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Sample records for a2 strain replicates

  1. Convergent Replication of Mouse Synthetic Prion Strains

    PubMed Central

    Ghaemmaghami, Sina; Colby, David W.; Nguyen, Hoang-Oanh B.; Hayashi, Shigenari; Oehler, Abby; DeArmond, Stephen J.; Prusiner, Stanley B.

    2014-01-01

    Prion diseases are neurodegenerative disorders characterized by the aberrant folding of endogenous proteins into self-propagating pathogenic conformers. Prion disease can be initiated in animal models by inoculation with amyloid fibrils formed from bacterially derived recombinant prion protein. The synthetic prions that accumulated in infected organisms are structurally distinct from the amyloid preparations used to initiate their formation and change conformationally on repeated passage. To investigate the nature of synthetic prion transformation, we infected mice with a conformationally diverse set of amyloids and serially passaged the resulting prion strains. At each passage, we monitored changes in the biochemical and biological properties of the adapting strain. The physicochemical properties of each synthetic prion strain gradually changed on serial propagation until attaining a common adapted state with shared physicochemical characteristics. These results indicate that synthetic prions can assume multiple intermediate conformations before converging into one conformation optimized for in vivo propagation. PMID:23438476

  2. Strain-Specific Role of RNAs in Prion Replication

    PubMed Central

    Sferrazza, Gian Franco; Ottenberg, Gregory; Oelschlegel, Anja M.; Dorsey, Kerri

    2012-01-01

    Several lines of evidence suggest that various cofactors may be required for prion replication. PrP binds to polyanions, and RNAs were shown to promote the conversion of PrPC into PrPSc in vitro. In the present study, we investigated strain-specific differences in RNA requirement during in vitro conversion and the potential role of RNA as a strain-specifying component of infectious prions. We found that RNase treatment impairs PrPSc-converting activity of 9 murine prion strains by protein misfolding cyclic amplification (PMCA) in a strain-specific fashion. While the addition of RNA restored PMCA conversion efficiency, the effect of synthetic polynucleotides or DNA was strain dependent, showing a different promiscuity of prion strains in cofactor utilization. The biological properties of RML propagated by PMCA under RNA-depleted conditions were compared to those of brain-derived and PMCA material generated in the presence of RNA. Inoculation of RNA-depleted RML in Tga20 mice resulted in an increased incidence of a distinctive disease phenotype characterized by forelimb paresis. However, this abnormal phenotype was not conserved in wild-type mice or upon secondary transmission. Immunohistochemical and cell panel assay analyses of mouse brains did not reveal significant differences between mice injected with the different RML inocula. We conclude that replication under RNA-depleted conditions did not modify RML prion strain properties. Our study cannot, however, exclude small variations of RML properties that would explain the abnormal clinical phenotype observed. We hypothesize that RNA molecules may act as catalysts of prion replication and that variable capacities of distinct prion strains to utilize different cofactors may explain strain-specific dependency upon RNA. PMID:22811520

  3. Conserved genes and pathways in primary human fibroblast strains undergoing replicative and radiation induced senescence.

    PubMed

    Marthandan, Shiva; Menzel, Uwe; Priebe, Steffen; Groth, Marco; Guthke, Reinhard; Platzer, Matthias; Hemmerich, Peter; Kaether, Christoph; Diekmann, Stephan

    2016-07-28

    Cellular senescence is induced either internally, for example by replication exhaustion and cell division, or externally, for example by irradiation. In both cases, cellular damages accumulate which, if not successfully repaired, can result in senescence induction. Recently, we determined the transcriptional changes combined with the transition into replicative senescence in primary human fibroblast strains. Here, by γ-irradiation we induced premature cellular senescence in the fibroblast cell strains (HFF and MRC-5) and determined the corresponding transcriptional changes by high-throughput RNA sequencing. Comparing the transcriptomes, we found a high degree of similarity in differential gene expression in replicative as well as in irradiation induced senescence for both cell strains suggesting, in each cell strain, a common cellular response to error accumulation. On the functional pathway level, "Cell cycle" was the only pathway commonly down-regulated in replicative and irradiation-induced senescence in both fibroblast strains, confirming the tight link between DNA repair and cell cycle regulation. However, "DNA repair" and "replication" pathways were down-regulated more strongly in fibroblasts undergoing replicative exhaustion. We also retrieved genes and pathways in each of the cell strains specific for irradiation induced senescence. We found the pathways associated with "DNA repair" and "replication" less stringently regulated in irradiation induced compared to replicative senescence. The strong regulation of these pathways in replicative senescence highlights the importance of replication errors for its induction.

  4. Effect of Mycoplasma gallinarum on the replication in vitro of goose parvovirus strain "B".

    PubMed

    Kisary, J; Stipkovits, L

    1975-01-01

    Replication in vitro of the goose parovirus strain "B" was inhibited by Mycoplasma gallinarum co-infection. This effect could be prevented by continuous supplementation of cell cultures with arginine. The infection of cell cultures with Acholeplasma axanthum did not influence virus replication.

  5. Differential Replication of Pathogenic and Nonpathogenic Strains of West Nile Virus within Astrocytes

    PubMed Central

    Hussmann, Katherine L.; Samuel, Melanie A.; Kim, Kwang S.; Diamond, Michael S.

    2013-01-01

    The severity of West Nile virus (WNV) infection in immunocompetent animals is highly strain dependent, ranging from avirulent to highly neuropathogenic. Here, we investigate the nature of this strain-specific restriction by analyzing the replication of avirulent (WNV-MAD78) and highly virulent (WNV-NY) strains in neurons, astrocytes, and microvascular endothelial cells, which comprise the neurovascular unit within the central nervous system (CNS). We demonstrate that WNV-MAD78 replicated in and traversed brain microvascular endothelial cells as efficiently as WNV-NY. Likewise, similar levels of replication were detected in neurons. Thus, WNV-MAD78's nonneuropathogenic phenotype is not due to an intrinsic inability to replicate in key target cells within the CNS. In contrast, replication of WNV-MAD78 was delayed and reduced compared to that of WNV-NY in astrocytes. The reduced susceptibility of astrocytes to WNV-MAD78 was due to a delay in viral genome replication and an interferon-independent reduction in cell-to-cell spread. Together, our data suggest that astrocytes regulate WNV spread within the CNS and therefore are an attractive target for ameliorating WNV-induced neuropathology. PMID:23269784

  6. Variable Inhibition of Zika Virus Replication by Different Wolbachia Strains in Mosquito Cell Cultures.

    PubMed

    Schultz, Michaela J; Isern, Sharon; Michael, Scott F; Corley, Ronald B; Connor, John H; Frydman, Horacio M

    2017-07-15

    Mosquito-borne arboviruses are a major source of human disease. One strategy to reduce arbovirus disease is to reduce the mosquito's ability to transmit virus. Mosquito infection with the bacterial endosymbiont Wolbachia pipientis wMel is a novel strategy to reduce Aedes mosquito competency for flavivirus infection. However, experiments investigating cyclic environmental temperatures have shown a reduction in maternal transmission of wMel, potentially weakening the integration of this strain into a mosquito population relative to that of other Wolbachia strains. Consequently, it is important to investigate additional Wolbachia strains. All Zika virus (ZIKV) suppression studies are limited to the wMel Wolbachia strain. Here we show ZIKV inhibition by two different Wolbachia strains: wAlbB (isolated from Aedes albopictus mosquitoes) and wStri (isolated from the planthopper Laodelphax striatellus) in mosquito cells. Wolbachia strain wStri inhibited ZIKV most effectively. Single-cycle infection experiments showed that ZIKV RNA replication and nonstructural protein 5 translation were reduced below the limits of detection in wStri-containing cells, demonstrating early inhibition of virus replication. ZIKV replication was rescued when Wolbachia was inhibited with a bacteriostatic antibiotic. We observed a partial rescue of ZIKV growth when Wolbachia-infected cells were supplemented with cholesterol-lipid concentrate, suggesting competition for nutrients as one of the possible mechanisms of Wolbachia inhibition of ZIKV. Our data show that wAlbB and wStri infection causes inhibition of ZIKV, making them attractive candidates for further in vitro mechanistic and in vivo studies and future vector-centered approaches to limit ZIKV infection and spread.IMPORTANCE Zika virus (ZIKV) has swiftly spread throughout most of the Western Hemisphere. This is due in large part to its replication in and spread by a mosquito vector host. There is an urgent need for approaches that limit

  7. Smooth Brucella strains invade and replicate in human lung epithelial cells without inducing cell death.

    PubMed

    Ferrero, Mariana C; Fossati, Carlos A; Baldi, Pablo C

    2009-04-01

    Inhalation is a common route for Brucella infection. We investigated whether Brucella species can invade and replicate within alveolar(A549) and bronchial (Calu-6 and 16HBE14o-) human epithelial cells. The number of adherent and intracellular bacteria was higher for rough strains (Brucella canis and Brucella abortus RB51) than for smooth strains (B. abortus 2308 and Brucella suis 1330). Only smooth strains exhibited efficient intracellular replication (1.5-3.5 log increase at 24 h p.i.). A B. abortus mutant with defective expression of the type IV secretion system did not replicate. B. abortus internalization was inhibited by specific inhibitors of microfilaments, microtubules and PI3-kinase activity. As assessed with fluorescent probes, B. abortus infection did not affect the viability of A549 and 16HBE14o- cells, but increased the percentage of injured cells (both strains) and dead cells (RB51) in Calu-6 cultures. LDH levels were increased in supernatants of Calu-6 and 16HBE14o- cells infected with B. abortus RB51, and to a lower extent in Calu-6 infected with B. abortus 2308. No apoptosis was detected by TUNEL upon infection with smooth or rough B. abortus. This study shows that smooth brucellae can infect and replicate in human respiratory epithelial cells inducing minimal or null cytotoxicity. (c) 2009 Elsevier Masson SAS. All rights reserved.

  8. Role of Prion Replication in the Strain-dependent Brain Regional Distribution of Prions.

    PubMed

    Hu, Ping Ping; Morales, Rodrigo; Duran-Aniotz, Claudia; Moreno-Gonzalez, Ines; Khan, Uffaf; Soto, Claudio

    2016-06-10

    One intriguing feature of prion diseases is their strain variation. Prion strains are differentiated by the clinical consequences they generate in the host, their biochemical properties, and their potential to infect other animal species. The selective targeting of these agents to specific brain structures have been extensively used to characterize prion strains. However, the molecular basis dictating strain-specific neurotropism are still elusive. In this study, isolated brain structures from animals infected with four hamster prion strains (HY, DY, 139H, and SSLOW) were analyzed for their content of protease-resistant PrP(Sc) Our data show that these strains have different profiles of PrP deposition along the brain. These patterns of accumulation, which were independent of regional PrP(C) production, were not reproduced by in vitro replication when different brain regions were used as substrate for the misfolding-amplification reaction. On the contrary, our results show that in vitro replication efficiency depended exclusively on the amount of PrP(C) present in each part of the brain. Our results suggest that the variable regional distribution of PrP(Sc) in distinct strains is not determined by differences on prion formation, but on other factors or cellular pathways. Our findings may contribute to understand the molecular mechanisms of prion pathogenesis and strain diversity.

  9. An Archaeal Chromosomal Autonomously Replicating Sequence Element from an Extreme Halophile, Halobacterium sp. Strain NRC-1

    PubMed Central

    Berquist, Brian R.; DasSarma, Shiladitya

    2003-01-01

    We report on the identification and first cloning of an autonomously replicating sequence element from the chromosome of an archaeon, the extreme halophile Halobacterium strain NRC-1. The putative replication origin was identified by association with the orc7 gene and replication ability in the host strain, demonstrated by cloning into a nonreplicating plasmid. Deletion analysis showed that sequences located up to 750 bp upstream of the orc7 gene translational start, plus the orc7 gene and 50 bp downstream, are sufficient to endow the plasmid with replication ability, as judged by expression of a plasmid-encoded mevinolin resistance selectable marker and plasmid recovery after transformation. Sequences located proximal to the two other chromosomally carried haloarchaeal orc genes (orc6 and orc8) are not able to promote efficient autonomous replication. Located within the 750-bp region upstream of orc7 is a nearly perfect inverted repeat of 31 bp, which flanks an extremely AT-rich (44%) stretch of 189 bp. The replication ability of the plasmid was lost when one copy of the inverted repeat was deleted. Additionally, the inverted repeat structure near orc7 homologs in the genomic sequences of two other halophiles, Haloarcula marismortui and Haloferax volcanii, is highly conserved. Our results indicate that, in halophilic archaea, a chromosomal origin of replication is physically linked to orc7 homologs and that this element is sufficient to promote autonomous replication. We discuss the finding of a functional haloarchaeal origin in relation to the large number of orc1-cdc6 homologs identified in the genomes of all haloarchaea to date. PMID:14526006

  10. In vivo replication of pathogenic and attenuated strains of Junin virus in different cell populations of lymphatic tissue.

    PubMed Central

    Laguens, M; Chambó, J G; Laguens, R P

    1983-01-01

    Lymphatic tissue is one of the main sites for replication of Junin virus. To characterize which cells are involved in that replication, the presence of Junin virus in purified populations of macrophages and dendritic cells from the spleens of guinea pigs infected with pathogenic and attenuated strains was investigated by immunofluorescence and intracerebral inoculation into newborn mice. The pathogenic strain was present both in macrophages and in dendritic cells, but the attenuated strain selectively infected dendritic cells. These observations suggest that the pathogenic behavior and replication efficiency of these two strains of Junin virus may be related to a difference in cell targets. Images PMID:6309667

  11. Influenza virus neuraminidase contributes to the dextran sulfate-dependent suppressive replication of some influenza A virus strains.

    PubMed

    Yamada, Hiroshi; Moriishi, Eiko; Haredy, Ahmad M; Takenaka, Nobuyuki; Mori, Yasuko; Yamanishi, Koichi; Okamoto, Shigefumi

    2012-12-01

    Dextran sulfate (DS), a negatively charged, sulfated polysaccharide, suppresses the replication of an influenza A virus strain, and this suppression is associated with inhibition of the hemagglutinin (HA)-dependent fusion activity. However, it remains unknown whether the replication of all or just some influenza A virus strains is suppressed by DS, or whether HA is the only target for the replication suppression. In the present study, we found that DS inhibited the replication of some, but not all influenza A virus strains. The suppression in the DS-sensitive strains was dose-dependent and neutralized by diethylaminoethyl-dextran (DD), which has a positive charge. The suppression by DS was observed not only at the initial stage of viral infection, which includes viral attachment and entry, but also at the late stage, which includes virus assembly and release from infected cells. Electron microscopy revealed that the DS induced viral aggregation at the cell surface. The neuraminidase (NA) activity of the strains whose viral replication was inhibited at the late stage was also more suppressed by DS than that of the strains whose replication was not inhibited, and this inhibition of NA activity was also neutralized by adding positively charged DD. Furthermore, we found that replacing the NA gene of a strain in which viral replication was inhibited by DS at the late stage with the NA gene from a strain in which viral replication was not inhibited, eliminated the DS-dependent suppression. These results suggest that the influenza virus NA contributes to the DS-suppressible virus release from infected cells at the late stage, and the suppression may involve the inhibition of NA activity by DS's negative charge.

  12. Methods for intense aeration, growth, storage, and replication of bacterial strains in microtiter plates.

    PubMed

    Duetz, W A; Rüedi, L; Hermann, R; O'Connor, K; Büchs, J; Witholt, B

    2000-06-01

    Miniaturized growth systems for heterogeneous culture collections are not only attractive in reducing demands for incubation space and medium but also in making the parallel handling of large numbers of strains more practicable. We report here on the optimization of oxygen transfer rates in deep-well microtiter plates and the development of a replication system allowing the simultaneous and reproducible sampling of 96 frozen glycerol stock cultures while the remaining culture volume remains frozen. Oxygen transfer rates were derived from growth curves of Pseudomonas putida and from rates of oxygen disappearance due to the cobalt-catalyzed oxidation of sulfite. Maximum oxygen transfer rates (38 mmol liter(-1) h(-1), corresponding to a mass transfer coefficient of 188 h(-1)) were measured during orbital shaking at 300 rpm at a shaking diameter of 5 cm and a culture volume of 0.5 ml. A shaking diameter of 2.5 cm resulted in threefold-lower values. These high oxygen transfer rates allowed P. putida to reach a cell density of approximately 9 g (dry weight) liter(-1) during growth on a glucose mineral medium at culture volumes of up to 1 ml. The growth-and-replication system was evaluated for a culture collection consisting of aerobic strains, mainly from the genera Pseudomonas, Rhodococcus, and Alcaligenes, using mineral media and rich media. Cross-contamination and excessive evaporation during vigorous aeration were adequately prevented by the use of a sandwich cover of spongy silicone and cotton wool on top of the microtiter plates.

  13. Positive correlation between replication rate and pathotype of Marek’s disease virus strains in maternal antibody negative chickens

    USDA-ARS?s Scientific Manuscript database

    Pathotyping of new field strains of MDV requires both a long period of time and a large number of birds. Confirming a positive correlation of virus replication and pathotype may lead to faster and cheaper alternative pathotyping methods or as a screening assay for choosing isolates to be pathotyped....

  14. Multiple replication origins of Halobacterium sp. strain NRC-1: properties of the conserved orc7-dependent oriC1.

    PubMed

    Coker, James A; DasSarma, Priya; Capes, Melinda; Wallace, Tammitia; McGarrity, Karen; Gessler, Rachael; Liu, Jingfang; Xiang, Hua; Tatusov, Roman; Berquist, Brian R; DasSarma, Shiladitya

    2009-08-01

    The eukaryote-like DNA replication system of the model haloarchaeon Halobacterium NRC-1 is encoded within a circular chromosome and two large megaplasmids or minichromosomes, pNRC100 and pNRC200. We previously showed by genetic analysis that 2 (orc2 and orc10) of the 10 genes coding for Orc-Cdc6 replication initiator proteins were essential, while a third (orc7), located near a highly conserved autonomously replicating sequence, oriC1, was nonessential for cell viability. Here we used whole-genome marker frequency analysis (MFA) and found multiple peaks, indicative of multiple replication origins. The largest chromosomal peaks were located proximal to orc7 (oriC1) and orc10 (oriC2), and the largest peaks on the extrachromosomal elements were near orc9 (oriP1) in both pNRC100 and -200 and near orc4 (oriP2) in pNRC200. MFA of deletion strains containing different combinations of chromosomal orc genes showed that replication initiation at oriC1 requires orc7 but not orc6 and orc8. The initiation sites at oriC1 were determined by replication initiation point analysis and found to map divergently within and near an AT-rich element flanked by likely Orc binding sites. The oriC1 region, Orc binding sites, and orc7 gene orthologs were conserved in all sequenced haloarchaea. Serial deletion of orc genes resulted in the construction of a minimal strain containing not only orc2 and orc10 but also orc9. Our results suggest that replication in this model system is intriguing and more complex than previously thought. We discuss these results from the perspective of the replication strategy and evolution of haloarchaeal genomes.

  15. Enhanced Replication of Hepatitis E Virus Strain 47832c in an A549-Derived Subclonal Cell Line.

    PubMed

    Schemmerer, Mathias; Apelt, Silke; Trojnar, Eva; Ulrich, Rainer G; Wenzel, Jürgen J; Johne, Reimar

    2016-09-29

    Hepatitis E virus (HEV) is a human pathogen with increasing importance. The lack of efficient cell culture systems hampers systematic studies on its replication cycle, virus neutralization and inactivation. Here, several cell lines were inoculated with the HEV genotype 3c strain 47832c, previously isolated from a chronically infected transplant patient. At 14 days after inoculation the highest HEV genome copy numbers were found in A549 cells, followed by PLC/PRF/5 cells, whereas HepG2/C3A, Huh-7 Lunet BLR and MRC-5 cells only weakly supported virus replication. Inoculation of A549-derived subclone cell lines resulted in most cases in reduced HEV replication. However, the subclone A549/D3 was susceptible to lower virus concentrations and resulted in higher virus yields as compared to parental A549 cells. Transcriptome analysis indicated a downregulation of genes for carcinoembryonic antigen-related cell adhesion molecules (CEACAM) 5 and 6, and an upregulation of the syndecan 2 (SDC2) gene in A549/D3 cells compared to A549 cells. However, treatment of A549/D3 cells or A549 cells with CEACAM- or syndecan 2-specific antisera did not influence HEV replication. The results show that cells supporting more efficient HEV replication can be selected from the A549 cell line. The specific mechanisms responsible for the enhanced replication remain unknown.

  16. Enhanced Replication of Hepatitis E Virus Strain 47832c in an A549-Derived Subclonal Cell Line

    PubMed Central

    Schemmerer, Mathias; Apelt, Silke; Trojnar, Eva; Ulrich, Rainer G.; Wenzel, Jürgen J.; Johne, Reimar

    2016-01-01

    Hepatitis E virus (HEV) is a human pathogen with increasing importance. The lack of efficient cell culture systems hampers systematic studies on its replication cycle, virus neutralization and inactivation. Here, several cell lines were inoculated with the HEV genotype 3c strain 47832c, previously isolated from a chronically infected transplant patient. At 14 days after inoculation the highest HEV genome copy numbers were found in A549 cells, followed by PLC/PRF/5 cells, whereas HepG2/C3A, Huh-7 Lunet BLR and MRC-5 cells only weakly supported virus replication. Inoculation of A549-derived subclone cell lines resulted in most cases in reduced HEV replication. However, the subclone A549/D3 was susceptible to lower virus concentrations and resulted in higher virus yields as compared to parental A549 cells. Transcriptome analysis indicated a downregulation of genes for carcinoembryonic antigen-related cell adhesion molecules (CEACAM) 5 and 6, and an upregulation of the syndecan 2 (SDC2) gene in A549/D3 cells compared to A549 cells. However, treatment of A549/D3 cells or A549 cells with CEACAM- or syndecan 2-specific antisera did not influence HEV replication. The results show that cells supporting more efficient HEV replication can be selected from the A549 cell line. The specific mechanisms responsible for the enhanced replication remain unknown. PMID:27690085

  17. Replication and persistence of different strains of bovine viral diarrhea virus in an in vitro embryo production system.

    PubMed

    Givens, M D; Galik, P K; Riddell, K P; Brock, K V; Stringfellow, D A

    2000-10-15

    Recent studies have shown that exposed, in vitro-derived embryos remain contaminated with bovine viral diarrhea virus (BVDV) after washing. However, introduction of a Genotype II versus Genotype I strain of BVDV into an IVF system was reported to provide greater potential for transmission of disease. The primary objective of this study was to compare the potentials for different strains of noncytopathic BVDV to replicate in an IVF system, associate with IVF embryos and infect co-cultured cells via association with washed embryos. The secondary objective was to compare the effect of different strains of BVDV on embryonic development. Two Genotype I (SD-1 and NY-1) and 2 Genotype II (CD-87 and PA-131) strains of BVDV were evaluated. After IVM and IVF of oocytes, presumptive zygotes were washed and transferred into in vitro cultures containing uterine tubal cells (UTC) and medium that was free of BVDV-neutralizing activity. Immediately before addition of zygotes, the cultures were inoculated with 10(5) cell culture infective doses (50%, CCID50) of a strain of BVDV or maintained as a negative control. Cultures of zygotes were then incubated for 7 d. Embryonic development was observed on Days 3 and 7, and attempts were made to isolate BVDV from UTC and medium on Day 7. Also on Day 7, groups of intact, washed blastocysts were either transferred into virus-free secondary cultures containing UTC or sonicated with sonicate fluid assayed by both virus isolation and single-closed-tube reverse transcription nested polymerase chain reaction (RT-nPCR). After 3 d in secondary culture, hatched embryos were enumerated, and medium from the cultures, washed UTC and embryos were tested for BVDV by virus isolation. In addition, washed UTC and embryos were tested for BVDV using RT-nPCR. All strains of BVDV persisted and replicated in the embryo culture environment, but cleavage beyond the 4-cell stage, blastocyst development and hatching varied among cultures contaminated with different

  18. Secretion Chaperones PrsA2 and HtrA Are Required for Listeria monocytogenes Replication following Intracellular Induction of Virulence Factor Secretion

    PubMed Central

    Ahmed, Jana K.

    2016-01-01

    The Gram-positive bacterium Listeria monocytogenes transitions from an environmental organism to an intracellular pathogen following its ingestion by susceptible mammalian hosts. Bacterial replication within the cytosol of infected cells requires activation of the central virulence regulator PrfA followed by a PrfA-dependent induction of secreted virulence factors. The PrfA-induced secreted chaperone PrsA2 and the chaperone/protease HtrA contribute to the folding and stability of select proteins translocated across the bacterial membrane. L. monocytogenes strains that lack both prsA2 and htrA exhibit near-normal patterns of growth in broth culture but are severely attenuated in vivo. We hypothesized that, in the absence of PrsA2 and HtrA, the increase in PrfA-dependent protein secretion that occurs following bacterial entry into the cytosol results in misfolded proteins accumulating at the bacterial membrane with a subsequent reduction in intracellular bacterial viability. Consistent with this hypothesis, the introduction of a constitutively activated allele of prfA (prfA*) into ΔprsA2 ΔhtrA strains was found to essentially inhibit bacterial growth at 37°C in broth culture. ΔprsA2 ΔhtrA strains were additionally found to be defective for cell invasion and vacuole escape in selected cell types, steps that precede full PrfA activation. These data establish the essential requirement for PrsA2 and HtrA in maintaining bacterial growth under conditions of PrfA activation. In addition, chaperone function is required for efficient bacterial invasion and rapid vacuole lysis within select host cell types, indicating roles for PrsA2/HtrA prior to cytosolic PrfA activation and the subsequent induction of virulence factor secretion. PMID:27481256

  19. Isolation and characterization of highly replicable hepatitis C virus genotype 1a strain HCV-RMT.

    PubMed

    Arai, Masaaki; Tokunaga, Yuko; Takagi, Asako; Tobita, Yoshimi; Hirata, Yuichi; Ishida, Yuji; Tateno, Chise; Kohara, Michinori

    2013-01-01

    Multiple genotype 1a clones have been reported, including the very first hepatitis C virus (HCV) clone called H77. The replication ability of some of these clones has been confirmed in vitro and in vivo, although this ability is somehow compromised. We now report a newly isolated genotype 1a clone, designated HCV-RMT, which has the ability to replicate efficiently in patients, chimeric mice with humanized liver, and cultured cells. An authentic subgenomic replicon cell line was established from the HCV-RMT sequence with spontaneous introduction of three adaptive mutations, which were later confirmed to be responsible for efficient replication in HuH-7 cells as both subgenomic replicon RNA and viral genome RNA. Following transfection, the HCV-RMT RNA genome with three adaptive mutations was maintained for more than 2 months in HuH-7 cells. One clone selected from the transfected cells had a high copy number, and its supernatant could infect naïve HuH-7 cells. Direct injection of wild-type HCV-RMT RNA into the liver of chimeric mice with humanized liver resulted in vigorous replication, similar to inoculation with the parental patient's serum. A study of virus replication using HCV-RMT derivatives with various combinations of adaptive mutations revealed a clear inversely proportional relationship between in vitro and in vivo replication abilities. Thus, we suggest that HCV-RMT and its derivatives are important tools for HCV genotype 1a research and for determining the mechanism of HCV replication in vitro and in vivo.

  20. Comparison of the replication characteristics of vaccinia virus strains Guang 9 and Tian Tan in vivo and in vitro.

    PubMed

    Zhu, Rong; Liu, Qiang; Huang, Weijin; Yu, Yongxin; Wang, Youchun

    2014-10-01

    Vaccinia virus is widely used as a vector in the development of recombinant vaccines. Vaccinia virus strain Guang 9 (VG9), which was derived from vaccinia virus strain Tian Tan (VTT) by successive plaque-cloning purification, was more attenuated than VTT. In this study, the host cell range and the growth and replication of VG9 were compared with those of VTT. The results showed that both VG9 and VTT could infect permissive cells (Vero, TK-143 and CEF) and semipermissive cells PK (15) and induced a visible cytopathic effect (CPE). Both strains could infect nonpermissive CHO-K1 cells but neither was able to reproduce. The replicative ability of VG9 was a little lower than that of VTT. Additionally, recombinant vaccinia viruses containing a firefly luciferase gene (VG9-L and VTT-L) were constructed, and their expression in vitro and replication and spread in vivo were compared. The expression ability of VG9-L was lower than that of VTT-L. Whole-animal imaging data indicated that VG9-L could reproduce quickly and express the exogenous protein at the site of inoculation, regardless of whether the intramuscular, intracutaneous, subcutaneous or celiac inoculation route was used. VG9-L was better in its ability to express a foreign protein than VTT-L, but the time during which expression occurred was shorter. There was no dissemination of virus in mice inoculated with either strain. In summary, this study demonstrates the possibility of using VG9 for the production of smallpox vaccines or the construction of recombinant vaccinia virus vaccines.

  1. Genetic and Phenotypic Characterization of a Salmonella enterica serovar Enteritidis Emerging Strain with Superior Intra-macrophage Replication Phenotype

    PubMed Central

    Shomer, Inna; Avisar, Alon; Desai, Prerak; Azriel, Shalhevet; Smollan, Gill; Belausov, Natasha; Keller, Nathan; Glikman, Daniel; Maor, Yasmin; Peretz, Avi; McClelland, Michael; Rahav, Galia; Gal-Mor, Ohad

    2016-01-01

    Salmonella enterica serovar Enteritidis (S. Enteritidis) is one of the ubiquitous Salmonella serovars worldwide and a major cause of food-born outbreaks, which are often associated with poultry and poultry derivatives. Here we report a nation-wide S. Enteritidis clonal outbreak that occurred in Israel during the last third of 2015. Pulsed field gel electrophoresis and whole genome sequencing identified genetically related strains that were circulating in Israel as early as 2008. Global comparison linked this outbreak strain to several clinical and marine environmental isolates that were previously isolated in California and Canada, indicating that similar strains are prevalent outside of Israel. Phenotypic comparison between the 2015 outbreak strain and other clinical and reference S. Enteritidis strains showed only limited intra-serovar phenotypic variation in growth in rich medium, invasion into Caco-2 cells, uptake by J774.1A macrophages, and host cell cytotoxicity. In contrast, significant phenotypic variation was shown among different S. Enteritidis isolates when biofilm-formation, motility, invasion into HeLa cells and uptake by THP-1 human macrophages were studied. Interestingly, the 2015 outbreak clone was found to possess superior intra-macrophage replication ability within both murine and human macrophages in comparison to the other S. Enteritidis strains studied. This phenotype is likely to play a role in the virulence and host-pathogen interactions of this emerging clone. PMID:27695450

  2. Brucella spp. of amphibians comprise genomically diverse motile strains competent for replication in macrophages and survival in mammalian hosts

    PubMed Central

    Al Dahouk, Sascha; Köhler, Stephan; Occhialini, Alessandra; Jiménez de Bagüés, María Pilar; Hammerl, Jens Andre; Eisenberg, Tobias; Vergnaud, Gilles; Cloeckaert, Axel; Zygmunt, Michel S.; Whatmore, Adrian M.; Melzer, Falk; Drees, Kevin P.; Foster, Jeffrey T.; Wattam, Alice R.; Scholz, Holger C.

    2017-01-01

    Twenty-one small Gram-negative motile coccobacilli were isolated from 15 systemically diseased African bullfrogs (Pyxicephalus edulis), and were initially identified as Ochrobactrum anthropi by standard microbiological identification systems. Phylogenetic reconstructions using combined molecular analyses and comparative whole genome analysis of the most diverse of the bullfrog strains verified affiliation with the genus Brucella and placed the isolates in a cluster containing B. inopinata and the other non-classical Brucella species but also revealed significant genetic differences within the group. Four representative but molecularly and phenotypically diverse strains were used for in vitro and in vivo infection experiments. All readily multiplied in macrophage-like murine J774-cells, and their overall intramacrophagic growth rate was comparable to that of B. inopinata BO1 and slightly higher than that of B. microti CCM 4915. In the BALB/c murine model of infection these strains replicated in both spleen and liver, but were less efficient than B. suis 1330. Some strains survived in the mammalian host for up to 12 weeks. The heterogeneity of these novel strains hampers a single species description but their phenotypic and genetic features suggest that they represent an evolutionary link between a soil-associated ancestor and the mammalian host-adapted pathogenic Brucella species. PMID:28300153

  3. Ellagitannins as synergists of ACV on the replication of ACV-resistant strains of HSV 1 and 2.

    PubMed

    Vilhelmova-Ilieva, N; Jacquet, R; Quideau, S; Galabov, A S

    2014-10-01

    The plant-derived polyphenolic compounds castalagin, vescalagin and grandinin (C-glucosidic ellagitannins containing nonahydroxyterphenoyl) manifested a strong inhibitory effect on the replication of acyclovir-resistant strains of herpes simplex viruses (HSV) type 1 and 2 in MDBK cells in focus forming units (i.e., microscopically registered microplaques) reduction assay and in two variants of cytopathic effect inhibition test. The effect on the acyclovir (ACV)-resistant herpes simplex virus type 1 (HSV-1) strain was markedly higher compared to that on the ACV-resistant herpes simplex virus type 2 (HSV-2). The three compounds showed comparable levels of antiviral activity against ACV-resistant HSV strains, in contrast with previous results where castalagin exerted the highest degree of activity against wild type HSV strains (Vilhelmova et al., 2011). Combinations of ellagitannins and ACV were tested on the ACV-resistant strains of both HSV-1 and 2 and produced synergistic effects that were revealed by applying the three-dimensional approach of Prichard and Shipman (1990). The ellagitannin(s)-ACV combination applied against ACV-resistant HSV-1 produced a much stronger synergistic effect compared to the effect observed against ACV-resistant HSV-2. The study of the effects of the combination ellagitannin(s)-ACF on intact cell monolayers did not show any toxicity resulting from interaction between the two substances. Altogether, the results obtained in this study demonstrate the highly promising potential of these plant polyphenols as antiherpetic agents.

  4. Flavone enhances dengue virus type-2 (NGC strain) infectivity and replication in vero cells.

    PubMed

    Zandi, Keivan; Lani, Rafidah; Wong, Pooi-Fong; Teoh, Boon-Teong; Sam, Sing-Sin; Johari, Jefree; Mustafa, Mohd Rais; AbuBakar, Sazaly

    2012-02-28

    This study investigates the effects of 2-phenyl-1-benzopyran-4-one (flavone) on DENV-2 infectivity in Vero cells. Virus adsorption and attachment and intracellular virus replication were investigated using a foci forming unit assay (FFUA) and quantitative RT-PCR, respectively. Addition of flavone (100 μg/mL) significantly increased the number of DENV-2 foci by 35.66% ± 1.52 and 49.66% ± 2.51 when added during and after virus adsorption to the Vero cells, respectively. The average foci size after 4 days of infection increased by 33% ± 2.11 and 89% ± 2.13. The DENV-2 specific RNA copy number in the flavone-treated infected cells increased by 6.41- and 23.1-fold when compared to the mock-treated infected cells. Flavone (100 μg/mL) did not promote or inhibit Vero cell proliferation. The CC₅₀ value of flavone against Vero cells was 446 µg/mL. These results suggest that flavone might enhance dengue virus replication by acting antagonistically towards flavonoids known to inhibit dengue virus replication.

  5. Potent inhibition of respiratory syncytial virus replication using a 2-5A-antisense chimera targeted to signals within the virus genomic RNA

    PubMed Central

    Player, Mark R.; Barnard, Dale L.; Torrence, Paul F.

    1998-01-01

    The 2-5A system is a recognized mechanistic component of the antiviral action of interferon. Interferon-induced 2-5A synthetase generates 2-5A, which, in turn, activates the latent constitutive RNase L that degrades viral RNA. Chemical conjugation of 2-5A to an antisense oligonucleotide can target the 2-5A-dependent RNase L to the antisense-specified RNA and effect its selective destruction. Such a 2-5A-antisense chimera (NIH351) has been developed that targets a consensus sequence within the respiratory syncytial virus (RSV) genomic RNA. NIH351 was 50- to 90-fold more potent against RSV strain A2 than was ribavirin, the presently approved drug for clinical management of RSV infection. It was similarly active against a variety of RSV strains of both A and B subgroups and possessed a cell culture selectivity index comparable to ribavirin. In addition, the anti-RSV activity of NIH351 was shown to be virus-specific and a result of a true antisense effect, because a scrambled nucleotide sequence in the antisense domain of NIH351 caused a significant decrease in antiviral activity. The 2-5A system’s RNase L was implicated in the mechanism of action of NIH351 because a congener with a disabled 2-5A moiety was of greatly reduced anti-RSV effectiveness. These findings represent an innovative approach to the control of RSV replication. PMID:9671772

  6. Brucella suis Vaccine Strain 2 Induces Endoplasmic Reticulum Stress that Affects Intracellular Replication in Goat Trophoblast Cells In vitro

    PubMed Central

    Wang, Xiangguo; Lin, Pengfei; Li, Yang; Xiang, Caixia; Yin, Yanlong; Chen, Zhi; Du, Yue; Zhou, Dong; Jin, Yaping; Wang, Aihua

    2016-01-01

    Brucella has been reported to impair placental trophoblasts, a cellular target where Brucella efficiently replicates in association with the endoplasmic reticulum (ER), and ultimately trigger abortion in pregnant animals. However, the precise effects of Brucella on trophoblast cells remain unclear. Here, we describe the infection and replication of Brucella suis vaccine strain 2 (B.suis.S2) in goat trophoblast cells (GTCs) and the cellular and molecular responses induced in vitro. Our studies demonstrated that B.suis.S2 was able to infect and proliferate to high titers, hamper the proliferation of GTCs and induce apoptosis due to ER stress. Tunicamycin (Tm), a pharmacological chaperone that strongly mounts ER stress-induced apoptosis, inhibited B.suis.S2 replication in GTCs. In addition, 4 phenyl butyric acid (4-PBA), a pharmacological chaperone that alleviates ER stress-induced apoptosis, significantly enhanced B.suis.S2 replication in GTCs. The Unfolded Protein Response (UPR) chaperone molecule GRP78 also promoted B.suis.S2 proliferation in GTCs by inhibiting ER stress-induced apoptosis. We also discovered that the IRE1 pathway, but not the PERK or ATF6 pathway, was activated in the process. However, decreasing the expression of phosphoIRE1α and IRE1α proteins with Irestatin 9389 (IRE1 antagonist) in GTCs did not affect the proliferation of B.suis.S2. Although GTC implantation was not affected upon B.suis.S2 infection, progesterone secretion was suppressed, and prolactin and estrogen secretion increased; these effects were accompanied by changes in the expression of genes encoding key steroidogenic enzymes. This study systematically explored the mechanisms of abortion in Brucella infection from the viewpoint of pathogen invasion, ER stress and reproductive endocrinology. Our findings may provide new insight for understanding the mechanisms involved in goat abortions caused by Brucella infection. PMID:26904517

  7. Brucella suis Vaccine Strain 2 Induces Endoplasmic Reticulum Stress that Affects Intracellular Replication in Goat Trophoblast Cells In vitro.

    PubMed

    Wang, Xiangguo; Lin, Pengfei; Li, Yang; Xiang, Caixia; Yin, Yanlong; Chen, Zhi; Du, Yue; Zhou, Dong; Jin, Yaping; Wang, Aihua

    2016-01-01

    Brucella has been reported to impair placental trophoblasts, a cellular target where Brucella efficiently replicates in association with the endoplasmic reticulum (ER), and ultimately trigger abortion in pregnant animals. However, the precise effects of Brucella on trophoblast cells remain unclear. Here, we describe the infection and replication of Brucella suis vaccine strain 2 (B.suis.S2) in goat trophoblast cells (GTCs) and the cellular and molecular responses induced in vitro. Our studies demonstrated that B.suis.S2 was able to infect and proliferate to high titers, hamper the proliferation of GTCs and induce apoptosis due to ER stress. Tunicamycin (Tm), a pharmacological chaperone that strongly mounts ER stress-induced apoptosis, inhibited B.suis.S2 replication in GTCs. In addition, 4 phenyl butyric acid (4-PBA), a pharmacological chaperone that alleviates ER stress-induced apoptosis, significantly enhanced B.suis.S2 replication in GTCs. The Unfolded Protein Response (UPR) chaperone molecule GRP78 also promoted B.suis.S2 proliferation in GTCs by inhibiting ER stress-induced apoptosis. We also discovered that the IRE1 pathway, but not the PERK or ATF6 pathway, was activated in the process. However, decreasing the expression of phosphoIRE1α and IRE1α proteins with Irestatin 9389 (IRE1 antagonist) in GTCs did not affect the proliferation of B.suis.S2. Although GTC implantation was not affected upon B.suis.S2 infection, progesterone secretion was suppressed, and prolactin and estrogen secretion increased; these effects were accompanied by changes in the expression of genes encoding key steroidogenic enzymes. This study systematically explored the mechanisms of abortion in Brucella infection from the viewpoint of pathogen invasion, ER stress and reproductive endocrinology. Our findings may provide new insight for understanding the mechanisms involved in goat abortions caused by Brucella infection.

  8. Protective efficacy of multivalent replication-abortive vaccine strains in horses against African horse sickness virus challenge.

    PubMed

    Lulla, Valeria; Losada, Andres; Lecollinet, Sylvie; Kerviel, Adeline; Lilin, Thomas; Sailleau, Corinne; Beck, Cecile; Zientara, Stephan; Roy, Polly

    2017-07-24

    African horse sickness virus (AHSV) is an orbivirus, a member of the Reoviridae family. Nine different serotypes have been described so far. AHSV is vectored by Culicoides spp. to equids, causing high mortality, particularly in horses, with considerable economic impacts. For development of a safe attenuated vaccine, we previously established an efficient reverse genetics (RG) system to generate Entry Competent Replication-Abortive (ECRA) virus strains, for all nine serotypes and demonstrated the vaccine potential of these strains in type I interferon receptor (IFNAR)-knockout mice. Here, we evaluated the protective efficacies of these ECRA viruses in AHSV natural hosts. One monoserotype (ECRA.A4) vaccine and one multivalent cocktail (ECRA.A1/4/6/8) vaccine were tested in ponies and subsequently challenged with a virulent AHSV4. In contrast to control animals, all vaccinated ponies were protected and did not develop severe clinical symptoms of AHS. Furthermore, the multivalent cocktail vaccinated ponies produced neutralizing antibodies against all serotypes present in the cocktail, and a foal born during the trial was healthy and had no viremia. These results validate the suitability of these ECRA strains as a new generation of vaccines for AHSV. Copyright © 2017. Published by Elsevier Ltd.

  9. Replication of two influenza virus strains and a recombinant in HEF and LEP cells.

    PubMed

    Ghendon, Y; Tucková, E; Vonka, V; Klimov, A; Ginzburg, V; Markushin, S

    1979-07-01

    The replication of influenza viruses A/NWS-D, A/WS-MK and their r12 recombinant in human embryo fibroblast (HEF) and human diploid fibroblast (LEP) cell lines was studied. In HEF cells virus NWS-D and recombinant r12 induced synthesis of virus-specific macromolecules and produced infectious virions; virus WS-MK induced synthesis of virus complementary RNA (cRNA), virion RNA (vRNA), protein, RNP and non-infectious virions, but haemagglutinin cleavage was impaired and the virions formed contained uncleaved haemagglutinin. In LEP cells, infectious virions were formed only by virus NWS-D; viruses WS-MK and r12 induced synthesis of virus cRNA, vRNA, proteins and RNP; virus r12 had the haemagglutinin cleaved, whereas in virus WS-MK this process was impaired; neither virus WS-MK nor r12 was capable of forming virions. Analysis of the recombinant r12 genome showed that it had only inherited a single gene from NWS-D, the one coding for neuraminidase, having inherited all others (P1, P2, P3, HA, NP, M, NS) from WS-MK. The data obtained suggested that the inability of virus WS-MK to form infectious virions in HEF cells is due to the character of its neuraminidase, which is incapable of participating in haemagglutinin cleavage. The deficient reproduction of this virus in the other host-cell system (LEP) is apparently associated with some characteristics of another protein (other proteins) of this virus.

  10. Human immunodeficiency virus type 1 strains of subtypes B and E replicate in cutaneous dendritic cell-T-cell mixtures without displaying subtype-specific tropism.

    PubMed Central

    Pope, M; Frankel, S S; Mascola, J R; Trkola, A; Isdell, F; Birx, D L; Burke, D S; Ho, D D; Moore, J P

    1997-01-01

    A report that genetic subtype E human immunodeficiency virus type 1 (HIV-1) strains display a preferential tropism for Langerhans cells (epidermal dendritic cells [DCs]) compared to genetic subtype B strains suggested a possible explanation for the rapid heterosexual spread of subtype E strains in Thailand (L. E. Soto-Ramirez et al., Science 271:1291-1293, 1996). In an independent system, we applied subtype E and B isolates to skin leukocytes, since skin is a relevant model for the histologically comparable surfaces of the vagina and ectocervix. Isolates of both HIV-1 subtypes infected DC-T-cell mixtures, and no subtype-specific pattern of infection was observed. Purified DCs did not support the replication of strains of either subtype B or E. Our findings do not support the conclusion that subtype E strains have a preferential tropism for DCs, suggesting that other explanations for the rapid heterosexual spread of subtype E strains in Asia should be considered. PMID:9311895

  11. Multiple Replication Origins of Halobacterium sp. Strain NRC-1: Properties of the Conserved orc7-Dependent oriC1▿

    PubMed Central

    Coker, James A.; DasSarma, Priya; Capes, Melinda; Wallace, Tammitia; McGarrity, Karen; Gessler, Rachael; Liu, Jingfang; Xiang, Hua; Tatusov, Roman; Berquist, Brian R.; DasSarma, Shiladitya

    2009-01-01

    The eukaryote-like DNA replication system of the model haloarchaeon Halobacterium NRC-1 is encoded within a circular chromosome and two large megaplasmids or minichromosomes, pNRC100 and pNRC200. We previously showed by genetic analysis that 2 (orc2 and orc10) of the 10 genes coding for Orc-Cdc6 replication initiator proteins were essential, while a third (orc7), located near a highly conserved autonomously replicating sequence, oriC1, was nonessential for cell viability. Here we used whole-genome marker frequency analysis (MFA) and found multiple peaks, indicative of multiple replication origins. The largest chromosomal peaks were located proximal to orc7 (oriC1) and orc10 (oriC2), and the largest peaks on the extrachromosomal elements were near orc9 (oriP1) in both pNRC100 and -200 and near orc4 (oriP2) in pNRC200. MFA of deletion strains containing different combinations of chromosomal orc genes showed that replication initiation at oriC1 requires orc7 but not orc6 and orc8. The initiation sites at oriC1 were determined by replication initiation point analysis and found to map divergently within and near an AT-rich element flanked by likely Orc binding sites. The oriC1 region, Orc binding sites, and orc7 gene orthologs were conserved in all sequenced haloarchaea. Serial deletion of orc genes resulted in the construction of a minimal strain containing not only orc2 and orc10 but also orc9. Our results suggest that replication in this model system is intriguing and more complex than previously thought. We discuss these results from the perspective of the replication strategy and evolution of haloarchaeal genomes. PMID:19502403

  12. Replication kinetics of neurovirulent versus non-neurovirulent equine herpesvirus type 1 strains in equine nasal mucosal explants.

    PubMed

    Vandekerckhove, Annelies P; Glorieux, S; Gryspeerdt, A C; Steukers, L; Duchateau, L; Osterrieder, N; Van de Walle, G R; Nauwynck, H J

    2010-08-01

    Equine herpesvirus type 1 (EHV-1) is the causative agent of equine herpes myeloencephalopathy, of which outbreaks are reported with increasing frequency throughout North America and Europe. This has resulted in its classification as a potentially emerging disease by the US Department of Agriculture. Recently, it was found that a single nucleotide polymorphism (SNP) in the viral DNA polymerase gene (ORF30) at aa 752 (N-->D) is associated with the neurovirulent potential of EHV-1. In the present study, equine respiratory mucosal explants were inoculated with several Belgian isolates typed in their ORF30 as D(752) or N(752), to evaluate a possible difference in replication in the upper respiratory tract. In addition, to evaluate whether any observed differences could be attributed to the SNP associated with neurovirulence, the experiments were repeated with parental Ab4 (reference neurovirulent strain), parental NY03 (reference non-neurovirulent strain) and their N/D revertant recombinant viruses. The salient findings were that EHV-1 spreads plaquewise in the epithelium, but plaques never cross the basement membrane (BM). However, single EHV-1-infected cells could be observed below the BM at 36 h post-inoculation (p.i.) for all N(752) isolates and at 24 h p.i. for all D(752) isolates, and were identified as monocytic cells and T lymphocytes. Interestingly, the number of infected cells was two to five times higher for D(752) isolates compared with N(752) isolates at every time point analysed. Finally, this study showed that equine respiratory explants are a valuable and reproducible model to study EHV-1 neurovirulence in vitro, thereby reducing the need for horses as experimental animals.

  13. Infection with equine infectious anemia virus vaccine strain EIAVDLV121 causes no visible histopathological lesions in target organs in association with restricted viral replication and unique cytokine response.

    PubMed

    Liu, Qiang; Ma, Jian; Wang, Xue-Feng; Xiao, Fei; Li, Li-Jia; Zhang, Jiao-Er; Lin, Yue-Zhi; Du, Cheng; He, Xi-Jun; Wang, Xiaojun; Zhou, Jian-Hua

    2016-02-01

    The live equine infectious anemia virus (EIAV) vaccine strain EIAVDLV121 was developed by in vitro attenuation of a virulent strain, EIAVLN40, in the 1970s, and it has been demonstrated to induce protective immunity under laboratory and natural EIAV infection conditions. The detailed biological features of this attenuated virus remain to be further investigated. Experimental inoculation with EIAVDLV121 did not result in clinical symptoms even with immunosuppressive treatment in our previous studies. Here, we further investigated whether the replication of the vaccine strain EIAVDLV121 in experimentally infected horses causes histopathological lesions to develop in the targeted organs. Both the lungs and the spleen have been demonstrated to support EIAV replication. By evaluating the gross macroscopic and histological changes, we found that EIAVDLV121 did not cause detectable histopathological lesions and that it replicated several hundred times more slowly than its parental virulent strain, EIAVLN40, in tissues. Immunochemical assays of these tissues indicated that the primary target cells of EIAVDLV121 were monocytes/macrophages, but that EIAVLN40 also infected alveolar epithelial cells and vascular endothelial cells. In addition, both of these viral strains promoted the up- and down-regulation of the expression of various cytokines and chemokines, implicating the potential involvement of these cellular factors in the pathological outcomes of EIAV infection and host immune responses. Taken together, these results demonstrate that the EIAV vaccine strain does not cause obvious histopathological lesions or clinical symptoms and that it induces a unique cytokine response profile. These features are considered essential for EIAVDLV121 to function as an effective live vaccine. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. A small plasmid, pCA2.4, from the cyanobacterium Synechocystis sp. strain PCC 6803 encodes a rep protein and replicates by a rolling circle mechanism.

    PubMed Central

    Yang, X; McFadden, B A

    1993-01-01

    Different cryptic plasmids are widely distributed in many strains of cyanobacteria. A small cryptic plasmid, pCA2.4, from Synechocystis strain PCC 6803 was completely sequenced, and its replication mode was determined. pCA2.4 contained 2,378 bp and encoded a replication (Rep) protein, designated RepA. An analysis of the deduced amino acid sequence revealed that RepA of pCA2.4 has significant homology with Rep proteins of pKYM from Shigella sonnei, a pUB110 plasmid family from gram-positive bacteria, and with a protein corresponding to an open reading frame in a Nostoc plasmid and open reading frame C of Plectonema plasmid pRF1. pKYM and pUB110 family plasmids replicate by a rolling circle mechanism in which a Rep protein nicks the origin of replication to allow the generation of a single-stranded plasmid as a replication intermediate. RepA encoded by pC2.4 was expressed in Escherichia coli cells harboring a vector, pCRP336, containing the entire repA gene. The observed molecular weight of RepA was consistent with the value of 39,200 calculated from its deduced amino acid sequence, as was the N-terminal sequence analysis done through the 12th residue. Single-stranded plasmid DNA of pCA2.4 that was specifically degraded by S1 nuclease was detected in Synechocystis cells by Southern hybridization. These observations suggest that pCA2.4 replicates by a rolling circle mechanism in Synechocystis cells. Images PMID:8320214

  15. Previous infection with virulent strains of Newcastle disease virus reduces highly pathogenic avian influenza virus replication, disease, and mortality in chickens.

    PubMed

    Costa-Hurtado, Mar; Afonso, Claudio L; Miller, Patti J; Shepherd, Eric; Cha, Ra Mi; Smith, Diane; Spackman, Erica; Kapczynski, Darrell R; Suarez, David L; Swayne, David E; Pantin-Jackwood, Mary J

    2015-09-23

    Highly pathogenic avian influenza virus (HPAIV) and Newcastle disease virus (NDV) are two of the most important viruses affecting poultry worldwide and produce co-infections especially in areas of the world where both viruses are endemic; but little is known about the interactions between these two viruses. The objective of this study was to determine if co-infection with NDV affects HPAIV replication in chickens. Only infections with virulent NDV strains (mesogenic Pigeon/1984 or velogenic CA/2002), and not a lentogenic NDV strain (LaSota), interfered with the replication of HPAIV A/chicken/Queretaro/14588-19/95 (H5N2) when the H5N2 was given at a high dose (10(6.9) EID50) two days after the NDV inoculation, but despite this interference, mortality was still observed. However, chickens infected with the less virulent mesogenic NDV Pigeon/1984 strain three days prior to being infected with a lower dose (10(5.3-5.5) EID50) of the same or a different HPAIV, A/chicken/Jalisco/CPA-12283-12/2012 (H7N3), had reduced HPAIV replication and increased survival rates. In conclusion, previous infection of chickens with virulent NDV strains can reduce HPAIV replication, and consequently disease and mortality. This interference depends on the titer of the viruses used, the virulence of the NDV, and the timing of the infections. The information obtained from these studies helps to understand the possible interactions and outcomes of infection (disease and virus shedding) when HPAIV and NDV co-infect chickens in the field.

  16. Influence of Foot-and-Mouth Disease Virus O/CHN/Mya98/33-P Strain Leader Protein on Viral Replication and Host Innate Immunity.

    PubMed

    Jiang, Shaodong; Bai, Xingwen; Li, Pinghua; Zhang, Meng; Bao, Huifang; Sun, Pu; Lu, Zengjun; Cao, Yimei; Chen, Yingli; Li, Dong; Fu, Yuanfang; Liu, Zaixin

    2015-09-01

    Foot-and-mouth disease virus (FMDV) O/CHN/Mya98/33-P strain was isolated from the esophageal-pharyngeal fluid sample of cattle, and was shown to cause persistent infection. Its leader protein contains 200 amino acids with one amino acid deletion, which is upstream and next to the second initiation codon compared with the majority of FMDV Mya98 strains. The FMDV genome includes two initiation codons that can produce two different leader proteins, Lab (from the first AUG) and Lb (from the second AUG). For convenience, the inter-AUG region was named as La. Previously, it was found that a recombinant virus with Lab of FMDV O/CHN/Mya98/33-P strain had higher proliferation efficiency, and better ability to inhibit the host innate immune response. Three full-length infectious cDNA clones-rHN33-Lb, rHN33-La, and rHNGSLX-Lb-containing the FMDV O/CHN/Mya98/33-P strain leader proteins Lb, La, or the FMDV O/GSLX/2010 strain leader protein Lb, respectively, were constructed based on an established infectious clone r-HN rescued from FMDV O/HN/CHN/93 strain. After infecting pig kidney primary cells, rHN33-La showed higher replication efficiency than r-HN, and rHN33-Lb displayed better ability to resist host innate immunity than rHNGSLX-Lb. These results demonstrated that the inter-AUG region of FMDV strain O/CHN/Mya98/33-P leader protein must be involved in increasing viral replication efficiency. Additionally, the Lb of FMDV O/CHN/Mya98/33-P must be involve in increasing its ability to inhibit host innate immune response, and the distinctive amino acids G56 and/or R118 of FMDV leader protein may play essential roles in it.

  17. Cellular Response of Sinorhizobium sp. Strain A2 during Arsenite Oxidation

    PubMed Central

    Fukushima, Koh; Huang, He; Hamamura, Natsuko

    2015-01-01

    Arsenic (As) is a widely distributed toxic element in the environment and microorganisms have developed resistance mechanisms in order to tolerate it. The cellular response of the chemoorganotrophic arsenite (As[III])-oxidizing α-Proteobacteria, Sinorhizobium sp. strain A2, to arsenic was examined in the present study. Several proteins associated with arsenite oxidase and As resistance were shown to be accumulated in the presence of As(III). A shift in central carbon metabolism from the tricarboxylic acid pathway to glyoxylate pathway was also observed in response to oxidative stress. Our results revealed the strategy of the As(III)-oxidizing Sinorhizobium strain to mitigate arsenic toxicity and oxidative damage by multiple metabolic adaptations. PMID:26477790

  18. Attenuated and Replication-Competent Vaccinia Virus Strains M65 and M101 with Distinct Biology and Immunogenicity as Potential Vaccine Candidates against Pathogens

    PubMed Central

    Sánchez-Sampedro, Lucas; Gómez, Carmen Elena; Mejías-Pérez, Ernesto; Pérez-Jiménez, Eva; Oliveros, Juan Carlos

    2013-01-01

    Replication-competent poxvirus vectors with an attenuation phenotype and with a high immunogenic capacity of the foreign expressed antigen are being pursued as novel vaccine vectors against different pathogens. In this investigation, we have examined the replication and immunogenic characteristics of two vaccinia virus (VACV) mutants, M65 and M101. These mutants were generated after 65 and 101 serial passages of persistently infected Friend erythroleukemia (FEL) cells. In cultured cells of different origins, the mutants are replication competent and have growth kinetics similar to or slightly reduced in comparison with those of the parental Western Reserve (WR) virus strain. In normal and immune-suppressed infected mice, the mutants showed different levels of attenuation and pathogenicity in comparison with WR and modified vaccinia Ankara (MVA) strains. Wide genome analysis after deep sequencing revealed selected genomic deletions and mutations in a number of viral open reading frames (ORFs). Mice immunized in a DNA prime/mutant boost regimen with viral vectors expressing the LACK (Leishmania homologue for receptors of activated C kinase) antigen of Leishmania infantum showed protection or a delay in the onset of cutaneous leishmaniasis. Protection was similar to that triggered by MVA-LACK. In immunized mice, both polyfunctional CD4+ and CD8+ T cells with an effector memory phenotype were activated by the two mutants, but the DNA-LACK/M65-LACK protocol preferentially induced CD4+ whereas DNA-LACK/M101-LACK preferentially induced CD8+ T cell responses. Altogether, our findings showed the adaptive changes of the WR genome during long-term virus-host cell interaction and how the replication competency of M65 and M101 mutants confers distinct biological properties and immunogenicity in mice compared to those of the MVA strain. These mutants could have applicability for understanding VACV biology and as potential vaccine vectors against pathogens and tumors. PMID

  19. Construction of a subgenomic CV-B3 replicon expressing emerald green fluorescent protein to assess viral replication of a cardiotropic enterovirus strain in cultured human cells.

    PubMed

    Wehbe, Michel; Huguenin, Antoine; Leveque, Nicolas; Semler, Bert L; Hamze, Monzer; Andreoletti, Laurent; Bouin, Alexis

    2016-04-01

    Coxsackieviruses B (CV-B) (Picornaviridae) are a common infectious cause of acute myocarditis in children and young adults, a disease, which is a precursor to 10-20% of chronic myocarditis and dilated cardiomyopathy (DCM) cases. The mechanisms involved in the disease progression from acute to chronic myocarditis phase and toward the DCM clinical stage are not fully understood but are influenced by both viral and host factors. Subgenomic replicons of CV-B can be used to assess viral replication mechanisms in human cardiac cells and evaluate the effects of potential antiviral drugs on viral replication activities. Our objectives were to generate a reporter replicon from a cardiotropic prototype CV-B3/28 strain and to characterize its replication properties into human cardiac primary cells. To obtain this replicon, a cDNA plasmid containing the full CV-B3/28 genome flanked by a hammerhead ribozyme sequence and an MluI restriction site was generated and used as a platform for the insertion of sequences encoding emerald green fluorescent protein (EmGFP) in place of those encoding VP3. In vitro transcribed RNA from this plasmid was transfected into HeLa cells and human primary cardiac cells and was able to produce EmGFP and VP1-containing polypeptides. Moreover, non-structural protein biological activity was assessed by the specific cleavage of eIF4G1 by viral 2A(pro). Viral RNA replication was indirectly demonstrated by inhibition assays, fluoxetine was added to cell culture and prevented the EmGFP synthesis. Our results indicated that the EmGFP CV-B3 replicon was able to replicate and translate as well as the CV-B3/28 prototype strain. Our EmGFP CV-B3 replicon will be a valuable tool to readily investigate CV-B3 replication activities in human target cell models. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. A naturally occurring prfA truncation in a Listeria monocytogenes field strain contributes to reduced replication and cell-to-cell spread.

    PubMed

    Rupp, Sebastian; Aguilar-Bultet, Lisandra; Jagannathan, Vidhya; Guldimann, Claudia; Drögemüller, Cord; Pfarrer, Christiane; Vidondo, Beatriz; Seuberlich, Torsten; Frey, Joachim; Oevermann, Anna

    2015-08-31

    Listeria (L.) monocytogenes is an environmental bacterium that may become an intracellular pathogen upon ingestion to cause gastroenteritis, septicaemia, abortions, and/or fatal infections of the central nervous system. We here describe a L. monocytogenes field strain (JF5171) isolated from a bovine placenta in the context of abortion, which exhibited attenuation in bovine brain-slice cultures. The whole genome of strain JF5171 was sequenced, and the invasion, replication, and intercellular spread of JF5171 were further analyzed by quantification of colony forming units and immunofluorescence studies. Phospholipase and hemolysis activity of JF5171 were also quantified along with transcription levels of actA, hly and prfA. The data obtained were compared to those of the widely used L. monocytogenes reference strain, EGD-e. JF5171 exhibited reduced replication and lower levels of phospholipase and hemolysis activity. Invasion and cell-to-cell spread was strongly decreased compared to EGD-e, and actin polymerization was absent. A frame shift deletion was identified in the JF5171 coding region of the major regulator for virulence, prfA. This resulted in a truncated C-terminus sequence (WEN* vs. WGKLN*). In addition, a point mutation resulted in a lysine to arginine substitution at amino acid position 197. Complementation with prfA from EGD-e and with (EGD-e) prfA-K197N increased the replication and spread efficiency of JF5171. In contrast, complementation with the truncated version of prfA had no effect. Taken together, these results suggest that the truncated C-terminus of prfA considerably contributes to the strongly attenuated phenotype observed in vitro. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Genetic diversity among Clostridium botulinum strains harboring bont/A2 and bont/A3 genes.

    PubMed

    Lúquez, Carolina; Raphael, Brian H; Joseph, Lavin A; Meno, Sarah R; Fernández, Rafael A; Maslanka, Susan E

    2012-12-01

    Clostridium botulinum type A strains are known to be genetically diverse and widespread throughout the world. Genetic diversity studies have focused mainly on strains harboring one type A botulinum toxin gene, bont/A1, although all reported bont/A gene variants have been associated with botulism cases. Our study provides insight into the genetic diversity of C. botulinum type A strains, which contain bont/A2 (n = 42) and bont/A3 (n = 4) genes, isolated from diverse samples and geographic origins. Genetic diversity was assessed by using bont nucleotide sequencing, content analysis of the bont gene clusters, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). Sequences of bont genes obtained in this study showed 99.9 to 100% identity with other bont/A2 or bont/A3 gene sequences available in public databases. The neurotoxin gene clusters of the subtype A2 and A3 strains analyzed in this study were similar in gene content. C. botulinum strains harboring bont/A2 and bont/A3 genes were divided into six and two MLST profiles, respectively. Four groups of strains shared a similarity of at least 95% by PFGE; the largest group included 21 out of 46 strains. The strains analyzed in this study showed relatively limited genetic diversity using either MLST or PFGE.

  2. Strain-induced Fermi contour anisotropy of GaAs (311)A 2D holes

    NASA Astrophysics Data System (ADS)

    Shabani, Javad; Shayegan, Mansour; Winkler, Roland

    2008-03-01

    There is considerable current interest in electronic properties of two-dimensional (2D) carriers whose energy bands are spin-split at finite values of in-plane wave vector, thanks to the spin-orbit interaction and the lack of inversion symmetry. We report experimental and theoretical results revealing that the spin-subband Fermi contours of the heavy and light heavy-holes (HHh and HHl) can be tuned in high mobility GaAs (311)A 2D hole systems via the application of symmetry-breaking in-plane strain. Our calculations show that the HHl spin-subband Fermi contour is circular but the HHh spin-subband Fermi contour is distorted. Experimentally, we probe the Fermi contour anisotropy by measuring the magneto-resistance commensurability peaks induced by square arrays of antidots. When the spin splitting is sufficiently large, the magneto-resistance trace exhibits two peaks, providing clear evidence for spin-resolved ballistic transport. The experimental results are in good agreement with the calculations, and confirm that the majority spin-subband (HHh) has a severely distorted Fermi contour whose anisotropy can be tuned with strain while Fermi contour of the minority spin-subband (HHl) remains nearly isotropic.

  3. Host-cell-dependent role of actin cytoskeleton during the replication of a human strain of influenza A virus.

    PubMed

    Arcangeletti, M C; De Conto, F; Ferraglia, F; Pinardi, F; Gatti, R; Orlandini, G; Covan, S; Motta, F; Rodighiero, I; Dettori, G; Chezzi, C

    2008-01-01

    This study was aimed at investigating the possible involvement of the actin cytoskeleton in the modulation of host permissiveness to A/NWS/33 human influenza virus infection in two mammalian (MDCK and LLC-MK2) cell lines in vitro. During the early stages of infection, no appreciable association between incoming NWS/33 virions and cortical actin was detectable in the permissive MDCK model by confocal microscopy, while extensive colocalization and a slower infection progression were observed in LLC-MK2 cells. In the latter model, we also demonstrated the inability of the virus to carry out multiple replication cycles, irrespective of the presence of cleaved HA subunits in the released virions. Treatment with the actin-depolymerizing agent cytochalasin D significantly increased the infection efficiency in LLC-MK2 cells, while a detrimental effect was observed in the MDCK cell line. Our data suggest a selective role of the actin network in inducing a restriction to influenza virus replication, mostly depending on its molecular organization, the host cell type and virus replication phase.

  4. Infection of monocyte-derived macrophages with human immunodeficiency virus type 1 (HIV-1). Monocyte-tropic and lymphocyte-tropic strains of HIV-1 show distinctive patterns of replication in a panel of cell types.

    PubMed

    Collman, R; Hassan, N F; Walker, R; Godfrey, B; Cutilli, J; Hastings, J C; Friedman, H; Douglas, S D; Nathanson, N

    1989-10-01

    To characterize the host range of different strains of HIV-1, we have used four types of cells, primary monocyte-derived macrophages (MDM), primary PBL, a promonocyte cell line (U937), and a CD4+ T cell line (SUP-T1). These cells were infected with three prototype strains of HIV-1, a putative lymphocyte-tropic strain (IIIB), and two putative monocyte-tropic strains (SF162 and DV). Infections were monitored by assays for infectious virus, for cell-free and cell-associated viral antigen (p24), and for the proportion of cells infected by immunohistochemical staining. It was concluded that: (a) the use of four different cell types provides a useful biological matrix for distinguishing the tropism of different strains of HIV-1; this matrix yields more information than the infection of any single cell type. (b) A monocyte-tropic strain of HIV-1, such as strain SF162, shows a reciprocal host range when compared with a lymphocyte-tropic strain such as IIIB; strain SF162 replicates well in primary MDM but not in U937 or SUP-T1 cells, while strain IIIB replicates well in both U937 and SUP-T1 cells but not in MDM. (c) Both lymphocyte-tropic and monocyte-tropic strains of HIV-1 replicate well in PBL. (d) The promonocyte cell line, U937, and the T cell line, SUP-T1, differ markedly from primary cells, such as MDM and PBL, in their ability to support the replication of different strains of HIV-1; these cell lines cannot be used as surrogates for primary cells in host range studies of HIV-1 strains.

  5. Replication-Competent Influenza Virus and Respiratory Syncytial Virus Luciferase Reporter Strains Engineered for Co-Infections Identify Antiviral Compounds in Combination Screens

    PubMed Central

    Yan, Dan; Weisshaar, Marco; Lamb, Kristen; Chung, Hokyung K; Lin, Michael Z; Plemper, Richard K

    2016-01-01

    Myxoviruses such as influenza A virus (IAV) and respiratory syncytial virus (RSV) are major human pathogens, mandating the development of novel therapeutics. To establish a high-throughput screening protocol for the simultaneous identification of pathogen- and host-targeted hit candidates against either or both pathogens, we have attempted coinfection of cells with IAV and RSV. However, viral replication kinetics were incompatible, RSV signal window was low, and an IAV-driven minireplicon reporter assay used in initial screens narrowed the host cell range and restricted to single-cycle infections. To overcome these limitations, we developed an RSV strain carrying firefly luciferase fused to an innovative universal small-molecule assisted shut-off domain, which boosted assay signal window, and a hyperactive fusion protein that synchronized IAV and RSV reporter expression kinetics and suppresses the identification of RSV entry inhibitors sensitive to a recently reported RSV pan-resistance mechanism. Combined with a replication-competent recombinant IAV strain harboring nano-luciferase, the assay performed well on a human respiratory cell line and supports multi-cycle infections. Miniaturized to 384-well format, the protocol was validated through screening of a set of the NIH Clinical Collection (NCC) in quadruplicate. These test screens demonstrated favorable assay parameters and reproducibility. Application to a LOPAC library of bioactive compounds in a proof-of-concept campaign detected licensed anti-myxovirus therapeutics, ribavirin and the neuraminidase inhibitor zanamivir, and identified two unexpected RSV-specific hit candidates, Fenretinide and the opioid receptor antagonist BNTX-7. Hits were evaluated in direct and orthogonal dose-response counterscreens using a standard recRSV reporter strain expressing renilla luciferase. PMID:26307636

  6. Signal strains that can detect certain DNA replication and membrane mutants of Escherichia coli: isolation of a new ssb allele, ssb-3.

    PubMed Central

    Schmellik-Sandage, C S; Tessman, E S

    1990-01-01

    Mutations in several dna genes of Escherichia coli, when introduced into a strain with a lac fusion in the SOS gene sulA, resulted in formation of blue colonies on plates containing 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-Gal). Unexpectedly, several lines of evidence indicated that the blue colony color was not primarily due to induction of the SOS system but rather was due to a membrane defect, along with the replication defect, making the cell X-Gal extrasensitive (phenotypically Xgx), possibly because of enhanced permeability to X-Gal or leakage of beta-galactosidase. (i) In most cases, beta-galactosidase specific activity increased only two- to threefold. (ii) Mutations conferring tolerance to colicin E1 resulted in blue colony color with no increase in beta-galactosidase specific activity. (iii) Mutations in either the dnaA, dnaB, dnaC, dnaE, dnaG, or ssb gene, when introduced into a strain containing a bioA::lac fusion, produced a blue colony color without an increase in beta-galactosidase synthesis. These lac fusion strains can serve as signal strains to detect dna mutations as well as membrane mutations. By localized mutagenesis of the 92-min region of the chromosome of the sulA::lac signal strain and picking blue colonies, we isolated a novel ssb allele that confers the same extreme UV sensitivity as a delta recA allele, which is a considerably greater sensitivity than that conferred by the two well-studied ssb alleles, ssb-1 and ssb-113. The technique also yielded dnaB mutants; fortuitously, uvrA mutants were also found. PMID:2142938

  7. Monoclonal antibodies against Aleutian disease virus distinguish virus strains and differentiate sites of virus replication from sites of viral antigen sequestration.

