 3130XL 96-well plate sequencer and MWG 4204 or Aviso Theonyx liquid handling robots, but is readily adaptable for use with other combinations of robots. PIMS has been extended to provide a user-friendly and integrated data management solution for DNA sequencing facilities that is accessed through a normal web browser and allows simultaneous access by multiple users as well as facility managers. The system integrates sequencing and liquid handling robots, manages the data flow, and provides remote access to the sequencing results. The software is freely available, for academic users, from http://www.pims-lims.org/.

  12. A novel color image encryption scheme using fractional-order hyperchaotic system and DNA sequence operations

    International Nuclear Information System (INIS)

    Zhang Li-Min; Sun Ke-Hui; Liu Wen-Hao; He Shao-Bo

    2017-01-01

    In this paper, Adomian decomposition method (ADM) with high accuracy and fast convergence is introduced to solve the fractional-order piecewise-linear (PWL) hyperchaotic system. Based on the obtained hyperchaotic sequences, a novel color image encryption algorithm is proposed by employing a hybrid model of bidirectional circular permutation and DNA masking. In this scheme, the pixel positions of image are scrambled by circular permutation, and the pixel values are substituted by DNA sequence operations. In the DNA sequence operations, addition and substraction operations are performed according to traditional addition and subtraction in the binary, and two rounds of addition rules are used to encrypt the pixel values. The simulation results and security analysis show that the hyperchaotic map is suitable for image encryption, and the proposed encryption algorithm has good encryption effect and strong key sensitivity. It can resist brute-force attack, statistical attack, differential attack, known-plaintext, and chosen-plaintext attacks. (paper)

  13. Computational Approach for Studying Optical Properties of DNA Systems in Solution

    DEFF Research Database (Denmark)

    Nørby, Morten Steen; Svendsen, Casper Steinmann; Olsen, Jógvan Magnus Haugaard

    2016-01-01

    In this paper we present a study of the methodological aspects regarding calculations of optical properties for DNA systems in solution. Our computational approach will be built upon a fully polarizable QM/MM/Continuum model within a damped linear response theory framework. In this approach...... the environment is given a highly advanced description in terms of the electrostatic potential through the polarizable embedding model. Furthermore, bulk solvent effects are included in an efficient manner through a conductor-like screening model. With the aim of reducing the computational cost we develop a set...... of averaged partial charges and distributed isotropic dipole-dipole polarizabilities for DNA suitable for describing the classical region in ground-state and excited-state calculations. Calculations of the UV-spectrum of the 2-aminopurine optical probe embedded in a DNA double helical structure are presented...

  14. Role of the inhibitors of angiotensin renin system on the DNA integrity of irradiated spermatozoids

    International Nuclear Information System (INIS)

    Spadella, Maria A.; Mansano, Naira S.; Schwarz, Franciele C.; Viani, Gustavo A.; Chies, Agnaldo B.

    2016-01-01

    Radiation action in the testes can significantly affect the reproductive capacity due to oxidative stress generated; phenomenon in which there is evidence of involvement of the Renin Angiotensin System (RAS). This study evaluated the role of AT1 receptor inhibitors, in mitigating the radioinduced DNA damage sperm from semen samples left vas deferens. Male Wistar rats were divided into six experimental groups: Control, 5Gy, Telmisartan (12mg/kg/day) and Losartan (34mg/kg/2x/day), 5 Gy + Telmisartan and 5 Gy + Losartan. The results showed increase in the percentage of sperm with fragmented DNA in irradiated groups when compared to controls, which was not reversed in the irradiated and treated groups. The radiation of 5Gy (single dose) affected the DNA-protein complex of the sperm and the treatments did not influence in reversing this damage, considering the experimental protocol used. (author)

  15. On Integrating Unmanned Aircraft Systems into the National Airspace System Issues, Challenges, Operational Restrictions, Certification, and Recommendations

    CERN Document Server

    Dalamagkidis, Konstantinos; Piegl, Les A

    2012-01-01

    This book presents, in a comprehensive way, current unmanned aviation regulation, airworthiness certification, special aircraft categories, pilot certification, federal aviation requirements, operation rules, airspace classes and regulation development models. It discusses unmanned aircraft systems levels of safety derived mathematically based on the corresponding levels for manned aviation. It provides an overview of the history and current status of UAS airworthiness and operational regulation worldwide. Existing regulations have been developed considering the need for a complete regulatory framework for UAS. It focuses on UAS safety assessment and functional requirements, achieved in terms of defining an “Equivalent Level of Safety”, or ELOS, with that of manned aviation, specifying what the ELOS requirement entails for UAS regulations. To accomplish this, the safety performance of manned aviation is first evaluated, followed by a novel model to derive reliability requirements for achieving target lev...

  16. Detection of mitochondrial DNA with the compact bead array sensor system (cBASS)

    Science.gov (United States)

    Mulvaney, Shawn P.; Ibe, Carol N.; Caldwell, Jane M.; Levine, Jay F.; Whitman, Lloyd J.; Tamanaha, Cy R.

    2009-02-01

    Enteric pathogens are a significant contaminant in surface waters used for recreation, fish and shellfish harvesting, crop irrigation, and human consumption. The need for water monitoring becomes more pronounced when industrial, agricultural, and residential lands are found in close proximity. Fecal contamination is particularly problematic and identification of the pollution source essential to remediation efforts. Standard monitoring for fecal contamination relies on indicator organisms, but the technique is too broad to identify the source of contamination. Instead, real-time PCR of mitochondrial DNA (mtDNA) is an emerging method for identification of the contamination source. Presented herein, we evaluate an alternative technology, the compact Bead Array Sensor System (cBASS®) and its assay approach Fluidic Force Discrimination (FFD), for the detection of mtDNA. Previously, we achieved multiplexed, attomolar detection of toxins and femtomolar detection of nucleic acids in minutes with FFD assays. More importantly, FFD assays are compatible with a variety of complex matrices and therefore potentially applicable for samples where the matrix would interfere with PCR amplification. We have designed a triplex assay for the NADH gene found in human, swine, and bovine mtDNA and demonstrated the specific detection of human mtDNA spiked into a waste water sample.

  17. FBIS: A regional DNA barcode archival & analysis system for Indian fishes

    Science.gov (United States)

    Nagpure, Naresh Sahebrao; Rashid, Iliyas; Pathak, Ajey Kumar; Singh, Mahender; Singh, Shri Prakash; Sarkar, Uttam Kumar

    2012-01-01

    DNA barcode is a new tool for taxon recognition and classification of biological organisms based on sequence of a fragment of mitochondrial gene, cytochrome c oxidase I (COI). In view of the growing importance of the fish DNA barcoding for species identification, molecular taxonomy and fish diversity conservation, we developed a Fish Barcode Information System (FBIS) for Indian fishes, which will serve as a regional DNA barcode archival and analysis system. The database presently contains 2334 sequence records of COI gene for 472 aquatic species belonging to 39 orders and 136 families, collected from available published data sources. Additionally, it contains information on phenotype, distribution and IUCN Red List status of fishes. The web version of FBIS was designed using MySQL, Perl and PHP under Linux operating platform to (a) store and manage the acquisition (b) analyze and explore DNA barcode records (c) identify species and estimate genetic divergence. FBIS has also been integrated with appropriate tools for retrieving and viewing information about the database statistics and taxonomy. It is expected that FBIS would be useful as a potent information system in fish molecular taxonomy, phylogeny and genomics. Availability The database is available for free at http://mail.nbfgr.res.in/fbis/ PMID:22715304

  18. A Novel Image Encryption Algorithm Based on a Fractional-Order Hyperchaotic System and DNA Computing

    Directory of Open Access Journals (Sweden)

    Taiyong Li

    2017-01-01

    Full Text Available In the era of the Internet, image encryption plays an important role in information security. Chaotic systems and DNA operations have been proven to be powerful for image encryption. To further enhance the security of image, in this paper, we propose a novel algorithm that combines the fractional-order hyperchaotic Lorenz system and DNA computing (FOHCLDNA for image encryption. Specifically, the algorithm consists of four parts: firstly, we use a fractional-order hyperchaotic Lorenz system to generate a pseudorandom sequence that will be utilized during the whole encryption process; secondly, a simple but effective diffusion scheme is performed to spread the little change in one pixel to all the other pixels; thirdly, the plain image is encoded by DNA rules and corresponding DNA operations are performed; finally, global permutation and 2D and 3D permutation are performed on pixels, bits, and acid bases. The extensive experimental results on eight publicly available testing images demonstrate that the encryption algorithm can achieve state-of-the-art performance in terms of security and robustness when compared with some existing methods, showing that the FOHCLDNA is promising for image encryption.

  19. The effects of exercise training and caloric restriction on the cardiac oxytocin natriuretic peptide system in the diabetic mouse

    Directory of Open Access Journals (Sweden)

    Broderick TL

    2017-01-01

    Full Text Available Tom L Broderick,1 Marek Jankowski,2 Jolanta Gutkowska2 1Department of Physiology, Laboratory of Diabetes and Exercise Metabolism, Midwestern University, Glendale, AZ, USA; 2Department of Medicine, Laboratory of Cardiovascular Biochemistry, Centre Hospitalier de l‘Université de Montréal-Hôtel-Dieu, Montréal, QC, Canada Background: Regular exercise training (ET and caloric restriction (CR are the frontline strategies in the treatment of type 2 diabetes mellitus with the aim at reducing cardiometabolic risk. ET and CR improve body weight and glycemic control, and experimental studies indicate that these paradigms afford cardioprotection. In this study, the effects of combined ET and CR on the cardioprotective oxytocin (OT–natriuretic peptide (NP system were determined in the db/db mouse, a model of type 2 diabetes associated with insulin resistance, hyperglycemia, and obesity. Methods: Five-week-old male db/db mice were assigned to the following groups: sedentary, ET, and ET + CR. Nonobese heterozygote littermates served as controls. ET was performed on a treadmill at moderate intensity, and CR was induced by reducing food intake by 30% of that consumed by sedentary db/db mice for a period of 8 weeks. Results: After 8 weeks, only ET + CR, but not ET, slightly improved body weight compared to sedentary db/db mice. Regardless of the treatment, db/db mice remained hyperglycemic. Hearts from db/db mice demonstrated reduced expression of genes linked to the cardiac OT–NP system. In fact, compared to control mice, mRNA expression of GATA binding protein 4 (GATA4, OT receptor, OT, brain NP, NP receptor type C, and endothelial nitric oxide synthase (eNOS was decreased in hearts from sedentary db/db mice. Both ET alone and ET + CR increased the mRNA expression of GATA4 compared to sedentary db/db mice. Only ET combined with CR produced increased eNOS mRNA and protein expression. Conclusion: Our data indicate that enhancement of eNOS by combined

  20. Localization of radiolabeled anti-DNA monoclonal antibodies in murine systemic lupus erythematosus (SLE)

    International Nuclear Information System (INIS)

    Wahl, R.; Hahn, B.; Ebling, F.

    1984-01-01

    The diagnosis of SLE can be extremely difficult. This multi-system disease is characterized by the deposition of DNA-anti-DNA antibody (Ab) complexes in many tissues, producing glomerulonephritis and systemic vasculitis. This study evaluates an IGG monoclonal (Mo) Ab directe3d against DNA (MrSSl) for potential radioimmunodiagnosis of SLE. Six 15 wk. old F-1 female hybrids of NZB+NZW mice (an animal SLE model that develops vasculitis and nephritis) were injected with 50 μCl of I-131 MrSSl and 15 μCl of I-125 isotype-matched control mouse myeloma (LPC-1) (non-reactive with DNA). Imaging and tissue distribution were studied. Two animals were also imaged using I-131 LPC Ab. Images at 2 and 9 days showed no clear differences in scan patterns using MrSSl or LPC-1 Ab. Tissue distribution studies at six days, however, showed a significantly higher accumulation of MrSSl in the kidneys vs. control Ab (2.7% vs. 1.8% of injected dose) (p < .04). Similarly, higher levels of MrSS were also seen in the spleen, liver and lungs (p < .03). Blood levels tended to be higher with the specific antibody as well. These differences were not apparent at 3 days post injection. The increased concentration of MrSSl present at 9 days in several organs may be secondary to MrSSl binding to DNA containing immune complexes present in diseased tissues. Blocked clearance by immune complexes or DNA, or differences in electrical charges of the antibodies could be contributing to the higher MrSSl levels seen. Images did not suggest deiodination as responsible. Further studies are necessary to determine if the amount of MrSSl retained by diseased animals is indicative of SLE disease activity

  1. Pitfalls of establishing DNA barcoding systems in protists: the cryptophyceae as a test case.

    Science.gov (United States)

    Hoef-Emden, Kerstin

    2012-01-01

    A DNA barcode is a preferrably short and highly variable region of DNA supposed to facilitate a rapid identification of species. In many protistan lineages, a lack of species-specific morphological characters hampers an identification of species by light or electron microscopy, and difficulties to perform mating experiments in laboratory cultures also do not allow for an identification of biological species. Thus, testing candidate barcode markers as well as establishment of accurately working species identification systems are more challenging than in multicellular organisms. In cryptic species complexes the performance of a potential barcode marker can not be monitored using morphological characters as a feedback, but an inappropriate choice of DNA region may result in artifactual species trees for several reasons. Therefore a priori knowledge of the systematics of a group is required. In addition to identification of known species, methods for an automatic delimitation of species with DNA barcodes have been proposed. The Cryptophyceae provide a mixture of systematically well characterized as well as badly characterized groups and are used in this study to test the suitability of some of the methods for protists. As species identification method the performance of blast in searches against badly to well-sampled reference databases has been tested with COI-5P and 5'-partial LSU rDNA (domains A to D of the nuclear LSU rRNA gene). In addition the performance of two different methods for automatic species delimitation, fixed thresholds of genetic divergence and the general mixed Yule-coalescent model (GMYC), have been examined. The study demonstrates some pitfalls of barcoding methods that have to be taken care of. Also a best-practice approach towards establishing a DNA barcode system in protists is proposed.

  2. Pitfalls of establishing DNA barcoding systems in protists: the cryptophyceae as a test case.

    Directory of Open Access Journals (Sweden)

    Kerstin Hoef-Emden

    Full Text Available A DNA barcode is a preferrably short and highly variable region of DNA supposed to facilitate a rapid identification of species. In many protistan lineages, a lack of species-specific morphological characters hampers an identification of species by light or electron microscopy, and difficulties to perform mating experiments in laboratory cultures also do not allow for an identification of biological species. Thus, testing candidate barcode markers as well as establishment of accurately working species identification systems are more challenging than in multicellular organisms. In cryptic species complexes the performance of a potential barcode marker can not be monitored using morphological characters as a feedback, but an inappropriate choice of DNA region may result in artifactual species trees for several reasons. Therefore a priori knowledge of the systematics of a group is required. In addition to identification of known species, methods for an automatic delimitation of species with DNA barcodes have been proposed. The Cryptophyceae provide a mixture of systematically well characterized as well as badly characterized groups and are used in this study to test the suitability of some of the methods for protists. As species identification method the performance of blast in searches against badly to well-sampled reference databases has been tested with COI-5P and 5'-partial LSU rDNA (domains A to D of the nuclear LSU rRNA gene. In addition the performance of two different methods for automatic species delimitation, fixed thresholds of genetic divergence and the general mixed Yule-coalescent model (GMYC, have been examined. The study demonstrates some pitfalls of barcoding methods that have to be taken care of. Also a best-practice approach towards establishing a DNA barcode system in protists is proposed.

  3. Pitfalls of Establishing DNA Barcoding Systems in Protists: The Cryptophyceae as a Test Case

    Science.gov (United States)

    Hoef-Emden, Kerstin

    2012-01-01

    A DNA barcode is a preferrably short and highly variable region of DNA supposed to facilitate a rapid identification of species. In many protistan lineages, a lack of species-specific morphological characters hampers an identification of species by light or electron microscopy, and difficulties to perform mating experiments in laboratory cultures also do not allow for an identification of biological species. Thus, testing candidate barcode markers as well as establishment of accurately working species identification systems are more challenging than in multicellular organisms. In cryptic species complexes the performance of a potential barcode marker can not be monitored using morphological characters as a feedback, but an inappropriate choice of DNA region may result in artifactual species trees for several reasons. Therefore a priori knowledge of the systematics of a group is required. In addition to identification of known species, methods for an automatic delimitation of species with DNA barcodes have been proposed. The Cryptophyceae provide a mixture of systematically well characterized as well as badly characterized groups and are used in this study to test the suitability of some of the methods for protists. As species identification method the performance of blast in searches against badly to well-sampled reference databases has been tested with COI-5P and 5′-partial LSU rDNA (domains A to D of the nuclear LSU rRNA gene). In addition the performance of two different methods for automatic species delimitation, fixed thresholds of genetic divergence and the general mixed Yule-coalescent model (GMYC), have been examined. The study demonstrates some pitfalls of barcoding methods that have to be taken care of. Also a best-practice approach towards establishing a DNA barcode system in protists is proposed. PMID:22970104

  4. Molecular motion in restricted geometries

    Indian Academy of Sciences (India)

    Molecular dynamics in restricted geometries is known to exhibit anomalous behaviour. Diffusion, translational or rotational, of molecules is altered significantly on confinement in restricted geometries. Quasielastic neutron scattering (QENS) offers a unique possibility of studying molecular motion in such systems. Both time ...

  5. Integrated DNA walking system to characterize a broad spectrum of GMOs in food/feed matrices.

    Science.gov (United States)

    Fraiture, Marie-Alice; Herman, Philippe; Lefèvre, Loic; Taverniers, Isabel; De Loose, Marc; Deforce, Dieter; Roosens, Nancy H

    2015-08-14

    In order to provide a system fully integrated with qPCR screening, usually used in GMO routine analysis, as well as being able to detect, characterize and identify a broad spectrum of GMOs in food/feed matrices, two bidirectional DNA walking methods targeting p35S or tNOS, the most common transgenic elements found in GM crops, were developed. These newly developed DNA walking methods are completing the previously implemented DNA walking method targeting the t35S pCAMBIA element. Food/feed matrices containing transgenic crops (Bt rice or MON863 maize) were analysed using the integrated DNA walking system. First, the newly developed DNA walking methods, anchored on the sequences used for the p35S or tNOS qPCR screening, were tested on Bt rice that contains these two transgenic elements. Second, the methods were assessed on a maize sample containing a low amount of the GM MON863 event, representing a more complex matrix in terms of genome size and sensitivity. Finally, to illustrate its applicability in GMO routine analysis by enforcement laboratories, the entire workflow of the integrated strategy, including qPCR screening to detect the potential presence of GMOs and the subsequent DNA walking methods to characterize and identify the detected GMOs, was applied on a GeMMA Scheme Proficiency Test matrix. Via the characterization of the transgene flanking region between the transgenic cassette and the plant genome as well as of a part of the transgenic cassette, the presence of GMOs was properly confirmed or infirmed in all tested samples. Due to their simple procedure and their short time-frame to get results, the developed DNA walking methods proposed here can be easily implemented in GMO routine analysis by the enforcement laboratories. In providing crucial information about the transgene flanking regions and/or the transgenic cassettes, this DNA walking strategy is a key molecular tool to prove the presence of GMOs in any given food/feed matrix.

  6. Prototype Systems Containing Human Cytochrome P450 for High-Throughput Real-Time Detection of DNA Damage by Compounds That Form DNA-Reactive Metabolites.

    Science.gov (United States)

    Brito Palma, Bernardo; Fisher, Charles W; Rueff, José; Kranendonk, Michel

    2016-05-16

    The formation of reactive metabolites through biotransformation is the suspected cause of many adverse drug reactions. Testing for the propensity of a drug to form reactive metabolites has increasingly become an integral part of lead-optimization strategy in drug discovery. DNA reactivity is one undesirable facet of a drug or its metabolites and can lead to increased risk of cancer and reproductive toxicity. Many drugs are metabolized by cytochromes P450 in the liver and other tissues, and these reactions can generate hard electrophiles. These hard electrophilic reactive metabolites may react with DNA and may be detected in standard in vitro genotoxicity assays; however, the majority of these assays fall short due to the use of animal-derived organ extracts that inadequately represent human metabolism. The current study describes the development of bacterial systems that efficiently detect DNA-damaging electrophilic reactive metabolites generated by human P450 biotransformation. These assays use a GFP reporter system that detects DNA damage through induction of the SOS response and a GFP reporter to control for cytotoxicity. Two human CYP1A2-competent prototypes presented here have appropriate characteristics for the detection of DNA-damaging reactive metabolites in a high-throughput manner. The advantages of this approach include a short assay time (120-180 min) with real-time measurement, sensitivity to small amounts of compound, and adaptability to a microplate format. These systems are suitable for high-throughput assays and can serve as prototypes for the development of future enhanced versions.

  7. Impact of DNA mismatch repair system alterations on human fertility and related treatments.

    Science.gov (United States)

    Hu, Min-hao; Liu, Shu-yuan; Wang, Ning; Wu, Yan; Jin, Fan

    2016-01-01

    DNA mismatch repair (MMR) is one of the biological pathways, which plays a critical role in DNA homeostasis, primarily by repairing base-pair mismatches and insertion/deletion loops that occur during DNA replication. MMR also takes part in other metabolic pathways and regulates cell cycle arrest. Defects in MMR are associated with genomic instability, predisposition to certain types of cancers and resistance to certain therapeutic drugs. Moreover, genetic and epigenetic alterations in the MMR system demonstrate a significant relationship with human fertility and related treatments, which helps us to understand the etiology and susceptibility of human infertility. Alterations in the MMR system may also influence the health of offspring conceived by assisted reproductive technology in humans. However, further studies are needed to explore the specific mechanisms by which the MMR system may affect human infertility. This review addresses the physiological mechanisms of the MMR system and associations between alterations of the MMR system and human fertility and related treatments, and potential effects on the next generation.

  8. The Microbial DNA Index System (MiDIS): A tool for microbial pathogen source identification

    Energy Technology Data Exchange (ETDEWEB)

    Velsko, S. P. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

    2010-08-09

    The microbial DNA Index System (MiDIS) is a concept for a microbial forensic database and investigative decision support system that can be used to help investigators identify the sources of microbial agents that have been used in a criminal or terrorist incident. The heart of the proposed system is a rigorous method for calculating source probabilities by using certain fundamental sampling distributions associated with the propagation and mutation of microbes on disease transmission networks. This formalism has a close relationship to mitochondrial and Y-chromosomal human DNA forensics, and the proposed decision support system is somewhat analogous to the CODIS and SWGDAM mtDNA databases. The MiDIS concept does not involve the use of opportunistic collections of microbial isolates and phylogenetic tree building as a basis for inference. A staged approach can be used to build MiDIS as an enduring capability, beginning with a pilot demonstration program that must meet user expectations for performance and validation before evolving into a continuing effort. Because MiDIS requires input from a a broad array of expertise including outbreak surveillance, field microbial isolate collection, microbial genome sequencing, disease transmission networks, and laboratory mutation rate studies, it will be necessary to assemble a national multi-laboratory team to develop such a system. The MiDIS effort would lend direction and focus to the national microbial genetics research program for microbial forensics, and would provide an appropriate forensic framework for interfacing to future national and international disease surveillance efforts.

  9. From head to heart; : the effects of fetal growth restriction and preterm birth on the cerebral and systemic circulation

    NARCIS (Netherlands)

    Cohen, Emily

    2017-01-01

    Fetal growth restriction (FGR) is the condition where a fetus does not grow according to its genetic growth potential. It is estimated that 3-7% of pregnancies are complicated by FGR. FGR has been associated with many adverse outcomes, including an increased risk of perinatal and neonatal morbidity

  10. Developing a Bacteroides System for Function-Based Screening of DNA from the Human Gut Microbiome.

    Science.gov (United States)

    Lam, Kathy N; Martens, Eric C; Charles, Trevor C

    2018-01-01

    Functional metagenomics is a powerful method that allows the isolation of genes whose role may not have been predicted from DNA sequence. In this approach, first, environmental DNA is cloned to generate metagenomic libraries that are maintained in Escherichia coli, and second, the cloned DNA is screened for activities of interest. Typically, functional screens are carried out using E. coli as a surrogate host, although there likely exist barriers to gene expression, such as lack of recognition of native promoters. Here, we describe efforts to develop Bacteroides thetaiotaomicron as a surrogate host for screening metagenomic DNA from the human gut. We construct a B. thetaiotaomicron-compatible fosmid cloning vector, generate a fosmid clone library using DNA from the human gut, and show successful functional complementation of a B. thetaiotaomicron glycan utilization mutant. Though we were unable to retrieve the physical fosmid after complementation, we used genome sequencing to identify the complementing genes derived from the human gut microbiome. Our results demonstrate that the use of B. thetaiotaomicron to express metagenomic DNA is promising, but they also exemplify the challenges that can be encountered in the development of new surrogate hosts for functional screening. IMPORTANCE Human gut microbiome research has been supported by advances in DNA sequencing that make it possible to obtain gigabases of sequence data from metagenomes but is limited by a lack of knowledge of gene function that leads to incomplete annotation of these data sets. There is a need for the development of methods that can provide experimental data regarding microbial gene function. Functional metagenomics is one such method, but functional screens are often carried out using hosts that may not be able to express the bulk of the environmental DNA being screened. We expand the range of current screening hosts and demonstrate that human gut-derived metagenomic libraries can be

  11. A novel Listeria monocytogenes-based DNA delivery system for cancer gene therapy.

    LENUS (Irish Health Repository)

    van Pijkeren, Jan Peter

    2012-01-31

    Bacteria-mediated transfer of plasmid DNA to mammalian cells (bactofection) has been shown to have significant potential as an approach to express heterologous proteins in various cell types. This is achieved through entry of the entire bacterium into cells, followed by release of plasmid DNA. In a murine model, we show that Listeria monocytogenes can invade and spread in tumors, and establish the use of Listeria to deliver genes to tumors in vivo. A novel approach to vector lysis and release of plasmid DNA through antibiotic administration was developed. Ampicillin administration facilitated both plasmid transfer and safety control of vector. To further improve on the gene delivery system, we selected a Listeria monocytogenes derivative that is more sensitive to ampicillin, and less pathogenic than the wild-type strain. Incorporation of a eukaryotic-transcribed lysin cassette in the plasmid further increased bacterial lysis. Successful gene delivery of firefly luciferase to growing tumors in murine models and to patient breast tumor samples ex vivo was achieved. The model described encompasses a three-phase treatment regimen, involving (1) intratumoral administration of vector followed by a period of vector spread, (2) systemic ampicillin administration to induce vector lysis and plasmid transfer, and (3) systemic administration of combined moxifloxacin and ampicillin to eliminate systemic vector. For the first time, our results reveal the potential of Listeria monocytogenes for in vivo gene delivery.

  12. A portable pulmonary delivery system for nano engineered DNA vaccines driven by surface acoustic wave devices

    International Nuclear Information System (INIS)

    Rajapaksa, A.E.; Qi, Aisha; Yeo, L.; Friend, J.

