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Sample records for extrachromosomal circular dna

  1. Extrachromosomal circular DNA is common in yeast

    PubMed Central

    Møller, Henrik D.; Parsons, Lance; Jørgensen, Tue S.; Botstein, David; Regenberg, Birgitte

    2015-01-01

    Examples of extrachromosomal circular DNAs (eccDNAs) are found in many organisms, but their impact on genetic variation at the genome scale has not been investigated. We mapped 1,756 eccDNAs in the Saccharomyces cerevisiae genome using Circle-Seq, a highly sensitive eccDNA purification method. Yeast eccDNAs ranged from an arbitrary lower limit of 1 kb up to 38 kb and covered 23% of the genome, representing thousands of genes. EccDNA arose both from genomic regions with repetitive sequences ≥15 bases long and from regions with short or no repetitive sequences. Some eccDNAs were identified in several yeast populations. These eccDNAs contained ribosomal genes, transposon remnants, and tandemly repeated genes (HXT6/7, ENA1/2/5, and CUP1-1/-2) that were generally enriched on eccDNAs. EccDNAs seemed to be replicated and 80% contained consensus sequences for autonomous replication origins that could explain their maintenance. Our data suggest that eccDNAs are common in S. cerevisiae, where they might contribute substantially to genetic variation and evolution. PMID:26038577

  2. 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

  3. 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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  5. Formation of extrachromosomal circles from telomeric DNA in Xenopus laevis.

    PubMed

    Cohen, Sarit; Méchali, Marcel

    2002-12-01

    Instability and plasticity of telomeric DNA, which includes extrachromosomal DNA, are usually correlated with the absence of telomerase and with abnormal growth of mammalian cells. Here, we show the formation of extrachromosomal circular DNA of telomeric repeats (tel-eccDNA) during the development of Xenopus laevis. Tel-eccDNA is double-stranded relaxed circles composed of the vertebrate consensus telomeric repeats [TTAGGG](n). Its size varies from <2 to >20 kb and it comprises up to 10% of the total cellular telomere content of the early embryo (pre-MBT stage). The amount of tel-eccDNA is reduced in later developmental stages and in adult tissues. Using a cell-free system derived from Xenopus egg extracts, we show that tel-eccDNA can be formed de novo from the telomere chromosomal tracts of sperm nuclei and naked DNA in a replication-independent manner. These results reveal an unusual plasticity of telomeric DNA during normal development of Xenopus. PMID:12446568

  6. 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

  7. 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. PMID:25662602

  8. 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.

  9. 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.

  10. Tandem Repeats in Extrachromosomal Ribosomal DNA of Dictyostelium Discoideum, Resulting from Chromosomal Mutations

    PubMed Central

    Cole, R. A.; Williams, K. L.

    1992-01-01

    Extrachromosomal ribosomal DNA in the simple eukaryote Dictyostelium discoideum is readily separated from chromosomal DNA by orthogonal field electrophoresis (OFAGE), forming a prominent band in the 110-kb region of the gel. Here we show that mutations in at least two chromosomal genes give rise to a ladder of rDNA bands increasing in size up to about 300 kb. One of these mutations, the rrcA350 allele, which is recessive to wild type and maps to the centromere-proximal region of linkage group II, has an unstable phenotype; spontaneous revertants, which no longer exhibit the rDNA ladder, have been recovered. Another mutation rrc-351, provisionally mapped to linkage group IV, is dominant to wild type. The rDNA ladder is caused by concatamerization of a 34-kb fragment in the nontranscribed central spacer region of the 88-kb linear rDNA palindrome. Restriction enzyme analysis has revealed that each concatamer is generated by crossovers between two rDNA molecules. PMID:1582557

  11. Resistance of Spiroplasma citri Lines to the Virus SVTS2 Is Associated with Integration of Viral DNA Sequences into Host Chromosomal and Extrachromosomal DNA

    PubMed Central

    Sha, Y.; Melcher, U.; Davis, R. E.; Fletcher, J.

    1995-01-01

    Spiroplasmavirus SVTS2, isolated from Spiroplasma melliferum TS2, produces plaques when inoculated onto lawns of Spiroplasma citri M200H, a derivative of the type strain Maroc R8A2. S. citri strains MR2 and MR3, originally selected as colonies growing within plaques on a lawn of M200H inoculated with SVTS2, were resistant to SVTS2. Genomic DNA fingerprints and electrophoretic protein profiles of M200H, MR2, and MR3 were similar, but three proteins present in M200H were missing or significantly reduced in both resistant lines. None of these three polypeptides reacted with antiserum against S. citri membrane proteins, indicating that they probably are not surface-located virus receptors. Electroporation with SVTS2 DNA produced 1.5 x 10(sup5) transfectants per (mu)g of DNA in M200H but none in MR2 or MR3, suggesting that resistance may result from inhibition of viral replication. The digestion patterns of the extrachromosomal double-stranded (ds) DNA of these lines were similar. Three TaqI fragments of MR2 extrachromosomal DNA that were not present in M200H extrachromosomal DNA hybridized strongly to an SVTS2 probe, and two of these fragments plus an additional one hybridized with the MR3 extrachromosomal DNA, indicating that a fragment of SVTS2 DNA was present in the extrachromosomal ds DNA of MR2 and MR3 but not of M200H. When the restricted genomes of all three lines were probed with SVTS2 DNA, strong hybridization to two EcoRI fragments of chromosomal MR2 and MR3 DNA but not M200H DNA indicated that SVTS2 DNA had integrated into the genomes of MR2 and MR3 but not of M200H. When MR3 extrachromosomal ds DNA containing a 2.1-kb SVTS2 DNA fragment was transfected into M200H, the transformed spiroplasmas were resistant to SVTS2. These results suggest that SVTS2 DNA fragments, possibly integrated into the chromosomal or extrachromosomal DNA of a previously susceptible spiroplasma, may function as viral incompatibility elements, providing resistance to superinfection by

  12. Condensation of circular DNA

    NASA Astrophysics Data System (ADS)

    Starostin, E. L.

    2013-04-01

    A simple model of a circularly closed double-stranded DNA in a poor solvent is considered as an example of a semi-flexible polymer with self-attraction. To find the ground states, the conformational energy is computed as a sum of the bending and torsional elastic components and the effective self-attraction energy. The model includes a relative orientation or sequence dependence of the effective attraction forces between different pieces of the polymer chain. Two series of conformations are analysed: a multicovered circle (a toroid) and a multifold two-headed racquet. The results are presented as a diagram of state. It is suggested that the stability of particular conformations may be controlled by proper adjustment of the primary structure. Application of the model to other semi-flexible polymers is considered.

  13. Argonaute Proteins Affect siRNA Levels and Accumulation of a Novel Extrachromosomal DNA from the Dictyostelium Retrotransposon DIRS-1*

    PubMed Central

    Boesler, Benjamin; Meier, Doreen; Förstner, Konrad U.; Friedrich, Michael; Hammann, Christian; Sharma, Cynthia M.; Nellen, Wolfgang

    2014-01-01

    The retrotransposon DIRS-1 is the most abundant retroelement in Dictyostelium discoideum and constitutes the pericentromeric heterochromatin of the six chromosomes in D. discoideum. The vast majority of cellular siRNAs is derived from DIRS-1, suggesting that the element is controlled by RNAi-related mechanisms. We investigated the role of two of the five Argonaute proteins of D. discoideum, AgnA and AgnB, in DIRS-1 silencing. Deletion of agnA resulted in the accumulation of DIRS-1 transcripts, the expression of DIRS-1-encoded proteins, and the loss of most DIRS-1-derived secondary siRNAs. Simultaneously, extrachromosomal single-stranded DIRS-1 DNA accumulated in the cytoplasm of agnA− strains. These DNA molecules appear to be products of reverse transcription and thus could represent intermediate structures before transposition. We further show that transitivity of endogenous siRNAs is impaired in agnA− strains. The deletion of agnB alone had no strong effect on DIRS-1 transposon regulation. However, in agnA−/agnB− double mutant strains strongly reduced accumulation of extrachromosomal DNA compared with the single agnA− strains was observed. PMID:25352599

  14. Detection of circular and linear herpesvirus DNA molecules in mammalian cells by gel electrophoresis.

    PubMed Central

    Gardella, T; Medveczky, P; Sairenji, T; Mulder, C

    1984-01-01

    A simple gel technique is described for the detection of large, covalently closed, circular DNA molecules in eucaryotic cells. The procedure is based on the electrophoretic technique of Eckhardt (T. Eckhardt, Plasmid 1:584-588, 1978) for detecting bacterial plasmids and has been modified for the detection of circular and linear extrachromosomal herpesvirus genomes in mammalian cells. Gentle lysis of suspended cells in the well of an agarose gel followed by high-voltage electrophoresis allows separation of extrachromosomal DNA from the bulk of cellular DNA. Circular viral DNA from cells which carry the genomes of Epstein-Barr virus, Herpesvirus saimiri, and Herpesvirus ateles can be detected in these gels as sharp bands which comigrate with bacterial plasmid DNA of 208 kilobases. Epstein-Barr virus producer cell lines also show a sharp band of linear 160-kilobase DNA. The kinetics of the appearance of this linear band after induction of viral replication after temperature shift parallels the known kinetics of Epstein-Barr virus production in these cell lines. Hybridization of DNA after transfer to filters shows that the circular and linear DNA bands are virus specific and that as little as 0.25 Epstein-Barr virus genome per cell can be detected. The technique is simple, rapid, and sensitive and requires relatively low amounts of cells (0.5 X 10(6) to 2.5 X 10(6)). Images PMID:6321792

  15. 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

  16. 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. PMID:23138713

  17. Induction of circles of heterogeneous sizes in carcinogen-treated cells: two-dimensional gel analysis of circular DNA molecules.

    PubMed Central

    Cohen, S; Lavi, S

    1996-01-01

    Extrachromosomal circular DNA molecules are associated with genomic instability, and circles containing inverted repeats were suggested to be the early amplification products. Here we present for the first time the use of neutral-neutral two-dimensional (2D) gel electrophoresis as a technique for the identification, isolation, and characterization of heterogeneous populations of circular molecules. Using this technique, we demonstrated that in N-methyl-N'-nitro-N-nitrosoguanidine-treated simian virus 40-transformed Chinese hamster cells (CO60 cells), the viral sequences are amplified as circular molecules of various sizes. The supercoiled circular fraction was isolated and was shown to contain molecules with inverted repeats. 2D gel analysis of extrachromosomal DNA from CHO cells revealed circular molecules containing highly repetitive DNA which are similar in size to the simian virus 40-amplified molecules. Moreover, enhancement of the amount of circular DNA was observed upon N-methyl-N'-nitro-N-nitrosoguanidine treatment of CHO cells. The implications of these findings regarding the processes of gene amplification and genomic instability and the possible use of the 2D gel technique to study these phenomena are discussed. PMID:8628266

  18. 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

  19. Circular DNA Molecules in the Genus Drosophila

    PubMed Central

    Travaglini, E. C.; Schultz, J.

    1972-01-01

    The satellite DNA's from the embryos of five species of Drosophila (D. melanogaster, D. simulans, D. nasuta, D. virilis and D. hydei) have been analyzed for the presence of closed circular duplex DNA molecules, as determined by CsCl-EBr gradients. Circular DNA molecules were found in every species but D. melanogaster. Analyses of cell fractions from adult Drosophila and organ fractions from Drosophila larvae show that fractions containing mitochondria are highly enriched in these molecules. PMID:4643820

  20. A novel cell-free system reveals a mechanism of circular DNA formation from tandem repeats.

    PubMed

    Cohen, S; Mechali, M

    2001-06-15

    One characteristic of genomic plasticity is the presence of extrachromosomal circular DNA (eccDNA). High levels of eccDNA are associated with genomic instability, exposure to carcinogens and aging. We have recently reported developmentally regulated formation of eccDNA that occurs preferentially in pre-blastula Xenopus laevis embryos. Multimers of tandemly repeated sequences were over-represented in the circle population while dispersed sequences were not detected, indicating that circles were not formed at random from any chromosomal sequence. Here we present detailed mechanistic studies of eccDNA formation in a cell-free system derived from Xenopus egg extracts. We show that naked chromosomal DNA from sperm or somatic tissues serves as a substrate for direct tandem repeat circle formation. Moreover, a recombinant bacterial tandem repeat can generate eccDNA in the extract through a de novo mechanism which is independent of DNA replication. These data suggest that the presence of a high level of any direct tandem repeat can confer on DNA the ability to be converted into circular multimers in the early embryo irrespective of its sequence and that homologous recombination is involved in this process. PMID:11410662

  1. Extra-chromosomal DNA maintenance in Bacillus subtilis, dependence on flagellation factor FliF and moonlighting mediator EdmS.

    PubMed

    Hakumai, Yuichi; Shimomoto, Kouko; Ashiuchi, Makoto

    2015-05-15

    Extra-chromosomal DNA maintenance (EDM) as an important process in the propagation and genetic engineering of microbes. Bacillus subtilis EdmS (formerly PgsE), a protein comprising 55 amino acids, is a mediator of the EDM process. In this study, the effect of mutation of global regulators on B. subtilis EDM was examined. Mutation of the swrA gene abolished EdmS-mediated EDM. It is known that swrA predominantly regulates expression of the fla/che operon in B. subtilis. We therefore performed EDM analysis using fla/che-deletion mutants and identified an EDM-mediated EDM cooperator in the flgB-fliL region. Further genetic investigation identified the flagellation factor FliF is a crucial EDM cooperator. To our knowledge, this is the first observation of the moonlighting function of FliF in DNA maintenance. PMID:25843804

  2. Beyond the chromosome: the prevalence of unique extra-chromosomal bacteriophages with integrated virulence genes in pathogenic Staphylococcus aureus.

    PubMed

    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

  3. 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

  4. Topologically non-linked circular duplex DNA.

    PubMed

    Biegeleisen, Ken

    2002-05-01

    The discovery of circular DNA, over 30 years ago, introduced an element of uneasiness in what had been, up to that point, the almost picture-perfect story of the elucidation of the molecular biology of heredity. If DNA indeed has the Watson-Crick right-handed helical secondary structure, then in circular DNA, thousands, or perhaps even millions of twists must be removed in each generation, and re-wound in the next generation. Although enzyme systems adequate for this task have long since been found and characterized, there have nevertheless arisen a number of proposals for alternative DNA structures in which the strands are topologically non-linked, so that they might separate during replication without having to be unwound. These structures have generally been put forth as theory only, and have been largely unaccompanied by experimental evidence to support their applicability to native DNA from living systems. Recently, however, a report has emerged suggesting that it might be possible to separate, intact, the individual single-stranded circular half-chromosomes which constitute the double-stranded circular chromosomes of certain plasmids. This would not be possible unless the chromosomes had one of the alternative, topologically non-linked structures. It is widely believed that after a half-century of worldwide DNA research, any significant change to the Watson-Crick structure is unlikely to stand up to scrutiny. Nevertheless, the present author has found that in many instances in which the behavior of circular duplex DNA is considered to be explicable only in terms of the topologically linked helical model, it is also possible to explain that same behavior in terms of a topologically non-linked model. It is necessary, in these instances, to make certain logical assumptions which cannot be conclusively proven at the present time. The author herein offers an example of one such instance, namely an examination of the behavior of circular duplex DNA in an alkaline

  5. Entropic penalties in circular DNA assembly

    NASA Astrophysics Data System (ADS)

    Zoli, Marco

    2014-11-01

    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.

  6. Entropic penalties in circular DNA assembly.

    PubMed

    Zoli, Marco

    2014-11-01

    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. PMID:25381507

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. Purification of circular DNA using benzoylated naphthoylated DEAE-cellulose.

    PubMed

    Gamper, H; Lehman, N; Piette, J; Hearst, J E

    1985-04-01

    Un-nicked circular DNA can be separated from protein, RNA, and other DNA in a simple three-step protocol consisting of exonuclease III digestion, extraction with benzoylated naphthoylated DEAE-cellulose (BND cellulose) in 1 M NaCl, and alcohol precipitation of the remaining supercoiled DNA. Exonuclease III treatment introduces single-stranded regions into contaminating linear and nicked circular DNA. This DNA, together with most RNA and protein, is adsorbed onto BND cellulose leaving form I DNA in solution. The protocol can be used to purify analytical as well as preparative amounts of supercoiled DNA. This procedure is a substitute for cesium chloride-ethidium bromide gradient ultracentrifugation and gives a comparable yield of pure form I DNA. Other classes of DNA can be isolated by changing the pretreatment step. Selective digestion of linear DNA with lambda exonuclease permits the isolation of both nicked circular and supercoiled DNA while brief heat-induced or alkali-induced denaturation leads to the recovery of rapidly reannealing DNA. In large-scale purifications, the basic protocol is usually preceded by one or more BND cellulose extractions in 1 M NaCl to remove contaminants absorbing UV or inhibiting exonuclease III. PMID:3996184

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  13. CSA: An efficient algorithm to improve circular DNA multiple alignment

    PubMed Central

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

    2009-01-01

    Background 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. Results 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. Conclusion 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

  14. Isolation of Circular DNA from a Mitochondrial Fraction from Yeast

    PubMed Central

    Clark-Walker, G. D.

    1972-01-01

    Breakage and fractionation of respiratory competent yeast in the presence of ethidium bromide, and subsequent centrifugation of a detergent lysate of the mitochondrial fraction by the dye-buoyant-density technique, results in the isolation of closed-circular DNA. After removal of bound dye, this DNA has two components when analyzed by equilibrium buoyant density in the analytical ultracentrifuge. A minor component has a buoyant density of 1.684 g/cm3, which is characteristic of mitochondrial DNA, but the major component has a buoyant density of 1.701 g/cm3. This species of DNA is also present in yeast that have been mutagenized to respiratory deficiency in the presence of the highest concentration of ethidium bromide compatible with cell growth. The closed-circular DNA of buoyant density 1.701 g/cm3, and free of linear DNA, is associated with the sole particulate band obtained on sucrose gradient centrifugation of a mitochondrial preparation from respiratory-deficient cells. Two particulate bands are obtained on sucrose gradient centrifugation of a mitochondrial preparation from respiratory-competent cells, the upper band containing DNA of buoyant density 1.701 g/cm3 and the lower band DNA of buoyant density 1.684 g/cm3. The suggestion is advanced, in view of the reputed sedimentation behaviour of yeast peroxisomes, that the closed-circular DNA of buoyant density 1.701 g/cm3 may be located in peroxisomes. Images PMID:4551142

  15. Analysis of extrachromosomal Ac/Ds transposable elements.

    PubMed Central

    Gorbunova, V; Levy, A A

    2000-01-01

    The mechanism of transposition of the maize Ac/Ds elements is not well understood. The true transposition intermediates are not known and it has not been possible to distinguish between excision models involving 8-bp staggered cuts or 1-bp staggered cuts followed by hairpin formation. In this work, we have analyzed extrachromosomal excision products to gain insight into the excision mechanism. Plasmid rescue was used to demonstrate that Ds excision is associated with the formation of circular molecules. In addition, we present evidence for the formation of linear extrachromosomal species during Ds excision. Sequences found at the termini of circular and linear elements showed a broad range of nucleotide additions or deletions, suggesting that these species are not true intermediates. Additional nucleotides adjacent to the termini in extrachromosomal elements were compared to the sequence of the original donor site. This analysis showed that: (1) the first nucleotide adjacent to the transposon end was significantly more similar to the first nucleotide flanking the element in the donor site than to a random sequence and (2) the second and farther nucleotides did not resemble the donor site. The implications of these findings for excision models are discussed. PMID:10790408

  16. Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences.