    PubMed Central

    Race, R E; Chesebro, B; Bloom, M E; Aasted, B; Wolfinbarger, J

    1986-01-01

    Monoclonal antibodies (mAbs) were used to study antigenic differences among strains of Aleutian disease virus (ADV) and to characterize viral proteins in vitro and in vivo. A number of ADV field strains could be discriminated, and highly virulent Utah I ADV was clearly delineated from the tissue culture-adapted avirulent ADV-G strain. This specificity could be demonstrated by indirect immunofluorescence against infected cultures of Crandell feline kidney cells or against tissues of Utah I ADV-infected mink. Viral antigens were demonstrated in both the nuclei and the cytoplasm of infected tissue culture cells. However, in mink mesenteric lymph node, spleen, and liver, viral antigen was observed only in the cytoplasm. Absence of nuclear fluorescence suggested that the detected antigen represented phagocytized viral antigens rather than replicating virus. This conclusion was supported by the finding that mAbs reactive only against low-molecular-weight polypeptides derived from intact viral proteins gave the same pattern of in vivo fluorescence as mAbs with broad reactivity for large or small (or both) viral polypeptides. The distribution of infected cells was the same as that described for macrophages in these tissues and suggested that cells of the reticuloendothelial system had sequestered viral antigens. Images PMID:3001352

  8. Pattern of disease after murine hepatitis virus strain 3 infection correlates with macrophage activation and not viral replication.

    PubMed Central

    Pope, M; Rotstein, O; Cole, E; Sinclair, S; Parr, R; Cruz, B; Fingerote, R; Chung, S; Gorczynski, R; Fung, L

    1995-01-01

    Murine hepatitis virus strain (MHV-3) produces a strain-dependent pattern of disease which has been used as a model for fulminant viral hepatitis. This study was undertaken to examine whether there was a correlation between macrophage activation and susceptibility or resistance to MHV-3 infection. Peritoneal macrophages were isolated from resistant A/J and susceptible BALB/cJ mice and, following stimulation with MHV-3 or lipopolysaccharide (LPS), analyzed for transcription of mRNA and production of interleukin-1 (IL-1), tumor necrosis factor alpha (TNF-alpha), transforming growth factor beta (TGF-beta), mouse fibrinogen-like protein (musfiblp), tissue factor (TF), leukotriene B4, and prostaglandin E2 (PGE2). Macrophages from BALB/cJ mice produced greater amounts of IL-1, TNF-alpha, TGF-beta, leukotriene B4, and musfiblp following MHV-3 infection than macrophages from resistant A/J mice, whereas in response to LPS, equivalent amounts of IL-1, TNF-alpha, TGF-beta, and TF were produced by macrophages from both strains of mice. Levels of mRNA of IL-1, TNF-alpha, and musfiblp were greater and more persistent in BALB/cJ than in A/J macrophages, whereas the levels and kinetics of IL-1, TNF-alpha, and TF mRNA following LPS stimulation were identical in macrophages from both strains of mice. Levels of production of PGE2 by MHV-3-stimulated macrophages from resistant and susceptible mice were equivalent; however, the time course for induction of PGE2, differed, but the total quantity of PGE2 produced was insufficient to inhibit induction of musfiblp, a procoagulant known to correlate with development of fulminant hepatic necrosis in susceptible mice. These results demonstrate marked differences in production of inflammatory mediators to MHV-3 infection in macrophages from resistant A/J and susceptible BALB/cJ mice, which may explain the marked hepatic necrosis and fibrin deposition and account for the lethality of MHV-3 in susceptible mice. PMID:7636967

  9. An unusual gene arrangement for the putative chromosome replication origin and circadian expression of dnaN in Synechococcus sp. strain PCC 7942.

    PubMed

    Liu, Y; Tsinoremas, N F

    1996-06-12

    In eubacteria, the clustering of DnaA boxes around the dnaN (beta subunit of DNA polymerase III) and dnaA genes usually defines the chromosome replication origin (oriC). In this study, the dnaN locus from the cyanobacterium Synechococcus sp. strain PCC 7942 was sequenced. The gene order in this region is cbbZp-dnaN-orf288-purL-purF which contrasts with other eubacteria. A cluster of eleven DnaA boxes (consensus sequence: TTTTCCACA) was found in the intergenic region between dnaN and cbbZp. We also found a 41-bp sequence within this region that is 80% identical to the proposed oriC of Streptomyces coelicolor. Therefore, we propose that this intergenic region may serve as an oriC in Synechococcus. Using bacterial luciferase as a reporter, we also showed that dnaN is rhythmically expressed, suggesting that DNA replication could be under circadian control in this organism.

  10. Comparative replication capacity of raltegravir-resistant strains and antiviral activity of the new-generation integrase inhibitor dolutegravir in human primary macrophages and lymphocytes.

    PubMed

    Pollicita, Michela; Surdo, Matteo; Di Santo, Fabiola; Cortese, Maria Francesca; Fabeni, Lavinia; Fedele, Valentina; Malet, Isabelle; Marcelin, Anne-Genevieve; Calvez, Vincent; Ceccherini-Silberstein, Francesca; Perno, Carlo Federico; Svicher, Valentina

    2014-09-01

    To evaluate the replication capacity and phenotypic susceptibility to dolutegravir and raltegravir of wild-type and raltegravir-resistant HIV-1 strains in several cellular systems. The antiviral activities of dolutegravir and raltegravir were evaluated in human primary monocyte-derived macrophages (MDMs), peripheral blood mononuclear cells (PBMCs) and C8166 T lymphocytic cells. The following raltegravir resistance mutations were analysed: N155H, Y143C, N155H + Y143C and G140S+Q148H. In the absence of drug, the replication capacity of raltegravir-resistant viruses was strongly reduced compared with wild-type in all cellular models analysed. In MDMs and PBMCs, a dramatic decrease in viral replication was observed for the double mutants N155H + Y143C and G140S + Q148H (ranging from 0.1% to 2.5% compared with wild-type). In MDMs, dolutegravir exhibited high potency, with EC50 and EC90 values of 1.1 ± 0.9 and 5.5 ± 3.4 nM, respectively (comparable to raltegravir). These values (particularly for EC90) were significantly lower than those observed in PBMCs (EC50: 2.7 ± 1.5 nM; EC90: 14.8 ± 0.9 nM) and C8166 cells (EC50: 5.5 ± 0.8 nM; EC90: 64.8 ± 5.8 nM). In all cellular models analysed, dolutegravir showed full efficacy against N155H and Y143C mutants (dolutegravir fold-change resistance ranging from 0.1 to 1.4; raltegravir fold-change resistance ranging from 0.1 to 10.3). In C8166 (the only cell model in which replication capacity was sufficient to perform the test) dolutegravir showed full efficacy against mutations N155H + Y143C (dolutegravir fold-change resistance: 0.6) and a slightly lower activity against G140S+Q148H (dolutegravir fold-change resistance: 2.1). Dolutegravir is effective in different HIV cellular targets and against raltegravir-resistant mutants. The high efficacy of dolutegravir in MDMs (cells with limited metabolism) has relevant clinical implications in light of the role of MDMs in the transmission of HIV infection and

  11. Complete genome sequence of Spiroplasma citri strain R8-A2T, causal agent of stubborn disease in Citrus spp

    USDA-ARS?s Scientific Manuscript database

    Spiroplasma citri is the causal agent of stubborn disease in Citrus spp., as well as the cause of diseases in numerous other plant genera. Here we report the nucleotide sequence of the 1,599,709 bp circular chromosome and two plasmids of strain R8-A2T. This information will facilitate comparative ...

  12. A 2D strain estimator with numerical optimization method for soft-tissue elastography.

    PubMed

    Liu, Ke; Zhang, Pengfei; Shao, Jinhua; Zhu, Xinjian; Zhang, Yun; Bai, Jing

    2009-12-01

    Elastography is a bioelasticity-based imaging modality which has been proved to be a potential evaluation tool to detect the tissue abnormalities. Conventional method for elastography is to estimate the displacement based on cross-correlation technique firstly, then strain profile is calculated as the gradient of the displacement. The main problem of this method arises from the fact that the cross-correlation between pre- and post-compression signals will be decreased because of the signal's compression-to-deformation. It may constrain the estimation of the displacement. Numerical optimization, as an efficient tool to estimate the non-rigid deformation in image registration, has its potential to achieve the elastogram. This paper incorporates the idea of image registration into elastography and proposes a radio frequency (RF) signal registration strain estimator based on the minimization of a cost function using numerical optimization method with Powell algorithm (NOMPA). To evaluate the proposed scheme, the simulation data with a hard inclusion embedded in the homogeneous background is produced for analysis. NOMPA can obtain the displacement profiles and strain profiles simultaneously. When compared with the cross-correlation based method, NOMPA presents better signal-to-noise ratio (SNR, 32.6+/-1.5 dB vs. 23.8+/-1.1 dB) and contrast-to-noise ratio (CNR, 28.8+/-1.8 dB vs. 21.7+/-0.9 dB) in axial normal strain estimation. The in vitro experiment of porcine liver with ethanol-induced lesion is also studied. The statistic results of SNR and CNR indicate that strain profiles by NOMPA performs better anti-noise and target detectability than that by cross-correlation based method. Though NOMPA carry a heavier computational burden than cross-correlation based method, it may be an useful method to obtain 2D strains in elastography.

  13. Pravastatin and simvastatin inhibit the adhesion, replication and proliferation of Toxoplasma gondii (RH strain) in HeLa cells.

    PubMed

    Sanfelice, Raquel Arruda; da Silva, Suelen Santos; Bosqui, Larissa Rodrigues; Miranda-Sapla, Milena Menegazzo; Barbosa, Bellisa Freitas; Silva, Rafaela José; Ferro, Eloísa A Vieira; Panagio, Luciano Aparecido; Navarro, Italmar Teodorico; Bordignon, Juliano; Conchon-Costa, Ivete; Pavanelli, Wander Rogerio; Almeida, Ricardo Sergio; Costa, Idessania Nazareth

    2017-03-01

    The conventional treatment for toxoplasmosis with pyrimethamine and sulfadiazine shows toxic effects to the host, and it is therefore necessary to search for new drugs. Some studies suggest the use of statins, which inhibit cholesterol synthesis in humans and also the initial processes of isoprenoid biosynthesis in the parasite. Thus, the objective of this study was to evaluate the activity of the statins pravastatin and simvastatin in HeLa cells infected in vitro with the RH strain of T. gondii. HeLa cells (1×10(5)) were infected with T. gondii tachyzoites (5×10(5)) following two different treatment protocols. In the first protocol, T. gondii tachyzoites were pretreated with pravastatin (50 and 100μg/mL) and simvastatin (1.56 and 3.125μg/mL) for 30min prior to infection. In the second, HeLa cells were first infected (5×10(5)) with tachyzoites and subsequently treated with pravastatin and simvastatin for 24h at the concentrations noted above. Initially, we evaluated the cytotoxicity of drugs by the MTT assay, number of tachyzoites adhered to cells, number of infected cells, and viability of tachyzoites by trypan blue exclusion. The supernatant of the cell cultures was collected post-treatment for determination of the pattern of Th1/Th2/Th17 cytokines by cytometric bead array. There was no cytotoxicity to HeLa cells with 50 and 100μg/mL pravastatin and 1.56 and 3.125μg/mL simvastatin. There was no change in the viability of tachyzoites that received pretreatment. Regarding the pre- and post-treatment of the cells with pravastatin and simvastatin alone, there was a reduction in adhesion, invasion and proliferation of cells to T. gondii. As for the production of cytokines, we found that IL-6 and IL-17 were significantly reduced in cells infected with T. gondii and treated with pravastatin and simvastatin, when compared to control. Based on these results, we can infer that pravastatin and simvastatin alone possess antiproliferative effects on tachyzoites forms

  14. Mucosal immunization induces a higher level of lasting neutralizing antibody response in mice by a replication-competent smallpox vaccine: vaccinia Tiantan strain.

    PubMed

    Lu, Bin; Yu, Wenbo; Huang, Xiaoxing; Wang, Haibo; Liu, Li; Chen, Zhiwei

    2011-01-01

    The possible bioterrorism threat using the variola virus, the causative agent of smallpox, has promoted us to further investigate the immunogenicity profiles of existing vaccines. Here, we study for the first time the immunogenicity profile of a replication-competent smallpox vaccine (vaccinia Tiantan, VTT strain) for inducing neutralizing antibodies (Nabs) through mucosal vaccination, which is noninvasive and has a critical implication for massive vaccination programs. Four different routes of vaccination were tested in parallel including intramuscular (i.m.), intranasal (i.n.), oral (i.o.), and subcutaneous (s.c.) inoculations in mice. We found that one time vaccination with an optimal dose of VTT was able to induce anti-VTT Nabs via each of the four routes. Higher levels of antiviral Nabs, however, were induced via the i.n. and i.o. inoculations when compared with the i.m. and s.c. routes. Moreover, the i.n. and i.o. vaccinations also induced higher sustained levels of Nabs overtime, which conferred better protections against homologous or alternating mucosal routes of viral challenges six months post vaccination. The VTT-induced immunity via all four routes, however, was partially effective against the intramuscular viral challenge. Our data have implications for understanding the potential application of mucosal smallpox vaccination and for developing VTT-based vaccines to overcome preexisting antivaccinia immunity.

  15. Mucosal Immunization Induces a Higher Level of Lasting Neutralizing Antibody Response in Mice by a Replication-Competent Smallpox Vaccine: Vaccinia Tiantan Strain

    PubMed Central

    Lu, Bin; Yu, Wenbo; Huang, Xiaoxing; Wang, Haibo; Liu, Li; Chen, Zhiwei

    2011-01-01

    The possible bioterrorism threat using the variola virus, the causative agent of smallpox, has promoted us to further investigate the immunogenicity profiles of existing vaccines. Here, we study for the first time the immunogenicity profile of a replication-competent smallpox vaccine (vaccinia Tiantan, VTT strain) for inducing neutralizing antibodies (Nabs) through mucosal vaccination, which is noninvasive and has a critical implication for massive vaccination programs. Four different routes of vaccination were tested in parallel including intramuscular (i.m.), intranasal (i.n.), oral (i.o.), and subcutaneous (s.c.) inoculations in mice. We found that one time vaccination with an optimal dose of VTT was able to induce anti-VTT Nabs via each of the four routes. Higher levels of antiviral Nabs, however, were induced via the i.n. and i.o. inoculations when compared with the i.m. and s.c. routes. Moreover, the i.n. and i.o. vaccinations also induced higher sustained levels of Nabs overtime, which conferred better protections against homologous or alternating mucosal routes of viral challenges six months post vaccination. The VTT-induced immunity via all four routes, however, was partially effective against the intramuscular viral challenge. Our data have implications for understanding the potential application of mucosal smallpox vaccination and for developing VTT-based vaccines to overcome preexisting antivaccinia immunity. PMID:21765641

  16. ROR1/RPA2A, a Putative Replication Protein A2, Functions in Epigenetic Gene Silencing and in Regulation of Meristem Development in ArabidopsisW⃞

    PubMed Central

    Xia, Ran; Wang, Junguo; Liu, Chunyan; Wang, Yu; Wang, Youqun; Zhai, Jixian; Liu, Jun; Hong, Xuhui; Cao, Xiaofeng; Zhu, Jian-Kang; Gong, Zhizhong

    2006-01-01

    We screened for suppressors of repressor of silencing1 (ros1) using the silenced 35S promoter-neomycin phosphotransferase II (Pro35S:NPTII) gene as a marker and identified two allelic mutants, ror1-1 and ror1-2 (for suppressor of ros1). Map-based cloning revealed that ROR1 encodes a 31-kD protein similar to DNA replication protein A2 (RPA2A). Mutations in ROR1 reactivate the silenced Pro35S:NPTII gene but not RD29A promoter-luciferase in the ros1 mutant. DNA methylation in rDNA, centromeric DNA, and RD29A promoter regions is not affected by ror1. However, chromatin immunoprecipitation data suggest that histone H3 acetylation is increased and histone H3K9 dimethylation is decreased in the 35S promoter in the ror1 ros1 mutant compared with ros1. These results indicate that release of silenced Pro35S:NPTII by ror1 mutations is independent of DNA methylation. ROR1/RPA2A is strongly expressed in shoot and root meristems. Mutations in ROR1/RPA2A affect cell division in meristems but not final cell sizes. Our work suggests important roles of ROR1/RPA2A in epigenetic gene silencing and in the regulation of plant development. PMID:16326925

  17. Probing localized strain in solution-derived YB a2C u3O7 -δ nanocomposite thin films

    NASA Astrophysics Data System (ADS)

    Guzman, Roger; Gazquez, Jaume; Mundet, Bernat; Coll, Mariona; Obradors, Xavier; Puig, Teresa

    2017-07-01

    Enhanced pinning due to nanoscale strain is unique to the high-Tc cuprates, where pairing may be modified with lattice distortion. Therefore a comprehensive understanding of the defect landscape is required for a broad range of applications. However, determining the type and distribution of defects and their associated strain constitutes a critical task, and for this aim, real-space techniques for atomic resolution characterization are necessary. Here, we use scanning transmission electron microscopy (STEM) to study the atomic structure of individual defects of solution-derived YB a2C u3O7 (YBCO) nanocomposites, where the inclusion of incoherent secondary phase nanoparticles within the YBCO matrix dramatically increases the density of Y1B a2C u4O8 (Y124) intergrowths, the commonest defect in YBCO thin films. The formation of the Y124 is found to trigger a concatenation of strain-derived interactions with other defects and the concomitant nucleation of intrinsic defects, which weave a web of randomly distributed nanostrained regions that profoundly transform the vortex-pinning landscape of the YBCO nanocomposite thin films.

  18. Higher level of replication efficiency of 2009 (H1N1) pandemic influenza virus than those of seasonal and avian strains: kinetics from epithelial cell culture and computational modeling.

    PubMed

    Mitchell, Hugh; Levin, Drew; Forrest, Stephanie; Beauchemin, Catherine A A; Tipper, Jennifer; Knight, Jennifer; Donart, Nathaniel; Layton, R Colby; Pyles, John; Gao, Peng; Harrod, Kevin S; Perelson, Alan S; Koster, Frederick

    2011-01-01

    The pathogenicity and transmission of influenza A viruses are likely determined in part by replication efficiency in human cells, which is the net effect of complex virus-host interactions. H5N1 avian, H1N1 seasonal, and H1N1 2009 pandemic influenza virus strains were compared by infecting human differentiated bronchial epithelial cells in air-liquid interface cultures at relatively low virus particle/cell ratios. Differential equation and computational models were used to characterize the in vitro kinetic behaviors of the three strains. The models were calibrated by fitting experimental data in order to estimate difficult-to-measure parameters. Both models found marked differences in the relative values of p, the virion production rate per cell, and R(0), an index of the spread of infection through the monolayer, with the values for the strains in the following rank order (from greatest to least): pandemic strain, followed by seasonal strain, followed by avian strain, as expected. In the differential equation model, which treats virus and cell populations as well mixed, R(0) and p varied proportionately for all 3 strains, consistent with a primary role for productivity. In the spatially explicit computational model, R(0) and p also varied proportionately except that R(0) derived for the pandemic strain was reduced, consistent with constrained viral spread imposed by multiple host defenses, including mucus and paracrine antiviral effects. This synergistic experimental-computational strategy provides relevant parameters for identifying and phenotyping potential pandemic strains.

  19. babA2- and cagA-positive Helicobacter pylori strains are associated with duodenal ulcer and gastric carcinoma in Brazil.

    PubMed

    Oliveira, Adriana Gonçalves; Santos, Adriana; Guerra, Juliana Becattini; Rocha, Gifone Aguiar; Rocha, Andreia Maria Camargos; Oliveira, Celso Affonso; Cabral, Mônica Maria Demas Alvares; Nogueira, Ana Margarida Miguel Ferreira; Queiroz, Dulciene Maria Magalhães

    2003-08-01

    The babA2 and cagA genes were investigated in 208 Brazilian Helicobacter pylori strains. A strong association between babA2 and duodenal ulcer or gastric carcinoma was observed, even after adjusting for confounding factors, such as age, gender, and cagA status. cagA-positive strains were also independently associated with H. pylori-related diseases.

  20. High correlation of babA 2-positive strains of Helicobacter pylori with the presence of gastric cancer.

    PubMed

    Talebi Bezmin Abadi, Amin; Taghvaei, Tarang; Mohabbati Mobarez, Ashraf; Vaira, Giuseppina; Vaira, Dino

    2013-09-01

    Helicobacter pylori (H. pylori) is a Gram-negative bacterium that is well known in the involvement of chronic inflammation in the gastric mucosa of the human stomach. Several studies have investigated the possible role of H. pylori presence in different gastroduodenal disorders with conflicting results. This study aimed to further investigate such a field. Helicobacter pylori strains were cultured from 160 patients (mean age of 42 years; range 15-75; 90 were male, and 70 were female) [40 gastric cancer (GC), 55 duodenal ulcer (DU) and 65 non-ulcer dyspepsia (NUD)]. In this study, allelic variants of iceA 1, iceA 2 and babA 2 were identified by polymerase chain reaction. The overall prevalence of babA 2 gene was 40.6% (65/160). The prevalence of babA 2 gene was 95% with gastric cancer, 18.1% with duodenal ulcer and 26.1% with non ulcer dyspepsia, respectively. The prevalence of babA 2 in GC patients was significantly higher as compared to either NUD or UD patients (P = 0.0004), while no statistical significance was found between the latter two patient groups. Our study finds that babA 2 and iceA 1 genes are more prevalent in GC compared to either NUD or DU patients in Iran.

  1. Characterization of a chelator-resistant proteinase from Thermus strain Rt4A2.

    PubMed Central

    Freeman, S A; Peek, K; Prescott, M; Daniel, R

    1993-01-01

    The Thermus isolate Rt4A2 was found to produce an extracellular chelator-resistant proteinase. The proteinase was purified to homogeneity by (NH4)2SO4 precipitation, cation-exchange chromatography, gel-filtration chromatography, and weak anion-exchange chromatography. The Rt4A2 proteinase was found to have properties typical of an alkaline serine proteinase. It had a pH optimum of 9.0 and was specifically inhibited by phenylmethanesulphonyl fluoride. Its isoelectric point was greater than 10.25. Its molecular-mass was 31.6 kDa as determined by SDS/PAGE. N-terminal sequencing has shown it to have high sequence similarity with other serine proteinases from Thermus species. The proteinase hydrolysed a number of substrates including fibrin, casein, haemoglobin, collagen, albumin and the synthetic chromogenic peptide substrate Suc-Ala-Ala-Pro-Phe-NH-Np. The specific activity of the purified proteinase using azocasein as substrate was 313 units/mg. Substrate inhibition was observed above an azocasein concentration of 0.05% (w/v). Esterase activity was directed mainly towards those substrates containing the aliphatic or aromatic residues of alanine, glycine, tryptophan, tyrosine and phenylalanine. Thermostability half-lives of greater than 7 days at 70 degrees C, 43 h at 80 degrees C and 90 min at 90 degrees C were found in the presence of 5 mM CaCl2. At 90 degrees C increasing the CaCl2 concentration 100-fold (0.5 mM to 50 mM) caused a 4.3-fold increase in the half-life of the enzyme from 30 to 130 min. Half-lives of 19.4 min at 100 degrees C and 4.4 min at 105 degrees C were found in the presence of 50 mM CaCl2. The metal chelators EGTA and EDTA reduced the stability at higher temperatures but had no effect on the activity of the proteinase. Activity was not stimulated by common metal activators such as Ca2+, Mg2+ and Zn2+. PMID:8240244

  2. hnRNP A2/B1 interacts with influenza A viral protein NS1 and inhibits virus replication potentially through suppressing NS1 RNA/protein levels and NS1 mRNA nuclear export

    SciTech Connect

    Wang, Yimeng; Zhou, Jianhong; Du, Yuchun

    2014-01-20

    The NS1 protein of influenza viruses is a major virulence factor and exerts its function through interacting with viral/cellular RNAs and proteins. In this study, we identified heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) as an interacting partner of NS1 proteins by a proteomic method. Knockdown of hnRNP A2/B1 by small interfering RNA (siRNA) resulted in higher levels of NS vRNA, NS1 mRNA, and NS1 protein in the virus-infected cells. In addition, we demonstrated that hnRNP A2/B1 proteins are associated with NS1 and NS2 mRNAs and that knockdown of hnRNP A2/B1 promotes transport of NS1 mRNA from the nucleus to the cytoplasm in the infected cells. Lastly, we showed that knockdown of hnRNP A2/B1 leads to enhanced virus replication. Our results suggest that hnRNP A2/B1 plays an inhibitory role in the replication of influenza A virus in host cells potentially through suppressing NS1 RNA/protein levels and NS1 mRNA nucleocytoplasmic translocation. - Highlights: • Cellular protein hnRNP A2/B1 interacts with influenza viral protein NS1. • hnRNP A2/B1 suppresses the levels of NS1 protein, vRNA and mRNA in infected cells. • hnRNP A2/B1 protein is associated with NS1 and NS2 mRNAs. • hnRNP A2/B1 inhibits the nuclear export of NS1 mRNAs. • hnRNP A2/B1 inhibits influenza virus replication.

  3. Whole-Genome Sequence of Clostridium botulinum A2B3 87, a Highly Virulent Strain Involved in a Fatal Case of Foodborne Botulism in Italy

    PubMed Central

    Giordani, Francesco; Fillo, Silvia; Anselmo, Anna; Palozzi, Anna Maria; Fortunato, Antonella; Gentile, Bernardina; Pittiglio, Valentina; Spagnolo, Ferdinando; Anniballi, Fabrizio; Fiore, Alfonsina; Auricchio, Bruna; De Medici, Dario

    2015-01-01

    Here, we report the genome sequence of a rare bivalent strain of Clostridium botulinum, A2B3 87. The strain was isolated from a foodborne botulism case that occurred in Italy in 1995. The case was characterized by rapid evolution of the illness and failure of conventional treatments. PMID:25814616

  4. The InhA2 Metalloprotease of Bacillus thuringiensis Strain 407 Is Required for Pathogenicity in Insects Infected via the Oral Route

    PubMed Central

    Fedhila, Sinda; Nel, Patricia; Lereclus, Didier

    2002-01-01

    The entomopathogenic bacterium Bacillus thuringiensis is known to secrete a zinc metalloprotease (InhA) that specifically cleaves antibacterial peptides produced by insect hosts. We identified a second copy of the inhA gene, named inhA2, in B. thuringiensis strain 407 Cry−. The inhA2 gene encodes a putative polypeptide showing 66.2% overall identity with the InhA protein and harboring the zinc-binding domain (HEXXH), which is characteristic of the zinc-requiring metalloproteases. We used a transcriptional inhA2′-lacZ fusion to show that inhA2 expression is induced at the onset of the stationary phase and is overexpressed in a Spo0A minus background. The presence of a reverse Spo0A box in the promoter region of inhA2 suggests that Spo0A directly regulates the transcription of inhA2. To determine the role of the InhA and InhA2 metalloproteases in pathogenesis, we used allelic exchange to isolate single and double mutant strains for the two genes. Spores and vegetative cells of the mutant strains were as virulent as those of the parental strain in immunized Bombyx mori larvae infected by the intrahemocoelic route. Exponential phase cells of all the strains displayed the same in vitro potential for colonizing the vaccinated hemocoel. We investigated the synergistic effect of the mutant strain spores on the toxicity of Cry1C proteins against Galleria mellonella larvae infected via the oral pathway. The spores of ΔinhA2 mutant strain were ineffective in providing synergism whereas those of the ΔinhA mutant strain were not. These results indicate that the B. thuringiensis InhA2 zinc metalloprotease has a vital role in virulence when the host is infected via the oral route. PMID:12029046

  5. hnRNP A2/B1 interacts with influenza A viral protein NS1 and inhibits virus replication potentially through suppressing NS1 RNA/protein levels and NS1 mRNA nuclear export.

    PubMed

    Wang, Yimeng; Zhou, Jianhong; Du, Yuchun

    2014-01-20

    The NS1 protein of influenza viruses is a major virulence factor and exerts its function through interacting with viral/cellular RNAs and proteins. In this study, we identified heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) as an interacting partner of NS1 proteins by a proteomic method. Knockdown of hnRNP A2/B1 by small interfering RNA (siRNA) resulted in higher levels of NS vRNA, NS1 mRNA, and NS1 protein in the virus-infected cells. In addition, we demonstrated that hnRNP A2/B1 proteins are associated with NS1 and NS2 mRNAs and that knockdown of hnRNP A2/B1 promotes transport of NS1 mRNA from the nucleus to the cytoplasm in the infected cells. Lastly, we showed that knockdown of hnRNP A2/B1 leads to enhanced virus replication. Our results suggest that hnRNP A2/B1 plays an inhibitory role in the replication of influenza A virus in host cells potentially through suppressing NS1 RNA/protein levels and NS1 mRNA nucleocytoplasmic translocation.

  6. Replicating vaccines

    USDA-ARS?s Scientific Manuscript database

    Early work on fish immunology and disease resistance demonstrated fish (like animals and humans) that survived infection were typically resistant to re-infection with the same pathogen. The concepts of resistance upon reinfection lead to the research and development of replicating (live) vaccines in...

  7. Development of plaque assays for hepatitis C virus-JFH1 strain and isolation of mutants with enhanced cytopathogenicity and replication capacity.

    PubMed

    Sekine-Osajima, Yuko; Sakamoto, Naoya; Mishima, Kako; Nakagawa, Mina; Itsui, Yasuhiro; Tasaka, Megumi; Nishimura-Sakurai, Yuki; Chen, Cheng-Hsin; Kanai, Takanori; Tsuchiya, Kiichiro; Wakita, Takaji; Enomoto, Nobuyuki; Watanabe, Mamoru

    2008-02-05

    HCV culture in vitro results in massive cell death, which suggests the presence of HCV-induced cytopathic effects. Therefore, we investigated its mechanisms and viral nucleotide sequences involved in this effect using HCV-JFH1 cell culture and a newly developed HCV plaque assay technique. The plaque assay developed cytopathic plaques, depending on the titer of the inoculum. In the virus-infected cells, the ER stress markers, GRP78 and phosphorylated eIF2-alpha, were overexpressed. Cells in the plaques were strongly positive for an apoptosis marker, annexin V. Isolated virus subclones from individual plaque showed greater replication efficiency and cytopathogenicity than the parental virus. The plaque-purified virus had 9 amino acid substitutions, of which 5 were clustered in the C terminal of the NS5B region. Taken together, the cytopathic effect of HCV infection involves ER-stress-induced apoptotic cell death. Certain HCV genomic structures may determine the viral replication capacity and cytopathogenicity.

  8. Analysis of a new strain of Euphorbia mosaic virus with distinct replication specificity unveils a lineage of begomoviruses with short Rep sequences in the DNA-B intergenic region

    PubMed Central

    2010-01-01

    Background Euphorbia mosaic virus (EuMV) is a member of the SLCV clade, a lineage of New World begomoviruses that display distinctive features in their replication-associated protein (Rep) and virion-strand replication origin. The first entirely characterized EuMV isolate is native from Yucatan Peninsula, Mexico; subsequently, EuMV was detected in weeds and pepper plants from another region of Mexico, and partial DNA-A sequences revealed significant differences in their putative replication specificity determinants with respect to EuMV-YP. This study was aimed to investigate the replication compatibility between two EuMV isolates from the same country. Results A new isolate of EuMV was obtained from pepper plants collected at Jalisco, Mexico. Full-length clones of both genomic components of EuMV-Jal were biolistically inoculated into plants of three different species, which developed symptoms indistinguishable from those induced by EuMV-YP. Pseudorecombination experiments with EuMV-Jal and EuMV-YP genomic components demonstrated that these viruses do not form infectious reassortants in Nicotiana benthamiana, presumably because of Rep-iteron incompatibility. Sequence analysis of the EuMV-Jal DNA-B intergenic region (IR) led to the unexpected discovery of a 35-nt-long sequence that is identical to a segment of the rep gene in the cognate viral DNA-A. Similar short rep sequences ranging from 35- to 51-nt in length were identified in all EuMV isolates and in three distinct viruses from South America related to EuMV. These short rep sequences in the DNA-B IR are positioned downstream to a ~160-nt non-coding domain highly similar to the CP promoter of begomoviruses belonging to the SLCV clade. Conclusions EuMV strains are not compatible in replication, indicating that this begomovirus species probably is not a replicating lineage in nature. The genomic analysis of EuMV-Jal led to the discovery of a subgroup of SLCV clade viruses that contain in the non-coding region of

  9. 180-Nucleotide Duplication in the G Gene of Human metapneumovirus A2b Subgroup Strains Circulating in Yokohama City, Japan, since 2014

    PubMed Central

    Saikusa, Miwako; Kawakami, Chiharu; Nao, Naganori; Takeda, Makoto; Usuku, Shuzo; Sasao, Tadayoshi; Nishimoto, Kimiko; Toyozawa, Takahiro

    2017-01-01

    Human metapneumovirus (HMPV), a member of the family Paramyxoviridae, was first isolated in 2001. Seroepidemiological studies have shown that HMPV has been a major etiological agent of acute respiratory infections in humans for more than 50 years. Molecular epidemiological, genetic, and antigenetic evolutionary studies of HMPV will strengthen our understanding of the epidemic behavior of the virus and provide valuable insight for the control of HMPV and the development of vaccines and antiviral drugs against HMPV infection. In this study, the nucleotide sequence of and genetic variations in the G gene were analyzed in HMPV strains prevalent in Yokohama City, in the Kanto area, Japan, between January 2013 and June 2016. As a part of the National Epidemiological Surveillance of Infectious Diseases, Japan, 1308 clinical specimens (throat swabs, nasal swabs, nasal secretions, and nasal aspirate fluids) collected at 24 hospitals or clinics in Yokohama City were screened for 15 major respiratory viruses with a multiplex reverse transcription–PCR assay. HMPV was detected in 91 specimens, accounting for 7.0% of the total specimens, and the nucleotide sequences of the G genes of 84 HMPV strains were determined. Among these 84 strains, 6, 43, 10, and 25 strains were classified into subgroups A2a, A2b, B1, and B2, respectively. Approximately half the HMPV A2b subgroup strains detected since 2014 had a 180-nucleotide duplication (180nt-dup) in the G gene and clustered on a phylogenic tree with four classical 180nt-dup-lacking HMPV A2b strains prevalent between 2014 and 2015. The 180nt-dup causes a 60-amino-acid duplication (60aa-dup) in the G protein, creating 23–25 additional potential acceptor sites for O-linked sugars. Our data suggest that 180nt-dup occurred between 2011 and 2013 and that HMPV A2b strains with 180nt-dup (A2b180nt-dup HMPV) became major epidemic strains within 3 years. The detailed mechanism by which the A2b180nt-dup HMPV strains gained an advantage

  10. Replicated spectrographs in astronomy

    NASA Astrophysics Data System (ADS)

    Hill, Gary J.

    2014-06-01

    As telescope apertures increase, the challenge of scaling spectrographic astronomical instruments becomes acute. The next generation of extremely large telescopes (ELTs) strain the availability of glass blanks for optics and engineering to provide sufficient mechanical stability. While breaking the relationship between telescope diameter and instrument pupil size by adaptive optics is a clear path for small fields of view, survey instruments exploiting multiplex advantages will be pressed to find cost-effective solutions. In this review we argue that exploiting the full potential of ELTs will require the barrier of the cost and engineering difficulty of monolithic instruments to be broken by the use of large-scale replication of spectrographs. The first steps in this direction have already been taken with the soon to be commissioned MUSE and VIRUS instruments for the Very Large Telescope and the Hobby-Eberly Telescope, respectively. MUSE employs 24 spectrograph channels, while VIRUS has 150 channels. We compare the information gathering power of these replicated instruments with the present state of the art in more traditional spectrographs, and with instruments under development for ELTs. Design principles for replication are explored along with lessons learned, and we look forward to future technologies that could make massively-replicated instruments even more compelling.

  11. Role of trypsin in the replication of Avian metapneumovirus subtype C (strain MN-2a) and its entry into the Vero cells.

    PubMed

    Paudel, Sarita; Shin, Hyun-Jin

    2015-12-01

    To understand the molecular mechanisms of Avian metapneumovirus (aMPV) and the requirements involved in the infection and fusion, trypsin treatment was done in the different stages of virus; before infection, during entry and after virus infection followed by aMPV infection. The growth kinetics of aMPV was compared in time dependent manner. The effect of trypsin was found in the later stage of aMPV infection increasing the numbers of infected cells with the significant higher titer of infectious virions to that of trypsin treated before infection, during entry and aMPV. A serine protease inhibitor reduced aMPV replication in a significant way, whereas cysteine peptidase (E-64), aspartic protease (pepstatin A), and metalloprotease (phosphoramidon) inhibitors had no effect on aMPV replication. Inoculation of aMPV on Vero cells expressing the membrane-associated protease TMPRSS2 resulted in higher virus titers than that inoculated on normal Vero cells and is statistically significant (p < 0.05). Also, an inhibitor of clathrin/caveolae-mediated endocytosis had no effect on virus progeny, indicating that aMPV does not use the endocytic pathway for entry but undergoes direct fusion. The effect of lysosomotropic agents was not significant, suggesting that aMPV does not require low-pH environment in endosomes to fuse its envelope with the plasma membrane.

  12. Expression of a single siRNA against a conserved region of NP gene strongly inhibits in vitro replication of different Influenza A virus strains of avian and swine origin.

    PubMed

    Stoppani, Elena; Bassi, Ivan; Dotti, Silvia; Lizier, Michela; Ferrari, Maura; Lucchini, Franco

    2015-08-01

    Influenza A virus is the principal agent responsible of the respiratory tract's infections in humans. Every year, highly pathogenic and infectious strains with new antigenic assets appear, making ineffective vaccines so far developed. The discovery of RNA interference (RNAi) opened the way to the progress of new promising drugs against Influenza A virus and also to the introduction of disease resistance traits in genetically modified animals. In this paper, we show that Madin-Darby Canine Kidney (MDCK) cell line expressing short hairpin RNAs (shRNAs) cassette, designed on a specific conserved region of the nucleoprotein (NP) viral genome, can strongly inhibit the viral replication of four viral strains sharing the target sequence, reducing the viral mRNA respectively to 2.5×10(-4), 7.5×10(-5), 1.7×10(-3), 1.9×10(-4) compared to the control, as assessed by real-time PCR. Moreover, we demonstrate that during the challenge with a viral strain bearing a single mismatch on the target sequence, although a weaker inhibition is observed, viral mRNA is still lowered down to 1.2×10(-3) folds in the shRNA-expressing clone compared to the control, indicating a broad potential use of this approach. In addition, we developed a highly predictive and fast screening test of siRNA sequences based on dual-luciferase assay, useful for the in vitro prediction of the potential effect of viral inhibition. In conclusion, these findings reveal new siRNA sequences able to inhibit Influenza A virus replication and provide a basis for the development of siRNAs as prophylaxis and therapy for influenza infection both in humans and animals. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Factors influencing the attenuation of serotype 1 Marek's disease virus by serial cell culture passage, and evaluation of attenuated strains for protection and replication

    USDA-ARS?s Scientific Manuscript database

    This study was to better understand factors that influenced the process of attenuation of Marek’s disease (MD) virus by serial passage in cell cultures. Three virulent (v) pathotype and 3 very virulent plus (vv+) pathotype strains were passed by 3 techniques up to 131 times and the passage level at...

  14. The DNA damage response and checkpoint adaptation in Saccharomyces cerevisiae: distinct roles for the replication protein A2 (Rfa2) N-terminus.

    PubMed

    Ghospurkar, Padmaja L; Wilson, Timothy M; Severson, Amber L; Klein, Sarah J; Khaku, Sakina K; Walther, André P; Haring, Stuart J

    2015-03-01

    In response to DNA damage, two general but fundamental processes occur in the cell: (1) a DNA lesion is recognized and repaired, and (2) concomitantly, the cell halts the cell cycle to provide a window of opportunity for repair to occur. An essential factor for a proper DNA-damage response is the heterotrimeric protein complex Replication Protein A (RPA). Of particular interest is hyperphosphorylation of the 32-kDa subunit, called RPA2, on its serine/threonine-rich amino (N) terminus following DNA damage in human cells. The unstructured N-terminus is often referred to as the phosphorylation domain and is conserved among eukaryotic RPA2 subunits, including Rfa2 in Saccharomyces cerevisiae. An aspartic acid/alanine-scanning and genetic interaction approach was utilized to delineate the importance of this domain in budding yeast. It was determined that the Rfa2 N-terminus is important for a proper DNA-damage response in yeast, although its phosphorylation is not required. Subregions of the Rfa2 N-terminus important for the DNA-damage response were also identified. Finally, an Rfa2 N-terminal hyperphosphorylation-mimetic mutant behaves similarly to another Rfa1 mutant (rfa1-t11) with respect to genetic interactions, DNA-damage sensitivity, and checkpoint adaptation. Our data indicate that post-translational modification of the Rfa2 N-terminus is not required for cells to deal with "repairable" DNA damage; however, post-translational modification of this domain might influence whether cells proceed into M-phase in the continued presence of unrepaired DNA lesions as a "last-resort" mechanism for cell survival. Copyright © 2015 by the Genetics Society of America.

  15. The DNA Damage Response and Checkpoint Adaptation in Saccharomyces cerevisiae: Distinct Roles for the Replication Protein A2 (Rfa2) N-Terminus

    PubMed Central

    Ghospurkar, Padmaja L.; Wilson, Timothy M.; Severson, Amber L.; Klein, Sarah J.; Khaku, Sakina K.; Walther, André P.; Haring, Stuart J.

    2015-01-01

    In response to DNA damage, two general but fundamental processes occur in the cell: (1) a DNA lesion is recognized and repaired, and (2) concomitantly, the cell halts the cell cycle to provide a window of opportunity for repair to occur. An essential factor for a proper DNA-damage response is the heterotrimeric protein complex Replication Protein A (RPA). Of particular interest is hyperphosphorylation of the 32-kDa subunit, called RPA2, on its serine/threonine-rich amino (N) terminus following DNA damage in human cells. The unstructured N-terminus is often referred to as the phosphorylation domain and is conserved among eukaryotic RPA2 subunits, including Rfa2 in Saccharomyces cerevisiae. An aspartic acid/alanine-scanning and genetic interaction approach was utilized to delineate the importance of this domain in budding yeast. It was determined that the Rfa2 N-terminus is important for a proper DNA-damage response in yeast, although its phosphorylation is not required. Subregions of the Rfa2 N-terminus important for the DNA-damage response were also identified. Finally, an Rfa2 N-terminal hyperphosphorylation-mimetic mutant behaves similarly to another Rfa1 mutant (rfa1-t11) with respect to genetic interactions, DNA-damage sensitivity, and checkpoint adaptation. Our data indicate that post-translational modification of the Rfa2 N-terminus is not required for cells to deal with “repairable” DNA damage; however, post-translational modification of this domain might influence whether cells proceed into M-phase in the continued presence of unrepaired DNA lesions as a “last-resort” mechanism for cell survival. PMID:25595672

  16. Identification of novel subgroup A variants with enhanced receptor binding and replicative capacity in primary isolates of anaemogenic strains of feline leukaemia virus.

    PubMed

    Stewart, Hazel; Adema, Karen W; McMonagle, Elizabeth L; Hosie, Margaret J; Willett, Brian J

    2012-05-31

    The development of anaemia in feline leukaemia virus (FeLV)-infected cats is associated with the emergence of a novel viral subgroup, FeLV-C. FeLV-C arises from the subgroup that is transmitted, FeLV-A, through alterations in the amino acid sequence of the receptor binding domain (RBD) of the envelope glycoprotein that result in a shift in the receptor usage and the cell tropism of the virus. The factors that influence the transition from subgroup A to subgroup C remain unclear, one possibility is that a selective pressure in the host drives the acquisition of mutations in the RBD, creating A/C intermediates with enhanced abilities to interact with the FeLV-C receptor, FLVCR. In order to understand further the emergence of FeLV-C in the infected cat, we examined primary isolates of FeLV-C for evidence of FeLV-A variants that bore mutations consistent with a gradual evolution from FeLV-A to FeLV-C. Within each isolate of FeLV-C, we identified variants that were ostensibly subgroup A by nucleic acid sequence comparisons, but which bore mutations in the RBD. One such mutation, N91D, was present in multiple isolates and when engineered into a molecular clone of the prototypic FeLV-A (Glasgow-1), enhanced replication was noted in feline cells. Expression of the N91D Env on murine leukaemia virus (MLV) pseudotypes enhanced viral entry mediated by the FeLV-A receptor THTR1 while soluble FeLV-A Env bearing the N91D mutation bound more efficiently to mouse or guinea pig cells bearing the FeLV-A and -C receptors. Long-term in vitro culture of variants bearing the N91D substitution in the presence of anti-FeLV gp70 antibodies did not result in the emergence of FeLV-C variants, suggesting that additional selective pressures in the infected cat may drive the subsequent evolution from subgroup A to subgroup C. Our data support a model in which variants of FeLV-A, bearing subtle differences in the RBD of Env, may be predisposed towards enhanced replication in vivo and subsequent

  17. CENTRIOLE REPLICATION

    PubMed Central

    Gall, Joseph G.

    1961-01-01

    This paper describes the replication of centrioles during spermatogenesis in the Prosobranch snail, Viviparus malleatus Reeve. Sections for electron microscopy were cut from pieces of testis fixed in OsO4 and embedded in the polyester resin Vestopal W. Two kinds of spermatocytes are present. These give rise to typical uniflagellate sperm carrying the haploid number of 9 chromosomes, and atypical multiflagellate sperm with only one chromosome. Two centrioles are present in the youngest typical spermatocyte. Each is a hollow cylinder about 160 mµ in diameter and 330 mµ long. The wall consists of 9 sets of triplet fibers arranged in a characteristic pattern. Sometime before pachytene an immature centriole, or procentriole as it will be called, appears next to each of the mature centrioles. The procentriole resembles a mature centriole in most respects except length: it is more annular than tubular. The daughter procentriole lies with its axis perpendicular to that of its parent. It presumably grows to full size during the late prophase, although the maturation stages have not been observed with the electron microscope. It is suggested that centrioles possess a constant polarization. The distal end forms the flagellum or other centriole products, while the proximal end represents the procentriole and is concerned with replication. The four centrioles of prophase (two parents and two daughters) are distributed by the two meiotic divisions to the four typical spermatids, in which they function as the basal bodies of the flagella. Atypical spermatocytes at first contain two normal centrioles. Each of these becomes surrounded by a cluster of procentrioles, which progressively elongate during the late prophase. After two aberrant meiotic divisions the centriole clusters give rise to the basal bodies of the multiflagellate sperm. These facts are discussed in the light of the theory, first proposed by Pollister, that the supernumerary centrioles in the atypical cells are

  18. A novel actinomycete strain de-replication approach based on the diversity of polyketide synthase and nonribosomal peptide synthetase biosynthetic pathways.

    PubMed

    Ayuso, Angel; Clark, Desmond; González, Ignacio; Salazar, Oscar; Anderson, Annaliesa; Genilloud, Olga

    2005-06-01

    The actinomycetes traditionally represent one of the most important sources for the discovery of new metabolites with biological activity; and many of these are described as being produced by polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS). We present a strain characterization system based on the metabolic potential of microbial strains by targeting these biosynthetic genes. After an initial evaluation of the existing bias derived from the PCR detection in a well defined biosynthetic systems, we developed a new fingerprinting approach based on the restriction analysis of these PKS and NRPS amplified sequences. This method was applied to study the distribution of PKS and NRPS biosynthetic systems in a collection of wild-type actinomycetes isolated from tropical soil samples that were evaluated for the production of antimicrobial activities. We discuss the application of this tool as an alternative characterization approach for actinomycetes and we comment on the relationship observed between the presence of PKS-I, PKS-II and NRPS sequences and the antimicrobial activities observed in some of the microbial groups tested.

  19. Expression of a recombinant protein, A2 family, from Leishmania infantum (Jaboticabal strain) and its evaluation in Canine Visceral Leishmaniasis serological test.

    PubMed

    Jusi, Márcia Mariza Gomes; Oliveira, Trícia Maria Ferreira de Sousa; Nakaghi, Andréa Cristina Higa; André, Marcos Rogério; Machado, Rosangela Zacarias

    2015-01-01

    This study aimed to express a recombinant A2 family protein of Leishmania chagasi, Jaboticabal strain; test this protein as an antigen in serological assays; and investigate its antigenicity and immunogenicity. A protein coded by an allele of the A2 gene isolated from L. chagasi was expressed in three different strains of Escherichia coli. We used 29 sera samples from Leishmune-vaccinated dogs, 482 sera samples from dogs from endemic areas (positive controls), and 170 sera samples from dogs from non-endemic areas (negative controls) in ELISA tests using soluble Leishmaniaantigen (SLA) and His-A2 as antigen. Expressed proteins showed, by western blotting, the expression of an 11 KDa protein. Sixty-three percent (303/482) of the samples from endemic areas were positive by ELISA His-A2, whereas 93.1% (27/29) of Leishmune®-vaccinated animals were negative by His-A2-ELISA. Anti-A2 antibodies from mice inoculated with the A2 protein were detected in slides containing amastigote forms, but not in slides containing promastigote forms. The A2 recombinant protein from L. chagasi may be a useful tool in the diagnosis of CVL, and further tests regarding the infection stage and the specie of parasite at which the dogs are sampled should provide a better understanding of our results.

  20. Transverse Strains in Muscle Fascicles during Voluntary Contraction: A 2D Frequency Decomposition of B-Mode Ultrasound Images

    PubMed Central

    Wakeling, James M.

    2014-01-01

    When skeletal muscle fibres shorten, they must increase in their transverse dimensions in order to maintain a constant volume. In pennate muscle, this transverse expansion results in the fibres rotating to greater pennation angle, with a consequent reduction in their contractile velocity in a process known as gearing. Understanding the nature and extent of this transverse expansion is necessary to understand the mechanisms driving the changes in internal geometry of whole muscles during contraction. Current methodologies allow the fascicle lengths, orientations, and curvatures to be quantified, but not the transverse expansion. The purpose of this study was to develop and validate techniques for quantifying transverse strain in skeletal muscle fascicles during contraction from B-mode ultrasound images. Images were acquired from the medial and lateral gastrocnemii during cyclic contractions, enhanced using multiscale vessel enhancement filtering and the spatial frequencies resolved using 2D discrete Fourier transforms. The frequency information was resolved into the fascicle orientations that were validated against manually digitized values. The transverse fascicle strains were calculated from their wavelengths within the images. These methods showed that the transverse strain increases while the longitudinal fascicle length decreases; however, the extent of these strains was smaller than expected. PMID:25328509

  1. Molecular replication

    NASA Technical Reports Server (NTRS)

    Orgel, L. E.

    1986-01-01

    The object of our research program is to understand how polynucleotide replication originated on the primitive Earth. This is a central issue in studies of the origins of life, since a process similar to modern DNA and RNA synthesis is likely to have formed the basis for the most primitive system of genetic information transfer. The major conclusion of studies so far is that a preformed polynucleotide template under many different experimental conditions will facilitate the synthesis of a new oligonucleotide with a sequence complementary to that of the template. It has been shown, for example, that poly(C) facilitates the synthesis of long oligo(G)s and that the short template CCGCC facilities the synthesis of its complement GGCGG. Very recently we have shown that template-directed synthesis is not limited to the standard oligonucleotide substrates. Nucleic acid-like molecules with a pyrophosphate group replacing the phosphate of the standard nucleic acid backbone are readily synthesized from deoxynucleotide 3'-5'-diphosphates on appropriate templates.

  2. Molecular replication

    NASA Technical Reports Server (NTRS)

    Orgel, L. E.

    1986-01-01

    The object of our research program is to understand how polynucleotide replication originated on the primitive Earth. This is a central issue in studies of the origins of life, since a process similar to modern DNA and RNA synthesis is likely to have formed the basis for the most primitive system of genetic information transfer. The major conclusion of studies so far is that a preformed polynucleotide template under many different experimental conditions will facilitate the synthesis of a new oligonucleotide with a sequence complementary to that of the template. It has been shown, for example, that poly(C) facilitates the synthesis of long oligo(G)s and that the short template CCGCC facilities the synthesis of its complement GGCGG. Very recently we have shown that template-directed synthesis is not limited to the standard oligonucleotide substrates. Nucleic acid-like molecules with a pyrophosphate group replacing the phosphate of the standard nucleic acid backbone are readily synthesized from deoxynucleotide 3'-5'-diphosphates on appropriate templates.

  3. COMPARISON OF OVERALL METABOLISM OF 1, 2, 7, 8-PECDD IN CYP1A2(-L-) KNOCKOUT AND C57BL/6N PARENTAL STRAINS OF MICE

    EPA Science Inventory

    COMPARISON OF OVERALL METABOLISM OF 1,2,3,7,8-PeCDD
    IN CYP1A2 (-/-) KNOCKOUT AND C57BL/6N PARENTAL
    STRAINS OF MICE

    Heldur Hakk1 and Janet J. Diliberto2

    1 USDA-ARS, Biosciences Research Laboratory, P.O. Box 5674, Fargo, ND, USA
    2 US EPA, ORD, National Heal...

  4. Complete Genome Sequence of the Multiresistant Acinetobacter baumannii Strain AbH12O-A2, Isolated during a Large Outbreak in Spain

    PubMed Central

    Merino, M.; Alvarez-Fraga, L.; Gómez, M. J.; Aransay, A. M.; Lavín, J. L.; Chaves, F.

    2014-01-01

    We report the complete genome sequence of Acinetobacter baumannii strain AbH12O-A2, isolated during a large outbreak in Spain. The genome has 3,875,775 bp and 3,526 coding sequences, with 39.4% G+C content. The availability of this genome will facilitate the study of the pathogenicity of the Acinetobacter species. PMID:25395646

  5. COMPARISON OF OVERALL METABOLISM OF 1, 2, 7, 8-PECDD IN CYP1A2(-L-) KNOCKOUT AND C57BL/6N PARENTAL STRAINS OF MICE

    EPA Science Inventory

    COMPARISON OF OVERALL METABOLISM OF 1,2,3,7,8-PeCDD
    IN CYP1A2 (-/-) KNOCKOUT AND C57BL/6N PARENTAL
    STRAINS OF MICE

    Heldur Hakk1 and Janet J. Diliberto2

    1 USDA-ARS, Biosciences Research Laboratory, P.O. Box 5674, Fargo, ND, USA
    2 US EPA, ORD, National Heal...

  6. Replication of Vibrio cholerae classical CTX phage.

    PubMed

    Kim, Eun Jin; Yu, Hyun Jin; Lee, Je Hee; Kim, Jae-Ouk; Han, Seung Hyun; Yun, Cheol-Heui; Chun, Jongsik; Nair, G Balakrish; Kim, Dong Wook

    2017-02-28

    The toxigenic classical and El Tor biotype Vibrio cholerae serogroup O1 strains are generated by lysogenization of host-type-specific cholera toxin phages (CTX phages). Experimental evidence of the replication and transmission of an El Tor biotype-specific CTX phage, CTX-1, has explained the evolution of V. cholerae El Tor biotype strains. The generation of classical biotype strains has not been demonstrated in the laboratory, and the classical biotype-specific CTX phage, CTX-cla, is considered to be defective with regard to replication. However, the identification of atypical El Tor strains that contain CTX-cla-like phage, CTX-2, indicates that CTX-cla and CTX-2 replicate and can be transmitted to V. cholerae strains. The replication of CTX-cla and CTX-2 phages and the transduction of El Tor biotype strains by various CTX phages under laboratory conditions are demonstrated in this report. We have established a plasmid-based CTX phage replication system that supports the replication of CTX-1, CTX-cla, CTX-2, and CTX-O139. The replication of CTX-2 from the tandem repeat of lysogenic CTX-2 in Wave 2 El Tor strains is also presented. El Tor biotype strains can be transduced by CTX phages in vitro by introducing a point mutation in toxT, the transcriptional activator of the tcp (toxin coregulated pilus) gene cluster and the cholera toxin gene. This mutation also increases the expression of cholera toxin in El Tor strains in a sample single-phase culture. Our results thus constitute experimental evidence of the genetic mechanism of the evolution of V. cholerae.

  7. Frequency of vacA, cagA and babA2 virulence markers in Helicobacter pylori strains isolated from Mexican patients with chronic gastritis

    PubMed Central

    Paniagua, Gloria Luz; Monroy, Eric; Rodríguez, Raymundo; Arroniz, Salvador; Rodríguez, Cristina; Cortés, José Luis; Camacho, Ausencio; Negrete, Erasmo; Vaca, Sergio

    2009-01-01

    Background Helicobacter pylori has been strongly associated with chronic gastritis, peptic and duodenal ulcers, and it is a risk factor for gastric cancer. Three major virulence factors of H. pylori have been described: the vacuolating toxin (VacA), the cytotoxin-associated gene product (CagA) and the adhesion protein BabA2. Since considerable geographic diversity in the prevalence of H. pylori virulence factors has been reported, the aim of this work was to establish the H. pylori and vacA, cagA and babA2 gene status in 238 adult patients, from a marginal urban area of Mexico, with chronic gastritis. Methods H. pylori was identified in cultures of gastric biopsies by nested PCR. vacA and cagA genes were detected by multiplex PCR, whereas babA2 gene was identified by conventional PCR. Results H. pylori-positive biopsies were 143 (60.1%). All H. pylori strains were vacA+; 39.2% were cagA+; 13.3% were cagA+ babA2+ and 8.4% were babA2+. Mexican strains examined possessed the vacA s1, m1 (43.4%), s1, m2 (24.5%), s2, m1 (20.3%) and s2, m2 (11.9%) genotypes. Conclusion These results show that the Mexican patients suffering chronic gastritis we have studied had a high incidence of infection by H. pylori. Forty four percent (63/143) of the H. pylori strains analyzed in this work may be considered as highly virulent since they possessed two or three of the virulence markers analyzed: vacA s1 cagA babA2 (9.8%, 14/143), vacA s1 babA2 (4.9%, 7/143), and vacA s1 cagA (29.4%, 42/143). However, a statistically significant correlation was not observed between vacAs1, cagA and babA2 virulence markers (χ2 test; P > 0.05). PMID:19405980

  8. Frequency of vacA, cagA and babA2 virulence markers in Helicobacter pylori strains isolated from Mexican patients with chronic gastritis.

    PubMed

    Paniagua, Gloria Luz; Monroy, Eric; Rodríguez, Raymundo; Arroniz, Salvador; Rodríguez, Cristina; Cortés, José Luis; Camacho, Ausencio; Negrete, Erasmo; Vaca, Sergio

    2009-04-30

    Helicobacter pylori has been strongly associated with chronic gastritis, peptic and duodenal ulcers, and it is a risk factor for gastric cancer. Three major virulence factors of H. pylori have been described: the vacuolating toxin (VacA), the cytotoxin-associated gene product (CagA) and the adhesion protein BabA2. Since considerable geographic diversity in the prevalence of H. pylori virulence factors has been reported, the aim of this work was to establish the H. pylori and vacA, cagA and babA2 gene status in 238 adult patients, from a marginal urban area of Mexico, with chronic gastritis. H. pylori was identified in cultures of gastric biopsies by nested PCR. vacA and cagA genes were detected by multiplex PCR, whereas babA2 gene was identified by conventional PCR. H. pylori-positive biopsies were 143 (60.1%). All H. pylori strains were vacA+; 39.2% were cagA+; 13.3% were cagA+ babA2+ and 8.4% were babA2+. Mexican strains examined possessed the vacA s1, m1 (43.4%), s1, m2 (24.5%), s2, m1 (20.3%) and s2, m2 (11.9%) genotypes. These results show that the Mexican patients suffering chronic gastritis we have studied had a high incidence of infection by H. pylori. Forty four percent (63/143) of the H. pylori strains analyzed in this work may be considered as highly virulent since they possessed two or three of the virulence markers analyzed: vacA s1 cagA babA2 (9.8%, 14/143), vacA s1 babA2 (4.9%, 7/143), and vacA s1 cagA (29.4%, 42/143). However, a statistically significant correlation was not observed between vacAs1, cagA and babA2 virulence markers (chi2 test; P > 0.05).

  9. Archaeal DNA replication.

    PubMed

    Kelman, Lori M; Kelman, Zvi

    2014-01-01

    DNA replication is essential for all life forms. Although the process is fundamentally conserved in the three domains of life, bioinformatic, biochemical, structural, and genetic studies have demonstrated that the process and the proteins involved in archaeal DNA replication are more similar to those in eukaryal DNA replication than in bacterial DNA replication, but have some archaeal-specific features. The archaeal replication system, however, is not monolithic, and there are some differences in the replication process between different species. In this review, the current knowledge of the mechanisms governing DNA replication in Archaea is summarized. The general features of the replication process as well as some of the differences are discussed.

  10. Cytoplasmic Viral Replication Complexes

    PubMed Central

    den Boon, Johan A.; Diaz, Arturo; Ahlquist, Paul

    2010-01-01

    Many viruses that replicate in the cytoplasm compartmentalize their genome replication and transcription in organelle-like structures that enhance replication efficiency and protection from host defenses. In particular, recent studies with diverse positive-strand RNA viruses have further elucidated the ultrastructure of membrane-bounded RNA replication complexes and their close coordination with virion assembly and budding. The structure, function and assembly of some positive-strand RNA virus replication complexes have parallels and potential evolutionary links with the replicative cores of double-strand RNA virus and retrovirus virions, and more general similarities with the replication factories of cytoplasmic DNA viruses. PMID:20638644

  11. cis Expression of the F12 Human Immunodeficiency Virus (HIV) Nef Allele Transforms the Highly Productive NL4-3 HIV Type 1 to a Replication-Defective Strain: Involvement of both Env gp41 and CD4 Intracytoplasmic Tails

    PubMed Central

    Olivetta, Eleonora; Pugliese, Katherina; Bona, Roberta; D'Aloja, Paola; Ferrantelli, Flavia; Santarcangelo, Anna Claudia; Mattia, Gianfranco; Verani, Paola; Federico, Maurizio

    2000-01-01

    F12 human immunodeficiency virus type 1 (HIV-1) nef is a naturally occurring nef mutant cloned from the provirus of a nonproductive, nondefective, and interfering HIV-1 variant (F12-HIV). We have already shown that cells stably transfected with a vector expressing the F12-HIV nef allele do not downregulate CD4 receptors and, more peculiarly, become resistant to the replication of wild type (wt) HIV. In order to investigate the mechanism of action of such an HIV inhibition, the F12-HIV nef gene was expressed in the context of the NL4-3 HIV-1 infectious molecular clone by replacing the wt nef gene (NL4-3/chi). Through this experimental approach we established the following. First, NL4-3/chi and nef-defective (Δnef) NL4-3 viral particles behave very similarly in terms of viral entry and HIV protein production during the first replicative cycle. Second, no viral particles were produced from cells infected with NL4-3/chi virions, whatever the multiplicity of infection used. The viral inhibition apparently occurs at level of viral assembling and/or release. Third, this block could not be relieved by in-trans expression of wt nef. Finally, NL4-3/chi reverts to a producer HIV strain when F12-HIV Nef is deprived of its myristoyl residue. Through a CD4 downregulation competition assay, we demonstrated that F12-HIV Nef protein potently inhibits the CD4 downregulation induced by wt Nef. Moreover, we observed a redistribution of CD4 receptors at the cell margin induced by F12-HIV Nef. These observations strongly suggest that F12-HIV Nef maintains the ability to interact with the intracytoplasmic tail of the CD4 receptor molecule. Remarkably, we distinguished the intracytoplasmic tails of Env gp41 and CD4 as, respectively, viral and cellular targets of the F12-HIV Nef-induced viral retention. For the first time, the inhibition of the viral life cycle by means of in-cis expression of a Nef mutant is here reported. Delineation of the F12-HIV Nef mechanism of action may offer

  12. Pulsed-field gel electrophoresis (PFGE) typing of Listeria strains isolated from a meat processing plant over a 2-year period.

    PubMed

    Senczek, D; Stephan, R; Untermann, F

    2000-12-05

    As part of a hygiene monitoring program in a meat processing plant a total of 131 Listeria isolates were detected by sampling different processing areas and meat products within a 2-year period. The isolates were differentiated by means of phenotypic characteristics. Furthermore, the genomic ApaI and SmaI fragment patterns of all isolates were examined by pulsed-field gel electrophoresis (PFGE). PFGE using SmaI and ApaI yielded 15 (Listeria monocytogenes), 20 (Listeria innocua) and six (Listeria welshimeri) pulsotypes. Of the environmental Listeria monocytogenes isolates the predominating PFGE-type B was clearly associated with processing area A whereas PFGE-type E predominated in the meat products. Moreover, the study showed the persistence of closely related Listeria strains over a 2-year period in the environment of this meat processing plant.

  13. Replication of Tobamovirus RNA.

    PubMed

    Ishibashi, Kazuhiro; Ishikawa, Masayuki

    2016-08-04

    Tobacco mosaic virus and other tobamoviruses have served as models for studying the mechanisms of viral RNA replication. In tobamoviruses, genomic RNA replication occurs via several steps: (a) synthesis of viral replication proteins by translation of the genomic RNA; (b) translation-coupled binding of the replication proteins to a 5'-terminal region of the genomic RNA; (c) recruitment of the genomic RNA by replication proteins onto membranes and formation of a complex with host proteins TOM1 and ARL8; (d) synthesis of complementary (negative-strand) RNA in the complex; and (e) synthesis of progeny genomic RNA. This article reviews current knowledge on tobamovirus RNA replication, particularly regarding how the genomic RNA is specifically selected as a replication template and how the replication proteins are activated. We also focus on the roles of the replication proteins in evading or suppressing host defense systems.

  14. Genetically engineered Oenococcus oeni strains to highlight the impact of estA2 and estA7 esterase genes on wine ester profile.

    PubMed

    Darsonval, M; Alexandre, H; Grandvalet, C

    2016-12-01

    Besides deacidifying wine, Oenococcus oeni bring significant changes in the chemical composition of wine by releasing esters by the action of their own esterases. The impact of O. oeni esterases remains relatively unexplored. Four esterase genes were identified from O. oeni genome (estA2, estA7, estC, and estB). The dual objective of this study was, first to use a genetic tool enabling the expression of esterase genes in enological conditions and, second, to investigate the impact of O. oeni esterase gene expression during winemaking on wine aromatic profile. Both estA2 and estA7 genes were successfully cloned and expressed in O. oeni and recombinant strains were inoculated in Aligoté wine to initiate malolactic fermentation (MLF). Ester profile of experimental wine was established by SPME-GC-MS. EstA2 caused significant decreases in the concentrations of isoamyl acetate, ethyl hexanoate, isobutyl acetate, and hexyl acetate, by 42.7%, 23.4%, 51.5%, and 28.9%, respectively. EstA2 has preferential hydrolytic activity toward acetate esters from higher alcohols. EstA7 has synthetic activity toward hexyl acetate with a significant 22.7% increase. This study reports the first efficient expression system enabling the production of a functional protein in O. oeni in enological conditions.

  15. Replication Restart in Bacteria.

    PubMed

    Michel, Bénédicte; Sandler, Steven J

    2017-07-01

    In bacteria, replication forks assembled at a replication origin travel to the terminus, often a few megabases away. They may encounter obstacles that trigger replisome disassembly, rendering replication restart from abandoned forks crucial for cell viability. During the past 25 years, the genes that encode replication restart proteins have been identified and genetically characterized. In parallel, the enzymes were purified and analyzed in vitro, where they can catalyze replication initiation in a sequence-independent manner from fork-like DNA structures. This work also revealed a close link between replication and homologous recombination, as replication restart from recombination intermediates is an essential step of DNA double-strand break repair in bacteria and, conversely, arrested replication forks can be acted upon by recombination proteins and converted into various recombination substrates. In this review, we summarize this intense period of research that led to the characterization of the ubiquitous replication restart protein PriA and its partners, to the definition of several replication restart pathways in vivo, and to the description of tight links between replication and homologous recombination, responsible for the importance of replication restart in the maintenance of genome stability. Copyright © 2017 American Society for Microbiology.

  16. Novel frame-shift mutation in Slc5a2 encoding SGLT2 in a strain of senescence-accelerated mouse SAMP10.

    PubMed

    Unno, Keiko; Yamamoto, Hiroyuki; Toda, Masateru; Hagiwara, Shiori; Iguchi, Kazuaki; Hoshino, Minoru; Takabayashi, Fumiyo; Hasegawa-Ishii, Sanae; Shimada, Atsuyoshi; Hosokawa, Masanori; Higuchi, Keiichi; Mori, Masayuki

    2014-11-07

    The senescence-accelerated mouse prone10 (SAMP10) strain, a model of aging, exhibits cognitive impairments and cerebral atrophy. We noticed that SAMP10/TaSlc mice, a SAMP10 substrain, have developed persistent glucosuria over the past few years. In the present study, we characterized SAMP10/TaSlc mice and further identified a spontaneous mutation in the Slc5a2 gene encoding sodium-glucose co-transporter (SGLT) 2. The mean concentration of urine glucose was high in SAMP10/TaSlc mice and increased further with advancing age, whereas other strains of senescence-accelerated mice, including SAMP1/SkuSlc, SAMP6/TaSlc and SAMP8/TaSlc or normal aging control SAMR1/TaSlc mice, exhibited no detectable glucose in urine. SAMP10/TaSlc mice consumed increasing amounts of food and water compared to SAMR1/TaSlc mice, suggesting the compensation of polyuria and the loss of glucose. Oral glucose tolerance tests showed decreased glucose reabsorption in the kidney of SAMP10/TaSlc mice. In addition, blood glucose levels decreased in an age-dependent fashion. The kidney was innately larger than that of control mice with no histological alterations. We examined the expression levels of glucose transporters in the kidney. Among SGLT1, SGLT2, glucose transporter (GLUT) 1 and GLUT2, we found a significant decrease only in the level of SGLT2. DNA sequencing of SGLT2 in SAMP10/TaSlc mice revealed a single nucleotide deletion of guanine at 1236, which resulted in a frameshift mutation that produced a truncated protein. We designate this strain as SAMP10/TaSlc-Slc5a2(slc) (SAMP10-ΔSglt2). Recently, SGLT2 inhibitors have been demonstrated to be effective for the treatment of patients with type 2 diabetes (T2D). SAMP10-ΔSglt2 mice may serve as a unique preclinical model to study the link between aging-related neurodegenerative disorders and T2D. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Ultima Replicated Optics Research

    NASA Technical Reports Server (NTRS)

    Hadaway, James; Engelhaupt, Darell

    1997-01-01

    Designs are reviewed incorporating processes suitable for replication of precision spherical segments of very large (greater than 20 meter diameter) telescopes combining ultra-lightweight and high precision. These designs must be amenable to assembly and alignment after deployment . The methods considered lie outside the present scope of fabrication, deployment and alignment considered to date. Design guidelines for reducing the weight and low frequency resonance in low G environment were given by The Serius Group, Dr. Glenn Zeiders, and are considered baseline for this activity. The goal of a rigid design of 10 Kg/sq M is being persued for the Next Generation Space Telescope (NGST) and is not likely adequate for advanced efforts. Flexures have been considered for maintaining the figure of many lightweight structures by control loop processes. This adds to the complexity and weight to the extent that it becomes difficult to recover the benefits. Two fabrication guidelines lead to a stiffer and concurrently lighter structure. First the use of thin vertical wall triangular structural reinforcements to increase the resistance to bending is preferred over hexagonal or square similar sections. Secondly, the incorporation of a similar back sheet on a cellular structure markedly improves the geometric stiffness. Neither improves the short range stiffness. Also often overlooked is that selected material properties must include high microyield and low hysteresis in addition to high elastic modulus to weight (stiffness). The fabrication steps can easily exceed the strain requirement.

  18. Salivaricin A2 and the novel lantibiotic salivaricin B are encoded at adjacent loci on a 190-kilobase transmissible megaplasmid in the oral probiotic strain Streptococcus salivarius K12.

