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Sample records for extrachromosomal circular dna

  1. Formation of Extrachromosomal Circular DNA from Long Terminal Repeats of Retrotransposons in Saccharomyces cerevisiae.

    PubMed

    Møller, Henrik D; Larsen, Camilla E; Parsons, Lance; Hansen, Anders Johannes; Regenberg, Birgitte; Mourier, Tobias

    2015-12-17

    Extrachromosomal circular DNA (eccDNA) derived from chromosomal Ty retrotransposons in yeast can be generated in multiple ways. Ty eccDNA can arise from the circularization of extrachromosomal linear DNA during the transpositional life cycle of retrotransposons, or from circularization of genomic Ty DNA. Circularization may happen through nonhomologous end-joining (NHEJ) of long terminal repeats (LTRs) flanking Ty elements, by Ty autointegration, or by LTR-LTR recombination. By performing an in-depth investigation of sequence reads stemming from Ty eccDNAs obtained from populations of Saccharomyces cerevisiae S288c, we find that eccDNAs predominantly correspond to full-length Ty1 elements. Analyses of sequence junctions reveal no signs of NHEJ or autointegration events. We detect recombination junctions that are consistent with yeast Ty eccDNAs being generated through recombination events within the genome. This opens the possibility that retrotransposable elements could move around in the genome without an RNA intermediate directly through DNA circularization.

  2. Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells.

    PubMed

    Møller, Henrik D; Bojsen, Rasmus K; Tachibana, Chris; Parsons, Lance; Botstein, David; Regenberg, Birgitte

    2016-04-04

    Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods for detecting eccDNA are needed to clarify how these elements affect genome stability and how environmental and biological factors induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA, removal of remaining linear chromosomal DNA, rolling-circle amplification of eccDNA, deep sequencing, and mapping. Extensive exonuclease treatment was required for sufficient linear chromosomal DNA degradation. The rolling-circle amplification step by φ29 polymerase enriched for circular DNA over linear DNA. Validation of the Circle-Seq method on three S. cerevisiae CEN.PK populations of 10(10) cells detected hundreds of eccDNA profiles in sizes larger than 1 kilobase. Repeated findings of ASP3-1, COS111, CUP1, RSC30, HXT6, HXT7 genes on circular DNA in both S288c and CEN.PK suggests that DNA circularization is conserved between strains at these loci. In sum, the Circle-Seq method has broad applicability for genome-scale screening for eccDNA in eukaryotes as well as for detecting specific eccDNA types.

  3. Amplification of a Zygosaccharomyces bailii DNA segment in wine yeast genomes by extrachromosomal circular DNA formation.

    PubMed

    Galeote, Virginie; Bigey, Frédéric; Beyne, Emmanuelle; Novo, Maite; Legras, Jean-Luc; Casaregola, Serge; Dequin, Sylvie

    2011-03-10

    We recently described the presence of large chromosomal segments resulting from independent horizontal gene transfer (HGT) events in the genome of Saccharomyces cerevisiae strains, mostly of wine origin. We report here evidence for the amplification of one of these segments, a 17 kb DNA segment from Zygosaccharomyces bailii, in the genome of S. cerevisiae strains. The copy number, organization and location of this region differ considerably between strains, indicating that the insertions are independent and that they are post-HGT events. We identified eight different forms in 28 S. cerevisiae strains, mostly of wine origin, with up to four different copies in a single strain. The organization of these forms and the identification of an autonomously replicating sequence functional in S. cerevisiae, strongly suggest that an extrachromosomal circular DNA (eccDNA) molecule serves as an intermediate in the amplification of the Z. bailii region in yeast genomes. We found little or no sequence similarity at the breakpoint regions, suggesting that the insertions may be mediated by nonhomologous recombination. The diversity between these regions in S. cerevisiae represents roughly one third the divergence among the genomes of wine strains, which confirms the recent origin of this event, posterior to the start of wine strain expansion. This is the first report of a circle-based mechanism for the expansion of a DNA segment, mediated by nonhomologous recombination, in natural yeast populations.

  4. Amplification of a Zygosaccharomyces bailii DNA Segment in Wine Yeast Genomes by Extrachromosomal Circular DNA Formation

    PubMed Central

    Galeote, Virginie; Bigey, Frédéric; Beyne, Emmanuelle; Novo, Maite; Legras, Jean-Luc; Casaregola, Serge; Dequin, Sylvie

    2011-01-01

    We recently described the presence of large chromosomal segments resulting from independent horizontal gene transfer (HGT) events in the genome of Saccharomyces cerevisiae strains, mostly of wine origin. We report here evidence for the amplification of one of these segments, a 17 kb DNA segment from Zygosaccharomyces bailii, in the genome of S. cerevisiae strains. The copy number, organization and location of this region differ considerably between strains, indicating that the insertions are independent and that they are post-HGT events. We identified eight different forms in 28 S. cerevisiae strains, mostly of wine origin, with up to four different copies in a single strain. The organization of these forms and the identification of an autonomously replicating sequence functional in S. cerevisiae, strongly suggest that an extrachromosomal circular DNA (eccDNA) molecule serves as an intermediate in the amplification of the Z. bailii region in yeast genomes. We found little or no sequence similarity at the breakpoint regions, suggesting that the insertions may be mediated by nonhomologous recombination. The diversity between these regions in S. cerevisiae represents roughly one third the divergence among the genomes of wine strains, which confirms the recent origin of this event, posterior to the start of wine strain expansion. This is the first report of a circle-based mechanism for the expansion of a DNA segment, mediated by nonhomologous recombination, in natural yeast populations. PMID:21423766

  5. Identification of extrachromosomal circular DNA in hop via rolling circle amplification

    USDA-ARS?s Scientific Manuscript database

    During a survey looking for viruses affecting hop plants in the Willamette Valley of Oregon, a circular DNA molecule was identified via rolling circle amplification (RCA) and later characterized. The 5.7 kb long molecule only matched in a minor cover to a microsatellite region in the Humulus lupulus...

  6. Rapid evolution of recombinant Saccharomyces cerevisiae for Xylose fermentation through formation of extra-chromosomal circular DNA.

    PubMed

    Demeke, Mekonnen M; Foulquié-Moreno, María R; Dumortier, Françoise; Thevelein, Johan M

    2015-03-01

    Circular DNA elements are involved in genome plasticity, particularly of tandem repeats. However, amplifications of DNA segments in Saccharomyces cerevisiae reported so far involve pre-existing repetitive sequences such as ribosomal DNA, Ty elements and Long Terminal Repeats (LTRs). Here, we report the generation of an eccDNA, (extrachromosomal circular DNA element) in a region without any repetitive sequences during an adaptive evolution experiment. We performed whole genome sequence comparison between an efficient D-xylose fermenting yeast strain developed by metabolic and evolutionary engineering, and its parent industrial strain. We found that the heterologous gene XylA that had been inserted close to an ARS sequence in the parent strain has been amplified about 9 fold in both alleles of the chromosomal locus of the evolved strain compared to its parent. Analysis of the amplification process during the adaptive evolution revealed formation of a XylA-carrying eccDNA, pXI2-6, followed by chromosomal integration in tandem arrays over the course of the evolutionary adaptation. Formation of the eccDNA occurred in the absence of any repetitive DNA elements, probably using a micro-homology sequence of 8 nucleotides flanking the amplified sequence. We isolated the pXI2-6 eccDNA from an intermediate strain of the evolutionary adaptation process, sequenced it completely and showed that it confers high xylose fermentation capacity when it is transferred to a new strain. In this way, we have provided clear evidence that gene amplification can occur through generation of eccDNA without the presence of flanking repetitive sequences and can serve as a rapid means of adaptation to selection pressure.

  7. Rapid Evolution of Recombinant Saccharomyces cerevisiae for Xylose Fermentation through Formation of Extra-chromosomal Circular DNA

    PubMed Central

    Demeke, Mekonnen M.; Foulquié-Moreno, María R.; Dumortier, Françoise; Thevelein, Johan M.

    2015-01-01

    Circular DNA elements are involved in genome plasticity, particularly of tandem repeats. However, amplifications of DNA segments in Saccharomyces cerevisiae reported so far involve pre-existing repetitive sequences such as ribosomal DNA, Ty elements and Long Terminal Repeats (LTRs). Here, we report the generation of an eccDNA, (extrachromosomal circular DNA element) in a region without any repetitive sequences during an adaptive evolution experiment. We performed whole genome sequence comparison between an efficient D-xylose fermenting yeast strain developed by metabolic and evolutionary engineering, and its parent industrial strain. We found that the heterologous gene XylA that had been inserted close to an ARS sequence in the parent strain has been amplified about 9 fold in both alleles of the chromosomal locus of the evolved strain compared to its parent. Analysis of the amplification process during the adaptive evolution revealed formation of a XylA-carrying eccDNA, pXI2-6, followed by chromosomal integration in tandem arrays over the course of the evolutionary adaptation. Formation of the eccDNA occurred in the absence of any repetitive DNA elements, probably using a micro-homology sequence of 8 nucleotides flanking the amplified sequence. We isolated the pXI2-6 eccDNA from an intermediate strain of the evolutionary adaptation process, sequenced it completely and showed that it confers high xylose fermentation capacity when it is transferred to a new strain. In this way, we have provided clear evidence that gene amplification can occur through generation of eccDNA without the presence of flanking repetitive sequences and can serve as a rapid means of adaptation to selection pressure. PMID:25738959

  8. Sequencing the extrachromosomal circular mobilome reveals retrotransposon activity in plants

    PubMed Central

    Llauro, Christel; Jobet, Edouard; Robakowska-Hyzorek, Dagmara; Lasserre, Eric; Ghesquière, Alain; Panaud, Olivier

    2017-01-01

    Retrotransposons are mobile genetic elements abundant in plant and animal genomes. While efficiently silenced by the epigenetic machinery, they can be reactivated upon stress or during development. Their level of transcription not reflecting their transposition ability, it is thus difficult to evaluate their contribution to the active mobilome. Here we applied a simple methodology based on the high throughput sequencing of extrachromosomal circular DNA (eccDNA) forms of active retrotransposons to characterize the repertoire of mobile retrotransposons in plants. This method successfully identified known active retrotransposons in both Arabidopsis and rice material where the epigenome is destabilized. When applying mobilome-seq to developmental stages in wild type rice, we identified PopRice as a highly active retrotransposon producing eccDNA forms in the wild type endosperm. The mobilome-seq strategy opens new routes for the characterization of a yet unexplored fraction of plant genomes. PMID:28212378

  9. Excision and replication of extrachromosomal DNA of pea (Pisum sativum)

    SciTech Connect

    Hof, J.V.; Bjerknes, C.A.; Delihas, N.C.

    1983-02-01

    Experiments with cultured pea roots were conducted to determine (i) whether extrachromosomal DNA was produced by cells in the late S phase or in the G2 phase of the cell cycle, (ii) whether the maturation of nascent DNA replicated by these cells achieved chromosomal size, (iii) when extrachromosomal DNA was removed from the chromosomal duplex, and (iv) the replication of nascent chains by the extrachromosomal DNA after its release from the chromosomal duplex. Autoradiography and cytophotometry of cells of carbohydrate-starved root tips revealed that extrachromosomal DNA was produced by a small fraction of cells accumulated in the late S phase after they had replicated about 80% of their DNA. Velocity sedimentation of nascent chromosomal DNA in alkaline sucrose gradients indicated that the DNA of cells in the late S phase failed to achieve chromosomal size. After reaching sizes of 70 x 10/sup 6/ to 140 x 10/sup 6/ daltons, some of the nascent chromosomal molecules were broken, presumably releasing extrachromosomal DNA several hours later. Sedimentation of selectively extracted extrachromosomal DNA either from dividing cells or from those in the late S phase showed that it replicated two nascent chains, one of 3 x 10/sup 6/ daltons and another of 7 x 10/sup 6/ daltons. Larger molecules of extrachromosomal DNA were detectable after cells were labeled for 24 h. These two observations were compatible with the idea that the extrachromosomal DNA was first replicated as an integral part of the chromosomal duplex, was cut from the duplex, and then, once free of the chromosome, replicated two smaller chains of 3 x 10/sup 6/ and 7 x 10/sup 6/ daltons. 24 references.

  10. Linear extrachromosomal DNA in the morel Morchella conica.

    PubMed

    Meinhardt, F; Esser, K

    1984-01-01

    Altogether 18 different strains of the genus Morchella were assayed for the presence of extrachromosomal genetic elements. It was shown that 8 out of 13 strains of the Morchella conica group contain plasmids of comparable size (6 kb and 8 kb respectively). The 5 representatives of Morchella esculenta were not found to contain extrachromosomal DNA. The plasmid of one strain (nr. 3) was further analysed. By restriction analyses and electron microscopy it was confirmed that the plasmid is linear having a molecular weight of 6 kb. It was further shown that it carries at both ends inverted repeats of 0.75 kb.

  11. Visualization and quantitative analysis of extrachromosomal telomere-repeat DNA in individual human cells by Halo-FISH.

    PubMed

    Komosa, Martin; Root, Heather; Meyn, M Stephen

    2015-02-27

    Current methods for characterizing extrachromosomal nuclear DNA in mammalian cells do not permit single-cell analysis, are often semi-quantitative and frequently biased toward the detection of circular species. To overcome these limitations, we developed Halo-FISH to visualize and quantitatively analyze extrachromosomal DNA in single cells. We demonstrate Halo-FISH by using it to analyze extrachromosomal telomere-repeat (ECTR) in human cells that use the Alternative Lengthening of Telomeres (ALT) pathway(s) to maintain telomere lengths. We find that GM847 and VA13 ALT cells average ∼80 detectable G/C-strand ECTR DNA molecules/nucleus, while U2OS ALT cells average ∼18 molecules/nucleus. In comparison, human primary and telomerase-positive cells contain <5 ECTR DNA molecules/nucleus. ECTR DNA in ALT cells exhibit striking cell-to-cell variations in number (<20 to >300), range widely in length (<1 to >200 kb) and are composed of primarily G- or C-strand telomere-repeat DNA. Halo-FISH enables, for the first time, the simultaneous analysis of ECTR DNA and chromosomal telomeres in a single cell. We find that ECTR DNA comprises ∼15% of telomere-repeat DNA in GM847 and VA13 cells, but <4% in U2OS cells. In addition to its use in ALT cell analysis, Halo-FISH can facilitate the study of a wide variety of extrachromosomal DNA in mammalian cells.

  12. Visualization and quantitative analysis of extrachromosomal telomere-repeat DNA in individual human cells by Halo-FISH

    PubMed Central

    Komosa, Martin; Root, Heather; Meyn, M. Stephen

    2015-01-01

    Current methods for characterizing extrachromosomal nuclear DNA in mammalian cells do not permit single-cell analysis, are often semi-quantitative and frequently biased toward the detection of circular species. To overcome these limitations, we developed Halo-FISH to visualize and quantitatively analyze extrachromosomal DNA in single cells. We demonstrate Halo-FISH by using it to analyze extrachromosomal telomere-repeat (ECTR) in human cells that use the Alternative Lengthening of Telomeres (ALT) pathway(s) to maintain telomere lengths. We find that GM847 and VA13 ALT cells average ∼80 detectable G/C-strand ECTR DNA molecules/nucleus, while U2OS ALT cells average ∼18 molecules/nucleus. In comparison, human primary and telomerase-positive cells contain <5 ECTR DNA molecules/nucleus. ECTR DNA in ALT cells exhibit striking cell-to-cell variations in number (<20 to >300), range widely in length (<1 to >200 kb) and are composed of primarily G- or C-strand telomere-repeat DNA. Halo-FISH enables, for the first time, the simultaneous analysis of ECTR DNA and chromosomal telomeres in a single cell. We find that ECTR DNA comprises ∼15% of telomere-repeat DNA in GM847 and VA13 cells, but <4% in U2OS cells. In addition to its use in ALT cell analysis, Halo-FISH can facilitate the study of a wide variety of extrachromosomal DNA in mammalian cells. PMID:25662602

  13. Characterization of extrachromosomal DNA in the flesh fly Sarcophaga bullata.

    PubMed

    Samols, D; Swift, H

    1979-11-01

    The polytene pupal foot pad cells of the flesh fly Sarcophaga bullata contain numerous extrachromosomal DNA containing granules. We have determined both the origin and the nature of the DNA sequences present in these granules. Studies done with quinacrine staining of seven day old pupal foot-pad polytene nuclei showed that the granules fluoresced very brightly while the chromosomal bands to which the granules were attached did not. The only other highly fluorescent regions of the polytene karyotype were the centromeric heterochromatin of chromosomes C and E and several bands associated with the nucleolus of Chromsome A. When polytene nuclei were hybridized in situ with cRNA made from highly repetitive DNA, many of the granules positively labeled. Most of the label on these slides was concentrated on the centromeric heterochromatin of chromosomes C and E. Quinacrine staining of the foot-pad cells at very early stages of pupal development showed that when granules were present, they were always closely associated with the same two centromeric regions, those of chromosomes C and E. Since the highly repetitive DNA located in these centromeric regions is underreplicated, we conclude that the granules result from an extrusion process which takes place early during the polytenization of these cells. The chromosomal integrity of the centromeric heterochromatin of chromosomes C and E is apparently disrupted and repetitive sequences are dissociated from the chromosomes as DNA granules which then secondarily become associated with chromosomal bands throughout the nucleus.

  14. Mismatch repair of heteroduplex DNA intermediates of extrachromosomal recombination in mammalian cells.

    PubMed Central

    Deng, W P; Nickoloff, J A

    1994-01-01

    Previous work indicated that extrachromosomal recombination in mammalian cells could be explained by the single-strand annealing (SSA) model. This model predicts that extrachromosomal recombination leads to nonconservative crossover products and that heteroduplex DNA (hDNA) is formed by annealing of complementary single strands. Mismatched bases in hDNA may subsequently be repaired to wild-type or mutant sequences, or they may remain unrepaired and segregate following DNA replication. We describe a system to examine the formation and mismatch repair of hDNA in recombination intermediates. Our results are consistent with extrachromosomal recombination occurring via SSA and producing crossover recombinant products. As predicted by the SSA model, hDNA was present in double-strand break-induced recombination intermediates. By placing either silent or frameshift mutations in the predicted hDNA region, we have shown that mismatches are efficiently repaired prior to DNA replication. Images PMID:8264607

  15. Extrachromosomal DNA and cell differentiation in cultured pea roots (Pisum sativum)

    SciTech Connect

    Van't Hof, J.

    1986-01-01

    Histological, cytological and molecular analyses of DNA replication and differentiation and meristematic precursors of vascular parenchyma in cultured pea roots show that the initial steps in the transition from a dividing to a differentiated cell involve retardation of DNA replication in late S phase and production of extrachromosomal molecules by excision from late replicating chromosomal DNA. Portions of the extrachromosomal DNA are displaced by a strand displacement mechanism thereby producing free single-stranded molecules. That extrachromosomal molecules have rDNA reflects the fact that in pea roots replication of the ribosomal genes is maximal during late S phase (unpublished results). It is likely, therefore, that the excised late replicating DNA contains some rDNA sequences. 30 refs., 3 figs.

  16. Isolation and characterization of extrachromosomal circular DNAs in mouse heart, brain and liver tissues at various ages

    SciTech Connect

    Flores, S.C.

    1988-01-01

    Eucaryotic cells contains extrachromosomal circular (eccDNAs) which can be separated and distinguished from chromosomal DNA. Using alkaline denaturation-renaturation, exonuclease III digestion and density gradient centrifugations, covalently closed circular DNA (cc-cDNA) molecules were isolated from 1-, 8-, 16-, and 24-month C57BL/6 mouse heart, brain and liver organs. Restriction enzyme analyses and other enzymatic treatments established the covalently closed nature of the isolated molecules. Electron microscopic analyses of heart eccDNAs showed similar size distributions at all ages, but more discrete size classes and slightly larger circles were observed in 24-month heart eccDNA preparations. Heart contained more circles per cell than either liver or brain, which contained approximately the same amount of eccDNAs per genome. Furthermore, ({sup 3}H)-pBR322 recovery studies revealed no endogenous factors that might have affected the yields of eccDNAs from young and old tissues. To determine if there were any age-related or tissue-specific differences in repetitive sequences in eccDNAs, heart, brain and liver eccDNAs were probed with B1, B2, IAP, L1 and satellite sequences of the mouse genome. The hybridization results showed that these sequence families were differentially represented at all ages in eccDNAs. B2 sequences were the highest in heart, while satellite sequences were the highest in liver and brain. In heart, very little age-related change was observed in the quantity of repetitive sequences. Nevertheless, a tendency to decrease for B1 and B2 sequences at 24 months was observed. In liver, repetitive sequences decreased from 1 to 8 months of age, with very little change beyond that time point. Brain eccDNA repetitive sequences did not change significantly with age.

  17. Cells of pea (Pisum sativum) that differentiate from G2 phase have extrachromosomal DNA

    SciTech Connect

    Van't Hof, J.; Bjerknes, C.A.

    1982-04-01

    Velocity sedimentation in an alkaline sucrose gradient of newly replicated chromosomal DNA revealed the presence of extrachromosomal DNA that was not replicated by differentiating cells in the elongation zone. The extrachromosomal DNA had a number average molecular weight of 12 x 10/sup 6/ to 15 x 10/sup 6/ and a weight average molecular weight of 25 x 10/sup 6/, corresponding to about 26 x 10/sup 6/ and 50 x 10/sup 6/ daltons, respectively, of double-stranded DNA. The molecules were stable, lasting at least 72 h after being formed. Concurrent measurements by velocity sedimentation, autoradiography, and cytophotometry of isolated nuclei indicated that the extrachromosomal molecules were associated with root-tip-cells that stopped dividing and differentiated from G2 phase but not with those that stopped dividing and differentiated from G1 phase.

  18. Extrachromosomal DNA of pea (Pisum sativum) root-tip cells replicates by strand displacement

    SciTech Connect

    Krimer, D.B.; Van't Hof, J.

    1983-04-01

    In cultured pea roots there is extrachromosomal DNA associated with cells that differentiate from the G/sub 2/ phase of the cell cycle that is absent from those that differentiate from the G/sub 1/ phase. The authors examined this extrachromosomal DNA by electron microscopy and found that it consisted of three types: (i) double-stranded linear molecules with single-stranded branches (74%), (ii) double-stranded molecules without branches (26%), and (iii) free single-stranded molecules. The double-stranded molecules with or without branches were similar in length, having a modal length of 10-15 ..mu..m. The free single-stranded molecules were shorter and had a mean length of 3.8 ..mu..m. The length of the branches attached to the duplex molecules was only slightly less than that of the free form. The duplex molecules with branches were interpreted as configurations reflecting an ongoing strand-displacement process that results in free single-stranded molecules. Finally, measurements on duplex molecules with multiple branches suggested that the extrachromosomal DNA may exist in the form of tandemly repeated sequences. 8 references, 8 figures.

  19. Selective Entrapment of Extrachromosomally Amplified DNA by Nuclear Budding and Micronucleation during S Phase

    PubMed Central

    Shimizu, Noriaki; Itoh, Nobuo; Utiyama, Hiroyasu; Wahl, Geoffrey M.

    1998-01-01

    Acentric, autonomously replicating extrachromosomal structures called double-minute chromosomes (DMs) frequently mediate oncogene amplification in human tumors. We show that DMs can be removed from the nucleus by a novel micronucleation mechanism that is initiated by budding of the nuclear membrane during S phase. DMs containing c-myc oncogenes in a colon cancer cell line localized to and replicated at the nuclear periphery. Replication inhibitors increased micronucleation; cell synchronization and bromodeoxyuridine–pulse labeling demonstrated de novo formation of buds and micronuclei during S phase. The frequencies of S-phase nuclear budding and micronucleation were increased dramatically in normal human cells by inactivating p53, suggesting that an S-phase function of p53 minimizes the probability of producing the broken chromosome fragments that induce budding and micronucleation. These data have implications for understanding the behavior of acentric DNA in interphase nuclei and for developing chemotherapeutic strategies based on this new mechanism for DM elimination. PMID:9508765

  20. Extrachromosomal HPV-16 LCR transcriptional activation by HDACi opposed by cellular differentiation and DNA integration

    PubMed Central

    Bojilova, Ekaterina Dimitrova; Weyn, Christine; Antoine, Marie-Hélène; Fontaine, Véronique

    2016-01-01

    Histone deacetylase inhibitors (HDACi) have been shown to render HPV-carrying cells susceptible to intrinsic and extrinsic apoptotic signals. As such, these epigenetic drugs have entered clinical trials in the effort to treat cervical cancer. Here, we studied the effect of common HDACi, with an emphasis on Trichostatin A (TSA), on the transcriptional activity of the HPV-16 Long Control Region (LCR) in order to better understand the impact of these agents in the context of the HPV life cycle and infection. HDACi strongly induced transcription of the firefly luciferase reporter gene under the control of the HPV-16 LCR in a variety of cell lines. In the HaCaT keratinocyte cell line undergoing differentiation induced by TSA, we observed a reduction in LCR-controlled transcription. Three major AP-1 binding sites in the HPV-16 LCR are involved in the regulation by TSA. However, whatever the status of differentiation of the HaCaT cells, TSA induced integration of extra-chromosomal transfected DNA into the cellular genome. Although these data suggest caution using HDACi in the treatment of HR HPV infection, further in vivo studies are necessary to better assess the risk. PMID:27705914

  1. Campoletis sonorensis Endoparasitic Wasps Contain Forms of C. sonorensis Virus DNA Suggestive of Integrated and Extrachromosomal Polydnavirus DNAs

    PubMed Central

    Fleming, Jo-Ann G. W.; Summers, Max D.

    1986-01-01

    Campoletis sonorensis virus (CsV) (Polydnaviridae) previously was detected only in the calyx epithelial cells and lumen of the oviducts from female C. sonorensis (Ichneumonidae) endoparasitic wasps (Norton et al., Cell Tissue Res. 162:195-208, 1975). Using dot-blot hybridizations, we detected low amounts of CsV DNA in male and female wasp head and thorax tissues and in male abdominal tissues. Low amounts of extrachromosomal viral DNA were detected in Southern blots of undigested male wasp DNA and in male DNA purified by isopycnic centrifugation. High-molecular-weight male wasp DNA digested with any of several restriction endonucleases and hybridized with cloned viral DNAs from CsV superhelices B and Q under stringent conditions contained CsV-specific DNA fragments that differed significantly in size and number from the hybridizing fragments detected in comparably digested viral DNA. Identical offsize restriction fragments were detected in digested female head and thorax DNA. These data suggest that at least CsV DNAs B and Q are integrated in C. sonorensis cellular DNA and that the virus may be transmitted through the germline. Images PMID:16789255

  2. The linear extrachromosomal DNA of Physarum polycephalum replicates and is maintained under non-selective conditions in two different lower eukaryotes.

    PubMed

    Künzler, P

    1985-03-25

    The slime mould Physarum polycephalum contains 100 to 200 molecules of extrachromosomal linear DNA (PeDNA). Two sets of the 19S and 26S ribosomal genes are located on each molecule of PeDNA. In the nonmitotic phase of the cell cycle PeDNA is localised in the nucleolus. The molecules are maintained throughout vegetative growth. In order to study the signals responsible for its maintenance, PeDNA was purified and introduced into the two distantly related yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. Surprisingly, intact PeDNA transforms both yeasts with high frequency and PeDNA sequences are maintained in the absence of selective pressure.

  3. Extrachromosomal sequences of hepatitis B virus DNA in peripheral blood mononuclear cells of acquired immune deficiency syndrome patients.

    PubMed

    Noonan, C A; Yoffe, B; Mansell, P W; Melnick, J L; Hollinger, F B

    1986-08-01

    The primary etiologic agent of the acquired immune deficiency syndrome (AIDS) is a human T-lymphotropic retrovirus (the AIDS virus). However, the pathogenesis of this virus suggests that other cofactors may contribute to the development of clinically overt disease. The hepatitis B virus (HBV) has been implicated as a potential cofactor because HBV and AIDS virus infections frequently coexist, striking similarities exist in their epidemiologic patterns, and recent data indicate that HBV is lymphotropic. To establish the prevalence of HBV infections in lymphoid cells from individuals with AIDS-related disorders, sera and peripheral blood mononuclear cells (PBMC) from 16 males with AIDS virus infections were examined for the presence of HBV DNA by DNA X DNA blot hybridization. Fifteen (94%) of these individuals had serologic evidence of a recent or prior HBV infection. HBV DNA was detected in the PBMC of all of these patients, regardless of existing HBV serology. Among the 36 control individuals without AIDS-related symptomatology, PBMC-associated HBV DNA was detected in 8 of 14 carriers of hepatitis B surface antigen (HBsAg) and in 3 of 10 individuals immune to HBV, but it was absent from the PBMC of 12 individuals without HBV infection. In all instances, the HBV DNA was extrachromosomal and existed as replicative intermediates or high molecular weight oligomers of the viral genome. Replicative intermediates and serum-associated HBV DNA were detected in all hepatitis B e antigen-positive carriers, regardless of their clinical status. In contrast, the high molecular weight oligomers of HBV DNA were detected in the PBMC of all of the AIDS virus-infected patients examined, but in only 33% of those in the control group who had evidence of HBV infection. This finding suggests that a unique and complex HBV-host-cell interaction exists in patients infected with the AIDS virus.

  4. Extrachromosomal homologous DNA recombination in plant cells is fast and is not affected by CpG methylation.

    PubMed Central

    Puchta, H; Kocher, S; Hohn, B

    1992-01-01

    Using a sensitive transient assay, we investigated extrachromosomal homologous DNA recombination (ECR) in plant cells. As the plant genome is highly C methylated, we addressed the question of whether CpG methylation has an influence on DNA recombination efficiencies. Whereas the expression level of the fully CpG-methylated DNA molecules was reduced drastically, we found no significant changes in ECR efficiencies between two partly CpG-methylated plasmids or between one fully CpG-methylated and one nonmethylated plasmid. Using a modified polymerase chain reaction analysis, we were able to detect recombination between two fully CpG-methylated plasmids. Furthermore, we characterized the kinetics of the ECR reaction. Cotransfection of plasmids carrying truncated copies of the beta-glucuronidase (GUS) gene resulted in enzyme activity with a delay of only half an hour compared with that of the plasmid carrying the functional marker gene. This indicates that the ECR reaction itself requires no more than 30 min. By polymerase chain reaction, we were able to detect the recombined GUS gene as early as 2 h after transfection. This result and the time course of the transient GUS activity indicate that ECR occurs mainly early after transfection. The biological significance of this finding is discussed, and properties of ECR and intrachromosomal recombination are compared. Images PMID:1630452

  5. Extrachromosomal genetic elements in Micrococcus.

    PubMed

    Dib, Julián Rafael; Liebl, Wolfgang; Wagenknecht, Martin; Farías, María Eugenia; Meinhardt, Friedhelm

    2013-01-01

    Micrococci are Gram-positive G + C-rich, nonmotile, nonspore-forming actinomycetous bacteria. Micrococcus comprises ten members, with Micrococcus luteus being the type species. Representatives of the genus play important roles in the biodegradation of xenobiotics, bioremediation processes, production of biotechnologically important enzymes or bioactive compounds, as test strains in biological assays for lysozyme and antibiotics, and as infective agents in immunocompromised humans. The first description of plasmids dates back approximately 28 years, when several extrachromosomal elements ranging in size from 1.5 to 30.2 kb were found in Micrococcus luteus. Up to the present, a number of circular plasmids conferring antibiotic resistance, the ability to degrade aromatic compounds, and osmotolerance are known, as well as cryptic elements with unidentified functions. Here, we review the Micrococcus extrachromosomal traits reported thus far including phages and the only quite recently described large linear extrachromosomal genetic elements, termed linear plasmids, which range in size from 75 kb (pJD12) to 110 kb (pLMA1) and which confer putative advantageous capabilities, such as antibiotic or heavy metal resistances (inferred from sequence analyses and curing experiments). The role of the extrachromosomal elements for the frequently proven ecological and biotechnological versatility of the genus will be addressed as well as their potential for the development and use as genetic tools.

  6. Small RNA-based feedforward loop with AND-gate logic regulates extrachromosomal DNA transfer in Salmonella.

    PubMed

    Papenfort, Kai; Espinosa, Elena; Casadesús, Josep; Vogel, Jörg

    2015-08-25

    Horizontal gene transfer via plasmid conjugation is a major driving force in microbial evolution but constitutes a complex process that requires synchronization with the physiological state of the host bacteria. Although several host transcription factors are known to regulate plasmid-borne transfer genes, RNA-based regulatory circuits for host-plasmid communication remain unknown. We describe a posttranscriptional mechanism whereby the Hfq-dependent small RNA, RprA, inhibits transfer of pSLT, the virulence plasmid of Salmonella enterica. RprA employs two separate seed-pairing domains to activate the mRNAs of both the sigma-factor σ(S) and the RicI protein, a previously uncharacterized membrane protein here shown to inhibit conjugation. Transcription of ricI requires σ(S) and, together, RprA and σ(S) orchestrate a coherent feedforward loop with AND-gate logic to tightly control the activation of RicI synthesis. RicI interacts with the conjugation apparatus protein TraV and limits plasmid transfer under membrane-damaging conditions. To our knowledge, this study reports the first small RNA-controlled feedforward loop relying on posttranscriptional activation of two independent targets and an unexpected role of the conserved RprA small RNA in controlling extrachromosomal DNA transfer.

  7. Small RNA-based feedforward loop with AND-gate logic regulates extrachromosomal DNA transfer in Salmonella

    PubMed Central

    Papenfort, Kai; Espinosa, Elena; Casadesús, Josep; Vogel, Jörg

    2015-01-01

    Horizontal gene transfer via plasmid conjugation is a major driving force in microbial evolution but constitutes a complex process that requires synchronization with the physiological state of the host bacteria. Although several host transcription factors are known to regulate plasmid-borne transfer genes, RNA-based regulatory circuits for host–plasmid communication remain unknown. We describe a posttranscriptional mechanism whereby the Hfq-dependent small RNA, RprA, inhibits transfer of pSLT, the virulence plasmid of Salmonella enterica. RprA employs two separate seed-pairing domains to activate the mRNAs of both the sigma-factor σS and the RicI protein, a previously uncharacterized membrane protein here shown to inhibit conjugation. Transcription of ricI requires σS and, together, RprA and σS orchestrate a coherent feedforward loop with AND-gate logic to tightly control the activation of RicI synthesis. RicI interacts with the conjugation apparatus protein TraV and limits plasmid transfer under membrane-damaging conditions. To our knowledge, this study reports the first small RNA-controlled feedforward loop relying on posttranscriptional activation of two independent targets and an unexpected role of the conserved RprA small RNA in controlling extrachromosomal DNA transfer. PMID:26307765

  8. The linear extrachromosomal DNA of Physarum polycephalum replicates and is maintained under non-selective conditions in two different lower eukaryotes.

    PubMed Central

    Künzler, P

    1985-01-01

    The slime mould Physarum polycephalum contains 100 to 200 molecules of extrachromosomal linear DNA (PeDNA). Two sets of the 19S and 26S ribosomal genes are located on each molecule of PeDNA. In the nonmitotic phase of the cell cycle PeDNA is localised in the nucleolus. The molecules are maintained throughout vegetative growth. In order to study the signals responsible for its maintenance, PeDNA was purified and introduced into the two distantly related yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. Surprisingly, intact PeDNA transforms both yeasts with high frequency and PeDNA sequences are maintained in the absence of selective pressure. Images PMID:3889843

  9. TRE5-A retrotransposition profiling reveals putative RNA polymerase III transcription complex binding sites on the Dictyostelium extrachromosomal rDNA element.

    PubMed

    Spaller, Thomas; Groth, Marco; Glöckner, Gernot; Winckler, Thomas

    2017-01-01

    The amoeba Dictyostelium discoideum has a haploid genome in which two thirds of the DNA encodes proteins. Consequently, the space available for selfish mobile elements to expand without excess damage to the host genome is limited. The non-long terminal repeat retrotransposon TRE5-A maintains an active population in the D. discoideum genome and apparently adapted to this gene-dense environment by targeting positions ~47 bp upstream of tRNA genes that are devoid of protein-coding regions. Because only ~24% of tRNA genes are associated with a TRE5-A element in the reference genome, we evaluated whether TRE5-A retrotransposition is limited to this subset of tRNA genes. We determined that a tagged TRE5-A element (TRE5-Absr) integrated at 384 of 405 tRNA genes, suggesting that expansion of the current natural TRE5-A population is not limited by the availability of targets. We further observed that TRE5-Absr targets the ribosomal 5S gene on the multicopy extrachromosomal DNA element that carries the ribosomal RNA genes, indicating that TRE5-A integration may extend to the entire RNA polymerase III (Pol III) transcriptome. We determined that both natural TRE5-A and cloned TRE5-Absr retrotranspose to locations on the extrachromosomal rDNA element that contain tRNA gene-typical A/B box promoter motifs without displaying any other tRNA gene context. Based on previous data suggesting that TRE5-A targets tRNA genes by locating Pol III transcription complexes, we propose that A/B box loci reflect Pol III transcription complex assembly sites that possess a function in the biology of the extrachromosomal rDNA element.

  10. Transposable elements and circular DNAs

    PubMed Central

    2016-01-01

    ABSTRACT Circular DNAs are extra-chromosomal fragments that become circularized by genomic recombination events. We have recently shown that yeast LTR elements generate circular DNAs through recombination events between their flanking long terminal repeats (LTRs). Similarly, circular DNAs can be generated by recombination between LTRs residing at different genomic loci, in which case the circular DNA will contain the intervening sequence. In yeast, this can result in gene copy number variations when circles contain genes and origins of replication. Here, I speculate on the potential and implications of circular DNAs generated through recombination between human transposable elements. PMID:28090380

  11. Archaeal Extrachromosomal Genetic Elements

    PubMed Central

    Wang, Haina; Peng, Nan; Shah, Shiraz A.

    2015-01-01

    SUMMARY Research on archaeal extrachromosomal genetic elements (ECEs) has progressed rapidly in the past decade. To date, over 60 archaeal viruses and 60 plasmids have been isolated. These archaeal viruses exhibit an exceptional diversity in morphology, with a wide array of shapes, such as spindles, rods, filaments, spheres, head-tails, bottles, and droplets, and some of these new viruses have been classified into one order, 10 families, and 16 genera. Investigation of model archaeal viruses has yielded important insights into mechanisms underlining various steps in the viral life cycle, including infection, DNA replication and transcription, and virion egression. Many of these mechanisms are unprecedented for any known bacterial or eukaryal viruses. Studies of plasmids isolated from different archaeal hosts have also revealed a striking diversity in gene content and innovation in replication strategies. Highly divergent replication proteins are identified in both viral and plasmid genomes. Genomic studies of archaeal ECEs have revealed a modular sequence structure in which modules of DNA sequence are exchangeable within, as well as among, plasmid families and probably also between viruses and plasmids. In particular, it has been suggested that ECE-host interactions have shaped the coevolution of ECEs and their archaeal hosts. Furthermore, archaeal hosts have developed defense systems, including the innate restriction-modification (R-M) system and the adaptive CRISPR (clustered regularly interspaced short palindromic repeats) system, to restrict invasive plasmids and viruses. Together, these interactions permit a delicate balance between ECEs and their hosts, which is vitally important for maintaining an innovative gene reservoir carried by ECEs. In conclusion, while research on archaeal ECEs has just started to unravel the molecular biology of these genetic entities and their interactions with archaeal hosts, it is expected to accelerate in the next decade. PMID

  12. Beyond the Chromosome: The Prevalence of Unique Extra-Chromosomal Bacteriophages with Integrated Virulence Genes in Pathogenic Staphylococcus aureus

    PubMed Central

    Utter, Bryan; Deutsch, Douglas R.; Schuch, Raymond; Winer, Benjamin Y.; Verratti, Kathleen; Bishop-Lilly, Kim; Sozhamannan, Shanmuga; Fischetti, Vincent A.

    2014-01-01

    In Staphylococcus aureus, the disease impact of chromosomally integrated prophages on virulence is well described. However, the existence of extra-chromosomal prophages, both plasmidial and episomal, remains obscure. Despite the recent explosion in bacterial and bacteriophage genomic sequencing, studies have failed to specifically focus on extra-chromosomal elements. We selectively enriched and sequenced extra-chromosomal DNA from S. aureus isolates using Roche-454 technology and uncovered evidence for the widespread distribution of multiple extra-chromosomal prophages (ExPΦs) throughout both antibiotic-sensitive and -resistant strains. We completely sequenced one such element comprised of a 43.8 kbp, circular ExPΦ (designated ФBU01) from a vancomycin-intermediate S. aureus (VISA) strain. Assembly and annotation of ФBU01 revealed a number of putative virulence determinants encoded within a bacteriophage immune evasion cluster (IEC). Our identification of several potential ExPΦs and mobile genetic elements (MGEs) also revealed numerous putative virulence factors and antibiotic resistance genes. We describe here a previously unidentified level of genetic diversity of stealth extra-chromosomal elements in S. aureus, including phages with a larger presence outside the chromosome that likely play a prominent role in pathogenesis and strain diversity driven by horizontal gene transfer (HGT). PMID:24963913

  13. Temperature dependence of circular DNA topological states

    NASA Astrophysics Data System (ADS)

    Chen, Hu; Liu, Yanhui; Zhou, Zhen; Hu, Lin; Ou-Yang, Zhong-Can; Yan, Jie

    2009-04-01

    Circular double-stranded DNA has different topological states which are defined by their linking numbers. Equilibrium distribution of linking numbers can be obtained by closing a linear DNA into a circle by ligase. Using Monte Carlo simulation, we predict the temperature dependence of the linking number distribution of small circular DNAs. Our predictions are based on flexible defect excitations that resulted from local melting or unstacking of DNA base pairs. We found that the reduced bending rigidity alone can lead to measurable changes of the variance of linking number distribution of short circular DNAs. If the defect is accompanied by local unwinding, the effect becomes much more prominent. The predictions can be easily investigated in experiments, providing a new method to study the micromechanics of sharply bent DNAs and the thermal stability of specific DNA sequences. Furthermore, the predictions are directly applicable to the studies of binding of DNA-distorting proteins that can locally reduce DNA rigidity, form DNA kinks, or introduce local unwinding.

  14. Temperature dependence of circular DNA topological states.

    PubMed

    Chen, Hu; Liu, Yanhui; Zhou, Zhen; Hu, Lin; Ou-Yang, Zhong-Can; Yan, Jie

    2009-04-01

    Circular double-stranded DNA has different topological states which are defined by their linking numbers. Equilibrium distribution of linking numbers can be obtained by closing a linear DNA into a circle by ligase. Using Monte Carlo simulation, we predict the temperature dependence of the linking number distribution of small circular DNAs. Our predictions are based on flexible defect excitations that resulted from local melting or unstacking of DNA base pairs. We found that the reduced bending rigidity alone can lead to measurable changes of the variance of linking number distribution of short circular DNAs. If the defect is accompanied by local unwinding, the effect becomes much more prominent. The predictions can be easily investigated in experiments, providing a new method to study the micromechanics of sharply bent DNAs and the thermal stability of specific DNA sequences. Furthermore, the predictions are directly applicable to the studies of binding of DNA-distorting proteins that can locally reduce DNA rigidity, form DNA kinks, or introduce local unwinding.

  15. Widespread Horizontal Gene Transfer from Circular Single-stranded DNA Viruses to Eukaryotic Genomes

    PubMed Central

    2011-01-01

    Background In addition to vertical transmission, organisms can also acquire genes from other distantly related species or from their extra-chromosomal elements (plasmids and viruses) via horizontal gene transfer (HGT). It has been suggested that phages represent substantial forces in prokaryotic evolution. In eukaryotes, retroviruses, which can integrate into host genome as an obligate step in their replication strategy, comprise approximately 8% of the human genome. Unlike retroviruses, few members of other virus families are known to transfer genes to host genomes. Results Here we performed a systematic search for sequences related to circular single-stranded DNA (ssDNA) viruses in publicly available eukaryotic genome databases followed by comprehensive phylogenetic analysis. We conclude that the replication initiation protein (Rep)-related sequences of geminiviruses, nanoviruses and circoviruses have been frequently transferred to a broad range of eukaryotic species, including plants, fungi, animals and protists. Some of the transferred viral genes were conserved and expressed, suggesting that these genes have been coopted to assume cellular functions in the host genomes. We also identified geminivirus-like and parvovirus-like transposable elements in genomes of fungi and lower animals, respectively, and thereby provide direct evidence that eukaryotic transposons could derive from ssDNA viruses. Conclusions Our discovery extends the host range of circular ssDNA viruses and sheds light on the origin and evolution of these viruses. It also suggests that ssDNA viruses act as an unforeseen source of genetic innovation in their hosts. PMID:21943216

  16. Hundreds of Circular Novel Plasmids and DNA Elements Identified in a Rat Cecum Metamobilome

    PubMed Central

    Jørgensen, Tue Sparholt; Xu, Zhuofei; Hansen, Martin Asser; Sørensen, Søren Johannes; Hansen, Lars Hestbjerg

    2014-01-01

    Metagenomic approaches are widespread in microbiological research, but so far, the knowledge on extrachromosomal DNA diversity and composition has largely remained dependant on cultivating host organisms. Even with the emergence of metagenomics, complete circular sequences are rarely identified, and have required manual curation. We propose a robust in silico procedure for identifying complete small plasmids in metagenomic datasets from whole genome shotgun sequencing. From one very pure and exhaustively sequenced metamobilome from rat cecum, we identified a total of 616 circular sequences, 160 of which were carrying a gene with plasmid replication domain. Further homology analyses indicated that the majority of these plasmid sequences are novel. We confirmed the circularity of the complete plasmid candidates using an inverse-type PCR approach on a subset of sequences with 95% success, confirming the existence and length of discrete sequences. The implication of these findings is a broadened understanding of the traits of circular elements in nature and the possibility of massive data mining in existing metagenomic datasets to discover novel pools of complete plasmids thus vastly expanding the current plasmid database. PMID:24503942

  17. A Real Time PCR Platform for the Simultaneous Quantification of Total and Extrachromosomal HIV DNA Forms in Blood of HIV-1 Infected Patients

    PubMed Central

    Canovari, Benedetta; Scotti, Maddalena; Acetoso, Marcello; Valentini, Massimo; Petrelli, Enzo; Magnani, Mauro

    2014-01-01

    Background The quantitative measurement of various HIV-1 DNA forms including total, unintegrated and integrated provirus play an increasingly important role in HIV-1 infection monitoring and treatment-related research. We report the development and validation of a SYBR Green real time PCR (TotUFsys platform) for the simultaneous quantification of total and extrachromosomal HIV-1 DNA forms in patients. This innovative technique makes it possible to obtain both measurements in a single PCR run starting from frozen blood employing the same primers and standard curve. Moreover, due to identical amplification efficiency, it allows indirect estimation of integrated level. To specifically detect 2-LTR a qPCR method was also developed. Methodology/Findings Primers used for total HIV-1 DNA quantification spanning a highly conserved region were selected and found to detect all HIV-1 clades of group M and the unintegrated forms of the same. A total of 195 samples from HIV-1 patients in a wide range of clinical conditions were analyzed with a 100% success rate, even in patients with suppressed plasma viremia, regardless of CD4+ or therapy. No significant correlation was observed between the two current prognostic markers, CD4+ and plasma viremia, while a moderate or high inverse correlation was found between CD4+ and total HIV DNA, with strong values for unintegrated HIV DNA. Conclusions/Significance Taken together, the results support the use of HIV DNA as another tool, in addition to traditional assays, which can be used to estimate the state of viral infection, the risk of disease progression and to monitor the effects of ART. The TotUFsys platform allowed us to obtain a final result, expressed as the total and unintegrated HIV DNA copy number per microgram of DNA or 104 CD4+, for 12 patients within two working days. PMID:25364909

  18. Isolation of bacterial extrachromosomal DNA from human dental plaque associated with periodontal disease,using transposonaided capture (TRACA)

    PubMed Central

    Warburton, Philip J; Allan, Elaine; Hunter, Stephanie; Ward, John; Booth, Veronica; Wade, William G; Mullany, Peter

    2011-01-01

    The human oral cavity is host to a complex microbial community estimated to comprise > 700 bacterial species, of which at least half are thought to be not yet cultivable in vitro. To investigate the plasmids present in this community, we used a transposon-aided capture system, which allowed the isolation of plasmids from human oral supra- and subgingival plaque samples. Thirty-two novel plasmids and a circular molecule that could be an integrase-generated circular intermediate were isolated. PMID:21711368

  19. Extrachromosomal oncogene amplification drives tumour evolution and genetic heterogeneity.

    PubMed

    Turner, Kristen M; Deshpande, Viraj; Beyter, Doruk; Koga, Tomoyuki; Rusert, Jessica; Lee, Catherine; Li, Bin; Arden, Karen; Ren, Bing; Nathanson, David A; Kornblum, Harley I; Taylor, Michael D; Kaushal, Sharmeela; Cavenee, Webster K; Wechsler-Reya, Robert; Furnari, Frank B; Vandenberg, Scott R; Rao, P Nagesh; Wahl, Geoffrey M; Bafna, Vineet; Mischel, Paul S

    2017-03-02

    Human cells have twenty-three pairs of chromosomes. In cancer, however, genes can be amplified in chromosomes or in circular extrachromosomal DNA (ecDNA), although the frequency and functional importance of ecDNA are not understood. We performed whole-genome sequencing, structural modelling and cytogenetic analyses of 17 different cancer types, including analysis of the structure and function of chromosomes during metaphase of 2,572 dividing cells, and developed a software package called ECdetect to conduct unbiased, integrated ecDNA detection and analysis. Here we show that ecDNA was found in nearly half of human cancers; its frequency varied by tumour type, but it was almost never found in normal cells. Driver oncogenes were amplified most commonly in ecDNA, thereby increasing transcript level. Mathematical modelling predicted that ecDNA amplification would increase oncogene copy number and intratumoural heterogeneity more effectively than chromosomal amplification. We validated these predictions by quantitative analyses of cancer samples. The results presented here suggest that ecDNA contributes to accelerated evolution in cancer.

  20. Entropic penalties in circular DNA assembly

    SciTech Connect

    Zoli, Marco

    2014-11-07

    The thermodynamic properties of DNA circular molecules are investigated by a new path integral computational method which treats in the real space the fundamental forces stabilizing the molecule. The base pair and stacking contributions to the classical action are evaluated separately by simulating a broad ensemble of twisted conformations. We obtain, for two short sequences, a free energy landscape with multiple wells corresponding to the most convenient values of helical repeat. Our results point to a intrinsic flexibility of the circular structures in which the base pair fluctuations move the system from one well to the next thus causing the local unwinding of the helix. The latter is more pronounced in the shorter sequence whose cyclization causes a higher bending stress. The entropic reductions associated to the formation of the ordered helicoidal structure are estimated.

  1. Role of DNA damage and repair in the function of eukaryotic genes: radiation-induced single-strand breaks and their rejoining in chromosomal and extrachromosomal ribosomal DNA of Tetrahymena

    SciTech Connect

    Chiu, S.M.; Oleinick, N.L.

    1980-04-01

    The production and rejoining of single-strand breaks (SSB) in chromosomal DNA and extrachromosomal ribosomal DNA (rDNA) were investigated after sublethal doses of ..gamma.. radiation to exponentially growing Tetrahymena. Hydrogen-3-labeled total nuclear DNA isolated from either control or irradiated cells was heat denatured and electrophoresed in agarose gels containing formaldehyde. Ribosomal DNA was identified by hybridization to (/sup 32/P)rRNA after transferring the DNA from the gels to nitrocellulose strips. It was found that (a) approximately 0.68 SSB is produced in each strand of rDNA exposed to 40 krad; (b) greater than 80% of SSB were rejoined within the first 20 min after irradiation in both chromosomal and rDNA; and (c) the rejoining process in both chromosomal and rDNA proceeded in the presence of inhibitors of protein synthesis, RNA synthesis, or oxidative metabolism. While the majority of SSB induced by 40 krad is rejoined within 20 min after irradiation, the resumption of rRNA synthesis does not occur until 30 min thereafter; it is concluded that the restoration of the normal size of the rDNA template is probably necessary but not sufficient for the resumption of rRNA synthesis.

  2. Construction of DNA Hemicatenanes from Two Small Circular DNA Molecules

    PubMed Central

    Gaillard, Claire; Strauss, François

    2015-01-01

    DNA hemicatenanes, one of the simplest possible junctions between two double stranded DNA molecules, have frequently been mentioned in the literature for their possible function in DNA replication, recombination, repair, and organization in chromosomes. They have been little studied experimentally, however, due to the lack of an appropriate method for their preparation. Here we have designed a method to build hemicatenanes from two small circular DNA molecules. The method involves, first, the assembly of two linear single strands and their circularization to form a catenane of two single stranded circles, and, second, the addition and base-pairing of the two single stranded circles complementary to the first ones, followed by their annealing using DNA topoisomerase I. The product was purified by gel electrophoresis and characterized. The arrangement of strands was as expected for a hemicatenane and clearly distinct from a full catenane. In addition, each circle was unwound by an average of half a double helical turn, also in excellent agreement with the structure of a hemicatenane. It was also observed that hemicatenanes are quickly destabilized by a single cut on either of the two strands passing inside the junction, strongly suggesting that DNA strands are able to slide easily inside the hemicatenane. This method should make it possible to study the biochemical properties of hemicatenanes and to test some of the hypotheses that have been proposed about their function, including a possible role for this structure in the organization of complex genomes in loops and chromosomal domains. PMID:25799010

  3. Ty3 integrase mutants defective in reverse transcription or 3'-end processing of extrachromosomal Ty3 DNA.

    PubMed Central

    Kirchner, J; Sandmeyer, S B

    1996-01-01

    Ty3, a retroviruslike element in Saccharomyces cerevisiae, encodes an integrase (IN) which is essential for position-specific transposition. The Ty3 integrase contains the highly conserved His-Xaa(3-7)-His-Xaa(23-32)-Cys-Xaa(2)-Cys and Asp, Asp-Xaa(35)-Glu [D,D(35)E] motifs found in retroviral integrases. Mutations were introduced into the coding region for the Ty3 integrase to determine the effects in vivo of changes in conserved residues of the putative catalytic triad D,D(35)E and the nonconserved carboxyl-terminal region. Ty3 viruslike particles were found to be associated with significant amounts of linear DNA of the approximate size expected for a full-length reverse transcription product and with plus-strand strong-stop DNA. The full-length, preintegrative DNA has at each 3' end 2 bp that are removed prior to or during integration. Such 3'-end processing has not been observed for other retroviruslike elements. A mutation at either D-225 or E-261 of the Ty3 integrase blocked transposition and prevented processing of the 3' ends of Ty3 DNA in vivo, suggesting that the D,D(35)E region is part of the catalytic domain of Ty3 IN. Carboxyl-terminal deletions of integrase caused a dramatic reduction in the amount of Ty3 DNA in vivo and a decrease in reverse transcriptase activity in vitro but did not affect the apparent size or amount of the 55-kDa reverse transcriptase in viruslike particles. The 115-kDa viruslike particle protein, previously shown to react with antibodies to Ty3 integrase, was shown to be a reverse transcriptase-IN fusion protein. These results are consistent with a role for the integrase domain either in proper folding of reverse transcriptase or as part of a heterodimeric reverse transcriptase molecule. PMID:8676501

  4. Unstable amplification of two extrachromosomal elements in alpha-difluoromethylornithine-resistant Leishmania donovani.

    PubMed Central

    Hanson, S; Beverley, S M; Wagner, W; Ullman, B

    1992-01-01

    We describe the first example of unstable gene amplification consisting of linear extrachromosomal DNAs in drug-resistant eukaryotic cells. alpha-Difluoromethylornithine (DFMO)-resistant Leishmania donovani with an amplified ornithine decarboxylase (ODC) gene copy number contained two new extrachromosomal DNAs, both present in 10 to 20 copies. One of these was a 140-kb linear DNA (ODC140-L) on which all of the amplified copies of the odc gene were located. The second was a 70-kb circular DNA (ODC70-C) containing an inverted repeat but lacking the odc gene. Both ODC140-L and ODC70-C were derived from a preexisting wild-type chromosome, probably by a conservative amplification mechanism. Both elements were unstable in the absence of DFMO, and their disappearance coincided with a decrease in ODC activity and an increase in DFMO growth sensitivity. These results suggest the possibility that ODC70-C may play a role in DFMO resistance. These data expand the diversity of known amplification mechanisms in eukaryotes to include the simultaneous unstable amplification of both linear and circular DNAs. Further characterization of these molecules will provide insights into the molecular mechanisms underlying gene amplification, including the ability of linear amplified DNAs to acquire telomeres and the determinants of chromosomal stability. Images PMID:1448081

  5. Z-DNA: vacuum ultraviolet circular dichroism

    SciTech Connect

    Sutherland, J.C.; Griffin, K.P.; Keck, P.C.; Takacs, P.Z.

    1981-08-01

    In concentrated salt or ethanolic solutions, the self-complementary copolymer poly(dG-dC)-poly(dG-dC) forms a left-handed double-helical structure that has been termed Z-DNA. The first evidence for this structure came from changes observed in the circular dichroism (CD) spectrum between 230 and 300 nm for low- and high-salt solutions. In 3 M NaCl, the CD spectrum is approximately inverted compared to the B-form spectrum observed in low salt solution. We measured the vacuum ultraviolet CD spectrum of poly(dG-dC)-poly(dG-dC) down to 180 nm under conditions in which the 230- to 300-nm spectrum is inverted. Below 200 nm, where the B form exhibits the large positive peak at 187 nm that is characteristic of right-handed double-helical DNAs, the Z form exhibits a large negative peak at 194 nm and a positive band below 186 nm. Therefore, the Z-form vacuum ultraviolet CD spectrum resembles an inverted and red-shifted B-form spectrum. The magnitudes of the differences observed between the B and Z forms in the CD spectrum below 200 nm are about 10 times greater than those observed between 230 and 300 nm. The vacuum ultraviolet CD spectrum of poly(dG-dC)-poly(dG-dC) is 3 M C/sub 2/O/sub 4/ also is inverted compared to the B-form spectrum; however, between 230 and 300 nm, it is nonconservative with a negative maximum at 290 nm and a weak positive CD signal above 300 nm, presumably reflecting differential light scattering and indicating the existence of molecular aggregates. Our results suggest that the vacuum ultraviolet CD spectrum is sensitive to the handedness of doublehelical DNA structures.

  6. Z-DNA Vacuum ultraviolet circular dichroism

    SciTech Connect

    Sutherland, J.C.; Griffin, K.P.; Keck, P.C.; Takacs, P.Z.

    1981-08-01

    In concentrated salt or ethanolic solutions, the self-complementary copolymer poly(dG-dC).poly(dG-dC) forms a left-handed double-helical structure that has been termed ZDNA. The first evidence for this structure came from changes observed in the circular dichroism (CD) spectrum between 230 and 300 nm for low- and high-salt solutions (Pohl, F.M. and Jovin, T.M. (1972) J. Mol. Biol. 67, 675-696). In 3 M NaCl, the CD spectrum is approximately inverted compared to the B-form spectrum observed in low-salt solution. We measured the vacuum ultraviolet CD spectrum of poly(dG-dC).poly(dG-dC) down to 180 nm under conditions in which the 230 to 300 nm spectrum is inverted. Below 200 nm, where the B form exhibits the large positive peak at 187 nm that is characteristic of right-handed double-helical DNAs, the Z form exhibits a large negative peak at 194 nm and a positive band below 186 nm. Therefore, the Z-form vacuum ultraviolet CD spectrum resembles an inverted and red-shifted B-form spectrum. The magnitudes of the differences observed between the Band Z forms in the CD spectrum below 200 nm are about 10 times greater than those observed between 230 and 300 nm. The vacuum ultraviolet CD spectrum of poly(dG-dC).poly(dG-dC) in 3 M Cs/sub 2/SO/sub 4/ also is inverted compared to the B-form spectrum; however, between 230 and 300 nm, it is nonconservative with a negative maximum at 290 nm and a weak positive CD signal above 300 nm, presumably reflecting differential light scattering and indicating the existence of molecular aggregates. Our results suggest that the vacuum ultraviolet CD spectrum is sensitive to the handedness of double-helical DNA structures.

  7. Effect of Temperature on Topological States of Circular DNA

    NASA Astrophysics Data System (ADS)

    Fan, Yang-Tao; Li, Xiu-Yan; Liu, Yan-Hui; Chen, Hu

    2017-07-01

    The different topological states of circular double-stranded DNA can be defined by their linking number. The equilibrium distribution of linking number can be obtained by circularizing a linear DNA into a circle by ligase. Based on the recent experimental results that the DNA bending rigidity and twist rigidity strongly depend on temperature, the reduced bending rigidity can be approximated by g=(3.19× {10}-19-T\\cdot 4.14× {10}-22) {erg}\\cdot {cm} over the temperature interval (5 ∼ 53) °C, and the temperature dependence of twist rigidity can be fitted by C(T)=(4588.89{exp} (-T/117.04)-251.33) nm. The temperature dependence of the linking number distribution of circular DNAs can be predicted by using Monte Carlo simulation. The variance of linking number distribution on temperature is in accordance with the previous experimental results. Compared with the temperature dependence of bending rigidity, the temperature dependence of twist rigidity causes a noticeable fluctuation in linking number distribution and mainly contribute towards the variance change of linking number distribution of circular DNA. The variance of the writhe number and twist number in the equation < {({{Δ }}{Lk})}2> =< {({{Δ }}{Tw})}2> +< {({Wr})}2> depends on the length of circular DNA. When the length of circular DNA is less than 230 nm, the variance of twist number < {({{Δ }}{Tw})}2> is dominant over the variance of writhe number (< {({Wr})}2> ), whereas for the condition that the length of the circular DNA is larger than 370 nm. Supported by the National Natural Science Foundation of China under Grant Nos. 11047022, 11204045, and 11464004, Guizhou Provincial Tracking Key Program of Social Development (SY20123089, SZ20113069), the General Financial Grant from the China Postdoctoral Science Foundation (2014M562341), the Research Foundation for Young University Teachers from Guizhou University (201311), and College Innovation Talent Team of Guizhou Province (2014)32

  8. Identification of a novel circular DNA virus in pig feces

    USDA-ARS?s Scientific Manuscript database

    Metagenomic analysis of fecal samples collected from a swine with diarrhea detected sequences encoding a replicase (Rep) protein typically found in small circular Rep-encoding ssDNA (CRESS-DNA) viruses. The complete 3,062 nucleotide genome was generated and found to encode two bi-directionally trans...

  9. Isolation of Circular DNA Molecules from Whole Cellular DNA by Use of ATP-Dependent Deoxyribonuclease

    PubMed Central

    Mukai, Tsunehiro; Matsubara, Kenichi; Takagi, Yasuyuki

    1973-01-01

    A technique is described for isolation of plasmid DNA in closed and open circular double-stranded forms from bacterial cells, by use of ATP-dependent deoxyribonuclease purified from Micrococcus luteus. This DNase, acting only upon linear DNA molecules, degrades all bacterial chromosomal DNA extracted in the linear form. Circular plasmid DNAs are left intact, and are then separated by sedimentation through a sucrose gradient. Unlike previous techniques for analysis of plasmid DNA, this technique can be used to isolate not only closed circular DNA but also open circular DNA. Several plasmids, such as those from phage (λdv1 and λdv21), a colicinogenic factor (Col E2), a sex factor (F8′ gal), and “minicircles” in Escherichia coli 15, in both the open and closed circular forms, were well separated from chromosomal DNA by this technique. PMID:4355370

  10. CSA: an efficient algorithm to improve circular DNA multiple alignment.

    PubMed

    Fernandes, Francisco; Pereira, Luísa; Freitas, Ana T

    2009-07-23

    The comparison of homologous sequences from different species is an essential approach to reconstruct the evolutionary history of species and of the genes they harbour in their genomes. Several complete mitochondrial and nuclear genomes are now available, increasing the importance of using multiple sequence alignment algorithms in comparative genomics. MtDNA has long been used in phylogenetic analysis and errors in the alignments can lead to errors in the interpretation of evolutionary information. Although a large number of multiple sequence alignment algorithms have been proposed to date, they all deal with linear DNA and cannot handle directly circular DNA. Researchers interested in aligning circular DNA sequences must first rotate them to the "right" place using an essentially manual process, before they can use multiple sequence alignment tools. In this paper we propose an efficient algorithm that identifies the most interesting region to cut circular genomes in order to improve phylogenetic analysis when using standard multiple sequence alignment algorithms. This algorithm identifies the largest chain of non-repeated longest subsequences common to a set of circular mitochondrial DNA sequences. All the sequences are then rotated and made linear for multiple alignment purposes.To evaluate the effectiveness of this new tool, three different sets of mitochondrial DNA sequences were considered. Other tests considering randomly rotated sequences were also performed. The software package Arlequin was used to evaluate the standard genetic measures of the alignments obtained with and without the use of the CSA algorithm with two well known multiple alignment algorithms, the CLUSTALW and the MAVID tools, and also the visualization tool SinicView. The results show that a circularization and rotation pre-processing step significantly improves the efficiency of public available multiple sequence alignment algorithms when used in the alignment of circular DNA sequences

  11. Supercoiled circular DNA of an insect granulosis virus

    PubMed Central

    Tweeten, Kathleen A.; Bulla, Lee A.; Consigli, Richard A.

    1977-01-01

    The DNA of the granulosis virus of the Indian meal moth, Plodia interpunctella, was characterized by physical chemical and electron microscopic techniques. Twenty-five percent of the DNA extracted from purified virus was isolated as supercoiled circular molecules. The remaining 75% consisted of relaxed circular molecules. These molecular forms were indicated by the production of two radioactive bands during sedimentation of 3H-labeled granulosis virus DNA in alkaline sucrose gradients or in equilibrium density gradients of neutral cesium chloride/propidium iodide. Electron microscopic visualization of the DNA that banded at the higher density in the latter gradients revealed supercoiled structures whereas that of DNA that banded at the lower density demonstrated relaxed circular molecules. The superhelical molecules were converted to relaxed circles by treatment with pancreatic DNase. The molecular weight of the viral DNA was calculated to be 81 × 106 by sedimentation in neutral sucrose and 78 × 106 by sedimentation in alkaline sucrose. The molecular weight estimated from length measurements in electron micrographs was 76 × 106. The buoyant density of the granulosis virus DNA was 1.703 g/cm3 and that of its insect host DNA was 1.697 g/cm3. Equilibrium sedimentation in cesium chloride and thermal denaturation indicated G + C contents of 44% and 39% for the viral and host DNA, respectively. Images PMID:198791

  12. Fluorescently labeled circular DNA molecules for DNA topology and topoisomerases

    PubMed Central

    Gu, Maxwell; Berrido, Andrea; Gonzalez, Walter G.; Miksovska, Jaroslava; Chambers, Jeremy W.; Leng, Fenfei

    2016-01-01

    DNA topology plays essential roles in several fundamental biological processes, such as DNA replication, recombination, and transcription. Typically agarose gel electrophoresis is employed to study DNA topology. Since gel electrophoresis is time-consuming and labor intensive, it is desirable to develop other methods, such as fluorescence-based methods, for such studies. In this paper we report the synthesis of a type of unique fluorescence-labeled DNA molecules that can be used to study DNA topology and topoisomerases by fluorescence resonance energy transfer (FRET). Specifically, we inserted an 82 nt. synthetic DNA oligomer FL905 carrying a 42 nt. AT sequence with fluorescein and dabcyl labels into a gapped DNA molecule to generate relaxed and supercoiled pAB1_FL905. Since the fluorescence intensity of pAB1_FL905 is dependent on its supercoiling status, pAB1_FL905 is a powerful tool to study DNA topology and topoisomerases by FRET. pAB1_FL905 can also be developed into rapid and efficient high-throughput screening assays to identify inhibitors that target various DNA topoisomerases. PMID:27796331

  13. Diverse small circular DNA viruses circulating amongst estuarine molluscs.

    PubMed

    Dayaram, Anisha; Goldstien, Sharyn; Argüello-Astorga, Gerardo R; Zawar-Reza, Peyman; Gomez, Christopher; Harding, Jon S; Varsani, Arvind

    2015-04-01

    Our understanding of the diversity and abundance of circular replication associated protein (Rep) - encoding single stranded (CRESS) DNA viruses has increased considerably over the last few years due to a combination of modern sequencing technologies and new molecular tools. Studies have used these to identify and recover CRESS DNA viruses from a range of different marine organisms, including copepods, shrimp and molluscs. In our study we identified 79 novel CRESS DNA viruses from three mollusc species (Austrovenus stutchburyi, Paphies subtriangulata and Amphibola crenata) and benthic sediments from the Avon-Heathcote estuary in Christchurch, New Zealand. The genomes recovered have varying genome architectures, with all encoding at least two major ORFs that have either unidirectional or bidirectional organisation. Analysis of the Reps of the viral genomes showed they are all highly diverse, with only one Rep sequence sharing 65% amino acid identity with the Rep of gastropod-associated circular DNA virus (GaCSV). Our study adds significantly to the wealth of CRESS DNA viruses recovered from freshwater and marine environments and extends our knowledge of the distribution of these viruses. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Small polydispersed circular DNA contains strains of mobile genetic elements and occurs more frequently in permanent cell lines of malignant tumors than in normal lymphocytes.