    2010-01-01

    Full text: The increase in the need for effective delivery of potelll vaccines against infectious diseases, require robust yet straightforward pro duction of encapsulated DNA-laden aerosols. Aerosol delivery of drugs represents the next generation of vaccine delivery where the drug is deposited into the lung, which provides an ideal, non-invasive route. Moreover, several features of D A vaccines make them more attractive than conventional vaccines; thus, DNA vaccines have gained global interest for a variety of applications. However, several limitations such as ineffective cellular uptake and intracellular delivery, and degradation of DNA need to be overcome before clin ical applications. In this study, a novel and scalable engineered technique has been developed to create a biodegradable polymer system, which enables controlled delivery of a well designed DNA vaccine for immuno-therapeutics. Surface Acoustic Wave (SAW) atomisation has been found as useful mechanism for atomising fluid samples for medical and industrial devices. It is a straightforward method for synthesising un-agglomerated biodegradable nanoparti cles (<250 nm) in the absence of organic solvents which would represent a major breakthrough for biopharmaceutical encapsulation and delivery. Nano-scale polymer particles for DNA vaccines deliv ery were obtained through an evaporative process of the initial aerosol created by surface acoustic waves at 8-150 MHz, the final size of which could be controlled by modifying the initial polymer concen tration and solid contents. Thus, SAW atomiser represents a promising alternative for the development of a low power device for producing nano-engineered vaccines with a controlled and narrow size distribution as delivery system for genetic immuno-therapeutics.

  13. Mechanism for iron control of the Vibrio fischeri luminescence system: involvement of cyclic AMP and cyclic AMP receptor protein and modulation of DNA level.

    Science.gov (United States)

    Dunlap, P V

    1992-07-01

    Iron controls luminescence in Vibrio fischeri by an indirect but undefined mechanism. To gain insight into that mechanism, the involvement of cyclic AMP (cAMP) and cAMP receptor protein (CRP) and of modulation of DNA levels in iron control of luminescence were examined in V. fischeri and in Escherichia coli containing the cloned V. fischeri lux genes on plasmids. For V. fischeri and E. coli adenylate cyclase (cya) and CRP (crp) mutants containing intact lux genes (luxR luxICDABEG), presence of the iron chelator ethylenediamine-di(o-hydroxyphenyl acetic acid) (EDDHA) increased expression of the luminescence system like in the parent strains only in the cya mutants in the presence of added cAMP. In the E. coli strains containing a plasmid with a Mu dl(lacZ) fusion in luxR, levels of beta-galactosidase activity (expression from the luxR promoter) and luciferase activity (expression from the lux operon promoter) were both 2-3-fold higher in the presence of EDDHA in the parent strain, and for the mutants this response to EDDHA was observed only in the cya mutant in the presence of added cAMP. Therefore, cAMP and CRP are required for the iron restriction effect on luminescence, and their involvement in iron control apparently is distinct from the known differential control of transcription from the luxR and luxICDABEG promoters by cAMP-CRP. Furthermore, plasmid and chromosomal DNA levels were higher in E. coli and V. fischeri in the presence of EDDHA. The higher DNA levels correlated with an increase in expression of chromosomally encoded beta-galactosidase in E. coli and with a higher level of autoinducer in cultures of V. fischeri. These results implicate cAMP-CRP and modulation of DNA levels in the mechanism of iron control of the V. fischeri luminescence system.

  14. DataDotDNA: an alternative marking system for tortoises of genus Testudo

    Directory of Open Access Journals (Sweden)

    Luca Brugnola

    2013-12-01

    Full Text Available It was analyzed the effectiveness of one method of individual and unique marking, alternative to the application of microchip, on 17 specimens of Testudo hermanni through DataDotDNA technology. This technology has proven to be an effective system of marking of Testudo spp., answering the need for unambiguous identification of individuals. Further advantages are the easy application and reading, the long-term resistance as well as the difficulties of possible fraudulent tampering.

  15. NmeSI restriction-modification system identified by representational difference analysis of a hypervirulent Neisseria meningitidis strain

    NARCIS (Netherlands)

    Bart, A.; Pannekoek, Y.; Dankert, J.; van der Ende, A.

    2001-01-01

    Neisseria meningitidis is a gram-negative bacterium that may cause meningitis, sepsis, or both. The increase in the incidence of meningococcal disease in various countries in the past 2 decades is mainly due the genotypically related lineage III meningococci. The chromosomal DNA differences between

  16. Computer-assisted design for scaling up systems based on DNA reaction networks.

    Science.gov (United States)

    Aubert, Nathanaël; Mosca, Clément; Fujii, Teruo; Hagiya, Masami; Rondelez, Yannick

    2014-04-06

    In the past few years, there have been many exciting advances in the field of molecular programming, reaching a point where implementation of non-trivial systems, such as neural networks or switchable bistable networks, is a reality. Such systems require nonlinearity, be it through signal amplification, digitalization or the generation of autonomous dynamics such as oscillations. The biochemistry of DNA systems provides such mechanisms, but assembling them in a constructive manner is still a difficult and sometimes counterintuitive process. Moreover, realistic prediction of the actual evolution of concentrations over time requires a number of side reactions, such as leaks, cross-talks or competitive interactions, to be taken into account. In this case, the design of a system targeting a given function takes much trial and error before the correct architecture can be found. To speed up this process, we have created DNA Artificial Circuits Computer-Assisted Design (DACCAD), a computer-assisted design software that supports the construction of systems for the DNA toolbox. DACCAD is ultimately aimed to design actual in vitro implementations, which is made possible by building on the experimental knowledge available on the DNA toolbox. We illustrate its effectiveness by designing various systems, from Montagne et al.'s Oligator or Padirac et al.'s bistable system to new and complex networks, including a two-bit counter or a frequency divider as well as an example of very large system encoding the game Mastermind. In the process, we highlight a variety of behaviours, such as enzymatic saturation and load effect, which would be hard to handle or even predict with a simpler model. We also show that those mechanisms, while generally seen as detrimental, can be used in a positive way, as functional part of a design. Additionally, the number of parameters included in these simulations can be large, especially in the case of complex systems. For this reason, we included the

  17. Intermolecular G-quadruplex structure-based fluorescent DNA detection system.

    Science.gov (United States)

    Zhou, Hui; Wu, Zai-Sheng; Shen, Guo-Li; Yu, Ru-Qin

    2013-03-15

    Adopting multi-donors to pair with one acceptor could improve the performance of fluorogenic detection probes. However, common dyes (e.g., fluorescein) in close proximity to each other would self-quench the fluorescence, and the fluorescence is difficult to restore. In this contribution, we constructed a novel "multi-donors-to-one acceptor" fluorescent DNA detection system by means of the intermolecular G-quadruplex (IGQ) structure-based fluorescence signal enhancement combined with the hairpin oligonucleotide. The novel IGQ-hairpin system was characterized using the p53 gene as the model target DNA. The proposed system showed an improved assay performance due to the introduction of IGQ-structure into fluorescent signaling probes, which could inhibit the background fluorescence and increase fluorescence restoration amplitude of fluoresceins upon target DNA hybridization. The proof-of-concept scheme is expected to provide new insight into the potential of G-quadruplex structure and promote the application of fluorescent oligonucleotide probes in fundamental research, diagnosis, and treatment of genetic diseases. Copyright © 2012 Elsevier B.V. All rights reserved.

  18. The HIrisPlex-S system for eye, hair and skin colour prediction from DNA: Introduction and forensic developmental validation.

    Science.gov (United States)

    Chaitanya, Lakshmi; Breslin, Krystal; Zuñiga, Sofia; Wirken, Laura; Pośpiech, Ewelina; Kukla-Bartoszek, Magdalena; Sijen, Titia; Knijff, Peter de; Liu, Fan; Branicki, Wojciech; Kayser, Manfred; Walsh, Susan

    2018-07-01

    Forensic DNA Phenotyping (FDP), i.e. the prediction of human externally visible traits from DNA, has become a fast growing subfield within forensic genetics due to the intelligence information it can provide from DNA traces. FDP outcomes can help focus police investigations in search of unknown perpetrators, who are generally unidentifiable with standard DNA profiling. Therefore, we previously developed and forensically validated the IrisPlex DNA test system for eye colour prediction and the HIrisPlex system for combined eye and hair colour prediction from DNA traces. Here we introduce and forensically validate the HIrisPlex-S DNA test system (S for skin) for the simultaneous prediction of eye, hair, and skin colour from trace DNA. This FDP system consists of two SNaPshot-based multiplex assays targeting a total of 41 SNPs via a novel multiplex assay for 17 skin colour predictive SNPs and the previous HIrisPlex assay for 24 eye and hair colour predictive SNPs, 19 of which also contribute to skin colour prediction. The HIrisPlex-S system further comprises three statistical prediction models, the previously developed IrisPlex model for eye colour prediction based on 6 SNPs, the previous HIrisPlex model for hair colour prediction based on 22 SNPs, and the recently introduced HIrisPlex-S model for skin colour prediction based on 36 SNPs. In the forensic developmental validation testing, the novel 17-plex assay performed in full agreement with the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines, as previously shown for the 24-plex assay. Sensitivity testing of the 17-plex assay revealed complete SNP profiles from as little as 63 pg of input DNA, equalling the previously demonstrated sensitivity threshold of the 24-plex HIrisPlex assay. Testing of simulated forensic casework samples such as blood, semen, saliva stains, of inhibited DNA samples, of low quantity touch (trace) DNA samples, and of artificially degraded DNA samples as well as

  19. Design of a Clinical Information Management System to Support DNA Analysis Laboratory Operation

    Science.gov (United States)

    Dubay, Christopher J.; Zimmerman, David; Popovich, Bradley

    1995-01-01

    The LabDirector system has been developed at the Oregon Health Sciences University to support the operation of our clinical DNA analysis laboratory. Through an iterative design process which has spanned two years, we have produced a system that is both highly tailored to a clinical genetics production laboratory and flexible in its implementation, to support the rapid growth and change of protocols and methodologies in use in the field. The administrative aspects of the system are integrated with an enterprise schedule management system. The laboratory side of the system is driven by a protocol modeling and execution system. The close integration between these two aspects of the clinical laboratory facilitates smooth operations, and allows management to accurately measure costs and performance. The entire application has been designed and documented to provide utility to a wide range of clinical laboratory environments.

  20. Comparison of QIAsymphony Automated and QIAamp Manual DNA Extraction Systems for Measuring Epstein-Barr Virus DNA Load in Whole Blood Using Real-Time PCR

    OpenAIRE

    Laus, Stella; Kingsley, Lawrence A.; Green, Michael; Wadowsky, Robert M.

    2011-01-01

    Automated and manual extraction systems have been used with real-time PCR for quantification of Epstein-Barr virus [human herpesvirus 4 (HHV-4)] DNA in whole blood, but few studies have evaluated relative performances. In the present study, the automated QIAsymphony and manual QIAamp extraction systems (Qiagen, Valencia, CA) were assessed using paired aliquots derived from clinical whole-blood specimens and an in-house, real-time PCR assay. The detection limits using the QIAsymphony and QIAam...

  1. Enhancing Targeted Genomic DNA Editing in Chicken Cells Using the CRISPR/Cas9 System

    Science.gov (United States)

    Wang, Ling; Yang, Likai; Guo, Yijie; Du, Weili; Yin, Yajun; Zhang, Tao; Lu, Hongzhao

    2017-01-01

    The CRISPR/Cas9 system has enabled highly efficient genome targeted editing for various organisms. However, few studies have focused on CRISPR/Cas9 nuclease-mediated chicken genome editing compared with mammalian genomes. The current study combined CRISPR with yeast Rad52 (yRad52) to enhance targeted genomic DNA editing in chicken DF-1 cells. The efficiency of CRISPR/Cas9 nuclease-induced targeted mutations in the chicken genome was increased to 41.9% via the enrichment of the dual-reporter surrogate system. In addition, the combined effect of CRISPR nuclease and yRad52 dramatically increased the efficiency of the targeted substitution in the myostatin gene using 50-mer oligodeoxynucleotides (ssODN) as the donor DNA, resulting in a 36.7% editing efficiency after puromycin selection. Furthermore, based on the effect of yRad52, the frequency of exogenous gene integration in the chicken genome was more than 3-fold higher than that without yRad52. Collectively, these results suggest that ssODN is an ideal donor DNA for targeted substitution and that CRISPR/Cas9 combined with yRad52 significantly enhances chicken genome editing. These findings could be extensively applied in other organisms. PMID:28068387

  2. Non-electrostatic complexes with DNA: towards novel synthetic gene delivery systems.

    Science.gov (United States)

    Soto, J; Bessodes, M; Pitard, B; Mailhe, P; Scherman, D; Byk, G

    2000-05-01

    We have developed new DNA complexing amphiphile based on Hoechst 33258 interaction with DNA grooves. The synthesis and physicochemical characterisation of lipid/DNA complexes, as compared to that of classical lipopolyamine for gene delivery, are described and discussed.

  3. An image encryption scheme based on the MLNCML system using DNA sequences

    Science.gov (United States)

    Zhang, Ying-Qian; Wang, Xing-Yuan; Liu, Jia; Chi, Ze-Lin

    2016-07-01

    We propose a new image scheme based on the spatiotemporal chaos of the Mixed Linear-Nonlinear Coupled Map Lattices (MLNCML). This spatiotemporal chaotic system has more cryptographic features in dynamics than the system of Coupled Map Lattices (CML). In the proposed scheme, we employ the strategy of DNA computing and one time pad encryption policy, which can enhance the sensitivity to the plaintext and resist differential attack, brute-force attack, statistical attack and plaintext attack. Simulation results and theoretical analysis indicate that the proposed scheme has superior high security.

  4. Enzyme-free colorimetric detection systems based on the DNA strand displacement competition reaction

    DEFF Research Database (Denmark)

    Zhang, Zhao; Birkedal, Victoria; Gothelf, Kurt Vesterager

    2016-01-01

    The strand displacement competition assay is based on the dynamic equilibrium of the competitive hybridization of two oligonucleotides (A and B) to a third oligonucleotide (S). In the presence of an analyte that binds to a specific affinity-moiety conjugated to strand B, the equilibrium shifts, w...... G-quadruplex DNAzyme for colorimetric readout of the detection of streptavidin by the naked eye. Finally, we integrate the whole G-quadruplex DNAzyme system in a single DNA strand and show that it is applicable to colorimetric detection......., which can be detected by a shift in the fluorescence resonance energy transfer signal between dyes attached to the DNA strands. In the present study we have integrated an ATP aptamer in the strand B and demonstrated the optical detection of ATP. Furthermore we explore a new readout method using a split...

  5. Enzyme-free colorimetric detection systems based on the DNA strand displacement competition reaction

    Science.gov (United States)

    Zhang, Z.; Birkedal, V.; Gothelf, K. V.

    2016-05-01

    The strand displacement competition assay is based on the dynamic equilibrium of the competitive hybridization of two oligonucleotides (A and B) to a third oligonucleotide (S). In the presence of an analyte that binds to a specific affinity-moiety conjugated to strand B, the equilibrium shifts, which can be detected by a shift in the fluorescence resonance energy transfer signal between dyes attached to the DNA strands. In the present study we have integrated an ATP aptamer in the strand B and demonstrated the optical detection of ATP. Furthermore we explore a new readout method using a split G-quadruplex DNAzyme for colorimetric readout of the detection of streptavidin by the naked eye. Finally, we integrate the whole G-quadruplex DNAzyme system in a single DNA strand and show that it is applicable to colorimetric detection.

  6. Dna fingerprinting - review paper

    OpenAIRE

    Blundell, Renald

    2006-01-01

    Before the Polymerase Chain Reaction (PCR) was established, DNA fingerprinting technology has relied for years on Restriction Fragment Length Polymorphism (RFLP) and Variable Number of Tandom Repeats (VNTR) analysis, a very efficient technique but quite laborious and not suitable for high throughput mapping. Since its, development, PCR has provided a new and powerful tool for DNA fingerprinting.

  7. Reversible thermal transition in GrpE, the nucleotide exchange factor of the DnaK heat-shock system.

    Science.gov (United States)

    Grimshaw, J P; Jelesarov, I; Schönfeld, H J; Christen, P

    2001-03-02

    DnaK, a Hsp70 acting in concert with its co-chaperones DnaJ and GrpE, is essential for Escherichia coli to survive environmental stress, including exposure to elevated temperatures. Here we explored the influence of temperature on the structure of the individual components and the functional properties of the chaperone system. GrpE undergoes extensive but fully reversible conformational changes in the physiologically relevant temperature range (transition midpoint at approximately 48 degrees C), as observed with both circular dichroism measurements and differential scanning calorimetry, whereas no thermal transitions occur in DnaK and DnaJ between 15 degrees C and 48 degrees C. The conformational changes in GrpE appear to be important in controlling the interconversion of T-state DnaK (ATP-liganded, low affinity for polypeptide substrates) and R-state DnaK (ADP-liganded, high affinity for polypeptide substrates). The rate of the T --> R conversion of DnaK due to DnaJ-triggered ATP hydrolysis follows an Arrhenius temperature dependence. In contrast, the rate of the R --> T conversion due to GrpE-catalyzed ADP/ATP exchange increases progressively less with increasing temperature and even decreases at temperatures above approximately 40 degrees C, indicating a temperature-dependent reversible inactivation of GrpE. At heat-shock temperatures, the reversible structural changes of GrpE thus shift DnaK toward its high-affinity R state.

  8. Performance Characteristics of Different Anti-Double-Stranded DNA Antibody Assays in the Monitoring of Systemic Lupus Erythematosus

    Directory of Open Access Journals (Sweden)

    Michael Mahler

    2017-01-01

    Full Text Available Objective. We sought to evaluate different anti-double-stranded DNA assays for their performance characteristics in monitoring disease activity fluctuations in systemic lupus erythematosus (SLE. Methods. 36 active SLE patients were followed monthly. At each study visit (total n=371, blood was collected and disease activity was scored using the SELENA-SLEDAI (excluding anti-dsDNA or complement components and by a physician’s global assessment (PGA. Four anti-dsDNA tests were compared. Linear mixed-effects models with random intercept and fixed slopes were used to evaluate the relationship between the longitudinal fluctuations of disease activity and anti-dsDNA titers. Results. At enrollment, positivity for QUANTA Lite and high-avidity anti-dsDNA assay was both 64% and significantly lower than anti-dsDNA positivity by QUANTA Flash (83% and CLIFT (96%. Linear mixed-effects modeling indicated that the change in clinical SELENA-SLEDAI scores was associated with the titers of all anti-dsDNA with QUANTA Flash yielding the highest marginal R2 (0.15; p<0.01. QUANTA Flash was the only anti-dsDNA assay significantly associated with the change in PGA (marginal R2=0.05; p<0.01. Conclusion. These data indicate that anti-dsDNA antibodies determined by QUANTA Flash have a value in monitoring SLE disease activity.

  9. An att site-based recombination reporter system for genome engineering and synthetic DNA assembly.

    Science.gov (United States)

    Bland, Michael J; Ducos-Galand, Magaly; Val, Marie-Eve; Mazel, Didier

    2017-07-14

    Direct manipulation of the genome is a widespread technique for genetic studies and synthetic biology applications. The tyrosine and serine site-specific recombination systems of bacteriophages HK022 and ΦC31 are widely used for stable directional exchange and relocation of DNA sequences, making them valuable tools in these contexts. We have developed site-specific recombination tools that allow the direct selection of recombination events by embedding the attB site from each system within the β-lactamase resistance coding sequence (bla). The HK and ΦC31 tools were developed by placing the attB sites from each system into the signal peptide cleavage site coding sequence of bla. All possible open reading frames (ORFs) were inserted and tested for recombination efficiency and bla activity. Efficient recombination was observed for all tested ORFs (3 for HK, 6 for ΦC31) as shown through a cointegrate formation assay. The bla gene with the embedded attB site was functional for eight of the nine constructs tested. The HK/ΦC31 att-bla system offers a simple way to directly select recombination events, thus enhancing the use of site-specific recombination systems for carrying out precise, large-scale DNA manipulation, and adding useful tools to the genetics toolbox. We further show the power and flexibility of bla to be used as a reporter for recombination.

  10. Planning model for hydraulic and thermic generation systems expansion under uncertainly conditions and financial restrictions; Modelo de planejamento da expansao de sistemas hidrotermicos sob incertezas e restricoes financeiras

    Energy Technology Data Exchange (ETDEWEB)

    Gorenstin, B.G.; Costa, J.P. da [CEPEL, Rio de Janeiro, RJ (Brazil); Pereira, M.V.F.; Campodonico, N.M.

    1993-12-31

    This issue presents a methodology for planning the systems expansion of hydraulic and thermic power generation associated considering several uncertainly factors, such as: demand growing, fuel costs, delays on the work construction, financial restrictions, etc. The solution focus is based on stock optimize techniques, decision analysis. This work is being developed by the Brazilian Electrical Centre (ELETROBRAS) and rely on the Energy Latin-American Organization (OLADE), Development Inter-American Bank (BID), World Bank (BIRD) and International Energy Agency (IAEA) support. An example case with Costa Rica system is also discussed 19 refs., 4 figs., 2 tabs.

  11. Polarized DNA Ejection from the Herpesvirus Capsid

    Science.gov (United States)

    Newcomb, William W.; Cockrell, Shelley K.; Homa, Fred L.; Brown, Jay C.

    2009-01-01

    Ejection of DNA from the capsid is an early step in infection by all herpesviruses. Ejection or DNA uncoating occurs after a parental capsid has entered the host cell cytoplasm, migrated to the nucleus and bound to a nuclear pore. DNA exits the capsid through the portal vertex and proceeds by way of the nuclear pore complex into the nucleoplasm where it is transcribed and replicated. Here we describe use of an in vitro uncoating system to determine which genome end exits first from the herpes simplex virus (HSV-1) capsid. Purified DNA-containing capsids were bound to a solid surface and warmed under conditions in which some, but not all, of the DNA was ejected. Restriction endonuclease digestion was then used to identify the genomic origin of the ejected DNA. The results support the view that the S segment end exits the capsid first. Preferential release at the S end demonstrates that herpesvirus DNA uncoating conforms to the paradigm in dsDNA bacteriophage where the last end packaged is the first to be ejected. Release of HSV-1 DNA beginning at the S end causes the first gene to enter the host cell nucleus to be α4, a transcription factor required for expression of early genes. PMID:19631662

  12. Ter-dependent stress response systems: novel pathways related to metal sensing, production of a nucleoside-like metabolite, and DNA-processing.

    Science.gov (United States)

    Anantharaman, Vivek; Iyer, Lakshminarayan M; Aravind, L

    2012-10-30

    , and are likely to comprise membrane-associated sensory complexes that might additionally contain periplasmic binding-protein-II and OmpA domains. We also show that the TerD and TerB domains and the TerY-associated phosphorylation system are functionally linked to many distinct DNA-processing complexes, which feature proteins with SWI2/SNF2 and RecQ-like helicases, multiple AAA+ ATPases, McrC-N-terminal domain proteins, several restriction endonuclease fold DNases, DNA-binding domains and a type-VII/Esx-like system, which is at the center of a predicted DNA transfer apparatus. These DNA-processing modules and associated genes are predicted to be involved in restriction or suicidal action in response to phages and possibly repairing xenobiotic-induced DNA damage. In some eukaryotes, certain components of the ter system appear to be recruited to function in conjunction with the ubiquitin system and calcium-signaling pathways.

  13. Molecular Genetic Characterization of Mutagenesis Using a Highly Sensitive Single-Stranded DNA Reporter System in Budding Yeast.

    Science.gov (United States)

    Chan, Kin

    2018-01-01

    Mutations are permanent alterations to the coding content of DNA. They are starting material for the Darwinian evolution of species by natural selection, which has yielded an amazing diversity of life on Earth. Mutations can also be the fundamental basis of serious human maladies, most notably cancers. In this chapter, I describe a highly sensitive reporter system for the molecular genetic analysis of mutagenesis, featuring controlled generation of long stretches of single-stranded DNA in budding yeast cells. This system is ~100- to ~1000-fold more susceptible to mutation than conventional double-stranded DNA reporters, and is well suited for generating large mutational datasets to investigate the properties of mutagens.

  14. 49 CFR 383.95 - Restrictions.

    Science.gov (United States)

    2010-10-01

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

  15. Systems Biology of Saccharomyces cerevisiae Physiology and its DNA Damage Response

    DEFF Research Database (Denmark)

    Fazio, Alessandro

    The yeast Saccharomyces cerevisiae is a model organism in biology, being widely used in fundamental research, the first eukaryotic organism to be fully sequenced and the platform for the development of many genomics techniques. Therefore, it is not surprising that S. cerevisiae has also been widely...... used in the field of systems biology during the last decade. This thesis investigates S. cerevisiae growth physiology and DNA damage response by using a systems biology approach. Elucidation of the relationship between growth rate and gene expression is important to understand the mechanisms regulating...... set of growth dependent genes by using a multi-factorial experimental design. Moreover, new insights into the metabolic response and transcriptional regulation of these genes have been provided by using systems biology tools (Chapter 3). One of the prerequisite of systems biology should...

  16. Integration of the Reconfigurable Self-Healing eDNA Architecture in an Embedded System

    DEFF Research Database (Denmark)

    Boesen, Michael Reibel; Keymeulen, Didier; Madsen, Jan

    2011-01-01

    In this work we describe the first real world case study for the self-healing eDNA (electronic DNA) architecture by implementing the control and data processing of a Fourier Transform Spectrometer (FTS) on an eDNA prototype. For this purpose the eDNA prototype has been ported from a Xilinx Virtex 5...

  17. Effects of Dietary Sodium Restriction in Kidney Transplant Recipients Treated With Renin-Angiotensin-Aldosterone System Blockade: A Randomized Clinical Trial.

    Science.gov (United States)

    de Vries, Laura V; Dobrowolski, Linn C; van den Bosch, Jacqueline J O N; Riphagen, Ineke J; Krediet, C T Paul; Bemelman, Frederike J; Bakker, Stephan J L; Navis, Gerjan

    2016-06-01

    In patients with chronic kidney disease receiving renin-angiotensin-aldosterone system (RAAS) blockade, dietary sodium restriction is an often-used treatment strategy to reduce blood pressure (BP) and albuminuria. Whether these effects extend to kidney transplant recipients is unknown. We therefore studied the effects of dietary sodium restriction on BP and urinary albumin excretion (UAE) in kidney transplant recipients receiving RAAS blockade. Two-center randomized crossover trial. Stable outpatient kidney transplant recipients with creatinine clearance > 30mL/min, BP ≥120/80mmHg, receiving stable RAAS blockade therapy. 6-week regular-sodium diet (target, 150mmol/24 h) and a 6-week low-sodium diet (target, 50mmol/24 h). Main outcome parameters were systolic and diastolic BP, UAE, and estimated glomerular filtration rate (eGFR) at the end of each diet period. Dietary adherence was assessed by 24-hour urinary sodium excretion. We randomly assigned 23 kidney transplant recipients, of whom 22 (mean age, 58±8 [SD] years; 50% men; mean eGFR, 51±21mL/min/1.73m(2)) completed the study. One patient withdrew from the study because of concerns regarding orthostatic hypotension on the low-sodium diet. Sodium excretion decreased from 164±50mmol/24 h during the regular-sodium diet to 87±55mmol/24 h during the low-sodium diet (mean difference, -77 [95% CI, -110 to -44] mmol/24 h; Padherence to sodium diet was achieved in 86% of patients. In stable kidney transplant recipients receiving RAAS blockade, dietary sodium restriction effectively reduces BP without affecting eGFR. Dietary sodium restriction is relevant to BP management in kidney transplant recipients receiving RAAS blockade. Copyright © 2016 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.