    PubMed

    Hyink, Otto; Wescombe, Philip A; Upton, Mathew; Ragland, Nancy; Burton, Jeremy P; Tagg, John R

    2007-02-01

    The commercial probiotic Streptococcus salivarius strain K12 is the prototype of those S. salivarius strains that are the most strongly inhibitory in a standardized test of streptococcal bacteriocin production and has been shown to produce the 2,368-Da salivaricin A2 (SalA2) and the 2,740-Da salivaricin B (SboB) lantibiotics. The previously uncharacterized SboB belongs to the type AII class of lantibiotic bacteriocins and is encoded by an eight-gene cluster. The genetic loci encoding SalA2 and SboB in strain K12 have been fully characterized and are localized to nearly adjacent sites on pSsal-K12, a 190-kb megaplasmid. Of 61 strongly inhibitory strains of S. salivarius, 19 (31%) were positive for the sboB structural gene. All but one (strain NR) of these 19 strains were also positive for salA2, and in each of these cases of double positivity, the two loci were separated by fewer than 10 kb. This is the first report of a single streptococcus strain producing two distinct lantibiotics.

  19. The evolution of replicators.

    PubMed

    Szathmáry, E

    2000-11-29

    Replicators of interest in chemistry, biology and culture are briefly surveyed from a conceptual point of view. Systems with limited heredity have only a limited evolutionary potential because the number of available types is too low. Chemical cycles, such as the formose reaction, are holistic replicators since replication is not based on the successive addition of modules. Replicator networks consisting of catalytic molecules (such as reflexively autocatalytic sets of proteins, or reproducing lipid vesicles) are hypothetical ensemble replicators, and their functioning rests on attractors of their dynamics. Ensemble replicators suffer from the paradox of specificity: while their abstract feasibility seems to require a high number of molecular types, the harmful effect of side reactions calls for a small system size. No satisfactory solution to this problem is known. Phenotypic replicators do not pass on their genotypes, only some aspects of the phenotype are transmitted. Phenotypic replicators with limited heredity include genetic membranes, prions and simple memetic systems. Memes in human culture are unlimited hereditary, phenotypic replicators, based on language. The typical path of evolution goes from limited to unlimited heredity, and from attractor-based to modular (digital) replicators.

  20. The evolution of replicators.

    PubMed Central

    Szathmáry, E

    2000-01-01

    Replicators of interest in chemistry, biology and culture are briefly surveyed from a conceptual point of view. Systems with limited heredity have only a limited evolutionary potential because the number of available types is too low. Chemical cycles, such as the formose reaction, are holistic replicators since replication is not based on the successive addition of modules. Replicator networks consisting of catalytic molecules (such as reflexively autocatalytic sets of proteins, or reproducing lipid vesicles) are hypothetical ensemble replicators, and their functioning rests on attractors of their dynamics. Ensemble replicators suffer from the paradox of specificity: while their abstract feasibility seems to require a high number of molecular types, the harmful effect of side reactions calls for a small system size. No satisfactory solution to this problem is known. Phenotypic replicators do not pass on their genotypes, only some aspects of the phenotype are transmitted. Phenotypic replicators with limited heredity include genetic membranes, prions and simple memetic systems. Memes in human culture are unlimited hereditary, phenotypic replicators, based on language. The typical path of evolution goes from limited to unlimited heredity, and from attractor-based to modular (digital) replicators. PMID:11127914

  1. Enhanced Viral Replication by Cellular Replicative Senescence

    PubMed Central

    Kim, Ji-Ae; Seong, Rak-Kyun

    2016-01-01

    Cellular replicative senescence is a major contributing factor to aging and to the development and progression of aging-associated diseases. In this study, we sought to determine viral replication efficiency of influenza virus (IFV) and Varicella Zoster Virus (VZV) infection in senescent cells. Primary human bronchial epithelial cells (HBE) or human dermal fibroblasts (HDF) were allowed to undergo numbers of passages to induce replicative senescence. Induction of replicative senescence in cells was validated by positive senescence-associated β-galactosidase staining. Increased susceptibility to both IFV and VZV infection was observed in senescent HBE and HDF cells, respectively, resulting in higher numbers of plaque formation, along with the upregulation of major viral antigen expression than that in the non-senescent cells. Interestingly, mRNA fold induction level of virus-induced type I interferon (IFN) was attenuated by senescence, whereas IFN-mediated antiviral effect remained robust and potent in virus-infected senescent cells. Additionally, we show that a longevity-promoting gene, sirtuin 1 (SIRT1), has antiviral role against influenza virus infection. In conclusion, our data indicate that enhanced viral replication by cellular senescence could be due to senescence-mediated reduction of virus-induced type I IFN expression. PMID:27799874

  2. Prelife catalysts and replicators

    PubMed Central

    Ohtsuki, Hisashi; Nowak, Martin A.

    2009-01-01

    Life is based on replication and evolution. But replication cannot be taken for granted. We must ask what there was prior to replication and evolution. How does evolution begin? We have proposed prelife as a generative system that produces information and diversity in the absence of replication. We model prelife as a binary soup of active monomers that form random polymers. ‘Prevolutionary’ dynamics can have mutation and selection prior to replication. Some sequences might have catalytic activity, thereby enhancing the rates of certain prelife reactions. We study the selection criteria for these prelife catalysts. Their catalytic efficiency must be above certain critical values. We find a maintenance threshold and an initiation threshold. The former is a linear function of sequence length, and the latter is an exponential function of sequence length. Therefore, it is extremely hard to select for prelife catalysts that have long sequences. We compare prelife catalysis with a simple model for replication. Assuming fast template-based elongation reactions, we can show that replicators have selection thresholds that are independent of their sequence length. Our calculation demonstrates the efficiency of replication and provides an explanation of why replication was selected over other forms of prelife catalysis. PMID:19692408

  3. CHARACTERIZATION AND NUCLEOTIDE SEQUENCE DETERMINATION OF A REPEAT ELEMENT ISOLATED FROM A 2,4,5,-T DEGRADING STRAIN OF PSEUDOMONAS CEPACIA

    EPA Science Inventory

    Pseudomonas cepacia strain AC1100, capable of growth on 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), was mutated to the 2,4,5-T− strain PT88 by a ColE1 :: Tn5 chromosomal insertion. Using cloned DNA from the region flanking the insertion, a 1477-bp sequence (designated RS1100) wa...

  4. CHARACTERIZATION AND NUCLEOTIDE SEQUENCE DETERMINATION OF A REPEAT ELEMENT ISOLATED FROM A 2,4,5,-T DEGRADING STRAIN OF PSEUDOMONAS CEPACIA

    EPA Science Inventory

    Pseudomonas cepacia strain AC1100, capable of growth on 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), was mutated to the 2,4,5-T− strain PT88 by a ColE1 :: Tn5 chromosomal insertion. Using cloned DNA from the region flanking the insertion, a 1477-bp sequence (designated RS1100) wa...

  5. Who Needs Replication?

    ERIC Educational Resources Information Center

    Porte, Graeme

    2013-01-01

    In this paper, the editor of a recent Cambridge University Press book on research methods discusses replicating previous key studies to throw more light on their reliability and generalizability. Replication research is presented as an accepted method of validating previous research by providing comparability between the original and replicated…

  6. Project New Pride: Replication.

    ERIC Educational Resources Information Center

    National Inst. for Juvenile Justice and Delinquency Prevention (Dept. of Justice/LEAA), Washington, DC.

    The Office of Juvenile Justice and Delinquency Prevention, Law Enforcement Assistance Administration, is establishing a new discretionary grant program entitled Replication of Project New Pride: A Serious Offender Youth Treatment Program. Project New Pride was chosen for replication because of its demonstrated effectiveness in Denver, Colorado,…

  7. Thermal Replication Trap

    NASA Astrophysics Data System (ADS)

    Braun, Dieter

    2011-03-01

    The hallmark of living matter is the replication of genetic molecules and their active storage against diffusion. We have argued in the past that thermal convection can host the million-fold accumulation even of single nucleotides and at the same time trigger exponential replication. Accumulation is driven by thermophoresis and convection in elongated chambers, replication by the inherent temperature cycling in convection. Optothermal pumping [2,3] allows to implement the thermal trap efficiently in a toroidal or linear geometry. Based on this method, we were in a position to combine accumulation and replication of DNA in the same chamber. As we are missing a solid chemistry of prebiotic replication, we used as a proxy reaction for to replication the polymerase chain reaction. Convective flow both drives the DNA replicating polymerase chain reaction (PCR) while concurrent thermophoresis accumulates the replicated 143 base pair DNA in bulk solution. The time constant for accumulation is 92 s while DNA is doubled every 50 s. The length of the amplified DNA is checked with thermophoresis. Finite element simulations confirm the findings. The experiments explore conditions in pores of hydrothermal rock which can serve as a model environment for the origin of life and has prospects towards the first autonomous evolution, hosting the Darwin process by molecular selection using the thermophoretic trap. On the other side, the implemented continuous evolution will be able to breed well specified DNA or RNA molecules in the future.

  8. DNA Virus Replication Compartments

    PubMed Central

    Schmid, Melanie; Speiseder, Thomas; Dobner, Thomas

    2014-01-01

    Viruses employ a variety of strategies to usurp and control cellular activities through the orchestrated recruitment of macromolecules to specific cytoplasmic or nuclear compartments. Formation of such specialized virus-induced cellular microenvironments, which have been termed viroplasms, virus factories, or virus replication centers, complexes, or compartments, depends on molecular interactions between viral and cellular factors that participate in viral genome expression and replication and are in some cases associated with sites of virion assembly. These virus-induced compartments function not only to recruit and concentrate factors required for essential steps of the viral replication cycle but also to control the cellular mechanisms of antiviral defense. In this review, we summarize characteristic features of viral replication compartments from different virus families and discuss similarities in the viral and cellular activities that are associated with their assembly and the functions they facilitate for viral replication. PMID:24257611

  9. Accelerated growth in the absence of DNA replication origins.

    PubMed

    Hawkins, Michelle; Malla, Sunir; Blythe, Martin J; Nieduszynski, Conrad A; Allers, Thorsten

    2013-11-28

    DNA replication initiates at defined sites called origins, which serve as binding sites for initiator proteins that recruit the replicative machinery. Origins differ in number and structure across the three domains of life and their properties determine the dynamics of chromosome replication. Bacteria and some archaea replicate from single origins, whereas most archaea and all eukaryotes replicate using multiple origins. Initiation mechanisms that rely on homologous recombination operate in some viruses. Here we show that such mechanisms also operate in archaea. We use deep sequencing to study replication in Haloferax volcanii and identify four chromosomal origins of differing activity. Deletion of individual origins results in perturbed replication dynamics and reduced growth. However, a strain lacking all origins has no apparent defects and grows significantly faster than wild type. Origin-less cells initiate replication at dispersed sites rather than at discrete origins and have an absolute requirement for the recombinase RadA, unlike strains lacking individual origins. Our results demonstrate that homologous recombination alone can efficiently initiate the replication of an entire cellular genome. This raises the question of what purpose replication origins serve and why they have evolved.

  10. The Temporal Program of Chromosome Replication: Genomewide Replication in clb5Δ Saccharomyces cerevisiae

    PubMed Central

    McCune, Heather J.; Danielson, Laura S.; Alvino, Gina M.; Collingwood, David; Delrow, Jeffrey J.; Fangman, Walton L.; Brewer, Bonita J.; Raghuraman, M. K.

    2008-01-01

    Temporal regulation of origin activation is widely thought to explain the pattern of early- and late-replicating domains in the Saccharomyces cerevisiae genome. Recently, single-molecule analysis of replication suggested that stochastic processes acting on origins with different probabilities of activation could generate the observed kinetics of replication without requiring an underlying temporal order. To distinguish between these possibilities, we examined a clb5Δ strain, where origin firing is largely limited to the first half of S phase, to ask whether all origins nonspecifically show decreased firing (as expected for disordered firing) or if only some origins (“late” origins) are affected. Approximately half the origins in the mutant genome show delayed replication while the remainder replicate largely on time. The delayed regions can encompass hundreds of kilobases and generally correspond to regions that replicate late in wild-type cells. Kinetic analysis of replication in wild-type cells reveals broad windows of origin firing for both early and late origins. Our results are consistent with a temporal model in which origins can show some heterogeneity in both time and probability of origin firing, but clustering of temporally like origins nevertheless yields a genome that is organized into blocks showing different replication times. PMID:18832352

  11. Recombination and Replication

    PubMed Central

    Syeda, Aisha H.; Hawkins, Michelle; McGlynn, Peter

    2014-01-01

    The links between recombination and replication have been appreciated for decades and it is now generally accepted that these two fundamental aspects of DNA metabolism are inseparable: Homologous recombination is essential for completion of DNA replication and vice versa. This review focuses on the roles that recombination enzymes play in underpinning genome duplication, aiding replication fork movement in the face of the many replisome barriers that challenge genome stability. These links have many conserved features across all domains of life, reflecting the conserved nature of the substrate for these reactions, DNA. PMID:25341919

  12. Poxvirus DNA Replication

    PubMed Central

    Moss, Bernard

    2013-01-01

    Poxviruses are large, enveloped viruses that replicate in the cytoplasm and encode proteins for DNA replication and gene expression. Hairpin ends link the two strands of the linear, double-stranded DNA genome. Viral proteins involved in DNA synthesis include a 117-kDa polymerase, a helicase–primase, a uracil DNA glycosylase, a processivity factor, a single-stranded DNA-binding protein, a protein kinase, and a DNA ligase. A viral FEN1 family protein participates in double-strand break repair. The DNA is replicated as long concatemers that are resolved by a viral Holliday junction endonuclease. PMID:23838441

  13. Modeling DNA Replication.

    ERIC Educational Resources Information Center

    Bennett, Joan

    1998-01-01

    Recommends the use of a model of DNA made out of Velcro to help students visualize the steps of DNA replication. Includes a materials list, construction directions, and details of the demonstration using the model parts. (DDR)

  14. Eukaryotic DNA Replication Fork.

    PubMed

    Burgers, Peter M J; Kunkel, Thomas A

    2017-06-20

    This review focuses on the biogenesis and composition of the eukaryotic DNA replication fork, with an emphasis on the enzymes that synthesize DNA and repair discontinuities on the lagging strand of the replication fork. Physical and genetic methodologies aimed at understanding these processes are discussed. The preponderance of evidence supports a model in which DNA polymerase ε (Pol ε) carries out the bulk of leading strand DNA synthesis at an undisturbed replication fork. DNA polymerases α and δ carry out the initiation of Okazaki fragment synthesis and its elongation and maturation, respectively. This review also discusses alternative proposals, including cellular processes during which alternative forks may be utilized, and new biochemical studies with purified proteins that are aimed at reconstituting leading and lagging strand DNA synthesis separately and as an integrated replication fork.

  15. Modeling DNA Replication.

    ERIC Educational Resources Information Center

    Bennett, Joan

    1998-01-01

    Recommends the use of a model of DNA made out of Velcro to help students visualize the steps of DNA replication. Includes a materials list, construction directions, and details of the demonstration using the model parts. (DDR)

  16. COMPARISON OF OVERALL METABOLISM OF 2, 3, 7, 8-TETRACHLORODIBENZO-P-DIOXIN IN CYP1A2(-/-) KNOCKOUT AND C57BL/6N PARENTAL STRAINS OF MICE

    EPA Science Inventory

    Comparison of Overall Metabolism of 2,3,7,8-TCDD
    in CYP1A2 (-/-) Knockout and C57BL/6N Parental Strains of Mice

    Heldur Hakk* and Janet J. Diliberto**

    * USDA-ARS Biosciences Research Laboratory, P.O. Box 5674, Fargo, ND, USA
    ** US-EPA ORD, National Health Eff...

  17. COMPARISON OF OVERALL METABOLISM OF 2, 3, 7, 8-TETRACHLORODIBENZO-P-DIOXIN IN CYP1A2(-/-) KNOCKOUT AND C57BL/6N PARENTAL STRAINS OF MICE

    EPA Science Inventory

    Comparison of Overall Metabolism of 2,3,7,8-TCDD
    in CYP1A2 (-/-) Knockout and C57BL/6N Parental Strains of Mice

    Heldur Hakk* and Janet J. Diliberto**

    * USDA-ARS Biosciences Research Laboratory, P.O. Box 5674, Fargo, ND, USA
    ** US-EPA ORD, National Health Eff...

  18. Replication-Competent Controlled Herpes Simplex Virus.

    PubMed

    Bloom, David C; Feller, Joyce; McAnany, Peterjon; Vilaboa, Nuria; Voellmy, Richard

    2015-10-01

    We present the development and characterization of a replication-competent controlled herpes simplex virus 1 (HSV-1). Replication-essential ICP4 and ICP8 genes of HSV-1 wild-type strain 17syn+ were brought under the control of a dually responsive gene switch. The gene switch comprises (i) a transactivator that is activated by a narrow class of antiprogestins, including mifepristone and ulipristal, and whose expression is mediated by a promoter cassette that comprises an HSP70B promoter and a transactivator-responsive promoter and (ii) transactivator-responsive promoters that drive the ICP4 and ICP8 genes. Single-step growth experiments in different cell lines demonstrated that replication of the recombinant virus, HSV-GS3, is strictly dependent on an activating treatment consisting of administration of a supraphysiological heat dose in the presence of an antiprogestin. The replication-competent controlled virus replicates with an efficiency approaching that of the wild-type virus from which it was derived. Essentially no replication occurs in the absence of activating treatment or if HSV-GS3-infected cells are exposed only to heat or antiprogestin. These findings were corroborated by measurements of amounts of viral DNA and transcripts of the regulated ICP4 gene and the glycoprotein C (gC) late gene, which was not regulated. Similar findings were made in experiments with a mouse footpad infection model. The alphaherpesviruses have long been considered vectors for recombinant vaccines and oncolytic therapies. The traditional approach uses vector backbones containing attenuating mutations that restrict replication to ensure safety. The shortcoming of this approach is that the attenuating mutations tend to limit both the immune presentation and oncolytic properties of these vectors. HSV-GS3 represents a novel type of vector that, when activated, replicates with the efficiency of a nonattenuated virus and whose safety is derived from deliberate, stringent regulation of

  19. Replication-Competent Controlled Herpes Simplex Virus

    PubMed Central

    Bloom, David C.; Feller, Joyce; McAnany, Peterjon; Vilaboa, Nuria

    2015-01-01

    ABSTRACT We present the development and characterization of a replication-competent controlled herpes simplex virus 1 (HSV-1). Replication-essential ICP4 and ICP8 genes of HSV-1 wild-type strain 17syn+ were brought under the control of a dually responsive gene switch. The gene switch comprises (i) a transactivator that is activated by a narrow class of antiprogestins, including mifepristone and ulipristal, and whose expression is mediated by a promoter cassette that comprises an HSP70B promoter and a transactivator-responsive promoter and (ii) transactivator-responsive promoters that drive the ICP4 and ICP8 genes. Single-step growth experiments in different cell lines demonstrated that replication of the recombinant virus, HSV-GS3, is strictly dependent on an activating treatment consisting of administration of a supraphysiological heat dose in the presence of an antiprogestin. The replication-competent controlled virus replicates with an efficiency approaching that of the wild-type virus from which it was derived. Essentially no replication occurs in the absence of activating treatment or if HSV-GS3-infected cells are exposed only to heat or antiprogestin. These findings were corroborated by measurements of amounts of viral DNA and transcripts of the regulated ICP4 gene and the glycoprotein C (gC) late gene, which was not regulated. Similar findings were made in experiments with a mouse footpad infection model. IMPORTANCE The alphaherpesviruses have long been considered vectors for recombinant vaccines and oncolytic therapies. The traditional approach uses vector backbones containing attenuating mutations that restrict replication to ensure safety. The shortcoming of this approach is that the attenuating mutations tend to limit both the immune presentation and oncolytic properties of these vectors. HSV-GS3 represents a novel type of vector that, when activated, replicates with the efficiency of a nonattenuated virus and whose safety is derived from deliberate

  20. DNA Replication Origins

    PubMed Central

    Leonard, Alan C.; Méchali, Marcel

    2013-01-01

    The onset of genomic DNA synthesis requires precise interactions of specialized initiator proteins with DNA at sites where the replication machinery can be loaded. These sites, defined as replication origins, are found at a few unique locations in all of the prokaryotic chromosomes examined so far. However, replication origins are dispersed among tens of thousands of loci in metazoan chromosomes, thereby raising questions regarding the role of specific nucleotide sequences and chromatin environment in origin selection and the mechanisms used by initiators to recognize replication origins. Close examination of bacterial and archaeal replication origins reveals an array of DNA sequence motifs that position individual initiator protein molecules and promote initiator oligomerization on origin DNA. Conversely, the need for specific recognition sequences in eukaryotic replication origins is relaxed. In fact, the primary rule for origin selection appears to be flexibility, a feature that is modulated either by structural elements or by epigenetic mechanisms at least partly linked to the organization of the genome for gene expression. PMID:23838439

  1. Replication of lightweight mirrors

    NASA Astrophysics Data System (ADS)

    Chen, Ming Y.; Matson, Lawrence E.; Lee, Heedong; Chen, Chenggang

    2009-08-01

    The fabrication of lightweight mirror assemblages via a replication technique offers great potential for eliminating the high cost and schedule associated with the grinding and polishing steps needed for conventional glass or SiC mirrors. A replication mandrel is polished to an inverse figure shape and to the desired finish quality. It is then, coated with a release layer, the appropriate reflective layer, and followed by a laminate for coefficient of thermal expansion (CTE) tailorability and strength. This optical membrane is adhered to a mirror structural substrate with a low shrinkage, CTE tailored adhesive. Afterwards, the whole assembly is separated from the mandrel. The mandrel is then cleaned and reused for the next replication run. The ultimate goal of replication is to preserve the surface finish and figure of the optical membrane upon its release from the mandrel. Successful replication requires a minimization of the residual stresses within the optical coating stack, the curing stresses from the adhesive and the thermal stress resulting from CTE mismatch between the structural substrate, the adhesive, and the optical membrane. In this paper, the results on replicated trials using both metal/metal and ceramic/ceramic laminates adhered to light weighted structural substrates made from syntactic foams (both inorganic and organic) will be discussed.

  2. Glycoprotein 5 of porcine reproductive and respiratory syndrome virus strain SD16 inhibits viral replication and causes G2/M cell cycle arrest, but does not induce cellular apoptosis in Marc-145 cells

    SciTech Connect

    Mu, Yang; Li, Liangliang; Zhang, Beibei; Huang, Baicheng; Gao, Jiming; and others

    2015-10-15

    Cell apoptosis is common after infection with porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV GP5 has been reported to induce cell apoptosis. To further understand the role of GP5 in PRRSV induced cell apoptosis, we established Marc-145 cell lines stably expressing full-length GP5, GP5{sup Δ84-96} (aa 84-96 deletion), and GP5{sup Δ97-119} (aa 97-119 deletion). Cell proliferation, cell cycle progression, cell apoptosis and virus replication in these cell lines were evaluated. Neither truncated nor full-length GP5 induced cell apoptosis in Marc-145 cells. However, GP5{sup Δ97-119}, but not full-length or GP5{sup Δ84-96}, induced a cell cycle arrest at the G2/M phase resulting in a reduction in the growth of Marc-145 cells. Additionally, GP5{sup Δ84-96} inhibited the replication of PRRSV in Marc-145 cells through induction of IFN-β. These findings suggest that PRRSV GP5 is not responsible for inducing cell apoptosis in Marc-145 cells under these experimental conditions; however it has other important roles in virus/host cell biology. - Highlights: • Marc-145 cell lines stable expression PRRSV GP5 or truncated GP5 were constructed. • GP5{sup Δ97-119} expression in Marc-145 cell induced cell cycle arrest at G2/M phase. • Expression of GP5 and truncated GP5 could not induce Marc-145 cells apoptosis. • PRRSV replication in Marc-145-GP5{sup Δ84-96} was significantly inhibited.

  3. Genetic stability of A/Ann Arbor/6/60 cold-mutant (temperature-sensitive) live influenza virus genes: analysis by oligonucleotide mapping of recombinant vaccine strains before and after replication in volunteers.

    PubMed

    Cox, N J; Kendal, A P

    1984-02-01

    Ten cold-adapted (ca) recombinant influenza viruses that had been derived by reassortment between the ca temperature-sensitive (ts) A/Ann Arbor/6/60 mutant and selected wild-type viruses were examined by RNA oligonucleotide mapping for the presence or absence of several previously identified oligonucleotide markers. These markers comprise oligonucleotides that appeared only in the mutant donor strain after cold adaptation and other oligonucleotides present only in the parent A/Ann Arbor/6/60 wild-type virus. The markers in the mutant donor strain were transferred without change to the recombinant ca vaccine strains, and reversion causing a reappearance of markers for the wild-type A/Ann Arbor/6/60 virus did not occur. Furthermore, examination by oligonucleotide mapping of 10 isolates from vaccinated, seronegative children revealed a high degree of genetic stability of the viral genome; an oligonucleotide change was detected in only one of the 10 isolates examined.

  4. Continuous Passaging of a Recombinant C-Strain Virus in PK-15 Cells Selects Culture-Adapted Variants that Showed Enhanced Replication but Failed to Induce Fever in Rabbits.

    PubMed

    Tong, Chao; Chen, Ning; Liao, Xun; Yuan, Xuemei; Sun, Mengjiao; Li, Xiaoliang; Fang, Weihuan

    2017-09-28

    Classical swine fever virus (CSFV) is the etiologic agent of classical swine fever, a highly contagious disease that causes significant economic losses to the swine industry. The lapinized C-strain, a widely used vaccine strain against CSFV, has low growth efficiency in cell culture, which limits the productivity in the vaccine industry. In this study, a recombinant virus derived from C-strain was constructed and subjected to continuous passaging in PK-15 cells with the goal of acquiring a high progeny virus yield. A cell-adapted virus variant, RecCpp80, had nearly 1,000-fold higher titer than its parent C-strain but lost the ability to induce fever in rabbits. Sequence analysis of cell-adapted RecC variants indicated that at least six nucleotide changes were fixed in RecCpp80. Further adaption of RecCpp80 variant in swine testicle cells led to a higher virus yield without additional mutations. Introduction of each of these residues into the wild-type RecC backbone showed that one mutation, M979R (T3310G), located in the C-terminal region of E2 might be closely related to the cell-adapted phenotype. Rabbit inoculation revealed that RecCpp80+10 failed to induce fever in rabbits, whereas RecCpp40+10 caused a fever response similar to the commercial C-strain vaccine. In conclusion, the C-strain can be adapted to cell culture by introducing specific mutations in its E2 protein. The mutations in RecCpp80 that led to the loss of fever response in rabbits require further investigation. Continuous passaging of the C-strain-based recombinant viruses in PK-15 cells could enhance its in vitro adaption. The non-synonymous mutations at 3310 and 3531 might play major roles in the enhanced capacity of general virus reproduction. Such findings may help design a modified C-strain for improved productivity of commercial vaccines at reduced production cost.

  5. Modeling DNA Replication Intermediates

    SciTech Connect

    Broyde, S.; Roy, D.; Shapiro, R.

    1997-06-01

    While there is now available a great deal of information on double stranded DNA from X-ray crystallography, high resolution NMR and computer modeling, very little is known about structures that are representative of the DNA core of replication intermediates. DNA replication occurs at a single strand/double strand junction and bulged out intermediates near the junction can lead to frameshift mutations. The single stranded domains are particularly challenging. Our interest is focused on strategies for modeling the DNA of these types of replication intermediates. Modeling such structures presents special problems in addressing the multiple minimum problem and in treating the electrostatic component of the force field. We are testing a number of search strategies for locating low energy structures of these types and we are also investigating two different distance dependent dielectric functions in the coulombic term of the force field. We are studying both unmodified DNA and DNA damaged by aromatic amines, carcinogens present in the environment in tobacco smoke, barbecued meats and automobile exhaust. The nature of the structure adopted by the carcinogen modified DNA at the replication fork plays a key role in determining whether the carcinogen will cause a mutation during replication that can initiate the carcinogenic process. In the present work results are presented for unmodified DNA.

  6. DNA Replication Timing

    PubMed Central

    Rhind, Nicholas; Gilbert, David M.

    2013-01-01

    Patterns of replication within eukaryotic genomes correlate with gene expression, chromatin structure, and genome evolution. Recent advances in genome-scale mapping of replication kinetics have allowed these correlations to be explored in many species, cell types, and growth conditions, and these large data sets have allowed quantitative and computational analyses. One striking new correlation to emerge from these analyses is between replication timing and the three-dimensional structure of chromosomes. This correlation, which is significantly stronger than with any single histone modification or chromosome-binding protein, suggests that replication timing is controlled at the level of chromosomal domains. This conclusion dovetails with parallel work on the heterogeneity of origin firing and the competition between origins for limiting activators to suggest a model in which the stochastic probability of individual origin firing is modulated by chromosomal domain structure to produce patterns of replication. Whether these patterns have inherent biological functions or simply reflect higher-order genome structure is an open question. PMID:23838440

  7. Psychology, replication & beyond.

    PubMed

    Laws, Keith R

    2016-06-01

    Modern psychology is apparently in crisis and the prevailing view is that this partly reflects an inability to replicate past findings. If a crisis does exists, then it is some kind of 'chronic' crisis, as psychologists have been censuring themselves over replicability for decades. While the debate in psychology is not new, the lack of progress across the decades is disappointing. Recently though, we have seen a veritable surfeit of debate alongside multiple orchestrated and well-publicised replication initiatives. The spotlight is being shone on certain areas and although not everyone agrees on how we should interpret the outcomes, the debate is happening and impassioned. The issue of reproducibility occupies a central place in our whig history of psychology.

  8. Replicated Composite Optics Development

    NASA Technical Reports Server (NTRS)

    Engelhaupt, Darell

    1997-01-01

    Advanced optical systems for applications such as grazing incidence Wolter I x-ray mirror assemblies require extraordinary mirror surfaces in ten-ns of fine surface finish and figure. The impeccable mirror surface is on the inside of the rotational mirror form. One practical method of producing devices with these requirements is to first fabricate an exterior surface for the optical device then replicate that surface to have the inverse component with lightweight characteristics. The replicate optic is not better than the master or mandrel from which it is made. This task is a continuance of previous studies to identify methods and materials for forming these extremely low roughness optical components.

  9. Activation of new replication foci under conditions of replication stress

    PubMed Central

    Rybak, P; Waligórska, A; Bujnowicz, Ł; Hoang, A; Dobrucki, JW

    2015-01-01

    DNA damage, binding of drugs to DNA or a shortage of nucleotides can decrease the rate or completely halt the progress of replication forks. Although the global rate of replication decreases, mammalian cells can respond to replication stress by activating new replication origins. We demonstrate that a moderate level of stress induced by inhibitors of topoisomerase I, commencing in early, mid or late S-phase, induces activation of new sites of replication located within or in the immediate vicinity of the original replication factories; only in early S some of these new sites are also activated at a distance greater than 300 nm. Under high stress levels very few new replication sites are activated; such sites are located within the original replication regions. There is a large variation in cellular response to stress – while in some cells the number of replication sites increases even threefold, it decreases almost twofold in other cells. Replication stress results in a loss of PCNA from replication factories and a twofold increase in nuclear volume. These observations suggest that activation of new replication origins from the pool of dormant origins within replication cluster under conditions of mild stress is generally restricted to the original replication clusters (factories) active at a time of stress initiation, while activation of distant origins and new replication factories is suppressed. PMID:26212617

  10. Relationships and Evolution of Double-Stranded RNA Totiviruses of Yeasts Inferred from Analysis of L-A-2 and L-BC Variants in Wine Yeast Strain Populations.

    PubMed

    Rodríguez-Cousiño, Nieves; Esteban, Rosa

    2017-02-15

    Saccharomyces cerevisiae killer strains secrete a protein toxin active on nonkiller strains of the same (or other) yeast species. Different killer toxins, K1, K2, K28, and Klus, have been described. Each toxin is encoded by a medium-size (1.5- to 2.3-kb) M double-stranded RNA (dsRNA) located in the cytoplasm. M dsRNAs require L-A helper virus for maintenance. L-A belongs to the Totiviridae family, and its dsRNA genome of 4.6 kb codes for the major capsid protein Gag and a minor Gag-Pol protein, which form the virions that separately encapsidate L-A or the M satellites. Different L-A variants exist in nature; on average, 24% of their nucleotides are different. Previously, we reported that L-A-lus was specifically associated with Mlus, suggesting coevolution, and proposed a role of the toxin-encoding M dsRNAs in the appearance of new L-A variants. Here we confirm this by analyzing the helper virus in K2 killer wine strains, which we named L-A-2. L-A-2 is required for M2 maintenance, and neither L-A nor L-A-lus shows helper activity for M2 in the same genetic background. This requirement is overcome when coat proteins are provided in large amounts by a vector or in ski mutants. The genome of another totivirus, L-BC, frequently accompanying L-A in the same cells shows a lower degree of variation than does L-A (about 10% of nucleotides are different). Although L-BC has no helper activity for M dsRNAs, distinct L-BC variants are associated with a particular killer strain. The so-called L-BC-lus (in Klus strains) and L-BC-2 (in K2 strains) are analyzed.

  11. Wolbachia modulates Chikungunya replication in Aedes albopictus.

    PubMed

    Mousson, L; Martin, E; Zouache, K; Madec, Y; Mavingui, P; Failloux, A B

    2010-05-01

    The Aedes albopictus mosquito has been involved as the principal vector of recent major outbreaks due to the chikungunya virus (CHIKV). The species is naturally infected by two strains of Wolbachia (wAlbA and wAlbB). Wolbachia infections are thought to have spread by manipulating the reproduction of their hosts; cytoplasmic incompatibility is the mechanism used by Wolbachia to invade natural populations of many insects including Ae. albopictus. Here, we report a study on the effects of removing Wolbachia from Ae. albopictus on CHIKV replication and examine the consequences of CHIKV infection on some life-history traits (survival and reproduction) of Wolbachia-free Ae. albopictus. We found that Wolbachia-free mosquitoes maintained a highly heterogeneous CHIKV replication compared to Wolbachia-infected individuals. In Wolbachia-infected Ae. albopictus, the regular increase of CHIKV followed by a steady viral load from day 4 post-infection onwards was concomitant with a decline in Wolbachia density. This profile was also detected when examining the two key organs for viral transmission, the midgut and the salivary glands. Moreover, Wolbachia-free Ae. albopictus was not altered in life-history traits such as survival, oviposition and hatching characteristics whether infected or not with CHIKV. We found that Wolbachia is not essential for viral replication, its presence could lead to optimize replication from day 4 post-infection onwards, coinciding with a decrease in Wolbachia density. Wolbachia may regulate viral replication in Ae. albopictus, with consequences on survival and reproduction.

  12. Coronavirus Attachment and Replication

    DTIC Science & Technology

    1988-03-28

    synthesis during RNA replication of vesicular stomatitis virus. J. Virol. 49:303-309. Pedersen, N.C. 1976a. Feline infectious peritonitis: Something old...receptors on intestinal brush border membranes from normal host species were developed for canine (CCV), feline (FIPV), porcine (TGEV), human (HCV...gastroenteritis receptor on pig BBMs ...... ................. ... 114 Feline infectious peritonitis virus receptor on cat BBMs ... .............. 117 Human

  13. Evolution of Replication Machines

    PubMed Central

    Yao, Nina Y.; O'Donnell, Mike E.

    2016-01-01

    The machines that decode and regulate genetic information require the translation, transcription and replication pathways essential to all living cells. Thus, it might be expected that all cells share the same basic machinery for these pathways that were inherited from the primordial ancestor cell from which they evolved. A clear example of this is found in the translation machinery that converts RNA sequence to protein. The translation process requires numerous structural and catalytic RNAs and proteins, the central factors of which are homologous in all three domains of life, bacteria, archaea and eukarya. Likewise, the central actor in transcription, RNA polymerase, shows homology among the catalytic subunits in bacteria, archaea and eukarya. In contrast, while some “gears” of the genome replication machinery are homologous in all domains of life, most components of the replication machine appear to be unrelated between bacteria and those of archaea and eukarya. This review will compare and contrast the central proteins of the “replisome” machines that duplicate DNA in bacteria, archaea and eukarya, with an eye to understanding the issues surrounding the evolution of the DNA replication apparatus. PMID:27160337

  14. Human Mitochondrial DNA Replication

    PubMed Central

    Holt, Ian J.; Reyes, Aurelio

    2012-01-01

    Elucidation of the process of DNA replication in mitochondria is in its infancy. For many years, maintenance of the mitochondrial genome was regarded as greatly simplified compared to the nucleus. Mammalian mitochondria were reported to lack all DNA repair systems, to eschew DNA recombination, and to possess but a single DNA polymerase, polymerase γ. Polγ was said to replicate mitochondrial DNA exclusively via one mechanism, involving only two priming events and a handful of proteins. In this “strand-displacement model,” leading strand DNA synthesis begins at a specific site and advances approximately two-thirds of the way around the molecule before DNA synthesis is initiated on the “lagging” strand. Although the displaced strand was long-held to be coated with protein, RNA has more recently been proposed in its place. Furthermore, mitochondrial DNA molecules with all the features of products of conventional bidirectional replication have been documented, suggesting that the process and regulation of replication in mitochondria is complex, as befits a genome that is a core factor in human health and longevity. PMID:23143808

  15. Neonatal neosporosis in a 2-week-old Bernese mountain dog infected with multiple Neospora caninum strains based on MS10 microsatellite analysis.

    PubMed

    Prandini da Costa Reis, Rodrigo; Crisman, Robin; Roser, Margie; Malik, Richard; Šlapeta, Jan

    2016-05-15

    Neonatal neosporosis is a challenging disease to diagnose in neonatal and young puppies because the first signs of this condition may not be strongly suggestive of an infectious aetiology. Within two weeks of birth, three of four pups died with a subacute clinical course, some with dyspnea, some with diarrhoea and some with neurologic signs. Neosporosis was diagnosed post-mortem, but only after microscopic examination of tissues collected at necropsy. Histological findings consisted of (i) necrotizing, diffuse interstitial pneumonia associated with intralesional protozoa and (ii) necrotizing multifocal myocarditis with mineralization and intralesional protozoa. No significant alterations were found in the cerebrum or cerebellum (spinal cord was not examined). Immunohistochemistry confirmed protozoal stages and cysts were Neospora caninum. Immunohistochemistry for Toxoplasma gondii was negative. Lung and heart were the most severely affected tissues with large numbers of free zoites, BAG5 positive bradyzoites and tissue cysts of N. caninum further confirmed by N. caninum-specific quantitative real-time PCR. One affected pup which displayed knuckling, ataxia and diarrhoea were treated with trimethoprim sulfadiazine and clindamycin, and made a complete recovery. This surviving pup (at 8 weeks-of-age) and dam were both positive for N. caninum antibody (reciprocal titres 4096 and 256, respectively). Three other intact bitches on the same property were seropositive for N. caninum, suggesting horizontal transmission and a common source of infection, possibly due to consumption of infected meat. Analysis using microsatellite-10 (MS10) demonstrated that multiple strains of N. caninum were present. It was likely that all MS10 N. caninum strains were transplacentally transmitted from dam to pups. This is the first time that multiple N. caninum strains have been demonstrated to be vertically transmitted in dogs. N. caninum should be considered in the differential diagnosis for

  16. Replication and preferential inheritance of hypersuppressive petite mitochondrial DNA

    PubMed Central

    MacAlpine, David M.; Kolesar, Jill; Okamoto, Koji; Butow, Ronald A.; Perlman, Philip S.

    2001-01-01

    Wild-type yeast mitochondrial DNA (mtDNA) is inherited biparentally, whereas mtDNA of hypersuppressive petite mutants is inherited uniparentally in crosses to strains with wild-type mtDNA. Genomes of hypersuppressive petites contain a conserved ori sequence that includes a promoter, but it is unclear whether the ori confers a segregation or replication advantage. Fluorescent in situ hybridization analysis of wild-type and petite mtDNAs in crosses reveals no preferential segregation of hypersuppressive petite mtDNA to first zygotic buds. We identify single-stranded DNA circles and RNA-primed DNA replication intermediates in hypersuppressive petite mtDNA that are absent from non-hypersuppressive petites. Mutating the promoter blocks hypersuppressiveness in crosses to wild-type strains and eliminates the distinctive replication intermediates. We propose that promoter-dependent RNA-primed replication accounts for the uniparental inheritance of hypersuppressive petite mtDNA. PMID:11285243

  17. A New Saccharomyces cerevisiae Strain with a Mutant Smt3-Deconjugating Ulp1 Protein Is Affected in DNA Replication and Requires Srs2 and Homologous Recombination for Its Viability

    PubMed Central

    Soustelle, Christine; Vernis, Laurence; Fréon, Karine; Reynaud-Angelin, Anne; Chanet, Roland; Fabre, Francis; Heude, Martine

    2004-01-01

    The Saccharomyces cerevisiae Srs2 protein is involved in DNA repair and recombination. In order to gain better insight into the roles of Srs2, we performed a screen to identify mutations that are synthetically lethal with an srs2 deletion. One of them is a mutated allele of the ULP1 gene that encodes a protease specifically cleaving Smt3-protein conjugates. This allele, ulp1-I615N, is responsible for an accumulation of Smt3-conjugated proteins. The mutant is unable to grow at 37°C. At permissive temperatures, it still shows severe growth defects together with a strong hyperrecombination phenotype and is impaired in meiosis. Genetic interactions between ulp1 and mutations that affect different repair pathways indicated that the RAD51-dependent homologous recombination mechanism, but not excision resynthesis, translesion synthesis, or nonhomologous end-joining processes, is required for the viability of the mutant. Thus, both Srs2, believed to negatively control homologous recombination, and the process of recombination per se are essential for the viability of the ulp1 mutant. Upon replication, mutant cells accumulate single-stranded DNA interruptions. These structures are believed to generate different recombination intermediates. Some of them are fixed by recombination, and others require Srs2 to be reversed and fixed by an alternate pathway. PMID:15169880

  18. Reversible Switching of Cooperating Replicators

    NASA Astrophysics Data System (ADS)

    Urtel, Georg C.; Rind, Thomas; Braun, Dieter

    2017-02-01

    How can molecules with short lifetimes preserve their information over millions of years? For evolution to occur, information-carrying molecules have to replicate before they degrade. Our experiments reveal a robust, reversible cooperation mechanism in oligonucleotide replication. Two inherently slow replicating hairpin molecules can transfer their information to fast crossbreed replicators that outgrow the hairpins. The reverse is also possible. When one replication initiation site is missing, single hairpins reemerge from the crossbreed. With this mechanism, interacting replicators can switch between the hairpin and crossbreed mode, revealing a flexible adaptation to different boundary conditions.

  19. HIV-1 replication.

    PubMed

    Freed, E O

    2001-11-01

    In general terms, the replication cycle of lentiviruses, including HIV-1, closely resembles that of other retroviruses. There are, however, a number of unique aspects of HIV replication; for example, the HIVs and SIVs target receptors and coreceptors distinct from those used by other retroviruses. Lentiviruses encode a number of regulatory and accessory proteins not encoded by the genomes of the prototypical "simple" retroviruses. Of particular interest from the gene therapy perspective, lentiviruses possess the ability to productively infect some types of non-dividing cells. This chapter, while reiterating certain points discussed in Chapter 1, will attempt to focus on issues unique to HIV-1 replication. The HIV-1 genome encodes the major structural and non-structural proteins common to all replication-competent retroviruses (Fig. 1, and Chapter 1). From the 5'- to 3'-ends of the genome are found the gag (for group-specific antigen), pol (for polymerase), and env (for envelope glycoprotein) genes. The gag gene encodes a polyprotein precursor whose name, Pr55Gag, is based on its molecular weight. Pr55Gag is cleaved by the viral protease (PR) to the mature Gag proteins matrix (also known as MA or p17), capsid (CA or p24), nucleocapsid (NC or p7), and p6. Two spacer peptides, p2 and p1, are also generated upon Pr55Gag processing. The pol-encoded enzymes are initially synthesized as part of a large polyprotein precursor, Pr160GagPol, whose synthesis results from a rare frameshifting event during Pr55Gag translation. The individual pol-encoded enzymes, PR, reverse transcriptase (RT), and integrase (IN), are cleaved from Pr160GagPol by the viral PR. The envelope (Env) glycoproteins are also synthesized as a polyprotein precursor (Fig. 1). Unlike the Gag and Pol precursors, which are cleaved by the viral PR, the Env precursor, known as gp160, is processed by a cellular protease during Env trafficking to the cell surface, gp160 processing results in the generation of the

  20. DNA replication in thermophiles.

    PubMed

    Majerník, A I; Jenkinson, E R; Chong, J P J

    2004-04-01

    DNA replication enzymes in the thermophilic Archaea have previously attracted attention due to their obvious use in methods such as PCR. The proofreading ability of the Pyrococcus furiosus DNA polymerase has resulted in a commercially successful product (Pfu polymerase). One of the many notable features of the Archaea is the fact that their DNA processing enzymes appear on the whole to be more like those found in eukaryotes than bacteria. These proteins also appear to be simpler versions of those found in eukaryotes. For these reasons, archaeal organisms make potentially interesting model systems to explore the molecular mechanisms of processes such as DNA replication, repair and recombination. Why archaeal DNA-manipulation systems were adopted over bacterial systems by eukaryotic cells remains a most interesting question that we suggest may be linked to thermophily.

  1. Analysis of Pupil Replication

    NASA Astrophysics Data System (ADS)

    Spaan, F. H. P.; Greenaway, A. H.

    2007-04-01

    Pupil replication and hypertelescope systems for imaging telluric exoplanets in scattered light are treated. Analytic expressions for the spread functions in one and two dimensions and in the presence of various forms of error are given. Error effects considered include aperture misalignment, tilts, piston, pointing errors, and unequal beam amplitude. The performance of the two approaches is contrasted, and the analytic results are compared with simulation results.

  2. A New Replicator: A theoretical framework for analysing replication

    PubMed Central

    2010-01-01

    Background Replicators are the crucial entities in evolution. The notion of a replicator, however, is far less exact than the weight of its importance. Without identifying and classifying multiplying entities exactly, their dynamics cannot be determined appropriately. Therefore, it is importance to decide the nature and characteristics of any multiplying entity, in a detailed and formal way. Results Replication is basically an autocatalytic process which enables us to rest on the notions of formal chemistry. This statement has major implications. Simple autocatalytic cycle intermediates are considered as non-informational replicators. A consequence of which is that any autocatalytically multiplying entity is a replicator, be it simple or overly complex (even nests). A stricter definition refers to entities which can inherit acquired changes (informational replicators). Simple autocatalytic molecules (and nests) are excluded from this group. However, in turn, any entity possessing copiable information is to be named a replicator, even multicellular organisms. In order to deal with the situation, an abstract, formal framework is presented, which allows the proper identification of various types of replicators. This sheds light on the old problem of the units and levels of selection and evolution. A hierarchical classification for the partition of the replicator-continuum is provided where specific replicators are nested within more general ones. The classification should be able to be successfully applied to known replicators and also to future candidates. Conclusion This paper redefines the concept of the replicator from a bottom-up theoretical approach. The formal definition and the abstract models presented can distinguish between among all possible replicator types, based on their quantity of variable and heritable information. This allows for the exact identification of various replicator types and their underlying dynamics. The most important claim is that

  3. Caffeine Reverts Memory But Not Mood Impairment in a Depression-Prone Mouse Strain with Up-Regulated Adenosine A2A Receptor in Hippocampal Glutamate Synapses.

    PubMed

    Machado, Nuno J; Simões, Ana Patrícia; Silva, Henrique B; Ardais, Ana Paula; Kaster, Manuella P; Garção, Pedro; Rodrigues, Diana I; Pochmann, Daniela; Santos, Ana Isabel; Araújo, Inês M; Porciúncula, Lisiane O; Tomé, Ângelo R; Köfalvi, Attila; Vaugeois, Jean-Marie; Agostinho, Paula; El Yacoubi, Malika; Cunha, Rodrigo A; Gomes, Catarina A

    2017-03-01

    Caffeine prophylactically prevents mood and memory impairments through adenosine A2A receptor (A2AR) antagonism. A2AR antagonists also therapeutically revert mood and memory impairments, but it is not known if caffeine is also therapeutically or only prophylactically effective. Since depression is accompanied by mood and memory alterations, we now explored if chronic (4 weeks) caffeine consumption (0.3 g/L) reverts mood and memory impairment in helpless mice (HM, 12 weeks old), a bred-based model of depression. HM displayed higher immobility in the tail suspension and forced swimming tests, greater anxiety in the elevated plus maze, and poorer memory performance (modified Y-maze and object recognition). HM also had reduced density of synaptic (synaptophysin, SNAP-25), namely, glutamatergic (vGluT1; -22 ± 7 %) and GABAergic (vGAT; -23 ± 8 %) markers in the hippocampus. HM displayed higher A2AR density (72 ± 6 %) in hippocampal synapses, an enhanced facilitation of hippocampal glutamate release by the A2AR agonist, CGS21680 (30 nM), and a larger LTP amplitude (54 ± 8 % vs. 21 ± 5 % in controls) that was restored to control levels (30 ± 10 %) by the A2AR antagonist, SCH58261 (50 nM). Notably, caffeine intake reverted memory deficits and reverted the loss of hippocampal synaptic markers but did not affect helpless or anxiety behavior. These results reinforce the validity of HM as an animal model of depression by showing that they also display reference memory deficits. Furthermore, caffeine intake selectively reverted memory but not mood deficits displayed by HM, which are associated with an increased density and functional impact of hippocampal A2AR controlling synaptic glutamatergic function.

  4. Roles of Long and Short Replication Initiation Proteins in the Fate of IncP-1 Plasmids

    PubMed Central

    Yano, Hirokazu; Deckert, Gail E.; Rogers, Linda M.

    2012-01-01

    Broad-host-range IncP-1 plasmids generally encode two replication initiation proteins, TrfA1 and TrfA2. TrfA2 is produced from an internal translational start site within trfA1. While TrfA1 was previously shown to be essential for replication in Pseudomonas aeruginosa, its role in other bacteria within its broad host range has not been established. To address the role of TrfA1 and TrfA2 in other hosts, efficiency of transformation, plasmid copy number (PCN), and plasmid stability were first compared between a mini-IncP-1β plasmid and its trfA1 frameshift variant in four phylogenetically distant hosts: Escherichia coli, Pseudomonas putida, Sphingobium japonicum, and Cupriavidus necator. TrfA2 was sufficient for replication in these hosts, but the presence of TrfA1 enhanced transformation efficiency and PCN. However, TrfA1 did not contribute to, and even negatively affected, long-term plasmid persistence. When trfA genes were cloned under a constitutive promoter in the chromosomes of the four hosts, strains expressing either both TrfA1 and TrfA2 or TrfA1 alone, again, generally elicited a higher PCN of an IncP1-β replicon than strains expressing TrfA2 alone. When a single species of TrfA was produced at different concentrations in E. coli cells, TrfA1 maintained a 3- to 4-fold higher PCN than TrfA2 at the same TrfA concentrations, indicating that replication mediated by TrfA1 is more efficient than that by TrfA2. These results suggest that the broad-host-range properties of IncP-1 plasmids are essentially conferred by TrfA2 and the intact replication origin alone but that TrfA1 is nonetheless important to efficiently establish plasmid replication upon transfer into a broad range of hosts. PMID:22228734

  5. The sub-cellular localization of Sulfolobus DNA replication.

    PubMed

    Gristwood, Tamzin; Duggin, Iain G; Wagner, Michaela; Albers, Sonja V; Bell, Stephen D

    2012-07-01

    Analyses of the DNA replication-associated proteins of hyperthermophilic archaea have yielded considerable insight into the structure and biochemical function of these evolutionarily conserved factors. However, little is known about the regulation and progression of DNA replication in the context of archaeal cells. In the current work, we describe the generation of strains of Sulfolobus solfataricus and Sulfolobus acidocaldarius that allow the incorporation of nucleoside analogues during DNA replication. We employ this technology, in conjunction with immunolocalization analyses of replisomes, to investigate the sub-cellular localization of nascent DNA and replisomes. Our data reveal a peripheral localization of replisomes in the cell. Furthermore, while the two replication forks emerging from any one of the three replication origins in the Sulfolobus chromosome remain in close proximity, the three origin loci are separated.

  6. Ultrastructural Characterization of Zika Virus Replication Factories.

    PubMed

    Cortese, Mirko; Goellner, Sarah; Acosta, Eliana Gisela; Neufeldt, Christopher John; Oleksiuk, Olga; Lampe, Marko; Haselmann, Uta; Funaya, Charlotta; Schieber, Nicole; Ronchi, Paolo; Schorb, Martin; Pruunsild, Priit; Schwab, Yannick; Chatel-Chaix, Laurent; Ruggieri, Alessia; Bartenschlager, Ralf

    2017-02-28

    A global concern has emerged with the pandemic spread of Zika virus (ZIKV) infections that can cause severe neurological symptoms in adults and newborns. ZIKV is a positive-strand RNA virus replicating in virus-induced membranous replication factories (RFs). Here we used various imaging techniques to investigate the ultrastructural details of ZIKV RFs and their relationship with host cell organelles. Analyses of human hepatic cells and neural progenitor cells infected with ZIKV revealed endoplasmic reticulum (ER) membrane invaginations containing pore-like openings toward the cytosol, reminiscent to RFs in Dengue virus-infected cells. Both the MR766 African strain and the H/PF/2013 Asian strain, the latter linked to neurological diseases, induce RFs of similar architecture. Importantly, ZIKV infection causes a drastic reorganization of microtubules and intermediate filaments forming cage-like structures surrounding the viral RF. Consistently, ZIKV replication is suppressed by cytoskeleton-targeting drugs. Thus, ZIKV RFs are tightly linked to rearrangements of the host cell cytoskeleton.

  7. Mechanisms of DNA replication termination.

    PubMed

    Dewar, James M; Walter, Johannes C

    2017-08-01

    Genome duplication is carried out by pairs of replication forks that assemble at origins of replication and then move in opposite directions. DNA replication ends when converging replication forks meet. During this process, which is known as replication termination, DNA synthesis is completed, the replication machinery is disassembled and daughter molecules are resolved. In this Review, we outline the steps that are likely to be common to replication termination in most organisms, namely, fork convergence, synthesis completion, replisome disassembly and decatenation. We briefly review the mechanism of termination in the bacterium Escherichia coli and in simian virus 40 (SV40) and also focus on recent advances in eukaryotic replication termination. In particular, we discuss the recently discovered E3 ubiquitin ligases that control replisome disassembly in yeast and higher eukaryotes, and how their activity is regulated to avoid genome instability.

  8. T-antigen-independent replication of polyomavirus DNA in murine embryonal carcinoma cells

    SciTech Connect

    Dandolo, L.; Aghion, J.; Blangy, D.

    1984-02-01

    Expression of wild-type polyomavirus (Py) is restricted in murine embryonal carcinoma (EC) cells. The block appears to be located at the level of early transcription. Since no T antigen is produced, the authors investigated the fate of viral DNA upon infection of these cells; they showed that wild-type Py DNA replicates efficiently in all EC cells, probably via a T-antigen-independent mechanism. Furthermore, they studied, at permissive and restrictive temperatures, the replication of tsa (thermosensitive for T antigen) viral DNA of an in vitro-constructed deletion mutant lacking part of the early region coding sequences and of a double mutant carrying both the tsa mutation and the PyEC F9 mutation (allowing expression of early and late viral functions in EC cells). The results imply that replication of wild-type A2 strain Py DNA can occur in EC cells in the absence of a functional T antigen. However, this protein clearly enhances viral DNA replication and is absolutely required in differentiated cells.

  9. Relationships and Evolution of Double-Stranded RNA Totiviruses of Yeasts Inferred from Analysis of L-A-2 and L-BC Variants in Wine Yeast Strain Populations

    PubMed Central

    Rodríguez-Cousiño, Nieves

    2016-01-01

    ABSTRACT Saccharomyces cerevisiae killer strains secrete a protein toxin active on nonkiller strains of the same (or other) yeast species. Different killer toxins, K1, K2, K28, and Klus, have been described. Each toxin is encoded by a medium-size (1.5- to 2.3-kb) M double-stranded RNA (dsRNA) located in the cytoplasm. M dsRNAs require L-A helper virus for maintenance. L-A belongs to the Totiviridae family, and its dsRNA genome of 4.6 kb codes for the major capsid protein Gag and a minor Gag-Pol protein, which form the virions that separately encapsidate L-A or the M satellites. Different L-A variants exist in nature; on average, 24% of their nucleotides are different. Previously, we reported that L-A-lus was specifically associated with Mlus, suggesting coevolution, and proposed a role of the toxin-encoding M dsRNAs in the appearance of new L-A variants. Here we confirm this by analyzing the helper virus in K2 killer wine strains, which we named L-A-2. L-A-2 is required for M2 maintenance, and neither L-A nor L-A-lus shows helper activity for M2 in the same genetic background. This requirement is overcome when coat proteins are provided in large amounts by a vector or in ski mutants. The genome of another totivirus, L-BC, frequently accompanying L-A in the same cells shows a lower degree of variation than does L-A (about 10% of nucleotides are different). Although L-BC has no helper activity for M dsRNAs, distinct L-BC variants are associated with a particular killer strain. The so-called L-BC-lus (in Klus strains) and L-BC-2 (in K2 strains) are analyzed. IMPORTANCE Killer strains of S. cerevisiae secrete protein toxins that kill nonkiller yeasts. The “killer phenomenon” depends on two dsRNA viruses: L-A and M. M encodes the toxin, and L-A, the helper virus, provides the capsids for both viruses. Different killer toxins exist: K1, K2, K28, and Klus, encoded on different M viruses. Our data indicate that each M dsRNA depends on a specific helper virus; these

  10. Replication of grazing incidence optics

    NASA Technical Reports Server (NTRS)

    Ulmer, Melville P.

    1986-01-01

    The replication of grazing incidence optics is reviewed. Electroform and epoxy replication are described and compared. It is concluded that for light weight and deep nesting, replication has a distinct advantage over direct production. The resolution of optics produced in this manner is however, limited to about 10 arc seconds; a typical value is 40 arc seconds. Epoxy replicated pieces tend to have better optical figures than electroformed optics, but the latter can be made thinner to make more deeply nested systems.

  11. Replication of grazing incidence optics

    NASA Technical Reports Server (NTRS)

    Ulmer, Melville P.

    1986-01-01

    The replication of grazing incidence optics is reviewed. Electroform and epoxy replication are described and compared. It is concluded that for light weight and deep nesting, replication has a distinct advantage over direct production. The resolution of optics produced in this manner is however, limited to about 10 arc seconds; a typical value is 40 arc seconds. Epoxy replicated pieces tend to have better optical figures than electroformed optics, but the latter can be made thinner to make more deeply nested systems.

  12. Replication Research and Special Education

    ERIC Educational Resources Information Center

    Travers, Jason C.; Cook, Bryan G.; Therrien, William J.; Coyne, Michael D.

    2016-01-01

    Replicating previously reported empirical research is a necessary aspect of an evidence-based field of special education, but little formal investigation into the prevalence of replication research in the special education research literature has been conducted. Various factors may explain the lack of attention to replication of special education…

  13. Replication Research and Special Education

    ERIC Educational Resources Information Center

    Travers, Jason C.; Cook, Bryan G.; Therrien, William J.; Coyne, Michael D.

    2016-01-01

    Replicating previously reported empirical research is a necessary aspect of an evidence-based field of special education, but little formal investigation into the prevalence of replication research in the special education research literature has been conducted. Various factors may explain the lack of attention to replication of special education…

  14. Replication data collection highlights value in diversity of replication attempts

    PubMed Central

    DeSoto, K. Andrew; Schweinsberg, Martin

    2017-01-01

    Researchers agree that replicability and reproducibility are key aspects of science. A collection of Data Descriptors published in Scientific Data presents data obtained in the process of attempting to replicate previously published research. These new replication data describe published and unpublished projects. The different papers in this collection highlight the many ways that scientific replications can be conducted, and they reveal the benefits and challenges of crucial replication research. The organizers of this collection encourage scientists to reuse the data contained in the collection for their own work, and also believe that these replication examples can serve as educational resources for students, early-career researchers, and experienced scientists alike who are interested in learning more about the process of replication. PMID:28291224

  15. Self-Replicating RNA.

    PubMed

    Tews, Birke Andrea; Meyers, Gregor

    2017-01-01

    Self-replicating RNA derived from the genomes of positive strand RNA viruses represents a powerful tool for both molecular studies on virus biology and approaches to novel safe and effective vaccines. The following chapter summarizes the principles how such RNAs can be established and used for design of vaccines. Due to the large variety of strategies needed to circumvent specific pitfalls in the design of such constructs the technical details of the experiments are not described here but can be found in the cited literature.

  16. Anatomy of Mammalian Replication Domains

    PubMed Central

    Takebayashi, Shin-ichiro; Ogata, Masato; Okumura, Katsuzumi

    2017-01-01

    Genetic information is faithfully copied by DNA replication through many rounds of cell division. In mammals, DNA is replicated in Mb-sized chromosomal units called “replication domains.” While genome-wide maps in multiple cell types and disease states have uncovered both dynamic and static properties of replication domains, we are still in the process of understanding the mechanisms that give rise to these properties. A better understanding of the molecular basis of replication domain regulation will bring new insights into chromosome structure and function. PMID:28350365

  17. DNA replication origins in archaea.

    PubMed

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

    2014-01-01

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

  18. Regulatory parameters of DNA replication.

    PubMed

    Zannis-Hadjopoulos, M; Price, G B

    1998-01-01

    One of the fundamental characteristics that help define life is the ability to propagate. At the basest level in the act of propagation is replication of the genetic information as the databank and architectural plans for each particular life form. Thus propagation of life requires the replication of the genome--for the purposes of our review, eukaryotic DNA replication. In this critical review, we have chosen to present the issues and supporting experimental evidence in question-and-answer format. Over the past 3 to 4 years, the research domain of eukaryotic DNA replication has developed a new dynamism. This new force in discovery of the fundamental elements and mechanisms for DNA replication in higher eukaryotes has been propelled by accepted methodologies for mapping (identification) of origins of DNA replication, applicable to mammalian DNA replication, and by the discovery of the origin recognition complex (ORC) in yeast, which has served as a model in the search for the mammalian equivalent.

  19. Chromatin and DNA Replication

    PubMed Central

    MacAlpine, David M.; Almouzni, Geneviève

    2013-01-01

    The size of a eukaryotic genome presents a unique challenge to the cell: package and organize the DNA to fit within the confines of the nucleus while at the same time ensuring sufficient dynamics to allow access to specific sequences and features such as genes and regulatory elements. This is achieved via the dynamic nucleoprotein organization of eukaryotic DNA into chromatin. The basic unit of chromatin, the nucleosome, comprises a core particle with 147 bp of DNA wrapped 1.7 times around an octamer of histones. The nucleosome is a highly versatile and modular structure, both in its composition, with the existence of various histone variants, and through the addition of a series of posttranslational modifications on the histones. This versatility allows for both short-term regulatory responses to external signaling, as well as the long-term and multigenerational definition of large functional chromosomal domains within the nucleus, such as the centromere. Chromatin organization and its dynamics participate in essentially all DNA-templated processes, including transcription, replication, recombination, and repair. Here we will focus mainly on nucleosomal organization and describe the pathways and mechanisms that contribute to assembly of this organization and the role of chromatin in regulating the DNA replication program. PMID:23751185

  20. Chromatin and DNA replication.

    PubMed

    MacAlpine, David M; Almouzni, Geneviève

    2013-08-01

    The size of a eukaryotic genome presents a unique challenge to the cell: package and organize the DNA to fit within the confines of the nucleus while at the same time ensuring sufficient dynamics to allow access to specific sequences and features such as genes and regulatory elements. This is achieved via the dynamic nucleoprotein organization of eukaryotic DNA into chromatin. The basic unit of chromatin, the nucleosome, comprises a core particle with 147 bp of DNA wrapped 1.7 times around an octamer of histones. The nucleosome is a highly versatile and modular structure, both in its composition, with the existence of various histone variants, and through the addition of a series of posttranslational modifications on the histones. This versatility allows for both short-term regulatory responses to external signaling, as well as the long-term and multigenerational definition of large functional chromosomal domains within the nucleus, such as the centromere. Chromatin organization and its dynamics participate in essentially all DNA-templated processes, including transcription, replication, recombination, and repair. Here we will focus mainly on nucleosomal organization and describe the pathways and mechanisms that contribute to assembly of this organization and the role of chromatin in regulating the DNA replication program.

  1. Chk1 promotes replication fork progression by controlling replication initiation.

    PubMed

    Petermann, Eva; Woodcock, Mick; Helleday, Thomas

    2010-09-14

    DNA replication starts at initiation sites termed replication origins. Metazoan cells contain many more potential origins than are activated (fired) during each S phase. Origin activation is controlled by the ATR checkpoint kinase and its downstream effector kinase Chk1, which suppresses origin firing in response to replication blocks and during normal S phase by inhibiting the cyclin-dependent kinase Cdk2. In addition to increased origin activation, cells deficient in Chk1 activity display reduced rates of replication fork progression. Here we investigate the causal relationship between increased origin firing and reduced replication fork progression. We use the Cdk inhibitor roscovitine or RNAi depletion of Cdc7 to inhibit origin firing in Chk1-inhibited or RNAi-depleted cells. We report that Cdk inhibition and depletion of Cdc7 can alleviate the slow replication fork speeds in Chk1-deficient cells. Our data suggest that increased replication initiation leads to slow replication fork progression and that Chk1 promotes replication fork progression during normal S phase by controlling replication origin activity.

  2. Modeling inhomogeneous DNA replication kinetics.

    PubMed

    Gauthier, Michel G; Norio, Paolo; Bechhoefer, John

    2012-01-01

    In eukaryotic organisms, DNA replication is initiated at a series of chromosomal locations called origins, where replication forks are assembled proceeding bidirectionally to replicate the genome. The distribution and firing rate of these origins, in conjunction with the velocity at which forks progress, dictate the program of the replication process. Previous attempts at modeling DNA replication in eukaryotes have focused on cases where the firing rate and the velocity of replication forks are homogeneous, or uniform, across the genome. However, it is now known that there are large variations in origin activity along the genome and variations in fork velocities can also take place. Here, we generalize previous approaches to modeling replication, to allow for arbitrary spatial variation of initiation rates and fork velocities. We derive rate equations for left- and right-moving forks and for replication probability over time that can be solved numerically to obtain the mean-field replication program. This method accurately reproduces the results of DNA replication simulation. We also successfully adapted our approach to the inverse problem of fitting measurements of DNA replication performed on single DNA molecules. Since such measurements are performed on specified portion of the genome, the examined DNA molecules may be replicated by forks that originate either within the studied molecule or outside of it. This problem was solved by using an effective flux of incoming replication forks at the model boundaries to represent the origin activity outside the studied region. Using this approach, we show that reliable inferences can be made about the replication of specific portions of the genome even if the amount of data that can be obtained from single-molecule experiments is generally limited.

  3. DNA double-strand breaks caused by replication arrest.

    PubMed Central

    Michel, B; Ehrlich, S D; Uzest, M

    1997-01-01

    We report here that DNA double-strand breaks (DSBs) form in Escherichia coli upon arrest of replication forks due to a defect in, or the inhibition of, replicative DNA helicases. The formation of DSBs was assessed by the appearance of linear DNA detected by pulse-field gel electrophoresis. Processing of DSBs by recombination repair or linear DNA degradation was abolished by mutations in recBCD genes. Two E. coli replicative helicases were tested, Rep, which is essential in recBC mutants, and DnaB. The proportion of linear DNA increased up to 50% upon shift of rep recBTS recCTS cells to restrictive temperature. No increase in linear DNA was observed in the absence of replicating chromosomes, indicating that the formation of DSBs in rep strains requires replication. Inhibition of the DnaB helicase either by a strong replication terminator or by a dnaBTS mutation led to the formation of linear DNA, showing that blocked replication forks are prone to DSB formation. In wild-type E. coli, linear DNA was detected in the absence of RecBC or of both RecA and RecD. This reveals the existence of a significant amount of spontaneous DSBs. We propose that some of them may also result from the impairment of replication fork progression. PMID:9029161

  4. In vitro replication of cyanobacterial plasmids from Synechocystis PCC 6803.

    PubMed

    Yang, X; Daniell, H; McFadden, B

    1994-09-01

    Little knowledge of DNA replication in cyanobacteria is available. In this study, we report the development and characterization of an in vitro system for studies of replication of the endogenous plasmids from the unicellular cyanobacterium Synechocystis 6803. This system (fraction III) was isolated at high salt concentrations and partially purified on a heparin-agarose column. DNA polymerases in Synechocystis 6803 appeared to be associated with membranes and could be released by the addition of ammonium sulfate to 20% saturation. DNA synthesis in fraction III was dependent on the addition of cyanobacterial plasmids isolated from the same strain. The in vitro replication products consist mostly of the supercoiled form of the plasmids. Unlike replication of many Escherichia coli plasmids, replication of cyanobacterial plasmids did not require added ATP, was not inhibited by omission of the ribonucleotides, and was insensitive to the RNA polymerase inhibitor rifampicin and the gyrase inhibitor novobiocin, but was inhibited by ethidium bromide. These data suggest that RNA may not be involved in the initiation of replication of cyanobacterial plasmids from Synechocystis 6803. In addition, intermediates of replication have been detected by two-dimensional gel electrophoresis. Density labeling experiments also indicate that cyanobacterial plasmid synthesis in vitro occurs by a semiconservative replication.

  5. Replication fork progression is paused in two large chromosomal zones flanking the DNA replication origin in Escherichia coli.

    PubMed

    Akiyama, Masahiro Tatsumi; Oshima, Taku; Chumsakul, Onuma; Ishikawa, Shu; Maki, Hisaji

    2016-08-01

    Although the speed of nascent DNA synthesis at individual replication forks is relatively uniform in bacterial cells, the dynamics of replication fork progression on the chromosome are hampered by a variety of natural impediments. Genome replication dynamics can be directly measured from an exponentially growing cell population by sequencing newly synthesized DNA strands that were specifically pulse-labeled with the thymidine analogue 5-bromo-2'-deoxyuridine (BrdU). However, a short pulse labeling with BrdU is impracticable for bacteria because of poor incorporation of BrdU into the cells, and thus, the genomewide dynamics of bacterial DNA replication remain undetermined. Using a new thymidine-requiring Escherichia coli strain, eCOMB, and high-throughput sequencing, we succeeded in determining the genomewide replication profile in bacterial cells. We also found that fork progression is paused in two ~200-kb chromosomal zones that flank the replication origin in the growing cells. This origin-proximal obstruction to fork progression was overcome by an increased thymidine concentration in the culture medium and enhanced by inhibition of transcription. These indicate that DNA replication near the origin is sensitive to the impediments to fork progression, namely a scarcity of the DNA precursor deoxythymidine triphosphate and probable conflicts between replication and transcription machineries.

  6. 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

  7. Replicative Aging in Yeast

    PubMed Central

    Steinkraus, K.A.; Kaeberlein, M.; Kennedy, B.K.