    PubMed

    Schmidt, Hannelore; Taubert, Helge; Lange, Heidemarie; Kriese, Karen; Schmitt, Wolfgang Daniel; Hoffmann, Steve; Bartel, Frank; Hauptmann, Steffen

    2009-08-01

    Small polydispersed circular DNA (spcDNA) belongs to the extrachromosomal pool of DNA and is composed of heterogeneous DNA circles. Whether spcDNA has a special function is currently unclear but their occurrence was suggested to be linked to genetic instability. In this study we investigated as to whether human lymphocytes from healthy volunteers also harbour spcDNA and whether spcDNA is present in all permanent cell lines from human normal and malignant tissues. Moreover, we were interested to see whether spcDNA contains sequences of mobile genetic elements. Our results show that spcDNA is present in all samples investigated yet the amount is lower in normal lymphocytes when compared to cancer cell lines (5.4 vs. 17.8%). Alu sequences were present in 12/16 cancer cell lines whereas LINE-1 (L1) sequences were present in 15 of them. Six tumor cell lines also contained telomeric sequences. In contrast to that, spcDNA of normal lymphocytes contains Alu and L1 sequences only in 3/16 cases and no telomeric sequences at all. Our findings suggest a direct dependency of the amount of Alu and L1 sequences on that of spcDNA. Beside these repetitive sequences, sequencing of spcDNA revealed in most cases chromosomal sequences of almost all chromosomes without an increased frequency of single regions. We suggest that the whole spcDNA including retrotranspositional elements and telomeric sequences may play a role for chromosomal rearrangements and genomic instability.

  15. Small circular DNA molecules act as rigid motifs to build DNA nanotubes.

    PubMed

    Zheng, Hongning; Xiao, Minyu; Yan, Qin; Ma, Yinzhou; Xiao, Shou-Jun

    2014-07-23

    Small circular DNA molecules with designed lengths, for example 64 and 96 nucleotides (nt), after hybridization with a few 32-nt staple strands respectively, can act as rigid motifs for the construction of DNA nanotubes with excellent uniformity in ring diameter. Unlike most native DNA nanotubes, which consist of longitudinal double helices, nanotubes assembled from circular DNAs are constructed from lateral double helices. Of the five types of DNA nanotubes designed here, four are built by alternating two different rings of the same ring size, while one is composed of all the same 96-nt rings. Nanotubes constructed from the same 96-nt rings are 10-100 times shorter than those constructed from two different 96-nt rings, because there are fewer hinge joints on the rings.

  16. Quantitative analysis of the flexibility effect of cisplatin on circular DNA

    NASA Astrophysics Data System (ADS)

    Ji, Chao; Zhang, Lingyun; Wang, Peng-Ye

    2013-10-01

    We study the effects of cisplatin on the circular configuration of DNA using atomic force microscopy (AFM) and observe that the DNA gradually transforms to a complex configuration with an intersection and interwound structures from a circlelike structure. An algorithm is developed to extract the configuration profiles of circular DNA from AFM images and the radius of gyration is used to describe the flexibility of circular DNA. The quantitative analysis of the circular DNA demonstrates that the radius of gyration gradually decreases and two processes on the change of flexibility of circular DNA are found as the cisplatin concentration increases. Furthermore, a model is proposed and discussed to explain the mechanism for understanding the complicated interaction between DNA and cisplatin.

  17. Quantitative analysis of the flexibility effect of cisplatin on circular DNA.

    PubMed

    Ji, Chao; Zhang, Lingyun; Wang, Peng-Ye

    2013-10-01

    We study the effects of cisplatin on the circular configuration of DNA using atomic force microscopy (AFM) and observe that the DNA gradually transforms to a complex configuration with an intersection and interwound structures from a circlelike structure. An algorithm is developed to extract the configuration profiles of circular DNA from AFM images and the radius of gyration is used to describe the flexibility of circular DNA. The quantitative analysis of the circular DNA demonstrates that the radius of gyration gradually decreases and two processes on the change of flexibility of circular DNA are found as the cisplatin concentration increases. Furthermore, a model is proposed and discussed to explain the mechanism for understanding the complicated interaction between DNA and cisplatin.

  18. ELECTRON MICROSCOPY OF SINGLE-STRANDED DNA: CIRCULARITY OF DNA OF BACTERIOPHAGE PHI-X174.

    PubMed

    FREIFELDER, D; KLEINSCHMIDT, A K; SINSHEIMER, R L

    1964-10-09

    The single-stranded DNA of coliphage X174 has been examined with the electron microscope by a modification of the protein-monolayer-adsorption technique. The molecules were found to be circular with a total length of 1.77 +/- 0.13 microns.

  19. Molecular modeling of closed circular DNA thermodynamic ensembles.

    PubMed

    Sprous, D; Tan, R K; Harvey, S C

    1996-08-01

    Many modeling studies of supercoiled DNA are based on equilibrium structures from theoretical calculations or energy minimization. Since closed circular DNAs are flexible, it is possible that errors are introduced by calculating properties from a single minimum energy structure, rather than from a complete thermodynamic ensemble. We have investigated this question using molecular dynamics simulations on a low resolution molecular mechanics model in which each base pair is represented by three points (a plane). This allows the inclusion of sequence-dependent variations of tip, inclination, and twist. Three kinds of sequences were tested: (1) homogeneous DNA, in which all base pairs have the helicoidal parameters of an ideal, average B-DNA; (2) random sequence DNA; and (3) curved DNA. We examined the rate of convergence of various structural parameters. Convergence for most of these is slowest for homogeneous sequences, more rapid for random sequences, and most rapid for curved sequences. The most slowly converging parameter is the antipodes profile. In a plasmid with N base pairs (bp), the antipodes distance is the distance dij from base pair i to base pair j halfway around the plasmid, j = i + N/2. The antipodes profile at time tau is a plot of dij over the range i = 1, N/2. In a homogeneous plasmid, convergence requires that the antipodes profile averaged over time must be flat. Even in the small plasmids examined here, the average properties of the ensembles were found to differ from those of static equilibrium structures. These effects will be even more dramatic for larger plasmids. Further, average and dynamic properties are affected by both plasmid size and sequence.

  20. Atomic force microscopy studies on circular DNA structural changes by vincristine and aspirin.

    PubMed

    Xiao, Zhongdang; Cao, Lili; Zhu, Dan; Lu, Zuhong

    2011-01-01

    In this chapter, we have presented materials and methods to study the interaction between DNA and small molecule drugs by AFM. The detailed AFM imaging of the circular DNA after incubation with -various concentrations of vincristine and aspirin have been demonstrated. The immobilization of DNA fragments on mica surface as well as the force between tip and sample plays an important role for successful imaging of DNA-drug complexes. How to quantitatively describe the conformations and structures of circular DNA molecules and their changes is also introduced. Our work indicates that the AFM is a powerful tool in studying the interaction between DNA and small molecules.

  1. Spectroscopic detection of DNA quadruplexes by vibrational circular dichroism.

    PubMed

    Andrushchenko, Valery; Tsankov, Dimiter; Krasteva, Maria; Wieser, Helmut; Bour, Petr

    2011-09-28

    The four-stranded G-quadruplex motif is a conformation frequently adopted by guanine-rich nucleic acids that plays an important role in biology, medicine, and nanotechnology. Although vibrational spectroscopy has been widely used to investigate nucleic acid structure, association of particular spectral features with the quadruplex structure has to date been ambiguous. In this work, experimental IR absorption and vibrational circular dichroism (VCD) spectra of the model quadruplex systems d(G)(8) and deoxyguanosine-5'-monophosphate (5'-dGMP) were analyzed using molecular dynamics (MD) and quantum-chemical modeling. The experimental spectra were unambiguously assigned to the quadruplex DNA arrangement, and several IR and VCD bands related to this structural motif were determined. Involvement of MD in the modeling was essential for realistic simulation of the spectra. The VCD signal was found to be more sensitive to dynamical structural variations than the IR signal. The combination of the spectroscopic techniques with multiscale simulations provides extended information about nucleic acid conformations and their dynamics.

  2. Circular Dichroism for the Analysis of Protein-DNA Interactions.

    PubMed

    Scarlett, Garry; Siligardi, Giuliano; Kneale, Geoffrey G

    2015-01-01

    The aim of this chapter is to provide information on the practical aspects of circular dichroism (CD) and synchrotron radiation circular dichroism (SRCD) in protein-nucleic acids interaction solution studies. The chapter will describe the guidelines appropriate to designing experiments and conducting correct data interpretation, the use of both benchtop and synchrotron CD approaches is discussed and the advantages of SRCD outlined. Further information and a good general review of the field a can be found in Gray (Circular Dichroism of protein-nucleic acid interactions. In: Fasman GD (ed) Circular dichroism and the conformational analysis of biomolecules. Plenum Press, New York. pp 469-500, 1996).

  3. Validation of DNA probes for molecular cytogenetics by mapping onto immobilized circular DNA

    PubMed Central

    Greulich-Bode, Karin M; Wang, Mei; Rhein, Andreas P; Weier, Jingly F; Weier, Heinz-Ulli G

    2008-01-01

    Background Fluorescence in situ hybridization (FISH) is a sensitive and rapid procedure to detect gene rearrangements in tumor cells using non-isotopically labeled DNA probes. Large insert recombinant DNA clones such as bacterial artificial chromosome (BAC) or P1/PAC clones have established themselves in recent years as preferred starting material for probe preparations due to their low rates of chimerism and ease of use. However, when developing probes for the quantitative analysis of rearrangements involving genomic intervals of less than 100 kb, careful probe selection and characterization are of paramount importance. Results We describe a sensitive approach to quality control probe clones suspected of carrying deletions or for measuring clone overlap with near kilobase resolution. The method takes advantage of the fact that P1/PAC/BAC's can be isolated as circular DNA molecules, stretched out on glass slides and fine-mapped by multicolor hybridization with smaller probe molecules. Two examples demonstrate the application of this technique: mapping of a gene-specific ~6 kb plasmid onto an unusually small, ~55 kb circular P1 molecule and the determination of the extent of overlap between P1 molecules homologous to the human NF-κB2 locus. Conclusion The relatively simple method presented here does not require specialized equipment and may thus find widespread applications in DNA probe preparation and characterization, the assembly of physical maps for model organisms or in studies on gene rearrangements. PMID:19108707

  4. Validation of DNA probes for molecular cytogenetics by mapping onto immobilized circular DNA

    SciTech Connect

    Greulich-Bode, Karin; Wang, Mei; Rhein, Andreas; Weier, Jingly; Weier, Heinz-Ulli

    2008-12-16

    Fluorescence in situ hybridization (FISH) is a sensitive and rapid procedure to detect gene rearrangements in tumor cells using non-isotopically labeled DNA probes. Large insert recombinant DNA clones such as bacterial artificial chromosome (BAC) or P1/PAC clones have established themselves in recent years as preferred starting material for probe preparations due to their low rates of chimerism and ease of use. However, when developing probes for the quantitative analysis of rearrangements involving genomic intervals of less than 100kb, careful probe selection and characterization are of paramount importance. We describe a sensitive approach to quality control probe clones suspected of carrying deletions or for measuring clone overlap with near kilobase resolution. The method takes advantage of the fact that P1/PAC/BAC's can be isolated as circular DNA molecules, stretched out on glass slides and fine-mapped by multicolor hybridization with smaller probe molecules. Two examples demonstrate the application of this technique: mapping of a gene-specific {approx}6kb plasmid onto an unusually small, {approx}55kb circular P1 molecule and the determination of the extent of overlap between P1 molecules homologous to the human NF-?B2 locus. The relatively simple method presented here does not require specialized equipment and may thus find widespread applications in DNA probe preparation and characterization, the assembly of physical maps for model organisms or in studies on gene rearrangements.

  5. Validation of DNA probes for molecular cytogenetics by mapping onto immobilized circular DNA

    SciTech Connect

    Greulich-Bode, Karin M.; Wang, Mei; Rhein, Andreas P.; Weier, Jingly F.; Weier, Heinz-Ulli G.

    2008-12-04

    Fluorescence in situ hybridization (FISH) is a sensitive and rapid procedure to detect gene rearrangements in tumor cells using non-isotopically labeled DNA probes. Large insert recombinant DNA clones such as bacterial artificial chromosome (BAC) or P1/PAC clones have established themselves in recent years as preferred starting material for probe preparations due to their low rates of chimerism and ease of use. However, when developing probes for the quantitative analysis of rearrangements involving genomic intervals of less than 100kb, careful probe selection and characterization are of paramount importance. We describe a sensitive approach to quality control probe clones suspected of carrying deletions or for measuring clone overlap with near kilobase resolution. The method takes advantage of the fact that P1/PAC/BAC's can be isolated as circular DNA molecules, stretched out on glass slides and fine-mapped by multicolor hybridization with smaller probe molecules. Two examples demonstrate the application of this technique: mapping of a gene-specific {approx}6kb plasmid onto an unusually small, {approx}55kb circular P1 molecule and the determination of the extent of overlap between P1 molecules homologous to the human NF-{kappa}B2 locus. The relatively simple method presented here does not require specialized equipment and may thus find widespread applications in DNA probe preparation and characterization, the assembly of physical maps for model organisms or in studies on gene rearrangements.

  6. Apparent circular dichroism signature of stirring-oriented DNA and drug-DNA complexes.

    PubMed

    Zsila, Ferenc

    2015-01-01

    It is shown that in water with no added salts calf-thymus DNA and its drug-loaded forms exhibit peculiar, completely reversible circular dichroism (CD) spectroscopic changes upon mechanical rotatory stirring. Due to the stirring-induced spatial alignment of the helices, the CD spectra are overwhelmed by the much more intense linear dichroism contribution. This apparent chiroptical response can be generated and detected without using any sophisticated attachment. It is a technically simple complement to existing methods suitable to obtain additional structural information which can not be derived from isotropic spectra. Drug-DNA interactions generating no or very weak CD spectroscopic changes under isotropic conditions become easily detectable upon stirring the sample solution. Stirring-induced changes of the CD profile also enable to clarify the DNA binding mode of various compounds (e.g., imatinib, thioflavin T) which would remain ambiguous considering isotropic spectral data only. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Novel circular DNA viruses in stool samples of wild-living chimpanzees

    PubMed Central

    Blinkova, Olga; Victoria, Joseph; Li, Yingying; Keele, Brandon F.; Sanz, Crickette; Ndjango, Jean-Bosco N.; Peeters, Martine; Travis, Dominic; Lonsdorf, Elizabeth V.; Wilson, Michael L.; Pusey, Anne E.; Hahn, Beatrice H.; Delwart, Eric L.

    2010-01-01

    Viral particles in stool samples from wild-living chimpanzees were analysed using random PCR amplification and sequencing. Sequences encoding proteins distantly related to the replicase protein of single-stranded circular DNA viruses were identified. Inverse PCR was used to amplify and sequence multiple small circular DNA viral genomes. The viral genomes were related in size and genome organization to vertebrate circoviruses and plant geminiviruses but with a different location for the stem–loop structure involved in rolling circle DNA replication. The replicase genes of these viruses were most closely related to those of the much smaller (∼1 kb) plant nanovirus circular DNA chromosomes. Because the viruses have characteristics of both animal and plant viruses, we named them chimpanzee stool-associated circular viruses (ChiSCV). Further metagenomic studies of animal samples will greatly increase our knowledge of viral diversity and evolution. PMID:19759238

  8. Novel circular DNA viruses in stool samples of wild-living chimpanzees.

    PubMed

    Blinkova, Olga; Victoria, Joseph; Li, Yingying; Keele, Brandon F; Sanz, Crickette; Ndjango, Jean-Bosco N; Peeters, Martine; Travis, Dominic; Lonsdorf, Elizabeth V; Wilson, Michael L; Pusey, Anne E; Hahn, Beatrice H; Delwart, Eric L

    2010-01-01

    Viral particles in stool samples from wild-living chimpanzees were analysed using random PCR amplification and sequencing. Sequences encoding proteins distantly related to the replicase protein of single-stranded circular DNA viruses were identified. Inverse PCR was used to amplify and sequence multiple small circular DNA viral genomes. The viral genomes were related in size and genome organization to vertebrate circoviruses and plant geminiviruses but with a different location for the stem-loop structure involved in rolling circle DNA replication. The replicase genes of these viruses were most closely related to those of the much smaller (approximately 1 kb) plant nanovirus circular DNA chromosomes. Because the viruses have characteristics of both animal and plant viruses, we named them chimpanzee stool-associated circular viruses (ChiSCV). Further metagenomic studies of animal samples will greatly increase our knowledge of viral diversity and evolution.

  9. A localized transition in the size variation of circular DNA in nanoslits

    NASA Astrophysics Data System (ADS)

    Strychalski, Elizabeth A.; Stavis, Samuel M.; Geist, Jon

    2013-03-01

    We observe a localized transition in the size variation of circular DNA between strong and moderate regimes of nanofluidic slitlike confinement. We applied a rigorous statistical analysis to our recent experimental measurements of DNA size for linear and circular topologies in nanoslits with depths around ~2p, where p is the DNA persistence length [E. A. Strychalski, J. Geist, M. Gaitan, L. E. Locascio, S. M. Stavis. Macromolecules, 45, 1602-1611 (2012)]. Our empirical approach revealed a localized transition between confinement regimes for circular DNA at a nanoslit depth of ~3p but detected no such transition for linear DNA with a similar contour length. These results provide the first indication of the localized influence of polymer topology on size variation across changing nanoslit depths. Improved understanding of differences in polymer behavior due to topology in this controversial system is of fundamental importance in polymer science and will inform new nanofluidic methods for biopolymer analysis.

  10. Self-assembly of aptamer-circular DNA nanostructures for controlled biocatalysis.

    PubMed

    Wang, Zhen-Gang; Wilner, Ofer I; Willner, Itamar

    2009-12-01

    Two kinds of circular DNA components are generated by the hybridization of short nucleic acids with the 3' and 5' ends of single-stranded DNA chains. The circular DNA components include, each, complementary domains for the anticocaine aptamer subunits, and sequence-specific domains for the auxiliary hybridization of programmed nucleic acid-functionalized proteins. The circular DNA components are self-assembled, in the presence of cocaine, into DNA nanowires (micrometer-long nanowires exhibiting heights of ca. 1.6-3.0 nm). Nucleic acids functionalized with glucose oxidase (GOx) and horseradish peroxidase (HRP) are hybridized with the circular DNA components to yield nanostructures consisting of HRP and GOx on the DNA scaffold. A biocatalytic cascade, where the GOx-catalyzed oxidization of glucose by O(2) yields H(2)O(2), and the resulting H(2)O(2) oxidizes 2,2'-azino-bis[3-ethylbenzthiazoline-6-sulfonic acid] (ABTS(2-)), in the presence of HRP, is activated by the system. The biocatalyzed oxidization of ABTS(2-) on the DNA scaffold is 6-fold enhanced as compared to a nonbridged homogeneous system of the two biocatalysts. The enhanced biocatalytic cascade on the DNA scaffold is attributed to high local concentrations of the reactive components in the vicinity of biocatalysts.

  11. The Infection Efficiency and Replication Ability of Circularized HBV DNA Optimized the Linear HBV DNA in Vitro and in Vivo

    PubMed Central

    Li, Xiaosong; Zhu, Junke; Lai, Guoqi; Yan, Lei; Hu, Jieli; Chen, Juan; Tang, Ni; Huang, Ailong

    2015-01-01

    Studies on molecular mechanisms of the persist infection of hepatitis B virus have been hampered by a lack of a robust animal model. We successfully established a simple, versatile, and reproducible HBV persist infection model in vitro and in vivo with the circularized HBV DNA. The cells and mice were transfected or injected with circularized HBV DNA and pAAV/HBV1.2, respectively. At the indicated time, the cells, supernatants, serum samples, and liver tissues were collected for virological and serological detection. Both in vitro and in vivo, the circularized HBV DNA and pAAV/HBV1.2 could replicate and transcribe efficiently, but the infection effect of the former was superior to the latter (p < 0.05). The injection of circularized HBV genome DNA into the mice robustly supported HBV infection and approximately 80% of HBV infected mice established persistent infection for at least 10 weeks. This study demonstrated that the infection efficiency and replication ability of the circularized structure of HBV DNA overmatched that of the expression plasmid containing the linear structure of HBV DNA in vitro and in vivo. Meanwhile, this research results could provide useful tools and methodology for further study of pathogenic mechanisms and potential antiviral treatments of human chronic HBV infection in vitro and in vivo. PMID:25751726

  12. Characterization of Quadruplex DNA Structure by Circular Dichroism.

    PubMed

    Del Villar-Guerra, Rafael; Gray, Robert D; Chaires, Jonathan B

    2017-03-02

    Circular dichroism (CD) is a phenomenon that arises from the differential absorption of left- and right-handed circularly polarized light, and may be seen with optically active molecules. CD spectroscopy provides useful spectral signatures for biological macromolecules in solution, and provides low-resolution structural information about macromolecular conformation. CD spectroscopy is particularly useful for monitoring conformational changes in macromolecules upon environmental perturbations. G-quadruplex structures show unique CD spectral signatures, and CD is an important tool for characterizing their formation and global structure. This protocol offers step-by-step methods for determining reliable and reproducible CD spectra of quadruplex structures and normalizing the spectra for presentation. CD spectra properly normalized with respect to quadruplex concentration and path length are required to facilitate accurate comparison of results among laboratories. The standard operating procedures proposed are recommended to make such comparison accurate and informative. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  13. Characterization of the host factors required for hepadnavirus covalently closed circular (ccc) DNA formation.

    PubMed

    Guo, Haitao; Xu, Chunxiao; Zhou, Tianlun; Block, Timothy M; Guo, Ju-Tao

    2012-01-01

    Synthesis of the covalently closed circular (ccc) DNA is a critical, but not well-understood step in the life cycle of hepadnaviruses. Our previous studies favor a model that removal of genome-linked viral DNA polymerase occurs in the cytoplasm and the resulting deproteinized relaxed circular DNA (DP-rcDNA) is subsequently transported into the nucleus and converted into cccDNA. In support of this model, our current study showed that deproteinization of viral double-stranded linear (dsl) DNA also took place in the cytoplasm. Furthermore, we demonstrated that Ku80, a component of non-homologous end joining DNA repair pathway, was essential for synthesis of cccDNA from dslDNA, but not rcDNA. In an attempt to identify additional host factors regulating cccDNA biosynthesis, we found that the DP-rcDNA was produced in all tested cell lines that supported DHBV DNA replication, but cccDNA was only synthesized in the cell lines that accumulated high levels of DP-rcDNA, except for NCI-H322M and MDBK cells, which failed to synthesize cccDNA despite of the existence of nuclear DP-rcDNA. The results thus imply that while removal of the genome-linked viral DNA polymerase is most likely catalyzed by viral or ubiquitous host function(s), nuclear factors required for the conversion of DP-rcDNA into cccDNA and/or its maintenance are deficient in the above two cell lines, which could be useful tools for identification of the elusive host factors essential for cccDNA biosynthesis or maintenance.

  14. Modeling chain folding in protein-constrained circular DNA.

    PubMed Central

    Martino, J A; Olson, W K

    1998-01-01

    An efficient method for sampling equilibrium configurations of DNA chains binding one or more DNA-bending proteins is presented. The technique is applied to obtain the tertiary structures of minimal bending energy for a selection of dinucleosomal minichromosomes that differ in degree of protein-DNA interaction, protein spacing along the DNA chain contour, and ring size. The protein-bound portions of the DNA chains are represented by tight, left-handed supercoils of fixed geometry. The protein-free regions are modeled individually as elastic rods. For each random spatial arrangement of the two nucleosomes assumed during a stochastic search for the global minimum, the paths of the flexible connecting DNA segments are determined through a numerical solution of the equations of equilibrium for torsionally relaxed elastic rods. The minimal energy forms reveal how protein binding and spacing and plasmid size differentially affect folding and offer new insights into experimental minichromosome systems. PMID:9591675

  15. Rapid purification of circular DNA by triplex-mediated affinity capture

    DOEpatents

    Ji, Huamin; Smith, Lloyd M.

    1997-01-01

    A single-step capture of a target supercoiled double-stranded DNA molecule is accomplished by forming a local triple-helix among two strands of the supercoiled circular DNA and an oligonucleotide probe. The oligonucleotide is bound to an immobilizing support which facilitates the immobilization and purification of target DNA molecules. Non-target DNA molecules and other contaminating cellular material are easily removed by washing. The triple-helical structure is destabilized by raising the pH, leaving purified target DNA in the supernatant and reusable affinity capture oligonucleotide secured to the immobilizing support.

  16. Rapid purification of circular DNA by triplex-mediated affinity capture

    DOEpatents

    Ji, H.; Smith, L.M.

    1997-01-07

    A single-step capture of a target supercoiled double-stranded DNA molecule is accomplished by forming a local triple-helix among two strands of the supercoiled circular DNA and an oligonucleotide probe. The oligonucleotide is bound to an immobilizing support which facilitates the immobilization and purification of target DNA molecules. Non-target DNA molecules and other contaminating cellular material are easily removed by washing. The triple-helical structure is destabilized by raising the pH, leaving purified target DNA in the supernatant and reusable affinity capture oligonucleotide secured to the immobilizing support. 3 figs.

  17. PicoGreen assay of circular DNA for radiation biodosimetry.

    PubMed

    Zhang, Steven B; Yang, Shanmin; Vidyasagar, Sadasivan; Zhang, Mei; Casey-Sawicki, Katherine; Liu, Chaomei; Yin, Liangjie; Zhang, Lei; Cao, Yongbing; Tian, Yeping; Swarts, Steven; Fenton, Bruce M; Keng, Peter; Zhang, Lurong; Okunieff, Paul

    2015-02-01

    We developed a simple, rapid and quantitative assay using the fluorescent probe PicoGreen to measure the concentration of ionizing radiation-induced double-stranded DNA (dsDNA) in mouse plasma, and we correlated this concentration with the radiation dose. With 70 μl of blood obtained by fingerstick, this 30 min assay reduces protein interference without extending sample processing time. Plasma from nonirradiated mice (BALB/c and NIH Swiss) was pooled, diluted and spiked with dsDNA to establish sensitivity and reproducibility of the assay to quantify plasma dsDNA. The assay was then used to directly quantify dsDNA in plasma at 0-48 h after mice received 0-10 Gy total-body irradiation (TBI). There are three optimal conditions for this assay: 1:10 dilution of plasma in water; 1:200 dilution of PicoGreen reagent in water; and calibration of radiation-induced dsDNA concentration through a standard addition method using serial spiking of samples with genomic dsDNA. Using the internal standard calibration curve of the spiked samples method, the signal developed within 5 min, exhibiting a linear signal (r(2) = 0.997). The radiation-induced elevation of plasma DNA in mice started at 1-3 h, peaked at 9 h and gradually returned to baseline at 24 h after TBI (6 Gy). DNA levels in plasma collected from mice 9 h after 0-10 Gy TBI correlated strongly with dose (r(2) = 0.991 and 0.947 for BALB/c and NIH Swiss, respectively). Using the PicoGreen assay, we observed a radiation dose-dependent response in extracellular plasma DNA 9 h after irradiation with an assay time ≤ 30 min.

  18. PicoGreen Assay of Circular DNA for Radiation Biodosimetry

    PubMed Central

    Zhang, Steven B.; Yang, Shanmin; Vidyasagar, Sadasivan; Zhang, Mei; Casey-Sawicki, Katherine; Liu, Chaomei; Yin, Liangjie; Zhang, Lei; Cao, Yongbing; Tian, Yeping; Swarts, Steven; Fenton, Bruce M.; Keng, Peter; Zhang, Lurong; Okunieff, Paul

    2015-01-01

    We developed a simple, rapid and quantitative assay using the fluorescent probe PicoGreen to measure the concentration of ionizing radiation-induced double-stranded DNA (dsDNA) in mouse plasma, and we correlated this concentration with the radiation dose. With 70 μl of blood obtained by fingerstick, this 30 min assay reduces protein interference without extending sample processing time. Plasma from nonirradiated mice (BALB/c and NIH Swiss) was pooled, diluted and spiked with dsDNA to establish sensitivity and reproducibility of the assay to quantify plasma dsDNA. The assay was then used to directly quantify dsDNA in plasma at 0–48 h after mice received 0–10 Gy total-body irradiation (TBI). There are three optimal conditions for this assay: 1:10 dilution of plasma in water; 1:200 dilution of PicoGreen reagent in water; and calibration of radiation-induced dsDNA concentration through a standard addition method using serial spiking of samples with genomic dsDNA. Using the internal standard calibration curve of the spiked samples method, the signal developed within 5 min, exhibiting a linear signal (r2 0.997). The radiation-induced elevation of plasma DNA in mice started at 1–3 h, peaked at 9 h and gradually returned to baseline at 24 h after TBI (6 Gy). DNA levels in plasma collected from mice 9 h after 0–10 Gy TBI correlated strongly with dose (r2 0.991 and 0.947 for BALB/c and NIH Swiss, respectively). Using the PicoGreen assay, we observed a radiation dose-dependent response in extracellular plasma DNA 9 h after irradiation with an assay time ≤30 min. PMID:25574588

  19. Discovery of a novel circular DNA virus in the Forbes sea star, Asterias forbesi.

    PubMed

    Fahsbender, Elizabeth; Hewson, Ian; Rosario, Karyna; Tuttle, Allison D; Varsani, Arvind; Breitbart, Mya

    2015-09-01

    A single-stranded DNA (ssDNA) virus, Asterias forbesi-associated circular virus (AfaCV), was discovered in a Forbes sea star displaying symptoms of sea star wasting disease (SSWD). The AfaCV genome organization is typical of circular Rep-encoding ssDNA (CRESS-DNA) viruses and is similar to that of members of the family Circoviridae. PCR-based surveys indicate that AfaCV is not clearly associated with SSWD, whereas the sea star-associated densovirus (SSaDV), recently implicated in SSWD in the Pacific, was prevalent in symptomatic specimens. AfaCV represents the first CRESS-DNA virus detected in echinoderms, adding to the growing diversity of these viruses recently recovered from invertebrates.

  20. Kaposi's sarcoma-associated herpesvirus stably clusters its genomes across generations to maintain itself extrachromosomally.

    PubMed

    Chiu, Ya-Fang; Sugden, Arthur U; Fox, Kathryn; Hayes, Mitchell; Sugden, Bill

    2017-09-04

    Genetic elements that replicate extrachromosomally are rare in mammals; however, several human tumor viruses, including the papillomaviruses and the gammaherpesviruses, maintain their plasmid genomes by tethering them to cellular chromosomes. We have uncovered an unprecedented mechanism of viral replication: Kaposi's sarcoma-associated herpesvirus (KSHV) stably clusters its genomes across generations to maintain itself extrachromosomally. To identify and characterize this mechanism, we developed two complementary, independent approaches: live-cell imaging and a predictive computational model. The clustering of KSHV requires the viral protein, LANA1, to bind viral genomes to nucleosomes arrayed on both cellular and viral DNA. Clustering affects both viral partitioning and viral genome numbers of KSHV. The clustering of KSHV plasmids provides it with an effective evolutionary strategy to rapidly increase copy numbers of genomes per cell at the expense of the total numbers of cells infected. © 2017 Chiu et al.

  1. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin.

    PubMed

    Bolotin, Alexander; Quinquis, Benoit; Sorokin, Alexei; Ehrlich, S Dusko

    2005-08-01

    Numerous prokaryote genomes contain structures known as clustered regularly interspaced short palindromic repeats (CRISPRs), composed of 25-50 bp repeats separated by unique sequence spacers of similar length. CRISPR structures are found in the vicinity of four genes named cas1 to cas4. In silico analysis revealed another cluster of three genes associated with CRISPR structures in many bacterial species, named here as cas1B, cas5 and cas6, and also revealed a certain number of spacers that have homology with extant genes, most frequently derived from phages, but also derived from other extrachromosomal elements. Sequence analysis of CRISPR structures from 24 strains of Streptococcus thermophilus and Streptococcus vestibularis confirmed the homology of spacers with extrachromosomal elements. Phage sensitivity of S. thermophilus strains appears to be correlated with the number of spacers in the CRISPR locus the strain carries. The authors suggest that the spacer elements are the traces of past invasions by extrachromosomal elements, and hypothesize that they provide the cell immunity against phage infection, and more generally foreign DNA expression, by coding an anti-sense RNA. The presence of gene fragments in CRISPR structures and the nuclease motifs in cas genes of both cluster types suggests that CRISPR formation involves a DNA degradation step.

  2. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin.

    PubMed

    Bolotin, Alexander; Quinquis, Benoit; Sorokin, Alexei; Ehrlich, S Dusko

    2005-08-01

    Numerous prokaryote genomes contain structures known as clustered regularly interspaced short palindromic repeats (CRISPRs), composed of 25-50 bp repeats separated by unique sequence spacers of similar length. CRISPR structures are found in the vicinity of four genes named cas1 to cas4. In silico analysis revealed another cluster of three genes associated with CRISPR structures in many bacterial species, named here as cas1B, cas5 and cas6, and also revealed a certain number of spacers that have homology with extant genes, most frequently derived from phages, but also derived from other extrachromosomal elements. Sequence analysis of CRISPR structures from 24 strains of Streptococcus thermophilus and Streptococcus vestibularis confirmed the homology of spacers with extrachromosomal elements. Phage sensitivity of S. thermophilus strains appears to be correlated with the number of spacers in the CRISPR locus the strain carries. The authors suggest that the spacer elements are the traces of past invasions by extrachromosomal elements, and hypothesize that they provide the cell immunity against phage infection, and more generally foreign DNA expression, by coding an anti-sense RNA. The presence of gene fragments in CRISPR structures and the nuclease motifs in cas genes of both cluster types suggests that CRISPR formation involves a DNA degradation step.

  3. New Type of Papillomavirus and Novel Circular Single Stranded DNA Virus Discovered in Urban Rattus norvegicus Using Circular DNA Enrichment and Metagenomics

    PubMed Central

    Hansen, Thomas Arn; Fridholm, Helena; Frøslev, Tobias Guldberg; Kjartansdóttir, Kristín Rós; Willerslev, Eske; Nielsen, Lars Peter; Hansen, Anders Johannes

    2015-01-01

    Rattus norvegicus (R. norvegicus) are ubiquitous and their presence has several effects on the human populations in our urban areas on a global scale. Both historically and presently, this close interaction has facilitated the dissemination of many pathogens to humans, making screening for potentially zoonotic and emerging viruses in rats highly relevant. We have investigated faecal samples from R. norvegicus collected from urban areas using a protocol based on metagenomic enrichment of circular DNA genomes and subsequent sequencing. We found a new type of papillomavirus, with a L1 region 82% identical to that of the known R. norvegicus Papillomavirus 2. Additionally, we found 20 different circular replication associated protein (Rep)-encoding single stranded DNA (CRESS-DNA) virus-like genomes, one of which has homology to the replication-associated gene of Beak and feather disease virus. Papillomaviruses are a group of viruses known for their carcinogenic potential, and although they are known to infect several different vertebrates, they are mainly studied and characterised in humans. CRESS-DNA viruses are found in many different environments and tissue types. Both papillomaviruses and CRESS-DNA viruses are known to have pathogenic potential and screening for novel and known viruses in R. norvegicus could help identify viruses with pathogenic potential. PMID:26559957

  4. New Type of Papillomavirus and Novel Circular Single Stranded DNA Virus Discovered in Urban Rattus norvegicus Using Circular DNA Enrichment and Metagenomics.

    PubMed

    Hansen, Thomas Arn; Fridholm, Helena; Frøslev, Tobias Guldberg; Kjartansdóttir, Kristín Rós; Willerslev, Eske; Nielsen, Lars Peter; Hansen, Anders Johannes

    2015-01-01

    Rattus norvegicus (R. norvegicus) are ubiquitous and their presence has several effects on the human populations in our urban areas on a global scale. Both historically and presently, this close interaction has facilitated the dissemination of many pathogens to humans, making screening for potentially zoonotic and emerging viruses in rats highly relevant. We have investigated faecal samples from R. norvegicus collected from urban areas using a protocol based on metagenomic enrichment of circular DNA genomes and subsequent sequencing. We found a new type of papillomavirus, with a L1 region 82% identical to that of the known R. norvegicus Papillomavirus 2. Additionally, we found 20 different circular replication associated protein (Rep)-encoding single stranded DNA (CRESS-DNA) virus-like genomes, one of which has homology to the replication-associated gene of Beak and feather disease virus. Papillomaviruses are a group of viruses known for their carcinogenic potential, and although they are known to infect several different vertebrates, they are mainly studied and characterised in humans. CRESS-DNA viruses are found in many different environments and tissue types. Both papillomaviruses and CRESS-DNA viruses are known to have pathogenic potential and screening for novel and known viruses in R. norvegicus could help identify viruses with pathogenic potential.

  5. Epigenetic Repeat-Induced Gene Silencing in the Chromosomal and Extrachromosomal Contexts in Human Cells

    PubMed Central

    Mitsuda, Sho-hei; Shimizu, Noriaki

    2016-01-01

    A plasmid bearing both a replication initiation region and a matrix attachment region is spontaneously amplified in transfected mammalian cells and generates plasmid repeats in the extrachromosomal double minutes (DMs) or the chromosomal homogeneously staining region (HSR). Generally, the repeat sequences are subject to repeat-induced gene silencing, the mechanism of which remains to be elucidated. Previous research showed that gene expression from the same plasmid repeat was higher from repeats located at DMs than at the HSR, which may reflect the extrachromosomal environment of the DMs. In the current study, plasmid repeats in both DMs and HSR were associated with repressive histone modifications (H3K9me3, H3K9me2), and the levels of repressive chromatin markers were higher in HSR than in DMs. Inactive chromatin is known to spread to neighboring regions in chromosome arm. Here, we found that such spreading also occurs in extrachromosomal DMs. Higher levels of active histone modifications (H3K9Ac, H3K4me3, and H3K79me2) were detected at plasmid repeats in DMs than in HSR. The level of DNA CpG methylation was generally low in both DMs and HSR; however, there were some hypermethylated copies within the population of repeated sequences, and the frequency of such copies was higher in DMs than in HSR. Together, these data suggest a “DNA methylation-core and chromatin-spread” model for repeat-induced gene silencing. The unique histone modifications at the extrachromosomal context are discussed with regard to the model. PMID:27525955

  6. Diverse circular ssDNA viruses discovered in dragonflies (Odonata: Epiprocta).

    PubMed

    Rosario, Karyna; Dayaram, Anisha; Marinov, Milen; Ware, Jessica; Kraberger, Simona; Stainton, Daisy; Breitbart, Mya; Varsani, Arvind

    2012-12-01

    Viruses with circular ssDNA genomes that encode a replication initiator protein (Rep) are among the smallest viruses known to infect both eukaryotic and prokaryotic organisms. In the past few years an overwhelming diversity of novel circular Rep-encoding ssDNA (CRESS-DNA) viruses has been unearthed from various hosts and environmental sources. Since there is limited information regarding CRESS-DNA viruses in invertebrates, this study explored the diversity of CRESS-DNA viruses circulating among insect populations by targeting dragonflies (Epiprocta), top insect predators that accumulate viruses from their insect prey over space and time. Using degenerate PCR and rolling circle amplification coupled with restriction digestion, 17 CRESS-DNA viral genomes were recovered from eight different dragonfly species collected in tropical and temperate regions. Nine of the genomes are similar to cycloviruses and represent five species within this genus, suggesting that cycloviruses are commonly associated with insects. Three of the CRESS-DNA viruses share conserved genomic features with recently described viruses similar to the mycovirus Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1, leading to the proposal of the genus Gemycircularvirus. The remaining viruses are divergent species representing four novel CRESS-DNA viral genera, including a gokushovirus-like prokaryotic virus (microphage) and three eukaryotic viruses with Reps similar to circoviruses. The novelty of CRESS-DNA viruses identified in dragonflies using simple molecular techniques indicates that there is an unprecedented diversity of ssDNA viruses among insect populations.

  7. DNA Polymerase κ Is a Key Cellular Factor for the Formation of Covalently Closed Circular DNA of Hepatitis B Virus

    PubMed Central

    Qi, Yonghe; Gao, Zhenchao; Peng, Bo; Yan, Huan; Tang, Dingbin; Song, Zilin; He, Wenhui; Sun, Yinyan; Guo, Ju-Tao; Li, Wenhui

    2016-01-01

    Hepatitis B virus (HBV) infection of hepatocytes begins by binding to its cellular receptor sodium taurocholate cotransporting polypeptide (NTCP), followed by the internalization of viral nucleocapsid into the cytoplasm. The viral relaxed circular (rc) DNA genome in nucleocapsid is transported into the nucleus and converted into covalently closed circular (ccc) DNA to serve as a viral persistence reservoir that is refractory to current antiviral therapies. Host DNA repair enzymes have been speculated to catalyze the conversion of rcDNA to cccDNA, however, the DNA polymerase(s) that fills the gap in the plus strand of rcDNA remains to be determined. Here we conducted targeted genetic screening in combination with chemical inhibition to identify the cellular DNA polymerase(s) responsible for cccDNA formation, and exploited recombinant HBV with capsid coding deficiency which infects HepG2-NTCP cells with similar efficiency of wild-type HBV to assure cccDNA synthesis is exclusively from de novo HBV infection. We found that DNA polymerase κ (POLK), a Y-family DNA polymerase with maximum activity in non-dividing cells, substantially contributes to cccDNA formation during de novo HBV infection. Depleting gene expression of POLK in HepG2-NTCP cells by either siRNA knockdown or CRISPR/Cas9 knockout inhibited the conversion of rcDNA into cccDNA, while the diminished cccDNA formation in, and hence the viral infection of, the knockout cells could be effectively rescued by ectopic expression of POLK. These studies revealed that POLK is a crucial host factor required for cccDNA formation during a de novo HBV infection and suggest that POLK may be a potential target for developing antivirals against HBV. PMID:27783675

  8. PCR mediated recombination impacts the analysis of hepatitis B Virus covalently closed circular DNA.

    PubMed

    Suspène, Rodolphe; Thiers, Valérie; Vartanian, Jean-Pierre; Wain-Hobson, Simon

    2016-12-20

    The replication of HBV involves the production of covalently closed circular DNA (cccDNA) from the HBV genome through the repair of virion relaxed circular DNA (rcDNA) in the virion. As cccDNA is the transcription template for HBV genomes, it needs to be eliminated from hepatocytes if the eradication of chronic HBV infection is to be achieved. PCR quantitation of cccDNA copy number is the technique of choice for evaluating the efficiency of treatment regimens. The PCR target commonly used to identify cccDNA spans the gapped region of rcDNA and is considered to accurately distinguish between cccDNA and rcDNA. There is however, a potentially confounding issue in that PCR can generate larger targets from collections of small DNA fragments, a phenomenon known as PCR recombination. The impact of PCR recombination towards the amplification of this cccDNA specific target was explored by mixing three marked, yet overlapping HBV DNA fragments. Thirteen of sixteen possible recombinants were identified by sequencing with frequencies ranging from 0.6 to 23%. To confirm this finding in vivo, HBV positive sera were treated with DNase I and submitted to quantitative real-time PCR. Under these conditions, it was possible to amplify the cccDNA specific segment without difficulty. As the virion contains uniquely rcDNA, amplification of the cccDNA target resulted from PCR recombination. PCR quantitation of cccDNA may be more difficult than hitherto thought. Current detection protocols need to be investigated so as to help in the management of chronic HBV infection.

  9. HIV 2-Long Terminal Repeat Circular DNA is Stable in Primary CD4+ T Cells

    PubMed Central

    Pace, Matthew J; Graf, Erin H.; O’Doherty, Una

    2013-01-01

    Treatment resistant latent reservoirs remain a barrier to curing HIV, but the maintenance and properties of these reservoirs are not completely understood. 2-LTR circular HIV DNA has been used to assess ongoing viral replication in HAART treated patients. However, the half-life of this DNA form is still debated with conflicting in vivo and in vitro data. Prior in vitro studies have focused on cell lines or short lived activated cells in cultures of brief duration, while in vivo studies have the added complications of cell migration, division, and death. Therefore, we monitored the stability of 2-LTR circles in primary CD4+T cells in a month long culture and compared it to the stability of integrated HIV DNA and T cell receptor excision circles (TRECs), another circular DNA form that is thought to be stable. We found that 2-LTRs, along with TRECs, were stable, suggesting 2-LTRs do not necessarily indicate ongoing replication. PMID:23537959

  10. Characterization of Circular ssDNA Viruses within the Echinoderm Nanobiome

    NASA Astrophysics Data System (ADS)

    Jackson, E.; Bistolas, K. S.; Hewson, I.

    2016-02-01

    Viral metagenomics has revealed a great diversity and presence of circular single-stranded(ss) DNA viruses most similar to the viral family Circoviridae in various environments both ambient and host. These viruses are an emerging paradigm in viral discovery amongst aquatic invertebrates mainly from the sub-phlya Crustacea and to a lesser extent the phylum Echinodermata. This parasite-host relationship is furthered here with the discovery of circo-like viruses extracted from the tissue of members from the family Holothuroidea (sea cucumbers). Verification and presence of these viruses within the tissue of the host was confirmed through rigorous genome architecture screening and PCR amplification of the rep gene from unamplified viral DNA extracts. Phylogenetic analysis of the rep gene reveals high similarity to circular ssDNA viruses from environmental metagenomic surveys of marine habitats. The significance of these findings is changing the perception and understanding of circular ssDNA viruses by broadening the known host range and blurring certain defining characteristics established by their pathogenic counterparts. Aside from discover and characterization, the potential ecological impacts of ssDNA viruses upon their host remains relatively unknown and further investigations should aim to determine the pathology, route of infection, and ecological implications of viral infection.

  11. Amplified DNAs in laboratory stocks of Leishmania tarentolae: extrachromosomal circles structurally and functionally similar to the inverted-H-region amplification of methotrexate-resistant Leishmania major

    SciTech Connect

    Petrillo-Peixoto, M.L.; Beverley, S.M. )

    1988-12-01

    We describe the structure of amplified DNA that was discovered in two laboratory stocks of the protozoan parasite Leishmania tarentolae. Restriction mapping and molecular cloning revealed that a region of 42 kilobases was amplified 8- to 30-fold in these lines. Southern blot analyses of digested DNAs or chromosomes separated by pulsed-field electrophoresis showed that the amplified DNA corresponded to the H region, a locus defined originally by its amplification in methotrexate-resistant Leishmania major. Similarities between the amplified DNA of the two species included (i) extensive cross-hybridization; (ii) approximate conservation of sequence order; (iii) extrachromosomal localization; (iv) an overall inverted, head-to-head configuration as a circular 140-kilobase tetrameric molecule; (v) two regions of DNA sequence rearrangement, each of which was closely associated with the two centers of the inverted repeats; (vi) association with methotrexate resistance; and (vii) phenotypically conservative amplification, in which the wild-type chromosomal arrangement was retained without apparent modification. Our data showed that amplified DNA mediating drug resistance arose in unselected L. tarentolae, although the pressures leading to apparently spontaneous amplification and maintenance of the H region are not known. The simple structure and limited extent of DNA amplified in these and other Leishmania lines suggests that the study of gene amplification in Leishmania spp. offers an attractive model system for the study of amplification in cultured mammalian cells and tumors. We also introduced a method for measuring the size of large circular DNAs, using gamma-irradiation to introduce limited double-strand breaks followed by sizing of the linear DNAs by pulsed-field electrophoresis.

  12. Novel Circular Single-Stranded DNA Viruses among an Asteroid, Echinoid and Holothurian (Phylum: Echinodermata).

    PubMed

    Jackson, Elliot W; Bistolas, Kalia S I; Button, Jason B; Hewson, Ian

    2016-01-01

    Echinoderms are prone to large population fluctuations that can be mediated by pervasive disease events. For the majority of echinoderm disease events the causative pathogen is unknown. Viruses have only recently been explored as potential pathogens using culture-independent techniques though little information currently exists on echinoderm viruses. In this study, ten circular ssDNA viruses were discovered in tissues among an asteroid (Asterias forbesi), an echinoid (Strongylocentrotus droebachiensis) and a holothurian (Parastichopus californicus) using viral metagenomics. Genome architecture and sequence similarity place these viruses among the rapidly expanding circular rep-encoding single stranded (CRESS) DNA viral group. Multiple genomes from the same tissue were no more similar in sequence identity to each other than when compared to other known CRESS DNA viruses. The results from this study are the first to describe a virus from a holothurian and continue to show the ubiquity of these viruses among aquatic invertebrates.

  13. Novel Circular Single-Stranded DNA Viruses among an Asteroid, Echinoid and Holothurian (Phylum: Echinodermata)

    PubMed Central

    Jackson, Elliot W.; Bistolas, Kalia S. I.; Button, Jason B.; Hewson, Ian

    2016-01-01

    Echinoderms are prone to large population fluctuations that can be mediated by pervasive disease events. For the majority of echinoderm disease events the causative pathogen is unknown. Viruses have only recently been explored as potential pathogens using culture-independent techniques though little information currently exists on echinoderm viruses. In this study, ten circular ssDNA viruses were discovered in tissues among an asteroid (Asterias forbesi), an echinoid (Strongylocentrotus droebachiensis) and a holothurian (Parastichopus californicus) using viral metagenomics. Genome architecture and sequence similarity place these viruses among the rapidly expanding circular rep-encoding single stranded (CRESS) DNA viral group. Multiple genomes from the same tissue were no more similar in sequence identity to each other than when compared to other known CRESS DNA viruses. The results from this study are the first to describe a virus from a holothurian and continue to show the ubiquity of these viruses among aquatic invertebrates. PMID:27855181

  14. Circular dimers of a lambda DNA in infected, nonlysogenic Escherichia coli.

    PubMed

    Freifelder, D; Baran, N; Folkmanis, A; Freifelder, D L

    1977-09-01

    Covalently closed circular dimers of phage lambda DNA have been found in Escherichia coli infected with lambda. These dimers can be formed by either the lambda Red or Int systems, by a nonrecombinational replicative mechanism requiring the activity of the lambda O and P genes or by joining of the cohesive ends. Dimers mediated by the E. coli Rec system have not been observed. Those formed by the Int system often result from recombination between different DNA molecules; however, the Red-mediated dimers may be a result of replicative extension of a single DNA molecule. Trimers have also been observed but studied only briefly.

  15. The effect of intrinsic curvature on conformational properties of circular DNA.

    PubMed Central

    Katritch, V; Vologodskii, A

    1997-01-01

    Both thermal fluctuations and the intrinsic curvature of DNA contribute to conformations of the DNA axis. We looked for a way to estimate the relative contributions of these two components of the double-helix curvature for DNA with a typical sequence. We developed a model and Monte Carlo procedure to simulate the Boltzmann distribution of DNA conformations with a specific intrinsic curvature. Two steps were used to construct the equilibrium conformation of the model chain. We first specified the equilibrium DNA conformation at the base pair level of resolution, using a set of the equilibrium dinucleotide angles and DNA sequence. This conformation was then approximated by the conformation of the model chain consisting of a reduced number of longer, straight cylindrical segments. Each segment of the chain corresponded to a certain number of DNA base pairs. We simulated conformational properties of nicked circular DNA for different sets of equilibrium dinucleotide angles, different random DNA sequences, and lengths. Only random sequences of DNA generated with equal probability of appearance for all types of bases at any site of the sequence were used. The results showed that for a broad range of intrinsic curvature parameters, the radius of gyration of DNA circles should be nearly independent of DNA sequence for all DNA lengths studied. We found, however, a DNA properly that should strongly depend on DNA sequence if the double helix has essential intrinsic curvature. This property is the equilibrium distribution of the linking number for DNA circles that are 300-1000 bp in length. We found that a large fraction of the distributions corresponding to random DNA sequences should have two separate maxima. The physical nature of this unexpected effect is discussed. This finding opens new opportunities for joined experimental and theoretical studies of DNA intrinsic curvature. Images FIGURE 1 PMID:9138556

  16. Hepatitis B Virus Covalently Closed Circular DNA Formation in Immortalized Mouse Hepatocytes Associated with Nucleocapsid Destabilization.

    PubMed

    Cui, Xiuji; Guo, Ju-Tao; Hu, Jianming

    2015-09-01

    Hepatitis B virus (HBV) infects hundreds of millions of people worldwide and causes acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HBV is an enveloped virus with a relaxed circular (RC) DNA genome. In the nuclei of infected human hepatocytes, conversion of RC DNA from the incoming virion or cytoplasmic mature nucleocapsid (NC) to the covalently closed circular (CCC) DNA, which serves as the template for producing all viral transcripts, is essential to establish and sustain viral replication. For reasons yet to be understood, HBV is apparently unable to make CCC DNA in normal mouse hepatocytes in the liver. We report here that HBV CCC DNA was formed efficiently in an immortalized mouse hepatocyte cell line, AML12HBV10, and this is associated with destabilization of mature NCs in these cells. These results suggest that destabilization of mature HBV NCs in AML12HBV10 cells facilitates efficient NC uncoating and subsequent CCC DNA formation. They further implicate NC uncoating as an important step in CCC DNA formation that is subject to host regulation and potentially a critical determinant of host range and/or cell tropism of HBV. IMPORTANCE Persistent infection by hepatitis B virus (HBV), afflicting hundreds of millions worldwide, is sustained by the episomal viral covalently closed circular (CCC) DNA in the nuclei of infected hepatocytes. CCC DNA is converted from the viral genomic (precursor) DNA contained in cytoplasmic viral nucleocapsids. The conversion process remains ill defined, but host cell factors are thought to play an essential role. In particular, HBV fails to make CCC DNA in normal mouse hepatocytes despite the presence of large amounts of nucleocapsids containing the precursor viral DNA. We have found that in an immortalized mouse hepatocyte cell line, HBV is able to make abundant amounts of CCC DNA. This ability correlates with increased instability of viral nucleocapsids in these cells, which likely facilitates nucleocapsid

  17. Discovery of a novel circular single-stranded DNA virus from porcine faeces.

    PubMed

    Sikorski, Alyssa; Argüello-Astorga, Gerardo R; Dayaram, Anisha; Dobson, Renwick C J; Varsani, Arvind

    2013-01-01

    A large number of novel single-stranded DNA (ssDNA) viruses have been characterised from various environmental sources in the last 5 years. The bulk of these have been from faecal sources, and faecal sampling is an ideal non-invasive pathogen sampling method. We characterised a novel ssDNA from a porcine faecal sample from Cass Basin of the South Island of New Zealand. The novel viral genome has two large open reading frames (ORFs), which are bidirectionally transcribed and separated by intergenic regions. The largest ORF has some degree of similarity (<30 %) to the putative capsid protein of chimpanzee stool-associated circular ssDNA virus (ChiSCV) and pig stool-associated single-stranded DNA virus (PigSCV), whereas the second-largest ORF has high similarity to the putative replication-associated protein (Rep) of ChiSCV (~50 %) and bovine stool-associated circular DNA virus (BoSCV; ~30 %). Based on genome architecture, location of putative stem-loop like elements, and maximum-likelihood phylogenetic analysis of the gene encoding the Rep protein, the novel isolate belongs to the same family of ssDNA viruses as ChiSCV and BoSCV.

  18. Sizing of single globular DNA molecules by using a circular acceleration technique with laser trapping.

    PubMed

    Hirano, Ken; Nagata, Hideya; Ishido, Tomomi; Tanaka, Yoshio; Baba, Yoshinobu; Ishikawa, Mitsuru

    2008-07-01

    We describe a method for in situ sizing individual huge DNA molecules by laser trapping. Single DNA molecules are reversibly transformed, without mechanical fragmentation of fragile huge-sized DNA, from their random coil state into their globular state induced by condensing agents poly(ethylene glycol) and Mg(2+). With the use of a globular DNA molecule folded by condensation, the critical velocity of the circularly accelerated single globular DNA molecule by laser trapping was found to be proportional to the size of the DNA. Yeast, Saccharomyces cerevisiae, chromosome III (285 kbp) was successfully sized (281 +/- 40 kbp) from a calibration curve scaled using lambda, T4, and yeast chromosome VI (48.5, 166, and 385 kbp, respectively). The use of critical velocity as a sizing parameter makes it possible to size single DNA molecules without prior conformational information, i.e., the radius of a single globular huge DNA molecule as a nanoparticle. A sized single globular DNA molecule could be trapped again for subsequent manipulation, such as transportation of it anywhere. We also investigated a possibility of reusing the globular DNA molecules condensed by PEG and Mg(2+) for PCR and found that PCR efficiency was not deteriorated in the presence of the condensation agents.

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

    PubMed

    Marotti, K R; Tomich, C S

    1989-01-01

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

  20. Hexamminecobalt(III)-induced condensation of calf thymus DNA: circular dichroism and hydration measurements.

    PubMed

    Kankia, B I; Buckin, V; Bloomfield, V A

    2001-07-01

    The interaction of hexamminecobalt(III), Co(NH(3))(6)(3+), with 160 and 3000-8000 bp length calf thymus DNA has been investigated by circular dichroism, acoustic and densimetric techniques. The acoustic titration curves of 160 bp DNA revealed three stages of interaction: (i) Co(NH(3))(6)(3+) binding up to the molar ratio [Co(NH(3))(6)(3+)]/[P] = 0.25, prior to DNA condensation; (ii) a condensation process between [Co(NH(3))(6)(3+)]/[P] = 0.25 and 0.30; and (iii) precipitation after [Co(NH(3))(6)(3+)]/[P] = 0.3. In the case of 3000-8000 bp DNA only two processes were observed: (i) binding up to [Co(NH(3))(6)(3+)]/[P] = 0.3; and (ii) precipitation after this point. In agreement with earlier observations, long DNA aggregates without changes in its B-form circular dichroism spectrum, while short DNA demonstrates a positive B-->Psi transition after [Co(NH(3))(6)(3+)]/[P] = 0.25. From ultrasonic and densimetric measurements the effects of Co(NH(3))(6)(3+) binding on volume and compressibility have been obtained. The binding of Co(NH(3))(6)(3+) to both short and long DNA is characterized by similar changes in volume and compressibility calculated per mole Co(NH(3))(6)(3+): DeltaV = 9 cm(3) mol(-1) and Deltakappa = 33 x 10(-4) cm(3) mol(-1) bar(-1). The positive sign of the parameters indicates dehydration, i.e. water release from Co(NH(3))(6)(3+) and the atomic groups of DNA. This extent of water displacement would be consistent with the formation of two direct, hydrogen bonded contacts between the cation and the phosphates of DNA.

  1. Metabolic stability of the extrachromosomal ribosomal RNA genes in the slime mould Physarum polycephalum.

    PubMed

    Hall, L; Gubler, U; Braun, R

    1978-05-01

    The rRNA genes of the slime mould Physarum polycephalum are located on free, linear DNA molecules of a discrete size, Mr=38X10(6). Using an isotope dilution technique we have examined the metabolic stability of these extrachromosomal genes during active, balanced growth. Microplasmodia, prelabelled with [3H]thymidine, were used to prepare synchronous surface plasmodial cultures which were subsequently grown on unlabelled medium. The gross synthesis of ribosomal DNA was then determined over three consecutive mitotic divisions from the ratio of 3H to 14C in a hybrid formed between the extracted ribosomal [3H]DNA and a [14C]rRNA probe. It was found that ribosomal DNA, like chromosomal DNA, is completely stable during active growth.

  2. Single-molecule measurements of trapped and migrating circular DNA during electrophoresis in agarose gels.

    PubMed

    Cole, Kenneth D; Gaigalas, Adolfas; Akerman, Björn

    2006-11-01

    The effect of agarose gel concentration and field strength on the electrophoretic trapping of open (relaxed) circular DNA was investigated using microscopic measurements of individual molecules stained with a fluorescent dye. Three open circles with sizes of 52.5, 115, and 220 kbp were trapped by the electric field (6 V/cm) and found to be predominately fixed and stretched at a single point in the gel. The length of the stretched circles did not significantly change with agarose concentration of the gels (mass fractions of 0.0025, 0.01, and 0.02). The relaxation kinetics of the trapped circles was also measured in the gels. The relaxation of the large open circles was found to be a slow process, taking several seconds. The velocity and average length of the 52.5 kbp open circles and 48.5 kbp linear DNA were measured during electrophoresis in the agarose gels. The velocity increased when the agarose concentrations were lowered, but the average length of the open-circle DNA (during electrophoresis) did not significantly change with agarose gel concentrations. The circles move through the gels by cycles of stretching and relaxation during electrophoresis. Linear dichroism was also used to investigate the trapping and alignment of the 52.5 kbp open circles. The results in this study provide information that can be used to improve electrophoretic separations of circular DNA, an important form of genetic material and commonly used to clone DNA.

  3. Bending and circularization of site-specific and stereoisomeric carcinogen-DNA adducts.

    PubMed

    Xu, R; Mao, B; Amin, S; Geacintov, N E

    1998-01-13

    The potent tumorigen and mutagen (+)-7(R),8(S)-dihydroxy-9(S), 10(R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene ((+)-anti-BPDE) is a metabolite of benzo[a]pyrene that binds predominantly to the exocyclic amino group of guanine residues in DNA in vivo and in vitro. While the (-)-7S,8R,9R,10Senantiomer, (-)-anti-BPDE, also reacts with DNA to form similar covalent N2-deoxyguanosyl adducts, this diol epoxide is nontumorigenic and its mutagenic activities are different from those of (+)-anti-BPDE. In this work, T4 ligase-induced cyclization methods have been employed to demonstrate that the (+)-anti-[BP]-N2-dG lesions (G*) cause significantly greater amounts of bending and circularization of the one-base overhang undecamer duplex 5'-d(CACAT[G*]TACAC).d(TGTACATGTGG) than the stereoisomeric oligonucleotide duplex with G* = (-)-anti-[BP]-N2-dG. In the case of the (+)-anti-BPDE-modified oligonucleotides, the ratio of circular to linear DNA multimers reaches values of 8-9 for circle contour sizes of 99-121 base pairs, while for the (-)-anti-[BP]-N2-dG-modified DNA this ratio reaches a maximum value of only approximately 1 at 154-176 base pairs. Assuming a planar circle DNA model, the inferred bending angles for 90-92% of the observed circular ligation products range from 30 to 51 degrees per (+)-trans-anti-[BP]-N2-dG lesion and from 20 to 40 degrees per (-)-trans-anti-[BP]-N2-dG lesion. In the case of unmodified DNA, the probability of circular product formation is at least 1 order of magnitude less efficient than in the BPDE-modified sequences and about 90% of the circular products exhibit bending angles in the range of 14 -19 degrees . In the most abundant circular products observed experimentally, the bending angles are 40 degrees and 26 +/- 2 degrees per (+)-anti-[BP]- or (-)-anti-[BP]-modified 11-mer; these values correspond to a net contribution of 21-26 degrees and 5-19 degrees , respectively, to the observed overall bending per lesion. The coexistence of circular DNA

  4. Relaxed circular SV40 DNA as cleavage intermediate of two restriction endonucleases.

    PubMed Central

    Ruben, G; Spielman, P; Tu, C D; Jay, E; Siegel, B; Wu, R

    1977-01-01

    We have determined the mode of cleavage of superhelical SV40 DNA (Form I) by restriction endonucleases EcoRI and HpaII at 37 degrees C. By analysis with agarose gel electrophoresis and direct examination with dark field electron microscopy, we found that a large amount of the single-nicked circular DNA (Form II) was produced before the linear SV40 DNA (Form III) appeared. Thus, both restriction enzymes cleave only one strand of the superhelical DNA first. The second cleavage on the complementary strand occurred after a lag period. The first order rate constant for the second cleavage by EcoRI endonuclease was determined and a kinetic reaction scheme for both enzymes is proposed. Images PMID:197493

  5. Assessment of amsacrine binding with DNA using UV-visible, circular dichroism and Raman spectroscopic techniques.

    PubMed

    Jangir, Deepak Kumar; Dey, Sanjay Kumar; Kundu, Suman; Mehrotra, Ranjana

    2012-09-03

    Proper understanding of the mechanism of binding of drugs to their targets in cell is a fundamental requirement to develop new drug therapy regimen. Amsacrine is a rationally designed anticancer drug, used to treat leukemia and lymphoma. Binding with cellular DNA is a crucial step in its mechanism of cytotoxicity. Despite numerous studies, DNA binding properties of amsacrine are poorly understood. Its reversible binding with DNA does not permit X-ray crystallography or NMR spectroscopic evaluation of amsacrine-DNA complexes. In the present work, interaction of amsacrine with calf thymus DNA is investigated at physiological conditions. UV-visible, FT-Raman and circular dichroism spectroscopic techniques were employed to determine the binding mode, binding constant, sequence specificity and conformational effects of amsacrine binding to native calf thymus DNA. Our results illustrate that amsacrine interacts with DNA by and large through intercalation between base pairs. Binding constant of the amsacrine-DNA complex was found to be K=1.2±0.1×10(4) M(-1) which is indicative of moderate type of binding of amsacrine to DNA. Raman spectroscopic results suggest that amsacrine has a binding preference of intercalation between AT base pairs of DNA. Minor groove binding is also observed in amsacrine-DNA complexes. These results are in good agreement with in silico investigation of amsacrine binding to DNA and thus provide detailed insight into DNA binding properties of amsacrine, which could ultimately, renders its cytotoxic efficacy. Copyright © 2012 Elsevier B.V. All rights reserved.

  6. Long Terminal Repeat Circular DNA as Markers of Active Viral Replication of Human T Lymphotropic Virus-1 in Vivo

    PubMed Central

    Fox, James M; Hilburn, Silva; Demontis, Maria-Antonietta; Brighty, David W; Rios Grassi, Maria Fernanda; Galvão-Castro, Bernardo; Taylor, Graham P; Martin, Fabiola

    2016-01-01

    Clonal expansion of human T-lymphotropic virus type-1 (HTLV-1) infected cells in vivo is well documented. Unlike human immunodeficiency virus type 1 (HIV-1), HTLV-1 plasma RNA is sparse. The contribution of the “mitotic” spread of HTLV-1 compared with infectious spread of the virus to HTLV-1 viral burden in established infection is uncertain. Since extrachromosomal long terminal repeat (LTR) DNA circles are indicators of viral replication in HIV-1 carriers with undetectable plasma HIV RNA, we hypothesised that HTLV-1 LTR circles could indicate reverse transcriptase (RT) usage and infectious activity. 1LTR and 2LTR DNA circles were measured in HTLV-1 cell lines and peripheral blood mononuclear cells (PBMC) of asymptomatic carriers (ACs) and patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) or adult T cell leukaemia/lymphoma (ATLL). 1LTR DNA circles were detected in 14/20 patients at a mean of 1.38/100 PBMC but did not differentiate disease status nor correlate with HTLV-1 DNA copies. 2LTR DNA circles were detected in 30/31 patients and at higher concentrations in patients with HTLV-1-associated diseases, independent of HTLV-1 DNA load. In an incident case the 2LTR DNA circle concentration increased 2.1 fold at the onset of HAM/TSP compared to baseline. Detectable and fluctuating levels of HTLV-1 DNA circles in patients indicate viral RT usage and virus replication. Our results indicate HTLV-1 viral replication capacity is maintained in chronic infection and may be associated with disease onset. PMID:26985903

  7. Novel circular DNA viruses identified in Procordulia grayi and Xanthocnemis zealandica larvae using metagenomic approaches.

    PubMed

    Dayaram, Anisha; Galatowitsch, Mark; Harding, Jon S; Argüello-Astorga, Gerardo R; Varsani, Arvind

    2014-03-01

    Recent advances in sequencing and metagenomics have enabled the discovery of many novel single stranded DNA (ssDNA) viruses from various environments. We have previously demonstrated that adult dragonflies, as predatory insects, are useful indicators of ssDNA viruses in terrestrial ecosystems. Here we recover and characterise 13 viral genomes which represent 10 novel and diverse circular replication associated protein (Rep)-encoding single stranded (CRESS) DNA viruses (1628-2668nt) from Procordulia grayi and Xanthocnemis zealandica dragonfly larvae collected from four high-country lakes in the South Island of New Zealand. The dragonfly larvae associated CRESS DNA viruses have different genome architectures, however, they all encode two major open reading frames (ORFs) which either have bidirectional or unidirectional arrangement. The 13 viral genomes have a conserved NAGTATTAC nonanucleotide motif and in their predicted Rep proteins we identified the rolling circle replication (RCR) motif 1, 2 and 3, as well as superfamily 3 (SF3) helicase motifs. Maximum likelihood phylogenetic and pairwise identity analysis of the Rep amino acid sequences reveal that the dragonfly larvae novel CRESS DNA viruses share <63% pairwise amino acid identity to the Reps of other CRESS DNA viruses whose complete genomes have been determined and available in public databases and that these viruses are novel. CRESS DNA viruses are circulating in larval dragonfly populations; however, we are unable to ascertain whether these viruses are infecting the larvae directly or are transient within dragonflies via their diet. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Circular DNA by "Bis-Click" Ligation: Template-Independent Intramolecular Circularization of Oligonucleotides with Terminal Alkynyl Groups Utilizing Bifunctional Azides.

    PubMed

    Yang, Haozhe; Seela, Frank

    2016-01-22

    A highly effective and convenient "bis-click" strategy was developed for the template-independent circularization of single-stranded oligonucleotides by employing copper(I)-assisted azide-alkyne cycloaddition. Terminal triple bonds were incorporated at both ends of linear oligonucleotides. Alkynylated 7-deaza-2'-deoxyadenosine and 2'-deoxyuridine residues with different side chains were used in solid-phase synthesis with phosphoramidite chemistry. The bis-click ligation of linear 9- to 36-mer oligonucleotides with 1,4-bis(azidomethyl)benzene afforded circular DNA in a simple and selective way; azido modification of the oligonucleotide was not necessary. Short ethynyl side chains were compatible with the circularization of longer oligonucleotides, whereas octadiynyl residues were used for short 9-mers. Compared with linear duplexes, circular bis-click constructs exhibit a significantly increased duplex stability over their linear counterparts. The intramolecular bis-click ligation protocol is not limited to DNA, but may also be suitable for the construction of other macrocycles, such as circular RNAs, peptides, or polysaccharides. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. A novel transcriptional element in circular DNA monomers of the duck hepatitis B virus.