  18. CCR6+ Th cell distribution differentiates systemic lupus erythematosus patients based on anti-dsDNA antibody status.

    Science.gov (United States)

    Zhong, Wei; Jiang, Zhenyu; Wu, Jiang; Jiang, Yanfang; Zhao, Ling

    2018-01-01

    Systemic lupus erythematosus (SLE) disease has been shown to be associated with the generation of multiple auto-antibodies. Among these, anti-dsDNA antibodies (anti-DNAs) are specific and play a pathogenic role in SLE. Indeed, anti-DNA + SLE patients display a worse disease course. The generation of these pathogenic anti-DNAs has been attributed to the interaction between aberrant T helper (Th) cells and autoimmune B cells. Thus, in this study we have investigated whether CCR6 + Th cells have the ability to differentiate SLE patients based on anti-DNA status, and if their distribution has any correlation with disease activity. We recruited 25 anti-DNA + and 25 anti-DNA - treatment-naive onset SLE patients, matched for various clinical characteristics in our nested matched case-control study. CCR6 + Th cells and their additional subsets were analyzed in each patient by flow cytometry. Anti-DNA + SLE patients specifically had a higher percentage of Th cells expressing CCR6 and CXCR3. Further analysis of CCR6 + Th cell subsets showed that anti-DNA + SLE patients had elevated proportions of Th9, Th17, Th17.1 and CCR4/CXCR3 double-negative (DN) cells. However, the proportions of CCR6 - Th subsets, including Th1 and Th2 cells, did not show any association with anti-DNA status. Finally, we identified a correlation between CCR6 + Th subsets and clinical indicators, specifically in anti-DNA + SLE patients. Our data indicated that CCR6 + Th cells and their subsets were elevated and correlated with disease activity in anti-DNA + SLE patients. We speculated that CCR6 + Th cells may contribute to distinct disease severity in anti-DNA + SLE patients.

  19. An ultra-high discrimination Y chromosome short tandem repeat multiplex DNA typing system.

    Directory of Open Access Journals (Sweden)

    Erin K Hanson

    Full Text Available In forensic casework, Y chromosome short tandem repeat markers (Y-STRs are often used to identify a male donor DNA profile in the presence of excess quantities of female DNA, such as is found in many sexual assault investigations. Commercially available Y-STR multiplexes incorporating 12-17 loci are currently used in forensic casework (Promega's PowerPlex Y and Applied Biosystems' AmpFlSTR Yfiler. Despite the robustness of these commercial multiplex Y-STR systems and the ability to discriminate two male individuals in most cases, the coincidence match probabilities between unrelated males are modest compared with the standard set of autosomal STR markers. Hence there is still a need to develop new multiplex systems to supplement these for those cases where additional discriminatory power is desired or where there is a coincidental Y-STR match between potential male participants. Over 400 Y-STR loci have been identified on the Y chromosome. While these have the potential to increase the discrimination potential afforded by the commercially available kits, many have not been well characterized. In the present work, 91 loci were tested for their relative ability to increase the discrimination potential of the commonly used 'core' Y-STR loci. The result of this extensive evaluation was the development of an ultra high discrimination (UHD multiplex DNA typing system that allows for the robust co-amplification of 14 non-core Y-STR loci. Population studies with a mixed African American and American Caucasian sample set (n = 572 indicated that the overall discriminatory potential of the UHD multiplex was superior to all commercial kits tested. The combined use of the UHD multiplex and the Applied Biosystems' AmpFlSTR Yfiler kit resulted in 100% discrimination of all individuals within the sample set, which presages its potential to maximally augment currently available forensic casework markers. It could also find applications in human evolutionary

  20. A chronic increase of corticosterone age-dependently reduces systemic DNA damage from oxidation in rats

    DEFF Research Database (Denmark)

    Jorgensen, Anders; Kalliokoski, Otto; Forsberg, Kristin

    2017-01-01

    Stress and depression are associated with an acceleration of brain and bodily aging; effects which have been attributed to chronic elevations of glucocorticoids. We tested the hypothesis that a three week administration of stress-associated levels of corticosterone (CORT, the principal rodent...... glucocorticoid) would increase systemic and CNS DNA and RNA damage from oxidation; a phenomenon known to be centrally involved in the aging process. We also hypothesized that older individuals would be more sensitive to this effect and that the chronic CORT administration would exacerbate age-related memory...

  1. DNA Delivery and Genomic Integration into Mammalian Target Cells through Type IV A and B Secretion Systems of Human Pathogens

    Directory of Open Access Journals (Sweden)

    Dolores L. Guzmán-Herrador

    2017-08-01

    Full Text Available We explore the potential of bacterial secretion systems as tools for genomic modification of human cells. We previously showed that foreign DNA can be introduced into human cells through the Type IV A secretion system of the human pathogen Bartonella henselae. Moreover, the DNA is delivered covalently attached to the conjugative relaxase TrwC, which promotes its integration into the recipient genome. In this work, we report that this tool can be adapted to other target cells by using different relaxases and secretion systems. The promiscuous relaxase MobA from plasmid RSF1010 can be used to deliver DNA into human cells with higher efficiency than TrwC. MobA also promotes DNA integration, albeit at lower rates than TrwC. Notably, we report that DNA transfer to human cells can also take place through the Type IV secretion system of two intracellular human pathogens, Legionella pneumophila and Coxiella burnetii, which code for a distantly related Dot/Icm Type IV B secretion system. This suggests that DNA transfer could be an intrinsic ability of this family of secretion systems, expanding the range of target human cells. Further analysis of the DNA transfer process showed that recruitment of MobA by Dot/Icm was dependent on the IcmSW chaperone, which may explain the higher DNA transfer rates obtained. Finally, we observed that the presence of MobA negatively affected the intracellular replication of C. burnetii, suggesting an interference with Dot/Icm translocation of virulence factors.

  2. Light emitting diode, photodiode-based fluorescence detection system for DNA analysis with microchip electrophoresis.

    Science.gov (United States)

    Hall, Gordon H; Glerum, D Moira; Backhouse, Christopher J

    2016-02-01

    Electrophoretic separation of fluorescently end-labeled DNA after a PCR serves as a gold standard in genetic diagnostics. Because of their size and cost, instruments for this type of analysis have had limited market uptake, particularly for point-of-care applications. This might be changed through a higher level of system integration and lower instrument costs that can be realized through the use of LEDs for excitation and photodiodes for detection--if they provide sufficient sensitivity. Here, we demonstrate an optimized microchip electrophoresis instrument using polymeric fluidic chips with fluorescence detection of end-labeled DNA with a LOD of 0.15 nM of Alexa Fluor 532. This represents orders of magnitude improvement over previously reported instruments of this type. We demonstrate the system with an electrophoretic separation of two PCR products and their respective primers. We believe that this is the first LED-induced fluorescence microchip electrophoresis system with photodiode-based detection that could be used for standard applications of PCR and electrophoresis. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Growth of the parvovirus minute virus of mice MVMp3 in EL4 lymphocytes is restricted after cell entry and before viral DNA amplification: cell-specific differences in virus uncoating in vitro.

    Science.gov (United States)

    Previsani, N; Fontana, S; Hirt, B; Beard, P

    1997-10-01

    Two murine parvoviruses with genomic sequences differing only in 33 nucleotides (8 amino acids) in the region coding for the capsid proteins show different host cell specificities: MVMi grows in EL4 T lymphocytes and MVMp3 grows in A9 fibroblasts. In this study we compared the courses of infections with these two viruses in EL4 cells in order to investigate at which step(s) the infection process of MVMp3 is interrupted. The two viruses bound equally well to EL4 cells, and similar amounts of MVMi and MVMp3 input virion DNA appeared in the nuclear fractions of EL4 cells 1 h after infection. However, double-stranded replicative-form (RF) DNA of the two viruses appeared at different times, at 10 h postinfection with MVMi and at 24 h postinfection with MVMp3. The amount of MVMp3 RF DNA detected at 24 h was very small because it was produced only in a tiny subset of the population of EL4 cells that proved to be permissive for MVMp3. Replication of double-stranded viral DNA in EL4 cells was measured after transfection of purified RF DNA, cloned viral DNA, and cloned viral DNA with a mutation preventing synthesis of the capsid proteins. In each of these cases, DNA replication was comparable for MVMi and MVMp3. Production of virus particles also appeared to be similar after transfection of the two types of RF DNA into EL4 cells. Conversion of incoming 32P-labeled single-stranded MVM DNA to 32P-labeled double-stranded RF DNA was detected only after RF DNA amplification, indicating that few molecules serve as templates for viral DNA amplification. We showed that extracts of EL4 cells contain a factor which can destabilize MVMi virions but not MVMp3 by testing the sensitivity of viral DNA to DNase and by CsCl gradient analyses of viral particles. We therefore conclude that the MVMp3 life cycle is arrested after the transport of virions to the nucleus and prior to the replication of RF DNA, most likely at the stage of viral decapsidation.

  4. Flow-Induced Dispersion Analysis for Probing Anti-dsDNA Antibody Binding Heterogeneity in Systemic Lupus Erythematosus Patients

    DEFF Research Database (Denmark)

    Poulsen, Nicklas N; Pedersen, Morten E; Østergaard, Jesper

    2016-01-01

    Detection of immune responses is important in the diagnosis of many diseases. For example, the detection of circulating autoantibodies against double-stranded DNA (dsDNA) is used in the diagnosis of Systemic Lupus Erythematosus (SLE). It is, however, difficult to reach satisfactory sensitivity......, the patient antibodies bound DNA sequences with different affinities, suggesting pronounced heterogeneity among autoantibodies produced in SLE. The FIDA based methodology is a new approach for autoantibody detection and holds promise for being used for patient stratification and monitoring of disease activity....

  5. GENOMIC DNA-FINGERPRINTING OF CLINICAL HAEMOPHILUS-INFLUENZAE ISOLATES BY POLYMERASE CHAIN-REACTION AMPLIFICATION - COMPARISON WITH MAJOR OUTER-MEMBRANE PROTEIN AND RESTRICTION-FRAGMENT-LENGTH-POLYMORPHISM ANALYSIS

    NARCIS (Netherlands)

    VANBELKUM, A; DUIM, B; REGELINK, A; MOLLER, L; QUINT, W; VANALPHEN, L

    Non-capsulate strains of Haemophilus influenzae were genotyped by analysis of variable DNA segments obtained by amplification of genomic DNA with the polymerase chain reaction (PCR fingerprinting). Discrete fragments of 100-2000 bp were obtained. The reproducibility of the procedure was assessed by

  6. Synthesis of a multi-functional DNA nanosphere barcode system for direct cell detection.

    Science.gov (United States)

    Han, Sangwoo; Lee, Jae Sung; Lee, Jong Bum

    2017-09-28

    Nucleic acid-based technologies have been applied to numerous biomedical applications. As a novel material for target detection, DNA has been used to construct a barcode system with a range of structures. This paper reports multi-functionalized DNA nanospheres (DNANSs) by rolling circle amplification (RCA) with several functionalized nucleotides. DNANSs with a barcode system were designed to exhibit fluorescence for coding enhanced signals and contain biotin for more functionalities, including targeting through the biotin-streptavidin (biotin-STA) interaction. Functionalized deoxynucleotide triphosphates (dNTPs) were mixed in the RCA process and functional moieties can be expressed on the DNANSs. The anti-epidermal growth factor receptor antibodies (anti-EGFR Abs) can be conjugated on DNANSs for targeting cancer cells specifically. As a proof of concept, the potential of the multi-functional DNANS barcode was demonstrated by direct cell detection as a simple detection method. The DNANS barcode provides a new route for the simple and rapid selective recognition of cancer cells.

  7. The Chern-Simons Current in Systems of DNA-RNA Transcriptions

    Science.gov (United States)

    Capozziello, Salvatore; Pincak, Richard; Kanjamapornkul, Kabin; Saridakis, Emmanuel N.

    2018-04-01

    A Chern-Simons current, coming from ghost and anti-ghost fields of supersymmetry theory, can be used to define a spectrum of gene expression in new time series data where a spinor field, as alternative representation of a gene, is adopted instead of using the standard alphabet sequence of bases $A, T, C, G, U$. After a general discussion on the use of supersymmetry in biological systems, we give examples of the use of supersymmetry for living organism, discuss the codon and anti-codon ghost fields and develop an algebraic construction for the trash DNA, the DNA area which does not seem active in biological systems. As a general result, all hidden states of codon can be computed by Chern-Simons 3 forms. Finally, we plot a time series of genetic variations of viral glycoprotein gene and host T-cell receptor gene by using a gene tensor correlation network related to the Chern-Simons current. An empirical analysis of genetic shift, in host cell receptor genes with separated cluster of gene and genetic drift in viral gene, is obtained by using a tensor correlation plot over time series data derived as the empirical mode decomposition of Chern-Simons current.

  8. Simple system for isothermal DNA amplification coupled to lateral flow detection.

    Directory of Open Access Journals (Sweden)

    Kristina Roskos

    Full Text Available Infectious disease diagnosis in point-of-care settings can be greatly improved through integrated, automated nucleic acid testing devices. We have developed an early prototype for a low-cost system which executes isothermal DNA amplification coupled to nucleic acid lateral flow (NALF detection in a mesofluidic cartridge attached to a portable instrument. Fluid handling inside the cartridge is facilitated through one-way passive valves, flexible pouches, and electrolysis-driven pumps, which promotes a compact and inexpensive instrument design. The closed-system disposable prevents workspace amplicon contamination. The cartridge design is based on standard scalable manufacturing techniques such as injection molding. Nucleic acid amplification occurs in a two-layer pouch that enables efficient heat transfer. We have demonstrated as proof of principle the amplification and detection of Mycobacterium tuberculosis (M.tb genomic DNA in the cartridge, using either Loop Mediated Amplification (LAMP or the Exponential Amplification Reaction (EXPAR, both coupled to NALF detection. We envision that a refined version of this cartridge, including upstream sample preparation coupled to amplification and detection, will enable fully-automated sample-in to answer-out infectious disease diagnosis in primary care settings of low-resource countries with high disease burden.

  9. Cell-based DNA demethylation detection system for screening of epigenetic drugs in 2D, 3D, and xenograft models

    Czech Academy of Sciences Publication Activity Database

    Agrawal, K.; Das, V.; Otmar, Miroslav; Krečmerová, Marcela; Džubák, P.; Hajdúch, M.

    91A, č. 2 (2017), s. 133-143 ISSN 1552-4922 R&D Projects: GA MZd(CZ) NV15-31984A; GA MŠk(CZ) LO1304; GA MŠk(CZ) LM2015064; GA TA ČR(CZ) TE01020028 Institutional support: RVO:61388963 Keywords : DNA methylation * DNA methylation inhibitors * demethylation detection system * epigenetic drugs * high content screening Subject RIV: CC - Organic Chemistry OBOR OECD: Organic chemistry Impact factor: 3.222, year: 2016

  10. Design and construction of a DNA origami drug delivery system based on MPT64 antibody aptamer for tuberculosis treatment.

    Science.gov (United States)

    Ranjbar, Reza; Hafezi-Moghadam, Mohammad Sadegh

    2016-02-01

    With all of the developments on infectious diseases, tuberculosis (TB) remains a cause of death among people. One of the most promising assembly techniques in nano-technology is "scaffolded DNA origami" to design and construct a nano-scale drug delivery system. Because of the global health problems of tuberculosis, the development of potent new anti-tuberculosis drug delivery system without cross-resistance with known anti-mycobacterial agents is urgently needed. The aim of this study was to design a nano-scale drug delivery system for TB treatment using the DNA origami method. In this study, we presented an experimental research on a DNA drug delivery system for treating Tuberculosis. TEM images were visualized with an FEI Tecnai T12 BioTWIN at 120 kV. The model was designed by caDNAno software and computational prediction of the 3D solution shape and its flexibility was calculated with a CanDo server. Synthesizing the product was imaged using transmission electron microscopy after negative-staining by uranyl formate. We constructed a multilayer 3D DNA nanostructure system by designing square lattice geometry with the scaffolded-DNA-origami method. With changes in the lock and key sequences, we recommend that this system be used for other infectious diseases to target the pathogenic bacteria.

  11. 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

  12. 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.

  13. Efficient systemic DNA delivery to the tumor by self-assembled nanoparticle

    Science.gov (United States)

    Tang, Hailin; Xie, Xinhua; Guo, Jiaoli; Wei, Weidong; Wu, Minqing; Liu, Peng; Kong, Yanan; Yang, Lu; Hung, Mien-Chie; Xie, Xiaoming

    2014-01-01

    There are few delivery agents that could deliver gene with high efficiency and low toxicity, especially for animal experiments. Therefore, creating vectors with good delivery efficiency and safety profile is a meaningful work. We have developed a self-assembled gene delivery system (XM001), which can more efficiently deliver DNA to multiple cell lines and breast tumor, as compared to commercial delivery agents. In addition, systemically administrated XM001-BikDD (BikDD is a mutant form of proapoptotic gene Bik) significantly inhibited the growth of human breast cancer cells and prolonged the life span in implanted nude mice. This study demonstrates that XM001 is an efficient and widespread transfection agent, which could be a promising tumor delivery vector for cancer targeted therapy.

  14. Present status of DNA repair mechanisms in uv irradiated yeast taken as a model eukaryotic system

    International Nuclear Information System (INIS)

    Moustacchi, E.; Waters, R.; Heude, M.; Chanet, R.

    1975-01-01

    The repair mechanisms of altered DNA are generally less well understood for eukaryotes than they are for prokaryotes and bacteriophages. For mammalian cell lines cultured in vitro the specific labelling of DNA has allowed the biochemical analysis of some of the steps of the repair processes whereas the determination of their genetic controls is, with a few exceptions, obviously difficult. On the other hand, with fungi and more specifically with yeast taken as a model unicellular eukaryotic system, the genetic approach has been extensively explored: radiosensitive mutants are readily detected and genetically analyzed, double and multiple mutants can be constructed and from their responses to irradiation the number of repair pathways involved can be suggested. The lack of thymidine kinase in these organisms has hampered for a certain time the biochemical analysis of repair. However, the recent isolation of yeast strains capable of taking up and incorporating thymidine 5'-monophosphate into their DNA opens new possibilities for the future. In spite of this difficulty, attempts to measure the induction and removal of uv-induced pyrimidine dimers were performed by several groups during the last three years. The two main repair pathways described for E. coli, i.e., the excision-resynthesis and post-replicative recombinational repair pathways, do exist in yeast. The existence of the former pathway is supported not only by indirect evidence but also by biochemical analysis. The rad 1 and rad 2 mutants for instance have been shown to be blocked in the excision of uv-induced pyrimidine dimers. Other loci are epistatic to rad 1 and rad 2 (rad 3 , rad 4 ) and are likely to act on this excision pathway. The genetic control of the mitochondrial response to a uv treatment involves nuclear genes and mitochondrial determinants

  15. Simulation model of converging-diverging (CD) nozzle to improve particle delivery system of deoxyribonucleic acid (DNA)

    Science.gov (United States)

    Sumarsono, Danardono A.; Ibrahim, Fera; Santoso, Satria P.; Sari, Gema P.

    2018-02-01

    Gene gun is a mechanical device which has been used to deliver DNA vaccine into the cells and tissues by increasing the uptake of DNA plasmid so it can generate a high immune response with less amount of DNA. Nozzle is an important part of the gene gun which used to accelerate DNA in particle form with a gas flow to reach adequate momentum to enter the epidermis of human skin and elicit immune response. We developed new designs of nozzle for gene gun to make DNA uptake more efficient in vaccination. We used Computational Fluid Dynamics (CFD) by Autodesk® Simulation 2015 to simulate static fluid pressure and velocity contour of supersonic wave and parametric distance to predict the accuracy of the new nozzle. The result showed that the nozzle could create a shockwave at the distance parametric to the object from 4 to 5 cm using fluid pressure varied between 0.8-1.2 MPa. This is indication a possibility that the DNA particle could penetrate under the mammalian skin. For the future research step, this new nozzle model could be considered for development the main component of the DNA delivery system in vaccination in vivo

  16. RNA and DNA Targeting by a Reconstituted Thermus thermophilus Type III-A CRISPR-Cas System.

    Science.gov (United States)

    Liu, Tina Y; Iavarone, Anthony T; Doudna, Jennifer A

    2017-01-01

    CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) systems are RNA-guided adaptive immunity pathways used by bacteria and archaea to defend against phages and plasmids. Type III-A systems use a multisubunit interference complex called Csm, containing Cas proteins and a CRISPR RNA (crRNA) to target cognate nucleic acids. The Csm complex is intriguing in that it mediates RNA-guided targeting of both RNA and transcriptionally active DNA, but the mechanism is not well understood. Here, we overexpressed the five components of the Thermus thermophilus (T. thermophilus) Type III-A Csm complex (TthCsm) with a defined crRNA sequence, and purified intact TthCsm complexes from E. coli cells. The complexes were thermophilic, targeting complementary ssRNA more efficiently at 65°C than at 37°C. Sequence-independent, endonucleolytic cleavage of single-stranded DNA (ssDNA) by TthCsm was triggered by recognition of a complementary ssRNA, and required a lack of complementarity between the first 8 nucleotides (5' tag) of the crRNA and the 3' flanking region of the ssRNA. Mutation of the histidine-aspartate (HD) nuclease domain of the TthCsm subunit, Cas10/Csm1, abolished DNA cleavage. Activation of DNA cleavage was dependent on RNA binding but not cleavage. This leads to a model in which binding of an ssRNA target to the Csm complex would stimulate cleavage of exposed ssDNA in the cell, such as could occur when the RNA polymerase unwinds double-stranded DNA (dsDNA) during transcription. Our findings establish an amenable, thermostable system for more in-depth investigation of the targeting mechanism using structural biology methods, such as cryo-electron microscopy and x-ray crystallography.

  17. RNA and DNA Targeting by a Reconstituted Thermus thermophilus Type III-A CRISPR-Cas System.

    Directory of Open Access Journals (Sweden)

    Tina Y Liu

    Full Text Available CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated systems are RNA-guided adaptive immunity pathways used by bacteria and archaea to defend against phages and plasmids. Type III-A systems use a multisubunit interference complex called Csm, containing Cas proteins and a CRISPR RNA (crRNA to target cognate nucleic acids. The Csm complex is intriguing in that it mediates RNA-guided targeting of both RNA and transcriptionally active DNA, but the mechanism is not well understood. Here, we overexpressed the five components of the Thermus thermophilus (T. thermophilus Type III-A Csm complex (TthCsm with a defined crRNA sequence, and purified intact TthCsm complexes from E. coli cells. The complexes were thermophilic, targeting complementary ssRNA more efficiently at 65°C than at 37°C. Sequence-independent, endonucleolytic cleavage of single-stranded DNA (ssDNA by TthCsm was triggered by recognition of a complementary ssRNA, and required a lack of complementarity between the first 8 nucleotides (5' tag of the crRNA and the 3' flanking region of the ssRNA. Mutation of the histidine-aspartate (HD nuclease domain of the TthCsm subunit, Cas10/Csm1, abolished DNA cleavage. Activation of DNA cleavage was dependent on RNA binding but not cleavage. This leads to a model in which binding of an ssRNA target to the Csm complex would stimulate cleavage of exposed ssDNA in the cell, such as could occur when the RNA polymerase unwinds double-stranded DNA (dsDNA during transcription. Our findings establish an amenable, thermostable system for more in-depth investigation of the targeting mechanism using structural biology methods, such as cryo-electron microscopy and x-ray crystallography.

  18. Biophysics of DNA-Protein Interactions From Single Molecules to Biological Systems

    CERN Document Server

    Williams, Mark C

    2011-01-01

    This book presents a concise overview of current research on the biophysics of DNA-protein interactions. A wide range of new and classical methods are presented by authors investigating physical mechanisms by which proteins interact with DNA. For example, several chapters address the mechanisms by which proteins search for and recognize specific binding sites on DNA, a process critical for cellular function. Single molecule methods such as force spectroscopy as well as fluorescence imaging and tracking are described in these chapters as well as other parts of the book that address the dynamics of protein-DNA interactions. Other important topics include the mechanisms by which proteins engage DNA sequences and/or alter DNA structure. These simple but important model interactions are then placed in the broader biological context with discussion of larger protein-DNA complexes . Topics include replication forks, recombination complexes, DNA repair interactions, and ultimately, methods to understand the chromatin...

  19. A System Architecture for Efficient Transmission of Massive DNA Sequencing Data.

    Science.gov (United States)

    Sağiroğlu, Mahmut Şamİl; Külekcİ, M Oğuzhan

    2017-11-01

    The DNA sequencing data analysis pipelines require significant computational resources. In that sense, cloud computing infrastructures appear as a natural choice for this processing. However, the first practical difficulty in reaching the cloud computing services is the transmission of the massive DNA sequencing data from where they are produced to where they will be processed. The daily practice here begins with compressing the data in FASTQ file format, and then sending these data via fast data transmission protocols. In this study, we address the weaknesses in that daily practice and present a new system architecture that incorporates the computational resources available on the client side while dynamically adapting itself to the available bandwidth. Our proposal considers the real-life scenarios, where the bandwidth of the connection between the parties may fluctuate, and also the computing power on the client side may be of any size ranging from moderate personal computers to powerful workstations. The proposed architecture aims at utilizing both the communication bandwidth and the computing resources for satisfying the ultimate goal of reaching the results as early as possible. We present a prototype implementation of the proposed architecture, and analyze several real-life cases, which provide useful insights for the sequencing centers, especially on deciding when to use a cloud service and in what conditions.

  20. Alginate and DNA Gels Are Suitable Delivery Systems for Diabetic Wound Healing.

    Science.gov (United States)

    Tellechea, Ana; Silva, Eduardo A; Min, Jianghong; Leal, Ermelindo C; Auster, Michael E; Pradhan-Nabzdyk, Leena; Shih, William; Mooney, David J; Veves, Aristidis

    2015-06-01

    Diabetic foot ulcers (DFU) represent a severe health problem and an unmet clinical challenge. In this study, we tested the efficacy of novel biomaterials in improving wound healing in mouse models of diabetes mellitus (DM). The biomaterials are composed of alginate- and deoxyribonucleic acid (DNA)-based gels that allow incorporation of effector cells, such as outgrowth endothelial cells (OEC), and provide sustained release of bioactive factors, such as neuropeptides and growth factors, which have been previously validated in experimental models of DM wound healing or hind limb ischemia. We tested these biomaterials in mice and demonstrate that they are biocompatible and can be injected into the wound margins without major adverse effects. In addition, we show that the combination of OEC and the neuropeptide Substance P has a better healing outcome than the delivery of OEC alone, while subtherapeutic doses of vascular endothelial growth factor (VEGF) are required for the transplanted cells to exert their beneficial effects in wound healing. In summary, alginate and DNA scaffolds could serve as potential delivery systems for the next-generation DFU therapies. © The Author(s) 2015.

  1. Applications of Engineered DNA-Binding Molecules Such as TAL Proteins and the CRISPR/Cas System in Biology Research

    Directory of Open Access Journals (Sweden)

    Toshitsugu Fujita

    2015-09-01

    Full Text Available Engineered DNA-binding molecules such as transcription activator-like effector (TAL or TALE proteins and the clustered regularly interspaced short palindromic repeats (CRISPR and CRISPR-associated proteins (Cas (CRISPR/Cas system have been used extensively for genome editing in cells of various types and species. The sequence-specific DNA-binding activities of these engineered DNA-binding molecules can also be utilized for other purposes, such as transcriptional activation, transcriptional repression, chromatin modification, visualization of genomic regions, and isolation of chromatin in a locus-specific manner. In this review, we describe applications of these engineered DNA-binding molecules for biological purposes other than genome editing.