    2009-01-01

    Progress in aging research is now rapid, and surprisingly, studies in a single-celled eukaryote are a driving force. The genetic modulators of replicative life span in yeast are being identified, the molecular events that accompany aging are being discovered, and the extent to which longevity pathways are conserved between yeast and multicellular eukaryotes is being tested. In this review, we provide a brief retrospective view on the development of yeast as a model for aging and then turn to recent discoveries that have pushed aging research into novel directions and also linked aging in yeast to well-developed hypotheses in mammals. Although the question of what causes aging still cannot be answered definitively, that day may be rapidly approaching. PMID:18616424

  8. Survival and replication of Rhodococcus equi in macrophages.

    PubMed Central

    Hondalus, M K; Mosser, D M

    1994-01-01

    Rhodococcus equi is a facultative intracellular bacterium of macrophages that can cause serious pneumonia in both young horses and immunocompromised people. Essential to understanding rhodococcus pathogenesis is a quantitative documentation of the intracellular events that follow macrophage phagocytosis of the organism. By using a bacterial immunofluorescence staining assay, we verified the intracellular survival and replicative potential of R. equi in both murine peritoneal macrophages and equine alveolar macrophages in vitro. Following an initial lag period of 6 to 12 h, the intracellular numbers of R. equi begin to rise, often reaching macrophage-compromising levels by 48 h. A quantitative determination of bacterial growth by a novel image analysis cytometry technique confirmed our fluorescence microscopic results. By 48 h postinfection, bacterial numbers had increased by more than fivefold, and the majority of infected macrophages in the monolayer contained 10 or more bacteria per cell. The intracellular organisms were viable, as evidenced by the ability to incorporate radiolabeled uracil. The use of these techniques has identified differences in the in vitro replicative capacities of a virulent strain and an avirulent strain of R. equi. A clinical isolate of R. equi expressing a 17-kDa virulence-associated plasmid-encoded antigen was able to survive and replicate within macrophages, whereas an avirulent, non-plasmid-containing strain replicated poorly. These results suggest that plasmid-encoded bacterial virulence factors may contribute to the ability of R. equi to replicate within its host cell, the macrophage. Images PMID:7927672

  9. Regulation of DNA replication licensing.

    PubMed

    Niida, Hiroyuki; Kitagawa, Masatoshi

    2012-12-01

    In eukaryotic cells, DNA replication is tightly regulated to occur only once per cell cycle. DNA licensing is a mechanism to guarantee this aim; that is, licensing of replication initiation is permitted during late M phase to G1 phase. The license is canceled by the start of DNA replication. Once DNA replication begins, the license is never given until the next late M phase. The licensing corresponds to the process of assembling components of the pre-replication complex (pre-RC) on the replication origin DNA. This pre-RC is the target of several different regulation systems to prevent rereplication of DNA during a single cell cycle. In this review, the regulation mechanisms mainly in mammals to control assembling components of the pre-RC will be discussed.

  10. The effect of dose on 2,3,7,8-TCDD tissue distribution, metabolism and elimination in CYP1A2 (-/-) knockout and C57BL/6N parental strains of mice

    SciTech Connect

    Hakk, Heldur; Diliberto, Janet J.; Birnbaum, Linda S.

    2009-11-15

    Numerous metabolism studies have demonstrated that the toxic contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is poorly metabolized. A hallmark feature of TCDD exposure is induction of hepatic CYP1A2 and subsequent sequestration leading to high liver-to-fat concentration ratios. This study was initiated to determine whether TCDD was inherently poorly metabolized or unavailable for metabolism because of sequestration to CYP1A2. [{sup 3}H]TCDD was administered as a single, oral dose (0.1 and 10 mug/kg) to 12 male C57BL/6N mice or 12 CYP1A2 (-/-) mice. At 96 h, less than 5% of the dose was eliminated in the urine of all groups, and TCDD detected in urine was bound to mouse major urinary protein (mMUP). Feces were the major elimination pathway (24-31% of dose), and fecal extracts and non-extractables were quantitated by HPLC for metabolites. No great differences in urinary or fecal elimination (% dose) were observed between the high and low dose treatments. TCDD concentrations were the highest in adipose tissue for CYP1A2 knockout mice but in liver for C57BL/6N mice supporting the role of hepatic CYP1A2 in the sequestration of TCDD. Overall metabolism between parental and knockout strains showed no statistical differences at either the high or low doses. The data suggested that metabolism of TCDD is inherently slow, due principally to CYP1A1, and that hepatic CYP1A2 is not an active participant in the metabolism of TCDD in male mice. Rather, CYP1A2 governs the pharmacokinetics of TCDD by making it unavailable for hepatic CYP1A1 through sequestration and attenuating extrahepatic tissue disposition.

  11. Plant virus replication and movement.

    PubMed

    Heinlein, Manfred

    2015-05-01

    Replication and intercellular spread of viruses depend on host mechanisms supporting the formation, transport and turnover of functional complexes between viral genomes, virus-encoded products and cellular factors. To enhance these processes, viruses assemble and replicate in membrane-associated complexes that may develop into "virus factories" or "viroplasms" in which viral components and host factors required for replication are concentrated. Many plant viruses replicate in association with the cortical ER-actin network that is continuous between cells through plasmodesmata. The replication complexes can be highly organized and supported by network interactions between the viral genome and the virus-encoded proteins. Intracellular PD targeting of replication complexes links the process of movement to replication and provides specificity for transport of the viral genome by the virus-encoded movement proteins. The formation and trafficking of replication complexes and also the development and anchorage of replication factories involves important roles of the cortical cytoskeleton and associated motor proteins. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Mechanisms of Theta Plasmid Replication.

    PubMed

    Lilly, Joshua; Camps, Manel

    2015-02-01

    Plasmids are autonomously replicating pieces of DNA. This article discusses theta plasmid replication, which is a class of circular plasmid replication that includes ColE1-like origins of replication popular with expression vectors. All modalities of theta plasmid replication initiate synthesis with the leading strand at a predetermined site and complete replication through recruitment of the host's replisome, which extends the leading strand continuously while synthesizing the lagging strand discontinuously. There are clear differences between different modalities of theta plasmid replication in mechanisms of DNA duplex melting and in priming of leading- and lagging-strand synthesis. In some replicons duplex melting depends on transcription, while other replicons rely on plasmid-encoded trans-acting proteins (Reps); primers for leading-strand synthesis can be generated through processing of a transcript or in other replicons by the action of host- or plasmid-encoded primases. None of these processes require DNA breaks. The frequency of replication initiation is tightly regulated to facilitate establishment in permissive hosts and to achieve a steady state. The last section of the article reviews how plasmid copy number is sensed and how this feedback modulates the frequency of replication.

  13. Solving the Telomere Replication Problem

    PubMed Central

    Maestroni, Laetitia; Matmati, Samah; Coulon, Stéphane

    2017-01-01

    Telomeres are complex nucleoprotein structures that protect the extremities of linear chromosomes. Telomere replication is a major challenge because many obstacles to the progression of the replication fork are concentrated at the ends of the chromosomes. This is known as the telomere replication problem. In this article, different and new aspects of telomere replication, that can threaten the integrity of telomeres, will be reviewed. In particular, we will focus on the functions of shelterin and the replisome for the preservation of telomere integrity. PMID:28146113

  14. Chromatin dynamics during DNA replication

    PubMed Central

    Bar-Ziv, Raz; Voichek, Yoav; Barkai, Naama

    2016-01-01

    Chromatin is composed of DNA and histones, which provide a unified platform for regulating DNA-related processes, mostly through their post-translational modification. During DNA replication, histone arrangement is perturbed, first to allow progression of DNA polymerase and then during repackaging of the replicated DNA. To study how DNA replication influences the pattern of histone modification, we followed the cell-cycle dynamics of 10 histone marks in budding yeast. We find that histones deposited on newly replicated DNA are modified at different rates: While some marks appear immediately upon replication (e.g., H4K16ac, H3K4me1), others increase with transcription-dependent delays (e.g., H3K4me3, H3K36me3). Notably, H3K9ac was deposited as a wave preceding the replication fork by ∼5–6 kb. This replication-guided H3K9ac was fully dependent on the acetyltransferase Rtt109, while expression-guided H3K9ac was deposited by Gcn5. Further, topoisomerase depletion intensified H3K9ac in front of the replication fork and in sites where RNA polymerase II was trapped, suggesting supercoiling stresses trigger H3K9 acetylation. Our results assign complementary roles for DNA replication and gene expression in defining the pattern of histone modification. PMID:27225843

  15. Break-Induced DNA Replication

    PubMed Central

    Anand, Ranjith P.; Lovett, Susan T.; Haber, James E.

    2013-01-01

    Recombination-dependent DNA replication, often called break-induced replication (BIR), was initially invoked to explain recombination events in bacteriophage but it has recently been recognized as a fundamentally important mechanism to repair double-strand chromosome breaks in eukaryotes. This mechanism appears to be critically important in the restarting of stalled and broken replication forks and in maintaining the integrity of eroded telomeres. Although BIR helps preserve genome integrity during replication, it also promotes genome instability by the production of loss of heterozygosity and the formation of nonreciprocal translocations, as well as in the generation of complex chromosomal rearrangements. PMID:23881940

  16. Replication patterns and organization of replication forks in Vibrio cholerae.

    PubMed

    Stokke, Caroline; Waldminghaus, Torsten; Skarstad, Kirsten

    2011-03-01

    We have investigated the replication patterns of the two chromosomes of the bacterium Vibrio cholerae grown in four different media. By combining flow cytometry and quantitative real-time PCR with computer simulations, we show that in rich media, V. cholerae cells grow with overlapping replication cycles of both the large chromosome (ChrI) and the small chromosome (ChrII). In Luria-Bertani (LB) medium, initiation occurs at four copies of the ChrI origin and two copies of the ChrII origin. Replication of ChrII was found to occur at the end of the ChrI replication period in all four growth conditions. Novel cell-sorting experiments with marker frequency analysis support these conclusions. Incubation with protein synthesis inhibitors indicated that the potential for initiation of replication of ChrII was present at the same time as that of ChrI, but was actively delayed until much of ChrI was replicated. Investigations of the localization of SeqA bound to new DNA at replication forks indicated that the forks were co-localized in pairs when cells grew without overlapping replication cycles and in higher-order structures during more rapid growth. The increased degree of fork organization during rapid growth may be a means by which correct segregation of daughter molecules is facilitated.

  17. In Vitro Studies Show that Sequence Variability Contributes to Marked Variation in Hepatitis B Virus Replication, Protein Expression, and Function Observed across Genotypes.

    PubMed

    Sozzi, Vitina; Walsh, Renae; Littlejohn, Margaret; Colledge, Danni; Jackson, Kathy; Warner, Nadia; Yuen, Lilly; Locarnini, Stephen A; Revill, Peter A

    2016-11-15

    The hepatitis B virus (HBV) exists as 9 major genotypes (A to I), one minor strain (designated J) and multiple subtypes. Marked differences in HBV natural history, disease progression and treatment response are exhibited by many of these genotypes and subtypes. For example, HBV genotype C is associated with later hepatitis B e antigen (HBeAg) seroconversion and high rates of liver cancer compared to other HBV genotypes, whereas genotype A2 is rarely associated with HBeAg-negative disease or liver cancer. The reasons for these and other differences in HBV natural history are yet to be determined but could in part be due to sequence differences in the HBV genome that alter replicative capacity and/or gene expression. Direct comparative studies on HBV replication and protein expression have been limited to date due largely to the absence of infectious HBV cDNA clones for each of the HBV genotypes present in the same genetic arrangement. We have produced replication-competent infectious cDNA clones of the most common subtypes of genotypes A to D, namely, A2, B2, C2, D3, and the minor strain J, and compared their HBV replication phenotype using transient-transfection models. We identified striking differences in HBV replicative capacity as well as HBeAg and surface (HBsAg) protein expression across genotypes, which may in part be due to sequence variability in regulatory regions of the HBV genome. Functional analysis showed that sequence differences in the major upstream regulatory region across genotypes impacted promoter activity. There have been very few studies directly comparing the replication phenotype of different HBV genotypes, for which there are marked differences in natural history and disease progression worldwide. We have generated replication-competent 1.3-mer cDNA clones of the major genotypes A2, B2, C2, and D3, as well as a recently identified strain J, and identified striking differences in replicative capacity and protein expression that may

  18. In Vitro Studies Show that Sequence Variability Contributes to Marked Variation in Hepatitis B Virus Replication, Protein Expression, and Function Observed across Genotypes

    PubMed Central

    Sozzi, Vitina; Walsh, Renae; Littlejohn, Margaret; Colledge, Danni; Jackson, Kathy; Warner, Nadia; Yuen, Lilly; Locarnini, Stephen A.

    2016-01-01

    ABSTRACT The hepatitis B virus (HBV) exists as 9 major genotypes (A to I), one minor strain (designated J) and multiple subtypes. Marked differences in HBV natural history, disease progression and treatment response are exhibited by many of these genotypes and subtypes. For example, HBV genotype C is associated with later hepatitis B e antigen (HBeAg) seroconversion and high rates of liver cancer compared to other HBV genotypes, whereas genotype A2 is rarely associated with HBeAg-negative disease or liver cancer. The reasons for these and other differences in HBV natural history are yet to be determined but could in part be due to sequence differences in the HBV genome that alter replicative capacity and/or gene expression. Direct comparative studies on HBV replication and protein expression have been limited to date due largely to the absence of infectious HBV cDNA clones for each of the HBV genotypes present in the same genetic arrangement. We have produced replication-competent infectious cDNA clones of the most common subtypes of genotypes A to D, namely, A2, B2, C2, D3, and the minor strain J, and compared their HBV replication phenotype using transient-transfection models. We identified striking differences in HBV replicative capacity as well as HBeAg and surface (HBsAg) protein expression across genotypes, which may in part be due to sequence variability in regulatory regions of the HBV genome. Functional analysis showed that sequence differences in the major upstream regulatory region across genotypes impacted promoter activity. IMPORTANCE There have been very few studies directly comparing the replication phenotype of different HBV genotypes, for which there are marked differences in natural history and disease progression worldwide. We have generated replication-competent 1.3-mer cDNA clones of the major genotypes A2, B2, C2, and D3, as well as a recently identified strain J, and identified striking differences in replicative capacity and protein

  19. Affinity Purification of an Archaeal DNA Replication Protein Network

    PubMed Central

    Li, Zhuo; Santangelo, Thomas J.; Čuboňová, Ľubomíra; Reeve, John N.; Kelman, Zvi

    2010-01-01

    Nineteen Thermococcus kodakarensis strains have been constructed, each of which synthesizes a different His6-tagged protein known or predicted to be a component of the archaeal DNA replication machinery. Using the His6-tagged proteins, stable complexes assembled in vivo have been isolated directly from clarified cell lysates and the T. kodakarensis proteins present have been identified by mass spectrometry. Based on the results obtained, a network of interactions among the archaeal replication proteins has been established that confirms previously documented and predicted interactions, provides experimental evidence for previously unrecognized interactions between proteins with known functions and with unknown functions, and establishes a firm experimental foundation for archaeal replication research. The proteins identified and their participation in archaeal DNA replication are discussed and related to their bacterial and eukaryotic counterparts. PMID:20978540

  20. The asymmetry of telomere replication contributes to replicative senescence heterogeneity

    PubMed Central

    Bourgeron, Thibault; Xu, Zhou; Doumic, Marie; Teresa Teixeira, Maria

    2015-01-01

    In eukaryotes, the absence of telomerase results in telomere shortening, eventually leading to replicative senescence, an arrested state that prevents further cell divisions. While replicative senescence is mainly controlled by telomere length, the heterogeneity of its onset is not well understood. This study proposes a mathematical model based on the molecular mechanisms of telomere replication and shortening to decipher the causes of this heterogeneity. Using simulations fitted on experimental data obtained from individual lineages of senescent Saccharomyces cerevisiae cells, we decompose the sources of senescence heterogeneity into interclonal and intraclonal components, and show that the latter is based on the asymmetry of the telomere replication mechanism. We also evidence telomere rank-switching events with distinct frequencies in short-lived versus long-lived lineages, revealing that telomere shortening dynamics display important variations. Thus, the intrinsic heterogeneity of replicative senescence and its consequences find their roots in the asymmetric structure of telomeres. PMID:26468778

  1. Replication of Mu prophages lacking the central strong gyrase site.

    PubMed

    Pato, Martin L

    2004-09-01

    Replication of Mu prophages lacking the central strong gyrase site (SGS) is severely slowed. To study details of the replication of these prophages, an assay was developed for determining the rate and extent of introduction of nicks at the 3'-ends of a Mu prophage, an early step in Mu replicative transposition. The maximal level of end-nicking of a prophage with the SGS, about 70-90% depending upon the host strain, was achieved within about 15 min after induction, whereas at that time less than 5% nicking was observed with a prophage lacking the SGS. The amount of nicking at the end of the SGS(-) prophage increased with time, and approx. 30% nicking of the SGS(-) prophage was achieved by 60 min post-induction. Nicking kinetics were identical at each end of the prophages, and no nicking was observed at the 5'-ends of the prophages, verifying in vivo the earlier results with in vitro systems. To determine if prophage location affects the kinetics of replication, we examined prophages at numerous chromosomal locations. SGS(+) prophages at different chromosomal locations showed essentially identical replication kinetics. SGS(-) prophages showed a range of delays in replication and host lysis. A gradient of delays was apparent, with prophages further from the chromosomal origin of replication, oriC, showing longer delays than ones nearer to oriC. However, there were also exceptions to this overall gradient. Possible explanations for the differences in the delays observed with SGS(-) prophages are discussed.

  2. Ease of articulation: A replication.

    PubMed

    Shuster, Linda I; Cottrill, Claire

    2015-01-01

    Researchers, as well as the lay public and the popular press, have become increasingly concerned about the lack of reproducibility of research findings. Despite this concern, research has shown that replications of previously published work comprise a very small proportion of published studies. Moreover, there are fewer published direct replications of research studies by independent investigators, and this type of replication is much less likely to confirm the results of the original research than are replications by the original investigator or conceptual replications. A search of the communication disorders research literature reveals that direct replications by independent investigators are virtually non-existent. The purpose of this project was to describe the major issues related to research reproducibility and report the results of a direct replication of a study by Locke (1972) regarding ease of articulation. Two methods for rating ease of articulation were employed. We were able to reproduce the results of the original study for the first method, obtaining a moderate positive correlation between our rankings of phoneme difficulty and Locke's rankings. We obtained a very high positive correlation between our phoneme rankings and rankings obtained in the original study for the second method. Moreover, we found a higher correlation between difficulty rankings and order of speech sound acquisition for American English than was found in the original study. Direct replication is not necessary for all studies in communication disorders, but should be considered for high impact studies, treatment studies, and those that provide data to support models and theories. The reader will be able to: (1) describe the major concerns related to the replicability of research findings; (2) describe the status of research replications in communication disorders; (3) describe how ease of articulation may relate to the order of speech sound acquisition in children; (4) list some

  3. Rapid mold replication

    SciTech Connect

    Heestand, G.M.; Beeler, R.G. Jr.; Brown, D.L.

    1995-06-01

    The desire to reduce tooling costs have driven manufacturers to investigate new manufacturing methods and materials. In the plastics injection molding industry replicating molds to meet production needs is time consuming (up to 6 months) and costly in terms of lost business. We have recently completed a feasibility study demonstrating the capability of high rate Electron Beam Physical Vapor Deposition (EBPVD) in producing mold inserts in days, not months. In the current practice a graphite mandrel, in the shape of the insert`s negative image, was exposed to a jet of metal vapor atoms emanating from an electron beam heated source of an aluminum-bronze alloy. The condensation rate of the metal atoms on the mandrel was sufficient to allow the deposit to grow at over 30 {mu}m/min or 1.2 mils per minute. The vaporization process continued for approximately 14 hours after which the mandrel and deposit were removed from the EBPVD vacuum chamber. The mandrel and condensate were easily separated resulting in a fully dense aluminum-bronze mold insert about 2.5 cm or one inch thick. This mold was subsequently cleaned and drilled for water cooling passages and mounted on a fixture for operation in an actual injection molding machine. Results of the mold`s operation were extremely successful showing great promise for this technique. This paper describes the EBPVD feasibility demonstration in more detail and discusses future development work needed to bring this technique into practice.

  4. Analysis of viroid replication.

    PubMed

    Flores, Ricardo; Gas, Marída-Eugenia; Molina, Diego; Hernández, Carmen; Darós, José-Antonio

    2008-01-01

    Viroids, as a consequence of not encoding any protein, are extremely dependent on their hosts. Replication of these minimal genomes, composed exclusively by a circular RNA of 246-401 nt, occurs in the nucleus (family Pospiviroidae) or in the chloroplast (family Avsunviroidae) by an RNA-based rolling-circle mechanism with three steps: (1) synthesis of longer-than-unit strands catalyzed by host DNA-dependent RNA polymerases recruited and redirected to transcribe RNA templates, (2) cleavage to unit-length, which in family Avsunviroidae is mediated by hammerhead ribozymes, and (3) circularization through an RNA ligase or autocatalytically. This consistent but still fragmentary picture has emerged from a combination of studies with in vitro systems (analysis of RNA preparations from infected plants, transcription assays with nuclear and chloroplastic fractions, characterization of enzymes and ribozymes mediating cleavage and ligation of viroid strands, dissection of 5' terminal groups of viroid strands, and in situ hybridization and microscopy of subcellular fractions and tissues), and in vivo systems (tissue infiltration studies, protoplasts, studies in planta and use of transgenic plants expressing viroid RNAs).

  5. Archaeology of Eukaryotic DNA Replication

    PubMed Central

    Makarova, Kira S.; Koonin, Eugene V.

    2013-01-01

    Recent advances in the characterization of the archaeal DNA replication system together with comparative genomic analysis have led to the identification of several previously uncharacterized archaeal proteins involved in replication and currently reveal a nearly complete correspondence between the components of the archaeal and eukaryotic replication machineries. It can be inferred that the archaeal ancestor of eukaryotes and even the last common ancestor of all extant archaea possessed replication machineries that were comparable in complexity to the eukaryotic replication system. The eukaryotic replication system encompasses multiple paralogs of ancestral components such that heteromeric complexes in eukaryotes replace archaeal homomeric complexes, apparently along with subfunctionalization of the eukaryotic complex subunits. In the archaea, parallel, lineage-specific duplications of many genes encoding replication machinery components are detectable as well; most of these archaeal paralogs remain to be functionally characterized. The archaeal replication system shows remarkable plasticity whereby even some essential components such as DNA polymerase and single-stranded DNA-binding protein are displaced by unrelated proteins with analogous activities in some lineages. PMID:23881942

  6. Effect of dam methylation on the activity of the E. coli replication origin, oriC.

    PubMed Central

    Messer, W; Bellekes, U; Lother, H

    1985-01-01

    Methylation of GATC sites by the dam methylase is required for efficient initiation of DNA replication at the replication origin, oriC, of Escherichia coli. This is demonstrated by the inability of minichromosomes to be maintained in dam mutant strains. The requirement for methylated GATC sites is less stringent in vitro than in vivo. The time required for complete methylation of the origin region apparently determines the minimal spacing of replication forks on the chromosome. PMID:3891330

  7. Regulating DNA Replication in Plants

    PubMed Central

    Sanchez, Maria de la Paz; Costas, Celina; Sequeira-Mendes, Joana; Gutierrez, Crisanto

    2012-01-01

    Chromosomal DNA replication in plants has requirements and constraints similar to those in other eukaryotes. However, some aspects are plant-specific. Studies of DNA replication control in plants, which have unique developmental strategies, can offer unparalleled opportunities of comparing regulatory processes with yeast and, particularly, metazoa to identify common trends and basic rules. In addition to the comparative molecular and biochemical studies, genomic studies in plants that started with Arabidopsis thaliana in the year 2000 have now expanded to several dozens of species. This, together with the applicability of genomic approaches and the availability of a large collection of mutants, underscores the enormous potential to study DNA replication control in a whole developing organism. Recent advances in this field with particular focus on the DNA replication proteins, the nature of replication origins and their epigenetic landscape, and the control of endoreplication will be reviewed. PMID:23209151

  8. LHCb experience with LFC replication

    NASA Astrophysics Data System (ADS)

    Bonifazi, F.; Carbone, A.; Perez, E. D.; D'Apice, A.; dell'Agnello, L.; Duellmann, D.; Girone, M.; Re, G. L.; Martelli, B.; Peco, G.; Ricci, P. P.; Sapunenko, V.; Vagnoni, V.; Vitlacil, D.

    2008-07-01

    Database replication is a key topic in the framework of the LHC Computing Grid to allow processing of data in a distributed environment. In particular, the LHCb computing model relies on the LHC File Catalog, i.e. a database which stores information about files spread across the GRID, their logical names and the physical locations of all the replicas. The LHCb computing model requires the LFC to be replicated at Tier-1s. The LCG 3D project deals with the database replication issue and provides a replication service based on Oracle Streams technology. This paper describes the deployment of the LHC File Catalog replication to the INFN National Center for Telematics and Informatics (CNAF) and to other LHCb Tier-1 sites. We performed stress tests designed to evaluate any delay in the propagation of the streams and the scalability of the system. The tests show the robustness of the replica implementation with performance going much beyond the LHCb requirements.

  9. Efficient SIVcpz replication in human lymphoid tissue requires viral matrix protein adaptation

    PubMed Central

    Bibollet-Ruche, Frederic; Heigele, Anke; Keele, Brandon F.; Easlick, Juliet L.; Decker, Julie M.; Takehisa, Jun; Learn, Gerald; Sharp, Paul M.; Hahn, Beatrice H.; Kirchhoff, Frank

    2012-01-01

    SIVs infecting wild-living apes in west central Africa have crossed the species barrier to humans on at least four different occasions, one of which spawned the AIDS pandemic. Although the chimpanzee precursor of pandemic HIV-1 strains must have been able to infect humans, the capacity of SIVcpz strains to replicate in human lymphoid tissues (HLTs) is not known. Here, we show that SIVcpz strains from two chimpanzee subspecies are capable of replicating in human tonsillary explant cultures, albeit only at low titers. However, SIVcpz replication in HLT was significantly improved after introduction of a previously identified human-specific adaptation at position 30 in the viral Gag matrix protein. An Arg or Lys at this position significantly increased SIVcpz replication in HLT, while the same mutation reduced viral replication in chimpanzee-derived CD4+ T cells. Thus, naturally occurring SIVcpz strains are capable of infecting HLTs, the major site of HIV-1 replication in vivo. However, efficient replication requires the acquisition of a host-specific adaptation in the viral matrix protein. These results identify Gag matrix as a major determinant of SIVcpz replication fitness in humans and suggest a critical role in the emergence of HIV/AIDS. PMID:22505456

  10. Genomic Diversity of Phages Infecting Probiotic Strains of Lactobacillus paracasei

    PubMed Central

    Rousseau, Geneviève M.; Capra, María L.; Quiberoni, Andrea; Tremblay, Denise M.; Labrie, Simon J.

    2015-01-01

    Strains of the Lactobacillus casei group have been extensively studied because some are used as probiotics in foods. Conversely, their phages have received much less attention. We analyzed the complete genome sequences of five L. paracasei temperate phages: CL1, CL2, iLp84, iLp1308, and iA2. Only phage iA2 could not replicate in an indicator strain. The genome lengths ranged from 34,155 bp (iA2) to 39,474 bp (CL1). Phages iA2 and iLp1308 (34,176 bp) possess the smallest genomes reported, thus far, for phages of the L. casei group. The GC contents of the five phage genomes ranged from 44.8 to 45.6%. As observed with many other phages, their genomes were organized as follows: genes coding for DNA packaging, morphogenesis, lysis, lysogeny, and replication. Phages CL1, CL2, and iLp1308 are highly related to each other. Phage iLp84 was also related to these three phages, but the similarities were limited to gene products involved in DNA packaging and structural proteins. Genomic fragments of phages CL1, CL2, iLp1308, and iLp84 were found in several genomes of L. casei strains. Prophage iA2 is unrelated to these four phages, but almost all of its genome was found in at least four L. casei strains. Overall, these phages are distinct from previously characterized Lactobacillus phages. Our results highlight the diversity of L. casei phages and indicate frequent DNA exchanges between phages and their hosts. PMID:26475105

  11. Genomic Diversity of Phages Infecting Probiotic Strains of Lactobacillus paracasei.

    PubMed

    Mercanti, Diego J; Rousseau, Geneviève M; Capra, María L; Quiberoni, Andrea; Tremblay, Denise M; Labrie, Simon J; Moineau, Sylvain

    2015-10-16

    Strains of the Lactobacillus casei group have been extensively studied because some are used as probiotics in foods. Conversely, their phages have received much less attention. We analyzed the complete genome sequences of five L. paracasei temperate phages: CL1, CL2, iLp84, iLp1308, and iA2. Only phage iA2 could not replicate in an indicator strain. The genome lengths ranged from 34,155 bp (iA2) to 39,474 bp (CL1). Phages iA2 and iLp1308 (34,176 bp) possess the smallest genomes reported, thus far, for phages of the L. casei group. The GC contents of the five phage genomes ranged from 44.8 to 45.6%. As observed with many other phages, their genomes were organized as follows: genes coding for DNA packaging, morphogenesis, lysis, lysogeny, and replication. Phages CL1, CL2, and iLp1308 are highly related to each other. Phage iLp84 was also related to these three phages, but the similarities were limited to gene products involved in DNA packaging and structural proteins. Genomic fragments of phages CL1, CL2, iLp1308, and iLp84 were found in several genomes of L. casei strains. Prophage iA2 is unrelated to these four phages, but almost all of its genome was found in at least four L. casei strains. Overall, these phages are distinct from previously characterized Lactobacillus phages. Our results highlight the diversity of L. casei phages and indicate frequent DNA exchanges between phages and their hosts.

  12. Centromere Stability: The Replication Connection

    PubMed Central

    Forsburg, Susan L.; Shen, Kuo-Fang

    2017-01-01

    The fission yeast centromere, which is similar to metazoan centromeres, contains highly repetitive pericentromere sequences that are assembled into heterochromatin. This is required for the recruitment of cohesin and proper chromosome segregation. Surprisingly, the pericentromere replicates early in the S phase. Loss of heterochromatin causes this domain to become very sensitive to replication fork defects, leading to gross chromosome rearrangements. This review examines the interplay between components of DNA replication, heterochromatin assembly, and cohesin dynamics that ensures maintenance of genome stability and proper chromosome segregation. PMID:28106789

  13. Cytosolic clearance of replication-deficient mutants reveals Francisella tularensis interactions with the autophagic pathway.

    PubMed

    Chong, Audrey; Wehrly, Tara D; Child, Robert; Hansen, Bryan; Hwang, Seungmin; Virgin, Herbert W; Celli, Jean

    2012-09-01

    Cytosolic bacterial pathogens must evade intracellular innate immune recognition and clearance systems such as autophagy to ensure their survival and proliferation. The intracellular cycle of the bacterium Francisella tularensis is characterized by rapid phagosomal escape followed by extensive proliferation in the macrophage cytoplasm. Cytosolic replication, but not phagosomal escape, requires the locus FTT0369c, which encodes the dipA gene (deficient in intracellular replication A). Here, we show that a replication-deficient, ∆dipA mutant of the prototypical SchuS4 strain is eventually captured from the cytosol of murine and human macrophages into double-membrane vacuoles displaying the late endosomal marker, LAMP1, and the autophagy-associated protein, LC3, coinciding with a reduction in viable intracellular bacteria. Capture of SchuS4ΔdipA was not dipA-specific as other replication-deficient bacteria, such as chloramphenicol-treated SchuS4 and a purine auxotroph mutant SchuS4ΔpurMCD, were similarly targeted to autophagic vacuoles. Vacuoles containing replication-deficient bacteria were labeled with ubiquitin and the autophagy receptors SQSTM1/p62 and NBR1, and their formation was decreased in macrophages from either ATG5-, LC3B- or SQSTM1-deficient mice, indicating recognition by the ubiquitin-SQSTM1-LC3 pathway. While a fraction of both the wild-type and the replication-impaired strains were ubiquitinated and recruited SQSTM1, only the replication-defective strains progressed to autophagic capture, suggesting that wild-type Francisella interferes with the autophagic cascade. Survival of replication-deficient strains was not restored in autophagy-deficient macrophages, as these bacteria died in the cytosol prior to autophagic capture. Collectively, our results demonstrate that replication-impaired strains of Francisella are cleared by autophagy, while replication-competent bacteria seem to interfere with autophagic recognition, therefore ensuring survival

  14. The Psychology of Replication and Replication in Psychology.

    PubMed

    Francis, Gregory

    2012-11-01

    Like other scientists, psychologists believe experimental replication to be the final arbiter for determining the validity of an empirical finding. Reports in psychology journals often attempt to prove the validity of a hypothesis or theory with multiple experiments that replicate a finding. Unfortunately, these efforts are sometimes misguided because in a field like experimental psychology, ever more successful replication does not necessarily ensure the validity of an empirical finding. When psychological experiments are analyzed with statistics, the rules of probability dictate that random samples should sometimes be selected that do not reject the null hypothesis, even if an effect is real. As a result, it is possible for a set of experiments to have too many successful replications. When there are too many successful replications for a given set of experiments, a skeptical scientist should be suspicious that null or negative findings have been suppressed, the experiments were run improperly, or the experiments were analyzed improperly. This article describes the implications of this observation and demonstrates how to test for too much successful replication by using a set of experiments from a recent research paper.

  15. Sabin-to-Mahoney Transition Model of Quasispecies Replication

    SciTech Connect

    2009-05-31

    Qspp is an agent-based stochastic simulation model of the Poliovirus Sabin-to-Mahoney transition. This code simulates a cell-to-cell model of Poliovirus replication. The model tracks genotypes (virus genomes) as they are replicated in cells, and as the cells burst and release particles into the medium of a culture dish. An inoculum is then taken from the pool of virions and is used to inoculate cells on a new dish. This process repeats. The Sabin genotype comprises the initial inoculum. Nucleotide positions that match the Sabin1 (vaccine strain) and Mahoney (wild type) genotypes, as well as the neurovirulent phenotype (from the literature) are enumerated as constants.

  16. Self-replication: Nanostructure evolution

    NASA Astrophysics Data System (ADS)

    Simmel, Friedrich C.

    2017-10-01

    DNA origami nanostructures were utilized to replicate a seed pattern that resulted in the growth of populations of nanostructures. Exponential growth could be controlled by environmental conditions depending on the preferential requirements of each population.

  17. DNA recombination: the replication connection.

    PubMed

    Haber, J E

    1999-07-01

    Chromosomal double-strand breaks (DSBs) arise after exposure to ionizing radiation or enzymatic cleavage, but especially during the process of DNA replication itself. Homologous recombination plays a critical role in repair of such DSBs. There has been significant progress in our understanding of two processes that occur in DSB repair: gene conversion and recombination-dependent DNA replication. Recent evidence suggests that gene conversion and break-induced replication are related processes that both begin with the establishment of a replication fork in which both leading- and lagging-strand synthesis occur. There has also been much progress in characterization of the biochemical roles of recombination proteins that are highly conserved from yeast to humans.

  18. Regulating DNA Replication in Eukarya

    PubMed Central

    Siddiqui, Khalid; On, Kin Fan; Diffley, John F.X.

    2013-01-01

    DNA replication is tightly controlled in eukaryotic cells to ensure that an exact copy of the genetic material is inherited by both daughter cells. Oscillating waves of cyclin-dependent kinase (CDK) and anaphase-promoting complex/cyclosome (APC/C) activities provide a binary switch that permits the replication of each chromosome exactly once per cell cycle. Work from several organisms has revealed a conserved strategy whereby inactive replication complexes are assembled onto DNA during periods of low CDK and high APC activity but are competent to execute genome duplication only when these activities are reversed. Periods of high CDK and low APC/C serve an essential function by blocking reassembly of replication complexes, thereby preventing rereplication. Higher eukaryotes have evolved additional CDK-independent mechanisms for preventing rereplication. PMID:23838438

  19. Nucleotide Metabolism and DNA Replication.

    PubMed

    Warner, Digby F; Evans, Joanna C; Mizrahi, Valerie

    2014-10-01

    The development and application of a highly versatile suite of tools for mycobacterial genetics, coupled with widespread use of "omics" approaches to elucidate the structure, function, and regulation of mycobacterial proteins, has led to spectacular advances in our understanding of the metabolism and physiology of mycobacteria. In this article, we provide an update on nucleotide metabolism and DNA replication in mycobacteria, highlighting key findings from the past 10 to 15 years. In the first section, we focus on nucleotide metabolism, ranging from the biosynthesis, salvage, and interconversion of purine and pyrimidine ribonucleotides to the formation of deoxyribonucleotides. The second part of the article is devoted to DNA replication, with a focus on replication initiation and elongation, as well as DNA unwinding. We provide an overview of replication fidelity and mutation rates in mycobacteria and summarize evidence suggesting that DNA replication occurs during states of low metabolic activity, and conclude by suggesting directions for future research to address key outstanding questions. Although this article focuses primarily on observations from Mycobacterium tuberculosis, it is interspersed, where appropriate, with insights from, and comparisons with, other mycobacterial species as well as better characterized bacterial models such as Escherichia coli. Finally, a common theme underlying almost all studies of mycobacterial metabolism is the potential to identify and validate functions or pathways that can be exploited for tuberculosis drug discovery. In this context, we have specifically highlighted those processes in mycobacterial DNA replication that might satisfy this critical requirement.

  20. Plasmid Rolling-Circle Replication.

    PubMed

    Ruiz-Masó, J A; MachóN, C; Bordanaba-Ruiseco, L; Espinosa, M; Coll, M; Del Solar, G

    2015-02-01

    Plasmids are DNA entities that undergo controlled replication independent of the chromosomal DNA, a crucial step that guarantees the prevalence of the plasmid in its host. DNA replication has to cope with the incapacity of the DNA polymerases to start de novo DNA synthesis, and different replication mechanisms offer diverse solutions to this problem. Rolling-circle replication (RCR) is a mechanism adopted by certain plasmids, among other genetic elements, that represents one of the simplest initiation strategies, that is, the nicking by a replication initiator protein on one parental strand to generate the primer for leading-strand initiation and a single priming site for lagging-strand synthesis. All RCR plasmid genomes consist of a number of basic elements: leading strand initiation and control, lagging strand origin, phenotypic determinants, and mobilization, generally in that order of frequency. RCR has been mainly characterized in Gram-positive bacterial plasmids, although it has also been described in Gram-negative bacterial or archaeal plasmids. Here we aim to provide an overview of the RCR plasmids' lifestyle, with emphasis on their characteristic traits, promiscuity, stability, utility as vectors, etc. While RCR is one of the best-characterized plasmid replication mechanisms, there are still many questions left unanswered, which will be pointed out along the way in this review.

  1. The Biology of Replicative Senescence

    SciTech Connect

    Campisi, J.

    1996-12-04

    Most cells cannot divide indefinitely due to a processtermed cellular or replicative senescence. Replicative senescence appearsto be a fundamental feature of somatic cells, with the exception of mosttumour cells and possibly certain stem cells. How do cells sense thenumber of divisions they have completed? Although it has not yet beencritically tested, the telomere shortening hypothesis is currentlyperhaps the best explanation for a cell division 'counting' mechanism.Why do cells irreversibly cease proliferation after completing a finitenumber of divisions? It is now known that replicative senescence altersthe expression of a few crucial growth-regulatory genes. It is not knownhow these changes in growth-regulatory gene expression are related totelomere shortening in higher eukaryotes. However, lower eukaryotes haveprovided several plausible mechanisms. Finally, what are thephysiological consequences of replicative senescence? Several lines ofevidence suggest that, at least in human cells, replicative senescence isa powerful tumour suppressive mechanism. There is also indirect evidencethat replicative senescence contributes to ageing. Taken together,current findings suggest that, at least in mammals, replicativesenescence may have evolved to curtail tumorigenesis, but may also havethe unselected effect of contributing to age-related pathologies,including cancer.

  2. Pathways of mammalian replication fork restart.

    PubMed

    Petermann, Eva; Helleday, Thomas

    2010-10-01

    Single-molecule analyses of DNA replication have greatly advanced our understanding of mammalian replication restart. Several proteins that are not part of the core replication machinery promote the efficient restart of replication forks that have been stalled by replication inhibitors, suggesting that bona fide fork restart pathways exist in mammalian cells. Different models of replication fork restart can be envisaged, based on the involvement of DNA helicases, nucleases, homologous recombination factors and the importance of DNA double-strand break formation.

  3. Replication stress: getting back on track

    PubMed Central

    Berti, Matteo; Vindigni, Alessandro

    2016-01-01

    The replication stress response enables the DNA replication machinery to overcome DNA lesions or intrinsic replication fork obstacles, and is essential to ensure faithful transmission of genetic information to daughter cells. Multiple replication stress response pathways have been identified in recent years, raising questions about their specific and possibly redundant functions. Here, we review the emerging mechanisms of the replication stress response in mammalian cells, and consider how they may influence the dynamics of the core DNA replication complex. PMID:26840898

  4. Natural Strain

    NASA Technical Reports Server (NTRS)

    Freed, Alan D.

    1995-01-01

    The purpose of this paper is to present a consistent and thorough development of the strain and strain-rate measures affiliated with Hencky. Natural measures for strain and strain-rate, as I refer to them, are first expressed in terms of of the fundamental body-metric tensors of Lodge. These strain and strain-rate measures are mixed tensor fields. They are mapped from the body to space in both the Eulerian and Lagrangian configurations, and then transformed from general to Cartesian fields. There they are compared with the various strain and strain-rate measures found in the literature. A simple Cartesian description for Hencky strain-rate in the Lagrangian state is obtained.

  5. Modeling the Control of DNA Replication in Fission Yeast

    NASA Astrophysics Data System (ADS)

    Novak, Bela; Tyson, John J.

    1997-08-01

    A central event in the eukaryotic cell cycle is the decision to commence DNA replication (S phase). Strict controls normally operate to prevent repeated rounds of DNA replication without intervening mitoses (``endoreplication'') or initiation of mitosis before DNA is fully replicated (``mitotic catastrophe''). Some of the genetic interactions involved in these controls have recently been identified in yeast. From this evidence we propose a molecular mechanism of ``Start'' control in Schizosaccharomyces pombe. Using established principles of biochemical kinetics, we compare the properties of this model in detail with the observed behavior of various mutant strains of fission yeast: wee1- (size control at Start), cdc13Δ and rum1OP (endoreplication), and wee1- rum1Δ (rapid division cycles of diminishing cell size). We discuss essential features of the mechanism that are responsible for characteristic properties of Start control in fission yeast, to expose our proposal to crucial experimental tests.

  6. Shell Separation for Mirror Replication

    NASA Technical Reports Server (NTRS)

    1999-01-01

    NASA's Space Optics Manufacturing Center has been working to expand our view of the universe via sophisticated new telescopes. The Optics Center's goal is to develop low-cost, advanced space optics technologies for the NASA program in the 21st century - including the long-term goal of imaging Earth-like planets in distant solar systems. To reduce the cost of mirror fabrication, Marshall Space Flight Center (MSFC) has developed replication techniques, the machinery, and materials to replicate electro-formed nickel mirrors. Optics replication uses reusable forms, called mandrels, to make telescope mirrors ready for final finishing. MSFC optical physicist Bill Jones monitors a device used to chill a mandrel, causing it to shrink and separate from the telescope mirror without deforming the mirror's precisely curved surface.

  7. Norovirus gene expression and replication.

    PubMed

    Thorne, Lucy G; Goodfellow, Ian G

    2014-02-01

    Noroviruses are small, positive-sense RNA viruses within the family Caliciviridae, and are now accepted widely as a major cause of acute gastroenteritis in both developed and developing countries. Despite their impact, our understanding of the life cycle of noroviruses has lagged behind that of other RNA viruses due to the inability to culture human noroviruses (HuNVs). Our knowledge of norovirus biology has improved significantly over the past decade as a result of numerous technological advances. The use of a HuNV replicon, improved biochemical and cell-based assays, combined with the discovery of a murine norovirus capable of replication in cell culture, has improved greatly our understanding of the molecular mechanisms of norovirus genome translation and replication, as well as the interaction with host cell processes. In this review, the current state of knowledge of the intracellular life of noroviruses is discussed with particular emphasis on the mechanisms of viral gene expression and viral genome replication.

  8. Autonomous replicating sequences from mouse cells which can replicate in mouse cells in vivo and in vitro.

    PubMed Central

    Ariga, H; Itani, T; Iguchi-Ariga, S M

    1987-01-01

    We have already reported that the cloned mouse DNA fragment (pMU65) could replicate in a simian virus 40 T antigen-dependent system in vivo and in vitro (H. Ariga, Z. Tsuchihashi, M. Naruto, and M. Yamada, Mol. Cell. Biol. 5:563-568, 1985). The plasmid p65-tk, containing the thymidine kinase (tk) gene of herpes simplex virus and the BglII-EcoRI region of pMU65 homologous to the simian virus 40 origin of DNA replication, was constructed. The p65-tk persisted episomally in tk+ transformants after the transfection of p65-tk into mouse FM3Atk- cells. The copy numbers of p65-tk in FM3Atk+ cells were 100 to 200 copies per cell. Furthermore, the p65-tk replicated semiconservatively, and the initiation of DNA replication started from the mouse DNA sequences when the replicating activity of p65-tk was tested in the in vitro DNA replication system developed from the FM3A cells. These results show that a 2.5-kilobase fragment of mouse DNA contains the autonomously replicating sequences. Images PMID:3031448

  9. Mismatch Repair Balances Leading and Lagging Strand DNA Replication Fidelity

    DTIC Science & Technology

    2012-10-11

    Pol e mutator variant to confirm that Pol e is the primary leading strand replicase in Saccharomyces cerevisiae . We then use polymerase-specific error...variables using Saccharomyces cerevisiae strains containing mutant alleles of the POL1 (Pol a), POL2 (Pol e) and POL3 (Pol d) genes. These mutant...AL, Johnston LH, Sugino A (1993) Pathway correcting DNA replication errors in Saccharomyces cerevisiae . Embo J 12: 1467–1473. 17. Morrison A, Sugino A

  10. A novel benzonitrile analogue inhibits rhinovirus replication.

    PubMed

    Lacroix, Céline; Querol-Audí, Jordi; Roche, Manon; Franco, David; Froeyen, Mathy; Guerra, Pablo; Terme, Thierry; Vanelle, Patrice; Verdaguer, Núria; Neyts, Johan; Leyssen, Pieter

    2014-10-01

    To study the characteristics and the mode of action of the anti-rhinovirus compound 4-[1-hydroxy-2-(4,5-dimethoxy-2-nitrophenyl)ethyl]benzonitrile (LPCRW_0005). The antiviral activity of LPCRW_0005 was evaluated in a cytopathic effect reduction assay against a panel of human rhinovirus (HRV) strains. To unravel its precise molecular mechanism of action, a time-of-drug-addition study, resistance selection and thermostability assays were performed. The crystal structure of the HRV14/LPCRW_0005 complex was elucidated as well. LPCRW_0005 proved to be a selective inhibitor of the replication of HRV14 (EC(50) of 2 ± 1 μM). Time-of-drug-addition studies revealed that LPCRW_0005 interferes with the earliest stages of virus replication. Phenotypic drug-resistant virus variants were obtained (≥30-fold decrease in susceptibility to the inhibitory effect of LPCRW_0005), which carried either an A150T or A150V amino acid substitution in the VP1 capsid protein. The link between the mutant genotype and drug-resistant phenotype was confirmed by reverse genetics. Cross-resistance studies and thermostability assays revealed that LPCRW_0005 has a similar mechanism of action to the capsid binder pleconaril. Elucidation of the crystal structure of the HRV14/LPCRW_0005 complex revealed the existence of multiple hydrophobic and polar interactions between the VP1 pocket and LPCRW_0005. LPCRW_0005 is a novel inhibitor of HRV14 replication that acts as a capsid binder. The compound has a chemical structure that is markedly smaller than that of other capsid binders. Structural studies show that LPCRW_0005, in contrast to pleconaril, leaves the toe end of the pocket in VP1 empty. This suggests that extended analogues of LPCRW_0005 that fill the full cavity could be more potent inhibitors of rhinovirus replication. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e

  11. Personality and Academic Motivation: Replication, Extension, and Replication

    ERIC Educational Resources Information Center

    Jones, Martin H.; McMichael, Stephanie N.

    2015-01-01

    Previous work examines the relationships between personality traits and intrinsic/extrinsic motivation. We replicate and extend previous work to examine how personality may relate to achievement goals, efficacious beliefs, and mindset about intelligence. Approximately 200 undergraduates responded to the survey with a 150 participants replicating…

  12. Replicating systems concepts: Self-replicating lunar factory and demonstration

    NASA Technical Reports Server (NTRS)

    1982-01-01

    Automation of lunar mining and manufacturing facility maintenance and repair is addressed. Designing the factory as an automated, multiproduct, remotely controlled, reprogrammable Lunar Manufacturing Facility capable of constructing duplicates of itself which would themselves be capable of further replication is proposed.

  13. A Replication of Failure, Not a Failure to Replicate

    ERIC Educational Resources Information Center

    Holden, Gary; Barker, Kathleen; Kuppens, Sofie; Rosenberg, Gary; LeBreton, Jonathan

    2015-01-01

    Purpose: The increasing role of systematic reviews in knowledge production demands greater rigor in the literature search process. The performance of the Social Work Abstracts (SWA) database has been examined multiple times over the past three decades. The current study is a replication within this line of research. Method: Issue-level coverage…

  14. Exploiting replication in distributed systems

    NASA Technical Reports Server (NTRS)

    Birman, Kenneth P.; Joseph, T. A.

    1989-01-01

    Techniques are examined for replicating data and execution in directly distributed systems: systems in which multiple processes interact directly with one another while continuously respecting constraints on their joint behavior. Directly distributed systems are often required to solve difficult problems, ranging from management of replicated data to dynamic reconfiguration in response to failures. It is shown that these problems reduce to more primitive, order-based consistency problems, which can be solved using primitives such as the reliable broadcast protocols. Moreover, given a system that implements reliable broadcast primitives, a flexible set of high-level tools can be provided for building a wide variety of directly distributed application programs.

  15. Discovery and Optimization of Benzotriazine Di-N-Oxides Targeting Replicating and Non-replicating Mycobacterium tuberculosis

    PubMed Central

    Chopra, Sidharth; Koolpe, Gary A.; Tambo-ong, Arlyn A.; Matsuyama, Karen N.; Ryan, Kenneth J.; Tran, Tran B.; Doppalapudi, Rupa S.; Riccio, Edward S.; Iyer, Lalitha V.; Green, Carol E.; Wan, Baojie; Franzblau, Scott G.; Madrid, Peter B.

    2012-01-01

    Compounds bactericidal against both replicating and non-replicating Mtb may shorten the length of TB treatment regimens by eliminating infections more rapidly. Screening of a panel of antimicrobial and anticancer drug classes that are bioreduced into cytotoxic species revealed that 1,2,4-benzotriazine di-N-oxides (BTOs) are potently bactericidal against replicating and non-replicating Mtb. Medicinal chemistry optimization, guided by semi-empirical molecular orbital calculations, identified a new lead compound (20q) from this series with an MIC of 0.31 μg/mL against H37Rv and a cytotoxicity (CC50) against Vero cells of 25 μg/mL. 20q also had equivalent potency against a panel of single-drug resistant strains of Mtb and remarkably selective activity for Mtb over a panel of other pathogenic bacterial strains. 20q was also negative in a L5178Y MOLY assay, indicating low potential for genetic toxicity. These data along with measurements of the physiochemical properties and pharmacokinetic profile demonstrate that BTOs have the potential to be developed into a new class of antitubercular drugs. PMID:22691154

  16. Arenavirus Infection Induces Discrete Cytosolic Structures for RNA Replication

    PubMed Central

    Baird, Nicholas L.; York, Joanne

    2012-01-01

    Arenaviruses are responsible for acute hemorrhagic fevers with high mortality and pose significant threats to public health and biodefense. These enveloped negative-sense RNA viruses replicate in the cell cytoplasm and express four proteins. To better understand how these proteins insinuate themselves into cellular processes to orchestrate productive viral replication, we have identified and characterized novel cytosolic structures involved in arenavirus replication and transcription. In cells infected with the nonpathogenic Tacaribe virus or the attenuated Candid#1 strain of Junín virus, we find that newly synthesized viral RNAs localize to cytosolic puncta containing the nucleoprotein (N) of the virus. Density gradient centrifugation studies reveal that these replication-transcription complexes (RTCs) are associated with cellular membranes and contain full-length genomic- and antigenomic-sense RNAs. Viral mRNAs segregate at a higher buoyant density and are likewise scant in immunopurified RTCs, consistent with their translation on bulk cellular ribosomes. In addition, confocal microscopy analysis reveals that RTCs contain the lipid phosphatidylinositol-4-phosphate and proteins involved in cellular mRNA metabolism, including the large and small ribosomal subunit proteins L10a and S6, the stress granule protein G3BP1, and a subset of translation initiation factors. Elucidating the structure and function of RTCs will enhance our understanding of virus-cell interactions that promote arenavirus replication and mitigate against host cell immunity. This knowledge may lead to novel intervention strategies to limit viral virulence and pathogenesis. PMID:22875974

  17. Phenotype switching is a natural consequence of Staphylococcus aureus replication.

    PubMed

    Edwards, Andrew M

    2012-10-01

    The pathogen Staphylococcus aureus undergoes phenotype switching in vivo from its normal colony phenotype (NCP) to a slow-growing, antibiotic-resistant small-colony-variant (SCV) phenotype that is associated with persistence in host cells and tissues. However, it is not clear whether phenotype switching is the result of a constitutive process that is selected for under certain conditions or is triggered by particular environmental stimuli. Examination of cultures of diverse S. aureus strains in the absence of selective pressure consistently revealed a small gentamicin-resistant SCV subpopulation that emerged during exponential-phase NCP growth and increased in number until NCP stationary phase. Treatment of replicating bacteria with the antibiotic gentamicin, which inhibited NCP but not SCV replication, resulted in an initial decrease in SCV numbers, demonstrating that SCVs arise as a consequence of NCP replication. However, SCV population expansion in the presence of gentamicin was reestablished by selection of phenotype-stable SCVs and subsequent SCV replication. In the absence of selective pressure, however, phenotype switching was bidirectional and occurred at a high frequency during NCP replication, resulting in SCV turnover. In summary, these data demonstrate that S. aureus phenotype switching occurs via a constitutive mechanism that generates a dynamic, antibiotic-resistant subpopulation of bacteria that can revert to the parental phenotype. The emergence of SCVs can therefore be considered a normal part of the S. aureus life cycle and provides an insurance policy against exposure to antibiotics that would otherwise eliminate the entire population.

  18. Thermosensitive replication of a kanamycin resistance factor.

    PubMed

    Terawaki, Y; Takayasu, H; Akiba, T

    1967-09-01

    A strain of Proteus vulgaris isolated from the urinary tract of a patient with postoperative pyelonephritis and resistant to sulfonamide, streptomycin, tetracycline, and kanamycin (KM) was found to transfer only KM resistance by cell-to-cell conjugation. The genetic determinant controlling the transferable KM resistance was considered to be an R factor and was designated R (KM). Successive transfer of KM resistance was demonstrated also from Escherichia coli 20S0, which received the R (KM) factor, to other substrains of E. coli K-12 or Salmonella typhimurium LT-2. The transfer of the R (KM) factor was strongly affected by the temperature at which the mating culture was kept. The transfer frequency of R (KM) at 25 C was about 10(5) times higher than at 37 C. The R (KM) factor was spontaneously eliminated from the host bacterial cells when P. vulgaris was cultured at 42 C, but no elimination occurred at 25 C. This elimination of the R (KM) factor at elevated temperature was also observed when the R (KM) factor infected E. coli and S. typhimurium. On the other hand, a normal R factor could not be eliminated from the same E. coli host strain by cultivation at the higher temperature. We consider the thermosensitive transfer and the spontaneous elimination of the R (KM) factor at higher temperature to depend upon thermosensitive replication of the R (KM) factor.

  19. Cellular Aspects of Prion Replication In Vitro

    PubMed Central

    Grassmann, Andrea; Wolf, Hanna; Hofmann, Julia; Graham, James; Vorberg, Ina

    2013-01-01

    Prion diseases or transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative disorders in mammals that are caused by unconventional agents predominantly composed of aggregated misfolded prion protein (PrP). Prions self-propagate by recruitment of host-encoded PrP into highly ordered β-sheet rich aggregates. Prion strains differ in their clinical, pathological and biochemical characteristics and are likely to be the consequence of distinct abnormal prion protein conformers that stably replicate their alternate states in the host cell. Understanding prion cell biology is fundamental for identifying potential drug targets for disease intervention. The development of permissive cell culture models has greatly enhanced our knowledge on entry, propagation and dissemination of TSE agents. However, despite extensive research, the precise mechanism of prion infection and potential strain effects remain enigmatic. This review summarizes our current knowledge of the cell biology and propagation of prions derived from cell culture experiments. We discuss recent findings on the trafficking of cellular and pathologic PrP, the potential sites of abnormal prion protein synthesis and potential co-factors involved in prion entry and propagation. PMID:23340381

  20. Sustaining Interferon Induction by a High-Passage Atypical Porcine Reproductive and Respiratory Syndrome Virus Strain

    PubMed Central

    Ma, Zexu; Yu, Ying; Xiao, Yueqiang; Opriessnig, Tanja; Wang, Rong; Yang, Liping; Nan, Yuchen; Samal, Siba K.; Halbur, Patrick G.; Zhang, Yan-Jin

    2016-01-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) strain A2MC2 induces type I interferons in cultured cells. The objective of this study was to attenuate this strain by serial passaging in MARC-145 cells and assess its virulence and immunogenicity in pigs. The A2MC2 serially passaged 90 times (A2MC2-P90) retains the feature of interferon induction. The A2MC2-P90 replicates faster with a higher virus yield than wild type A2MC2 virus. Infection of primary pulmonary alveolar macrophages (PAMs) also induces interferons. Sequence analysis showed that the A2MC2-P90 has genomic nucleic acid identity of 99.8% to the wild type but has a deletion of 543 nucleotides in nsp2. The deletion occurred in passage 60. The A2MC2-P90 genome has a total of 35 nucleotide variations from the wild type, leading to 26 amino acid differences. Inoculation of three-week-old piglets showed that A2MC2-P90 is avirulent and elicits immune response. Compared with Ingelvac PRRS® MLV strain, A2MC2-P90 elicits higher virus neutralizing antibodies. The attenuated IFN-inducing A2MC2-P90 should be useful for development of an improved PRRSV vaccine. PMID:27805024

  1. Specific DNA replication mutations affect telomere length in Saccharomyces cerevisiae.

    PubMed Central

    Adams, A K; Holm, C

    1996-01-01

    To investigate the relationship between the DNA replication apparatus and the control of telomere length, we examined the effects of several DNA replication mutations on telomere length in Saccharomyces cerevisiae. We report that a mutation in the structural gene for the large subunit of DNA replication factor C (cdc44/rfc1) causes striking increases in telomere length. A similar effect is seen with mutations in only one other DNA replication gene: the structural gene for DNA polymerase alpha (cdc17/pol1) (M.J. Carson and L. Hartwell, Cell 42:249-257, 1985). For both genes, the telomere elongation phenotype is allele specific and appears to correlate with the penetrance of the mutations. Furthermore, fluorescence-activated cell sorter analysis reveals that those alleles that cause elongation also exhibit a slowing of DNA replication. To determine whether elongation is mediated by telomerase or by slippage of the DNA polymerase, we created cdc17-1 mutants carrying deletions of the gene encoding the RNA component of telomerase (TLC1). cdc17-1 strains that would normally undergo telomere elongation failed to do so in the absence of telomerase activity. This result implies that telomere elongation in cdc17-1 mutants is mediated by the action of telomerase. Since DNA replication involves transfer of the nascent strand from polymerase alpha to replication factor C (T. Tsurimoto and B. Stillman, J. Biol. Chem. 266:1950-1960, 1991; T. Tsurimoto and B. Stillman, J. Biol. Chem. 266:1961-1968, 1991; S. Waga and B. Stillman, Nature [London] 369:207-212, 1994), one possibility is that this step affects the regulation of telomere length. PMID:8756617

  2. Strain patterns and strain accumulation along plate margins

    NASA Technical Reports Server (NTRS)

    Savage, J. C.

    1978-01-01

    Observations of strain accumulation along plate margins in Japan, New Zealand, and the United States indicate that: (1) a typical maximum rate of secular strain accumulation is on the order of 0.3 ppm/a, (2) a substantial part of the strain accumulation process can be attributed to slip at depth on the major plate boundary faults, and (3) some plastic deformation in a zone 100 km or more in width is apparently involved in the strain accumulation process.

  3. Differential Host Response, Rather Than Early Viral Replication Efficiency, Correlates with Pathogenicity Caused by Influenza Viruses

    PubMed Central

    Askovich, Peter S.; Sanders, Catherine J.; Rosenberger, Carrie M.; Diercks, Alan H.; Dash, Pradyot; Navarro, Garnet; Vogel, Peter; Doherty, Peter C.; Thomas, Paul G.; Aderem, Alan

    2013-01-01

    Influenza viruses exhibit large, strain-dependent differences in pathogenicity in mammalian hosts. Although the characteristics of severe disease, including uncontrolled viral replication, infection of the lower airway, and highly inflammatory cytokine responses have been extensively documented, the specific virulence mechanisms that distinguish highly pathogenic strains remain elusive. In this study, we focused on the early events in influenza infection, measuring the growth rate of three strains of varying pathogenicity in the mouse airway epithelium and simultaneously examining the global host transcriptional response over the first 24 hours. Although all strains replicated equally rapidly over the first viral life-cycle, their growth rates in both lung and tracheal tissue strongly diverged at later times, resulting in nearly 10-fold differences in viral load by 24 hours following infection. We identified separate networks of genes in both the lung and tracheal tissues whose rapid up-regulation at early time points by specific strains correlated with a reduced viral replication rate of those strains. The set of early-induced genes in the lung that led to viral growth restriction is enriched for both NF-κB binding site motifs and members of the TREM1 and IL-17 signaling pathways, suggesting that rapid, NF-κB –mediated activation of these pathways may contribute to control of viral replication. Because influenza infection extending into the lung generally results in severe disease, early activation of these pathways may be one factor distinguishing high- and low-pathogenicity strains. PMID:24073225

  4. Differential host response, rather than early viral replication efficiency, correlates with pathogenicity caused by influenza viruses.

    PubMed

    Askovich, Peter S; Sanders, Catherine J; Rosenberger, Carrie M; Diercks, Alan H; Dash, Pradyot; Navarro, Garnet; Vogel, Peter; Doherty, Peter C; Thomas, Paul G; Aderem, Alan

    2013-01-01

    Influenza viruses exhibit large, strain-dependent differences in pathogenicity in mammalian hosts. Although the characteristics of severe disease, including uncontrolled viral replication, infection of the lower airway, and highly inflammatory cytokine responses have been extensively documented, the specific virulence mechanisms that distinguish highly pathogenic strains remain elusive. In this study, we focused on the early events in influenza infection, measuring the growth rate of three strains of varying pathogenicity in the mouse airway epithelium and simultaneously examining the global host transcriptional response over the first 24 hours. Although all strains replicated equally rapidly over the first viral life-cycle, their growth rates in both lung and tracheal tissue strongly diverged at later times, resulting in nearly 10-fold differences in viral load by 24 hours following infection. We identified separate networks of genes in both the lung and tracheal tissues whose rapid up-regulation at early time points by specific strains correlated with a reduced viral replication rate of those strains. The set of early-induced genes in the lung that led to viral growth restriction is enriched for both NF-κB binding site motifs and members of the TREM1 and IL-17 signaling pathways, suggesting that rapid, NF-κB -mediated activation of these pathways may contribute to control of viral replication. Because influenza infection extending into the lung generally results in severe disease, early activation of these pathways may be one factor distinguishing high- and low-pathogenicity strains.

  5. Covert Reinforcement: A Partial Replication.

    ERIC Educational Resources Information Center

    Ripstra, Constance C.; And Others

    A partial replication of an investigation of the effect of covert reinforcement on a perceptual estimation task is described. The study was extended to include an extinction phase. There were five treatment groups: covert reinforcement, neutral scene reinforcement, noncontingent covert reinforcement, and two control groups. Each subject estimated…

  6. Chameleon Chasing II: A Replication.

    ERIC Educational Resources Information Center

    Newsom, Doug A.; And Others

    1993-01-01

    Replicates a 1972 survey of students, educators, and Public Relations Society of America members regarding who the public relations counselor really serves. Finds that, in 1992, most respondents thought primary responsibility was to the client, then to the client's relevant publics, then to self, then to society, and finally to media. Compares…

  7. Replication and Reporting: A Commentary.

    ERIC Educational Resources Information Center

    Polio, Charlene; Gass, Susan

    1997-01-01

    Addresses the need for replication studies in the field of second-language acquisition and discusses the problems surrounding standards of reporting research. Notes a lack of uniform standards in reporting second-language learners' proficiency levels and proposes ways to achieve more thorough reporting of research that will allow others to engage…

  8. Hyperthermia stimulates HIV-1 replication.

    PubMed

    Roesch, Ferdinand; Meziane, Oussama; Kula, Anna; Nisole, Sébastien; Porrot, Françoise; Anderson, Ian; Mammano, Fabrizio; Fassati, Ariberto; Marcello, Alessandro; Benkirane, Monsef; Schwartz, Olivier

    2012-01-01

    HIV-infected individuals may experience fever episodes. Fever is an elevation of the body temperature accompanied by inflammation. It is usually beneficial for the host through enhancement of immunological defenses. In cultures, transient non-physiological heat shock (42-45°C) and Heat Shock Proteins (HSPs) modulate HIV-1 replication, through poorly defined mechanisms. The effect of physiological hyperthermia (38-40°C) on HIV-1 infection has not been extensively investigated. Here, we show that culturing primary CD4+ T lymphocytes and cell lines at a fever-like temperature (39.5°C) increased the efficiency of HIV-1 replication by 2 to 7 fold. Hyperthermia did not facilitate viral entry nor reverse transcription, but increased Tat transactivation of the LTR viral promoter. Hyperthermia also boosted HIV-1 reactivation in a model of latently-infected cells. By imaging HIV-1 transcription, we further show that Hsp90 co-localized with actively transcribing provirus, and this phenomenon was enhanced at 39.5°C. The Hsp90 inhibitor 17-AAG abrogated the increase of HIV-1 replication in hyperthermic cells. Altogether, our results indicate that fever may directly stimulate HIV-1 replication, in a process involving Hsp90 and facilitation of Tat-mediated LTR activity.

  9. Replication of kinetoplast minicircle DNA

    SciTech Connect

    Sheline, C.T.

    1989-01-01

    These studies describe the isolation and characterization of early minicircle replication intermediates from Crithidia fasciculata, and Leishmania tarentolae, the mitochondrial localization of a type II topoisomerase (TIImt) in C. fasciculata, and the implication of the aforementioned TIImt in minicircle replication in L. tarentolae. Early minicircle replication intermediates from C. fasciculata were identified and characterized using isolated kinetoplasts to incorporate radiolabeled nucleotides into its DNA. The pulse-label in an apparent theta-type intermediate chase into two daughter molecules. A uniquely gapped, ribonucleotide primed, knotted molecule represents the leading strand in the model proposed, and a highly gapped molecule represents the lagging strand. This theta intermediate is repaired in vitro to a doubly nicked catenated dimer which was shown to result from the replication of a single parental molecule. Very similar intermediates were found in the heterogeneous population of minicircles of L. tarentolae. The sites of the Leishmania specific discontinuities were mapped and shown to lie within the universally conserved sequence blocks in identical positions as compared to C. fasciculata and Trypanosoma equiperdum.

  10. Hyperthermia Stimulates HIV-1 Replication

    PubMed Central

    Roesch, Ferdinand; Meziane, Oussama; Kula, Anna; Nisole, Sébastien; Porrot, Françoise; Anderson, Ian; Mammano, Fabrizio; Fassati, Ariberto; Marcello, Alessandro; Benkirane, Monsef; Schwartz, Olivier

    2012-01-01

    HIV-infected individuals may experience fever episodes. Fever is an elevation of the body temperature accompanied by inflammation. It is usually beneficial for the host through enhancement of immunological defenses. In cultures, transient non-physiological heat shock (42–45°C) and Heat Shock Proteins (HSPs) modulate HIV-1 replication, through poorly defined mechanisms. The effect of physiological hyperthermia (38–40°C) on HIV-1 infection has not been extensively investigated. Here, we show that culturing primary CD4+ T lymphocytes and cell lines at a fever-like temperature (39.5°C) increased the efficiency of HIV-1 replication by 2 to 7 fold. Hyperthermia did not facilitate viral entry nor reverse transcription, but increased Tat transactivation of the LTR viral promoter. Hyperthermia also boosted HIV-1 reactivation in a model of latently-infected cells. By imaging HIV-1 transcription, we further show that Hsp90 co-localized with actively transcribing provirus, and this phenomenon was enhanced at 39.5°C. The Hsp90 inhibitor 17-AAG abrogated the increase of HIV-1 replication in hyperthermic cells. Altogether, our results indicate that fever may directly stimulate HIV-1 replication, in a process involving Hsp90 and facilitation of Tat-mediated LTR activity. PMID:22807676

  11. Replication and Reporting: A Commentary.

    ERIC Educational Resources Information Center

    Polio, Charlene; Gass, Susan

    1997-01-01

    Addresses the need for replication studies in the field of second-language acquisition and discusses the problems surrounding standards of reporting research. Notes a lack of uniform standards in reporting second-language learners' proficiency levels and proposes ways to achieve more thorough reporting of research that will allow others to engage…

  12. Mcm2 phosphorylation and the response to replicative stress

    PubMed Central

    2012-01-01

    Background The replicative helicase in eukaryotic cells is comprised of minichromosome maintenance (Mcm) proteins 2 through 7 (Mcm2-7) and is a key target for regulation of cell proliferation. In addition, it is regulated in response to replicative stress. One of the protein kinases that targets Mcm2-7 is the Dbf4-dependent kinase Cdc7 (DDK). In a previous study, we showed that alanine mutations of the DDK phosphorylation sites at S164 and S170 in Saccharomyces cerevisiae Mcm2 result in sensitivity to caffeine and methyl methanesulfonate (MMS) leading us to suggest that DDK phosphorylation of Mcm2 is required in response to replicative stress. Results We show here that a strain with the mcm2 allele lacking DDK phosphorylation sites (mcm2AA) is also sensitive to the ribonucleotide reductase inhibitor, hydroxyurea (HU) and to the base analogue 5-fluorouracil (5-FU) but not the radiomimetic drug, phleomycin. We screened the budding yeast non-essential deletion collection for synthetic lethal interactions with mcm2AA and isolated deletions that include genes involved in the control of genome integrity and oxidative stress. In addition, the spontaneous mutation rate, as measured by mutations in CAN1, was increased in the mcm2AA strain compared to wild type, whereas with a phosphomimetic allele (mcm2EE) the mutation rate was decreased. These results led to the idea that the mcm2AA strain is unable to respond properly to DNA damage. We examined this by screening the deletion collection for suppressors of the caffeine sensitivity of mcm2AA. Deletions that decrease spontaneous DNA damage, increase homologous recombination or slow replication forks were isolated. Many of the suppressors of caffeine sensitivity suppressed other phenotypes of mcm2AA including sensitivity to genotoxic drugs, the increased frequency of cells with RPA foci and the increased mutation rate. Conclusions Together these observations point to a role for DDK-mediated phosphorylation of Mcm2 in the

  13. Combination therapy with conditionally replicating adenovirus and replication defective adenovirus.

    PubMed

    Lee, Choon-Taek; Park, Kyung-Ho; Yanagisawa, Kiyoshi; Adachi, Yasushi; Ohm, Joyce E; Nadaf, Sorena; Dikov, Mikhail M; Curiel, David T; Carbone, David P

    2004-09-15

    Low gene transfer rate is the most substantial hurdle in the practical application of gene therapy. One strategy to improve transfer efficiency is the use of a conditionally replicating adenovirus (CRAD) that can selectively replicate in tumor cells. We hypothesized that conventional E1-deleted adenoviruses (ad) can become replication-competent when cotransduced with a CRAD to selectively supply E1 in trans in tumors. The resulting selective production of large numbers of the E1-deleted ad within the tumor mass will increase the transduction efficiency. We used a CRAD (Delta24RGD) that produces a mutant E1 without the ability to bind retinoblastoma but retaining viral replication competence in cancer cells with a defective pRb/p16. Ad-lacZ, adenovirus-luciferase (ad-luc), and adenovirus insulin-like growth factor-1R/dominant-negative (ad-IGF-1R/dn; 482, 950) are E1-deleted replication-defective adenoviruses. The combination of CRAD and ad-lacZ increased the transduction efficiency of lacZ to 100% from 15% observed with ad-lacZ alone. Transfer of media of CRAD and ad-lacZ cotransduced cells induced the transfer of lacZ (media transferable bystander effect). Combination of CRAD and ad-IGF-1R/dn increased the production of truncated IGF-1R or soluble IGF-1R > 10 times compared with transduction with ad-IGF-1R/dn alone. Combined intratumoral injection of CRAD and ad-luc increased the luciferase expression about 70 times compared with ad-luc alone without substantial systemic spread. Combined intratumoral injection of CRAD and ad-IGF-1R/482 induced stronger growth suppression of established lung cancer xenografts than single injections. The combination of CRAD and E1-deleted ad induced tumor-specific replication of CRAD and E1-deleted ad and increased the transduction rate and therapeutic efficacy of these viruses in model tumors.