    PubMed Central

    Beckel-Mitchener, A; Summers, J

    1997-01-01

    We report the presence of two elements, pet and net, that are required for proper transcription of the duck hepatitis B virus (DHBV). These regions were previously identified by using plasmid clones of the virus in transient expression assays (M. Huang and J. Summers, J. Virol. 68:1564-1572, 1994). In this study, we further analyzed these regions by using in vitro-synthesized circular DHBV DNA monomers to mimic the authentic transcriptional template. We observed that pet was required for pregenome transcription from circular viral monomers, and in the absence of pet-dependent transcription, expression of the viral envelope genes was increased. We found that deletion of net in circularized DNA monomers led to the production of abnormally long transcripts due to a failure to form 3' ends during transcription. In addition, we report the presence of a net-like region in the mammalian hepadnavirus woodchuck hepatitis virus. These results are consistent with a model that net is a region involved in transcription termination and that in DHBV, pet is required for transcription complexes to read through this region during the first pass through net. PMID:9311882

  10. Duck hepatitis B virus covalently closed circular DNA appears to survive hepatocyte mitosis in the growing liver

    SciTech Connect

    Reaiche-Miller, Georget Y.; Thorpe, Michael; Low, Huey Chi; Qiao, Qiao; Scougall, Catherine A.; Mason, William S.; Litwin, Samuel; Jilbert, Allison R.

    2013-11-15

    Nucleos(t)ide analogues that inhibit hepatitis B virus (HBV) DNA replication are typically used as monotherapy for chronically infected patients. Treatment with a nucleos(t)ide analogue eliminates most HBV DNA replication intermediates and produces a gradual decline in levels of covalently closed circular DNA (cccDNA), the template for viral RNA synthesis. It remains uncertain if levels of cccDNA decline primarily through hepatocyte death, or if loss also occurs during hepatocyte mitosis. To determine if cccDNA survives mitosis, growing ducklings infected with duck hepatitis B virus (DHBV) were treated with the nucleoside analogue, Entecavir. Viremia was suppressed at least 10{sup 5}-fold, during a period when average liver mass increased 23-fold. Analysis of the data suggested that if cccDNA synthesis was completely inhibited, at least 49% of cccDNA survived hepatocyte mitosis. However, there was a large duck-to-duck variation in cccDNA levels, suggesting that low level cccDNA synthesis may contribute to this apparent survival through mitosis. - Highlights: • The hepatitis B virus nuclear template is covalently closed circular DNA (cccDNA). • cccDNA was studied during liver growth in duck hepatitis B virus infected ducks. • Virus DNA replication and new cccDNA synthesis were inhibited with Entecavir. • At least 49% of cccDNA appeared to survive hepatocyte mitosis. • Low level virus DNA synthesis may contribute to survival of cccDNA through mitosis.

  11. Recombinant covalently closed circular hepatitis B virus DNA induces prolonged viral persistence in immunocompetent mice.

    PubMed

    Qi, Zhihua; Li, Gaiyun; Hu, Hao; Yang, Chunhui; Zhang, Xiaoming; Leng, Qibin; Xie, Youhua; Yu, Demin; Zhang, Xinxin; Gao, Yueqiu; Lan, Ke; Deng, Qiang

    2014-07-01

    It remains crucial to develop a laboratory model for studying hepatitis B virus (HBV) chronic infection. We hereby produced a recombinant covalently closed circular DNA (rcccDNA) in view of the key role of cccDNA in HBV persistence. A loxP-chimeric intron was engineered into a monomeric HBV genome in a precursor plasmid (prcccDNA), which was excised using Cre/loxP-mediated DNA recombination into a 3.3-kb rcccDNA in the nuclei of hepatocytes. The chimeric intron was spliced from RNA transcripts without interrupting the HBV life cycle. In cultured hepatoma cells, cotransfection of prcccDNA and pCMV-Cre (encoding Cre recombinase) resulted in accumulation of nuclear rcccDNA that was heat stable and epigenetically organized as a minichromosome. A mouse model of HBV infection was developed by hydrodynamic injection of prcccDNA. In the presence of Cre recombinase, rcccDNA was induced in the mouse liver with effective viral replication and expression, triggering a compromised T-cell response against HBV. Significant T-cell hyporesponsiveness occurred in mice receiving 4 μg prcccDNA, resulting in prolonged HBV antigenemia for up to 9 weeks. Persistent liver injury was observed as elevated alanine transaminase activity in serum and sustained inflammatory infiltration in the liver. Although a T-cell dysfunction was induced similarly, mice injected with a plasmid containing a linear HBV replicon showed rapid viral clearance within 2 weeks. Collectively, our study provides an innovative approach for producing a cccDNA surrogate that established HBV persistence in immunocompetent mice. It also represents a useful model system in vitro and in vivo for evaluating antiviral treatments against HBV cccDNA. Importance: (i) Unlike plasmids that contain a linear HBV replicon, rcccDNA established HBV persistence with sustained liver injury in immunocompetent mice. This method could be a prototype for developing a mouse model of chronic HBV infection. (ii) An exogenous intron was

  12. Circular rapid amplification of cDNA ends for high-throughput extension cloning of partial genes.

    PubMed

    Fu, Glenn K; Wang, Jonathan T; Yang, Junming; Au-Young, Janice; Stuve, Laura L

    2004-07-01

    The rapid amplification of cDNA ends (RACE) procedure is a widely used PCR-based method to clone the cDNA ends of mRNA transcripts. Current RACE methods often produce a high background of nonspecific PCR products, which can exclude the identification of the target cDNA of interest. We describe here an improved RACE procedure using circular cDNA templates and demonstrate the successful extension cloning of 4406 cDNAs.

  13. Amplicon structure in multidrug-resistant murine cells: a nonrearranged region of genomic DNA corresponding to large circular DNA.

    PubMed Central

    Ståhl, F; Wettergren, Y; Levan, G

    1992-01-01

    Multidrug resistance (MDR) in tumor cell lines is frequently correlated with amplification of one or more mdr genes. Usually the amplified domain also includes several neighboring genes. Using pulsed-field gel electrophoresis, we have established a restriction map covering approximately 2,200 kb in the drug-sensitive mouse tumor cell line TC13K. The mapped region is located on mouse chromosome 5 and includes the three mdr genes, the gene for the calcium-binding sorcin protein, and a gene with unknown function designated class 5. Long-range maps of the amplified DNA sequences in five of six MDR sublines that had been independently derived from TC13K generally displayed the same pattern as did the parental cell line. All six MDR sublines exhibited numerous double minutes, and one of them displayed a homogeneously staining region in a subpopulation. Large circular molecules, most likely identical to one chromatid of the double minutes, were detected in four of the sublines by linearization with gamma irradiation. The size of the circles was about 2,500 kb, which correlated to a single unit of the amplified domain. We therefore propose that in four independent instances of MDR development, a single unit of about 2,500 kb has been amplified in the form of circular DNA molecules. The restriction enzyme map of the amplified unit is unchanged compared with that of the parental cell line, whereas the joining sites of the circular DNA molecules are not identical but are in the same region. Images PMID:1545798

  14. Kaposi’s sarcoma–associated herpesvirus stably clusters its genomes across generations to maintain itself extrachromosomally

    DOE PAGES

    Chiu, Ya-Fang; Sugden, Arthur U.; Fox, Kathryn; ...

    2017-07-10

    Genetic elements that replicate extrachromosomally are rare in mammals; however, several human tumor viruses, including the papillomaviruses and the gammaherpesviruses, maintain their plasmid genomes by tethering them to cellular chromosomes. We have uncovered an unprecedented mechanism of viral replication: Kaposi’s sarcoma–associated herpesvirus (KSHV) stably clusters its genomes across generations to maintain itself extrachromosomally. To identify and characterize this mechanism, we developed two complementary, independent approaches: live-cell imaging and a predictive computational model. The clustering of KSHV requires the viral protein, LANA1, to bind viral genomes to nucleosomes arrayed on both cellular and viral DNA. Clustering affects both viral partitioning andmore » viral genome numbers of KSHV. The clustering of KSHV plasmids provides it with an effective evolutionary strategy to rapidly increase copy numbers of genomes per cell at the expense of the total numbers of cells infected.« less

  15. Circular Dichroism of DNA G-Quadruplexes: Combining Modeling and Spectroscopy To Unravel Complex Structures.

    PubMed

    Gattuso, Hugo; Spinello, Angelo; Terenzi, Alessio; Assfeld, Xavier; Barone, Giampaolo; Monari, Antonio

    2016-03-31

    We report on the comparison between the computational and experimental determination of electronic circular dichroism spectra of different guanine quadruplexes obtained from human telomeric sequences. In particular the difference between parallel, antiparallel, and hybrid structures is evidenced, as well as the induction of transitions between the polymorphs depending on the solution environment. Extensive molecular dynamics simulations (MD) are used to probe the conformational space of the different quadruplexes, and subsequently state-of-the-art hybrid quantum mechanics/molecular mechanics (QM/MM) techniques coupled with excitonic semiempirical Hamiltonian are used to simulate the macromolecular induced circular dichroism. The coupling of spectroscopy and molecular simulation allows an efficient one-to-one mapping between structures and optical properties, offering a way to disentangle the rich, yet complicated, quantity of information embedded in circular dichroism spectra. We show that our methodology is robust and efficient and allows us to take into account subtle conformational changes. As such, it could be used as an efficient tool to investigate structural modification upon DNA/drug interactions.

  16. A label-free amplified fluorescence DNA detection based on isothermal circular strand-displacement polymerization reaction and graphene oxide.

    PubMed

    Li, Zhen; Zhu, Wenping; Zhang, Jinwen; Jiang, Jianhui; Shen, Guoli; Yu, Ruqin

    2013-07-07

    A label-free fluorescent DNA biosensor has been presented based on isothermal circular strand-displacement polymerization reaction (ICSDPR) combined with graphene oxide (GO) binding. The proposed method is simple and cost-effective with a low detection limit of 4 pM, which compares favorably with other GO-based homogenous DNA detection methods.

  17. Characterization of a complete genome of a circular single-stranded DNA virus from porcine stools in Korea.

    PubMed

    Kim, A Reum; Chung, Hee Chun; Kim, Hye Kwon; Kim, Eun Ok; Nguyen, Van Giap; Choi, Min Gyung; Yang, Hye Jung; Kim, Jung Ah; Park, Bong Kyun

    2014-02-01

    Porcine circular single-stranded DNA viruses have been just identified from swine feces in Korea. This virus was mentioned as bovine stool-associated circular DNA virus (BoSCV)-like virus discovered from porcine stools. However, the thorough characteristics of the virus were not identified. Therefore, this research focuses on finding a full genome sequence and analyzing the genetic features of the virus. The virus, now called porcine stool-associated circular DNA virus in Korea (PoSCV Kor), consists of 2,589 bases forming circular structure. It has two major ORFs inversely encoding replicase and capsid protein, with each stem-loop structure between 5' ends and 3' ends of the two putative ORFs. This characteristics is the same as PoSCV in New Zealand, but different from chimpanzee stool-associated circular virus (ChiSCVs) and BoSCV, which have one stem-loop structure. Therefore, it would be sure that PoSCV Kor is very similar to PoSCV in respect to the genetic aspect; the same number of nucleotide bases and the amino acid identity of replicase and capsid protein (96 and 93 %, respectively). This fact could be certified through the finding that PoSCV Kor and PoSCV are in the same cluster by phylogenetic analysis based on the comparison with full-sequences of other circular ssDNA viruses.

  18. Effects of Circular DNA Length on Transfection Efficiency by Electroporation into HeLa Cells

    PubMed Central

    Hornstein, Benjamin D.; Roman, Dany; Arévalo-Soliz, Lirio M.; Engevik, Melinda A.

    2016-01-01

    The ability to produce extremely small and circular supercoiled vectors has opened new territory for improving non-viral gene therapy vectors. In this work, we compared transfection of supercoiled DNA vectors ranging from 383 to 4,548 bp, each encoding shRNA against GFP under control of the H1 promoter. We assessed knockdown of GFP by electroporation into HeLa cells. All of our vectors entered cells in comparable numbers when electroporated with equal moles of DNA. Despite similar cell entry, we found length-dependent differences in how efficiently the vectors knocked down GFP. As vector length increased up to 1,869 bp, GFP knockdown efficiency per mole of transfected DNA increased. From 1,869 to 4,257 bp, GFP knockdown efficiency per mole was steady, then decreased with increasing vector length. In comparing GFP knockdown with equal masses of vectors, we found that the shorter vectors transfect more efficiently per nanogram of DNA transfected. Our results rule out cell entry and DNA mass as determining factors for gene knockdown efficiency via electroporation. The length-dependent effects we have uncovered are likely explained by differences in nuclear translocation or transcription. These data add an important step towards clinical applications of non-viral vector delivery. PMID:27918590

  19. Fabrication of a microarray using a combination of the large circular sense and antisense DNA.

    PubMed

    Doh, Kyung-Oh; Lee, Yun-Han; Han, Kil-Hwan; Uhm, Seok-Yong; Kim, Jong-Pil; Bae, Yun-Ui; Park, Jeong-Hoh; Moon, Ik-Jae; Park, Jong-Gu

    2010-01-01

    In the present study, single-stranded large circular (LC)-sense molecules were utilized as probes for DNA microarrays and showed stronger binding signals than those of PCR-amplified cDNA probes. A microarray experiment using 284 LC-sense DNA probes found 6 upregulated and 7 downregulated genes in A549 cells as compared to WI38VA13 cells. Repeated experiments showed largely consistent results, and microarray data strongly correlated with data acquired from quantitative real-time RT-PCR. A large array comprising 5,079 LC-sense DNA was prepared, and analysis of the mean differential expression from dye-swap experiments revealed 332 upregulated and 509 downregulated genes in A549 cells compared to WI38VA13 cells. Subsequent functional analysis using an LC-antisense library of overexpressed genes identified 28 genes involved in A549 cell growth. These experiments demonstrated the proper features of LC-sense molecules as probe DNA for microarray and the potential utility of the combination of LC-sense and -antisense libraries for an effective functional validation of genes.

  20. Genomic Characterization of Novel Circular ssDNA Viruses from Insectivorous Bats in Southern Brazil

    PubMed Central

    Lima, Francisco Esmaile de Sales; Cibulski, Samuel Paulo; dos Santos, Helton Fernandes; Teixeira, Thais Fumaco; Varela, Ana Paula Muterle; Roehe, Paulo Michel; Delwart, Eric; Franco, Ana Cláudia

    2015-01-01

    Circoviruses are highly prevalent porcine and avian pathogens. In recent years, novel circular ssDNA genomes have recently been detected in a variety of fecal and environmental samples using deep sequencing approaches. In this study the identification of genomes of novel circoviruses and cycloviruses in feces of insectivorous bats is reported. Pan-reactive primers were used targeting the conserved rep region of circoviruses and cycloviruses to screen DNA bat fecal samples. Using this approach, partial rep sequences were detected which formed five phylogenetic groups distributed among the Circovirus and the recently proposed Cyclovirus genera of the Circoviridae. Further analysis using inverse PCR and Sanger sequencing led to the characterization of four new putative members of the family Circoviridae with genome size ranging from 1,608 to 1,790 nt, two inversely arranged ORFs, and canonical nonamer sequences atop a stem loop. PMID:25688970

  1. Circular DNA genomics (circomics) exemplified for geminiviruses in bean crops and weeds of northeastern Brazil.

    PubMed

    Wyant, Patricia Soares; Strohmeier, Stephan; Schäfer, Benjamin; Krenz, Björn; Assunção, Iraildes Pereira; Lima, Gaus Silvestre de Andrade; Jeske, Holger

    2012-06-05

    Circomics was coined to describe the combination of rolling circle amplification (RCA), restriction fragment length polymorphism (RFLP) and pyro-sequencing to investigate the genome structures of small circular DNAs. A batch procedure is described using 61 plant samples from Asia, South America and Central America which revealed 83 contig sequences of geminiviral DNA components and 4 contig sequences of DNA satellites. The usefulness of this approach is validated for the Brazilian begomoviruses, and the sequence fidelity is determined by comparing the results with those of conventional cloning and sequencing of Bolivian begomoviruses reported recently. Therefore, circomics has been proven to be a major step forward to economize costs and labor and to characterize reliably geminiviral genomes in their population structure of the quasispecies.

  2. Effects of supercoiling in electrophoretic trapping of circular DNA in polyacrylamide gels.

    PubMed Central

    Akerman, B

    1998-01-01

    Electrophoretic velocity and orientation have been used to study the electric-field-induced trapping of supercoiled and relaxed circular DNA (2926 and 5386 bp) in polyacrylamide gels (5% T, 3.3% C) at 7.5-22.5 V/cm, using as controls linear molecules of either the same contour length or the same radius of gyration. The circle-specific trapping is reversible. From the duration of the reverse pulse needed to detrap the molecules, the average trap depth is estimated to be 90 A, which is consistent with the molecular charge and the field strengths needed to keep molecules trapped. Trapped circles exhibit a strong field alignment compared to the linear form, and there is a good correlation between the enhanced field alignment for the circles and the onset of trapping in both constant and pulsed fields. The circles do not exhibit the orientation overshoot response to a field pulse seen with linear DNA, and the rate of orientation growth scales as E(-2+/-0.1) with the field, as opposed to E(-1.1+/-0.1) for the linear form. These results show that the linear form migrates by cyclic reptation, whereas the circles most likely are trapped by impalement on gel fibers. This proposal is supported by very similar velocity and orientation behavior of circular DNA in agarose gels, where impalement has been deemed more likely because of stiffer gel fibers. The trapping efficiency is sensitive to DNA topology, as expected for impalement. In polyacrylamide the supercoiled form (superhelical density sigma = -0.05) has a two- to fourfold lower probability of trapping than the corresponding relaxed species, whereas in agarose gels the supercoiled form is not trapped at all. These results are consistent with existing data on the average holes in the plectonemic supercoiled structures and the fiber thicknesses in the two gel types. On the basis of the topology effect, it is argued that impalement during pulsed-field electrophoresis in polyacrylamide gels may be useful for the separation of

  3. Distinct circular single-stranded DNA viruses exist in different soil types.

    PubMed

    Reavy, Brian; Swanson, Maud M; Cock, Peter J A; Dawson, Lorna; Freitag, Thomas E; Singh, Brajesh K; Torrance, Lesley; Mushegian, Arcady R; Taliansky, Michael

    2015-06-15

    The potential dependence of virus populations on soil types was examined by electron microscopy, and the total abundance of virus particles in four soil types was similar to that previously observed in soil samples. The four soil types examined differed in the relative abundances of four morphological groups of viruses. Machair, a unique type of coastal soil in western Scotland and Ireland, differed from the others tested in having a higher proportion of tailed bacteriophages. The other soils examined contained predominantly spherical and thin filamentous virus particles, but the Machair soil had a more even distribution of the virus types. As the first step in looking at differences in populations in detail, virus sequences from Machair and brown earth (agricultural pasture) soils were examined by metagenomic sequencing after enriching for circular Rep-encoding single-stranded DNA (ssDNA) (CRESS-DNA) virus genomes. Sequences from the family Microviridae (icosahedral viruses mainly infecting bacteria) of CRESS-DNA viruses were predominant in both soils. Phylogenetic analysis of Microviridae major coat protein sequences from the Machair viruses showed that they spanned most of the diversity of the subfamily Gokushovirinae, whose members mainly infect obligate intracellular parasites. The brown earth soil had a higher proportion of sequences that matched the morphologically similar family Circoviridae in BLAST searches. However, analysis of putative replicase proteins that were similar to those of viruses in the Circoviridae showed that they are a novel clade of Circoviridae-related CRESS-DNA viruses distinct from known Circoviridae genera. Different soils have substantially different taxonomic biodiversities even within ssDNA viruses, which may be driven by physicochemical factors.

  4. Comparative analysis of inosine-substituted duplex DNA by circular dichroism and X-ray crystallography.

    PubMed

    Peters, Justin P; Kowal, Ewa A; Pallan, Pradeep S; Egli, Martin; Maher, L James

    2017-09-04

    Leveraging structural biology tools, we report the results of experiments seeking to determine if the different mechanical properties of DNA polymers with base analog substitutions can be attributed, at least in part, to induced changes from classical B-form DNA. The underlying hypothesis is that different inherent bending and twisting flexibilities may characterize non-canonical B-DNA, so that it is inappropriate to interpret mechanical changes caused by base analog substitution as resulting simply from 'electrostatic' or 'base stacking' influences without considering the larger context of altered helical geometry. Circular dichroism spectra of inosine-substituted oligonucleotides and longer base-substituted DNAs in solution indicated non-canonical helical conformations, with the degree of deviation from a standard B-form geometry depending on the number of I⋅C pairs. X-ray diffraction of a highly inosine-substituted DNA decamer crystal (eight I⋅C and two A⋅T pairs) revealed an A-tract-like conformation with a uniformly narrow minor groove, reduced helical rise, and the majority of sugars adopting a C1'-exo (southeastern) conformation. This contrasts with the standard B-DNA geometry with C2'-endo sugar puckers (south conformation). In contrast, the crystal structure of a decamer with only four I⋅C pairs has a geometry similar to that of the reference duplex with eight G⋅C and two A⋅T pairs. The unique crystal geometry of the inosine-rich duplex is noteworthy given its unusual CD signature in solution and the altered mechanical properties of some inosine-containing DNAs.

  5. Vacuum-ultraviolet circular dichroism reveals DNA duplex formation between short strands of adenine and thymine.

    PubMed

    Nielsen, Lisbeth Munksgaard; Hoffmann, Søren Vrønning; Brøndsted Nielsen, Steen

    2012-11-21

    Absorbance spectroscopy is used extensively to tell when two DNA single strands come together and form a double strand. Here we show that circular dichroism in the vacuum ultraviolet region provides an even stronger indication for duplex formation in the case of short strands of adenine and thymine (4 to 16 bases in each strand). Indeed, our results show that a strong positive CD band appears at 179 nm when double strands are formed. Melting experiments were done in aqueous solution with and without added Na(+) counter ions. With additional salt present a huge increase in the 179 nm CD band was observed when lowering the temperature. A 179 nm CD marker band for duplex formation can be used to measure the kinetics for the association of two single strands. Such experiments rely on large changes at one particular wavelength since it is too time-consuming to record a full-wavelength spectrum.

  6. Circular DNA Intermediate in the Duplication of Nile Tilapia vasa Genes

    PubMed Central

    Fujimura, Koji; Conte, Matthew A.; Kocher, Thomas D.

    2011-01-01

    vasa is a highly conserved RNA helicase involved in animal germ cell development. Among vertebrate species, it is typically present as a single copy per genome. Here we report the isolation and sequencing of BAC clones for Nile tilapia vasa genes. Contrary to a previous report that Nile tilapia have a single copy of the vasa gene, we find evidence for at least three vasa gene loci. The vasa gene locus was duplicated from the original site and integrated into two distant novel sites. For one of these insertions we find evidence that the duplication was mediated by a circular DNA intermediate. This mechanism of gene duplication may explain the origin of isolated gene duplicates during the evolution of fish genomes. These data provide a foundation for studying the role of multiple vasa genes in the development of tilapia gonads, and will contribute to investigations of the molecular mechanisms of sex determination and evolution in cichlid fishes. PMID:22216289

  7. Evidence for a partial RNA transcript of the small circular component of kinetoplast DNA of Crithidia acanthocephali.

    PubMed Central

    Fouts, D L; Wolstenholme, D R

    1979-01-01

    The major component of kinetoplast DNA (kDNA) in the protozoan Crithidia acanthocephali is an association of approximately 27,000, 0.8 micrometers (1.58 x 10(6) dalton) circular molecules apparently held together in a particular structural configuration by topological interlocking. We have carried out hybridization experiments between kDNA samples containing one or the other of the two complementary (H and L) strands of purified 0.8 micrometers molecules derived from mechanically disrupted associations and RNA samples prepared either from whole C. acanthocephali cells or from a mitochondrion-enriched fraction. The results of experiments involving cesium sulfate buoyant density centrifugation indicate that whole cell RNA contains a component(s) complementary to all kDNA H strands, but none complementary to kDNA L strands. Similar results were obtained using mitochondrion-associated RNA. Digestion of RNA/DNA hybrids and suitable controls with the single-strand-specific nuclease S1 indicated that 10% of the kDNA H strand is involved in hybrid formation. Visualization of RNA/DNA hybrids stained with bacteriophage T4 gene 32 protein revealed that hybridation involves a single region of each kDNA H strand, equal to approximately 10% of the molecule length. These data suggest that at least 10% of the small circular component of kDNA of Crithidia acanthocephali is transcribed. Images PMID:493124

  8. Involvement of the host DNA-repair enzyme TDP2 in formation of the covalently closed circular DNA persistence reservoir of hepatitis B viruses

    PubMed Central

    Königer, Christian; Wingert, Ida; Marsmann, Moritz; Rösler, Christine; Beck, Jürgen; Nassal, Michael

    2014-01-01

    Hepatitis B virus (HBV), the causative agent of chronic hepatitis B and prototypic hepadnavirus, is a small DNA virus that replicates by protein-primed reverse transcription. The product is a 3-kb relaxed circular DNA (RC-DNA) in which one strand is linked to the viral polymerase (P protein) through a tyrosyl–DNA phosphodiester bond. Upon infection, the incoming RC-DNA is converted into covalently closed circular (ccc) DNA, which serves as a viral persistence reservoir that is refractory to current anti-HBV treatments. The mechanism of cccDNA formation is unknown, but the release of P protein is one mandatory step. Structural similarities between RC-DNA and cellular topoisomerase–DNA adducts and their known repair by tyrosyl-DNA-phosphodiesterase (TDP) 1 or TDP2 suggested that HBV may usurp these enzymes for its own purpose. Here we demonstrate that human and chicken TDP2, but only the yeast ortholog of TDP1, can specifically cleave the Tyr–DNA bond in virus-adapted model substrates and release P protein from authentic HBV and duck HBV (DHBV) RC-DNA in vitro, without prior proteolysis of the large P proteins. Consistent with TPD2’s having a physiological role in cccDNA formation, RNAi-mediated TDP2 depletion in human cells significantly slowed the conversion of RC-DNA to cccDNA. Ectopic TDP2 expression in the same cells restored faster conversion kinetics. These data strongly suggest that TDP2 is a first, although likely not the only, host DNA-repair factor involved in HBV cccDNA biogenesis. In addition to establishing a functional link between hepadnaviruses and DNA repair, our results open new prospects for directly targeting HBV persistence. PMID:25201958

  9. Nonselective Persistence of a Rickettsia conorii Extrachromosomal Plasmid during Mammalian Infection

    PubMed Central

    Riley, Sean P.; Fish, Abigail I.; Garza, Daniel A.; Banajee, Kaikhushroo H.; Harris, Emma K.; del Piero, Fabio

    2016-01-01

    Scientific analysis of the genus Rickettsia is undergoing a rapid period of change with the emergence of viable genetic tools. The development of these tools for the mutagenesis of pathogenic bacteria will permit forward genetic analysis of Rickettsia pathogenesis. Despite these advances, uncertainty still remains regarding the use of plasmids to study these bacteria in in vivo mammalian models of infection, namely, the potential for virulence changes associated with the presence of extrachromosomal DNA and nonselective persistence of plasmids in mammalian models of infection. Here, we describe the transformation of Rickettsia conorii Malish 7 with the plasmid pRam18dRGA[AmTrCh]. Transformed R. conorii stably maintains this plasmid in infected cell cultures, expresses the encoded fluorescent proteins, and exhibits growth kinetics in cell culture similar to those of nontransformed R. conorii. Using a well-established murine model of fatal Mediterranean spotted fever, we demonstrate that R. conorii(pRam18dRGA[AmTrCh]) elicits the same fatal outcomes in animals as its untransformed counterpart and, importantly, maintains the plasmid throughout infection in the absence of selective antibiotic pressure. Interestingly, plasmid-transformed R. conorii was readily observed both in endothelial cells and within circulating leukocytes. Together, our data demonstrate that the presence of an extrachromosomal DNA element in a pathogenic rickettsial species does not affect either in vitro proliferation or in vivo infectivity in models of disease and that plasmids such as pRam18dRGA[AmTrCh] are valuable tools for the further genetic manipulation of pathogenic rickettsiae. PMID:26755154

  10. Production of extrachromosomal microDNAs is linked to mismatch repair pathways and transcriptional activity

    PubMed Central

    Dillon, Laura W.; Kumar, Pankaj; Shibata, Yoshiyuki; Wang, Yuh-Hwa; Willcox, Smaranda; Griffith, Jack D.; Pommier, Yves; Takeda, Shunichi; Dutta, Anindya

    2015-01-01

    SUMMARY MicroDNAs are <400-base extrachromosomal circles found in mammalian cells. Tens of thousands of microDNAs have been found in all tissue types, including sperm. MicroDNAs arise preferentially from areas with high gene density, GC content, and exon density, from promoters with activating chromatin modifications and in sperm from the 5'-UTR of full-length LINE-1 elements, but are depleted from lamin-associated heterochromatin. Analysis of microDNAs from a set of human cancer cell lines revealed lineage-specific patterns of microDNA origins. A survey of microDNAs from chicken cells defective in various DNA repair proteins reveal that homologous recombination and nonhomologous end joining repair pathways are not required for microDNA production. Deletion of the MSH3 DNA mismatch repair protein results in a significant decrease in microDNA abundance, specifically from non-CpG genomic regions. Thus, microDNAs arise as part of normal cellular physiology; either from DNA breaks associated with RNA metabolism or from replication slippage followed by mismatch repair. PMID:26051933

  11. Production of Extrachromosomal MicroDNAs Is Linked to Mismatch Repair Pathways and Transcriptional Activity.

    PubMed

    Dillon, Laura W; Kumar, Pankaj; Shibata, Yoshiyuki; Wang, Yuh-Hwa; Willcox, Smaranda; Griffith, Jack D; Pommier, Yves; Takeda, Shunichi; Dutta, Anindya

    2015-06-23

    MicroDNAs are <400-base extrachromosomal circles found in mammalian cells. Tens of thousands of microDNAs have been found in all tissue types, including sperm. MicroDNAs arise preferentially from areas with high gene density, GC content, and exon density from promoters with activating chromatin modifications and in sperm from the 5'-UTR of full-length LINE-1 elements, but are depleted from lamin-associated heterochromatin. Analysis of microDNAs from a set of human cancer cell lines revealed lineage-specific patterns of microDNA origins. A survey of microDNAs from chicken cells defective in various DNA repair proteins reveals that homologous recombination and non-homologous end joining repair pathways are not required for microDNA production. Deletion of the MSH3 DNA mismatch repair protein results in a significant decrease in microDNA abundance, specifically from non-CpG genomic regions. Thus, microDNAs arise as part of normal cellular physiology—either from DNA breaks associated with RNA metabolism or from replication slippage followed by mismatch repair.

  12. Allele-specific polymerase chain reaction for detection of a mutation in the relax circular DNA and the covalently closed circular DNA of hepatitis B virus.

    PubMed

    Pan, Wan-Long; Hu, Jie-Li; Fang, Yan; Luo, Qiang; Xu, Ge; Xu, Lei; Jing, Zhou-Hong; Shan, Xue-Feng; Zhu, Yan-Ling; Huang, Ai-Long

    2013-12-01

    The relax circle DNA (rcDNA) sequence and the covalently closed circle DNA (cccDNA) sequence in hepatitis B virus (HBV) are crucial regions for HBV infections. To analyze mutations in rcDNA and cccDNA, DNA sequencing is often used, although it is time-consuming and expensive. Herein, we report a simple, economic, albeit accurate allele-specific polymerase chain reaction (AS-PCR) to detect mutations in these regions of HBV. This method can be extensively used to screen for mutations at specific positions of HBV genome.

  13. Genomics of clostridial pathogens: implication of extrachromosomal elements in pathogenicity.

    PubMed

    Brüggemann, Holger

    2005-10-01

    The recently decoded genomes of the major clostridial toxin-producing pathogens Clostridium perfringens, Clostridium tetani, Clostridium botulinum and Clostridium difficile have provided a huge amount of new sequence data. Recent studies have focused on the identification and investigation of pathogenic determinants and the regulatory events governing their expression. The sequence data revealed also the genomic background of virulence genes, as well as the contribution of extrachromosomal elements to a pathogenic phenotype. This has generated new insights in clostridial pathogenesis - and will continue to do so in the future - and has deepened our understanding of the anaerobic lifestyle of clostridial species.

  14. Epstein-Barr-based episomal chromosomes shuttle 100 kb of self-replicating circular human DNA in mouse cells

    SciTech Connect

    Kelleher, Z.T.; Fu, H.; Livanos, E.; Wendelburg, B.; Gulino, S.; Vos, J.M.

    1998-08-01

    The authors describe the microcell fusion transfer of 100--200 kb self-replicating circular human minichromosomes from human into mouse cells. This experimental approach is illustrated through the shuttling of the latent 170 kb double-stranded DNA genome from the human herpesvirus, Epstein-Barr virus, into nonpermissive rodent cells. Using this interspecies transfer strategy, circular episomes carrying 95--105 kb of human DNA were successfully established at low copy number in mouse A9 cells. Selected episomes were stably maintained for 6 months, and unselected episomes were characterized by a 95% episomal retention per cell division. The establishment of a mouse artificial episomal chromosome system should facilitate evolutionary and therapeutic studies of large human DNA in rodent genetic backgrounds.

  15. Roles of the Envelope Proteins in the Amplification of Covalently Closed Circular DNA and Completion of Synthesis of the Plus-Strand DNA in Hepatitis B Virus ▿

    PubMed Central

    Lentz, Thomas B.; Loeb, Daniel D.

    2011-01-01

    Covalently closed circular DNA (cccDNA), the nuclear form of hepatitis B virus (HBV), is synthesized by repair of the relaxed circular (RC) DNA genome. Initially, cccDNA is derived from RC DNA from the infecting virion, but additional copies of cccDNA are derived from newly synthesized RC DNA molecules in a process termed intracellular amplification. It has been shown that the large viral envelope protein limits the intracellular amplification of cccDNA for duck hepatitis B virus. The role of the envelope proteins in regulating the amplification of cccDNA in HBV is not well characterized. The present report demonstrates regulation of synthesis of cccDNA by the envelope proteins of HBV. Ablation of expression of the envelope proteins led to an increase (>6-fold) in the level of cccDNA. Subsequent restoration of envelope protein expression led to a decrease (>50%) in the level of cccDNA, which inversely correlated with the level of the envelope proteins. We found that the expression of L protein alone or in combination with M and/or S proteins led to a decrease in cccDNA levels, indicating that L contributes to the regulation of cccDNA. Coexpression of L and M led to greater regulation than either L alone or L and S. Coexpression of all three envelope proteins was also found to limit completion of plus-strand DNA synthesis, and the degree of this effect correlated with the level of the proteins and virion secretion. PMID:21900164

  16. The Invariance of Order Parameter and Temperature Redefinition in Helix-Coil Transition Theory of Circular Closed DNA

    SciTech Connect

    Grigoryan, A.V.; Badasyan, A.V.; Mamasakhlisov, E.Sh.; Morozov, V.F.

    2005-10-01

    The order-disorder (helix-coil) transition in circular closed DNA (ccDNA) is described on the basis of the open chain DNA (ocDNA) model, proposed earlier, which considers the transition as loop formation. The Hamiltonian of the ccDNA model is constructed on the basis of the open chain model taking into account topological restrictions. These restrictions are taken into account through hydrogen bond reduced energy dependence on the fraction of broken hydrogen bonds in the macromolecule. The invariance of the order parameter (helicity degree) has been shown for ocDNA and ccDNA. This invariance results in the interdependence between temperatures of ocDNA and ccDNA with the same value of helicity degree. The dependence can be obtained with the help of the derivative of reduced energy of hydrogen bonding dependence on instantaneous denaturation degree. Thus, it has been shown that the melting curve of ccDNA can be obtained from the consequent curve of ocDNA through the redefinition of temperature scale. The calculated and experimentally measured melting curves have been compared under inversion conditions and qualitative agreement between them is found.

  17. Generation of Covalently Closed Circular DNA of Hepatitis B Viruses via Intracellular Recycling Is Regulated in a Virus Specific Manner

    PubMed Central

    Köck, Josef; Rösler, Christine; Zhang, Jing-Jing; Blum, Hubert E.; Nassal, Michael; Thoma, Christian

    2010-01-01

    Persistence of hepatitis B virus (HBV) infection requires covalently closed circular (ccc)DNA formation and amplification, which can occur via intracellular recycling of the viral polymerase-linked relaxed circular (rc) DNA genomes present in virions. Here we reveal a fundamental difference between HBV and the related duck hepatitis B virus (DHBV) in the recycling mechanism. Direct comparison of HBV and DHBV cccDNA amplification in cross-species transfection experiments showed that, in the same human cell background, DHBV but not HBV rcDNA converts efficiently into cccDNA. By characterizing the distinct forms of HBV and DHBV rcDNA accumulating in the cells we find that nuclear import, complete versus partial release from the capsid and complete versus partial removal of the covalently bound polymerase contribute to limiting HBV cccDNA formation; particularly, we identify genome region-selectively opened nuclear capsids as a putative novel HBV uncoating intermediate. However, the presence in the nucleus of around 40% of completely uncoated rcDNA that lacks most if not all of the covalently bound protein strongly suggests a major block further downstream that operates in the HBV but not DHBV recycling pathway. In summary, our results uncover an unexpected contribution of the virus to cccDNA formation that might help to better understand the persistence of HBV infection. Moreover, efficient DHBV cccDNA formation in human hepatoma cells should greatly facilitate experimental identification, and possibly inhibition, of the human cell factors involved in the process. PMID:20824087

  18. Duck hepatitis B virus covalently closed circular DNA appears to survive hepatocyte mitosis in the growing liver.

    PubMed

    Reaiche-Miller, Georget Y; Thorpe, Michael; Low, Huey Chi; Qiao, Qiao; Scougall, Catherine A; Mason, William S; Litwin, Samuel; Jilbert, Allison R

    2013-11-01

    Nucleos(t)ide analogues that inhibit hepatitis B virus (HBV) DNA replication are typically used as monotherapy for chronically infected patients. Treatment with a nucleos(t)ide analogue eliminates most HBV DNA replication intermediates and produces a gradual decline in levels of covalently closed circular DNA (cccDNA), the template for viral RNA synthesis. It remains uncertain if levels of cccDNA decline primarily through hepatocyte death, or if loss also occurs during hepatocyte mitosis. To determine if cccDNA survives mitosis, growing ducklings infected with duck hepatitis B virus (DHBV) were treated with the nucleoside analogue, Entecavir. Viremia was suppressed at least 10(5)-fold, during a period when average liver mass increased 23-fold. Analysis of the data suggested that if cccDNA synthesis was completely inhibited, at least 49% of cccDNA survived hepatocyte mitosis. However, there was a large duck-to-duck variation in cccDNA levels, suggesting that low level cccDNA synthesis may contribute to this apparent survival through mitosis.

  19. Pathways of Transformation in Ustilago Maydis Determined by DNA Conformation

    PubMed Central

    Fotheringham, S.; Holloman, W. K.

    1990-01-01

    Ustilago maydis was transformed by plasmids bearing a cloned, selectable gene but lacking an autonomously replicating sequence. Transformation was primarily through integration at nonhomologous loci when the plasmid DNA was circular. When the DNA was made linear by cleavage within the cloned gene, the spectrum of integration events shifted from random to targeted recombination at the resident chromosomal allele. In a large fraction of the transformants obtained using linear DNA, the plasmid DNA was not integrated but was maintained in an extrachromosomal state composed of a concatameric array of plasmid units joined end-to-end. The results suggest the operation of several pathways for transformation in U. maydis, and that DNA conformation at the time of transformation governs choice of pathways. PMID:2323553

  20. A divergent clade of circular single-stranded DNA viruses from pig feces

    USDA-ARS?s Scientific Manuscript database

    Using metagenomics and molecular cloning methods, we characterized five novel small circular viral genomes from pig feces distantly related to chimpanzee and porcine stool-associated circular viruses, (ChiSCV and PoSCV1). Phylogenetic analysis placed these viruses into a new, highly divergent, clade...

  1. Circular dichroism microscopy of compact forms of DNA and chromatin in vivo and in vitro: cholesteric liquid-crystalline phases of DNA and single dinoflagellate nuclei

    SciTech Connect

    Livolant, F.; Maestre, M.E.

    1988-04-19

    Two highly condensed structures of DNA have been analyzed in the circular dichroism (CD) microscope: the cholesteric liquid-crystalline phase of DNA and the nucleus of a dinoflagellate (Prorocentrum micans). In both cases, the DNA shows a helical cholesteric organization, but the helical pitch equals about 2500 nm in the first case and 250 nm in the second one. Since the absorption band of DNA is located at 260 nm, the reflection and absorption bands are well separated in the cholesteric phase of DNA and are overlapping in the dinoflagellate nucleus. However, both structures give a very strong negative CD signal at 265 nm. The authors show that this very strong signal cannot correspond to a Borrmann effect, i.e., to a superposition of the absorption and reflection bands, but is a differential absorption of left versus right circularly polarized light. This anomalous differential absorption is probably due to a significant scattering of light, inside of the structure, which produces a resonance phenomenon in the absorption band of the chromophore. Intercalative dyes were inserted in the dinoflagellate chromatin. The CD signal recorded in their absorption bands mimics the signal observed in the absorption band of DNA. In both structures, the negative sign of the CD at 265 nm indicates that the twist occurring between DNA molecules is left-handed, and they show that this situation is the more frequently encountered in vivo and in vitro.

  2. Analysis of Telomere-Homologous DNA with Different Conformations Using 2D Agarose Electrophoresis and In-Gel Hybridization.

    PubMed

    Zhang, Zepeng; Hu, Qian; Zhao, Yong

    2017-01-01

    In mammalian cells, in addition to double-stranded telomeric DNA at chromosome ends, extra telomere-homologous DNA is present that adopts different conformations, including single-stranded G- or C-rich DNA, extrachromosomal circular DNA (T-circle), and telomeric complex (T-complex) with an unidentified structure. The formation of such telomere-homologous DNA is closely related to telomeric DNA metabolism and chromosome end protection by telomeres. Conventional agarose gel electrophoresis is unable to separate DNA based on conformation. Here, we introduce the method of two-dimensional (2D) agarose electrophoresis in combination with in-gel native/denatured hybridization to determine different conformations formed by telomere-homologous DNA.

  3. Isothermal circular-strand-displacement polymerization of DNA and microRNA in digital microfluidic devices.

    PubMed

    Giuffrida, Maria Chiara; Zanoli, Laura Maria; D'Agata, Roberta; Finotti, Alessia; Gambari, Roberto; Spoto, Giuseppe

    2015-02-01

    Nucleic-acid amplification is a crucial step in nucleic-acid-sequence-detection assays. The use of digital microfluidic devices to miniaturize amplification techniques reduces the required sample volume and the analysis time and offers new possibilities for process automation and integration in a single device. The recently introduced droplet polymerase-chain-reaction (PCR) amplification methods require repeated cycles of two or three temperature-dependent steps during the amplification of the nucleic-acid target sequence. In contrast, low-temperature isothermal-amplification methods have no need for thermal cycling, thus requiring simplified microfluidic-device features. Here, the combined use of digital microfluidics and molecular-beacon (MB)-assisted isothermal circular-strand-displacement polymerization (ICSDP) to detect microRNA-210 sequences is described. MicroRNA-210 has been described as the most consistently and predominantly upregulated hypoxia-inducible factor. The nmol L(-1)-pmol L(-1) detection capabilities of the method were first tested by targeting single-stranded DNA sequences from the genetically modified Roundup Ready soybean. The ability of the droplet-ICSDP method to discriminate between full-matched, single-mismatched, and unrelated sequences was also investigated. The detection of a range of nmol L(-1)-pmol L(-1) microRNA-210 solutions compartmentalized in nanoliter-sized droplets was performed, establishing the ability of the method to detect as little as 10(-18) mol of microRNA target sequences compartmentalized in 20 nL droplets. The suitability of the method for biological samples was tested by detecting microRNA-210 from transfected K562 cells.

  4. Thermodynamics of the first transition in writhe of a small circular DNA by Monte Carlo simulation.

    PubMed

    Gebe, J A; Schurr, J M

    1996-04-01

    Monte Carlo simulations are employed to investigate the thermodynamics of the first transition in writhe of a circular model filament corresponding to a 468 base-pair DNA. Parameters employed in these simulations are the torsional rigidity, C = 2.0 x 10(-19) dyne cm2, and persistence length, P = 500 A. Intersubunit interactions are modeled by a screened Coulomb potential. For a straight line of subunits this accurately approximates the nonlinear Poisson-Boltzmann potential of a cylinder with the linear charge density of DNA. Curves of relative free energy vs writhe at fixed linking difference (delta l) exhibit two minima, one corresponding to slightly writhed circles and one to slightly underwrithed figure-8's, whenever delta l lies in the transition region. The free energies of the two minima are equal when delta lc = 1.35, which defines the midpoint of the transition. At this midpoint, the free energy barrier between the two minima is found to be delta Gbar = (0.20) kBT at 298 K. Curves of mean potential energy vs writhe at fixed linking difference similarly exhibit two minima for delta l values in the transition region, and the two minimum mean potential energies are equal when delta l = 1.50. At the midpoint writhe, delta lc = 1.35, the difference in mean potential energy between the minimum free energy figure-8 and circle states is (1.3) kBT, and the difference in their entropies is 1.3 kB. Thus, the entropy of the minimum free energy figure-8 state significantly exceeds that of the circle at the midpoint of the transition. The first transition in writhe is found to occur over a rather broad range of delta l values from 0.85 to 1.85. The twist energy parameter (ET), which governs the overall free energy of supercoiling, undergoes a sigmoidal decrease, while the translational diffusion coefficient undergoes a sigmoidal increase, over this same range. The static structure factor exhibits an increase, which reflects a decrease in radius of gyration associated

  5. Gene editing activity on extrachromosomal arrays in C. elegans transgenics.

    PubMed

    Falgowski, Kerry A; Kmiec, Eric B

    2011-04-15

    Gene editing by modified single-stranded oligonucleotides is a strategy aimed at inducing single base changes into the genome, generating a permanent genetic change. The work presented here explores gene editing capabilities in the model organism Caenorhabditis elegans. Current approaches to gene mutagenesis in C. elegans have been plagued by non-specificity and thus the ability to induce precise, directed alterations within the genome of C. elegans would offer a platform upon which structure/function analyses can be carried out. As such, several in vivo assay systems were developed to evaluate gene editing capabilities in C. elegans. Fluorescence was chosen as the selectable endpoint as fluorescence can be easily detected through the transparent worm body even from minimal expression. Two tissue specific fluorescent expression vectors containing either a GFP or mCherry transgene were mutagenized to create a single nonsense mutation within the open reading frame of each respective fluorescent gene. These served as the target site to evaluate the frequency of gene editing on extrachromosomal array transgenic lines. Extrachromosomal arrays can carry hundreds of copies of the transgene, therefore low frequency events (like those in the gene editing reaction) may be detected. Delivery of the oligonucleotide was accomplished by microinjection into the gonads of young adult worms in an effort to induce repair of the mutated fluorescent gene in the F1 progeny. Despite many microinjections on the transgenic strains with varying concentrations of ODNs, no gene editing events were detected. This result is consistent with the previous research, demonstrating the difficulties encountered in targeting embryonic stem cells and the pronuclei of single-celled embryos. Copyright © 2011 Elsevier B.V. All rights reserved.

  6. The persistence in the liver of residual duck hepatitis B virus covalently closed circular DNA is not dependent upon new viral DNA synthesis.

    PubMed

    Reaiche, Georget Y; Le Mire, Marc F; Mason, William S; Jilbert, Allison R

    2010-10-25

    Residual hepatitis B virus (HBV) DNA can be detected following the resolution of acute HBV infection. Our previous work using duck hepatitis B virus (DHBV) infected ducks, indicated that ~80% of residual DHBV DNA in the liver is in the covalently closed circular DNA (cccDNA) form, suggesting that viral DNA synthesis is suppressed. The current study asked more directly if maintenance of residual DHBV cccDNA is dependent upon ongoing viral DNA synthesis. Ducks that recovered from acute DHBV infection were divided into 2 groups and treated with the antiviral drug, Entecavir (ETV), or placebo. No major differences in the stability of cccDNA or levels of residual cccDNA were observed in liver biopsy tissues taken 95 days apart from ETV treated and placebo control ducks. The data suggest that residual DHBV cccDNA is highly stable and present in a cell population with a rate of turnover similar to normal, uninfected hepatocytes. Copyright © 2010 Elsevier Inc. All rights reserved.

  7. DNA Electronic Circular Dichroism on the Inter-Base Pair Scale: An Experimental-Theoretical Case Study of the AT Homo-Oligonucleotide.

    PubMed

    Di Meo, Florent; Pedersen, Morten N; Rubio-Magnieto, Jenifer; Surin, Mathieu; Linares, Mathieu; Norman, Patrick

    2015-02-05

    A successful elucidation of the near-ultraviolet electronic circular dichroism spectrum of a short double-stranded DNA is reported. Time-dependent density functional theory methods are shown to accurately predict spectra and assign bands on the microscopic base-pair scale, a finding that opens the field for using circular dichroism spectroscopy as a sensitive nanoscale probe of DNA to reveal its complex interactions with the environment.

  8. A sensitive method for the quantification of virion-sense and complementary-sense DNA strands of circular single-stranded DNA viruses

    PubMed Central

    Rodríguez-Negrete, Edgar A.; Sánchez-Campos, Sonia; Cañizares, M. Carmen; Navas-Castillo, Jesús; Moriones, Enrique; Bejarano, Eduardo R.; Grande-Pérez, Ana

    2014-01-01

    Circular single-stranded DNA (ssDNA) viruses are the smallest viruses known to infect eukaryotes. High recombination and mutation rates have conferred these viruses with an evolutionary potential that has facilitated their emergence. Their damaging effects on livestock (circoviruses) and crops (geminiviruses and nanoviruses), and the ubiquity of anelloviruses in human populations and other mammalian species, have resulted in increased interest in better understanding their epidemiology and infection mechanisms. Circular ssDNA viral replication involves the synthesis of dsDNA intermediates containing complementary-sense (CS) and virion-sense (VS) strands. Precise quantification of VS and CS accumulation during viral infections can provide insights into the molecular mechanisms underlying viral replication and the host invasion process. Although qPCR protocols for quantifying viral molecules exist, none of them discriminate VS and CS strands. Here, using a two-step qPCR protocol we have quantified VS and CS molecule accumulation during the infection process of Tomato yellow leaf curl virus (TYLCV) and Tomato yellow leaf curl Sardinia virus (TYLCSV) (genus Begomovirus, family Geminiviridae). Our results show that the VS/CS strand ratio and overall dsDNA amounts vary throughout the infection process. Moreover, we show that these values depend on the virus-host combination, and that most CS strands are present as double-stranded molecules. PMID:25241765

  9. TTV, a new human virus with single stranded circular DNA genome.

    PubMed

    Hino, Shigeo

    2002-01-01

    TT virus (TTV) was found in 1997 from a hepatitis patient without virus markers. However, the real impact of TTV on liver diseases remains uncertain to date. Due to the lack of suitable cell systems to support the growth of TTV, the biology of TTV is still obscure. This review tries to summarise the current status of TTV on aspects other than the taxonomic diversity of TTV. TTV was the first human virus with a single stranded circular DNA genome. TTV was considered to be a member of Circoviridae, but others suggested it conformed to a new family. TTV is distinct from ambisense viruses in the genus Circovirus, since the former genome is negative stranded. The genome structure of TTV is more related to chicken anaemia virus in the genus Gyrovirus, however, the sequence similarity is minimal except for a short stretch at 3816-3851 of TA278. Currently the working group is proposing the full name for TTV as TorqueTenoVirus and the TTV-like mini virus as TorqueTenoMiniVirus (TTMV) in a new genus Anellovirus (ring). TTVs are prevalent in non-human primates and human TTV can cross-infect chimpanzees. Furthermore, TTV sequences have been detected in chickens, pigs, cows and sheep. TTV can be transmitted by mother-to-child infection. However, within a year after birth, the prevalence reaches the same level for children born to both TTV-positive and TTV-negative mothers even without breast-feeding. The non-coding region surrounding a short 113 nt GC-rich stretch and occupying approximately one-third of the genome is considered to contain the putative replication origin. Three mRNAs are expressed by TTV, 3.0 and 1.2 and 1.0 kb species. A protein translated from the 3.0 kb mRNA is considered to be the major capsid protein as well as replicase. The nature of the proteins translated by the other two mRNAs are still putative.

  10. Detection of the hepatitis B virus (HBV) covalently-closed-circular DNA (cccDNA) in mice transduced with a recombinant AAV-HBV vector.

    PubMed

    Lucifora, Julie; Salvetti, Anna; Marniquet, Xavier; Mailly, Laurent; Testoni, Barbara; Fusil, Floriane; Inchauspé, Aurore; Michelet, Maud; Michel, Marie-Louise; Levrero, Massimo; Cortez, Pierre; Baumert, Thomas F; Cosset, François-Loic; Challier, Cécile; Zoulim, Fabien; Durantel, David

    2017-09-01

    Hepatitis B Virus (HBV) persists in infected hepatocytes as an episomal covalently-closed-circular DNA mini-chromosome, called cccDNA. As the main nuclear transcription template, HBV cccDNA is a key replication intermediate in the viral life cycle. Little is known about the mechanisms involved in its formation, maintenance and fate under antiviral therapies. This is mainly due to the lack of small immune-competent animal models able to recapitulate the entire HBV replication cycle, including formation of HBV cccDNA. Here we report that HBV cccDNA can be detected by Southern blot analyses in the liver of C57BL6 mice transduced with AAV-HBV. HBV cccDNA persists in the liver of these animals together with the AAV-HBV episome. We also set up a PCR strategy to distinguish the HBV cccDNA from the AAV-HBV episome. These suggest that the AAV-HBV/mouse model might be relevant to test drugs targeting HBV cccDNA regulation and persistence. Copyright © 2017. Published by Elsevier B.V.

  11. Identification of several clades of novel single-stranded circular DNA viruses with conserved stem-loop structures in pig feces

    USDA-ARS?s Scientific Manuscript database

    Metagenomic analysis of fecal samples collected from swine with diarrhea detected sequences encoding a replication initiator protein (Rep) typically found in small circular Rep-encoding single-stranded DNA (CRESS-DNA) viruses. Complete nucleotide sequences of ten novel genomes were cloned and determ...

  12. Molecular cloning of circular unintegrated DNA of two types of the SEATO strain of gibbon ape leukemia virus.

    PubMed Central

    Gelmann, E P; Trainor, C D; Wong-Staal, F; Reitz, M S

    1982-01-01

    Closed circular unintegrated DNA of the SEATO strain of gibbon ape leukemia virus (GaLV-S) was isolated from canine thymus fibroblasts after cocultivation with chronically infected bat lung fibroblasts. Restriction endonuclease HindIII cleaves GaLV-S DNA once, thus allowing isolation and cloning of HindIII-digested unintegrated DNA in a permitted form. Two clones isolated in the vector, Charon 21A, were nearly identical by restriction enzyme mapping to each of the two types of GaLV-S previously observed. These two types differ at a single SalI site. Unlike previous maps of GaLV-S proviral DNA, however, both clones lack SstI sites in the long-terminal-repeat units. Both the GaLV-S clones and the major species of GaLV-S proviral DNA contain an EcoRI site in the long-terminal-repeat units. The presence of this EcoRI site and the absence of an SstI site in the GaLV-S long-terminal-repeat units differentiate it from all other known GaLV strains and from the closely related nononcogenic simian sarcoma-associated virus. Heteroduplex comparisons of each of the two clones to clones of simian sarcoma-associated virus show no obvious deletion or substitution loops. This suggests that the ability of GaLV-S to induce myeloid leukemia in gibbon apes in not due to an acquired onc gene. Images PMID:6292490

  13. Serum Hepatitis B Virus DNA, RNA, and HBsAg: Which Correlated Better with Intrahepatic Covalently Closed Circular DNA before and after Nucleos(t)ide Analogue Treatment?

    PubMed

    Gao, Yuhua; Li, Yutang; Meng, Qinghua; Zhang, Zhanqing; Zhao, Ping; Shang, Qinghua; Li, Yue; Su, Mingze; Li, Tong; Liu, Xueen; Zhuang, Hui

    2017-10-01

    The study was designed to investigate whether serum hepatitis B virus (HBV) RNA is a strong surrogate marker for intrahepatic HBV covalently closed circular DNA (cccDNA) compared with serum HBV DNA, hepatitis B surface antigen (HBsAg), and hepatitis B e antigen (HBeAg) in HBeAg-positive chronic hepatitis B (CHB) patients. Serum HBV RNA, HBV DNA, HBsAg, HBeAg, and intrahepatic cccDNA were quantitatively detected at baseline (n = 82) and 96 weeks (n = 62) after treatment with nucleos(t)ide analogue (NUC) in HBeAg-positive CHB patients. The correlations among serum HBV RNA, HBV DNA, HBsAg, HBeAg, and intrahepatic cccDNA levels were then statistically analyzed. The results showed that pretreatment intrahepatic cccDNA levels correlated better with serum HBV DNA levels (r = 0.36, P < 0.01) than with serum HBV RNA levels (r = 0.25, P = 0.02), whereas no correlations were found between pretreatment intrahepatic cccDNA levels and HBsAg (r = 0.15, P = 0.17) or HBeAg (r = 0.07, P = 0.56) levels. At 96 weeks after NUC treatment, intrahepatic cccDNA levels correlated well with HBsAg levels (r = 0.39, P < 0.01) but not with serum HBV RNA, HBV DNA, and HBeAg levels (all P > 0.05). Besides, the decline in the intrahepatic cccDNA level from baseline to week 96 correlated better with the reduction in the serum HBsAg levels than with the decreases in the levels of the other markers (for the HBsAg decline, r = 0.38, P < 0.01; for the HBV DNA decline, r = 0.35, P = 0.01; for the HBV RNA decline, r = 0.28, P < 0.05; for the HBeAg decline, r = 0.18, P = 0.19). In conclusion, the baseline serum HBV RNA level or its decline after 96 weeks of NUC therapy correlated with the corresponding intrahepatic cccDNA level, while it was less than that seen with serum HBV DNA at baseline and HBsAg (or its decline) at 96 weeks after treatment, respectively. Copyright © 2017 American Society for Microbiology.

  14. Interaction between adenovirus DNA-binding protein and single-stranded polynucleotides studied by circular dichroism and ultraviolet absorption.

    PubMed

    van Amerongen, H; van Grondelle, R; van der Vliet, P C

    1987-07-28

    The adenovirus DNA-binding protein (AdDBP) is a multifunctional protein required for viral DNA replication and control of transcription. We have studied the binding of AdDBP to single-stranded M13 DNA and to the homopolynucleotides poly(rA), poly(dA), and poly(dT) by means of circular dichroism (CD) and optical density (OD) measurements. The binding to all these polynucleotides was strong and nearly stoichiometric. Titration experiments showed that the size of the binding site is 9-11 nucleotides long for M13 DNA, poly(dA), and poly(rA). A higher value (15.0 +/- 0.8) was found for poly(dT). Pronounced changes in the circular dichroism and optical density spectra were observed upon binding of AdDBP. In general, both the positive peak around 260-270 nm and the negative peak around 240-250 nm in the CD spectra decreased in intensity, and a shift of the crossover point to longer wavelengths was found. The OD spectra observed upon binding of AdDBP are remarkably similar to those obtained with prokaryotic helix-destabilizing proteins like bacteriophage T4 gene 32 protein and fd gene 5 protein. The data can best be interpreted by assuming that the AdDBP-polynucleotide complex has a regular, rigid, and extended configuration that satifies two criteria: (1) a considerable tilt of the bases in combination with (2) a small rotation per base and/or a shift of the bases closer to the helix axis.

  15. Thermostable DNA ligase-mediated PCR production of circular plasmid (PPCP) and its application in directed evolution via in situ error-prone PCR.

    PubMed

    Le, Yilin; Chen, Huayou; Zagursky, Robert; Wu, J H David; Shao, Weilan

    2013-08-01

    Polymerase chain reaction (PCR) is a powerful method to produce linear DNA fragments. Here we describe the Tma thermostable DNA ligase-mediated PCR production of circular plasmid (PPCP) and its application in directed evolution via in situ error-prone PCR. In this thermostable DNA ligase-mediated whole-plasmid amplification method, the resultant DNA nick between the 5' end of the PCR primer and the extended newly synthesized DNA 3' end of each PCR cycle is ligated by Tma DNA ligase, resulting in circular plasmid DNA product that can be directly transformed. The template plasmid DNA is eliminated by 'selection marker swapping' upon transformation. When performed under an error-prone condition with Taq DNA polymerase, PPCP allows one-step construction of mutagenesis libraries based on in situ error-prone PCR so that random mutations are introduced into the target gene without altering the expression vector plasmid. A significant difference between PPCP and previously published methods is that PPCP allows exponential amplification of circular DNA. We used this method to create random mutagenesis libraries of a xylanase gene and two cellulase genes. Screening of these libraries resulted in mutant proteins with desired properties, demonstrating the usefulness of in situ error-prone PPCP for creating random mutagenesis libraries for directed evolution.

  16. Thermostable DNA Ligase-Mediated PCR Production of Circular Plasmid (PPCP) and Its Application in Directed Evolution via In situ Error-Prone PCR

    PubMed Central

    Le, Yilin; Chen, Huayou; Zagursky, Robert; Wu, J.H. David; Shao, Weilan

    2013-01-01

    Polymerase chain reaction (PCR) is a powerful method to produce linear DNA fragments. Here we describe the Tma thermostable DNA ligase-mediated PCR production of circular plasmid (PPCP) and its application in directed evolution via in situ error-prone PCR. In this thermostable DNA ligase-mediated whole-plasmid amplification method, the resultant DNA nick between the 5′ end of the PCR primer and the extended newly synthesized DNA 3′ end of each PCR cycle is ligated by Tma DNA ligase, resulting in circular plasmid DNA product that can be directly transformed. The template plasmid DNA is eliminated by ‘selection marker swapping’ upon transformation. When performed under an error-prone condition with Taq DNA polymerase, PPCP allows one-step construction of mutagenesis libraries based on in situ error-prone PCR so that random mutations are introduced into the target gene without altering the expression vector plasmid. A significant difference between PPCP and previously published methods is that PPCP allows exponential amplification of circular DNA. We used this method to create random mutagenesis libraries of a xylanase gene and two cellulase genes. Screening of these libraries resulted in mutant proteins with desired properties, demonstrating the usefulness of in situ error-prone PPCP for creating random mutagenesis libraries for directed evolution. PMID:23633530

  17. Hepatitis B virus covalently closed circular DNA homeostasis is independent of the lymphotoxin pathway during chronic HBV infection.

    PubMed

    Meier, M-A; Suslov, A; Ketterer, S; Heim, M H; Wieland, S F

    2017-08-01

    Current treatment options for patients with chronic hepatitis B virus (HBV) infection are not curative as they are not effective in eliminating covalently closed circular DNA (cccDNA). cccDNA is a stable template for HBV transcription in the nucleus of hepatocytes and is thought to be one of the main factors responsible for HBV persistence. Recently, activation of the lymphotoxin beta receptor (LTβR) has been shown to trigger degradation of cccDNA through induction of cytidine deaminases of the APOBEC3 family in HBV cell culture model systems. To assess the presence and relevance of such mechanisms in the liver of chronically HBV-infected patients, we compared intrahepatic cccDNA levels with the expression levels of lymphotoxins and some of their target genes (eg APOBEC deaminases) in liver biopsy tissue. Our results confirm elevated gene expression levels of components of the lymphotoxin pathway including lymphotoxin alpha (LTα), lymphotoxin beta (LTβ), APOBEC3B (A3B) and APOBEC3G (A3G) in the chronically HBV-infected liver compared to uninfected liver. Furthermore, expression levels of the genes of the APOBEC deaminase family were correlated with those of LTα and LTβ gene expression, consistent with lymphotoxin-mediated upregulation of APOBEC gene expression. However, intrahepatic cccDNA and HBV replication levels were not correlated with LTα, LTβ and APOBEC gene expression. In conclusion, these results suggest that although the lymphotoxin pathway is activated in the chronically HBV-infected liver, it has no major impact on HBV cccDNA metabolism in chronic HBV infection. © 2017 John Wiley & Sons Ltd.

  18. Discovery, Prevalence, and Persistence of Novel Circular Single-Stranded DNA Viruses in the Ctenophores Mnemiopsis leidyi and Beroe ovata.

    PubMed

    Breitbart, Mya; Benner, Bayleigh E; Jernigan, Parker E; Rosario, Karyna; Birsa, Laura M; Harbeitner, Rachel C; Fulford, Sidney; Graham, Carina; Walters, Anna; Goldsmith, Dawn B; Berger, Stella A; Nejstgaard, Jens C

    2015-01-01

    Gelatinous zooplankton, such as ctenophores and jellyfish, are important components of marine and brackish ecosystems and play critical roles in aquatic biogeochemistry. As voracious predators of plankton, ctenophores have key positions in aquatic food webs and are often successful invaders when introduced to new areas. Gelatinous zooplankton have strong impacts on ecosystem services, particularly in coastal environments. However, little is known about the factors responsible for regulating population dynamics of gelatinous organisms, including biological interactions that may contribute to bloom demise. Ctenophores are known to contain specific bacterial communities and a variety of invertebrate parasites and symbionts; however, no previous studies have examined the presence of viruses in these organisms. Building upon recent studies demonstrating a diversity of single-stranded DNA viruses that encode a replication initiator protein (Rep) in aquatic invertebrates, this study explored the presence of circular, Rep-encoding single-stranded DNA (CRESS-DNA) viruses in the ctenophores Mnemiopsis leidyi and Beroe ovata collected from the Skidaway River Estuary and Savannah River in Georgia, USA. Using rolling circle amplification followed by restriction enzyme digestion, this study provides the first evidence of viruses in ctenophores. Investigation of four CRESS-DNA viruses over an 8-month period using PCR demonstrated temporal trends in viral prevalence and indicated that some of the viruses may persist in ctenophore populations throughout the year. Although future work needs to examine the ecological roles of these ctenophore-associated viruses, this study indicates that viral infection may play a role in population dynamics of gelatinous zooplankton.

  19. A new model mimicking persistent HBV e antigen-negative infection using covalently closed circular DNA in immunocompetent mice.

    PubMed

    Wang, Lei; Cao, Min; Wei, Qing Lu; Zhao, Zhong Hua; Xiang, Qin; Wang, Hui Juan; Zhang, Hua Tang; Lai, Guo Qi

    2017-01-01

    Despite the availability of an effective vaccine, hepatitis B virus (HBV) infection remains a major health problem. HBV e antigen (HBeAg)-negative strains have become prevalent. Previously, no animal model mimicked the clinical course of HBeAg-negative HBV infection. To establish an HBeAg-negative HBV infection model, the 3.2-kb full-length genome of HBeAg-negative HBV was cloned from a clinical sample and then circularized to form covalently closed circular (cccDNA). The resulting cccDNA was introduced into the liver of C57BL/6J mice through hydrodynamic injection. Persistence of the HBeAg-negative infection was monitored at predetermined time points using HBV-specific markers including HBV surface antigen (HBsAg), HBeAg, and HBV core antigen (HBcAg) as well as DNA copies. Throughout the study, pAAV-HBV1.2 was used as a control. In mice injected with HBeAg-negative cccDNA, the HBV infection rate was 100% at the initial stage. HBsAg levels increased up to 1 week, at which point levels peaked and dropped quickly thereafter. In 60% of injected mice, HBsAg and HBcAg persisted for more than 10 weeks. High numbers of HBV DNA copies were detected in the serum and liver. Moreover, cccDNA persisted in the liver tissue of HBeAg-negative mice. In contrast to the pAAV-HBV 1.2 injected mice, no HBeAg was found in mice injected with HBeAg-negative HBV throughout the study period. These results demonstrate the first successful establishment of a model of HBeAg-negative HBV-persistent infection in immunocompetent mice. Compared to pAAV-HBV1.2-injected mice, the infection persistence and levels of serum virological and biochemical markers were approximately equal in the model mice. This model will be useful for mechanistic studies on HBeAg-negative HBV infection and will facilitate the evaluation of new antiviral drugs.

  20. Elite suppressors harbor low levels of integrated HIV DNA and high levels of 2-LTR circular HIV DNA compared to HIV+ patients on and off HAART.