  2. Ancient DNA

    DEFF Research Database (Denmark)

    Willerslev, Eske; Cooper, Alan

    2004-01-01

    ancient DNA, palaeontology, palaeoecology, archaeology, population genetics, DNA damage and repair......ancient DNA, palaeontology, palaeoecology, archaeology, population genetics, DNA damage and repair...

  3. Evaluation of Refrigerating and Air Conditioning Devices in Energy Cascade Systems under the Restriction of Carbon Dioxide Emissions

    Science.gov (United States)

    Shimazaki, Yoichi; Akisawa, Atsushi; Kashiwagi, Takao

    It is necessary to introduce energy cascade systems into the industrial sector in Japan to reduce carbon dioxide emissions. The aim of this study is to evaluate the refrigerating and air conditioning devices in cases of introducing both energy cascade systems and thermal recycling systems in industries located around urban areas. The authors have developed an energy cascade model based on linear programming so as to minimize the total system costs with carbon taxes. Five cases are investigated. Limitation of carbon dioxide emissions results in the enhancement of heat cascading, where high temperature heat is supplied for process heating while low temperature one is shifted to refrigeration. It was found that increasing the amount of garbage combustor waste heat can reduce electric power for the turbo refrigerator by promoting waste heat driven ammonia absorption refrigerator.

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

    Science.gov (United States)

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

    2009-01-01

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

  5. A Traffic Restriction Scheme for Enhancing Carpooling

    Directory of Open Access Journals (Sweden)

    Dong Ding

    2017-01-01

    Full Text Available For the purpose of alleviating traffic congestion, this paper proposes a scheme to encourage travelers to carpool by traffic restriction. By a variational inequity we describe travelers’ mode (solo driving and carpooling and route choice under user equilibrium principle in the context of fixed demand and detect the performance of a simple network with various restriction links, restriction proportions, and carpooling costs. Then the optimal traffic restriction scheme aiming at minimal total travel cost is designed through a bilevel program and applied to a Sioux Fall network example with genetic algorithm. According to various requirements, optimal restriction regions and proportions for restricted automobiles are captured. From the results it is found that traffic restriction scheme is possible to enhance carpooling and alleviate congestion. However, higher carpooling demand is not always helpful to the whole network. The topology of network, OD demand, and carpooling cost are included in the factors influencing the performance of the traffic system.

  6. Impact of hydroxychloroquine on preterm delivery and intrauterine growth restriction in pregnant women with systemic lupus erythematosus: a descriptive cohort study.

    Science.gov (United States)

    Leroux, M; Desveaux, C; Parcevaux, M; Julliac, B; Gouyon, J B; Dallay, D; Pellegrin, J L; Boukerrou, M; Blanco, P; Lazaro, E

    2015-11-01

    The aim of this study was to evaluate the effect of hydroxychloroquine (HCQ) on fetal preterm delivery and intrauterine growth restriction (IUGR) in a cohort of pregnant women with systemic lupus erythematosus (SLE). Over an 11-year period (January 1, 2001 to December 31, 2011), all women with SLE and admitted to deliver after 22 weeks of gestation to Bordeaux University Hospital (France), were retrospectively enrolled in the present study. The population was then split into two groups based on the treatment they received: HCQ exposed (HCQ+) versus HCQ non-exposed (HCQ-) group. 118 pregnancies were included, 41 in the HCQ+ group and 77 in the HCQ- group. The rate of adverse fetal outcome was significantly lower in the HCQ+ group (p = 0.001), particularly in terms of preterm delivery, 15.8% versus 44.2% (p = 0.006), and IUGR, 10.5% versus 28.6% (p = 0.03). No adverse outcomes were reported in the HCQ+ group. HCQ reduces neonatal morbidity in women with SLE by significantly decreasing the rate of prematurity and intrauterine growth restriction. © The Author(s) 2015.

  7. A Micro Polymerase Chain Reaction Module for Integrated and Portable DNA Analysis Systems

    Directory of Open Access Journals (Sweden)

    Elisa Morganti

    2011-01-01

    Full Text Available This work deals with the design, fabrication, and thermal characterization of a disposable miniaturized Polymerase Chain Reaction (PCR module that will be integrated in a portable and fast DNA analysis system. It is composed of two independent parts: a silicon substrate with embedded heater and thermometers and a PDMS (PolyDiMethylSiloxane chamber reactor as disposable element; the contact between the two parts is assured by a mechanical clamping obtained using a Plastic Leaded Chip Carrier (PLCC. This PLCC is also useful, avoid the PCR mix evaporation during the thermal cycles. Finite Element Analysis was used to evaluate the thermal requirements of the device. The thermal behaviour of the device was characterized revealing that the temperature can be controlled with a precision of ±0.5°C. Different concentrations of carbon nanopowder were mixed to the PDMS curing agent in order to increase the PDMS thermal conductivity and so the temperature control accuracy.

  8. Fluorescence correlation spectroscopy analysis for accurate determination of proportion of doubly labeled DNA in fluorescent DNA pool for quantitative biochemical assays.

    Science.gov (United States)

    Hou, Sen; Sun, Lili; Wieczorek, Stefan A; Kalwarczyk, Tomasz; Kaminski, Tomasz S; Holyst, Robert

    2014-01-15

    Fluorescent double-stranded DNA (dsDNA) molecules labeled at both ends are commonly produced by annealing of complementary single-stranded DNA (ssDNA) molecules, labeled with fluorescent dyes at the same (3' or 5') end. Because the labeling efficiency of ssDNA is smaller than 100%, the resulting dsDNA have two, one or are without a dye. Existing methods are insufficient to measure the percentage of the doubly-labeled dsDNA component in the fluorescent DNA sample and it is even difficult to distinguish the doubly-labeled DNA component from the singly-labeled component. Accurate measurement of the percentage of such doubly labeled dsDNA component is a critical prerequisite for quantitative biochemical measurements, which has puzzled scientists for decades. We established a fluorescence correlation spectroscopy (FCS) system to measure the percentage of doubly labeled dsDNA (PDL) in the total fluorescent dsDNA pool. The method is based on comparative analysis of the given sample and a reference dsDNA sample prepared by adding certain amount of unlabeled ssDNA into the original ssDNA solution. From FCS autocorrelation functions, we obtain the number of fluorescent dsDNA molecules in the focal volume of the confocal microscope and PDL. We also calculate the labeling efficiency of ssDNA. The method requires minimal amount of material. The samples have the concentration of DNA in the nano-molar/L range and the volume of tens of microliters. We verify our method by using restriction enzyme Hind III to cleave the fluorescent dsDNA. The kinetics of the reaction depends strongly on PDL, a critical parameter for quantitative biochemical measurements. Copyright © 2013 Elsevier B.V. All rights reserved.

  9. A Standardized DNA Variant Scoring System for Pathogenicity Assessments in Mendelian Disorders.

    Science.gov (United States)

    Karbassi, Izabela; Maston, Glenn A; Love, Angela; DiVincenzo, Christina; Braastad, Corey D; Elzinga, Christopher D; Bright, Alison R; Previte, Domenic; Zhang, Ke; Rowland, Charles M; McCarthy, Michele; Lapierre, Jennifer L; Dubois, Felicita; Medeiros, Katelyn A; Batish, Sat Dev; Jones, Jeffrey; Liaquat, Khalida; Hoffman, Carol A; Jaremko, Malgorzata; Wang, Zhenyuan; Sun, Weimin; Buller-Burckle, Arlene; Strom, Charles M; Keiles, Steven B; Higgins, Joseph J

    2016-01-01

    We developed a rules-based scoring system to classify DNA variants into five categories including pathogenic, likely pathogenic, variant of uncertain significance (VUS), likely benign, and benign. Over 16,500 pathogenicity assessments on 11,894 variants from 338 genes were analyzed for pathogenicity based on prediction tools, population frequency, co-occurrence, segregation, and functional studies collected from internal and external sources. Scores were calculated by trained scientists using a quantitative framework that assigned differential weighting to these five types of data. We performed descriptive and comparative statistics on the dataset and tested interobserver concordance among the trained scientists. Private variants defined as variants found within single families (n = 5,182), were either VUS (80.5%; n = 4,169) or likely pathogenic (19.5%; n = 1,013). The remaining variants (n = 6,712) were VUS (38.4%; n = 2,577) or likely benign/benign (34.7%; n = 2,327) or likely pathogenic/pathogenic (26.9%, n = 1,808). Exact agreement between the trained scientists on the final variant score was 98.5% [95% confidence interval (CI) (98.0, 98.9)] with an interobserver consistency of 97% [95% CI (91.5, 99.4)]. Variant scores were stable and showed increasing odds of being in agreement with new data when re-evaluated periodically. This carefully curated, standardized variant pathogenicity scoring system provides reliable pathogenicity scores for DNA variants encountered in a clinical laboratory setting. © 2015 The Authors. **Human Mutation published by Wiley Periodicals, Inc.

  10. Evolutionary genomics and HIV restriction factors.

    Science.gov (United States)

    Pyndiah, Nitisha; Telenti, Amalio; Rausell, Antonio

    2015-03-01

    To provide updated insights into innate antiviral immunity and highlight prototypical evolutionary features of well characterized HIV restriction factors. Recently, a new HIV restriction factor, Myxovirus resistance 2, has been discovered and the region/residue responsible for its activity identified using an evolutionary approach. Furthermore, IFI16, an innate immunity protein known to sense several viruses, has been shown to contribute to the defense to HIV-1 by causing cell death upon sensing HIV-1 DNA. Restriction factors against HIV show characteristic signatures of positive selection. Different patterns of accelerated sequence evolution can distinguish antiviral strategies--offense or defence--as well as the level of specificity of the antiviral properties. Sequence analysis of primate orthologs of restriction factors serves to localize functional domains and sites responsible for antiviral action. We use recent discoveries to illustrate how evolutionary genomic analyses help identify new antiviral genes and their mechanisms of action.

  11. Patterns of exchange of forensic DNA data in the European Union through the Pr?m system

    OpenAIRE

    Santos, Filipe; Machado, Helena

    2017-01-01

    Article in press This paper presents a study of the 5-year operation (2011–2015) of the transnational exchange of forensic DNA data between Member States of the European Union (EU) for the purpose of combating cross-border crime and terrorism within the so-called Prüm system. This first systematisation of the full official statistical dataset provides an overall assessment of the match figures and patterns of operation of the Prüm system for DNA exchange. These figures and patterns are ana...

  12. Locality and nonlocality of classical restrictions of quantum spin systems with applications to quantum large deviations and entanglement

    Czech Academy of Sciences Publication Activity Database

    De Roeck, W.; Maes, C.; Netočný, Karel; Schütz, M.

    2015-01-01

    Roč. 56, č. 2 (2015), "023301-1"-"023301-30" ISSN 0022-2488 Institutional support: RVO:68378271 Keywords : quantum systems * quantum large deviations * entanglement * cluster expansions Subject RIV: BE - Theoretical Physics Impact factor: 1.234, year: 2015

  13. An automated quantitative DNA image cytometry system detects abnormal cells in cervical cytology with high sensitivity.

    Science.gov (United States)

    Wong, O G; Ho, M W; Tsun, O K; Ng, A K; Tsui, E Y; Chow, J N; Ip, P P; Cheung, A N

    2018-03-26

    To evaluate the performance of an automated DNA-image-cytometry system as a tool to detect cervical carcinoma. Of 384 liquid-based cervical cytology samples with available biopsy follow-up were analyzed by both the Imager System and a high-risk HPV test (Cobas). The sensitivity and specificity of Imager System for detecting biopsy proven high-grade squamous intraepithelial lesion (HSIL, cervical intraepithelial neoplasia [CIN]2-3) and carcinoma were 89.58% and 56.25%, respectively, compared to 97.22% and 23.33% of HPV test but additional HPV 16/18 genotyping increased the specificity to 69.58%. The sensitivity and specificity of the Imager System for predicting HSIL+ (CIN2-3+) lesions among atypical squamous cells of undetermined significance samples were 80.00% and 70.53%, respectively, compared to 100% and 11.58% of HPV test whilst the HPV 16/18 genotyping increased the specificity to 77.89%. Among atypical squamous cells-cannot exclude HSIL, the sensitivity and specificity of Imager System for predicting HSIL+ (CIN2-3+) lesions upon follow up were 82.86% and 33.33%%, respectively, compared to 97.14% and 4.76% of HPV test and the HPV 16/18 genotyping increased the specificity to 19.05%. Among low-grade squamous intraepithelial lesion cases, the sensitivity and specificity of the Imager System for predicting HSIL+ (CIN2-3+) lesions were 66.67% and 35.71%%, respectively, compared to 66.67% and 29.76% of HPV test while HPV 16/18 genotyping increased the specificity to 79.76%. The overall results of imager and high-risk HPV test agreed in 69.43% (268) of all samples. The automated imager system and HPV 16/18 genotyping can enhance the specificity of detecting HSIL+ (CIN2-3+) lesions. © 2018 John Wiley & Sons Ltd.

  14. Gentile statistics and restricted partitions

    Indian Academy of Sciences (India)

    In a recent paper (Tran et al, Ann. Phys. 311, 204 (2004)), some asymptotic number theoretical results on the partitioning of an integer were derived exploiting its connection to the quantum density of states of a many-particle system. We generalise these results to obtain an asymptotic formula for the restricted or coloured ...

  15. A maize root tip system to study DNA replication programmes in somatic and endocycling nuclei during plant development.

    Science.gov (United States)

    Bass, Hank W; Wear, Emily E; Lee, Tae-Jin; Hoffman, Gregg G; Gumber, Hardeep K; Allen, George C; Thompson, William F; Hanley-Bowdoin, Linda

    2014-06-01

    The progress of nuclear DNA replication is complex in both time and space, and may reflect several levels of chromatin structure and 3-dimensional organization within the nucleus. To understand the relationship between DNA replication and developmental programmes, it is important to examine replication and nuclear substructure in different developmental contexts including natural cell-cycle progressions in situ. Plant meristems offer an ideal opportunity to analyse such processes in the context of normal growth of an organism. Our current understanding of large-scale chromosomal DNA replication has been limited by the lack of appropriate tools to visualize DNA replication with high resolution at defined points within S phase. In this perspective, we discuss a promising new system that can be used to visualize DNA replication in isolated maize (Zea mays L.) root tip nuclei after in planta pulse labelling with the thymidine analogue, 5-ethynyl-2'-deoxyuridine (EdU). Mixed populations of EdU-labelled nuclei are then separated by flow cytometry into sequential stages of S phase and examined directly using 3-dimensional deconvolution microscopy to characterize spatial patterns of plant DNA replication. Combining spatiotemporal analyses with studies of replication and epigenetic inheritance at the molecular level enables an integrated experimental approach to problems of mitotic inheritance and cellular differentiation. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  16. Influence of oxygen on the repair of direct radiation damage to DNA by thiols in model systems

    International Nuclear Information System (INIS)

    Becker, D.; Summerfield, S.; Gillich, S.; Sevilla, M.D.

    1994-01-01

    Here the reactions of thiols with DNA primary radical intermediates formed after γ-irradiation of frozen (77K) anoxic and oxic solutions of DNA/thiol mixtures are investigated. Through analysis of the experimental composite spectra at each annealing temperature, the relative concentrations of individual radicals present are estimated and reaction sequences inferred. In all samples the primary DNA radical anions and cations (DNA · + and DNA · - ) are suggested to be the predominant radicals at low temperatures. In anoxic samples, TH · (5,6-dihydrothym-5-yl radical), RSSR · - and, in glutathione samples, · GSH [γ-glu-NHC(CH 2 SH) CO-gly] radicals are observed as the temperature is increased. The presence of oxygen efficiently suppresses the formation of RSSR · - and · GSH; instead, in oxic samples, O 2 · - , DNAOO · , RSOO · and RSO · are observed at higher temperatures. The photolytic conversion of RSOO · to RSO 2 · is used to verify the presence of RSOO · in γ-irradiated DNA/thiol systems and confirm that the computer analysis employed yields reasonable estimates of the relative DNAOO · and RSOO · concentrations. (Author)

  17. DNA nanotechnology

    Science.gov (United States)

    Seeman, Nadrian C.; Sleiman, Hanadi F.

    2018-01-01

    DNA is the molecule that stores and transmits genetic information in biological systems. The field of DNA nanotechnology takes this molecule out of its biological context and uses its information to assemble structural motifs and then to connect them together. This field has had a remarkable impact on nanoscience and nanotechnology, and has been revolutionary in our ability to control molecular self-assembly. In this Review, we summarize the approaches used to assemble DNA nanostructures and examine their emerging applications in areas such as biophysics, diagnostics, nanoparticle and protein assembly, biomolecule structure determination, drug delivery and synthetic biology. The introduction of orthogonal interactions into DNA nanostructures is discussed, and finally, a perspective on the future directions of this field is presented.

  18. Restrictions and Proportionality

    DEFF Research Database (Denmark)

    Werlauff, Erik

    2009-01-01

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

  19. Comparison of QIAsymphony automated and QIAamp manual DNA extraction systems for measuring Epstein-Barr virus DNA load in whole blood using real-time PCR.

    Science.gov (United States)

    Laus, Stella; Kingsley, Lawrence A; Green, Michael; Wadowsky, Robert M

    2011-11-01

    Automated and manual extraction systems have been used with real-time PCR for quantification of Epstein-Barr virus [human herpesvirus 4 (HHV-4)] DNA in whole blood, but few studies have evaluated relative performances. In the present study, the automated QIAsymphony and manual QIAamp extraction systems (Qiagen, Valencia, CA) were assessed using paired aliquots derived from clinical whole-blood specimens and an in-house, real-time PCR assay. The detection limits using the QIAsymphony and QIAamp systems were similar (270 and 560 copies/mL, respectively). For samples estimated as having ≥10,000 copies/mL, the intrarun and interrun variations were significantly lower using QIAsymphony (10.0% and 6.8%, respectively), compared with QIAamp (18.6% and 15.2%, respectively); for samples having ≤1000 copies/mL, the two variations ranged from 27.9% to 43.9% and were not significantly different between the two systems. Among 68 paired clinical samples, 48 pairs yielded viral loads ≥1000 copies/mL under both extraction systems. Although the logarithmic linear correlation from these positive samples was high (r(2) = 0.957), the values obtained using QIAsymphony were on average 0.2 log copies/mL higher than those obtained using QIAamp. Thus, the QIAsymphony and QIAamp systems provide similar EBV DNA load values in whole blood. Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  20. Real-time DNA Amplification and Detection System Based on a CMOS Image Sensor.

    Science.gov (United States)

    Wang, Tiantian; Devadhasan, Jasmine Pramila; Lee, Do Young; Kim, Sanghyo

    2016-01-01

    In the present study, we developed a polypropylene well-integrated complementary metal oxide semiconductor (CMOS) platform to perform the loop mediated isothermal amplification (LAMP) technique for real-time DNA amplification and detection simultaneously. An amplification-coupled detection system directly measures the photon number changes based on the generation of magnesium pyrophosphate and color changes. The photon number decreases during the amplification process. The CMOS image sensor observes the photons and converts into digital units with the aid of an analog-to-digital converter (ADC). In addition, UV-spectral studies, optical color intensity detection, pH analysis, and electrophoresis detection were carried out to prove the efficiency of the CMOS sensor based the LAMP system. Moreover, Clostridium perfringens was utilized as proof-of-concept detection for the new system. We anticipate that this CMOS image sensor-based LAMP method will enable the creation of cost-effective, label-free, optical, real-time and portable molecular diagnostic devices.

  1. Expression of renin-angiotensin system signalling compounds in maternal protein-restricted rats: effect on renal sodium excretion and blood pressure.

    Science.gov (United States)

    Mesquita, Flávia Fernandes; Gontijo, José Antonio Rocha; Boer, Patrícia Aline

    2010-02-01

    Intrauterine growth restriction due to low maternal dietary protein during pregnancy is associated with retardation of foetal growth, renal alterations and adult hypertension. The renin-angiotensin system (RAS) is a coordinated hormonal cascade in the control of cardiovascular, renal and adrenal function that governs body fluid and electrolyte balance, as well as arterial pressure. In the kidney, all the components of the renin-angiotensin system including angiotensin II type 1 (AT1) and type 2 (AT2) receptors are expressed locally during nephrogenesis. Hence, we investigated whether low protein diet intake during pregnancy altered kidney and adrenal expression of AT1(R) and AT2(R) receptors, their pathways and if the modified expression of the RAS compounds occurs associated with changes in urinary sodium and in arterial blood pressure in sixteen-week-old males' offspring of the underfed group. The pregnancy dams were divided in two groups: with normal protein diet (pups named NP) (17% protein) or low protein diet (pups LP) (6% protein) during all pregnancy. The present data confirm a significant enhancement in arterial pressure in the LP group. Furthermore, the study showed a significantly decreased expression of RAS pathway protein and Ang II receptors in the kidney and an increased expression in the adrenal of LP rats. The detailed immunohistochemical analysis of RAS signalling proteins in the kidney confirm the immunoblotting results for both groups. The present investigation also showed a pronounced decrease in fractional urinary sodium excretion in maternal protein-restricted offspring, compared with the NP age-matched group. This occurred despite unchanged creatinine clearance. The current data led us to hypothesize that foetal undernutrition could be associated with decreased kidney expression of AT(R) resulting in the inability of renal tubules to handle the hydro-electrolyte balance, consequently causing arterial hypertension.

  2. Rolling replication of UV-irradiated duplex DNA in the phi X174 replicative-form----single-strand replication system in vitro

    International Nuclear Information System (INIS)

    Shavitt, O.; Livneh, Z.

    1989-01-01

    Cloning of the phi X174 viral origin of replication into phage M13mp8 produced an M13-phi X174 chimera, the DNA of which directed efficient replicative-form----single-strand rolling replication in vitro. This replication assay was performed with purified phi X174-encoded gene A protein, Escherichia coli rep helicase, single-stranded DNA-binding protein, and DNA polymerase III holoenzyme. The nicking of replicative-form I (RFI) DNA by gene A protein was essentially unaffected by the presence of UV lesions in the DNA. However, unwinding of UV-irradiated DNA by the rep helicase was inhibited twofold as compared with unwinding of the unirradiated substrate. UV irradiation of the substrate DNA caused a strong inhibition in its ability to direct DNA synthesis. However, even DNA preparations that contained as many as 10 photodimers per molecule still supported the synthesis of progeny full-length single-stranded DNA. The appearance of full-length radiolabeled products implied at least two full rounds of replication, since the first round released the unlabeled plus viral strand of the duplex DNA. Pretreatment of the UV-irradiated DNA substrate with purified pyrimidine dimer endonuclease from Micrococcus luteus, which converted photodimer-containing supercoiled RFI DNA into relaxed, nicked RFII DNA and thus prevented its replication, reduced DNA synthesis by 70%. Analysis of radiolabeled replication products by agarose gel electrophoresis followed by autoradiography revealed that this decrease was due to a reduction in the synthesis of progeny full-length single-stranded DNA. This implies that 70 to 80% of the full-length DNA products produced in this system were synthesized on molecules that carried photodimers

  3. Agarose electrophoresis of DNA in discontinuous buffers, using a horizontal slab apparatus and a buffer system with improved properties.

    Science.gov (United States)

    Zsolnai, A; Orbán, L; Chrambach, A

    1993-03-01

    Using a horizontal slab apparatus with a buffer in the reservoirs at the level of the gel ("sea-level electrophoresis"), the retrograde discontinuous buffer system reported by Wiltfang et al. for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of proteins was applied to DNA electrophoresis. This application yielded the advantages of an increased displacement rate of the moving boundary front and a decrease in the concentration of the counterion base in the resolving phase, which yielded reduced relative mobility values at equivalent gel concentrations and practicable low buffer concentrations. The change of relative mobilities (Rf) with a variation of field strength is decreased compared to that of the migration rate in the continuous Tris-boric-acid-EDTA (TBE) buffer and thus the robustness of the system is improved, as well as the efficiency of separation. The system of Wiltfang et al. has in common with previously described discontinuous DNA system, that it is able to stack DNA from dilute samples and is insensitive to sample components with lower net mobilities than DNA, such as acetate. However, the variance of Rf at constant current density in the discontinuous buffer system is not improved over that of the migration rate at constant field strength in the continuous TBE buffer.

  4. Restricting wolves risks escape

    Science.gov (United States)

    Mech, L. David; Ballard, Warren; Bangs, Ed; Ream, Bob

    2010-01-01

    Implementing the proposal set forth by Licht and colleagues (BioScience 60: 147–153) requires restricting wolves to tiny "islands," areas that are magnitudes smaller than the ranges of most wolf populations. Wolves naturally have large ranges; restricting their spatial needs increases the risk of wolves escaping, exacerbating public relations and political and legal problems.

  5. OligArch: A software tool to allow artificially expanded genetic information systems (AEGIS to guide the autonomous self-assembly of long DNA constructs from multiple DNA single strands

    Directory of Open Access Journals (Sweden)

    Kevin M. Bradley

    2014-08-01

    Full Text Available Synthetic biologists wishing to self-assemble large DNA (L-DNA constructs from small DNA fragments made by automated synthesis need fragments that hybridize predictably. Such predictability is difficult to obtain with nucleotides built from just the four standard nucleotides. Natural DNA's peculiar combination of strong and weak G:C and A:T pairs, the context-dependence of the strengths of those pairs, unimolecular strand folding that competes with desired interstrand hybridization, and non-Watson–Crick interactions available to standard DNA, all contribute to this unpredictability. In principle, adding extra nucleotides to the genetic alphabet can improve the predictability and reliability of autonomous DNA self-assembly, simply by increasing the information density of oligonucleotide sequences. These extra nucleotides are now available as parts of artificially expanded genetic information systems (AEGIS, and tools are now available to generate entirely standard DNA from AEGIS DNA during PCR amplification. Here, we describe the OligArch (for "oligonucleotide architecting" software, an application that permits synthetic biologists to engineer optimally self-assembling DNA constructs from both six- and eight-letter AEGIS alphabets. This software has been used to design oligonucleotides that self-assemble to form complete genes from 20 or more single-stranded synthetic oligonucleotides. OligArch is therefore a key element of a scalable and integrated infrastructure for the rapid and designed engineering of biology.

  6. Performance verification and comparison of TianLong automatic hypersensitive hepatitis B virus DNA quantification system with Roche CAP/CTM system.