  14. More precise mapping of the replication origin in Escherichia coli K-12.

    PubMed

    Louarn, J; Funderburgh, M; Bird, R E

    1974-10-01

    The origin of replication in Escherichia coli K-12 was mapped by determining the rate of marker replication during a synchronous round of replication. Four isogenic strains were made lysogenic for lambdaind(-) and for phage Mu-1, with Mu-1 integrated into a different chromosomal location in each strain. Cultures were starved for amino acids to allow completion of chromosome replication cycles and then starved for thymine in the presence of amino acids, and a synchronous cycle of replication was initiated by the addition of thymine. Samples were exposed to radioactive thymidine at intervals, deoxyribonucleic acid was extracted, and the rate of marker replication was determined by deoxyribonucleic acid-deoxyribonucleic acid hybridization to filters containing Mu-1, lambda, and E. coli deoxyribonucleic acid. The results confirm that the origin of replication is near ilv. The travel times of the replication forks, calculated from the data obtained for cultures with doubling times of approximately 40 and 61 min, are 40 and 52 min, respectively.

  15. Replication of Chinese hamster embryo cells transformed by temperature-sensitive T-antigen mutants of simian virus 40.

    PubMed Central

    Robinson, C C; Swartzendruber, D E; Lehman, J M

    1980-01-01

    Chinese hamster embryo cells transformed by simian virus 40 temperature-sensitive T-antigen mutants replicated when confluent at 40.5 degrees C, regardless of the selection method, selection temperature, or virus strain used. Images PMID:6251272

  16. The hemagglutinin protein of highly pathogenic H5N1 influenza viruses overcomes an early block in the replication cycle to promote productive replication in macrophages.

    PubMed

    Cline, Troy D; Karlsson, Erik A; Seufzer, Bradley J; Schultz-Cherry, Stacey

    2013-02-01

    Macrophages are known to be one of the first lines of defense against influenza virus infection. However, they may also contribute to severe disease caused by the highly pathogenic avian (HPAI) H5N1 influenza viruses. One reason for this may be the ability of certain influenza virus strains to productively replicate in macrophages. However, studies investigating the productive replication of influenza viruses in macrophages have been contradictory, and the results may depend on both the type of macrophages used and the specific viral strain. In this work, we investigated the ability of H1 to H16 viruses to productively replicate in primary murine alveolar macrophages and RAW264.7 macrophages. We show that only a subset of HPAI H5N1 viruses, those that cause high morbidity and mortality in mammals, can productively replicate in macrophages, as measured by the release of newly synthesized virus particles into the cell supernatant. Mechanistically, we found that these H5 strains can overcome a block early in the viral life cycle leading to efficient nuclear entry, viral transcription, translation, and ultimately replication. Studies with reassortant viruses demonstrated that expression of the hemagglutinin gene from an H5N1 virus rescued replication of H1N1 influenza virus in macrophages. This study is the first to characterize H5N1 influenza viruses as the only subtype of influenza virus capable of productive replication in macrophages and establishes the viral gene that is required for this characteristic. The ability to productively replicate in macrophages is unique to H5N1 influenza viruses and may contribute to their increased pathogenesis.

  17. Replication of nanoscale DNA patterns

    NASA Astrophysics Data System (ADS)

    Maass, Corinna; Wang, Tong; Sha, Ruojie; Leunissen, Mirjam; Dreyfus, Remi; Seeman, Nadrian; Chaikin, Paul

    2011-03-01

    We present an artificial supramolecular system mimicking self- replication and information transmission strategies in nature, but without the aid of enzymes or equivalent biological mechanisms. Using DNA nanotechnology techniques, we can make DNA tiles with selective interactions based on complementary single-strand connections. A linear tile pattern distinguished by their connector sequences is transmitted to a subsequent generation of copies by connector hybridisation. Longitudinal pattern formation and transverse copy attachment are well separated by different melting temperatures. We have achieved a faithful transmission of the pattern information to the second replication generation. We use AFM imaging to test for pattern fidelity and gel electrophoresis for quantitative yield analysis. supported by a DAAD postdoc grant.

  18. Neuropathogenic Capacity of Lentogenic, Mesogenic, and Velogenic Newcastle Disease Virus Strains in Day-Old Chickens.

    PubMed

    Moura, V M B D; Susta, L; Cardenas-Garcia, S; Stanton, J B; Miller, P J; Afonso, C L; Brown, C C

    2016-01-01

    Strains of Newcastle disease virus (NDV) have different abilities to elicit neurologic signs. To determine the capacity of different NDV strains to replicate and cause lesions in the brain, independently of their peripheral replication, 1-day-old chickens were inoculated in the subdural space with 7 NDV strains of different virulence (4 velogenic, 2 mesogenic, 1 lentogenic). Velogenic strains induced severe necrotizing and heterophilic ventriculitis and meningitis, as well as edema of the neuroparenchyma, and replicated extensively in the nervous tissue by day 2 postinfection, as demonstrated by immunohistochemistry, when all infected birds died. Clinical signs, microscopic lesions, and viral replication were delayed (days 3 and 4 postinfection) with mesogenic strains. Velogenic and mesogenic NDV strains replicated mainly in neurons, and immunolabeling was first detected in surface-oriented areas (periventricular and submeningeal), possibly as a reflection of the inoculation route. The lentogenic NDV strain did not cause death of infected birds; replication was confined to the epithelium of the ependyma and choroid plexuses; and lesions consisted of lymphoid aggregates limited to the choroid plexuses. Results show that extensive NDV replication in the brain is typical of velogenic and mesogenic, but not lentogenic, NDV strains. In addition, this study suggests that differences in the rate of NDV replication in nervous tissue, not differences in neurotropism, differentiate velogenic from mesogenic NDV strains. This study indicates that intracerebral inoculation might be used as an effective method to study the mechanisms of NDV neuropathogenesis.

  19. Alphavirus polymerase and RNA replication.

    PubMed

    Pietilä, Maija K; Hellström, Kirsi; Ahola, Tero

    2017-01-16

    Alphaviruses are typically arthropod-borne, and many are important pathogens such as chikungunya virus. Alphaviruses encode four nonstructural proteins (nsP1-4), initially produced as a polyprotein P1234. nsP4 is the core RNA-dependent RNA polymerase but all four nsPs are required for RNA synthesis. The early replication complex (RC) formed by the polyprotein P123 and nsP4 synthesizes minus RNA strands, and the late RC composed of fully processed nsP1-nsP4 is responsible for the production of genomic and subgenomic plus strands. Different parts of nsP4 recognize the promoters for minus and plus strands but the binding also requires the other nsPs. The alphavirus polymerase has been purified and is capable of de novo RNA synthesis only in the presence of the other nsPs. The purified nsP4 also has terminal adenylyltransferase activity, which may generate the poly(A) tail at the 3' end of the genome. Membrane association of the nsPs is vital for replication, and alphaviruses induce membrane invaginations called spherules, which form a microenvironment for RNA synthesis by concentrating replication components and protecting double-stranded RNA intermediates. The RCs isolated as crude membrane preparations are active in RNA synthesis in vitro, but high-resolution structure of the RC has not been achieved, and thus the arrangement of viral and possible host components remains unknown. For some alphaviruses, Ras-GTPase-activating protein (Src-homology 3 (SH3) domain)-binding proteins (G3BPs) and amphiphysins have been shown to be essential for RNA replication and are present in the RCs. Host factors offer an additional target for antivirals, as only few alphavirus polymerase inhibitors have been described.

  20. In vitro replication of poliovirus

    SciTech Connect

    Lubinski, J.M.

    1986-01-01

    Poliovirus is a member of the Picornaviridae whose genome is a single stranded RNA molecule of positive polarity surrounded by a proteinaceous capsid. Replication of poliovirus occurs via negative strand intermediates in infected cells using a virally encoded RNA-dependent RNA polymerase and host cell proteins. The authors have exploited the fact that complete cDNA copies of the viral genome when transfected onto susceptible cells generate virus. Utilizing the bacteriophage SP6 DNA dependent RNA polymerase system to synthesize negative strands in vitro and using these in an in vitro reaction the authors have generated full length infectious plus strands. Mutagenesis of the 5' and 3' ends of the negative and positive strands demonstrated that replication could occur either de novo or be extensions of the templates from their 3' ends or from nicks occurring during replication. The appearance of dimeric RNA molecules generated in these reactions was not dependent upon the same protein required for de novo initiation. Full length dimeric RNA molecules using a 5' /sup 32/P end-labelled oligo uridylic acid primer and positive strand template were demonstrated in vitro containing only the 35,000 Mr host protein and the viral RNA-dependent RNA polymerase. A model for generating positive strands without protein priming by cleavage of dimeric RNA molecules was proposed.

  1. A novel replicating circular DNAzyme

    PubMed Central

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

    2004-01-01

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

  2. Links between genome replication and chromatin landscapes.

    PubMed

    Sequeira-Mendes, Joana; Gutierrez, Crisanto

    2015-07-01

    Post-embryonic organogenesis in plants requires the continuous production of cells in the organ primordia, their expansion and a coordinated exit to differentiation. Genome replication is one of the most important processes that occur during the cell cycle, as the maintenance of genomic integrity is of primary relevance for development. As it is chromatin that must be duplicated, a strict coordination occurs between DNA replication, the deposition of new histones, and the introduction of histone modifications and variants. In turn, the chromatin landscape affects several stages during genome replication. Thus, chromatin accessibility is crucial for the initial stages and to specify the location of DNA replication origins with different chromatin signatures. The chromatin landscape also determines the timing of activation during the S phase. Genome replication must occur fully, but only once during each cell cycle. The re-replication avoidance mechanisms rely primarily on restricting the availability of certain replication factors; however, the presence of specific histone modifications are also revealed as contributing to the mechanisms that avoid re-replication, in particular for heterochromatin replication. We provide here an update of genome replication mostly focused on data from Arabidopsis, and the advances that genomic approaches are likely to provide in the coming years. The data available, both in plants and animals, point to the relevance of the chromatin landscape in genome replication, and require a critical evaluation of the existing views about the nature of replication origins, the mechanisms of origin specification and the relevance of epigenetic modifications for genome replication.

  3. Recombination-dependent concatemeric viral DNA replication.

    PubMed

    Lo Piano, Ambra; Martínez-Jiménez, María I; Zecchi, Lisa; Ayora, Silvia

    2011-09-01

    The initiation of viral double stranded (ds) DNA replication involves proteins that recruit and load the replisome at the replication origin (ori). Any block in replication fork progression or a programmed barrier may act as a factor for ori-independent remodelling and assembly of a new replisome at the stalled fork. Then replication initiation becomes dependent on recombination proteins, a process called recombination-dependent replication (RDR). RDR, which is recognized as being important for replication restart and stability in all living organisms, plays an essential role in the replication cycle of many dsDNA viruses. The SPP1 virus, which infects Bacillus subtilis cells, serves as a paradigm to understand the links between replication and recombination in circular dsDNA viruses. SPP1-encoded initiator and replisome assembly proteins control the onset of viral replication and direct the recruitment of host-encoded replisomal components at viral oriL. SPP1 uses replication fork reactivation to switch from ori-dependent θ-type (circle-to-circle) replication to σ-type RDR. Replication fork arrest leads to a double strand break that is processed by viral-encoded factors to generate a D-loop into which a new replisome is assembled, leading to σ-type viral replication. SPP1 RDR proteins are compared with similar proteins encoded by other viruses and their possible in vivo roles are discussed.

  4. Replication stress caused by low MCM expression limits fetal erythropoiesis and hematopoietic stem cell functionality

    PubMed Central

    Alvarez, Silvia; Díaz, Marcos; Flach, Johanna; Rodriguez-Acebes, Sara; López-Contreras, Andrés J.; Martínez, Dolores; Cañamero, Marta; Fernández-Capetillo, Oscar; Isern, Joan; Passegué, Emmanuelle; Méndez, Juan

    2015-01-01

    Replicative stress during embryonic development influences ageing and predisposition to disease in adults. A protective mechanism against replicative stress is provided by the licensing of thousands of origins in G1 that are not necessarily activated in the subsequent S-phase. These ‘dormant' origins provide a backup in the presence of stalled forks and may confer flexibility to the replication program in specific cell types during differentiation, a role that has remained unexplored. Here we show, using a mouse strain with hypomorphic expression of the origin licensing factor mini-chromosome maintenance (MCM)3 that limiting origin licensing in vivo affects the functionality of hematopoietic stem cells and the differentiation of rapidly-dividing erythrocyte precursors. Mcm3-deficient erythroblasts display aberrant DNA replication patterns and fail to complete maturation, causing lethal anemia. Our results indicate that hematopoietic progenitors are particularly sensitive to replication stress, and full origin licensing ensures their correct differentiation and functionality. PMID:26456157

  5. Replication stress caused by low MCM expression limits fetal erythropoiesis and hematopoietic stem cell functionality.

    PubMed

    Alvarez, Silvia; Díaz, Marcos; Flach, Johanna; Rodriguez-Acebes, Sara; López-Contreras, Andrés J; Martínez, Dolores; Cañamero, Marta; Fernández-Capetillo, Oscar; Isern, Joan; Passegué, Emmanuelle; Méndez, Juan

    2015-10-12

    Replicative stress during embryonic development influences ageing and predisposition to disease in adults. A protective mechanism against replicative stress is provided by the licensing of thousands of origins in G1 that are not necessarily activated in the subsequent S-phase. These 'dormant' origins provide a backup in the presence of stalled forks and may confer flexibility to the replication program in specific cell types during differentiation, a role that has remained unexplored. Here we show, using a mouse strain with hypomorphic expression of the origin licensing factor mini-chromosome maintenance (MCM)3 that limiting origin licensing in vivo affects the functionality of hematopoietic stem cells and the differentiation of rapidly-dividing erythrocyte precursors. Mcm3-deficient erythroblasts display aberrant DNA replication patterns and fail to complete maturation, causing lethal anemia. Our results indicate that hematopoietic progenitors are particularly sensitive to replication stress, and full origin licensing ensures their correct differentiation and functionality.

  6. Low-molecular-weight DNA replication intermediates in Escherichia coli: mechanism of formation and strand specificity.

    PubMed

    Amado, Luciana; Kuzminov, Andrei

    2013-11-15

    Chromosomal DNA replication intermediates, revealed in ligase-deficient conditions in vivo, are of low molecular weight (LMW) independently of the organism, suggesting discontinuous replication of both the leading and the lagging DNA strands. Yet, in vitro experiments with purified enzymes replicating sigma-structured substrates show continuous synthesis of the leading DNA strand in complete absence of ligase, supporting the textbook model of semi-discontinuous DNA replication. The discrepancy between the in vivo and in vitro results is rationalized by proposing that various excision repair events nick continuously synthesized leading strands after synthesis, producing the observed LMW intermediates. Here, we show that, in an Escherichia coli ligase-deficient strain with all known excision repair pathways inactivated, new DNA is still synthesized discontinuously. Furthermore, hybridization to strand-specific targets demonstrates that the LMW replication intermediates come from both the lagging and the leading strands. These results support the model of discontinuous leading strand synthesis in E. coli.

  7. Saikosaponin A inhibits influenza A virus replication and lung immunopathology

    PubMed Central

    Zhao, Yaqin; Ling, Fangfang; Xiao, Kun; Li, Qian; Li, Bin; Lu, Chunni; Qi, Wenbao; Zeng, Zhenling; Liao, Ming; Liu, Yahong; Chen, Weisan

    2015-01-01

    Fatal influenza outcomes result from a combination of rapid virus replication and collateral lung tissue damage caused by exaggerated pro-inflammatory host immune cell responses. There are few therapeutic agents that target both biological processes for the attenuation of influenza-induced lung pathology. We show that Saikosaponin A, a bioactive triterpene saponin with previouslyestablished anti-inflammatory effects, demonstrates both in vitro and in vivo anti-viral activity against influenza A virus infections. Saikosaponin A attenuated the replication of three different influenza A virus strains, including a highly pathogenic H5N1 strain, in human alveolar epithelial A549 cells. This anti-viral activity occurred through both downregulation of NF-κB signaling and caspase 3-dependent virus ribonucleoprotein nuclear export as demonstrated by NF-κB subunit p65 and influenza virus nucleoprotein nuclear translocation studies in influenza virus infected A549 cells. Critically, Saikosaponin A also attenuated viral replication, aberrant pro-inflammatory cytokine production and lung histopathology in the widely established H1N1 PR8 model of influenza A virus lethality in C57BL/6 mice. Flow cytometry studies of mouse bronchoalveolar lavage cells revealed that SSa exerted immunomodulatory effects through a selective attenuation of lung neutrophil and monocyte recruitment during the early peak of the innate immune response to PR8 infection. Altogether, our results indicate that Saikosaponin A possesses novel therapeutic potential for the treatment of pathological influenza virus infections. PMID:26637810

  8. Strain Gage

    NASA Technical Reports Server (NTRS)

    1995-01-01

    HITEC Corporation developed a strain gage application for DanteII, a mobile robot developed for NASA. The gage measured bending forces on the robot's legs and warned human controllers when acceptable forces were exceeded. HITEC further developed the technology for strain gage services in creating transducers out of "Indy" racing car suspension pushrods, NASCAR suspension components and components used in motion control.

  9. 36 CFR 910.64 - Replication.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... GUIDELINES AND UNIFORM STANDARDS FOR URBAN PLANNING AND DESIGN OF DEVELOPMENT WITHIN THE PENNSYLVANIA AVENUE DEVELOPMENT AREA Glossary of Terms § 910.64 Replication. Replication means the process of using modern...

  10. 36 CFR 910.64 - Replication.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... DEVELOPMENT AREA Glossary of Terms § 910.64 Replication. Replication means the process of using modern methods and materials to reproduce the exact form and details of a vanished building, structure, object,...

  11. Suppression of feline calicivirus replication using small interfering RNA targeted to its polymerase gene.

    PubMed

    Taharaguchi, Satoshi; Matsuhiro, Takahisa; Harima, Hayato; Sato, Atsuko; Ohe, Kyoko; Sakai, Sachi; Takahashi, Toshikazu; Hara, Motonobu

    2012-06-01

    Feline calicivirus (FCV) is a pathogenic microorganism that causes upper respiratory diseases in cats. Recently, an FCV infection with a high mortality rate has been confirmed, and there is need to develop a treatment for cases of acute infection. We evaluated whether the replication of FCV could be prevented by RNA interference. For this study, we designed an siRNA targeted to the polymerase region of the strain FCV-B isolated from a cat that died after exhibiting neurological symptoms. Cells transfected with siR-pol dose-dependently suppressed the replication of FCV-B. siR-pol suppressed its replication by suppressing the target viral RNA.

  12. Cytosolic Replication of Group A Streptococcus in Human Macrophages

    PubMed Central

    O’Neill, Alan M.; Thurston, Teresa L. M.

    2016-01-01

    ABSTRACT As key components of innate immune defense, macrophages are essential in controlling bacterial pathogens, including group A Streptococcus (GAS). Despite this, only a limited number of studies have analyzed the recovery of GAS from within human neutrophils and macrophages. Here, we determined the intracellular fate of GAS in human macrophages by using several quantitative approaches. In both U937 and primary human macrophages, the appearance over time of long GAS chains revealed that despite GAS-mediated cytotoxicity, replication occurred in viable, propidium iodide-negative macrophages. Whereas the major virulence factor M1 did not contribute to bacterial growth, a GAS mutant strain deficient in streptolysin O (SLO) was impaired for intracellular replication. SLO promoted bacterial escape from the GAS-containing vacuole (GCV) into the macrophage cytosol. Up to half of the cytosolic GAS colocalized with ubiquitin and p62, suggesting that the bacteria were targeted by the autophagy machinery. Despite this, live imaging of U937 macrophages revealed proficient replication of GAS after GCV rupture, indicating that escape from the GCV is important for growth of GAS in macrophages. Our results reveal that GAS can replicate within viable human macrophages, with SLO promoting GCV escape and cytosolic growth, despite the recruitment of autophagy receptors to bacteria. PMID:27073088

  13. Minimal region necessary for autonomous replication of pTAR.

    PubMed

    Gallie, D R; Kado, C I

    1988-07-01

    The native 44-kilobase-pair plasmid pTAR, discovered in a grapevine strain of Agrobacterium tumefaciens, contains a single origin of DNA replication confined to a 1.0-kilobase-pair region of the macromolecule. This region (ori) confers functions sufficient for replication in Agrobacterium and Rhizobium species but not in Pseudomonas solanacearum, Pseudomonas glumae, Pseudomonas syringae pv. savastanoi, Xanthomonas campestris pv. campestris, and Escherichia coli. ori contains a repA gene that encodes a 28,000-dalton protein required for replication. Nucleotide sequencing of repA and its promoter region revealed four 8-base-pair palindromic repeats upstream of the repA coding region. Deletion of these repeats alters repA expression and plasmid copy number. Downstream of repA are three additional repeats in a region essential for replication. A locus responsible for plasmid partitioning (parA) and a putative second locus regulating plasmid copy number are part of the origin region and are required for stable plasmid maintenance.

  14. Multiple replication origins with diverse control mechanisms in Haloarcula hispanica

    PubMed Central

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

    2014-01-01

    The use of multiple replication origins in archaea is not well understood. In particular, little is known about their specific control mechanisms. Here, we investigated the active replication origins in the three replicons of a halophilic archaeon, Haloarcula hispanica, by extensive gene deletion, DNA mutation and genome-wide marker frequency analyses. We revealed that individual origins are specifically dependent on their co-located cdc6 genes, and a single active origin/cdc6 pairing is essential and sufficient for each replicon. Notably, we demonstrated that the activities of oriC1 and oriC2, the two origins on the main chromosome, are differently controlled. A G-rich inverted repeat located in the internal region between the two inverted origin recognition boxes (ORBs) plays as an enhancer for oriC1, whereas the replication initiation at oriC2 is negatively regulated by an ORB-rich region located downstream of oriC2-cdc6E, likely via Cdc6E-titrating. The oriC2 placed on a plasmid is incompatible with the wild-type (but not the ΔoriC2) host strain, further indicating that strict control of the oriC2 activity is important for the cell. This is the first report revealing diverse control mechanisms of origins in haloarchaea, which has provided novel insights into the use and coordination of multiple replication origins in the domain of Archaea. PMID:24271389

  15. [Helper viruses of adeno-associated virus type 4 replication].

    PubMed

    Dreĭzin, R S; Zhuravel', T F; Shalunova, N V; Klenova, A V; Zolotarskaia, E E

    1979-01-01

    In replication of adeno-associated virus type 4 (AAV-4) the helper function may be performed by a non-defective virus from the same group of parvoviruses (Kilham virus). The synthesis of AAV-4 antigen was observed in a pig embryo kidney cell line, SPEV, chronically infected with Kilham virus, strain RV-13, 45--52 passages. A one-day-old SPEV-Kilham culture was infected with AAV-4. The AAV-4 antigen was detected by immunofluorescence at 6, 8, 12, 18 hours, 2, 3, 4, and 5 days after inoculation. During the first 2--4 days after inoculation the AAV-4 antigen was found in the nucleus and perinuclear zone, later in the cytoplasm. A "new" helper virus for AAV-4 replication has been found: simiancytomegalovirus in human embryo fibroblast cell culture permissive for the helper virus. In the systems where AAV-4 replicates, its antigen can be detected in the nucleus and perinuclear zone by IF. AAV-4 did not replicate in a system insensitive to the helper virus or under non-permissive conditions: at the time, the AAV-4 antigen localized only in the cell cytoplasm was detected.

  16. DNA Replication in Mycobacterium tuberculosis

    PubMed Central

    DITSE, ZANELE; LAMERS, MEINDERT H.; WARNER, DIGBY F.

    2017-01-01

    Faithful replication and maintenance of the genome are essential to the ability of any organism to survive and propagate. For an obligate pathogen such as Mycobacterium tuberculosis that has to complete successive cycles of transmission, infection, and disease in order to retain a foothold in the human population, this requires that genome replication and maintenance must be accomplished under the metabolic, immune, and antibiotic stresses encountered during passage through variable host environments. Comparative genomic analyses have established that chromosomal mutations enable M. tuberculosis to adapt to these stresses: the emergence of drug-resistant isolates provides direct evidence of this capacity, so too the well-documented genetic diversity among M. tuberculosis lineages across geographic loci, as well as the microvariation within individual patients that is increasingly observed as whole-genome sequencing methodologies are applied to clinical samples and tuberculosis (TB) disease models. However, the precise mutagenic mechanisms responsible for M. tuberculosis evolution and adaptation are poorly understood. Here, we summarize current knowledge of the machinery responsible for DNA replication in M. tuberculosis, and discuss the potential contribution of the expanded complement of mycobacterial DNA polymerases to mutagenesis. We also consider briefly the possible role of DNA replication—in particular, its regulation and coordination with cell division—in the ability of M. tuberculosis to withstand antibacterial stresses, including host immune effectors and antibiotics, through the generation at the population level of a tolerant state, or through the formation of a subpopulation of persister bacilli—both of which might be relevant to the emergence and fixation of genetic drug resistance. PMID:28361736

  17. Full-Length Genomic Analysis of Korean Porcine Sapelovirus Strains

    PubMed Central

    Kwon, Joseph; Choi, Jong-Soon; Kang, Mun-Il; Belsham, Graham J.; Cho, Kyoung-Oh

    2014-01-01

    Porcine sapelovirus (PSV), a species of the genus Sapelovirus within the family Picornaviridae, is associated with diarrhea, pneumonia, severe neurological disorders, and reproductive failure in pigs. However, the structural features of the complete PSV genome remain largely unknown. To analyze the structural features of PSV genomes, the full-length nucleotide sequences of three Korean PSV strains were determined and analyzed using bioinformatic techniques in comparison with other known PSV strains. The Korean PSV genomes ranged from 7,542 to 7,566 nucleotides excluding the 3′ poly(A) tail, and showed the typical picornavirus genome organization; 5′untranslated region (UTR)-L-VP4-VP2-VP3-VP1-2A-2B-2C-3A-3B-3C-3D-3′UTR. Three distinct cis-active RNA elements, the internal ribosome entry site (IRES) in the 5′UTR, a cis-replication element (CRE) in the 2C coding region and 3′UTR were identified and their structures were predicted. Interestingly, the structural features of the CRE and 3′UTR were different between PSV strains. The availability of these first complete genome sequences for PSV strains will facilitate future investigations of the molecular pathogenesis and evolutionary characteristics of PSV. PMID:25229940

  18. pBR322 vectors having tetracycline-dependent replication.

    PubMed

    Cronan, John E

    2016-01-01

    Few Escherichia coli cloning vectors are available that can both be stably maintained and efficiently cured. One such vector is pAM34, a pBR332 derivative constructed by Gil and Bouché (1991). Replication of this plasmid is driven by the lacZYA promoter under control of a gratuitous inducer. However, lac operator-repressor interactions are also used to regulate many expression systems which limit the utility of pAM34. In this report pAM34 has been modified by replacement of the lac regulatory elements with those of the transposon Tn10 tetracycline resistance module. This resulted in medium copy number plasmids that are dependent on the presence of tetracycline (or less satisfactorily, anhydrotetracycline) for replication. The tetracycline-dependent plasmids are rapidly lost in the absence of tetracycline and plasmid loss is markedly accelerated when the host strain expresses a tetracycline efflux pump. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Optimal Placement of Origins for DNA Replication

    NASA Astrophysics Data System (ADS)

    Karschau, Jens; Blow, J. Julian; de Moura, Alessandro P. S.

    2012-02-01

    DNA replication is an essential process in biology and its timing must be robust so that cells can divide properly. Random fluctuations in the formation of replication starting points, called origins, and the subsequent activation of proteins lead to variations in the replication time. We analyze these stochastic properties of DNA and derive the positions of origins corresponding to the minimum replication time. We show that under some conditions the minimization of replication time leads to the grouping of origins, and relate this to experimental data in a number of species showing origin grouping.

  20. Optimal placement of origins for DNA replication.

    PubMed

    Karschau, Jens; Blow, J Julian; de Moura, Alessandro P S

    2012-02-03

    DNA replication is an essential process in biology and its timing must be robust so that cells can divide properly. Random fluctuations in the formation of replication starting points, called origins, and the subsequent activation of proteins lead to variations in the replication time. We analyze these stochastic properties of DNA and derive the positions of origins corresponding to the minimum replication time. We show that under some conditions the minimization of replication time leads to the grouping of origins, and relate this to experimental data in a number of species showing origin grouping.

  1. Thermal, Autonomous Replicator Made from Transfer RNA

    NASA Astrophysics Data System (ADS)

    Krammer, Hubert; Möller, Friederike M.; Braun, Dieter

    2012-06-01

    Evolving systems rely on the storage and replication of genetic information. Here we present an autonomous, purely thermally driven replication mechanism. A pool of hairpin molecules, derived from transfer RNA replicates the succession of a two-letter code. Energy is first stored thermally in metastable hairpins. Thereafter, energy is released by a highly specific and exponential replication with a duplication time of 30 s, which is much faster than the tendency to produce false positives in the absence of template. Our experiments propose a physical rather than a chemical scenario for the autonomous replication of protein encoding information in a disequilibrium setting.

  2. Causes and Consequences of Replication Stress

    PubMed Central

    Zeman, Michelle K.; Cimprich, Karlene A.

    2015-01-01

    Replication stress is a complex phenomenon which has serious implications for genome stability, cell survival, and human disease. Generation of aberrant replication fork structures containing single-stranded DNA activates the replication stress response, primarily mediated by the kinase ATM- and Rad3-related (ATR). ATR and its downstream effectors stabilize and help to restart stalled replication forks, avoiding the generation of DNA damage and genome instability. Understanding these pathways may be key to diagnosis and treatment of human diseases caused by defective responses to replication stress. PMID:24366029

  3. Replication of prions in differentiated muscle cells.

    PubMed

    Herbst, Allen; Aiken, Judd M; McKenzie, Debbie

    2014-01-01

    We have demonstrated that prions accumulate to high levels in non-proliferative C2C12 myotubes. C2C12 cells replicate as myoblasts but can be differentiated into myotubes. Earlier studies indicated that C2C12 myoblasts are not competent for prion replication. (1) We confirmed that observation and demonstrated, for the first time, that while replicative myoblasts do not accumulate PrP(Sc), differentiated post-mitotic myotube cultures replicate prions robustly. Here we extend our observations and describe the implication and utility of this system for replicating prions.

  4. Programming DNA replication origins and chromosome organization.

    PubMed

    Cayrou, Christelle; Coulombe, Philippe; Méchali, Marcel

    2010-01-01

    During each cell cycle, thousands of DNA replication origins are activated in each cell of a metazoan organism. Although they appear site-specific, their usage and organization are rather plastic. Moreover, no strict sequence specificity has been observed in contrast to bacterial or Saccharomyces cerevisiae DNA replication origins. Epigenetic regulation linked to chromatin structure, chromosome organization, and transcription has been suggested to explain how DNA replication origins are selected and recognized by replication initiation factors. In this paper, we review these epigenetic features and discuss how, during the previous mitosis, chromosomal architecture might prepare DNA replication origins for a new cell cycle.

  5. DNA replication timing influences gene expression level

    PubMed Central

    2017-01-01

    Eukaryotic genomes are replicated in a reproducible temporal order; however, the physiological significance is poorly understood. We compared replication timing in divergent yeast species and identified genomic features with conserved replication times. Histone genes were among the earliest replicating loci in all species. We specifically delayed the replication of HTA1-HTB1 and discovered that this halved the expression of these histone genes. Finally, we showed that histone and cell cycle genes in general are exempt from Rtt109-dependent dosage compensation, suggesting the existence of pathways excluding specific loci from dosage compensation mechanisms. Thus, we have uncovered one of the first physiological requirements for regulated replication time and demonstrated a direct link between replication timing and gene expression. PMID:28539386

  6. Regulation of mouse satellite DNA replication time.

    PubMed

    Selig, S; Ariel, M; Goitein, R; Marcus, M; Cedar, H

    1988-02-01

    The satellite DNA sequences located near the centromeric regions of mouse chromosomes replicate very late in S in both fibroblast and lymphocyte cells and are heavily methylated at CpG residues. F9 teratocarcinoma cells, on the other hand, contain satellite sequences which are undermethylated and replicate much earlier in S. DNA methylation probably plays some role in the control of satellite replication time since 5-azacytidine treatment of RAG fibroblasts causes a dramatic temporal shift of replication to mid S. In contrast to similar changes accompanying the inactivation of the X-chromosome, early replication of satellite DNA is not associated with an increase in local chromosomal DNase I sensitivity. Fusion of F9 with mouse lymphocytes caused a dramatic early shift in the timing of the normally late replicating lymphocyte satellite heterochromatin, suggesting that trans-activating factors may be responsible for the regulation of replication timing.

  7. How dormant origins promote complete genome replication

    PubMed Central

    Blow, J. Julian; Ge, Xin Quan; Jackson, Dean A.

    2012-01-01

    Many replication origins that are licensed by loading MCM2-7 complexes in G1 are not normally used. Activation of these dormant origins during S phase provides a first line of defence for the genome if replication is inhibited. When replication forks fail, dormant origins are activated within regions of the genome currently engaged in replication. At the same time, DNA damage response kinases activated by the stalled forks preferentially suppress the assembly of new replication factories, thereby ensuring that chromosomal regions experiencing replicative stress complete synthesis before new regions of the genome are replicated. Mice expressing reduced levels of MCM2-7 have fewer dormant origins, are cancer prone and are genetically unstable, thus demonstrating the importance of dormant origins for preserving genome integrity. Here we review the function of dormant origins, the molecular mechanism of their regulation and their physiological implications. PMID:21641805

  8. DNA replication stress: causes, resolution and disease.

    PubMed

    Mazouzi, Abdelghani; Velimezi, Georgia; Loizou, Joanna I

    2014-11-15

    DNA replication is a fundamental process of the cell that ensures accurate duplication of the genetic information and subsequent transfer to daughter cells. Various pertubations, originating from endogenous or exogenous sources, can interfere with proper progression and completion of the replication process, thus threatening genome integrity. Coordinated regulation of replication and the DNA damage response is therefore fundamental to counteract these challenges and ensure accurate synthesis of the genetic material under conditions of replication stress. In this review, we summarize the main sources of replication stress and the DNA damage signaling pathways that are activated in order to preserve genome integrity during DNA replication. We also discuss the association of replication stress and DNA damage in human disease and future perspectives in the field.

  9. A model for DNA replication showing how dormant origins safeguard against replication fork failure

    PubMed Central

    Blow, J Julian; Ge, Xin Quan

    2009-01-01

    Replication origins are ‘licensed' for a single initiation event before entry into S phase; however, many licensed replication origins are not used, but instead remain dormant. The use of these dormant origins helps cells to survive replication stresses that block replication fork movement. Here, we present a computer model of the replication of a typical metazoan origin cluster in which origins are assigned a certain initiation probability per unit time and are then activated stochastically during S phase. The output of this model is in good agreement with experimental data and shows how inefficient dormant origins can be activated when replication forks are inhibited. The model also shows how dormant origins can allow replication to complete even if some forks stall irreversibly. This provides a simple explanation for how replication origin firing is regulated, which simultaneously provides protection against replicative stress while minimizing the cost of using large numbers of replication forks. PMID:19218919

  10. How to securely replicate services

    NASA Technical Reports Server (NTRS)

    Reiter, Michael; Birman, Kenneth

    1992-01-01

    A method is presented for constructing replicated services that retain their availability and integrity despite several servers and clients corrupted by an intruder, in addition to others failing benignly. More precisely, a service is replicated by n servers in such a way that a correct client will accept a correct server's response if, for some prespecified parameter k, at least k servers are correct and fewer than k servers are corrupt. The issue of maintaining causality among client requests is also addressed. A security breach resulting from an intruder's ability to effect a violation of causality in the sequence of requests processed by the service is illustrated. An approach to counter this problem is proposed that requires fewer than k servers to be corrupt and that is live if at least k+b servers are correct, where b is the assumed maximum total number of corrupt servers in any system run. An important and novel feature of these schemes is that the client need not be able to identify or authenticate even a single server. Instead, the client is required only to possess at most two public keys for the service. The practicality of these schemes is illustrated through a discussion of several issues pertinent to their implementation and use, and their intended role in a secure version of the Isis system is also described.

  11. Tracking replication enzymology in vivo by genome-wide mapping of ribonucleotide incorporation.

    PubMed

    Clausen, Anders R; Lujan, Scott A; Burkholder, Adam B; Orebaugh, Clinton D; Williams, Jessica S; Clausen, Maryam F; Malc, Ewa P; Mieczkowski, Piotr A; Fargo, David C; Smith, Duncan J; Kunkel, Thomas A

    2015-03-01

    Ribonucleotides are frequently incorporated into DNA during replication in eukaryotes. Here we map genome-wide distribution of these ribonucleotides as markers of replication enzymology in budding yeast, using a new 5' DNA end-mapping method, hydrolytic end sequencing (HydEn-seq). HydEn-seq of DNA from ribonucleotide excision repair-deficient strains reveals replicase- and strand-specific patterns of ribonucleotides in the nuclear genome. These patterns support the roles of DNA polymerases α and δ in lagging-strand replication and of DNA polymerase ɛ in leading-strand replication. They identify replication origins, termination zones and variations in ribonucleotide incorporation frequency across the genome that exceed three orders of magnitude. HydEn-seq also reveals strand-specific 5' DNA ends at mitochondrial replication origins, thus suggesting unidirectional replication of a circular genome. Given the conservation of enzymes that incorporate and process ribonucleotides in DNA, HydEn-seq can be used to track replication enzymology in other organisms.

  12. Regulatory interactions between phospholipid synthesis and DNA replication in Caulobacter crescentus.

    PubMed Central

    Loewy, B; Marczynski, G T; Dingwall, A; Shapiro, L

    1990-01-01

    Several Caulobacter crescentus mutants with lesions in phospholipid biosynthesis have DNA replication phenotypes. A C. crescentus mutant deficient in glycerol 3-phosphate dehydrogenase activity (gpsA) blocks phospholipid synthesis, ceases DNA replication, and loses viability in the absence of a glycerol phosphate supplement. To investigate the interaction between membrane synthesis and DNA replication during a single cell cycle, we moved the gpsA mutation into a synchronizable, but otherwise wild-type, strain. The first effect of withholding supplement was the cessation of synthesis of phosphatidylglycerol, a major component of the C. crescentus membrane. In the absence of glycerol 3-phosphate, DNA replication was initiated in the stalked cell at the correct time in the cell cycle and at the correct site on the chromosome. However, after replication proceeded bidirectionally for a short time, DNA synthesis dropped to a low level. The cell cycle blocked at a distinct middivision stalked cell, and this was followed by cell death. The "glycerol-less" death of the gpsA mutant could be prevented if the cells were treated with novobiocin to prevent the initiation of DNA replication. Our observations suggest that the processivity of C. crescentus replication requires concomitant phospholipid synthesis and that cell death results from incomplete replication of the chromosome. Images PMID:2211495

  13. Screening of the Pan-African Natural Product Library Identifies Ixoratannin A-2 and Boldine as Novel HIV-1 Inhibitors

    PubMed Central

    Tietjen, Ian; Ntie-Kang, Fidele; Mwimanzi, Philip; Onguéné, Pascal Amoa; Scull, Margaret A.; Idowu, Thomas Oyebode; Ogundaini, Abiodun Oguntuga; Meva’a, Luc Mbaze; Abegaz, Berhanu M.; Rice, Charles M.; Andrae-Marobela, Kerstin; Brockman, Mark A.; Brumme, Zabrina L.; Fedida, David

    2015-01-01

    The continued burden of HIV in resource-limited regions such as parts of sub-Saharan Africa, combined with adverse effects and potential risks of resistance to existing antiretroviral therapies, emphasize the need to identify new HIV inhibitors. Here we performed a virtual screen of molecules from the pan-African Natural Product Library, the largest collection of medicinal plant-derived pure compounds on the African continent. We identified eight molecules with structural similarity to reported interactors of Vpu, an HIV-1 accessory protein with reported ion channel activity. Using in vitro HIV-1 replication assays with a CD4+ T cell line and peripheral blood mononuclear cells, we confirmed antiviral activity and minimal cytotoxicity for two compounds, ixoratannin A-2 and boldine. Notably, ixoratannin A-2 retained inhibitory activity against recombinant HIV-1 strains encoding patient-derived mutations that confer resistance to protease, non-nucleoside reverse transcriptase, or integrase inhibitors. Moreover, ixoratannin A-2 was less effective at inhibiting replication of HIV-1 lacking Vpu, supporting this protein as a possible direct or indirect target. In contrast, boldine was less effective against a protease inhibitor-resistant HIV-1 strain. Both ixoratannin A-2 and boldine also inhibited in vitro replication of hepatitis C virus (HCV). However, BIT-225, a previously-reported Vpu inhibitor, demonstrated antiviral activity but also cytotoxicity in HIV-1 and HCV replication assays. Our work identifies pure compounds derived from African plants with potential novel activities against viruses that disproportionately afflict resource-limited regions of the world. PMID:25830320

  14. Screening of the Pan-African natural product library identifies ixoratannin A-2 and boldine as novel HIV-1 inhibitors.

    PubMed

    Tietjen, Ian; Ntie-Kang, Fidele; Mwimanzi, Philip; Onguéné, Pascal Amoa; Scull, Margaret A; Idowu, Thomas Oyebode; Ogundaini, Abiodun Oguntuga; Meva'a, Luc Mbaze; Abegaz, Berhanu M; Rice, Charles M; Andrae-Marobela, Kerstin; Brockman, Mark A; Brumme, Zabrina L; Fedida, David

    2015-01-01

    The continued burden of HIV in resource-limited regions such as parts of sub-Saharan Africa, combined with adverse effects and potential risks of resistance to existing antiretroviral therapies, emphasize the need to identify new HIV inhibitors. Here we performed a virtual screen of molecules from the pan-African Natural Product Library, the largest collection of medicinal plant-derived pure compounds on the African continent. We identified eight molecules with structural similarity to reported interactors of Vpu, an HIV-1 accessory protein with reported ion channel activity. Using in vitro HIV-1 replication assays with a CD4+ T cell line and peripheral blood mononuclear cells, we confirmed antiviral activity and minimal cytotoxicity for two compounds, ixoratannin A-2 and boldine. Notably, ixoratannin A-2 retained inhibitory activity against recombinant HIV-1 strains encoding patient-derived mutations that confer resistance to protease, non-nucleoside reverse transcriptase, or integrase inhibitors. Moreover, ixoratannin A-2 was less effective at inhibiting replication of HIV-1 lacking Vpu, supporting this protein as a possible direct or indirect target. In contrast, boldine was less effective against a protease inhibitor-resistant HIV-1 strain. Both ixoratannin A-2 and boldine also inhibited in vitro replication of hepatitis C virus (HCV). However, BIT-225, a previously-reported Vpu inhibitor, demonstrated antiviral activity but also cytotoxicity in HIV-1 and HCV replication assays. Our work identifies pure compounds derived from African plants with potential novel activities against viruses that disproportionately afflict resource-limited regions of the world.

  15. A polysaccharide produced by a mucoid strain of Moraxella nonliquefaciens with a 2-acetamido-2-deoxy-5-O-(3-deoxy-beta-D-manno-octulopyranosyl)-beta-D- galactopyranosyl repeating unit.

    PubMed

    Reistad, R; Zähringer, U; Bryn, K; Alstad, J; Bøvre, K; Jantzen, E

    1993-07-05

    A capsular polysaccharide, isolated from the mucoid Moraxella nonliquefaciens strain 3828/60, has been investigated by component analyses, periodate oxidation, methylation analyses, mass spectrometry, 1H and 13C NMR spectroscopy, and hydrolysis to give a disaccharide that was isolated and characterised. The results showed that the polysaccharide has the repeating unit-->3)-beta-D- GalpNAc-(1-->5)-beta-Kdo p-(2-->, with approximately 40% of O-8 of Kdo being acetylated.

  16. [Searching for new antibiotics--inhibitors of bacterial chromosome replication].

    PubMed

    Trojanowski, Damian; Skut, Patrycja; Hołówka, Joanna; Szafran, Marcin Jan

    2014-06-03

    The excessive and often unreasonable use of antibacterial drugs leads to rise of antibioticresistant strains. To overcome this problem, new antibiotics are searched and the new drug targets are investigated. The proteins involved in replication of bacterial chromosomes seem to be promising candidates for drug targets since they are involved in crucial life pathways and are structurally and/or functionally different from the eukaryotic homologues. Within last few years, a large number of newly developed methods allowed to search among thousands of molecules for the ones that specifically inhibit DNA synthesis in the prokaryotic cell. In this review, we present some of these methods.

  17. Mechanisms of strain-mediated mesenchymal stem cell apoptosis.

    PubMed

    Kearney, E M; Prendergast, P J; Campbell, V A

    2008-12-01

    Mechanical conditioning of mesenchymal stem cells (MSCs) has been adopted widely as a biophysical signal to aid tissue engineering applications. The replication of in vivo mechanical signaling has been used in in vitro environments to regulate cell differentiation, and extracellular matrix synthesis, so that both the chemical and mechanical properties of the tissue-engineered construct are compatible with the implant site. While research in these areas contributes to tissue engineering, the effects of mechanical strain on MSC apoptosis remain poorly defined. To evaluate the effects of uniaxial cyclic tensile strain on MSC apoptosis and to investigate mechanotransduction associated with strain-mediated cell death, MSCs seeded on a 2D silicone membrane were stimulated by a range of strain magnitudes for 3 days. Mechanotransduction was investigated using the stretch-activated cation channel blocker gadolinium chloride, the L-type voltage-activated calcium channel blocker nicardipine, the c-jun NH(2)-terminal kinase (JNK) blocker D-JNK inhibitor 1, and the calpain inhibitor MDL 28170. Apoptosis was assessed through DNA fragmentation using the terminal deoxynucleotidyl transferase mediated-UTP-end nick labeling method. Results demonstrated that tensile strains of 7.5% or greater induce apoptosis in MSCs. L-type voltage-activated calcium channels coupled mechanical stress to activation of calpain and JNK, which lead to apoptosis through DNA fragmentation. The definition of the in vitro boundary conditions for tensile strain and MSCs along with a proposed mechanism for apoptosis induced by mechanical events positively contributes to the development of MSC biology, bioreactor design for tissue engineering, and development of computational methods for mechanobiology.

  18. Rolling-circle replication of bacterial plasmids.

    PubMed Central

    Khan, S A

    1997-01-01

    Many bacterial plasmids replicate by a rolling-circle (RC) mechanism. Their replication properties have many similarities to as well as significant differences from those of single-stranded DNA (ssDNA) coliphages, which also replicate by an RC mechanism. Studies on a large number of RC plasmids have revealed that they fall into several families based on homology in their initiator proteins and leading-strand origins. The leading-strand origins contain distinct sequences that are required for binding and nicking by the Rep proteins. Leading-strand origins also contain domains that are required for the initiation and termination of replication. RC plasmids generate ssDNA intermediates during replication, since their lagging-strand synthesis does not usually initiate until the leading strand has been almost fully synthesized. The leading- and lagging-strand origins are distinct, and the displaced leading-strand DNA is converted to the double-stranded form by using solely the host proteins. The Rep proteins encoded by RC plasmids contain specific domains that are involved in their origin binding and nicking activities. The replication and copy number of RC plasmids, in general, are regulated at the level of synthesis of their Rep proteins, which are usually rate limiting for replication. Some RC Rep proteins are known to be inactivated after supporting one round of replication. A number of in vitro replication systems have been developed for RC plasmids and have provided insight into the mechanism of plasmid RC replication. PMID:9409148

  19. Self-replication of DNA rings

    NASA Astrophysics Data System (ADS)

    Kim, Junghoon; Lee, Junwye; Hamada, Shogo; Murata, Satoshi; Ha Park, Sung

    2015-06-01

    Biology provides numerous examples of self-replicating machines, but artificially engineering such complex systems remains a formidable challenge. In particular, although simple artificial self-replicating systems including wooden blocks, magnetic systems, modular robots and synthetic molecular systems have been devised, such kinematic self-replicators are rare compared with examples of theoretical cellular self-replication. One of the principal reasons for this is the amount of complexity that arises when you try to incorporate self-replication into a physical medium. In this regard, DNA is a prime candidate material for constructing self-replicating systems due to its ability to self-assemble through molecular recognition. Here, we show that DNA T-motifs, which self-assemble into ring structures, can be designed to self-replicate through toehold-mediated strand displacement reactions. The inherent design of these rings allows the population dynamics of the systems to be controlled. We also analyse the replication scheme within a universal framework of self-replication and derive a quantitative metric of the self-replicability of the rings.

  20. Recombination-dependent concatemeric plasmid replication.

    PubMed Central

    Viret, J F; Bravo, A; Alonso, J C

    1991-01-01

    The replication of covalently closed circular supercoiled (form I) DNA in prokaryotes is generally controlled at the initiation level by a rate-limiting effector. Once initiated, replication proceeds via one of two possible modes (theta or sigma replication) which do not rely on functions involved in DNA repair and general recombination. Recently, a novel plasmid replication mode, leading to the accumulation of linear multigenome-length plasmid concatemers in both gram-positive and gram-negative bacteria, has been described. Unlike form I DNA replication, an intermediate recombination step is most probably involved in the initiation of concatemeric plasmid DNA replication. On the basis of structural and functional studies, we infer that recombination-dependent plasmid replication shares important features with phage late replication modes and, in several aspects, parallels the synthesis of plasmid concatemers in phage-infected cells. The characterization of the concatemeric plasmid replication mode has allowed new insights into the mechanisms of DNA replication and recombination in prokaryotes. PMID:1779931

  1. A large inversion in the linear chromosome of Streptomyces griseus caused by replicative transposition of a new Tn3 family transposon.

    PubMed

    Murata, M; Uchida, T; Yang, Y; Lezhava, A; Kinashi, H

    2011-04-01

    We have comprehensively analyzed the linear chromosomes of Streptomyces griseus mutants constructed and kept in our laboratory. During this study, macrorestriction analysis of AseI and DraI fragments of mutant 402-2 suggested a large chromosomal inversion. The junctions of chromosomal inversion were cloned and sequenced and compared with the corresponding target sequences in the parent strain 2247. Consequently, a transposon-involved mechanism was revealed. Namely, a transposon originally located at the left target site was replicatively transposed to the right target site in an inverted direction, which generated a second copy and at the same time caused a 2.5-Mb chromosomal inversion. The involved transposon named TnSGR was grouped into a new subfamily of the resolvase-encoding Tn3 family transposons based on its gene organization. At the end, terminal diversity of S. griseus chromosomes is discussed by comparing the sequences of strains 2247 and IFO13350.

  2. Cascade replication of dissipative solitons

    NASA Astrophysics Data System (ADS)

    Kochetov, Bogdan A.; Tuz, Vladimir R.

    2017-07-01

    We report a new effect of a cascade replication of dissipative solitons from a single one. It is discussed in the framework of a common model based on the one-dimensional cubic-quintic complex Ginzburg-Landau equation in which an additional linear term is introduced to account the perturbation from a particular potential of externally applied force. The effect is demonstrated on the light beams propagating through a planar waveguide. The waveguide consists of a nonlinear layer able to guide dissipative solitons and a magneto-optic substrate. In the waveguide an externally applied force is considered to be an inhomogeneous magnetic field which is induced by modulated electric currents flowing along a set of conducting wires adjusted on the top of the waveguide.

  3. Therapeutic targeting of replicative immortality.

    PubMed

    Yaswen, Paul; MacKenzie, Karen L; Keith, W Nicol; Hentosh, Patricia; Rodier, Francis; Zhu, Jiyue; Firestone, Gary L; Matheu, Ander; Carnero, Amancio; Bilsland, Alan; Sundin, Tabetha; Honoki, Kanya; Fujii, Hiromasa; Georgakilas, Alexandros G; Amedei, Amedeo; Amin, Amr; Helferich, Bill; Boosani, Chandra S; Guha, Gunjan; Ciriolo, Maria Rosa; Chen, Sophie; Mohammed, Sulma I; Azmi, Asfar S; Bhakta, Dipita; Halicka, Dorota; Niccolai, Elena; Aquilano, Katia; Ashraf, S Salman; Nowsheen, Somaira; Yang, Xujuan

    2015-12-01

    One of the hallmarks of malignant cell populations is the ability to undergo continuous proliferation. This property allows clonal lineages to acquire sequential aberrations that can fuel increasingly autonomous growth, invasiveness, and therapeutic resistance. Innate cellular mechanisms have evolved to regulate replicative potential as a hedge against malignant progression. When activated in the absence of normal terminal differentiation cues, these mechanisms can result in a state of persistent cytostasis. This state, termed "senescence," can be triggered by intrinsic cellular processes such as telomere dysfunction and oncogene expression, and by exogenous factors such as DNA damaging agents or oxidative environments. Despite differences in upstream signaling, senescence often involves convergent interdependent activation of tumor suppressors p53 and p16/pRB, but can be induced, albeit with reduced sensitivity, when these suppressors are compromised. Doses of conventional genotoxic drugs required to achieve cancer cell senescence are often much lower than doses required to achieve outright cell death. Additional therapies, such as those targeting cyclin dependent kinases or components of the PI3K signaling pathway, may induce senescence specifically in cancer cells by circumventing defects in tumor suppressor pathways or exploiting cancer cells' heightened requirements for telomerase. Such treatments sufficient to induce cancer cell senescence could provide increased patient survival with fewer and less severe side effects than conventional cytotoxic regimens. This positive aspect is countered by important caveats regarding senescence reversibility, genomic instability, and paracrine effects that may increase heterogeneity and adaptive resistance of surviving cancer cells. Nevertheless, agents that effectively disrupt replicative immortality will likely be valuable components of new combinatorial approaches to cancer therapy.

  4. Therapeutic targeting of replicative immortality

    PubMed Central

    Yaswen, Paul; MacKenzie, Karen L.; Keith, W. Nicol; Hentosh, Patricia; Rodier, Francis; Zhu, Jiyue; Firestone, Gary L.; Matheu, Ander; Carnero, Amancio; Bilsland, Alan; Sundin, Tabetha; Honoki, Kanya; Fujii, Hiromasa; Georgakilas, Alexandros G.; Amedei, Amedeo; Amin, Amr; Helferich, Bill; Boosani, Chandra S.; Guha, Gunjan; Ciriolo, Maria Rosa; Chen, Sophie; Mohammed, Sulma I.; Azmi, Asfar S.; Bhakta, Dipita; Halicka, Dorota; Niccolai, Elena; Aquilano, Katia; Ashraf, S. Salman; Nowsheen, Somaira; Yang, Xujuan

    2015-01-01

    One of the hallmarks of malignant cell populations is the ability to undergo continuous proliferation. This property allows clonal lineages to acquire sequential aberrations that can fuel increasingly autonomous growth, invasiveness, and therapeutic resistance. Innate cellular mechanisms have evolved to regulate replicative potential as a hedge against malignant progression. When activated in the absence of normal terminal differentiation cues, these mechanisms can result in a state of persistent cytostasis. This state, termed “senescence,” can be triggered by intrinsic cellular processes such as telomere dysfunction and oncogene expression, and by exogenous factors such as DNA damaging agents or oxidative environments. Despite differences in upstream signaling, senescence often involves convergent interdependent activation of tumor suppressors p53 and p16/pRB, but can be induced, albeit with reduced sensitivity, when these suppressors are compromised. Doses of conventional genotoxic drugs required to achieve cancer cell senescence are often much lower than doses required to achieve outright cell death. Additional therapies, such as those targeting cyclin dependent kinases or components of the PI3K signaling pathway, may induce senescence specifically in cancer cells by circumventing defects in tumor suppressor pathways or exploiting cancer cells’ heightened requirements for telomerase. Such treatments sufficient to induce cancer cell senescence could provide increased patient survival with fewer and less severe side effects than conventional cytotoxic regimens. This positive aspect is countered by important caveats regarding senescence reversibility, genomic instability, and paracrine effects that may increase heterogeneity and adaptive resistance of surviving cancer cells. Nevertheless, agents that effectively disrupt replicative immortality will likely be valuable components of new combinatorial approaches to cancer therapy. PMID:25869441

  5. Directionality of replication fork movement determined by two-dimensional native-native DNA agarose gel electrophoresis.

    PubMed

    Ivessa, Andreas S

    2013-01-01

    The analysis of replication intermediates by the neutral-neutral two-dimensional agarose gel technique allows determining the chromosomal positions where DNA replication initiates, whether replication forks pause or stall at specific sites, or whether two DNA molecules undergo DNA recombination events. This technique does not, however, immediately tell in which direction replication forks migrate through the DNA region under investigation. Here, we describe the procedure to determine the direction of replication fork progression by carrying out a restriction enzyme digest of DNA imbedded in agarose after the completion of the first dimension of a 2D gel.

  6. Replication efficiency of soil-bound prions varies with soil type.

    PubMed

    Saunders, Samuel E; Shikiya, Ronald A; Langenfeld, Katie; Bartelt-Hunt, Shannon L; Bartz, Jason C

    2011-06-01

    Prion sorption to soil is thought to play an important role in the transmission of scrapie and chronic wasting disease (CWD) via the environment. Sorption of PrP to soil and soil minerals is influenced by the strain and species of PrP(Sc) and by soil characteristics. However, the ability of soil-bound prions to convert PrP(c) to PrP(Sc) under these wide-ranging conditions remains poorly understood. We developed a semiquantitative protein misfolding cyclic amplification (PMCA) protocol to evaluate replication efficiency of soil-bound prions. Binding of the hyper (HY) strain of transmissible mink encephalopathy (TME) (hamster) prions to a silty clay loam soil yielded a greater-than-1-log decrease in PMCA replication efficiency with a corresponding 1.3-log reduction in titer. The increased binding of PrP(Sc) to soil over time corresponded with a decrease in PMCA replication efficiency. The PMCA efficiency of bound prions varied with soil type, where prions bound to clay and organic surfaces exhibited significantly lower replication efficiencies while prions bound to sand exhibited no apparent difference in replication efficiency compared to unbound controls. PMCA results from hamster and CWD agent-infected elk prions yielded similar findings. Given that PrP(Sc) adsorption affinity varies with soil type, the overall balance between prion adsorption affinity and replication efficiency for the dominant soil types of an area may be a significant determinant in the environmental transmission of prion diseases.

  7. Character relations and replication identities in 2d Conformal Field Theory

    NASA Astrophysics Data System (ADS)

    Bantay, P.

    2016-10-01

    We study replication identities satisfied by conformal characters of a 2D CFT, providing a natural framework for a physics interpretation of the famous Hauptmodul property of Monstrous Moonshine, and illustrate the underlying ideas in simple cases.

  8. Regulation of Unperturbed DNA Replication by Ubiquitylation.

    PubMed

    Moreno, Sara Priego; Gambus, Agnieszka

    2015-06-25

    Posttranslational modification of proteins by means of attachment of a small globular protein ubiquitin (i.e., ubiquitylation) represents one of the most abundant and versatile mechanisms of protein regulation employed by eukaryotic cells. Ubiquitylation influences almost every cellular process and its key role in coordination of the DNA damage response is well established. In this review we focus, however, on the ways ubiquitylation controls the process of unperturbed DNA replication. We summarise the accumulated knowledge showing the leading role of ubiquitin driven protein degradation in setting up conditions favourable for replication origin licensing and S-phase entry. Importantly, we also present the emerging major role of ubiquitylation in coordination of the active DNA replication process: preventing re-replication, regulating the progression of DNA replication forks, chromatin re-establishment and disassembly of the replisome at the termination of replication forks.

  9. Self-replicating devices with dipolar colloids

    NASA Astrophysics Data System (ADS)

    Dempster, Joshua; Zhang, Rui; Olvera de La Cruz, Monica

    2014-03-01

    Ubiquitous in nature, self-replication on the nano-scale has been challenging to produce in the laboratory. Recent efforts with DNA tiles have shown great success in correctly replicating tile-sequence templates but require frequent manipulation by the experimenter. We propose a scheme for achieving self-replication with dipolar colloids. Dimers in these systems replicate exponentially over millisecond time scales with no intervention other than periodic energy pulses supplied by external fields. We develop a general formalism governing the rate of self-replication as a function of the interval between pulses. Results from kinetic Monte Carlo simulations show good agreement with the growth rates predicted by simple models of the replication process. We thank the Office of the Director of Defense Research and Engineering (DDR&E) and Air Force Office of Scientific Research (AFOSR) for their support under Award FA9550-10-1-0167.

  10. The ABCs of plasmid replication and segregation.

    PubMed

    Pinto, Uelinton M; Pappas, Katherine M; Winans, Stephen C

    2012-11-01

    To ensure faithful transmission of low-copy plasmids to daughter cells, these plasmids must replicate once per cell cycle and distribute the replicated DNA to the nascent daughter cells. RepABC family plasmids are found exclusively in alphaproteobacteria and carry a combined replication and partitioning locus, the repABC cassette, which is also found on secondary chromosomes in this group. RepC and a replication origin are essential for plasmid replication, and RepA, RepB and the partitioning sites distribute the replicons to predivisional cells. Here, we review our current understanding of the transcriptional and post-transcriptional regulation of the Rep proteins and of their functions in plasmid replication and partitioning.

  11. Replication-selective viruses for cancer therapy.

    PubMed

    Biederer, Carola; Ries, Stefan; Brandts, Christian H; McCormick, Frank

    2002-03-01

    Advances in our understanding of the molecular basis of cancer and the availability of technology to genetically engineer viruses have led to the development of replication-competent viruses to treat cancer. In theory, replication-selective viruses offer several appealing properties as biological agents for cancer therapy: they kill tumor cells selectively, and their replication leads to amplification of their oncolytic potential. Most preclinical experiments in tissue culture and in animal models support this notion. Clinical data on the first generation of replication-selective viruses are now rapidly accruing. The therapeutic index, and ultimately the clinical outcome, will depend on a complex balance between host and viral factors. This review discusses strategies to kill cancer cells based on our understanding of their molecular defects and the progress being made using replication-competent viruses for tumor therapy. We focus our discussion on a replication-selective adenovirus called ONYX-015 that has recently demonstrated encouraging results in clinical trials

  12. Mapping origins of DNA replication in eukaryotes.

    PubMed

    Gerbi, Susan A

    2005-01-01

    Methods are described here to map an origin of replication in eukaryotes. Replicating DNA is enriched by BND cellulose column chromatography and by lambda-exonuclease digestion; this approach has largely superceded enrichment by BrdU incorporation. The general area in which replication begins can be deciphered by neutral/neutral 2D gel electrophoresis: a restriction fragment containing the replication bubble will form a bubble arc on these gels. A more sensitive method employs PCR analysis of nascent strands that are size-fractionated. Once the general area containing the origin of bidirectional replication has been mapped, a finer level of resolution can be obtained by replication initiation point (RIP) mapping, in which start sites of DNA synthesis are identified at the nucleotide level.

  13. Replication of UV-irradiated DNA in human cell extracts: Evidence for mutagenic bypass of pyrimidine dimers

    SciTech Connect

    Thomas, D.C.; Kunkel, T.A. )

    1993-08-15

    The authors have examined the efficiency and fidelity of simian virus 40-origin-dependent replication of UV-irradiated double-stranded DNA in extracts of human cells. Using as a mutational target the [alpha]-complementation domain of the Escherichia coli lacZ gene in bacteriophage M13mp2DNA, replication of undamaged DNA in HeLa cell extracts was highly accurate, whereas replication of DNA irradiated with UV light (280-320 nm) was both less efficient and less accurate. Replication was inhibited by irradiation in a dose-dependent manner. Nonetheless, covalently closed, monomer-length circular products were generated that were resistant to digestion by Dpn I, showing that they resulted from semiconservative replication. These products were incised by T4 endonuclease V, whereas the undamaged replication products were not, suggesting that pyrimidine dimers were bypassed during replication. When replicated, UV-irradiated DNA was used to transfect an E. coli [alpha]-complementation host strain to score mutant M13mp2 plaques, the mutant plaque frequency was substantially higher than that obtained with either unirradiated, replicated DNA, or unreplicated, UV-irradiated DNA. Both the increased mutagenicity and the inhibition of replication associated with UV irradiation were reversed by treatment of the irradiated DNA with photolyase before replication. Sequence analysis of mutants resulting from replication of UV-irradiated DNA demonstrated that most mutants contained C [yields] T transition errors at dipyrimidine sites. A few mutants contained 1-nt frameshift errors or tandem double CC [yields] TT substitutions. The data are consistent with the interpretation that pyrimidine dimers are bypassed during replication by the multiprotein replication apparatus in human cell extracts and that this bypass is mutagenic primarily via misincorporation of dAMP opposite a cytosine (or uracil) in the dimer. 56 refs., 2 figs., 3 tabs.

  14. Semiconservative replication in the quasispecies model

    NASA Astrophysics Data System (ADS)

    Tannenbaum, Emmanuel; Deeds, Eric J.; Shakhnovich, Eugene I.

    2004-06-01

    This paper extends Eigen’s quasispecies equations to account for the semiconservative nature of DNA replication. We solve the equations in the limit of infinite sequence length for the simplest case of a static, sharply peaked fitness landscape. We show that the error catastrophe occurs when μ , the product of sequence length and per base pair mismatch probability, exceeds 2 ln [2/ ( 1+1/k ) ] , where k>1 is the first-order growth rate constant of the viable “master” sequence (with all other sequences having a first-order growth rate constant of 1 ). This is in contrast to the result of ln k for conservative replication. In particular, as k→∞ , the error catastrophe is never reached for conservative replication, while for semiconservative replication the critical μ approaches 2 ln 2 . Semiconservative replication is therefore considerably less robust than conservative replication to the effect of replication errors. We also show that the mean equilibrium fitness of a semiconservatively replicating system is given by k ( 2 e-μ/2 -1 ) below the error catastrophe, in contrast to the standard result of k e-μ for conservative replication (derived by Kimura and Maruyama in 1966). From this result it is readily shown that semiconservative replication is necessary to account for the observation that, at sufficiently high mutagen concentrations, faster replicating cells will die more quickly than more slowly replicating cells. Thus, in contrast to Eigen’s original model, the semiconservative quasispecies equations are able to provide a mathematical basis for explaining the efficacy of mutagens as chemotherapeutic agents.

  15. Replication Licensing and Cancer - a Fatal Entanglement?

    PubMed Central

    Blow, J. Julian; Gillespie, Peter J.

    2008-01-01

    Preface Correct regulation of the replication licensing system ensures that chromosomal DNA is precisely duplicated in each cell division cycle. Licensing proteins are inappropriately expressed at an early stage of tumorigenesis in a wide variety of cancers. Here we discuss evidence that misregulation of replication licensing is a consequence of oncogene-induced cell proliferation. This misregulation can cause either under- or over-replication of chromosomal DNA, and could explain the genetic instability commonly seen in cancer cells. PMID:18756287

  16. Serratia marcescens internalization and replication in human bladder epithelial cells

    PubMed Central

    Hertle, Ralf; Schwarz, Heinz

    2004-01-01

    Background Serratia marcescens, a frequent agent of catheterization-associated bacteriuria, strongly adheres to human bladder epithelial cells in culture. The epithelium normally provides a barrier between lumal organisms and the interstitium; the tight adhesion of bacteria to the epithelial cells can lead to internalization and subsequent lysis. However, internalisation was not shown yet for S. marcescens strains. Methods Elektronmicroscopy and the common gentamycin protection assay was used to assess intracellular bacteria. Via site directed mutagenesis, an hemolytic negative isogenic Serratia strain was generated to point out the importance of hemolysin production. Results We identified an important bacterial factor mediating the internalization of S. marcescens, and lysis of epithelial cells, as the secreted cytolysin ShlA. Microtubule filaments and actin filaments were shown to be involved in internalization. However, cytolysis of eukaryotic cells by ShlA was an interfering factor, and therefore hemolytic-negative mutants were used in subsequent experiments. Isogenic hemolysin-negative mutant strains were still adhesive, but were no longer cytotoxic, did not disrupt the cell culture monolayer, and were no longer internalized by HEp-2 and RT112 bladder epithelial cells under the conditions used for the wild-type strain. After wild-type S. marcescens became intracellular, the infected epithelial cells were lysed by extended vacuolation induced by ShlA. In late stages of vacuolation, highly motile S. marcescens cells were observed in the vacuoles. S. marcescens was also able to replicate in cultured HEp-2 cells, and replication was not dependent on hemolysin production. Conclusion The results reported here showed that the pore-forming toxin ShlA triggers microtubule-dependent invasion and is the main factor inducing lysis of the epithelial cells to release the bacteria, and therefore plays a major role in the development of S. marcescens infections. PMID:15189566

  17. Characterization of the replication and stability regions of Agrobacterium tumefaciens plasmid pTAR.

    PubMed

    Gallie, D R; Zaitlin, D; Perry, K L; Kado, C I

    1984-03-01

    A 5.4-kilobase region containing the origin of replication and stability maintenance of the 44-kilobase Agrobacterium tumefaciens plasmid pTAR has been mapped and characterized. Within this region is a 1.3-kilobase segment that is capable of directing autonomous replication. The remaining segment contains the stability locus for maintenance of pTAR during nonselective growth. Approximately 35% of pTAR shares sequence homology with pAg119, a 44-kilobase cryptic plasmid in grapevine strain 1D1119. However, no homology was detected between pTAR DNA and several Ti plasmids or several other small cryptic plasmids in many A. tumefaciens strains. A recombinant plasmid containing the origin of replication and stability maintenance region of pTAR was compatible with pTiC58, pTi15955, and pTi119 and incompatible with pAg119. A new compatibility group, Inc Ag-1, is discussed.

  18. Quantifying the Antiviral Effect of IFN on HIV-1 Replication in Cell Culture

    PubMed Central

    Ikeda, Hiroki; Godinho-Santos, Ana; Rato, Sylvie; Vanwalscappel, Bénédicte; Clavel, François; Aihara, Kazuyuki; Iwami, Shingo; Mammano, Fabrizio

    2015-01-01

    Type-I interferons (IFNs) induce the expression of hundreds of cellular genes, some of which have direct antiviral activities. Although IFNs restrict different steps of HIV replication cycle, their dominant antiviral effect remains unclear. We first quantified the inhibition of HIV replication by IFN in tissue culture, using viruses with different tropism and growth kinetics. By combining experimental and mathematical analyses, we determined quantitative estimates for key parameters of HIV replication and inhibition, and demonstrate that IFN mainly inhibits de novo infection (33% and 47% for a X4- and a R5-strain, respectively), rather than virus production (15% and 6% for the X4 and R5 strains, respectively). This finding is in agreement with patient-derived data analyses. PMID:26119462

  19. Quantifying the Antiviral Effect of IFN on HIV-1 Replication in Cell Culture

    NASA Astrophysics Data System (ADS)

    Ikeda, Hiroki; Godinho-Santos, Ana; Rato, Sylvie; Vanwalscappel, Bénédicte; Clavel, François; Aihara, Kazuyuki; Iwami, Shingo; Mammano, Fabrizio

    2015-06-01

    Type-I interferons (IFNs) induce the expression of hundreds of cellular genes, some of which have direct antiviral activities. Although IFNs restrict different steps of HIV replication cycle, their dominant antiviral effect remains unclear. We first quantified the inhibition of HIV replication by IFN in tissue culture, using viruses with different tropism and growth kinetics. By combining experimental and mathematical analyses, we determined quantitative estimates for key parameters of HIV replication and inhibition, and demonstrate that IFN mainly inhibits de novo infection (33% and 47% for a X4- and a R5-strain, respectively), rather than virus production (15% and 6% for the X4 and R5 strains, respectively). This finding is in agreement with patient-derived data analyses.