    PubMed Central

    Mello, C C; Kramer, J M; Stinchcomb, D; Ambros, V

    1991-01-01

    We describe a dominant behavioral marker, rol-6(su-1006), and an efficient microinjection procedure which facilitate the recovery of Caenorhabditis elegans transformants. We use these tools to study the mechanism of C.elegans DNA transformation. By injecting mixtures of genetically marked DNA molecules, we show that large extrachromosomal arrays assemble directly from the injected molecules and that homologous recombination drives array assembly. Appropriately placed double-strand breaks stimulated homologous recombination during array formation. Our data indicate that the size of the assembled transgenic structures determines whether or not they will be maintained extrachromosomally or lost. We show that low copy number extrachromosomal transformation can be achieved by adjusting the relative concentration of DNA molecules in the injection mixture. Integration of the injected DNA, though relatively rare, was reproducibly achieved when single-stranded oligonucleotide was co-injected with the double-stranded DNA. Images PMID:1935914

  17. Chiral plasmonic DNA nanostructures with switchable circular dichroism

    NASA Astrophysics Data System (ADS)

    Schreiber, Robert; Luong, Ngoc; Fan, Zhiyuan; Kuzyk, Anton; Nickels, Philipp C.; Zhang, Tao; Smith, David M.; Yurke, Bernard; Kuang, Wan; Govorov, Alexander O.; Liedl, Tim

    2013-12-01

    Circular dichroism spectra of naturally occurring molecules and also of synthetic chiral arrangements of plasmonic particles often exhibit characteristic bisignate shapes. Such spectra consist of peaks next to dips (or vice versa) and result from the superposition of signals originating from many individual chiral objects oriented randomly in solution. Here we show that by first aligning and then toggling the orientation of DNA-origami-scaffolded nanoparticle helices attached to a substrate, we are able to reversibly switch the optical response between two distinct circular dichroism spectra corresponding to either perpendicular or parallel helix orientation with respect to the light beam. The observed directional circular dichroism of our switchable plasmonic material is in good agreement with predictions based on dipole approximation theory. Such dynamic metamaterials introduce functionality into soft matter-based optical devices and may enable novel data storage schemes or signal modulators.

  18. Sound Packing DNA: packing open circular DNA with low-intensity ultrasound

    NASA Astrophysics Data System (ADS)

    Park, Donghee; Jung, Bong-Kwang; Park, Hyunjin; Lee, Hyungbeen; Lee, Gyudo; Park, Jingam; Shin, Unchul; Won, Jong Ho; Jo, Yong Jun; Chang, Jin Woo; Lee, Sangwoo; Yoon, Daesung; Seo, Jongbum; Kim, Chul-Woo

    2015-04-01

    Supercoiling DNA (folding DNA into a more compact molecule) from open circular forms requires significant bending energy. The double helix is coiled into a higher order helix form; thus it occupies a smaller footprint. Compact packing of DNA is essential to improve the efficiency of gene delivery, which has broad implications in biology and pharmaceutical research. Here we show that low-intensity pulsed ultrasound can pack open circular DNA into supercoil form. Plasmid DNA subjected to 5.4 mW/cm2 intensity ultrasound showed significant (p-values <0.001) supercoiling compared to DNA without exposure to ultrasound. Radiation force induced from ultrasound and dragging force from the fluid are believed to be the main factors that cause supercoiling. This study provides the first evidence to show that low-intensity ultrasound can directly alter DNA topology. We anticipate our results to be a starting point for improved non-viral gene delivery.

  19. Sound Packing DNA: packing open circular DNA with low-intensity ultrasound

    PubMed Central

    Park, DongHee; Jung, Bong-Kwang; Park, Hyunjin; Lee, Hyungbeen; Lee, Gyudo; Park, Jingam; Shin, Unchul; Won, Jong Ho; Jo, Yong Jun; Chang, Jin Woo; Lee, Sangwoo; Yoon, Daesung; Seo, Jongbum; Kim, Chul-Woo

    2015-01-01

    Supercoiling DNA (folding DNA into a more compact molecule) from open circular forms requires significant bending energy. The double helix is coiled into a higher order helix form; thus it occupies a smaller footprint. Compact packing of DNA is essential to improve the efficiency of gene delivery, which has broad implications in biology and pharmaceutical research. Here we show that low-intensity pulsed ultrasound can pack open circular DNA into supercoil form. Plasmid DNA subjected to 5.4 mW/cm2 intensity ultrasound showed significant (p-values <0.001) supercoiling compared to DNA without exposure to ultrasound. Radiation force induced from ultrasound and dragging force from the fluid are believed to be the main factors that cause supercoiling. This study provides the first evidence to show that low-intensity ultrasound can directly alter DNA topology. We anticipate our results to be a starting point for improved non-viral gene delivery. PMID:25892035

  20. Uncovering novel mobile genetic elements and their dynamics through an extra-chromosomal sequencing approach.

    PubMed

    Deutsch, Douglas R; Utter, Bryan; Fischetti, Vincent A

    2016-01-01

    Staphylococcus aureus is a major clinically important pathogen with well-studied phage contributions to its virulence potential. In this commentary, we describe our method to enrich and sequence stealth extra-chromosomal DNA elements in the bacterial cell, allowing the identification of novel extra-chromosomal prophages in S. aureus clinical strains. Extra-chromosomal sequencing is a useful and broadly applicable tool to study bacterial genomics, giving a temporal glance at the extra-chromosomal compartment of the cell and allowing researchers to uncover lower-copy plasmidial elements (e.g., prophages) as well as gain a greater understanding of mobile genetic elements that shuffle on and off the chromosome. Here, we describe how episomal and plasmidial DNA elements can have profound downstream effects on the host cell and surrounding bacterial population, and discuss specific examples of their importance in Gram-positive bacteria. We also offer potential avenues of future research where extra-chromosomal sequencing may play a key role in our understanding of the complete virulence potential of infectious bacteria. PMID:27581613

  1. Circular Herpesvirus sylvilagus DNA in spleen cells of experimentally infected cottontail rabbits.

    PubMed

    Medveczky, P; Kramp, W J; Sullivan, J L

    1984-11-01

    Cottontail rabbits (Sylvilagus floridanus) were infected with Herpesvirus sylvilagus, and spleen cells were analyzed for the presence of virus-specific, covalently closed circular, and linear DNA molecules by a simple electrophoretic technique, followed by transfer to nitrocellulose filters and hybridization with cloned viral DNA (Gardella et al., J. Virol. 50:248-254, 1984). Approximately 0.2 copies per cell of circular DNA and 0.2 copies per cell of linear DNA were detected by hybridization with a cloned viral DNA fragment. The size of the viral DNA was estimated at ca. 158 kilobase pairs. Restriction endonuclease patterns suggested structural similarities to cottontail herpesvirus DNA. PMID:6092696

  2. Programmed pH-Driven Reversible Association and Dissociation of Interconnected Circular DNA Dimer Nanostructures.

    PubMed

    Hu, Yuwei; Ren, Jiangtao; Lu, Chun-Hua; Willner, Itamar

    2016-07-13

    The switchable pH-driven reversible assembly and dissociation of interlocked circular DNA dimers is presented. The circular DNA dimers are interconnected by pH-responsive nucleic acid bridges. In one configuration, the two-ring nanostructure is separated at pH = 5.0 to individual rings by reconfiguring the interlocking bridges into C-G·C(+) triplex units, and the two-ring assembly is reformed at pH = 7.0. In the second configuration, the dimer of circular DNAs is bridged at pH = 7.0 by the T-A·T triplex bridging units that are separated at pH = 10.0, leading to the dissociation of the dimer to single circular DNA nanostructures. The two circular DNA units are also interconnected by two pH-responsive locks. The pH-programmed opening of the locks at pH = 5.0 or pH = 10.0 yields two isomeric dimer structures composed of two circular DNAs. The switchable reconfigured states of the circular DNA nanostructures are followed by time-dependent fluorescence changes of fluorophore/quencher labeled systems and by complementary gel electrophoresis experiments. The dimer circular DNA structures are further implemented as scaffolds for the assembly of Au nanoparticle dimers exhibiting controlled spatial separation. PMID:27225955

  3. 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.

  4. 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.

  5. [Electron microscopic research on the extrachromosomal genetic elements of Escherichia coli].

    PubMed

    Kiseleva, E V; Vorob'eva, N V; Romashchenko, A G; Serdiukova, N A; Khristoliubova, N B

    1986-08-01

    The structural organization of extrachromosomal genetic elements were studied in a subfraction obtained after centrifugation of the lysate of E. coli spheroplasts. With this method of isolation, the tertiary structure of the extrachromosomal genetic elements was preserved. The majority of DNA macromolecules were released in the form of single and connected rosettes. Typical rosettes composed of radial loops of DNA clustered around the central dense core (the diameter is about 60 nm). The mean length of the rosette loops was 1.06 +/- 0.4 micron. Both relaxed folded and supercoiled folded forms of DNA were observed on the preparation. Sometimes the rosettes were connected with large aggregates of DNA (possibly the material of bacterial chromosomes) and had the appearance of thick fibers with numerous lateral loops. Linear, cyclic and various replicative forms of DNA have also been observed. It is assumed that rosettes of the extrachromosomal elements of E. coli reflect one of the levels of organization of prokaryotic genetic material. PMID:3535183

  6. Characterization of the Host Factors Required for Hepadnavirus Covalently Closed Circular (ccc) DNA Formation

    PubMed Central

    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. PMID:22912842

  7. Identification of novel Bromus- and Trifolium-associated circular DNA viruses.

    PubMed

    Kraberger, Simona; Farkas, Kata; Bernardo, Pauline; Booker, Cameron; Argüello-Astorga, Gerardo R; Mesléard, François; Martin, Darren P; Roumagnac, Philippe; Varsani, Arvind

    2015-05-01

    The genomes of a large number of highly diverse novel circular DNA viruses from a wide range of sources have been characterised in recent years, including circular single-stranded DNA (ssDNA) viruses that share similarities with plant-infecting ssDNA viruses of the family Geminiviridae. Here, we describe six novel circular DNA viral genomes that encode replication-associated (Rep) proteins that are most closely related to those of either geminiviruses or gemycircularviruses (a new group of ssDNA viruses that are closely related to geminiviruses). Four possible viral genomes were recovered from Bromus hordeaceus sampled in New Zealand, and two were recovered from B. hordeaceus and Trifolium resupinatum sampled in France. Two of the viral genomes from New Zealand (one from the North Island and one from the South Island each) share >99 % sequence identity, and two genomes recovered from B. hordeaceus and T. resupinatum sampled in France share 74 % identity. All of the viral genomes that were recovered were found to have a major open reading frame on both their complementary and virion-sense strands, one of which likely encodes a Rep and the other a capsid protein. Although future infectivity studies are needed to identify the host range of these viruses, this is the first report of circular DNA viruses associated with grasses in New Zealand. PMID:25701210

  8. Estimation of circular DNA size using gamma-irradiation and pulsed-field gel electrophoresis

    SciTech Connect

    Beverley, S.M. )

    1989-02-15

    A method is described for estimating the size of large circular DNAs found within complex chromosomal DNA preparations. DNAs are treated with low levels of gamma-irradiation, sufficient to introduce a single double-stranded break per circle, and the resulting linear DNA is sized by pulsed-field electrophoresis and blot hybridization. The method is fast, reproducible, and very conveniently applied to the agarose-enclosed chromosomal DNA preparations commonly used in pulsed field electrophoresis.

  9. 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.

  10. 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.

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

    PubMed

    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

  12. 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

  13. 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

  14. Evaluation of circular DNA substrates for whole genome amplification prior to forensic analysis.

    PubMed

    Tate, Courtney M; Nuñez, Ada N; Goldstein, Cori A; Gomes, Iva; Robertson, James M; Kavlick, Mark F; Budowle, Bruce

    2012-03-01

    Forensic biological evidence often contains low quantities of DNA or substantially degraded DNA which makes samples refractory to genotype analysis. One approach that shows promise to overcome the limited quantity of DNA is whole genome amplification (WGA). One WGA technique, termed rolling circle amplification (RCA), involves the amplification of circular DNA fragments and this study evaluates a single-stranded (ss) DNA ligase enzyme for generating circular DNA templates for RCA WGA. Fast, efficient ligation of several sizes of ssDNA templates was achieved. The enzyme also ligated double-stranded (ds) DNA templates, a novel activity not previously reported. Adapter sequences containing optimal terminal nucleotide ends for increased ligation efficiency were designed and ligation of adapters to template DNA was optimized. Increased amplification of DNA templates was observed following WGA; however, no amplification advantage for ssDNA ligase treatment of templates was evident compared to linear templates. A multi-step process to utilize ssDNA ligase prior to WGA was developed and short tandem repeat (STR) analysis of simulated low template (LT) and fragmented DNA was evaluated. The process resulted in the loss of template DNA and failed STR analysis whereas input of linear genomic DNA template directly into WGA prior to STR analysis improved STR genotyping results compared to non-WGA treated samples. Inclusion of an extreme thermostable single-stranded DNA binding protein (SSB) during WGA also increased DNA yields. While STR artifacts such as peak imbalance, drop-in, and dropout persisted, WGA shows potential for successful genetic profiling of LT and fragmented DNA samples. Further research and development is warranted prior to use of WGA in forensic casework. PMID:21570374

  15. 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. PMID:26112764

  16. 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

  17. Induction of differentiation in HL60 cells by the reduction of extrachromosomally amplified c-myc.

    PubMed Central

    Eckhardt, S G; Dai, A; Davidson, K K; Forseth, B J; Wahl, G M; Von Hoff, D D

    1994-01-01

    Oncogene amplification in tumor cells results in the overexpression of proteins that confer a growth advantage in vitro and in vivo. Amplified oncogenes can reside intrachromosomally, within homogeneously staining regions (HSRs), or extrachromosomally, within double minute chromosomes (DMs). Since previous studies have shown that low concentrations of hydroxyurea (HU) can eliminate DMs, we studied the use of HU as a gene-targeting agent in tumor cells containing extrachromosomally amplified oncogenes. In a neuroendocrine cell line (COLO 320), we have shown that HU can eliminate amplified copies of c-myc located on DMs, leading to a reduction in tumorigenicity in vitro and in vivo. To determine whether the observed reduction in tumorigenicity was due to differentiation, we next investigated whether HU could induce differentiation in HL60 cells containing extrachromosomally amplified c-myc. We compared the effects of HU, as well as two other known differentiating agents (dimethyl sulfoxide and retinoic acid), on c-myc gene copy number, c-myc expression, and differentiation in HL60 cells containing amplified c-myc genes either on DMs or HSRs. We discovered that HU and dimethyl sulfoxide reduced both c-myc gene copy number and expression and induced differentiation in cells containing c-myc amplified on DMs. These agents failed to have similar effects on HL60 cells with amplified c-myc in HSRs. By contrast, retinoic acid induced differentiation independent of the localization of amplified c-myc. These data illustrate the utility of targeting extrachromosomal DNA to modulate tumor phenotype and reveal that both HU and dimethyl sulfoxide induce differentiation in HL60 cells through DM elimination. Images PMID:8022834

  18. [Circular dichroism of DNA liquid-crystalline dispersion particles].

    PubMed

    Semenov, S V; Yevdokimov, Yu M

    2015-01-01

    The optical activity of DNA liquid-crystalline dispersions is being investigated based on a theory for absorption of electromagnetic waves by large molecular aggregates. The impact on the dispersions-optical properties, exerted by the interaction between the complexes of nucleic acid molecules and nanoparticles, is being considered. PMID:26016021

  19. Convergent DNA synthesis: a non-enzymatic dimerization approach to circular oligodeoxynucleotides.

    PubMed Central

    Rubin, E; Rumney, S; Wang, S; Kool, E T

    1995-01-01

    We report a novel convergent approach to the construction of circular DNA oligonucleotides from two smaller linear precursors. Circular DNAs 34-74 nucleotides (nt) in size are constructed non-enzymatically in a single step from two half-length oligomers. A DNA template is used to assemble the constituent parts into a triple helical complex which brings the four reactive ends together for chemical ligation with BrCN/imidazole/Ni2+. A homodimerization reaction strategy is successfully used on a small scale to construct circles 42, 58 and 74 nt in size. In addition, a heterodimerization strategy is successfully used in two cases to construct circular 34mers from different 16mer and 18mer precursors. Measurement of preparative yields for one biologically active 34mer circle shows that the dimerization strategy gives a yield higher than that from conventional cyclization and nearly as high as that for a normally synthesized linear DNA, establishing that there is not necessarily a yield penalty for circle construction. Six additional preparative circle constructions, giving conversions of approximately 33-85% from precursors to circular product, are also described. Convergent strategies allow the construction of medium and large size DNA molecules in higher yields than can be achieved by standard linear synthesis alone. Images PMID:7567468

  20. 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.

  1. The circular dichroism and X-ray diffraction of DNA condensed from ethanolic solutions.

    PubMed Central

    Gray, D M; Edmondson, S P; Lang, D; Vaughan, M

    1979-01-01

    It is known that DNA in aqueous-ethanol solutions undergoes a B to A conformational change between 60% and 80% (w/w) ethanol. We have found that precipitates formed by adding salt to DNA in 60% and 80% ethanolic solutions can be very different. DNA precipitated from 60% ethanol forms a fine condensate that only slowly settles out of suspension and shows a characteristic differential scattering of circularly polarized light at long wavelengths. DNA precipitated from 80% ethanol forms a flocculent aggregate that exhibits the CD spectral features of the A conformation. Data from circular dichroism spectra of natural and synthetic nucleic acids and from X-ray diffraction patterns of the precipitates show that DNA molecules precipitated from 60% and 80% ethanol are, respectively, in the B and A conformation. Therefore, the different secondary conformations of DNA in ethanolic solutions are maintained during precipitation under these conditions. These results are of general importance for the preparation and study of condensed forms of DNA, since a relatively small change in the extent of dehydration can change the secondary conformation of DNA and markedly affect the character of a subsequent precipitate. Images PMID:572544

  2. Circular dichroic properties and average dimensions of DNA-containing reverse micellar aggregates.

    PubMed

    Pietrini, Adriana V; Luisi, Pier Luigi

    2002-05-01

    With the aim of investigating the compartmentation of nucleic acids and surfactant aggregates, we have studied the circular dichroic properties of DNA solubilized in reverse micelles. DNA incorporated in AOT/isooctane reverse micelles (AOT=bis-2-ethyl-hexyl sodium sulfosuccinate) assumes an anomalous circular dichroism (CD) spectrum with the characteristic features of a psi spectrum. Older literature observations could therefore be confirmed that attribute these spectral changes to the fact that the reverse micelles induce the formation of a condensed form of DNA. A dynamic light scattering (DLS) characterization of the DNA-containing micellar solutions was carried out, and three populations of aggregates in a polar solvent are observed, with an average radius centered at 5, 100 and 1000 nm, respectively, all three containing DNA. Several forms of DNA, including a plasmid, have been investigated. The formation of 1 microm-large aggregates depends on the DNA concentration and such aggregates disappear in the course of a few hours. Conversely, the 100 nm aggregates are stable for at least 1 day and contain DNA in a normal spectral state at low concentration and in a condensed form-it is the characteristic psi spectrum-in a higher concentration range. The solubilization of DNA in reverse micelles brings about unexpected larger structures in hydrocarbon solution, and whereas the very large component can be with all likelihood be attributed to clusters of smaller reverse micelles, the components at 100 nm radius appear to be a quite stable and characteristic feature of DNA-containing reverse micelles. PMID:11988222

  3. Identification of a Novel Circular DNA Virus in New Zealand Fur Seal (Arctocephalus forsteri) Fecal Matter

    PubMed Central

    Sikorski, Alyssa; Dayaram, Anisha

    2013-01-01

    Fur seal feces-associated circular DNA virus (FSfaCV) is a novel virus isolated from the fecal matter of New Zealand fur seals. FSfaCV has two main open reading frames in its 2,925-nucleotide (nt) genome. The replication-associated protein (Rep) of FSfaCV has similarity to Rep-like sequences in the Giardia intestinalis genome. PMID:23929471

  4. Identification of a Novel Circular DNA Virus in New Zealand Fur Seal (Arctocephalus forsteri) Fecal Matter.

    PubMed

    Sikorski, Alyssa; Dayaram, Anisha; Varsani, Arvind

    2013-01-01

    Fur seal feces-associated circular DNA virus (FSfaCV) is a novel virus isolated from the fecal matter of New Zealand fur seals. FSfaCV has two main open reading frames in its 2,925-nucleotide (nt) genome. The replication-associated protein (Rep) of FSfaCV has similarity to Rep-like sequences in the Giardia intestinalis genome. PMID:23929471

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

    PubMed Central

    Kankia, Besik I.; Buckin, Vitaly; Bloomfield, Victor A.