    Science.gov (United States)

    Li, Ming; Chen, Lin; Liu, Li-Ming; Li, Yong-Li; Li, Bo-An; Li, Bo; Mao, Yuan-Li; Xia, Li-Fang; Wang, Tong; Liu, Ya-Nan; Li, Zheng; Guo, Tong-Sheng

    2017-10-07

    To investigate and compare the analytical and clinical performance of TianLong automatic hypersensitive hepatitis B virus (HBV) DNA quantification system and Roche CAP/CTM system. Two hundred blood samples for HBV DNA testing, HBV-DNA negative samples and high-titer HBV-DNA mixture samples were collected and prepared. National standard materials for serum HBV and a worldwide HBV DNA panel were employed for performance verification. The analytical performance, such as limit of detection, limit of quantification, accuracy, precision, reproducibility, linearity, genotype coverage and cross-contamination, was determined using the TianLong automatic hypersensitive HBV DNA quantification system (TL system). Correlation and Bland-Altman plot analyses were carried out to compare the clinical performance of the TL system assay and the CAP/CTM system. The detection limit of the TL system was 10 IU/mL, and its limit of quantification was 30 IU/mL. The differences between the expected and tested concentrations of the national standards were less than ± 0.4 Log 10 IU/mL, which showed high accuracy of the system. Results of the precision, reproducibility and linearity tests showed that the multiple test coefficient of variation (CV) of the same sample was less than 5% for 10 2 -10 6 IU/mL; and for 30-10 8 IU/mL, the linear correlation coefficient r 2 = 0.99. The TL system detected HBV DNA (A-H) genotypes and there was no cross-contamination during the "checkerboard" test. When compared with the CAP/CTM assay, the two assays showed 100% consistency in both negative and positive sample results (15 negative samples and 185 positive samples). No statistical differences between the two assays in the HBV DNA quantification values were observed ( P > 0.05). Correlation analysis indicated a significant correlation between the two assays, r 2 = 0.9774. The Bland-Altman plot analysis showed that 98.9% of the positive data were within the 95% acceptable range, and the maximum difference

  7. High-resolution genotyping of Listeria monocytogenes by fluorescent amplified fragment length polymorphism analysis compared to pulsed-field gel electrophoresis, random amplified polymorphic DNA analysis, ribotyping, and PCR-restriction fragment length polymorphism analysis

    DEFF Research Database (Denmark)

    Vogel, Birte Fonnesbech; Fussing, V.; Ojeniyi, B.

    2004-01-01

    . Isolates with identical DNA profiles were distributed across the spectrum of origin. It was not possible to associate certain types with specific food sectors or clinical cases, which is indicative of the spread of L. monocytogenes clones across species. Overall, AFLP fingerprinting was suitable...

  8. Anti-double stranded DNA antibodies in systemic lupus erythematosus : Detection and clinical relevance of IgM-class antibodies

    NARCIS (Netherlands)

    Bootsma, H; Spronk, PE; Hummel, EJ; deBoer, G; terBorg, EJ; Limburg, PC; Kallenberg, CGM

    1996-01-01

    We determined the discriminative value of the Farr assay in comparison to ELISA and Crithidia luciliae immunofluorescence assay (IFT) for detecting anti-dsDNA antibodies as a diagnostic tool for systemic lupus erythematosus (SLE). Special attention was paid to the diagnostic significance of

  9. Characterization of Recombinant Thermococcus kodakaraensis (KOD) DNA Polymerases Produced Using Silkworm-Baculovirus Expression Vector System

    KAUST Repository

    Yamashita, Mami; Xu, Jian; Morokuma, Daisuke; Hirata, Kazuma; Hino, Masato; Mon, Hiroaki; Takahashi, Masateru; Hamdan, Samir; Sakashita, Kosuke; Iiyama, Kazuhiro; Banno, Yutaka; Kusakabe, Takahiro; Lee, Jae Man

    2017-01-01

    The KOD DNA polymerase from Thermococcus kodakarensis (Tkod-Pol) has been preferred for PCR due to its rapid elongation rate, extreme thermostability and outstanding fidelity. Here in this study, we utilized silkworm-baculovirus expression vector system (silkworm-BEVS) to express the recombinant Tkod-Pol (rKOD) with N-terminal (rKOD-N) or C-terminal (rKOD-C) tandem fusion tags. By using BEVS, we produced functional rKODs with satisfactory yields, about 1.1 mg/larva for rKOD-N and 0.25 mg/larva for rKOD-C, respectively. Interestingly, we found that rKOD-C shows higher thermostability at 95 °C than that of rKOD-N, while that rKOD-N is significantly unstable after exposing to long period of heat-shock. We also assessed the polymerase activity as well as the fidelity of purified rKODs under various conditions. Compared with commercially available rKOD, which is expressed in E. coli expression system, rKOD-C exhibited almost the same PCR performance as the commercial rKOD did, while rKOD-N did lower performance. Taken together, our results suggested that silkworm-BEVS can be used to express and purify efficient rKOD in a commercial way.

  10. Characterization of Recombinant Thermococcus kodakaraensis (KOD) DNA Polymerases Produced Using Silkworm-Baculovirus Expression Vector System

    KAUST Repository

    Yamashita, Mami

    2017-05-08

    The KOD DNA polymerase from Thermococcus kodakarensis (Tkod-Pol) has been preferred for PCR due to its rapid elongation rate, extreme thermostability and outstanding fidelity. Here in this study, we utilized silkworm-baculovirus expression vector system (silkworm-BEVS) to express the recombinant Tkod-Pol (rKOD) with N-terminal (rKOD-N) or C-terminal (rKOD-C) tandem fusion tags. By using BEVS, we produced functional rKODs with satisfactory yields, about 1.1 mg/larva for rKOD-N and 0.25 mg/larva for rKOD-C, respectively. Interestingly, we found that rKOD-C shows higher thermostability at 95 °C than that of rKOD-N, while that rKOD-N is significantly unstable after exposing to long period of heat-shock. We also assessed the polymerase activity as well as the fidelity of purified rKODs under various conditions. Compared with commercially available rKOD, which is expressed in E. coli expression system, rKOD-C exhibited almost the same PCR performance as the commercial rKOD did, while rKOD-N did lower performance. Taken together, our results suggested that silkworm-BEVS can be used to express and purify efficient rKOD in a commercial way.

  11. Efficient silkworm expression of human GPCR (nociceptin receptor) by a Bombyx mori bacmid DNA system

    International Nuclear Information System (INIS)

    Kajikawa, Mizuho; Sasaki, Kaori; Wakimoto, Yoshitaro; Toyooka, Masaru; Motohashi, Tomoko; Shimojima, Tsukasa; Takeda, Shigeki; Park, Enoch Y.; Maenaka, Katsumi

    2009-01-01

    Guanine nucleotide-binding protein (G protein) coupled receptors (GPCRs) are frequently expressed by a baculovirus expression vector system (BEVS). We recently established a novel BEVS using the bacmid system of Bombyx mori nucleopolyhedrovirus (BmNPV), which is directly applicable for protein expression in silkworms. Here, we report the first example of GPCR expression in silkworms by the simple injection of BmNPV bacmid DNA. Human nociceptin receptor, an inhibitory GPCR, and its fusion protein with inhibitory G protein alpha subunit (G i α) were both successfully expressed in the fat bodies of silkworm larvae as well as in the BmNPV viral fraction. Its yield was much higher than that from Sf9 cells. The microsomal fractions including the nociceptin receptor fusion, which are easily prepared by only centrifugation steps, exhibited [ 35 S]GTPγS-binding activity upon specific stimulation by nociceptin. Therefore, this rapid method is easy-to-use and has a high expression level, and thus will be an important tool for human GPCR production.

  12. Efficient silkworm expression of human GPCR (nociceptin receptor) by a Bombyx mori bacmid DNA system

    Energy Technology Data Exchange (ETDEWEB)

    Kajikawa, Mizuho; Sasaki, Kaori [Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Wakimoto, Yoshitaro; Toyooka, Masaru [Department of Chemistry and Chemical Biology, Graduate School of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515 (Japan); Motohashi, Tomoko; Shimojima, Tsukasa [National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540 (Japan); Takeda, Shigeki [Department of Chemistry and Chemical Biology, Graduate School of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515 (Japan); Park, Enoch Y. [Laboratory of Biotechnology, Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University, 836 Oya, Suruga-ku, Shizuoka, Shizuoka 422-8529 (Japan); Maenaka, Katsumi, E-mail: kmaenaka-umin@umin.net [Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan)

    2009-07-31

    Guanine nucleotide-binding protein (G protein) coupled receptors (GPCRs) are frequently expressed by a baculovirus expression vector system (BEVS). We recently established a novel BEVS using the bacmid system of Bombyx mori nucleopolyhedrovirus (BmNPV), which is directly applicable for protein expression in silkworms. Here, we report the first example of GPCR expression in silkworms by the simple injection of BmNPV bacmid DNA. Human nociceptin receptor, an inhibitory GPCR, and its fusion protein with inhibitory G protein alpha subunit (G{sub i}{alpha}) were both successfully expressed in the fat bodies of silkworm larvae as well as in the BmNPV viral fraction. Its yield was much higher than that from Sf9 cells. The microsomal fractions including the nociceptin receptor fusion, which are easily prepared by only centrifugation steps, exhibited [{sup 35}S]GTP{gamma}S-binding activity upon specific stimulation by nociceptin. Therefore, this rapid method is easy-to-use and has a high expression level, and thus will be an important tool for human GPCR production.

  13. DNA targeting by the type I-G and type I-A CRISPR–Cas systems of Pyrococcus furiosus

    Science.gov (United States)

    Elmore, Joshua; Deighan, Trace; Westpheling, Jan; Terns, Rebecca M.; Terns, Michael P.

    2015-01-01

    CRISPR–Cas systems silence plasmids and viruses in prokaryotes. CRISPR–Cas effector complexes contain CRISPR RNAs (crRNAs) that include sequences captured from invaders and direct CRISPR-associated (Cas) proteins to destroy corresponding invader nucleic acids. Pyrococcus furiosus (Pfu) harbors three CRISPR–Cas immune systems: a Cst (Type I-G) system with an associated Cmr (Type III-B) module at one locus, and a partial Csa (Type I-A) module (lacking known invader sequence acquisition and crRNA processing genes) at another locus. The Pfu Cmr complex cleaves complementary target RNAs, and Csa systems have been shown to target DNA, while the mechanism by which Cst complexes silence invaders is unknown. In this study, we investigated the function of the Cst as well as Csa system in Pfu strains harboring a single CRISPR–Cas system. Plasmid transformation assays revealed that the Cst and Csa systems both function by DNA silencing and utilize similar flanking sequence information (PAMs) to identify invader DNA. Silencing by each system specifically requires its associated Cas3 nuclease. crRNAs from the 7 shared CRISPR loci in Pfu are processed for use by all 3 effector complexes, and Northern analysis revealed that individual effector complexes dictate the profile of mature crRNA species that is generated. PMID:26519471

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

    International Nuclear Information System (INIS)

    Thoms, B.; Wackernagel, W.

    1983-01-01

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

  15. Global solutions of restricted open-shell Hartree-Fock theory from semidefinite programming with applications to strongly correlated quantum systems.

    Science.gov (United States)

    Veeraraghavan, Srikant; Mazziotti, David A

    2014-03-28

    We present a density matrix approach for computing global solutions of restricted open-shell Hartree-Fock theory, based on semidefinite programming (SDP), that gives upper and lower bounds on the Hartree-Fock energy of quantum systems. While wave function approaches to Hartree-Fock theory yield an upper bound to the Hartree-Fock energy, we derive a semidefinite relaxation of Hartree-Fock theory that yields a rigorous lower bound on the Hartree-Fock energy. We also develop an upper-bound algorithm in which Hartree-Fock theory is cast as a SDP with a nonconvex constraint on the rank of the matrix variable. Equality of the upper- and lower-bound energies guarantees that the computed solution is the globally optimal solution of Hartree-Fock theory. The work extends a previously presented method for closed-shell systems [S. Veeraraghavan and D. A. Mazziotti, Phys. Rev. A 89, 010502-R (2014)]. For strongly correlated systems the SDP approach provides an alternative to the locally optimized Hartree-Fock energies and densities with a certificate of global optimality. Applications are made to the potential energy curves of C2, CN, Cr2, and NO2.

  16. Population genetic data for six non-combined DNA index system ...

    African Journals Online (AJOL)

    The respective values for the combined power of exclusion in these populations were 0.94 and 0.99. The allele frequency data generated can be used for estimating DNA profile frequencies for the studied populations residing in South Africa. Key words: Allele frequencies, MiniSTR, DNA typing, population data, South Africa ...

  17. Elucidation of the antibacterial mechanism of the Curvularia haloperoxidase system by DNA microarray profiling

    DEFF Research Database (Denmark)

    Hansen, E.H.; Schembri, Mark; Klemm, Per

    2004-01-01

    was the wild type. Our results demonstrate that DNA microarray technology cannot be used as the only technique to investigate the mechanisms of action of new antimicrobial compounds. However, by combining DNA microarray analysis with the subsequent creation of knockout mutants, we were able to pinpoint one...

  18. Effect of Interannual Variability on the Ocean Acidification-induced Habitat Restriction of the Humboldt Current System.

    Science.gov (United States)

    Franco, A. C.; Gruber, N.; Munnich, M.

    2016-02-01

    The Humboldt Current System (HCS) is one of the most productive ecosystems in the world. This high productivity is supported by a large input of nutrients from the subsurface layers to the surface due to year-round upwelling. However, upwelling also supplies waters with low pH and low aragonite saturation state potentially affecting many organisms, especially those that calcify. The influence, extent and source of upwelled water varies substantially on interannual timescales in association with the El Niño/Southern Oscillation (ENSO) phenomenon, accentuating productivity during La Niña events and dampening it during El Niño, altering the dynamics of the whole ecosystem. On top of this natural variability, the continuing acidification of the upper ocean in response to raising atmospheric CO2 may decrease pH further and increase the volume of water corrosive to aragonite in this region, leading to a progressively smaller suitable habitat for sensitive organisms. Here we use an eddy-resolving basin-scale ocean model that covers the whole Pacific Ocean with higher resolution near the coast off South America ( 6 km) to investigate the role of ENSO events on low aragonite saturation episodes and productivity variations. We compare 2 simulations: a hindcast simulation that spans the last 30 years and a future scenario that represents year 2090 (following IPCC's "business-as-usual" scenario). We found that in the region off Peru, the sole effect of increasing atmospheric CO2 to 840 matm shoals the annual average aragonite saturation depth to 30 m, creating a year round presence of aragonite undersaturated water in the euphotic zone. We then contrast the effect on primary productivity and the aragonite saturation state of at least eight El Niño and eight La Niña episodes that have been reported for the past 30 years, in an attempt to answer the question: does habitat availability under future ocean acidification will resemble a pervasive La Niña-like state?

  19. Involvement of the DNA mismatch repair system in cisplatin sensitivity of testicular germ cell tumours.

    Science.gov (United States)

    Rudolph, Christiane; Melau, Cecilie; Nielsen, John E; Vile Jensen, Kristina; Liu, Dekang; Pena-Diaz, Javier; Rajpert-De Meyts, Ewa; Rasmussen, Lene Juel; Jørgensen, Anne

    2017-08-01

    Testicular germ cell tumours (TGCT) are highly sensitive to cisplatin-based chemotherapy, but patients with tumours containing differentiated teratoma components are less responsive to this treatment. The cisplatin sensitivity in TGCT has previously been linked to the embryonic phenotype in the majority of tumours, although the underlying mechanism largely remains to be elucidated. The aim of this study was to investigate the role of the DNA mismatch repair (MMR) system in the cisplatin sensitivity of TGCT. The expression pattern of key MMR proteins, including MSH2, MSH6, MLH1 and PMS2, were investigated during testis development and in the pathogenesis of TGCT, including germ cell neoplasia in situ (GCNIS). The TGCT-derived cell line NTera2 was differentiated using retinoic acid (10 μM, 6 days) after which MMR protein expression and activity, as well as cisplatin sensitivity, were investigated in both undifferentiated and differentiated cells. Finally, the expression of MSH2 was knocked down by siRNA in NTera2 cells after which the effect on cisplatin sensitivity was examined. MMR proteins were expressed in proliferating cells in the testes, while in malignant germ cells MMR protein expression was found to coincide with the expression of the pluripotency factor OCT4, with no or low expression in the more differentiated yolk sac tumours, choriocarcinomas and teratomas. In differentiated NTera2 cells we found a significantly (p cisplatin sensitivity, compared to undifferentiated NTera2 cells. Also, we found that partial knockdown of MSH2 expression in undifferentiated NTera2 cells resulted in a significantly (p cisplatin sensitivity. This study reports, for the first time, expression of the MMR system in fetal gonocytes, from which GCNIS cells are derived. Our findings in primary TGCT specimens and TGCT-derived cells suggest that a reduced sensitivity to cisplatin in differentiated TGCT components could result from a reduced expression of MMR proteins, in

  20. Radiation damage to DNA constituents

    International Nuclear Information System (INIS)

    Bergene, R.

    1977-01-01

    The molecular changes of the DNA molecule, in various systems exposed to inoizing radiation, have been the subject of a great number of studies. In the present work electron spin resonance spectroscopy (ESR) has been applied to irradiated crystalline systems, in particular single crystals of DNA subunits and their derivatives. The main conclusions about the molecular damage are based on this technique in combination with molecular orbital calculations. It should be emphasized that the ESR technique is restricted to damage containing unpaired electrons. These unstable intermediates called free radicals seem, however, to be involved in all molecular models describing the action of radiation on DNA. One of the premises for a detailed theory of the radiation induced reactions at the physico-chemical level seems to involve exact knowledge of the induced free radicals as well as the modes of their formation and fate. For DNA, as such, it is hardly possible to arrive at such a level of knowledge since the molecular complexity prevents selective studies of the many different radiation induced products. One possible approach is to study the free radicals formed in the constituents of DNA. In the present work three lines of approach should be mentioned. The first is based on the observation that radical formation in general causes only minor structural alterations to the molecule in question. The use of isotopes with different spin and magnetic moment (in particular deuterium) may also serve a source of information. Deuteration leads to a number of protons, mainly NH - and OH, becoming substituted, and if any of these are involved in interactions with unpaired protons the resonance pattern is influeneed. The third source of information is molecular orbital calculation. The electron spin density distribution is a function in the three dimensional space based on the system's electronic wave functions. This constitutes the basis for the idea that ESR data can be correlated with

  1. Induction of a systemic lupus erythematosus-like disease in mice by a common human anti-DNA idiotype

    International Nuclear Information System (INIS)

    Mendlovic, S.; Brocke, S.; Meshorer, A.; Mozes, E.; Shoenfeld, Y.; Bakimer, R.; Ben-Bassat, M.

    1988-01-01

    Systemic lupus erythematosus (SLE) is considered to be the quintessential autoimmune disease. It has not been possible to induce SLE in animal models by DNA immunization or by challenge with anti-DNA antibodies. The authors report a murine model of SLE-like disease induced by immunization of C3H.SW female mice with a common human monoclonal anti-DNA idiotype (16/6 idiotype). Following a booster injection with the 16/6 idiotype, high levels of murine anti-16/6 and anti-anti-16/6 antibodies (associated with anti-DNA activity) were detected in the sera of the immunized mice. Elevated titers of autoantibodies reacting with DNA, poly(I), poly(dT), ribonucleoprotein, autoantigens [Sm, SS-A (Ro), and SS-B (La)], and cardiolipin were noted. The serological findings were associated with increased erythrocyte sedimentation rate, leukopenia, proteinuria, immune complex deposition in the glomerular mesangium, and sclerosis of the glomeruli. The immune complexes in the kidneys were shown to contain the 16/6 idiotype. This experimental SLE-like model may be used to elucidate the mechanisms underlying SLE

  2. The Influence of Hepatitis C Virus Therapy on the DNA Base Excision Repair System of Peripheral Blood Mononuclear Cells.

    Science.gov (United States)

    Czarny, Piotr; Merecz-Sadowska, Anna; Majchrzak, Kinga; Jabłkowski, Maciej; Szemraj, Janusz; Śliwiński, Tomasz; Karwowski, Bolesław

    2017-07-01

    Hepatitis C virus (HCV) can infect extrahepatic tissues, including lymphocytes, creating reservoir of the virus. Moreover, HCV proteins can interact with DNA damage response proteins of infected cells. In this article we investigated the influence of the virus infection and a new ombitasvir/paritaprevir/ritonavir ± dasabuvir ± ribavirin (OBV/PTV/r ± DSV ± RBV) anti-HCV therapy on the PBMCs (peripheral blood mononuclear cells, mainly lymphocytes) DNA base excision repair (BER) system. BER protein activity was analyzed in the nuclear and mitochondrial extracts (NE and ME) of PBMC isolated from patients before and after therapy, and from subjects without HCV, using modeled double-strand DNA, with 2'-deoxyuridine substitution as the DNA damage. The NE and ME obtained from patients before therapy demonstrated lower efficacy of 2'-deoxyuridine removal and DNA repair polymerization than those of the control group or patients after therapy. Moreover, the extracts from the patients after therapy had similar activity to those from the control group. However, the efficacy of apurinic/apyrimidinic site excision in NE did not differ between the studied groups. We postulate that infection of lymphocytes by the HCV can lead to a decrease in the activity of BER enzymes. However, the use of novel therapy results in the improvement of glycosylase activity as well as the regeneration of endonuclease and other crucial repair enzymes.

  3. DNA vaccination for cervical cancer: Strategic optimisation of RALA mediated gene delivery from a biodegradable microneedle system.

    Science.gov (United States)

    Cole, Grace; Ali, Ahlam A; McCrudden, Cian M; McBride, John W; McCaffrey, Joanne; Robson, Tracy; Kett, Vicky L; Dunne, Nicholas J; Donnelly, Ryan F; McCarthy, Helen O

    2018-03-03

    Dissolvable microneedles can be employed to deliver DNA to antigen presenting cells within the skin. However, this technology faces two main challenges: the poor transfection efficacy of pDNA following release from the microneedle matrix, and the limited loading capacity of the micron-scale devices. Two-tier delivery systems combining microneedle platforms and DNA delivery vectors have increased efficacy but the challenge of increasing the loading capacity remains. This study utilised lyophilisation to increase the loading of RALA/pDNA nanoparticles within dissolvable PVA microneedles. As a result, delivery was significantly enhanced in vivo into an appropriate range for DNA vaccination (∼50 μg per array). Furthermore, modifying the manufacturing process was not detrimental to the microneedle mechanical properties or cargo functionality. It was demonstrated that arrays retained mechanical and functional stability over short term storage, and were able to elicit gene expression in vitro and in vivo. Finally, treatment with this novel formulation significantly retarded the growth of established tumours, and proved superior to standard intramuscular injection in a preclinical model of cervical cancer. Copyright © 2018. Published by Elsevier B.V.

  4. Chip-Oriented Fluorimeter Design and Detection System Development for DNA Quantification in Nano-Liter Volumes

    Directory of Open Access Journals (Sweden)

    Da-Sheng Lee

    2009-12-01

    Full Text Available The chip-based polymerase chain reaction (PCR system has been developed in recent years to achieve DNA quantification. Using a microstructure and miniature chip, the volume consumption for a PCR can be reduced to a nano-liter. With high speed cycling and a low reaction volume, the time consumption of one PCR cycle performed on a chip can be reduced. However, most of the presented prototypes employ commercial fluorimeters which are not optimized for fluorescence detection of such a small quantity sample. This limits the performance of DNA quantification, especially low experiment reproducibility. This study discusses the concept of a chip-oriented fluorimeter design. Using the analytical model, the current study analyzes the sensitivity and dynamic range of the fluorimeter to fit the requirements for detecting fluorescence in nano-liter volumes. Through the optimized processes, a real-time PCR on a chip system with only one nano-liter volume test sample is as sensitive as the commercial real-time PCR machine using the sample with twenty micro-liter volumes. The signal to noise (S/N ratio of a chip system for DNA quantification with hepatitis B virus (HBV plasmid samples is 3 dB higher. DNA quantification by the miniature chip shows higher reproducibility compared to the commercial machine with respect to samples of initial concentrations from 103 to 105 copies per reaction.

  5. Ternary polyplex micelles with PEG shells and intermediate barrier to complexed DNA cores for efficient systemic gene delivery.

    Science.gov (United States)

    Li, Junjie; Chen, Qixian; Zha, Zengshi; Li, Hui; Toh, Kazuko; Dirisala, Anjaneyulu; Matsumoto, Yu; Osada, Kensuke; Kataoka, Kazunori; Ge, Zhishen

    2015-07-10

    Simultaneous achievement of prolonged retention in blood circulation and efficient gene transfection activity in target tissues has always been a major challenge hindering in vivo applications of nonviral gene vectors via systemic administration. Herein, we constructed novel rod-shaped ternary polyplex micelles (TPMs) via complexation between the mixed block copolymers of poly(ethylene glycol)-b-poly{N'-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} (PEG-b-PAsp(DET)) and poly(N-isopropylacrylamide)-b-PAsp(DET) (PNIPAM-b-PAsp(DET)) and plasmid DNA (pDNA) at room temperature, exhibiting distinct temperature-responsive formation of a hydrophobic intermediate layer between PEG shells and pDNA cores through facile temperature increase from room temperature to body temperature (~37 °C). As compared with binary polyplex micelles of PEG-b-PAsp(DET) (BPMs), TPMs were confirmed to condense pDNA into a more compact structure, which achieved enhanced tolerability to nuclease digestion and strong counter polyanion exchange. In vitro gene transfection results demonstrated TPMs exhibiting enhanced gene transfection efficiency due to efficient cellular uptake and endosomal escape. Moreover, in vivo performance evaluation after intravenous injection confirmed that TPMs achieved significantly prolonged blood circulation, high tumor accumulation, and promoted gene expression in tumor tissue. Moreover, TPMs loading therapeutic pDNA encoding an anti-angiogenic protein remarkably suppressed tumor growth following intravenous injection into H22 tumor-bearing mice. These results suggest TPMs with PEG shells and facilely engineered intermediate barrier to inner complexed pDNA have great potentials as systemic nonviral gene vectors for cancer gene therapy. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Detection of herpes simplex virus-specific DNA sequences in latently infected mice and in humans.

    Science.gov (United States)

    Efstathiou, S; Minson, A C; Field, H J; Anderson, J R; Wildy, P

    1986-02-01

    Herpes simplex virus-specific DNA sequences have been detected by Southern hybridization analysis in both central and peripheral nervous system tissues of latently infected mice. We have detected virus-specific sequences corresponding to the junction fragment but not the genomic termini, an observation first made by Rock and Fraser (Nature [London] 302:523-525, 1983). This "endless" herpes simplex virus DNA is both qualitatively and quantitatively stable in mouse neural tissue analyzed over a 4-month period. In addition, examination of DNA extracted from human trigeminal ganglia has shown herpes simplex virus DNA to be present in an "endless" form similar to that found in the mouse model system. Further restriction enzyme analysis of latently infected mouse brainstem and human trigeminal DNA has shown that this "endless" herpes simplex virus DNA is present in all four isomeric configurations.

  7. The essential component in DNA-based information storage system: robust error-tolerating module

    Directory of Open Access Journals (Sweden)

    Aldrin Kay-Yuen eYim

    2014-11-01

    Full Text Available The size of digital data is ever increasing and is expected to grow to 40,000EB by 2020, yet the estimated global information storage capacity in 2011 is less than 300EB, indicating that most of the data are transient. DNA, as a very stable nano-molecule, is an ideal massive storage device for long-term data archive. The two most notable illustrations are from Church et al. and Goldman et al., whose approaches are well-optimized for most sequencing platforms – short synthesized DNA fragments without homopolymer. Here we suggested improvements on error handling methodology that could enable the integration of DNA-based computational process, e.g. algorithms based on self-assembly of DNA. As a proof of concept, a picture of size 438 bytes was encoded to DNA with Low-Density Parity-Check error-correction code. We salvaged a significant portion of sequencing reads with mutations generated during DNA synthesis and sequencing and successfully reconstructed the entire picture. A modular-based programming framework - DNAcodec with a XML-based data format was also introduced. Our experiments demonstrated the practicability of long DNA message recovery with high error-tolerance, which opens the field to biocomputing and synthetic biology.