  20. Primer removal during mammalian mitochondrial DNA replication.

    PubMed

    Uhler, Jay P; Falkenberg, Maria

    2015-10-01

    The small circular mitochondrial genome in mammalian cells is replicated by a dedicated replisome, defects in which can cause mitochondrial disease in humans. A fundamental step in mitochondrial DNA (mtDNA) replication and maintenance is the removal of the RNA primers needed for replication initiation. The nucleases RNase H1, FEN1, DNA2, and MGME1 have been implicated in this process. Here we review the role of these nucleases in the light of primer removal pathways in mitochondria, highlight associations with disease, as well as consider the implications for mtDNA replication initiation. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  1. Comparison of three replication strategies in complex multicellular organisms: Asexual replication, sexual replication with identical gametes, and sexual replication with distinct sperm and egg gametes

    NASA Astrophysics Data System (ADS)

    Tannenbaum, Emmanuel

    2008-01-01

    This paper studies the mutation-selection balance in three simplified replication models. The first model considers a population of organisms replicating via the production of asexual spores. The second model considers a sexually replicating population that produces identical gametes. The third model considers a sexually replicating population that produces distinct sperm and egg gametes. All models assume diploid organisms whose genomes consist of two chromosomes, each of which is taken to be functional if equal to some master sequence, and defective otherwise. In the asexual population, the asexual diploid spores develop directly into adult organisms. In the sexual populations, the haploid gametes enter a haploid pool, where they may fuse with other haploids. The resulting immature diploid organisms then proceed to develop into mature organisms. Based on an analysis of all three models, we find that, as organism size increases, a sexually replicating population can only outcompete an asexually replicating population if the adult organisms produce distinct sperm and egg gametes. A sexual replication strategy that is based on the production of large numbers of sperm cells to fertilize a small number of eggs is found to be necessary in order to maintain a sufficiently low cost for sex for the strategy to be selected for over a purely asexual strategy. We discuss the usefulness of this model in understanding the evolution and maintenance of sexual replication as the preferred replication strategy in complex, multicellular organisms.

  2. Precise replication of antireflective nanostructures from biotemplates

    NASA Astrophysics Data System (ADS)

    Gao, Hongjun; Liu, Zhongfan; Zhang, Jin; Zhang, Guoming; Xie, Guoyong

    2007-03-01

    The authors report herein a new type of nanonipple structures on the cicada's eye and the direct structural replication of the complex micro- and nanostructures for potential functional emulation. A two-step direct molding process is developed to replicate these natural micro- and nanostructures using epoxy resin with high fidelity, which demonstrates a general way of fabricating functional nanostructures by direct replication of natural biotemplates via a suitable physicochemical process. Measurements of spectral reflectance showed that this kind of replicated nanostructure has remarkable antireflective property, suggestive of its potential applications to optical devices.

  3. Replication Terminator Protein-Based Replication Fork-Arrest Systems in Various Bacillus Species

    PubMed Central

    Griffiths, A. A.; Andersen, P. A.; Wake, R. G.

    1998-01-01

    The replication terminator protein (RTP) of Bacillus subtilis interacts with its cognate DNA terminators to cause replication fork arrest, thereby ensuring that the forks approaching one another at the conclusion of a round of replication meet within a restricted terminus region. A similar situation exists in Escherichia coli, but it appears that the fork-arrest systems in these two organisms have evolved independently of one another. In the present work, RTP homologs in four species closely related to B. subtilis (B. atrophaeus, B. amyloliquefaciens, B. mojavensis, and B. vallismortis) have been identified and characterized. An RTP homolog could not be identified in another closely related species, B. licheniformis. The nucleotide and amino acid changes from B. subtilis among the four homologs are consistent with the recently established phylogenetic tree for these species. The GC contents of the rtp genes raise the possibility that these organisms arose within this branch of the tree by horizontal transfer into a common ancestor after their divergence from B. licheniformis. Only 5 amino acid residue positions were changed among the four homologs, despite an up to 17.2% change in the nucleotide sequence, a finding that highlights the importance of the precise folded structure to the functioning of RTP. The absence of any significant change in the proposed DNA-binding region of RTP emphasizes the importance of its high affinity for the DNA terminator in its functioning. By coincidence, the single change (E30K) found in the B. mojavensis RTP corresponds exactly to that purposefully introduced by others into B. subtilis RTP to implicate a crucial role for E30 in the fork-arrest mechanism. The natural occurrence of this variant is difficult to reconcile with such an implication, and it was shown directly that RTP.E30K functions normally in fork arrest in B. subtilis in vivo. Additional DNA terminators were identified in the new RTP homolog-containing strains, allowing

  4. A Natural Polymorphism in rDNA Replication Origins Links Origin Activation with Calorie Restriction and Lifespan

    PubMed Central

    Kwan, Elizabeth X.; Foss, Eric J.; Tsuchiyama, Scott; Alvino, Gina M.; Kruglyak, Leonid; Kaeberlein, Matt; Raghuraman, M. K.; Brewer, Bonita J.; Kennedy, Brian K.; Bedalov, Antonio

    2013-01-01

    Aging and longevity are complex traits influenced by genetic and environmental factors. To identify quantitative trait loci (QTLs) that control replicative lifespan, we employed an outbred Saccharomyces cerevisiae model, generated by crossing a vineyard and a laboratory strain. The predominant QTL mapped to the rDNA, with the vineyard rDNA conferring a lifespan increase of 41%. The lifespan extension was independent of Sir2 and Fob1, but depended on a polymorphism in the rDNA origin of replication from the vineyard strain that reduced origin activation relative to the laboratory origin. Strains carrying vineyard rDNA origins have increased capacity for replication initiation at weak plasmid and genomic origins, suggesting that inability to complete genome replication presents a major impediment to replicative lifespan. Calorie restriction, a conserved mediator of lifespan extension that is also independent of Sir2 and Fob1, reduces rDNA origin firing in both laboratory and vineyard rDNA. Our results are consistent with the possibility that calorie restriction, similarly to the vineyard rDNA polymorphism, modulates replicative lifespan through control of rDNA origin activation, which in turn affects genome replication dynamics. PMID:23505383

  5. Evaluation of porcine reproductive and respiratory syndrome virus replication in laboratory rodents

    PubMed Central

    Rosenfeld, Paul; Turner, Patricia V.; MacInnes, Janet I.; Nagy, Éva; Yoo, Dongwan

    2009-01-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is a major cause of economic losses in the swine industry. The disease is widespread worldwide, and so PRRSV-negative pigs are often difficult to find for the study of PRRSV in vivo. To determine if a small animal model could be developed for PRRSV, 3 strains of laboratory rodent were examined for their susceptibility to the virus. No virus replication was detected in BALB/c or SCID (severe combined immunodeficiency) mice after intraperitoneal inoculation. Moderate replication of PRRSV was detected in primary cotton rat lung cell cultures, but no viral replication was detected following intranasal or intraperitoneal inoculation. Following intratracheal inoculation, viral transcripts were detected in the lungs of cotton rats, but only for 1 day. This study indicates that PRRSV replication in common laboratory rodent species is inefficient, and suggests that a rodent model for this virus is not appropriate. PMID:20046635

  6. Generation of Food-Grade Recombinant Lactic Acid Bacterium Strains by Site-Specific Recombination

    PubMed Central

    Martín, M. Cruz; Alonso, Juan C.; Suárez, Juan E.; Alvarez, Miguel A.

    2000-01-01

    The construction of a delivery and clearing system for the generation of food-grade recombinant lactic acid bacterium strains, based on the use of an integrase (Int) and a resolvo-invertase (β-recombinase) and their respective target sites (attP-attB and six, respectively) is reported. The delivery system contains a heterologous replication origin and antibiotic resistance markers surrounded by two directly oriented six sites, a multiple cloning site where passenger DNA could be inserted (e.g., the cI gene of bacteriophage A2), the int gene, and the attP site of phage A2. The clearing system provides a plasmid-borne gene encoding β-recombinase. The nonreplicative vector-borne delivery system was transformed into Lactobacillus casei ATCC 393 and, by site-specific recombination, integrated as a single copy in an orientation- and Int-dependent manner into the attB site present in the genome of the host strain. The transfer of the clearing system into this strain, with the subsequent expression of the β-recombinase, led to site-specific DNA resolution of the non-food-grade DNA. These methods were validated by the construction of a stable food-grade L. casei ATCC 393-derived strain completely immune to phage A2 infection during milk fermentation. PMID:10831443

  7. Replicative Intermediates of Human Papillomavirus Type 11 in Laryngeal Papillomas: Site of Replication Initiation and Direction of Replication

    NASA Astrophysics Data System (ADS)

    Auborn, K. J.; Little, R. D.; Platt, T. H. K.; Vaccariello, M. A.; Schildkraut, C. L.

    1994-07-01

    We have examined the structures of replication intermediates from the human papillomavirus type 11 genome in DNA extracted from papilloma lesions (laryngeal papillomas). The sites of replication initiation and termination utilized in vivo were mapped by using neutral/neutral and neutral/alkaline two-dimensional agarose gel electrophoresis methods. Initiation of replication was detected in or very close to the upstream regulatory region (URR; the noncoding, regulatory sequences upstream of the open reading frames in the papillomavirus genome). We also show that replication forks proceed bidirectionally from the origin and converge 180circ opposite the URR. These results demonstrate the feasibility of analysis of replication of viral genomes directly from infected tissue.

  8. [Recombinations occurring in the process of DNA replication in Escherichia coli].

    PubMed

    Sukhodolets, V V

    2006-07-01

    In a number of works dealing with the relationship between replication and recombination in bacteria, it is assumed that recombinations permit the replication forks to resume moving after having stopped at the damage sites of the template DNA. As an evidence for recombination occurring during DNA replication, the involvement in this process of proteins RuvABC and RecG, providing processing of the Holliday junctions after recombination, is considered. However, it has been shown that these proteins are not essential for resuming DNA synthesis after an exposure of bacteria to UV light. These data cast doubt on the necessity of recombination for reactivation of replication initiated in the oriC region. Studying recombination in tandem duplications in Escherichia coli showed that during replication, unequal crossing over occurs between direct DNA repeats of sister chromosomes. In wild strains, this crossing over results in tandem duplications, thereby enhancing the expression of certain genes. Thus, recombination of two types occurs during DNA replication: unequal crossing over leading to duplications and homologous exchange, responsible for post-replication DNA repair. The unequal exchange constitutes a component of SOS response of the cell to deterioration of the environment.

  9. Iron modulates the replication of virulent Mycobacterium bovis in resting and activated bovine and possum macrophages.

    PubMed

    Denis, Michel; Buddle, Bryce M

    2005-09-15

    Bovine and possum macrophages were infected in vitro with a virulent strain of Mycobacterium bovis, and mycobacterial replication was measured in the infected macrophages cultured under a variety of conditions. Virulent M. bovis replicated substantially in alveolar possum macrophages as well as in bovine blood monocyte-derived macrophages. Addition of recombinant bovine interferon-gamma (IFN-gamma) with low concentrations of lipopolysaccharide (LPS) rendered bovine macrophages significantly more resistant to M. bovis replication. Disruption of iron levels in infected macrophages by addition of apotransferrin or bovine lactoferrin blocked replication of M. bovis in both bovine and possum macrophages. On the other hand, addition of exogenous iron, either in the form of iron citrate or iron-saturated transferrin, rendered macrophages of both species much more permissive for the replication of M. bovis. The impact of iron deprivation/loading on the mycobacteriostatic activity of cells was independent of nitric-oxide release, as well as independent of the generation of oxygen radical species in both possum and bovine macrophages. Exogenous iron was shown to reverse the ability of IFN-gamma/LPS pulsed bovine macrophages to restrict M. bovis replication. When autologous possum lymphocytes from animals vaccinated with M. bovis strain BCG were added to infected macrophages, they rendered the macrophages less permissive for virulent M. bovis replication. Loading the cells with iron prior to this macrophage-lymphocyte interaction, reversed this immune effect induced by sensitized cells. We conclude that, in two important animal species, intracellular iron level plays an important role in M. bovis replication in macrophages, irrespective of their activation status.

  10. Chromosome replication in Myxococcus xanthus.

    PubMed Central

    Zusman, D R; Krotoski, D M; Cumsky, M

    1978-01-01

    The rates of DNA synthesis during the cell-division cycle were measured in Myxococcus xanthus growing in three different media permitting a twofold variation in doubling time. In all three media, simple DNA cycles were observed. Synthesis of DNA occurred during 85% of the cell-division cycle, independent of generation time, from 5 to 11 h. Cells were observed to contain one bacterial nucleoid at birth that later divided synchronously midway through the cell cycle. Nucleoid segregation appeared to begin before chromosome replication was completed. The DNA content of exponential-phase bacteria was determined to be about 20 +/- 3 X 10(-9) microgram per cell; newborn bacteria contained about 14 +/- 2 X 10(-9) microgram of DNA per cell. Exponential-phase bacteria showed about a 50% increase in DNA in the presence of chloramphenicol (50 microgram/ml). The number of randomly segregating chromosomes present in exponential-phase bacteria was determined by following the fate of prelabeled DNA during outgrowth in nonradioactive media. The results are consistent with a model in which cells are born with exactly one complete unreplicated chromosome. The molecular weight of such a chromosome is about 8.4 +/- 1.2 X 10(9). PMID:412830

  11. How frog embryos replicate their DNA reliably

    NASA Astrophysics Data System (ADS)

    Bechhoefer, John; Marshall, Brandon

    2007-03-01

    Frog embryos contain three billion base pairs of DNA. In early embryos (cycles 2-12), DNA replication is extremely rapid, about 20 min., and the entire cell cycle lasts only 25 min., meaning that mitosis (cell division) takes place in about 5 min. In this stripped-down cell cycle, there are no efficient checkpoints to prevent the cell from dividing before its DNA has finished replication - a disastrous scenario. Even worse, the many origins of replication are laid down stochastically and are also initiated stochastically throughout the replication process. Despite the very tight time constraints and despite the randomness introduced by origin stochasticity, replication is extremely reliable, with cell division failing no more than once in 10,000 tries. We discuss a recent model of DNA replication that is drawn from condensed-matter theories of 1d nucleation and growth. Using our model, we discuss different strategies of replication: should one initiate all origins as early as possible, or is it better to hold back and initiate some later on? Using concepts from extreme-value statistics, we derive the distribution of replication times given a particular scenario for the initiation of origins. We show that the experimentally observed initiation strategy for frog embryos meets the reliability constraint and is close to the one that requires the fewest resources of a cell.

  12. Completion of DNA replication in Escherichia coli.

    PubMed

    Wendel, Brian M; Courcelle, Charmain T; Courcelle, Justin

    2014-11-18

    The mechanism by which cells recognize and complete replicated regions at their precise doubling point must be remarkably efficient, occurring thousands of times per cell division along the chromosomes of humans. However, this process remains poorly understood. Here we show that, in Escherichia coli, the completion of replication involves an enzymatic system that effectively counts pairs and limits cellular replication to its doubling point by allowing converging replication forks to transiently continue through the doubling point before the excess, over-replicated regions are incised, resected, and joined. Completion requires RecBCD and involves several proteins associated with repairing double-strand breaks including, ExoI, SbcDC, and RecG. However, unlike double-strand break repair, completion occurs independently of homologous recombination and RecA. In some bacterial viruses, the completion mechanism is specifically targeted for inactivation to allow over-replication to occur during lytic replication. The results suggest that a primary cause of genomic instabilities in many double-strand-break-repair mutants arises from an impaired ability to complete replication, independent from DNA damage.

  13. Replicating Milgram Would People Still Obey Today?

    ERIC Educational Resources Information Center

    Burger, Jerry M.

    2009-01-01

    The author conducted a partial replication of Stanley Milgram's (1963, 1965, 1974) obedience studies that allowed for useful comparisons with the original investigations while protecting the well-being of participants. Seventy adults participated in a replication of Milgram's Experiment 5 up to the point at which they first heard the learner's…

  14. Using Replication Projects in Teaching Research Methods

    ERIC Educational Resources Information Center

    Standing, Lionel G.; Grenier, Manuel; Lane, Erica A.; Roberts, Meigan S.; Sykes, Sarah J.

    2014-01-01

    It is suggested that replication projects may be valuable in teaching research methods, and also address the current need in psychology for more independent verification of published studies. Their use in an undergraduate methods course is described, involving student teams who performed direct replications of four well-known experiments, yielding…

  15. Replication and Robustness in Developmental Research

    ERIC Educational Resources Information Center

    Duncan, Greg J.; Engel, Mimi; Claessens, Amy; Dowsett, Chantelle J.

    2014-01-01

    Replications and robustness checks are key elements of the scientific method and a staple in many disciplines. However, leading journals in developmental psychology rarely include explicit replications of prior research conducted by different investigators, and few require authors to establish in their articles or online appendices that their key…

  16. DNA replication: failures and inverted fusions.

    PubMed

    Carr, Antony M; Paek, Andrew L; Weinert, Ted

    2011-10-01

    DNA replication normally follows the rules passed down from Watson and Crick: the chromosome duplicates as dictated by its antiparallel strands, base-pairing and leading and lagging strand differences. Real-life replication is more complicated, fraught with perils posed by chromosome damage for one, and by transcription of genes and by other perils that disrupt progress of the DNA replication machinery. Understanding the replication fork, including DNA structures, associated replisome and its regulators, is key to understanding how cells overcome perils and minimize error. Replication fork error leads to genome rearrangements and, potentially, cell death. Interest in the replication fork and its errors has recently gained added interest by the results of deep sequencing studies of human genomes. Several pathologies are associated with sometimes-bizarre genome rearrangements suggestive of elaborate replication fork failures. To try and understand the links between the replication fork, its failure and genome rearrangements, we discuss here phases of fork behavior (stall, collapse, restart and fork failures leading to rearrangements) and analyze two examples of instability from our own studies; one in fission yeast and the other in budding yeast.

  17. The origin of replicators and reproducers

    PubMed Central

    Szathmáry, Eörs

    2006-01-01

    Replicators are fundamental to the origin of life and evolvability. Their survival depends on the accuracy of replication and the efficiency of growth relative to spontaneous decay. Infrabiological systems are built of two coupled autocatalytic systems, in contrast to minimal living systems that must comprise at least a metabolic subsystem, a hereditary subsystem and a boundary, serving respective functions. Some scenarios prefer to unite all these functions into one primordial system, as illustrated in the lipid world scenario, which is considered as a didactic example in detail. Experimentally produced chemical replicators grow parabolically owing to product inhibition. A selection consequence is survival of everybody. The chromatographized replicator model predicts that such replicators spreading on surfaces can be selected for higher replication rate because double strands are washed away slower than single strands from the surface. Analysis of real ribozymes suggests that the error threshold of replication is less severe by about one order of magnitude than thought previously. Surface-bound dynamics is predicted to play a crucial role also for exponential replicators: unlinked genes belonging to the same genome do not displace each other by competition, and efficient and accurate replicases can spread. The most efficient form of such useful population structure is encapsulation by reproducing vesicles. The stochastic corrector model shows how such a bag of genes can survive, and what the role of chromosome formation and intragenic recombination could be. Prebiotic and early evolution cannot be understood without the models of dynamics. PMID:17008217

  18. Replication and Robustness in Developmental Research

    ERIC Educational Resources Information Center

    Duncan, Greg J.; Engel, Mimi; Claessens, Amy; Dowsett, Chantelle J.

    2014-01-01

    Replications and robustness checks are key elements of the scientific method and a staple in many disciplines. However, leading journals in developmental psychology rarely include explicit replications of prior research conducted by different investigators, and few require authors to establish in their articles or online appendices that their key…

  19. Chromatin signatures of the Drosophila replication program

    PubMed Central

    Eaton, Matthew L.; Prinz, Joseph A.; MacAlpine, Heather K.; Tretyakov, George; Kharchenko, Peter V.; MacAlpine, David M.

    2011-01-01

    DNA replication initiates from thousands of start sites throughout the Drosophila genome and must be coordinated with other ongoing nuclear processes such as transcription to ensure genetic and epigenetic inheritance. Considerable progress has been made toward understanding how chromatin modifications regulate the transcription program; in contrast, we know relatively little about the role of the chromatin landscape in defining how start sites of DNA replication are selected and regulated. Here, we describe the Drosophila replication program in the context of the chromatin and transcription landscape for multiple cell lines using data generated by the modENCODE consortium. We find that while the cell lines exhibit similar replication programs, there are numerous cell line-specific differences that correlate with changes in the chromatin architecture. We identify chromatin features that are associated with replication timing, early origin usage, and ORC binding. Primary sequence, activating chromatin marks, and DNA-binding proteins (including chromatin remodelers) contribute in an additive manner to specify ORC-binding sites. We also generate accurate and predictive models from the chromatin data to describe origin usage and strength between cell lines. Multiple activating chromatin modifications contribute to the function and relative strength of replication origins, suggesting that the chromatin environment does not regulate origins of replication as a simple binary switch, but rather acts as a tunable rheostat to regulate replication initiation events. PMID:21177973

  20. Replicating Milgram Would People Still Obey Today?

    ERIC Educational Resources Information Center

    Burger, Jerry M.

    2009-01-01

    The author conducted a partial replication of Stanley Milgram's (1963, 1965, 1974) obedience studies that allowed for useful comparisons with the original investigations while protecting the well-being of participants. Seventy adults participated in a replication of Milgram's Experiment 5 up to the point at which they first heard the learner's…

  1. Chromatin signatures of the Drosophila replication program.

    PubMed

    Eaton, Matthew L; Prinz, Joseph A; MacAlpine, Heather K; Tretyakov, George; Kharchenko, Peter V; MacAlpine, David M

    2011-02-01

    DNA replication initiates from thousands of start sites throughout the Drosophila genome and must be coordinated with other ongoing nuclear processes such as transcription to ensure genetic and epigenetic inheritance. Considerable progress has been made toward understanding how chromatin modifications regulate the transcription program; in contrast, we know relatively little about the role of the chromatin landscape in defining how start sites of DNA replication are selected and regulated. Here, we describe the Drosophila replication program in the context of the chromatin and transcription landscape for multiple cell lines using data generated by the modENCODE consortium. We find that while the cell lines exhibit similar replication programs, there are numerous cell line-specific differences that correlate with changes in the chromatin architecture. We identify chromatin features that are associated with replication timing, early origin usage, and ORC binding. Primary sequence, activating chromatin marks, and DNA-binding proteins (including chromatin remodelers) contribute in an additive manner to specify ORC-binding sites. We also generate accurate and predictive models from the chromatin data to describe origin usage and strength between cell lines. Multiple activating chromatin modifications contribute to the function and relative strength of replication origins, suggesting that the chromatin environment does not regulate origins of replication as a simple binary switch, but rather acts as a tunable rheostat to regulate replication initiation events.

  2. Microarray analysis of DNA replication timing.

    PubMed

    Karnani, Neerja; Taylor, Christopher M; Dutta, Anindya

    2009-01-01

    Although all of the DNA in an eukaryotic cell replicates during the S-phase of cell cycle, there is a significant difference in the actual time in S-phase when a given chromosomal segment replicates. Methods are described here for generation of high-resolution temporal maps of DNA replication in synchronized human cells. This method does not require amplification of DNA before microarray hybridization and so avoids errors introduced during PCR. A major advantage of using this procedure is that it facilitates finer dissection of replication time in S-phase. Also, it helps delineate chromosomal regions that undergo biallelic or asynchronous replication, which otherwise are difficult to detect at a genome-wide scale by existing methods. The continuous TR50 (time of completion of 50% replication) maps of replication across chromosomal segments identify regions that undergo acute transitions in replication timing. These transition zones can play a significant role in identifying insulators that separate chromosomal domains with different chromatin modifications.

  3. Ribosome biogenesis in replicating cells: Integration of experiment and theory.

    PubMed

    Earnest, Tyler M; Cole, John A; Peterson, Joseph R; Hallock, Michael J; Kuhlman, Thomas E; Luthey-Schulten, Zaida

    2016-10-01

    Ribosomes-the primary macromolecular machines responsible for translating the genetic code into proteins-are complexes of precisely folded RNA and proteins. The ways in which their production and assembly are managed by the living cell is of deep biological importance. Here we extend a recent spatially resolved whole-cell model of ribosome biogenesis in a fixed volume [Earnest et al., Biophys J 2015, 109, 1117-1135] to include the effects of growth, DNA replication, and cell division. All biological processes are described in terms of reaction-diffusion master equations and solved stochastically using the Lattice Microbes simulation software. In order to determine the replication parameters, we construct and analyze a series of Escherichia coli strains with fluorescently labeled genes distributed evenly throughout their chromosomes. By measuring these cells' lengths and number of gene copies at the single-cell level, we could fit a statistical model of the initiation and duration of chromosome replication. We found that for our slow-growing (120 min doubling time) E. coli cells, replication was initiated 42 min into the cell cycle and completed after an additional 42 min. While simulations of the biogenesis model produce the correct ribosome and mRNA counts over the cell cycle, the kinetic parameters for transcription and degradation are lower than anticipated from a recent analytical time dependent model of in vivo mRNA production. Describing expression in terms of a simple chemical master equation, we show that the discrepancies are due to the lack of nonribosomal genes in the extended biogenesis model which effects the competition of mRNA for ribosome binding, and suggest corrections to parameters to be used in the whole-cell model when modeling expression of the entire transcriptome. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 735-751, 2016.

  4. Recommendations for Replication Research in Special Education: A Framework of Systematic, Conceptual Replications

    ERIC Educational Resources Information Center

    Coyne, Michael D.; Cook, Bryan G.; Therrien, William J.

    2016-01-01

    Special education researchers conduct studies that can be considered replications. However, they do not often refer to them as replication studies. The purpose of this article is to consider the potential benefits of conceptualizing special education intervention research within a framework of systematic, conceptual replication. Specifically, we…

  5. A Replication by Any Other Name: A Systematic Review of Replicative Intervention Studies

    ERIC Educational Resources Information Center

    Cook, Bryan G.; Collins, Lauren W.; Cook, Sara C.; Cook, Lysandra

    2016-01-01

    Replication research is essential to scientific knowledge. Reviews of replication studies often electronically search for "replicat*" as a textword, which does not identify studies that replicate previous research but do not self-identify as such. We examined whether the 83 intervention studies published in six non-categorical research…

  6. Recommendations for Replication Research in Special Education: A Framework of Systematic, Conceptual Replications

    ERIC Educational Resources Information Center

    Coyne, Michael D.; Cook, Bryan G.; Therrien, William J.

    2016-01-01

    Special education researchers conduct studies that can be considered replications. However, they do not often refer to them as replication studies. The purpose of this article is to consider the potential benefits of conceptualizing special education intervention research within a framework of systematic, conceptual replication. Specifically, we…

  7. A Replication by Any Other Name: A Systematic Review of Replicative Intervention Studies

    ERIC Educational Resources Information Center

    Cook, Bryan G.; Collins, Lauren W.; Cook, Sara C.; Cook, Lysandra

    2016-01-01

    Replication research is essential to scientific knowledge. Reviews of replication studies often electronically search for "replicat*" as a textword, which does not identify studies that replicate previous research but do not self-identify as such. We examined whether the 83 intervention studies published in six non-categorical research…

  8. Replication efficiency of oncolytic vaccinia virus in cell cultures prognosticates the virulence and antitumor efficacy in mice

    PubMed Central

    2011-01-01

    Background We have shown that insertion of the three vaccinia virus (VACV) promoter-driven foreign gene expression cassettes encoding Renilla luciferase-Aequorea GFP fusion protein, β-galactosidase, and β-glucuronidase into the F14.5L, J2R, and A56R loci of the VACV LIVP genome, respectively, results in a highly attenuated mutant strain GLV-1h68. This strain shows tumor-specific replication and is capable of eradicating tumors with little or no virulence in mice. This study aimed to distinguish the contribution of added VACV promoter-driven transcriptional units as inserts from the effects of insertional inactivation of three viral genes, and to determine the correlation between replication efficiency of oncolytic vaccinia virus in cell cultures and the virulence and antitumor efficacy in mice Methods A series of recombinant VACV strains was generated by replacing one, two, or all three of the expression cassettes in GLV-1h68 with short non-coding DNA sequences. The replication efficiency and tumor cell killing capacity of these newly generated VACV strains were compared with those of the parent virus GLV-1h68 in cell cultures. The virus replication efficiency in tumors and antitumor efficacy as well as the virulence were evaluated in nu/nu (nude) mice bearing human breast tumor xenografts. Results we found that virus replication efficiency increased with removal of each of the expression cassettes. The increase in virus replication efficiency was proportionate to the strength of removed VACV promoters linked to foreign genes. The replication efficiency of the new VACV strains paralleled their cytotoxicity in cell cultures. The increased replication efficiency in tumor xenografts resulted in enhanced antitumor efficacy in nude mice. Similarly, the enhanced virus replication efficiency was indicative of increased virulence in nude mice. Conclusions These data demonstrated that insertion of VACV promoter-driven transcriptional units into the viral genome for the

  9. Histone lysine methylation and chromatin replication.

    PubMed

    Rivera, Carlos; Gurard-Levin, Zachary A; Almouzni, Geneviève; Loyola, Alejandra

    2014-12-01

    In eukaryotic organisms, the replication of the DNA sequence and its organization into chromatin are critical to maintain genome integrity. Chromatin components, such as histone variants and histone post-translational modifications, along with the higher-order chromatin structure, impact several DNA metabolic processes, including replication, transcription, and repair. In this review we focus on lysine methylation and the relationships between this histone mark and chromatin replication. We first describe studies implicating lysine methylation in regulating early steps in the replication process. We then discuss chromatin reassembly following replication fork passage, where the incorporation of a combination of newly synthesized histones and parental histones can impact the inheritance of lysine methylation marks on the daughter strands. Finally, we elaborate on how the inheritance of lysine methylation can impact maintenance of the chromatin landscape, using heterochromatin as a model chromatin domain, and we discuss the potential mechanisms involved in this process.

  10. Rescue from replication stress during mitosis.

    PubMed

    Fragkos, Michalis; Naim, Valeria

    2017-04-03

    Genomic instability is a hallmark of cancer and a common feature of human disorders, characterized by growth defects, neurodegeneration, cancer predisposition, and aging. Recent evidence has shown that DNA replication stress is a major driver of genomic instability and tumorigenesis. Cells can undergo mitosis with under-replicated DNA or unresolved DNA structures, and specific pathways are dedicated to resolving these structures during mitosis, suggesting that mitotic rescue from replication stress (MRRS) is a key process influencing genome stability and cellular homeostasis. Deregulation of MRRS following oncogene activation or loss-of-function of caretaker genes may be the cause of chromosomal aberrations that promote cancer initiation and progression. In this review, we discuss the causes and consequences of replication stress, focusing on its persistence in mitosis as well as the mechanisms and factors involved in its resolution, and the potential impact of incomplete replication or aberrant MRRS on tumorigenesis, aging and disease.

  11. Multiple pathways process stalled replication forks.

    PubMed

    Michel, Bénédicte; Grompone, Gianfranco; Florès, Maria-Jose; Bidnenko, Vladimir

    2004-08-31

    Impairment of replication fork progression is a serious threat to living organisms and a potential source of genome instability. Studies in prokaryotes have provided evidence that inactivated replication forks can restart by the reassembly of the replication machinery. Several strategies for the processing of inactivated replication forks before replisome reassembly have been described. Most of these require the action of recombination proteins, with different proteins being implicated, depending on the cause of fork arrest. The action of recombination proteins at blocked forks is not necessarily accompanied by a strand-exchange reaction and may prevent rather than repair fork breakage. These various restart pathways may reflect different structures at stalled forks. We review here the different strategies of fork processing elicited by different kinds of replication impairments in prokaryotes and the variety of roles played by recombination proteins in these processes.

  12. Rescue from replication stress during mitosis

    PubMed Central

    Naim, Valeria

    2017-01-01

    ABSTRACT Genomic instability is a hallmark of cancer and a common feature of human disorders, characterized by growth defects, neurodegeneration, cancer predisposition, and aging. Recent evidence has shown that DNA replication stress is a major driver of genomic instability and tumorigenesis. Cells can undergo mitosis with under-replicated DNA or unresolved DNA structures, and specific pathways are dedicated to resolving these structures during mitosis, suggesting that mitotic rescue from replication stress (MRRS) is a key process influencing genome stability and cellular homeostasis. Deregulation of MRRS following oncogene activation or loss-of-function of caretaker genes may be the cause of chromosomal aberrations that promote cancer initiation and progression. In this review, we discuss the causes and consequences of replication stress, focusing on its persistence in mitosis as well as the mechanisms and factors involved in its resolution, and the potential impact of incomplete replication or aberrant MRRS on tumorigenesis, aging and disease. PMID:28166452

  13. A replication clock for Mycobacterium tuberculosis

    PubMed Central

    Gill, Wendy P; Harik, Nada S; Whiddon, Molly R; Liao, Reiling P; Mittler, John E; Sherman, David R

    2009-01-01

    Few tools exist to assess replication of chronic pathogens during infection. This has been a considerable barrier to understanding latent tuberculosis, and efforts to develop new therapies generally assume that the bacteria are very slowly replicating or nonreplicating during latency1–3. To monitor Mycobacterium tuberculosis replication within hosts, we exploit an unstable plasmid that is lost at a steady, quantifiable rate from dividing cells in the absence of antibiotic selection. By applying a mathematical model, we calculate bacterial growth and death rates during infection of mice. We show that during chronic infection the cumulative bacterial burden—enumerating total live, dead and removed organisms encountered by the mouse lung—is substantially higher than estimates from colony forming units. Our data show that M. tuberculosis replicates throughout the course of chronic infection of mice and is restrained by the host immune system. This approach may also shed light on the replication dynamics of other chronic pathogens. PMID:19182798

  14. Replication in genome-wide association studies

    PubMed Central

    Kraft, Peter; Zeggini, Eleftheria; Ioannidis, John P. A.

    2009-01-01

    Summary Replication helps ensure that a genotype-phenotype association observed in a genome-wide association (GWA) study represents a credible association and is not a chance finding or an artifact due to uncontrolled biases. We discuss prerequisites for exact replication; issues of heterogeneity; advantages and disadvantages of different methods of data synthesis across multiple studies; frequentist vs. Bayesian inferences for replication; and challenges that arise from multi-team collaborations. While consistent replication can greatly improve the credibility of a genotype-phenotype association, it may not eliminate spurious associations due to biases shared by many studies. Conversely, lack of replication in well-powered follow-up studies usually invalidates the initially proposed association, although occasionally it may point to differences in linkage disequilibrium or effect modifiers across studies. PMID:20454541

  15. Intrapulmonary Hartmannella vermiformis: a potential niche for Legionella pneumophila replication in a murine model of legionellosis.

    PubMed Central

    Brieland, J; McClain, M; LeGendre, M; Engleberg, C

    1997-01-01

    The potential role of inhaled protozoa as a niche for intrapulmonary replication of Legionella pneumophila was investigated in vivo with mutant strains of L. pneumophila which have reduced virulence for the amoeba Hartmannella vermiformis. L. pneumophila AA488 and AA502 were derived from wild-type strain AA100 after transposon mutagenesis. These mutants have reduced virulence for H. vermiformis but are fully virulent for mononuclear phagocytic cells. A/J mice, which are susceptible to replicative L. pneumophila lung infections, were inoculated intratracheally with L. pneumophila AA100, AA488, or AA502 (10[6] bacteria per mouse) or were coinoculated with one of the L. pneumophila strains (10[6] bacteria per mouse) and uninfected H. vermiformis (10[6] amoebae per mouse). The effect of coinoculation with H. vermiformis on intrapulmonary growth of each L. pneumophila strain was subsequently assessed. In agreement with our previous studies, coinoculation with H. vermiformis significantly enhanced intrapulmonary growth of the parent L. pneumophila strain (AA100). In contrast, intrapulmonary growth of L. pneumophila AA488 or AA502 was not significantly enhanced by coinoculation of mice with H. vermiformis. These studies demonstrate that L. pneumophila virulence for amoebae is required for maximal intrapulmonary growth of the bacteria in mice coinoculated with H. vermiformis and support the hypothesis that inhaled amoebae may potentiate intrapulmonary growth of L. pneumophila by providing a niche for bacterial replication. PMID:9353084

  16. Influence of Plasmid Type on the Replication of Rhodococcus equi in Host Macrophages

    PubMed Central

    Willingham-Lane, Jennifer M.; Berghaus, Londa J.; Giguère, Steeve

    2016-01-01

    ABSTRACT The soil-dwelling, saprophytic actinomycete Rhodococcus equi is a multihost, facultative intracellular pathogen of macrophages. When inhaled by susceptible foals, it causes severe bronchopneumonia. It is also a pathogen of pigs, which may develop submaxillary lymphadenitis upon exposure. R. equi isolates obtained from foals and pigs possess conjugative plasmids housing a pathogenicity island (PAI) containing a novel family of genes of unknown function called the virulence-associated protein or vap family. The PAI regions of the equine and swine plasmids differ in vap gene composition, with equine isolates possessing six vap genes, including the major virulence determinant vapA, while the PAIs of swine isolates house vapB and five other unique vap genes. Possession of the pVAPA-type virulence plasmid by equine isolates bestows the capacity for intramacrophage replication essential for disease development in vivo. Swine isolates of R. equi are largely unstudied. Here, we show that R. equi isolates from pigs, carrying pVAPB-type plasmids, are able to replicate in a plasmid-dependent manner in macrophages obtained from a variety of species (murine, swine, and equine) and anatomical locations. Similarly, equine isolates carrying pVAPA-type plasmids are capable of replication in swine macrophages. Plasmid swapping between equine and swine strains through conjugation did not alter the intracellular replication capacity of the parental strain, indicating that coevolution of the plasmid and chromosome is not crucial for this attribute. These results demonstrate that while distinct plasmid types exist among R. equi isolates obtained from equine and swine sources, this tropism is not determined by host species-specific intramacrophage replication capabilities. IMPORTANCE This work greatly advances our understanding of the opportunistic pathogen Rhodococcus equi, a disease agent of animals and immunocompromised people. Clinical isolates from diseased foals carry a

  17. Influence of Plasmid Type on the Replication of Rhodococcus equi in Host Macrophages.

    PubMed

    Willingham-Lane, Jennifer M; Berghaus, Londa J; Giguère, Steeve; Hondalus, Mary K

    2016-01-01

    The soil-dwelling, saprophytic actinomycete Rhodococcus equi is a multihost, facultative intracellular pathogen of macrophages. When inhaled by susceptible foals, it causes severe bronchopneumonia. It is also a pathogen of pigs, which may develop submaxillary lymphadenitis upon exposure. R. equi isolates obtained from foals and pigs possess conjugative plasmids housing a pathogenicity island (PAI) containing a novel family of genes of unknown function called the virulence-associated protein or vap family. The PAI regions of the equine and swine plasmids differ in vap gene composition, with equine isolates possessing six vap genes, including the major virulence determinant vapA, while the PAIs of swine isolates house vapB and five other unique vap genes. Possession of the pVAPA-type virulence plasmid by equine isolates bestows the capacity for intramacrophage replication essential for disease development in vivo. Swine isolates of R. equi are largely unstudied. Here, we show that R. equi isolates from pigs, carrying pVAPB-type plasmids, are able to replicate in a plasmid-dependent manner in macrophages obtained from a variety of species (murine, swine, and equine) and anatomical locations. Similarly, equine isolates carrying pVAPA-type plasmids are capable of replication in swine macrophages. Plasmid swapping between equine and swine strains through conjugation did not alter the intracellular replication capacity of the parental strain, indicating that coevolution of the plasmid and chromosome is not crucial for this attribute. These results demonstrate that while distinct plasmid types exist among R. equi isolates obtained from equine and swine sources, this tropism is not determined by host species-specific intramacrophage replication capabilities. IMPORTANCE This work greatly advances our understanding of the opportunistic pathogen Rhodococcus equi, a disease agent of animals and immunocompromised people. Clinical isolates from diseased foals carry a

  18. Persistent HIV-1 replication during antiretroviral therapy

    PubMed Central

    Martinez-Picado, Javier; Deeks, Steven G.

    2016-01-01

    Purpose of review The present review will highlight some of the recent findings regarding the capacity of HIV-1 to replicate during antiretroviral therapy (ART). Recent findings Although ART is highly effective at inhibiting HIV replication, it is not curative. Several mechanisms contribute to HIV persistence during ART, including HIV latency, immune dysfunction, and perhaps persistent low-level spread of the virus to uninfected cells (replication). The success in curing HIV will depend on efficiently targeting these three aspects. The degree to which HIV replicates during ART remains controversial. Most studies have failed to find any evidence of HIV evolution in blood, even with samples collected over many years, although a recent very intensive study of three individuals suggested that the virus population does shift, at least during the first few months of therapy. Stronger but still not definitive evidence for replication comes from a series of studies in which standard regimens were intensified with an integration inhibitor, resulting in changes in episomal DNA (blood) and cell-associated RNA (tissue). Limited drug penetration within tissues and the presence of immune sanctuaries have been argued as potential mechanisms allowing HIV to spread during ART. Mathematical models suggest that HIV replication and evolution is possible even without the selection of fully drug-resistant variants. As persistent HIV replication could have clinical consequences and might limit the efficacy of curative interventions, determining if HIV replicates during ART and why, should remain a key focus of the HIV research community. Summary Residual viral replication likely persists in lymphoid tissues, at least in a subset of individuals. Abnormal levels of immune activation might contribute to sustain virus replication. PMID:27078619

  19. The effect of dose on 2,3,7,8-TCDD tissue distribution, metabolism and elimination in CYP1A2(-/_) knockout and C57BL/6N parental strains of mice

    EPA Science Inventory

    Numerous metabolism studies have demonstrated that the toxic contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is poorly metabolized. A hallmark feature of TCDD exposure is induction of hepatic CYP1A2 and subsequent sequestration leading to high liver-to-fat concentration ra...

  20. The Effect of Dose on 2,3,7,8-TCDD Tissue Distribution, Metabolism and Elimination in CYP1A2 (-/-) Knockout and C57BL/6N Parental Strains of Mice

    USDA-ARS?s Scientific Manuscript database

    Numerous metabolism studies have demonstrated that the highly toxic 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is poorly metabolized. A hallmark feature of TCDD exposure is induction of hepatic CYP1A2 and subsequent sequestration leading to high liver to fat concentration ratios. This study was in...

  1. Inferring Where and When Replication Initiates from Genome-Wide Replication Timing Data

    NASA Astrophysics Data System (ADS)

    Baker, A.; Audit, B.; Yang, S. C.-H.; Bechhoefer, J.; Arneodo, A.

    2012-06-01

    Based on an analogy between DNA replication and one dimensional nucleation-and-growth processes, various attempts to infer the local initiation rate I(x,t) of DNA replication origins from replication timing data have been developed in the framework of phase transition kinetics theories. These works have all used curve-fit strategies to estimate I(x,t) from genome-wide replication timing data. Here, we show how to invert analytically the Kolmogorov-Johnson-Mehl-Avrami model and extract I(x,t) directly. Tests on both simulated and experimental budding-yeast data confirm the location and firing-time distribution of replication origins.

  2. Mechanism of DNA Replication in Drosophila Chromosomes: Structure of Replication Forks and Evidence for Bidirectionality

    PubMed Central

    Kriegstein, Henry J.; Hogness, David S.

    1974-01-01

    The replicating chromosomal DNA in Drosophila melanogaster cleavage nuclei has been visualized in the electron microscope as a serial array of closely spaced replicated regions created by pairs of diverging replication forks. The fine structure of the forks is very similar to that observed for the replication forks of bidirectionally replicating bacteriophage DNAs. However, the mean length of the single-stranded gaps in Drosophila forks is less than 200 nucleotide residues, much shorter than the gaps in phage forks. This difference in gap length corresponds to the observed difference in the size of Okazaki fragments from Drosophila and phage. Images PMID:4204203

  3. Role of multifunctional autonomously replicating sequence binding factor 1 in the initiation of DNA replication and transcriptional control in Saccharomyces cerevisiae.

    PubMed Central

    Rhode, P R; Elsasser, S; Campbell, J L

    1992-01-01

    Autonomously replicating sequence (ARS) binding factor 1 (ABF1) is an abundant DNA-binding protein that specifically recognizes the motif RTCRYN5ACG at many sites in the yeast genome, including promoter elements, mating-type silencers, and ARSs. Mutational analysis of these sites suggests that ABF1 is involved in constitutive and carbon source-regulated transcriptional activation, transcriptional silencing, and ARS activity. To better assess the role of ABF1 in DNA replication and transcriptional control, temperature-sensitive lethal mutations in the ABF1 gene were isolated. Several of the abf1(Ts) strains show rapid growth arrest at the nonpermissive temperature. At the semipermissive temperature, these strains show an ARS-specific defect in the mitotic stability of ARS-CEN plasmids, such that the abf1 mutants show defects in ARS function identical to those of mutants bearing the mutations in the cis-acting ABF1 binding sites analyzed previously by numerous investigators. Flow cytometric analysis and in vivo DNA labeling experiments on an alpha-factor synchronized abf1(Ts) strain showed that at the nonpermissive temperature, these cells fail to progress efficiently from G1 through S phase and synthesize DNA at 25% of the level seen in the isogenic ABF1 strain. RNA synthesis is also reduced in the abf1(Ts) strains. In addition, transcriptional activation by an ABF1 binding site upstream activation sequence is completely defective in an abf1(Ts) strain at the semipermissive temperature. These phenotypes provide evidence that the same protein, ABF1, functions in the initiation of DNA replication and transcriptional activation. Images PMID:1545789

  4. Replication of HIV-1 deleted Nef mutants in chronically immune activated human T cells.

    PubMed

    Shapira-Nahor, Orit; Maayan, Shlomo; Peden, Keith W C; Rabinowitz, Ruth; Schlesinger, Michael; Alian, Akram; Panet, Amos

    2002-11-10

    Lymphocytes (PBMC) obtained from blood of HIV-sera negative Ethiopian immigrants (ETH) were highly susceptible to HIV-1 infection in vitro with no need for stimulation by mitogens. As the HIV nef gene product has been shown to enhance viral replication in stimulated primary lymphocytes, we investigated in this work the role of Nef in viral replication in the ETH cells. Lymphocytes obtained from ETH individuals supported high replication of wild-type HIV-1 and low but significant replication level of the two deleted Nef mutants (encode truncated Nef proteins consisting only of either the first 35 or the first 86 amino acids of Nef). In contrast, no replication was observed in nonactivated cells obtained from non-ETH individuals. After activation of the PBMC from ETH individuals with PHA, replication of both wild-type strains and the two deleted Nef mutant viruses further increased. The CD4(+) T cells of ETH individuals exhibited elevated levels of the surface activation markers CD45RO and HLA-DR, compared with T cells derived from non-ETH group. Likewise, expression of the chemokine receptors CCR5 and CXCR4 on these cells was higher in the ETH group than in the non-ETH group. Replication of HIV-1 wild-type and the isogenic-deleted Nef mutants was significantly correlated with the proportion of ETH cells expressing CD45RO and the chemokine receptors. This study suggests that HIV-1 may respond differently to several activation states characteristic of T cells. One activation state, defined by chronically activated lymphocytes from ETH individuals, is permissive to the wild-type HIV-1 and, to a lesser degree, to the Nef mutants. Further activation of these cells by exogenous stimuli enhances replication of the virus. Our results support the notion that Nef enhances the basal level of T cell activation and consequently, viral replication.

  5. [Determination of genomic and replicative RNA of hepatitis C virus in patients treated with interferon].

    PubMed

    Zeilicoff, R; Ameigeiras, B; Ojeda, E; Isla Rodríguez, R; Grünbaum, S; Genero, M; Cappelletti, C; Tielli, G; Roatta, R; Koch, O

    1995-01-01

    We have investigated the presence of genomic and replicative RNA strands of hepatitis C virus in liver and serum. Eleven patients with proven chronic hepatitis C, received Interferon a2a 4,5 MU, three times a week during six months. RT-PCR was used with sense primer to detect the replicative strand and an antisense primer to identify genomic strand. Before treatment, genomic strands were present in liver and serum of all patients. Replicative strands were present in liver and serum in five and six cases, respectively. Seven out of eleven responded to treatment. In responders, genomic strands were absent in liver of 3 cases (43%) and replicative strands in liver of 4 (57%). In plasma genomic and replicative strands were absent in 5 (71%) and 7 (100%), respectively. In all non responders, genomic strands in liver and plasma remained present. Replicative strands in liver and plasma were present in 100% and 25%, respectively. Knodell score improved in 5 out of 7 responders and remained unchanged in 3 out of 4 non responders. In 2 out of 4 responders with genomic and replicative strands in liver, Knodell score remained unchanged or worse. In all non responders, genomic and replicative strands in liver were present and Knodell score remained unchanged or worse. Genomic and replicative strands in plasma tended to be negative after treatment in responders. Genomic strands in plasma remained present in non responders. Conversely, genomic and replicative strands in liver were present in all non responders. It seems to exist a relationship between genomic and replicative strands in liver and the same or worse Knodell score. After a follow up, it will be possible to determined whether responders who still present viral RNA in liver would be prone to a relapse.

  6. Replication forks reverse at high frequency upon replication stress in Physarum polycephalum.

    PubMed

    Maric, Chrystelle; Bénard, Marianne

    2014-12-01

    The addition of hydroxyurea after the onset of S phase allows replication to start and permits the successive detecting of replication-dependent joint DNA molecules and chicken foot structures in the synchronous nuclei of Physarum polycephalum. We find evidence for a very high frequency of reversed replication forks upon replication stress. The formation of these reversed forks is dependent on the presence of joint DNA molecules, the impediment of the replication fork progression by hydroxyurea, and likely on the propensity of some replication origins to reinitiate replication to counteract the action of this compound. As hydroxyurea treatment enables us to successively detect the appearance of joint DNA molecules and then of reversed replication forks, we propose that chicken foot structures are formed both from the regression of hydroxyurea-frozen joint DNA molecules and from hydroxyurea-stalled replication forks. These experiments underscore the transient nature of replication fork regression, which becomes detectable due to the hydroxyurea-induced slowing down of replication fork progression.

  7. Phosphorylated SIRT1 associates with replication origins to prevent excess replication initiation and preserve genomic stability

    PubMed Central

    Utani, Koichi; Fu, Haiqing; Jang, Sang-Min; Marks, Anna B.; Smith, Owen K.; Zhang, Ya; Redon, Christophe E.; Shimizu, Noriaki

    2017-01-01

    Abstract Chromatin structure affects DNA replication patterns, but the role of specific chromatin modifiers in regulating the replication process is yet unclear. We report that phosphorylation of the human SIRT1 deacetylase on Threonine 530 (T530-pSIRT1) modulates DNA synthesis. T530-pSIRT1 associates with replication origins and inhibits replication from a group of ‘dormant’ potential replication origins, which initiate replication only when cells are subject to replication stress. Although both active and dormant origins bind T530-pSIRT1, active origins are distinguished from dormant origins by their unique association with an open chromatin mark, histone H3 methylated on lysine 4. SIRT1 phosphorylation also facilitates replication fork elongation. SIRT1 T530 phosphorylation is essential to prevent DNA breakage upon replication stress and cells harboring SIRT1 that cannot be phosphorylated exhibit a high prevalence of extrachromosomal elements, hallmarks of perturbed replication. These observations suggest that SIRT1 phosphorylation modulates the distribution of replication initiation events to insure genomic stability. PMID:28549174

  8. Replication potentials of HIV-1/HSIV in PBMCs from northern pig-tailed macaque (Macaca leonina).

    PubMed

    Lei, Ai-Hua; Zhang, Gao-Hong; Tian, Ren-Rong; Zhu, Jia-Wu; Zheng, Hong-Yi; Pang, Wei; Zheng, Yong-Tang

    2014-05-01

    The northern pig-tailed macaque (Macaca leonina) has been identified as an independent species of Old World monkey, and we previously found that PBMCs from M. leonina were susceptible to human immunodeficiency virus type 1 (HIV-1), which may be due to the absence of a TRIM5 protein restricting HIV-1 replication. Here we investigated the infection potentials of six laboratory adapted HIV-1 strains and three primary HIV-1 isolates in PBMCs from M. leonina. The results indicate that these strains are characterized by various but low replication levels, and among which, HIV-1NL4-3 shows the highest replication ability. Based on the abundant evidence of species-specific interactions between restriction factors APOBEC3 and HIV/SIV-derived Vif protein, we subsequently examined the replication potentials of vif-substituted HIV-1 (HSIV) in M. leonina PBMCs. Notably, HSIV-vifmac and stHIV-1SV chimeras, two HIV-1NL4-3-derived viruses encoding the viral infectivity factor (Vif) protein from SIVmac239, replicated robustly in cells from M. leonina, which suggests that HSIV could effectively antagonize the antiviral activity of APOBEC3 proteins expressed in cells of M. leonina. Therefore, our data demonstrate that M. leonina has the potential to be developed into a promising animal model for human AIDS.

  9. Replication potentials of HIV-1/HSIV in PBMCs from northern pigtailed macaque (Macaca leonina)

    PubMed Central

    LEI, Ai-Hua; ZHANG, Gao-Hong; TIAN, Ren-Rong; ZHU, Jia-Wu; ZHENG, Hong-Yi; PANG, Wei; ZHENG, Yong-Tang

    2014-01-01

    The northern pig-tailed macaque (Macaca leonina) has been identified as an independent species of Old World monkey, and we previously found that PBMCs from M. leonina were susceptible to human immunodeficiency virus type 1 (HIV-1), which may be due to the absence of a TRIM5 protein restricting HIV-1 replication. Here we investigated the infection potentials of six laboratory adapted HIV-1 strains and three primary HIV-1 isolates in PBMCs from M. leonina. The results indicate that these strains are characterized by various but low replication levels, and among which, HIV-1NL4-3 shows the highest replication ability. Based on the abundant evidence of species-specific interactions between restriction factors APOBEC3 and HIV/SIV-derived Vif protein, we subsequently examined the replication potentials of vif-substituted HIV-1 (HSIV) in M. leonina PBMCs. Notably, HSIV-vifmac and stHIV-1SV chimeras, two HIV-1NL4-3-derived viruses encoding the viral infectivity factor (Vif) protein from SIVmac239, replicated robustly in cells from M. leonina, which suggests that HSIV could effectively antagonize the antiviral activity of APOBEC3 proteins expressed in cells of M. leonina. Therefore, our data demonstrate that M. leonina has the potential to be developed into a promising animal model for human AIDS. PMID:24866489

  10. Molecular analysis of Porcine Circovirus Type 2 strains from Uruguay: evidence for natural occurring recombination.

    PubMed

    Ramos, Natalia; Mirazo, Santiago; Castro, Gustavo; Arbiza, Juan

    2013-10-01

    Porcine Circovirus Type 2 (PCV2) is a worldwide distributed virus and is considered an important emerging pathogen related to several distinct disease syndromes in pigs. Genomic structure consists of three major open reading frames (ORFs). ORF1 (rep gene) encodes replication-related proteins, ORF2 (cap gene) encodes the capsid protein and ORF3 encodes a protein putatively involved in virus-induced apoptosis. Based on cap gene sequences, PCV2 strains are classified into two main genotypes, PCV2a with five clusters (2A-2E) and PCV2b with three clusters (1A-1C). According to previous theoretical studies, PCV2 strains can eventually undergo intra and inter-genotype recombination, mainly within the rep gene. Ever since, several evidences of recombination in the field have been reported and confirmed this hypothesis. In South America, data regarding molecular characterization of PCV2 strains is still scant. Genotyping studies in the region have concluded that PCV2b is the predominant circulating genotype in the region and till now, no recombinant strains have ever been reported. In this work we thoroughly characterized at the molecular level Uruguayan PCV2 strains by extensive sequence data analysis. Moreover, recombination software tools were applied to explore and characterize eventual occurrence of natural recombination events. Two recombinant PCV2 strains were detected in this study, as a consequence of an inter-genotype recombination event between PCV2b-1A and PCV2a-2D, as the major and minor parent, respectively. According to recombination software analysis, in both cases the event occurred within the ORF1. Herein, extensive viral sequence dataset is provided, including the characterization of the first PCV2 recombinant strains ever reported in South America. Additionally, our results suggested a multi-centered source of PCV2 infection in Uruguay, which probably involved Brazilian and European origins.

  11. Recovery from the DNA Replication Checkpoint.

    PubMed

    Chaudhury, Indrajit; Koepp, Deanna M

    2016-10-28

    Checkpoint recovery is integral to a successful checkpoint response. Checkpoint pathways monitor progress during cell division so that in the event of an error, the checkpoint is activated to block the cell cycle and activate repair pathways. Intrinsic to this process is that once repair has been achieved, the checkpoint signaling pathway is inactivated and cell cycle progression resumes. We use the term "checkpoint recovery" to describe the pathways responsible for the inactivation of checkpoint signaling and cell cycle re-entry after the initial stress has been alleviated. The DNA replication or S-phase checkpoint monitors the integrity of DNA synthesis. When replication stress is encountered, replication forks are stalled, and the checkpoint signaling pathway is activated. Central to recovery from the S-phase checkpoint is the restart of stalled replication forks. If checkpoint recovery fails, stalled forks may become unstable and lead to DNA breaks or unusual DNA structures that are difficult to resolve, causing genomic instability. Alternatively, if cell cycle resumption mechanisms become uncoupled from checkpoint inactivation, cells with under-replicated DNA might proceed through the cell cycle, also diminishing genomic stability. In this review, we discuss the molecular mechanisms that contribute to inactivation of the S-phase checkpoint signaling pathway and the restart of replication forks during recovery from replication stress.

  12. Diversity of DNA Replication in the Archaea.

    PubMed

    Ausiannikava, Darya; Allers, Thorsten

    2017-01-31

    DNA replication is arguably the most fundamental biological process. On account of their shared evolutionary ancestry, the replication machinery found in archaea is similar to that found in eukaryotes. DNA replication is initiated at origins and is highly conserved in eukaryotes, but our limited understanding of archaea has uncovered a wide diversity of replication initiation mechanisms. Archaeal origins are sequence-based, as in bacteria, but are bound by initiator proteins that share homology with the eukaryotic origin recognition complex subunit Orc1 and helicase loader Cdc6). Unlike bacteria, archaea may have multiple origins per chromosome and multiple Orc1/Cdc6 initiator proteins. There is no consensus on how these archaeal origins are recognised- some are bound by a single Orc1/Cdc6 protein while others require a multi- Orc1/Cdc6 complex. Many archaeal genomes consist of multiple parts-the main chromosome plus several megaplasmids-and in polyploid species these parts are present in multiple copies. This poses a challenge to the regulation of DNA replication. However, one archaeal species (Haloferax volcanii) can survive without replication origins; instead, it uses homologous recombination as an alternative mechanism of initiation. This diversity in DNA replication initiation is all the more remarkable for having been discovered in only three groups of archaea where in vivo studies are possible.

  13. Diversity of DNA Replication in the Archaea

    PubMed Central

    Ausiannikava, Darya; Allers, Thorsten

    2017-01-01

    DNA replication is arguably the most fundamental biological process. On account of their shared evolutionary ancestry, the replication machinery found in archaea is similar to that found in eukaryotes. DNA replication is initiated at origins and is highly conserved in eukaryotes, but our limited understanding of archaea has uncovered a wide diversity of replication initiation mechanisms. Archaeal origins are sequence-based, as in bacteria, but are bound by initiator proteins that share homology with the eukaryotic origin recognition complex subunit Orc1 and helicase loader Cdc6). Unlike bacteria, archaea may have multiple origins per chromosome and multiple Orc1/Cdc6 initiator proteins. There is no consensus on how these archaeal origins are recognised—some are bound by a single Orc1/Cdc6 protein while others require a multi- Orc1/Cdc6 complex. Many archaeal genomes consist of multiple parts—the main chromosome plus several megaplasmids—and in polyploid species these parts are present in multiple copies. This poses a challenge to the regulation of DNA replication. However, one archaeal species (Haloferax volcanii) can survive without replication origins; instead, it uses homologous recombination as an alternative mechanism of initiation. This diversity in DNA replication initiation is all the more remarkable for having been discovered in only three groups of archaea where in vivo studies are possible. PMID:28146124

  14. Mechanisms of Post-Replication DNA Repair.

    PubMed

    Gao, Yanzhe; Mutter-Rottmayer, Elizabeth; Zlatanou, Anastasia; Vaziri, Cyrus; Yang, Yang

    2017-02-08

    Accurate DNA replication is crucial for cell survival and the maintenance of genome stability. Cells have developed mechanisms to cope with the frequent genotoxic injuries that arise from both endogenous and environmental sources. Lesions encountered during DNA replication are often tolerated by post-replication repair mechanisms that prevent replication fork collapse and avert the formation of DNA double strand breaks. There are two predominant post-replication repair pathways, trans-lesion synthesis (TLS) and template switching (TS). TLS is a DNA damage-tolerant and low-fidelity mode of DNA synthesis that utilizes specialized 'Y-family' DNA polymerases to replicate damaged templates. TS, however, is an error-free 'DNA damage avoidance' mode of DNA synthesis that uses a newly synthesized sister chromatid as a template in lieu of the damaged parent strand. Both TLS and TS pathways are tightly controlled signaling cascades that integrate DNA synthesis with the overall DNA damage response and are thus crucial for genome stability. This review will cover the current knowledge of the primary mediators of post-replication repair and how they are regulated in the cell.

  15. Plasmid R6K replication control.

    PubMed

    Rakowski, Sheryl A; Filutowicz, Marcin

    2013-05-01

    The focus of this minireview is the replication control of the 39.9-kb plasmid R6K and its derivatives. Historically, this plasmid was thought to have a narrow host range but more recent findings indicate that its derivatives can replicate in a variety of enteric and non-enteric bacterial species (Wild et al., 2004). In the four-plus decades since it was first described, R6K has proven to be an excellent model for studies of plasmid DNA replication. In part this is because of its similarities to other systems in which replication is activated and regulated by Rep protein and iteron-containing DNA. However its apparent idiosynchracies have also added to its significance (e.g., independent and co-dependent replication origins, and Rep dimers that stably bind iterons). Here, we survey the current state of knowledge regarding R6K replication and place individual regulatory elements into a proposed homeostatic model with implications for the biological significance of R6K and its multiple origins of replication.

  16. Mechanisms of Post-Replication DNA Repair

    PubMed Central

    Gao, Yanzhe; Mutter-Rottmayer, Elizabeth; Zlatanou, Anastasia; Vaziri, Cyrus; Yang, Yang

    2017-01-01

    Accurate DNA replication is crucial for cell survival and the maintenance of genome stability. Cells have developed mechanisms to cope with the frequent genotoxic injuries that arise from both endogenous and environmental sources. Lesions encountered during DNA replication are often tolerated by post-replication repair mechanisms that prevent replication fork collapse and avert the formation of DNA double strand breaks. There are two predominant post-replication repair pathways, trans-lesion synthesis (TLS) and template switching (TS). TLS is a DNA damage-tolerant and low-fidelity mode of DNA synthesis that utilizes specialized ‘Y-family’ DNA polymerases to replicate damaged templates. TS, however, is an error-free ‘DNA damage avoidance’ mode of DNA synthesis that uses a newly synthesized sister chromatid as a template in lieu of the damaged parent strand. Both TLS and TS pathways are tightly controlled signaling cascades that integrate DNA synthesis with the overall DNA damage response and are thus crucial for genome stability. This review will cover the current knowledge of the primary mediators of post-replication repair and how they are regulated in the cell. PMID:28208741

  17. Phage P4 DNA replication in vitro.

    PubMed Central

    Díaz Orejas, R; Ziegelin, G; Lurz, R; Lanka, E

    1994-01-01

    Phage P4 DNA is replicated in cell-free extracts of Escherichia coli in the presence of partially purified P4 alpha protein [Krevolin and Calendar (1985), J. Mol. Biol. 182, 507-517]. Using a modified in vitro replication assay, we have further characterized this process. Analysis by agarose gel electrophoresis and autoradiography of in vitro replicated molecules demonstrates that the system yields supercoiled monomeric DNA as the main product. Electron microscopic analysis of in vitro generated intermediates indicates that DNA synthesis initiates in vitro mainly at ori, the origin of replication used in vivo. Replication proceeds from this origin bidirectionally, resulting in theta-type molecules. In contrast to the in vivo situation, no extensive single-stranded regions were found in these intermediates. The initiation proteins of the host, DnaB and DnaG, and the chaperones DnaJ and DnaK are not required for P4 replication, because polyclonal antibodies against those polypeptides do not inhibit the process. The reaction is inhibited by antibodies against the SSB protein, and by ara-CTP, a specific inhibitor of DNA polymerase III holoenzyme. Consistent with previous reports, P4 in vitro replication is independent of transcription by host RNA polymerase. Novobiocin, a DNA gyrase inhibitor, strongly inhibits P4 DNA synthesis, indicating that form I DNA is the required substrate. Images PMID:8029013

  18. Targeting DNA Replication Stress for Cancer Therapy

    PubMed Central

    Zhang, Jun; Dai, Qun; Park, Dongkyoo; Deng, Xingming

    2016-01-01

    The human cellular genome is under constant stress from extrinsic and intrinsic factors, which can lead to DNA damage and defective replication. In normal cells, DNA damage response (DDR) mediated by various checkpoints will either activate the DNA repair system or induce cellular apoptosis/senescence, therefore maintaining overall genomic integrity. Cancer cells, however, due to constitutive growth signaling and defective DDR, may exhibit “replication stress” —a phenomenon unique to cancer cells that is described as the perturbation of error-free DNA replication and slow-down of DNA synthesis. Although replication stress has been proven to induce genomic instability and tumorigenesis, recent studies have counterintuitively shown that enhancing replicative stress through further loosening of the remaining checkpoints in cancer cells to induce their catastrophic failure of proliferation may provide an alternative therapeutic approach. In this review, we discuss the rationale to enhance replicative stress in cancer cells, past approaches using traditional radiation and chemotherapy, and emerging approaches targeting the signaling cascades induced by DNA damage. We also summarize current clinical trials exploring these strategies and propose future research directions including the use of combination therapies, and the identification of potential new targets and biomarkers to track and predict treatment responses to targeting DNA replication stress. PMID:27548226

  19. Recovery from the DNA Replication Checkpoint

    PubMed Central

    Chaudhury, Indrajit; Koepp, Deanna M.

    2016-01-01

    Checkpoint recovery is integral to a successful checkpoint response. Checkpoint pathways monitor progress during cell division so that in the event of an error, the checkpoint is activated to block the cell cycle and activate repair pathways. Intrinsic to this process is that once repair has been achieved, the checkpoint signaling pathway is inactivated and cell cycle progression resumes. We use the term “checkpoint recovery” to describe the pathways responsible for the inactivation of checkpoint signaling and cell cycle re-entry after the initial stress has been alleviated. The DNA replication or S-phase checkpoint monitors the integrity of DNA synthesis. When replication stress is encountered, replication forks are stalled, and the checkpoint signaling pathway is activated. Central to recovery from the S-phase checkpoint is the restart of stalled replication forks. If checkpoint recovery fails, stalled forks may become unstable and lead to DNA breaks or unusual DNA structures that are difficult to resolve, causing genomic instability. Alternatively, if cell cycle resumption mechanisms become uncoupled from checkpoint inactivation, cells with under-replicated DNA might proceed through the cell cycle, also diminishing genomic stability. In this review, we discuss the molecular mechanisms that contribute to inactivation of the S-phase checkpoint signaling pathway and the restart of replication forks during recovery from replication stress. PMID:27801838

  20. Oncogene v-jun modulates DNA replication.

    PubMed

    Wasylyk, C; Schneikert, J; Wasylyk, B

    1990-07-01

    Cell transformation leads to alterations in both transcription and DNA replication. Activation of transcription by the expression of a number of transforming oncogenes is mediated by the transcription factor AP1 (Herrlich & Ponta, 1989; Imler & Wasylyk, 1989). AP1 is a composite transcription factor, consisting of members of the jun and fos gene-families. c-jun and c-fos are progenitors of oncogenes, suggestion that an important transcriptional event in cell transformation is altered activity of AP1, which may arise either indirectly by oncogene expression or directly by structural modification of AP1. We report here that the v-jun oncogene and its progenitor c-jun, as fusion proteins with the lex-A-repressor DNA binding domain, can activate DNA replication from the Polyoma virus (Py) origin of replication, linked to the lex-A operator. The transcription-activation region of v-jun is required for activation of replication. When excess v-jun is expressed in the cell, replication is inhibited or 'squelched'. These results suggest that one consequence of deregulated jun activity could be altered DNA replication and that there are similarities in the way v-jun activates replication and transcription.

  1. Understanding HIV resistance, fitness, replication capacity and compensation: targeting viral fitness as a therapeutic strategy.

    PubMed

    Buckheit, Robert W

    2004-08-01

    The increasingly prevalent emergence of drug-resistant virus strains in patients being treated with highly active antiretroviral regimens and the increasing rates of transmission of drug-resistant virus strains have focused attention on the critical need for additional antiretroviral agents with novel mechanisms of action and enhanced potency. Furthermore, novel means of employing highly active antiretroviral therapy are needed to reduce or eliminate the virological treatment failures that currently occur. Over the past several years, evidence has mounted supporting the fact that the emergence of resistant strains is associated with reductions in viral fitness, yielding decreases in plasma virus load in treated patients harbouring resistant populations of the virus. Additional mutations that serve to modify fitness (compensatory mutations) and mutations that impact the viral replication capacity also emerge under the selective pressure of drug treatment, and have both negative and positive effects on virus growth. Fitness is generally accepted to refer to the ability of HIV to replicate in a defined environment and thus is used to describe the viral replication potential in the absence of the drug. Although viral fitness and replication capacity are related in some ways, it is important to recognise that viral fitness is not the same as viral replication capacity. This review will assess the recent literature on antiviral drug resistance, viral fitness and viral replication capacity, and discuss means by which the adaptability of HIV to respond rapidly to antiviral treatment through mutation may be used against it. This would be done by treating patients with an aim to lock the deleterious mutations into the resistant virus genome, resulting in a positive therapeutic outcome despite the presence of resistance to the selecting agents. The review will specifically discuss the literature on nucleoside and non-nucleoside reverse transcriptase inhibitors, protease

  2. Hip flexor strain - aftercare

    MedlinePlus

    ... flexor - aftercare; Hip flexor injury - aftercare; Hip flexor tear - aftercare; Iliopsoas strain - aftercare; Strained iliopsoas muscle - aftercare; Torn iliopsoas muscle - aftercare; Psoas strain - aftercare

  3. MYC and the Control of DNA Replication

    PubMed Central

    Dominguez-Sola, David; Gautier, Jean

    2014-01-01

    The MYC oncogene is a multifunctional protein that is aberrantly expressed in a significant fraction of tumors from diverse tissue origins. Because of its multifunctional nature, it has been difficult to delineate the exact contributions of MYC’s diverse roles to tumorigenesis. Here, we review the normal role of MYC in regulating DNA replication as well as its ability to generate DNA replication stress when overexpressed. Finally, we discuss the possible mechanisms by which replication stress induced by aberrant MYC expression could contribute to genomic instability and cancer. PMID:24890833

  4. DNA replication in yeast is stochastic

    NASA Astrophysics Data System (ADS)

    Cheng-Hsin Yang, Scott; Rhind, Nicholas; Bechhoefer, John

    2010-03-01

    Largely on the basis of a simple --- perhaps too simple --- analysis of microarray-chip experiments, people have concluded that DNA replication in budding yeast (S. cerevisiae) is a nearly deterministic process, in which the position and activation time of each origin of replication is pre-determined. In this talk, we introduce a more quantitative approach to the analysis of microarray data. Applying our new methods to budding yeast, we show that the microarray data imply a picture of replication where the timing of origin activation is highly stochastic. We then propose a physical model (the ``multiple-initiator model") to account for the observed probability distributions of origin- activation timing.