    2001-01-01

    The interaction of hexamminecobalt(III), Co(NH3)63+, 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(NH3)63+ binding up to the molar ratio [Co(NH3)63+]/[P] = 0.25, prior to DNA condensation; (ii) a condensation process between [Co(NH3)63+]/[P] = 0.25 and 0.30; and (iii) precipitation after [Co(NH3)63+]/[P] = 0.3. In the case of 3000–8000 bp DNA only two processes were observed: (i) binding up to [Co(NH3)63+]/[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→Ψ transition after [Co(NH3)63+]/[P] = 0.25. From ultrasonic and densimetric measurements the effects of Co(NH3)63+ binding on volume and compressibility have been obtained. The binding of Co(NH3)63+ to both short and long DNA is characterized by similar changes in volume and compressibility calculated per mole Co(NH3)63+: ΔV = 9 cm3 mol–1 and Δκ = 33 × 10–4 cm3 mol–1 bar–1. The positive sign of the parameters indicates dehydration, i.e. water release from Co(NH3)63+ 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. PMID:11433025

  6. Novel, reversible, benzo[a]pyrene metabolite binding sites in closed-circular, single-stranded DNA

    SciTech Connect

    Price, H.L.

    1991-01-01

    The formation of reversible hydrocarbon epoxide-DNA complexes and the simultaneous increase in the reactivity of these bound epoxides is a general phenomenon. The relationship between physical binding, catalysis of epoxide reactions, and the formation of covalent adducts is not clearly understood at present. In these investigations it was found that linear, double-stranded DNA and supercoiled plasmid DNA both bind and enhance the reactivity of epoxide metabolites of benzo[a]pyrene to similar extents. The results of experiments carried out with closed-circular, single-stranded DNA however, indicate that this DNA possesses a unique binding site not present in either linear, double-stranded DNA or supercoiled DNA. Results of kinetic studies revealed that closed-circular, single-stranded DNA was less efficient at catalyzing epoxide reactions that double-stranded DNA. The results of these kinetic studies are explained in terms of a model which takes into account the polyelectrolyte nature of DNA.

  7. 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

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

    PubMed

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

    2016-03-01

    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

  9. 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.

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

    PubMed

    Beckel-Mitchener, A; Summers, J

    1997-10-01

    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

  11. 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. PMID:26943487

  12. Genomic characterization of novel circular ssDNA viruses from insectivorous bats in Southern Brazil.

    PubMed

    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

  13. 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

  14. Isolation of an IgH gene circular DNA clone from human bone marrow.

    PubMed Central

    Abe, M; Shiku, H

    1989-01-01

    Circular DNA was obtained from human bone marrow. Then a phage library was prepared and screened by use of two probes of the IgH gene; 5'-DHQ52, containing the 5' flanking region of DHQ52, and JH4.3, containing the sequence from JH3 to the 3' flanking region of JH6. One clone, HBMC-1, that was DHQ52+JH4.3- was obtained. HBMC-1 had the germline IgH region upstream of JH1 and the 3' flanking region of DXP1. A recombination signal sequence flanking the 5' side of the JH1 segment was attached to the recombination signal sequence flanking the 3' side of DXP1 forming a head-to-head structure of two 7mers with 10 nucleotides in-between. HBMC-1 is thus considered to be a circular DNA deleted as a consequence of DXP1-JH1 joining of the IgH gene. Images PMID:2492093

  15. Genetic diversity of novel circular ssDNA viruses in bats in China.

    PubMed

    Ge, Xingyi; Li, Jialu; Peng, Cheng; Wu, Lijun; Yang, Xinglou; Wu, Yongquan; Zhang, Yunzhi; Shi, Zhengli

    2011-11-01

    Novel circular ssDNA genomes have recently been detected in animals and in the environment using metagenomic and high-throughput sequencing approaches. In this study, five full-length circular ssDNA genomes were recovered from bat faecal samples using inverse PCR with sequences designed based on circovirus-related sequences obtained from Solexa sequencing data derived from a random amplification method. These five sequences shared a similar genomic organization to circovirus or the recently proposed cyclovirus of the family Circoviridae. The newly obtained circovirus/cyclovirus-like genomes ranged from 1741 to 2177 bp, and each consisted of two major ORFs, ORF1 and ORF2, encoding putative replicase (Rep) and capsid (Cap) proteins, respectively. The potential stem-loop region was predicted in all five genomes, and three of them had the typical conserved nonanucleotide motif of cycloviruses. A set of primers targeting the conserved Rep region was designed and used to detect the prevalence of circovirus/cyclovirus sequences in individual bats. Among 199 samples tested, 47 were positive (23.6%) for the circovirus genome and two (1.0%) were positive for the cyclovirus genome. In total, 48 partial Rep sequences plus the five full-length genomes were obtained in this study. Detailed analysis indicated that these sequences are distantly related to known circovirus/cyclovirus genomes and may represent 22 novel species that belong to the family Circoviridae. PMID:21795473

  16. Portable and sensitive quantitative detection of DNA based on personal glucose meters and isothermal circular strand-displacement polymerization reaction.

    PubMed

    Xu, Xue-tao; Liang, Kai-yi; Zeng, Jia-ying

    2015-02-15

    A portable and sensitive quantitative DNA detection method based on personal glucose meters and isothermal circular strand-displacement polymerization reaction was developed. The target DNA triggered target recycling process, which opened capture DNA. The released target then found another capture DNA to trigger another polymerization cycle, which was repeated for many rounds, resulting in the multiplication of the DNA-invertase conjugation on the surface of Streptavidin-MNBs. The DNA-invertase was used to catalyze the hydrolysis of sucrose into glucose for PGM readout. There was a liner relationship between the signal of PGM and the concentration of target DNA in the range of 5.0 to 1000 fM, which is lower than some DNA detection method. In addition, the method exhibited excellent sequence selectivity and there was almost no effect of biological complex to the detection performance, which suggested our method can be successfully applied to DNA detection in real biological samples. PMID:25441417

  17. Distinct Circular Single-Stranded DNA Viruses Exist in Different Soil Types

    PubMed Central

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

    2015-01-01

    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. PMID:25841004

  18. 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. PMID:25841004

  19. Identification of Disubstituted Sulfonamide Compounds as Specific Inhibitors of Hepatitis B Virus Covalently Closed Circular DNA Formation

    PubMed Central

    Cai, Dawei; Mills, Courtney; Yu, Wenquan; Yan, Ran; Aldrich, Carol E.; Saputelli, Jeffry R.; Mason, William S.; Xu, Xiaodong; Guo, Ju-Tao; Block, Timothy M.

    2012-01-01

    Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) plays a central role in viral infection and persistence and is the basis for viral rebound after the cessation of therapy, as well as the elusiveness of a cure even after extended treatment. Therefore, there is an urgent need for the development of novel therapeutic agents that directly target cccDNA formation and maintenance. By employing an innovative cell-based cccDNA assay in which secreted HBV e antigen is a cccDNA-dependent surrogate, we screened an in-house small-molecule library consisting of 85,000 drug-like compounds. Two structurally related disubstituted sulfonamides (DSS), termed CCC-0975 and CCC-0346, emerged and were confirmed as inhibitors of cccDNA production, with low micromolar 50% effective concentrations (EC50s) in cell culture. Further mechanistic studies demonstrated that DSS compound treatment neither directly inhibited HBV DNA replication in cell culture nor reduced viral polymerase activity in the in vitro endogenous polymerase assay but synchronously reduced the levels of HBV cccDNA and its putative precursor, deproteinized relaxed circular DNA (DP-rcDNA). However, DSS compounds did not promote the intracellular decay of HBV DP-rcDNA and cccDNA, suggesting that the compounds interfere primarily with rcDNA conversion into cccDNA. In addition, we demonstrated that CCC-0975 was able to reduce cccDNA biosynthesis in duck HBV-infected primary duck hepatocytes. This is the first attempt, to our knowledge, to identify small molecules that target cccDNA formation, and DSS compounds thus potentially serve as proof-of-concept drug candidates for development into therapeutics to eliminate cccDNA from chronic HBV infection. PMID:22644022

  20. Suppression of rolling circle amplification by nucleotide analogs in circular template for three DNA polymerases.

    PubMed

    Tang, Suming; Wei, Hua; Hu, Tianyu; Jiang, Jiquan; Chang, Jinglin; Guan, Yifu; Zhao, Guojie

    2016-08-01

    Among wide applications of nucleotide analogs, their roles in enzyme catalytic reactions are significant in both fundamental and medical researches. By introducing analogs into circular templates, we succeeded in determining effects of four analogs on RCA efficiency for three different DNA polymerases. Results showed an obvious suppression effect for 2'-OMeRNA modification, which might be due to the size of the C2'-modified moieties. 2'-F RNA, LNA and PS had little interference, suggesting good analog candidates for application in RCA. Different polymerases and nucleobases made a little difference according to analogs we used. These results are useful for understanding polymerase catalytic mechanism and analogs applications in RCA reaction. PMID:27151504

  1. Biochemical Method for Inserting New Genetic Information into DNA of Simian Virus 40: Circular SV40 DNA Molecules Containing Lambda Phage Genes and the Galactose Operon of Escherichia coli

    PubMed Central

    Jackson, David A.; Symons, Robert H.; Berg, Paul

    1972-01-01

    We have developed methods for covalently joining duplex DNA molecules to one another and have used these techniques to construct circular dimers of SV40 DNA and to insert a DNA segment containing lambda phage genes and the galactose operon of E. coli into SV40 DNA. The method involves: (a) converting circular SV40 DNA to a linear form, (b) adding single-stranded homodeoxypolymeric extensions of defined composition and length to the 3′ ends of one of the DNA strands with the enzyme terminal deoxynucleotidyl transferase (c) adding complementary homodeoxypolymeric extensions to the other DNA strand, (d) annealing the two DNA molecules to form a circular duplex structure, and (e) filling the gaps and sealing nicks in this structure with E. coli DNA polymerase and DNA ligase to form a covalently closed-circular DNA molecule. PMID:4342968

  2. Biochemical method for inserting new genetic information into DNA of Simian Virus 40: circular SV40 DNA molecules containing lambda phage genes and the galactose operon of Escherichia coli.

    PubMed

    Jackson, D A; Symons, R H; Berg, P

    1972-10-01

    We have developed methods for covalently joining duplex DNA molecules to one another and have used these techniques to construct circular dimers of SV40 DNA and to insert a DNA segment containing lambda phage genes and the galactose operon of E. coli into SV40 DNA. The method involves: (a) converting circular SV40 DNA to a linear form, (b) adding single-stranded homodeoxypolymeric extensions of defined composition and length to the 3' ends of one of the DNA strands with the enzyme terminal deoxynucleotidyl transferase (c) adding complementary homodeoxypolymeric extensions to the other DNA strand, (d) annealing the two DNA molecules to form a circular duplex structure, and (e) filling the gaps and sealing nicks in this structure with E. coli DNA polymerase and DNA ligase to form a covalently closed-circular DNA molecule. PMID:4342968

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

    PubMed

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

    2016-03-01

    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

  4. Isolation of bacterial plasmid-related replication-associated circular DNA from a serum sample of a multiple sclerosis patient.

    PubMed

    Gunst, Karin; Zur Hausen, Harald; de Villiers, Ethel-Michele

    2014-01-01

    Psychrobacter species are considered to be opportunistic human pathogens. We report here the isolation of a circular DNA molecule, MSSI1.162, from a serum sample taken from a multiple sclerosis patient during relapse. This isolate is distantly related to known Psychrobacter species and their plasmids. PMID:25169857

  5. Isolation of Bacterial Plasmid-Related Replication-Associated Circular DNA from a Serum Sample of a Multiple Sclerosis Patient

    PubMed Central

    Gunst, Karin; zur Hausen, Harald

    2014-01-01

    Psychrobacter species are considered to be opportunistic human pathogens. We report here the isolation of a circular DNA molecule, MSSI1.162, from a serum sample taken from a multiple sclerosis patient during relapse. This isolate is distantly related to known Psychrobacter species and their plasmids. PMID:25169857

  6. Radiation damage to a DNA-binding protein. Combined circular dichroism and molecular dynamics simulation analysis.

    PubMed

    Mazier, S; Villette, S; Goffinont, S; Renouard, S; Maurizot, J C; Genest, D; Spotheim-Maurizot, M

    2008-11-01

    The E. coli lactose operon, the paradigm of gene expression regulation systems, is the best model for studying the effect of radiation on such systems. The operon function requires the binding of a protein, the repressor, to a specific DNA sequence, the operator. We have previously shown that upon irradiation the repressor loses its operator binding ability. The main radiation-induced lesions of the headpiece have been identified by mass spectrometry. All tyrosine residues are oxidized into 3,4-dihydroxyphenylalanine (DOPA). In the present study we report a detailed characterization of the headpiece radiation-induced modification. An original approach combining circular dichroism measurements and the analysis of molecular dynamics simulation of headpieces bearing DOPA-s instead of tyrosines has been applied. The CD measurements reveal an irreversible modification of the headpiece structure and stability. The molecular dynamics simulation shows a loss of stability shown by an increase in internal dynamics and allows the estimation of the modifications due to tyrosine oxidation for each structural element of the protein. The changes in headpiece structure and stability can explain at least in part the radiation-induced loss of binding ability of the repressor to the operator. This conclusion should hold for all proteins containing radiosensitive amino acids in their DNA-binding site. PMID:18959464

  7. Yeast RNA polymerase II transcription of circular DNA at different degrees of supercoiling.

    PubMed Central

    Pedone, F; Filetici, P; Ballario, P

    1982-01-01

    Purified yeast RNA polymerase II was tested for transcriptional activity as a function of the degree of circular DNA supercoiling. Chimaeric plasmids P30 and P31 both containing inserts from the yeast transposable element TY1 cloned in pBR322 and the vector pBR322 were used as templates. For pBR322 the transcriptional activity increases about 4 fold from the fully relaxed covalently closed circles to the native supercoiled forms, further supercoiling having no effect on transcription. P30 shows a 5 fold increase of transcriptional activity reaching a plateau at the native supercoiled conformation. However, at an intermediate degree of supercoiling (sigma = 0.024), transcription decreases to a value close to zero. P31 too exhibits a conformation (sigma = 0.014) in which there is a drop of transcriptional activity. Furthermore, a 10 fold increase of transcription is obtained at the higher values of superhelix density. Both kinetic and autoradiographic experiments confirm the existence of DNA conformations that can inhibit "in vitro" transcription. Images PMID:6292834

  8. The fecal virome of South and Central American children with diarrhea includes small circular DNA viral genomes of unknown origin.

    PubMed

    Phan, Tung Gia; da Costa, Antonio Charlys; Del Valle Mendoza, Juana; Bucardo-Rivera, Filemon; Nordgren, Johan; O'Ryan, Miguel; Deng, Xutao; Delwart, Eric

    2016-04-01

    Viral metagenomics of feces collected from 58 Peruvian children with unexplained diarrhea revealed several small circular ssDNA genomes. Two genomes related to sequences previously reported in feces from chimpanzees and other mammals and recently named smacoviruses were characterized and then detected by PCR in 1.7 % (1/58) and 19 % (11/58) of diarrheal samples, respectively. Another three genomes from a distinct small circular ssDNA viral group provisionally called pecoviruses encoded Cap and Rep proteins with <35 % identity to those in related genomes reported in human, seal, porcine and dromedary feces. Pecovirus DNA was detected in 15.5 % (9/58), 5.9 % (3/51) and 3 % (3/100) of fecal samples from unexplained diarrhea in Peru, Nicaragua and Chile, respectively. Feces containing these ssDNA genomes also contained known human enteric viral pathogens. The cellular origins of these circular ssDNA viruses, whether human cells, ingested plants, animals or fungal foods, or residents of the gut microbiome, are currently unknown. PMID:26780893

  9. 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.

  10. PENICILLINASE PLASMID DNA FROM Staphylococcus aureus*

    PubMed Central

    Rush, Mark G.; Gordon, C. N.; Novick, Richard P.; Warner, Robert C.

    1969-01-01

    A penicillinase plasmid from Staphylococcus aureus and three of its derivatives, all previously identified as extrachromosomal genetic elements, have been isolated in high yield as circular duplex DNA molecules. The wild-type plasmid was found by contour-length measurements of electron micrographs to have a molecular weight of 18.6 × 106 daltons. Two plasmids with deletions encompassing six and eight of the eleven known plasmid cistrons had molecular weights of 16.4 × 106 and 15.3 × 106 daltons, respectively. This information was used to establish approximate physical distances for the genetic map. A high-frequency transducing element also derived from the plasmid had a molecular weight of approximately 24 × 106 daltons. Although each plasmid preparation appeared homogeneous by ultracentrifugal analysis, electron micrographs always revealed the presence of a low percentage of complex oligomeric forms, particularly circular and catenated dimers. Images PMID:5260933

  11. Circular replication-associated protein encoding DNA viruses identified in the faecal matter of various animals in New Zealand.

    PubMed

    Steel, Olivia; Kraberger, Simona; Sikorski, Alyssa; Young, Laura M; Catchpole, Ryan J; Stevens, Aaron J; Ladley, Jenny J; Coray, Dorien S; Stainton, Daisy; Dayaram, Anisha; Julian, Laurel; van Bysterveldt, Katherine; Varsani, Arvind

    2016-09-01

    In recent years, innovations in molecular techniques and sequencing technologies have resulted in a rapid expansion in the number of known viral sequences, in particular those with circular replication-associated protein (Rep)-encoding single-stranded (CRESS) DNA genomes. CRESS DNA viruses are present in the virome of many ecosystems and are known to infect a wide range of organisms. A large number of the recently identified CRESS DNA viruses cannot be classified into any known viral families, indicating that the current view of CRESS DNA viral sequence space is greatly underestimated. Animal faecal matter has proven to be a particularly useful source for sampling CRESS DNA viruses in an ecosystem, as it is cost-effective and non-invasive. In this study a viral metagenomic approach was used to explore the diversity of CRESS DNA viruses present in the faeces of domesticated and wild animals in New Zealand. Thirty-eight complete CRESS DNA viral genomes and two circular molecules (that may be defective molecules or single components of multicomponent genomes) were identified from forty-nine individual animal faecal samples. Based on shared genome organisations and sequence similarities, eighteen of the isolates were classified as gemycircularviruses and twelve isolates were classified as smacoviruses. The remaining eight isolates lack significant sequence similarity with any members of known CRESS DNA virus groups. This research adds significantly to our knowledge of CRESS DNA viral diversity in New Zealand, emphasising the prevalence of CRESS DNA viruses in nature, and reinforcing the suggestion that a large proportion of CRESS DNA viruses are yet to be identified. PMID:27211884

  12. Conformational state of DNA in chromatin subunits. Circular dichroism, melting, and ethidium bromide binding analysis.

    PubMed Central

    Lawrence, J J; Chan, D C; Piette, L H

    1976-01-01

    This study compares some physical properties of DNA in native chromatin and mono-, di-, trinucleosomes obtained after mild micrococcal nuclease digestion. Melting curves and derivatives are shown to be very similar from one sample to another although a shift from 79 to 82 degrees C is observed between the mainly monophasic peak of multimers and chromatin. Careful analysis of the positive band of the circular dichroism spectra shows the appearance of a shoulder at 275nm, the intensity of which increases from the mono- to the di- and trinucleosome. This shoulder is maximum for native chromatin. At the same time binding isotherms of ethidium - bromide are characterized by two highly fluorescent binding sites for all the samples but the product KN of the apparent binding constant of the higher affinity binding sites by the apparent number of those sites increases from the mono- to the di- and trinucleosome. There again the valus is maximum for native chromatin. Such results strongly suggest that the native state of chromatin requires something more than the indefinite repeat of an elementary subunit. Images PMID:1005108

  13. Mercury-induced DNA polymorphism: Probing the conformation of Hg(II)-DNA via Staphylococcal nuclease digestion and circular dichroism measurements

    SciTech Connect

    Gruenwedel, D.W.; Cruikshank, M.K. )

    1990-02-27

    Exposing native calf thymus DNA to increasing concentrations of Hg(ClO{sub 4}){sub 2} not only produces dramatic changes in its circular dichroism (CD) but results also in the decrease, and ultimate cessation, of endonucleolytic DNA cleavage by staphylococcal nuclease. DNA cleavage proceeds at or near the rates exhibited by untreated DNA. At Hg(II) levels of 0.08 < r < 0.5, the rate of DNA hydrolysis decreases monotonically with increasing Hg(II) concentrations, and at r > 0.4, DNA cleavage ceases. Both the CD changes and the changes in the rate of DNA digestion are totally reversible upon the removal of Hg(II). For comparison purposes, native calf thymus DNA was also treated with methylmercury (CH{sub 3}Hg(II)), an agent known to disrupt the secondary structure of DNA. The treatment yielded single-stranded methylmercurated DNA with preserved right-handed helix screwness. The authors interpret the Hg(II)-induced alterations in the CD of native calf thymus DNA, and the hydrolysis rate changes observed with staphylococcal nuclease, to indicate that Hg(II) either produces in DNA reversible B {leftrightarrow} Z transitions, passing transiently through C-like conformations, or generates non-B-conformational structures of presumably left-handed geometry.