  8. Chromatin Constrains the Initiation and Elongation of DNA Replication.

    Science.gov (United States)

    Devbhandari, Sujan; Jiang, Jieqing; Kumar, Charanya; Whitehouse, Iestyn; Remus, Dirk

    2017-01-05

    Eukaryotic chromosomal DNA is faithfully replicated in a complex series of cell-cycle-regulated events that are incompletely understood. Here we report the reconstitution of DNA replication free in solution with purified proteins from the budding yeast Saccharomyces cerevisiae. The system recapitulates regulated bidirectional origin activation; synthesis of leading and lagging strands by the three replicative DNA polymerases Pol α, Pol δ, and Pol ε; and canonical maturation of Okazaki fragments into continuous daughter strands. We uncover a dual regulatory role for chromatin during DNA replication: promoting origin dependence and determining Okazaki fragment length by restricting Pol δ progression. This system thus provides a functional platform for the detailed mechanistic analysis of eukaryotic chromosome replication. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Tyramine Hydrochloride Based Label-Free System for Operating Various DNA Logic Gates and a DNA Caliper for Base Number Measurements.

    Science.gov (United States)

    Fan, Daoqing; Zhu, Xiaoqing; Dong, Shaojun; Wang, Erkang

    2017-07-05

    DNA is believed to be a promising candidate for molecular logic computation, and the fluorogenic/colorimetric substrates of G-quadruplex DNAzyme (G4zyme) are broadly used as label-free output reporters of DNA logic circuits. Herein, for the first time, tyramine-HCl (a fluorogenic substrate of G4zyme) is applied to DNA logic computation and a series of label-free DNA-input logic gates, including elementary AND, OR, and INHIBIT logic gates, as well as a two to one encoder, are constructed. Furthermore, a DNA caliper that can measure the base number of target DNA as low as three bases is also fabricated. This DNA caliper can also perform concatenated AND-AND logic computation to fulfil the requirements of sophisticated logic computing. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Establishment of a non-radioactive cleavage assay to assess the DNA repair capacity towards oxidatively damaged DNA in subcellular and cellular systems and the impact of copper

    International Nuclear Information System (INIS)

    Hamann, Ingrit; Schwerdtle, Tanja; Hartwig, Andrea

    2009-01-01

    Oxidative stress is involved in many diseases, and the search for appropriate biomarkers is one major focus in molecular epidemiology. 8-Oxoguanine (8-oxoG), a potentially mutagenic DNA lesion, is considered to be a sensitive biomarker for oxidative stress. Another approach consists in assessing the repair capacity towards 8-oxoG, mediated predominantly by the human 8-oxoguanine DNA glycosylase 1 (hOGG1). With respect to the latter, during the last few years so-called cleavage assays have been described, investigating the incision of 32 P-labelled and 8-oxoG damaged oligonucleotides by cell extracts. Within the present study, a sensitive non-radioactive test system based on a Cy5-labelled oligonucleotide has been established. Sources of incision activity are isolated proteins or extracts prepared from cultured cells and peripheral blood mononuclear cells (PBMC). After comparing different oligonucleotide structures, a hairpin-like structure was selected which was not degraded by cell extracts. Applying this test system the impact of copper on the activity of isolated hOGG1 and on hOGG activity in A549 cells was examined, showing a distinct inhibition of the isolated protein at low copper concentration as compared to a modest inhibition of hOGG activity in cells at beginning cytotoxic concentrations. For investigating PBMC, all reaction conditions, including the amounts of oligonucleotide and cell extract as well as the reaction time have been optimized. The incision activities of PBMC protein extracts obtained from different donors have been investigated, and inter-individual differences have been observed. In summary, the established method is as sensitive and even faster than the radioactive technique, and additionally, offers the advantage of reduced costs and low health risk.

  11. Evidence from Animal Models: Is a Restricted or Conventional Intestinal Microbiota Composition Predisposing to Risk for High-LET Radiation Injury?

    Science.gov (United States)

    Maier, Irene; Schiestl, Robert H

    2015-06-01

    Intestinal microbiota affect cell responses to ionizing radiation at the molecular level and can be linked to the development of the immune system, controlled cell death or apoptosis. We have developed a microbiota mouse model and report here that high-linear energy transfer (LET) radiation induced the repair of chromosomal DNA lesions more efficiently in conventional than in restricted intestinal microbiota mice. Based on different phylotype densities after whole-body irradiation, bacterial indicator phylotypes were found to be more abundant in restricted in microbiota than in conventional microbiota. Genotoxic phenotypes of irradiated restricted and conventional microbiota mice were compared with ataxia telangiectasia-deficient restricted and conventional microbiota mice, respectively. Those indicator phylotypes, including Bacteroides (Gram-negative bacterium cTPY-13), Barnesiella intestinihominis and others, which were identified in nonirradiated restricted microbiota mice, increase in radiation-exposed conventional microbiota along with a reduction of persistent DNA double-strand breaks in blood lymphocytes. The dynamic change of phylotype abundances elucidated a feedback mechanism and effect of intestinal microbiota composition on the adaptive response to high-LET radiation. Several other bacterial phylotypes ( Helicobacter hepaticus , Helicobacter spp and others) were found to be more abundant in conventional than restricted microbiota. In this commentary, mouse models used in cancer research and radiotherapy for the study on the effects of intestinal microbiota composition on normal tissue radiation response are characterized and discussed. Highlights of this commentary: 1. Restricted microbiota phylotypes were correlated with persistent DNA double-strand breaks (DSBs) and were found to orchestrate onco-protective controlled cell death after radiation; 2. Restricted microbiota composition reduced proinflammatory extracellular-stimulated immune responses, but

  12. An archaeal CRISPR type III-B system exhibiting distinctive RNA targeting features and mediating dual RNA and DNA interference

    DEFF Research Database (Denmark)

    Peng, Wenfang; Feng, Mingxia; Feng, Xu

    2015-01-01

    CRISPR-Cas systems provide a small RNA-based mechanism to defend against invasive genetic elements in archaea and bacteria. To investigate the in vivo mechanism of RNA interference by two type III-B systems (Cmr-α and Cmr-β) in Sulfolobus islandicus, a genetic assay was developed using plasmids...... carrying an artificial mini-CRISPR (AC) locus with a single spacer. After pAC plasmids were introduced into different strains, Northern analyses confirmed that mature crRNAs were produced from the plasmid-borne CRISPR loci, which then guided gene silencing to target gene expression. Spacer mutagenesis....... islandicus Cmr-α mediated transcription-dependent DNA interference, the Cmr-α constitutes the first CRISPR system exhibiting dual targeting of RNA and DNA....

  13. A DNA-based registry for all animal species: the barcode index number (BIN system.

    Directory of Open Access Journals (Sweden)

    Sujeevan Ratnasingham

    Full Text Available Because many animal species are undescribed, and because the identification of known species is often difficult, interim taxonomic nomenclature has often been used in biodiversity analysis. By assigning individuals to presumptive species, called operational taxonomic units (OTUs, these systems speed investigations into the patterning of biodiversity and enable studies that would otherwise be impossible. Although OTUs have conventionally been separated through their morphological divergence, DNA-based delineations are not only feasible, but have important advantages. OTU designation can be automated, data can be readily archived, and results can be easily compared among investigations. This study exploits these attributes to develop a persistent, species-level taxonomic registry for the animal kingdom based on the analysis of patterns of nucleotide variation in the barcode region of the cytochrome c oxidase I (COI gene. It begins by examining the correspondence between groups of specimens identified to a species through prior taxonomic work and those inferred from the analysis of COI sequence variation using one new (RESL and four established (ABGD, CROP, GMYC, jMOTU algorithms. It subsequently describes the implementation, and structural attributes of the Barcode Index Number (BIN system. Aside from a pragmatic role in biodiversity assessments, BINs will aid revisionary taxonomy by flagging possible cases of synonymy, and by collating geographical information, descriptive metadata, and images for specimens that are likely to belong to the same species, even if it is undescribed. More than 274,000 BIN web pages are now available, creating a biodiversity resource that is positioned for rapid growth.

  14. Characterization of soil nematode communities in three cropping systems through morphological and DNA metabarcoding approaches

    Science.gov (United States)

    Communities of soil nematodes impact ecosystem functions, including plant growth, decomposition, and nutrient cycling, all of which are vital processes in agriculture. We used complementary morphological and DNA metabarcoding analyses to characterize soil nematode communities in three cropping syste...

  15. Type I-E CRISPR-cas systems discriminate target from non-target DNA through base pairing-independent PAM recognition

    NARCIS (Netherlands)

    Westra, E.R.; Semenova, E.; Datsenko, K.A.; Jackson, R.N.; Wiedenheft, B.; Severinov, K.; Brouns, S.J.J.

    2013-01-01

    Discriminating self and non-self is a universal requirement of immune systems. Adaptive immune systems in prokaryotes are centered around repetitive loci called CRISPRs (clustered regularly interspaced short palindromic repeat), into which invader DNA fragments are incorporated. CRISPR transcripts

  16. Detection of polyomavirus simian virus 40 tumor antigen DNA in AIDS-related systemic non-Hodgkin lymphoma

    Science.gov (United States)

    Vilchez, Regis A.; Lednicky, John A.; Halvorson, Steven J.; White, Zoe S.; Kozinetz, Claudia A.; Butel, Janet S.

    2002-01-01

    Systemic non-Hodgkin lymphoma (S-NHL) is a common malignancy during HIV infection, and it is hypothesized that infectious agents may be involved in the etiology. Epstein-Barr virus DNA is found in <40% of patients with AIDS-related S-NHL, suggesting that other oncogenic viruses, such as polyomaviruses, may play a role in pathogenesis. We analyzed AIDS-related S-NHL samples, NHL samples from HIV-negative patients, peripheral blood leukocytes from HIV-infected and -uninfected patients without NHL, and lymph nodes without tumors from HIV-infected patients. Specimens were examined by polymerase chain reaction analysis with use of primers specific for an N-terminal region of the oncoprotein large tumor antigen ( T-ag ) gene conserved among all three polyomaviruses (simian virus 40 [SV40], JC virus, and BK virus). Polyomavirus T-ag DNA sequences, proven to be SV40-specific, were detected more frequently in AIDS-related S-NHL samples (6 of 26) than in peripheral blood leukocytes from HIV-infected patients (6 of 26 vs. 0 of 69; p =.0001), NHL samples from HIV-negative patients (6 of 26 vs. 0 of 10; p =.09), or lymph nodes (6 of 26 vs. 0 of 7; p =.16). Sequences of C-terminal T-ag DNA from SV40 were amplified from two AIDS-related S-NHL samples. Epstein-Barr virus DNA sequences were detected in 38% (10 of 26) AIDS-related S-NHL samples, 50% (5 of 10) HIV-negative S-NHL samples, and 57% (4 of 7) lymph nodes. None of the S-NHL samples were positive for both Epstein-Barr virus DNA and SV40 DNA. Further studies of the possible role of SV40 in the pathogenesis of S-NHL are warranted.

  17. Sequential action of ATPase, ATP, ADP, Pi and dsDNA in procapsid-free system to enlighten mechanism in viral dsDNA packaging.

    Science.gov (United States)

    Schwartz, Chad; Fang, Huaming; Huang, Lisa; Guo, Peixuan

    2012-03-01

    Many cells and double-stranded DNA (dsDNA) viruses contain an AAA(+) ATPase that assembles into oligomers, often hexamers, with a central channel. The dsDNA packaging motor of bacteriophage phi29 also contains an ATPase to translocate dsDNA through a dodecameric channel. The motor ATPase has been investigated substantially in the context of the entire procapsid. Here, we report the sequential action between the ATPase and additional motor components. It is suggested that the contact of ATPase to ATP resulted in its conformational change to a higher binding affinity toward dsDNA. It was found that ATP hydrolysis led to the departure of dsDNA from the ATPase/dsDNA complex, an action that is speculated to push dsDNA to pass the connector channel. Our results suggest that dsDNA packaging goes through a combined effort of both the gp16 ATPase for pushing and the channel as a one-way valve to control the dsDNA translocation direction. Many packaging models have previously been proposed, and the packaging mechanism has been contingent upon the number of nucleotides packaged per ATP relative to the 10.5 bp per helical turn for B-type dsDNA. Both 2 and 2.5 bp per ATP have been used to argue for four, five or six discrete steps of dsDNA translocation. Combination of the two distinct roles of gp16 and connector renews the perception of previous dsDNA packaging energy calculations and provides insight into the discrepancy between 2 and 2.5 bp per ATP.

  18. A Single-Molecule Barcoding System using Nanoslits for DNA Analysis

    Science.gov (United States)

    Jo, Kyubong; Schramm, Timothy M.; Schwartz, David C.

    Single DNA molecule approaches are playing an increasingly central role in the analytical genomic sciences because single molecule techniques intrinsically provide individualized measurements of selected molecules, free from the constraints of bulk techniques, which blindly average noise and mask the presence of minor analyte components. Accordingly, a principal challenge that must be addressed by all single molecule approaches aimed at genome analysis is how to immobilize and manipulate DNA molecules for measurements that foster construction of large, biologically relevant data sets. For meeting this challenge, this chapter discusses an integrated approach for microfabricated and nanofabricated devices for the manipulation of elongated DNA molecules within nanoscale geometries. Ideally, large DNA coils stretch via nanoconfinement when channel dimensions are within tens of nanometers. Importantly, stretched, often immobilized, DNA molecules spanning hundreds of kilobase pairs are required by all analytical platforms working with large genomic substrates because imaging techniques acquire sequence information from molecules that normally exist in free solution as unrevealing random coils resembling floppy balls of yarn. However, nanoscale devices fabricated with sufficiently small dimensions fostering molecular stretching make these devices impractical because of the requirement of exotic fabrication technologies, costly materials, and poor operational efficiencies. In this chapter, such problems are addressed by discussion of a new approach to DNA presentation and analysis that establishes scaleable nanoconfinement conditions through reduction of ionic strength; stiffening DNA molecules thus enabling their arraying for analysis using easily fabricated devices that can also be mass produced. This new approach to DNA nanoconfinement is complemented by the development of a novel labeling scheme for reliable marking of individual molecules with fluorochrome labels

  19. Clustered DNA lesions containing 5-formyluracil and AP site: repair via the BER system.

    Directory of Open Access Journals (Sweden)

    Ekaterina A Belousova

    Full Text Available Lesions in the DNA arise under ionizing irradiation conditions or various chemical oxidants as a single damage or as part of a multiply damaged site within 1-2 helical turns (clustered lesion. Here, we explored the repair opportunity of the apurinic/apyrimidinic site (AP site composed of the clustered lesion with 5-formyluracil (5-foU by the base excision repair (BER proteins. We found, that if the AP site is shifted relative to the 5-foU of the opposite strand, it could be repaired primarily via the short-patch BER pathway. In this case, the cleavage efficiency of the AP site-containing DNA strand catalyzed by human apurinic/apyrimidinic endonuclease 1 (hAPE1 decreased under AP site excursion to the 3'-side relative to the lesion in the other DNA strand. DNA synthesis catalyzed by DNA polymerase lambda was more accurate in comparison to the one catalyzed by DNA polymerase beta. If the AP site was located exactly opposite 5-foU it was expected to switch the repair to the long-patch BER pathway. In this situation, human processivity factor hPCNA stimulates the process.

  20. Life forms employ different repair strategies of repair single- and double strand DNA breaks caused by different qualities of radiation: criticality of RecA mediated repair system

    International Nuclear Information System (INIS)

    Sharan, R.N.

    2013-01-01

    Different qualities of radiation, either through direct or indirect pathway, induce qualitative different spectrum of damages in DNA, which are also different in in vitro and in vivo systems. The single- and double strand breaks of DNA are of special interest as they lead to serious biological consequences. The implications of such damage to DNA and their processing by various inherent repair pathways together decide the fate of the living form

  1. Is the kisspeptin system involved in responses to food restriction in order to preserve reproduction in pubertal male sea bass (Dicentrarchus labrax)?

    Science.gov (United States)

    Escobar, Sebastián; Felip, Alicia; Zanuy, Silvia; Carrillo, Manuel

    2016-09-01

    Previous works on European sea bass have determined that long-term exposure to restrictive feeding diets alters the rhythms of some reproductive/metabolic hormones, delaying maturation and increasing apoptosis during gametogenesis. However, exactly how these diets affect key genes and hormones on the brain-pituitary-gonad (BPG) axis to trigger puberty is still largely unknown. We may hypothesize that all these signals could be integrated, at least in part, by the kisspeptin system. In order to capture a glimpse of these regulatory mechanisms, kiss1 and kiss2 mRNA expression levels and those of their kiss receptors (kiss1r, kiss2r) were analyzed in different areas of the brain and in the pituitary of pubertal male sea bass during gametogenesis. Furthermore, other reproductive hormones and factors as well as the percentage of males showing full spermiation were also analyzed. Treated fish fed maintenance diets provided evidence of overexpression of the kisspeptin system in the main hypophysiotropic regions of the brain throughout the entire sexual cycle. Conversely, Gnrh1 and gonadotropin pituitary content and plasma sexual steroid levels were downregulated, except for Fsh levels, which were shown to increase during spermiation. Treated fish exhibited lower rates of spermiation as compared to control group and a delay in its accomplishment. These results demonstrate how the kisspeptin system and plasma Fsh levels are differentially affected by maintenance diets, causing a retardation, but not a full blockage of the reproductive process in the teleost fish European sea bass. This suggests that a hormonal adaptive strategy may be operating in order to preserve reproductive function in this species. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Diversity of ribosomal 16S DNA- and RNA-based bacterial community in an office building drinking water system.

    Science.gov (United States)

    Inkinen, J; Jayaprakash, B; Santo Domingo, J W; Keinänen-Toivola, M M; Ryu, H; Pitkänen, T

    2016-06-01

    Next-generation sequencing of 16S ribosomal RNA genes (rDNA) and ribosomal RNA (rRNA) was used to characterize water and biofilm microbiome collected from a drinking water distribution system of an office building after its first year of operation. The total bacterial community (rDNA) and active bacterial members (rRNA) sequencing databases were generated by Illumina MiSeq PE250 platform. As estimated by Chao1 index, species richness in cold water system was lower (180-260) in biofilms (Sphingomonas spp., Methylobacterium spp., Limnohabitans spp., Rhizobiales order) than in waters (250-580), (also Methylotenera spp.) (P = 0·005, n = 20). Similarly species richness (Chao1) was slightly higher (210-580) in rDNA libraries compared to rRNA libraries (150-400; P = 0·054, n = 24). Active Mycobacterium spp. was found in cross-linked polyethylene (PEX), but not in corresponding copper pipeline biofilm. Nonpathogenic Legionella spp. was found in rDNA libraries but not in rRNA libraries. Microbial communities differed between water and biofilms, between cold and hot water systems, locations in the building and between water rRNA and rDNA libraries, as shown by clear clusters in principal component analysis (PcoA). By using the rRNA method, we found that not all bacterial community members were active (e.g. Legionella spp.), whereas other members showed increased activity in some locations; for example, Pseudomonas spp. in hot water circulations' biofilm and order Rhizobiales and Limnohabitans spp. in stagnated locations' water and biofilm. rRNA-based methods may be better than rDNA-based methods for evaluating human health implications as rRNA methods can be used to describe the active bacterial fraction. This study indicates that copper as a pipeline material might have an adverse impact on the occurrence of Mycobacterium spp. The activity of Legionella spp. maybe questionable when detected solely by using DNA-based methods. © 2016 The Society for Applied

  3. DNA repair

    International Nuclear Information System (INIS)

    Setlow, R.

    1978-01-01

    Some topics discussed are as follows: difficulty in extrapolating data from E. coli to mammalian systems; mutations caused by UV-induced changes in DNA; mutants deficient in excision repair; other postreplication mechanisms; kinds of excision repair systems; detection of repair by biochemical or biophysical means; human mutants deficient in repair; mutagenic effects of UV on XP cells; and detection of UV-repair defects among XP individuals

  4. Assessment of time-dependent density functional theory with the restricted excitation space approximation for excited state calculations of large systems

    Science.gov (United States)

    Hanson-Heine, Magnus W. D.; George, Michael W.; Besley, Nicholas A.

    2018-06-01

    The restricted excitation subspace approximation is explored as a basis to reduce the memory storage required in linear response time-dependent density functional theory (TDDFT) calculations within the Tamm-Dancoff approximation. It is shown that excluding the core orbitals and up to 70% of the virtual orbitals in the construction of the excitation subspace does not result in significant changes in computed UV/vis spectra for large molecules. The reduced size of the excitation subspace greatly reduces the size of the subspace vectors that need to be stored when using the Davidson procedure to determine the eigenvalues of the TDDFT equations. Furthermore, additional screening of the two-electron integrals in combination with a reduction in the size of the numerical integration grid used in the TDDFT calculation leads to significant computational savings. The use of these approximations represents a simple approach to extend TDDFT to the study of large systems and make the calculations increasingly tractable using modest computing resources.

  5. Amino acid substitutions within the heptad repeat domain 1 of murine coronavirus spike protein restrict viral antigen spread in the central nervous system

    International Nuclear Information System (INIS)

    Tsai, Jean C.; Groot, Linda de; Pinon, Josefina D.; Iacono, Kathryn T.; Phillips, Joanna J.; Seo, Suhun; Lavi, Ehud; Weiss, Susan R.

    2003-01-01

    Targeted recombination was carried out to select mouse hepatitis viruses (MHVs) in a defined genetic background, containing an MHV-JHM spike gene encoding either three heptad repeat 1 (HR1) substitutions (Q1067H, Q1094H, and L1114R) or L1114R alone. The recombinant virus, which expresses spike with the three substitutions, was nonfusogenic at neutral pH. Its replication was significantly inhibited by lysosomotropic agents, and it was highly neuroattenuated in vivo. In contrast, the recombinant expressing spike with L1114R alone mediated cell-to-cell fusion at neutral pH and replicated efficiently despite the presence of lysosomotropic agents; however, it still caused only subclinical morbidity and no mortality in animals. Thus, both recombinant viruses were highly attenuated and expressed viral antigen which was restricted to the olfactory bulbs and was markedly absent from other regions of the brains at 5 days postinfection. These data demonstrate that amino acid substitutions, in particular L1114R, within HR1 of the JHM spike reduced the ability of MHV to spread in the central nervous system. Furthermore, the requirements for low pH for fusion and viral entry are not prerequisites for the highly attenuated phenotype

  6. Capillary column switching restricted-access media-liquid chromatography-electrospray ionization-tandem mass spectrometry system for simultaneous and direct analysis of drugs in biofluids.

    Science.gov (United States)

    Santos-Neto, Alvaro J; Markides, Karin E; Sjöberg, Per J R; Bergquist, Jonas; Lancas, Fernando M

    2007-08-15

    Capillary online restricted-access media-liquid chromatography-electrospray ionization-tandem mass spectrometry (RAM-LC-ESI-MS/MS) for direct analysis of drugs and metabolites spiked in biological fluids was developed. Using a column switching setup it was possible to perform effective sample preparation and analysis of raw biological fluids (plasma and urine) without matrix effects in the electrospray mass spectrometric detection step. The peak focusing efficiency of the extraction column was more effective in backflush compared to foreflush mode. The system was able to concentrate diminished samples of polar drugs and their metabolites reaching quantifiable results as low as 1 ng/mL utilizing a sample volume of only 333 nL of biofluids. New column hardware was developed to circumvent clogging problems experienced with plasma injections. The glass fiber filter frit, which is commonly used, was replaced with a short piece of 20 microm i.d. fused silica capillary. The extraction columns were able to handle up to 60 injections and showed a high loading capacity, making the saturation of the MS detector the limiting factor on the linear dynamic range. The simultaneous separation and detection of 10 drugs and metabolites was obtained in 8 min of analysis, including the online sample preparation and enrichment step.

  7. New detection systems of bacteria using highly selective media designed by SMART: selective medium-design algorithm restricted by two constraints.

    Directory of Open Access Journals (Sweden)

    Takeshi Kawanishi

    Full Text Available Culturing is an indispensable technique in microbiological research, and culturing with selective media has played a crucial role in the detection of pathogenic microorganisms and the isolation of commercially useful microorganisms from environmental samples. Although numerous selective media have been developed in empirical studies, unintended microorganisms often grow on such media probably due to the enormous numbers of microorganisms in the environment. Here, we present a novel strategy for designing highly selective media based on two selective agents, a carbon source and antimicrobials. We named our strategy SMART for highly Selective Medium-design Algorithm Restricted by Two constraints. To test whether the SMART method is applicable to a wide range of microorganisms, we developed selective media for Burkholderia glumae, Acidovorax avenae, Pectobacterium carotovorum, Ralstonia solanacearum, and Xanthomonas campestris. The series of media developed by SMART specifically allowed growth of the targeted bacteria. Because these selective media exhibited high specificity for growth of the target bacteria compared to established selective media, we applied three notable detection technologies: paper-based, flow cytometry-based, and color change-based detection systems for target bacteria species. SMART facilitates not only the development of novel techniques for detecting specific bacteria, but also our understanding of the ecology and epidemiology of the targeted bacteria.

  8. DNA apoptosis and stability in B-cell chronic lymphoid leukaemia: implication of the DNA double-strand breaks repair system by non homologous recombination

    International Nuclear Information System (INIS)

    Deriano, L.

    2005-01-01

    After an introduction presenting the diagnosis and treatment of chronic lymphoid leukaemia, its molecular and genetic characteristics, and its cellular origin and clonal evolution, this research thesis describes the apoptosis (definition and characteristics, cancer and chemotherapy, apoptotic ways induced by gamma irradiation), the genotoxic stresses, the different repair mechanisms for different damages, and the DNA repair processes. It reports how human chronic lymphocytic leukaemia B cells can escape DNA damage-induced apoptosis through the non-homologous end-joining DNA repair pathway, and presents non-homologous end-joining DNA repair as a potent mutagenic process in human chronic lymphocytic leukaemia B cells

  9. Termination of DNA synthesis in vitro at apurinic sites but not at ethyl adducts of the template

    Energy Technology Data Exchange (ETDEWEB)

    Lockhart, M.L.; Deutsch, J.F.; Yamaura, I.; Cavalieri, L.F.; Rosenberg, B.H.

    1982-01-01

    The effects of DNA lesions produced by the carcinogenic alkylating agents ethylnitrosourea and diethylsulfate on the extent of DNA synthesis have been studied in a system utilizing circular single-stranded phi X174 DNA as template and a 392-base restriction fragment as primer with E. coli polymerase I (Klenow fragment). Apurinic sites produced by loss of unstable ethylated bases from the template terminate DNA synthesis at the first such site encountered, but ethyl adducts at most, if not all, locations permit readthrough. 22 references, 3 figures, 1 table.

  10. Pursuing the quest for better understanding the taxonomic distribution of the system of doubly uniparental inheritance of mtDNA

    Directory of Open Access Journals (Sweden)

    Arthur Gusman

    2016-12-01

    Full Text Available There is only one exception to strict maternal inheritance of mitochondrial DNA (mtDNA in the animal kingdom: a system named doubly uniparental inheritance (DUI, which is found in several bivalve species. Why and how such a radically different system of mitochondrial transmission evolved in bivalve remains obscure. Obtaining a more complete taxonomic distribution of DUI in the Bivalvia may help to better understand its origin and function. In this study we provide evidence for the presence of sex-linked heteroplasmy (thus the possible presence of DUI in two bivalve species, i.e., the nuculanoid Yoldia hyperborea(Gould, 1841and the veneroid Scrobicularia plana(Da Costa,1778, increasing the number of families in which DUI has been found by two. An update on the taxonomic distribution of DUI in the Bivalvia is also presented.