    PubMed

    Graf, Erin H; Mexas, Angela M; Yu, Jianqing J; Shaheen, Farida; Liszewski, Megan K; Di Mascio, Michele; Migueles, Stephen A; Connors, Mark; O'Doherty, Una

    2011-02-01

    Elite suppressors (ES) are a rare population of HIV-infected individuals that are capable of naturally controlling the infection without the use of highly active anti-retroviral therapy (HAART). Patients on HAART often achieve viral control to similar (undetectable) levels. Accurate and sensitive methods to measure viral burden are needed to elucidate important differences between these two patient populations in order to better understand their mechanisms of control. Viral burden quantification in ES patients has been limited to measurements of total DNA in PBMC, and estimates of Infectious Units per Million cells (IUPM). There appears to be no significant difference in the level of total HIV DNA between cells from ES patients and patients on HAART. However, recovering infectious virus from ES patient samples is much more difficult, suggesting their reservoir size should be much smaller than that in patients on HAART. Here we find that there is a significant difference in the level of integrated HIV DNA in ES patients compared to patients on HAART, providing an explanation for the previous results. When comparing the level of total to integrated HIV DNA in these samples we find ES patients have large excesses of unintegrated HIV DNA. To determine the composition of unintegrated HIV DNA in these samples, we measured circular 2-LTR HIV DNA forms and found ES patients frequently have high levels of 2-LTR circles in PBMC. We further show that these high levels of 2-LTR circles are not the result of inefficient integration in ES cells, since HIV integrates with similar efficiency in ES and normal donor cells. Our findings suggest that measuring integration provides a better surrogate of viral burden than total HIV DNA in ES patients. Moreover, they add significantly to our understanding of the mechanisms that allow viral control and reservoir maintenance in this unique patient population.

  1. Circular dichroism anisotrophy of DNA with different modifications at N7 of guanine.

    PubMed

    Zavriev, S K; Minchenkova, L E; Vorlícková, M; Kolchinsky, A M; Volkenstein, M V; Ivanov, V I

    1979-09-27

    The complexex DNA-Ag1+, DNA-Cu1+, protonated DNA and DNA methylated at N7 of guanine were oriented by pumping the solutions through a multicapillary cell in the direction of a light beam. The CD components along the DNA axis, delta epsilon parallel, and normal to it, 2 delta epsilon perpendicular, were calculated from the CD spectra of the oriented samples by the method of Chung and Holzwarth, (1975) J. Mol. Biol. 92, 449--466. It was shown that in most cases, except that of the protonated DNA, the degree of orientation was only slightly less than that for pure DNA. This demonstrated the absence of aggregation and of appreciable denaturation. In all cases the modifications of DNA give rise to a negative component 2 delta epsilon perpendicular, whose magnitude increased as the extent of modification increased. From both the CD spectra of non-oriented samples and the absorption spectra, an inference is drawn that Ag1+ and Cu1+ are attached to the same site as CH3 groups i.e., to the N7 atom of guanine. Proton transfer along the H-bond from the N1 atom of G to the N3 atom of the complementary cytosine is suggested to be a result of the modifications, although the case of H+-DNA may differ from the others. Based on the CD spectra for the anisotropic components, delta epsilon parallel and 2 delta epsilon perpendicular, it is proposed that ligand binding is accompanied by winding of the DNA helix.

  2. A set of mini-Mu transposons for versatile cloning of circular DNA and novel dual-transposon strategy for increased efficiency.

    PubMed

    Pulkkinen, Elsi; Haapa-Paananen, Saija; Turakainen, Hilkka; Savilahti, Harri

    2016-07-01

    Mu transposition-based cloning of DNA circles employs in vitro transposition reaction to deliver both the plasmid origin of replication and a selectable marker into the target DNA of interest. We report here the construction of a platform for the purpose that contains ten mini-Mu transposons with five different replication origins, enabling a variety of research approaches for the discovery and study of circular DNA. We also demonstrate that the simultaneous use of two transposons, one with the origin of replication and the other with selectable marker, is beneficial as it improves the cloning efficiency by reducing the fraction of autointegration-derived plasmid clones. The constructed transposons now provide a set of new tools for the studies on DNA circles and widen the applicability of Mu transposition based approaches to clone circular DNA from various sources.

  3. Production of DNA minicircles less than 250 base pairs through a novel concentrated DNA circularization assay enabling minicircle design with NF-κB inhibition activity.

    PubMed

    Thibault, Thomas; Degrouard, Jeril; Baril, Patrick; Pichon, Chantal; Midoux, Patrick; Malinge, Jean-Marc

    2017-03-17

    Double-stranded DNA minicircles of less than 1000 bp in length have great interest in both fundamental research and therapeutic applications. Although minicircles have shown promising activity in gene therapy thanks to their good biostability and better intracellular trafficking, minicircles down to 250 bp in size have not yet been investigated from the test tube to the cell for lack of an efficient production method. Herein, we report a novel versatile plasmid-free method for the production of DNA minicircles comprising fewer than 250 bp. We designed a linear nicked DNA double-stranded oligonucleotide blunt-ended substrate for efficient minicircle production in a ligase-mediated and bending protein-assisted circularization reaction at high DNA concentration of 2 μM. This one pot multi-step reaction based-method yields hundreds of micrograms of minicircle with sequences of any base composition and position and containing or not a variety of site-specifically chemical modifications or physiological supercoiling. Biochemical and cellular studies were then conducted to design a 95 bp minicircle capable of binding in vitro two NF-κB transcription factors per minicircle and to efficiently inhibiting NF-κB-dependent transcriptional activity in human cells. Therefore, our production method could pave the way for the design of minicircles as new decoy nucleic acids. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  4. Single primer-mediated circular polymerase chain reaction for hairpin DNA cloning and plasmid editing.

    PubMed

    Huang, Jiansheng; Khan, Inamullah; Liu, Rui; Yang, Yan; Zhu, Naishuo

    2016-05-01

    We developed and validated a universal polymerase chain reaction (PCR) method, single primer circular (SPC)-PCR, using single primer to simultaneously insert and amplify a short hairpin sequence into a vector with a high success rate. In this method, the hairpin structure is divided into two parts and fused into a vector by PCR. Then, a single primer is used to cyclize the chimera into a mature short hairpin RNA (shRNA) expression vector. It is not biased by loop length or palindromic structures. Six hairpin DNAs with short 4-nucleotide loops were successfully cloned. Moreover, SPC-PCR was also applied to plasmid editing within 3 h with a success rate higher than 95%.

  5. Improved DNA equilibrium binding affinity determinations of platinum(II) complexes using synchrotron radiation circular dichroism.

    PubMed

    Ang, Dale L; Jones, Nykola C; Stootman, Frank; Ghadirian, Bahman; Aldrich-Wright, Janice R

    2015-06-21

    The binding affinity of a series of square planar platinum(II) compounds of the type [Pt(A(L))(I(L))](2+), where A(L) is 1,2-diaminoethane and I(L) are 1,10-phenanthroline (phen), 4-methyl-1,10-phenanthroline (4Mephen), 5-methyl-1,10-phenanthroline (5Mephen), 4,7-dimethyl-1,10-phenanthroline (47Me2phen), 5,6-dimethyl-1,10-phenanthroline (56Me2phen) or 3,4,7,8-tetramethyl-1,10-phenanthroline (3478Me4phen) has been reinvestigated using Synchrotron Radiation Circular Dichroism (SRCD) spectroscopy. The additional peaks exhibited considerably greater intensity than those observed between 200 and 400 nm affording additional binding affinity determinations. In addition, the authors have reviewed the various mathematical approaches used to estimate equilibrium binding constants and thereby demonstrate that their mathematical approach, implemented with Wolfram Mathematica, has merit over other methods.

  6. Improved rolling circle amplification (RCA) of hepatitis B virus (HBV) relaxed-circular serum DNA (RC-DNA).

    PubMed

    Martel, Nora; Gomes, Selma A; Chemin, Isabelle; Trépo, Christian; Kay, Alan

    2013-11-01

    For functional analysis of HBV isolates, epidemiological studies and correct identification of recombinant genomes, the amplification of complete genomes is necessary. A method for completely in vitro amplification of full-length HBV genomes starting from serum RC-DNA is described. This uses in vitro completion/ligation of plus-strand HBV RC-DNA and amplification using Rolling-Circle Amplification, eventually followed by a genomic PCR. The method can amplify complete HBV genomes from sera with viral loads ranging from >1.0E+8 IU/ml down to 1.0E+3 IU/ml. The method can be applied to archived sera that have undergone long-term storage or to archived DNA serum extracts. The genomes can easily be cloned. HBV genotypes A-G can all be amplified with no apparent problems. A recombinant subgenotype A3/genotype E genome was identified and fully sequenced.

  7. Formation of AAV single stranded DNA genome from a circular plasmid in Saccharomyces cerevisiae.

    PubMed

    Cervelli, Tiziana; Backovic, Ana; Galli, Alvaro

    2011-01-01

    Adeno-associated virus (AAV)-based vectors are promising tools for targeted transfer in gene therapy studies. Many efforts have been accomplished to improve production and purification methods. We thought to develop a simple eukaryotic system allowing AAV replication which could provide an excellent opportunity for studying AAV biology and, more importantly, for AAV vector production. It has been shown that yeast Saccharomyces cerevisiae is able to replicate and form the capsid of many viruses. We investigated the ability of the yeast Saccharomyces cerevisiae to carry out the replication of a recombinant AAV (rAAV). When a plasmid containing a rAAV genome in which the cap gene was replaced with the S. cerevisiae URA3 gene, was co-transformed in yeast with a plasmid expressing Rep68, a significant number of URA3(+) clones were scored (more than 30-fold over controls). Molecular analysis of low molecular weight DNA by Southern blotting revealed that single stranded DNA is formed and that the plasmid is entirely replicated. The ssDNA contains the ITRs, URA3 gene and also vector sequences suggesting the presence of two distinct molecules. Its formation was dependent on Rep68 expression and ITR. These data indicate that DNA is not obtained by the canonical AAV replication pathway.

  8. Formation of AAV Single Stranded DNA Genome from a Circular Plasmid in Saccharomyces cerevisiae

    PubMed Central

    Cervelli, Tiziana; Backovic, Ana; Galli, Alvaro

    2011-01-01

    Adeno-associated virus (AAV)-based vectors are promising tools for targeted transfer in gene therapy studies. Many efforts have been accomplished to improve production and purification methods. We thought to develop a simple eukaryotic system allowing AAV replication which could provide an excellent opportunity for studying AAV biology and, more importantly, for AAV vector production. It has been shown that yeast Saccharomyces cerevisiae is able to replicate and form the capsid of many viruses. We investigated the ability of the yeast Saccharomyces cerevisiae to carry out the replication of a recombinant AAV (rAAV). When a plasmid containing a rAAV genome in which the cap gene was replaced with the S. cerevisiae URA3 gene, was co-transformed in yeast with a plasmid expressing Rep68, a significant number of URA3+ clones were scored (more than 30-fold over controls). Molecular analysis of low molecular weight DNA by Southern blotting revealed that single stranded DNA is formed and that the plasmid is entirely replicated. The ssDNA contains the ITRs, URA3 gene and also vector sequences suggesting the presence of two distinct molecules. Its formation was dependent on Rep68 expression and ITR. These data indicate that DNA is not obtained by the canonical AAV replication pathway. PMID:21853137

  9. Discovery, Prevalence, and Persistence of Novel Circular Single-Stranded DNA Viruses in the Ctenophores Mnemiopsis leidyi and Beroe ovata

    PubMed Central

    Breitbart, Mya; Benner, Bayleigh E.; Jernigan, Parker E.; Rosario, Karyna; Birsa, Laura M.; Harbeitner, Rachel C.; Fulford, Sidney; Graham, Carina; Walters, Anna; Goldsmith, Dawn B.; Berger, Stella A.; Nejstgaard, Jens C.

    2015-01-01

    Gelatinous zooplankton, such as ctenophores and jellyfish, are important components of marine and brackish ecosystems and play critical roles in aquatic biogeochemistry. As voracious predators of plankton, ctenophores have key positions in aquatic food webs and are often successful invaders when introduced to new areas. Gelatinous zooplankton have strong impacts on ecosystem services, particularly in coastal environments. However, little is known about the factors responsible for regulating population dynamics of gelatinous organisms, including biological interactions that may contribute to bloom demise. Ctenophores are known to contain specific bacterial communities and a variety of invertebrate parasites and symbionts; however, no previous studies have examined the presence of viruses in these organisms. Building upon recent studies demonstrating a diversity of single-stranded DNA viruses that encode a replication initiator protein (Rep) in aquatic invertebrates, this study explored the presence of circular, Rep-encoding single-stranded DNA (CRESS-DNA) viruses in the ctenophores Mnemiopsis leidyi and Beroe ovata collected from the Skidaway River Estuary and Savannah River in Georgia, USA. Using rolling circle amplification followed by restriction enzyme digestion, this study provides the first evidence of viruses in ctenophores. Investigation of four CRESS-DNA viruses over an 8-month period using PCR demonstrated temporal trends in viral prevalence and indicated that some of the viruses may persist in ctenophore populations throughout the year. Although future work needs to examine the ecological roles of these ctenophore-associated viruses, this study indicates that viral infection may play a role in population dynamics of gelatinous zooplankton. PMID:26733971

  10. Nudiviruses and other large, double-stranded circular DNA viruses of invertebrates: new insights on an old topic.

    PubMed

    Wang, Yongjie; Jehle, Johannes A

    2009-07-01

    Nudiviruses (NVs) are a highly diverse group of large, circular dsDNA viruses pathogenic for invertebrates. They have rod-shaped and enveloped nucleocapsids, replicate in the nucleus of infected host cells, and possess interesting biological and molecular properties. The unassigned viral genus Nudivirus has been proposed for classification of nudiviruses. Currently, the nudiviruses comprise five different viruses: the palm rhinoceros beetle virus (Oryctes rhinoceros NV, OrNV), the Hz-1 virus (Heliothis zea NV-1, HzNV-1), the cricket virus (Gryllus bimaculatus NV, GbNV), the corn earworm moth Hz-2 virus (HzNV-2), and the occluded shrimp Monodon Baculovirus reassigned as Penaeus monodon NV (PmNV). Thus far, the genomes of OrNV, GbNV, HzNV-1 and HzNV-2 have been completely sequenced. They vary between 97 and 230kbp in size and encode between 98 and 160 open reading frames (ORFs). All sequenced nudiviruses have 33 ORFs in common. Strikingly, 20 of them are homologous to baculovirus core genes involved in RNA transcription, DNA replication, virion structural components and other functions. Another nine conserved ORFs are likely associated with DNA replication, repair and recombination, and nucleotide metabolism; one is homologous to baculovirus iap-3 gene; two are nudivirus-specific ORFs of unknown function. Interestingly, one nudivirus ORF is similar to polh/gran gene, encoding occlusion body protein matrix and being conserved in Alpha- Beta- and Gammabaculoviruses. Members of nudiviruses are closely related and form a monophyletic group consisting of two sister clades of OrNV/GbNV and HzNVs/PmNV. It is proposed that nudiviruses and baculoviruses derived from a common ancestor and are evolutionarily related to other large DNA viruses such as the insect-specific salivary gland hypertrophy virus (SGHV) and the marine white spot syndrome virus (WSSV).

  11. Rapidly expanding genetic diversity and host range of the Circoviridae viral family and other Rep encoding small circular ssDNA genomes.

    PubMed

    Delwart, Eric; Li, Linlin

    2012-03-01

    The genomes of numerous circoviruses and distantly related circular ssDNA viruses encoding a rolling circle replication initiator protein (Rep) have been characterized from the tissues of mammals, fish, insects, plants (geminivirus and nanovirus), in human and animal feces, in an algae cell, and in diverse environmental samples. We review the genome organization, phylogenetic relationships and initial prevalence studies of cycloviruses, a proposed new genus in the Circoviridae family. Viral fossil rep sequences were also recently identified integrated on the chromosomes of mammals, frogs, lancelets, crustaceans, mites, gastropods, roundworms, placozoans, hydrozoans, protozoans, land plants, fungi, algae, and phytoplasma bacterias and their plasmids, reflecting the very wide past host range of rep bearing viruses. An ancient origin for viruses with Rep-encoding small circular ssDNA genomes, predating the diversification of eukaryotes, is discussed. The cellular hosts and pathogenicity of many recently described rep-containing circular ssDNA genomes remain to be determined. Future studies of the virome of single cell and multi-cellular eukaryotes are likely to further extend the known diversity and host-range of small rep-containing circular ssDNA viral genomes. Copyright © 2011 Elsevier B.V. All rights reserved.

  12. X-ray scattering from randomly oriented superhelices. Circular superhelical DNA.

    PubMed Central

    Benham, C J; Brady, G W; Fein, D B

    1980-01-01

    The scattering functions of randomly oriented filaments of finite length exhibiting two orders of helicity have been calculated. It is shown to a good approximation that each order scatters as if present alone as a first order helix of the same contour length and pitch angle. These results show that the measured scattering pattern from dissolved superhelical DNA molecules is consistent with the scattering pattern calculated for a coiled coil geometry. PMID:7028148

  13. Identification of regions essential for extrachromosomal replication and maintenance of an endogenous plasmid in Dictyostelium.

    PubMed Central

    Ahern, K G; Howard, P K; Firtel, R A

    1988-01-01

    Initial experiments with the endogenous 12.3 kb Dictyostelium discoideum plasmid Ddp1 led to the generation of a large shuttle vector, Ddp1-20. In addition to Ddp1, this vector contains pBR322 and a gene fusion that confers G418 resistance in Dictyostelium cells. We have shown that Ddp1-20 replicates extrachromosomally in Dictyostelium cells and can be grown in Escherichia coli cells (1). We have now examined deletions within this vector to identify the elements essential for extrachromosomal replication and stable maintenance of the plasmid. We find that a 2.2 kb fragment is sufficient to confer stable, extrachromosomal replication with a reduction in copy number from about 40 to approximately 10-15 copies per cell. Vectors containing additional Ddp1 sequences have a higher copy number. The 2.2 kb region contains none of the complete, previously identified transcription units on Ddp1 expressed during vegetative growth or development. These results suggest that gene products expressed by Ddp1 are not essential for replication, stability, or partitioning of the plasmid between daughter cells. Vectors carrying only the 2.2 kb fragment plus the gene fusion conferring G418 resistance transform Dictyostelium cells with high efficiency using either calcium phosphate mediated transformation or electroporation. Finally, we have examined the relative levels of expression of actin promoters driving neoR genes when in extrachromosomal or integrating vectors. Images PMID:3405751

  14. Differential salt-induced dissociation of the p53 protein complexes with circular and linear plasmid DNA substrates suggest involvement of a sliding mechanism.

    PubMed

    Šebest, Peter; Brázdová, Marie; Fojta, Miroslav; Pivoňková, Hana

    2015-01-30

    A study of the effects of salt conditions on the association and dissociation of wild type p53 with different ~3 kbp long plasmid DNA substrates (supercoiled, relaxed circular and linear, containing or lacking a specific p53 binding site, p53CON) using immunoprecipitation at magnetic beads is presented. Salt concentrations above 200 mM strongly affected association of the p53 protein to any plasmid DNA substrate. Strikingly different behavior was observed when dissociation of pre-formed p53-DNA complexes in increased salt concentrations was studied. While contribution from the p53CON to the stability of the p53-DNA complexes was detected between 100 and 170 mM KCl, p53 complexes with circular DNAs (but not linear) exhibited considerable resistance towards salt treatment for KCl concentrations as high as 2 M provided that the p53 basic C-terminal DNA binding site (CTDBS) was available for DNA binding. On the contrary, when the CTDBS was blocked by antibody used for immunoprecipitation, all p53-DNA complexes were completely dissociated from the p53 protein in KCl concentrations≥200 mM under the same conditions. These observations suggest: (a) different ways for association and dissociation of the p53-DNA complexes in the presence of the CTDBS; and (b) a critical role for a sliding mechanism, mediated by the C-terminal domain, in the dissociation process.

  15. Detection of Short-Range DNA Interactions in Mammalian Cells Using High-Resolution Circular Chromosome Conformation Capture Coupled to Deep Sequencing.

    PubMed

    Millau, Jean-François; Gaudreau, Luc

    2015-01-01

    DNA interactions shape the genome to physically and functionally connect regulatory elements to their target genes. Studying these interactions is crucial to understanding the molecular mechanisms that regulate gene expression. In this chapter, we present a protocol for high-resolution circular chromosome conformation capture coupled to deep sequencing. This methodology allows to investigate short-range DNA interactions (<100 kbp) and to obtain high-resolution DNA interaction maps of loci. It is a powerful tool to explore how regulatory elements and genes are connected together.

  16. Enhanced production of hepatitis B surface antigen in NIH 3T3 mouse fibroblasts by using extrachromosomally replicating bovine papillomavirus vector.

    PubMed Central

    Wang, Y; Stratowa, C; Schaefer-Ridder, M; Doehmer, J; Hofschneider, P H

    1983-01-01

    We have constructed a recombinant pBR322 plasmid composed of a subgenomic transforming fragment of bovine papillomavirus DNA and the hepatitis B surface antigen gene from cloned hepatitis B virus DNA and used it for transfection of NIH 3T3 mouse fibroblasts. The transformed cells retain the plasmids in extrachromosomal form with a copy number of about 50 to 100 per cell. Expression of the hepatitis B surface antigen gene linked to bovine papillomavirus DNA is independent of its orientation relative to the bovine papillomavirus vector. Cell lines continuously secreting high amounts of hepatitis B surface antigen into the medium could be established. The antigen is released into the culture medium as 22-nm particles, having the same physical properties and constituent polypeptides as those found in the serum of hepatitis B virus-infected patients. Images PMID:6308420

  17. Discovery of a novel mastrevirus and alphasatellite-like circular DNA in dragonflies (Epiprocta) from Puerto Rico.

    PubMed

    Rosario, Karyna; Padilla-Rodriguez, Marco; Kraberger, Simona; Stainton, Daisy; Martin, Darren P; Breitbart, Mya; Varsani, Arvind

    2013-01-01

    Geminiviruses have emerged as serious agricultural pathogens. Despite all the species that have been already catalogued, new molecular techniques continue to expand the diversity and geographical ranges of these single-stranded DNA viruses and their associated satellite molecules. Since all geminiviruses are insect-transmitted, examination of insect vector populations through vector-enabled metagenomics (VEM) has been recently used to investigate the diversity of geminiviruses transmitted by a specific vector in a given region. Here we used a more comprehensive adaptation of the VEM approach by surveying small circular DNA viruses found within top insect predators, specifically dragonflies (Epiprocta). This 'predator-enabled' approach is not limited to viral groups transmitted by specific vectors since dragonflies can accumulate the wide range of viruses transmitted by their diverse insect prey. Analysis of six dragonflies collected from an agricultural field in Puerto Rico culminated in the discovery of the first mastrevirus (Dragonfly-associated mastrevirus; DfasMV) and alphasatellite molecule (Dragonfly-associated alphasatellite; Dfas-alphasatellite) from the Caribbean. Since DfasMV and Dfas-alphasatellite are divergent from the limited number of sequences that have been reported from the Americas, this study unequivocally demonstrates that there have been at least two independent past introductions of both mastreviruses and alphasatellites to the New World. Overall, the use of predacious insects as sampling tools can profoundly alter our views of natural plant virus diversity and biogeography by allowing the discovery of novel geminiviruses and associated satellite molecules without a priori knowledge of the types of viruses or insect vectors in a given area.

  18. A diverse group of small circular ssDNA viral genomes in human and non-human primate stools

    PubMed Central

    Ng, Terry Fei Fan; Zhang, Wen; Sachsenröder, Jana; Kondov, Nikola O.; da Costa, Antonio Charlys; Vega, Everardo; Holtz, Lori R.; Wu, Guang; Wang, David; Stine, Colin O.; Antonio, Martin; Mulvaney, Usha S.; Muench, Marcus O.; Deng, Xutao; Ambert-Balay, Katia; Pothier, Pierre; Vinjé, Jan; Delwart, Eric

    2015-01-01

    Viral metagenomics sequencing of fecal samples from outbreaks of acute gastroenteritis from the US revealed the presence of small circular ssDNA viral genomes encoding a replication initiator protein (Rep). Viral genomes were ∼2.5 kb in length, with bi-directionally oriented Rep and capsid (Cap) encoding genes and a stem loop structure downstream of Rep. Several genomes showed evidence of recombination. By digital screening of an in-house virome database (1.04 billion reads) using BLAST, we identified closely related sequences from cases of unexplained diarrhea in France. Deep sequencing and PCR detected such genomes in 7 of 25 US (28 percent) and 14 of 21 French outbreaks (67 percent). One of eighty-five sporadic diarrhea cases in the Gambia was positive by PCR. Twenty-two complete genomes were characterized showing that viruses from patients in the same outbreaks were closely related suggesting common origins. Similar genomes were also characterized from the stools of captive chimpanzees, a gorilla, a black howler monkey, and a lemur that were more diverse than the human stool-associated genomes. The name smacovirus is proposed for this monophyletic viral clade. Possible tropism include mammalian enteric cells or ingested food components such as infected plants. No evidence of viral amplification was found in immunodeficient mice orally inoculated with smacovirus-positive stool supernatants. A role for smacoviruses in diarrhea, if any, remains to be demonstrated. PMID:27774288

  19. The R-Type Pyocin of Pseudomonas aeruginosa C Is a Bacteriophage Tail-Like Particle That Contains Single-Stranded DNA

    PubMed Central

    Lee, Frank K. N.; Dudas, Kathleen C.; Hanson, Julie A.; Nelson, M. Bud; LoVerde, Philip T.; Apicella, Michael A.

    1999-01-01

    Pseudomonas aeruginosa R-type pyocin particles have been described as bacteriocins that resemble bacteriophage tail-like structures. Because of their unusual structure, we reexamined whether they contained nucleic acids. Our data indicated that pyocin particles isolated from P. aeruginosa C (pyocin C) contain DNA. Probes generated from this DNA by the random-primer extension method hybridized to distinct bands in restriction endonuclease-digested P. aeruginosa C genomic DNA. These probes also hybridized to genomic DNA from 6 of 18 P. aeruginosa strains that produced R-type pyocins. Asymmetric PCR, complementary oligonucleotide hybridization, and electron microscopy indicated that pyocin C particles contained closed circular single-stranded DNA, approximately 4.0 kb in length. Examination of total intracellular DNA from mitomycin C-induced cultures revealed the presence of two extrachromosomal DNA molecules, a double-stranded molecule and a single-stranded molecule, which hybridized to pyocin DNA. Sequence analysis of 7,480 nucleotides of P. aeruginosa C chromosomal DNA containing the pyocin DNA indicated the presence of pyocin open reading frames with similarities to open reading frames from filamentous phages and cryptic phage elements. We did not observe any similarities to known phage structural proteins or previously characterized pseudomonal prt genes expressing R-type pyocin structural proteins. These studies demonstrate that pyocin particles from P. aeruginosa C are defective phages that contain a novel closed circular single-stranded DNA and that this DNA was derived from the chromosome of P. aeruginosa C. PMID:9916082

  20. A Sequence-Independent Strategy for Detection and Cloning of Circular DNA Virus Genomes by Using Multiply Primed Rolling-Circle Amplification

    PubMed Central

    Rector, Annabel; Tachezy, Ruth; Van Ranst, Marc

    2004-01-01

    The discovery of novel viruses has often been accomplished by using hybridization-based methods that necessitate the availability of a previously characterized virus genome probe or knowledge of the viral nucleotide sequence to construct consensus or degenerate PCR primers. In their natural replication cycle, certain viruses employ a rolling-circle mechanism to propagate their circular genomes, and multiply primed rolling-circle amplification (RCA) with φ29 DNA polymerase has recently been applied in the amplification of circular plasmid vectors used in cloning. We employed an isothermal RCA protocol that uses random hexamer primers to amplify the complete genomes of papillomaviruses without the need for prior knowledge of their DNA sequences. We optimized this RCA technique with extracted human papillomavirus type 16 (HPV-16) DNA from W12 cells, using a real-time quantitative PCR assay to determine amplification efficiency, and obtained a 2.4 × 104-fold increase in HPV-16 DNA concentration. We were able to clone the complete HPV-16 genome from this multiply primed RCA product. The optimized protocol was subsequently applied to a bovine fibropapillomatous wart tissue sample. Whereas no papillomavirus DNA could be detected by restriction enzyme digestion of the original sample, multiply primed RCA enabled us to obtain a sufficient amount of papillomavirus DNA for restriction enzyme analysis, cloning, and subsequent sequencing of a novel variant of bovine papillomavirus type 1. The multiply primed RCA method allows the discovery of previously unknown papillomaviruses, and possibly also other circular DNA viruses, without a priori sequence information. PMID:15113879

  1. Denaturation of circular or linear DNA facilitates targeted integrative transformation of Streptomyces coelicolor A3(2): possible relevance to other organisms.

    PubMed Central

    Oh, S H; Chater, K F

    1997-01-01

    Using Streptomyces coelicolor A3(2) protoplasts, the number of transformants obtained by homologous recombination of incoming double-stranded circular DNA with the recipient chromosome was greatly stimulated by simple denaturation of the donor DNA. This procedure was very effective with inserts over a ca. 100-fold size range, the largest tested being ca. 40-kb inserts in cosmids. These observations led to transformation experiments with linearized cloned DNA and randomly sheared genomic DNA. In both cases, DNA denaturation led to significant levels of transformation. Most of the transformants had resulted from the predicted homologous recombination events. A number of genetic manipulations will be made easier or possible by these procedures. PMID:8981988

  2. Novel circular single-stranded DNA viruses identified in marine invertebrates reveal high sequence diversity and consistent predicted intrinsic disorder patterns within putative structural proteins

    PubMed Central

    Rosario, Karyna; Schenck, Ryan O.; Harbeitner, Rachel C.; Lawler, Stephanie N.; Breitbart, Mya

    2015-01-01

    Viral metagenomics has recently revealed the ubiquitous and diverse nature of single-stranded DNA (ssDNA) viruses that encode a conserved replication initiator protein (Rep) in the marine environment. Although eukaryotic circular Rep-encoding ssDNA (CRESS-DNA) viruses were originally thought to only infect plants and vertebrates, recent studies have identified these viruses in a number of invertebrates. To further explore CRESS-DNA viruses in the marine environment, this study surveyed CRESS-DNA viruses in various marine invertebrate species. A total of 27 novel CRESS-DNA genomes, with Reps that share less than 60.1% identity with previously reported viruses, were recovered from 21 invertebrate species, mainly crustaceans. Phylogenetic analysis based on the Rep revealed a novel clade of CRESS-DNA viruses that included approximately one third of the marine invertebrate associated viruses identified here and whose members may represent a novel family. Investigation of putative capsid proteins (Cap) encoded within the eukaryotic CRESS-DNA viral genomes from this study and those in GenBank demonstrated conserved patterns of predicted intrinsically disordered regions (IDRs), which can be used to complement similarity-based searches to identify divergent structural proteins within novel genomes. Overall, this study expands our knowledge of CRESS-DNA viruses associated with invertebrates and explores a new tool to evaluate divergent structural proteins encoded by these viruses. PMID:26217327

  3. Novel circular single-stranded DNA viruses identified in marine invertebrates reveal high sequence diversity and consistent predicted intrinsic disorder patterns within putative structural proteins.

    PubMed

    Rosario, Karyna; Schenck, Ryan O; Harbeitner, Rachel C; Lawler, Stephanie N; Breitbart, Mya

    2015-01-01

    Viral metagenomics has recently revealed the ubiquitous and diverse nature of single-stranded DNA (ssDNA) viruses that encode a conserved replication initiator protein (Rep) in the marine environment. Although eukaryotic circular Rep-encoding ssDNA (CRESS-DNA) viruses were originally thought to only infect plants and vertebrates, recent studies have identified these viruses in a number of invertebrates. To further explore CRESS-DNA viruses in the marine environment, this study surveyed CRESS-DNA viruses in various marine invertebrate species. A total of 27 novel CRESS-DNA genomes, with Reps that share less than 60.1% identity with previously reported viruses, were recovered from 21 invertebrate species, mainly crustaceans. Phylogenetic analysis based on the Rep revealed a novel clade of CRESS-DNA viruses that included approximately one third of the marine invertebrate associated viruses identified here and whose members may represent a novel family. Investigation of putative capsid proteins (Cap) encoded within the eukaryotic CRESS-DNA viral genomes from this study and those in GenBank demonstrated conserved patterns of predicted intrinsically disordered regions (IDRs), which can be used to complement similarity-based searches to identify divergent structural proteins within novel genomes. Overall, this study expands our knowledge of CRESS-DNA viruses associated with invertebrates and explores a new tool to evaluate divergent structural proteins encoded by these viruses.

  4. Circular Coinduction

    NASA Technical Reports Server (NTRS)

    Rosu, Grigore; Goguen, Joseph; Norvig, Peter (Technical Monitor)

    2001-01-01

    Circular coinduction is a technique for behavioral reasoning that extends cobasis coinduction to specifications with circularities. Because behavioral satisfaction is not recursively enumerable, no algorithm can work for every behavioral statement. However. algorithms using circular coinduction can prove every practical behavioral result that we know. This paper proves the correctness of circular coinduction and some consequences.

  5. I. RNA A-to-Z transitions and DNA B-to-Z transitions. II. Circular intensity differential scattering of cholesteric liquid crystals

    SciTech Connect

    Hall, K.B.

    1984-12-01

    The thesis is composed of two parts: Part I describes the conformational transitions of DNA and RNA from right-handed helices to left-handed helices. These transitions, referred to as B-to-Z for DNA and A-to-Z for RNA, are effected by specific solvents and temperatures. Various spectroscopic methods show the details of the transition, with near ultraviolet circular dichroism (CD) indicating the differences in the transition moments between the DNA and RNA, and nuclear magnetic resonance (NMR) and vacuum ultraviolet CD indicating that the structures are very similar. Thermodynamic parameters for the DNA polymer transition are calculated, indicating that the transition is not isoenthalpic as previously postulated. A tetramer of RNA, rCGCG, is used for obtaining thermodynamic data on the RNA A-to-Z transition. This short sequence also appears to adopt a left-handed structure, but thermodynamic data indicates that the A-form is more favorable. Part II describes the circular differential scattering (CIDS) of cholesteric liquid crystals. These compounds have a natural helical order, with enormous scattering power. The scattering patterns obtained from the liquid crystals are influenced by the helical parameters such as pitch and handedness, as predicted by the theory. It is hoped that these model compounds will provide information for the interpretation of the CIDS spectra obtained for biological samples, such as gels of DNA or oriented viruses.

  6. Timing of Developmentally Programmed Excision and Circularization of Paramecium Internal Eliminated Sequences

    PubMed Central

    Bétermier, Mireille; Duharcourt, Sandra; Seitz, Hervé; Meyer, Eric

    2000-01-01

    Paramecium internal eliminated sequences (IESs) are short AT-rich DNA elements that are precisely eliminated from the germ line genome during development of the somatic macronucleus. They are flanked by one 5′-TA-3′ dinucleotide on each side, a single copy of which remains at the donor site after excision. The timing of their excision was examined in synchronized conjugating cells by quantitative PCR. Significant amplification of the germ line genome was observed prior to IES excision, which starts 12 to 14 h after initiation of conjugation and extends over a 2- to 4-h period. Following excision, two IESs were shown to form extrachromosomal circles that can be readily detected on Southern blots of genomic DNA from cells undergoing macronuclear development. On these circular molecules, covalently joined IES ends are separated by one copy of the flanking 5′-TA-3′ repeat. The similar structures of the junctions formed on the excised and donor molecules point to a central role for this dinucleotide in IES excision. PMID:10669733

  7. Rapidly expanding genetic diversity and host range of the Circoviridae viral family and other Rep encoding small circular ssDNA genomes

    PubMed Central

    Delwart, Eric; Li, Linlin

    2011-01-01

    The genomes of numerous circoviruses and distantly related circular DNA viruses encoding a rolling circle replication initiator protein (Rep) have been characterized from the tissues of mammals, fish, insects, and plants (geminivirus and nanovirus), human and animal feces, in an algae cell, and in diverse environmental samples. We review the genome organization, phylogenetic relationships and initial prevalence studies of cycloviruses, a proposed new genus in the Circoviridae family. Viral fossil rep sequences were also identified integrated on the chromosomes of mammals, frogs, lancelets, crustaceans, mites, gastropods, roundworms, placozoans, hydrozoans, protozoans, land plants, fungi, algae, and phytoplasma bacterias and their plasmids, reflecting their past host range. An ancient origin for viruses with rep-encoding single stranded small circular genomes, predating the diversification of eukaryotes, is discussed. The cellular hosts and pathogenicity of many recently described rep-containing circular genomes remain to be determined. Future studies of the virome of single cell and multi-cellular eukaryotes are likely to further extend the known diversity and host-range of small rep-containing circular viral genomes. PMID:22155583

  8. Repercussions of DNA tracking by the type IC restriction endonuclease EcoR124I on linear, circular and catenated substrates.

    PubMed

    Szczelkun, M D; Dillingham, M S; Janscak, P; Firman, K; Halford, S E

    1996-11-15

    Type I restriction endonucleases such as EcoR124I cleave DNA at undefined loci, distant from their recognition sequences, by a mechanism that involves the enzyme tracking along the DNA between recognition and cleavage sites. This mechanism was examined on plasmids that carried recognition sites for EcoR124I and recombination sites for resolvase, the latter to create DNA catenanes. Supercoiled substrates with either one or two restriction sites were linearized by EcoR124I at similar rates, although the two-site molecule underwent further cleavage more readily than the one-site DNA. The catenane from the plasmid with one EcoR124I site, carrying the site on the smaller of the two rings, was cleaved by EcoR124I exclusively in the small ring, and this underwent multiple cleavage akin to the two-site plasmid. Linear substrates derived from the plasmids were cleaved by EcoR124I at very slow rates. The communication between recognition and cleavage sites therefore cannot stem from random looping. Instead, it must follow the DNA contour between the sites. On a circular DNA, the translocation of non-specific DNA past the specifically bound protein should increase negative supercoiling in one domain and decrease it in the other. The ensuing topological barrier may be the trigger for DNA cleavage.

  9. Ultrasensitive electrochemical biosensor for specific detection of DNA based on molecular beacon mediated circular strand displacement polymerization and hyperbranched rolling circle amplification.

    PubMed

    Li, Xiaolu; Guo, Jing; Zhai, Qian; Xia, Jing; Yi, Gang

    2016-08-31

    Using a cascade signal amplification strategy, an ultrasensitive electrochemical biosensor for specific detection of DNA based on molecular beacon (MB) mediated circular strand displacement polymerization (CSDP) and hyperbranched rolling circle amplification (HRCA) was proposed. The hybridization of MB probe to target DNA resulted in a conformational change of the MB and triggered the CSDP in the presence of bio-primer and Klenow fragment (KF exo(-)), leading to multiple biotin-tagged DNA duplex. Furthermore, the HRCA was implemented to product amounts of double-stranded DNA (ds-DNA) fragments using phi29 DNA polymerase via biotin-streptavidin interaction. After the product of HRCA binded numerous biotinylated detection probes, an ultrasensitive electrochemical readout by further employing the streptavidin-alkaline phosphatase. The proposed biosensor exhibited excellent detection sensitivity and specificity with a log-linear response to target DNA from 0.01 fM to 10 pM as low as 8.9 aM. The proposed method allowed DNA detection with simplicity, rapidness, low cost and high specificity, which might have the potential for application in clinical molecular diagnostics and environmental monitoring.

  10. Serum hepatitis B core-related antigen is a satisfactory surrogate marker of intrahepatic covalently closed circular DNA in chronic hepatitis B.

    PubMed

    Chen, En-Qiang; Feng, Shu; Wang, Meng-Lan; Liang, Ling-Bo; Zhou, Ling-Yun; Du, Ling-Yao; Yan, Li-Bo; Tao, Chuan-Min; Tang, Hong

    2017-12-01

    Recently, hepatitis B core-related antigen (HBcrAg) has been suggested as an additional marker of hepatitis B virus (HBV) infection. This study aimed to investigate whether serum quantitative HBcrAg (qHBcrAg) was a satisfactory surrogate marker of intrahepatic covalently closed circular DNA (cccDNA). A total of 139 patients with liver biopsy were enrolled, consisting of 59 patients in immune tolerance (IT) phase, 52 patients in immune clearance (IC) phase, 18 patients in low-replication (LR) phase, and 10 patients in reactivation phase. All patients in IC phase have received entecavir (ETV) therapy, and 32 of them undergone a second liver biopsy at 24 months. Among those patients, qHBcrAg was strongly correlated with intrahepatic cccDNA, which is superior to that of qHBsAg and HBV DNA. And similar findings were also observed in patients in IT, IC, LR and reactivation phases. Among the 32 ETV-treated patients with a second liver biopsy in IC phase, the decline of intrahepatic cccDNA was accompanied by changes in both qHBcrAg and qHBsAg. However, as compared to qHBsAg, the change of qHBcrAg was more strongly associated with intrahepatic cccDNA-decline. In summary, serum qHBcrAg should be a satisfactory surrogate of intrahepatic HBV cccDNA in CHB patients.

  11. Alteration of Mature Nucleocapsid and Enhancement of Covalently Closed Circular DNA Formation by Hepatitis B Virus Core Mutants Defective in Complete-Virion Formation.

    PubMed

    Cui, Xiuji; Luckenbaugh, Laurie; Bruss, Volker; Hu, Jianming

    2015-10-01

    Assembly of hepatitis B virus (HBV) begins with packaging of the pregenomic RNA (pgRNA) into immature nucleocapsids (NC), which are converted to mature NCs containing the genomic relaxed circular (RC) DNA as a result of reverse transcription. Mature NCs have two alternative fates: (i) envelopment by viral envelope proteins, leading to secretion extracellularly as virions, or (ii) disassembly (uncoating) to deliver their RC DNA content into the host cell nucleus for conversion to the covalently closed circular (CCC) DNA, the template for viral transcription. How these two alternative fates are regulated remains to be better understood. The NC shell is composed of multiple copies of a single viral protein, the HBV core (HBc) protein. HBc mutations located on the surface of NC have been identified that allow NC maturation but block its envelopment. The potential effects of some of these mutations on NC uncoating and CCC DNA formation have been analyzed by transfecting HBV replication constructs into hepatoma cells. All envelopment-defective HBc mutations tested were competent for CCC DNA formation, indicating that core functions in envelopment and uncoating/nuclear delivery of RC DNA were genetically separable. Some of the envelopment-defective HBc mutations were found to alter specifically the integrity of mature, but not immature, NCs such that RC DNA became susceptible to nuclease digestion. Furthermore, CCC DNA formation could be enhanced by NC surface mutations that did or did not significantly affect mature NC integrity, indicating that the NC surface residues may be closely involved in NC uncoating and/or nuclear delivery of RC DNA. Hepatitis B virus (HBV) infection is a major health issue worldwide. HBV assembly begins with the packaging into immature nucleocapsids (NCs) of a viral RNA pregenome, which is converted to the DNA genome in mature NCs. Mature NCs are then selected for envelopment and secretion as complete-virion particles or, alternatively, can

  12. Characterisation of a diverse range of circular replication-associated protein encoding DNA viruses recovered from a sewage treatment oxidation pond.

    PubMed

    Kraberger, Simona; Argüello-Astorga, Gerardo R; Greenfield, Laurence G; Galilee, Craig; Law, Donald; Martin, Darren P; Varsani, Arvind

    2015-04-01

    Our knowledge of circular replication-associated protein encoding single-stranded (CRESS) DNA virus diversity has increased dramatically in recent years, largely due to advances in high-throughput sequencing technologies. These viruses are apparently major virome components in most terrestrial and aquatic environments and it is therefore of interest to determine their diversity at the interfaces between these environments. Treated sewage water is a particularly interesting interface between terrestrial and aquatic viromes in that it is directly pumped into waterways and is likely to contain virus populations that have been strongly impacted by humans. We used a combination of high-throughput sequencing, full genome PCR amplification, cloning and Sanger sequencing to investigate the diversity of CRESS DNA viruses present in a sewage oxidation pond. Using this approach, we recovered 50 putatively complete novel CRESS viral genomes (it remains possible that some are components of multipartite viral genomes) and 11 putatively sub-genome-length circular DNA molecules which may be either defective genomes or components of multipartite genomes. Thirteen of the genomes have bidirectional genome organisations and share similar conserved replication-associated protein (Rep) motifs to those of the gemycircularviruses: a group that in turn is most closely related to the geminiviruses. The remaining 37 viral genomes share very low degrees of Rep similarity to those of all other known CRESS DNA viruses. This number of highly divergent CRESS DNA virus genomes within a single sewage treatment pond further reinforces the notion that there likely exist hundreds of completely unknown genus/family level CRESS DNA virus groupings. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Effects of antiviral agents and HBV genotypes on intrahepatic covalently closed circular DNA in HBeAg-positive chronic hepatitis B patients

    PubMed Central

    Lu, Hai-Ying; Zhuang, Li-Wei; Yu, Yan-Yan; Si, Chong-Wen; Li, Jun; Zhang, Jian-Jun; Zeng, Zheng; Chen, Xin-Yue; Han, Zhong-Hou; Chen, Yong

    2008-01-01

    AIM: To evaluate the effects of antiviral agents and HBV genotypes on intrahepatic covalently closed circular DNA (ccc DNA) in HBeAg-positive chronic hepatitis B patients. METHODS: Seventy-one patients received lamivudine (n = 35), or sequential therapy with lamivudine- interferon alpha 2b (IFN-α 2b, n = 24) for 48 wk, or IFN-α 2b (n = 12) for 24 wk. All subjects were followed up for 24 wk. Intrahepatic ccc DNA was measured quantitatively by PCR. HBV genotypes were analyzed by PCR-RFLP. RESULTS: Sequential lamivudine- INF-α therapy, lamivudine and INF-α monotherapy reduced ccc DNA of 1.7 log, 1.4 log and 0.8 log, respectively (P < 0.05). Seventeen out of the 71 patients developed HBeAg seroconversion, the reduction of ccc DNA in the HBeAg seroconversion patients was more significant than that in the HBeAg positive patients (3.0 log vs 1.6 log, P = 0.0407). Twenty-four weeks after antiviral therapy withdrawal, 16 patients had a sustained virological response, the baseline intrahepatic ccc DNA in the patients with a sustained virological response was significantly lower than that in the patients with virological rebound (4.6 log vs 5.4 log, P = 0.0472). HBV genotype C accounted for 85.9% (n = 61), and genotype B for 14.1% (n = 10), respectively, in the 71 patients. There was no significant difference in the change of ccc DNA level between HBV genotypes C and B (2.1 log vs 1.9 log). CONCLUSION: Forty-eight week sequential lamivudine-INF-α therapy and lamivudine monotherapy reduce ccc DNA more significantly than 24-wk INF-α monotherapy. Low baseline intrahepatic ccc DNA level may predict the long-term efficacy of antiviral treatment. HBV genotypes C and B have no obvious influence on ccc DNA load. PMID:18300356

  14. Detection of Hepatitis B Virus Covalently Closed Circular DNA in the Plasma of Iranian HBeAg-Negative Patients With Chronic Hepatitis B

    PubMed Central

    Tajik, Zahra; Keyvani, Hossein; Bokharaei-Salim, Farah; Zolfaghari, Mohammad Reza; Fakhim, Shahin; Keshvari, Maryam; Alavian, Seyed Moayed

    2015-01-01

    Background: Covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is a marker of HBV replication in the liver of patients infected with HBV. Objectives: This study aimed to investigate the association between the presence of cccDNA in the plasma samples of Iranian treatment-naive patients with chronic hepatitis B infection and HBV viral load and HBsAg levels. Patients and Methods: From April 2012 to May 2015, 106 treatment-naive patients with chronic hepatitis B infection were enrolled in this cross-sectional study. The HBsAg titer was measured by the Roche HBsAg II assay on the Cobas e411 system, and HBV DNA quantitation was performed using the COBAS TaqMan 48 kit. Real-time polymerase chain reaction was performed for the detection of HBV cccDNA. Results: The mean (SD) age of the patients was 41.1 ± 12.4 years (range, 20 - 62 years). From a total of 106 study participants, 67 (63.2%) were males. The HBV cccDNA was detected in plasma specimens in 19 (17.9%) out of the total 106 patients, and a significant relationship was found between the presence of cccDNA in plasma sample of males (23.9%) and females (7.7%) (P = 0.039). Also, a significant correlation was found between the presence of cccDNA in plasma sample of the patients and HBV viral load level (P < 0.0001) and HBsAg titer (P = 0.0043). Conclusions: This study showed that cccDNA can be detected in the plasma specimen of 17.9% of Iranian treatment-naive patients with chronic hepatitis B infection. Therefore, designing prospective studies focusing on the detection of cccDNA in these patients would provide more information. PMID:26504471

  15. Identification of diverse circular single-stranded DNA viruses in adult dragonflies and damselflies (Insecta: Odonata) of Arizona and Oklahoma, USA.

    PubMed

    Dayaram, Anisha; Potter, Kristen A; Pailes, Roberta; Marinov, Milen; Rosenstein, Dana D; Varsani, Arvind

    2015-03-01

    Next generation sequencing and metagenomic approaches are commonly used for the identification of circular replication associated protein (Rep)-encoding single stranded (CRESS) DNA viruses circulating in various environments. These approaches have enabled the discovery of some CRESS DNA viruses associated with insects. In this study we identified and recovered 31 viral genomes which represent 24 distinct CRESS DNA viruses from seven dragonfly species (Rhionaeschna multicolor, Erythemis simplicicollis, Erythrodiplax fusca, Libellula quadrimaculata, Libellula saturata, Pachydiplax longipennis, and Pantala hymenaea) and two damselfly species (Ischnura posita, Ischnura ramburii) sampled in various locations in the states of Arizona and Oklahoma of the United States of America (USA). We also identified Sclerotinia sclerotiorum hypovirulence-associated DNA virus-1 (SsHADV-1) in P. hymenaea, E. simplicicollis and I. ramburii sampled in Oklahoma, which is the first report of SsHADV-1 in the New World. The genome architectures of the CRESS DNA viruses recovered vary, but they all have at least two major open reading frames (ORFs) that have either a bidirectional or unidirectional arrangement. Four of the viral genomes recovered, in addition to the three isolates of SsHADV-1, show similarities to viruses of the proposed gemycircularvirus group. Analysis of the Rep encoded by the remaining 24 viral genomes reveals that these are highly diverse and allude to the fact that they represent novel CRESS DNA viruses.

  16. DNA replication origins in archaea.

    PubMed

    Wu, Zhenfang; Liu, Jingfang; Yang, Haibo; Xiang, Hua

    2014-01-01

    DNA replication initiation, which starts at specific chromosomal site (known as replication origins), is the key regulatory stage of chromosome replication. Archaea, the third domain of life, use a single or multiple origin(s) to initiate replication of their circular chromosomes. The basic structure of replication origins is conserved among archaea, typically including an AT-rich unwinding region flanked by several conserved repeats (origin recognition box, ORB) that are located adjacent to a replication initiator gene. Both the ORB sequence and the adjacent initiator gene are considerably diverse among different replication origins, while in silico and genetic analyses have indicated the specificity between the initiator genes and their cognate origins. These replicator-initiator pairings are reminiscent of the oriC-dnaA system in bacteria, and a model for the negative regulation of origin activity by a downstream cluster of ORB elements has been recently proposed in haloarchaea. Moreover, comparative genomic analyses have revealed that the mosaics of replicator-initiator pairings in archaeal chromosomes originated from the integration of extrachromosomal elements. This review summarizes the research progress in understanding of archaeal replication origins with particular focus on the utilization, control and evolution of multiple replication origins in haloarchaea.

  17. DNA replication origins in archaea

    PubMed Central

    Wu, Zhenfang; Liu, Jingfang; Yang, Haibo; Xiang, Hua

    2014-01-01

    DNA replication initiation, which starts at specific chromosomal site (known as replication origins), is the key regulatory stage of chromosome replication. Archaea, the third domain of life, use a single or multiple origin(s) to initiate replication of their circular chromosomes. The basic structure of replication origins is conserved among archaea, typically including an AT-rich unwinding region flanked by several conserved repeats (origin recognition box, ORB) that are located adjacent to a replication initiator gene. Both the ORB sequence and the adjacent initiator gene are considerably diverse among different replication origins, while in silico and genetic analyses have indicated the specificity between the initiator genes and their cognate origins. These replicator–initiator pairings are reminiscent of the oriC-dnaA system in bacteria, and a model for the negative regulation of origin activity by a downstream cluster of ORB elements has been recently proposed in haloarchaea. Moreover, comparative genomic analyses have revealed that the mosaics of replicator-initiator pairings in archaeal chromosomes originated from the integration of extrachromosomal elements. This review summarizes the research progress in understanding of archaeal replication origins with particular focus on the utilization, control and evolution of multiple replication origins in haloarchaea. PMID:24808892

  18. Biomolecular and structural analyses of cauliflower-like DNAs by ultraviolet, circular dichroism, and fluorescence spectroscopies in comparison with natural DNA.

    PubMed

    Gill, Pooria; Ranjbar, Bijan; Saber, Reza; Khajeh, Khosro; Mohammadian, Mehdi

    2011-07-01

    Cauliflower-like DNAs are stem-loop DNAs that are fabricated periodically in inverted repetitions from deoxyribonucleic acid phosphates (dNTPs) by loop-mediated isothermal amplification (LAMP). Cauliflower-like DNAs have ladder-shape behaviors on gel electrophoresis, and increasing the time of LAMP leads to multiplying the repetitions, stem-loops, and electrophoretic bands. Cauliflower-like DNAs were fabricated via LAMP using two loop primers, two bumper primers, dNTPs, a λ-phage DNA template, and a Bst DNA polymerase in 75- and 90-min periods. These times led to manufacturing two types of cauliflower-like DNAs with different contents of inverted repetitions and stem-loops, which were clearly indicated by two comparable electrophoresis patterns in agarose gel. LAMP-fabricated DNAs and natural dsB-DNA (salmon genomic DNA) were dialyzed in Gomori phosphate buffer (10 mM, pH 7.4) to be isolated from salts, nucleotides, and primers. Dialyzed DNAs were studied using UV spectroscopy, circular dichroism spectropolarimetry, and fluorescence spectrophotometry. Structural analyses indicated reduction of the molecular ellipticity and extinction coefficients in comparison with B-DNA. Also, cauliflower-like DNAs demonstrated less intrinsic and more extrinsic fluorescence in comparison with natural DNA. The overwinding and lengthening of the cauliflower-like configurations of LAMP DNAs led to changes in physical parameters of this type of DNA in comparison with natural DNA. The results obtained introduced new biomolecular characteristics of DNA macromolecules fabricated within a LAMP process and show the effects of more inverted repeats and stem-loops, which are manufactured by lengthening the process.

  19. Biomolecular and Structural Analyses of Cauliflower-like DNAs by Ultraviolet, Circular Dichroism, and Fluorescence Spectroscopies in Comparison with Natural DNA

    PubMed Central

    Gill, Pooria; Ranjbar, Bijan; Saber, Reza; Khajeh, Khosro; Mohammadian, Mehdi

    2011-01-01

    Cauliflower-like DNAs are stem-loop DNAs that are fabricated periodically in inverted repetitions from deoxyribonucleic acid phosphates (dNTPs) by loop-mediated isothermal amplification (LAMP). Cauliflower-like DNAs have ladder-shape behaviors on gel electrophoresis, and increasing the time of LAMP leads to multiplying the repetitions, stem-loops, and electrophoretic bands. Cauliflower-like DNAs were fabricated via LAMP using two loop primers, two bumper primers, dNTPs, a λ-phage DNA template, and a Bst DNA polymerase in 75- and 90-min periods. These times led to manufacturing two types of cauliflower-like DNAs with different contents of inverted repetitions and stem-loops, which were clearly indicated by two comparable electrophoresis patterns in agarose gel. LAMP-fabricated DNAs and natural dsB-DNA (salmon genomic DNA) were dialyzed in Gomori phosphate buffer (10 mM, pH 7.4) to be isolated from salts, nucleotides, and primers. Dialyzed DNAs were studied using UV spectroscopy, circular dichroism spectropolarimetry, and fluorescence spectrophotometry. Structural analyses indicated reduction of the molecular ellipticity and extinction coefficients in comparison with B-DNA. Also, cauliflower-like DNAs demonstrated less intrinsic and more extrinsic fluorescence in comparison with natural DNA. The overwinding and lengthening of the cauliflower-like configurations of LAMP DNAs led to changes in physical parameters of this type of DNA in comparison with natural DNA. The results obtained introduced new biomolecular characteristics of DNA macromolecules fabricated within a LAMP process and show the effects of more inverted repeats and stem-loops, which are manufactured by lengthening the process. PMID:21738438

  20. A triplex-mediated knot between separated polypurine-polypyrimidine tracts in circular DNA blocks transcription by Escherichia coli RNA polymerase.

    PubMed

    Ashley, C; Lee, J S

    2000-04-01

    Polypurine-polypyrimidine tracts are overrepresented in eukaryotes and many have the potential to form triplex DNA. Transmolecular triplexes form between separated but complementary polypurine-polypyrimidine tracts in duplex DNA. Transmolecular triplexes (T-loops) were studied previously using a circular plasmid containing a pair of separated polypurine-polypyrimidine tracts designed to able to form a triplex with each other. T-Loops formed when the nicked plasmid was incubated at low pH in the presence of spermine. When the pH was raised to 8, the T-loops were constrained by a hydrogen-bonded knot composed of multistranded and single-stranded regions. The present experiments used T-loops as a model system to investigate the influence of transmolecular triplex formation on transcription. T-Loops and control open circular, linear, and supercoiled plasmid forms were isolated from bands on agarose gels. Transcription assays were carried out with the isolated plasmid forms and Escherichia coli RNA polymerase holoenzyme and the core enzyme, which lacked sigma70. Transcription was significantly inhibited in T-loop forms compared with control plasmid forms. There was no evidence that the single-stranded regions of T-loops facilitated nonspecific initiation of transcription. Instead, the multistranded component of the hydrogen-bonded knot at the root of the T-loop structure inhibited transcription.

  1. Analysis of the VPE sequences in the Caenorhabditis elegans vit-2 promoter with extrachromosomal tandem array-containing transgenic strains.

    PubMed Central

    MacMorris, M; Spieth, J; Madej, C; Lea, K; Blumenthal, T

    1994-01-01

    The Caenorhabditis elegans vit genes, encoding vitellogenins, are abundantly expressed in the adult hermaphrodite intestine. Two repeated elements, vit promoter element 1 (VPE1 [TGTCAAT]) and VPE2 (CTGATAA), have been identified in the 5' flanking DNA of each of the vit genes of C. elegans and Caenorhabditis briggsae. These elements have previously been shown to be needed for correctly regulated expression of a vit-2/vit-6 fusion gene in low-copy-number, integrated transgenes. Here we extend the analysis of the function of VPE1 and VPE2 by using transgenic lines carrying large, extrachromosomal arrays of the test genes. The results validate the use of such arrays for transgenic analysis of gene regulation in C. elegans, by confirming previous findings showing that the VPE1 at -45 and both VPE2s are sites of activation. Additional experiments now indicate that when the -45 VPE1 is inverted or replaced by a VPE2, nearly total loss of promoter function results, suggesting that the highly conserved -45 VPE1 plays a unique role in vit-2 promoter function. In contrast, single mutations eliminating the three upstream VPE1s are without effect. However, in combination in double and triple mutants, these upstream VPE1 mutations cause drastic reductions in expression levels. The -150 VPE2 can be replaced by a XhoI site (CTCGAG), and the -90 VPE2 can be eliminated, as long as the overlapping VPE1 is left intact, but when these two replacements are combined, activity is lost. Thus, the promoter must have at least one VPE2 and it must have at least two VPE1s, one at -45 and one additional upstream element. Images PMID:8264616

  2. Characterization of the microDNA through the response to chemotherapeutics in lymphoblastoid cell lines.

    PubMed

    Mehanna, Pamela; Gagné, Vincent; Lajoie, Mathieu; Spinella, Jean-François; St-Onge, Pascal; Sinnett, Daniel; Brukner, Ivan; Krajinovic, Maja

    2017-01-01

    Recently, a new class of extrachromosomal circular DNA, called microDNA, was identified. They are on average 100 to 400 bp long and are derived from unique non-repetitive genomic regions with high gene density. MicroDNAs are thought to arise from DNA breaks associated with RNA metabolism or replication slippage. Given the paucity of information on this entirely novel phenomenon, we aimed to get an additional insight into microDNA features by performing the microDNA analysis in 20 independent human lymphoblastoid cell lines (LCLs) prior and after treatment with chemotherapeutic drugs. The results showed non-random genesis of microDNA clusters from the active regions of the genome. The size periodicity of 190 bp was observed, which matches DNA fragmentation typical for apoptotic cells. The chemotherapeutic drug-induced apoptosis of LCLs increased both number and size of clusters further suggesting that part of microDNAs could result from the programmed cell death. Interestingly, proportion of identified microDNA sequences has common loci of origin when compared between cell line experiments. While compatible with the original observation that microDNAs originate from a normal physiological process, obtained results imply complementary source of its production. Furthermore, non-random genesis of microDNAs depicted by redundancy between samples makes these entities possible candidates for new biomarker generation.

  3. Characterization of the microDNA through the response to chemotherapeutics in lymphoblastoid cell lines

    PubMed Central

    Mehanna, Pamela; Gagné, Vincent; Lajoie, Mathieu; Spinella, Jean-François; St-Onge, Pascal; Sinnett, Daniel; Brukner, Ivan

    2017-01-01

    Recently, a new class of extrachromosomal circular DNA, called microDNA, was identified. They are on average 100 to 400 bp long and are derived from unique non-repetitive genomic regions with high gene density. MicroDNAs are thought to arise from DNA breaks associated with RNA metabolism or replication slippage. Given the paucity of information on this entirely novel phenomenon, we aimed to get an additional insight into microDNA features by performing the microDNA analysis in 20 independent human lymphoblastoid cell lines (LCLs) prior and after treatment with chemotherapeutic drugs. The results showed non-random genesis of microDNA clusters from the active regions of the genome. The size periodicity of 190 bp was observed, which matches DNA fragmentation typical for apoptotic cells. The chemotherapeutic drug-induced apoptosis of LCLs increased both number and size of clusters further suggesting that part of microDNAs could result from the programmed cell death. Interestingly, proportion of identified microDNA sequences has common loci of origin when compared between cell line experiments. While compatible with the original observation that microDNAs originate from a normal physiological process, obtained results imply complementary source of its production. Furthermore, non-random genesis of microDNAs depicted by redundancy between samples makes these entities possible candidates for new biomarker generation. PMID:28877255

  4. Specific recognition of human telomeric G-quadruplex DNA with small molecules and the conformational analysis by ESI mass spectrometry and circular dichroism spectropolarimetry.

    PubMed

    Zhou, Jiang; Yuan, Gu

    2007-01-01

    Electrospray ionization mass spectrometry (ESI-MS) was utilized to investigate the binding affinity and stoichiometry of small molecules with human telomeric G-quadruplex DNA. The binding-affinity order obtained for the (AGGGTT)(4) quadruplex was: Tel01>ImImImbetaDp>PyPyPygammaImImImbetaDp. The specific binding of Tel01 and PyPyPygammaImImImbetaDp in one system consisting of human telomeric G-quadruplex and duplex DNA was observed directly for the first time. This revealed that PyPyPygammaImImImbetaDp has a binding specificity for the duplex DNA, whereas Tel01 selectively recognizes the G-quadruplex DNA. Moreover, both ESI-MS and circular dichroism (CD) spectra indicated that Tel01 favored the formation and stabilization of the antiparallel G-quadruplex, and a structural transition of the (AGGGTT)(4) sequence from a coexistence of parallel and antiparallel G-quadruplexes to a parallel G-quadruplex induced by annealing.

  5. Hepatitis B virus (HBV)-specific short hairpin RNA is capable of reducing the formation of HBV covalently closed circular (CCC) DNA but has no effect on established CCC DNA in vitro.

    PubMed

    Starkey, Jason L; Chiari, Estelle F; Isom, Harriet C

    2009-01-01

    Hepatitis B virus (HBV) covalently closed circular (CCC) DNA is the source of HBV transcripts and persistence in chronically infected patients. The novel aspect of this study was to determine the effect of RNA interference (RNAi) on HBV CCC DNA when administered prior to establishment of HBV replication or during chronic HBV infection. HBV replication was initiated in HepG2 cells by transduction with HBV baculovirus. Subculture of HBV-expressing HepG2 cells at 10 days post-transduction generates a system in which HBV replication is ongoing and HBV is expressed largely from CCC DNA, thus simulating chronic HBV infection. HepG2 cells were transduced with short hairpin RNA (shRNA)-expressing baculovirus prior to initiation of HBV replication or during chronic HBV replication, and the levels of HBV RNA, HBV surface antigens (HBsAg) and replicative intermediates (RI), extracellular (EC) and CCC DNA species were measured. HBsAg, HBV RNA and DNA levels were markedly reduced until day 8 whether cells were transduced with shRNA prior to or during a chronic infection; however, the CCC DNA species were only affected when shRNA was administered prior to initiation of infection. We conclude that RNAi may have a therapeutic value for controlling HBV replication at the level of RI and EC DNA and for reducing establishment of CCC DNA during HBV infection. Our data support previous findings demonstrating the stability of HBV CCC DNA following antiviral therapy. This study also reports the development of a novel HBV baculovirus subculture system that can be used to evaluate antiviral effects on chronic HBV replication.

  6. Gene expression of benthic amphipods (genus: Diporeia) in relation to a circular ssDNA virus across two Laurentian Great Lakes.

    PubMed

    Bistolas, Kalia S I; Rudstam, Lars G; Hewson, Ian

    2017-01-01

    Circular rep-encoding ssDNA (CRESS-DNA) viruses are common constituents of invertebrate viral consortia. Despite their ubiquity and sequence diversity, the effects of CRESS-DNA viruses on invertebrate biology and ecology remain largely unknown. This study assessed the relationship between the transcriptional profile of benthic amphipods of genus Diporeia and the presence of the CRESS-DNA virus, LM29173, in the Laurentian Great Lakes to provide potential insight into the influence of these viruses on invertebrate gene expression. Twelve transcriptomes derived from Diporeia were compared, representing organisms from two amphipod haplotype clades (Great Lakes Michigan and Superior, defined by COI barcode sequencing) with varying viral loads (up to 3 × 10(6) genome copies organism(-1)). Read recruitment to de novo assembled transcripts revealed 2,208 significantly over or underexpressed contigs in transcriptomes with above average LM29173 load. Of these contigs, 31.5% were assigned a putative function. The greatest proportion of annotated, differentially expressed transcripts were associated with functions including: (1) replication, recombination, and repair, (2) cell structure/biogenesis, and (3) post-translational modification, protein turnover, and chaperones. Contigs putatively associated with innate immunity displayed no consistent pattern of expression, though several transcripts were significantly overexpressed in amphipods with high viral load. Quantitation (RT-qPCR) of target transcripts, non-muscular myosin heavy chain, β-actin, and ubiquitin-conjugating enzyme E2, corroborated transcriptome analysis and indicated that Lake Michigan and Lake Superior amphipods with high LM29173 load exhibit lake-specific trends in gene expression. While this investigation provides the first comparative survey of the transcriptional profile of invertebrates of variable CRESS-DNA viral load, additional inquiry is required to define the scope of host-specific responses to

  7. Circular Motion.

    ERIC Educational Resources Information Center

    Lee, Paul D.

    1995-01-01

    Provides a period-long activity using battery powered cars rolling in a circular motion on a tile floor. Students measure the time and distance as the car moves to derive the equation for centripetal acceleration. (MVL)

  8. Circular Motion.

    ERIC Educational Resources Information Center

    Lee, Paul D.

    1995-01-01

    Provides a period-long activity using battery powered cars rolling in a circular motion on a tile floor. Students measure the time and distance as the car moves to derive the equation for centripetal acceleration. (MVL)

  9. Characterization of Rhizobium grahamii extrachromosomal replicons and their transfer among rhizobia.

    PubMed

    Althabegoiti, María Julia; Ormeño-Orrillo, Ernesto; Lozano, Luis; Torres Tejerizo, Gonzalo; Rogel, Marco Antonio; Mora, Jaime; Martínez-Romero, Esperanza

    2014-01-08

    Rhizobium grahamii belongs to a new phylogenetic group of rhizobia together with Rhizobium mesoamericanum and other species. R. grahamii has a broad-host-range that includes Leucaena leucocephala and Phaseolus vulgaris, although it is a poor competitor for P. vulgaris nodulation in the presence of Rhizobium etli or Rhizobium phaseoli strains. This work analyzed the genome sequence and transfer properties of R. grahamii plasmids. Genome sequence was obtained from R. grahamii CCGE502 type strain isolated from Dalea leporina in Mexico. The CCGE502 genome comprises one chromosome and two extrachromosomal replicons (ERs), pRgrCCGE502a and pRgrCCGE502b. Additionally, a plasmid integrated in the CCGE502 chromosome was found. The genomic comparison of ERs from this group showed that gene content is more variable than average nucleotide identity (ANI). Well conserved nod and nif genes were found in R. grahamii and R. mesoamericanum with some differences. R. phaseoli Ch24-10 genes expressed in bacterial cells in roots were found to be conserved in pRgrCCGE502b. Regarding conjugative transfer we were unable to transfer the R. grahamii CCGE502 symbiotic plasmid and its megaplasmid to other rhizobial hosts but we could transfer the symbiotic plasmid to Agrobacterium tumefaciens with transfer dependent on homoserine lactones. Variable degrees of nucleotide identity and gene content conservation were found among the different R. grahamii CCGE502 replicons in comparison to R. mesoamericanum genomes. The extrachromosomal replicons from R. grahamii were more similar to those found in phylogenetically related Rhizobium species. However, limited similarities of R. grahamii CCGE502 symbiotic plasmid and megaplasmid were observed in other more distant Rhizobium species. The set of conserved genes in R. grahamii comprises some of those that are highly expressed in R. phaseoli on plant roots, suggesting that they play an important role in root colonization.