  14. Cleavage of supercoiled circular double-stranded DNA induced by a eukaryotic cambialistic superoxide dismutase from Cinnamomum camphora.

    PubMed

    Wang, Bao-Zhong; Wei, Xu-Bin; Liu, Wang-Yi

    2004-09-01

    A eukaryotic cambialistic superoxide dismutase (SOD) has been purified to homogeneity from mature seeds of the disease- and insect-resistant camphor tree (Cinnamomum camphora). Besides the known role of this SOD in protecting cells against oxidative stress, it can induce the cleavage of supercoiled double-stranded DNA into nicked and linear DNA. It can not cleave linear DNA or RNA, demonstrating there is no DNase or RNase in the purified cambialistic SOD. Furthermore, the SOD can linearize circular pGEM-4Z DNA that is relaxed by topoisomerase I. This result indicates that the DNA-cleaving activity requires substrates being topologically constrained. The supercoiled DNA-cleaving activity of the cambialistic SOD can be inhibited by either SOD inhibitor (azide) or catalase and hydroxyl radical scavengers (ethanol and mannitol). The chelator of iron, diethylenetriaminepentaacetic acid (DTPA), also inhibits the supercoiled DNA-cleaving activity. These results show that the dismutation activity is crucial for the supercoiled DNA cleavage. The modification of tryptophan residue of the cambialistic SOD with N-bromosuccinimide (NBS) shows that these two activities are structurally correlative. The reaction mechanism is proposed that the hydroxyl radical formed in a transition-metal-catalyzing Fenton-type reaction contributes to the DNA-cleaving activity. In addition, the cleavage sites in supercoiled pGEM-4Z DNA are random. PMID:15346198

  15. Development of an extrachromosomal cloning vector system for use in Borrelia burgdorferi.

    PubMed

    Sartakova, M; Dobrikova, E; Cabello, F C

    2000-04-25

    Molecular genetic analysis of Borrelia burgdorferi, the cause of Lyme disease, has been hampered by the absence of any means of efficient generation, identification, and complementation of chromosomal and plasmid null gene mutants. The similarity of borrelial G + C content to that of Gram-positive organisms suggested that a wide-host-range plasmid active in Gram-positive bacteria might also be recognized by borrelial DNA replication machinery. One such plasmid, pGK12, is able to propagate in both Gram-positive and Gram-negative bacteria and carries erythromycin and chloramphenicol resistance markers. pGK12 propagated extrachromosomally in B. burgdorferi B31 after electroporation but conferred only erythromycin resistance. pGK12 was used to express enhanced green fluorescent protein in B31 under the control of the flaB promoter. Escherichia coli transformed with pGK12 DNA extracted from B31 expressing only erythromycin resistance developed both erythromycin and chloramphenicol resistance, and plasmid DNA isolated from these transformed E. coli had a restriction pattern similar to the original pGK12. Our data indicate that the replicons of pGK12 can provide the basis to continue developing efficient genetic systems for B. burgdorferi together with the erythromycin resistance and reporter egfp genes. PMID:10781091

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  17. MuA-mediated in vitro cloning of circular DNA: transpositional autointegration and the effect of MuB.

    PubMed

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

    2016-06-01

    Transposons provide useful tools for genetics and genomics studies, as they can be modified easily for a variety of purposes. In this study, a strategy to clone circular DNA was developed on the basis of an efficient Mu in vitro transposition reaction catalyzed by MuA transposase. The transposon used contains a selectable marker as well as an origin of replication, and in vitro integration of the transposon into circular DNA generates a plasmid that can replicate in E. coli. We show that the substrate stoichiometry plays an important role in the profile of intermolecular versus intramolecular transposition reaction products. Increasing the relative amount of target DNA reduced the frequency of intramolecular products that are non-productive with regard to the developed cloning application. Such autointegration was also reduced in the reactions containing phage Mu-encoded MuB, indicating that this protein can be used for cloning in combination with MuA, and it is particularly useful with a limited amount of target DNA. The developed strategy can now be utilized to clone DNA circles regardless of their origin as long as their size is not prohibitive for transformation. PMID:26847688

  18. 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. PMID:25579461

  19. Amplicon rearrangements during the extrachromosomal and intrachromosomal amplification process in a glioma

    PubMed Central

    Vogt, Nicolas; Gibaud, Anne; Lemoine, Frédéric; de la Grange, Pierre; Debatisse, Michelle; Malfoy, Bernard

    2014-01-01

    The mechanisms of gene amplification in tumour cells are poorly understood and the relationship between extrachromosomal DNA molecules, named double minutes (dmins), and intrachromosomal homogeneously staining regions (hsr) is not documented at nucleotide resolution. Using fluorescent in situ hybridization and whole genome sequencing, we studied a xenografted human oligodendroglioma where the co-amplification of the EGFR and MYC loci was present in the form of dmins at early passages and of an hsr at later passages. The amplified regions underwent multiple rearrangements and deletions during the formation of the dmins and their transformation into hsr. In both forms of amplification, non-homologous end-joining and microhomology-mediated end-joining rather than replication repair mechanisms prevailed in fusions. Small fragments, some of a few tens of base pairs, were associated in contigs. They came from clusters of breakpoints localized hundreds of kilobases apart in the amplified regions. The characteristics of some pairs of junctions suggest that at least some fragments were not fused randomly but could result from the concomitant repair of neighbouring breakpoints during the interaction of remote DNA sequences. This characterization at nucleotide resolution of the transition between extra- and intrachromosome amplifications highlights a hitherto uncharacterized organization of the amplified regions suggesting the involvement of new mechanisms in their formation. PMID:25378339

  20. 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

    2015-02-01

    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. PMID:26261947

  1. Actinophage R4 integrase-based site-specific chromosomal integration of non-replicative closed circular DNA.

    PubMed

    Miura, Takamasa; Nishizawa, Akito; Nishizawa, Tomoyasu; Asayama, Munehiko; Shirai, Makoto

    2016-06-01

    The actinophage R4 integrase (Sre)-based molecular genetic engineering system was developed for the chromosomal integration of multiple genes in Escherichia coli. A cloned DNA fragment containing two attP sites, green fluorescent protein (gfp) as a first transgene, and an antibiotic resistance gene as a selection marker was self-ligated to generate non-replicative closed circular DNA (nrccDNA) for integration. nrccDNA was introduced into attB-inserted E. coli cells harboring the plasmid expressing Sre by electroporation. The expressed Sre catalyzed site-specific integration between one of the two attP sites on nrccDNA and the attB site on the E. coli chromosome. The integration frequency was affected by the chromosomal location of the target site. A second nrccDNA containing two attB sites, lacZα encoding the alpha fragment of β-galactosidase as a transgene, and another antibiotic resistance gene was integrated into the residual attP site on the gfp-integrated E. coli chromosome via one of the two attB sites according to reiterating site-specific recombination. The integrants clearly exhibited β-galactosidase activity and green fluorescence, suggesting the simultaneous expression of multiple recombinant proteins in E. coli. The results of the present study showed that a step-by-step integration procedure using nrccDNA achieved the chromosomal integration of multiple genes. PMID:26870903

  2. Co-existence of circular and multiple linear amplicons in methotrexate-resistant Leishmania.

    PubMed Central

    Olmo, A; Arrebola, R; Bernier, V; González-Pacanowska, D; Ruiz-Pérez, L M

    1995-01-01

    Circular and linear amplicons were analyzed in detail in Leishmania tropica cells resistant to methotrexate (MTX). Both types of elements presented sequences related to the H locus and coexisted in resistant cells. The linear amplicons appeared first during the selection process (at 10 microM MTX) and varied with regard to size and structure in cells exposed to increasing concentrations of drug. The circular element was evident at higher concentrations (50 microMs) but was the major amplified DNA in cells resistant to 1000 microM MTX while the level of amplification of the linear elements remained low. The extrachromosomal DNAs were unstable in the absence of drug and their disappearance coincided with an increase in sensitivity to MTX. Mapping of the minichromosomes and the circular element showed that they were all constituted by inverted duplications. The circular amplicon contained an inverted repeat derived from the H locus that encompassed the pteridine reductase gene (PTR1) responsible for MTX resistance. The amplified segment in the linear amplicons was longer and included the pgpB and pgpC genes that encode P-glycoproteins of unknown function previously characterized in different Leishmania species. Images PMID:7659507

  3. 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. PMID:11987140

  4. Chromosomally and Extrachromosomally Mediated High-Level Gentamicin Resistance in Streptococcus agalactiae.

    PubMed

    Sendi, Parham; Furitsch, Martina; Mauerer, Stefanie; Florindo, Carlos; Kahl, Barbara C; Shabayek, Sarah; Berner, Reinhard; Spellerberg, Barbara

    2016-03-01

    Streptococcus agalactiae (group B Streptococcus [GBS]) is a leading cause of sepsis in neonates. The rate of invasive GBS disease in nonpregnant adults also continues to climb. Aminoglycosides alone have little or no effect on GBS, but synergistic killing with penicillin has been shown in vitro. High-level gentamicin resistance (HLGR) in GBS isolates, however, leads to the loss of a synergistic effect. We therefore performed a multicenter study to determine the frequency of HLGR GBS isolates and to elucidate the molecular mechanisms leading to gentamicin resistance. From eight centers in four countries, 1,128 invasive and colonizing GBS isolates were pooled and investigated for the presence of HLGR. We identified two strains that displayed HLGR (BSU1203 and BSU452), both of which carried the aacA-aphD gene, typically conferring HLGR. However, only one strain (BSU1203) also carried the previously described chromosomal gentamicin resistance transposon designated Tn3706. For the other strain (BSU452), plasmid purification and subsequent DNA sequencing resulted in the detection of plasmid pIP501 carrying a remnant of a Tn3 family transposon. Its ability to confer HLGR was proven by transfer into an Enterococcus faecalis isolate. Conversely, loss of HLGR was documented after curing both GBS BSU452 and the transformed E. faecalis strain from the plasmid. This is the first report showing plasmid-mediated HLGR in GBS. Thus, in our clinical GBS isolates, HLGR is mediated both chromosomally and extrachromosomally. PMID:26729498

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  6. Experimental Analysis of a Paternally Inherited Extrachromosomal Factor

    PubMed Central

    Werren, John H.; van den Assem, Johannes

    1986-01-01

    Virtually all known cases of extrachromosomal inheritance involve cytoplasmic inheritance through the maternal line. Recently, a paternally transmitted factor that causes the production of all-male families has been discovered in a parasitic wasp. The wasp has haplodiploid sex determination: male offspring are haploid and usually develop from unfertilized eggs, whereas females are diploid and usually develop from fertilized eggs. It has been postulated that this paternal sex-ratio factor (psr) is either (1) an infectious agent (a venereal disease) that is transmitted to the female reproductive tract during copulation with an infected male and, subsequently, causes all-male families or (2) a male cytoplasmic factor that is transmitted by sperm to eggs upon egg fertilization and, somehow, causes loss of the paternal set of chromosomes.—Experimental evidence is presented which shows that the factor requires egg fertilization for transmission to the next generation; therefore, it is likely to be a cytoplasmic factor. Significant potential intragenomic conflict results from the presence of this factor and two other sex-ratio distorters in this wasp species. PMID:17246344

  7. Soma to germline inheritance of extrachromosomal genetic information via a LINE-1 reverse transcriptase-based mechanism.

    PubMed

    Spadafora, Corrado

    2016-08-01

    Mature spermatozoa are permeable to foreign DNA and RNA molecules. Here I propose a model, whereby extrachromosomal genetic information, mostly encoded in the form of RNA in somatic cells, can cross the Weismann barrier and reach epididymal spermatozoa. LINE-1 retrotransposon-derived reverse transcriptase (RT) can play key roles in the process by expanding the RNA-encoded information. Retrotransposon-encoded RT is stored in mature gametes, is highly expressed in early embryos and undifferentiated cells, and becomes downregulated in differentiated cells. In turn, RT plays a role in developmental control, as its inhibition arrests developmental progression of early embryos with globally altered transcriptomic profiles. Thus, sperm cells act as recipients, and transgenerational vectors of somatically derived genetic information which they pass to the next generation with the potential to modify the fate of the developing embryos. PMID:27315018

  8. Small circular single stranded DNA viral genomes in unexplained cases of human encephalitis, diarrhea, and in untreated sewage.

    PubMed

    Phan, Tung Gia; Mori, Daisuke; Deng, Xutao; Rajindrajith, Shaman; Ranawaka, Udaya; Fan Ng, Terry Fei; Bucardo-Rivera, Filemon; Orlandi, Patricia; Ahmed, Kamruddin; Delwart, Eric

    2015-08-01

    Viruses with small circular ssDNA genomes encoding a replication initiator protein can infect a wide range of eukaryotic organisms ranging from mammals to fungi. The genomes of two such viruses, a cyclovirus (CyCV-SL) and gemycircularvirus (GemyCV-SL) were detected by deep sequencing of the cerebrospinal fluids of Sri Lankan patients with unexplained encephalitis. One and three out of 201 CSF samples (1.5%) from unexplained encephalitis patients tested by PCR were CyCV-SL and GemyCV-SL DNA positive respectively. Nucleotide similarity searches of pre-existing metagenomics datasets revealed closely related genomes in feces from unexplained cases of diarrhea from Nicaragua and Brazil and in untreated sewage from Nepal. Whether the tropism of the cyclovirus and gemycircularvirus reported here include humans or other cellular sources in or on the human body remains to be determined. PMID:25839169

  9. Differential replication of circular DNA molecules co-injected into early Xenopus laevis embryos.

    PubMed Central

    Marini, N J; Hiriyanna, K T; Benbow, R M

    1989-01-01

    Replication of co-injected supercoiled DNA molecules in fertilized Xenopus eggs was monitored through the blastula stage of development. The extent of replication, as measured by 32P-dTMP incorporation into form I DNA, was directly proportional to the number of molecules, rather than the size, of the plasmid injected. Although only a small fraction of molecules of either template was replicated, incorporation was predominantly into full length daughter molecules. Over at least a 20-fold concentration range of microinjected DNA, injection of equal masses of DNA resulted in greater incorporation into the smaller form I DNA present in molar excess. The extent of incorporation into supercoiled DNA for a particular plasmid was apparently independent of the concentration of a second, co-injected plasmid. The relative extents of replication of co-injected supercoiled templates could be altered simply by changing the molar ratios of the templates. Images PMID:2762153

  10. In-Phase Assembly of Slim DNA Lattices with Small Circular DNA Motifs via Short Connections of 11 and 16 Base Pairs.

    PubMed

    Wang, Meng; Guo, Xin; Jiang, Chuan; Wang, Xuemei; Xiao, Shou-Jun

    2016-06-16

    Two kinds of stable motif were constructed: SAE (semi-crossover, antiparallel, even half-turns) tile from one small circular DNA molecule (42 or 64 nt) and two linear oligonucleotides; and DAE (double-crossover, antiparallel, even half-turns) tile from one small circular DNA molecule (42 or 64 nt) and four linear oligonucleotides. With the SAE tiles, in-phase assembly of SAE-E (SAE tiles with even half-turns as connections (-E)) with the shortest -E of 11 base pairs (bp) generated homogeneous nanotubes with an average length of over 14 μm and a diameter of 16-20 nm; with the DAE tiles, in-phase assembly of DAE-O (DAE tiles with odd half-turns as connections (-O)) with the shortest -O of 16 bp produced slim monolayer nanoyarns (25-30 nm wide), nanoscarfs (100-300 nm wide), and nanoribbons (∼100 nm wide). Interestingly, a phenomenon we term "knitting nanoyarns" into nanoscarfs was observed. Finally a curvature mechanism according to the ring rotation directions is suggested to explain the formation of nanotubes, wavy nanoyarns, nanoscarfs, and nanoribbons. PMID:27187004

  11. 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. PMID:27387339

  12. Isolation of protein-associated circular DNA from healthy cattle serum.

    PubMed

    Funk, Mathis; Gunst, Karin; Lucansky, Vincent; Müller, Hermann; Zur Hausen, Harald; de Villiers, Ethel-Michele

    2014-01-01

    Three replication-competent single-stranded DNA molecules sharing nucleotide similarity to transmissible spongiform encephalopathy (TSE)-associated isolate Sphinx 2.36 were isolated from healthy bovine serum. PMID:25169856

  13. Isolation of Protein-Associated Circular DNA from Healthy Cattle Serum

    PubMed Central

    Funk, Mathis; Gunst, Karin; Lucansky, Vincent; Müller, Hermann; zur Hausen, Harald

    2014-01-01

    Three replication-competent single-stranded DNA molecules sharing nucleotide similarity to transmissible spongiform encephalopathy (TSE)-associated isolate Sphinx 2.36 were isolated from healthy bovine serum. PMID:25169856

  14. Acinetobacter phage genome is similar to Sphinx 2.36, the circular DNA copurified with TSE infected particles

    PubMed Central

    Longkumer, Toshisangba; Kamireddy, Swetha; Muthyala, Venkateswar Reddy; Akbarpasha, Shaikh; Pitchika, Gopi Krishna; Kodetham, Gopinath; Ayaluru, Murali; Siddavattam, Dayananda

    2013-01-01

    While analyzing plasmids of Acinetobacter sp. DS002 we have detected a circular DNA molecule pTS236, which upon further investigation is identified as the genome of a phage. The phage genome has shown sequence similarity to the recently discovered Sphinx 2.36 DNA sequence co-purified with the Transmissible Spongiform Encephalopathy (TSE) particles isolated from infected brain samples collected from diverse geographical regions. As in Sphinx 2.36, the phage genome also codes for three proteins. One of them codes for RepA and is shown to be involved in replication of pTS236 through rolling circle (RC) mode. The other two translationally coupled ORFs, orf106 and orf96, code for coat proteins of the phage. Although an orf96 homologue was not previously reported in Sphinx 2.36, a closer examination of DNA sequence of Sphinx 2.36 revealed its presence downstream of orf106 homologue. TEM images and infection assays revealed existence of phage AbDs1 in Acinetobacter sp. DS002. PMID:23867905

  15. Acinetobacter phage genome is similar to Sphinx 2.36, the circular DNA copurified with TSE infected particles.

    PubMed

    Longkumer, Toshisangba; Kamireddy, Swetha; Muthyala, Venkateswar Reddy; Akbarpasha, Shaikh; Pitchika, Gopi Krishna; Kodetham, Gopinath; Ayaluru, Murali; Siddavattam, Dayananda

    2013-01-01

    While analyzing plasmids of Acinetobacter sp. DS002 we have detected a circular DNA molecule pTS236, which upon further investigation is identified as the genome of a phage. The phage genome has shown sequence similarity to the recently discovered Sphinx 2.36 DNA sequence co-purified with the Transmissible Spongiform Encephalopathy (TSE) particles isolated from infected brain samples collected from diverse geographical regions. As in Sphinx 2.36, the phage genome also codes for three proteins. One of them codes for RepA and is shown to be involved in replication of pTS236 through rolling circle (RC) mode. The other two translationally coupled ORFs, orf106 and orf96, code for coat proteins of the phage. Although an orf96 homologue was not previously reported in Sphinx 2.36, a closer examination of DNA sequence of Sphinx 2.36 revealed its presence downstream of orf106 homologue. TEM images and infection assays revealed existence of phage AbDs1 in Acinetobacter sp. DS002. PMID:23867905

  16. Circularly permuted viral pRNA active and specific in the packaging of bacteriophage phi 29 DNA.

    PubMed

    Zhang, C; Trottier, M; Guo, P

    1995-03-10

    A viral-encoded 120-base pRNA has been shown to have an essential role in the packaging of bacteriophage phi 29 DNA. The finding that both the 5'- and 3'-termini of the pRNA are proximate and crucial for biological function (C. Zhang, C. Lee, and P. Guo, 1994, Virology, 201, 77-85) prompted investigation of the activity of circularly permuted pRNAs (cpRNA) and of the expandability and essentiality of bases extending from the termini. A 117-base pRNA with a deletion of three bases downstream of the proximal terminus was active in DNA packaging. Concatemeric DNAs containing two tandem pRNA genes separated by a short or a long loop sequence were constructed. The cpRNAs from these DNA templates were transcribed in vitro and shown to be active in phi 29 DNA packaging, with activity comparable to the parental (noncircularly permuted) pRNA, indicating that neither of the loops tested affected the activity and folding of the cpRNA. As few as four bases were sufficient to serve as a loop for the terminal 180 degree turn, and a loop as long as 27 bases did not affect the cpRNA structure and function. Eight cpRNAs were constructed to assess the effect of openings within the wild-type pRNA structure. Opening of the bulge at residue 38 did not affect cpRNA activity, but opening the bulge at residue 55 greatly reduced it. Although the sequence of the 5',3'-terminal loop was not important for the folding and activity of the cpRNA, the activities of cpRNAs with openings at individual bulges or hairpins were different, indicating that each region plays a different role in pRNA folding and function. Our results indicate that it is possible to generate active circularly permuted pRNA by assigning internal sites of the pRNA as new 3'- and 5'-termini. The creation of new variable ends makes the labeling of internal bases of the pRNA molecule possible and will facilitate the analysis of pRNA secondary and tertiary structure. PMID:7533964

  17. 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%. PMID:26792375

  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. PMID:26733971

  19. 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

  20. 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

  1. 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. PMID:21853137

  2. Novel replication-competent circular DNA molecules from healthy cattle serum and milk and multiple sclerosis-affected human brain tissue.