  5. Prebiotic chemistry and nucleic acid replication

    NASA Technical Reports Server (NTRS)

    Orgel, L. E.; Lohrmann, R.

    1974-01-01

    Recent work is reviewed on some reactions that could have occurred on the primitive earth and that could have played a part in the evolution of a self-replicating system. The transition from the primitive atmosphere to the simplest replicating molecules is considered in four stages: (1) the formation of a 'prebiotic soup' of organic precursors, including the purine and pyrimidine bases and the pentose sugars; (2) the condensation of these precursors and inorganic phosphate to form monomeric nucleotides and activated nucleotide derivatives; (3) the polymerization of nucleotide derivatives to oligonucleotides; and (4) the complementary replication of oligonucleotides in a template-directed process that depends on Watson-Crick base pairing.

  6. Top 10 Replicated Findings from Behavioral Genetics

    PubMed Central

    Plomin, Robert; DeFries, John C.; Knopik, Valerie S.; Neiderhiser, Jenae M.

    2015-01-01

    In the context of current concerns about replication in psychological science, we describe 10 findings from behavioral genetic research that have robustly replicated. These are ‘big’ findings, both in terms of effect size and potential impact on psychological science, such as linearly increasing heritability of intelligence from infancy (20%) through adulthood (60%). Four of our top-10 findings involve the environment, discoveries that could only have been found using genetically sensitive research designs. We also consider reasons specific to behavioral genetics that might explain why these findings replicate. PMID:26817721

  7. Replication-transcription conflicts in bacteria

    PubMed Central

    Merrikh, Houra; Zhang, Yan; Grossman, Alan D.; Wang, Jue D.

    2012-01-01

    Preface DNA replication and transcription use the same template and occur concurrently in bacteria. The lack of temporal and spatial separations of these two processes leads to their conflict. Failure to deal with these conflicts can result in genome alterations and reduced fitness. In recent years, significant advances have been made in understanding how cells avoid conflicts between replication and transcription, and how conflicts are resolved when they do occur. In this review, we summarize these findings, which shed light on the significance of the problem and on how cells deal with unwanted encounters between the replication and transcription machineries. PMID:22669220

  8. Topological locking restrains replication fork reversal

    PubMed Central

    Fierro-Fernández, Marta; Hernández, Pablo; Krimer, Dora B.; Stasiak, Andrzej; Schvartzman, Jorge B.

    2007-01-01

    Two-dimensional agarose gel electrophoresis, psoralen cross-linking, and electron microscopy were used to study the effects of positive supercoiling on fork reversal in isolated replication intermediates of bacterial DNA plasmids. The results obtained demonstrate that the formation of Holliday-like junctions at both forks of a replication bubble creates a topological constraint that prevents further regression of the forks. We propose that this topological locking of replication intermediates provides a biological safety mechanism that protects DNA molecules against extensive fork reversals. PMID:17242356

  9. Mapping Replication Origin Sequences in Eukaryotic Chromosomes

    PubMed Central

    Fu, Haiqing; Besnard, Emilie; Desprat, Romain; Ryan, Michael; Kahli, Malik; Lemaitre, Jean-Marc; Aladjem, Mirit I.

    2014-01-01

    Recent advances in genome sequencing technology have led towards the complete mapping of DNA replication initiation sites in the human genome. This thorough origin mapping facilitates the understanding of the relationship between replication initiation events, transcription and chromatin modifications and allows the characterization of consensus sequences of potential replication origins. This unit provides a detailed protocol for isolation and sequence analyses of nascent DNA strands. Two variations of the protocol based on non-overlapping assumptions are described below, addressing potential bias issues for whole genome analyses. PMID:25447077

  10. Mechanisms for Initiating Cellular DNA Replication

    PubMed Central

    Costa, Alessandro; Hood, Iris V.; Berger, James M.

    2015-01-01

    The initiation of DNA replication represents a committing step to cell proliferation. Appropriate replication onset depends on multiprotein complexes that help properly distinguish origin regions, generate nascent replication bubbles, and promote replisome formation. This review describes initiation systems employed by bacteria, archaea, and eukaryotes, with a focus on comparing and contrasting molecular mechanisms among organisms. Although commonalities can be found in the functional domains and strategies used to carry out and regulate initiation, many key participants have markedly different activities and appear to have evolved convergently. Despite significant advances in the field, major questions still persist in understanding how initiation programs are executed at the molecular level. PMID:23746253

  11. [Analysis of the mechanisms of tobamovirus RNA replication].

    PubMed

    Ishikawa, Masayuki

    2006-06-01

    The replication of eukaryotic positive-strand RNA virus genomes occurs in membrane-bound replication complexes. Currently, little is known about the process of replication complex formation and the molecular structure of the replication complexes. We are trying to understand how eukaryotic positive-strand RNA viruses replicate using tobamoviruses as models. Here, I describe our approaches to this end.

  12. Analysis of human cytomegalovirus replication in primary cultured human corneal endothelial cells

    PubMed Central

    Hosogai, Mayumi; Shima, Nobuyuki; Nakatani, Yoko; Inoue, Teruki; Iso, Tatsuya; Yokoo, Hideaki; Yorifuji, Hiroshi; Akiyama, Hideo; Kishi, Shoji; Isomura, Hiroki

    2015-01-01

    Background/aims Since the first case of human cytomegalovirus (HCMV)-induced corneal endotheliitis in which HCMV DNA was detected from the patient's aqueous humour using PCR, the clinical evidence for HCMV endotheliitis has been accumulating. However, it remains to be confirmed whether HCMV can efficiently replicate in corneal endothelial cells. We, therefore, sought to determine whether primary cultured human corneal endothelial cells (HCECs) could support HCMV replication. Methods Human foreskin fibroblasts (HFFs) have been shown to be fully permissive for HCMV replication, and are commonly used as an in vitro model for HCMV lytic replication. Therefore, primary cultured HCECs or HFFs were infected with the vascular endotheliotropic HCMV strain TB40/E or laboratory strain Towne. We then compared viral mRNA and protein expression, genome replication and growth between the TB40/E-infected and Towne-infected HCECs and HFFs. Results When HCECs were infected with TB40/E or Towne, rounded cells resembling owl's eyes as well as viral antigens were detected. Viral mRNA synthesis and protein expression proceeded efficiently in the HCECs and HFFs infected with TB40/E or Towne at a high multiplicity of infection (MOI). Similarly, the viral genome was also effectively replicated, with UL44—a viral DNA polymerase processivity factor—foci observed in the nuclei of HCECs. HCECs produced a substantial number of infectious virions after infection with TB40/E at both a high and low MOI. Conclusions Primary cultured HCECs could efficiently support HCMV replication after infection at both a high and low MOI. PMID:26261231

  13. Accessory genes confer a high replication rate to virulent feline immunodeficiency virus.

    PubMed

    Troyer, Ryan M; Thompson, Jesse; Elder, John H; VandeWoude, Sue

    2013-07-01

    Feline immunodeficiency virus (FIV) is a lentivirus that causes AIDS in domestic cats, similar to human immunodeficiency virus (HIV)/AIDS in humans. The FIV accessory protein Vif abrogates the inhibition of infection by cat APOBEC3 restriction factors. FIV also encodes a multifunctional OrfA accessory protein that has characteristics similar to HIV Tat, Vpu, Vpr, and Nef. To examine the role of vif and orfA accessory genes in FIV replication and pathogenicity, we generated chimeras between two FIV molecular clones with divergent disease potentials: a highly pathogenic isolate that replicates rapidly in vitro and is associated with significant immunopathology in vivo, FIV-C36 (referred to here as high-virulence FIV [HV-FIV]), and a less-pathogenic strain, FIV-PPR (referred to here as low-virulence FIV [LV-FIV]). Using PCR-driven overlap extension, we produced viruses in which vif, orfA, or both genes from virulent HV-FIV replaced equivalent genes in LV-FIV. The generation of these chimeras is more straightforward in FIV than in primate lentiviruses, since FIV accessory gene open reading frames have very little overlap with other genes. All three chimeric viruses exhibited increased replication kinetics in vitro compared to the replication kinetics of LV-FIV. Chimeras containing HV-Vif or Vif/OrfA had replication rates equivalent to those of the virulent HV-FIV parental virus. Furthermore, small interfering RNA knockdown of feline APOBEC3 genes resulted in equalization of replication rates between LV-FIV and LV-FIV encoding HV-FIV Vif. These findings demonstrate that Vif-APOBEC interactions play a key role in controlling the replication and pathogenicity of this immunodeficiency-inducing virus in its native host species and that accessory genes act as mediators of lentiviral strain-specific virulence.

  14. A Differential Fluorescence-Based Genetic Screen Identifies Listeria monocytogenes Determinants Required for Intracellular Replication

    PubMed Central

    Perry, Kyle J.

    2013-01-01

    Listeria monocytogenes is a Gram-positive, facultative intracellular pathogen capable of causing severe invasive disease with high mortality rates in humans. While previous studies have largely elucidated the bacterial and host cell mechanisms necessary for invasion, vacuolar escape, and subsequent cell-to-cell spread, the L. monocytogenes factors required for rapid replication within the restrictive environment of the host cell cytosol are poorly understood. In this report, we describe a differential fluorescence-based genetic screen utilizing fluorescence-activated cell sorting (FACS) and high-throughput microscopy to identify L. monocytogenes mutants defective in optimal intracellular replication. Bacteria harboring deletions within the identified gene menD or pepP were defective for growth in primary murine macrophages and plaque formation in monolayers of L2 fibroblasts, thus validating the ability of the screening method to identify intracellular replication-defective mutants. Genetic complementation of the menD and pepP deletion strains rescued the in vitro intracellular infection defects. Furthermore, the menD deletion strain displayed a general extracellular replication defect that could be complemented by growth under anaerobic conditions, while the intracellular growth defect of this strain could be complemented by the addition of exogenous menaquinone. As prior studies have indicated the importance of aerobic metabolism for L. monocytogenes infection, these findings provide further evidence for the importance of menaquinone and aerobic metabolism for L. monocytogenes pathogenesis. Lastly, both the menD and pepP deletion strains were attenuated during in vivo infection of mice. These findings demonstrate that the differential fluorescence-based screening approach provides a powerful tool for the identification of intracellular replication determinants in multiple bacterial systems. PMID:23687268

  15. A Novel System for Identification of Inhibitors of Rift Valley Fever Virus Replication

    PubMed Central

    Piper, Mary E.; Gerrard, Sonja R.

    2010-01-01

    Rift Valley fever virus (RVFV) is a human and livestock pathogen endemic to sub-Saharan Africa. We have developed a T7-dependent system for the efficient production of RVFV-like particles (RVF-VLPs) based on the virulent ZH-501 strain of RVFV. The RVF-VLPs are capable of performing a single round of infection, allowing for the study of viral replication, assembly, and infectivity. We demonstrate that these RVF-VLPs are antigenically indistinguishable from authentic RVFV and respond similarly to a wide array of known and previously unknown chemical inhibitors. This system should be useful for screening for small molecule inhibitors of RVFV replication. PMID:21994655

  16. Effect of Wolbachia on replication of West Nile virus in a mosquito cell line and adult mosquitoes.

    PubMed

    Hussain, Mazhar; Lu, Guangjin; Torres, Shessy; Edmonds, Judith H; Kay, Brian H; Khromykh, Alexander A; Asgari, Sassan

    2013-01-01

    Wolbachia as an endosymbiont is widespread in insects and other arthropods and is best known for reproductive manipulations of the host. Recently, it has been shown that wMelpop and wMel strains of Wolbachia inhibit the replication of several RNA viruses, including dengue virus, and other vector-borne pathogens (e.g., Plasmodium and filarial nematodes) in mosquitoes, providing an alternative approach to limit the transmission of vector-borne pathogens. In this study, we tested the effect of Wolbachia on the replication of West Nile Virus (WNV). Surprisingly, accumulation of the genomic RNA of WNV for all three strains of WNV tested (New York 99, Kunjin, and New South Wales) was enhanced in Wolbachia-infected Aedes aegypti cells (Aag2). However, the amount of secreted virus was significantly reduced in the presence of Wolbachia. Intrathoracic injections showed that replication of WNV in A. aegypti mosquitoes infected with wMel strain of Wolbachia was not inhibited, whereas wMelPop strain of Wolbachia significantly reduced the replication of WNV in mosquitoes. Further, when wMelPop mosquitoes were orally fed with WNV, virus infection, transmission, and dissemination rates were very low in Wolbachia-free mosquitoes and were completely inhibited in the presence of Wolbachia. The results suggest that (i) despite the enhancement of viral genomic RNA replication in the Wolbachia-infected cell line the production of secreted virus was significantly inhibited, (ii) the antiviral effect in intrathoracically infected mosquitoes depends on the strain of Wolbachia, and (iii) replication of the virus in orally fed mosquitoes was completely inhibited in wMelPop strain of Wolbachia.

  17. A transcription and translation-coupled DNA replication system using rolling-circle replication.

    PubMed

    Sakatani, Yoshihiro; Ichihashi, Norikazu; Kazuta, Yasuaki; Yomo, Tetsuya

    2015-05-27

    All living organisms have a genome replication system in which genomic DNA is replicated by a DNA polymerase translated from mRNA transcribed from the genome. The artificial reconstitution of this genome replication system is a great challenge in in vitro synthetic biology. In this study, we attempted to construct a transcription- and translation-coupled DNA replication (TTcDR) system using circular genomic DNA encoding phi29 DNA polymerase and a reconstituted transcription and translation system. In this system, phi29 DNA polymerase was translated from the genome and replicated the genome in a rolling-circle manner. When using a traditional translation system composition, almost no DNA replication was observed, because the tRNA and nucleoside triphosphates included in the translation system significantly inhibited DNA replication. To minimize these inhibitory effects, we optimized the composition of the TTcDR system and improved replication by approximately 100-fold. Using our system, genomic DNA was replicated up to 10 times in 12 hours at 30 °C. This system provides a step toward the in vitro construction of an artificial genome replication system, which is a prerequisite for the construction of an artificial cell.

  18. Is psychology suffering from a replication crisis? What does "failure to replicate" really mean?

    PubMed

    Maxwell, Scott E; Lau, Michael Y; Howard, George S

    2015-09-01

    Psychology has recently been viewed as facing a replication crisis because efforts to replicate past study findings frequently do not show the same result. Often, the first study showed a statistically significant result but the replication does not. Questions then arise about whether the first study results were false positives, and whether the replication study correctly indicates that there is truly no effect after all. This article suggests these so-called failures to replicate may not be failures at all, but rather are the result of low statistical power in single replication studies, and the result of failure to appreciate the need for multiple replications in order to have enough power to identify true effects. We provide examples of these power problems and suggest some solutions using Bayesian statistics and meta-analysis. Although the need for multiple replication studies may frustrate those who would prefer quick answers to psychology's alleged crisis, the large sample sizes typically needed to provide firm evidence will almost always require concerted efforts from multiple investigators. As a result, it remains to be seen how many of the recently claimed failures to replicate will be supported or instead may turn out to be artifacts of inadequate sample sizes and single study replications. (PsycINFO Database Record (c) 2015 APA, all rights reserved).

  19. DNA replication origins, ORC/DNA interaction, and assembly of pre-replication complex in eukaryotes.

    PubMed

    Sun, Jingya; Kong, Daochun

    2010-07-01

    Chromosomal DNA replication in eukaryotic cells is highly complicated and sophisticatedly regulated. Owing to its large size, a typical eukaryotic genome contains hundreds to tens of thousands of initiation sites called DNA replication origins where DNA synthesis takes place. Multiple initiation sites remove the constraint of a genome size because only a certain amount of DNA can be replicated from a single origin in a limited time. The activation of these multiple origins must be coordinated so that each segment of chromosomal DNA is precisely duplicated only once per cell cycle. Although DNA replication is a vital process for cell growth and its mechanism is highly conserved, recent studies also reveal significant diversity in origin structure, assembly of pre-replication complex (pre-RC) and regulation of replication initiation along evolutionary lines. The DNA replication origins in the fission yeast Schizosaccharomyces pombe are found to contain a second essential element that is bound by Sap1 protein besides the essential origin recognition complex-binding site. Sap1 is recently demonstrated to be a novel replication initiation protein that plays an essential role in loading the initiation protein Cdc18 to origins and thus directly participates in pre-RC formation. In this review, we summarize the recent advance in understanding how DNA replication origins are organized, how pre-RC is assembled and how DNA replication is initiated and regulated in yeast and metazoans.

  20. Mechanism of chromosomal DNA replication initiation and replication fork stabilization in eukaryotes.

    PubMed

    Wu, LiHong; Liu, Yang; Kong, DaoChun

    2014-05-01

    Chromosomal DNA replication is one of the central biological events occurring inside cells. Due to its large size, the replication of genomic DNA in eukaryotes initiates at hundreds to tens of thousands of sites called DNA origins so that the replication could be completed in a limited time. Further, eukaryotic DNA replication is sophisticatedly regulated, and this regulation guarantees that each origin fires once per S phase and each segment of DNA gets duplication also once per cell cycle. The first step of replication initiation is the assembly of pre-replication complex (pre-RC). Since 1973, four proteins, Cdc6/Cdc18, MCM, ORC and Cdt1, have been extensively studied and proved to be pre-RC components. Recently, a novel pre-RC component called Sap1/Girdin was identified. Sap1/Girdin is required for loading Cdc18/Cdc6 to origins for pre-RC assembly in the fission yeast and human cells, respectively. At the transition of G1 to S phase, pre-RC is activated by the two kinases, cyclindependent kinase (CDK) and Dbf4-dependent kinase (DDK), and subsequently, RPA, primase-polα, PCNA, topoisomerase, Cdc45, polδ, and polɛ are recruited to DNA origins for creating two bi-directional replication forks and initiating DNA replication. As replication forks move along chromatin DNA, they frequently stall due to the presence of a great number of replication barriers on chromatin DNA, such as secondary DNA structures, protein/DNA complexes, DNA lesions, gene transcription. Stalled forks must require checkpoint regulation for their stabilization. Otherwise, stalled forks will collapse, which results in incomplete DNA replication and genomic instability. This short review gives a concise introduction regarding the current understanding of replication initiation and replication fork stabilization.

  1. Using autonomous replication to physically and genetically define human origins of replication

    SciTech Connect

    Krysan, P.J.

    1993-01-01

    The author previously developed a system for studying autonomous replication in human cells involving the use of sequences from the Epstein-Barr virus (EBV) genome to provide extrachromosomal plasmids with a nuclear retention function. Using this system, it was demonstrated that large fragments of human genomic DNA could be isolated which replicate autonomously in human cells. In this study the DNA sequences which function as origins of replication in human cells are defined physically and genetically. These experiments demonstrated that replication initiates at multiple locations distributed throughout the plasmid. Another line of experiments addressed the DNA sequence requirements for autonomous replication in human cells. These experiments demonstrated that human DNA fragments have a higher replication activity than bacterial fragments do. It was also found, however, that the bacterial DNA sequence could support efficient replication if enough copies of it were present on the plasmid. These findings suggested that autonomous replication in human cells does not depend on extensive, specific DNA sequences. The autonomous replication system which the author has employed for these experiments utilizes a cis-acting sequence from the EBV origin and the trans-acting EBNA-1 protein to provide plasmids with a nuclear retention function. It was therefore relevant to verify that the autonomous replication of human DNA fragments did not depend on the replication activity associated with the EBV sequences utilized for nuclear retention. To accomplish this goal, the author demonstrated that plasmids carrying the EBV sequences and large fragments of human DNA could support long-term autonomous replication in hamster cells, which are not permissive for EBV replication.

  2. Molecular Mechanisms of DNA Replication Checkpoint Activation

    PubMed Central

    Recolin, Bénédicte; van der Laan, Siem; Tsanov, Nikolay; Maiorano, Domenico

    2014-01-01

    The major challenge of the cell cycle is to deliver an intact, and fully duplicated, genetic material to the daughter cells. To this end, progression of DNA synthesis is monitored by a feedback mechanism known as replication checkpoint that is untimely linked to DNA replication. This signaling pathway ensures coordination of DNA synthesis with cell cycle progression. Failure to activate this checkpoint in response to perturbation of DNA synthesis (replication stress) results in forced cell division leading to chromosome fragmentation, aneuploidy, and genomic instability. In this review, we will describe current knowledge of the molecular determinants of the DNA replication checkpoint in eukaryotic cells and discuss a model of activation of this signaling pathway crucial for maintenance of genomic stability. PMID:24705291

  3. Replicated Electro-Formed Nickel Alloy Mirror

    NASA Technical Reports Server (NTRS)

    1999-01-01

    NASA's Space Optics Manufacturing Center has been working to expand our view of the universe via sophisticated new telescopes. The Optics Center's goal is to develop low-cost, advanced space optics technologies for the NASA program in the 21st century - including the long-term goal of imaging Earth-like planets in distant solar systems. To reduce the cost of mirror fabrication, Marshall Space Flight Center (MSFC) has developed replication techniques, the machinery, and materials to replicate electro-formed nickel mirrors. The process allows fabricating precisely shaped mandrels to be used and reused as masters for replicating high-quality mirrors. Dr. Joe Ritter examines a replicated electro-formed nickel-alloy mirror which exemplifies the improvements in mirror fabrication techniques, with benefits such as dramtic weight reduction that have been achieved at the Marshall Space Flight Center's Space Optics Manufacturing Technology Center (SOMTC).

  4. Statistical physics of self-replication

    NASA Astrophysics Data System (ADS)

    England, Jeremy L.

    2013-09-01

    Self-replication is a capacity common to every species of living thing, and simple physical intuition dictates that such a process must invariably be fueled by the production of entropy. Here, we undertake to make this intuition rigorous and quantitative by deriving a lower bound for the amount of heat that is produced during a process of self-replication in a system coupled to a thermal bath. We find that the minimum value for the physically allowed rate of heat production is determined by the growth rate, internal entropy, and durability of the replicator, and we discuss the implications of this finding for bacterial cell division, as well as for the pre-biotic emergence of self-replicating nucleic acids.

  5. Dynamic lipid landscape of picornavirus replication organelles.

    PubMed

    Belov, George A

    2016-08-01

    Picornavirus infection induces rapid reorganization of the cellular membrane architecture and appearance of novel membranous structures associated with the viral RNA replication and virion assembly-replication organelles. Recent studies significantly advanced our understanding of their lipid composition and cellular mechanisms involved in their development. Picornaviruses activate synthesis of both structural and signaling lipids and reroute cellular cholesterol trafficking pathways to create unique membranous domains favoring viral replication. Rapidly replicating picornaviruses rely on posttranslational activation and/or specific recruitment of cellular proteins rather than on modulation of expression of cellular genes to create favorable membrane microenvironment. At the same time picornaviruses demonstrate remarkable adaptability to changes in the lipid landscape which should be taken into account when developing novel antiviral strategies. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Statistical physics of self-replication.

    PubMed

    England, Jeremy L

    2013-09-28

    Self-replication is a capacity common to every species of living thing, and simple physical intuition dictates that such a process must invariably be fueled by the production of entropy. Here, we undertake to make this intuition rigorous and quantitative by deriving a lower bound for the amount of heat that is produced during a process of self-replication in a system coupled to a thermal bath. We find that the minimum value for the physically allowed rate of heat production is determined by the growth rate, internal entropy, and durability of the replicator, and we discuss the implications of this finding for bacterial cell division, as well as for the pre-biotic emergence of self-replicating nucleic acids.

  7. Cryptoporic acid E from Cryptoporus volvatus inhibits influenza virus replication in vitro.

    PubMed

    Gao, Li; Han, Jiayuan; Si, Jianyong; Wang, Junchi; Wang, Hexiang; Sun, Yipeng; Bi, Yuhai; Liu, Jinhua; Cao, Li

    2017-07-01

    Influenza virus infection is a global public health issue. The efficacy of antiviral agents for influenza virus has been limited by the emergence of drug-resistant virus strains. Thus, there is an urgent need to identify novel antiviral therapies. Our previous studies have found that Cryptoporus volvatus extract can potently inhibit influenza virus replication in vitro and in vivo. However, the effective component of Cryptoporus volvatus, which mediates the antiviral activity, hasn't been identified. Here, we identified a novel anti-influenza virus molecule, Cryptoporic acid E (CAE), from Cryptoporus volvatus. Our results showed that CAE had broad-spectrum anti-influenza activity against 2009 pandemic strain A/Beijing/07/2009 (H1N1/09pdm), seasonal strain A/Beijing/CAS0001/2007(H3N2), mouse adapted strains A/WSN/33 (H1N1), and A/PR8/34 (H1N1). We further investigated the mode of CAE action. Time-course-analysis indicated that CAE exerted its inhibition mainly at the middle stages of the replication cycle of influenza virus. Subsequently, we confirmed that CAE inhibited influenza virus RNA polymerase activity and blocked virus RNA replication and transcription in MDCK cells. In addition, we found that CAE also impaired influenza virus infectivity by directly targeting virus particles. Our data suggest that CAE is a major effective component of Cryptoporus volvatus. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Mycobacterium tuberculosis replicates within necrotic human macrophages

    PubMed Central

    Lerner, Thomas R.; Repnik, Urska; Herbst, Susanne; Collinson, Lucy M.; Griffiths, Gareth

    2017-01-01

    Mycobacterium tuberculosis modulation of macrophage cell death is a well-documented phenomenon, but its role during bacterial replication is less characterized. In this study, we investigate the impact of plasma membrane (PM) integrity on bacterial replication in different functional populations of human primary macrophages. We discovered that IFN-γ enhanced bacterial replication in macrophage colony-stimulating factor–differentiated macrophages more than in granulocyte–macrophage colony-stimulating factor–differentiated macrophages. We show that permissiveness in the different populations of macrophages to bacterial growth is the result of a differential ability to preserve PM integrity. By combining live-cell imaging, correlative light electron microscopy, and single-cell analysis, we found that after infection, a population of macrophages became necrotic, providing a niche for M. tuberculosis replication before escaping into the extracellular milieu. Thus, in addition to bacterial dissemination, necrotic cells provide first a niche for bacterial replication. Our results are relevant to understanding the environment of M. tuberculosis replication in the host. PMID:28242744

  9. Nucleosomes influence multiple steps during replication initiation

    PubMed Central

    Azmi, Ishara F; Watanabe, Shinya; Maloney, Michael F; Kang, Sukhyun; Belsky, Jason A; MacAlpine, David M; Peterson, Craig L; Bell, Stephen P

    2017-01-01

    Eukaryotic replication origin licensing, activation and timing are influenced by chromatin but a mechanistic understanding is lacking. Using reconstituted nucleosomal DNA replication assays, we assessed the impact of nucleosomes on replication initiation. To generate distinct nucleosomal landscapes, different chromatin-remodeling enzymes (CREs) were used to remodel nucleosomes on origin-DNA templates. Nucleosomal organization influenced two steps of replication initiation: origin licensing and helicase activation. Origin licensing assays showed that local nucleosome positioning enhanced origin specificity and modulated helicase loading by influencing ORC DNA binding. Interestingly, SWI/SNF- and RSC-remodeled nucleosomes were permissive for origin licensing but showed reduced helicase activation. Specific CREs rescued replication of these templates if added prior to helicase activation, indicating a permissive chromatin state must be established during origin licensing to allow efficient origin activation. Our studies show nucleosomes directly modulate origin licensing and activation through distinct mechanisms and provide insights into the regulation of replication initiation by chromatin. DOI: http://dx.doi.org/10.7554/eLife.22512.001 PMID:28322723

  10. The Inherent Asymmetry of DNA Replication.

    PubMed

    Snedeker, Jonathan; Wooten, Matthew; Chen, Xin

    2017-08-11

    Semiconservative DNA replication has provided an elegant solution to the fundamental problem of how life is able to proliferate in a way that allows cells, organisms, and populations to survive and replicate many times over. Somewhat lost, however, in our admiration for this elegant mechanism is an appreciation for the asymmetries that occur in the process of DNA replication. As we discuss in this review, these asymmetries arise as a consequence of the structure of the DNA molecule and the enzymatic mechanism of DNA synthesis. Increasing evidence suggests that asymmetries inDNAreplication are able to play a central role in the processes of adaptation and evolution by shaping the mutagenic landscape of cells. Additionally, in eukaryotes, recent work has demonstrated that the inherent asymmetries in DNA replication may play an important role in the process of chromatin replication. As chromatin plays an essential role in defining cell identity, asymmetries generated during the process of DNA replication may play critical roles in cell fate dicisions related to patterning and development. Expected final online publication date for the Annual Review of Cell and Developmental Biology Volume 33 is October 6, 2017. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

  11. Overcoming a nucleosomal barrier to replication

    PubMed Central

    Chang, Han-Wen; Pandey, Manjula; Kulaeva, Olga I.; Patel, Smita S.; Studitsky, Vasily M.

    2016-01-01

    Efficient overcoming and accurate maintenance of chromatin structure and associated histone marks during DNA replication are essential for normal functioning of the daughter cells. However, the molecular mechanisms of replication through chromatin are unknown. We have studied traversal of uniquely positioned mononucleosomes by T7 replisome in vitro. Nucleosomes present a strong, sequence-dependent barrier for replication, with particularly strong pausing of DNA polymerase at the +(31–40) and +(41–65) regions of the nucleosomal DNA. The exonuclease activity of T7 DNA polymerase increases the overall rate of progression of the replisome through a nucleosome, likely by resolving nonproductive complexes. The presence of nucleosome-free DNA upstream of the replication fork facilitates the progression of DNA polymerase through the nucleosome. After replication, at least 50% of the nucleosomes assume an alternative conformation, maintaining their original positions on the DNA. Our data suggest a previously unpublished mechanism for nucleosome maintenance during replication, likely involving transient formation of an intranucleosomal DNA loop. PMID:27847876

  12. Possible applications for replicating HIV 1 vectors

    PubMed Central

    Das, Atze T; Jeeninga, Rienk E; Berkhout, Ben

    2010-01-01

    Since its discovery some 25 years ago, much has been learned about HIV type 1 and the molecular details of its replication cycle. This insight has been used to develop lentiviral vector systems that have advantages over conventional retroviral vector systems. For safety reasons, the lentiviral vector systems are replication incompetent and the risk of generating a replication competent virus has been minimized. Nevertheless, there may be certain applications for replication competent HIV based vector systems, and we will review our activities in this particular field. This includes the generation of a conditionally replicating HIV 1 variant as a safe live attenuated virus vaccine, the construction of mini HIV variants as cancer selective viruses for virotherapy against leukemia, and the use of a conditionally live anti HIV gene therapy vector. Although safety concerns will undoubtedly remain for the use of replication competent HIV based vector systems, some of the results in cell culture systems are very promising and warrant further testing in appropriate animal models. PMID:20582153

  13. Possible applications for replicating HIV 1 vectors.

    PubMed

    Das, Atze T; Jeeninga, Rienk E; Berkhout, Ben

    2010-05-01

    Since its discovery some 25 years ago, much has been learned about HIV type 1 and the molecular details of its replication cycle. This insight has been used to develop lentiviral vector systems that have advantages over conventional retroviral vector systems. For safety reasons, the lentiviral vector systems are replication incompetent and the risk of generating a replication competent virus has been minimized. Nevertheless, there may be certain applications for replication competent HIV based vector systems, and we will review our activities in this particular field. This includes the generation of a conditionally replicating HIV 1 variant as a safe live attenuated virus vaccine, the construction of mini HIV variants as cancer selective viruses for virotherapy against leukemia, and the use of a conditionally live anti HIV gene therapy vector. Although safety concerns will undoubtedly remain for the use of replication competent HIV based vector systems, some of the results in cell culture systems are very promising and warrant further testing in appropriate animal models.

  14. Replication and robustness in developmental research.

    PubMed

    Duncan, Greg J; Engel, Mimi; Claessens, Amy; Dowsett, Chantelle J

    2014-11-01

    Replications and robustness checks are key elements of the scientific method and a staple in many disciplines. However, leading journals in developmental psychology rarely include explicit replications of prior research conducted by different investigators, and few require authors to establish in their articles or online appendices that their key results are robust across estimation methods, data sets, and demographic subgroups. This article makes the case for prioritizing both explicit replications and, especially, within-study robustness checks in developmental psychology. It provides evidence on variation in effect sizes in developmental studies and documents strikingly different replication and robustness-checking practices in a sample of journals in developmental psychology and a sister behavioral science-applied economics. Our goal is not to show that any one behavioral science has a monopoly on best practices, but rather to show how journals from a related discipline address vital concerns of replication and generalizability shared by all social and behavioral sciences. We provide recommendations for promoting graduate training in replication and robustness-checking methods and for editorial policies that encourage these practices. Although some of our recommendations may shift the form and substance of developmental research articles, we argue that they would generate considerable scientific benefits for the field. (PsycINFO Database Record (c) 2014 APA, all rights reserved).

  15. Commercial Building Partnerships Replication and Diffusion

    SciTech Connect

    Antonopoulos, Chrissi A.; Dillon, Heather E.; Baechler, Michael C.

    2013-09-16

    This study presents findings from survey and interview data investigating replication efforts of Commercial Building Partnership (CBP) partners that worked directly with the Pacific Northwest National Laboratory (PNNL). PNNL partnered directly with 12 organizations on new and retrofit construction projects, which represented approximately 28 percent of the entire U.S. Department of Energy (DOE) CBP program. Through a feedback survey mechanism, along with personal interviews, PNNL gathered quantitative and qualitative data relating to replication efforts by each organization. These data were analyzed to provide insight into two primary research areas: 1) CBP partners’ replication efforts of technologies and approaches used in the CBP project to the rest of the organization’s building portfolio (including replication verification), and, 2) the market potential for technology diffusion into the total U.S. commercial building stock, as a direct result of the CBP program. The first area of this research focused specifically on replication efforts underway or planned by each CBP program participant. Factors that impact replication include motivation, organizational structure and objectives firms have for implementation of energy efficient technologies. Comparing these factors between different CBP partners revealed patterns in motivation for constructing energy efficient buildings, along with better insight into market trends for green building practices. The second area of this research develops a diffusion of innovations model to analyze potential broad market impacts of the CBP program on the commercial building industry in the United States.

  16. Structural organization of human replication timing domains.

    PubMed

    Boulos, Rasha E; Drillon, Guénola; Argoul, Françoise; Arneodo, Alain; Audit, Benjamin

    2015-10-07

    Recent analysis of genome-wide epigenetic modification data, mean replication timing (MRT) profiles and chromosome conformation data in mammals have provided increasing evidence that flexibility in replication origin usage is regulated locally by the epigenetic landscape and over larger genomic distances by the 3D chromatin architecture. Here, we review the recent results establishing some link between replication domains and chromatin structural domains in pluripotent and various differentiated cell types in human. We reconcile the originally proposed dichotomic picture of early and late constant timing regions that replicate by multiple rather synchronous origins in separated nuclear compartments of open and closed chromatins, with the U-shaped MRT domains bordered by "master" replication origins specified by a localized (∼200-300 kb) zone of open and transcriptionally active chromatin from which a replication wave likely initiates and propagates toward the domain center via a cascade of origin firing. We discuss the relationships between these MRT domains, topologically associated domains and lamina-associated domains. This review sheds a new light on the epigenetically regulated global chromatin reorganization that underlies the loss of pluripotency and the determination of differentiation properties.

  17. Design and Implementation of Replicated Object Layer

    NASA Technical Reports Server (NTRS)

    Koka, Sudhir

    1996-01-01

    One of the widely used techniques for construction of fault tolerant applications is the replication of resources so that if one copy fails sufficient copies may still remain operational to allow the application to continue to function. This thesis involves the design and implementation of an object oriented framework for replicating data on multiple sites and across different platforms. Our approach, called the Replicated Object Layer (ROL) provides a mechanism for consistent replication of data over dynamic networks. ROL uses the Reliable Multicast Protocol (RMP) as a communication protocol that provides for reliable delivery, serialization and fault tolerance. Besides providing type registration, this layer facilitates distributed atomic transactions on replicated data. A novel algorithm called the RMP Commit Protocol, which commits transactions efficiently in reliable multicast environment is presented. ROL provides recovery procedures to ensure that site and communication failures do not corrupt persistent data, and male the system fault tolerant to network partitions. ROL will facilitate building distributed fault tolerant applications by performing the burdensome details of replica consistency operations, and making it completely transparent to the application.Replicated databases are a major class of applications which could be built on top of ROL.

  18. Abnormal regulation of DNA replication and increased lethality in ataxia telangiectasia cells exposed to carcinogenic agents

    SciTech Connect

    Jaspers, N.G.; de Wit, J.; Regulski, M.R.; Bootsma, D.

    1982-01-01

    The effect of different carcinogenic agents on the rate of semiconservative DNA replication in normal and ataxia telangiectasis (AT) cells was investigated. The rate of DNA synthesis in all AT cell strains tested was depressed to a significantly lesser extent than in normal cells after exposure to X-rays under oxia or hypoxia or to bleomycin, agents to which AT cells are hypersensitive. In contrast, inhibition of DNA replication in normal human and AT cells was similar after treatment with some DNA-methylating agents or mitomycin C. Colony-forming ability of AT cells treated with these agents was not different from normal cells. Treatment with 4-nitroquinoline 1-oxide elicited a variable response in both AT and normal cell strains. In some strains, including those shown to be hypersensitive to the drug by other workers, the inhibition of DNA synthesis was more pronounced than in other cell strains, but no significant difference between AT and normal cells could be detected. The rejoining of DNA strand breaks induced by X-rays, measured by DNA elution techniques, occurred within l2 hr after treatment and could not be correlated with the difference in DNA synthesis inhibition in AT and normal cells. After low doses of X-rays, AT cells rejoined single-strand breaks slightly more slowly than did normal cells. The rate of DNA replication in X-irradiation AT and normal cells was not affected by nicotinamide, an inhibitor of poly(adenosine diphosphate ribose) synthesis. These data indicate that the diminished inhibition of DNA replication in carcinogen-treated AT cells (a) is a general characteristic of all AT cell strains, (b) correlates with AT cellular hypersensitivity, (c) is not directly caused by the bulk of the DNA strand breaks produced by carcinogenic agents, and (d) is not based on differences in the induction of poly(adenosine diphosphate ribose) synthesis between X-irradiated AT and normal cells.

  19. The structure and function of replication protein A in DNA replication.

    PubMed

    Prakash, Aishwarya; Borgstahl, Gloria E O

    2012-01-01

    In all organisms from bacteria and archaea to eukarya, single-stranded DNA binding proteins play an essential role in most, if not all, nuclear metabolism involving single-stranded DNA (ssDNA). Replication protein A (RPA), the major eukaryotic ssDNA binding protein, has two important roles in DNA metabolism: (1) in binding ssDNA to protect it and to keep it unfolded, and (2) in coordinating the assembly and disassembly of numerous proteins and protein complexes during processes such as DNA replication. Since its discovery as a vital player in the process of replication, RPAs roles in recombination and DNA repair quickly became evident. This chapter summarizes the current understanding of RPA's roles in replication by reviewing the available structural data, DNA-binding properties, interactions with various replication proteins, and interactions with DNA repair proteins when DNA replication is stalled.

  20. Replication Checkpoint: Tuning and Coordination of Replication Forks in S Phase

    PubMed Central

    Hustedt, Nicole; Gasser, Susan M.; Shimada, Kenji

    2013-01-01

    Checkpoints monitor critical cell cycle events such as chromosome duplication and segregation. They are highly conserved mechanisms that prevent progression into the next phase of the cell cycle when cells are unable to accomplish the previous event properly. During S phase, cells also provide a surveillance mechanism called the DNA replication checkpoint, which consists of a conserved kinase cascade that is provoked by insults that block or slow down replication forks. The DNA replication checkpoint is crucial for maintaining genome stability, because replication forks become vulnerable to collapse when they encounter obstacles such as nucleotide adducts, nicks, RNA-DNA hybrids, or stable protein-DNA complexes. These can be exogenously induced or can arise from endogenous cellular activity. Here, we summarize the initiation and transduction of the replication checkpoint as well as its targets, which coordinate cell cycle events and DNA replication fork stability. PMID:24705211

  1. [The effects of TorR protein on initiation of DNA replication in Escherichia coli].

    PubMed

    Yuan, Yao; Jiaxin, Qiao; Jing, Li; Hui, Li; Morigen, Morigen

    2015-03-01

    The two-component systems, which could sense and respond to environmental changes, widely exist in bacteria as a signal transduction pathway. The bacterial CckA/CtrA, ArcA/ArcB and PhoP/PhoQ two-component systems are associated with initiation of DNA replication and cell division, however, the effects of the TorS/TorR system on cell cycle and DNA replication remains unknown. The TorS/TorR system in Escherichia coli can sense changes in trimethylamine oxide (TMAO) concentration around the cells. However, it is unknown if it also affects initiation of DNA replication. We detected DNA replication patterns in ΔtorS and ΔtorR mutant strains by flow cytometry. We found that the average number of replication origins (oriCs) per cell and doubling time in ΔtorS mutants were the same while the average number of oriCs in ΔtorR mutants was increased compared with that in wild-type cells. These results indicated that absence of TorR led to an earlier initiation of DNA replication than that in wild-type cells. Strangely, neither overexpression of TorR nor co-expression of TorR and TorS could restore ΔtorR mutant phenotype to the wild type. However, overexpression of SufD in both wild type and ΔtorR mutants promoted initiation of DNA replication, while mutation of SufD delayed it in ΔtorR mutants. Thus, TorR may affect initiation of DNA replication indirectly through regulating gene expression of sufD.

  2. Host cell variations resulting from F plasmid-controlled replication of the Escherichia coli chromosome.

    PubMed Central

    Tresguerres, E F; Nieto, C; Casquero, I; Cánovas, J L

    1986-01-01

    Cell size and DNA concentration were measured in Escherichia coli K-12 ET64. This strain carries a dnaA (Ts) mutation that has been suppressed by the insertion of the F plasmid into the chromosome. ET64 can grow in a balanced steady state of exponential growth at the restrictive temperature for its dnaA allele (39 degrees C), in which chromosome replication is controlled by the F plasmid, and at the permissive temperature (30 degrees C), in which chromosome replication is controlled by dnaA-oriC. When cells grown at the indicated temperatures were compared, it was observed that at 39 degrees C, the cell mass increased and the amount of cellular DNA decreased slightly; therefore, the DNA concentration was strongly reduced. These changes can neither be explained by the reduction of the generation time (which is only 10-15%) nor from observed changes in the replication time and in the time between DNA synthesis termination and cell division. Variations were mainly due to the increase in cell mass per origin of replication, at initiation, in cells grown at 39 degrees C. Control of chromosome replication by the F plasmid appears to be the reason for the increase in the initiation mass. Other possible causes, such as the modification of growth temperature, the generation time, or both, were discarded. These observations suggest that at one growth rate, the F plasmid replicates at a particular cell mass to F particle number ratio, and that this ratio is higher than the cell mass to oriC ratio at the initiation of chromosome replication. This fact might be significant to coordinate the replication of two different replicons in the same cell. PMID:3511032

  3. Combinatorial selection for replicable RNA by Qβ replicase while maintaining encoded gene function.

    PubMed

    Yumura, Mio; Yamamoto, Natsuko; Yokoyama, Katsushi; Mori, Hirotada; Yomo, Tetsuya; Ichihashi, Norikazu

    2017-01-01

    Construction of a complex artificial self-replication system is challenging in the field of in vitro synthetic biology. Recently, we developed a translation-coupled RNA replication system, wherein an artificial genomic RNA replicates with the Qβ RNA replicase gene encoded on itself. The challenge is to introduce additional genes into the RNA to develop a complex system that mimics natural living systems. However, most RNA sequence encoding genes are not replicable by the Qβ replicase owing to its requirement for strong secondary structures throughout the RNA sequence that are absent in most genes. In this study, we establish a new combinatorial selection method to find an RNA sequence with secondary structures and functional amino acid sequences of the encoded gene. We selected RNA sequences based on their in vitro replication and in vivo gene functions. First, we used the α-domain gene of β-galactosidase as a model-encoding gene, with functional selection based on blue-white screening. Through the combinatorial selection, we developed more replicable RNAs while maintaining the function of the encoded α-domain. The selected sequence improved the affinity between the minus strand RNA and Qβ replicase. Second, we established an in vivo selection method applicable to a broader range of genes by using an Escherichia coli strain with one of the essential genes complemented with a plasmid. We performed the combinatorial selection using an RNA encoding serS and obtained more replicable RNA encoding functional serS gene. These results suggest that combinatorial selection methods are useful for the development of RNA sequences replicable by Qβ replicase while maintaining the encoded gene function.

  4. Combinatorial selection for replicable RNA by Qβ replicase while maintaining encoded gene function

    PubMed Central

    Yumura, Mio; Yamamoto, Natsuko; Yokoyama, Katsushi; Mori, Hirotada; Yomo, Tetsuya

    2017-01-01

    Construction of a complex artificial self-replication system is challenging in the field of in vitro synthetic biology. Recently, we developed a translation-coupled RNA replication system, wherein an artificial genomic RNA replicates with the Qβ RNA replicase gene encoded on itself. The challenge is to introduce additional genes into the RNA to develop a complex system that mimics natural living systems. However, most RNA sequence encoding genes are not replicable by the Qβ replicase owing to its requirement for strong secondary structures throughout the RNA sequence that are absent in most genes. In this study, we establish a new combinatorial selection method to find an RNA sequence with secondary structures and functional amino acid sequences of the encoded gene. We selected RNA sequences based on their in vitro replication and in vivo gene functions. First, we used the α-domain gene of β-galactosidase as a model-encoding gene, with functional selection based on blue-white screening. Through the combinatorial selection, we developed more replicable RNAs while maintaining the function of the encoded α-domain. The selected sequence improved the affinity between the minus strand RNA and Qβ replicase. Second, we established an in vivo selection method applicable to a broader range of genes by using an Escherichia coli strain with one of the essential genes complemented with a plasmid. We performed the combinatorial selection using an RNA encoding serS and obtained more replicable RNA encoding functional serS gene. These results suggest that combinatorial selection methods are useful for the development of RNA sequences replicable by Qβ replicase while maintaining the encoded gene function. PMID:28328998

  5. Curcumin inhibits Rift Valley fever virus replication in human cells.

    PubMed

    Narayanan, Aarthi; Kehn-Hall, Kylene; Senina, Svetlana; Lundberg, Lindsay; Van Duyne, Rachel; Guendel, Irene; Das, Ravi; Baer, Alan; Bethel, Laura; Turell, Michael; Hartman, Amy Lynn; Das, Bhaskar; Bailey, Charles; Kashanchi, Fatah

    2012-09-28

    Rift Valley fever virus (RVFV) is an arbovirus that is classified as a select agent, an emerging infectious virus, and an agricultural pathogen. Understanding RVFV-host interactions is imperative to the design of novel therapeutics. Here, we report that an infection by the MP-12 strain of RVFV induces phosphorylation of the p65 component of the NFκB cascade. We demonstrate that phosphorylation of p65 (serine 536) involves phosphorylation of IκBα and occurs through the classical NFκB cascade. A unique, low molecular weight complex of the IKK-β subunit can be observed in MP-12-infected cells, which we have labeled IKK-β2. The IKK-β2 complex retains kinase activity and phosphorylates an IκBα substrate. Inhibition of the IKK complex using inhibitors impairs viral replication, thus alluding to the requirement of an active IKK complex to the viral life cycle. Curcumin strongly down-regulates levels of extracellular infectious virus. Our data demonstrated that curcumin binds to and inhibits kinase activity of the IKK-β2 complex in infected cells. Curcumin partially exerts its inhibitory influence on RVFV replication by interfering with IKK-β2-mediated phosphorylation of the viral protein NSs and by altering the cell cycle of treated cells. Curcumin also demonstrated efficacy against ZH501, the fully virulent version of RVFV. Curcumin treatment down-regulated viral replication in the liver of infected animals. Our data point to the possibility that RVFV infection may result in the generation of novel versions of host components (such as IKK-β2) that, by virtue of altered protein interaction and function, qualify as unique therapeutic targets.

  6. Curcumin Inhibits Rift Valley Fever Virus Replication in Human Cells*

    PubMed Central

    Narayanan, Aarthi; Kehn-Hall, Kylene; Senina, Svetlana; Lundberg, Lindsay; Van Duyne, Rachel; Guendel, Irene; Das, Ravi; Baer, Alan; Bethel, Laura; Turell, Michael; Hartman, Amy Lynn; Das, Bhaskar; Bailey, Charles; Kashanchi, Fatah

    2012-01-01

    Rift Valley fever virus (RVFV) is an arbovirus that is classified as a select agent, an emerging infectious virus, and an agricultural pathogen. Understanding RVFV-host interactions is imperative to the design of novel therapeutics. Here, we report that an infection by the MP-12 strain of RVFV induces phosphorylation of the p65 component of the NFκB cascade. We demonstrate that phosphorylation of p65 (serine 536) involves phosphorylation of IκBα and occurs through the classical NFκB cascade. A unique, low molecular weight complex of the IKK-β subunit can be observed in MP-12-infected cells, which we have labeled IKK-β2. The IKK-β2 complex retains kinase activity and phosphorylates an IκBα substrate. Inhibition of the IKK complex using inhibitors impairs viral replication, thus alluding to the requirement of an active IKK complex to the viral life cycle. Curcumin strongly down-regulates levels of extracellular infectious virus. Our data demonstrated that curcumin binds to and inhibits kinase activity of the IKK-β2 complex in infected cells. Curcumin partially exerts its inhibitory influence on RVFV replication by interfering with IKK-β2-mediated phosphorylation of the viral protein NSs and by altering the cell cycle of treated cells. Curcumin also demonstrated efficacy against ZH501, the fully virulent version of RVFV. Curcumin treatment down-regulated viral replication in the liver of infected animals. Our data point to the possibility that RVFV infection may result in the generation of novel versions of host components (such as IKK-β2) that, by virtue of altered protein interaction and function, qualify as unique therapeutic targets. PMID:22847000

  7. Bridging from Replication to Translation with a Thermal, Autonomous Replicator Made from Transfer RNA

    NASA Astrophysics Data System (ADS)

    Braun, Dieter; Möller, Friederike M.; Krammer, Hubert

    2013-03-01

    Central to the understanding of living systems is the interplay between DNA/RNA and proteins. Known as Eigen paradox, proteins require genetic information while proteins are needed for the replication of genes. RNA world scenarios focus on a base by base replication disconnected from translation. Here we used strategies from DNA machines to demonstrate a tight connection between a basic replication mechanism and translation. A pool of hairpin molecules replicate a two-letter code. The replication is thermally driven: the energy and negative entropy to drive replication is initially stored in metastable hairpins by kinetic cooling. Both are released by a highly specific and exponential replication reaction that is solely implemented by base hybridization. The duplication time is 30s. The reaction is monitored by fluorescence and described by a detailed kinetic model. The RNA hairpins usetransfer RNA sequences and the replication is driven by the simple disequilibrium setting of a thermal gradient The experiments propose a physical rather than a chemical scenario for the autonomous replication of protein encoding information. Supported by the NanoSystems Initiative Munich and ERC.

  8. Inhibition of adenovirus replication in vitro by trifluridine.

    PubMed

    Lennette, D A; Eiferman, R A

    1978-09-01

    At present, there is no effective chemotherapeutic agent available for the treatment of adenoviral keratoconjunctivitis. Recent evidence suggests that trifluridine (3FT) may effectively inhibit the replication of some adenovirus serotypes known to cause keratoconjunctivitis. The ability of 3FT to inhibit two reference strains of adenoviruses, type 8 and type 19, was examined using cell cultures. Two second-passage isolates of adenoviruses, identified as serotype 13, were also tested. Compared with untreated, virusinfected cell cultures, drug-treated cell cultures developed a lesser degree of cytopathic effect following infection with all three serotypes. Virus production was reduced in the drug-treated cell cultures: approximately tenfold for type 8, more than 1,000-fold for type 19, and 5,000-fold for the type 13 isolates.

  9. Digoxin Suppresses HIV-1 Replication by Altering Viral RNA Processing

    PubMed Central

    Wong, Raymond W.; Balachandran, Ahalya; Ostrowski, Mario A.; Cochrane, Alan

    2013-01-01

    To develop new approaches to control HIV-1 replication, we examined the capacity of recently described small molecular modulators of RNA splicing for their effects on viral RNA metabolism. Of the drugs tested, digoxin was found to induce a dramatic inhibition of HIV-1 structural protein synthesis, a response due, in part, to reduced accumulation of the corresponding viral mRNAs. In addition, digoxin altered viral RNA splice site use, resulting in loss of the essential viral factor Rev. Digoxin induced changes in activity of the CLK family of SR protein kinases and modification of several SR proteins, including SRp20 and Tra2β, which could account for the effects observed. Consistent with this hypothesis, overexpression of SRp20 elicited changes in HIV-1 RNA processing similar to those observed with digoxin. Importantly, digoxin was also highly active against clinical strains of HIV-1 in vitro, validating this novel approach to treatment of this infection. PMID:23555254

  10. Species-barrier-independent prion replication in apparently resistant species

    NASA Astrophysics Data System (ADS)

    Hill, Andrew F.; Joiner, Susan; Linehan, Jackie; Desbruslais, Melanie; Lantos, Peter L.; Collinge, John

    2000-08-01

    Transmission of prions between mammalian species is thought to be limited by a "species barrier," which depends on differences in the primary structure of prion proteins in the infecting inoculum and the host. Here we demonstrate that a strain of hamster prions thought to be nonpathogenic for conventional mice leads to prion replication to high levels in such mice but without causing clinical disease. Prions pathogenic in both mice and hamsters are produced. These results demonstrate the existence of subclinical forms of prion infection with important public health implications, both with respect to iatrogenic transmission from apparently healthy humans and dietary exposure to cattle and other species exposed to bovine spongiform encephalopathy prions. Current definitions of the species barrier, which have been based on clinical end-points, need to be fundamentally reassessed.

  11. Undesigned selection for replication termination of bacterial chromosomes.

    PubMed

    Kono, Nobuaki; Arakawa, Kazuharu; Sato, Mitsuru; Yoshikawa, Hirofumi; Tomita, Masaru; Itaya, Mitsuhiro

    2014-08-12

    The oriC DNA replication origin in bacterial chromosomes, the location of which appears to be physically identified, is genetically regulated by relevant molecular machinery. In contrast, the location of the terminus remains obscure for many bacterial replicons, except for terC, the proposed and well-studied chromosome termination site in certain bacteria. The terC locus, which is composed of specific sequences for its binding protein, is located at a site opposite from oriC, exhibiting a symmetric structure around the oriC-terC axis. Here, we investigated Bacillus subtilis 168 strains whose axes were hindered and found that the native terC function was robust. However, eradication of terminus region specific binding protein resulted in the natural terC sites not being used for termination; instead, new termini were selected at a location exactly opposite to oriC. We concluded that replication generally terminates at the loci where the two approaching replisomes meet. This site was automatically selected, and two replisomes moving at the same rate supported symmetrical chromosome structures relative to oriC. The rule, which was even validated by artificial chromosomes irrespective of oriC, should be general for replicons administered by two replisomes. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

  12. Strain and sex based characterization of behavioral expressions in non-induced compulsive-like mice.

    PubMed

    Mitra, Swarup; Bastos, Cristiane P; Chesworth, Savanna; Frye, Cheryl; Bult-Ito, Abel

    2017-01-01

    There is currently a lack of understanding how genetic background and sex differences attribute to the heterogeneity of obsessive-compulsive disorder (OCD). An animal model of compulsive-like behaviors has been developed through bidirectional selection of house mice (Mus musculus) for high (big cotton nests; BIG mice) and low levels (small nests; SMALL mice) of nest-building behavior. The BIG male strains have predictive and face validity as a spontaneous animal model of OCD. Here, we evaluated compulsive-, anxiety-, cognitive-, and depression-like behaviors among male and proestrus female replicate strains each of BIG (BIG1, BIG2) and SMALL (SML1, SML2) nest-builders, and randomly-bred Controls (C1, C2). BIG1 and BIG2 males and females had higher nesting scores when compared to SMALL and Control strains. Male BIG1 and BIG2 strains showed more compulsive-like nesting than BIG1 and BIG2 proestrus females, which was not observed among the other strains. Nesting scores were also different between BIG replicate male strains. A similar pattern was observed in the compulsive-like marble burying behavior with BIG strains burying more marbles than SMALL and Control strains. Significant replicate and sex differences were also observed in marble burying among the BIG strains. The open field test revealed replicate effects while the BIG strains showed less anxiety-like behavior in the elevated plus maze test compared to the SMALL strains. For novel object recognition only the Control strains showed replicate and sex differences. In the depression-like forced swim test proestrus females demonstrated less depression-like behavior than males. BIG and SMALL nest-building strains had a higher corticosterone stress response than the Control strains. Together these results indicate a strong interplay of genetic background and sex in influencing expression of behaviors in our compulsive-like mouse model. These results are in congruence with the clinical heterogeneity of OCD.

  13. Lagomorphs (rabbits, pikas and hares) do not use telomere-directed replicative aging in vitro.

    PubMed

    Forsyth, Nicholas R; Elder, Frederick F B; Shay, Jerry W; Wright, Woodring E

    2005-01-01

    Telomere shortening is used for replicative aging in primates and ungulates but not rodents. We examined telomere biology in rabbits to expand the comparative biology of telomere-directed replicative senescence within mammals. The order Lagomorpha consists of two families; Leporidae and Ochotonidae. We examined telomere biology in species representing three leporid genera (European White Rabbit, Black-tailed Jack Rabbit, and Swamp Rabbit) and the monotypic ochotonid genus (North American Pika). Of the leporids one species was a laboratory strain and the others were wild caught. The leporids neither exhibited cellular senescence after sustained periods in culture nor displayed detectable telomerase activity. Continued culture was possible because of their extremely long telomeric arrays. Immunofluorescence showed robust telomere signals at chromosome ends and significant internal chromosomal staining in some instances. Pika was unique in displaying endogenous telomerase activity throughout time in culture. These results show that it is unlikely that lagomorphs use telomere shortening and replicative senescence as a tumor protective mechanism.

  14. Suppression of gross chromosomal rearrangements by a new alternative replication factor C complex

    SciTech Connect

    Banerjee, Soma; Sikdar, Nilabja; Myung, Kyungjae

    2007-10-26

    Defects in DNA replication fidelity lead to genomic instability. Gross chromosomal rearrangement (GCR), a type of genomic instability, is highly enhanced by various initial mutations affecting DNA replication. Frequent observations of GCRs in many cancers strongly argue the importance of maintaining high fidelity of DNA replication to suppress carcinogenesis. Recent genome wide screens in Saccharomyces cerevisiae identified a new GCR suppressor gene, ELG1, enhanced level of genome instability gene 1. Its physical interaction with proliferating cell nuclear antigen (PCNA) and complex formation with Rfc2-5p proteins suggest that Elg1 functions to load/unload PCNA onto DNA during a certain DNA metabolism. High level of DNA damage accumulation and enhanced phenotypes with mutations in genes involved in cell cycle checkpoints, homologous recombination (HR), or chromatin assembly in the elg1 strain suggest that Elg1p-Rfc2-5p functions in a fundamental DNA metabolism to suppress genomic instability.

  15. Small animal model systems for studying hepatitis B virus replication and pathogenesis.

    PubMed

    Dandri, Maura; Lutgehetmann, Marc; Volz, Tassilo; Petersen, Jörg

    2006-05-01

    The lack of reliable in vitro infection systems and convenient animal models has hindered progress in hepatitis B virus (HBV) research and the development of new treatment options. Due to various restraints encountered using chimpanzees and other models of HBV infection that are based on HBV-related viruses, and due to the necessity to work with small and well-characterized animal systems, it is not surprising that most recent developments have focused on mice. Various strains of transgenic mice harboring the entire HBV genome, or selected viral genes, were developed and proved to be very useful to elucidate mechanisms of HBV replication and pathogenesis. Furthermore, novel, exciting alternative mouse models have been developed recently, which allow not only studies on viral replication, but also the investigation of mechanisms of viral entry and clearance. This article provides an overview and discussion of the different mouse models of HBV replication and infection that are currently available.

  16. A new method for culturing Plasmodium falciparum shows replication at the highest erythrocyte densities

    NASA Technical Reports Server (NTRS)

    Li, Tao; Glushakova, Svetlana; Zimmerberg, Joshua

    2003-01-01

    Plasmodium falciparum replicates poorly in erythrocyte densities greater than a hematocrit of 20%. A new method to culture the major malaria parasite was developed by using a hollow fiber bioreactor that preserves healthy erythrocytes at hematocrit up to 100%. P. falciparum replicated equally well at all densities studied. This method proved advantageous for large-scale preparation of parasitized erythrocytes (and potentially immunogens thereof), because high yields ( approximately 10(10) in 4 days) could be prepared with less cost and labor. Concomitantly, secreted proteins were concentrated by molecular sieving during culture, perhaps contributing to the parasitemic limit of 8%-12% with the 3D7 strain. The finding that P. falciparum can replicate at packed erythrocyte densities suggests that this system may be useful for study of the pathogenesis of fatal cerebral malaria, of which one feature is densely packed blood cells in brain microvasculature.

  17. The Consequences of Replicating in the Wrong Orientation: Bacterial Chromosome Duplication without an Active Replication Origin.

    PubMed

    Dimude, Juachi U; Stockum, Anna; Midgley-Smith, Sarah L; Upton, Amy L; Foster, Helen A; Khan, Arshad; Saunders, Nigel J; Retkute, Renata; Rudolph, Christian J

    2015-11-03

    Chromosome replication is regulated in all organisms at the assembly stage of the replication machinery at specific origins. In Escherichia coli, the DnaA initiator protein regulates the assembly of replication forks at oriC. This regulation can be undermined by defects in nucleic acid metabolism. In cells lacking RNase HI, replication initiates independently of DnaA and oriC, presumably at persisting R-loops. A similar mechanism was assumed for origin-independent synthesis in cells lacking RecG. However, recently we suggested that this synthesis initiates at intermediates resulting from replication fork fusions. Here we present data suggesting that in cells lacking RecG or RNase HI, origin-independent synthesis arises by different mechanisms, indicative of these two proteins having different roles in vivo. Our data support the idea that RNase HI processes R-loops, while RecG is required to process replication fork fusion intermediates. However, regardless of how origin-independent synthesis is initiated, a fraction of forks will proceed in an orientation opposite to normal. We show that the resulting head-on encounters with transcription threaten cell viability, especially if taking place in highly transcribed areas. Thus, despite their different functions, RecG and RNase HI are both important factors for maintaining replication control and orientation. Their absence causes severe replication problems, highlighting the advantages of the normal chromosome arrangement, which exploits a single origin to control the number of forks and their orientation relative to transcription, and a defined termination area to contain fork fusions. Any changes to this arrangement endanger cell cycle control, chromosome dynamics, and, ultimately, cell viability. Cell division requires unwinding of millions of DNA base pairs to generate the template for RNA transcripts as well as chromosome replication. As both processes use the same template, frequent clashes are unavoidable. To

  18. DNA Copy Number Control Through Inhibition of Replication Fork Progression

    PubMed Central

    Nordman, Jared T.; Kozhevnikova, Elena N.; Verrijzer, C. Peter; Pindyurin, Alexey V.; Andreyeva, Evgeniya N.; Shloma, Victor V.; Zhimulev, Igor F.; Orr-Weaver, Terry L.

    2014-01-01

    Summary Proper control of DNA replication is essential to ensure faithful transmission of genetic material and to prevent chromosomal aberrations that can drive cancer progression and developmental disorders. DNA replication is regulated primarily at the level of initiation and is under strict cell cycle regulation. Importantly, DNA replication is highly influenced by developmental cues. In Drosophila, specific regions of the genome are repressed for DNA replication during differentiation by the SNF2 domain-containing protein SUUR through an unknown mechanism. We demonstrate that SUUR is recruited to active replication forks and mediates repression of DNA replication by directly inhibiting replication fork progression instead of functioning as a replication fork barrier. Mass-spec identification of SUUR associated proteins identified the replicative helicase member CDC45 as a SUUR-associated protein, supporting a role for SUUR directly at replication forks. Our results reveal that control of eukaryotic DNA copy number can occur through inhibition of replication fork progression. PMID:25437540

  19. Differentially active origins of DNA replication in tumor versus normal cells.

    PubMed

    Di Paola, Domenic; Price, Gerald B; Zannis-Hadjopoulos, Maria

    2006-05-15

    Previously, a degenerate 36 bp human consensus sequence was identified as a determinant of autonomous replication in eukaryotic cells. Random mutagenesis analyses further identified an internal 20 bp of the 36 bp consensus sequence as sufficient for acting as a core origin element. Here, we have located six versions of the 20 bp consensus sequence (20mer) on human chromosome 19q13 over a region spanning approximately 211 kb and tested them for ectopic and in situ replication activity by transient episomal replication assays and nascent DNA strand abundance analyses, respectively. The six versions of the 20mer alone were capable of supporting autonomous replication of their respective plasmids, unlike random genomic sequence of the same length. Furthermore, comparative analyses of the endogenous replication activity of these 20mers at their respective chromosomal sites, in five tumor/transformed and two normal cell lines, done by in situ chromosomal DNA replication assays, involving preparation of nascent DNA by the lambda exonuclease method and quantification by real-time PCR, showed that these sites coincided with chromosomal origins of DNA replication in all cell lines. Moreover, a 2- to 3-fold higher origin activity in the tumor/transformed cells by comparison to the normal cells was observed, suggesting a higher activation of these origins in tumor/transformed cell lines.

  20. The ATP requirements of adenovirus type 5 DNA replication and cellular DNA replication.

    PubMed

    De Jong, P J; Kwant, M M; van Driel, W; Jansz, H S; van der Vliet, P C

    1983-01-15

    Several in vitro DNA replication systems were employed to characterize the ATP dependency of adenovirus type 5 (Ad5) DNA replication. Ad5 DNA synthesis in isolated nuclei, representing the elongation of nascent DNA chains, was slightly ATP dependent. Reduction of the ATP concentration from the optimum (8 mM) to the endogenous value (0.16 microM) reduced Ad5 DNA replication only to 70%. No change in the pattern of replication was observed as indicated by the analysis of replicative intermediates using agarose gel electrophoresis. ATP could be replaced by dATP, but not by GTP or other nucleoside triphosphates. By contrast, cellular DNA replication in isolated nuclei from HeLa cells was reduced to 12% by the omission of ATP. These differences could not be explained by different ATP pools or by effects of ATP on dNTP pools. Cellular DNA replication in contrast to viral DNA replication was sensitive to low concentrations of adenosine 5'-O-(3-thiotriphosphate). Inhibition by this ATP analog was competitive with ATP (Ki = 0.4 mM). Adenovirus DNA replication by DNA-free nuclear extracts, representing initiation plus elongation (Challberg and Kelly, Proc. Nat. Acad. Sci. USA 76, 655-659, 1979), exhibited a nearly absolute requirement for ATP. ATP could be substituted not only by dATP, but also by GTP and dGTP and to a lesser extent by pyrimidine triphosphates. Similar results were found when the formation of a covalent complex between dCTP and the precursor terminal protein was studied. This reaction is essential for the initiation of Ad5 DNA replication. The results indicate that different ATP-requiring functions are employed during the initiation and elongation stages of adenovirus DNA replication.

  1. Stable DNA replication: interplay between DNA replication, homologous recombination, and transcription.

    PubMed Central

    Kogoma, T

    1997-01-01

    Chromosome replication in Escherichia coli is normally initiated at oriC, the origin of chromosome replication. E. coli cells possess at least three additional initiation systems for chromosome replication that are normally repressed but can be activated under certain specific conditions. These are termed the stable DNA replication systems. Inducible stable DNA replication (iSDR), which is activated by SOS induction, is proposed to be initiated from a D-loop, an early intermediate in homologous recombination. Thus, iSDR is a form of recombination-dependent DNA replication (RDR). Analysis of iSDR and RDR has led to the proposal that homologous recombination and double-strand break repair involve extensive semiconservative DNA replication. RDR is proposed to play crucial roles in homologous recombination, double-strand break repair, restoration of collapsed replication forks, and adaptive mutation. Constitutive stable DNA replication (cSDR) is activated in mhA mutants deficient in RNase HI or in recG mutants deficient in RecG helicase. cSDR is proposed to be initiated from an R-loop that can be formed by the invasion of duplex DNA by an RNA transcript, which most probably is catalyzed by RecA protein. The third form of SDR is nSDR, which can be transiently activated in wild-type cells when rapidly growing cells enter the stationary phase. This article describes the characteristics of these alternative DNA replication forms and reviews evidence that has led to the formulation of the proposed models for SDR initiation mechanisms. The possible interplay between DNA replication, homologous recombination, DNA repair, and transcription is explored. PMID:9184011

  2. Optical tweezers reveal how proteins alter replication

    NASA Astrophysics Data System (ADS)

    Chaurasiya, Kathy

    Single molecule force spectroscopy is a powerful method that explores the DNA interaction properties of proteins involved in a wide range of fundamental biological processes such as DNA replication, transcription, and repair. We use optical tweezers to capture and stretch a single DNA molecule in the presence of proteins that bind DNA and alter its mechanical properties. We quantitatively characterize the DNA binding mechanisms of proteins in order to provide a detailed understanding of their function. In this work, we focus on proteins involved in replication of Escherichia coli (E. coli ), endogenous eukaryotic retrotransposons Ty3 and LINE-1, and human immunodeficiency virus (HIV). DNA polymerases replicate the entire genome of the cell, and bind both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) during DNA replication. The replicative DNA polymerase in the widely-studied model system E. coli is the DNA polymerase III subunit alpha (DNA pol III alpha). We use optical tweezers to determine that UmuD, a protein that regulates bacterial mutagenesis through its interactions with DNA polymerases, specifically disrupts alpha binding to ssDNA. This suggests that UmuD removes alpha from its ssDNA template to allow DNA repair proteins access to the damaged DNA, and to facilitate exchange of the replicative polymerase for an error-prone translesion synthesis (TLS) polymerase that inserts nucleotides opposite the lesions, so that bacterial DNA replication may proceed. This work demonstrates a biophysical mechanism by which E. coli cells tolerate DNA damage. Retroviruses and retrotransposons reproduce by copying their RNA genome into the nuclear DNA of their eukaryotic hosts. Retroelements encode proteins called nucleic acid chaperones, which rearrange nucleic acid secondary structure and are therefore required for successful replication. The chaperone activity of these proteins requires strong binding affinity for both single- and double-stranded nucleic

  3. Methadone enhances human influenza A virus replication.

    PubMed

    Chen, Yun-Hsiang; Wu, Kuang-Lun; Tsai, Ming-Ta; Chien, Wei-Hsien; Chen, Mao-Liang; Wang, Yun

    2017-01-01

    Growing evidence has indicated that opioids enhance replication of human immunodeficiency virus and hepatitis C virus in target cells. However, it is unknown whether opioids can enhance replication of other clinically important viral pathogens. In this study, the interaction of opioid agonists and human influenza A/WSN/33 (H1N1) virus was examined in human lung epithelial A549 cells. Cells were exposed to morphine, methadone or buprenorphine followed by human H1N1 viral infection. Exposure to methadone differentially enhanced viral propagation, consistent with an increase in virus adsorption, susceptibility to virus infection and viral protein synthesis. In contrast, morphine or buprenorphine did not alter H1N1 replication. Because A549 cells do not express opioid receptors, methadone-enhanced H1N1 replication in human lung cells may not be mediated through these receptors. The interaction of methadone and H1N1 virus was also examined in adult mice. Treatment with methadone significantly increased H1N1 viral replication in lungs. Our data suggest that use of methadone facilitates influenza A viral infection in lungs and might raise concerns regarding the possible consequence of an increased risk of serious influenza A virus infection in people who receive treatment in methadone maintenance programs. © 2015 Society for the Study of Addiction.