  10. Characterization of Rhizobium grahamii extrachromosomal replicons and their transfer among rhizobia

    PubMed Central

    2014-01-01

    Background Rhizobium grahamii belongs to a new phylogenetic group of rhizobia together with Rhizobium mesoamericanum and other species. R. grahamii has a broad-host-range that includes Leucaena leucocephala and Phaseolus vulgaris, although it is a poor competitor for P. vulgaris nodulation in the presence of Rhizobium etli or Rhizobium phaseoli strains. This work analyzed the genome sequence and transfer properties of R. grahamii plasmids. Results Genome sequence was obtained from R. grahamii CCGE502 type strain isolated from Dalea leporina in Mexico. The CCGE502 genome comprises one chromosome and two extrachromosomal replicons (ERs), pRgrCCGE502a and pRgrCCGE502b. Additionally, a plasmid integrated in the CCGE502 chromosome was found. The genomic comparison of ERs from this group showed that gene content is more variable than average nucleotide identity (ANI). Well conserved nod and nif genes were found in R. grahamii and R. mesoamericanum with some differences. R. phaseoli Ch24-10 genes expressed in bacterial cells in roots were found to be conserved in pRgrCCGE502b. Regarding conjugative transfer we were unable to transfer the R. grahamii CCGE502 symbiotic plasmid and its megaplasmid to other rhizobial hosts but we could transfer the symbiotic plasmid to Agrobacterium tumefaciens with transfer dependent on homoserine lactones. Conclusion Variable degrees of nucleotide identity and gene content conservation were found among the different R. grahamii CCGE502 replicons in comparison to R. mesoamericanum genomes. The extrachromosomal replicons from R. grahamii were more similar to those found in phylogenetically related Rhizobium species. However, limited similarities of R. grahamii CCGE502 symbiotic plasmid and megaplasmid were observed in other more distant Rhizobium species. The set of conserved genes in R. grahamii comprises some of those that are highly expressed in R. phaseoli on plant roots, suggesting that they play an important role in root colonization

  11. Self-catalyzed site-specific depurination of G residues mediated by cruciform extrusion in closed circular DNA plasmids.

    PubMed

    Amosova, Olga; Kumar, Veena; Deutsch, Aaron; Fresco, Jacques R

    2011-10-21

    A major variety of "spontaneous" genomic damage is endogenous generation of apurinic sites. Depurination rates vary widely across genomes, occurring with higher frequency at "depurination hot spots." Recently, we discovered a site-specific self-catalyzed depurinating activity in short (14-18 nucleotides) DNA stem-loop-forming sequences with a 5'-G(T/A)GG-3' loop and T·A or G·C as the first base pair at the base of the loop; the 5'-G residue of the loop self-depurinates at least 10(5)-fold faster than random "spontaneous" depurination at pH 5. Formation of the catalytic intermediate for self-depurination in double-stranded DNA requires a stem-loop to extrude as part of a cruciform. In this study, evidence is presented for self-catalyzed depurination mediated by cruciform formation in plasmid DNA in vitro. Cruciform extrusion was confirmed, and its extent was quantitated by digestion of the plasmid with single strand-specific mung bean endonuclease, followed by restriction digestion and sequencing of resulting mung bean-generated fragments. Appearance of the apurinic site in the self-depurinating stem-loop was confirmed by digestion of plasmid DNA with apurinic endonuclease IV, followed by primer extension and/or PCR amplification to detect the endonuclease-generated strand break and identify its location. Self-catalyzed depurination was contingent on the plasmid being supercoiled and was not observed in linearized plasmids, consistent with the presence of the extruded cruciform in the supercoiled plasmid and not in the linear one. These results indicate that self-catalyzed depurination is not unique to single-stranded DNA; rather, it can occur in stem-loop structures extruding from double-stranded DNA and therefore could, in principle, occur in vivo.

  12. Use of circular permutation to assess six bulges and four loops of DNA-packaging pRNA of bacteriophage phi29.

    PubMed Central

    Zhang, C; Tellinghuisen, T; Guo, P

    1997-01-01

    A 120-base phage phi29 encoded RNA (pRNA) has a novel role in DNA packaging. This pRNA possesses five single-base bulges, one three-base bulge, one bifurcation bulge, one bulge loop, and two stem loops. Circularly permuted pRNAs (cpRNA) were constructed to examine the function of these bulges and loops as well as their adjacent sequences. Each of the five single-base bulges was nonessential. The bifurcation bulge could be deleted and replaced with a new opening to provide flexibility for maintaining an overall correct folding in three-way junction. All of these nonessential bulges or their adjacent bases could be used as new termini for cpRNAs. The three-base (C18C19A20) bulge was dispensable for procapsid binding, but was indispensable for DNA packaging. The secondary structure around this CCA bulge and the phylogenetically conserved bases within or around it were investigated. Bases A14C15U16 were confirmed, by compensatory modification, to pair with U103G102A101. A99 was needed only to allow the proper folding of CCA bulge in the appropriate sequence order and distance constraints. Beyond these, the seemingly phylogenetic conservation of other bases has little role in pRNA activity. Each of the three stem loops was essential for procapsid binding, DNA packaging, and phage assembly. Disruption of the middle of any one of the loops resulted in dramatic reductions in procapsid binding, subsequent DNA packaging, and phage assembly activities. However, disruption of the loops at sequences that were close to double-stranded regions of the RNA did not interfere with pRNA activity significantly. Our results suggest that double-stranded helical regions near these loops were most likely not involved in interactions with components of the DNA-packaging machinery. Instead, these regions appear to be merely present to serve as a scaffolding to display the single-stranded loops that are important for pRNA tertiary structure or for interaction with the procapsid or other

  13. [Polyadenylated RNA and mRNA export factors in extrachromosomal nuclear domains of vitellogenic oocytes of the insect Tenebrio molitor].

    PubMed

    Bogoliubov, D S; Kiselev, A M; Shabel'nikov, S V; Parfenov, V N

    2012-01-01

    The nucleus ofvitellogenic oocytes of the yellow mealworm, Tenebrio molitor, contains a karyosphere that consists of the condensed chromatin embedded in an extrachromosomal fibrogranular material. Numerous nuclear bodies located freely in the nucleoplasm are also observed. Amongst these bodies, counterparts of nuclear speckles (= interchromatin granule clusters, IGCs) can be identified by the presence of the marker protein SC35. Microinjections of fluorescently tagged methyloligoribonucleotide probes 2'-O-Me(U)22, complementary to poly(A) tails of RNAs, revealed poly(A)+ RNA in the vast majority of IGCs. We found that all T. molitor oocyte IGCs contain heterogeneous ribonucleoprotein (hnRNP) core protein Al that localizes to IGCs in an RNA-dependent manner. The extrachromosomal material of the karyosphere and a part of nucleoplasmic IGCs also contain the adapter protein Aly that is known to provide a link between pre-mRNA splicing and mRNA export. The essential mRNA export factor/receptor NXF1 was observed to colocalize with Aly. In nucleoplasmic IGCs, NXF1 was found to localize in an RNA-dependent manner whereas it is RNA-independently located in the extrachromosomal material of the karyosphere. We believe our data suggest on a role of the nucleoplasmic IGCs in mRNA biogenesis and retention in a road to nuclear export.

  14. An enzyme system for replication of duplex circular DNA: the replicative form of phage phi X174.

    PubMed

    Eisenberg, S; Scott, J F; Kornberg, A

    1976-05-01

    Viral single strands (SS) are converted to the duplex from (RF) by a soluble enzyme fraction uninfected Escherichia coli [Schekman et al. (1975) J. Biol. Chem. 250, 5859-5865]. When reactions were supplemented with a soluble enzyme fraction from phi X174-infected cells, replication of phi X174 superhelical RF I DNA was observed. The activity supplied by infected cells was absent in cells treated with chloramphenicol or in cells infected with a phi X174 phage mutant in cistron A (cis A). A host function coded by the rep gene, essential in vivo for RF replication (but not for SS leads to RF), was supplied by enzyme fractions from either infected or uninfected cells. Based on complementation assays, the cisA-dependent and the rep-dependent proteins have each been purified about 1000-fold. The synthetic products of the enzymatic reaction were identified as RF I and RF II in which viral (+) and complementary (-) strands were newly synthesized.

  15. Circular dichroism spectra of twelve short DNA restriction fragments of known sequence: a comparison of measured and calculated spectra.

    PubMed Central

    Hillen, W; Goodman, T C; Wells, R D

    1981-01-01

    The CD spectra of twelve DNA restriction fragments ranging in size from 12 to 360 base pairs are reported. Since the sequences of these fragments are known, it is possible to calculate their CD spectra from a set of nearest neighbor contributions derived from a combination of synthetic polydeoxyribonucleotides. While the calculations lead to good agreement in the negative band at approximately 245 nm, they generally reproduce the positive band at approximately 270 nm only poorly. The experimentally observed positive band consists of two peaks centered around 270 and 285 nm. The comparison of calculated and measured spectra reveals that end effects lead to increased disagreement for fragments smaller than approximately 40 base pairs. The disagreement between calculated and measured spectra can be partially attributed to the fraction of next nearest neighbors in the DNAs, which are also in the spectral components. Thus, the sequence specific CD contributions in the long wavelength region of the spectra extend at least to next nearest neighbor nucleotides and may extend beyond. Images PMID:6269070

  16. Faecal virome of healthy chickens reveals a large diversity of the eukaryote viral community, including novel circular ssDNA viruses.

    PubMed

    Lima, Diane A; Cibulski, Samuel P; Finkler, Fabrine; Teixeira, Thais F; Varela, Ana Paula M; Cerva, Cristine; Loiko, Márcia R; Scheffer, Camila M; Dos Santos, Helton F; Mayer, Fabiana Q; Roehe, Paulo M

    2017-04-01

    This study is focused on the identification of the faecal virome of healthy chickens raised in high-density, export-driven poultry farms in Brazil. Following high-throughput sequencing, a total of 7743 de novo-assembled contigs were constructed and compared with known nucleotide/amino acid sequences from the GenBank database. Analyses with blastx revealed that 279 contigs (4 %) were related to sequences of eukaryotic viruses. Viral genome sequences (total or partial) indicative of members of recognized viral families, including Adenoviridae, Caliciviridae, Circoviridae, Parvoviridae, Picobirnaviridae, Picornaviridae and Reoviridae, were identified, some of those representing novel genotypes. In addition, a range of circular replication-associated protein encoding DNA viruses were also identified. The characterization of the faecal virome of healthy chickens described here not only provides a description of the viruses encountered in such niche but should also represent a baseline for future studies comparing viral populations in healthy and diseased chicken flocks. Moreover, it may also be relevant for human health, since chickens represent a significant proportion of the animal protein consumed worldwide.

  17. Regulation and targeting of recombination in extrachromosomal substrates carrying immunoglobulin switch region sequences.

    PubMed Central

    Leung, H; Maizels, N

    1994-01-01

    We have used extrachromosomal substrates carrying immunoglobulin heavy-chain S mu and S gamma 3 switch region sequences to study activation and targeting of recombination by a transcriptional enhancer element. Substrates are transiently introduced into activated primary murine B cells, in which recombination involving S-region sequences deletes a conditionally lethal marker, and recombination is measured by transformation of Escherichia coli in the second step of the assay. Previously we found that as many as 25% of replicated substrates recombined during 40-h transfection of lipopolysaccharide (LPS)-stimulated primary cells and that efficient recombination was dependent on the presence of S-region sequences as well as a transcriptional activator region in the constructs (H. Leung and N. Maizels, Proc. Natl. Acad. Sci. USA 89:4154-4158, 1992). Here we show that recombination of the switch substrates is threefold more efficient in LPS-cultured primary B cells than in the T-cell line EL4; the activities responsible for switch substrate recombination thus appear to be more abundant or more active in cells which can carry out chromosomal switch recombination. We test the role of the transcriptional activator region and show that the immunoglobulin heavy-chain intron enhancer (E mu) alone stimulates recombination as well as E mu combined with a heavy-chain promoter and that mutations that diminish enhancer-dependent transcription 500-fold diminish recombinational activation less than 2-fold. These observations suggest that the enhancer stimulates recombination by a mechanism that does not depend on transcript production or that is insensitive to the level of transcript production over a very broad range. Furthermore, we find that E mu stimulates recombination when located either upstream or downstream of S mu but that the position of the recombinational activator does affect the targeting of recombination junctions, suggesting that the relatively imprecise targeting of

  18. Pulsed-field gel electrophoresis analysis of the genome of Rhodococcus fascians: genome size and linear and circular replicon composition in virulent and avirulent strains.

    PubMed

    Pisabarro, A; Correia, A; Martín, J F

    1998-05-01

    Total DNA of virulent and avirulent strains of Rhodococcus fascians was resolved by pulsed-field gel electrophoresis (PFGE) into a discrete number of fragments by digestion with the endonucleases AseI and DraI. Restriction endonucleases PacI, PmeI, and SwaI yielded no fragments upon digestion of R. fascians genome, and all the other tested endonucleases recognizing 6 bp released too many fragments. The genome size was 5.6 megabases for the type strain R. fascians DSM 20669, and 5.8 megabases for the virulent R. fascians D188 strain. However the genome size of R. fascians CECT 3001 (NRRL B15096) was 8.0 megabases. No linear chromosome in the megabase range was observed under pulse conditions in which Saccharomyces cerevisiae and Schizosaccharomyces pombe chromosomes were perfectly resolved, suggesting that the R. fascians chromosome is circular. A new linear plasmid pIRN640 of 640 kb was found in the avirulent R. fascians CECT 3001 that did not hybridize with a probe internal to the fas region of pFiD188 known to be involved in plant pathogenicity in the virulent strain R. fascians D188. Virulence was correlated in all strains tested with the presence of the fas region. The AseI and DraI bands corresponding to the extrachromosomal elements were identified providing the basis for a physical map of this organism.

  19. Switch telomerase to ALT mechanism by inducing telomeric DNA damages and dysfunction of ATRX and DAXX.

    PubMed

    Hu, Yang; Shi, Guang; Zhang, Laichen; Li, Feng; Jiang, Yuanling; Jiang, Shuai; Ma, Wenbin; Zhao, Yong; Songyang, Zhou; Huang, Junjiu

    2016-08-31

    Activation of telomerase or alternative lengthening of telomeres (ALT) is necessary for tumours to escape from dysfunctional telomere-mediated senescence. Anti-telomerase drugs might be effective in suppressing tumour growth in approximately 85-90% of telomerase-positive cancer cells. However, there are still chances for these cells to bypass drug treatment after switching to the ALT mechanism to maintain their telomere integrity. But the mechanism underlying this switch is unknown. In this study, we used telomerase-positive cancer cells (HTC75) to discover the mechanism of the telomerase-ALT switch by inducing telomere-specific DNA damage, alpha-thalassemia X-linked syndrome protein (ATRX) knockdown and deletion of death associated protein (DAXX). Surprisingly, two important ALT hallmarks in the ALT-like HTC75 cells were observed after treatments: ALT-associated promyelocytic leukaemia bodies (APBs) and extrachromosomal circular DNA of telomeric repeats. Moreover, knocking out hTERT by utilizing the CRISPR/Cas9 technique led to telomere elongation in a telomerase-independent manner in ALT-like HTC75 cells. In summary, this is the first report to show that inducing telomeric DNA damage, disrupting the ATRX/DAXX complex and inhibiting telomerase activity in telomerase-positive cancer cells lead to the ALT switch.

  20. Protection of the genome and central protein-coding sequences by non-coding DNA against DNA damage from radiation.

    PubMed

    Qiu, Guo-Hua

    2015-01-01

    Non-coding DNA comprises a very large proportion of the total genomic content in higher organisms, but its function remains largely unclear. Non-coding DNA sequences constitute the majority of peripheral heterochromatin, which has been hypothesized to be the genome's 'bodyguard' against DNA damage from chemicals and radiation for almost four decades. The bodyguard protective function of peripheral heterochromatin in genome defense has been strengthened by the results from numerous recent studies, which are summarized in this review. These data have suggested that cells and/or organisms with a higher level of heterochromatin and more non-coding DNA sequences, including longer telomeric DNA and rDNAs, exhibit a lower frequency of DNA damage, higher radioresistance and longer lifespan after IR exposure. In addition, the majority of heterochromatin is peripherally located in the three-dimensional structure of genome organization. Therefore, the peripheral heterochromatin with non-coding DNA could play a protective role in genome defense against DNA damage from ionizing radiation by both absorbing the radicals from water radiolysis in the cytosol and reducing the energy of IR. However, the bodyguard protection by heterochromatin has been challenged by the observation that DNA damage is less frequently detected in peripheral heterochromatin than in euchromatin, which is inconsistent with the expectation and simulation results. Previous studies have also shown that the DNA damage in peripheral heterochromatin is rarely repaired and moves more quickly, broadly and outwardly to approach the nuclear pore complex (NPC). Additionally, it has been shown that extrachromosomal circular DNAs (eccDNAs) are formed in the nucleus, highly detectable in the cytoplasm (particularly under stress conditions) and shuttle between the nucleus and the cytoplasm. Based on these studies, this review speculates that the sites of DNA damage in peripheral heterochromatin could occur more

  1. Synthesis of circular double-stranded DNA having single-stranded recognition sequence as molecular-physical probe for nucleic acid hybridization detection based on atomic force microscopy imaging.

    PubMed

    Nakano, Koji; Matsunaga, Hideshi; Murata, Masaharu; Soh, Nobuaki; Imato, Toshihiko

    2009-08-01

    A new class of DNA probes having a mechanically detectable tag is reported. The DNA probe, which consists of a single-stranded recognition sequence and a double-stranded circular DNA entity, was prepared by polymerase reaction. M13mp18 single strand and a 32mer oligodeoxynucleotide whose 5'-end is decorated with the recognition sequence were used in combination as template and primer, respectively. We have successfully demonstrated that the DNA probe is useful for bioanalytical purposes: by deliberately attaching target DNA molecules onto Au(111) substrates and by mechanically reading out the tag-entity using a high-resolution microscopy including atomic force microscopy, visualization/detection of the individual target/probe DNA conjugate was possible simply yet straightforwardly. The present DNA probe can be characterized as a 100%-nucleic acid product material. It is simply available by one-pod synthesis. A surface topology parameter, image roughness, has witnessed its importance as a quantitative analysis index with particular usability in the present visualization/detection method.

  2. Rolling circle amplification, a powerful tool for genetic and functional studies of complete hepatitis B virus genomes from low-level infections and for directly probing covalently closed circular DNA.

    PubMed

    Margeridon, Séverine; Carrouée-Durantel, Sandra; Chemin, Isabelle; Barraud, Luc; Zoulim, Fabien; Trépo, Christian; Kay, Alan

    2008-09-01

    Complete characterization of the biological properties of hepatitis B virus (HBV) variants requires the generation of full-length genomes. The aim of this study was to develop new tools for the efficient full-length genome amplification of virus from samples with low viral loads. Rolling circle amplification (RCA) was used to amplify full-length HBV genomes from both sera and liver biopsy samples from chronic HBV carriers. Serum-derived relaxed circular HBV DNA could be amplified only after completion and ligation of plus-strand DNA. Covalently closed circular DNA (cccDNA) from liver biopsies could be amplified directly from as few as 13 copies, using RCA, followed by a full-length HBV PCR. Three serial liver biopsy samples were obtained from a lamivudine-resistant patient who cleared detectable serum HBV after adefovir dipivoxil was added to the lamivudine therapy and then seroconverted to anti-HBs. Only the genomes from the last biopsy specimen obtained after the emergence of lamivudine resistance contained the lamivudine resistance-associated mutations rtL180M and rtM204V ("rt" indicates reverse transcriptase domain). Defective genomes were also found in this biopsy sample. Genomes cloned from the liver biopsy specimens were transfected into HuH7 cells to study their replication competence and their susceptibility to lamivudine. RCA is a powerful tool for amplifying full-length HBV genomes and will be especially useful for the study of occult or inactive HBV infections and patients undergoing antiviral treatment. It can also be used to probe HBV cccDNA, the crucial intermediate in viral persistence and the archive of resistance mutations.

  3. Genome defense against exogenous nucleic acids in eukaryotes by non-coding DNA occurs through CRISPR-like mechanisms in the cytosol and the bodyguard protection in the nucleus.

    PubMed

    Qiu, Guo-Hua

    2016-01-01

    In this review, the protective function of the abundant non-coding DNA in the eukaryotic genome is discussed from the perspective of genome defense against exogenous nucleic acids. Peripheral non-coding DNA has been proposed to act as a bodyguard that protects the genome and the central protein-coding sequences from ionizing radiation-induced DNA damage. In the proposed mechanism of protection, the radicals generated by water radiolysis in the cytosol and IR energy are absorbed, blocked and/or reduced by peripheral heterochromatin; then, the DNA damage sites in the heterochromatin are removed and expelled from the nucleus to the cytoplasm through nuclear pore complexes, most likely through the formation of extrachromosomal circular DNA. To strengthen this hypothesis, this review summarizes the experimental evidence supporting the protective function of non-coding DNA against exogenous nucleic acids. Based on these data, I hypothesize herein about the presence of an additional line of defense formed by small RNAs in the cytosol in addition to their bodyguard protection mechanism in the nucleus. Therefore, exogenous nucleic acids may be initially inactivated in the cytosol by small RNAs generated from non-coding DNA via mechanisms similar to the prokaryotic CRISPR-Cas system. Exogenous nucleic acids may enter the nucleus, where some are absorbed and/or blocked by heterochromatin and others integrate into chromosomes. The integrated fragments and the sites of DNA damage are removed by repetitive non-coding DNA elements in the heterochromatin and excluded from the nucleus. Therefore, the normal eukaryotic genome and the central protein-coding sequences are triply protected by non-coding DNA against invasion by exogenous nucleic acids. This review provides evidence supporting the protective role of non-coding DNA in genome defense.

  4. Correlation between risk of hepatitis B virus recurrence and tissue expression of covalently closed circular DNA in living donor liver transplant recipients treated with high-dose hepatitis B immunoglobulin.

    PubMed

    Song, G-W; Ahn, C-S; Lee, S-G; Hwang, S; Kim, K-H; Moon, D-B; Ha, T-Y; Jung, D-H; Park, G-C; Kang, S-H; Jung, B-H; Kim, N

    2014-12-01

    Despite the application of prophylaxis, the risk of hepatitis B virus (HBV) recurrence remains. However, actual mechanism(s) and definite risk factor(s) are obscure. The present study examined the correlation between the HBV load in liver explants and post-liver transplant (OLT) HBV recurrence. HBV DNA was extracted from liver tissue taken from 50 living donor OLT (LDLT) patients using the QuickGene DNA Tissue Kit S (Fujifilm, Tokyo, Japan) and subjected to real-time polymerase chain reaction with the following primers: 5'-CACATGGCCTCCAAGGAGTAA-3' (forward primer) and 5'-TGAGGGTCTCTCTCTTCCTCTTGT-3' (reverse primer). To prevent HBV infection, patients were treated daily with high-dose (10,000 IU) hepatitis B immunoglobulin (HBIG) for the first week after LDLT. They then received weekly doses for the next month and then monthly doses for ≤1 year. If the anti-hepatitis surface antigen antibody titer was <1,000 IU/L, an antiviral agent (AVA) was added to the regimen. The mean (±SD) tissue HBV DNA and covalently closed circular DNA (cccDNA) loads were -0.8 ± 1.2 (range, -2.9 to 2.6) and -2.3 ± 1.1 (range, -4.6 to 0.6) log10 copies/cell, respectively. There was a significant correlation between serum and tissue HBV DNA (r = 0.65; P = .00) and cccDNA concentrations (r = 0.55; P = .00). Six patients suffered HBV recurrence and 9 required additional AVA. There was no direct correlation between HBV recurrence and tissue cccDNA concentration. However, the concentration of cccDNA was significantly greater those patients suffering recurrence and receiving AVA treatment (high-risk group). High tissue cccDNA concentrations may be a risk factor for HBV recurrence despite high-dose HBIG prophylaxis. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. Characterization of ABCG2 gene amplification manifesting as extrachromosomal DNA in mitoxantrone-selected SF295 human glioblastoma cells.

    PubMed

    Rao, V Koneti; Wangsa, Darawalee; Robey, Robert W; Huff, Lyn; Honjo, Yasumasa; Hung, Jeffrey; Knutsen, Turid; Ried, Thomas; Bates, Susan E

    2005-07-15

    The human ABCG2 gene, located on chromosome 4, encodes an ATP-binding cassette half-transporter that has been shown to confer resistance to chemotherapeutic agents. Relatively little is known about the mechanisms controlling expression of ABCG2. In previous studies, we had shown that overexpression of ABCG2 can result from rearrangement or gene amplification involving chromosome 4. To better characterize the mechanisms of ABCG2 overexpression, SF295 glioblastoma cells were exposed to increasing amounts of mitoxantrone to generate the SF295 MX50, MX100, MX250, and MX500 sublines, maintained in mitoxantrone concentrations ranging from 50 to 500 nmol/L. Northern blot analysis confirmed overexpression of ABCG2 mRNA, and immunoblot analysis demonstrated increased protein expression in the selected cell lines. Efflux of BODIPY-prazosin confirmed a functional protein. ABCG2 gene amplification was observed in all resistant sublines, as determined by Southern blot analysis. Fluorescence in situ hybridization (FISH) revealed amplification of ABCG2 via double minute chromosomes (dmins) detected in metaphase chromosome spreads in the SF295 MX50 and MX100 sublines. At higher levels of drug selection, in the MX250 and MX500 sublines, fewer dmins were observed but homogeneously staining regions (hsr) were visible with FISH analysis, revealing reintegration of the ABCG2 gene into multiple chromosomes. Spectral karyotyping (SKY) demonstrated multiple clonal and nonclonal rearrangements of chromosome 4, including hsrs. These results suggest that amplification of ABCG2 occurred initially in the form of dmins, followed by chromosomal reintegration of the amplicon at multiple sites. This occurred with increasing drug-selection pressure, generating a more stable genotype.

  6. Use of the hepatitis B virus recombinant baculovirus-HepG2 system to study the effects of (-)-beta-2',3'-dideoxy-3'-thiacytidine on replication of hepatitis B virus and accumulation of covalently closed circular DNA.

    PubMed

    Delaney, W E; Miller, T G; Isom, H C

    1999-08-01

    (-)-Beta-2',3'-Dideoxy-3'-thiacytidine (lamivudine [3TC]) is a nucleoside analog which effectively interferes with the replication of hepatitis B virus (HBV) DNA in vitro and in vivo. We have investigated the antiviral properties of 3TC in vitro in HepG2 cells infected with recombinant HBV baculovirus. Different types of information can be obtained with the HBV baculovirus-HepG2 system because (i) experiments can be carried out at various levels of HBV replication including levels significantly higher than those that can be obtained from conventional HBV-expressing cell lines, (ii) cultures can be manipulated and/or treated prior to or during the initiation of HBV expression, and (iii) high levels of HBV replication allow the rapid detection of HBV products including covalently closed circular (CCC) HBV DNA from low numbers of HepG2 cells. The treatment of HBV baculovirus-infected HepG2 cells with 3TC resulted in an inhibition of HBV replication, evidenced by reductions in the levels of both extracellular HBV DNA and intracellular replicative intermediates. The effect of 3TC on HBV replication was both dose and time dependent, and the reductions in extracellular HBV DNA that we observed agreed well with the previously reported efficacy of 3TC in vitro. As expected, levels of HBV transcripts and extracellular hepatitis B surface antigen and e antigen were not affected by 3TC. Importantly, the HBV baculovirus-HepG2 system made it possible to observe for the first time that CCC HBV DNA levels are lower in cells treated with 3TC than in control cells. We also observed that the treatment of HepG2 cells prior to HBV baculovirus infection resulted in a slight increase in the efficacy of 3TC compared to treatments starting 24 h postinfection. The treatment of HepG2 cells with the highest concentration of 3TC tested in this study (2 microM) prior to the initiation of HBV replication markedly inhibited the accumulation of CCC DNA, whereas treatment with the same

  7. Complete Genome Sequence of a Cylindrospermopsin-Producing Cyanobacterium, Cylindrospermopsis raciborskii CS505, Containing a Circular Chromosome and a Single Extrachromosomal Element

    PubMed Central

    Fuentes-Valdés, Juan J.; Plominsky, Alvaro M.; Allen, Eric E.; Tamames, Javier

    2016-01-01

    Cylindrospermopsis raciborskii is a freshwater cyanobacterium producing bloom events and toxicity in drinking water source reservoirs. We present the first genome sequence for C. raciborskii CS505 (Australia), containing one 4.1-Mbp chromosome and one 110-Kbp plasmid having G+C contents of 40.3% (3933 genes) and 39.3% (111 genes), respectively. PMID:27563040

  8. Complete Circular Genome Sequence of Successful ST8/SCCmecIV Community-Associated Methicillin-Resistant Staphylococcus aureus (OC8) in Russia: One-Megabase Genomic Inversion, IS256's Spread, and Evolution of Russia ST8-IV.

    PubMed

    Wan, Tsai-Wen; Khokhlova, Olga E; Iwao, Yasuhisa; Higuchi, Wataru; Hung, Wei-Chun; Reva, Ivan V; Singur, Olga A; Gostev, Vladimir V; Sidorenko, Sergey V; Peryanova, Olga V; Salmina, Alla B; Reva, Galina V; Teng, Lee-Jene; Yamamoto, Tatsuo

    2016-01-01

    ST8/SCCmecIV community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has been a common threat, with large USA300 epidemics in the United States. The global geographical structure of ST8/SCCmecIV has not yet been fully elucidated. We herein determined the complete circular genome sequence of ST8/SCCmecIVc strain OC8 from Siberian Russia. We found that 36.0% of the genome was inverted relative to USA300. Two IS256, oppositely oriented, at IS256-enriched hot spots were implicated with the one-megabase genomic inversion (MbIN) and vSaβ split. The behavior of IS256 was flexible: its insertion site (att) sequences on the genome and junction sequences of extrachromosomal circular DNA were all divergent, albeit with fixed sizes. A similar multi-IS256 system was detected, even in prevalent ST239 healthcare-associated MRSA in Russia, suggesting IS256's strong transmission potential and advantage in evolution. Regarding epidemiology, all ST8/SCCmecIVc strains from European, Siberian, and Far Eastern Russia, examined had MbIN, and geographical expansion accompanied divergent spa types and resistance to fluoroquinolones, chloramphenicol, and often rifampicin. Russia ST8/SCCmecIVc has been associated with life-threatening infections such as pneumonia and sepsis in both community and hospital settings. Regarding virulence, the OC8 genome carried a series of toxin and immune evasion genes, a truncated giant surface protein gene, and IS256 insertion adjacent to a pan-regulatory gene. These results suggest that unique single ST8/spa1(t008)/SCCmecIVc CA-MRSA (clade, Russia ST8-IVc) emerged in Russia, and this was followed by large geographical expansion, with MbIN as an epidemiological marker, and fluoroquinolone resistance, multiple virulence factors, and possibly a multi-IS256 system as selective advantages.

  9. Mosaic Analysis Using a Ncl-1 (+) Extrachromosomal Array Reveals That Lin-31 Acts in the Pn.p Cells during Caenorhabditis Elegans Vulval Development

    PubMed Central

    Miller, L. M.; Waring, D. A.; Kim, S. K.

    1996-01-01

    We describe a genetic mosaic analysis procedure in which Caenorhabditis elegans mosaics are generated by spontaneous loss of an extrachromosomal array. This technique allows almost any C. elegans gene that can be used in germline transformation experiments to be used in mosaic analysis experiments. We identified a cosmid clone that rescues the mutant phenotype of ncl-1, so that this cell-autonomous marker could be used to analyze mosaic animals. To determine the sites of action for unc-29 and lin-31, an extrachromosomal array was constructed containing the ncl-1(+) cosmid linked to lin-31(+) and unc-29(+) cosmids. This array is mitotically unstable and can be lost to produce a clone of mutant cells. The specific cell division at which the extrachromosomal array had been lost was deduced by scoring the Ncl phenotypes of individual cells in genetic mosaics. The Unc-29 and Lin-31 phenotypes were then scored in these animals to determine in which cells these genes are required. This analysis showed that unc-29, which encodes a subunit of the acetylcholine receptor, acts in the body muscle cells. Furthermore, lin-31, which specifies cell fates during vulval induction and encodes a putative transcription factor similar to HNF-3/fork head, acts in the Pn.p cells. PMID:8807292

  10. Regulated expression of the feline panleukopenia virus P38 promoter on extrachromosomal FPV/EBV chimeric plasmids.

    PubMed Central

    Clemens, D L; Carlson, J O

    1989-01-01

    Feline panleukopenia virus/Epstein-Barr virus (FPV/EBV) chimeric expression plasmids were constructed to study regulation of the structural protein gene of the parvovirus, FPV, in a homologous cell culture system. Detection and quantitation of activity from the native FPV promoter, P38, was facilitated by fusing the Escherichia coli lacZ gene with the FPV structural protein gene. Feline cell lines which stably maintained these plasmids extrachromosomally were established. Constitutive beta-galactosidase activity was low but increased up to 40-fold after infection with FPV. Expression of beta-galactosidase was only detected when the FPV/lacZ gene was oriented in the same transcriptional direction as the Epstein-Barr virus gene coding for EBNA-1. When a small open reading frame upstream of the FPV/lacZ initiation codon was deleted, beta-galactosidase expression increased another 4.7- to 26-fold. These changes in beta-galactosidase activity indicate that expression of the FPV structural protein gene is regulated both transcriptionally and posttranscriptionally. Images PMID:2542586

  11. [Circular migration in Indonesia].

    PubMed

    Mantra, I B

    1979-12-01

    The author examines circular migration in Indonesia, with primary focus on the 1970s. It is found that circular, or repeated return migration, generally occurs over short distances and for short periods and is more frequent than lifetime migration. The relationships between improvements in the national transport system, access to labor force opportunities in both the formal and informal sectors of the economy, and circular migration are discussed.

  12. Molecular DNA switches and DNA chips

    NASA Astrophysics Data System (ADS)

    Sabanayagam, Chandran R.; Berkey, Cristin; Lavi, Uri; Cantor, Charles R.; Smith, Cassandra L.

    1999-06-01

    We present an assay to detect single-nucleotide polymorphisms on a chip using molecular DNA switches and isothermal rolling- circle amplification. The basic principle behind the switch is an allele-specific oligonucleotide circularization, mediated by DNA ligase. A DNA switch is closed when perfect hybridization between the probe oligonucleotide and target DNA allows ligase to covalently circularize the probe. Mismatches around the ligation site prevent probe circularization, resulting in an open switch. DNA polymerase is then used to preferentially amplify the closed switches, via rolling-circle amplification. The stringency of the molecular switches yields 102 - 103 fold discrimination between matched and mismatched sequences.

  13. Toroidal circular dichroism

    NASA Astrophysics Data System (ADS)

    Raybould, T. A.; Fedotov, V. A.; Papasimakis, N.; Kuprov, I.; Youngs, I. J.; Chen, W. T.; Tsai, D. P.; Zheludev, N. I.

    2016-07-01

    We demonstrate that the induced toroidal dipole, represented by currents flowing on the surface of a torus, makes a distinct and indispensable contribution to circular dichroism. We show that toroidal circular dichroism supplements the well-known mechanism involving electric dipole and magnetic dipole transitions. We illustrate this with rigorous analysis of the experimentally measured polarization-sensitive transmission spectra of an artificial metamaterial, constructed from elements of toroidal symmetry. We argue that toroidal circular dichroism will be found in large biomolecules with elements of toroidal symmetry and should be taken into account in the interpretation of circular dichroism spectra of organics.

  14. Size distributions of circular molecules in plant mitochondrial DNAs.

    PubMed

    Bailey-Serres, J; Leroy, P; Jones, S S; Wahleithner, J A; Wolstenholme, D R

    1987-01-01

    Some physicochemical properties of the mitochondrial DNAs (mtDNA) from plants of flax, broad bean and mung bean, and from tissue culture cells of jimson weed, soybean, petunia and tobacco were determined. Circular molecules were observed in electron microscope preparations of each mtDNA. In soybean, petunia, broad bean and mung bean mtDNAs, the circular molecules had a continuous distribution of lengths (ranges between 1 to 36 kb, and 1 to 126 kb), heavily skewed toward smaller molecules. Eighty-six percent of the flax circular molecules were from 27 to 54 kb in size, and 78% of the jimson weed circular molecules were from 4 to 15 kb. Replicative forms of 1.2-1.6 kb circular molecules were observed in electron microscope preparations of broad bean mtDNA.

  15. A novel replicating circular DNAzyme

    PubMed Central

    Chen, Fei; Wang, Ruijian; Li, Zhe; Liu, Bin; Wang, Xiaoping; Sun, Yanhong; Hao, Dongyun; Zhang, Jin

    2004-01-01

    10–23 DNAzyme has the potential to suppress gene expressions through sequence-specific mRNA cleavage. However, the dependence on exogenous delivery limits its applications. The objective of this work is to establish a replicating DNAzyme in bacteria using a single-stranded DNA vector. By cloning the 10–23 DNAzyme into the M13mp18 vector, we constructed two circular DNAzymes, C-Dz7 and C-Dz482, targeting the β-lactamase mRNA. These circular DNAzymes showed in vitro catalytic efficiencies (kcat/KM) of 7.82 × 106 and 1.36 × 107 M–1·min–1, respectively. Their dependence on divalent metal ions is similar to that found with linear 10–23 DNAzyme. Importantly, the circular DNAzymes were not only capable of replicating in bacteria but also exhibited high activities in inhibiting β-lactamase and bacterial growth. This study thus provides a novel strategy to produce replicating DNAzymes which may find widespread applications. PMID:15115797

  16. Distribution of Twelve Linear Extrachromosomal DNAs in Natural Isolates of Lyme Disease Spirochetes

    PubMed Central

    Palmer, Nanette; Fraser, Claire; Casjens, Sherwood

    2000-01-01

    We have analyzed a panel of independent North American isolates of the Lyme disease agent spirochete, Borrelia burgdorferi (sensu stricto), for the presence of linear plasmids with sequence similarities to the 12 linear plasmids present in the B. burgdorferi type strain, isolate B31. The frequency of similarities to probes from each of the 12 B31 plasmids varied from 13 to 100% in the strain panel examined, and these similarities usually reside on plasmids similar in size to the cognate B31 plasmid. Sequences similar to 5 of the 12 B31 plasmids were found in all of the isolates examined, and >66% of the panel members hybridized to probes from 4 other plasmids. Sequences similar to most of the B. burgdorferi B31 plasmid-derived DNA probes used were also found on linear plasmids in the related Eurasian Lyme agents Borrelia garinii and Borrelia afzelii; however, some of these plasmids had uniform but substantially different sizes from their B. burgdorferi counterparts. PMID:10762248

  17. Phytochemical and Bioactive Potential of in vivo and in vitro Grown Plants of Centaurea ragusina L. - Detection of DNA/RNA Active Compounds in Plant Extracts via Thermal Denaturation and Circular Dichroism.

    PubMed

    Vujčić, Valerija; Radić Brkanac, Sandra; Radojčić Redovniković, Ivana; Ivanković, Siniša; Stojković, Ranko; Žilić, Irena; Radić Stojković, Marijana

    2017-07-25

    The phytochemical composition and biological activity of non-volatile components of Centaurea ragusina L. has not been studied previously. Our aim was to evaluate the phytochemical and bioactive potential (including interactions with polynucleotides) of C. ragusina L. depending on the origin of plant material (in vivo - leaves from natural habitats, ex vitro - leaves from plants acclimated from culture media, in vitro - leaves and calli from plants grown in culture media) and polarity of solvents used in extract preparation (80 and 96% ethanol and water combinations or single solvents). The polyphenol composition was determined by spectrophotometric and HPLC analysis. Biological activity of extracts was evaluated by following methods: 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) methods for antioxidative activity, 2,3,5-triphenyl tetrazolium chloride (TTC) microdilution method for antibacterial activity, crystal-violet test for cytotoxic activity and thermal denaturation (TD) and circular dichroism (CD) for DNA/RNA interactions. Conditions for the most efficient polyphenol extraction were determined: the 80% ethanol/water solvent system was the most suitable for callus and leaf ex vitro samples and 80 or 96% ethanol for leaf in vivo samples. Significantly higher levels of chlorogenic acid and naringenin were detected in callus tissue than in vivo plant. Ethanolic extracts exhibited the significant antibacterial activity against Staphylococcus aureus ATCC 25923. DNA/RNA active compounds in plant extracts were detected by TD and CD methods. Callus tissue and ex vitro leaves represent a valuable source of polyphenols as in vivo leaves. TD and CD can be applied for detection of DNA/RNA active compounds in extracts from natural resources. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  18. 2. Northwest circular bastion, seen from edge of southwest circular ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    2. Northwest circular bastion, seen from edge of southwest circular bastion wall. Metal roof beams extend up to form peak. World War II gun installation at right. - Fort Hamilton, Northwest Circular Bastion, Rose Island, Newport, Newport County, RI

  19. DNA-binding-protein inhomogeneity in E. coli modeled as biphasic facilitated diffusion.

    PubMed

    Kuhlman, Thomas E; Cox, Edward C

    2013-08-01

    We have recently shown that nonspecifically bound lac repressors are spatially inhomogeneous in E. coli cells and depends upon the location of its encoding gene and the DNA compaction state [Kuhlman and Cox, Mol. Syst. Biol. 8, 610 (2012)]. Here we model this inhomogeneity as a consequence of diffusion within and exchange between two distinct intracellular phases: the condensed chromosomal DNA and an extrachromosomal compartment, the cytoplasm. We discuss the consequences of this model for the target search process.

  20. The genome of Oryctes rhinoceros nudivirus provides novel insight into the evolution of nuclear arthropod-specific large circular double-stranded DNA viruses.

    PubMed

    Wang, Yongjie; Bininda-Emonds, Olaf R P; van Oers, Monique M; Vlak, Just M; Jehle, Johannes A

    2011-06-01

    The Oryctes rhinoceros nudivirus (OrNV) is a dsDNA virus with enveloped, rod-shaped virions. Its genome is 127,615 bp in size and contains 139 predicted protein-coding open reading frames (ORFs). In-depth genome sequence comparisons revealed a varying number of shared gene homologues, not only with other nudiviruses (NVs) and baculoviruses, but also with other arthropod-specific large dsDNA viruses, including the so-called Monodon baculovirus (MBV), the salivary gland hypertrophy viruses (SGHVs) and white spot syndrome virus (WSSV). Nudivirus genomes contain 20 baculovirus core gene homologues associated with transcription (p47, lef-8, lef-9, lef-4, vlf-1, and lef-5), replication (dnapol and helicase), virus structure (p74, pif-1, pif-2, pif-3, 19kda/pif-4, odv-e56/pif-5, vp91, vp39, and 38K), and unknown functions (ac68, ac81, and p33). Most strikingly, a set of homologous genes involved in peroral infection (p74, pif-1, pif-2, and pif-3) are common to baculoviruses, nudiviruses, SGHVs, and WSSV indicating an ancestral mode of infection in these highly diverged viruses. A gene similar to polyhedrin/granulin encoding the baculovirus occlusion body protein was identified in non-occluded NVs and in Musca domestica SGHV evoking the question of the evolutionary origin of the baculovirus polyhedrin/granulin gene. Based on gene homologies, we further propose that the shrimp MBV is an occluded member of the nudiviruses. We conclude that baculoviruses, NVs and the shrimp MBV, the SGHVs and WSSV share the significant number of conserved genetic functions, which may point to a common ancestry of these viruses.

  1. Circularly polarized Hankel vortices.

    PubMed

    Kotlyar, Victor V; Kovalev, Alexey A

    2017-04-03

    We discuss vector Hankel beams with circular polarization. These beams appear as a generalization of a spherical wave with an embedded optical vortex with topological charge n. Explicit analytical relations to describe all six projections of the E- and H-field are derived. The relations are shown to satisfy Maxwell's equations. Hankel beams with clockwise and anticlockwise circular polarization are shown to have peculiar features while propagating in free space. Relations for the Poynting vector projections and the angular momentum in the far field are also obtained. It is shown that a Hankel beam with clockwise circular polarization has radial divergence (ratio between the radial and longitudinal projections of the Poynting vector) similar to that of the spherical wave, while the beam with the anticlockwise circular polarization has greater radial dependence. At n = 0, the circularly polarized Hankel beam has non-zero spin angular momentum. At n = 1, power flow of the Hankel beam with anticlockwise polarization consists of two parts: right-handed helical flow near the optical axis and left-handed helical flow in periphery. At n ≥2, power flow is directed along the right-handed helix regardless of the direction of the circular polarization. Power flow along the optical axis is the same for the Hankel beams of both circular polarizations, if they have the same topological charge.

  2. Spontaneous HBsAg loss in Korean patients: relevance of viral genotypes, S gene mutations, and covalently closed circular DNA copy numbers

    PubMed Central

    Chang, Hye-Young; Park, Jun Yong; Park, Eun-Sook; Park, Yong Kwang; Han, Kwang-Hyub

    2014-01-01

    Background/Aims Occult HBV infection can persist following HBsAg loss and be transmitted, but the virological features are not well defined. Methods Here we investigated 25 Korean patients who lost HBsAg during follow up, either spontaneously or subsequent to therapy. Results Whereas subtype adr (genotype C) was found in 96% of HBsAg positive patients, 75 % of patients who lost HBsAg spontaneously were seemed to be infected with the ayw subtype with sequence similar to genotype D. Mutations in the major hydrophilic region (MHR) of HBsAg were found in 7 patients who lost HBsAg spontaneously. The mutations include T123S, M125I/N, C139R, D144E, V177A, L192F, and W196L, some of which have not been reported before. Functional analysis via transfection experiments indicate that the C139R and D144E mutations drastically reduced HBsAg antigenicity, while the Y225del mutation found in one interferon-treated patient impaired HBsAg secretion. Conclusions Lack of detectable HBsAg in patient serum could be explained by low level of ccc DNA in liver tissue, low antigenicity of the surface protein, or its secretion defect. PMID:25320728

  3. Complete Circular Genome Sequence of Successful ST8/SCCmecIV Community-Associated Methicillin-Resistant Staphylococcus aureus (OC8) in Russia: One-Megabase Genomic Inversion, IS256’s Spread, and Evolution of Russia ST8-IV

    PubMed Central

    Wan, Tsai-Wen; Higuchi, Wataru; Hung, Wei-Chun; Reva, Ivan V.; Singur, Olga A.; Gostev, Vladimir V.; Sidorenko, Sergey V.; Peryanova, Olga V.; Salmina, Alla B.; Reva, Galina V.; Teng, Lee-Jene; Yamamoto, Tatsuo

    2016-01-01

    ST8/SCCmecIV community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has been a common threat, with large USA300 epidemics in the United States. The global geographical structure of ST8/SCCmecIV has not yet been fully elucidated. We herein determined the complete circular genome sequence of ST8/SCCmecIVc strain OC8 from Siberian Russia. We found that 36.0% of the genome was inverted relative to USA300. Two IS256, oppositely oriented, at IS256-enriched hot spots were implicated with the one-megabase genomic inversion (MbIN) and vSaβ split. The behavior of IS256 was flexible: its insertion site (att) sequences on the genome and junction sequences of extrachromosomal circular DNA were all divergent, albeit with fixed sizes. A similar multi-IS256 system was detected, even in prevalent ST239 healthcare-associated MRSA in Russia, suggesting IS256’s strong transmission potential and advantage in evolution. Regarding epidemiology, all ST8/SCCmecIVc strains from European, Siberian, and Far Eastern Russia, examined had MbIN, and geographical expansion accompanied divergent spa types and resistance to fluoroquinolones, chloramphenicol, and often rifampicin. Russia ST8/SCCmecIVc has been associated with life-threatening infections such as pneumonia and sepsis in both community and hospital settings. Regarding virulence, the OC8 genome carried a series of toxin and immune evasion genes, a truncated giant surface protein gene, and IS256 insertion adjacent to a pan-regulatory gene. These results suggest that unique single ST8/spa1(t008)/SCCmecIVc CA-MRSA (clade, Russia ST8-IVc) emerged in Russia, and this was followed by large geographical expansion, with MbIN as an epidemiological marker, and fluoroquinolone resistance, multiple virulence factors, and possibly a multi-IS256 system as selective advantages. PMID:27741255

  4. Squaring a Circular Segment

    ERIC Educational Resources Information Center

    Gordon, Russell

    2008-01-01

    Consider a circular segment (the smaller portion of a circle cut off by one of its chords) with chord length c and height h (the greatest distance from a point on the arc of the circle to the chord). Is there a simple formula involving c and h that can be used to closely approximate the area of this circular segment? Ancient Chinese and Egyptian…

  5. Squaring a Circular Segment

    ERIC Educational Resources Information Center

    Gordon, Russell

    2008-01-01

    Consider a circular segment (the smaller portion of a circle cut off by one of its chords) with chord length c and height h (the greatest distance from a point on the arc of the circle to the chord). Is there a simple formula involving c and h that can be used to closely approximate the area of this circular segment? Ancient Chinese and Egyptian…

  6. DNA.

    ERIC Educational Resources Information Center

    Felsenfeld, Gary

    1985-01-01

    Structural form, bonding scheme, and chromatin structure of and gene-modification experiments with deoxyribonucleic acid (DNA) are described. Indicates that DNA's double helix is variable and also flexible as it interacts with regulatory and other molecules to transfer hereditary messages. (DH)

  7. DNA.

    ERIC Educational Resources Information Center

    Felsenfeld, Gary

    1985-01-01

    Structural form, bonding scheme, and chromatin structure of and gene-modification experiments with deoxyribonucleic acid (DNA) are described. Indicates that DNA's double helix is variable and also flexible as it interacts with regulatory and other molecules to transfer hereditary messages. (DH)

  8. Models for circular-linear and circular-circular data constructed from circular distributions based on nonnegative trigonometric sums.

    PubMed

    Fernández-Durán, J J

    2007-06-01

    Johnson and Wehrly (1978, Journal of the American Statistical Association 73, 602-606) and Wehrly and Johnson (1980, Biometrika 67, 255-256) show one way to construct the joint distribution of a circular and a linear random variable, or the joint distribution of a pair of circular random variables from their marginal distributions and the density of a circular random variable, which in this article is referred to as joining circular density. To construct flexible models, it is necessary that the joining circular density be able to present multimodality and/or skewness in order to model different dependence patterns. Fernández-Durán (2004, Biometrics 60, 499-503) constructed circular distributions based on nonnegative trigonometric sums that can present multimodality and/or skewness. Furthermore, they can be conveniently used as a model for circular-linear or circular-circular joint distributions. In the current work, joint distributions for circular-linear and circular-circular data constructed from circular distributions based on nonnegative trigonometric sums are presented and applied to two data sets, one for circular-linear data related to the air pollution patterns in Mexico City and the other for circular-circular data related to the pair of dihedral angles between consecutive amino acids in a protein.

  9. Circular free-electron laser

    DOEpatents

    Brau, Charles A.; Kurnit, Norman A.; Cooper, Richard K.

    1984-01-01

    A high efficiency, free electron laser utilizing a circular relativistic electron beam accelerator and a circular whispering mode optical waveguide for guiding optical energy in a circular path in the circular relativistic electron beam accelerator such that the circular relativistic electron beam and the optical energy are spatially contiguous in a resonant condition for free electron laser operation. Both a betatron and synchrotron are disclosed for use in the present invention. A free electron laser wiggler is disposed around the circular relativistic electron beam accelerator for generating a periodic magnetic field to transform energy from the circular relativistic electron beam to optical energy.

  10. Compact waveguide circular polarizer

    SciTech Connect

    Tantawi, Sami G.

    2016-08-16

    A multi-port waveguide is provided having a rectangular waveguide that includes a Y-shape structure with first top arm having a first rectangular waveguide port, a second top arm with second rectangular waveguide port, and a base arm with a third rectangular waveguide port for supporting a TE.sub.10 mode and a TE.sub.20 mode, where the end of the third rectangular waveguide port includes rounded edges that are parallel to a z-axis of the waveguide, a circular waveguide having a circular waveguide port for supporting a left hand and a right hand circular polarization TE.sub.11 mode and is coupled to a base arm broad wall, and a matching feature disposed on the base arm broad wall opposite of the circular waveguide for terminating the third rectangular waveguide port, where the first rectangular waveguide port, the second rectangular waveguide port and the circular waveguide port are capable of supporting 4-modes of operation.

  11. Identification of Prostate Cancer-Specific microDNAs

    DTIC Science & Technology

    2016-02-01

    extrachromosomal circular DNAs (eccDNAs), called microDNAs. Unlike previously reported eccDNAs, microDNAs are relatively small in length, map to unique DNA ...exonuclease than linear DNAs and can be stably present in the cells or even possibly in the circulating system. Therefore, overall goal of this application...N/a 1 Introduction MicroDNAs are a special group of extrachromosomal circular DNAs (eccDNAs) derived from chromosomal

  12. Prokaryotic DNA ligases unwind superhelical DNA.

    PubMed

    Ivanchenko, M; van Holde, K; Zlatanova, J

    1996-09-13

    We have studied the effect on DNA topology of binding of prokaryotic DNA ligases (T4 and E. coli) to superhelical or nicked circular DNA. Performing topoisomerase I-mediated relaxation in the presence of increasing amounts of T4 ligase led to a shift in the topoisomer distribution to increasingly more negative values. This result suggested that T4 ligase unwound the DNA and was further substantiated by ligation of nicked circular molecules by E. coli DNA ligase in the presence of increasing amounts of T4 ligase. Such an experiment was possible since the two DNA ligases require different cofactors for enzymatic activity. Performing a similar experiment with reverse partners, using E. coli DNA ligase as ligand, and T4 ligase as sealing agent, we observed that the E. coli enzyme also unwound the DNA. Thus, prokaryotic DNA ligases can be added to an ever-growing list of DNA-binding proteins that unwind the DNA upon binding.

  13. 76 FR 62148 - Title VI; Proposed Circular, Environmental Justice; Proposed Circular

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-10-06

    ... Federal Transit Administration Title VI; Proposed Circular, Environmental Justice; Proposed Circular...; Proposed Circular'' and ``Environmental Justice; Proposed Circular.'' FOR FURTHER INFORMATION CONTACT: For... Circular'' (76 FR 60593) and ``Environmental Justice; Proposed Circular'' (76 FR 60590). Corrections The...

  14. Circular Fibonacci gratings.

    PubMed

    Gao, Nan; Zhang, Yuchao; Xie, Changqing

    2011-11-01

    We introduce circular Fibonacci gratings (CFGs) that combine the concept of circular gratings and Fibonacci structures. Theoretical analysis shows that the diffraction pattern of CFGs is composed of fractal distributions of impulse rings. Numerical simulations are performed with two-dimensional fast Fourier transform to reveal the fractal behavior of the diffraction rings. Experimental results are also presented and agree well with the numerical results. The fractal nature of the diffraction field should be of great theoretical interest, and shows potential to be further developed into practical applications, such as in laser measurement with wideband illumination.

  15. Polydnavirus DNA is integrated in the DNA of its parasitoid wasp host.

    PubMed Central

    Fleming, J G; Summers, M D

    1991-01-01

    The polydnavirus Campoletis sonorensis virus (CsV) is present in the oviducts of all adult C. sonorensis female wasps and appears to be required for these wasps to parasitize hosts successfully. Physical mapping, Southern blot analysis, and nucleotide sequence analysis demonstrate that the viral DNA B-specific sequences in cloned wasp DNA are colinear with viral genomic segment DNA B from nucleocapsids and are covalently linked to nonviral wasp sequences. Integrated DNA B terminates in 59-nucleotide imperfect direct repeats, but a single repeat exists in the extrachromosomal superhelical viral DNA B. Sequences near each junction form imperfect inverted repeats with sequences near the ends of an internal viral 540-base-pair repeat element gene. CsV appears to be the first documented integrated, nonretroviral DNA virus of insects and probably is vertically transmitted as a provirus. Images PMID:1946402

  16. Wiimote Experiments: Circular Motion

    ERIC Educational Resources Information Center

    Kouh, Minjoon; Holz, Danielle; Kawam, Alae; Lamont, Mary

    2013-01-01

    The advent of new sensor technologies can provide new ways of exploring fundamental physics. In this paper, we show how a Wiimote, which is a handheld remote controller for the Nintendo Wii video game system with an accelerometer, can be used to study the dynamics of circular motion with a very simple setup such as an old record player or a…

  17. Wiimote Experiments: Circular Motion

    ERIC Educational Resources Information Center

    Kouh, Minjoon; Holz, Danielle; Kawam, Alae; Lamont, Mary

    2013-01-01

    The advent of new sensor technologies can provide new ways of exploring fundamental physics. In this paper, we show how a Wiimote, which is a handheld remote controller for the Nintendo Wii video game system with an accelerometer, can be used to study the dynamics of circular motion with a very simple setup such as an old record player or a…

  18. Copyright Basics. Circular 1.

    ERIC Educational Resources Information Center

    Library of Congress, Washington, DC. Copyright Office.

    This circular answers some of the questions that are frequently asked about copyright, a form of protection provided by the laws of the United States to authors of "original works of authorship" including library, dramatic musical, artistic, and certain other intellectual works. The Copyright Act of 1976 (title 17 of the United States…

  19. Wiimote Experiments: Circular Motion

    NASA Astrophysics Data System (ADS)

    Kouh, Minjoon; Holz, Danielle; Kawam, Alae; Lamont, Mary

    2013-03-01

    The advent of new sensor technologies can provide new ways of exploring fundamental physics. In this paper, we show how a Wiimote, which is a handheld remote controller for the Nintendo Wii video game system with an accelerometer, can be used to study the dynamics of circular motion with a very simple setup such as an old record player or a bicycle wheel.

  20. DNA

    ERIC Educational Resources Information Center

    Stent, Gunther S.

    1970-01-01

    This history for molecular genetics and its explanation of DNA begins with an analysis of the Golden Jubilee essay papers, 1955. The paper ends stating that the higher nervous system is the one major frontier of biological inquiry which still offers some romance of research. (Author/VW)

  1. DNA

    ERIC Educational Resources Information Center

    Stent, Gunther S.

    1970-01-01

    This history for molecular genetics and its explanation of DNA begins with an analysis of the Golden Jubilee essay papers, 1955. The paper ends stating that the higher nervous system is the one major frontier of biological inquiry which still offers some romance of research. (Author/VW)

  2. Genome sequence of the Leisingera aquimarina type strain (DSM 24565T), a member of the marine Roseobacter clade rich in extrachromosomal elements

    PubMed Central

    Riedel, Thomas; Teshima, Hazuki; Petersen, Jörn; Fiebig, Anne; Davenport, Karen; Daligault, Hajnalka; Erkkila, Tracy; Gu, Wei; Munk, Christine; Xu, Yan; Chen, Amy; Pati, Amrita; Ivanova, Natalia; Goodwin, Lynne A.; Chain, Patrick; Detter, John C.; Rohde, Manfred; Gronow, Sabine; Kyrpides, Nikos C.; Woyke, Tanja; Göker, Markus; Brinkhoff, Thorsten; Klenk, Hans-Peter

    2013-01-01

    Leisingera aquimarina Vandecandelaere et al. 2008 is a member of the genomically well characterized Roseobacter clade within the family Rhodobacteraceae. Representatives of the marine Roseobacter clade are metabolically versatile and involved in carbon fixation and biogeochemical processes. They form a physiologically heterogeneous group, found predominantly in coastal or polar waters, especially in symbiosis with algae, in microbial mats, in sediments or associated with invertebrates. Here we describe the features of L. aquimarina DSM 24565T together with the permanent-draft genome sequence and annotation. The 5,344,253 bp long genome consists of one chromosome and an unusually high number of seven extrachromosomal elements and contains 5,129 protein-coding and 89 RNA genes. It was sequenced as part of the DOE Joint Genome Institute Community Sequencing Program 2010 and of the activities of the Transregional Collaborative Research Centre 51 funded by the German Research Foundation (DFG). PMID:24501625

  3. Circular inferences in schizophrenia.

    PubMed

    Jardri, Renaud; Denève, Sophie

    2013-11-01

    A considerable number of recent experimental and computational studies suggest that subtle impairments of excitatory to inhibitory balance or regulation are involved in many neurological and psychiatric conditions. The current paper aims to relate, specifically and quantitatively, excitatory to inhibitory imbalance with psychotic symptoms in schizophrenia. Considering that the brain constructs hierarchical causal models of the external world, we show that the failure to maintain the excitatory to inhibitory balance results in hallucinations as well as in the formation and subsequent consolidation of delusional beliefs. Indeed, the consequence of excitatory to inhibitory imbalance in a hierarchical neural network is equated to a pathological form of causal inference called 'circular belief propagation'. In circular belief propagation, bottom-up sensory information and top-down predictions are reverberated, i.e. prior beliefs are misinterpreted as sensory observations and vice versa. As a result, these predictions are counted multiple times. Circular inference explains the emergence of erroneous percepts, the patient's overconfidence when facing probabilistic choices, the learning of 'unshakable' causal relationships between unrelated events and a paradoxical immunity to perceptual illusions, which are all known to be associated with schizophrenia.

  4. Efficient circular thresholding.

    PubMed

    Lai, Yu-Kun; Rosin, Paul L

    2014-03-01

    Otsu's algorithm for thresholding images is widely used, and the computational complexity of determining the threshold from the histogram is O(N) where N is the number of histogram bins. When the algorithm is adapted to circular rather than linear histograms then two thresholds are required for binary thresholding. We show that, surprisingly, it is still possible to determine the optimal threshold in O(N) time. The efficient optimal algorithm is over 300 times faster than traditional approaches for typical histograms and is thus particularly suitable for real-time applications. We further demonstrate the usefulness of circular thresholding using the adapted Otsu criterion for various applications, including analysis of optical flow data, indoor/outdoor image classification, and non-photorealistic rendering. In particular, by combining circular Otsu feature with other colour/texture features, a 96.9% correct rate is obtained for indoor/outdoor classification on the well known IITM-SCID2 data set, outperforming the state-of-the-art result by 4.3%.

  5. HBx-elevated MSL2 Modulates HBV cccDNA through Inducing Degradation of APOBEC3B to Enhance Hepatocarcinogenesis.

    PubMed

    Gao, Yuen; Feng, Jinyan; Yang, Guang; Zhang, Shuqin; Liu, Yunxia; Bu, Yanan; Sun, Mingming; Zhao, Man; Chen, Fuquan; Zhang, Weiying; Ye, Lihong; Zhang, Xiaodong

    2017-06-13

    Chronic hepatitis B virus (HBV) infection is a leading cause in the occurrence of hepatitis B, liver cirrhosis and liver cancer, in which nuclear HBV covalently closed circular DNA (cccDNA), the genomic form that templates viral transcription and sustains viral persistence, plays crucial roles. In the present study, we explored the hypothesis that HBV X protein (HBx)-elevated male-specific lethal 2 (MSL2) activated HBV replication via modulating cccDNA in hepatoma cells, leading to hepatocarcinogenesis. Immunohistochemical analysis revealed that the expression of MSL2 was positively associated with that of HBV and was increased in the liver tissues of HBV-transgenic mice and clinical HCC patients. Interestingly, microarray profiling identified that MSL2 was associated with those genes responding to virus. Mechanistically, MSL2 could maintain HBV cccDNA stability through degradation of APOBEC3B by ubiquitylation in hepatoma cells. Above all, HBx accounted for the up-regulation of MSL2 in stably HBx-transfected hepatoma cell lines and liver tissues of HBx-transgenic mice. Luciferase reporter gene assays revealed that the promoter region of MSL2 regulated by HBx was located at nt -1317/-1167 containing FoxA1 binding element. ChIP assay validated that HBx could enhance the binding property of FoxA1 to MSL2 promoter region. HBx up-regulated MSL2 via activating YAP/FoxA1 signaling. Functionally, silencing MSL2 was able to block the growth of hepatoma cells in vitro and in vivo. In conclusion, HBx-elevated MSL2 modulates HBV cccDNA in hepatoma cells to promote hepatocarcinogenesis, forming a positive feedback loop of HBx/MSL2/cccDNA/HBV. Our finding uncovers novel insights into the mechanism by which MSL2 as a promotion factor in host cells selectively activates extrachromosomal DNA. This article is protected by copyright. All rights reserved. © 2017 by the American Association for the Study of Liver Diseases.

  6. Episomal High Copy Number Maintenance of Hairpin-capped DNA Bearing a Replication Initiation Region in Human Cells*

    PubMed Central

    Harada, Seiyu; Uchida, Masafumi; Shimizu, Noriaki

    2009-01-01

    We previously found that a plasmid bearing a replication initiation region efficiently initiates gene amplification in mammalian cells and that it generates extrachromosomal double minutes and/or chromosomal homogeneously staining regions. During analysis of the underlying mechanism, we serendipitously found that hairpin-capped linear DNA was stably maintained as numerous extrachromosomal tiny episomes for more than a few months in a human cancer cell line. Generation of such episomes depended on the presence of the replication initiation region in the original plasmid. Despite extrachromosomal maintenance, episomal gene expression was epigenetically suppressed. The Southern blot analysis of the DNA of cloned cells revealed that the region around the hairpin end was diversified between the clones. Furthermore, the bisulfite-modified PCR and the sequencing analyses revealed that the palindrome sequence that derived from the original hairpin end or its end-resected structure were well preserved during clonal long term growth. From these data, we propose a model that explains the formation and maintenance of these episomes, in which replication of the hairpin-capped DNA and cruciform formation and its resolution play central roles. Our findings may be relevant for the dissection of mammalian replicator sequences. PMID:19617622

  7. CIRCULAR CAVITY SLOT ANTENNA

    DOEpatents

    Kerley, P.L.

    1959-01-01

    A small-size antenna having a doughnut-shaped field pattern and which can act both as an antenna and a resonant circuit is described. The antenna is of the slotted type and comprises a resonant cavity with a center hole. A circular slot is provided in one wall of the cavity concentric with the hole and a radio frequency source is connected across the slot. The pattern and loading of the antenna are adjusted by varying the position and shape of a center element slidably disposed within the hole and projecting from the slotted side of the resonant cavity. The disclosed structure may also be used to propagate the oscillator signal down a transniission line by replacing the center element with one leg of the transmission line in a spaced relation from the walls of the cavity.

  8. Circularly Polarized MHOHG with Bichromatic Circularly Polarized Laser Pulses

    NASA Astrophysics Data System (ADS)

    Bandrauk, Andre D.; Mauger, Francois; Uzer, Turgay

    2016-05-01

    Circularly polarized MHOHG-Molecular High Order Harmonic Generation is shown to occur efficiently with intense ultrashort bichromatic circularly polarized pulses due to frequent electron-parent -ion recollision with co-or counter-rotating incident circular pulses as predicted in 1995. We show in this context that molecules offer a very robust and efficient frameworkfor the production of circularly polarized harmonics for the generation of single circularly polarized ``attosecond'' pulses. The efficiency of such new MHOHG is shown to depend on the compatibility of the symmetry of the molecular medium with the net electric field generated by the combination of the laser pulses.Using a time-dependent symmetry analysis with concrete examples such as H 2 + vs H 3 + we show how all the features(harmonic order and ∧ polarization) of MHOHG can be explained and predicted.

  9. Time-lapse ultrashort pulse microscopy of infection in three-dimensional versus two-dimensional culture environments reveals enhanced extra-chromosomal virus replication compartment formation

    NASA Astrophysics Data System (ADS)

    Gibbs, Holly C.; Sing, Garwin; Armas, Juan Carlos González; Campbell, Colin J.; Ghazal, Peter; Yeh, Alvin T.

    2013-03-01

    The mechanisms that enable viruses to harness cellular machinery for their own survival are primarily studied in cell lines cultured in two-dimensional (2-D) environments. However, there are increasing reports of biological differences between cells cultured in 2-D versus three-dimensional (3-D) environments. Here we report differences in host-virus interactions based on differences in culture environment. Using ultrashort pulse microscopy (UPM), a form of two-photon microscopy that utilizes sub-10-fs pulses to efficiently excite fluorophores, we have shown that de novo development of extra-chromosomal virus replication compartments (VRCs) upon murine cytomegalovirus (mCMV) infection is markedly enhanced when host cells are cultured in 3-D collagen gels versus 2-D monolayers. In addition, time-lapse imaging revealed that mCMV-induced VRCs have the capacity to grow by coalescence. This work supports the future potential of 3-D culture as a useful bridge between traditional monolayer cultures and animal models to study host-virus interactions in a more physiologically relevant environment for the development of effective anti-viral therapeutics. These advances will require broader adoption of modalities, such as UPM, to image deep within scattering tissues.

  10. Circular chemiresistors for microchemical sensors

    DOEpatents

    Ho, Clifford K.

    2007-03-13

    A circular chemiresistor for use in microchemical sensors. A pair of electrodes is fabricated on an electrically insulating substrate. The pattern of electrodes is arranged in a circle-filling geometry, such as a concentric, dual-track spiral design, or a circular interdigitated design. A drop of a chemically sensitive polymer (i.e., chemiresistive ink) is deposited on the insulating substrate on the electrodes, which spreads out into a thin, circular disk contacting the pair of electrodes. This circularly-shaped electrode geometry maximizes the contact area between the pair of electrodes and the polymer deposit, which provides a lower and more stable baseline resistance than with linear-trace designs. The circularly-shaped electrode pattern also serves to minimize batch-to-batch variations in the baseline resistance due to non-uniform distributions of conductive particles in the chemiresistive polymer film.