    PubMed

    Whitley, Corinna; Gunst, Karin; Müller, Hermann; Funk, Mathis; Zur Hausen, Harald; de Villiers, Ethel-Michele

    2014-01-01

    Epidemiological data point to the involvement of a cow milk factor in the etiology of multiple sclerosis (MS). Eleven circular DNA molecules closely related to transmissible spongiform encephalopathy (TSE)-associated isolate Sphinx 1.76 were isolated from healthy cattle serum, cow milk, and serum and brain tissue from MS patients. PMID:25169859

  3. 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. PMID:22155583

  4. Novel Replication-Competent Circular DNA Molecules from Healthy Cattle Serum and Milk and Multiple Sclerosis-Affected Human Brain Tissue

    PubMed Central

    Whitley, Corinna; Gunst, Karin; Müller, Hermann; Funk, Mathis; zur Hausen, Harald

    2014-01-01

    Epidemiological data point to the involvement of a cow milk factor in the etiology of multiple sclerosis (MS). Eleven circular DNA molecules closely related to transmissible spongiform encephalopathy (TSE)-associated isolate Sphinx 1.76 were isolated from healthy cattle serum, cow milk, and serum and brain tissue from MS patients. PMID:25169859

  5. Elimination of extrachromosomally amplified MYC genes from human tumor cells reduces their tumorigenicity.

    PubMed Central

    Von Hoff, D D; McGill, J R; Forseth, B J; Davidson, K K; Bradley, T P; Van Devanter, D R; Wahl, G M

    1992-01-01

    Oncogene amplification has been observed in a broad spectrum of human tumors and has been associated with a poor prognosis for patients with several different types of malignancies. Importantly, at biopsy, the amplified genes localize to acentric extrachromosomal elements such as double-minute chromosomes (DMs) in the vast majority of cases. We show here that treatment of several human tumor cell lines with low concentrations of hydroxyurea accelerates the loss of their extrachromosomally amplified oncogenes. The decreases in MYC copy number in a human tumor cell line correlated with a dramatic reduction in cloning efficiency in soft agar and tumorigenicity in nude mice. No effect on gene copy number or tumorigenicity was observed for a closely related cell line containing the same number of chromosomally amplified MYC genes. One step involved in the accelerated loss of extrachromosomal elements is shown to involve their preferential entrapment of DMs within micronuclei. The data suggest that agents that accelerate the loss of extrachromosomally amplified genes could provide valuable tools for moderating the growth of a large number of human neoplasms. Images PMID:1518843

  6. 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. PMID:26404155

  7. Diverse small circular single-stranded DNA viruses identified in a freshwater pond on the McMurdo Ice Shelf (Antarctica).

    PubMed

    Zawar-Reza, Peyman; Argüello-Astorga, Gerardo R; Kraberger, Simona; Julian, Laurel; Stainton, Daisy; Broady, Paul A; Varsani, Arvind

    2014-08-01

    Antarctica has some of the harshest environmental conditions for existence of life on Earth. In this pilot study we recovered eight diverse circular single-stranded DNA (ssDNA) viral genome sequences (1904-3120 nts) from benthic mats dominated by filamentous cyanobacteria in a freshwater pond on the McMurdo Ice Shelf sampled in 1988. All genomes contain two to three major open reading frames (ORFs) that are uni- or bi-directionally transcribed and all have an ORF encoding a replication-associated protein (Rep). In one genome, the second ORF has similarity to a capsid protein (CP) of Nepavirus which is most closely related to geminiviruses. Additionally, all genomes have two intergenic regions that contain putative stem loop structures, six genomes have NANTATTAC as the nonanucleotide motif, while one has CCTTATTAC, and another has a non-canonical stem loop. In the large intergenic region, we identified iterative sequences flanking the putative stem-loop elements which are a hallmark of most circular ssDNA viruses encoding rolling circle replication (RCR) initiators of the HUH endonuclease superfamily. The Reps encoded by ssDNA viral genomes recovered in this study shared <38% pairwise identity to all other Reps of known ssDNA viruses. A previous study on Lake Limnopolar (Livingston Island, South Shetland Islands), using next-generation sequencing identified circular ssDNA viruses and their putative Reps share <35% pairwise identity to those from the viral genomes removed in this study. It is evident from our pilot study that the global diversity of ssDNA viruses is grossly underestimated and there is limited knowledge on ssDNA viruses in Antarctica. PMID:24859088

  8. 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. PMID:23116593

  9. 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

  10. 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.

  11. 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. PMID:27506343

  12. 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

  13. 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

  14. Chromosomal and extrachromosomal deoxyribonucleic acid from four bacterial endosymbionts derived from stock 51 of Paramecium tetraurelia.

    PubMed Central

    Dilts, J A

    1977-01-01

    Four variant lines of stock 51 kappa (Paramecium tetraurelia) were screened for the presence of covalently closed circular (CCC) deoxyribonucleic acid (DNA). Stock 51m43 kappa, a nonkiller resistant to 51 killing, contained four classes of CCC DNA: 2.9 X 10(7), 9.7 X 10(7), and 11.8 X 10(7) daltons. The buoyant densities of 51m43 kappa chromosomal and CCC DNA were 1.700 and 1.698 g/cm3, respectively. Stock 51m43 pi, a sensitive nonkiller, contained two CCC species: 0.3 X 10(7) and 4.4 X 10(7) daltons. The buoyant densities of both the chromosomal and CCC DNA were 1.694 to 1.695 g/cm3. Three sizes of CCC DNA were found in 51m1 pi: 0.3 X 10(7), 2.3 X 10(7), and 4.5 X 10(7) daltons. The buoyant densities of both the chromosoaml DNA and the CC DNA were 1.694 to 1.695 g/cm3. It is not known whether 51m1 kappa, a sensitive spinner killer, contains CCC DNA. The buoyant density of its chromosomal DNA was 1.703 g/cm3. Of the four variant lines, only 51m43 kappa appears to be a mutant of 51 kappa. The chromosomal and CCC DNAs of 51m43 kappa have the same buoyant densities as those of 51 kappa; in addition 51m43 kappa contain a CCC molecule the same size as that found in 51 kappa (2.8 x 10(7) daltons). The three other lines are probably bacterial species that are distinct from 51 kappa and which, at one time, were co-inhabitants with 51 kappa in stock 51 paramecia. PMID:838691

  15. Association of Hepatitis B Virus Covalently Closed Circular DNA and Human APOBEC3B in Hepatitis B Virus-Related Hepatocellular Carcinoma.

    PubMed

    Luo, Xuan; Huang, Yao; Chen, Yanmeng; Tu, Zeng; Hu, Jieli; Tavis, John E; Huang, Ailong; Hu, Yuan

    2016-01-01

    Chronic Hepatitis B Virus (HBV) infections can progresses to liver cirrhosis and hepatocellular carcinoma (HCC). The HBV covalently-closed circular DNA cccDNA is a key to HBV persistence, and its degradation can be induced by the cellular deaminase APOBEC3. This study aimed to measure the distribution of intrahepatic cccDNA levels and evaluate the association between levels of cccDNA and APOBEC3 in HCC patients. Among 49 HCC patients, 35 matched cancerous and contiguous noncancerous liver tissues had detectable cccDNA, and the median intrahepatic cccDNA in the cancerous tissues (CT) was significantly lower than in the contiguous noncancerous tissues (CNCT) (p = 0.0033). RCA (rolling circle amplification), followed by 3D-PCR identified positive amplification in 27 matched HCC patients. Sequence analysis indicated G to A mutations accumulated to higher levels in CT samples compared to CNCT samples, and the dinucleotide context showed preferred editing in the GpA context. Among 7 APOBEC3 genes, APOBEC3B was the only one up-regulated in cancerous tissues both at the transcriptional and protein levels (p < 0.05). This implies APOBEC3B may contribute to cccDNA editing and subsequent degradation in cancerous tissues. PMID:27310677

  16. Association of Hepatitis B Virus Covalently Closed Circular DNA and Human APOBEC3B in Hepatitis B Virus-Related Hepatocellular Carcinoma

    PubMed Central

    Chen, Yanmeng; Tu, Zeng; Hu, Jieli; Tavis, John E.; Huang, Ailong; Hu, Yuan

    2016-01-01

    Chronic Hepatitis B Virus (HBV) infections can progresses to liver cirrhosis and hepatocellular carcinoma (HCC). The HBV covalently-closed circular DNA cccDNA is a key to HBV persistence, and its degradation can be induced by the cellular deaminase APOBEC3. This study aimed to measure the distribution of intrahepatic cccDNA levels and evaluate the association between levels of cccDNA and APOBEC3 in HCC patients. Among 49 HCC patients, 35 matched cancerous and contiguous noncancerous liver tissues had detectable cccDNA, and the median intrahepatic cccDNA in the cancerous tissues (CT) was significantly lower than in the contiguous noncancerous tissues (CNCT) (p = 0.0033). RCA (rolling circle amplification), followed by 3D-PCR identified positive amplification in 27 matched HCC patients. Sequence analysis indicated G to A mutations accumulated to higher levels in CT samples compared to CNCT samples, and the dinucleotide context showed preferred editing in the GpA context. Among 7 APOBEC3 genes, APOBEC3B was the only one up-regulated in cancerous tissues both at the transcriptional and protein levels (p < 0.05). This implies APOBEC3B may contribute to cccDNA editing and subsequent degradation in cancerous tissues. PMID:27310677

  17. 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.

  18. 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

  19. One-step cloning and chromosomal integration of DNA.

    PubMed

    St-Pierre, François; Cui, Lun; Priest, David G; Endy, Drew; Dodd, Ian B; Shearwin, Keith E

    2013-09-20

    We describe "clonetegration", a method for integrating DNA into prokaryotic chromosomes that approaches the simplicity of cloning DNA within extrachromosomal vectors. Compared to existing techniques, clonetegration drastically decreases the time and effort needed for integration of single or multiple DNA fragments. Additionally, clonetegration facilitates cloning and expression of genetic elements that are impossible to propagate within typical multicopy plasmids. PMID:24050148

  20. Sub-femtomolar electrochemical detection of DNA using surface circular strand-replacement polymerization and gold nanoparticle catalyzed silver deposition for signal amplification.

    PubMed

    Gao, Fenglei; Zhu, Zhu; Lei, Jianping; Geng, Yao; Ju, Huangxian

    2013-01-15

    A highly sensitive method was developed for detection of target DNA. This method combined circular strand-displacement polymerization (CSRP) with silver enhancement to achieve dual signal amplification. After molecular beacon (MB) hybridized with target DNA, the reporter gold nanoparticle (Au NPs) was attached to an electrode surface by hybridization between Au NP labeled primer and stem part of the MB to initiate a polymerization of DNA strand, which led to the release of target and another polymerization cycle. Thus the CSRP produced the multiplication of target-related reporter Au NPs on the surface. The Au NPs then catalyzed silver deposition for subsequent stripping analysis of silver. The dual signal amplification offered a dramatic enhancement of the stripping response. This signal could discriminate perfect matched target DNA from 1-base mismatch DNA. The dynamic range of the sequence-specific DNA detection was from 10(-16) to 10(-12)mol L(-1) with a detection limit down to sub-femtomolar level. This proposed method exhibited an efficient amplification performance, and would open new opportunities for sensitive detection of other biorecognition events. PMID:22883748

  1. Metagenomic identification of a nodavirus and a circular ssDNA virus in semi-purified viral nucleic acids from the hepatopancreas of healthy Farfantepenaeus duorarum shrimp.

    PubMed

    Ng, Terry Fei; Alavandi, Shankar; Varsani, Arvind; Burghart, Scott; Breitbart, Mya

    2013-09-01

    Fisheries and aquaculture are impacted sporadically by newly emerged viral diseases. In most cases, searches for a causative pathogen only occur after a serious disease has emerged. As random shotgun sequencing (metagenomics) offers opportunities to identify novel viruses preemptively, the method was tested on nucleic acids extracted from the hepatopancreas of 12 healthy northern pink shrimp Farfantepenaeus duorarum captured from the Gulf of Mexico. Among the sequences, a nodavirus (Farfantepenaeus duorarum nodavirus, FdNV) and a virus with similarities to circoviruses and cycloviruses that possess circular single-stranded DNA (ssDNA) genomes, were identified. The FdNV genome sequence was most closely related phylogenetically to nodaviruses causing white tail disease in Macrobrachium rosenbergii and muscle necrosis disease in Litopenaeus vannamei. While the circular ssDNA virus represents the third to be detected in association with a marine invertebrate, transmission trials are needed to confirm its infectivity for F. duorarum. This study highlights the potential for using metagenomic approaches in fisheries and aquaculture industries to identify new potential pathogens in asymptomatic marine invertebrates, uncharacterized pathogens causing a new disease, or multiple pathogens associated with disease syndromes. PMID:23999707

  2. 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

  3. Kaposi's sarcoma-associated herpesvirus LANA recruits the DNA polymerase clamp loader to mediate efficient replication and virus persistence

    PubMed Central

    Sun, Qiming; Tsurimoto, Toshiki; Juillard, Franceline; Li, Lin; Li, Shijun; De León Vázquez, Erika; Chen, She; Kaye, Kenneth

    2014-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) latently infects tumor cells and persists as a multiple-copy, extrachromosomal, circular episome. To persist, the viral genome must replicate with each cell cycle. The KSHV latency-associated nuclear antigen (LANA) mediates viral DNA replication and persistence, but little is known regarding the underlying mechanisms. We find that LANA recruits replication factor C (RFC), the DNA polymerase clamp [proliferating cell nuclear antigen (PCNA)] loader, to drive DNA replication efficiently. Mutated LANA lacking RFC interaction was deficient for LANA-mediated DNA replication and episome persistence. RFC depletion had a negative impact on LANA’s ability to replicate and maintain viral DNA in cells containing artificial KSHV episomes or in infected cells, leading to loss of virus. LANA substantially increased PCNA loading onto DNA in vitro and recruited RFC and PCNA to KSHV DNA in cells. These findings suggest that PCNA loading is a rate-limiting step in DNA replication that is incompatible with viral survival. LANA enhancement of PCNA loading permits efficient virus replication and persistence, revealing a previously unidentified mechanism for KSHV latency. PMID:25071216

  4. Single molecule analysis of DNA wrapping and looping by a circular 14mer wheel of the bacteriophage 186 CI repressor.

    PubMed

    Wang, Haowei; Dodd, Ian B; Dunlap, David D; Shearwin, Keith E; Finzi, Laura

    2013-06-01

    The lytic-lysogenic decision in bacteriophage 186 is governed by the 186 CI repressor protein in a unique way. The 186 CI is proposed to form a wheel-like oligomer that can mediate either wrapped or looped nucleoprotein complexes to provide the cooperative and competitive interactions needed for regulation. Although consistent with structural, biochemical and gene expression data, many aspects of this model are based on inference. Here, we use atomic force microscopy (AFM) to reveal the various predicted wrapped and looped species, and new ones, for CI regulation of lytic and lysogenic transcription. Automated AFM analysis showed CI particles of the predicted dimensions on the DNA, with CI multimerization favoured by DNA binding. Measurement of the length of the wrapped DNA segments indicated that CI may move on the DNA, wrapping or releasing DNA on either side of the wheel. Tethered particle motion experiments were consistent with wrapping and looping of DNA by CI in solution, where in contrast to λ repressor, the looped species were exceptionally stable. The CI regulatory system provides an intriguing comparison with that of nucleosomes, which share the ability to wrap and release similar sized segments of DNA. PMID:23620280

  5. Temperate membrane-containing halophilic archaeal virus SNJ1 has a circular dsDNA genome identical to that of plasmid pHH205.

    PubMed

    Zhang, Ziqian; Liu, Ying; Wang, Shuai; Yang, Di; Cheng, Yichen; Hu, Jiani; Chen, Jin; Mei, Yunjun; Shen, Ping; Bamford, Dennis H; Chen, Xiangdong

    2012-12-20

    A temperate haloarchaeal virus, SNJ1, was induced from the lysogenic host, Natrinema sp. J7-1, with mitomycin C, and the virus produced plaques on lawns of Natrinema sp. J7-2. Optimization of the induction conditions allowed us to increase the titer from ~10(4) PFU/ml to ~10(11) PFU/ml. Single-step growth curves exhibited a burst size of ~100 PFU/cell. The genome of SNJ1 was observed to be a circular, double-stranded DNA (dsDNA) molecule (16,341 bp). Surprisingly, the sequence of SNJ1 was identical to that of a previously described plasmid, pHH205, indicating that this plasmid is the provirus of SNJ1. Several structural protein-encoding genes were identified in the viral genome. In addition, the comparison of putative packaging ATPase sequences from bacterial, archaeal and eukaryotic viruses, as well as the presence of lipid constituents from the host phospholipid pool, strongly suggest that SNJ1 belongs to the PRD1-type lineage of dsDNA viruses, which have an internal membrane. PMID:22784791

  6. Extrachromosomal recombination substrates recapitulate beyond 12/23 restricted VDJ recombination in nonlymphoid cells.