  11. A Fluorescent Tile DNA Diagnocode System for In Situ Rapid and Selective Diagnosis of Cytosolic RNA Cancer Markers

    Science.gov (United States)

    Park, Kyung Soo; Shin, Seung Won; Jang, Min Su; Shin, Woojung; Yang, Kisuk; Min, Junhong; Cho, Seung-Woo; Oh, Byung-Keun; Bae, Jong Wook; Jung, Sunghwan; Choi, Jeong-Woo; Um, Soong Ho

    2015-01-01

    Accurate cancer diagnosis often requires extraction and purification of genetic materials from cells, and sophisticated instrumentations that follow. Otherwise in order to directly treat the diagnostic materials to cells, multiple steps to optimize dose concentration and treatment time are necessary due to diversity in cellular behaviors. These processes may offer high precision but hinder fast analysis of cancer, especially in clinical situations that need rapid detection and characterization of cancer. Here we present a novel fluorescent tile DNA nanostructure delivered to cancer cytosol by employing nanoparticle technology. Its structural anisotropicity offers easy manipulation for multifunctionalities, enabling the novel DNA nanostructure to detect intracellular cancer RNA markers with high specificity within 30 minutes post treatment, while the nanoparticle property bypasses the requirement of treatment optimization, effectively reducing the complexity of applying the system for cancer diagnosis. Altogether, the system offers a precise and rapid detection of cancer, suggesting the future use in the clinical fields. PMID:26678430

  12. Direct extraction of genomic DNA from maize with aqueous ionic liquid buffer systems for applications in genetically modified organisms analysis.

    Science.gov (United States)

    Gonzalez García, Eric; Ressmann, Anna K; Gaertner, Peter; Zirbs, Ronald; Mach, Robert L; Krska, Rudolf; Bica, Katharina; Brunner, Kurt

    2014-12-01

    To date, the extraction of genomic DNA is considered a bottleneck in the process of genetically modified organisms (GMOs) detection. Conventional DNA isolation methods are associated with long extraction times and multiple pipetting and centrifugation steps, which makes the entire procedure not only tedious and complicated but also prone to sample cross-contamination. In recent times, ionic liquids have emerged as innovative solvents for biomass processing, due to their outstanding properties for dissolution of biomass and biopolymers. In this study, a novel, easily applicable, and time-efficient method for the direct extraction of genomic DNA from biomass based on aqueous-ionic liquid solutions was developed. The straightforward protocol relies on extraction of maize in a 10 % solution of ionic liquids in aqueous phosphate buffer for 5 min at room temperature, followed by a denaturation step at 95 °C for 10 min and a simple filtration to remove residual biopolymers. A set of 22 ionic liquids was tested in a buffer system and 1-ethyl-3-methylimidazolium dimethylphosphate, as well as the environmentally benign choline formate, were identified as ideal candidates. With this strategy, the quality of the genomic DNA extracted was significantly improved and the extraction protocol was notably simplified compared with a well-established method.

  13. PMS2 gene mutation results in DNA mismatch repair system failure in a case of adult granulosa cell tumor

    OpenAIRE

    Wang, Wen-Chung; Lee, Ya-Ting; Lai, Yen-Chein

    2017-01-01

    Background Granulosa cell tumors are rare ovarian malignancies. Their characteristics include unpredictable indolent growth with malignant potential and late recurrence. Approximately 95% are of adult type. Recent molecular studies have characterized the FOXL2 402C?>?G mutation in adult granulosa cell tumor. Our previous case report showed that unique FOXL2 402C?>?G mutation and defective DNA mismatch repair system are associated with the development of adult granulosa cell tumor. Findings In...

  14. Protein restriction and cancer.

    Science.gov (United States)

    Yin, Jie; Ren, Wenkai; Huang, Xingguo; Li, Tiejun; Yin, Yulong

    2018-03-26

    Protein restriction without malnutrition is currently an effective nutritional intervention known to prevent diseases and promote health span from yeast to human. Recently, low protein diets are reported to be associated with lowered cancer incidence and mortality risk of cancers in human. In murine models, protein restriction inhibits tumor growth via mTOR signaling pathway. IGF-1, amino acid metabolic programing, FGF21, and autophagy may also serve as potential mechanisms of protein restriction mediated cancer prevention. Together, dietary intervention aimed at reducing protein intake can be beneficial and has the potential to be widely adopted and effective in preventing and treating cancers. Copyright © 2018 Elsevier B.V. All rights reserved.

  15. Patterns of exchange of forensic DNA data in the European Union through the Prüm system.

    Science.gov (United States)

    Santos, Filipe; Machado, Helena

    2017-07-01

    This paper presents a study of the 5-year operation (2011-2015) of the transnational exchange of forensic DNA data between Member States of the European Union (EU) for the purpose of combating cross-border crime and terrorism within the so-called Prüm system. This first systematisation of the full official statistical dataset provides an overall assessment of the match figures and patterns of operation of the Prüm system for DNA exchange. These figures and patterns are analysed in terms of the differentiated contributions by participating EU Member States. The data suggest a trend for West and Central European countries to concentrate the majority of Prüm matches, while DNA databases of Eastern European countries tend to contribute with profiles of people that match stains in other countries. In view of the necessary transparency and accountability of the Prüm system, more extensive and informative statistics would be an important contribution to the assessment of its functioning and societal benefits. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  16. Performance Characteristics and Comparison of Abbott and artus Real-Time Systems for Hepatitis B Virus DNA Quantification ▿

    Science.gov (United States)

    Ismail, Ashrafali M.; Sivakumar, Jayashree; Anantharam, Raghavendran; Dayalan, Sujitha; Samuel, Prasanna; Fletcher, Gnanadurai J.; Gnanamony, Manu; Abraham, Priya

    2011-01-01

    Virological monitoring of hepatitis B virus (HBV) DNA is critical to the management of HBV infection. With several HBV DNA quantification assays available, it is important to use the most efficient testing system for virological monitoring. In this study, we evaluated the performance characteristics and comparability of three HBV DNA quantification systems: Abbott HBV real-time PCR (Abbott PCR), artus HBV real-time PCR with QIAamp DNA blood kit purification (artus-DB), and artus HBV real-time PCR with the QIAamp DSP virus kit purification (artus-DSP). The lower limits of detection of these systems were established against the WHO international standards for HBV DNA and were found to be 1.43, 82, and 9 IU/ml, respectively. The intra-assay and interassay coefficients of variation of plasma samples (1 to 6 log10 IU/ml) ranged between 0.05 to 8.34% and 0.16 to 3.48% for the Abbott PCR, 1.53 to 26.85% and 0.50 to 12.89% for artus-DB, and 0.29 to 7.42% and 0.94 to 3.01% for artus-DSP, respectively. Ninety HBV clinical samples were used for comparison of assays, and paired quantitative results showed strong correlation by linear regression analysis (artus-DB with Abbott PCR, r = 0.95; Abbott PCR with artus-DSP, r = 0.97; and artus-DSP with artus-DB, r = 0.94). Bland-Altman analysis showed a good level of agreement for Abbott PCR and artus-DSP, with a mean difference of 0.10 log10 IU/ml and limits of agreement of −0.91 to 1.11 log10 IU/ml. No genotype-specific bias was seen in all three systems for HBV genotypes A, C, and D, which are predominant in this region. This finding illustrates that the Abbott real-time HBV and artus-DSP systems show more comparable performance than the artus-DB system, meeting the current guidelines for assays to be used in the management of hepatitis B. PMID:21795507

  17. Performance characteristics and comparison of Abbott and artus real-time systems for hepatitis B virus DNA quantification.

    Science.gov (United States)

    Ismail, Ashrafali M; Sivakumar, Jayashree; Anantharam, Raghavendran; Dayalan, Sujitha; Samuel, Prasanna; Fletcher, Gnanadurai J; Gnanamony, Manu; Abraham, Priya

    2011-09-01

    Virological monitoring of hepatitis B virus (HBV) DNA is critical to the management of HBV infection. With several HBV DNA quantification assays available, it is important to use the most efficient testing system for virological monitoring. In this study, we evaluated the performance characteristics and comparability of three HBV DNA quantification systems: Abbott HBV real-time PCR (Abbott PCR), artus HBV real-time PCR with QIAamp DNA blood kit purification (artus-DB), and artus HBV real-time PCR with the QIAamp DSP virus kit purification (artus-DSP). The lower limits of detection of these systems were established against the WHO international standards for HBV DNA and were found to be 1.43, 82, and 9 IU/ml, respectively. The intra-assay and interassay coefficients of variation of plasma samples (1 to 6 log(10) IU/ml) ranged between 0.05 to 8.34% and 0.16 to 3.48% for the Abbott PCR, 1.53 to 26.85% and 0.50 to 12.89% for artus-DB, and 0.29 to 7.42% and 0.94 to 3.01% for artus-DSP, respectively. Ninety HBV clinical samples were used for comparison of assays, and paired quantitative results showed strong correlation by linear regression analysis (artus-DB with Abbott PCR, r = 0.95; Abbott PCR with artus-DSP, r = 0.97; and artus-DSP with artus-DB, r = 0.94). Bland-Altman analysis showed a good level of agreement for Abbott PCR and artus-DSP, with a mean difference of 0.10 log(10) IU/ml and limits of agreement of -0.91 to 1.11 log(10) IU/ml. No genotype-specific bias was seen in all three systems for HBV genotypes A, C, and D, which are predominant in this region. This finding illustrates that the Abbott real-time HBV and artus-DSP systems show more comparable performance than the artus-DB system, meeting the current guidelines for assays to be used in the management of hepatitis B.

  18. Pair Natural Orbital Restricted Open-Shell Configuration Interaction (PNO-ROCIS) Approach for Calculating X-ray Absorption Spectra of Large Chemical Systems.

    Science.gov (United States)

    Maganas, Dimitrios; DeBeer, Serena; Neese, Frank

    2018-02-08

    In this work, the efficiency of first-principles calculations of X-ray absorption spectra of large chemical systems is drastically improved. The approach is based on the previously developed restricted open-shell configuration interaction singles (ROCIS) method and its parametrized version, based on a density functional theory (DFT) ground-state determinant ROCIS/DFT. The combination of the ROCIS or DFT/ROCIS methods with the well-known machinery of the pair natural orbitals (PNOs) leads to the new PNO-ROCIS and PNO-ROCIS/DFT variants. The PNO-ROCIS method can deliver calculated metal K-, L-, and M-edge XAS spectra orders of magnitude faster than ROCIS while maintaining an accuracy with calculated spectral parameters better than 1% relative to the original ROCIS method (referred to as canonical ROCIS). The method is of a black box character, as it does not require any user adjustments, while it scales quadratically with the system size. It is shown that for large systems, the size of the virtual molecular orbital (MO) space is reduced by more than 90% with respect to the canonical ROCIS method. This allows one to compute the X-ray absorption spectra of a variety of large "real-life" chemical systems featuring hundreds of atoms using a first-principles wave-function-based approach. Examples chosen from the fields of bioinorganic and solid-state chemistry include the Co K-edge XAS spectrum of aquacobalamin [H 2 OCbl] + , the Fe L-edge XAS spectrum of deoxymyoglobin (DMb), the Ti L-edge XAS spectrum of rutile TiO 2 , and the Fe M-edge spectrum of α-Fe 2 O 3 hematite. In the largest calculations presented here, molecules with more than 700 atoms and cluster models with more than 50 metal centers were employed. In all the studied cases, very good to excellent agreement with experiment is obtained. It will be shown that the PNO-ROCIS method provides an unprecedented performance of wave-function-based methods in the field of computational X-ray spectroscopy.

  19. Molecular mechanism of short-patch repair of radiation-damaged DNA by in vitro reconstituted systems

    International Nuclear Information System (INIS)

    Matsumoto, Y.; Kim, K.; Biade, S.

    1995-01-01

    Objective: Short-patch excision repair is the major pathway to correct DNA damage such as modified bases, apurinic/apyrimidinic (AP) sites and single-strand breaks. Recently this repair reaction was demonstrated to proceed by two alternative pathways: DNA polymerase β (pol β)-dependent pathway and proliferating cell nuclear antigen (PCNA)-dependent pathway. In this work, we focused to compare substrate specificity of these two repair pathways and elucidate their roles in cellular responses to radiation damage. Materials and Methods: Three protein fractions, AP endonuclease, pol β, and BE-1B, which are required for the pol β-dependent pathway, and five protein fractions, AP endonuclease, BE-1B (these two are common to the pol β-dependent pathway), PCNA, pol δ, and BE-2, which are essential for the PCNA-dependent pathway were obtained from Xenopus laevis ovaries through column chromatography. The circular DNA containing either one of the following three lesions: a natural AP site, its synthetic analog, 3-hydroxy-2-hydroxymethyltetrahydrofuran (tetrahydrofuran), and 5-iododeoxyuridine (IdU), was prepared by in vitro ligation of oligonucleotides to a gapped circular DNA. The IdU-containing DNA was irradiated with 312 nm UV light prior to repair reaction. In addition, DNA carrying a single-strand break was obtained by Cs-137 irradiation. Repair reactions of these substrate DNAs were conducted with either the reconstituted system for the pol β-dependent pathway or the one for the PCNA-dependent pathway. After the reaction, repaired and unrepaired DNAs were separated by gel electrophoresis and quantitated. Results: The pol β-dependent reconstituted system was able to repair natural AP sites but not tetrahydrofuran sites or UV-irradiated IdU. The single-strand breaks generated by γ-irradiation were partially repaired by thepol β-dependent pathway. The PCNA-dependent system was able to repair natural AP sites, tetrahydrofuran sites, and most of the single

  20. Genetic characterization of cells of homocystinuria patients with disrupted DNA repair system

    International Nuclear Information System (INIS)

    Sinel'shchikova, T.A.; L'vova, G.N.; Shoniya, N.N.; Zasukhina, G.D.

    1986-01-01

    Fibroblasts obtained from biopsy material and lymphocytes of patients with homocystinuria were investigated for repair activity according to the following criteria: rejoined DNA breaks, induced by 4-nitroquinoline-1-oxide and γ-radiation; indices of reactivation and induced mutagenesis of smallpox vaccine virus treated with these mutagens. In lymphocytes a defect of DNA repair was observed according to all criteria investigated. During passage of fibroblast cultures, inhibition of repair activity of cells was preserved according to γ-type. Increase in the number of spontaneous and γ-induced mutations of virus was noted according to degree of passage of fibroblasts

  1. Comparison of the performance of the borax buffer-based HRP-enhanced reagent and the 'Lumi-Phos 530' chemiluminescence systems in the detection of biotinylated DNA.

    Science.gov (United States)

    Cercek, B; Roby, K; Siaw, M

    1995-01-01

    A comparison of two chemiluminescence methods, the borax buffer-based HRP-enhanced reagent and Lumi-Phos 530, applied to the detection of a biotinylated 30-mer DNA slot blotted onto a nylon membrane, is presented. A streptavidin-HRP and streptavidin-ALP mediated detection system was used. The HRP-enhanced system is up to 15-fold greater with respect to the signal/background ratios than the Lumi-Phos 530 system at 0.5 microgram biotinylated DNA with at least a two-fold improvement in detection sensitivity for 0.5 ng biotinylated DNA.

  2. DNA capture reveals transoceanic gene flow in endangered river sharks

    OpenAIRE

    Li, Chenhong; Corrigan, Shannon; Yang, Lei; Straube, Nicolas; Harris, Mark; Hofreiter, Michael; White, William T.; Naylor, Gavin J. P.

    2015-01-01

    The river sharks of the genus Glyphis, widely feared as man-eaters throughout India, remain very poorly known to science. The group constitutes five described species, all of which are considered highly endangered and restricted to freshwater systems in Australasia and Southeast Asia. DNA sequence data derived from 19th-century dried museum material augmented with contemporary samples indicates that only three of the five currently described species are valid; that there is a genetically dist...

  3. Intrauterine growth restriction

    Directory of Open Access Journals (Sweden)

    Bernardita Donoso Bernales

    2012-07-01

    Full Text Available It is estimated that the true prevalence of intrauterine growth restriction is 3-10% of all pregnancies, making this fetal condition one of the most frequent obstetric problems, together with premature labor and premature rupture of membranes. The article stresses the importance of early diagnosis because of the associated risks.

  4. Late gestational nutrient restriction

    DEFF Research Database (Denmark)

    Tygesen, Malin Plumhoff; Nielsen, Mette Olaf; Nørgaard, Peder

    2008-01-01

    We investigated the effect of 50% nutrient restriction during the last 6 weeks of gestation on twin-pregnant ewes' plasma glucose, non-esterified fatty acid, ß-hydroxybutyrate, insulin, IGF-1 and leptin concentrations and the effects on lamb birth weight and ewes' lactation performance. Plasma...

  5. Restricted Variance Interaction Effects

    DEFF Research Database (Denmark)

    Cortina, Jose M.; Köhler, Tine; Keeler, Kathleen R.

    2018-01-01

    Although interaction hypotheses are increasingly common in our field, many recent articles point out that authors often have difficulty justifying them. The purpose of this article is to describe a particular type of interaction: the restricted variance (RV) interaction. The essence of the RV int...

  6. Kinetics of DNA tile dimerization.

    Science.gov (United States)

    Jiang, Shuoxing; Yan, Hao; Liu, Yan

    2014-06-24

    Investigating how individual molecular components interact with one another within DNA nanoarchitectures, both in terms of their spatial and temporal interactions, is fundamentally important for a better understanding of their physical behaviors. This will provide researchers with valuable insight for designing more complex higher-order structures that can be assembled more efficiently. In this report, we examined several spatial factors that affect the kinetics of bivalent, double-helical (DH) tile dimerization, including the orientation and number of sticky ends (SEs), the flexibility of the double helical domains, and the size of the tiles. The rate constants we obtained confirm our hypothesis that increased nucleation opportunities and well-aligned SEs accelerate tile-tile dimerization. Increased flexibility in the tiles causes slower dimerization rates, an effect that can be reversed by introducing restrictions to the tile flexibility. The higher dimerization rates of more rigid tiles results from the opposing effects of higher activation energies and higher pre-exponential factors from the Arrhenius equation, where the pre-exponential factor dominates. We believe that the results presented here will assist in improved implementation of DNA tile based algorithmic self-assembly, DNA based molecular robotics, and other specific nucleic acid systems, and will provide guidance to design and assembly processes to improve overall yield and efficiency.

  7. Genomic and functional integrity of the hematopoietic system requires tolerance of oxidative DNA lesions

    DEFF Research Database (Denmark)

    Martín-Pardillos, Ana; Tsaalbi-Shtylik, Anastasia; Chen, Si

    2017-01-01

    -distorting nucleotide lesions, resulted in the perinatal loss of hematopoietic stem cells, progressive loss of bone marrow, and fatal aplastic anemia between 3 and 4 months of age. This was associated with replication stress, genomic breaks, DNA damage signaling, senescence, and apoptosis in bone marrow. Surprisingly...

  8. Design and Test of an Oscillation-based System Architecture for DNA Sensor Arrays

    NARCIS (Netherlands)

    Liu, Hongyuan; Kerkhoff, Hans G.; Richardson, Andrew; Zhang, X.; Nouet, Pascal; Azais, Florence

    2005-01-01

    A DfT strategy for MEMS-based DNA sensors is investigated in this paper. Based on a fault-free and defect model developed for a single sensing element and the VHDL-AMS simulation results, it is implied that an oscillation-based interface might be a potential solution for both testing and read out of

  9. DNA damage, metabolism and aging in pro-inflammatory T cells: Rheumatoid arthritis as a model system.

    Science.gov (United States)

    Li, Yinyin; Goronzy, Jörg J; Weyand, Cornelia M

    2018-05-01

    The aging process is the major driver of morbidity and mortality, steeply increasing the risk to succumb to cancer, cardiovascular disease, infection and neurodegeneration. Inflammation is a common denominator in age-related pathologies, identifying the immune system as a gatekeeper in aging overall. Among immune cells, T cells are long-lived and exposed to intense replication pressure, making them sensitive to aging-related abnormalities. In successful T cell aging, numbers of naïve cells, repertoire diversity and activation thresholds are preserved as long as possible; in maladaptive T cell aging, protective T cell functions decline and pro-inflammatory effector cells are enriched. Here, we review in the model system of rheumatoid arthritis (RA) how maladaptive T cell aging renders the host susceptible to chronic, tissue-damaging inflammation. In T cells from RA patients, known to be about 20years pre-aged, three interconnected functional domains are altered: DNA damage repair, metabolic activity generating energy and biosynthetic precursor molecules, and shaping of plasma membranes to promote T cell motility. In each of these domains, key molecules and pathways have now been identified, including the glycolytic enzymes PFKFB3 and G6PD; the DNA repair molecules ATM, DNA-PKcs and MRE11A; and the podosome marker protein TKS5. Some of these molecules may help in defining targetable pathways to slow the T cell aging process. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. PMS2 gene mutation results in DNA mismatch repair system failure in a case of adult granulosa cell tumor.

    Science.gov (United States)

    Wang, Wen-Chung; Lee, Ya-Ting; Lai, Yen-Chein

    2017-03-27

    Granulosa cell tumors are rare ovarian malignancies. Their characteristics include unpredictable indolent growth with malignant potential and late recurrence. Approximately 95% are of adult type. Recent molecular studies have characterized the FOXL2 402C > G mutation in adult granulosa cell tumor. Our previous case report showed that unique FOXL2 402C > G mutation and defective DNA mismatch repair system are associated with the development of adult granulosa cell tumor. In this study, the DNA sequences of four genes, MSH2, MLH1, MSH6, and PMS2, in the DNA mismatch repair system were determined via direct sequencing to elucidate the exact mechanism for the development of this granulosa cell tumor. The results showed that two missense germline mutations, T485K and N775L, inactivate the PMS2 gene. The results of this case study indicated that although FOXL2 402C > G mutation determines the development of granulosa cell tumor, PMS2 mutation may be the initial driver of carcinogenesis. Immunohistochemistry-based tumor testing for mismatch repair gene expression may be necessary for granulosa cell tumors to determine their malignant potential or if they are part of Lynch syndrome.

  11. DNA barcoding discriminates freshwater fishes from southeastern Nigeria and provides river system-level phylogeographic resolution within some species.

    Science.gov (United States)

    Nwani, Christopher D; Becker, Sven; Braid, Heather E; Ude, Emmanuel F; Okogwu, Okechukwu I; Hanner, Robert

    2011-10-01

    Fishes are the main animal protein source for human beings and play a vital role in aquatic ecosystems and food webs. Fish identification can be challenging, especially in the tropics (due to high diversity), and this is particularly true for larval forms or fragmentary remains. DNA barcoding, which uses the 5' region of the mitochondrial cytochrome c oxidase subunit I (COI) as a target gene, is an efficient method for standardized species-level identification for biodiversity assessment and conservation, pending the establishment of reference sequence libraries. In this study, fishes were collected from three rivers in southeastern Nigeria, identified morphologically, and imaged digitally. DNA was extracted, PCR-amplified, and the standard barcode region was bidirectionally sequenced for 363 individuals belonging to 70 species in 38 genera. All specimen provenance data and associated sequence information were recorded in the barcode of life data systems (BOLD; www.barcodinglife.org ). Analytical tools on BOLD were used to assess the performance of barcoding to identify species. Using neighbor-joining distance comparison, the average genetic distance was 60-fold higher between species than within species, as pairwise genetic distance estimates averaged 10.29% among congeners and only 0.17% among conspecifics. Despite low levels of divergence within species, we observed river system-specific haplotype partitioning within eight species (11.4% of all species). Our preliminary results suggest that DNA barcoding is very effective for species identification of Nigerian freshwater fishes.

  12. Force induced DNA melting

    International Nuclear Information System (INIS)

    Santosh, Mogurampelly; Maiti, Prabal K

    2009-01-01

    When pulled along the axis, double-strand DNA undergoes a large conformational change and elongates by roughly twice its initial contour length at a pulling force of about 70 pN. The transition to this highly overstretched form of DNA is very cooperative. Applying a force perpendicular to the DNA axis (unzipping), double-strand DNA can also be separated into two single-stranded DNA, this being a fundamental process in DNA replication. We study the DNA overstretching and unzipping transition using fully atomistic molecular dynamics (MD) simulations and argue that the conformational changes of double-strand DNA associated with either of the above mentioned processes can be viewed as force induced DNA melting. As the force at one end of the DNA is increased the DNA starts melting abruptly/smoothly above a critical force depending on the pulling direction. The critical force f m , at which DNA melts completely decreases as the temperature of the system is increased. The melting force in the case of unzipping is smaller compared to the melting force when the DNA is pulled along the helical axis. In the case of melting through unzipping, the double-strand separation has jumps which correspond to the different energy minima arising due to sequence of different base pairs. The fraction of Watson-Crick base pair hydrogen bond breaking as a function of force does not show smooth and continuous behavior and consists of plateaus followed by sharp jumps.

  13. DNA hosted and aligned in aqueous interstitia of a lamellar liquid crystal – a membrane–biomacromolecule interaction model system

    KAUST Repository

    Carlsson, Nils; Jonsson, Fabian; Wilhelmsson, L. Marcus; Nordé n, Bengt; Å kerman, Bjö rn

    2013-01-01

    We report that DNA molecules can be intercalated and macroscopically oriented in the aqueous interstitia of a lyotropic lamellar liquid crystal. Using UV-vis linear dichroism and fluorescence spectroscopy we show that double-stranded oligonucleotides (25 base pairs) in the water-octanoate-decanol system remain base-paired in the B conformation and are confined in two dimensions, with the helix axis preferentially parallel to the lipid bilayer surfaces but free to rotate within this plane. The degree of helix confinement and the corresponding 2-D orientation can be improved by decreasing the thickness of the water interstitia via the fraction of water in the ternary mixture. Not surprisingly, the corresponding single-stranded oligonucleotides are not aligned, with their persistence length being short in comparison to the lamellar interstitium thickness. We propose this as a model system for studying interactions of DNA-ligand complexes near a lipid bilayer membrane which we demonstrate by using dye probes that are either covalently attached to one end of the oligonucleotide or reversibly bound by intercalation between the base pairs. Three cationic dyes, all strongly bound by intercalation to DNA when free in solution, are found to not bind to DNA but to prefer the membrane surface. The covalently attached Cy5 also binds to the bilayer while Cy3 tends to end-stack to the oligonucleotide duplex. The orientation of Cy5 parallel to the membrane indicates that electrostatic surface binding predominates over insertion into the hydrophobic interior of the membrane. Anionic and zwitterionic dyes (FAM and ROX) are found to remain randomly oriented in the water between the lipid bilayer surfaces. © The Royal Society of Chemistry.

  14. DNA methylation as a dynamic regulator of development and disease processes: spotlight on the prostate.

    Science.gov (United States)

    Keil, Kimberly P; Vezina, Chad M

    2015-01-01

    Prostate development, benign hyperplasia and cancer involve androgen and growth factor signaling as well as stromal-epithelial interactions. We review how DNA methylation influences these and related processes in other organ systems such as how proliferation is restricted to specific cell populations during defined temporal windows, how androgens elicit their actions and how cells establish, maintain and remodel DNA methylation in a time and cell specific fashion. We also discuss mechanisms by which hormones and endocrine disrupting chemicals reprogram DNA methylation in the prostate and elsewhere and examine evidence for a reawakening of developmental epigenetic pathways as drivers of prostate cancer and benign prostate hyperplasia.