  11. Mode of degradation of plasmid DNA with ozone.

    PubMed

    Sawadaishi, K; Miura, K; Ohthuka, E; Ueda, T; Ishizaki, K; Shinriki, N

    1984-01-01

    The ozonization of pBR322 closed circular DNA showed the conversion to open circular DNA. The damaged site was investigated by restriction mapping. The results showed the damage and subsequent cleavage of the DNA strand of ccDNA by ozonization may occur at the region sensitive to nuclease S1.

  12. Nuclear spin circular dichroism

    SciTech Connect

    Vaara, Juha; Rizzo, Antonio; Kauczor, Joanna; Norman, Patrick; Coriani, Sonia

    2014-04-07

    Recent years have witnessed a growing interest in magneto-optic spectroscopy techniques that use nuclear magnetization as the source of the magnetic field. Here we present a formulation of magnetic circular dichroism (CD) due to magnetically polarized nuclei, nuclear spin-induced CD (NSCD), in molecules. The NSCD ellipticity and nuclear spin-induced optical rotation (NSOR) angle correspond to the real and imaginary parts, respectively, of (complex) quadratic response functions involving the dynamic second-order interaction of the electron system with the linearly polarized light beam, as well as the static magnetic hyperfine interaction. Using the complex polarization propagator framework, NSCD and NSOR signals are obtained at frequencies in the vicinity of optical excitations. Hartree-Fock and density-functional theory calculations on relatively small model systems, ethene, benzene, and 1,4-benzoquinone, demonstrate the feasibility of the method for obtaining relatively strong nuclear spin-induced ellipticity and optical rotation signals. Comparison of the proton and carbon-13 signals of ethanol reveals that these resonant phenomena facilitate chemical resolution between non-equivalent nuclei in magneto-optic spectra.

  13. Disentangling DNA molecules

    NASA Astrophysics Data System (ADS)

    Vologodskii, Alexander

    2016-09-01

    The widespread circular form of DNA molecules inside cells creates very serious topological problems during replication. Due to the helical structure of the double helix the parental strands of circular DNA form a link of very high order, and yet they have to be unlinked before the cell division. DNA topoisomerases, the enzymes that catalyze passing of one DNA segment through another, solve this problem in principle. However, it is very difficult to remove all entanglements between the replicated DNA molecules due to huge length of DNA comparing to the cell size. One strategy that nature uses to overcome this problem is to create the topoisomerases that can dramatically reduce the fraction of linked circular DNA molecules relative to the corresponding fraction at thermodynamic equilibrium. This striking property of the enzymes means that the enzymes that interact with DNA only locally can access their topology, a global property of circular DNA molecules. This review considers the experimental studies of the phenomenon and analyzes the theoretical models that have been suggested in attempts to explain it. We describe here how various models of enzyme action can be investigated computationally. There is no doubt at the moment that we understand basic principles governing enzyme action. Still, there are essential quantitative discrepancies between the experimental data and the theoretical predictions. We consider how these discrepancies can be overcome.

  14. Disentangling DNA molecules.

    PubMed

    Vologodskii, Alexander

    2016-09-01

    The widespread circular form of DNA molecules inside cells creates very serious topological problems during replication. Due to the helical structure of the double helix the parental strands of circular DNA form a link of very high order, and yet they have to be unlinked before the cell division. DNA topoisomerases, the enzymes that catalyze passing of one DNA segment through another, solve this problem in principle. However, it is very difficult to remove all entanglements between the replicated DNA molecules due to huge length of DNA comparing to the cell size. One strategy that nature uses to overcome this problem is to create the topoisomerases that can dramatically reduce the fraction of linked circular DNA molecules relative to the corresponding fraction at thermodynamic equilibrium. This striking property of the enzymes means that the enzymes that interact with DNA only locally can access their topology, a global property of circular DNA molecules. This review considers the experimental studies of the phenomenon and analyzes the theoretical models that have been suggested in attempts to explain it. We describe here how various models of enzyme action can be investigated computationally. There is no doubt at the moment that we understand basic principles governing enzyme action. Still, there are essential quantitative discrepancies between the experimental data and the theoretical predictions. We consider how these discrepancies can be overcome. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Circular Scan Streak Tube Development

    NASA Technical Reports Server (NTRS)

    Nevin, S.

    1980-01-01

    A streak tube having circular scan was designed, built and tested. Continuous circular scan, easily derived from out of phase sine waves applied to the conventional deflection plates, permits the timing of pulses traveling long baselines. At the tube's output a circular array of 720 elements is scanned to provide 30 to 40 picosecond resolution. Initial difficulties with electron bombarded silicon arrays were circumvented by using microchannel plates within the streak tube to provide the needed electronic amplification and digital sensitivity and coupling the 720 element arrays to the electron beam by means of a phosphor on a fiber optics. Two ceramic body tubes with S-20 photocathodes were tested and delivered.

  16. Digitalizing the Circular Economy

    NASA Astrophysics Data System (ADS)

    Reuter, Markus A.

    2016-12-01

    Metallurgy is a key enabler of a circular economy (CE), its digitalization is the metallurgical Internet of Things (m-IoT). In short: Metallurgy is at the heart of a CE, as metals all have strong intrinsic recycling potentials. Process metallurgy, as a key enabler for a CE, will help much to deliver its goals. The first-principles models of process engineering help quantify the resource efficiency (RE) of the CE system, connecting all stakeholders via digitalization. This provides well-argued and first-principles environmental information to empower a tax paying consumer society, policy, legislators, and environmentalists. It provides the details of capital expenditure and operational expenditure estimates. Through this path, the opportunities and limits of a CE, recycling, and its technology can be estimated. The true boundaries of sustainability can be determined in addition to the techno-economic evaluation of RE. The integration of metallurgical reactor technology and systems digitally, not only on one site but linking different sites globally via hardware, is the basis for describing CE systems as dynamic feedback control loops, i.e., the m-IoT. It is the linkage of the global carrier metallurgical processing system infrastructure that maximizes the recovery of all minor and technology elements in its associated refining metallurgical infrastructure. This will be illustrated through the following: (1) System optimization models for multimetal metallurgical processing. These map large-scale m-IoT systems linked to computer-aided design tools of the original equipment manufacturers and then establish a recycling index through the quantification of RE. (2) Reactor optimization and industrial system solutions to realize the "CE (within a) Corporation—CEC," realizing the CE of society. (3) Real-time measurement of ore and scrap properties in intelligent plant structures, linked to the modeling, simulation, and optimization of industrial extractive process

  17. Towards future circular colliders

    NASA Astrophysics Data System (ADS)

    Benedikt, Michael; Zimmermann, Frank

    2016-09-01

    The Large Hadron Collider (LHC) at the European Organization for Nuclear Research (CERN) presently provides proton-proton collisions at a center-of-mass (c.m.) energy of 13 TeV. The LHC design was started more than 30 years ago, and its physics program will extend through the second half of the 2030's. The global Future Circular Collider (FCC) study is now preparing for a post-LHC project. The FCC study focuses on the design of a 100-TeV hadron collider (FCC-hh) in a new ˜100 km tunnel. It also includes the design of a high-luminosity electron-positron collider (FCCee) as a potential intermediate step, and a lepton-hadron collider option (FCC-he). The scope of the FCC study comprises accelerators, technology, infrastructure, detectors, physics, concepts for worldwide data services, international governance models, and implementation scenarios. Among the FCC core technologies figure 16-T dipole magnets, based on Nb3 S n superconductor, for the FCC-hh hadron collider, and a highly-efficient superconducting radiofrequency system for the FCC-ee lepton collider. Following the FCC concept, the Institute of High Energy Physics (IHEP) in Beijing has initiated a parallel design study for an e + e - Higgs factory in China (CEPC), which is to be succeeded by a high-energy hadron collider (SPPC). At present a tunnel circumference of 54 km and a hadron collider c.m. energy of about 70 TeV are being considered. After a brief look at the LHC, this article reports the motivation and the present status of the FCC study, some of the primary design challenges and R&D subjects, as well as the emerging global collaboration.

  18. Beam Rounders for Circular Colliders

    SciTech Connect

    A. Burov; S. Nagaitsev; Ya. Derbenev

    2001-07-01

    By means of linear optics, an arbitrary uncoupled beam can be locally transformed into a round (rotation-invariant) state and then back. This provides an efficient way to round beams in the interaction region of circular colliders.

  19. Beam rounders for circular colliders

    SciTech Connect

    A. Burov and S. Nagaitsev

    2002-12-10

    By means of linear optics, an arbitrary uncoupled beam can be locally transformed into a round (rotation-invariant) state and then back. This provides an efficient way to round beams in the interaction region of circular colliders.

  20. Fixed field circular accelerator designs

    SciTech Connect

    Johnstone, C.; Wan, W.; Garren, A.

    2000-01-06

    The rapid rate and cycle time required to efficiently accelerate muons precludes conventional circular accelerators. Recirculating linacs provide one option, but the separate return arcs per acceleration pass may prove costly. Recent work on muon acceleration schemes has concentrated on designing fixed-field circular accelerators whose strong superconducting fields can sustain a factor of 4 increase in energy from injection to extraction. A 4 to 16 GeV fixed-field circular accelerator has been designed which allows large orbit excursions and the tune to vary as a function of momentum. Acceleration is .6 GeV per turn so the entire cycle consists of only 20 turns. In addition, a 16 to 64 GeV fixed-field circular accelerator has been designed which is more in keeping with the traditional Fixed Field Alternating Gradient machines. In this work the two machine designs are described.

  1. Beach vortices near circular topography

    NASA Astrophysics Data System (ADS)

    Hinds, A. K.; Johnson, E. R.; McDonald, N. R.

    2016-10-01

    Finite-area monopolar vortices which propagate around topography without change in shape are computed for circular seamounts and wells including the limiting cases of each: islands and infinitely deep wells. The time-dependent behaviour of vortex pairs propagating toward circular topography is also examined. Trajectories of point-vortex pairs exterior to the topography are found and compared to trajectories of vortex patches computed using contour dynamics.

  2. Replication initiates at multiple dispersed sites in the ribosomal DNA plasmid of the protozoan parasite Entamoeba histolytica.

    PubMed Central

    Dhar, S K; Choudhury, N R; Mittal, V; Bhattacharya, A; Bhattacharya, S

    1996-01-01

    In the protozoan parasite Entamoeba histolytica (which causes amoebiasis in humans), the rRNA genes (rDNA) in the nucleus are carried on an extrachromosomal circular plasmid. For strain HM-1:IMSS, the size of the rDNA plasmid is 24.5 kb, and 200 copies per genome are present. Each circle contains two rRNA transcription units as inverted repeats separated by upstream and downstream spacers. We have studied the replication of this molecule by neutral/neutral two-dimensional gel electrophoresis and by electron microscopy. All restriction fragments analyzed by two-dimensional gel electrophoresis gave signals corresponding to simple Y's and bubbles. This showed that replication initiated in this plasmid at multiple, dispersed locations spread throughout the plasmid. On the basis of the intensity of the bubble arcs, initiations from the rRNA transcription units seemed to occur more frequently than those from intergenic spacers. Multiple, dispersed initiation sites were also seen in the rDNA plasmid of strain HK-9 when it was analyzed by two-dimensional gel electrophoresis. Electron microscopic visualization of replicating plasmid molecules in strain HM-1:IMISS showed multiple replication bubbles in the same molecule. The location of bubbles on the rDNA circle was mapped by digesting with PvuI or BsaHI, which linearize the molecule, and with SacII, which cuts the circle twice. The distance of the bubbles from one end of the molecule was measured by electron microscopy. The data corroborated those from two-dimensional gels and showed that replication bubbles were distributed throughout the molecule and that they appeared more frequently in rRNA transcription units. The same interpretation was drawn from electron microscopic analysis of the HK-9 plasmid. Direct demonstration of more than one bubble in the same molecule is clear evidence that replication of this plasmid initiates at multiple sites. Potential replication origins are distributed throughout the plasmid. Such a

  3. 78 FR 14620 - Joint Development: Proposed Circular

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-03-06

    ... Federal Transit Administration Joint Development: Proposed Circular AGENCY: Federal Transit Administration... the form of a circular, on joint development. This circular provides guidance to recipients of Federal... joint development. This circular: (1) Defines the term ``joint development''; ] (2) explains how to...

  4. Circular dichroism techniques: biomolecular and nanostructural analyses- a review.

    PubMed

    Ranjbar, Bijan; Gill, Pooria

    2009-08-01

    This paper reviews the best known techniques using circular dichroism spectroscopy such as conventional circular dichroism (i.e. electronic circular dichroism), magnetic circular dichroisms (magnetic vibrational circular dichroism, x-ray magnetic circular dichroism), fluorescence detected circular dichroism, near-infrared circular dichroism, vibrational circular dichroism, Fourier transform infrared circular dichroism, high pressure liquid chromatography circular dichroism, stopped-flow circular dichroism, and synchrotron radiation circular dichroism. Also, we have described here the most important applications of circular dichroism spectroscopy in structural biochemistry and nanoscience.

  5. Circular RNAs in heart failure.

    PubMed

    Devaux, Yvan; Creemers, Esther E; Boon, Reinier A; Werfel, Stanislas; Thum, Thomas; Engelhardt, Stefan; Dimmeler, Stefanie; Squire, Iain

    2017-06-01

    Cardiovascular disease, and particularly heart failure, is still a serious health care issue for which novel treatments and biomarkers are needed. The RNA family comprises different subgroups, among which the small-sized microRNAs and the larger long non-coding RNAs have shown some potential to aid in moving personalized health care of heart failure patients a step forward. Here, members of the Cardiolinc network review the recent findings suggesting that the less well-known circular RNAs may constitute a novel reservoir of therapeutic targets and biomarkers of heart failure. The knowledge of the mode of biogenesis of circular RNAs will first be reported, followed by a description of different features that make these RNA molecules of interest for the heart failure community. The functions of circular RNAs in the heart will be described, with some emphasis given to their regulation in the failing heart. Circulating in the bloodstream, circular RNAs have appeared as potential biomarkers and recent findings associated with the use of circular RNAs as heart failure biomarkers will be discussed. Finally, some directions for future research will be provided. © 2017 The Authors. European Journal of Heart Failure © 2017 European Society of Cardiology.

  6. Circular codes, symmetries and transformations.

    PubMed

    Fimmel, Elena; Giannerini, Simone; Gonzalez, Diego Luis; Strüngmann, Lutz

    2015-06-01

    Circular codes, putative remnants of primeval comma-free codes, have gained considerable attention in the last years. In fact they represent a second kind of genetic code potentially involved in detecting and maintaining the normal reading frame in protein coding sequences. The discovering of an universal code across species suggested many theoretical and experimental questions. However, there is a key aspect that relates circular codes to symmetries and transformations that remains to a large extent unexplored. In this article we aim at addressing the issue by studying the symmetries and transformations that connect different circular codes. The main result is that the class of 216 C3 maximal self-complementary codes can be partitioned into 27 equivalence classes defined by a particular set of transformations. We show that such transformations can be put in a group theoretic framework with an intuitive geometric interpretation. More general mathematical results about symmetry transformations which are valid for any kind of circular codes are also presented. Our results pave the way to the study of the biological consequences of the mathematical structure behind circular codes and contribute to shed light on the evolutionary steps that led to the observed symmetries of present codes.

  7. Photoelectron circular dichroism of isopropanolamine

    NASA Astrophysics Data System (ADS)

    Catone, D.; Turchini, S.; Contini, G.; Prosperi, T.; Stener, M.; Decleva, P.; Zema, N.

    2017-01-01

    Spectroscopies based on circular polarized light are sensitive to the electronic and structural properties of chiral molecules. Photoelectron circular dichroism (PECD) is a powerful technique that combines the chiral sensitivity of the circular polarized light and the electronic information obtained by photoelectron spectroscopy. An experimental and theoretical PECD study of the outer valence and C 1s core states of 1-amino-2-propanol in the gas phase is presented. The experimental dichroic dispersions in the photoelectron kinetic energy are compared with theoretical calculations employing a multicentric basis set of B-spline functions and a Kohn-Sham Hamiltonian. In order to understand analogies and differences in the dichroism of structural isomers bearing the same functional groups, a comparison with previous PECD study of valence band of 2-amino-1-propanol is carried out.

  8. Circular piezoelectric bender laser tuners

    NASA Technical Reports Server (NTRS)

    Mcelroy, J. H.; Thompson, P. E.; Walker, H. E.; Johnson, E. H.; Radecki, D. J.; Reynolds, R. S.

    1972-01-01

    The circular piezoelectric bender laser tuner to replace conventional laser tuners when mirror diameters up to 0.50 inch are sufficient is described. The circular piezoelectric bender laser tuner offers much higher displacements per applied volt and permits laser control circuits to be fabricated using standard operational amplifiers, rather than the expensive high-voltage amplifiers required by conventional tuners. The cost of the device is more than one order of magnitude lower than conventional tuners and the device is very rugged with all mechanical resonances easily designed to be greater than 3kHz. In addition to its use as a laser frequency tuner, the circular bender tuner should find many applications in interferometers and similar devices.

  9. Evolutionary Mobility of the Ribosomal DNA Array in Yeasts

    PubMed Central

    Proux-Wéra, Estelle; Byrne, Kevin P.; Wolfe, Kenneth H.

    2013-01-01

    The ribosomal DNA (rDNA) of eukaryotes is organized as large tandem arrays. Here, we compare the genomic locations of rDNA among yeast species and show that, despite its huge size (>1 Mb), the rDNA array has moved around the genome several times within the family Saccharomycetaceae. We identify an ancestral, nontelomeric, rDNA site that is conserved across many species including Saccharomyces cerevisiae. Within the genus Lachancea, however, the rDNA apparently transposed from the ancestral site to a new site internal to a different chromosome, becoming inserted into a short intergenic region beside a tRNA gene. In at least four other yeast lineages, the rDNA moved from the ancestral site to telomeric locations. Remarkably, both the ancestral rDNA site and the new site in Lachancea are adjacent to protein-coding genes whose products maintain the specialized chromatin structure of rDNA (HMO1 and CDC14, respectively). In almost every case where the rDNA was lost from the ancestral site, the entire array disappeared without any other rearrangements in the region, leaving just an intergenic spacer of less than 2 kb. The mechanism by which this large and complex locus moves around the genome is unknown, but we speculate that it may involve the formation of double-strand DNA breaks by Fob1 protein or the formation of extrachromosomal rDNA circles. PMID:23419706

  10. Proteolysis in plasmid DNA stable maintenance in bacterial cells.

    PubMed

    Karlowicz, Anna; Wegrzyn, Katarzyna; Dubiel, Andrzej; Ropelewska, Malgorzata; Konieczny, Igor

    2016-07-01

    Plasmids, as extrachromosomal genetic elements, need to work out strategies that promote independent replication and stable maintenance in host bacterial cells. Their maintenance depends on constant formation and dissociation of nucleoprotein complexes formed on plasmid DNA. Plasmid replication initiation proteins (Rep) form specific complexes on direct repeats (iterons) localized within the plasmid replication origin. Formation of these complexes along with a strict control of Rep protein cellular concentration, quaternary structure, and activity, is essential for plasmid maintenance. Another important mechanism for maintenance of low-copy-number plasmids are the toxin-antitoxin (TA) post-segregational killing (psk) systems, which prevent plasmid loss from the bacterial cell population. In this mini review we discuss the importance of nucleoprotein complex processing by energy-dependent host proteases in plasmid DNA replication and plasmid type II toxin-antitoxin psk systems, and draw attention to the elusive role of DNA in this process.

  11. Paired circularly polarized heterodyne ellipsometer

    SciTech Connect

    Yu, C.-J.; Lin, C.-E.; Yu, L.-P.; Chou, C

    2009-02-01

    We develop a paired circularly polarized heterodyne ellipsometer (PCPHE), in which a heterodyne interferometer based on a two-frequency circularly polarized laser beam is set up. It belongs to an amplitude-sensitive ellipsometer that is able to provide not only a wider dynamic range of polarization modulation frequency but also a higher detection sensitivity than that of a conventional photometric ellipsometer. A real-time and precise measurement of ellipsometric parameters, which demonstrated an accuracy of less than 1 nm on thickness measurement of SiO2 thin film deposited on silicon substrate, can be applied with the PCPHE.

  12. Logistic regression for circular data

    NASA Astrophysics Data System (ADS)

    Al-Daffaie, Kadhem; Khan, Shahjahan

    2017-05-01

    This paper considers the relationship between a binary response and a circular predictor. It develops the logistic regression model by employing the linear-circular regression approach. The maximum likelihood method is used to estimate the parameters. The Newton-Raphson numerical method is used to find the estimated values of the parameters. A data set from weather records of Toowoomba city is analysed by the proposed methods. Moreover, a simulation study is considered. The R software is used for all computations and simulations.

  13. Mobile units of DNA in phytoplasma genomes.

    PubMed

    Dickinson, Matt

    2010-09-01

    Phytoplasmas are obligate symbionts of plants and insects that are responsible for significant yield losses in diverse crops. Genome sequencing has revealed that many phytoplasma genomes appear to contain repeated genes organized in units of approximately 20 kb. These 'potential mobile units' (PMUs) resemble composite replicative transposons. PMUs contain several genes for recombination and some also contain putative 'virulence genes'. Genome alignments suggest that PMUs are involved in phytoplasma genome instability and recombination. In this edition of Molecular Microbiology, Hogenhout and colleagues report that one PMU from the aster yellows phytoplasma strain Witches' Broom (AY-WB) can exist as both a linear PMU within the chromosome and as an extrachromosomal circular form. The copy number of the circular form is much higher in the insect vector compared with the plant, and expression levels of genes present on the PMU are also higher in the insect. These observations suggest not only that this PMU could be a mobile element, but that it could also be involved in a phase-variation mechanism that allows the phytoplasma to adapt to its different hosts.

  14. Maximal dinucleotide and trinucleotide circular codes.

    PubMed

    Michel, Christian J; Pellegrini, Marco; Pirillo, Giuseppe

    2016-01-21

    We determine here the number and the list of maximal dinucleotide and trinucleotide circular codes. We prove that there is no maximal dinucleotide circular code having strictly less than 6 elements (maximum size of dinucleotide circular codes). On the other hand, a computer calculus shows that there are maximal trinucleotide circular codes with less than 20 elements (maximum size of trinucleotide circular codes). More precisely, there are maximal trinucleotide circular codes with 14, 15, 16, 17, 18 and 19 elements and no maximal trinucleotide circular code having less than 14 elements. We give the same information for the maximal self-complementary dinucleotide and trinucleotide circular codes. The amino acid distribution of maximal trinucleotide circular codes is also determined.

  15. Unitary circular code motifs in genomes of eukaryotes.

    PubMed

    El Soufi, Karim; Michel, Christian J

    A set X of 20 trinucleotides was identified in genes of bacteria, eukaryotes, plasmids and viruses, which has in average the highest occurrence in reading frame compared to its two shifted frames (Michel, 2015; Arquès and Michel, 1996). This set X has an interesting mathematical property as X is a circular code (Arquès and Michel, 1996). Thus, the motifs from this circular code X, called X motifs, have the property to always retrieve, synchronize and maintain the reading frame in genes. The origin of this circular code X in genes is an open problem since its discovery in 1996. Here, we first show that the unitary circular codes (UCC), i.e. sets of one word, allow to generate unitary circular code motifs (UCC motifs), i.e. a concatenation of the same motif (simple repeats) leading to low complexity DNA. Three classes of UCC motifs are studied here: repeated dinucleotides (D(+) motifs), repeated trinucleotides (T(+) motifs) and repeated tetranucleotides (T(+) motifs). Thus, the D(+), T(+) and T(+) motifs allow to retrieve, synchronize and maintain a frame modulo 2, modulo 3 and modulo 4, respectively, and their shifted frames (1 modulo 2; 1 and 2 modulo 3; 1, 2 and 3 modulo 4 according to the C(2), C(3) and C(4) properties, respectively) in the DNA sequences. The statistical distribution of the D(+), T(+) and T(+) motifs is analyzed in the genomes of eukaryotes. A UCC motif and its comp lementary UCC motif have the same distribution in the eukaryotic genomes. Furthermore, a UCC motif and its complementary UCC motif have increasing occurrences contrary to their number of hydrogen bonds, very significant with the T(+) motifs. The longest D(+), T(+) and T(+) motifs in the studied eukaryotic genomes are also given. Surprisingly, a scarcity of repeated trinucleotides (T(+) motifs) in the large eukaryotic genomes is observed compared to the D(+) and T(+) motifs. This result has been investigated and may be explained by two outcomes. Repeated trinucleotides (T(+) motifs

  16. Extrachromosomal elements in lower eukaryotes:

    SciTech Connect

    Wickner, R.B.; Hinnebusch, A.; Lambowitz, A.M.; Gunsalus, I.C.; Hollaender, A.

    1986-01-01

    While most genes are chromosomal, the nonchromosomal genes have played a disproportionate role in molecular biology, in part because of their easy accessibility and in part because they represent the most mobile portion of a cell's genome. Fungi, yeasts, protozoa, slime molds, algae, and other single-celled nucleated species, have recently gained dramatic popularity with the development of transformation methods for Saccharomyces, Neurospora, Schizosaccharomyces, Dictyostelium, and others of this group. The realization that Saccharomyces has oncogenes, RNA tumor viruses, intervening sequences, and all the mitotic, mitochondrial, and other structures typical of so-called ''higher'' eukaryotic organisms has confirmed the use of such organisms as model systems. Their use in biotechnology also shows great promise. The study in lower eukaryotes of mitochondria and chloroplasts has yielded many insights into similar structures in higher organisms as well as many unexpected finds, such as mechanisms of intron excision and the biology of introns, RNA catalysis, variation of the genetic code, and mechanisms of protein import across membranes.

  17. Class IIc or Circular Bacteriocins

    NASA Astrophysics Data System (ADS)

    Martin-Visscher, Leah A.; van Belkum, Marco J.; Vederas, John C.

    The circular bacteriocins produced by Gram-positive bacteria represent a diverse class of antimicrobial peptides. These bacteriocins display enhanced stability compared to linear bacteriocins, which arises from their characteristic circular backbone. Currently, eight unique circular bacteriocins have been identified, and analysis of their gene clusters indicates that they likely utilize complex mechanisms for maturation and secretion, as well as for immunity. These bacteriocins target the cytoplasmic membrane of sensitive cells, leading to pore formation that results in loss of ions, dissipation of membrane potential, and ultimately, cell death. Structural studies suggest that despite variation in their sequences, most of these bacteriocins likely adopt a common three-dimensional architecture, consisting of four or five tightly packed helices encompassing a hydrophobic core. There are many mysteries surrounding the biosynthesis of these peptides, particularly in regard to the mechanism by which they are cyclized. Elucidation of such a mechanism may provide exciting new approaches to the bioengineering of new, stable, and antimicrobially active circular peptides.

  18. Accelerator Science: Circular vs. Linear

    ScienceCinema

    Lincoln, Don

    2016-12-14

    Particle accelerator are scientific instruments that allow scientists to collide particles together at incredible energies to study the secrets of the universe. However, there are many manners in which particle accelerators can be constructed. In this video, Fermilab’s Dr. Don Lincoln explains the pros and cons of circular and linear accelerators.

  19. A Circular Education Organizational Structure.

    ERIC Educational Resources Information Center

    Hollander, Jim

    1993-01-01

    The circular nonhierarchical organization of the Hishkoonikun local education authority in Kashechewan, Ontario (Canada), makes students the central focus of educational decision making, provides equality and improves accountability and responsibility among various educational stakeholders, and reflects Native cultural values and traditions. (SV)

  20. Stress Analysis of Circular Frames

    NASA Technical Reports Server (NTRS)

    Fahlbusch, H; Wegner, W

    1941-01-01

    The stresses in circular frames of constant bending stiffnesses, as encountered in thin-wall shells, are investigated from the point of view of finite depth of sectional area of frame. The solution is carried out for four fundamental load conditions. The method is illustrated on a worked out example.

  1. Accelerator Science: Circular vs. Linear

    SciTech Connect

    Lincoln, Don

    2016-11-10

    Particle accelerator are scientific instruments that allow scientists to collide particles together at incredible energies to study the secrets of the universe. However, there are many manners in which particle accelerators can be constructed. In this video, Fermilab’s Dr. Don Lincoln explains the pros and cons of circular and linear accelerators.

  2. Food Analysis Using Organelle DNA and the Effects of Processing on Assays.

    PubMed

    Caldwell, Jane M

    2017-02-28

    Extrachromosomal DNA such as organelle DNA are increasingly targeted in molecular detection assays where samples have been degraded by physical or chemical means. Owing to multiple organelles per cell and greater copy numbers than nuclear genes, organelle gene targets provide a more robust signal in polymerase chain reaction (PCR), quantitative PCR (qPCR), and other emerging molecular technologies. Because of these advantages, direct analysis of organelle DNA in food matrices is used for detection of contaminants and identification and authentication of food ingredients and allergens. Non-nuclear DNA is also used as an assay normalizer for detection of genetically modified organisms (GMOs) in foods. This review describes these protocols plus the effects of processing on efficacy, with special emphasis on thermally produced DNA fragmentation. Future research may incorporate molecular techniques beyond detection, used instead as time-temperature indicators in thermal food processing or quality indicators in food fermentation or acidification.

  3. 21 CFR 606.122 - Instruction circular.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... instruction circular shall contain: (1) Instructions to administer a suitable plasma volume expander if Red... circular shall contain: (1) The approximate volume of plasma from which a sample unit of Platelets is... entering the container. (m) For Plasma, the instruction circular shall contain: (1) A warning against...

  4. Isolation of a complete circular virus genome sequence from an Alaskan black-capped chickadee (Poecile atricapillus) gastrointestinal tract sample.

    USGS Publications Warehouse

    Hanna, Zachary R.; Runckel, Charles; Fuchs, Jerome; DeRisi, Joseph L.; Mindell, David P.; Van Hemert, Caroline R.; Handel, Colleen M.; Dumbacher, John P.

    2015-01-01

    We report here the genome sequence of a circular virus isolated from samples of an Alaskan black-capped chickadee (Poecile atricapillus) gastrointestinal tract. The genome is 2,152 bp in length and is most similar (30 to 44.5% amino acid identity) to the genome sequences of other single-stranded DNA (ssDNA) circular viruses belonging to the gemycircularvirus group.

  5. Differentiating fragmented human and nonhuman long bone using osteon circularity.

    PubMed

    Crescimanno, Annamaria; Stout, Sam D

    2012-03-01

    Distinguishing between human and nonhuman bone is important in forensic anthropology and archeology when remains are fragmentary and DNA cannot be obtained. Histological examination of bone is affordable and practical in such situations. This study suggests using osteon circularity to distinguish human bone fragments and hypothesizes that osteons will more closely resemble a perfect circle in nonhumans than in humans. Standard histological methods were used, and circularity was determined using an image analysis program, where circularity was controlled for by Haversian canal measurements. Homogeneity was first tested for multiple variables within human and nonhuman samples. No significant differences were found between human sexes (p = 0.657) or among nonhuman species (p = 0.553). Significant differences were found among intraskeletal elements of both humans (p = 0.016) and nonhumans (p = 0.013) and between pooled samples of humans and nonhumans (p < 0.001). Results of this study indicate that osteon circularity can be used to distinguish between fragmented human and nonhuman long bone.

  6. Circular on planned parenthood, 1987.

    PubMed

    1987-01-01

    In 1987 fourteen units of the Government of Henan issued a Circular stating that: "Planned parenthood must be publicized deep into the grass roots and among the people, and importance must be attached to results." The Circular stresses: "In the propaganda drive, it is necessary to successfully grasp three key links: 1. It is necessary to disseminate intensively the important directive on population problems that is contained in the report of the 13th CPC National Congress and the seriousness of the population situation of our country and province so that the cadres and the masses can understand the relationship between population control and the achievement of the strategic target of the three big steps, understand the reason for carrying out planned parenthood, understand that the one-child policy is still advocated, and conscientiously carry out planned parenthood. 2. It is essential to succeed in propagating knowledge of contraception, sterilization, childbirth, and child care and in conducting ideological education for those who undergo operations and for their family members. 3. It is imperative to visit those who have undergone operations and to help them solve practically their difficulties in making a living." The Circular concludes by demanding that under the unified leadership of party committees and governments at all levels, the propaganda drive be carried out by relying on the efforts of all of society. In conjunction with their own work, departments, including the propaganda, education, public health, and cultural departments, must carry out propaganda and education for planned parenthood.

  7. Accuracy and efficiency define Bxb1 integrase as the best of fifteen candidate serine recombinases for the integration of DNA into the human genome

    PubMed Central

    2013-01-01

    Background Phage-encoded serine integrases, such as φC31 integrase, are widely used for genome engineering. Fifteen such integrases have been described but their utility for genome engineering has not been compared in uniform assays. Results We have compared fifteen serine integrases for their utility for DNA manipulations in mammalian cells after first demonstrating that all were functional in E. coli. Chromosomal recombination reporters were used to show that seven integrases were active on chromosomally integrated DNA in human fibroblasts and mouse embryonic stem cells. Five of the remaining eight enzymes were active on extra-chromosomal substrates thereby demonstrating that the ability to mediate extra-chromosomal recombination is no guide to ability to mediate site-specific recombination on integrated DNA. All the integrases that were active on integrated DNA also promoted DNA integration reactions that were not mediated through conservative site-specific recombination or damaged the recombination sites but the extent of these aberrant reactions varied over at least an order of magnitude. Bxb1 integrase yielded approximately two-fold more recombinants and displayed about two fold less damage to the recombination sites than the next best recombinase; φC31 integrase. Conclusions We conclude that the Bxb1 and φC31 integrases are the reagents of choice for genome engineering in vertebrate cells and that DNA damage repair is a major limitation upon the utility of this class of site-specific recombinase. PMID:24139482

  8. Blowing DNA bubbles.

    PubMed

    Severin, N; Zhuang, W; Ecker, C; Kalachev, A A; Sokolov, I M; Rabe, J P

    2006-11-01

    We report here experimental observations which indicate that topologically or covalently formed polymer loops embedded in an ultrathin liquid film on a solid substrate can be "blown" into circular "bubbles" during scanning force microscopy (SFM) imaging. In particular, supercoiled vector DNA has been unraveled, moved, stretched, and overstretched to two times its B-form length and then torn apart. We attribute the blowing of the DNA bubbles to the interaction of the tapping SFM tip with the ultrathin liquid film.

  9. Longitudinal motion in circular accelerators

    SciTech Connect

    Cole, F.T.

    1985-01-01

    A general description is given of the longitudinal motion of an idealized synchronous particle, exactly in step with the radiofrequency field of a circular accelerator, and a domain in phase and energy, called a bucket, around this particle within which particles are focused around the synchronous particle. This general picture is then made more precise and quantitative. The equations of longitudinal motion and their solutions and the resulting motion are discussed, followed by applications and amplication of the theory. 7 refs., 8 figs. (LEW)

  10. New branched DNA constructs.

    PubMed

    Chandra, Madhavaiah; Keller, Sascha; Gloeckner, Christian; Bornemann, Benjamin; Marx, Andreas

    2007-01-01

    The Watson-Crick base pairing of DNA is an advantageous phenomenon that can be exploited when using DNA as a scaffold for directed self-organization of nanometer-sized objects. Several reports have appeared in the literature that describe the generation of branched DNA (bDNA) with variable numbers of arms that self-assembles into predesigned architectures. These bDNA units are generated by using cleverly designed rigid crossover DNA molecules. Alternatively, bDNA can be generated by using synthetic branch points derived from either nucleoside or non-nucleoside building blocks. Branched DNA has scarcely been explored for use in nanotechnology or from self-assembling perspectives. Herein, we wish to report our results for the synthesis, characterization, and assembling properties of asymmetrical bDNA molecules that are able to generate linear and circular bDNA constructs. Our strategy for the generation of bDNA is based on a branching point that makes use of a novel protecting-group strategy. The bDNA units were generated by means of automated DNA synthesis methods and were used to generate novel objects by employing chemical and biological techniques. The entities generated might be useful building blocks for DNA-based nanobiotechnology.

  11. Segrosome Complex Formation during DNA Trafficking in Bacterial Cell Division.

    PubMed

    Oliva, María A

    2016-01-01

    Bacterial extrachromosomal DNAs often contribute to virulence in pathogenic organisms or facilitate adaptation to particular environments. The transmission of genetic information from one generation to the next requires sufficient partitioning of DNA molecules to ensure that at least one copy reaches each side of the division plane and is inherited by the daughter cells. Segregation of the bacterial chromosome occurs during or after replication and probably involves a strategy in which several protein complexes participate to modify the folding pattern and distribution first of the origin domain and then of the rest of the chromosome. Low-copy number plasmids rely on specialized partitioning systems, which in some cases use a mechanism that show striking similarity to eukaryotic DNA segregation. Overall, there have been multiple systems implicated in the dynamic transport of DNA cargo to a new cellular position during the cell cycle but most seem to share a common initial DNA partitioning step, involving the formation of a nucleoprotein complex called the segrosome. The particular features and complex topologies of individual segrosomes depend on both the nature of the DNA binding protein involved and on the recognized centromeric DNA sequence, both of which vary across systems. The combination of in vivo and in vitro approaches, with structural biology has significantly furthered our understanding of the mechanisms underlying DNA trafficking in bacteria. Here, I discuss recent advances and the molecular details of the DNA segregation machinery, focusing on the formation of the segrosome complex.

  12. Experimental Differential Light-Scattering Correction to the Circular Dichroism of Bacteriophage T2

    PubMed Central

    Dorman, Burton P.; Maestre, Marcos F.

    1973-01-01

    Experimental techniques are presented that can be used to assay and correct for differential light scattering effects in circular dichroism spectra of biological macrostructures. The assay is based upon use of variable detector geometries that collect light over large solid angles. Disrupted T2 virus suspensions and purified T2 phage DNA exhibit geometry-independent spectra; the spectrum of intact T2 virus is highly sensitive to detection geometry. On the basis of spectra obtained after light-scattering correction, the structure of T2 DNA in the phage particle is assigned to the C form. We conclude that: (i) The measured circular dichroism of a light-scattering specimen may be highly sensitive to light-detection geometry of the instrument. This effect is indicative of differential scattering intensity for left and right circularly polarized light. (ii) Some optically active particles, although they scatter light intensely, exhibit circular dichroism that is independent of detection geometry and, therefore, apparently uninfluenced by differential light scattering. We infer that whether differential light scattering arises may depend upon the presence or absence of ordered asymmetry in the organization of the scattering particle. (iii) The circular dichroism of any light-scattering specimen should be measured again in apparatus designed for differential light-scattering correction as a prerequisite to meaningful structural conclusions. (iv) Differential scattering effects in circular dichroism may be potentially useful as a probe for large-order organization of the scattering particle. PMID:4509658

  13. Dinucleotide circular codes and bijective transformations.

    PubMed

    Fimmel, Elena; Giannerini, Simone; Gonzalez, Diego Luis; Strüngmann, Lutz

    2015-12-07

    The presence of circular codes in mRNA coding sequences is postulated to be involved in informational mechanisms aimed at detecting and maintaining the normal reading frame during protein synthesis. Most of the recent research is focused on trinucleotide circular codes. However, also dinucleotide circular codes are important since dinucleotides are ubiquitous in genomes and associated to important biological functions. In this work we adopt the group theoretic approach used for trinucleotide codes in Fimmel et al. (2015) to study dinucleotide circular codes and highlight their symmetry properties. Moreover, we characterize such codes in terms of n-circularity and provide a graph representation that allows to visualize them geometrically. The results establish a theoretical framework for the study of the biological implications of dinucleotide circular codes in genomic sequences.

  14. The tra locus of streptomycete plasmid pIJ101 mediates efficient transfer of a circular but not a linear version of the same replicon.

    PubMed

    Wang, Jing; Pettis, Gregg S

    2010-09-01

    Conjugal transfer of circular plasmids in Streptomyces involves a unique mechanism employing few plasmid-encoded loci and the transfer of double-stranded DNA by an as yet uncharacterized intercellular route. Efficient transfer of the circular streptomycete plasmid pIJ101 requires only two plasmid loci: the pIJ101 tra gene, and as a cis-acting function known as clt. Here, we compared the ability of the pIJ101 transfer apparatus to promote conjugal transfer of circular versus linear versions of the same replicon. While the pIJ101 tra locus readily transferred the circular form of the replicon, the linear version was transferred orders of magnitude less efficiently and all plasmids isolated from the transconjugants were circular, regardless of their original configuration in the donor. Additionally, relatively rare circularization of linear plasmids was detectable in the donor cells, which is consistent with the notion that this event was a prerequisite for transfer by TraB(pIJ101). Linear versions of this same replicon did transfer efficiently, in that configuration, from strains containing the conjugative linear plasmid SLP2. Our data indicate that functions necessary and sufficient for transfer of circular DNA were insufficient for transfer of a related linear DNA molecule. The results here suggest that the conjugation mechanisms of linear versus circular DNA in Streptomyces spp. are inherently different and/or that efficient transfer of linear DNA requires additional components.

  15. Circular permutant GFP insertion folding reporters

    SciTech Connect

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2013-04-16

    Provided are methods of assaying and improving protein folding using circular permutants of fluorescent proteins, including circular permutants of GFP variants and combinations thereof. The invention further provides various nucleic acid molecules and vectors incorporating such nucleic acid molecules, comprising polynucleotides encoding fluorescent protein circular permutants derived from superfolder GFP, which polynucleotides include an internal cloning site into which a heterologous polynucleotide may be inserted in-frame with the circular permutant coding sequence, and which when expressed are capable of reporting on the degree to which a polypeptide encoded by such an inserted heterologous polynucleotide is correctly folded by correlation with the degree of fluorescence exhibited.

  16. Circular permutant GFP insertion folding reporters

    DOEpatents

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2008-06-24

    Provided are methods of assaying and improving protein folding using circular permutants of fluorescent proteins, including circular permutants of GFP variants and combinations thereof. The invention further provides various nucleic acid molecules and vectors incorporating such nucleic acid molecules, comprising polynucleotides encoding fluorescent protein circular permutants derived from superfolder GFP, which polynucleotides include an internal cloning site into which a heterologous polynucleotide may be inserted in-frame with the circular permutant coding sequence, and which when expressed are capable of reporting on the degree to which a polypeptide encoded by such an inserted heterologous polynucleotide is correctly folded by correlation with the degree of fluorescence exhibited.

  17. Circular permutant GFP insertion folding reporters

    DOEpatents

    Waldo, Geoffrey S; Cabantous, Stephanie

    2013-02-12

    Provided are methods of assaying and improving protein folding using circular permutants of fluorescent proteins, including circular permutants of GFP variants and combinations thereof. The invention further provides various nucleic acid molecules and vectors incorporating such nucleic acid molecules, comprising polynucleotides encoding fluorescent protein circular permutants derived from superfolder GFP, which polynucleotides include an internal cloning site into which a heterologous polynucleotide may be inserted in-frame with the circular permutant coding sequence, and which when expressed are capable of reporting on the degree to which a polypeptide encoded by such an inserted heterologous polynucleotide is correctly folded by correlation with the degree of fluorescence exhibited.

  18. Circular permutant GFP insertion folding reporters

    DOEpatents

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2011-06-14

    Provided are methods of assaying and improving protein folding using circular permutants of fluorescent proteins, including circular permutants of GFP variants and combinations thereof. The invention further provides various nucleic acid molecules and vectors incorporating such nucleic acid molecules, comprising polynucleotides encoding fluorescent protein circular permutants derived from superfolder GFP, which polynucleotides include an internal cloning site into which a heterologous polynucleotide may be inserted in-frame with the circular permutant coding sequence, and which when expressed are capable of reporting on the degree to which a polypeptide encoded by such an inserted heterologous polynucleotide is correctly folded by correlation with the degree of fluorescence exhibited.

  19. Inactivation of p53 Rescues the Maintenance of High Risk HPV DNA Genomes Deficient in Expression of E6

    PubMed Central

    Lorenz, Laurel D.; Rivera Cardona, Jessenia; Lambert, Paul F.

    2013-01-01

    The human papillomavirus DNA genome undergoes three distinct stages of replication: establishment, maintenance and amplification. We show that the HPV16 E6 protein is required for the maintenance of the HPV16 DNA genome as an extrachromosomal, nuclear plasmid in its natural host cell, the human keratinocyte. Based upon mutational analyses, inactivation of p53 by E6, but not necessarily E6-mediated degradation of p53, was found to correlate with the ability of E6 to support maintenance of the HPV16 genome as a nuclear plasmid. Inactivation of p53 with dominant negative p53 rescued the ability of HPV16 E6STOP and E6SAT mutant genomes to replicate as extrachromosomal genomes, though not to the same degree as observed for the HPV16 E6 wild-type (WT) genome. Inactivation of p53 also rescued the ability of HPV18 and HPV31 E6-deficient genomes to be maintained at copy numbers comparable to that of HPV18 and HPV31 E6WT genomes at early passages, though upon further passaging copy numbers for the HPV18 and 31 E6-deficient genomes lessened compared to that of the WT genomes. We conclude that inactivation of p53 is necessary for maintenance of HPV16 and for HPV18 and 31 to replicate at WT copy number, but that additional functions of E6 independent of inactivating p53 must also contribute to the maintenance of these genomes. Together these results suggest that re-activation of p53 may be a possible means for eradicating extrachromosomal HPV16, 18 or 31 genomes in the context of persistent infections. PMID:24204267

  20. The localization of ribosomal DNA in Sciaridae (Diptera: Nematocera) reassessed.

    PubMed

    Madalena, Christiane Rodriguez Gutierrez; Amabis, José Mariano; Stocker, Ann Jacob; Gorab, Eduardo

    2007-01-01

    The chromosomal localization of ribosomal DNA (rDNA) was studied in polytene and diploid tissues of four sciarid species, Trichosia pubescens, Rhynchosciara americana, R. milleri and Schwenkfeldina sp. While hybridization to mitotic chromosomes showed the existence of a single rDNA locus, ribosomal probes hybridized to more than one polytene chromosome region in all the species analyzed as a result of micronucleolar attachment to specific chromosome sites. Micronucleoli are small, round bodies containing transcriptionally active, probably extrachromosomal rDNA. In T. pubescens the rDNA is predominantly localized in chromosome sections X-10 and X-8. In R. americana the rDNA is frequently found associated with centromeric heterochromatin of the chromosomes X, C, B and A, and also with sections X-1 and B-13. Ribosomal probes in R. milleri hybridized with high frequency to pericentric and telomeric regions of its polytene complement. Schwfenkfeldina sp. displays a remarkably unusual distribution of rDNA in polytene nuclei, characterized by the attachment of micronucleoli to many chromosome regions. The results showed that micronucleoli preferentially associate with intercalary or terminal heterochromatin of all sciarid flies analyzed and, depending on the species, are attached to a few (Trichosia), moderate (Rhynchosciara) or a large (Schwenkfeldina sp.) number of polytene chromosome sites.

  1. Tests on Stiffened Circular Cylinders

    NASA Technical Reports Server (NTRS)

    Holt, Marshall

    1941-01-01

    Compressive tests were made of two series of stiffened circular cylindrical shells under axial load. All the shells were 16 inches in diameter by 24 inches in length and were made of aluminum-alloy sheet curved to the proper radius and welded with one longitudinal weld. The ratios of diameter to thickness of shell wall in the two series of specimens were 258 and 572. Strains were measured with Huggenberger tensometers at a number of gage lines on the stiffeners and shell. The results of these tests indicate that a spacing of circumferential stiffeners equal to 0.67 times the radius is too great to strengthen the shell wall appreciably. The results are not inclusive enough to show the optimum in stiffeners. Plain cylinders without stiffeners developed ultimate strengths approximately half as great as the buckling strengths computed by the equation resulting from the classical theory and slightly greater than those computed by Donnell's large deflection theory.

  2. Respiratory-deficient mutants of Torulopsis glabrata, a yeast with circular mitochondrial deoxyribonucleic acid of 6 mu m.

    PubMed Central

    O'Connor, R M; McArthur, C R; Clark-Walker, G D

    1976-01-01

    Purified mitochondria from the petite positive yeast Torulopsis glabrata contain a circular deoxyribonucleic acid (DNA) with a length of 6 mum and a buoyant density of 1.686 g/cm3. This DNA is absent from ethidium bromide induced respiratory-deficient mutants. Images PMID:944184

  3. Bijective transformation circular codes and nucleotide exchanging RNA transcription.

    PubMed

    Michel, Christian J; Seligmann, Hervé

    2014-04-01

    The C(3) self-complementary circular code X identified in genes of prokaryotes and eukaryotes is a set of 20 trinucleotides enabling reading frame retrieval and maintenance, i.e. a framing code (Arquès and Michel, 1996; Michel, 2012, 2013). Some mitochondrial RNAs correspond to DNA sequences when RNA transcription systematically exchanges between nucleotides (Seligmann, 2013a,b). We study here the 23 bijective transformation codes ΠX of X which may code nucleotide exchanging RNA transcription as suggested by this mitochondrial observation. The 23 bijective transformation codes ΠX are C(3) trinucleotide circular codes, seven of them are also self-complementary. Furthermore, several correlations are observed between the Reading Frame Retrieval (RFR) probability of bijective transformation codes ΠX and the different biological properties of ΠX related to their numbers of RNAs in GenBank's EST database, their polymerization rate, their number of amino acids and the chirality of amino acids they code. Results suggest that the circular code X with the functions of reading frame retrieval and maintenance in regular RNA transcription, may also have, through its bijective transformation codes ΠX, the same functions in nucleotide exchanging RNA transcription. Associations with properties such as amino acid chirality suggest that the RFR of X and its bijective transformations molded the origins of the genetic code's machinery. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  4. Circular permutation of a synthetic eukaryotic chromosome with the telomerator

    PubMed Central

    Mitchell, Leslie A.; Boeke, Jef D.

    2014-01-01

    Chromosome engineering is a major focus in the fields of systems biology, genetics, synthetic biology, and the functional analysis of genomes. Here, we describe the “telomerator,” a new synthetic biology device for use in Saccharomyces cerevisiae. The telomerator is designed to inducibly convert circular DNA molecules into mitotically stable, linear chromosomes replete with functional telomeres in vivo. The telomerator cassette encodes convergent yeast telomere seed sequences flanking the I-SceI homing endonuclease recognition site in the center of an intron artificially transplanted into the URA3 selectable/counterselectable auxotrophic marker. We show that inducible expression of the homing endonuclease efficiently generates linear molecules, identified by using a simple plate-based screening method. To showcase its functionality and utility, we use the telomerator to circularly permute a synthetic yeast chromosome originally constructed as a circular molecule, synIXR, to generate 51 linear variants. Many of the derived linear chromosomes confer unexpected phenotypic properties. This finding indicates that the telomerator offers a new way to study the effects of gene placement on chromosomes (i.e., telomere proximity). However, that the majority of synIXR linear derivatives support viability highlights inherent tolerance of S. cerevisiae to changes in gene order and overall chromosome structure. The telomerator serves as an important tool to construct artificial linear chromosomes in yeast; the concept can be extended to other eukaryotes. PMID:25378705

  5. DNA supercoiling and its role in DNA decatenation and unknotting

    PubMed Central

    Witz, Guillaume; Stasiak, Andrzej

    2010-01-01

    Chromosomal and plasmid DNA molecules in bacterial cells are maintained under torsional tension and are therefore supercoiled. With the exception of extreme thermophiles, supercoiling has a negative sign, which means that the torsional tension diminishes the DNA helicity and facilitates strand separation. In consequence, negative supercoiling aids such processes as DNA replication or transcription that require global- or local-strand separation. In extreme thermophiles, DNA is positively supercoiled which protects it from thermal denaturation. While the role of DNA supercoiling connected to the control of DNA stability, is thoroughly researched and subject of many reviews, a less known role of DNA supercoiling emerges and consists of aiding DNA topoisomerases in DNA decatenation and unknotting. Although DNA catenanes are natural intermediates in the process of DNA replication of circular DNA molecules, it is necessary that they become very efficiently decatenated, as otherwise the segregation of freshly replicated DNA molecules would be blocked. DNA knots arise as by-products of topoisomerase-mediated intramolecular passages that are needed to facilitate general DNA metabolism, including DNA replication, transcription or recombination. The formed knots are, however, very harmful for cells if not removed efficiently. Here, we overview the role of DNA supercoiling in DNA unknotting and decatenation. PMID:20026582

  6. 76 FR 60590 - Environmental Justice; Proposed Circular

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-09-29

    ... Federal Transit Administration Environmental Justice; Proposed Circular AGENCY: Federal Transit... guidance in the form of a Circular on incorporating environmental justice principles into plans, projects... recipients of FTA funds on how to fully engage environmental justice populations in the public transportation...

  7. A Random Walk on a Circular Path

    ERIC Educational Resources Information Center

    Ching, W.-K.; Lee, M. S.

    2005-01-01

    This short note introduces an interesting random walk on a circular path with cards of numbers. By using high school probability theory, it is proved that under some assumptions on the number of cards, the probability that a walker will return to a fixed position will tend to one as the length of the circular path tends to infinity.

  8. A Random Walk on a Circular Path

    ERIC Educational Resources Information Center

    Ching, W.-K.; Lee, M. S.

    2005-01-01

    This short note introduces an interesting random walk on a circular path with cards of numbers. By using high school probability theory, it is proved that under some assumptions on the number of cards, the probability that a walker will return to a fixed position will tend to one as the length of the circular path tends to infinity.

  9. Creation operators for Cartesian and circular beams.

    PubMed

    Siguenza-Torres, Anibal; Gutiérrez-Vega, Julio C

    2016-05-01

    Creation operators of fractional order, to derive the general Cartesian beams and circular beams from the lowest-order Gaussian beam, are introduced and discussed. Finding the creation operator for these general cases is a way to find the creation operator of all the special cases of Cartesian and circular beams.

  10. A lateral flow biosensor for detection of single nucleotide polymorphism by circular strand displacement reaction.

    PubMed

    Xiao, Zhuo; Lie, Puchang; Fang, Zhiyuan; Yu, Luxin; Chen, Junhua; Liu, Jie; Ge, Chenchen; Zhou, Xuemeng; Zeng, Lingwen

    2012-09-04

    A lateral flow biosensor for detection of single nucleotide polymorphism based on circular strand displacement reaction (CSDPR) has been developed. Taking advantage of high fidelity of T4 DNA ligase, signal amplification by CSDPR, and the optical properties of gold nanoparticles, this assay has reached a detection limit of 0.01 fM.

  11. Gauge-Invariant Formulation of Circular Dichroism.

    PubMed

    Raimbault, Nathaniel; de Boeij, Paul L; Romaniello, Pina; Berger, J A

    2016-07-12

    Standard formulations of magnetic response properties, such as circular dichroism spectra, are plagued by gauge dependencies, which can lead to unphysical results. In this work, we present a general gauge-invariant and numerically efficient approach for the calculation of circular dichroism spectra from the current density. First we show that in this formulation the optical rotation tensor, the response function from which circular dichroism spectra can be obtained, is independent of the origin of the coordinate system. We then demonstrate that its trace is independent of the gauge origin of the vector potential. We also show how gauge invariance can be retained in practical calculations with finite basis sets. As an example, we explain how our method can be applied to time-dependent current-density-functional theory. Finally, we report gauge-invariant circular dichroism spectra obtained using the adiabatic local-density approximation. The circular dichroism spectra we thus obtain are in good agreement with experiment.

  12. Circular motion in NUT space-time

    NASA Astrophysics Data System (ADS)

    Jefremov, Paul I.; Perlick, Volker

    2016-12-01

    We consider circular motion in the NUT (Newman-Unti-Tamburino) space-time. Among other things, we determine the location of circular time-like geodesic orbits, in particular of the innermost stable circular orbit (ISCO) and of the marginally bound circular orbit. Moreover, we discuss the von Zeipel cylinders with respect to the stationary observers and with respect to the zero angular momentum observers (ZAMOs). We also investigate the relation of von Zeipel cylinders to inertial forces, in particular in the ultra-relativistic limit. Finally, we generalise the construction of thick accretion tori (‘Polish doughnuts’) which are well known on the Schwarzschild or Kerr background to the case of the NUT metric. We argue that, in principle, an NUT source could be distinguished from a Schwarzschild or Kerr source by observing the features of circular matter flows in its neighbourhood.

  13. Local chromatin structure of heterochromatin regulates repeated DNA stability, nucleolus structure, and genome integrity

    SciTech Connect

    Peng, Jamy C.

    2007-01-01

    Heterochromatin constitutes a significant portion of the genome in higher eukaryotes; approximately 30% in Drosophila and human. Heterochromatin contains a high repeat DNA content and a low density of protein-encoding genes. In contrast, euchromatin is composed mostly of unique sequences and contains the majority of single-copy genes. Genetic and cytological studies demonstrated that heterochromatin exhibits regulatory roles in chromosome organization, centromere function and telomere protection. As an epigenetically regulated structure, heterochromatin formation is not defined by any DNA sequence consensus. Heterochromatin is characterized by its association with nucleosomes containing methylated-lysine 9 of histone H3 (H3K9me), heterochromatin protein 1 (HP1) that binds H3K9me, and Su(var)3-9, which methylates H3K9 and binds HP1. Heterochromatin formation and functions are influenced by HP1, Su(var)3-9, and the RNA interference (RNAi) pathway. My thesis project investigates how heterochromatin formation and function impact nuclear architecture, repeated DNA organization, and genome stability in Drosophila melanogaster. H3K9me-based chromatin reduces extrachromosomal DNA formation; most likely by restricting the access of repair machineries to repeated DNAs. Reducing extrachromosomal ribosomal DNA stabilizes rDNA repeats and the nucleolus structure. H3K9me-based chromatin also inhibits DNA damage in heterochromatin. Cells with compromised heterochromatin structure, due to Su(var)3-9 or dcr-2 (a component of the RNAi pathway) mutations, display severe DNA damage in heterochromatin compared to wild type. In these mutant cells, accumulated DNA damage leads to chromosomal defects such as translocations, defective DNA repair response, and activation of the G2-M DNA repair and mitotic checkpoints that ensure cellular and animal viability. My thesis research suggests that DNA replication, repair, and recombination mechanisms in heterochromatin differ from those in

  14. Characterization of Circular Plasmid Dimers in Borrelia burgdorferi

    PubMed Central

    Tilly, Kit; Lubke, Lori; Rosa, Patricia

    1998-01-01

    We have inactivated the ospC, oppAIV, and guaB genes on the 26-kb circular plasmid of Borrelia burgdorferi (cp26) by allelic exchange. On several occasions following such transformations, the cp26 of transformants had an aberrant mobility through agarose gels. Characterization of these cp26 molecules showed that the plasmid had dimerized. These dimers were quite stable during either selective or nonselective passage. Subsequent transformations with dimer DNA supported the hypothesis that in B. burgdorferi, transforming cp26 DNA most likely does not displace the resident homologous plasmid but rather must recombine in order to donate sequences that it carries. These serendipitous findings provide a mechanism for obtaining heterozygous complemented control strains when mutant phenotypes are characterized. PMID:9791118

  15. MARS: improving multiple circular sequence alignment using refined sequences.

    PubMed

    Ayad, Lorraine A K; Pissis, Solon P

    2017-01-14

    A fundamental assumption of all widely-used multiple sequence alignment techniques is that the left- and right-most positions of the input sequences are relevant to the alignment. However, the position where a sequence starts or ends can be totally arbitrary due to a number of reasons: arbitrariness in the linearisation (sequencing) of a circular molecular structure; or inconsistencies introduced into sequence databases due to different linearisation standards. These scenarios are relevant, for instance, in the process of multiple sequence alignment of mitochondrial DNA, viroid, viral or other genomes, which have a circular molecular structure. A solution for these inconsistencies would be to identify a suitable rotation (cyclic shift) for each sequence; these refined sequences may in turn lead to improved multiple sequence alignments using the preferred multiple sequence alignment program. We present MARS, a new heuristic method for improving Multiple circular sequence Alignment using Refined Sequences. MARS was implemented in the C++ programming language as a program to compute the rotations (cyclic shifts) required to best align a set of input sequences. Experimental results, using real and synthetic data, show that MARS improves the alignments, with respect to standard genetic measures and the inferred maximum-likelihood-based phylogenies, and outperforms state-of-the-art methods both in terms of accuracy and efficiency. Our results show, among others, that the average pairwise distance in the multiple sequence alignment of a dataset of widely-studied mitochondrial DNA sequences is reduced by around 5% when MARS is applied before a multiple sequence alignment is performed. Analysing multiple sequences simultaneously is fundamental in biological research and multiple sequence alignment has been found to be a popular method for this task. Conventional alignment techniques cannot be used effectively when the position where sequences start is arbitrary. We present

  16. Circular on family planning, 1988.

    PubMed

    1988-01-01

    This Hubei, China, Circular, issued near the end of 1988, provides the following: "The population growth situation in our country is grim. Since 1986, the natural population growth rate has risen continuously. To draw the prompt attention of the whole party and the entire people to the issue of our population, all localities must seriously unfold the activities of publicizing family planning (FP) this winter and next spring, in coordination with education in current affairs. It is necessary to publicize FP in an all-around way and with accuracy, and the activities of publicizing must be carried out effectively in a solid and deep-going way. In the rural areas, stress must be placed on areas where FP work is not carried out well and where there is a prevailing tendency toward early marriage, early child-bearing, and extra-budgetary births. In cities, publicity and education must be conducted especially among the transient population, individual households, and jobless households. During the period of publicity, large-scale street-corner publicity activities must be carried out in cities and towns so as to create strong public opinion and to combine the endeavor to publicize current affairs and policies with the effort to popularize knowledge about contraception and birth-control, to execute measures of contraception and birth control, and to establish FP associations in the countryside."

  17. Emerging Functions of Circular RNAs

    PubMed Central

    Cortés-López, Mariela; Miura, Pedro

    2016-01-01

    Many thousands of Circular RNAs (circRNAs) have recently been identified in metazoan genomes by transcriptome-wide sequencing. Most circRNAs are generated by back-splicing events from exons of protein-coding genes. A great deal of progress has recently been made in understanding the genome-wide expression patterns, biogenesis, and regulation of circRNAs. To date, however, few functions of circRNAs have been identified. CircRNAs are preferentially expressed in neural tissues and some are found at synapses, suggesting possible functions in the nervous system. Several circRNAs have been shown to function as microRNA “sponges” to counteract microRNA mediated repression of mRNA. New functions for circRNAs are arising, including protein sequestration, transcriptional regulation, and potential functions in cancer. Here, we highlight the recent progress made in understanding the biogenesis and regulation of circRNAs, discuss newly uncovered circRNA functions, and explain the methodological approaches that could reveal more exciting and unexpected roles for these RNAs. PMID:28018143

  18. Terahertz circular Airy vortex beams.

    PubMed

    Liu, Changming; Liu, Jinsong; Niu, Liting; Wei, Xuli; Wang, Kejia; Yang, Zhengang

    2017-06-20

    Vortex beams have received considerable research interests both in optical and millimeter-wave domain since its potential to be utilized in the wireless communications and novel imaging systems. Many well-known optical beams have been demonstrated to carry orbital angular momentum (OAM), such as Laguerre-Gaussian beams and high-order Bessel beams. Recently, the radially symmetric Airy beams that exhibit an abruptly autofocusing feature are also demonstrated to be capable of carrying OAM in the optical domain. However, due to the lack of efficient devices to manipulate terahertz (THz) beams, it could be a challenge to demonstrate the radially symmetric Airy beams in the THz domain. Here we demonstrate the THz circular Airy vortex beams (CAVBs) with a 0.3-THz continuous wave through 3D printing technology. Assisted by the rapidly 3D-printed phase plates, individual OAM states with topological charge l ranging from l = 0 to l = 3 and a multiplexed OAM state are successfully imposed into the radially symmetric Airy beams. We both numerically and experimentally investigate the propagation dynamics of the generated THz CAVBs, and the simulations agree well with the observations.

  19. Revival of circular variable filters

    NASA Astrophysics Data System (ADS)

    Cabib, Dario; Lavi, Moshe; Orr, Henry

    2010-10-01

    Up to about a decade or more ago Circular Variable Filters (CVF) were a commercial optical component useful in the visible and infrared wavelength ranges and were being produced and sold by the US company Optical Coating Laboratory, Inc. The CVF was used as a monochromator and its advantage was in its ease of use for spectroscopy especially in remote sensing applications. Based on the idea of multilayer interference coating with linearly varying thickness on the circumference of a rotating wheel, it worked as a continuously variable center-wavelength narrowbandpass filter with 1-2% spectral resolution. One spin of the wheel placed on the optical path of a light beam provided a full scan in the wavelength range and allowed a detector to record a full spectrum in that range. Since then this component was discontinued and companies used old remaining stocks from previous production batches. CI is now reconstructing the technology to be able to produce the CVF again and is building new radiometers for new applications based on it. In this paper we review the history of the CVF and its use by CI and we provide some details on the characteristics of the CVF as planned and as preliminarily tested.

  20. Circular photon drag effect in bulk semiconductors

    NASA Astrophysics Data System (ADS)

    Shalygin, V. A.; Moldavskaya, M. D.; Danilov, S. N.; Farbshtein, I. I.; Golub, L. E.

    2017-06-01

    We report on the observation of the circular photon drag effect in a bulk semiconductor. The photocurrent caused by a transfer of both linear and angular momenta of photons to charge carriers is detected in tellurium. Dependencies of the photocurrent on the light polarization and on the incidence angle agree with the symmetry analysis of the circular photon drag effect. Experimental spectral data on the photocurrent in mid-infrared range qualitatively agree with a microscopic model of the circular photon drag effect considering intersubband optical transitions of holes in tellurium.

  1. Toward larger DNA origami.

    PubMed

    Marchi, Alexandria N; Saaem, Ishtiaq; Vogen, Briana N; Brown, Stanley; LaBean, Thomas H

    2014-10-08

    Structural DNA nanotechnology, and specifically scaffolded DNA origami, is rapidly developing as a versatile method for bottom-up fabrication of novel nanometer-scale materials and devices. However, lengths of conventional single-stranded scaffolds, for example, 7,249-nucleotide circular genomic DNA from the M13mp18 phage, limit the scales of these uniquely addressable structures. Additionally, increasing DNA origami size generates the cost burden of increased staple-strand synthesis. We addressed this 2-fold problem by developing the following methods: (1) production of the largest to-date biologically derived single-stranded scaffold using a λ/M13 hybrid virus to produce a 51 466-nucleotide DNA in a circular, single-stranded form and (2) inexpensive DNA synthesis via an inkjet-printing process on a chip embossed with functionalized micropillars made from cyclic olefin copolymer. We have experimentally demonstrated very efficient assembly of a 51-kilobasepair origami from the λ/M13 hybrid scaffold folded by chip-derived staple strands. In addition, we have demonstrated two-dimensional, asymmetric origami sheets with controlled global curvature such that they land on a substrate in predictable orientations that have been verified by atomic force microscopy.

  2. Isolation and Characterization of Supercoiled Circular Deoxyribonucleic Acid from Beta-Hemolytic Strains of Escherichia coli

    PubMed Central

    Goebel, Werner; Schrempf, Hildgund

    1971-01-01

    Covalently closed circular deoxyribonucleic acid (DNA) molecules were isolated by cesium chloride centrifugation in the presence of ethidium bromide from a naturally occurring beta-hemolytic Escherichia coli strain (SC52). The open circular forms have contour lengths of 2.25 ± 0.1 μm, 24.0 ± 0.3 μm, and 29.5 ± 0.5 μm. The beta-hemolytic character of E. coli SC52 can be transferred by conjugation to a nonhemolytic recipient strain. Analysis of the supercoiled DNA of the hemolytic recipient demonstrated that the two large supercoiled DNA molecules of E. coli SC52 are transferred during this event, too. A beta-hemolytic laboratory E. coli strain and several of its derivatives have been shown to contain at least one circular DNA molecule, slightly larger in size than those isolated from E. coli SC52 and its conjugant. The possible significance of these DNA molecules for hemolysin production and transfer is discussed. Images PMID:4929855

  3. Circular Phonon Dichroism in Weyl Semimetals

    NASA Astrophysics Data System (ADS)

    Liu, Donghao; Shi, Junren

    2017-08-01

    We derive the phonon dynamics of magnetic metals in the presence of strong spin-orbit coupling. We show that both a dissipationless viscosity and a dissipative viscosity arise in the dynamics. While the dissipationless viscosity splits the dispersion of left-handed and right-handed circularly polarized phonons, the dissipative viscosity damps them differently, inducing circular phonon dichroism. The effect offers a new degree of manipulation of phonons, i.e., the control of the phonon polarization. We investigate the effect in Weyl semimetals. We find that there exists strong circular phonon dichroism in Weyl semimetals breaking both the time-reversal and the inversion symmetry, making them potential materials for realizing the acoustic circular polarizer.

  4. Large Circular Basin Flooded and then Cratered

    NASA Image and Video Library

    2000-08-05

    As NASA Mariner 10 passed by Mercury on its second encounter with the planet on Sept. 21, 1974, this picture of a large circular 350 kilometer, 220 mile diameter basin was obtained near the morning terminator.