    PubMed

    Jung, David; Bassing, Craig H; Fugmann, Sebastian D; Cheng, Hwei-Ling; Schatz, David G; Alt, Frederick W

    2003-01-01

    V(D)J recombination occurs efficiently only between gene segments flanked by recombination signals (RSs) containing 12 and 23 base pair spacers (the 12/23 rule). A further limitation "beyond the 12/23 rule" (B12/23) exists at the TCRbeta locus and ensures Dbeta usage. Herein, we show that extrachromosomal V(D)J recombination substrates recapitulate B12/23 restriction in nonlymphoid cells. We further demonstrate that the Vbeta coding flank, the 12-RS heptamer/nonamer, and the 23-RS spacer each can significantly influence B12/23 restriction. Finally, purified core RAG1 and RAG2 proteins (together with HMG2) also reproduce B12/23 restriction in a cell-free system. Our findings indicate that B12/23 restriction of V(D)J recombination is cemented at the level of interactions between the RAG proteins and TCRbeta RS sequences. PMID:12530976

  7. Oxidative stress and starvation in Dinoroseobacter shibae: the role of extrachromosomal elements

    PubMed Central

    Soora, Maya; Tomasch, Jürgen; Wang, Hui; Michael, Victoria; Petersen, Jörn; Engelen, Bert; Wagner-Döbler, Irene; Cypionka, Heribert

    2015-01-01

    Aerobic anoxygenic phototrophic bacteria (AAP) are abundant in the photic zone of the marine environment. Dinoroseobacter shibae, a representative of the Roseobacter group, converts light into additional energy that enhances its survival especially under starvation. However, light exposure results in the production of cytotoxic reactive oxygen species in AAPs. Here we investigated the response of D. shibae to starvation and oxidative stress, focusing on the role of extrachromosomal elements (ECRs). D. shibae possessing five ECRs (three plasmids and two chromids) was starved for 4 weeks either in the dark or under light/dark cycles and the survival was monitored. Transcriptomics showed that on the chromosome genes with a role in oxidative stress response and photosynthesis were differentially expressed during the light period. Most extrachromosomal genes in contrast showed a general loss of transcriptional activity, especially in dark-starved cells. The observed decrease of gene expression was not due to plasmid loss, as all five ECRs were maintained in the cells. Interestingly, the genes on the 72-kb chromid were the least downregulated, and one region with genes of the oxygen stress response and a light-dependent protochlorophyllide reductase of cyanobacterial origin was strongly activated under the light/dark cycle. A Δ72-kb curing mutant lost the ability to survive under starvation in a light/dark cycle demonstrating the essential role of this chromid for adaptation to starvation and oxidative stress. Our data moreover suggest that the other four ECRs of D. shibae have no vital function under the investigated conditions and therefore were transcriptionally silenced. PMID:25859246

  8. 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

  9. Characterization of a conserved extrachromosomal element isolated from the avian malarial parasite Plasmodium gallinaceum.

    PubMed Central

    Joseph, J T; Aldritt, S M; Unnasch, T; Puijalon, O; Wirth, D F

    1989-01-01

    We have identified a conserved, repeated, and highly transcribed DNA element from the avian malarial parasite Plasmodium gallinaceum. The element produced multiple transcripts in both zygotes and asexual blood stages of this parasite. It was found to be highly conserved in all of five malarial species tested and hybridized at reduced stringency to other members of the phylum Apicomplexa, including the genera Babesia, Eimeria, Toxoplasma, and Theileria. The copy number of the element was about 15, and it had a circularly permuted restriction map with a repeat unit length of about 6.2 kilobases. It could be separated from the main genomic DNA by using sucrose gradients and agarose gels, and it migrated separately from the recognized Plasmodium chromosomes on pulse-field gels. In the accompanying paper (S. M. Aldritt, J. T. Joseph, and D. F. Wirth, Mol. Cell. Biol. 9:3614-3620, 1989), evidence is presented that element contains the mitochondrial genes for the protein cytochrome b and a fragment of the large rRNA. We postulate that this element is an episome in the mitochondria of the obligate parasites belonging to the phylum Apicomplexa. Images PMID:2779561

  10. Detection of DNA damage induced in vivo by a cross-linking agent with a circular channel crucible oscillating viscometer.

    PubMed

    Balbi, C; Abelmoschi, M L; Roner, R; Giaretti, W; Parodi, S; Santi, L

    1985-11-01

    DNA damage induced in vivo by the cross-linking agent mitomycin C (MMC) was investigated with a new oscillating crucible viscometer. Viscosity was measured by lysing rat liver nuclei in an alkaline lysing solution (pH 12.5; 25 degrees C). In control samples the viscosity increased very slowly with time, reaching a plateau only after 10-12 h. The process was accelerated and the maximum viscosity was decreased by alkaline single-stranded breaks arising from methylation and subsequent depurination of DNA in vitro with dimethylsulphate (DMS). MMC, when given alone, had no evident effect on the time needed for reaching plateau viscosity but it induced a small increase in maximum viscosity. When MMC was given in association with DMS, the time of disentanglement remained unchanged (accelerated) but maximum viscosity was increased in a dose dependent way. We conclude that these data clearly confirm that the slow steady increase of the viscosity of control DNA with time reflects mainly the process of unwinding of the two strands. The speed of this process seems to depend only from the number of unwinding points in DNA (breaks). PMID:3935335

  11. Circular motion

    NASA Astrophysics Data System (ADS)

    Newton, Isaac; Henry, Richard Conn

    2000-07-01

    An extraordinarily simple and transparent derivation of the formula for the acceleration that occurs in uniform circular motion is presented, and is advocated for use in high school and college freshman physics textbooks.

  12. Alternative model for chromatin organization of the Saccharomyces cerevisiae chromosomal DNA plasmid TRP1 RI circle (YARp1).

    PubMed Central

    Long, C M; Brajkovich, C M; Scott, J F

    1985-01-01

    TRP1 RI circle (now designated YARp1, yeast acentric ring plasmid 1) is a 1,453-base-pair artificial plasmid composed exclusively of Saccharomyces cerevisiae chromosomal DNA. It contains both the TRP1 gene and ARS1 (a DNA sequence that permits extrachromosomal maintenance of recombinant plasmids). This high-copy-number, relatively stable plasmid was shown to be organized into nucleosomes comparable to typical yeast chromatin, containing a possible maximum of nine nucleosomes per circle. Therefore, YARp1 can be used to examine the structure of chromatin of both a chromosomally derived replicator and a functional gene. By mapping regions of micrococcal nuclease cleavage in chromatin versus purified DNA, we located the positions of protected regions on the circle with reference to six unique restriction sites. Measurements made on patterns of early digestion products indicated that a region of approximately 300 base pairs in the vicinity of ARS1 was strongly resistant to micrococcal nuclease. The remainder of the plasmid appeared to be associated with five positioned nucleosomes and two nonnucleosomal, partially protected regions on the bulk of the molecules. After similar extents of digestion, naked DNA did not exhibit an equivalent pattern, although some hypersensitive cleavage sites matched sites found in the chromatin. These results are consistent with the interpretation that the protected domains are aligned with respect to a specific site or sites on the small circular chromatin. Images PMID:3018502

  13. 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

    2016-01-01

    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. PMID:27578458

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

    PubMed Central

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

    2016-01-01

    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. PMID:27578458

  15. 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

  16. Son-Killer: A Third Extrachromosomal Factor Affecting the Sex Ratio in the Parasitoid Wasp, NASONIA (=MORMONIELLA) VITRIPENNIS

    PubMed Central

    Skinner, Samuel Way

    1985-01-01

    An extrachromosomal factor, termed son-killer (sk), affects the sex ratio in a parasitoid wasp, Nasonia (=Mormoniella ) vitripennis. The factor is maternally transmitted and alters the secondary sex ratio of an infected female through mortality of approximately 80% of the male embryos. No effect on the primary (zygotic) sex ratio is observed. Ninety-five percent of the daughters of an infected female inherit son-killer. The factor can also be transmitted contagiously when the progeny of infected and uninfected females develop simultaneously on a single host. In newly infected strains, the sex ratio effects are equivalent to those in the original. PMID:3988039

  17. Cervical keratinocytes containing stably replicating extrachromosomal HPV-16 are refractory to transformation by oncogenic H-Ras

    PubMed Central

    Berger, Kristi L.; Barriga, Felicia; Lace, Michael J.; Turek, Lubomir P.; Zamba, Gideon J.; Domann, Frederick E.; Lee, John H.; Klingelhutz, Aloysius J.

    2007-01-01

    Ras expression in human epithelial cells with integrated HPV genomes has been shown to cause tumorigenic transformation. The effects of Ras in cells representing early stage HPV-associated disease (i.e., when HPV is extrachromosomal and the oncogenes are under control of native promoters) have not been examined. Here, we used human cervical keratinocyte cell lines containing stably replicating extrachromosomal HPV-16 and present the novel finding that these cells resist transformation by oncogenic H-Ras. Ras expression consistently diminished anchorageindependent growth (AI), reduced E6 and E7 expression, and caused p53 induction in these cells. Conversely, AI was enhanced or maintained in Ras-transduced cervical cells that were immortalized with a 16E6/E7 retrovirus, and minimal effects on E6 and E7 expression were observed. Ras expression with either episomal HPV-16 or LXSN-E6/E7 was insufficient for tumorigenic growth suggesting that other events are needed for tumorigenic transformation. In conclusion, our results indicate that Ras-mediated transformation depends on the context of HPV oncogene expression and that this is an important point to address when developing HPV tumor models. PMID:16945398

  18. 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. PMID:27036064

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

    PubMed

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

    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

  20. 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

  1. Characterization of Circular RNAs.

    PubMed

    Zhang, Yang; Yang, Li; Chen, Ling-Ling

    2016-01-01

    Accumulated lines of evidence reveal that a large number of circular RNAs are produced in transcriptomes from fruit fly to mouse and human. Unlike linear RNAs shaped with 5' cap and 3' tail, circular RNAs are characterized by covalently closed loop structures without open terminals, thus requiring specific treatments for their identification and validation. Here, we describe a detailed pipeline for the characterization of circular RNAs. It has been successfully applied to the study of circular intronic RNAs derived from intron lariats (ciRNAs) and circular RNAs produced from back spliced exons (circRNAs) in human. PMID:26721494

  2. Molecular recognition principles and stationary-phase characteristics of topoisomer-selective chemoaffinity materials for chromatographic separation of circular plasmid DNA topoisomers.

    PubMed

    Mahut, Marek; Lindner, Wolfgang; Lämmerhofer, Michael

    2012-01-18

    We recently discovered the molecular recognition capability of a quinine carbamate ligand attached to silica as a powerful chemoaffinity material for the chromatographic separation of circular plasmid topoisomers of different linking numbers. In this paper we develop structure-selectivity relationship studies to figure out the essential structural features for topoisomer recognition. By varying different moieties of the original cinchonan-derived selector, it was shown that intercalation by the quinoline moiety of the ligand as assumed initially as the working hypothesis is not an essential feature for topoisomer recognition during chromatography. We found that the key elements for topoisomer selectivity are the presence of a rigid weak anion-exchange site and a H-donor site separated from each other in a defined distance by a 4-atom spacer. Additionally, incorporation of the weak anion-exchange site into a cyclic ring structure provides greater rigidity of the ligand molecule and turned out to be advantageous, if not mandatory, for (close to) baseline separation. PMID:22191385

  3. 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)

  4. 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.

  5. 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

  6. 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)

  7. A mechanism of gene amplification driven by small DNA fragments.

    PubMed

    Mukherjee, Kuntal; Storici, Francesca

    2012-01-01

    DNA amplification is a molecular process that increases the copy number of a chromosomal tract and often causes elevated expression of the amplified gene(s). Although gene amplification is frequently observed in cancer and other degenerative disorders, the molecular mechanisms involved in the process of DNA copy number increase remain largely unknown. We hypothesized that small DNA fragments could be the trigger of DNA amplification events. Following our findings that small fragments of DNA in the form of DNA oligonucleotides can be highly recombinogenic, we have developed a system in the yeast Saccharomyces cerevisiae to capture events of chromosomal DNA amplification initiated by small DNA fragments. Here we demonstrate that small DNAs can amplify a chromosomal region, generating either tandem duplications or acentric extrachromosomal DNA circles. Small fragment-driven DNA amplification (SFDA) occurs with a frequency that increases with the length of homology between the small DNAs and the target chromosomal regions. SFDA events are triggered even by small single-stranded molecules with as little as 20-nt homology with the genomic target. A double-strand break (DSB) external to the chromosomal amplicon region stimulates the amplification event up to a factor of 20 and favors formation of extrachromosomal circles. SFDA is dependent on Rad52 and Rad59, partially dependent on Rad1, Rad10, and Pol32, and independent of Rad51, suggesting a single-strand annealing mechanism. Our results reveal a novel molecular model for gene amplification, in which small DNA fragments drive DNA amplification and define the boundaries of the amplicon region. As DNA fragments are frequently found both inside cells and in the extracellular environment, such as the serum of patients with cancer or other degenerative disorders, we propose that SFDA may be a common mechanism for DNA amplification in cancer cells, as well as a more general cause of DNA copy number variation in nature. PMID

  8. 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…

  9. Tunable circular patch antennas

    NASA Astrophysics Data System (ADS)

    Lan, G.-L.; Sengupta, D. L.

    1985-10-01

    A method to control the resonant or operating frequencies of circular patch antennas has been investigated experimentally and theoretically. It consists of the placement of passive metallic or tuning posts at approximate locations within the input region of the antenna. Comparison of measured and analytical results seems to establish the validity of a theoretical model proposed to determine the input performance of such circular patch antennas.

  10. 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.

  11. Compact waveguide circular polarizer

    DOEpatents

    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.

  12. 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 Code), which…

  13. 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…

  14. 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.

  15. Compiling Multicopy Single-Stranded DNA Sequences from Bacterial Genome Sequences

    PubMed Central

    Yoo, Wonseok; Lim, Dongbin

    2016-01-01

    A retron is a bacterial retroelement that encodes an RNA gene and a reverse transcriptase (RT). The former, once transcribed, works as a template primer for reverse transcription by the latter. The resulting DNA is covalently linked to the upstream part of the RNA; this chimera is called multicopy single-stranded DNA (msDNA), which is extrachromosomal DNA found in many bacterial species. Based on the conserved features in the eight known msDNA sequences, we developed a detection method and applied it to scan National Center for Biotechnology Information (NCBI) RefSeq bacterial genome sequences. Among 16,844 bacterial sequences possessing a retron-type RT domain, we identified 48 unique types of msDNA. Currently, the biological role of msDNA is not well understood. Our work will be a useful tool in studying the distribution, evolution, and physiological role of msDNA. PMID:27103888

  16. Switchable circular beam deflectors

    NASA Astrophysics Data System (ADS)

    Shang, Xiaobing; Joshi, Pankaj; Tan, Jin-Yi; De Smet, Jelle; Cuypers, Dieter; Baghdasaryan, Tigran; Vervaeke, Michael; Thienpont, Hugo; De Smet, Herbert

    2016-04-01

    In this work, we report two types of electrically tunable photonic devices with circularly symmetric polarization independent beam steering performance (beam condensing resp. beam broadening). The devices consist of circular micro grating structures combined with nematic liquid crystal (LC) layers with anti-parallel alignment. A single beam deflector converts a polarized and monochromatic green laser beam (λ =543.5 nm) into a diffraction pattern, with the peak intensity appearing at the third order when 0~{{V}\\text{pp}} is applied and at the zeroth order (no deflection) for voltages above 30~{{V}\\text{pp}} . Depending on the shape of the grating structure (non-inverted or inverted), the deflection is inwards or outwards. Both grating types can be made starting from the same diamond-tooled master mold. A polarized white light beam is symmetrically condensed resp. broadened over 2° in the off state and is passed through unchanged in the on state. By stacking two such devices with mutually orthogonal LC alignment layers, polarization independent switchable circular beam deflectors are realized with a high transmittance (>80%), and with the same beam steering performance as the polarization dependent single devices.

  17. 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. PMID:24065721

  18. Sources of varieties and quality of circular Fructus Ligustri Lucidi.

    PubMed

    Li, Yan-Peng; Wang, Si-Jia; Zang, Yi-Mei; Hu, Zhong-Sheng; Liu, Chun-Sheng

    2016-03-01

    This study aimed to trace sources and quantitatively analyze the specnuezhenide content of circular Fructus Ligustri Lucidi for clinical use. Different specifications of Fructus Ligustri Lucidi were identified using DNA barcoding technology and the specnuezhenide content was analyzed by High Performance Liquid Chromatography (HPLC). The ITS sequence of circular Fructus Ligustri Lucidi was identical to that of standard privet, which was determined through botanical identification. ITS sequence similarity between circular Fructus Ligustri Lucidi and Fructus Ligustri Lucidi which was registered in NCBI ranged from 99.5% to 100%. The sequences of circular and other Fructus Ligustri Lucidi were clustered in a Neighbor-Joining tree with bootstrap value of 95, and these sequences could be distinguished from adulterants. Conforming to pharmacopoeia standard, the average specnuezhenide content of circular Fructus Ligustri Lucidi was higher than that of chicken waist Fructus Ligustri Lucidi. Circular Fructus Ligustri Lucidi derived from Ligustrum lucidum Ait. and the specnuezhenide content was higher in circular Fructus Ligustri Lucidi than that in chicken waist Fructus Ligustri Lucidi. PMID:27025372

  19. The Influence of Copy-Number of Targeted Extrachromosomal Genetic Elements on the Outcome of CRISPR-Cas Defense

    PubMed Central

    Severinov, Konstantin; Ispolatov, Iaroslav; Semenova, Ekaterina

    2016-01-01

    Prokaryotic type I CRISPR-Cas systems respond to the presence of mobile genetic elements such as plasmids and phages in two different ways. CRISPR interference efficiently destroys foreign DNA harboring protospacers fully matching CRISPR RNA spacers. In contrast, even a single mismatch between a spacer and a protospacer can render CRISPR interference ineffective but causes primed adaptation—efficient and specific acquisition of additional spacers from foreign DNA into the CRISPR array of the host. It has been proposed that the interference and primed adaptation pathways are mediated by structurally different complexes formed by the effector Cascade complex on matching and mismatched protospacers. Here, we present experimental evidence and present a simple mathematical model that shows that when plasmid copy number maintenance/phage genome replication is taken into account, the two apparently different outcomes of the CRISPR-Cas response can be accounted for by just one kind of effector complex on both targets. The results underscore the importance of consideration of targeted genome biology when considering consequences of CRISPR-Cas systems action.

  20. Circular RNA oligonucleotides. Synthesis, nucleic acid binding properties, and a comparison with circular DNAs.

    PubMed Central

    Wang, S; Kool, E T

    1994-01-01

    We report the synthesis and nucleic acid binding properties of two cyclic RNA oligonucleotides designed to bind single-stranded nucleic acids by pyr.pur.pyr-type triple helix formation. The circular RNAs are 34 nucleotides in size and were cyclized using a template-directed nonenzymatic ligation. To ensure isomeric 3'-5' purity in the ligation reaction, one nucleotide at the ligation site is a 2'-deoxyribose. One circle (1) is complementary to the sequence 5'-A12, and the second (2) is complementary to 5'-AAGAAAGAAAAG. Results of thermal denaturation experiments and mixing studies show that both circles bind complementary single-stranded DNA or RNA substrates by triple helix formation, in which two domains in a pyrimidine-rich circle sandwich a central purine-rich substrate. The affinities of these circles with their purine complements are much higher than the affinities of either the linear precursors or simple Watson-Crick DNA complements. For example, circle 1 binds rA12 (pH 7.0, 10 mM MgCl2, 100 mM NaCl) with a Tm of 48 degrees C and a Kd (37 degrees C) of 4.1 x 10(-9) M, while the linear precursor of the circle binds with a Tm of 34 degrees C and a Kd of 1.2 x 10(-6) M. The complexes of circle 2 are pH-dependent, as expected for triple helical complexes involving C(+)G.C triads, and mixing plots for both circles reveal one-to-one stoichiometry of binding either to RNA or DNA substrates. Comparison of circular RNAs with previously synthesized circular DNA oligonucleotides of the same sequence reveals similar behavior in the binding of DNA, but strikingly different behavior in the binding of RNA. The cyclic DNAs show high DNA-binding selectivity, giving relatively weaker duplex-type binding with complementary RNAs. The relative order of thermodynamic stability for the four types of triplex studied here is found to be DDD >> RRR > RDR >> DRD. The results are discussed in the context of recent reports of strong triplex dependence on RNA versus DNA backbones

  1. Circular RNA oligonucleotides. Synthesis, nucleic acid binding properties, and a comparison with circular DNAs.

    PubMed

    Wang, S; Kool, E T

    1994-06-25

    We report the synthesis and nucleic acid binding properties of two cyclic RNA oligonucleotides designed to bind single-stranded nucleic acids by pyr.pur.pyr-type triple helix formation. The circular RNAs are 34 nucleotides in size and were cyclized using a template-directed nonenzymatic ligation. To ensure isomeric 3'-5' purity in the ligation reaction, one nucleotide at the ligation site is a 2'-deoxyribose. One circle (1) is complementary to the sequence 5'-A12, and the second (2) is complementary to 5'-AAGAAAGAAAAG. Results of thermal denaturation experiments and mixing studies show that both circles bind complementary single-stranded DNA or RNA substrates by triple helix formation, in which two domains in a pyrimidine-rich circle sandwich a central purine-rich substrate. The affinities of these circles with their purine complements are much higher than the affinities of either the linear precursors or simple Watson-Crick DNA complements. For example, circle 1 binds rA12 (pH 7.0, 10 mM MgCl2, 100 mM NaCl) with a Tm of 48 degrees C and a Kd (37 degrees C) of 4.1 x 10(-9) M, while the linear precursor of the circle binds with a Tm of 34 degrees C and a Kd of 1.2 x 10(-6) M. The complexes of circle 2 are pH-dependent, as expected for triple helical complexes involving C(+)G.C triads, and mixing plots for both circles reveal one-to-one stoichiometry of binding either to RNA or DNA substrates. Comparison of circular RNAs with previously synthesized circular DNA oligonucleotides of the same sequence reveals similar behavior in the binding of DNA, but strikingly different behavior in the binding of RNA. The cyclic DNAs show high DNA-binding selectivity, giving relatively weaker duplex-type binding with complementary RNAs. The relative order of thermodynamic stability for the four types of triplex studied here is found to be DDD > RRR > RDR > DRD. The results are discussed in the context of recent reports of strong triplex dependence on RNA versus DNA backbones. Triplex

  2. DNA Knots: Theory and Experiments

    NASA Astrophysics Data System (ADS)

    Sumners, D. W.