  15. Making sense of the noise: The effect of hydrology on silver carp eDNA detection in the Chicago area waterway system.

    Science.gov (United States)

    Song, Jeffery W; Small, Mitchell J; Casman, Elizabeth A

    2017-12-15

    Environmental DNA (eDNA) sampling is an emerging tool for monitoring the spread of aquatic invasive species. One confounding factor when interpreting eDNA sampling evidence is that eDNA can be present in the water in the absence of living target organisms, originating from excreta, dead tissue, boats, or sewage effluent, etc. In the Chicago Area Waterway System (CAWS), electric fish dispersal barriers were built to prevent non-native Asian carp species from invading Lake Michigan, and yet Asian carp eDNA has been detected above the barriers sporadically since 2009. In this paper the influence of stream flow characteristics in the CAWS on the probability of invasive Asian carp eDNA detection in the CAWS from 2009 to 2012 was examined. In the CAWS, the direction of stream flow is mostly away from Lake Michigan, though there are infrequent reversals in flow direction towards Lake Michigan during dry spells. We find that the flow reversal volume into the Lake has a statistically significant positive relationship with eDNA detection probability, while other covariates, like gage height, precipitation, season, water temperature, dissolved oxygen concentration, pH and chlorophyll concentration do not. This suggests that stream flow direction is highly influential on eDNA detection in the CAWS and should be considered when interpreting eDNA evidence. We also find that the beta-binomial regression model provides a stronger fit for eDNA detection probability compared to a binomial regression model. This paper provides a statistical modeling framework for interpreting eDNA sampling evidence and for evaluating covariates influencing eDNA detection. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. DNA preservation in silk.

    Science.gov (United States)

    Liu, Yawen; Zheng, Zhaozhu; Gong, He; Liu, Meng; Guo, Shaozhe; Li, Gang; Wang, Xiaoqin; Kaplan, David L

    2017-06-27

    The structure of DNA is susceptible to alterations at high temperature and on changing pH, irradiation and exposure to DNase. Options to protect and preserve DNA during storage are important for applications in genetic diagnosis, identity authentication, drug development and bioresearch. In the present study, the stability of total DNA purified from human dermal fibroblast cells, as well as that of plasmid DNA, was studied in silk protein materials. The DNA/silk mixtures were stabilized on filter paper (silk/DNA + filter) or filter paper pre-coated with silk and treated with methanol (silk/DNA + PT-filter) as a route to practical utility. After air-drying and water extraction, 50-70% of the DNA and silk could be retrieved and showed a single band on electrophoretic gels. 6% silk/DNA + PT-filter samples provided improved stability in comparison with 3% silk/DNA + filter samples and DNA + filter samples for DNA preservation, with ∼40% of the band intensity remaining at 37 °C after 40 days and ∼10% after exposure to UV light for 10 hours. Quantitative analysis using the PicoGreen assay confirmed the results. The use of Tris/borate/EDTA (TBE) buffer enhanced the preservation and/or extraction of the DNA. The DNA extracted after storage maintained integrity and function based on serving as a functional template for PCR amplification of the gene for zinc finger protein 750 (ZNF750) and for transgene expression of red fluorescence protein (dsRed) in HEK293 cells. The high molecular weight and high content of a crystalline beta-sheet structure formed on the coated surfaces likely accounted for the preservation effects observed for the silk/DNA + PT-filter samples. Although similar preservation effects were also obtained for lyophilized silk/DNA samples, the rapid and simple processing available with the silk-DNA-filter membrane system makes it appealing for future applications.

  17. Microbial iron mats at the Mid-Atlantic Ridge and evidence that Zetaproteobacteria may be restricted to iron-oxidizing marine systems.

    Directory of Open Access Journals (Sweden)

    Jarrod J Scott

    Full Text Available Chemolithoautotrophic iron-oxidizing bacteria play an essential role in the global iron cycle. Thus far, the majority of marine iron-oxidizing bacteria have been identified as Zetaproteobacteria, a novel class within the phylum Proteobacteria. Marine iron-oxidizing microbial communities have been found associated with volcanically active seamounts, crustal spreading centers, and coastal waters. However, little is known about the presence and diversity of iron-oxidizing communities at hydrothermal systems along the slow crustal spreading center of the Mid-Atlantic Ridge. From October to November 2012, samples were collected from rust-colored mats at three well-known hydrothermal vent systems on the Mid-Atlantic Ridge (Rainbow, Trans-Atlantic Geotraverse, and Snake Pit using the ROV Jason II. The goal of these efforts was to determine if iron-oxidizing Zetaproteobacteria were present at sites proximal to black smoker vent fields. Small, diffuse flow venting areas with high iron(II concentrations and rust-colored microbial mats were observed at all three sites proximal to black smoker chimneys. A novel, syringe-based precision sampler was used to collect discrete microbial iron mat samples at the three sites. The presence of Zetaproteobacteria was confirmed using a combination of 16S rRNA pyrosequencing and single-cell sorting, while light micros-copy revealed a variety of iron-oxyhydroxide structures, indicating that active iron-oxidizing communities exist along the Mid-Atlantic Ridge. Sequencing analysis suggests that these iron mats contain cosmopolitan representatives of Zetaproteobacteria, but also exhibit diversity that may be uncommon at other iron-rich marine sites studied to date. A meta-analysis of publically available data encompassing a variety of aquatic habitats indicates that Zetaproteobacteria are rare if an iron source is not readily available. This work adds to the growing understanding of Zetaproteobacteria ecology and suggests

  18. Development of a calcium phosphate co-precipitate/poly(lactide-co-glycolide) DNA delivery system: release kinetics and cellular transfection studies.

    Science.gov (United States)

    Kofron, Michelle D; Laurencin, Cato T

    2004-06-01

    One of the most common non-viral methods for the introduction of foreign deoxyribonucleic acid (DNA) into cultured cells is calcium phosphate co-precipitate transfection. This technique involves the encapsulation of DNA within a calcium phosphate co-precipitate, particulate addition to in vitro cell culture, endocytosis of the co-precipitate, and exogenous DNA expression by the transfected cell. In this study, we fabricated a novel non-viral gene transfer system by adsorbing DNA, encapsulated in calcium phosphate (DNA/Ca-P) co-precipitates, to biodegradable two- and three-dimensional poly(lactide-co-glycolide) matrices (2D-DNA/Ca-P/PLAGA, 3D-DNA/Ca-P/PLAGA). Co-precipitate release studies demonstrated an initial burst release over the first 48 h. By day 7, approximately 96% of the initially adsorbed DNA/Ca-P co-precipitate had been released. This was followed by low levels of co-precipitate release for 42 days. Polymerase chain reaction was used to demonstrate the ability of the released DNA containing co-precipitates to transfect SaOS-2 cells cultured in vitro on the 3D-DNA/Ca-P/PLAGA matrix and maintenance of the structural integrity of the exogenous DNA. In summary, a promising system for the incorporation and controlled delivery of exogenous genes encapsulated within a calcium phosphate co-precipitate from biodegradable polymeric matrices has been developed and may have applicability to the delivery of therapeutic genes and the transfection of other cell types.

  19. Meta-analysis reveals an association of STAT4 polymorphisms with systemic autoimmune disorders and anti-dsDNA antibody.

    Science.gov (United States)

    Zheng, Junfeng; Yin, Junping; Huang, Renliang; Petersen, Frank; Yu, Xinhua

    2013-08-01

    Signal transducer and activator of transcription 4 (STAT4) has been recently identified as a susceptibility gene for multiple autoimmune diseases. Here we performed a comprehensive analysis of the association between STAT4 and several different autoimmune disorders to identify potential common inflammatory principles behind this association. Our meta-analysis revealed that the STAT4 rs7574865 polymorphism is associated with four autoimmune diseases with systemic pathology, including systemic lupus erythematosus (OR = 1.52; 95% CI = 1.48 - 1.56, Prs7574865 polymorphism is associated with the presence of autoantibodies with systemic reactivity (anti-ds-DNA antibodies) in SLE patients (OR = 1.37; 95% CI = 1.21 - 1.56, P = 1.12 × 10(-6)). However, no such specific association was seen in RA with regard to the presence of non-systemically reacting antibodies, including rheumatoid factor and anti-cyclic citrullinated peptide antibodies. Taken together, these results suggest that STAT4 polymorphisms are associated with autoimmune diseases which are characterized by a systemic pathology and anti-dsDNA antibody. Copyright © 2013 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

  20. Exposure to bacterial DNA before hemorrhagic shock strongly aggravates systemic inflammation and gut barrier loss via an IFN-gamma-dependent route

    NARCIS (Netherlands)

    Luyer, Misha D.; Buurman, Wim A.; Hadfoune, M.'hamed; Wolfs, T.; van't Veer, Cornelis; Jacobs, Jan A.; Dejong, Cornelis H.; Greve, Jan Willem M.

    2007-01-01

    OBJECTIVE: To investigate the role of bacterial DNA in development of an excessive inflammatory response and loss of gut barrier loss following systemic hypotension. SUMMARY BACKGROUND DATA: Bacterial infection may contribute to development of inflammatory complications following major surgery;

  1. 48 CFR 552.203-71 - Restriction on Advertising.

    Science.gov (United States)

    2010-10-01

    ... 48 Federal Acquisition Regulations System 4 2010-10-01 2010-10-01 false Restriction on Advertising... Restriction on Advertising. As prescribed in 503.570-2, insert the following clause: Restriction on Advertising (SEP 1999) The Contractor shall not refer to this contract in commercial advertising or similar...

  2. DNA-Catalyzed Henry Reaction in Pure Water and the Striking Influence of Organic Buffer Systems

    Directory of Open Access Journals (Sweden)

    Marleen Häring

    2015-03-01

    Full Text Available In this manuscript we report a critical evaluation of the ability of natural DNA to mediate the nitroaldol (Henry reaction at physiological temperature in pure water. Under these conditions, no background reaction took place (i.e., control experiment without DNA. Both heteroaromatic aldehydes (e.g., 2-pyridinecarboxaldehyde and aromatic aldehydes bearing strong or moderate electron-withdrawing groups reacted satisfactorily with nitromethane obeying first order kinetics and affording the corresponding β-nitroalcohols in good yields within 24 h. In contrast, aliphatic aldehydes and aromatic aldehydes having electron-donating groups either did not react or were poorly converted. Moreover, we discovered that a number of metal-free organic buffers efficiently promote the Henry reaction when they were used as reaction media without adding external catalysts. This constitutes an important observation because the influence of organic buffers in chemical processes has been traditionally underestimated.

  3. DNA repair systems and the pathogenesis of Mycobacterium tuberculosis: varying activities at different stages of infection

    OpenAIRE

    Gorna, AE; Bowater, RP; Dziadek, J

    2010-01-01

    Mycobacteria, including most of all MTB (Mycobacterium tuberculosis), cause pathogenic infections in humans and, during the infectious process, are exposed to a range of environmental insults, including the host's immune response. From the moment MTB is exhaled by infected individuals, through an active and latent phase in the body of the new host, until the time they reach the reactivation stage, MTB is exposed to many types of DNA-damaging agents. Like all cellular organisms, MTB has effici...

  4. Using Synthetic Nanopores for Single-Molecule Analyses: Detecting SNPs, Trapping DNA Molecules, and the Prospects for Sequencing DNA

    Science.gov (United States)

    Dimitrov, Valentin V.

    2009-01-01

    This work focuses on studying properties of DNA molecules and DNA-protein interactions using synthetic nanopores, and it examines the prospects of sequencing DNA using synthetic nanopores. We have developed a method for discriminating between alleles that uses a synthetic nanopore to measure the binding of a restriction enzyme to DNA. There exists…

  5. Epigenetic regulation of caloric restriction in aging

    Directory of Open Access Journals (Sweden)

    Daniel Michael

    2011-08-01

    Full Text Available Abstract The molecular mechanisms of aging are the subject of much research and have facilitated potential interventions to delay aging and aging-related degenerative diseases in humans. The aging process is frequently affected by environmental factors, and caloric restriction is by far the most effective and established environmental manipulation for extending lifespan in various animal models. However, the precise mechanisms by which caloric restriction affects lifespan are still not clear. Epigenetic mechanisms have recently been recognized as major contributors to nutrition-related longevity and aging control. Two primary epigenetic codes, DNA methylation and histone modification, are believed to dynamically influence chromatin structure, resulting in expression changes of relevant genes. In this review, we assess the current advances in epigenetic regulation in response to caloric restriction and how this affects cellular senescence, aging and potential extension of a healthy lifespan in humans. Enhanced understanding of the important role of epigenetics in the control of the aging process through caloric restriction may lead to clinical advances in the prevention and therapy of human aging-associated diseases.

  6. Detection of Wuchereria bancrofti DNA in paired serum and urine samples using polymerase chain reaction-based systems

    Directory of Open Access Journals (Sweden)

    Camila Ximenes

    2014-12-01

    Full Text Available The Global Program for the Elimination of Lymphatic Filariasis (GPELF aims to eliminate this disease by the year 2020. However, the development of more specific and sensitive tests is important for the success of the GPELF. The present study aimed to standardise polymerase chain reaction (PCR-based systems for the diagnosis of filariasis in serum and urine. Twenty paired biological urine and serum samples from individuals already known to be positive for Wuchereria bancrofti were collected during the day. Conventional PCR and semi-nested PCR assays were optimised. The detection limit of the technique for purified W. bancrofti DNA extracted from adult worms was 10 fg for the internal systems (WbF/Wb2 and 0.1 fg by using semi-nested PCR. The specificity of the primers was confirmed experimentally by amplification of 1 ng of purified genomic DNA from other species of parasites. Evaluation of the paired urine and serum samples by the semi-nested PCR technique indicated only two of the 20 tested individuals were positive, whereas the simple internal PCR system (WbF/Wb2, which has highly promising performance, revealed that all the patients were positive using both samples. This study successfully demonstrated the possibility of using the PCR technique on urine for the diagnosis of W. bancrofti infection.

  7. A Novel Recombinant DNA System for High Efficiency Affinity Purification of Proteins in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Brian H. Carrick

    2016-03-01

    Full Text Available Isolation of endogenous proteins from Saccharomyces cerevisiae has been facilitated by inserting encoding polypeptide affinity tags at the C-termini of chromosomal open reading frames (ORFs using homologous recombination of DNA fragments. Tagged protein isolation is limited by a number of factors, including high cost of affinity resins for bulk isolation and low concentration of ligands on the resin surface, leading to low isolation efficiencies and trapping of contaminants. To address this, we have created a recombinant “CelTag” DNA construct from which PCR fragments can be created to easily tag C-termini of S. cerevisiae ORFs using selection for a nat1 marker. The tag has a C-terminal cellulose binding module to be used in the first affinity step. Microgranular cellulose is very inexpensive and has an effectively continuous ligand on its surface, allowing rapid, highly efficient purification with minimal background. Cellulose-bound proteins are released by specific cleavage of an included site for TEV protease, giving nearly pure product. The tag can be lifted from the recombinant DNA construct either with or without a 13x myc epitope tag between the target ORF and the TEV protease site. Binding of CelTag protein fusions to cellulose is stable to high salt, nonionic detergents, and 1 M urea, allowing stringent washing conditions to remove loosely associated components, as needed, before specific elution. It is anticipated that this reagent could allow isolation of protein complexes from large quantities of yeast extract, including soluble, membrane-bound, or nucleic acid-associated assemblies.

  8. A comprehensive complex systems approach to the study and analysis of mammalian cell cycle control system in the presence of DNA damage stress.

    Science.gov (United States)

    Abroudi, Ali; Samarasinghe, Sandhya; Kulasiri, Don

    2017-09-21

    Not many models of mammalian cell cycle system exist due to its complexity. Some models are too complex and hard to understand, while some others are too simple and not comprehensive enough. Moreover, some essential aspects, such as the response of G1-S and G2-M checkpoints to DNA damage as well as the growth factor signalling, have not been investigated from a systems point of view in current mammalian cell cycle models. To address these issues, we bring a holistic perspective to cell cycle by mathematically modelling it as a complex system consisting of important sub-systems that interact with each other. This retains the functionality of the system and provides a clearer interpretation to the processes within it while reducing the complexity in comprehending these processes. To achieve this, we first update a published ODE mathematical model of cell cycle with current knowledge. Then the part of the mathematical model relevant to each sub-system is shown separately in conjunction with a diagram of the sub-system as part of this representation. The model sub-systems are Growth Factor, DNA damage, G1-S, and G2-M checkpoint signalling. To further simplify the model and better explore the function of sub-systems, they are further divided into modules. Here we also add important new modules of: chk-related rapid cell cycle arrest, p53 modules expanded to seamlessly integrate with the rapid arrest module, Tyrosine phosphatase modules that activate Cyc_Cdk complexes and play a crucial role in rapid and delay arrest at both G1-S and G2-M, Tyrosine Kinase module that is important for inactivating nuclear transport of CycB_cdk1 through Wee1 to resist M phase entry, Plk1-Related module that is crucial in activating Tyrosine phosphatases and inactivating Tyrosine kinase, and APC-Related module to show steps in CycB degradation. This multi-level systems approach incorporating all known aspects of cell cycle allowed us to (i) study, through dynamic simulation of an ODE model

  9. Identification of differentially expressed genes induced by energy restriction using annealing control primer system from the liver and adipose tissues of broilers.

    Science.gov (United States)

    Wang, J W; Chen, W; Kang, X T; Huang, Y Q; Tian, Y D; Wang, Y B

    2012-04-01

    Female Arbor Acre broilers were divided into 2 groups at 18 d of age. One group of chickens had free access to feed (AL), and the other group of chickens had 30% energy restriction (ER). Adipose and hepatic RNA samples were collected at 48 d of age. We employed an accurate reverse-transcription (RT) PCR method that involves annealing control primers to identify the differentially expressed genes (DEG) between ER and AL groups. Using 20 annealing control primers, 43 differentially expressed bands (40 downregulated and 3 upregulated in the ER group) were detected from the hepatic tissue, whereas no differentially expressed bands were detected from the adipose tissue. It seems that energy restriction could induce more DEG in hepatic tissue than that in adipose tissue and could result in more gene-expression downregulation in hepatic tissue. Eight DEG (6 known and 2 unknown genes) were gained from hepatic tissue and confirmed by RT-PCR, which were all supported by released expressed sequence tag sequences. Their expressions were all downregulated by energy restriction in hepatic tissues. Six known genes are RPL7, RPLP1, FBXL12, ND1, ANTXR2, and SLC22A18, respectively, which seem to play essential roles in the protein translation, energy metabolism, and tumor inhibition. The alterations of gene expression in 3 selected genes, including ND1 (P < 0.01), FBXL12 (P < 0.01), and RPLP1 (P < 0.05), were supported by real-time quantitative RT-PCR reaction. Our data provide new insights on the metabolic state of broilers changed by energy restriction.

  10. Anti-double strand (ds) DNA antibody formation by NZB/W (F1) spleen cells in a microculture system detected by solid phase radioimmunoassay.

    Science.gov (United States)

    Okudaira, H; Terada, E; Ogita, T; Aotsuka, S; Yokohari, R

    1981-01-01

    A solid-phase radioimmunoassay method was devised to detect mouse anti-double strand (ds) DNA antibody. This method could easily detect the anti-dsDNA antibody in 1 : 10,000 dilutions (1 unit) of pooled 9-10-month-old female NZB/W F1 sera. The sensitivity was about 10(3)- and 10(2)-fold higher than that of the modified Farr method and of the double antibody technique respectively. NZB/W mice developed high titer anti-dsDNA antibody as they grew older. Spleen cells brought to a microculture system using flat-bottomed polystyrene plates produced anti-dsDNA antibody clearly detectable by solid-phase radioimmunoassay. Anti-dsDNA antibody produced in vitro (y units) was in close correlation with the anti-dsDNA antibody titer of the spleen donor (x units) (y = 4.8 X 10(-2) x -65, gamma = 0.94, P less than 0.001). A combination of the microculture system and solid-phase radioimmunoassay was recommended for the characterization of anti-dsDNA antibody-forming cells.

  11. Anti-double strand (ds) DNA antibody formation by NZB/W (F1) spleen cells in a microculture system detected by solid-phase radioimmunoassay

    International Nuclear Information System (INIS)

    Okudaira, H.; Terada, E.; Ogita, T.; Aotsuka, S.; Yokohari, R.

    1981-01-01

    A solid-phase radioimmunoassay method was devised to detect mouse anti-double strand (ds) DNA antibody. This method could easily detect the anti-ds DNA antibody in 1 : 10,000 dilutions (1 unit) of pooled 9-10 month-old female NZB/W F1 sera. The sensitivity was about 10 3 and 10 2 -fold higher than that of the modified Farr method and of the double antibody technique respectively. NZB/W mice developed high titer anti-dsDNA antibody as they grew older. Spleen cells brought to a microculture system using flat-bottomed polystyrene plates produced anti-dsDNA antibody clearly detectable by solid-phase radioimmunoassay. Anti-dsDNA antibody produced in vitro (y units) was in close correlation with the anti-dsDNA antibody titer of the spleen donor (x units) (y = 4.8 X 10 -2 x-65, γ = 0.94, P < 0.001). A combination of the microculture system and solid-phase radioimmunoassay was recommended for the characterization of anti-dsDNA antibody-forming cells. (Auth.)

  12. Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system.

    Science.gov (United States)

    Sinkunas, Tomas; Gasiunas, Giedrius; Fremaux, Christophe; Barrangou, Rodolphe; Horvath, Philippe; Siksnys, Virginijus

    2011-04-06

    Clustered regularly interspaced short palindromic repeat (CRISPR) is a recently discovered adaptive prokaryotic immune system that provides acquired immunity against foreign nucleic acids by utilizing small guide crRNAs (CRISPR RNAs) to interfere with invading viruses and plasmids. In Escherichia coli, Cas3 is essential for crRNA-guided interference with virus proliferation. Cas3 contains N-terminal HD phosphohydrolase and C-terminal Superfamily 2 (SF2) helicase domains. Here, we provide the first report of the cloning, expression, purification and in vitro functional analysis of the Cas3 protein of the Streptococcus thermophilus CRISPR4 (Ecoli subtype) system. Cas3 possesses a single-stranded DNA (ssDNA)-stimulated ATPase activity, which is coupled to unwinding of DNA/DNA and RNA/DNA duplexes. Cas3 also shows ATP-independent nuclease activity located in the HD domain with a preference for ssDNA substrates. To dissect the contribution of individual domains, Cas3 separation-of-function mutants (ATPase(+)/nuclease(-) and ATPase(-)/nuclease(+)) were obtained by site-directed mutagenesis. We propose that the Cas3 ATPase/helicase domain acts as a motor protein, which assists delivery of the nuclease activity to Cascade-crRNA complex targeting foreign DNA.

  13. [Cell-penetrating chimeric apoptotic peptide AVPI-LMWP/DNA co-delivery system for cancer therapy].

    Science.gov (United States)

    Tan, Jiao; Wang, Ya-Ping; Wang, Hui-Xin; Liang, Jian-Ming; Zhang, Meng; Sun, Xun; Huang, Yong-Zhuo

    2014-12-01

    To develop a cell-penetrating chimeric apoptotic peptide AVPI-LMWP/DNA co-delivery system for cancer therapy, we prepared the AVPI-LMWP/pTRAIL self-assembled complexes containing a therapeutic combination of peptide drug AVPI and DNA drug TRAIL. The chimeric apoptotic peptide AVPI-LMWP was synthesized using the standard solid-phase synthesis. The cationic AVPI-LMWP could condense pTRAIL by electrostatic interaction. The physical-chemical properties of the AVPI-LMWP/pTRAIL complexes were characterized. The cellular uptake efficiency and the inhibitory activity of the AVPI-LMWP/pTRAIL complexes on tumor cell were also performed. The results showed that the AVPI-LMWP/pTRAIL complexes were successfully prepared by co-incubation. With the increase of mass ratio (AVPI-LMWP/DNA), the particle size was decreased and the zeta potential had few change. Agarose gel electrophoresis showed that AVPI-LMWP could fully bind and condense pTRAIL at a mass ratio above 15:1. Cellular uptake efficiency was improved along with the increased ratio of W(AVPI-LMWP)/WpTRAIL. The in vitro cytotoxicity experiments demonstrated that the AVPI-LMWP/pTRAIL (W:W = 20:1) complexes was significantly more effective than the pTRAIL, AVPI-LMWP alone or LMWP/pTRAIL complexes on inhibition of HeLa cell growth. Our studies indicated that the AVPI-LMWP/pTRAIL co-delivery system could deliver plasmid into HeLa cell and induce tumor cell apoptosis efficiently, which showed its potential in cancer therapy using combination of apoptoic peptide and gene drugs.

  14. Role of the inhibitors of angiotensin renin system on the DNA integrity of irradiated spermatozoids; Papel dos inibidores dos sistema renina angiotensina sobre a integridade do DNA de espermatozoides irradiados

    Energy Technology Data Exchange (ETDEWEB)

    Spadella, Maria A.; Mansano, Naira S.; Schwarz, Franciele C.; Viani, Gustavo A.; Chies, Agnaldo B. [Faculdade de Medicina de Marilia (FAMEMA), Marilia, SP (Brazil)

    2016-07-01

    Radiation action in the testes can significantly affect the reproductive capacity due to oxidative stress generated; phenomenon in which there is evidence of involvement of the Renin Angiotensin System (RAS). This study evaluated the role of AT1 receptor inhibitors, in mitigating the radioinduced DNA damage sperm from semen samples left vas deferens. Male Wistar rats were divided into six experimental groups: Control, 5Gy, Telmisartan (12mg/kg/day) and Losartan (34mg/kg/2x/day), 5 Gy + Telmisartan and 5 Gy + Losartan. The results showed increase in the percentage of sperm with fragmented DNA in irradiated groups when compared to controls, which was not reversed in the irradiated and treated groups. The radiation of 5Gy (single dose) affected the DNA-protein complex of the sperm and the treatments did not influence in reversing this damage, considering the experimental protocol used. (author)

  15. In vitro Repair of Oxidative DNA Damage by Human Nucleotide Excision Repair System: Possible Explanation for Neurodegeneration in Xeroderma Pigmentosum Patients

    Science.gov (United States)

    Reardon, Joyce T.; Bessho, Tadayoshi; Kung, Hsiang Chuan; Bolton, Philip H.; Sancar, Aziz

    1997-08-01

    Xeroderma pigmentosum (XP) patients fail to remove pyrimidine dimers caused by sunlight and, as a consequence, develop multiple cancers in areas exposed to light. The second most common sign, present in 20-30% of XP patients, is a set of neurological abnormalities caused by neuronal death in the central and peripheral nervous systems. Neural tissue is shielded from sunlight-induced DNA damage, so the cause of neurodegeneration in XP patients remains unexplained. In this study, we show that two major oxidative DNA lesions, 8-oxoguanine and thymine glycol, are excised from DNA in vitro by the same enzyme system responsible for removing pyrimidine dimers and other bulky DNA adducts. Our results suggest that XP neurological disease may be caused by defective repair of lesions that are produced in nerve cells by reactive oxygen species generated as by-products of an active oxidative metabolism.

  16. Directly Transforming PCR-Amplified DNA Fragments into Plant Cells