  5. Polarisation vision: beetles see circularly polarised light.

    PubMed

    Warrant, Eric J

    2010-07-27

    It has long been known that the iridescent cuticle of many scarab beetles reflects circularly polarised light. It now turns out that scarabs can also see this light, potentially using it as a covert visual signal.

  6. 17 CFR 230.253 - Offering circular.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    .... Repetition of information should be avoided; cross-referencing of information within the document is... COMPLETENESS OF ANY OFFERING CIRCULAR OR OTHER SELLING LITERATURE. THESE SECURITIES ARE OFFERED PURSUANT TO...

  7. 17 CFR 230.253 - Offering circular.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    .... Repetition of information should be avoided; cross-referencing of information within the document is... COMPLETENESS OF ANY OFFERING CIRCULAR OR OTHER SELLING LITERATURE. THESE SECURITIES ARE OFFERED PURSUANT TO...

  8. 17 CFR 230.253 - Offering circular.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    .... Repetition of information should be avoided; cross-referencing of information within the document is... COMPLETENESS OF ANY OFFERING CIRCULAR OR OTHER SELLING LITERATURE. THESE SECURITIES ARE OFFERED PURSUANT TO...

  9. 17 CFR 230.253 - Offering circular.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    .... Repetition of information should be avoided; cross-referencing of information within the document is... COMPLETENESS OF ANY OFFERING CIRCULAR OR OTHER SELLING LITERATURE. THESE SECURITIES ARE OFFERED PURSUANT TO...

  10. 17 CFR 230.253 - Offering circular.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    .... Repetition of information should be avoided; cross-referencing of information within the document is... COMPLETENESS OF ANY OFFERING CIRCULAR OR OTHER SELLING LITERATURE. THESE SECURITIES ARE OFFERED PURSUANT TO...

  11. Depth perception through circular movements of dots.

    PubMed

    Ito, Hiroyuki

    2010-01-01

    Elements that move with velocity gradients have been shown to give the impression of depth. In this study, it was found that dots in circular motion around a line of sight give a depth impression corresponding to the gradients of the angular velocities of circular motion on a screen. The results of two experiments show that depth perception through circular motion is as effective as that through expansion or spiral motion, but less effective than that through lateral motion parallax when the local speed distributions on the screen are matched. The present depth effect shows that expansion-contraction, spiral, and circular motions are a continuum in terms of producing depth effects; they differ from lateral motion parallax.

  12. Broadband Circularly Polarized Patch Antenna and Method

    DTIC Science & Technology

    2016-09-16

    300152 1 of 14 BROADBAND CIRCULARLY POLARIZED PATCH ANTENNA AND METHOD STATEMENT OF GOVERNMENT INTEREST [0001] The invention described herein may...thereon or therefor. CROSS REFERENCE TO OTHER PATENT APPLICATIONS [0002] None. BACKGROUND OF THE INVENTION (1) Field of the Invention [0003] The...present invention provides a method and apparatus for a broadband circularly polarized patch antenna. (2) Description of the Prior Art [0004] A

  13. CIRCULAR RIBBON FLARES AND HOMOLOGOUS JETS

    SciTech Connect

    Wang Haimin; Liu Chang

    2012-12-01

    Solar flare emissions in the chromosphere often appear as elongated ribbons on both sides of the magnetic polarity inversion line (PIL), which has been regarded as evidence of a typical configuration of magnetic reconnection. However, flares having a circular ribbon have rarely been reported, although it is expected in the fan-spine magnetic topology involving reconnection at a three-dimensional (3D) coronal null point. We present five circular ribbon flares with associated surges, using high-resolution and high-cadence H{alpha} blue wing observations obtained from the recently digitized films of Big Bear Solar Observatory. In all the events, a central parasitic magnetic field is encompassed by the opposite polarity, forming a circular PIL traced by filament material. Consequently, a flare kernel at the center is surrounded by a circular flare ribbon. The four homologous jet-related flares on 1991 March 17 and 18 are of particular interest, as (1) the circular ribbons brighten sequentially, with cospatial surges, rather than simultaneously, (2) the central flare kernels show an intriguing 'round-trip' motion and become elongated, and (3) remote brightenings occur at a region with the same magnetic polarity as the central parasitic field and are co-temporal with a separate phase of flare emissions. In another flare on 1991 February 25, the circular flare emission and surge activity occur successively, and the event could be associated with magnetic flux cancellation across the circular PIL. We discuss the implications of these observations combining circular flare ribbons, homologous jets, and remote brightenings for understanding the dynamics of 3D magnetic restructuring.

  14. Making A Circular-Harmonic Filter

    NASA Technical Reports Server (NTRS)

    Cheng, Yeou Yen

    1989-01-01

    Optical image-correlating system made rotationally invariant. Experiment shows circular-harmonic filter generated optically. Hologram made by phase-shifted-double-exposure procedure. Filter used in optical image-recognition system based on correlation between target (image to be recognized) and matched spatial filter. In addition to usual invariance under translation of target, circular harmonic filter invariant under rotation of target; peak intensity of illumination at origin of correlation plane does not vary when target rotated about optical axis.

  15. Circular Data Images for Directional Data

    NASA Technical Reports Server (NTRS)

    Morpet, William J.

    2004-01-01

    Directional data includes vectors, points on a unit sphere, axis orientation, angular direction, and circular or periodic data. The theoretical statistics for circular data (random points on a unit circle) or spherical data (random points on a unit sphere) are a recent development. An overview of existing graphical methods for the display of directional data is given. Cross-over occurs when periodic data are measured on a scale for the measurement of linear variables. For example, if angle is represented by a linear color gradient changing uniformly from dark blue at -180 degrees to bright red at +180 degrees, the color image will be discontinuous at +180 degrees and -180 degrees, which are the same location. The resultant color would depend on the direction of approach to the cross-over point. A new graphical method for imaging directional data is described, which affords high resolution without color discontinuity from "cross-over". It is called the circular data image. The circular data image uses a circular color scale in which colors repeat periodically. Some examples of the circular data image include direction of earth winds on a global scale, rocket motor internal flow, earth global magnetic field direction, and rocket motor nozzle vector direction vs. time.

  16. Biphasic DNA Synthesis in Spumaviruses

    PubMed Central

    Delelis, Olivier; Saïb, Ali; Sonigo, Pierre

    2003-01-01

    Spumaviruses are complex retroviruses whose replication cycle resembles that of hepadnaviruses, especially by a late-occurring reverse transcription step. The possible existence of an early reverse transcription as observed in other retroviruses was not documented. Using real-time quantitative PCR, we addressed directly the kinetics of DNA synthesis during spumavirus infection. An early phase of viral DNA synthesis developed until 3 h postinfection, followed by a second phase, culminating 10 h postinfection. Both phases were abolished by the reverse transcriptase inhibitor 3′-azido-3′-deoxythymidine. Similar to other retroviruses, circular forms of viral DNA harboring two long terminal repeats were mainly found in the nucleus of infected cells. Interestingly, a fraction of these circular forms were detected in the cytoplasm and in extracellular virions, a feature shared with hepadnaviruses. Combined with packaging of both viral DNA and RNA genomes in virions, early and late reverse transcription might allow spumavirus to maximize its genome replication. PMID:12829852

  17. DNA Technology in the Classroom.

    ERIC Educational Resources Information Center

    Williamson, John H.; Campbell, A. Malcolm

    1997-01-01

    Presents a protocol that gives students hands-on experience in generating a meaningful physical map of a circular molecule of DNA. Topics include agarose gel electrophoresis, logic of restriction maps, extracting data from an agarose gel, managing data from gels, experimental protocol, loading gels, electrophoresis, photographing gels, collecting…

  18. 5 CFR 1310.3 - Availability of circulars.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 5 Administrative Personnel 3 2013-01-01 2013-01-01 false Availability of circulars. 1310.3 Section 1310.3 Administrative Personnel OFFICE OF MANAGEMENT AND BUDGET OMB DIRECTIVES OMB CIRCULARS § 1310.3 Availability of circulars. Copies of individual circulars are available at OMB's Internet home page; you...

  19. 5 CFR 1310.3 - Availability of circulars.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 5 Administrative Personnel 3 2010-01-01 2010-01-01 false Availability of circulars. 1310.3 Section 1310.3 Administrative Personnel OFFICE OF MANAGEMENT AND BUDGET OMB DIRECTIVES OMB CIRCULARS § 1310.3 Availability of circulars. Copies of individual circulars are available at OMB's Internet home page; you...

  20. 24 CFR 574.605 - Applicability of OMB circulars.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 24 Housing and Urban Development 3 2011-04-01 2010-04-01 true Applicability of OMB circulars. 574... of 24 CFR part 85 (codified pursuant to OMB Circular No. A-102) and OMB Circular No. A-87 apply with... acceptance and use of funds under the program by private non-profit entities. (Copies of OMB Circulars may...

  1. Elastic Shapes of DNA

    NASA Astrophysics Data System (ADS)

    Fain, Boris; Rudnick, Joseph

    1997-03-01

    Short segments of DNA assume shapes that minimize their elastic energy. Modeling of the various mechanisms involving the molecule - replication, transcription, packaging, etc. - requires a description of the conformations of DNA under constraints. We develop a formalism that obtains analytic expressions for shape, link, twist and extension of a segment subject to sufficient number of constraints. We apply our technique to two particular cases: a) Stretched twisted linear DNA. This is an interesting test for our formalism, especially in light of recent experiments(Strick T.R., Allemand J.-F., Bensimon A., Croquette V. Science) 271 1835-1837, (1996). The molecule remains extended until a critical twist is reached, at which point it undergoes a plectonemic transition. b) Closed circular DNA. Describing the shapes of such molecules has been an outstanding problem for some time. We obtain a family of curves classified by their deviation in link from the plain circle.

  2. Bright circularly polarized soft X-ray high harmonics for X-ray magnetic circular dichroism

    DOE PAGES

    Fan, Tingting; Grychtol, Patrik; Knut, Ronny; ...

    2015-11-03

    Here, we demonstrate, to our knowledge, the first bright circularly polarized high-harmonic beams in the soft X-ray region of the electromagnetic spectrum, and use them to implement X-ray magnetic circular dichroism measurements in a tabletop-scale setup. Using counterrotating circularly polarized laser fields at 1.3 and 0.79 µm, we generate circularly polarized harmonics with photon energies exceeding 160 eV. The harmonic spectra emerge as a sequence of closely spaced pairs of left and right circularly polarized peaks, with energies determined by conservation of energy and spin angular momentum. We explain the single-atom and macroscopic physics by identifying the dominant electron quantummore » trajectories and optimal phase-matching conditions. The first advanced phase-matched propagation simulations for circularly polarized harmonics reveal the influence of the finite phase-matching temporal window on the spectrum, as well as the unique polarization-shaped attosecond pulse train. Finally, we use, to our knowledge, the first tabletop X-ray magnetic circular dichroism measurements at the N4,5 absorption edges of Gd to validate the high degree of circularity, brightness, and stability of this light source. These results demonstrate the feasibility of manipulating the polarization, spectrum, and temporal shape of high harmonics in the soft X-ray region by manipulating the driving laser waveform.« less

  3. Bright circularly polarized soft X-ray high harmonics for X-ray magnetic circular dichroism.

    PubMed

    Fan, Tingting; Grychtol, Patrik; Knut, Ronny; Hernández-García, Carlos; Hickstein, Daniel D; Zusin, Dmitriy; Gentry, Christian; Dollar, Franklin J; Mancuso, Christopher A; Hogle, Craig W; Kfir, Ofer; Legut, Dominik; Carva, Karel; Ellis, Jennifer L; Dorney, Kevin M; Chen, Cong; Shpyrko, Oleg G; Fullerton, Eric E; Cohen, Oren; Oppeneer, Peter M; Milošević, Dejan B; Becker, Andreas; Jaroń-Becker, Agnieszka A; Popmintchev, Tenio; Murnane, Margaret M; Kapteyn, Henry C

    2015-11-17

    We demonstrate, to our knowledge, the first bright circularly polarized high-harmonic beams in the soft X-ray region of the electromagnetic spectrum, and use them to implement X-ray magnetic circular dichroism measurements in a tabletop-scale setup. Using counterrotating circularly polarized laser fields at 1.3 and 0.79 µm, we generate circularly polarized harmonics with photon energies exceeding 160 eV. The harmonic spectra emerge as a sequence of closely spaced pairs of left and right circularly polarized peaks, with energies determined by conservation of energy and spin angular momentum. We explain the single-atom and macroscopic physics by identifying the dominant electron quantum trajectories and optimal phase-matching conditions. The first advanced phase-matched propagation simulations for circularly polarized harmonics reveal the influence of the finite phase-matching temporal window on the spectrum, as well as the unique polarization-shaped attosecond pulse train. Finally, we use, to our knowledge, the first tabletop X-ray magnetic circular dichroism measurements at the N4,5 absorption edges of Gd to validate the high degree of circularity, brightness, and stability of this light source. These results demonstrate the feasibility of manipulating the polarization, spectrum, and temporal shape of high harmonics in the soft X-ray region by manipulating the driving laser waveform.

  4. Bright circularly polarized soft X-ray high harmonics for X-ray magnetic circular dichroism

    PubMed Central

    Fan, Tingting; Grychtol, Patrik; Knut, Ronny; Hernández-García, Carlos; Hickstein, Daniel D.; Zusin, Dmitriy; Gentry, Christian; Dollar, Franklin J.; Mancuso, Christopher A.; Hogle, Craig W.; Kfir, Ofer; Legut, Dominik; Carva, Karel; Ellis, Jennifer L.; Dorney, Kevin M.; Chen, Cong; Shpyrko, Oleg G.; Fullerton, Eric E.; Cohen, Oren; Oppeneer, Peter M.; Milošević, Dejan B.; Becker, Andreas; Jaroń-Becker, Agnieszka A.; Popmintchev, Tenio; Murnane, Margaret M.; Kapteyn, Henry C.

    2015-01-01

    We demonstrate, to our knowledge, the first bright circularly polarized high-harmonic beams in the soft X-ray region of the electromagnetic spectrum, and use them to implement X-ray magnetic circular dichroism measurements in a tabletop-scale setup. Using counterrotating circularly polarized laser fields at 1.3 and 0.79 µm, we generate circularly polarized harmonics with photon energies exceeding 160 eV. The harmonic spectra emerge as a sequence of closely spaced pairs of left and right circularly polarized peaks, with energies determined by conservation of energy and spin angular momentum. We explain the single-atom and macroscopic physics by identifying the dominant electron quantum trajectories and optimal phase-matching conditions. The first advanced phase-matched propagation simulations for circularly polarized harmonics reveal the influence of the finite phase-matching temporal window on the spectrum, as well as the unique polarization-shaped attosecond pulse train. Finally, we use, to our knowledge, the first tabletop X-ray magnetic circular dichroism measurements at the N4,5 absorption edges of Gd to validate the high degree of circularity, brightness, and stability of this light source. These results demonstrate the feasibility of manipulating the polarization, spectrum, and temporal shape of high harmonics in the soft X-ray region by manipulating the driving laser waveform. PMID:26534992

  5. Environmental issues elimination through circular economy

    NASA Astrophysics Data System (ADS)

    Špirková, M.; Pokorná, E.; Šujanová, J.; Samáková, J.

    2016-04-01

    Environmental efforts of European Union are currently going towards circular economy. Tools like Extended Producer Responsibility and Eco-design were established. The circular economy deals with resources availability issue on one hand and waste management on the other hand. There are few pioneering companies all over the world with some kind of circular economy practice. Generally the concept is not very wide-spread. The paper aims to evaluate possibility of transition towards circular economy in Slovak industrial companies. They need to have an active approach to material treatment of their products after usage stage. Innovation is another important pre-condition for the transition. Main problem of current cradle to grave system is landfilling of valuable materials after one cycle of usage. Their potential value for next manufacturing cycles is lost. Companies may do not see connection between waste management and material resource prices and volatility of supplies. Municipalities are responsible for municipal waste collection and treatment in Slovakia. The circular economy operates by cradle to cradle principle. Company manages material flow until the material comes back to the beginning of manufacturing process by itself or by another partners. Stable material supplies with quite low costs are provided this way. It is necessary to deal with environmental problems in phase of product design. Questionnaire survey results show on one hand low involvement of industrial companies in waste management area, however on the other hand they are open to environmental innovations in future.

  6. Environmental issues elimination through circular economy

    SciTech Connect

    Špirková, M. Pokorná, E.; Šujanová, J.; Samáková, J.

    2016-04-21

    Environmental efforts of European Union are currently going towards circular economy. Tools like Extended Producer Responsibility and Eco-design were established. The circular economy deals with resources availability issue on one hand and waste management on the other hand. There are few pioneering companies all over the world with some kind of circular economy practice. Generally the concept is not very wide-spread. The paper aims to evaluate possibility of transition towards circular economy in Slovak industrial companies. They need to have an active approach to material treatment of their products after usage stage. Innovation is another important pre-condition for the transition. Main problem of current cradle to grave system is landfilling of valuable materials after one cycle of usage. Their potential value for next manufacturing cycles is lost. Companies may do not see connection between waste management and material resource prices and volatility of supplies. Municipalities are responsible for municipal waste collection and treatment in Slovakia. The circular economy operates by cradle to cradle principle. Company manages material flow until the material comes back to the beginning of manufacturing process by itself or by another partners. Stable material supplies with quite low costs are provided this way. It is necessary to deal with environmental problems in phase of product design. Questionnaire survey results show on one hand low involvement of industrial companies in waste management area, however on the other hand they are open to environmental innovations in future.

  7. A broadband, circular-polarization selective surface

    NASA Astrophysics Data System (ADS)

    Momeni Hasan Abadi, Seyed Mohamad Amin; Behdad, Nader

    2016-06-01

    We introduce a new technique for designing wideband circular-polarization selective surfaces (CPSSs) based on anisotropic miniaturized element frequency selective surfaces. The proposed structure is a combination of two linear-to-circular polarization converters sandwiching a linear polarizer. This CPSS consists of a number of metallic layers separated from each other by thin dielectric substrates. The metallic layers are in the form of two-dimensional arrays of subwavelength capacitive patches and inductive wire grids with asymmetric dimensions and a wire grid polarizer with sub-wavelength period. The proposed device is designed to offer a wideband circular-polarization selection capability allowing waves with left-hand circular polarization to pass through while rejecting those having right-hand circular polarization. A synthesis procedure is developed that can be used to design the proposed CPSS based on its desired band of operation. Using this procedure, a prototype of the proposed CPSS operating in the 12-18 GHz is designed. Full-wave electromagnetic simulations are used to predict the response of this structure. These simulation results confirm the validity of the proposed design concept and synthesis procedure and show that proposed CPSS operates within a fractional bandwidth of 40% with a co-polarization transmission discrimination of more than 15 dB. Furthermore, the proposed design is shown to be capable of providing an extremely wide field of view of ±60°.

  8. Circular and Elliptic Submerged Impinging Water Jets

    NASA Astrophysics Data System (ADS)

    Claudey, Eric; Benedicto, Olivier; Ravier, Emmanuel; Gutmark, Ephraim

    1999-11-01

    Experiments and CFD have been performed to study circular and elliptic jets in a submerged water jet facility. The tests included discharge coefficient measurement to evaluate pressure losses encountered in noncircular nozzles compared to circular ones. Three-dimensional pressure mappings on the impingement surface and PIV measurement of the jet mean and turbulent velocity have been performed at different compound impingement angles relative to the impingement surface and at different stand-off distances. The objective was to investigate the effect of the non-circular geometry on the flow field and on the impact region. The tests were performed in a close loop system in which the water was pumped through the nozzles into a clear Plexiglas tank. The Reynolds numbers were typically in the range of 250000. Discharge coefficients of the elliptic nozzle was somewhat lower than that of the circular jet but spreading rate and turbulence level were higher. Pressure mapping showed that the nozzle exit geometry had an effect on the pressure distribution in the impact region and that high-pressure zones were generated at specific impact points. PIV measurements showed that for a same total exit area, the elliptic jets affected a surface area that is 8the equivalent circular. The turbulence level in the elliptic jet tripled due to the nozzle design. Results of the CFD model were in good agreement with the experimental data.

  9. CircularLogo: A lightweight web application to visualize intra-motif dependencies.

    PubMed

    Ye, Zhenqing; Ma, Tao; Kalmbach, Michael T; Dasari, Surendra; Kocher, Jean-Pierre A; Wang, Liguo

    2017-05-22

    The sequence logo has been widely used to represent DNA or RNA motifs for more than three decades. Despite its intelligibility and intuitiveness, the traditional sequence logo is unable to display the intra-motif dependencies and therefore is insufficient to fully characterize nucleotide motifs. Many methods have been developed to quantify the intra-motif dependencies, but fewer tools are available for visualization. We developed CircularLogo, a web-based interactive application, which is able to not only visualize the position-specific nucleotide consensus and diversity but also display the intra-motif dependencies. Applying CircularLogo to HNF6 binding sites and tRNA sequences demonstrated its ability to show intra-motif dependencies and intuitively reveal biomolecular structure. CircularLogo is implemented in JavaScript and Python based on the Django web framework. The program's source code and user's manual are freely available at http://circularlogo.sourceforge.net . CircularLogo web server can be accessed from http://bioinformaticstools.mayo.edu/circularlogo/index.html . CircularLogo is an innovative web application that is specifically designed to visualize and interactively explore intra-motif dependencies.

  10. Diverse circular replication-associated protein encoding viruses circulating in invertebrates within a lake ecosystem.

    PubMed

    Dayaram, Anisha; Galatowitsch, Mark L; Argüello-Astorga, Gerardo R; van Bysterveldt, Katherine; Kraberger, Simona; Stainton, Daisy; Harding, Jon S; Roumagnac, Philippe; Martin, Darren P; Lefeuvre, Pierre; Varsani, Arvind

    2016-04-01

    Over the last five years next-generation sequencing has become a cost effective and efficient method for identifying known and unknown microorganisms. Access to this technique has dramatically changed the field of virology, enabling a wide range of environmental viral metagenome studies to be undertaken of organisms and environmental samples from polar to tropical regions. These studies have led to the discovery of hundreds of highly divergent single stranded DNA (ssDNA) virus-like sequences encoding replication-associated proteins. Yet, few studies have explored how viruses might be shared in an ecosystem through feeding relationships. Here we identify 169 circular molecules (160 CRESS DNA molecules, nine circular molecules) recovered from a New Zealand freshwater lake, that we have tentatively classified into 51 putatively novel species and five previously described species (DflaCV-3, -5, -6, -8, -10). The CRESS DNA viruses identified in this study were recovered from molluscs (Echyridella menzeisii, Musculium novaezelandiae, Potamopyrgus antipodarum and Physella acuta) and insect larvae (Procordulia grayi, Xanthocnemis zealandica, and Chironomus zealandicus) collected from Lake Sarah, as well as from the lake water and benthic sediments. Extensive diversity was observed across most CRESS DNA molecules recovered. The putative capsid protein of one viral species was found to be most similar to those of members of the Tombusviridae family, thus expanding the number of known RNA-DNA hybrid viruses in nature. We noted a strong association between the CRESS DNA viruses and circular molecules identified in the water and browser organisms (C. zealandicus, P. antipodarum and P. acuta), and between water sediments and undefended prey species (C. zealandicus). However, we were unable to find any significant correlation of viral assemblages to the potential feeding relationships of the host aquatic invertebrates. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. DISCO synchrotron-radiation circular-dichroism endstation at SOLEIL.

    PubMed

    Réfrégiers, Matthieu; Wien, Frank; Ta, Ha Phuong; Premvardhan, Lavanya; Bac, Stéphane; Jamme, Frederic; Rouam, Valerie; Lagarde, Bruno; Polack, François; Giorgetta, Jean Luc; Ricaud, Jean Paul; Bordessoule, Michel; Giuliani, Alexandre

    2012-09-01

    The new synchrotron-radiation circular-dichroism (SRCD) endstation on the UV-visible synchrotron beamline DISCO has been commissioned at the SOLEIL synchrotron. The design has been focused on preservation of a high degree of linear polarization at high flux and moderate resolving power covering the vacuum ultraviolet to visible spectral range (125-600 nm). The beam dimensions have been set to 4 mm × 4 mm at 1 nm bandwidth for lower sample degradation. The nitrogen-purged sample chamber fits three types of sample holders accommodating conventional round cell mounting, automated rotation of the samples, as well as a microfluidic set-up. Automated temperature-controlled data collection on microvolumes is now available to the biology and chemistry communities. Macromolecules including membrane proteins, soluble proteins, bio-nanotubes, sugars, DNA and RNAs are now routinely investigated.

  12. Chromatin Fractal Organization, Textural Patterns, and Circularity of Nuclear Envelope in Adrenal Zona Fasciculata Cells.

    PubMed

    Pantic, Igor; Nesic, Dejan; Basailovic, Milos; Cetkovic, Mila; Mazic, Sanja; Suzic-Lazic, Jelena; Popevic, Martin

    2016-12-01

    Despite previous research efforts in the fields of histology and cell physiology, the relationship between chromatin structural organization and nuclear shape remains unclear. The aim of this research was to test the existence and strength of correlations between mathematical parameters of chromatin microarchitecture and roundness of the nuclear envelope. On a sample of 240 nuclei of adrenal zona fasciculata cells stained using the DNA-specific Feulgen method, we quantified fractal parameters such as fractal dimension and lacunarity, as well as textural parameters such as angular second moment (ASM), entropy, inverse difference moment, contrast, and variance. Circularity of the nuclear envelope was determined from the nuclear area and perimeter. The results indicate that there is a statistically significant negative correlation between chromatin ASM and circularity. Moreover, there was a statistically significant positive correlation between chromatin fractal dimension and envelope circularity. This is the first study to demonstrate these relationships in adrenal tissue, and also one of the first studies to test the connection between circularity and fractal and gray-level co-occurrence matrix parameters in DNA-specific Feulgen stain. The results could be useful both as an addition to the current knowledge on chromatin/nuclear envelope interactions, and for design of future computer-assisted research software for evaluation of nuclear morphology.

  13. High-Throughput Sequencing Reveals Circular Substrates for an Archaeal RNA ligase.

    PubMed

    Becker, Hubert F; Heliou, Alice; Djaout, Kamel; Lestini, Roxane; Regnier, Mireille; Myllykallio, Hannu

    2017-03-09

    It is only recently that the abundant presence of circular RNAs (circRNAs) in all kingdoms of Life, including the hyperthermophilic archaeon Pyrococcus abyssi, has emerged. This led us to investigate the physiological significance of a previously observed weak intramolecular ligation activity of Pab1020 RNA ligase. Here we demonstrate that this enzyme, despite sharing significant sequence similarity with DNA ligases, is indeed an RNA-specific polynucleotide ligase efficiently acting on physiologically significant substrates. Using a combination of RNA immunoprecipitation assays and RNA-seq, our genome-wide studies revealed 133 individual circRNA loci in P. abyssi. The large majority of these loci interacted with Pab1020 in cells and circularization of selected C/D Box and 5S rRNA transcripts was confirmed biochemically. Altogether these studies revealed that Pab1020 is required for RNA circularization. Our results further suggest the functional speciation of an ancestral NTase domain and/or DNA ligase towards RNA ligase activity and prompt for further characterization of the widespread functions of circular RNAs in prokaryotes. Detailed insight into the cellular substrates of Pab1020 may facilitate the development of new biotechnological applications e.g. in ligation of preadenylated adaptors to RNA molecules.

  14. The Maximal C³ Self-Complementary Trinucleotide Circular Code X in Genes of Bacteria, Archaea, Eukaryotes, Plasmids and Viruses.

    PubMed

    Michel, Christian J

    2017-04-18

    In 1996, a set X of 20 trinucleotides was identified in genes of both prokaryotes and eukaryotes which has on average the highest occurrence in reading frame compared to its two shifted frames. Furthermore, this set X has an interesting mathematical property as X is a maximal C 3 self-complementary trinucleotide circular code. In 2015, by quantifying the inspection approach used in 1996, the circular code X was confirmed in the genes of bacteria and eukaryotes and was also identified in the genes of plasmids and viruses. The method was based on the preferential occurrence of trinucleotides among the three frames at the gene population level. We extend here this definition at the gene level. This new statistical approach considers all the genes, i.e., of large and small lengths, with the same weight for searching the circular code X . As a consequence, the concept of circular code, in particular the reading frame retrieval, is directly associated to each gene. At the gene level, the circular code X is strengthened in the genes of bacteria, eukaryotes, plasmids, and viruses, and is now also identified in the genes of archaea. The genes of mitochondria and chloroplasts contain a subset of the circular code X . Finally, by studying viral genes, the circular code X was found in DNA genomes, RNA genomes, double-stranded genomes, and single-stranded genomes.

  15. Circular Bacteriocins: Biosynthesis and Mode of Action

    PubMed Central

    Brede, Dag A.; Nes, Ingolf F.; Diep, Dzung B.

    2014-01-01

    Circular bacteriocins are a group of N-to-C-terminally linked antimicrobial peptides, produced by Gram-positive bacteria of the phylum Firmicutes. Circular bacteriocins generally exhibit broad-spectrum antimicrobial activity, including against common food-borne pathogens, such as Clostridium and Listeria spp. These peptides are further known for their high pH and thermal stability, as well as for resistance to many proteolytic enzymes, properties which make this group of bacteriocins highly promising for potential industrial applications and their biosynthesis of particular interest as a possible model system for the synthesis of highly stable bioactive peptides. In this review, we summarize the current knowledge on this group of bacteriocins, with emphasis on the recent progress in understanding circular bacteriocin genetics, biosynthesis, and mode of action; in addition, we highlight the current challenges and future perspectives for the application of these peptides. PMID:25172850

  16. Circular Proteins from Plants and Fungi*

    PubMed Central

    Göransson, Ulf; Burman, Robert; Gunasekera, Sunithi; Strömstedt, Adam A.; Rosengren, K. Johan

    2012-01-01

    Circular proteins, defined as head-to-tail cyclized polypeptides originating from ribosomal synthesis, represent a novel class of natural products attracting increasing interest. From a scientific point of view, these compounds raise questions of where and why they occur in nature and how they are formed. From a rational point of view, these proteins and their structural concept may be exploited for crop protection and novel pharmaceuticals. Here, we review the current knowledge of three protein families: cyclotides and circular sunflower trypsin inhibitors from the kingdom of plants and the Amanita toxins from fungi. A particular emphasis is placed on their biological origin, structure, and activity. In addition, the opportunity for discovery of novel circular proteins and recent insights into their mechanism of action are discussed. PMID:22700984

  17. Circular bacteriocins: biosynthesis and mode of action.

    PubMed

    Gabrielsen, Christina; Brede, Dag A; Nes, Ingolf F; Diep, Dzung B

    2014-11-01

    Circular bacteriocins are a group of N-to-C-terminally linked antimicrobial peptides, produced by Gram-positive bacteria of the phylum Firmicutes. Circular bacteriocins generally exhibit broad-spectrum antimicrobial activity, including against common food-borne pathogens, such as Clostridium and Listeria spp. These peptides are further known for their high pH and thermal stability, as well as for resistance to many proteolytic enzymes, properties which make this group of bacteriocins highly promising for potential industrial applications and their biosynthesis of particular interest as a possible model system for the synthesis of highly stable bioactive peptides. In this review, we summarize the current knowledge on this group of bacteriocins, with emphasis on the recent progress in understanding circular bacteriocin genetics, biosynthesis, and mode of action; in addition, we highlight the current challenges and future perspectives for the application of these peptides.

  18. Structure and genetics of circular bacteriocins.

    PubMed

    van Belkum, Marco J; Martin-Visscher, Leah A; Vederas, John C

    2011-08-01

    Circular bacteriocins are antimicrobial peptides produced by a variety of Gram-positive bacteria. They are part of a growing family of ribosomally synthesized peptides with a head-to-tail cyclization of their backbone that are found in mammals, plants, fungi and bacteria and are exceptionally stable. These bacteriocins permeabilize the membrane of sensitive bacteria, causing loss of ions and dissipation of the membrane potential. Most circular bacteriocins probably adopt a common 3D structure consisting of four or five α-helices encompassing a hydrophobic core. This review compares the various structures, as well as the gene clusters that encode circular bacteriocins, and discusses the biogenesis of this unique class of bacteriocins.

  19. Experimental evidence for circular inference in schizophrenia

    NASA Astrophysics Data System (ADS)

    Jardri, Renaud; Duverne, Sandrine; Litvinova, Alexandra S.; Denève, Sophie

    2017-01-01

    Schizophrenia (SCZ) is a complex mental disorder that may result in some combination of hallucinations, delusions and disorganized thinking. Here SCZ patients and healthy controls (CTLs) report their level of confidence on a forced-choice task that manipulated the strength of sensory evidence and prior information. Neither group's responses can be explained by simple Bayesian inference. Rather, individual responses are best captured by a model with different degrees of circular inference. Circular inference refers to a corruption of sensory data by prior information and vice versa, leading us to `see what we expect' (through descending loops), to `expect what we see' (through ascending loops) or both. Ascending loops are stronger for SCZ than CTLs and correlate with the severity of positive symptoms. Descending loops correlate with the severity of negative symptoms. Both loops correlate with disorganized symptoms. The findings suggest that circular inference might mediate the clinical manifestations of SCZ.

  20. Charged Particle Optics in Circular Higgs Factory

    SciTech Connect

    Cai, Yunhai

    2015-02-26

    Similar to a super B-factory, a circular Higgs factory will require strong focusing systems near the interaction points and a low-emittance lattice in arcs to achieve a factory luminosity. At electron beam energy of 120 GeV, beamstrahlung effects during the collision pose an additional challenge to the collider design. In particular, a large momentum acceptance at 2 percent level is necessary to retain an adequate beam lifetime. This turns out to be the most challenging aspect in the design of circular Higgs factory. In this paper, an example will be provided to illustrate the beam dynamics in circular Higgs factory, emphasizing on the chromatic optics. Basic optical modules and advanced analysis will be presented. Most important, we will show that 2% momentum aperture is achievable

  1. Reconnaissance with slant plane circular SAR imaging.

    PubMed

    Soumekh, M

    1996-01-01

    This paper presents a method for imaging from the slant plane data collected by a synthetic aperture radar (SAR) over the full rotation or a partial segment of a circular flight path. A Fourier analysis for the Green's function of the imaging system is provided. This analysis is the basis of an inversion for slant plane circular SAR data. The reconstruction algorithm and resolution for this SAR system are outlined. It is shown that the slant plane circular SAR, unlike the slant plane linear SAR, has the capability to extract three-dimensional imaging information of a target scene. The merits of the algorithm are demonstrated via a simulated target whose ultra wideband foliage penetrating (FOPEN) or ground penetrating (GPEN) ultrahigh frequency (UHF) radar signature varies with the radar's aspect angle.

  2. Experimental evidence for circular inference in schizophrenia

    PubMed Central

    Jardri, Renaud; Duverne, Sandrine; Litvinova, Alexandra S; Denève, Sophie

    2017-01-01

    Schizophrenia (SCZ) is a complex mental disorder that may result in some combination of hallucinations, delusions and disorganized thinking. Here SCZ patients and healthy controls (CTLs) report their level of confidence on a forced-choice task that manipulated the strength of sensory evidence and prior information. Neither group's responses can be explained by simple Bayesian inference. Rather, individual responses are best captured by a model with different degrees of circular inference. Circular inference refers to a corruption of sensory data by prior information and vice versa, leading us to ‘see what we expect' (through descending loops), to ‘expect what we see' (through ascending loops) or both. Ascending loops are stronger for SCZ than CTLs and correlate with the severity of positive symptoms. Descending loops correlate with the severity of negative symptoms. Both loops correlate with disorganized symptoms. The findings suggest that circular inference might mediate the clinical manifestations of SCZ. PMID:28139642

  3. Nanophotonic control of circular dipole emission

    NASA Astrophysics Data System (ADS)

    Le Feber, B.; Rotenberg, N.; Kuipers, L.

    2015-04-01

    Controlling photon emission by single emitters with nanostructures is crucial for scalable on-chip information processing. Nowadays, nanoresonators can affect the lifetime of linear dipole emitters, while nanoantennas can steer the emission direction. Expanding this control to the emission of orbital angular momentum-changing transitions would enable a future coupling between solid state and photonic qubits. As these transitions are associated with circular dipoles, such control requires knowledge of the interaction of a complex dipole with optical eigenstates containing local helicity. We experimentally map the coupling of classical, circular dipoles to photonic modes in a photonic crystal waveguide. We show that, depending on the combination of the local helicity of the mode and the dipole helicity, circular dipoles can couple to left- or rightwards propagating modes with a near-unity directionality. The experimental maps are in excellent agreement with calculations. Our measurements, therefore, demonstrate the possibility of coupling the spin to photonic pathway.

  4. Optics modules for circular accelerator design

    SciTech Connect

    Brown, K.L.; Servranckx, R.V.

    1986-05-01

    The first-order differential equations of motion for a single particle in a closed circular machine are solved, introducing the concepts of phase shift, beta functions, and the Courant-Snyder invariant. The transfer matrix between two points in the machine is derived as a function of the phase shift and the parameters contained in the Courant-Snyder invariant. Typical optical modules used in circular machine designs are introduced and related to their characteristic transfer matrix elements, the phase shift through them, and the Courant-Snyder-Twiss parameters. The systematics of some elementary phase ellipse matching problems between optical modules are discussed. Second-order optical modules are discussed, including how they are used to provide the momentum bandwidth needed for the design of a typical circular machine. (LEW)

  5. Interactions of Isophorone Derivatives with DNA: Spectroscopic Studies

    PubMed Central

    Deiana, Marco; Matczyszyn, Katarzyna; Massin, Julien; Olesiak-Banska, Joanna; Andraud, Chantal; Samoc, Marek

    2015-01-01

    Interactions of three new isophorone derivatives, Isoa Isob and Isoc with salmon testes DNA have been investigated using UV-Vis, fluorescence and circular dichroism spectroscopic methods. All the studied compounds interact with DNA through intercalative binding mode. The stoichiometry of the isophorone/DNA adducts was found to be 1:1. The fluorescence quenching data revealed a binding interaction with the base pairs of DNA. The CD data indicate that all the investigated isophorones induce DNA modifications. PMID:26069963

  6. Mechanism for CCC DNA synthesis in hepadnaviruses.

    PubMed

    Sohn, Ji A; Litwin, Samuel; Seeger, Christoph

    2009-11-30

    Hepadnavirus replication requires the synthesis of a covalently closed circular (CCC) DNA from the relaxed circular (RC) viral genome by an unknown mechanism. CCC DNA formation could require enzymatic activities of the viral reverse transcriptase (RT), or cellular DNA repair enzymes, or both. Physical mapping of the 5' and 3' ends of RC DNA and sequence analysis of CCC DNA revealed that CCC DNA synthesis requires the removal of the RT and an RNA oligomer from the 5' ends of minus and plus strand DNA, respectively, removal of sequences from the terminally redundant minus strand, completion of the less than full-length plus strand, and ligation of the ends. Two models have been proposed that could explain CCC DNA formation. The first (model 1) invokes a role for the RT to catalyze a cleavage-ligation reaction leading to the formation of a unit length minus strand in CCC DNA and a DNA repair reaction for the completion and ligation of plus strand DNA; the second (model 2) predicts that CCC DNA formation depends entirely on cellular DNA repair enzymes. To determine which mechanism is utilized, we developed cell lines expressing duck hepatitis B virus genomes carrying mutations permitting us to follow the fate of viral DNA sequences during their conversion from RC to CCC DNA. Our results demonstrated that the oligomer at the 5' end of minus strand DNA is completely or at least partially removed prior to CCC DNA synthesis. The results indicated that both RC DNA strands undergo DNA repair reactions carried out by the cellular DNA repair machinery as predicted by model 2. Thus, our study provided the basis for the identification of the cellular components required for CCC DNA formation.

  7. Rapid Detection and Identification of a Pathogen's DNA Using Phi29 DNA Polymerase

    SciTech Connect

    Xu, Y.; Dunn, J.; Gao, S.; Bruno, J. F.; Luft, B. J.

    2008-10-31

    Zoonotic pathogens including those transmitted by insect vectors are some of the most deadly of all infectious diseases known to mankind. A number of these agents have been further weaponized and are widely recognized as being potentially significant biothreat agents. We describe a novel method based on multiply-primed rolling circle in vitro amplification for profiling genomic DNAs to permit rapid, cultivation-free differential detection and identification of circular plasmids in infectious agents. Using Phi29 DNA polymerase and a two-step priming reaction we could reproducibly detect and characterize by DNA sequencing circular DNA from Borrelia burgdorferi B31 in DNA samples containing as little as 25 pg of Borrelia DNA amongst a vast excess of human DNA. This simple technology can ultimately be adapted as a sensitive method to detect specific DNA from both known and unknown pathogens in a wide variety of complex environments.

  8. Rotatable non-circular forebody flow controller

    NASA Technical Reports Server (NTRS)

    Moskovitz, Cary A. (Inventor)

    1991-01-01

    The invention is a rotatable, non-circular forebody flow controller. The apparatus comprises a small geometric device located at a nose of a forebody of an aircraft and a non-circular cross-sectional area that extends toward the apex of the aircraft. The device is symmetrical about a reference plane and preferably attaches to an axle which in turn attaches to a rotating motor. The motor rotates the device about an axis of rotation. Preferably, a control unit connected to an aircraft flight control computer signals to the rotating motor the proper rotational positioning of the geometric device.

  9. One-dimensional circular diffraction patterns

    NASA Astrophysics Data System (ADS)

    Daimon, Hiroshi; Ino, Shozo

    1989-11-01

    Circular diffraction patterns from a bulk crystal have been found in MEED patterns by using a newly developed two-dimensional spherical mirror analyzer. From the analysis of the energy dependence of their radii and from the fact that they are not associated with the tangential Kikuchi lines, the circles were interpreted by the concept of one-dimensional diffraction along the crystallographic axes. The hemi-circular patterns, which have been observed in RHEED patterns near superstructural spots from a surface structure, were also explained by this concept.

  10. Practical circular-polarization-maintaining optical fiber.

    PubMed

    Huang, H C

    1997-09-20

    The author describes a new idea for making circular-polarization-maintaining optical fiber with an existing fabrication technique. The method simply requires one to spin at a constant rate a special preform consisting of only one off-axis stress-applying element in addition to the on-axis core. Measurements taken with such a fiber specimen verify the existence of circular eigenmodes, the ease of joining or splicing two fiber segments, the tolerance to macrobending with a small radius, etc. Good agreement exists between the experimental data and the theoretical analysis. Prospective applications are discussed.

  11. Circularly Polarized Luminescence from Simple Organic Molecules.

    PubMed

    Sánchez-Carnerero, Esther M; Agarrabeitia, Antonia R; Moreno, Florencio; Maroto, Beatriz L; Muller, Gilles; Ortiz, María J; de la Moya, Santiago

    2015-09-21

    This article aims to show the identity of "circularly polarized luminescent active simple organic molecules" as a new concept in organic chemistry due to the potential interest of these molecules, as availed by the exponentially growing number of research articles related to them. In particular, it describes and highlights the interest and difficulty in developing chiral simple (small and non-aggregated) organic molecules able to emit left- or right-circularly polarized light efficiently, the efforts realized up to now to reach this challenging objective, and the most significant milestones achieved to date. General guidelines for the preparation of these interesting molecules are also presented.

  12. High-frequency asymptotics of circular dichroism

    NASA Astrophysics Data System (ADS)

    Pospelov, M. E.

    1996-07-01

    Circular dichroism of optically active isotropic media of chiral molecules Im(n+-n-)/Im(n++n-) falls off as ω-2 at frequencies Ry<<ω<>Z2Ry, where Z is a typical nuclear charge of atoms in the chiral group. The contribution of the spin of electron to the circular dichroism appears in the second order in spin-orbit perturbation only. The polarization of photoelectrons in the absorption of unpolarized light is connected with the chirality of molecule and constitutes Z2α3 from the degree of geometrical asymmetry.

  13. Identification of Intermediate in Hepatitis B Virus CCC DNA Formation and Sensitive and Selective CCC DNA Detection.

    PubMed

    Luo, Jun; Cui, Xiuji; Gao, Lu; Hu, Jianming

    2017-06-21

    The hepatitis B virus (HBV) covalently closed circular (CCC) DNA functions as the only viral template capable of coding for all the viral RNA species and is thus essential to initiate and sustain viral replication. CCC DNA is converted, in a multi-step and ill-understood process, from a relaxed circular (RC) DNA, in which neither of the two DNA strands is covalently closed. To detect putative intermediates during RC to CCC DNA conversion, two 3' exonucleases Exo I and Exo III, in combination were used to degrade all DNA strands with a free 3' end, which would nevertheless preserve closed circular DNA, either single-stranded (SS) or double-stranded (DS). Indeed, a RC DNA species with a covalently closed minus strand but an open plus strand (closed minus-strand RC DNA or cM-RC DNA) was detected by this approach. Further analyses indicated that at least some of the plus strands in such a putative intermediate likely still retained the RNA primer that is attached to the 5' end of the plus strand in RC DNA, suggesting that minus strand closing can occur before plus strand processing. Furthermore, the same nuclease treatment proved to be useful for sensitive and specific detection of CCC DNA by removing all DNA species other than closed circular DNA. Application of these and similar approaches may allow the identification of additional intermediates during CCC DNA formation and facilitate specific and sensitive detection of CCC DNA, which should help elucidate the pathways of CCC DNA formation and factors involved.IMPORTANCE The hepatitis B virus (HBV) covalently closed circular (CCC) DNA is the molecular basis of viral persistence, by serving as the viral transcriptional template. CCC DNA is converted, in a multi-step and ill-understood process, from a relaxed circular (RC) DNA. Little is currently understood about the pathways or factors involved in CCC DNA formation. We have now detected a likely intermediate during the conversion of RC to CCC DNA, thus providing

  14. Chiral DNA packaging in DNA-cationic liposome assemblies.

    PubMed

    Zuidam, N J; Barenholz, Y; Minsky, A

    1999-09-03

    Recent studies have indicated that the structural features of DNA-lipid assemblies, dictated by the lipid composition and cationic lipid-to-DNA ratio, critically affect the efficiency of these complexes in acting as vehicles for cellular delivery of genetic material. Using circular dichroism we find that upon binding DNA, positively-charged liposomes induce a secondary conformational transition of the DNA molecules from the native B form to the C motif. Liposomes composed of positively-charged and neutral 'helper' lipids, found to be particularly effective as transfecting agents, induce - in addition to secondary conformational changes - DNA condensation into a left-handed cholesteric-like phase. A structural model is presented according to which two distinct, yet inter-related modes of DNA packaging coexist within such assemblies. The results underline the notion that subtle changes in the components of a supramolecular assembly may substantially modulate the interplay of interactions which dictate its structure and functional properties.

  15. Chiroplasmonic DNA-based nanostructures

    NASA Astrophysics Data System (ADS)

    Cecconello, Alessandro; Besteiro, Lucas V.; Govorov, Alexander O.; Willner, Itamar

    2017-09-01

    Chiroplasmonic properties of nanoparticles, organized using DNA-based nanostructures, have attracted both theoretical and experimental interest. Theory suggests that the circular dichroism spectra accompanying chiroplasmonic nanoparticle assemblies are controlled by the sizes, shapes, geometries and interparticle distances of the nanoparticles. In this Review, we present different methods to assemble chiroplasmonic nanoparticle or nanorod systems using DNA scaffolds, and we discuss the operations of dynamically reconfigurable chiroplasmonic nanostructures. The chiroplasmonic properties of the different systems are characterized by circular dichroism and further supported by high-resolution transmission electron microscopy or cryo-transmission electron microscopy imaging and theoretical modelling. We also outline the applications of chiroplasmonic assemblies, including their use as DNA-sensing platforms and as functional systems for information processing and storage. Finally, future perspectives in applying chiroplasmonic nanoparticles as waveguides for selective information transfer and their use as ensembles for chiroselective synthesis are discussed. Specifically, we highlight the upscaling of the systems to device-like configurations.

  16. Plasmid segregation: how to survive as an extra piece of DNA.

    PubMed

    Salje, Jeanne

    2010-08-01

    Non-essential extra-chromosomal DNA elements such as plasmids are responsible for their own propagation in dividing host cells, and one means to ensure this is to carry a miniature active segregation system reminiscent of the mitotic spindle. Plasmids that are maintained at low numbers in prokaryotic cells have developed a range of such active partitioning systems, which are characterized by an impressive simplicity and efficiency and which are united by the use of dynamic, nucleotide-driven filaments to separate and position DNA molecules. A comparison of different plasmid segregation systems reveals (i) how unrelated filament-forming and DNA-binding proteins have been adopted and modified to create a range of simple DNA segregating complexes and (ii) how subtle changes in the few components of these DNA segregation machines has led to a remarkable diversity in the molecular mechanisms of closely related segregation systems. Here, our current understanding of plasmid segregation systems is reviewed and compared with other DNA segregation systems, and this is extended by a discussion of basic principles of plasmid segregation systems, evolutionary implications and the relationship between an autonomous DNA element and its host cell.

  17. Production of Double-stranded DNA Ministrings

    PubMed Central

    Wong, Shirley; Lam, Peggy; Nafissi, Nafiseh; Denniss, Steven; Slavcev, Roderick

    2016-01-01

    We constructed linear covalently closed (LCC) DNA minivectors as a non-viral gene-delivery vector alternative produced via a simple platform in vivo. DNA ministrings possess a heightened safety profile and also efficiently deliver DNA cargo to targeted cells. Conventional DNA vectors carry undesirable prokaryotic sequences, including antibiotic resistance genes, CpG motifs, and bacterial origins of replication, which may lead to the stimulation of host immunological responses. The bioavailability of conventional DNA vectors is also compromised due to their larger molecular size. Their circular nature may also impart chromosomal integration, leading to insertional mutagenesis. Bacterial sequences are excised from DNA minivectors, leaving only the gene of interest (GOI) and necessary eukaryotic expression elements. Our LCC DNA minivectors, or DNA ministrings, are devoid of immunogenic bacterial sequences; therefore improving their bioavailability and GOI expression. In the event of vector integration into the chromosome, the LCC DNA ministring will lethally disrupt the host chromosome, thereby removing the potentially dangerous mutant from the proliferating cell population. Consequently, DNA ministrings offer the benefits of 'minicircle' DNA while eliminating the potential for undesirable vector integration events. In comparison to conventional plasmids and their isogenic circular covalently closed (CCC) counterparts, DNA ministrings demonstrate superior bioavailability, transfection efficiency, and cytoplasmic kinetics - they thus require lower amounts of cationic surfactants for effective transfection of target cells. We have constructed a one-step inducible in vivo system for the production of DNA ministrings in Escherichia coli that is simple to use, rapid, and scalable. PMID:26967586

  18. Analysis of Transfer Maneuvers from Initial Circular Orbit to a Final Circular or Elliptic Orbit

    NASA Astrophysics Data System (ADS)

    Sharaf, M. A.; Saad, A. S.

    2016-10-01

    In the present paper an analysis of the transfer maneuvers from initial circular orbit to a final circular or elliptic orbit was developed to study the problem of impulsive transfers for space missions. It considers planar maneuvers using newly derived equations. With these equations, comparisons of circular and elliptic maneuvers are made. This comparison is important for the mission designers to obtain useful mappings showing where one maneuver is better than the other. In this aspect, we developed this comparison throughout ten results, together with some graphs to show their meaning.

  19. Condensed DNA in lipid microcompartments.

    PubMed

    Osfouri, Shahriar; Stano, Pasquale; Luisi, Pier Luigi

    2005-10-27

    DNA was studied in lipid reverse micelles with the aim of investigating the interactions of DNA with lipids in a restricted compartment with minimal water content. Circular dichroic (CD) spectra of DNA at low water content showed the characteristic polymer-salt-induced (psi) spectra of condensed DNA. Dynamic light scattering showed a peak around a radius of 400 nm (corresponding to DNA-containing micelles), and a peak around 2.5 nm (corresponding to "empty" micelles). Fourier Transform-IR (FT-IR) spectroscopy was carried out and analyzed in terms of three distinct states of water inside the micelle water pool, where the local concentration of DNA reached an estimated value of ca. 600 mg/mL, comparable to that found in restricted biological compartments.

  20. Combustor with non-circular head end

    SciTech Connect

    Kim, Won -Wook; McMahan, Kevin Weston

    2015-09-29

    The present application provides a combustor for use with a gas turbine engine. The combustor may include a head end with a non-circular configuration, a number of fuel nozzles positioned about the head end, and a transition piece extending downstream of the head end.

  1. Using Pesticides Safely. Special Circular 296.

    ERIC Educational Resources Information Center

    Hock, W. K.

    The use of pesticides is necessary to aid in the production of food, feed and fiber, and is equally important in the control of home garden and household pests, but correct use is critical in preventing injury to persons, animals, and plants. This circular contains information on State of Pennsylvania and federal pesticide laws; different types of…

  2. Circular polarisation characteristics of stacked microstrip antennas

    NASA Astrophysics Data System (ADS)

    Lee, R. Q.; Talty, T.; Lee, K. F.

    1990-12-01

    Experimental results on the circular polarization (CP) characteristics of a two-layer electromagnetically coupled (EMCP) antenna are presented. Compared to the single CP patch antenna, the two-layer EMCP antenna with proper spacings can provide better axial ratio and directivity.

  3. All-dielectric metasurface circular dichroism waveplate

    NASA Astrophysics Data System (ADS)

    Hu, Jingpei; Zhao, Xiaonan; Lin, Yu; Zhu, Aijiao; Zhu, Xiaojun; Guo, Peiji; Cao, Bing; Wang, Chinhua

    2017-01-01

    We propose and experimentally demonstrate a high efficient circularly polarizing dichroism waveplate (CPDW) using a Si-based all-dielectric 2Dchiral metasurface. We demonstrate that the CPDW exhibits a unique dichroism in that it functions as a transmissive quarter waveplate for one of either left-or right-handed circularly polarized incident lightand a reflective mirror for the opposite polarization. The circular polarization dichroism (CPD = IRCP ‑ ILCP) in transmission at wavelength ~1.5 μm reaches 97% and the extinction ratio (ER = IRCP/ILCP) is as high as 345:1. Experimental fabrications and measurements of the proposed all-dielectric metasurface are implemented and found to be in excellent agreement with the simulations. The proposed all-dielectric chiral metasurface is of advantages of high-dichroism, easy-fabrication and standard semiconductor fabrication techniques compatible, which could lead to enhanced security in fiber and free-space communications, as well as imaging and sensing applications for circularly polarized light with a highly integrated photonic platform.

  4. Circular tapered tape fabrication: Final report

    SciTech Connect

    Stockdale, D.A.

    1988-04-01

    A new approach to producing tapered tapes has been developed involving generating a tapered tape in a circular configuration on a lathe rather than a mill. This approach is more cost-effective and the redesigned slot configuration induced less stress on the tapes during installation and removal.

  5. Compliance Supplement. OMB Circular A-133.

    ERIC Educational Resources Information Center

    Office of Management and Budget, Washington, DC.

    This document is a supplement to Circular A-133 (1990) from the Office of Management and Budget (OMB), which extended the government's "single audit process" for agencies that administer federal financial assistance programs to higher education institutions and non-profit organizations. This supplement is based on the 1996 Amendments …

  6. All-dielectric metasurface circular dichroism waveplate

    PubMed Central

    Hu, Jingpei; Zhao, Xiaonan; Lin, Yu; Zhu, Aijiao; Zhu, Xiaojun; Guo, Peiji; Cao, Bing; Wang, Chinhua

    2017-01-01

    We propose and experimentally demonstrate a high efficient circularly polarizing dichroism waveplate (CPDW) using a Si-based all-dielectric 2Dchiral metasurface. We demonstrate that the CPDW exhibits a unique dichroism in that it functions as a transmissive quarter waveplate for one of either left-or right-handed circularly polarized incident lightand a reflective mirror for the opposite polarization. The circular polarization dichroism (CPD = IRCP − ILCP) in transmission at wavelength ~1.5 μm reaches 97% and the extinction ratio (ER = IRCP/ILCP) is as high as 345:1. Experimental fabrications and measurements of the proposed all-dielectric metasurface are implemented and found to be in excellent agreement with the simulations. The proposed all-dielectric chiral metasurface is of advantages of high-dichroism, easy-fabrication and standard semiconductor fabrication techniques compatible, which could lead to enhanced security in fiber and free-space communications, as well as imaging and sensing applications for circularly polarized light with a highly integrated photonic platform. PMID:28139753

  7. All-dielectric metasurface circular dichroism waveplate.

    PubMed

    Hu, Jingpei; Zhao, Xiaonan; Lin, Yu; Zhu, Aijiao; Zhu, Xiaojun; Guo, Peiji; Cao, Bing; Wang, Chinhua

    2017-01-31

    We propose and experimentally demonstrate a high efficient circularly polarizing dichroism waveplate (CPDW) using a Si-based all-dielectric 2Dchiral metasurface. We demonstrate that the CPDW exhibits a unique dichroism in that it functions as a transmissive quarter waveplate for one of either left-or right-handed circularly polarized incident lightand a reflective mirror for the opposite polarization. The circular polarization dichroism (CPD = IRCP - ILCP) in transmission at wavelength ~1.5 μm reaches 97% and the extinction ratio (ER = IRCP/ILCP) is as high as 345:1. Experimental fabrications and measurements of the proposed all-dielectric metasurface are implemented and found to be in excellent agreement with the simulations. The proposed all-dielectric chiral metasurface is of advantages of high-dichroism, easy-fabrication and standard semiconductor fabrication techniques compatible, which could lead to enhanced security in fiber and free-space communications, as well as imaging and sensing applications for circularly polarized light with a highly integrated photonic platform.

  8. Stability analysis of cylinders with circular cutouts

    NASA Technical Reports Server (NTRS)

    Almroth, B. O.; Brogan, F. A.; Marlowe, M. B.

    1973-01-01

    The stability of axially compressed cylinders with circular cutouts is analyzed numerically. An extension of the finite-difference method is used which removes the requirement that displacement components be defined in the directions of the grid lines. The results of this nonlinear analysis are found to be in good agreement with earlier experimental results.

  9. Cold Rydberg atoms in circular states

    NASA Astrophysics Data System (ADS)

    Anderson, David; Schwarzkopf, Andrew; Raithel, Georg

    2012-06-01

    Circular-state Rydberg atoms are interesting in that they exhibit a unique combination of extraordinary properties; long lifetimes (˜n^5), large magnetic moments (l=|m|=n-1) and no first order Stark shift. Circular states have found applications in cavity quantum electrodynamics and precision measurements [1,2], among other studies. In this work we present the production of circular states in an atom trapping apparatus using an adiabatic state-switching method (the crossed-field method [3]). To date, we have observed lifetimes of adiabatically prepared states of several milliseconds. Their relatively large ionization electric fields have been verified by time-of-flight signatures of ion trajectories. We intend to explore the magnetic trapping of circular state Rydberg atoms, as well as their production and interaction properties in ultra-cold and degenerate samples.[4pt] [1] P. Bertet et al., Phys. Rev. Lett., 88, 14 (2002)[0pt] [2] M. Brune et al., Phys. Rev. Lett., 72, 21 (1994)[0pt] [3] D. Delande and J.C. Gay, Europhys. Lett., 5, 303-308 (1988).

  10. 76 FR 60593 - Title VI; Proposed Circular

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-09-29

    ... Federal Transit Administration Title VI; Proposed Circular AGENCY: Federal Transit Administration (FTA... Federal Transit Administration (FTA) has placed in the docket and on its Web site, proposed guidance in... locations will be ADA- and transit-accessible. For details about the exact location of each...

  11. General Information on Copyright. Circular 1.

    ERIC Educational Resources Information Center

    Library of Congress, Washington, DC. Copyright Office.

    In this circular, the United State Copyright Office answers questions that are frequently asked about copyright. A definition of copyright is given and the items that can be copyrighted are listed. Four categories of items which cannot be copyrighted are also explained. Copyright procedures for unpublished and published works are outlined, and the…

  12. Non-Circular Wheels: Reuleaux and Squares

    ERIC Educational Resources Information Center

    Mills, Allan

    2011-01-01

    Circular wheels are so familiar on vehicles of all types that it is seldom realized that alternatives do exist. This short non-mathematical article describes Reuleaux and square wheels that, rolling along appropriate tracks, can maintain a moving platform at a constant height. Easily made working models lend themselves to demonstrations at science…

  13. 21 CFR 606.122 - Instruction circular.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... shall contain: (1) Instructions to administer a suitable plasma volume expander if Red Blood Cells are...: (1) The approximate volume of plasma from which a sample unit of Platelets is prepared. (2... container. (m) For Plasma, the instruction circular shall contain: (1) A warning against further processing...

  14. Non-Circular Wheels: Reuleaux and Squares

    ERIC Educational Resources Information Center

    Mills, Allan

    2011-01-01

    Circular wheels are so familiar on vehicles of all types that it is seldom realized that alternatives do exist. This short non-mathematical article describes Reuleaux and square wheels that, rolling along appropriate tracks, can maintain a moving platform at a constant height. Easily made working models lend themselves to demonstrations at science…

  15. Magnifying Devices: A Resource Guide. Reference Circular.

    ERIC Educational Resources Information Center

    Strauss, Carol, Comp.

    The devices listed in this reference circular are designed to assist people who have visual impairments by magnifying objects and print or graphic materials. Before buying a magnification device, one should consult a low-vision specialist to determine the level of usable vision, the power of magnification needed for a particular eye condition and…

  16. Home Sewage Disposal. Special Circular 212.

    ERIC Educational Resources Information Center

    Wooding, N. Henry

    This circular provides current information for homeowners who must repair or replace existing on-lot sewage disposal systems. Site requirements, characteristics and preparation are outlined for a variety of alternatives such as elevated sand mounds, sand-lined beds and trenches, and oversized absorption area. Diagrams indicating construction…

  17. Articulated Multimedia Physics, Lesson 10, Circular Motion.

    ERIC Educational Resources Information Center

    New York Inst. of Tech., Old Westbury.

    As the tenth lesson of the Articulated Multimedia Physics Course, instructional materials relating to circular motion are presented in this study guide. The topics are concerned with instantaneous velocity, centripetal force, centrifugal force, and satellite paths. The content is arranged in scrambled form, and the use of matrix transparencies is…

  18. Stability analysis of cylinders with circular cutouts

    NASA Technical Reports Server (NTRS)

    Almroth, B. O.; Brogan, F. A.; Marlowe, M. B.

    1973-01-01

    The stability of axially compressed cylinders with circular cutouts is analyzed numerically. An extension of the finite-difference method is used which removes the requirement that displacement components be defined in the directions of the grid lines. The results of this nonlinear analysis are found to be in good agreement with earlier experimental results.

  19. NOTE: Circular symmetry in topologically massive gravity

    NASA Astrophysics Data System (ADS)

    Deser, S.; Franklin, J.

    2010-05-01

    We re-derive, compactly, a topologically massive gravity (TMG) decoupling theorem: source-free TMG separates into its Einstein and Cotton sectors for spaces with a hypersurface-orthogonal Killing vector, here concretely for circular symmetry. We then generalize the theorem to include matter; surprisingly, the single Killing symmetry also forces conformal invariance, requiring the sources to be null.

  20. Using Pesticides Safely. Special Circular 296.

    ERIC Educational Resources Information Center

    Hock, W. K.

    The use of pesticides is necessary to aid in the production of food, feed and fiber, and is equally important in the control of home garden and household pests, but correct use is critical in preventing injury to persons, animals, and plants. This circular contains information on State of Pennsylvania and federal pesticide laws; different types of…

  1. Model of DNA topology simplification has come full (supercoiled) circle after two decades of research. Comment on "Disentangling DNA molecules" by Alexander Vologodskii

    NASA Astrophysics Data System (ADS)

    Stasiak, Andrzej

    2016-09-01

    Being a geek of DNA topology, I remember very well the stir caused by 1997 Science paper showing that DNA topoisomerases have the ability to simplify DNA topology below the topological equilibrium values [1]. In their seminal experiments Rybenkov et al. [1] started with linear double-stranded DNA molecules with cohesive ends. The mutual cohesiveness of DNA ends was due to mutual complementarity of single-stranded extensions at both ends of linear double-stranded DNA molecules. When such DNA molecules were heated up and then slowly cooled down the single-stranded ends eventually annealed with each other causing DNA circularization. This experimental protocol permitted the authors to establish topological/thermodynamic equilibrium within samples of circularized DNA molecules. Among simple unknotted circles one also observed knotted and catenated DNA molecules. The fraction of knotted molecules in DNA samples at topological equilibrium was increasing with the length of DNA molecules undergoing slow circularization. The fraction of catenated molecules was increasing with the length and the concentration of the molecules undergoing slow circularization. Rybenkov et al. incubated then such equilibrated DNA samples with type II DNA topoisomerases, which pass DNA duplex regions through each other, and observed that as the result of it the fraction of knotted and catenated DNA molecules was dramatically decreased (up to 80-fold). This elegant experiment indicated for the first time that type II DNA topoisomerases acting on knotted or catenated DNA molecules have the ability to select among many potential sites of DNA-DNA passages these that result in DNA unknotting or decatenation. Without such a selection topoisomerases could only maintain the original topological equilibrium obtained during the slow cyclization. The big question was how DNA topoisomerases can be directed to do DNA-DNA passages that preferentially result in DNA unknotting and decatenation.

  2. How Can Plant DNA Viruses Evade siRNA-Directed DNA Methylation and Silencing?

    PubMed Central

    Pooggin, Mikhail M.

    2013-01-01

    Plants infected with DNA viruses produce massive quantities of virus-derived, 24-nucleotide short interfering RNAs (siRNAs), which can potentially direct viral DNA methylation and transcriptional silencing. However, growing evidence indicates that the circular double-stranded DNA accumulating in the nucleus for Pol II-mediated transcription of viral genes is not methylated. Hence, DNA viruses most likely evade or suppress RNA-directed DNA methylation. This review describes the specialized mechanisms of replication and silencing evasion evolved by geminiviruses and pararetoviruses, which rescue viral DNA from repressive methylation and interfere with transcriptional and post-transcriptional silencing of viral genes. PMID:23887650

  3. A DNA-binding protein from Ustilago maydis prefers duplex DNA without chain interruptions.

    PubMed Central

    Rusche, J R; Holloman, W K

    1983-01-01

    Using a nitrocellulose filter binding assay, we have partially purified a protein from mitotic cells of Ustilago maydis that binds preferentially to covalently closed circular duplex DNA. DNA containing single- or double-strand breaks is bound poorly by the protein. Once formed, the DNA-protein complex is stable, resisting dissociation in high salt. However, when a DNA strand is broken, the complex appears to dissociate. The protein binds equally well to form I DNA of phi X174 or the plasmid pBR322, but has a higher affinity for a hybrid plasmid containing a cloned region of Drosophila melanogaster satellite DNA. Images PMID:6304499

  4. SMARCAL1 maintains telomere integrity during DNA replication.

    PubMed

    Poole, Lisa A; Zhao, Runxiang; Glick, Gloria G; Lovejoy, Courtney A; Eischen, Christine M; Cortez, David

    2015-12-01

    The SMARCAL1 (SWI/SNF related, matrix-associated, actin-dependent, regulator of chromatin, subfamily A-like 1) DNA translocase is one of several related enzymes, including ZRANB3 (zinc finger, RAN-binding domain containing 3) and HLTF (helicase-like transcription factor), that are recruited to stalled replication forks to promote repair and restart replication. These enzymes can perform similar biochemical reactions such as fork reversal; however, genetic studies indicate they must have unique cellular activities. Here, we present data showing that SMARCAL1 has an important function at telomeres, which present an endogenous source of replication stress. SMARCAL1-deficient cells accumulate telomere-associated DNA damage and have greatly elevated levels of extrachromosomal telomere DNA (C-circles). Although these telomere phenotypes are often found in tumor cells using the alternative lengthening of telomeres (ALT) pathway for telomere elongation, SMARCAL1 deficiency does not yield other ALT phenotypes such as elevated telomere recombination. The activity of SMARCAL1 at telomeres can be separated from its genome-maintenance activity in bulk chromosomal replication because it does not require interaction with replication protein A. Finally, this telomere-maintenance function is not shared by ZRANB3 or HLTF. Our results provide the first identification, to our knowledge, of an endogenous source of replication stress that requires SMARCAL1 for resolution and define differences between members of this class of replication fork-repair enzymes.

  5. Spectroscopic Evaluation of DNA-Borate Interactions.

    PubMed

    Ozdemir, Ayse; Sarioglu, Omer Faruk; Tekinay, Turgay

    2015-12-01

    We describe the binding characteristics of two natural borates (colemanite and ulexite) to calf thymus DNA by UV-vis absorbance spectroscopy, circular dichroism (CD) spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, and a competitive DNA binding assay. Our results suggest that colemanite and ulexite interact with calf thymus DNA under a non-intercalative mode of binding and do not alter the secondary structure of the DNA helix. The FT-IR spectroscopy results indicate that the two borates might interact with DNA through sugar-phosphate backbone binding.