    Cellular DNA is a long, thread-like molecule with remarkably complex topology. Enzymes that manipulate the geometry and topology of cellular DNA perform many vital cellular processes (including segregation of daughter chromosomes, gene regulation, DNA repair, and generation of antibody diversity). Some enzymes pass DNA through itself via enzyme-bridged transient breaks in the DNA; other enzymes break the DNA apart and reconnect it to different ends. In the topological approach to enzymology, circular DNA is incubated with an enzyme, producing an enzyme signature in the form of DNA knots and links. By observing the changes in DNA geometry (supercoiling) and topology (knotting and linking) due to enzyme action, the enzyme binding and mechanism can often be characterized. This paper will discuss some personal research history, and the tangle model for the analysis of site-specific recombination experiments on circular DNA.

  3. 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.

  4. Children's Sensitivity to Circular Explanations

    ERIC Educational Resources Information Center

    Baum, Laura A.; Danovitch, Judith H.; Keil, Frank C.

    2008-01-01

    The ability to evaluate the quality of explanations is an essential part of children's intellectual growth. Explanations can be faulty in structural ways such as when they are circular. A circular explanation reiterates the question as if it were an explanation rather than providing any new information. Two experiments (N=77) examined children's…

  5. Design of a titering assay for lentiviral vectors utilizing direct extraction of DNA from transduced cells in microtiter plates

    PubMed Central

    Murphy, Michele E; Vin, Chintan D; Slough, Megan M; Gombotz, Wayne R; Kelley-Clarke, Brenna

    2016-01-01

    Using lentiviral vector products in clinical applications requires an accurate method for measuring transduction titer. For vectors lacking a marker gene, quantitative polymerase chain reaction is used to evaluate the number of vector DNA copies in transduced target cells, from which a transduction titer is calculated. Immune Design previously described an integration-deficient lentiviral vector pseudotyped with a modified Sindbis virus envelope for use in cancer immunotherapy (VP02, of the ZVex platform). Standard protocols for titering integration-competent lentiviral vectors employ commercial spin columns to purify vector DNA from transduced cells, but such columns are not optimized for isolation of extrachromosomal (nonintegrated) DNA. Here, we describe a 96-well transduction titer assay in which DNA extraction is performed in situ in the transduction plate, yielding quantitative recovery of extrachromosomal DNA. Vector titers measured by this method were higher than when commercial spin columns were used for DNA isolation. Evaluation of the method’s specificity, linear range, and precision demonstrate that it is suitable for use as a lot release assay to support clinical trials with VP02. Finally, the method is compatible with titering both integrating and nonintegrating lentiviral vectors, suggesting that it may be used to evaluate the transduction titer for any lentiviral vector. PMID:26942209

  6. 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.

  7. 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.

  8. 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.

  9. Localization of a bidirectional DNA replication origin in the native locus and in episomally amplified murine adenosine deaminase loci.

    PubMed Central

    Carroll, S M; DeRose, M L; Kolman, J L; Nonet, G H; Kelly, R E; Wahl, G M

    1993-01-01

    Gene amplification is frequently mediated by the initial production of acentric, autonomously replicating extrachromosomal elements. The 4,000 extrachromosomal copies of the mouse adenosine deaminase (ADA) amplicon in B-1/50 cells initiate their replication remarkably synchronously in early S phase and at approximately the same time as the single-copy chromosomal locus from which they were derived. The abundance of ADA sequences and favorable replication timing characteristics in this system led us to determine whether DNA replication initiates in ADA episomes within a preferred region and whether this region is the same as that used at the corresponding chromosomal locus prior to amplification. This study reports the detection and localization of a discrete set of DNA fragments in the ADA amplicon which label soon after release of synchronized B-1/50 cells into S phase. A switch in template strand complementarity of Okazaki fragments, indicative of the initiation of bidirectional DNA replication, was found to lie within the same region. This putative replication origin is located approximately 28.5 kbp upstream of the 5' end of the ADA gene. The same region initiated DNA replication in the single-copy ADA locus of the parental cells. These analyses provide the first evidence that the replication of episomal intermediates involved in gene amplification initiates within a preferred region and that the same region is used to initiate DNA synthesis within the native locus. Images PMID:8474455

  10. 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.

  11. Circularly-Polarized Microstrip Antenna

    NASA Technical Reports Server (NTRS)

    Stanton, P. H.

    1985-01-01

    Microstrip construction compact for mobile applications. Circularly polarized microstrip antenna made of concentric cylindrical layers of conductive and dielectric materials. Coaxial cable feedlines connected to horizontal and vertical subelements from inside. Vertical subelement acts as ground for horizontal subelement.

  12. Circularization time of binary galaxies

    NASA Astrophysics Data System (ADS)

    Junqueira, S.; de Freitas Pacheco, J. A.

    1994-11-01

    We report the results of numerical experiments performed to study the orbital circularization time of binary galaxies. We find that the time scale is quite long (larger than the Hubble time), confirming earlier calculations. The results depend on the initial conditions. From our simulations we obtained a fitting formula for the circularization time as a function of the initial orbital parameters like the pericentric distance, mass ratio, and eccentricity.

  13. Circular polarization interferometry: circularly polarized modes of cholesteric liquid crystals.

    PubMed

    Sanchez-Castillo, A; Eslami, S; Giesselmann, F; Fischer, P

    2014-12-15

    We describe a novel polarization interferometer which permits the determination of the refractive indices for circularly-polarized light. It is based on a Jamin-Lebedeff interferometer, modified with waveplates, and permits us to experimentally determine the refractive indices nL and nR of the respectively left- and right-circularly polarized modes in a cholesteric liquid crystal. Whereas optical rotation measurements only determine the circular birefringence, i.e. the difference (nL - nR), the interferometer also permits the determination of their absolute values. We report refractive indices of a cholesteric liquid crystal in the region of selective (Bragg) reflection as a function of temperature. PMID:25607071

  14. Tiny telomere DNA

    PubMed Central

    Ren, Jinsong; Qu, Xiaogang; Trent, John O.; Chaires, Jonathan B.

    2002-01-01

    We describe the design, synthesis and biophysical characterization of a novel DNA construct in which a folded quadruplex structure is joined to a standard double helix. Circular dichroism, gel electrophoresis, three-dimensional UV melting and differential scanning calorimetry were all used to characterize the structure. Rigorous molecular dynamics simulations were used to build a plausible atomic-level structural model of the DNA construct. This novel DNA construct provides a model for the duplex–quadruplex junction region at the end of chromosomal DNA and offers a system for the study of structure-selective ligand binding. PMID:12034817

  15. Generalized bi-circular projections

    NASA Astrophysics Data System (ADS)

    Lin, Pei-Kee

    2008-04-01

    Recall that a projection P on a complex Banach space X is a generalized bi-circular projection if P+[lambda](I-P) is a (surjective) isometry for some [lambda] such that [lambda]=1 and [lambda][not equal to]1. It is easy to see that every hermitian projection is generalized bi-circular. A generalized bi-circular projection is said to be nontrivial if it is not hermitian. Botelho and Jamison showed that a projection P on C([0,1]) is a nontrivial generalized bi-circular projection if and only if P-(I-P) is a surjective isometry. In this article, we prove that if P is a projection such that P+[lambda](I-P) is a (surjective) isometry for some [lambda], then either P is hermitian or [lambda] is an nth unit root of unity. We also show that for any nth unit root [lambda] of unity, there are a complex Banach space X and a nontrivial generalized bi-circular projection P on X such that P+[lambda](I-P) is an isometry.

  16. 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. PMID:25008961

  17. Circular birefringence of banded spherulites.

    PubMed

    Cui, Xiaoyan; Shtukenberg, Alexander G; Freudenthal, John; Nichols, Shane; Kahr, Bart

    2014-04-01

    Crystal optical properties of banded spherulites of 21 different compounds--molecular crystals, polymers, and minerals--with helically twisted fibers were analyzed with Mueller matrix polarimetry. The well-established radial oscillations in linear birefringence of many polycrystalline ensembles is accompanied by oscillations in circular birefringence that cannot be explained by the natural optical activity of corresponding compounds, some of which are centrosymmetric in the crystalline state. The circular birefringence is shown to be a consequence of misoriented, overlapping anisotropic lamellae, a kind of optical activity associated with the mesoscale stereochemistry of the refracting components. Lamellae splay as a consequence of space constraints related to simultaneous twisting of anisometric lamellae. This mechanism is supported by quantitative simulations of circular birefringence arising from crystallite twisting and splaying under confinement. PMID:24625095

  18. Stable expression and replication of hepatitis B virus genome in an integrated state in a human hepatoma cell line transfected with the cloned viral DNA

    SciTech Connect

    Tsurimoto, T.; Fujiyama, A.; Matsubara, K.

    1987-01-01

    A human hepatocellular carcinoma cell line (Huh6-c15) was transfected with a recombinant DNA molecule that consists of tandemly arranged hepatitis B virus (HBV) genome and a neomycin-resistant gene. One clone resistant to G-418 produces and releases surface antigen and e antigen into medium at a high level and accumulates core particles intracellularly. This clone has a chromosomally integrated set of the original recombinant DNA and produces a 3.5-kilobase transcript corresponding to the pregenome RNA as well as HBV DNAs in an extrachromosomal form. Most of these DNAs were in single-stranded or partially double-stranded form and were packaged in the intracellular core particles. In the medium, particles were detected that contained HBV DNA and were morphologically indistinguishable from Dane particles. These results demonstrate that the HBV genome in an integrated state acted as a template for viral gene expression and replication. The cells were maintained for more than 6 months without losing the ability to produce the extrachromosomal HBV DNA and Dane-like particles. Thus, the cells can be used as a model system for analyses of gene expression and DNA replication of HBV in human hepatocytes.

  19. Podokinetic circular vection: characteristics and interaction with optokinetic circular vection.

    PubMed

    Becker, W; Kliegl, K; Kassubek, J; Jürgens, R

    2016-07-01

    Stabilising horizontal body orientation in space without sight on a rotating platform by holding to a stationary structure and circular 'treadmill' stepping in the opposite direction can elicit an illusion of self-turning in space (Bles and Kapteyn in Agressologie 18:325-328, 1977). Because this illusion is analogous to the well-known illusion of optokinetic circular vection (oCV), we call it 'podokinetic circular vection' (pCV) here. Previous studies using eccentric stepping on a path tangential to the rotation found that pCV was always contraversive relative to platform rotation. In contrast, when our subjects stepped at the centre of rotation about their vertical axis, we observed an inverted, ipsiversive pCV as a reproducible trait in many of our subjects. This ipCV occurred at the same latency as the pCV of subjects reporting the actually expected contraversive direction, but had lower gain. In contrast to pCV, the nystagmus accompanying circular treadmill stepping had the same direction in all individuals (slow phase in the direction of platform motion). The direction of an individual's pCV predicted the characteristics of the CV resulting from combined opto- and podokinetic stimulation (circular treadmill stepping while viewing a pattern rotating together with the platform): in individuals with contraversive pCV, latency shortened and both gain and felt naturalness increased in comparison with pure oCV, whereas the opposite (longer latency, reduced gain and naturalness) occurred in individuals with ipCV. Taken together, the reproducibility of ipCV, the constant direction of nystagmus and the fact that pCV direction predicts the outcome of combined stimulation suggest that ipCV is an individual trait of many subjects during compensatory stepping at the centre of rotation. A hypothetical model is presented of how ipCV possibly could arise. PMID:26965438

  20. 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. PMID:26382231

  1. Nanocrystal-based electrochemiluminescence sensor for cell detection with Au nanoparticles and isothermal circular double-assisted signal amplification.

    PubMed

    Dai, Pan-Pan; Li, Jin-Yi; Yu, Tao; Xu, Jing-Juan; Chen, Hong-Yuan

    2015-08-15

    Here we have developed a sensitive cancer cell amplified detection method which combined Au NPs enhanced electrochemiluminescence (ECL) of CdS nanocrystals (NCs) film, with isothermal circular amplification reaction of polymerase. In DNA circular amplification detection system, hairpin DNA beacon/Au NPs composite modified CdS NCs film was used as an ECL emitter. Messenger DNA is hybridized with the aptamer modified on magnetic beads (MBs) to form MB-Au bioconjugates. In the presence of HL-60 cell, the aptamer would conjugate with the glycoprotein at cell surface and messenger DNA sequence would be released. The released messenger DNA sequence was then introduced into the cycle amplification system to trigger circular polymerizations. This assay allows us to determine the released messenger DNA equivalent to 10 cells and exhibits a significant specificity for HL-60 cells. PMID:25966387

  2. Comparison of the Single Molecule Dynamics of Linear and Circular DNAs in Planar Extensional Flows

    NASA Astrophysics Data System (ADS)

    Li, Yanfei; Hsiao, Kai-Wen; Brockman, Christopher; Yates, Daniel; McKenna, Gregory; Schroeder, Charles; San Francisco, Michael; Kornfield, Julie; Anderson, Rae

    2015-03-01

    Chain topology has a profound impact on the flow behaviors of single macromolecules. The absence of free ends separates circular polymers from other chain architectures, i.e., linear, star, and branched. In the present work, we study the single chain dynamics of large circular and linear DNA molecules by comparing the relaxation dynamics, steady state coil-stretch transition, and transient molecular individualism behaviors for the two types of macromolecules. To this end, large circular DNA molecules were biologically synthesized and studied in a microfluidic device that has a cross-slot geometry to develop a stagnation point extensional flow. Although the relaxation time of rings scales in the same way as for the linear analog, the circular polymers show quantitatively different behaviors in the steady state extension and qualitatively different behaviors during a transient stretch. The existence of some commonality between these two topologies is proposed. Texas Tech University John R. Bradford Endowment.

  3. Circular RNA expands its territory.

    PubMed

    Bao, Chunyang; Lyu, Dongbin; Huang, Shenglin

    2016-03-01

    Circular RNAs (circRNAs) represent a novel class of widespread non-coding RNAs in eukaryotes. They are unusually stable RNA molecules with cell type-specific expression patterns, and are predominantly present in the cytoplasm. We recently demonstrated the existence of abundant circRNAs in exosomes and suggest a potential application of exosomal circRNAs for cancer detection. PMID:27308606

  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. 17 CFR 230.253 - Offering circular.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... in boldfaced type at least as large as that used generally in the body of such offering circular: THE... COMPLETENESS OF ANY OFFERING CIRCULAR OR OTHER SELLING LITERATURE. THESE SECURITIES ARE OFFERED PURSUANT TO...

  6. Local chromatin structure of heterochromatin regulates repeatedDNA stability, nucleolus structure, and genome integrity

    SciTech Connect

    Peng, Jamy C.

    2007-05-05

    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

  7. CpG methylation patterns of human mitochondrial DNA

    PubMed Central

    Liu, Baojing; Du, Qingqing; Chen, Lu; Fu, Guangping; Li, Shujin; Fu, Lihong; Zhang, Xiaojing; Ma, Chunling; Bin, Cong

    2016-01-01

    The epigenetic modification of mitochondrial DNA (mtDNA) is still in controversy. To clarify this point, we applied the gold standard method for DNA methylation, bisulfite pyrosequencing, to examine human mtDNA methylation status. Before bisulfite conversion, BamHI was used to digest DNA to open the loop of mtDNA. The results demonstrated that the linear mtDNA had significantly higher bisulfite conversion efficiency compared with circular mtDNA. Furthermore, the methylation values obtained from linear mtDNA were significantly lower than that of circular mtDNA, which was verified by SEQUENOM MassARRAY. The above impacts of circular structure were also observed in lung DNA samples but not in saliva DNA samples. Mitochondrial genome methylation of blood samples and saliva samples from 14 unrelated individuals was detected. The detected regions covered 83 CpG sites across mtDNA including D-loop, 12 S rRNA, 16 S rRNA, ND1, COXI, ND3, ND4, ND5, CYTB. We found that the average methylation levels of nine regions were all less than 2% for both sample types. In conclusion, our findings firstly show that the circular structure of mtDNA affects bisulfite conversion efficiency, which leads to overestimation of mtDNA methylation values. CpG methylation in human mtDNA is a very rare event at most DNA regions. PMID:26996456

  8. CpG methylation patterns of human mitochondrial DNA.

    PubMed

    Liu, Baojing; Du, Qingqing; Chen, Lu; Fu, Guangping; Li, Shujin; Fu, Lihong; Zhang, Xiaojing; Ma, Chunling; Bin, Cong

    2016-01-01

    The epigenetic modification of mitochondrial DNA (mtDNA) is still in controversy. To clarify this point, we applied the gold standard method for DNA methylation, bisulfite pyrosequencing, to examine human mtDNA methylation status. Before bisulfite conversion, BamHI was used to digest DNA to open the loop of mtDNA. The results demonstrated that the linear mtDNA had significantly higher bisulfite conversion efficiency compared with circular mtDNA. Furthermore, the methylation values obtained from linear mtDNA were significantly lower than that of circular mtDNA, which was verified by SEQUENOM MassARRAY. The above impacts of circular structure were also observed in lung DNA samples but not in saliva DNA samples. Mitochondrial genome methylation of blood samples and saliva samples from 14 unrelated individuals was detected. The detected regions covered 83 CpG sites across mtDNA including D-loop, 12 S rRNA, 16 S rRNA, ND1, COXI, ND3, ND4, ND5, CYTB. We found that the average methylation levels of nine regions were all less than 2% for both sample types. In conclusion, our findings firstly show that the circular structure of mtDNA affects bisulfite conversion efficiency, which leads to overestimation of mtDNA methylation values. CpG methylation in human mtDNA is a very rare event at most DNA regions. PMID:26996456

  9. 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. PMID:12346601

  10. The circular internal hydraulic jump

    NASA Astrophysics Data System (ADS)

    Thorpe, S. A.; Kavcic, I.