  4. Self-replication with magnetic dipolar colloids.

    PubMed

    Dempster, Joshua M; Zhang, Rui; Olvera de la Cruz, Monica

    2015-10-01

    Colloidal self-replication represents an exciting research frontier in soft matter physics. Currently, all reported self-replication schemes involve coating colloidal particles with stimuli-responsive molecules to allow switchable interactions. In this paper, we introduce a scheme using ferromagnetic dipolar colloids and preprogrammed external magnetic fields to create an autonomous self-replication system. Interparticle dipole-dipole forces and periodically varying weak-strong magnetic fields cooperate to drive colloid monomers from the solute onto templates, bind them into replicas, and dissolve template complexes. We present three general design principles for autonomous linear replicators, derived from a focused study of a minimalist sphere-dimer magnetic system in which single binding sites allow formation of dimeric templates. We show via statistical models and computer simulations that our system exhibits nonlinear growth of templates and produces nearly exponential growth (low error rate) upon adding an optimized competing electrostatic potential. We devise experimental strategies for constructing the required magnetic colloids based on documented laboratory techniques. We also present qualitative ideas about building more complex self-replicating structures utilizing magnetic colloids.

  5. The evolutionary ecology of molecular replicators

    PubMed Central

    2016-01-01

    By reasonable criteria, life on the Earth consists mainly of molecular replicators. These include viruses, transposons, transpovirons, coviruses and many more, with continuous new discoveries like Sputnik Virophage. Their study is inherently multidisciplinary, spanning microbiology, genetics, immunology and evolutionary theory, and the current view is that taking a unified approach has great power and promise. We support this with a new, unified, model of their evolutionary ecology, using contemporary evolutionary theory coupling the Price equation with game theory, studying the consequences of the molecular replicators' promiscuous use of each others' gene products for their natural history and evolutionary ecology. Even at this simple expository level, we can make a firm prediction of a new class of replicators exploiting viruses such as lentiviruses like SIVs, a family which includes HIV: these have been explicitly stated in the primary literature to be non-existent. Closely connected to this departure is the view that multicellular organism immunology is more about the management of chronic infections rather than the elimination of acute ones and new understandings emerging are changing our view of the kind of theatre we ourselves provide for the evolutionary play of molecular replicators. This study adds molecular replicators to bacteria in the emerging field of sociomicrobiology. PMID:27853598

  6. Self-replication with magnetic dipolar colloids

    NASA Astrophysics Data System (ADS)

    Dempster, Joshua M.; Zhang, Rui; Olvera de la Cruz, Monica

    2015-10-01

    Colloidal self-replication represents an exciting research frontier in soft matter physics. Currently, all reported self-replication schemes involve coating colloidal particles with stimuli-responsive molecules to allow switchable interactions. In this paper, we introduce a scheme using ferromagnetic dipolar colloids and preprogrammed external magnetic fields to create an autonomous self-replication system. Interparticle dipole-dipole forces and periodically varying weak-strong magnetic fields cooperate to drive colloid monomers from the solute onto templates, bind them into replicas, and dissolve template complexes. We present three general design principles for autonomous linear replicators, derived from a focused study of a minimalist sphere-dimer magnetic system in which single binding sites allow formation of dimeric templates. We show via statistical models and computer simulations that our system exhibits nonlinear growth of templates and produces nearly exponential growth (low error rate) upon adding an optimized competing electrostatic potential. We devise experimental strategies for constructing the required magnetic colloids based on documented laboratory techniques. We also present qualitative ideas about building more complex self-replicating structures utilizing magnetic colloids.

  7. The evolutionary ecology of molecular replicators.

    PubMed

    Nee, Sean

    2016-08-01

    By reasonable criteria, life on the Earth consists mainly of molecular replicators. These include viruses, transposons, transpovirons, coviruses and many more, with continuous new discoveries like Sputnik Virophage. Their study is inherently multidisciplinary, spanning microbiology, genetics, immunology and evolutionary theory, and the current view is that taking a unified approach has great power and promise. We support this with a new, unified, model of their evolutionary ecology, using contemporary evolutionary theory coupling the Price equation with game theory, studying the consequences of the molecular replicators' promiscuous use of each others' gene products for their natural history and evolutionary ecology. Even at this simple expository level, we can make a firm prediction of a new class of replicators exploiting viruses such as lentiviruses like SIVs, a family which includes HIV: these have been explicitly stated in the primary literature to be non-existent. Closely connected to this departure is the view that multicellular organism immunology is more about the management of chronic infections rather than the elimination of acute ones and new understandings emerging are changing our view of the kind of theatre we ourselves provide for the evolutionary play of molecular replicators. This study adds molecular replicators to bacteria in the emerging field of sociomicrobiology.

  8. Autophagy Negatively Regulates Transmissible Gastroenteritis Virus Replication.

    PubMed

    Guo, Longjun; Yu, Haidong; Gu, Weihong; Luo, Xiaolei; Li, Ren; Zhang, Jian; Xu, Yunfei; Yang, Lijun; Shen, Nan; Feng, Li; Wang, Yue

    2016-03-31

    Autophagy is an evolutionarily ancient pathway that has been shown to be important in the innate immune defense against several viruses. However, little is known about the regulatory role of autophagy in transmissible gastroenteritis virus (TGEV) replication. In this study, we found that TGEV infection increased the number of autophagosome-like double- and single-membrane vesicles in the cytoplasm of host cells, a phenomenon that is known to be related to autophagy. In addition, virus replication was required for the increased amount of the autophagosome marker protein LC3-II. Autophagic flux occurred in TGEV-infected cells, suggesting that TGEV infection triggered a complete autophagic response. When autophagy was pharmacologically inhibited by wortmannin or LY294002, TGEV replication increased. The increase in virus yield via autophagy inhibition was further confirmed by the use of siRNA duplexes, through which three proteins required for autophagy were depleted. Furthermore, TGEV replication was inhibited when autophagy was activated by rapamycin. The antiviral response of autophagy was confirmed by using siRNA to reduce the expression of gene p300, which otherwise inhibits autophagy. Together, the results indicate that TGEV infection activates autophagy and that autophagy then inhibits further TGEV replication.

  9. Calsyntenin-1 mediates hepatitis C virus replication.

    PubMed

    Awan, Zunaira; Tay, Enoch S E; Eyre, Nicholas S; Wu, Lindsay E; Beard, Michael R; Boo, Irene; Drummer, Heidi E; George, Jacob; Douglas, Mark W

    2016-08-01

    The hepatitis C virus (HCV) RNA genome of 9.6 kb encodes only 10 proteins, and so is highly dependent on host hepatocyte factors to facilitate replication. We aimed to identify host factors involved in the egress of viral particles. By screening the supernatant of HCV-infected Huh7 cells using SILAC-based proteomics, we identified the transmembrane protein calsyntenin-1 as a factor specifically secreted by infected cells. Calsyntenin-1 has previously been shown to mediate transport of endosomes along microtubules in neurons, through interactions with kinesin light chain-1. Here we demonstrate for the first time, we believe, a similar role for calsyntenin-1 in Huh7 cells, mediating intracellular transport of endosomes. In HCV-infected cells we show that calsyntenin-1 contributes to the early stages of the viral replication cycle and the formation of the replication complex. Importantly, we demonstrate in our model that silencing calsyntenin-1 disrupts the viral replication cycle, confirming the reliance of HCV on this protein as a host factor. Characterizing the function of calsyntenin-1 will increase our understanding of the HCV replication cycle and pathogenesis, with potential application to other viruses sharing common pathways.

  10. COPI Is Required for Enterovirus 71 Replication

    PubMed Central

    Wang, Jianmin; Wu, Zhiqiang; Jin, Qi

    2012-01-01

    Enterovirus 71 (EV71), a member of the Picornaviridae family, is found in Asian countries where it causes a wide range of human diseases. No effective therapy is available for the treatment of these infections. Picornaviruses undergo RNA replication in association with membranes of infected cells. COPI and COPII have been shown to be involved in the formation of picornavirus-induced vesicles. Replication of several picornaviruses, including poliovirus and Echovirus 11 (EV11), is dependent on COPI or COPII. Here, we report that COPI, but not COPII, is required for EV71 replication. Replication of EV71 was inhibited by brefeldin A and golgicide A, inhibitors of COPI activity. Furthermore, we found EV71 2C protein interacted with COPI subunits by co-immunoprecipitation and GST pull-down assay, indicating that COPI coatomer might be directed to the viral replication complex through viral 2C protein. Additionally, because the pathway is conserved among different species of enteroviruses, it may represent a novel target for antiviral therapies. PMID:22662263

  11. A Self-Replicating Ligase Ribozyme

    NASA Technical Reports Server (NTRS)

    Paul, Natasha; Joyce, Gerald F.

    2002-01-01

    A self-replicating molecule directs the covalent assembly of component molecules to form a product that is of identical composition to the parent. When the newly formed product also is able to direct the assembly of product molecules, the self-replicating system can be termed autocatalytic. A self-replicating system was developed based on a ribozyme that catalyzes the assembly of additional copies of Itself through an RNA-catalyzed RNA ligation reaction. The R3C ligase ribozyme was redesigned so that it would ligate two substrates to generate an exact copy of itself, which then would behave in a similar manner. This self-replicating system depends on the catalytic nature of the RNA for the generation of copies. A linear dependence was observed between the initial rate of formation of new copies and the starting concentration of ribozyme, consistent with exponential growth. The autocatalytic rate constant was 0.011 per min, whereas the initial rate of reaction in the absence of pre-existing ribozyme was only 3.3 x 10(exp -11) M per min. Exponential growth was limited, however, because newly formed ribozyme molecules had greater difficulty forming a productive complex with the two substrates. Further optimization of the system may lead to the sustained exponential growth of ribozymes that undergo self-replication.

  12. Transcription and replication of mitochondrial DNA.

    PubMed

    Clayton, D A

    2000-07-01

    The physical isolation of mammalian mitochondrial DNA (mtDNA) over 30 years ago marked the beginning of studies of its structure, replication and the expression of its genetic content. Such analyses have revealed a number of surprises: novel DNA structural features of the circular genome such as the displacement loop (D-loop); multiple sized and deleted forms of the circular genome; a minimal set of mitochondrially encoded rRNAs and tRNAs needed for translation; a bacteriophage-like, nuclear-encoded mitochondrial RNA polymerase for transcription; and a direct linkage between transcription and the commitment to replication of the leading mtDNA strand that centres on the nuclear encoded mitochondrial transcription factor A. One of the more recent revelations is the existence, near the D-loop, of an atypical, stable RNA-DNA hybrid (or R-loop) at the origin of mammalian leading-strand DNA replication, composed of the parent DNA strands and an RNA transcript. In mammalian mitochondrial systems, all of the proteins known to be involved in DNA replication are encoded in the nucleus. Thus alterations and deficiencies in mtDNA replication must arise from mutations in mtDNA regulatory sequences and nuclear gene defects. Further studies of the relationships between nuclear-encoded proteins and their mtDNA target sequences could result in strategies to manipulate genotypes within cellular mtDNA populations.

  13. A Self-Replicating Ligase Ribozyme

    NASA Technical Reports Server (NTRS)

    Paul, Natasha; Joyce, Gerald F.

    2002-01-01

    A self-replicating molecule directs the covalent assembly of component molecules to form a product that is of identical composition to the parent. When the newly formed product also is able to direct the assembly of product molecules, the self-replicating system can be termed autocatalytic. A self-replicating system was developed based on a ribozyme that catalyzes the assembly of additional copies of Itself through an RNA-catalyzed RNA ligation reaction. The R3C ligase ribozyme was redesigned so that it would ligate two substrates to generate an exact copy of itself, which then would behave in a similar manner. This self-replicating system depends on the catalytic nature of the RNA for the generation of copies. A linear dependence was observed between the initial rate of formation of new copies and the starting concentration of ribozyme, consistent with exponential growth. The autocatalytic rate constant was 0.011 per min, whereas the initial rate of reaction in the absence of pre-existing ribozyme was only 3.3 x 10(exp -11) M per min. Exponential growth was limited, however, because newly formed ribozyme molecules had greater difficulty forming a productive complex with the two substrates. Further optimization of the system may lead to the sustained exponential growth of ribozymes that undergo self-replication.

  14. Spacetime replication of continuous variable quantum information

    NASA Astrophysics Data System (ADS)

    Hayden, Patrick; Nezami, Sepehr; Salton, Grant; Sanders, Barry C.

    2016-08-01

    The theory of relativity requires that no information travel faster than light, whereas the unitarity of quantum mechanics ensures that quantum information cannot be cloned. These conditions provide the basic constraints that appear in information replication tasks, which formalize aspects of the behavior of information in relativistic quantum mechanics. In this article, we provide continuous variable (CV) strategies for spacetime quantum information replication that are directly amenable to optical or mechanical implementation. We use a new class of homologically constructed CV quantum error correcting codes to provide efficient solutions for the general case of information replication. As compared to schemes encoding qubits, our CV solution requires half as many shares per encoded system. We also provide an optimized five-mode strategy for replicating quantum information in a particular configuration of four spacetime regions designed not to be reducible to previously performed experiments. For this optimized strategy, we provide detailed encoding and decoding procedures using standard optical apparatus and calculate the recovery fidelity when finite squeezing is used. As such we provide a scheme for experimentally realizing quantum information replication using quantum optics.

  15. p38 (MAPK) stress signalling in replicative senescence in fibroblasts from progeroid and genomic instability syndromes.

    PubMed

    Tivey, Hannah S E; Brook, Amy J C; Rokicki, Michal J; Kipling, David; Davis, Terence

    2013-02-01

    Werner Syndrome (WS) is a human segmental progeria resulting from mutations in a DNA helicase. WS fibroblasts have a shortened replicative capacity, an aged appearance, and activated p38 MAPK, features that can be modulated by inhibition of the p38 pathway. Loss of the WRNp RecQ helicase has been shown to result in replicative stress, suggesting that a link between faulty DNA repair and stress-induced premature cellular senescence may lead to premature ageing in WS. Other progeroid syndromes that share overlapping pathophysiological features with WS also show defects in DNA processing, raising the possibility that faulty DNA repair, leading to replicative stress and premature cellular senescence, might be a more widespread feature of premature ageing syndromes. We therefore analysed replicative capacity, cellular morphology and p38 activation, and the effects of p38 inhibition, in fibroblasts from a range of progeroid syndromes. In general, populations of young fibroblasts from non-WS progeroid syndromes do not have a high level of cells with an enlarged morphology and F-actin stress fibres, unlike young WS cells, although this varies between strains. p38 activation and phosphorylated HSP27 levels generally correlate well with cellular morphology, and treatment with the p38 inhibitor SB203580 effects cellular morphology only in strains with enlarged cells and phosphorylated HSP27. For some syndromes fibroblast replicative capacity was within the normal range, whereas for others it was significantly shorter (e.g. HGPS and DKC). However, although in most cases SB203580 extended replicative capacity, with the exception of WS and DKC the magnitude of the effect was not significantly different from normal dermal fibroblasts. This suggests that stress-induced premature cellular senescence via p38 activation is restricted to a small subset of progeroid syndromes.

  16. Replication, Neurotropism, and Pathogenicity of Avian Paramyxovirus Serotypes 1–9 in Chickens and Ducks

    PubMed Central

    Kim, Shin-Hee; Xiao, Sa; Shive, Heather; Collins, Peter L.; Samal, Siba K.

    2012-01-01

    Avian paramyxovirus (APMV) serotypes 1–9 have been isolated from many different avian species. APMV-1 (Newcastle disease virus) is the only well-characterized serotype, because of the high morbidity, mortality, and economic loss caused by highly virulent strains. Very little is known about the pathogenesis, replication, virulence, and tropism of the other APMV serotypes. Here, this was evaluated for prototypes strains of APMV serotypes 2–9 in cell culture and in chickens and ducks. In cell culture, only APMV-1, -3 and -5 induced syncytium formation. In chicken DF1 cells, APMV-3 replicated with an efficiency approaching that of APMV-1, while APMV-2 and -5 replicated to lower, intermediate titers and the others were much lower. Mean death time (MDT) assay in chicken eggs and intracerebral pathogenicity index (ICPI) test in 1-day-old SPF chicks demonstrated that APMV types 2–9 were avirulent. Evaluation of replication in primary neuronal cells in vitro as well as in the brains of 1-day-old chicks showed that, among types 2–9, only APMV-3 was neurotropic, although this virus was not neurovirulent. Following intranasal infection of 1-day-old and 2-week-old chickens, replication of APMV types 2–9 was mostly restricted to the respiratory tract, although APMV-3 was neuroinvasive and neurotropic (but not neurovirulent) and also was found in the spleen. Experimental intranasal infection of 3-week-old mallard ducks with the APMVs did not produce any clinical signs (even for APMV-1) and exhibited restricted viral replication of the APMVs (including APMV-1) to the upper respiratory tract regardless of their isolation source, indicating avirulence of APMV types 1–9 in mallard ducks. The link between the presence of a furin cleavage site in the F protein, syncytium formation, systemic spread, and virulence that has been well-established with APMV-1 pathotypes was not evident with the other APMV serotypes. PMID:22558104

  17. High temperature strain gage apparent strain compensation

    NASA Technical Reports Server (NTRS)

    Holmes, Harlan K.; Moore, T. C., Sr.

    1992-01-01

    Once an installed strain gage is connected to a strain indicating device and the instrument is balanced, a subsequent change in temperature of the gage installation will generally produce a resistance change in the gage. This purely temperature-induced resistance will be registered by the indicating device as a strain and is referred to as 'apparent strain' to distinguish it from strain due to applied stress. One desirable technique for apparent strain compensation is to employ two identical gages with identical mounting procedures which are connected with a 'half bridge' configuration where gages see the same thermal environment but only one experiences a mechanical strain input. Their connection in adjacent arms of the bridge will then balance the thermally induced apparent strains and, in principle, only the mechanical strain remains. Two approaches that implement this technique are discussed.

  18. Replication of Human Noroviruses in Stem Cell-Derived Human Enteroids

    PubMed Central

    Ettayebi, Khalil; Crawford, Sue E.; Murakami, Kosuke; Broughman, James R.; Karandikar, Umesh; Tenge, Victoria R.; Neill, Frederick H.; Blutt, Sarah E.; Zeng, Xi-Lei; Qu, Lin; Kou, Baijun; Opekun, Antone R.; Burrin, Douglas; Graham, David Y.; Ramani, Sasirekha; Atmar, Robert L.

    2016-01-01

    The major barrier to research and development of effective interventions for human noroviruses (HuNoVs) has been the lack of a robust and reproducible in vitro cultivation system. HuNoVs are the leading cause of gastroenteritis worldwide. We report successful cultivation of multiple HuNoV strains in enterocytes in stem cell-derived, nontransformed human intestinal enteroid monolayer cultures. Bile, a critical factor of the intestinal milieu, is required for strain-dependent HuNoV replication. Lack of appropriate histoblood group antigen expression in intestinal cells restricts virus replication, and infectivity is abrogated by inactivation (e.g., irradiation, heating) and serum neutralization. This culture system recapitulates the human intestinal epithelium, permits human host-pathogen studies of previously noncultivatable pathogens, and allows the assessment of methods to prevent and treat HuNoV infections. PMID:27562956

  19. Pertussis toxin B-oligomer inhibits HIV infection and replication in hu-PBL-SCID mice.

    PubMed

    Lapenta, Caterina; Spada, Massimo; Santini, Stefano M; Racca, Sara; Dorigatti, Fernanda; Poli, Guido; Belardelli, Filippo; Alfano, Massimo

    2005-04-01

    Bordetella pertussis toxin B-oligomer (PTX-B) has been shown to inhibit HIV infection and replication in vitro. The potential anti-viral effect of PTX-B was tested here in an in vivo surrogate model of HIV infection, i.e. SCID mice reconstituted with human peripheral blood leukocytes (PBL) (hu-PBL-SCID) and infected with a CCR5-dependent (R5) HIV-1 strain. SCID mice inoculated intra-peritoneal (i.p.) with PTX-B and then infected with the R5 strain SF-162 were sacrificed 7 days later and analyzed for human PBL (hu-PBL) lymphoid tissue reconstitution, infection of hu-PBL, plasma viremia and viral rescue from ex vivo-cultivated i.p. hu-PBL. Unlike mice treated with 500 ng per animal of PTX-B showing no evidence of viral inhibition, daily administration of PTX-B (50 ng per mouse) strongly inhibited virus infection and replication, as determined by undetectable viremia, absence of infected hu-PBL and lack of rescue of infectious HIV in most animals. Furthermore, PTX-B injection 2 h before and twice after infection prevented HIV-1 infection and replication in all (10/10) tested animals. Thus, PTX-B potently inhibited virus infection and replication in hu-PBL-SCID mice, supporting the hypothesis that it may represent a new pharmacological agent against HIV-1 infection.

  20. The V protein of canine distemper virus is required for virus replication in human epithelial cells.

    PubMed

    Otsuki, Noriyuki; Nakatsu, Yuichiro; Kubota, Toru; Sekizuka, Tsuyoshi; Seki, Fumio; Sakai, Kouji; Kuroda, Makoto; Yamaguchi, Ryoji; Takeda, Makoto

    2013-01-01

    Canine distemper virus (CDV) becomes able to use human receptors through a single amino acid substitution in the H protein. In addition, CDV strains possessing an intact C protein replicate well in human epithelial H358 cells. The present study showed that CDV strain 007Lm, which was isolated from lymph node tissue of a dog with distemper, failed to replicate in H358 cells, although it possessed an intact C protein. Sequence analyses suggested that a cysteine-to-tyrosine substitution at position 267 of the V protein caused this growth defect. Analyses using H358 cells constitutively expressing the CDV V protein showed that the V protein with a cysteine, but not that with a tyrosine, at this position effectively blocked the interferon-stimulated signal transduction pathway, and supported virus replication of 007Lm in H358 cells. Thus, the V protein as well as the C protein appears to be functional and essential for CDV replication in human epithelial cells.

  1. Replication of poliovirus RNA and subgenomic RNA transcripts in transfected cells.

    PubMed Central

    Collis, P S; O'Donnell, B J; Barton, D J; Rogers, J A; Flanegan, J B

    1992-01-01

    Full-length and subgenomic poliovirus RNAs were transcribed in vitro and transfected into HeLa cells to study viral RNA replication in vivo. RNAs with deletion mutations were analyzed for the ability to replicate in either the absence or the presence of helper RNA by using a cotransfection procedure and Northern (RNA) blot analysis. An advantage of this approach was that viral RNA replication and genetic complementation could be characterized without first isolating conditional-lethal mutants. A subgenomic RNA with a large in-frame deletion in the capsid coding region (P1) replicated more efficiently than full-length viral RNA transcripts. In cotransfection experiments, both the full-length and subgenomic RNAs replicated at slightly reduced levels and appeared to interfere with each other's replication. In contrast, a subgenomic RNA with a similarly sized out-of-frame deletion in P1 did not replicate in transfected cells, either alone or in the presence of helper RNA. Similar results were observed with an RNA transcript containing a large in-frame deletion spanning the P1, P2, and P3 coding regions. A mutant RNA with an in-frame deletion in the P1-2A coding sequence was self-replicating but at a significantly reduced level. The replication of this RNA was fully complemented after cotransfection with a helper RNA that provided 2A in trans. A P1-2A-2B in-frame deletion, however, totally blocked RNA replication and was not complemented. Control experiments showed that all of the expected viral proteins were both synthesized and processed when the RNA transcripts were translated in vitro. Thus, our results indicated that 2A was a trans-acting protein and that 2B and perhaps other viral proteins were cis acting during poliovirus RNA replication in vivo. Our data support a model for poliovirus RNA replication which directly links the translation of a molecule of plus-strand RNA with the formation of a replication complex for minus-strand RNA synthesis. Images PMID

  2. Mycobacterial DNA Replication as a Target for Antituberculosis Drug Discovery.

    PubMed

    Płocinska, Renata; Korycka-Machala, Malgorzata; Plocinski, Przemyslaw; Dziadek, Jaroslaw

    2017-06-16

    Mycobacterium tuberculosis (M. tuberculosis), the causative agent of tuberculosis, is a leading infectious disease organism, causing millions of deaths each year. This serious pathogen has been greatly spread worldwide and recent years have observed an increase in the number of multi-drug resistant and totally drug resistant M. tuberculosis strains (WHO report, 2014). The danger of tuberculosis becoming an incurable disease has emphasized the need for the discovery of a new generation of antimicrobial agents. The development of novel alternative medical strategies, new drugs and the search for optimal drug targets are top priority areas of tuberculosis research. Key characteristics of mycobacteria include: slow growth, the ability to transform into a metabolically silent - latent state, intrinsic drug resistance and the relatively rapid development of acquired drug resistance. These factors make finding an ideal antituberculosis drug enormously challenging, even if it is designed to treat drug sensitive tuberculosis strains. A vast majority of canonical antibiotics including antituberculosis agents target bacterial cell wall biosynthesis or DNA/RNA processing. Novel therapeutic approaches are being tested to target mycobacterial cell division, twocomponent regulatory factors, lipid synthesis and the transition between the latent and actively growing states. This review discusses the choice of cellular targets for an antituberculosis therapy, describes putative drug targets evaluated in the recent literature and summarizes potential candidates under clinical and pre-clinical development. We focus on the key cellular process of DNA replication, as a prominent target for future antituberculosis therapy. We describe two main pathways: the biosynthesis of nucleic acids precursors - the nucleotides, and the synthesis of DNA molecules. We summarize data regarding replication associated proteins that are critical for nucleotide synthesis, initiation, unwinding and

  3. Chromatin Structure and Replication Origins: Determinants Of Chromosome Replication And Nuclear Organization

    PubMed Central

    Smith, Owen K.; Aladjem, Mirit I.

    2014-01-01

    The DNA replication program is, in part, determined by the epigenetic landscape that governs local chromosome architecture and directs chromosome duplication. Replication must coordinate with other biochemical processes occurring concomitantly on chromatin, such as transcription and remodeling, to insure accurate duplication of both genetic and epigenetic features and to preserve genomic stability. The importance of genome architecture and chromatin looping in coordinating cellular processes on chromatin is illustrated by two recent sets of discoveries. First, chromatin-associated proteins that are not part of the core replication machinery were shown to affect the timing of DNA replication. These chromatin-associated proteins could be working in concert, or perhaps in competition, with the transcriptional machinery and with chromatin modifiers to determine the spatial and temporal organization of replication initiation events. Second, epigenetic interactions are mediated by DNA sequences that determine chromosomal replication. In this review we summarize recent findings and current models linking spatial and temporal regulation of the replication program with epigenetic signaling. We discuss these issues in the context of the genome’s three-dimensional structure with an emphasis on events occurring during the initiation of DNA replication. PMID:24905010

  4. PTEN Controls the DNA Replication Process through MCM2 in Response to Replicative Stress.

    PubMed

    Feng, Jiawen; Liang, Jing; Li, Jiaju; Li, Yunqiao; Liang, Hui; Zhao, Xuyang; McNutt, Michael A; Yin, Yuxin

    2015-11-17

    PTEN is a tumor suppressor frequently mutated in human cancers. PTEN inhibits the phosphatidylinositol 3-kinase (PI3K)-AKT cascade, and nuclear PTEN guards the genome by multiple mechanisms. Here, we report that PTEN physically associates with the minichromosome maintenance complex component 2 (MCM2), which is essential for DNA replication. Specifically, PTEN dephosphorylates MCM2 at serine 41 (S41) and restricts replication fork progression under replicative stress. PTEN disruption results in unrestrained fork progression upon replication stalling, which is similar to the phenotype of cells expressing the phosphomimic MCM2 mutant S41D. Moreover, PTEN is necessary for prevention of chromosomal aberrations under replication stress. This study demonstrates that PTEN regulates DNA replication through MCM2 and loss of PTEN function leads to replication defects and genomic instability. We propose that PTEN plays a critical role in maintaining genetic stability through a replication-specific mechanism, and this is a crucial facet of PTEN tumor suppressor activity. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  5. Archaeal DNA Replication Origins and Recruitment of the MCM Replicative Helicase.

    PubMed

    Samson, R Y; Bell, S D

    2016-01-01

    DNA replication is fundamental to the propagation of all life on the planet. Remarkably, given the central importance for this process, two distinct core cellular DNA replication machineries have evolved. One is found in the bacterial domain of life and the other is present in Archaea and Eukarya. The archaeal machinery represents a simplified and presumably ancestral form of the eukaryotic DNA replication apparatus. As such, archaeal replication proteins have been studied extensively as models for their eukaryal counterparts. In addition, a number of archaea have been developed as model organisms. Accordingly, there has been a considerable increase in our knowledge of how archaeal chromosomes are replicated. It has become apparent that the majority of archaeal cells replicate their genomes from multiple origins per chromosome. Thus, at both organizational and mechanistic levels, archaeal DNA replication resembles that of eukarya. In this chapter, we will describe recent advances in our understanding of the basis of archaeal origin definition and how the archaeal initiator proteins recruit the replicative helicase to origins. © 2016 Elsevier Inc. All rights reserved.

  6. RMI1 Promotes DNA Replication Fork Progression and Recovery from Replication Fork Stress

    PubMed Central

    Yang, Jay; O'Donnell, Lara; Durocher, Daniel

    2012-01-01

    RMI1 is a member of an evolutionarily conserved complex composed of BLM and topoisomerase IIIα (TopoIIIα). This complex exhibits strand passage activity in vitro, which is likely important for DNA repair and DNA replication in vivo. The inactivation of RMI1 causes genome instability, including elevated levels of sister chromatid exchange and accelerated tumorigenesis. Using molecular combing to analyze DNA replication at the single-molecule level, we show that RMI1 is required to promote normal replication fork progression. The fork progression defect in RMI1-depleted cells is alleviated in cells lacking BLM, indicating that RMI1 functions downstream of BLM in promoting replication elongation. RMI1 localizes to subnuclear foci with BLM and TopoIIIα in response to replication stress. The proper localization of the complex requires a BLM-TopoIIIα-RMI1 interaction and is essential for RMI1 to promote recovery from replication stress. These findings reveal direct roles of RMI1 in DNA replication and the replication stress response, which could explain the molecular basis for its involvement in suppressing sister chromatid exchange and tumorigenesis. PMID:22645306

  7. Plasmid-like replicative intermediates of the Epstein-Barr virus lytic origin of DNA replication.

    PubMed Central

    Pfüller, R; Hammerschmidt, W

    1996-01-01

    During the lytic phase of herpesviruses, intermediates of viral DNA replication are found as large concatemeric molecules in the infected cells. It is not known, however, what the early events in viral DNA replication that yield these concatemers are. In an attempt to identify these early steps of DNA replication, replicative intermediates derived from the lytic origin of Epstein-Barr virus, oriLyt, were analyzed. As shown by density shift experiments with bromodeoxyuridine, oriLyt replicated semiconservatively soon after induction of the lytic cycle and oriLyt-containing DNA is amplified to yield monomeric plasmid progeny DNA (besides multimeric forms and high-molecular-weight DNA). A new class of plasmid progeny DNA which have far fewer negative supercoils than do plasmids extracted from uninduced cells is present only in cells undergoing the lytic cycle of Epstein-Barr virus. This finding is consistent with plasmid DNAs having fewer nucleosomes before extraction. The newly replicated plasmid DNAs are dependent on a functional oriLyt in cis and support an efficient marker transfer into Escherichia coli as monomeric plasmids. Multimeric forms of presumably circular progeny DNA of oriLyt, as well as detected recombination events, indicate that oriLyt-mediated DNA replication is biphasic: an early theta-like mode is followed by a complex pattern which could result from rolling-circle DNA replication. PMID:8648674

  8. Replication factory activation can be decoupled from the replication timing program by modulating Cdk levels

    PubMed Central

    Thomson, Alexander M.; Gillespie, Peter J.

    2010-01-01

    In the metazoan replication timing program, clusters of replication origins located in different subchromosomal domains fire at different times during S phase. We have used Xenopus laevis egg extracts to drive an accelerated replication timing program in mammalian nuclei. Although replicative stress caused checkpoint-induced slowing of the timing program, inhibition of checkpoint kinases in an unperturbed S phase did not accelerate it. Lowering cyclin-dependent kinase (Cdk) activity slowed both replication rate and progression through the timing program, whereas raising Cdk activity increased them. Surprisingly, modest alteration of Cdk activity changed the amount of DNA synthesized during different stages of the timing program. This was associated with a change in the number of active replication factories, whereas the distribution of origins within active factories remained relatively normal. The ability of Cdks to differentially effect replication initiation, factory activation, and progression through the timing program provides new insights into the way that chromosomal DNA replication is organized during S phase. PMID:20083602

  9. Portrait of replication stress viewed from telomeres.

    PubMed

    Ishikawa, Fuyuki

    2013-07-01

    Genetic instability is the driving force of the malignant progression of cancer cells. Recently, replication stress has attracted much attention as a source of genetic instability that gives rise to an accumulation of mutations during the lifespan of individuals. However, the molecular details of the process are not fully understood. Here, recent progress in understanding how genetic alterations accumulate at telomeres will be reviewed. In particular, two aspects of telomere replication will be discussed in this context, covering conventional semi-conservative replication, and DNA synthesis by telomerase plus the C-strand fill-in reactions. Although these processes are seemingly telomere-specific, I will emphasize the possibility that the molecular understanding of the telomere events may shed light on genetic instability at other genetic loci in general.

  10. Role of Chromatin Loops In DNA Replication

    NASA Astrophysics Data System (ADS)

    Bechhoefer, John; Jun, Suckjoon; Herrick, John; Bensimon, Aaron

    2003-03-01

    In eukaryotic organisms, DNA is packed together with proteins (histones) into a structure known as the 30-nm chromatin fiber, whose behavior can be modeled as a wormlike polymer chain. We have investigated the relationship between the distributions of chromatin loop sizes and DNA replication in Xenopus laevis egg extracts. We find that the loop-size distribution predicted from the worm-like chain model of chromatin agrees well with the reported spatial distribution of replication origins in this system and that loops can explain quantitatively the observed tendency for nearby origins to start synchronously. Thus, in Xenopus egg extracts, the persistence length of chromatin fiber determines the separation between and synchrony of DNA replication origins.

  11. Spatiotemporal visualization of DNA replication dynamics.

    PubMed

    Reinhart, Marius; Casas-Delucchi, Corella S; Cardoso, M Cristina

    2013-01-01

    The ability of cells to copy their DNA allows them to transmit their genetic information to their progeny. In such, this central biological process preserves the instructions that direct the entire development of a cell. Earlier biochemical analysis in vitro and genetic analysis in yeast laid the basis of our understanding of the highly conserved mechanism of DNA replication. Recent advances on labeling and live-cell microscopy permit now the dissection of this fundamental process in vivo within the context of intact cells. In this chapter, we describe in detail how to perform multiple DNA replication labeling and detection allowing high spatial resolution imaging, as well as how to follow DNA replication in living cells allowing high temporal resolution imaging.

  12. Evolution of Database Replication Technologies for WLCG

    NASA Astrophysics Data System (ADS)

    Baranowski, Zbigniew; Lobato Pardavila, Lorena; Blaszczyk, Marcin; Dimitrov, Gancho; Canali, Luca

    2015-12-01

    In this article we summarize several years of experience on database replication technologies used at WLCG and we provide a short review of the available Oracle technologies and their key characteristics. One of the notable changes and improvement in this area in recent past has been the introduction of Oracle GoldenGate as a replacement of Oracle Streams. We report in this article on the preparation and later upgrades for remote replication done in collaboration with ATLAS and Tier 1 database administrators, including the experience from running Oracle GoldenGate in production. Moreover, we report on another key technology in this area: Oracle Active Data Guard which has been adopted in several of the mission critical use cases for database replication between online and offline databases for the LHC experiments.

  13. BFT replication resistant to MAC attacks

    NASA Astrophysics Data System (ADS)

    Zbierski, Maciej

    2016-09-01

    Over the last decade numerous Byzantine fault-tolerant (BFT) replication protocols have been proposed in the literature. However, the vast majority of these solutions reuse the same authentication scheme, which makes them susceptible to a so called MAC attack. Such vulnerability enables malicious clients to undetectably prevent the replicated service from processing incoming client requests, and consequently making it permanently unavailable. While some BFT protocols attempted to address this issue by using different authentication mechanisms, they at the same time significantly degraded the performance achieved in correct environments. This article presents a novel adaptive authentication mechanism which can be combined with practically any Byzantine fault-tolerant replication protocol. Unlike previous solutions, the proposed scheme dynamically switches between two operation modes to combine high performance in correct environments and liveness during MAC attacks. The experiment results presented in the article demonstrate that the proposed mechanism can sufficiently tolerate MAC attacks without introducing any observable overhead whenever no faults are present.

  14. Diversification of self-replicating molecules.

    PubMed

    Sadownik, Jan W; Mattia, Elio; Nowak, Piotr; Otto, Sijbren

    2016-03-01

    How new species emerge in nature is still incompletely understood and difficult to study directly. Self-replicating molecules provide a simple model that allows us to capture the fundamental processes that occur in species formation. We have been able to monitor in real time and at a molecular level the diversification of self-replicating molecules into two distinct sets that compete for two different building blocks ('food') and so capture an important aspect of the process by which species may arise. The results show that the second replicator set is a descendant of the first and that both sets are kinetic products that oppose the thermodynamic preference of the system. The sets occupy related but complementary food niches. As diversification into sets takes place on the timescale of weeks and can be investigated at the molecular level, this work opens up new opportunities for experimentally investigating the process through which species arise both in real time and with enhanced detail.

  15. Patentability of Self-Replicating Technologies

    PubMed Central

    Shear, Richard H.

    2015-01-01

    Patents provide an incentive to inventors, investors, and entrepreneurs to conduct research and development, especially in risky (i.e., unpredictable) fields of technology. This review discusses whether self-replicating technologies are patentable within the United States and the issues surrounding them. Self-replicating technologies discussed include plants, bacteria, and genetic technology and the historical legal precedents that have led to the current status of the patent law. To clearly understand these issues, the review also discusses various U.S. Supreme Court cases that, although not related to self-replicating technologies, have an impact on these patentability issues. Finally, some thoughts regarding patent strategy are presented so as to maximize patent protection for these technologies. PMID:25256175

  16. Transitions: The Evolution of Linguistic Replicators

    NASA Astrophysics Data System (ADS)

    Kirby, Simon

    Maynard Smith and Szathmáry (1995) propose a series of major transitions in the evolutionary history of life. Their work provides a rich framework for thinking about replication. They identified the importance of language in this light, but language is a new system of replication in more than one sense: it is both an enabler of cultural replicators with unlimited heredity, and also a new kind of evolutionary system itself. Iterated learning is the process of linguistic transmission, and it drives both language change and the transitions to qualitatively new kinds of linguistic system. By seeing language as an evolutionary system, the biggest payoff we get may be the ability to take biologists' insights into the evolution of life and apply them to the evolution of language.

  17. Synchronization of DNA array replication kinetics

    NASA Astrophysics Data System (ADS)

    Manturov, Alexey O.; Grigoryev, Anton V.

    2016-04-01

    In the present work we discuss the features of the DNA replication kinetics at the case of multiplicity of simultaneously elongated DNA fragments. The interaction between replicated DNA fragments is carried out by free protons that appears at the every nucleotide attachment at the free end of elongated DNA fragment. So there is feedback between free protons concentration and DNA-polymerase activity that appears as elongation rate dependence. We develop the numerical model based on a cellular automaton, which can simulate the elongation stage (growth of DNA strands) for DNA elongation process with conditions pointed above and we study the possibility of the DNA polymerases movement synchronization. The results obtained numerically can be useful for DNA polymerase movement detection and visualization of the elongation process in the case of massive DNA replication, eg, under PCR condition or for DNA "sequencing by synthesis" sequencing devices evaluation.

  18. Genetic‐Genomic Replication to Identify Candidate Mouse Atherosclerosis Modifier Genes

    PubMed Central

    Hsu, Jeffrey; Smith, Jonathan D.

    2013-01-01

    Objective Genetics plays a large role in atherosclerosis susceptibility in humans and mice. We attempted to confirm previously determined mouse atherosclerosis‐associated loci and use bioinformatics and transcriptomics to create a catalog of candidate atherosclerosis modifier genes at these loci. Methods and Results A strain intercross was performed between AKR and DBA/2 mice on the apoE−/− background generating 166 F2 progeny. Using the phenotype log10 of the aortic root lesion area, we identified 3 suggestive atherosclerosis quantitative trait loci (Ath QTLs). When combined with our prior strain intercross, we confirmed 3 significant Ath QTLs on chromosomes 2, 15, and 17, with combined logarithm of odds scores of 5.9, 5.3, and 5.6, respectively, which each met the genome‐wide 5% false discovery rate threshold. We identified all of the protein coding differences between these 2 mouse strains within the Ath QTL intervals. Microarray gene expression profiling was performed on macrophages and endothelial cells from this intercross to identify expression QTLs (eQTLs), the loci that are associated with variation in the expression levels of specific transcripts. Cross tissue eQTLs and macrophage eQTLs that replicated from a prior strain intercross were identified. These bioinformatic and eQTL analyses produced a comprehensive list of candidate genes that may be responsible for the Ath QTLs. Conclusions Replication studies for clinical traits as well as gene expression traits are worthwhile in identifying true versus false genetic associations. We have replicated 3 loci on mouse chromosomes 2, 15, and 17 that are associated with atherosclerosis. We have also identified protein coding differences and multiple replicated eQTLs, which may be useful in the identification of atherosclerosis modifier genes. PMID:23525445

  19. The MG1363 and IL1403 Laboratory Strains of Lactococcus lactis and Several Dairy Strains Are Diploid▿ †

    PubMed Central

    Michelsen, Ole; Hansen, Flemming G.; Albrechtsen, Bjarne; Jensen, Peter Ruhdal

    2010-01-01

    Bacteria are normally haploid, maintaining one copy of their genome in one circular chromosome. We have examined the cell cycle of laboratory strains of Lactococcus lactis, and, to our surprise, we found that some of these strains were born with two complete nonreplicating chromosomes. We determined the cellular content of DNA by flow cytometry and by radioactive labeling of the DNA. These strains thus fulfill the criterion of being diploid. Several dairy strains were also found to be diploid while a nondairy strain and several other dairy strains were haploid in slow-growing culture. The diploid and haploid strains differed in their sensitivity toward UV light, in their cell size, and in their D period, the period between termination of DNA replication and cell division. PMID:20023021

  20. The psychophysiology of mixed emotional states: Internal and external replicability analysis of a direct replication study.

    PubMed

    Kreibig, Sylvia D; Samson, Andrea C; Gross, James J

    2015-07-01

    The replicability of emotion-related physiological changes constitutes a fundamental issue in affective science. We undertook a direct replication of the physiological differentiation of amusement, disgust, and a mixed emotional state as previously reported (Kreibig, Samson, & Gross, 2013). In the current study, 48 women watched 54 amusing, disgusting, and mixed emotional film clips while cardiovascular, electrodermal, and respiratory measures were obtained. Primary analyses indicated physiological differentiation of the mixed emotional state from amusement and disgust. We evaluated (a) the probability that future replications of the current study would yield similar results using bootstrapped confidence intervals of effect sizes, and (b) the stability of results of physiological reactivity between actual replications using correlation and regression analyses. Findings suggest replicable differentiation of amusement, disgust, and a mixed emotional state. © 2015 Society for Psychophysiological Research.

  1. The Replication Fork: Understanding the Eukaryotic Replication Machinery and the Challenges to Genome Duplication

    PubMed Central

    Leman, Adam R.; Noguchi, Eishi

    2013-01-01

    Eukaryotic cells must accurately and efficiently duplicate their genomes during each round of the cell cycle. Multiple linear chromosomes, an abundance of regulatory elements, and chromosome packaging are all challenges that the eukaryotic DNA replication machinery must successfully overcome. The replication machinery, the “replisome” complex, is composed of many specialized proteins with functions in supporting replication by DNA polymerases. Efficient replisome progression relies on tight coordination between the various factors of the replisome. Further, replisome progression must occur on less than ideal templates at various genomic loci. Here, we describe the functions of the major replisome components, as well as some of the obstacles to efficient DNA replication that the replisome confronts. Together, this review summarizes current understanding of the vastly complicated task of replicating eukaryotic DNA. PMID:23599899

  2. DciA is an ancestral replicative helicase operator essential for bacterial replication initiation

    PubMed Central

    Brézellec, Pierre; Vallet-Gely, Isabelle; Possoz, Christophe; Quevillon-Cheruel, Sophie; Ferat, Jean-Luc

    2016-01-01

    Delivery of the replicative helicase onto DNA is an essential step in the initiation of replication. In bacteria, DnaC (in Escherichia coli) and DnaI (in Bacillus subtilis) are representative of the two known mechanisms that assist the replicative helicase at this stage. Here, we establish that these two strategies cannot be regarded as prototypical of the bacterial domain since dnaC and dnaI (dna[CI]) are present in only a few bacterial phyla. We show that dna[CI] was domesticated at least seven times through evolution in bacteria and at the expense of one gene, which we rename dciA (dna[CI] antecedent), suggesting that DciA and Dna[CI] share a common function. We validate this hypothesis by establishing in Pseudomonas aeruginosa that DciA possesses the attributes of the replicative helicase-operating proteins associated with replication initiation. PMID:27830752

  3. The replication fork: understanding the eukaryotic replication machinery and the challenges to genome duplication.

    PubMed

    Leman, Adam R; Noguchi, Eishi

    2013-03-01

    Eukaryotic cells must accurately and efficiently duplicate their genomes during each round of the cell cycle. Multiple linear chromosomes, an abundance of regulatory elements, and chromosome packaging are all challenges that the eukaryotic DNA replication machinery must successfully overcome. The replication machinery, the "replisome" complex, is composed of many specialized proteins with functions in supporting replication by DNA polymerases. Efficient replisome progression relies on tight coordination between the various factors of the replisome. Further, replisome progression must occur on less than ideal templates at various genomic loci. Here, we describe the functions of the major replisome components, as well as some of the obstacles to efficient DNA replication that the replisome confronts. Together, this review summarizes current understanding of the vastly complicated task of replicating eukaryotic DNA.

  4. Plasmid Replication Control by Antisense RNAs.

    PubMed

    Brantl, Sabine

    2014-08-01

    Plasmids are selfish genetic elements that normally constitute a burden for the bacterial host cell. This burden is expected to favor plasmid loss. Therefore, plasmids have evolved mechanisms to control their replication and ensure their stable maintenance. Replication control can be either mediated by iterons or by antisense RNAs. Antisense RNAs work through a negative control circuit. They are constitutively synthesized and metabolically unstable. They act both as a measuring device and a regulator, and regulation occurs by inhibition. Increased plasmid copy numbers lead to increasing antisense-RNA concentrations, which, in turn, result in the inhibition of a function essential for replication. On the other hand, decreased plasmid copy numbers entail decreasing concentrations of the inhibiting antisense RNA, thereby increasing the replication frequency. Inhibition is achieved by a variety of mechanisms, which are discussed in detail. The most trivial case is the inhibition of translation of an essential replication initiator protein (Rep) by blockage of the rep-ribosome binding site. Alternatively, ribosome binding to a leader peptide mRNA whose translation is required for efficient Rep translation can be prevented by antisense-RNA binding. In 2004, translational attenuation was discovered. Antisense-RNA-mediated transcriptional attenuation is another mechanism that has, so far, only been detected in plasmids of Gram-positive bacteria. ColE1, a plasmid that does not need a plasmid-encoded replication initiator protein, uses the inhibition of primer formation. In other cases, antisense RNAs inhibit the formation of an activator pseudoknot that is required for efficient Rep translation.

  5. Entropy Involved in Fidelity of DNA Replication

    PubMed Central

    Arias-Gonzalez, J. Ricardo

    2012-01-01

    Information has an entropic character which can be analyzed within the framework of the Statistical Theory in molecular systems. R. Landauer and C.H. Bennett showed that a logical copy can be carried out in the limit of no dissipation if the computation is performed sufficiently slowly. Structural and recent single-molecule assays have provided dynamic details of polymerase machinery with insight into information processing. Here, we introduce a rigorous characterization of Shannon Information in biomolecular systems and apply it to DNA replication in the limit of no dissipation. Specifically, we devise an equilibrium pathway in DNA replication to determine the entropy generated in copying the information from a DNA template in the absence of friction. Both the initial state, the free nucleotides randomly distributed in certain concentrations, and the final state, a polymerized strand, are mesoscopic equilibrium states for the nucleotide distribution. We use empirical stacking free energies to calculate the probabilities of incorporation of the nucleotides. The copied strand is, to first order of approximation, a state of independent and non-indentically distributed random variables for which the nucleotide that is incorporated by the polymerase at each step is dictated by the template strand, and to second order of approximation, a state of non-uniformly distributed random variables with nearest-neighbor interactions for which the recognition of secondary structure by the polymerase in the resultant double-stranded polymer determines the entropy of the replicated strand. Two incorporation mechanisms arise naturally and their biological meanings are explained. It is known that replication occurs far from equilibrium and therefore the Shannon entropy here derived represents an upper bound for replication to take place. Likewise, this entropy sets a universal lower bound for the copying fidelity in replication. PMID:22912695

  6. Suppression of Adenovirus Replication by Cardiotonic Steroids.

    PubMed

    Grosso, Filomena; Stoilov, Peter; Lingwood, Clifford; Brown, Martha; Cochrane, Alan

    2017-02-01

    The dependence of adenovirus on the host pre-RNA splicing machinery for expression of its complete genome potentially makes it vulnerable to modulators of RNA splicing, such as digoxin and digitoxin. Both drugs reduced the yields of four human adenoviruses (HAdV-A31, -B35, and -C5 and a species D conjunctivitis isolate) by at least 2 to 3 logs by affecting one or more steps needed for genome replication. Immediate early E1A protein levels are unaffected by the drugs, but synthesis of the delayed protein E4orf6 and the major late capsid protein hexon is compromised. Quantitative reverse transcription-PCR (qRT-PCR) analyses revealed that both drugs altered E1A RNA splicing (favoring the production of 13S over 12S RNA) early in infection and partially blocked the transition from 12S and 13S to 9S RNA at late stages of virus replication. Expression of multiple late viral protein mRNAs was lost in the presence of either drug, consistent with the observed block in viral DNA replication. The antiviral effect was dependent on the continued presence of the drug and was rapidly reversible. RIDK34, a derivative of convallotoxin, although having more potent antiviral activity, did not show an improved selectivity index. All three drugs reduced metabolic activity to some degree without evidence of cell death. By blocking adenovirus replication at one or more steps beyond the onset of E1A expression and prior to genome replication, digoxin and digitoxin show potential as antiviral agents for treatment of serious adenovirus infections. Furthermore, understanding the mechanism(s) by which digoxin and digitoxin inhibit adenovirus replication will guide the development of novel antiviral therapies.

  7. Attenuated oncolytic Measles Virus strains as cancer therapeutics

    PubMed Central

    Msaouel, P.; Iankov, I.D.; Dispenzieri, A.; Galanis, E.

    2011-01-01

    Attenuated measles virus vaccine strains have emerged as a promising oncolytic vector platform, having shown significant anti-tumor activity against a broad range of malignant neoplasms. Measles virus strains derived from the attenuated Edmonston-B (MV-Edm) vaccine lineage have been shown to selectively infect, replicate in and lyse cancer cells while causing minimal cytopathic effect on normal tissues. This review summarizes the preclinical data that led to the rapid clinical translation of oncolytic measles vaccine strains and provides an overview of early clinical data using this oncolytic platform. Furthermore, novel approaches currently under development to further enhance the oncolytic efficacy of MV-Edm strains, including strategies to circumvent immunity or modulate immune system responses, combinatorial approaches with standard treatment modalities, virus retargeting as well as strategies for in vivo monitoring of viral replication are discussed. PMID:21740361

  8. On the processivity of DNA replication.

    PubMed

    Fairfield, F R; Newport, J W; Dolejsi, M K; von Hippel, P H

    1983-12-01

    In this paper we describe the nature and importance of processive enzymatic reactions in biological processes. A model is set up to describe the processive synthetic process in DNA replication, and experiments are presented to define and test the model, using the components of the T4 phage-coded five-protein (in vitro) DNA replication system of Alberts. Nossal and coworkers. These experiments are performed either with a homogeneous oligo dT-poly dA primer-template system, or with a natural primer-template system using phage M13 DNA. The results are used to define some molecular aspects of the microscopic "processivity cycle".

  9. Overexpression of multisubunit replication factors in yeast.

    PubMed

    Burgers, P M

    1999-07-01

    Facile genetic and biochemical manipulation coupled with rapid cell growth and low cost of growth media has established the yeast Saccharomyces cerevisiae as a versatile workhorse. This article describes the use of yeast expression systems for the overproduction of complex multipolypeptide replication factors. The regulated overexpression of these factors in yeast provides for a readily accessible and inexpensive source of these factors in large quantities. The methodology is illustrated with the five-subunit replication factor C. Whole-cell extracts are prepared by blending yeast cells with glass beads or frozen yeast with dry ice. Procedures are described that maximize the yield of these factors while minimizing proteolytic degradation.

  10. Signal replication in a DNA nanostructure

    NASA Astrophysics Data System (ADS)

    Mendoza, Oscar; Houmadi, Said; Aimé, Jean-Pierre; Elezgaray, Juan

    2017-01-01

    Logic circuits based on DNA strand displacement reaction are the basic building blocks of future nanorobotic systems. The circuits tethered to DNA origami platforms present several advantages over solution-phase versions where couplings are always diffusion-limited. Here we consider a possible implementation of one of the basic operations needed in the design of these circuits, namely, signal replication. We show that with an appropriate preparation of the initial state, signal replication performs in a reproducible way. We also show the existence of side effects concomitant to the high effective concentrations in tethered circuits, such as slow leaky reactions and cross-activation.

  11. Natural selection among replicators, interactors and transactors.

    PubMed

    Bergandi, Donato

    2013-01-01

    In evolutionary biology and ecology, ontological and epistemological perspectives based on the replicator and the interactor have become the background that makes it possible to transcend traditional biological levels of organization and to achieve a unified view of evolution in which replication and interaction are fundamental operating processes. Using the transactional perspective proposed originally by John Dewey and Arthur Fisher Bentley, a new ontological and methodological category is proposed here: the transactor. The transactional perspective, based on the concept of the transactor, bridges the dichotomy between organisms and environment that characterizes the interactional perspective on evolution and provides epistemological support for the emergentist, systemic view of evolutionary and developmental processes.

  12. Conserved nucleosome positioning defines replication origins

    PubMed Central

    Eaton, Matthew L.; Galani, Kyriaki; Kang, Sukhyun; Bell, Stephen P.; MacAlpine, David M.

    2010-01-01

    The origin recognition complex (ORC) specifies replication origin location. The Saccharomyces cerevisiae ORC recognizes the ARS (autonomously replicating sequence) consensus sequence (ACS), but only a subset of potential genomic sites are bound, suggesting other chromosomal features influence ORC binding. Using high-throughput sequencing to map ORC binding and nucleosome positioning, we show that yeast origins are characterized by an asymmetric pattern of positioned nucleosomes flanking the ACS. The origin sequences are sufficient to maintain a nucleosome-free origin; however, ORC is required for the precise positioning of nucleosomes flanking the origin. These findings identify local nucleosomes as an important determinant for origin selection and function. PMID:20351051

  13. The Replicative Consequences of Papillomavirus E2 Protein Binding to the Origin Replication Factor ORC2.

    PubMed

    DeSmet, Marsha; Kanginakudru, Sriramana; Rietz, Anne; Wu, Wai-Hong; Roden, Richard; Androphy, Elliot J

    2016-10-01

    The origin recognition complex (ORC) coordinates a series of events that lead to initiation of DNA strand duplication. As a nuclear double stranded DNA plasmid, the papillomavirus (PV) genome resembles a mini-chromosome in infected cells. To initiate its replication, the viral E2 protein binds to and recruits the E1 DNA helicase at the viral origin. PV genome replication program exhibits three stages: initial amplification from a single genome upon infection to a few copies per cell, a cell cycle linked maintenance phase, and a differentiation dependent late stage where the genome is amplified to thousands of copies. Involvement of ORC or other pre-replication complex (pre-RC) factors has not been described. We report that human PV (HPV) and bovine PV (BPV-1) E2 proteins bind to ORC2, however, ORC2 was not detected at the viral origin. Depletion of ORC2 enhanced PV replication in a transient replication model and in keratinocytes stably maintaining viral episomes, while there was no effect on copy number in a cell line with integrated HPV genomes. Consistent with this, occupancy of E1 and E2 at the viral origin increased following ORC2 silencing. These data imply that ORC2 is not necessary for activation of the PV origin by E1 and E2 but instead suppresses E2 replicative function. Furthermore, we observed that over-expression of HPV E2 decreased ORC2 occupation at two known mammalian origins of replication, suggesting that E2 restricts pre-ORC assembly that could otherwise compete for host replication complexes necessary for viral genome amplification. We infer that the ORC2 complex with E2 restricts viral replication in the maintenance phase of the viral replication program and that elevated levels of E2 that occur during the differentiation dependent amplification stage subvert ORC loading and hence DNA synthesis at cellular origins.

  14. Replication fork slowing and stalling are distinct, checkpoint-independent consequences of replicating damaged DNA

    PubMed Central

    2017-01-01

    In response to DNA damage during S phase, cells slow DNA replication. This slowing is orchestrated by the intra-S checkpoint and involves inhibition of origin firing and reduction of replication fork speed. Slowing of replication allows for tolerance of DNA damage and suppresses genomic instability. Although the mechanisms of origin inhibition by the intra-S checkpoint are understood, major questions remain about how the checkpoint regulates replication forks: Does the checkpoint regulate the rate of fork progression? Does the checkpoint affect all forks, or only those encountering damage? Does the checkpoint facilitate the replication of polymerase-blocking lesions? To address these questions, we have analyzed the checkpoint in the fission yeast Schizosaccharomyces pombe using a single-molecule DNA combing assay, which allows us to unambiguously separate the contribution of origin and fork regulation towards replication slowing, and allows us to investigate the behavior of individual forks. Moreover, we have interrogated the role of forks interacting with individual sites of damage by using three damaging agents—MMS, 4NQO and bleomycin—that cause similar levels of replication slowing with very different frequency of DNA lesions. We find that the checkpoint slows replication by inhibiting origin firing, but not by decreasing fork rates. However, the checkpoint appears to facilitate replication of damaged templates, allowing forks to more quickly pass lesions. Finally, using a novel analytic approach, we rigorously identify fork stalling events in our combing data and show that they play a previously unappreciated role in shaping replication kinetics in response to DNA damage. PMID:28806726

  15. Direct visualization of replication dynamics in early zebrafish embryos.

    PubMed

    Kuriya, Kenji; Higashiyama, Eriko; Avşar-Ban, Eriko; Okochi, Nanami; Hattori, Kaede; Ogata, Shin; Takebayashi, Shin-Ichiro; Ogata, Masato; Tamaru, Yutaka; Okumura, Katsuzumi

    2016-05-01

    We analyzed DNA replication in early zebrafish embryos. The replicating DNA of whole embryos was labeled with the thymidine analog 5-ethynyl-2'-deoxyuridine (EdU), and spatial regulation of replication sites was visualized in single embryo-derived cells. The results unveiled uncharacterized replication dynamics during zebrafish early embryogenesis.

  16. Geobacteraceae strains and methods

    DOEpatents

    Lovley, Derek R.; Nevin, Kelly P.; Yi, Hana

    2015-07-07

    Embodiments of the present invention provide a method of producing genetically modified strains of electricigenic microbes that are specifically adapted for the production of electrical current in microbial fuel cells, as well as strains produced by such methods and fuel cells using such strains. In preferred embodiments, the present invention provides genetically modified strains of Geobacter sulfurreducens and methods of using such strains.

  17. Brucella abortus Transits through the Autophagic Pathway and Replicates in the Endoplasmic Reticulum of Nonprofessional Phagocytes

    PubMed Central

    Pizarro-Cerdá, Javier; Méresse, Stéphane; Parton, Robert G.; van der Goot, Gisou; Sola-Landa, Alberto; Lopez-Goñi, Ignacio; Moreno, Edgardo; Gorvel, Jean-Pierre

    1998-01-01

    Brucella abortus is an intracellular pathogen that replicates within a membrane-bounded compartment. In this study, we have examined the intracellular pathway of the virulent B. abortus strain 2308 (S2308) and the attenuated strain 19 (S19) in HeLa cells. At 10 min after inoculation, both bacterial strains are transiently detected in phagosomes characterized by the presence of early endosomal markers such as the early endosomal antigen 1. At ∼1 h postinoculation, bacteria are located within a compartment positive for the lysosome-associated membrane proteins (LAMPs) and the endoplasmic reticulum (ER) marker sec61β but negative for the mannose 6-phosphate receptors and cathepsin D. Interestingly, this compartment is also positive for the autophagosomal marker monodansylcadaverin, suggesting that S2308 and S19 are located in autophagic vacuoles. At 24 h after inoculation, attenuated S19 is degraded in lysosomes, while virulent S2308 multiplies within a LAMP- and cathepsin D-negative but sec61β- and protein disulfide isomerase-positive compartment. Furthermore, treatment of infected cells with the pore-forming toxin aerolysin from Aeromonas hydrophila causes vacuolation of the bacterial replication compartment. These results are compatible with the hypothesis that pathogenic B. abortus exploits the autophagic machinery of HeLa cells to establish an intracellular niche favorable for its replication within the ER. PMID:9826346

  18. Spatial and Temporal Analysis of Alphavirus Replication and Assembly in Mammalian and Mosquito Cells

    PubMed Central

    Jose, Joyce; Taylor, Aaron B.

    2017-01-01

    ABSTRACT Sindbis virus (SINV [genus Alphavirus, family Togaviridae]) is an enveloped, mosquito-borne virus. Alphaviruses cause cytolytic infections in mammalian cells while establishing noncytopathic, persistent infections in mosquito cells. Mosquito vector adaptation of alphaviruses is a major factor in the transmission of epidemic strains of alphaviruses. Though extensive studies have been performed on infected mammalian cells, the morphological and structural elements of alphavirus replication and assembly remain poorly understood in mosquito cells. Here we used high-resolution live-cell imaging coupled with single-particle tracking and electron microscopy analyses to delineate steps in the alphavirus life cycle in both the mammalian host cell and insect vector cells. Use of dually labeled SINV in conjunction with cellular stains enabled us to simultaneously determine the spatial and temporal differences of alphavirus replication complexes (RCs) in mammalian and insect cells. We found that the nonstructural viral proteins and viral RNA in RCs exhibit distinct spatial organization in mosquito cytopathic vacuoles compared to replication organelles from mammalian cells. We show that SINV exploits filopodial extensions for virus dissemination in both cell types. Additionally, we propose a novel mechanism for replication complex formation around glycoprotein-containing vesicles in mosquito cells that produced internally released particles that were seen budding from the vesicles by live imaging. Finally, by characterizing mosquito cell lines that were persistently infected with fluorescent virus, we show that the replication and assembly machinery are highly modified, and this allows continuous production of alphaviruses at reduced levels. PMID:28196962

  19. Low molecular weight DNA replication intermediates in Escherichia coli: mechanism of formation and strand specificity

    PubMed Central

    Amado, Luciana; Kuzminov, Andrei

    2013-01-01

    Chromosomal DNA replication intermediates, revealed in ligase-deficient conditions in vivo, are of low molecular weight independently of the organism, suggesting discontinuous replication of both the leading and the lagging DNA strands. Yet, in vitro experiments with purified enzymes replicating sigma-structured substrates show continuous synthesis of the leading DNA strand in complete absence of ligase, supporting the textbook model of semi-discontinuous DNA replication. The discrepancy between the in vivo and in vitro results is rationalized by proposing that various excision repair events nick continuously-synthesized leading strands after synthesis, producing the observed low molecular weight intermediates. Here we show that, in an E. coli ligase-deficient strain with all known excision repair pathways inactivated, new DNA is still synthesized discontinuously. Furthermore, hybridization to strand-specific targets demonstrates that the low molecular weight replication intermediates come from both the lagging and the leading strands. These results support the model of discontinuous leading strand synthesis in E. coli. PMID:23876705

  20. Integration host factor is required for replication of pYGK-derived plasmids in Aggregatibacter actinomycetemcomitans.

    PubMed

    Torres-Escobar, Ascención; Juárez-Rodríguez, María D; Demuth, Donald R

    2014-08-01

    In this study, we show that integration host factor protein (IHF) is required for replication of pYGK plasmids in Aggregatibacter actinomycetemcomitans. YGK plasmids were not replicated in A. actinomycetemcomitans strains lacking either the α- or β- subunit of IHF. However, the deletion mutants were complemented, and plasmid replication was restored when the promoter region and gene for either ihfA or ihfB was cloned into pYGK. We also identified two motifs that resemble the consensus IHF-binding site in a 813-bp fragment containing the pYGK origin of replication. Using electrophoretic mobility shift assays, purified IHFα-IHFβ protein complex was shown to bind to probes containing either of these motifs. To our knowledge, this is the first report showing that plasmid replication is IHF-dependent in the family Pasteurellaceae. In addition, using site-direct mutagenesis, the XbaI and KpnI restriction sites in the suicide vector pJT1 were modified to generate plasmid pJT10. The introduction of these new unique sites in pJT10 facilitates the transfer of transcriptional or translational lacZ fusion constructs for the generation of single-copy chromosomal insertion of the reporter construct. Plasmid pJT10 and its derivatives will be useful for genetic studies in Aggregatibacter (Actinobacillus) and probably other genera of Pasteurellaceae, including Haemophilus, Pasteurella, and Mannheimia.

  1. Evaluation of the effect of pyrimethamine, an anti-malarial drug, on HIV-1 replication

    PubMed Central

    Oguariri, Raphael M.; Adelsberger, Joseph W.; Baseler, Michael W.; Imamichi, Tomozumi

    2010-01-01

    Co-infection of human immunodeficiency virus (HIV) with malaria is one of the pandemic problems in Africa and parts of Asia. Here we investigated the impact of PYR and two other clinical anti-malarial drugs (chloroquine [CQ] or artemisinin [ART]) on HIV-1 replication. Peripheral blood mononuclear cells (PBMCs) or MT-2 cells were infected with HIVNL4.3 strain and treated with different concentrations of the anti-malarial drugs. HIV-1 replication was measured using p24 ELISA. We show that 10 μM CQ and ART inhibited HIV-1 replication by 76% and 60% in PBMCs, respectively, but not in MT-2 cells. In contrast, 10 μM PYR enhanced HIV-1 replication in MT-2 cells by >10-fold. A series of molecular mechanism studies revealed that PYR increased intracellular HIV gag proteins without affecting the promoter or the reverse transcriptase activity. The effect of PYR was independent of HTLV-1 produced by MT-2 cells. Of interest, PYR treatment led to S-phase accumulation and increased AZT and d4T antiviral activity by ~4-fold. Taken together, we show that PYR significantly enhances HIV-1 replication by affecting the cellular machinery. Our results could be relevant for the management of malaria and HIV particularly in regions where HIV-1 and malaria epidemics overlap. PMID:20800626

  2. Experimental African swine fever: apoptosis of lymphocytes and virus replication in other cells.

    PubMed

    Gómez-Villamandos, J C; Hervás, J; Méndez, A; Carrasco, L; Martín de las Mulas, J; Villeda, C J; Wilkinson, P J; Sierra, M A

    1995-09-01

    In order to determine the cause of cellular death of lymphocytes in pigs with acute African swine fever and the relationships between African swine fever virus (ASFV) and interstitial cells, ten pigs were inoculated with a highly virulent strain of ASFV (Malawi '83) and samples taken for ultrastructural study of hepatic and renal interstitial tissues. We demonstrated death by apoptosis of lymphocytes and virus replication in fibroblasts, smooth muscle cells and endothelial cells in the interstitial tissues of pigs inoculated with ASFV. From day 5 onwards, apoptotic lymphocyte and intense virus replication in hepatic interstitial macrophages and fibroblasts were observed. By day 7, apoptotic lymphocytes and virus replication in macrophages, interstitial capillary endothelial cells and fibroblasts in the kidney were observed. Virus replication was also seen in smooth muscle cells of hepatic and renal arterioles and venules. Our results suggest that mononuclear phagocyte system (MPS) cell activation, and the resulting release of cytokines, could induce apoptosis of lymphocytes and virus replication in non-MPS cells.

  3. Legionella pneumophila CsrA is a pivotal repressor of transmission traits and activator of replication.

    PubMed

    Molofsky, Ari B; Swanson, Michele S

    2003-10-01

    Legionella pneumophila can replicate inside amoebae and also alveolar macrophages to cause Legionnaires' Disease in susceptible hosts. When nutrients become limiting, a stringent-like response coordinates the differentiation of L. pneumophila to a transmissive form, a process mediated by the two-component system LetA/S and the sigma factors RpoS and FliA. Here we demonstrate that the broadly conserved RNA binding protein CsrA is a global repressor of L. pneumophila transmission phenotypes and an essential activator of intracellular replication. By analysing csrA expression and the phenotypes of csrA single and double mutants and a strain that expresses csrA constitutively, we demonstrate that, during replication in broth, CsrA represses every post-exponential phase phenotype examined, including cell shape shortening, motility, pigmentation, stress resistance, sodium sensitivity, cytotoxicity and efficient macrophage infection. At the transition to the post-exponential phase, LetA/S relieves CsrA repression to induce transmission phenotypes by both FliA-dependent and -independent pathways. For L. pneumophila to avoid lysosomal degradation in macrophages, CsrA repression must be relieved by LetA/S before phagocytosis; conversely, before intracellular bacteria can replicate, CsrA repression must be restored. The reciprocal regulation of replication and transmission exemplified by CsrA likely enhances the fitness of microbes faced with fluctuating environments.

  4. Identification and functional characterization of Bet protein as a negative regulator of BFV3026 replication.

    PubMed

    Bing, Tiejun; Wu, Kai; Cui, Xiaoxu; Shao, Peng; Zhang, Qicheng; Bai, Xiaobo; Tan, Juan; Qiao, Wentao

    2014-06-01

    Foamy virus (FV) establishes persistent infection in the host without causing apparent disease. Besides the transactivator Tas protein, another auxiliary protein--Bet--has been reported in prototype foamy virus, equine foamy virus, and feline foamy virus. Here, we found the putative bbet gene in clone C74 from a cDNA library of bovine foamy virus strain 3026 (BFV3026) by comparison of gene localization, composition, and splicing features with other known bet genes. Subsequently, BBet protein was detected in BFV3026-infected cells by Western blot and immunofluorescence analyses. Analysis of the BBet mutant infectious clone (pBS-BFVdelBBet) revealed that BBet could inhibit BFV3026 replication. Consistent with this result, overexpression of BBet in Cf2Th cells reduced BFV replication by approximately threefold. Furthermore, virus replication levels similarly were reduced by approximately threefold in pBS-BFV-transfected and BFV3026-infected Cf2Th cells stably expressing BBet compared with control cells. After three passages, BFV3026 replicated more slowly in BBet-expressing cells. This study implicates BBet as a negative regulator of BFV replication and provides a resource for future studies on the function of this protein in the virus lifecycle.

  5. Enhanced replication of herpes simplex virus type 1 in human cells

    SciTech Connect

    Miller, C.S.; Smith, K.O. )

    1991-02-01

    The effects of DNA-damaging agents on the replication of herpes simplex virus type 1 (HSV-1) were assessed in vitro. Monolayers of human lung fibroblast cell lines were exposed to DNA-damaging agents (methyl methanesulfonate (MMS), methyl met