  6. Finding Optimal Alignment and Consensus of Circular Strings

    NASA Astrophysics Data System (ADS)

    Lee, Taehyung; Na, Joong Chae; Park, Heejin; Park, Kunsoo; Sim, Jeong Seop

    We consider the problem of finding the optimal alignment and consensus (string) of circular strings. Circular strings are different from linear strings in that the first (leftmost) symbol of a circular string is wrapped around next to the last (rightmost) symbol. In nature, for example, bacterial and mitochondrial DNAs typically form circular strings. The consensus string problem is finding a representative string (consensus) of a given set of strings, and it has been studied on linear strings extensively. However, only a few efforts have been made for the consensus problem for circular strings, even though circular strings are biologically important. In this paper, we introduce the consensus problem for circular strings and present novel algorithms to find the optimal alignment and consensus of circular strings under the Hamming distance metric. They are O(n 2logn)-time algorithms for three circular strings and an O(n 3logn)-time algorithm for four circular strings. Our algorithms are O(n/ logn) times faster than the naïve algorithm directly using the solutions for the linear consensus problems, which takes O(n 3) time for three circular strings and O(n 4) time for four circular strings. We achieved this speedup by adopting a convolution and a system of linear equations into our algorithms to reflect the characteristics of circular strings that we found.

  7. A simple circular-polarized antenna: Circular waveguide horn coated with lossy magnetic material

    NASA Technical Reports Server (NTRS)

    Lee, C. S.; Lee, S. W.; Justice, D. W.

    1986-01-01

    A circular waveguide horn coated with a lossy material in its interior wall can be used as an alternative to a corrugated waveguide for radiating a circularly polarized (CP) field. To achieve good CP radiation, the diameter of the structure must be larger than the free-space wavelength, and the coating material must be sufficiently lossy and magnetic. This device is cheaper and lighter in weight than the corrugated one.

  8. A simple circular-polarized antenna: Circular waveguide horn coated with lossy magnetic material

    NASA Technical Reports Server (NTRS)

    Lee, Choon S.; Justice, D. W.; Lee, Shung-Wu

    1988-01-01

    It is shown that a circular waveguide horn coated with a lossy material in its interior wall can be used as an alternative to a corrugated waveguide for radiating a circularly polarized (CP) field. To achieve good CP radiation, the diameter of the structure must be larger than the free-space wavelength, and the coating material must be sufficiently lossy and magnetic. The device is cheaper and lighter in weight than the corrugated one.

  9. Transcription and replication of mitochondrial DNA.

    PubMed

    Clayton, D A

    2000-07-01

    The physical isolation of mammalian mitochondrial DNA (mtDNA) over 30 years ago marked the beginning of studies of its structure, replication and the expression of its genetic content. Such analyses have revealed a number of surprises: novel DNA structural features of the circular genome such as the displacement loop (D-loop); multiple sized and deleted forms of the circular genome; a minimal set of mitochondrially encoded rRNAs and tRNAs needed for translation; a bacteriophage-like, nuclear-encoded mitochondrial RNA polymerase for transcription; and a direct linkage between transcription and the commitment to replication of the leading mtDNA strand that centres on the nuclear encoded mitochondrial transcription factor A. One of the more recent revelations is the existence, near the D-loop, of an atypical, stable RNA-DNA hybrid (or R-loop) at the origin of mammalian leading-strand DNA replication, composed of the parent DNA strands and an RNA transcript. In mammalian mitochondrial systems, all of the proteins known to be involved in DNA replication are encoded in the nucleus. Thus alterations and deficiencies in mtDNA replication must arise from mutations in mtDNA regulatory sequences and nuclear gene defects. Further studies of the relationships between nuclear-encoded proteins and their mtDNA target sequences could result in strategies to manipulate genotypes within cellular mtDNA populations.

  10. Rice HMGB1 protein recognizes DNA structures and bends DNA efficiently.

    PubMed

    Wu, Qiang; Zhang, Wensheng; Pwee, Keng-Hock; Kumar, Prakash P

    2003-03-01

    We analyzed the DNA-binding and DNA-bending properties of recombinant HMGB1 proteins based on a rice HMGB1 cDNA. Electrophoretic mobility shift assay demonstrated that rice HMGB1 can bind synthetic four-way junction (4H) DNA and DNA minicircles efficiently but the binding to 4H can be completed out by HMGA and histone H1. Conformational changes were detected by circular dichroism analysis with 4H DNA bound to various concentrations of HMGB1 or its truncated forms. T4 ligase-mediated circularization assays with short DNA fragments of 123 bp showed that the protein is capable of increasing DNA flexibility. The 123-bp DNA formed closed circular monomers efficiently in its presence, similar to that in an earlier study on maize HMG. Additionally, our results show for the first time that the basic N-terminal domain enhances the affinity of the plant HMGB1 protein for 4H DNA, while the acidic C-terminal domain has the converse effects.

  11. Efficient Sleeping Beauty DNA Transposition From DNA Minicircles

    PubMed Central

    Sharma, Nynne; Cai, Yujia; Bak, Rasmus O; Jakobsen, Martin R; Schrøder, Lisbeth Dahl; Mikkelsen, Jacob Giehm

    2013-01-01

    DNA transposon-based vectors have emerged as new potential delivery tools in therapeutic gene transfer. Such vectors are now showing promise in hematopoietic stem cells and primary human T cells, and clinical trials with transposon-engineered cells are on the way. However, the use of plasmid DNA as a carrier of the vector raises safety concerns due to the undesirable administration of bacterial sequences. To optimize vectors based on the Sleeping Beauty (SB) DNA transposon for clinical use, we examine here SB transposition from DNA minicircles (MCs) devoid of the bacterial plasmid backbone. Potent DNA transposition, directed by the hyperactive SB100X transposase, is demonstrated from MC donors, and the stable transfection rate is significantly enhanced by expressing the SB100X transposase from MCs. The stable transfection rate is inversely related to the size of circular donor, suggesting that a MC-based SB transposition system benefits primarily from an increased cellular uptake and/or enhanced expression which can be observed with DNA MCs. DNA transposon and transposase MCs are easily produced, are favorable in size, do not carry irrelevant DNA, and are robust substrates for DNA transposition. In accordance, DNA MCs should become a standard source of DNA transposons not only in therapeutic settings but also in the daily use of the SB system. PMID:23443502

  12. 1. Aerial view of southwest circular bastion. Rose Island lighthouse ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    1. Aerial view of southwest circular bastion. Rose Island lighthouse was built atop structure in 1869. - Fort Hamilton, Southwest Circular Bastion, Underneath Rose Island Lighthouse, Newport, Newport County, RI

  13. Achromatic circular polarization generation for ultra-intense lasers.

    SciTech Connect

    Atherton, Briggs W.; Schollmeier, Marius; Bennett, Guy R.; Rambo, Patrick K.; Schwarz, Jens; Kimmel, Mark W.

    2010-05-01

    Generating circular polarization for ultra-intense lasers requires solutions beyond traditional transmissive waveplates which have insufficient bandwidth and pose nonlinear phase (B-integral) problems. We demonstrate a reflective design employing 3 metallic mirrors to generate circular polarization.

  14. G-quadruplex-interacting compounds alter latent DNA replication and episomal persistence of KSHV

    PubMed Central

    Madireddy, Advaitha; Purushothaman, Pravinkumar; Loosbroock, Christopher P.; Robertson, Erle S.; Schildkraut, Carl L.; Verma, Subhash C.

    2016-01-01

    Kaposi's sarcoma associated herpesvirus (KSHV) establishes life-long latent infection by persisting as an extra-chromosomal episome in the infected cells and by maintaining its genome in dividing cells. KSHV achieves this by tethering its epigenome to the host chromosome by latency associated nuclear antigen (LANA), which binds in the terminal repeat (TR) region of the viral genome. Sequence analysis of the TR, a GC-rich DNA element, identified several potential Quadruplex G-Rich Sequences (QGRS). Since quadruplexes have the tendency to obstruct DNA replication, we used G-quadruplex stabilizing compounds to examine their effect on latent DNA replication and the persistence of viral episomes. Our results showed that these G-quadruplex stabilizing compounds led to the activation of dormant origins of DNA replication, with preferential bi-directional pausing of replications forks moving out of the TR region, implicating the role of the G-rich TR in the perturbation of episomal DNA replication. Over time, treatment with PhenDC3 showed a loss of viral episomes in the infected cells. Overall, these data show that G-quadruplex stabilizing compounds retard the progression of replication forks leading to a reduction in DNA replication and episomal maintenance. These results suggest a potential role for G-quadruplex stabilizers in the treatment of KSHV-associated diseases. PMID:26837574

  15. Amplitude-modulated circular-harmonic filter for pattern recognition.

    PubMed

    Chen, X W; Chen, Z P

    1995-02-10

    An amplitude-modulated circular-harmonic filter is proposed for rotation-invariant pattern recognition. We investigate the filter characteristics by varying two design parameters, A(ρ) and B(ρ), and select optimum values to design an amplitude-modulated circular-harmonic filter. When compared with the phase-only circular-harmonic filter, the amplitude-modulated circular-harmonic filter is found to yield a sharper correlation peak, a better noise tolerance, and an improved correlation discrimination.

  16. Circular magnetic x-ray dichroism in rare earth compounds

    SciTech Connect

    Lang, Jonathan

    1993-09-30

    This report discusses the following topics: Circular magnetic x-ray dichroism at the ER L3 Edge; angular dependence of circular magnetic x-ray dichroism in rare earth compounds: and circular magnetic x-ray dichroism in crystalline and amorphous GDFE2.

  17. Circular-Loop-Element Microwave Frequency-Selective Reflectors

    NASA Technical Reports Server (NTRS)

    Huang, John; Wu, Te-Kao; Lee, Shung-Wu

    1995-01-01

    Double or single planar arrays of circular loops laminated with dielectric sheets. Reflectors designed for use in multiplexing signals at several frequencies in microwave antenna system. Well suited to both circularly and linearly polarized radiation. Transmission and reflection characteristics relatively insensitive to angle of incidence. Similar device described in "Triband Circular-Loop Frequency-Selective Microwave Reflector" (NPO-18714).

  18. Photoelectron circular dichroism in different ionization regimes

    NASA Astrophysics Data System (ADS)

    Wollenhaupt, Matthias

    2016-12-01

    Photoelectron circular dichroism (PECD) describes an asymmetry in the photoelectron angular distribution (PAD) from photoionization of randomly oriented enantiomers with circularly polarized light. Beaulieu et al present a comprehensive set of measured PADs from multiphoton ionization of limonene and fenchone in different ionization regimes (multiphoton and tunneling) and analyze the resulting PECD (Beaulieu et al 2016 New J. Phys. 18 102002). From their observations the authors conclude that the PECD is universal in the sense that the molecular chirality is encoded in the PAD independent of the ionization regime. The analysis is supplemented by a classical model based on electron scattering in a chiral potential. The paper presents beautiful data and is an important step towards a more complete physical picture of PECD. The results and their interpretation stimulate the ongoing vivid debate on the role of resonances in multiphoton PECD.

  19. The circular migration of smallholders in Kenya.

    PubMed

    Bigsten, A

    1996-01-01

    Circular migration is a central phenomenon in the lives of smallholders in East Africa. Many migration decisions are not individual decisions, but rather household decisions in which the household allocates its labor force among activities to maximize household utility. A probit model which incorporates circular migration and takes into account contacts, information, and indivisibilities is used to analyze migration among 763 farm households in the Central and Nyanza provinces of Kenya. Study data are from a 1982 survey. The pull of high urban wages appears to be a far more important determinant of migration decision outcomes than the push of land scarcity, while a strong local nonagricultural economy does not seem to restrict migration. Networks of personal contacts were found to be highly significant determinants of migration. These findings suggest that rural development will probably not reduce the flow of migration.

  20. Impedance properties of circular microstrip antenna

    NASA Technical Reports Server (NTRS)

    Deshpande, M. D.; Bailey, M. C.

    1983-01-01

    A moment method solution to the input impedance of a circular microstrip antenna excited by either a microstrip feed or a coaxial probe is presented. Using the exact dyadic Green's function and the Fourier transform the problem is formulated in terms of Richmond's reaction integral equation from which the unknown patch current can be solved for. The patch current is expanded in terms of regular surface patch modes and an attachment mode (for probe excited case) which insures continuity of the current at probe/patch junction, proper polarization and p-dependance of patch current in the vicinity of the probe. The input impedance of a circular microstrip antenna is computed and compared with earlier results. Effect of attachment mode on the input impedance is also discussed.

  1. Quantized circular photogalvanic effect in Weyl semimetals.

    PubMed

    de Juan, Fernando; Grushin, Adolfo G; Morimoto, Takahiro; Moore, Joel E

    2017-07-06

    The circular photogalvanic effect (CPGE) is the part of a photocurrent that switches depending on the sense of circular polarization of the incident light. It has been consistently observed in systems without inversion symmetry and depends on non-universal material details. Here we find that in a class of Weyl semimetals (for example, SrSi2) and three-dimensional Rashba materials (for example, doped Te) without inversion and mirror symmetries, the injection contribution to the CPGE trace is effectively quantized in terms of the fundamental constants e, h, c and with no material-dependent parameters. This is so because the CPGE directly measures the topological charge of Weyl points, and non-quantized corrections from disorder and additional bands can be small over a significant range of incident frequencies. Moreover, the magnitude of the CPGE induced by a Weyl node is relatively large, which enables the direct detection of the monopole charge with current techniques.

  2. Quantized circular photogalvanic effect in Weyl semimetals

    PubMed Central

    de Juan, Fernando; Grushin, Adolfo G.; Morimoto, Takahiro; Moore, Joel E

    2017-01-01

    The circular photogalvanic effect (CPGE) is the part of a photocurrent that switches depending on the sense of circular polarization of the incident light. It has been consistently observed in systems without inversion symmetry and depends on non-universal material details. Here we find that in a class of Weyl semimetals (for example, SrSi2) and three-dimensional Rashba materials (for example, doped Te) without inversion and mirror symmetries, the injection contribution to the CPGE trace is effectively quantized in terms of the fundamental constants e, h, c and with no material-dependent parameters. This is so because the CPGE directly measures the topological charge of Weyl points, and non-quantized corrections from disorder and additional bands can be small over a significant range of incident frequencies. Moreover, the magnitude of the CPGE induced by a Weyl node is relatively large, which enables the direct detection of the monopole charge with current techniques. PMID:28681840

  3. Bilateral symmetry breaking in nonlinear circular cylinders.

    PubMed

    Yuan, Lijun; Lu, Ya Yan

    2014-12-01

    Symmetry breaking is a common phenomenon in nonlinear systems, it refers to the existence of solutions that do not preserve the original symmetries of the underlying system. In nonlinear optics, symmetry breaking has been previously investigated in a number of systems, usually based on simplified model equations or temporal coupled mode theories. In this paper, we analyze the scattering of an incident plane wave by one or two circular cylinders with a Kerr nonlinearity, and show the existence of solutions that break a lateral reflection symmetry. Although symmetry breaking is a known phenomenon in nonlinear optics, it is the first time that this phenomenon was rigorously studied in simple systems with one or two circular cylinders.

  4. Circular on early marriage, March 1988.

    PubMed

    1988-01-01

    This Circular calls on government at all levels in Hunan, China, to summon the departments concerned thoroughly to investigate the problem of early child-bearing so that they can do a good job in managing the problems of unregistered cohabiting and of unmarried mothers. The Circular recommends that: "It is necessary to criticize and educate, and even punish by discipline, those parents who connive with their sons and daughters in practicing unregistered cohabiting." It also states the following: "It is necessary to keep a strict check on registry personnel who do not carry out their duties properly. Those who violate laws and discipline, engage in malpractices for selfish ends, and practice bribery and corruption, resulting in early marriage and child-bearing, must be dealt with strictly. Legal sanctions must be enforced against those who break the law." full text

  5. Circular, confined distribution for charged particle beams

    DOEpatents

    Garnett, R.W.; Dobelbower, M.C.

    1995-11-21

    A charged particle beam line is formed with magnetic optics that manipulate the charged particle beam to form the beam having a generally rectangular configuration to a circular beam cross-section having a uniform particle distribution at a predetermined location. First magnetic optics form a charged particle beam to a generally uniform particle distribution over a square planar area at a known first location. Second magnetic optics receive the charged particle beam with the generally square configuration and affect the charged particle beam to output the charged particle beam with a phase-space distribution effective to fold corner portions of the beam toward the core region of the beam. The beam forms a circular configuration having a generally uniform spatial particle distribution over a target area at a predetermined second location. 26 figs.

  6. Circular, confined distribution for charged particle beams

    DOEpatents

    Garnett, Robert W.; Dobelbower, M. Christian

    1995-01-01

    A charged particle beam line is formed with magnetic optics that manipulate the charged particle beam to form the beam having a generally rectangular configuration to a circular beam cross-section having a uniform particle distribution at a predetermined location. First magnetic optics form a charged particle beam to a generally uniform particle distribution over a square planar area at a known first location. Second magnetic optics receive the charged particle beam with the generally square configuration and affect the charged particle beam to output the charged particle beam with a phase-space distribution effective to fold corner portions of the beam toward the core region of the beam. The beam forms a circular configuration having a generally uniform spatial particle distribution over a target area at a predetermined second location.

  7. Circular machine design techniques and tools

    SciTech Connect

    Servranckx, R.V.; Brown, K.L.

    1986-04-01

    Some of the basic optics principles involved in the design of circular accelerators such as Alternating Gradient Synchrotrons, Storage and Collision Rings, and Pulse Stretcher Rings are outlined. Typical problems facing a designer are defined, and the main references and computational tools are reviewed that are presently available. Two particular classes of problems that occur typically in accelerator design are listed - global value problems, which affect the control of parameters which are characteristic of the complete closed circular machine, and local value problems. Basic mathematical formulae are given that are considered useful for a first draft of a design. The basic optics building blocks that can be used to formulate an initial machine design are introduced, giving only the elementary properties and transfer matrices only in one transverse plane. Solutions are presented for some first-order and second-order design problems. (LEW)

  8. Shock structure in non-circular jets

    NASA Technical Reports Server (NTRS)

    Morris, Philip J.; Bhat, Thonse R. S.

    1989-01-01

    The shock-cell structure of supersonic jets with non-circular exit geometry is modeled using a linearized analysis. The model takes into account the finite thickness of the jet shear layer using realistic velocity and density profiles. The effects of the shear layer turbulence are included by incorporating eddy-viscosity terms. A finite-difference numerical method is used to solve the steady linearized equations of motion. A body-fitted coordinate system is used to describe the shear layer. The variation of the pressure fluctuation with downstream distance is given for circular jets and for an elliptic jet of aspect ratio 2.0. Comparisons with experimental data are made. Difficulties with the numerical technique are also discussed.

  9. Quantized circular photogalvanic effect in Weyl semimetals

    NASA Astrophysics Data System (ADS)

    de Juan, Fernando; Grushin, Adolfo G.; Morimoto, Takahiro; Moore, Joel E.

    2017-07-01

    The circular photogalvanic effect (CPGE) is the part of a photocurrent that switches depending on the sense of circular polarization of the incident light. It has been consistently observed in systems without inversion symmetry and depends on non-universal material details. Here we find that in a class of Weyl semimetals (for example, SrSi2) and three-dimensional Rashba materials (for example, doped Te) without inversion and mirror symmetries, the injection contribution to the CPGE trace is effectively quantized in terms of the fundamental constants e, h, c and with no material-dependent parameters. This is so because the CPGE directly measures the topological charge of Weyl points, and non-quantized corrections from disorder and additional bands can be small over a significant range of incident frequencies. Moreover, the magnitude of the CPGE induced by a Weyl node is relatively large, which enables the direct detection of the monopole charge with current techniques.

  10. A circular ferrofluid driven microchip for rapid polymerase chain reaction.

    PubMed

    Sun, Y; Kwok, Y C; Nguyen, N T

    2007-08-01

    In the past few years, much attention has been paid to the development of miniaturized polymerase chain reaction (PCR) devices. After a continuous flow (CF) PCR chip was introduced, several CFPCR systems employing various pumping mechanisms were reported. However, the use of pumps increases cost and imposes a high requirement on microchip bonding integrity due to the application of high pressure. Other significant limitations of CFPCR devices include the large footprint of the microchip and the fixed cycle number which is dictated by the channel layout. In this paper, we present a novel circular close-loop ferrofluid driven microchip for rapid PCR. A small ferrofluid plug, containing sub-domain magnetic particles in a liquid carrier, is driven by an external magnet along the circular microchannel, which in turn propels the PCR mixture through three temperature zones. Amplification of a 500 bp lambda DNA fragment has been demonstrated on the polymethyl methacrylate (PMMA) PCR microchip fabricated by CO(2) laser ablation and bonded by a low pressure, high temperature technique. Successful PCR was achieved in less than 4 min. Effects of cycle number and cycle time on PCR products were investigated. Using a magnet as the actuator eliminates the need for expensive pumps and provides advantages of low cost, small power consumption, low requirement on bonding strength and flexible number of PCR cycles. Furthermore, the microchip has a much simpler design and smaller footprint compared to the rectangular serpentine CFPCR devices. To demonstrate its application in forensics, a 16-loci short tandem repeat (STR) sample was successfully amplified using the PCR microchip.

  11. Acoustic characteristics of circular bends in pipes

    NASA Astrophysics Data System (ADS)

    Firth, D.; Fahy, F. J.

    1984-11-01

    The acoustic properties of circular bends in pipework systems are investigated by calculation of the mode shapes and propagation constants of the acoustic modes of the bend, the torus modes, and by evaluation of the transmission and reflection coefficients at a bend in an otherwise infinite straight pipe. The coefficients for the first three cylinder and torus modes are plotted against frequency for the case of a plane wave incident upon a 90° bend. The pipe walls are assumed to be rigid.

  12. Accelerator Considerations of Large Circular Colliders

    NASA Astrophysics Data System (ADS)

    Chao, Alex

    As we consider the tremendous physics reaches of the big future circular electron-positron and proton-proton colliders, it might be advisable to keep a close track of what accelerator challenges they face. Good progresses are being made, and yet it is reported here that substantial investments in funding, manpower, as well as a long sustained time to the R&D efforts will be required in preparation to realize these dream colliders.

  13. Accelerator considerations of large circular colliders

    NASA Astrophysics Data System (ADS)

    Chao, Alex

    2016-07-01

    As we consider the tremendous physics reaches of the big future circular electron-positron and proton-proton colliders, it might be advisable to keep a close track of what accelerator challenges they face. Good progresses are being made, and yet it is reported here that substantial investments in funding, manpower, as well as a long sustained time to the R&D efforts will be required in preparation to realize these dream colliders.

  14. Electromagnetic Scattering by Open Circular Waveguides.

    DTIC Science & Technology

    1980-01-01

    Camouflage Symposium , 18-20 November 1980 , sponsored by USAF Avionics Laboratory and Martin Marietta Corp. Pearson, J.D., (1953) "The Diffraction of...Huang, ( 1980 ) "An Analysis of the Electromag-: netic Fields Backscattered From a Jet Intake Configuration," Proceed- ings of the 1980 Radar ...8-AIBI 998 AIR FORCE INST OF TECH WRIGH4T-PATTERSON AFB ON F/6 21/B ELECTROMANETIC SCATTERING BY OPEN CIRCULAR WAVEUIDES.(U) 1980 T W JOHN4SON

  15. Peptide backbone circularization enhances antifreeze protein thermostability.

    PubMed

    Stevens, Corey A; Semrau, Joanna; Chiriac, Dragos; Litschko, Morgan; Campbell, Robert L; Langelaan, David N; Smith, Steven P; Davies, Peter L; Allingham, John S

    2017-10-01

    Antifreeze proteins (AFPs) are a class of ice-binding proteins that promote survival of a variety of cold-adapted organisms by decreasing the freezing temperature of bodily fluids. A growing number of biomedical, agricultural, and commercial products, such as organs, foods, and industrial fluids, have benefited from the ability of AFPs to control ice crystal growth and prevent ice recrystallization at subzero temperatures. One limitation of AFP use in these latter contexts is their tendency to denature and irreversibly lose activity at the elevated temperatures of certain industrial processing or large-scale AFP production. Using the small, thermolabile type III AFP as a model system, we demonstrate that AFP thermostability is dramatically enhanced via split intein-mediated N- and C-terminal end ligation. To engineer this circular protein, computational modeling and molecular dynamics simulations were applied to identify an extein sequence that would fill the 20-Å gap separating the free ends of the AFP, yet impose little impact on the structure and entropic properties of its ice-binding surface. The top candidate was then expressed in bacteria, and the circularized protein was isolated from the intein domains by ice-affinity purification. This circularized AFP induced bipyramidal ice crystals during ice growth in the hysteresis gap and retained 40% of this activity even after incubation at 100°C for 30 min. NMR analysis implicated enhanced thermostability or refolding capacity of this protein compared to the noncyclized wild-type AFP. These studies support protein backbone circularization as a means to expand the thermostability and practical applications of AFPs. © 2017 The Protein Society.

  16. Partially coherent sources with circular coherence: comment.

    PubMed

    Hyde, Milo W; Bose-Pillai, Santasri R

    2017-08-15

    In [Opt. Lett.42, 1512 (2017)OPLEDP0146-959210.1364/OL.42.001512], the authors present a new class of non-uniformly correlated sources with circular coherence. They also describe a basic experimental setup for synthesizing this class of sources, which uses the Van Cittert-Zernike theorem. Here, we present an alternative way to analyze these sources and a different way to generate them.

  17. Circular Metal/Semiconductor/Metal Photodetectors

    NASA Technical Reports Server (NTRS)

    Mcadoo, James A.; Towe, Elias; Bishop, William L.; Wang, Liang-Guo

    1995-01-01

    Metal/semiconductor/metal (MSM) photodetectors with multiple concentric circular electrodes developed. Some electrical characteristics expected superior to those of older MSM photodetectors containing interdigitated straight electrodes. Response times smaller and shorter, and breakdown voltages larger. Decrease in capacitance allows greater signal-detection bandwidth. Important advantage in fiber-optic telecommunication systems, in which photodectors central components in receiver circuits. Increasing bandwidth of such photodetector enables receiver to handle larger number of channels or increased information rate in each channel.

  18. Nuclear collisions at the Future Circular Collider

    NASA Astrophysics Data System (ADS)

    Armesto, N.; Dainese, A.; d'Enterria, D.; Masciocchi, S.; Roland, C.; Salgado, C. A.; van Leeuwen, M.; Wiedemann, U. A.

    2016-12-01

    The Future Circular Collider is a new proposed collider at CERN with centre-of-mass energies around 100 TeV in the pp mode. Ongoing studies aim at assessing its physics potential and technical feasibility. Here we focus on updates in physics opportunities accessible in pA and AA collisions not covered in previous Quark Matter contributions, including Quark-Gluon Plasma and gluon saturation studies, novel hard probes of QCD matter, and photon-induced collisions.

  19. Biological Sensing with Terahertz Circular Dichroism Spectroscopy

    DTIC Science & Technology

    2005-05-31

    S.J. and Plaxco, K.W. (2003) “Terahertz circular dichroism spectroscopy: a potential approach to unbiased, in situ life detection.” Astrobiology , 3...detection.” Astrobiology , 3, 489-504 Xu, J., Ramian, G.J., Galan, J.F., Savvidis, P.G., Scopatz, A.M., Birge, R.R., Allen, S.J. and Plaxco, K.W. (2004

  20. Circular Intensity Differential Scattering of chiral molecules

    SciTech Connect

    Bustamante, C.J.

    1980-12-01

    In this thesis a theory of the Circular Intensity Differential Scattering (CIDS) of chiral molecules as modelled by a helix oriented with respect to the direction of incidence of light is presented. It is shown that a necessary condition for the existence of CIDS is the presence of an asymmetric polarizability in the scatterer. The polarizability of the scatterer is assumed generally complex, so that both refractive and absorptive phenomena are taken into account.

  1. Circular zig-zag scan video format

    DOEpatents

    Peterson, C. Glen; Simmons, Charles M.

    1992-01-01

    A circular, ziz-zag scan for use with vidicon tubes. A sine wave is generated, rectified and its fourth root extracted. The fourth root, and its inverse, are used to generate horizontal ramp and sync signals. The fourth root is also used to generate a vertical sync signal, and the vertical sync signal, along with the horizontal sync signal, are used to generate the vertical ramp signal. Cathode blanking and preamplifier clamp signals are also obtained from the vertical sync signal.

  2. [Effect of supporting substrates on the structure of DNA and DNA-trivaline complexes studied by atomic force microscopy].

    PubMed

    Klinov, D V; Martynkina, L P; Iurchenko, V Iu; Demin, V V; Strel'tsov, S A; Gerasimov, Iu A; Vengerov, Iu Iu

    2003-01-01

    Linear DNA, circular DNA, and circular DNA complexes with trivaline (TV), a synthetic oligopeptide, were imaged by atomic force microscopy (AFM) using mica as a conventional supporting substrate and modified highly ordered pyrolytic graphite (HOPG) as an alternative substrate. A method of modifying the HOPG surface was developed that enabled the adsorption of DNA and DNA-TV complexes onto this surface. On mica, both purified DNA and DNA-TV complexes were shown to undergo significant structural distortions: DNA molecules decrease in height and DNA-TP displays substantial changes in the shape of its circular compact structures. Use of the HOPG support helps preserve the structural integrity of the complexes and increase the measured height of DNA molecules up to 2 nm. AFM with the HOPG support was shown to efficiently reveal the particular points of the complexes where, according to known models of their organization, a great number of bent DNA fibers meet. These results provide additional information on DNA organization in its complexes with TV and are also of methodological interest, since the use of the modified HOPG may widen the possibilities of AFM in studying DNA and its complexes with various ligands.

  3. Search by proteins for their DNA target site: 1. The effect of DNA conformation on protein sliding

    PubMed Central

    Bhattacherjee, Arnab; Levy, Yaakov

    2014-01-01

    The recognition of DNA-binding proteins (DBPs) to their specific site often precedes by a search technique in which proteins slide, hop along the DNA contour or perform inter-segment transfer and 3D diffusion to dissociate and re-associate to distant DNA sites. In this study, we demonstrated that the strength and nature of the non-specific electrostatic interactions, which govern the search dynamics of DBPs, are strongly correlated with the conformation of the DNA. We tuned two structural parameters, namely curvature and the extent of helical twisting in circular DNA. These two factors are mutually independent of each other and can modulate the electrostatic potential through changing the geometry of the circular DNA conformation. The search dynamics for DBPs on circular DNA is therefore markedly different compared with linear B-DNA. Our results suggest that, for a given DBP, the rotation-coupled sliding dynamics is precluded in highly curved DNA (as well as for over-twisted DNA) because of the large electrostatic energy barrier between the inside and outside of the DNA molecule. Under such circumstances, proteins prefer to hop in order to explore interior DNA sites. The change in the balance between sliding and hopping propensities as a function of DNA curvature or twisting may result in different search efficiency and speed. PMID:25324308

  4. Highly selective isolation of human DNAs from rodent–human hybrid cells as circular yeast artificial chromosomes by transformation-associated recombination cloning

    PubMed Central

    Larionov, Vladimir; Kouprina, Natalya; Graves, Joan; Resnick, Michael A.

    1996-01-01

    Transformation-associated recombination (TAR) can be exploited in yeast to clone human DNAs. TAR cloning was previously accomplished using one or two telomere-containing vectors with a common human repeat(s) that could recombine with human DNA during transformation to generate yeast artificial chromosomes (YACs). On basis of the proposal that broken DNA ends are more recombinogenic than internal sequences, we have investigated if TAR cloning could be applied to the generation of circular YACs by using a single centromere vector containing various human repeats at opposite ends. Transformation with these vectors along with human DNA led to the efficient isolation of circular YACs with a mean size of ≈150 kb. The circular YACs are stable and they can be easily separated from yeast chromosomes or moved into bacterial cells if the TAR vector contains an Escherichia coli F-factor cassette. More importantly, circular TAR cloning enabled the selective isolation of human DNAs from monochromosomal human–rodent hybrid cell lines. Although <2% of the DNA in the hybrid cells was human, as much as 80% of transformants had human DNA YACs when a TAR cloning vector contained Alu repeats. The level of enrichment of human DNA was nearly 3000-fold. A comparable level of enrichment was demonstrated with DNA isolated from a radiation hybrid cell line containing only 5 Mb of human DNA. A high selectivity of human DNA cloning was also observed for linear TAR cloning with two telomere vectors. No human–rodent chimeras were detected among YACs generated by TAR cloning. The results with a circular TAR cloning vector or two vectors differed from results with a single-telomere vector in that the latter often resulted in a series of terminal deletions in linear YACs. This could provide a means for physical mapping of cloned material. PMID:8943037

  5. Pulsed Accretion onto Eccentric and Circular Binaries

    NASA Astrophysics Data System (ADS)

    Muñoz, Diego J.; Lai, Dong

    2016-08-01

    We present numerical simulations of circumbinary accretion onto eccentric and circular binaries using the moving-mesh code AREPO. This is the first set of simulations to tackle the problem of binary accretion using a finite-volume scheme on a freely moving mesh, which allows for accurate measurements of accretion onto individual stars for arbitrary binary eccentricity. While accretion onto a circular binary shows bursts with period of ˜ 5 times the binary period P b, accretion onto an eccentric binary is predominantly modulated at the period ˜ 1{P}{{b}}. For an equal-mass circular binary, the accretion rates onto individual stars are quite similar to each other, following the same variable pattern in time. By contrast, for eccentric binaries, one of the stars can accrete at a rate 10-20 times larger than its companion. This “symmetry breaking” between the stars, however, alternates over timescales of order 200P b and can be attributed to a slowly precessing, eccentric circumbinary disk. Over longer timescales, the net accretion rates onto individual stars are the same, reaching a quasi-steady state with the circumbinary disk. These results have important implications for the accretion behavior of binary T Tauri stars and supermassive binary black holes.

  6. Continuous-wave circular polarization terahertz imaging

    NASA Astrophysics Data System (ADS)

    Martin, Jillian P.; Joseph, Cecil S.; Giles, Robert H.

    2016-07-01

    Biomedical applications of terahertz (THz) radiation are appealing because THz radiation is nonionizing and has the demonstrated ability to detect intrinsic contrasts between cancerous and normal tissue. A linear polarization-sensitive detection technique for tumor margin delineation has already been demonstrated; however, utilization of a circular polarization-sensitive detection technique has yet to be explored at THz frequencies. A reflective, continuous-wave THz imaging system capable of illuminating a target sample at 584 GHz with either linearly or circularly polarized radiation, and capable of collecting both cross- and copolarized signals remitted from the target, is implemented. To demonstrate the system's utility, a fresh ex vivo human skin tissue specimen containing nonmelanoma skin cancer was imaged. Both polarization-sensitive detection techniques showed contrast between tumor and normal skin tissue, although some differences in images were observed between the two techniques. Our results indicate that further investigation is required to explain the contrast mechanism, as well as to quantify the specificity and sensitivity of the circular polarization-sensitive detection technique.

  7. Functions and Regulation of Circular Dorsal Ruffles

    PubMed Central

    Hoon, Jing-Ling; Wong, Wai-Keung

    2012-01-01

    Cells construct a number of plasma membrane structures to meet a range of physiological demands. Driven by juxtamembrane actin machinery, these actin-based membrane protrusions are essential for the operation and maintenance of cellular life. They are required for diverse cellular functions, such as directed cell motility, cell spreading, adhesion, and substrate/matrix degradation. Circular dorsal ruffles (CDRs) are one class of such structures characterized as F-actin-rich membrane projections on the apical cell surface. CDRs commence their formation minutes after stimulation as flat, open, and immature ruffles and progressively develop into fully enclosed circular ruffles. These “rings” then mature and contract centrifugally before subsiding. Serving a critical function in receptor internalization and cell migration, CDRs are thus highly dynamic but transient formations. Here, we review the current state of knowledge concerning the regulation of circular dorsal ruffles. We focus specifically on the biochemical pathways leading to CDR formation in order to better define the roles and functions of these enigmatic structures. PMID:22927640

  8. Propulsion by Helical Strips in Circular Channels

    NASA Astrophysics Data System (ADS)

    Yesilyurt, Serhat; Demir, Ebru

    2016-11-01

    Progress in manufacturing techniques avails the production of artificial micro swimmers (AMS) in various shapes and sizes. There are numerous studies on the generation of efficient locomotion by means of helical tails with circular cross-sections. This work focuses on locomotion with helical strips in circular channels. A CFD model is used to analyze the effects of geometric parameters and the radius of the channel on swimming velocity of infinite helical-strips in circular channels. Results show that there is an optimum wavelength that depends on thickness to channel radius ratio, suggesting that these parameters need to be optimized simultaneously. With constant torque, thinner strips swim faster, whereas under constant angular velocity application, thicker strips (in radial direction) prevail. As width approaches the wavelength, velocity decreases under both conditions, unless a magnetically coated tail is simulated, for which width has an optimum value. Increasing channel radius to helix amplitude ratio increases the velocity up to a maximum and after a slight drop, saturation occurs as bulk swimming conditions are approached.

  9. Oxygen transfer in circular surface aeration tanks.

    PubMed

    Rao, Achanta Ramakrishna; Patel, Ajey Kumar; Kumar, Bimlesh

    2009-06-01

    Surface aeration systems employed in activated sludge plants are the most energy-intensive units of the plants and typically account for a higher percentage of the treatment facility's total energy use. The geometry of the aeration tank imparts a major effect on the system efficiency. It is said that at optimal geometric conditions, systems exhibits the maximum efficiency. Thus the quantification of the optimal geometric conditions in surface aeration tanks is needed. Optimal geometric conditions are also needed to scale up the laboratory result to the field installation. In the present work, experimental studies have been carried out on baffled and unbaffled circular surface aeration tanks to ascertain the optimal geometric conditions. It is found that no optimal geometric conditions exist for the liquid/water depth in circular surface aeration tanks; however, for design purposes, a standard value has been assumed. Based on the optimal geometric conditions, a scale-up equation has been developed for the baffled circular surface aeration tanks.

  10. Noise control of a circular saw

    NASA Astrophysics Data System (ADS)

    Okrzesik, Jeff

    2002-05-01

    Construction workers are subjected to high levels of noise pollution on a daily basis. Sound levels of about 85 dB(A) with exposures of 8 hours per day will result in permanent hearing loss after many years [Noise and Hearing Loss, NIH Consens Statement Online 8(1), 1-24 (1990)]. There are numerous sources contributing to the noise problems. Tools are of particular interest considering the amount of time the average worker uses them. A circular saw is a common tool used daily by many construction workers. The levels of noise produced by a circular saw will be tested in order to evaluate the problem. The noise levels as well as the locations of the noise sources will be evaluated. Several ideas regarding the reduction of sound emissions have been researched. There will be three initial focal points of the investigation. The blade properties, including the material, size and tooth design, are all key elements that will be studied closely. The numerous dynamic parts in the motor are also large sources of noise that may be reduced by increasing the efficiency of the motor. Another vital element to reducing noise involved with a circular saw is to study the interaction noise from the piece being worked on.

  11. Assembly of DNA Architectures in a Non-Aqueous Solution

    DTIC Science & Technology

    2012-08-31

    specific DNA sequence was synthesized by IDT and packaged separately as single - stranded DNA, dubbed “Wheel 1” (sequence: 5’-TCC ACG GTC TGC TAC TCG...C-3’). The reconstitution of the two single strands of DNA into the double- stranded , tertiary wheel structure was accomplished with the use of a...Kejnovska, I.; Renciuk, D.; Vorlickova, M. Circular dichroism and conformational polymorphism of DNA. Nucleic Acids Res. 2009, 37, 1713–1725. 32

  12. Navigation and vessel inspection circular No. 0-97. Index of Navigation and Vessel Inspection Circulars (NVICS). Final report

    SciTech Connect

    1996-11-21

    This Circular provides the current listing of Navigation and Vessel Inspection Circulars (NVICS). NVICs are issued by Coast Guard Headquarters in the form of duplicated circulars. Their purpose is to inform the public of Coast Guard guidance, requirements or information regarding marine safety activities.

  13. Intracellular Forms of Adenovirus DNA III. Integration of the DNA of Adenovirus Type 2 into Host DNA in Productively Infected Cells

    PubMed Central

    Burger, Harold; Doerfler, Walter

    1974-01-01

    KB cells productively infected with human adenovirus type 2 contain an alkalistable class of viral DNA sedimenting in a broad zone between 50 and 90S as compared to 34S for virion DNA. This type of DNA is characterized as viral by DNA-DNA hybridization. It is extremely sensitive to shear fragmentation. Extensive control experiments demonstrate that the fast-sedimenting viral DNA is not due to artifactual drag of viral DNA mechanically trapped in cellular DNA or to association of viral DNA with protein or RNA. Furthermore, the fast-sedimenting DNA is found after infection with multiplicities between 1 and 1,000 PFU/cell and from 6 to 8 h postinfection until very late in infection (24 h). Analysis in dye-buoyant density gradients eliminates the possibility that the fast-sedimenting viral DNA represents supercoiled circular molecules. Upon equilibrium centrifugation in alkaline CsCl density gradients, the fast-sedimenting viral DNA bands in a density stratum intermediate between that of cellular and viral DNA. In contrast, the 34S virion DNA isolated and treated in the same manner as the fast-sedimenting DNA cobands with viral marker DNA. After ultrasonic treatment of the fast-sedimenting viral DNA, it shifts to the density positions of viral DNA and to a lesser extent to that of cellular DNA. The evidence presented here demonstrates that the 50 to 90S viral DNA represents adenovirus DNA covalently integrated into cell DNA. PMID:4824714

  14. Determination of the folding of proteins as a function of denaturants, osmolytes or ligands using circular dichroism

    PubMed Central

    Greenfield, Norma J.

    2009-01-01

    Circular dichroism (CD) is an excellent tool for examining the interactions and stability of proteins. This protocol covers methods to obtain and analyze circular dichroism spectra to measure changes in the folding of proteins as a function of denaturants, osmolytes or ligands. Applications include determination of the free energy of folding of a protein, the effects of mutations on protein stability and the estimation of binding constants for the interactions of proteins with other proteins, DNA or ligands, such as substrates or inhibitors. The experiments take 2-5 h. PMID:17406529

  15. A Wideband Circularly Polarized Antenna with a Multiple-Circular-Sector Dielectric Resonator.

    PubMed

    Trinh-Van, Son; Yang, Youngoo; Lee, Kang-Yoon; Hwang, Keum Cheol

    2016-11-03

    This paper presents the design of a wideband circularly polarized antenna using a multiple-circular-sector dielectric resonator (DR). The DR is composed of twelve circular-sector DRs with identical central angles of 30 ∘ but with different radii. A genetic algorithm is utilized to optimize the radii of the twelve circular-sector DRs to realize wideband circular polarization. The proposed antenna is excited using an aperture-coupled feeding technique through a narrow rectangular slot etched onto the ground plane. An antenna prototype is experimentally verified. The measured -10 dB reflection and 3 dB axial ratio (AR) bandwidths are 31.39% (1.88-2.58 GHz) and 19.30% (2.06-2.50 GHz), respectively, covering the operating bands of the following systems: UMTS-2100 (2.145 GHz), WiMAX (2.3 GHz), and Wi-Fi (2.445 GHz). A measured peak gain of 7.65 dBic at 2.225 GHz and gain variation of less than 2.70 dBic within the measured 3 dB AR bandwidth are achieved. In addition, the radiation patterns of the proposed antenna are presented and discussed.

  16. A Wideband Circularly Polarized Antenna with a Multiple-Circular-Sector Dielectric Resonator

    PubMed Central

    Trinh-Van, Son; Yang, Youngoo; Lee, Kang-Yoon; Hwang, Keum Cheol

    2016-01-01

    This paper presents the design of a wideband circularly polarized antenna using a multiple-circular-sector dielectric resonator (DR). The DR is composed of twelve circular-sector DRs with identical central angles of 30∘ but with different radii. A genetic algorithm is utilized to optimize the radii of the twelve circular-sector DRs to realize wideband circular polarization. The proposed antenna is excited using an aperture-coupled feeding technique through a narrow rectangular slot etched onto the ground plane. An antenna prototype is experimentally verified. The measured −10 dB reflection and 3 dB axial ratio (AR) bandwidths are 31.39% (1.88–2.58 GHz) and 19.30% (2.06–2.50 GHz), respectively, covering the operating bands of the following systems: UMTS-2100 (2.145 GHz), WiMAX (2.3 GHz), and Wi-Fi (2.445 GHz). A measured peak gain of 7.65 dBic at 2.225 GHz and gain variation of less than 2.70 dBic within the measured 3 dB AR bandwidth are achieved. In addition, the radiation patterns of the proposed antenna are presented and discussed. PMID:27827881

  17. Circular dichroism and thermal denaturation studies of subnucleosomes and their relationships to nucleosome structure

    SciTech Connect

    Mencke, A.J.; Rill, R.L.

    1982-01-01

    Chicken erythrocyte chromatin moderately digested with micrococcal nuclease yields several species of nucleosomes and subnucleosomes that are resolved by electrophoresis in the presence of 3 M urea. This report compares the circulr dichroism spectra, thermal denaturation, and certain other properties of chromatosomes (trimmed nucleosomes cores, and four subnucleosomes. One subnucleosome is a partial core lacking an H2a-H2b pair and 40-50 DNA base pairs(bp) from one end. The stoichiometries of the other subnucleosomes, which contain homotypic histones associated with short DNA fragments, are (H3)(H4)/70-80 bp, (H1)/60-70 bp, and (H2a)(H2b)/50-60 bp. The latter subnucleosomes originate from the ends of nucleosome cores. All properties of partial and whole nucleosome cores were nearly identical, indicating that the terminal H2a-H2b pairs do not make binding contacts with the residual core DNA or histones that are critical to the conformation of the remaining core structure. Analyses of histone contributions of the far-UV circular dichroism of subnucleosomes showed that H2a-H2b pairs and H3-H4 pairs in nucleosomes are both nearly 50% ..cap alpha.. helical and that their helix contents do not depend on the nucleosome integrity. These and other results suggest that homotypic histone pairs and the DNA they tightly bind define quasi-independent conformational subdomains within nucleosomes. H3-H4 pairs stabilized and reduced the 275-nm circular dichroism intensity of short DNA fragments much more effectively than H2a-H2b pairs. In addition, H3-H4 pairs stabilized considerably more DNA than predicted for simple electrostatic interactions. H1 also thermally stabilized less effectively than H3-H4 pairs, and modestly increased the 275-nm CD intensity relative to protein-free DNA.

  18. DNA stability at temperatures typical for hyperthermophiles.

    PubMed Central

    Marguet, E; Forterre, P

    1994-01-01

    We have studied the fate of covalently-closed circular DNA in the temperature range from 95 to 107 degrees C. Supercoiled plasmid was not denatured up to the highest temperature tested. However, it was progressively transformed into open DNA by cleavage and then denatured. Thermodegradation was not dependent on the DNA supercoiling density. In particular, DNA made positively supercoiled by an archaeal reverse gyrase was not more resistant to depurination and thermodegradation than negatively supercoiled DNA. Thermodegradation was similar in aerobic or anaerobic conditions but strongly reduced in the presence of physiological concentrations of K+ or Mg2+. These results indicate that the major problem faced by covalently closed DNA in hyperthermophilic conditions is not thermodenaturation, but thermodegradation, and that intracellular salt concentration is important for stability of DNA primary structure. Our data suggest that reverse gyrase is not directly required to protect DNA against thermodegradation or thermodenaturation. Images PMID:8202372

  19. Covalently closed circular DNAs of murine type C retrovirus: depressed formation in cells treated with cycloheximide early after infection

    SciTech Connect

    Yang, W.K.; Yang, D.M.; Kiggans, J.O. Jr.

    1980-10-01

    Formation of viral closed circular supercoiled DNA duplexes and production of progeny virus were both inhibited in cultured mouse cells treated with cycloheximide in the first 4 h of type C retrovirus infection. With different doses of cycloheximide to cause different degrees of inhibition, the number of viral supercoiled DNA duplexes detected in the cells at 11 h showed an apparent correlation with the amount of progeny virus produced in the 12- to 22-h period of infection. These kinetic data suggest that a cycloheximide-sensitive metabolic process, probably early viral protein synthesis, is required for retrovirus replication and supercoiled viral DNA formation in the cell.

  20. Circular RNAs as Promising Biomarkers: A Mini-Review.

    PubMed

    Abu, Nadiah; Jamal, Rahman

    2016-01-01

    The interest in circular RNAs has resurfaced in the past few years. What was considered as "junk" for nearly two decades is now one of the most interesting molecules. Circular RNAs are non-coding RNAs that are formed by back-splicing events and have covalently closed loops with no poly-adenylated tails. The regulation of circular RNAs is distinctive and they are selectively abundant in different types of tissues. Based on the current knowledge of circular RNAs, these molecules have the potential to be the "next big thing" especially as biomarkers for different diseases. This mini-review attempts to concisely look at the biology of circular RNAs, the putative functional activities, the prevalence of circular RNAs, and the possible role of circular RNA as biomarkers for diagnosis or measuring drug response.

  1. The 68-kilodalton E1 protein of bovine papillomavirus is a DNA binding phosphoprotein which associates with the E2 transcriptional activator in vitro.

    PubMed Central

    Blitz, I L; Laimins, L A

    1991-01-01

    The E1 open reading frame of bovine papillomavirus type 1 encodes factors necessary for extrachromosomal maintenance of the viral genome in transformed cells. To facilitate biochemical characterization of the gene products encoded by this open reading frame, we have expressed the full-length E1 protein in a baculovirus-insect cell system. This protein was found to be phosphorylated and localized to the nucleus of infected cells. The E1 protein alone has affinity for DNA but appears to lack specificity for viral sequences. In addition, we present evidence that the E1 protein interacts with the virally encoded transcriptional activator E2 in vitro. These results are consistent with a model in which the E1 protein, as part of a complex with E2, interacts with specific DNA sequences in the viral genome. Images PMID:1846189

  2. Circular Dichroism Microscopy Free from Commingling Linear Dichroism via Discretely Modulated Circular Polarization.

    PubMed

    Narushima, Tetsuya; Okamoto, Hiromi

    2016-10-20

    In this work, we developed a circular dichroism (CD) imaging microscope with a device to suppress the commingling of linear birefringence (LB) and linear dichroism (LD) signals. CD signals are, in principle, free from the commingling influence of LD and LB if the sample is illuminated with pure circularly polarized light, with no linear polarization contribution. Based on this idea, we here propose a novel circular polarization modulation method to suppress the contribution of linear polarization, which enables high-sensitivity CD detection (10(-4) level in optical density unit or mdeg level in ellipticity) for microscopic imaging at a nearly diffraction limited spatial resolution (sub-μm level). The highly sensitive, diffraction-limited local CD detection will make direct analyses of chiral structures and spatial mappings of optical activity feasible for μm- to sub-μm-sized materials and may yield a number of applications as a unique optical imaging method.

  3. Circular and near-circular polarization states of evanescent monochromatic light fields in total internal reflection.

    PubMed

    Azzam, R M A

    2011-11-20

    Conditions for the production of near-circular polarization states of the evanescent field present in the rarer medium in total internal reflection of incident monochromatic p-polarized light at a dielectric-dielectric planar interface are determined. Such conditions are satisfied if high-index (>3.2) transparent prism materials (e.g., GaP and Ge) are used at angles of incidence well above the critical angle but sufficiently below grazing incidence. Furthermore, elliptical polarization of incident light with nonzero p and s components can be tailored to cause circular polarization of the resultant tangential electric field in the plane of the interface or circular polarization of the transverse electric field in a plane normal to the direction of propagation of the evanescent wave. Such polarization control of the evanescent field is significant, e.g., in the fluorescent excitation of molecules adsorbed at solid-liquid and solid-gas interfaces by total internal reflection.

  4. Circular Dichroism Microscopy Free from Commingling Linear Dichroism via Discretely Modulated Circular Polarization

    PubMed Central

    Narushima, Tetsuya; Okamoto, Hiromi

    2016-01-01

    In this work, we developed a circular dichroism (CD) imaging microscope with a device to suppress the commingling of linear birefringence (LB) and linear dichroism (LD) signals. CD signals are, in principle, free from the commingling influence of LD and LB if the sample is illuminated with pure circularly polarized light, with no linear polarization contribution. Based on this idea, we here propose a novel circular polarization modulation method to suppress the contribution of linear polarization, which enables high-sensitivity CD detection (10−4 level in optical density unit or mdeg level in ellipticity) for microscopic imaging at a nearly diffraction limited spatial resolution (sub-μm level). The highly sensitive, diffraction-limited local CD detection will make direct analyses of chiral structures and spatial mappings of optical activity feasible for μm- to sub-μm-sized materials and may yield a number of applications as a unique optical imaging method. PMID:27761022

  5. Computational simulations of hydrogen circular migration in protonated acetylene induced by circularly polarized light.

    PubMed

    Shi, Xuetao; Li, Wen; Schlegel, H Bernhard

    2016-08-28

    The hydrogens in protonated acetylene are very mobile and can easily migrate around the C2 core by moving between classical and non-classical structures of the cation. The lowest energy structure is the T-shaped, non-classical cation with a hydrogen bridging the two carbons. Conversion to the classical H2CCH(+) ion requires only 4 kcal/mol. The effect of circularly polarized light on the migration of hydrogens in oriented C2H3 (+) has been simulated by Born-Oppenheimer molecular dynamics. Classical trajectory calculations were carried out with the M062X/6-311+G(3df,2pd) level of theory using linearly and circularly polarized 32 cycle 7 μm cosine squared pulses with peak intensity of 5.6 × 10(13) W/cm(2) and 3.15 × 10(13) W/cm(2), respectively. These linearly and circularly polarized pulses transfer similar amounts of energy and total angular momentum to C2H3 (+). The average angular momentum vectors of the three hydrogens show opposite directions of rotation for right and left circularly polarized light, but no directional preference for linearly polarized light. This difference results in an appreciable amount of angular displacement of the three hydrogens relative to the C2 core for circularly polarized light, but only an insignificant amount for linearly polarized light. Over the course of the simulation with circularly polarized light, this corresponds to a propeller-like motion of the three hydrogens around the C2 core of protonated acetylene.

  6. Computational simulations of hydrogen circular migration in protonated acetylene induced by circularly polarized light

    NASA Astrophysics Data System (ADS)

    Shi, Xuetao; Li, Wen; Schlegel, H. Bernhard

    2016-08-01

    The hydrogens in protonated acetylene are very mobile and can easily migrate around the C2 core by moving between classical and non-classical structures of the cation. The lowest energy structure is the T-shaped, non-classical cation with a hydrogen bridging the two carbons. Conversion to the classical H2CCH+ ion requires only 4 kcal/mol. The effect of circularly polarized light on the migration of hydrogens in oriented C2H3+ has been simulated by Born-Oppenheimer molecular dynamics. Classical trajectory calculations were carried out with the M062X/6-311+G(3df,2pd) level of theory using linearly and circularly polarized 32 cycle 7 μm cosine squared pulses with peak intensity of 5.6 × 1013 W/cm2 and 3.15 × 1013 W/cm2, respectively. These linearly and circularly polarized pulses transfer similar amounts of energy and total angular momentum to C2H3+. The average angular momentum vectors of the three hydrogens show opposite directions of rotation for right and left circularly polarized light, but no directional preference for linearly polarized light. This difference results in an appreciable amount of angular displacement of the three hydrogens relative to the C2 core for circularly polarized light, but only an insignificant amount for linearly polarized light. Over the course of the simulation with circularly polarized light, this corresponds to a propeller-like motion of the three hydrogens around the C2 core of protonated acetylene.

  7. DNA binding studies of tartrazine food additive.

    PubMed

    Kashanian, Soheila; Zeidali, Sahar Heidary

    2011-07-01

    The interaction of native calf thymus DNA with tartrazine in 10 mM Tris-HCl aqueous solution at neutral pH 7.4 was investigated. Tartrazine is a nitrous derivative and may cause allergic reactions, with a potential of toxicological risk. Also, tartrazine induces oxidative stress and DNA damage. Its DNA binding properties were studied by UV-vis and circular dichroism spectra, competitive binding with Hoechst 33258, and viscosity measurements. Tartrazine molecules bind to DNA via groove mode as illustrated by hyperchromism in the UV absorption band of tartrazine, decrease in Hoechst-DNA solution fluorescence, unchanged viscosity of DNA, and conformational changes such as conversion from B-like to C-like in the circular dichroism spectra of DNA. The binding constants (K(b)) of DNA with tartrazine were calculated at different temperatures. Enthalpy and entropy changes were calculated to be +37 and +213 kJ mol(-1), respectively, according to the Van't Hoff equation, which indicated that the reaction is predominantly entropically driven. Also, tartrazine does not cleave plasmid DNA. Tartrazine interacts with calf thymus DNA via a groove interaction mode with an intrinsic binding constant of 3.75 × 10(4) M(-1).

  8. Primer removal during mammalian mitochondrial DNA replication.

    PubMed

    Uhler, Jay P; Falkenberg, Maria

    2015-10-01

    The small circular mitochondrial genome in mammalian cells is replicated by a dedicated replisome, defects in which can cause mitochondrial disease in humans. A fundamental step in mitochondrial DNA (mtDNA) replication and maintenance is the removal of the RNA primers needed for replication initiation. The nucleases RNase H1, FEN1, DNA2, and MGME1 have been implicated in this process. Here we review the role of these nucleases in the light of primer removal pathways in mitochondria, highlight associations with disease, as well as consider the implications for mtDNA replication initiation. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  9. Stabilization of circular Rydberg atoms by circularly polarized infrared laser fields

    SciTech Connect

    Askeland, S.; Soerngaard, S. A.; Nepstad, R.; Foerre, M.; Pilskog, I.

    2011-09-15

    The ionization dynamics of circular Rydberg states in strong circularly polarized infrared (800 nm) laser fields is studied by means of numerical simulations with the time-dependent Schroedinger equation. We find that at certain intensities, related to the radius of the Rydberg states, atomic stabilization sets in, and the ionization probability decreases as the intensity is further increased. Moreover, there is a strong dependence of the ionization probability on the rotational direction of the applied laser field, which can be understood from a simple classical analogy.

  10. A multi-split mapping algorithm for circular RNA, splicing, trans-splicing and fusion detection.

    PubMed

    Hoffmann, Steve; Otto, Christian; Doose, Gero; Tanzer, Andrea; Langenberger, David; Christ, Sabina; Kunz, Manfred; Holdt, Lesca M; Teupser, Daniel; Hackermüller, Jörg; Stadler, Peter F

    2014-02-10

    Numerous high-throughput sequencing studies have focused on detecting conventionally spliced mRNAs in RNA-seq data. However, non-standard RNAs arising through gene fusion, circularization or trans-splicing are often neglected. We introduce a novel, unbiased algorithm to detect splice junctions from single-end cDNA sequences. In contrast to other methods, our approach accommodates multi-junction structures. Our method compares favorably with competing tools for conventionally spliced mRNAs and, with a gain of up to 40% of recall, systematically outperforms them on reads with multiple splits, trans-splicing and circular products. The algorithm is integrated into our mapping tool segemehl (http://www.bioinf.uni-leipzig.de/Software/segemehl/).

  11. An Occult Hepatitis B-Derived Hepatoma Cell Line Carrying Persistent Nuclear Viral DNA and Permissive for Exogenous Hepatitis B Virus Infection

    PubMed Central

    Lin, Chih-Lang; Chien, Rong-Nan; Lin, Shi-Ming; Ke, Po-Yuan; Lin, Chen-Chun; Yeh, Chau-Ting

    2013-01-01

    Occult hepatitis B virus (HBV) infection is defined as persistence of HBV DNA in liver tissues, with or without detectability of HBV DNA in the serum, in individuals with negative serum HBV surface antigen (HBsAg). Despite accumulating evidence suggesting its important clinical roles, the molecular and virological basis of occult hepatitis B remains unclear. In an attempt to establish new hepatoma cell lines, we achieved a new cell line derived from a hepatoma patient with chronic hepatitis C virus (HCV) and occult HBV infection. Characterization of this cell line revealed previously unrecognized properties. Two novel human hepatoma cell lines were established. Hep-Y1 was derived from a male hepatoma patient negative for HCV and HBV infection. Hep-Y2 was derived from a female hepatoma patient suffering from chronic HCV and occult HBV infection. Morphological, cytogenetic and functional studies were performed. Permissiveness to HBV infection was assessed. Both cell lines showed typical hepatocyte-like morphology under phase-contrast and electron microscopy and expressed alpha-fetoprotein, albumin, transferrin, and aldolase B. Cytogenetic analysis revealed extensive chromosomal anomalies. An extrachromosomal form of HBV DNA persisted in the nuclear fraction of Hep-Y2 cells, while no HBsAg was detected in the medium. After treated with 2% dimethyl sulfoxide, both cell lines were permissive for exogenous HBV infection with transient elevation of the replication intermediates in the cytosol with detectable viral antigens by immunoflurescence analysis. In conclusions, we established two new hepatoma cell lines including one from occult HBV infection (Hep-Y2). Both cell lines were permissive for HBV infection. Additionally, Hep-Y2 cells carried persistent extrachromosomal HBV DNA in the nuclei. This cell line could serve as a useful tool to establish the molecular and virological basis of occult HBV infection. PMID:23734258

  12. An occult hepatitis B-derived hepatoma cell line carrying persistent nuclear viral DNA and permissive for exogenous hepatitis B virus infection.

    PubMed

    Lin, Chih-Lang; Chien, Rong-Nan; Lin, Shi-Ming; Ke, Po-Yuan; Lin, Chen-Chun; Yeh, Chau-Ting

    2013-01-01

    Occult hepatitis B virus (HBV) infection is defined as persistence of HBV DNA in liver tissues, with or without detectability of HBV DNA in the serum, in individuals with negative serum HBV surface antigen (HBsAg). Despite accumulating evidence suggesting its important clinical roles, the molecular and virological basis of occult hepatitis B remains unclear. In an attempt to establish new hepatoma cell lines, we achieved a new cell line derived from a hepatoma patient with chronic hepatitis C virus (HCV) and occult HBV infection. Characterization of this cell line revealed previously unrecognized properties. Two novel human hepatoma cell lines were established. Hep-Y1 was derived from a male hepatoma patient negative for HCV and HBV infection. Hep-Y2 was derived from a female hepatoma patient suffering from chronic HCV and occult HBV infection. Morphological, cytogenetic and functional studies were performed. Permissiveness to HBV infection was assessed. Both cell lines showed typical hepatocyte-like morphology under phase-contrast and electron microscopy and expressed alpha-fetoprotein, albumin, transferrin, and aldolase B. Cytogenetic analysis revealed extensive chromosomal anomalies. An extrachromosomal form of HBV DNA persisted in the nuclear fraction of Hep-Y2 cells, while no HBsAg was detected in the medium. After treated with 2% dimethyl sulfoxide, both cell lines were permissive for exogenous HBV infection with transient elevation of the replication intermediates in the cytosol with detectable viral antigens by immunoflurescence analysis. In conclusions, we established two new hepatoma cell lines including one from occult HBV infection (Hep-Y2). Both cell lines were permissive for HBV infection. Additionally, Hep-Y2 cells carried persistent extrachromosomal HBV DNA in the nuclei. This cell line could serve as a useful tool to establish the molecular and virological basis of occult HBV infection.

  13. Multipole Analysis of Circular Cylindircal Magnetic Systems

    SciTech Connect

    Selvaggi, Jerry P.

    2005-12-01

    This thesis deals with an alternate method for computing the external magnetic field from a circular cylindrical magnetic source. The primary objective is to characterize the magnetic source in terms of its equivalent multipole distribution. This multipole distribution must be valid at points close to the cylindrical source and a spherical multipole expansion is ill-equipped to handle this problem; therefore a new method must be introduced. This method, based upon the free-space Green's function in cylindrical coordinates, is developed as an alternative to the more familiar spherical harmonic expansion. A family of special functions, called the toroidal functions or Q-functions, are found to exhibit the necessary properties for analyzing circular cylindrical geometries. In particular, the toroidal function of zeroth order, which comes from the integral formulation of the free-space Green's function in cylindrical coordinates, is employed to handle magnetic sources which exhibit circular cylindrical symmetry. The toroidal functions, also called Q-functions, are the weighting coefficients in a ''Fourier series-like'' expansion which represents the free-space Green's function. It is also called a toroidal expansion. This expansion can be directly employed in electrostatic, magnetostatic, and electrodynamic problems which exhibit cylindrical symmetry. Also, it is shown that they can be used as an alternative to the Elliptic integral formulation. In fact, anywhere that an Elliptic integral appears, one can replace it with its corresponding Q-function representation. A number of problems, using the toroidal expansion formulation, are analyzed and compared to existing known methods in order to validate the results. Also, the equivalent multipole distribution is found for most of the solved problems along with its corresponding physical interpretation. The main application is to characterize the external magnetic field due to a six-pole permanent magnet motor in terms of

  14. Circular Polarization in AGNs: Polarity and Spectra

    NASA Astrophysics Data System (ADS)

    Aller, M. F.; Aller, H. D.; Plotkin, R. M.

    2005-12-01

    Circular polarization (Stokes V) observations potentially provide information on the nature and origin of the underlying magnetic fields in AGNs. We have been systematically monitoring a group of sources with detectable circular polarization (V>0.1 percent, a level set by the instrumental polarization of our system) in all 4 Stokes parameters at 8.0 and 4.8 GHz since 2000, and also at 14.5 GHz since November 2003, with the University of Michigan prime focus paraboloid antenna. These data are compared with historical observations obtained with the same instrument at 8.0 and 4.8 GHz extending back to 1978. Specific goals are to study the temporal spectral behavior of Stokes V and its relation to variability in total flux and linear polarization, and to investigate the question of polarity stability on decade-long time scales using data obtained with the same instrumentation and at the same frequencies. The data are consistent with linear-to-circular mode conversion in partially opaque regions of the source. We find examples of polarity changes with time at one or more frequencies associated with outbursts in total flux and linear polarization, and polarity differences within the 3 frequencies at a single epoch in one case, 3C 279. Such behavior argues against the notion that the sign of Stokes V is a simple tracer of the net flow of magnetic energy from the central engine to the jet or an indicator of the direction of rotation of the spinning central black hole/accretion disk via the winding up of the initial seed magnetic field. This work was supported in part by NSF grant AST-0307629 and by funds from the University of Michigan.

  15. Budgeted phylogenetic diversity on circular split systems.

    PubMed

    Minh, Bui Quang; Pardi, Fabio; Klaere, Steffen; von Haeseler, Arndt

    2009-01-01

    In the last 15 years, Phylogenetic Diversity (PD) has gained interest in the community of conservation biologists as a surrogate measure for assessing biodiversity. We have recently proposed two approaches to select taxa for maximizing PD, namely PD with budget constraints and PD on split systems. In this paper, we will unify these two strategies and present a dynamic programming algorithm to solve the unified framework of selecting taxa with maximal PD under budget constraints on circular split systems. An improved algorithm will also be given if the underlying split system is a tree.

  16. Steady viscous flow past a circular cylinder

    NASA Technical Reports Server (NTRS)

    Fornberg, B.

    1984-01-01

    Viscous flow past a circular cylinder becomes unstable around Reynolds number Re = 40. With a numerical technique based on Newton's method and made possible by the use of a supercomputer, steady (but unstable) solutions have been calculated up to Re = 400. It is found that the wake continues to grow in length approximately linearly with Re. However, in conflict with available asymptotic predictions, the width starts to increase very rapidly around Re = 300. All numerical calculations have been performed on the CDC CYBER 205 at the CDC Service Center in Arden Hills, Minnesota.

  17. Vortex motion behind a circular cylinder

    NASA Technical Reports Server (NTRS)

    Foeppl, L.

    1983-01-01

    Vortex motion behind a circular cylinder moving through water is discussed. It is shown that a pair of vortices form behind a moving cylinder and that their centers will move along a predictable curve. This curve represents an equilibrium condition which, however, is subject to perturbation. The stability of the vortex pair is investigated. Movement of the vortex pair away from the cylinder is calculated as an explanation of the resistance of the cylinder. Finally, the principles elaborated are applied to the flow around a flat plate.

  18. Chiral surface waves for enhanced circular dichroism

    NASA Astrophysics Data System (ADS)

    Pellegrini, Giovanni; Finazzi, Marco; Celebrano, Michele; Duò, Lamberto; Biagioni, Paolo

    2017-06-01

    We present a novel chiral sensing platform that combines a one-dimensional photonic crystal design with a birefringent surface defect. The platform sustains simultaneous transverse electric and transverse magnetic surface modes, which are exploited to generate chiral surface waves. The present design provides homogeneous and superchiral fields of both handednesses over arbitrarily large areas in a wide spectral range, resulting in the enhancement of the circular dichroism signal by more than two orders of magnitude, thus paving the road toward the successful combination of surface-enhanced spectroscopies and electromagnetic superchirality.

  19. Circular Waveguide Slotted Antenna with Inclined Beam

    NASA Astrophysics Data System (ADS)

    Sekretarov, S. S.; Vavriv, D. M.

    2009-03-01

    The novel design of a Ku-band circular waveguide slotted antenna is proposed. In contrast to standard antennas of this type, the main beam of the developed antenna is inclined from its surface normal by the value noticeably exceeding the beam width, which is necessary e.g. to reduce the radar cross section of the antenna in the direction towards an illuminated target. The design features of such antennas are considered. The practical desing of the antenna developed is presented along with the comparison of the simulation and experimental results.

  20. Poynting-Robertson effect - 'Circular' orbit

    NASA Astrophysics Data System (ADS)

    Klacka, J.; Kaufmannova, J.

    1992-11-01

    The study investigates time evolution of the interplanetary dust particle under the action of the solar electromagnetic radiation (Poynting-Robertson effect). The evolution of the initially circular orbit in terms of the orbital elements present in the standard equations for their secular changes is considered. It is shown that the osculating eccentricity is virtually constant during the motion in spite of the generally accepted opinion that the standard equations for the secular changes of orbital elements represent the time evolution of the osculating elements.