    Circular hydraulic jumps are familiar in single layers. Here we report the discovery of similar jumps in two-layer flows. A thin jet of fluid impinging vertically onto a rigid horizontal plane surface submerged in a deep layer of less-dense miscible fluid spreads radially, and a near-circular internal jump forms within a few centimetres from the point of impact with the plane surface. A jump is similarly formed as a jet of relatively less-dense fluid rises to the surface of a deep layer of fluid, but it appears less stable or permanent in form. Several experiments are made to examine the case of a downward jet onto a horizontal plate, the base of a square or circular container. The inlet Reynolds numbers, Re, of the jet range from 112 to 1790. Initially jumps have an undular, laminar form with typically 2-4 stationary waves on the interface between the dense and less-dense layers but, as the depth of the dense layer beyond the jump increases, the transitions become more abrupt and turbulent, resulting in mixing between the two layers. During the transition to a turbulent regime, single and sometimes moving multiple cusps are observed around the periphery of jumps. A semi-empirical model is devised that relates the parameters of the laboratory experiment, i.e. flow rate, inlet nozzle radius, kinematic viscosity and reduced gravity, to the layer depth beyond the jump and the radius at which an undular jump occurs. The experiments imply that surface tension is not an essential ingredient in the formation of circular hydraulic jumps and demonstrate that stationary jumps can exist in stratified shear flows which can be represented as two discrete layers. No stationary circular undular jumps are found, however, in the case of a downward jet of dense fluid when the overlying, less-dense, fluid is stratified, but a stationary turbulent transition is observed. This has implications for the existence of stationary jumps in continuously stratified geophysical flows: results

  11. Exon circularization requires canonical splice signals.

    PubMed

    Starke, Stefan; Jost, Isabelle; Rossbach, Oliver; Schneider, Tim; Schreiner, Silke; Hung, Lee-Hsueh; Bindereif, Albrecht

    2015-01-01

    Circular RNAs (circRNAs), an abundant class of noncoding RNAs in higher eukaryotes, are generated from pre-mRNAs by circularization of adjacent exons. Using a set of 15 circRNAs, we demonstrated their cell-type-specific expression and circular versus linear processing in mammalian cells. Northern blot analysis combined with RNase H cleavage conclusively proved a circular configuration for two examples, LPAR1 and HIPK3. To address the circularization mechanism, we analyzed the sequence requirements using minigenes derived from natural circRNAs. Both canonical splice sites are required for circularization, although they vary in flexibility and potential use of cryptic sites. Surprisingly, we found that no specific circRNA exon sequence is necessary and that potential flanking intron structures can modulate circularization efficiency. In combination with splice inhibitor assays, our results argue that the canonical spliceosomal machinery functions in circRNA biogenesis, constituting an alternative splicing mode. PMID:25543144

  12. Conformation-selective methylation of geminivirus DNA.

    PubMed

    Paprotka, T; Deuschle, K; Metzler, V; Jeske, H

    2011-11-01

    Geminiviruses with small circular single-stranded DNA genomes replicate in plant cell nuclei by using various double-stranded DNA (dsDNA) intermediates: distinct open circular and covalently closed circular as well as heterogeneous linear DNA. Their DNA may be methylated partially at cytosine residues, as detected previously by bisulfite sequencing and subsequent PCR. In order to determine the methylation patterns of the circular molecules, the DNAs of tomato yellow leaf curl Sardinia virus (TYLCSV) and Abutilon mosaic virus were investigated utilizing bisulfite treatment followed by rolling circle amplification. Shotgun sequencing of the products yielded a randomly distributed 50% rate of C maintenance after the bisulfite reaction for both viruses. However, controls with unmethylated single-stranded bacteriophage DNA resulted in the same level of C maintenance. Only one short DNA stretch within the C2/C3 promoter of TYLCSV showed hyperprotection of C, with the protection rate exceeding the threshold of the mean value plus 1 standard deviation. Similarly, the use of methylation-sensitive restriction enzymes suggested that geminiviruses escape silencing by methylation very efficiently, by either a rolling circle or recombination-dependent replication mode. In contrast, attempts to detect methylated bases positively by using methylcytosine-specific antibodies detected methylated DNA only in heterogeneous linear dsDNA, and methylation-dependent restriction enzymes revealed that the viral heterogeneous linear dsDNA was methylated preferentially. PMID:21835804

  13. Flexural Vibrations of Circular Beams

    NASA Astrophysics Data System (ADS)

    Silverman, I. K.

    1998-03-01

    For circular cylindrical beams, the Timoshenko theory, which includes shear and rotary effects, can be applied by introducing a constantK‧=0·9. The study presented here takes into account the actual configuration and thus makes it an integral part of the analysis. A variational approach previously used [7] is followed. A simplification of the three-dimensional problem is obtained using the inverse method due to St. Venant. A biquadratic equation yields numerical results for the natural frequencies of the first three modes for the following cases: simply supported, fixed-fixed, free-free and fixed-free.

  14. Conversion of a linear to a circular plasmid in the relapsing fever agent Borrelia hermsii.

    PubMed Central

    Ferdows, M S; Serwer, P; Griess, G A; Norris, S J; Barbour, A G

    1996-01-01

    Spirochetes of the genus Borrelia have genomes composed of both linear and circular replicons. We characterized the genomic organization of B. burgdorferi, B. hermsii, B. turicatae, and B. anserina with pulsed-field gel electrophoresis. All four species contained a linear chromosome approximately 1 Mb in size and multiple linear plasmids in the 16- to 200-kb size range. Plasmids 180 and 170 kb in size, present in the relapsing fever agents B. hermsii and B. turicatae but not in the other two species, behaved as linear duplex DNA molecules under different electrophoretic conditions. A variant of strain HSI of B. hermsii had a 180-kb circular instead of linear plasmid. There were no detectable differences in the growth rates or in the expression of cellular proteins between cells bearing linear forms and those bearing circular forms of the plasmid. The conversion to a circular conformation of monomeric length was demonstrated by the introduction of strand breaks with irradiation, restriction endonuclease analysis, and direct observation of the DNA molecules by fluorescent microscopy. Consideration of different models for the replication of linear DNA suggests that circular intermediates may be involved in the replication of linear replicons in Borrelia spp. PMID:8550515

  15. Method of oriented circular dichroism.

    PubMed Central

    Wu, Y; Huang, H W; Olah, G A

    1990-01-01

    We present a new method for determining the orientation of alpha-helical sections of proteins or peptides in membrane. To apply this method, membranes containing proteins must be prepared in a multilayer array. Circular dichroism (CD) spectra of the multilayer sample are then measured at the normal as well as oblique incident angles with respect to the bilayer planes; we call such spectra oriented circular dichroism (OCD). The procedure of OCD measurement, particularly the ways to avoid the spectral artifacts due to the effects of dielectric interfaces, linear dichroism and birefringence, and the method of data analysis are described in detail. To illustrate the method, we analyze the OCD of alamethicin in diphytanoylphosphatidylcholine multilayers. We conclude unambiguously that the helical section of alamethicin is parallel to the membrane normal when the sample is in the full-hydration state, but the helical section rotates to the plane of membrane when the sample is in a low-hydration state. We also obtained the parallel and perpendicular CD spectra of alpha-helix, and found them to be in agreement with previous theoretical calculations based on the exciton theory. These spectra are useful for analyzing protein orientations in future experiments. Images FIGURE 5 PMID:2344464

  16. Physics at Future Circular Colliders

    NASA Astrophysics Data System (ADS)

    Kotwal, Ashutosh

    2016-03-01

    The Large Hadron Collider has been a grand success with the discovery of the Higgs boson, with bright prospects for additional discoveries since the recent increase in collider energy and the anticipated large datasets. Big open questions such as the nature of dark matter, the origin of the matter-antimatter asymmetry in the Universe, and the theoretical puzzle of the finely-tuned parameters in the Higgs sector, demand new physics principles that extend the established Standard Model paradigm. Future circular colliders in a substantially larger tunnel can house both a high luminosity electron-positron collider for precision measurements of Higgs and electroweak parameters, as well as a very high energy proton-proton collider which can directly manifest particles associated with these new physics principles. We discuss the physics goals of these future circular colliders, and the prospects for elucidating fundamental new laws of nature that will significantly extend our understanding of the Universe. Detailed studies of the discovery potential in specific benchmark models will be presented, with implications for detector design.

  17. Amplification of target-specific, ligation-dependent circular probe.

    PubMed

    Zhang, D Y; Brandwein, M; Hsuih, T C; Li, H

    1998-05-12

    We describe a novel polymerase chain reaction (PCR)-based gene amplification method utilizing a circularizable oligodeoxyribonucleotide probe (C-probe). The C-probe contains two target complementary regions located at each terminus and an interposed generic PCR primer binding region. The hybridization of C-probe to a target brings two termini in direct apposition as the complementary regions of C-probe wind around the target to form a double helix. Subsequent ligation of the two termini results in a covalently linked C-probe that becomes 'locked on to' the target. The circular nature of the C-probe allows for the generation of a multimeric single-stranded DNA (ssDNA) via extension of the antisense primer by Taq DNA polymerase along the C-probe and displacement of downstream strand, analogous to 'rolling circle' replication of bacteriophage in vivo. This multimeric ssDNA then serves as a template for multiple sense primers to hybridize, extend, and displace downstream DNA, generating a large ramified (branching) DNA complex. Subsequent thermocycling denatures the dsDNA and initiates the next round of primer extension and ramification. This model results in significantly improved amplification kinetics (super-exponential) as compared to conventional PCR. Our results show that the C-probe was 1000 times more sensitive than the corresponding linear hemiprobes for detecting Epstein-Barr virus early RNA. The C-probe not only increases the power of amplification but also offers a means for decontaminating carryover amplicons. As the ligated C-probes possess no free termini, they are resistant to exonuclease digestion, whereas contaminated linear amplicons are susceptible to digestion. Treatment of the ligation reaction mixture with exonuclease prior to amplification eliminated the amplicon contaminant, which could also have been co-amplified with the same PCR primers; only the ligated C-probes were amplified. The combined advantages of the C-probe and thermocycling have a

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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. PMID:24565870

  3. Electrodynamics of circular dichroism and its application in the construction of a circular polaroid

    NASA Astrophysics Data System (ADS)

    Volobuev, A. N.

    2016-03-01

    Electrodynamic principles of circular dichroism are developed using a concept of conducting circular structures in matter. A simplified representation of material equations for an optically active medium is obtained in the absence of a transition to the complex domain. A dependence of the absorption coefficient of a circular polarization as a component of the linearly polarized radiation on material parameters is found. Such parameters are analyzed to reveal a possibility of construction of a circular polaroid.

  4. 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…

  5. 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.

  6. Microstrip Antenna Generates Circularly Polarized Beam

    NASA Technical Reports Server (NTRS)

    Huang, J.

    1986-01-01

    Circular microstrip antenna excited with higher order transverse magnetic (TM) modes generates circularly polarized, conical radiation patterns. Found both theoretically and experimentally that peak direction of radiation pattern is varied within wide angular range by combination of mode selection and loading substrate with materials of different dielectric constants.

  7. Circular RNAs: diversity of form and function.

    PubMed

    Lasda, Erika; Parker, Roy

    2014-12-01

    It is now clear that there is a diversity of circular RNAs in biological systems. Circular RNAs can be produced by the direct ligation of 5' and 3' ends of linear RNAs, as intermediates in RNA processing reactions, or by "backsplicing," wherein a downstream 5' splice site (splice donor) is joined to an upstream 3' splice site (splice acceptor). Circular RNAs have unique properties including the potential for rolling circle amplification of RNA, the ability to rearrange the order of genomic information, protection from exonucleases, and constraints on RNA folding. Circular RNAs can function as templates for viroid and viral replication, as intermediates in RNA processing reactions, as regulators of transcription in cis, as snoRNAs, and as miRNA sponges. Herein, we review the breadth of circular RNAs, their biogenesis and metabolism, and their known and anticipated functions. PMID:25404635

  8. 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. PMID:27295541

  9. Circular protecting female students, 1988.

    PubMed

    1988-01-01

    In 1988 the Ministry of Education of Benin issued a Circular designed to protect female students from sexual advances from male students and teachers. Teachers who are suspected of having sexual relations with a student are to receive a note in their file or can be suspended for a month. Teachers who have raped a student or are responsible for a student's pregnancy are to be suspended for three months if the student was not in their class, and six months if she was. Teachers who have incited their sexual partner to have an abortion are subject to demotion. Teachers who are repeat offenders or whose actions have led to an abortion resulting in death are to be fired. Students who have made a female student pregnant are to be suspended from school until delivery of the child and then are to be enrolled in a different school. The directors of schools are asked to put an end to the provocative clothes, make-up, and hairstyles of female students. PMID:12289212

  10. Circular RNAs in Eukaryotic Cells

    PubMed Central

    Chen, Liang; Huang, Chuan; Wang, Xiaolin; Shan, Ge

    2015-01-01

    Circular RNAs (circRNAs) are now recognized as large species of transcripts in eukaryotic cells. From model organisms such as C. elegans, Drosophila, mice to human beings, thousands of circRNAs formed from back-splicing of exons have been identified. The known complexity of transcriptome has been greatly expanded upon the discovery of these RNAs. Studies about the biogenesis and physiological functions have yielded substantial knowledge for the circRNAs, and they are now more likely to be viewed as regulatory elements coded by the genome rather than unavoidable noise of gene expression. Certain human diseases may also relate to circRNAs. These circRNAs show diversifications in features such as sequence composition and cellular localization, and thus we propose that they may be divided into subtypes such as cytoplasmic circRNAs, nuclear circRNAs, and exon-intron circRNAs (EIciRNAs). Here we summarize and discuss knowns and unknowns for these RNAs, and we need to keep in mind that the whole field is still at the beginning of exciting explorations. PMID:27047251

  11. 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." PMID:12289626

  12. 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.

  13. Entanglement of quantum circular states of light

    NASA Astrophysics Data System (ADS)

    Horoshko, D. B.; De Bièvre, S.; Kolobov, M. I.; Patera, G.

    2016-06-01

    We present a general approach to calculating the entanglement of formation for superpositions of two-mode coherent states, placed equidistantly on a circle in phase space. We show that in the particular case of rotationally invariant circular states the Schmidt decomposition of two modes, and therefore the value of their entanglement, are given by analytical expressions. We analyze the dependence of the entanglement on the radius of the circle and number of components in the superposition. We also show that the set of rotationally invariant circular states creates an orthonormal basis in the state space of the harmonic oscillator, and this basis is advantageous for representation of other circular states of light.

  14. Circular Polarization in PKS 1519-273

    NASA Astrophysics Data System (ADS)

    Bennett, W.; Macquart, J. P.; Johnston, H.; Jauncey, D.

    2005-12-01

    The intra-day variable BL Lac PKS 1519-273 exhibits variations at centimeter wavelengths in total intensity, linear polarization, and circular polarization. Their variability is caused by scintillation due to the interstellar medium. PKS 1519-273 displays 4% circular polarization at 4.8 GHz and the variability has persisted as long as the source has been observed. We present observations of this source over several years, showing that the circularly polarized emission is highly variable on micro-arcsecond scales. We determine properties of the structure of the emission by examining the light curves and associated scintillation theory.

  15. Generation of circular polarization of the CMB

    NASA Astrophysics Data System (ADS)

    Zarei, M.; Bavarsad, E.; Haghighat, M.; Mohammadi, R.; Motie, I.; Rezaei, Z.

    2010-04-01

    According to the standard cosmology, near the last scattering surface, the photons scattered via Compton scattering are just linearly polarized and then the primordial circular polarization of the cosmic microwave background (CMB) photons is zero. In this work we show that CMB polarization acquires a small degree of circular polarization when a background magnetic field is considered or the quantum electrodynamic sector of standard model is extended by Lorentz-noninvariant operators as well as noncommutativity. The existence of circular polarization for the CMB radiation may be verified during future observation programs, and it represents a possible new channel for investigating new physics effects.

  16. 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

  17. Production of Double-stranded DNA Ministrings.

    PubMed

    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. 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. PMID:26578802

  19. Recombination-Dependent Oligomerization of Human Papillomavirus Genomes upon Transient DNA Replication

    PubMed Central

    Orav, Marit; Henno, Liisi; Isok-Paas, Helen; Geimanen, Jelizaveta; Ustav, Mart

    2013-01-01

    We describe the extensive and progressive oligomerization of human papillomavirus (HPV) genomes after transfection into the U2OS cell line. The HPV genomic oligomers are extrachromosomal concatemeric molecules containing the viral genome in a head-to-tail orientation. The process of oligomerization does not depend on the topology of the input DNA, and it does not require any other viral factors besides replication proteins E1 and E2. We provide evidence that oligomerization of the HPV18 and HPV11 genomes involves homologous recombination. We also demonstrate oligomerization of the HPV18 and HPV11 genomes in SiHa, HeLa, and C-33 A cell lines and provide examples of oligomeric HPV genomes in clinical samples obtained from HPV-infected patients. PMID:23986589

  20. 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

  1. Dual frequency launcher for circularly polarized antenna

    NASA Astrophysics Data System (ADS)

    Chen, Ming H.

    1989-10-01

    A dual frequency antenna feed is formed from a central, circular waveguide connected to the flat boundry of circular, disk-shaped resonant cavity. A second circular waveguide is connected one end of a disk-shaped resonant cavity. Energy of one frequency enters and exits the cavity along the common axis of the waveguides. Energy of the second frequency is introduced to the same resonant cavity by way of a plurality of bandpass filters, also connected to the cavity. This energy enters by way of slots in the cylindrical walls of the cavity. The central circular waveguide is propagating at one frequency but cut off at the second frequency. These bandpass filters are at this pass band for the second frequency, but at the rejection band for the first frequency. Therefore, the isolation between these two input ports are obtained.

  2. Low energy aspects of circular accelerators

    SciTech Connect

    Holmes, S.D.

    1990-12-01

    Performance in circular accelerators can be limited by some of the same sorts of phenomena described by Miller and Wangler in their lectures on low energy behavior in linear accelerators. In general the strength of the perturbation required to degrade performance is reduced in circular accelerators due to the repetitive nature of the orbits. For example, we shall see that space-charge can severely limit performance in circular accelerators even when operating far from the space-charge dominated regime'' as defined in linear accelerators. We will be discussing two particular aspects of low energy operation in circular accelerators -- space-charge and transition. Low energy'' is defined within the context of these phenomena. We shall see that the phenomena are really only relevant in hadron accelerators.

  3. 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

  4. [The effect of DNA supercoiling DNA on nucleosome structure].

    PubMed

    Sivolob, A V; Khrapunov, S N

    1991-01-01

    The circular DNA which contains nucleosomes and additional supercoils has been considered theoretically. The different possible effect of increased negative supercoiling on the nucleosome structure have been studied. According to the model proposed all supercoils in the nucleosome-containing circular DNA are realized as torsional deformations of the double helix. The free energy of both supercoiling (torsional deformations) and nucleosome stabilization have been taken into consideration to obtain the equation for free energy of nucleosome-containing circular DNA. The analysis of this equation and the experimental data by Garner et al. (II Psoc. Natl. Acad. Sci. USA. 1987. P. 2620-2623) about the maximum amount of supercoiling obtained by DNA-topoisomerase II treatment of nucleosome-containing pBR322 plasmid has been performed. It has been shown that two possibilities are consistent with both the equation and experimental data. These are: (1) the increased supercoiling induces the torsional strains not only in linker regions but also in nucleosome DNA and thus supercoiling causes an instability on nucleosome structure; (2) increased supercoiling induces a structural change of nucleosome which is accompanied by nucleosome DNA unwinding and its transition into form with approximately 11 base pairs per turn of double helix. It has been evaluated that in the first case the average torsional rigidity of nucleosome DNA should be approximately 2.5 times as much and in the second case--much more than the rigidity of naked DNA. Both types of nucleosome structural changes may cause its transition to a potentially active state for transcription. It is suggested that increased supercoiling can be a switch mechanism of chromatin activation. PMID:1654518

  5. 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.

  6. Single particle dynamics in circular accelerators

    SciTech Connect

    Ruth, R.D.

    1986-10-01

    The purpose of this paper is to introduce the reader to the theory associated with the transverse dynamics of single particle, in circular accelerators. The discussion begins with a review of Hamiltonian dynamics and canonical transformations. The case of a single particle in a circular accelerator is considered with a discussion of non-linear terms and chromaticity. The canonical perturbation theory is presented and nonlinear resonances are considered. Finally, the concept of renormalization and residue criterion are examined. (FI)

  7. Circular polarization of sunlight reflected by clouds.

    NASA Technical Reports Server (NTRS)

    Hansen, J. E.

    1971-01-01

    Measurements of circular polarization of visible light from planets have recently been reported. It is pointed out that the values measured for the circular polarization for Jupiter and Venus are of the magnitude expected for sunlight reflected by a cloudy planetary atmosphere. The variations of the sense of the polarization with phase angle and with location on the planetary disk are also consistent with expectations for reflection by clouds.

  8. 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. PMID:25994227

  9. 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.

  10. 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